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  • 1
    ISSN: 1436-6215
    Keywords: Strontium ; oraler Strontium-Test ; Calcium ; Absorption ; gesunde Probanden ; Strontium ; oral strontium test ; calcium ; absorption ; healthy volunteers
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Medicine
    Description / Table of Contents: Summary Intestinal strontium absorption has been discussed recently as an indirect measure for calcium uptake. Prerequisite for the clinical use of an oral strontium test is the availability of a reliable procedure including controlled strontium supply, sample pretreatment and analysis as well as the assessment of normal values. In the present study, a group of young females (n=33; 24.0 ± 2.7 y; BMI 21.5 ± 1.9) received an oral dose of 2.27 mmol strontium in a standardized breakfast that contained 0.625 mmol calcium. Before and 220 min after the bolus serum strontium concentrations were determined by means of atomic absorption spectrophotometry (coefficient of variation: within day 4.8 %, n=10; day-to-day 9.5 %, n=8). The error of the method was 2.7 %. Calculation of the fractional strontium absorption rate considered the respective distribution volume (extracellular fluid; either estimated using body weight or determined by means of bioimpedance analysis [BIA]). Average absorption rates were 13.3 ± 3.1 % and, considering BIA measurement 13.6 ± 2.6 %, respectively. Smoking, exercise and, use of oral contraceptives showed no effects. Our oral strontium test is characterized by excellent reliability, easy handling and low costs and, thus, is suitable for routine use.
    Notes: Zusammenfassung Die Erfassung der Strontiumabsorption wird heute als indirektes Verfahren zur Beurteilung der intestinalen Calciumabsorption diskutiert. Voraussetzung für die klinische Anwendung ist ein vertrauenswürdiges Testverfahren inclusive kontrollierter Strontiumgabe, Probenaufarbeitung und -analyse sowie die Erfassung von Normalwerten. Für unsere Studien wurde ein Kollektiv junger Frauen (n=33, 24,0 ± 2,7 Jahre; BMI 21,5 ± 1,9) herangezogen. Die Probandinnen erhielten eine Bolusgabe von 2,27 mmol Strontium zusammen mit einem Standardfrühstück (ca. 0,625 mmol Calcium). Vor und 220 min nach der Bolusgabe erfolgte die Bestimmung des Serum-Strontiumgehaltes mittels Atomabsorptionsspektrometrie. Der Variationskoeffizient der Methode lag innerhalb eines Tages bei 4,8 % (n=10) und von Tag zu Tag 9,5 % (n=8). Der Fehler der Methode betrug 2,7 %. Die Berechnung der fraktionellen Strontiumabsorptionsrate erfolgte unter Berücksichtigung des entsprechenden Verteilungsraumes (Extrazellulärflüssigkeit; Schätzverfahren über Körpergewicht bzw. Bioimpedanz-Analyse [BIA]). Die Strontiumabsorptionsrate lag im Mittel bei 13,3 ± 3,1 %, unter Berücksichtigung der BIA-Werte bei 13,6 ± 2,6 %. Rauchen, sportliche Aktivität bzw. Einnahme oraler Kontrazeptiva zeigten keinen Einfluß. Das hier vorgestellte Testverfahren ist aufgrund seiner hohen Vertrauenswürdigkeit und relativ einfacher Handhabung für Routine-untersuchungen geeignet.
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  • 2
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    Cellular and molecular life sciences 46 (1990), S. 962-970 
    ISSN: 1420-9071
    Keywords: Acid deposition ; reproduction ; birds ; insects ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Acidification in aquatic habitats reduces the reproductive success of both piscivorous and non piscivorous birds, mainly by reducing the food supply. Piscivorous birds find some compensation in an increased transparency of the water, non piscivorous birds in less competition for invertebrate prey by fish. Acidification in forests often has large impacts on insect populations but how this affects forest birds is unknown. Some woodpeckers and nuthatches temporarily benefit from an increase in standing dead timber. In advanced stages of forest dieback the breeding density of forest birds is very much reduced, but species of open woodland increase. Calcium deficiency reduces the reproductive output of some passerine species, but the extent of this phenomenon is unknown. Increased exposure to toxic metals has reduced the reproductive success of some lake dwelling species. It is difficult to assess the effect of acid precipitation on birds since acidification affects ecosystems in many ways, the evidence is largely correlative and reliable estimates of the population size are often lacking. Future studies should concentrate on carefully selected indicator species suitable for detailed data collection.
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  • 3
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    Cellular and molecular life sciences 46 (1990), S. 1063-1065 
    ISSN: 1420-9071
    Keywords: Dictyostelium ; cell communication ; biological rhythms ; oscillations ; cAMP ; folate ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary With a light-scattering technique, two novel rhythms were discovered in cell suspensions ofDictyostelium discoideum. One is a damped oscillation with a period of 2 to 2.5 min (at 23°C) induced by folate in EDTA-dissociated undifferentiated cells. The other is a sinusoidal oscillation with a period of about 12 min occasionally observed with late differentiated cells. Obviously, the repertoire of rhythms of this simple eukaryotic organism is larger than previously assumed.
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  • 4
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    Cellular and molecular life sciences 46 (1990), S. 41-48 
    ISSN: 1420-9071
    Keywords: Cardiac muscle ; cell damage ; calcium ; calcium-paradox ; oxygen-paradox ; oxygen radicals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The O2− and Ca2+-paradoxes have a number of features in common and it is suggested that release of cytosolic proteins in both paradoxes is initiated by the activation of a sarcolemma NAD(P)H dehydrogenase which can generate a transmembrane flow of H+ and e− and also oxygen radicals or recox cycling which damage ion channels and membrane proteins (phase I). Entry of Ca2+ through the damaged ion channels then exacerbates the damage by further activating this system, either directly or indirectly, and the redox cycling and/or oxygen radicals cause further damage to integral and cytoskeletal proteins of the sarcolemma resulting in microdamage to the integrity of the membrane (phase II) and the consequent release or exocytosis of cytoplasmic proteins and, under specialised condition, the blebbing of the sarcolemma. The system may be primed either by removal of extracellular Ca2+ or by raising [Ca2+]i by a variety of measures, these two actions being synergistic. The system is initially activated in the Ca2+-paradox by the membrane perturbation associated with removal of extracellular Ca2+; prolonged anoxia in the metabolically active cardiac muscle causes a depletion of the ATP supply, particularly in the absence of glucose, and hence a rise in [Ca2+]i in phase I of the oxygen paradox with the consequent activation of the NAD(P)H oxidase at the sarcolemma. Oxygen radicals are probably generated in both paradoxes and may have a partial role in the genesis of damage, but are not essential in the Ca2+-paradox which continues under anoxia. Massive entry of Ca2+ also activates an intracellularly localised dehydrogenase (probably at the SR) which produces myofilament damage by redox cycling.
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  • 5
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    Cellular and molecular life sciences 46 (1990), S. 26-40 
    ISSN: 1420-9071
    Keywords: Opiomelanocortin ; cyclic adenosine 3′,5′-monophosphate (cAMP) ; calcium ; phosphatidylinositol (PI) ; glucocorticoids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Adrenocorticotropin (ACTH), an opiomelanocortin peptide, is secreted from anterior pituitary corticotrophs upon stimulation with corticotropin-releasing hormone (CRH), arginine vasopressin (AVP) and several other neuropeptides. CRH, the most potent secretagogue of ACTH, stimulates ACTH secretion and biosynthesis by increasing the production of cyclic adenosine 3′,5′-monophosphate (cAMP) within corticotrophs. AVP, which is a weak secretagogue of ACTH but strongly potentiates CRH-stimulated ACTH secretion, operates through the phosphatidylinositol (PI) transduction pathway. Both CRH and AVP increase cytosolic free [Ca2+] within normal corticotrophs indicating a role for Ca2+ in ACTH secretion. Glucocorticoids inhibit ACTH synthesis by suppressing transcription of the proopiomelanocortin (POMC) gene and attenuate ACTH release by decreasing cAMP accumulation stimulated by CRH. This review focuses on the roles of these intracellular messengers in ACTH secretion from normal anterior pituitary cells in vitro, and discusses the possible interactions between the cAMP, calcium and PI transduction pathways. Future areas of research are suggested such as identification of protein substrates of cAMP-dependent and Ca2+-dependent kinases within normal corticotrophs and evaluation of their role in ACTH biosynthesis and secretion.
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  • 6
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    Cellular and molecular life sciences 47 (1991), S. 224-226 
    ISSN: 1420-9071
    Keywords: aluminium ; snail ; shell-repair ; calcium ; phosphate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary In AlCl3-injected shell-repairing snails,Helix pomatia L., the Al-associated decrease of the weights of the shell-repair membranes was unrelated to the Al-concentration in the membranes. In the haemolymph the concentration of Al was related to the dose of injected Al, while the concentration of Ca was increased by the highest Al-dose only. No phosphate was detected in either controls or Al-injected snails. It is concluded that Al inhibits the growth of the CaCO3-crystals by mechanisms other than incorporation in, or adsorption to, the crystals.
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  • 7
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    Cellular and molecular life sciences 46 (1990), S. 1016-1017 
    ISSN: 1420-9071
    Keywords: In vitro absorption ; calcium ; wheat ; Bengal gram
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The in vitro absorption of calcium from the duodenum was significantly less in a group of rats fed on a wheat diet than in a group fed a wheat and Bengal gram (70∶30) diet.
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  • 8
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    Cellular and molecular life sciences 46 (1990), S. 1175-1179 
    ISSN: 1420-9071
    Keywords: Prostacyclin ; EDRF ; $$P_{O_2 } $$ ; calcium ; vascular oxygen sensitivity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Higher developed organisms are equipped with many central and local control mechanisms, which enable an adequate blood and oxygen supply to tissues over a wide range of demands. Global adaptive responses include changes in the circulatory and ventilatory system as well as increases in the oxygen carrying capacity of the blood. At the level of the specialized organs there exist additional control systems for the regulation of local blood flow. Most systems make use of highly specialized cells which are able to sense the oxygen partial pressure of the transport medium, blood, and within the tissues. In the past years, it has been shown that the vascular endothelium lining the entire circulatory system can actively modulate the vascular tone and platelet functions by the release of autacoids, among them prostacyclin and endothelium-derived nitric oxide (EDRF). Recent experiments demonstrate that the release of EDRF is $$P_{O_2 } $$ -dependent, which suggests that endothelial cells may act as functional local oxygen sensors within the vascular system.
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  • 9
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    Cellular and molecular life sciences 47 (1991), S. 1201-1208 
    ISSN: 1420-9071
    Keywords: Pancreatic islet ; β-cell ; calcium ; protein kinase C down-regulation ; sensitization ; desensitization ; insulin secretion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion was studied. A 22–24h exposure of mouse pancreatic islets to glucose (16.7 mmol/l) in TCM 199 culture medium, with 0.26 mmol/l or 1.26 mmol/l Ca2+, reduced total islet protein kinase C activity to approx. 85% and 60% of control values, respectively. At 0.26 mmol/l Ca2+ in TCM 199 medium, exposure to glucose (16.7 mmol/l) led to a potentiation of both phase 1 and phase 2 of glucose-induced insulin secretion, and caused a shift in the dose-response curve with 10 mmol/l and 16.7 mmol/l glucose exhibiting equipotent effects in stimulation of insulin secretion. In glucose-sensitized islets, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (0.16 μmol/l) did not further potentiate induction of secretion by 10 mmol/l or 16.7 mmol/l glucose. At 3.3 mmol/l glucose, however, phorbol ester-induced secretion was augmented, and was characterized by a faster onset of secretion in glucose-sensitized islets relative to control islets. In contrast, a partial reduction in arachidonic acid (100 μmol/l)-induced insulin release was observed in glucose-sensitized islets in the absence of extracellular Ca2+. Increasing the Ca2+ concentration to 1.26 mmol/l in TCM 199 during the 22–24h exposure to glucose (16.8 mmol/l) led to inhibition of phase 1 and abolition of phase 2 of glucose (10 mmol/l, 16.7 mmol/l)-induced insulin secretion. In addition, this treatment abolished phorbol ester-induced and arachidonic acid-induced insulin secretion at 3.3 mmol/l glucose. Altogether, these data suggest that sensitization of insulin secretion is caused by a preferential down-regulation of the inhibitory effects of protein kinase C, leading to an increased first phase, and an increased coupling of glucose to the stimulatory effects of protein kinase C during the second phase of glucose-induced insulin secretion. Desensitization of insulin secretion appears to be a consequence of sustained Ca2+ influx, inducing extensive down-regulation of protein kinase C and also causing deleterious effects on islet cell function in protein kinase C-deprived islets.
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  • 10
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    Cellular and molecular life sciences 49 (1993), S. 865-869 
    ISSN: 1420-9071
    Keywords: Zinc ions ; calcium ; ileal muscle
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Preincubation with 0.3 mM Zn2+ markedly inhibited both the tonic response and Ca2+ binding at low affinity sites induced by K+ (60 mM), with smaller effects on the phasic response and the high affinity Ca2+ sites, inTaenia coli. However, when the muscle was kept in Zn2+-containing medium following the first stimulation with the K+, the phasic response and the high affinity Ca2+ sites were more severely inhibited during the second stimulation with K+. This probably indicates that Zn2+ reduced the tonic tension response to K+ mainly by inhibiting Ca2+ influx at the cell membranes ofTaenia coli. However, when Zn2+ is continuously present, Ca2+ is not supplied at the storage sites and is not available for the phasic response to a second stimulation with K+.
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  • 11
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    Cellular and molecular life sciences 49 (1993), S. 1064-1072 
    ISSN: 1420-9071
    Keywords: Aminooxyacetic acid ; 1-methyl-4-phenylpyridinium ion ; malonic acid ; 3-nitropropionic acid ; rotenone ; sodium azide ; nitric oxide ; N-methyl-D-aspartate ; oxidative phosphorylation ; calcium ; cell death
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract It is thought that impairment, of energy metabolism that results in deterioration of membrane function, leading to loss of the Mg2+ block on NMDA receptors, and allowing persistent activation of these receptors by glutamate, might be a cause of neuronal death in neurodegenerative disorders. Studies in rodents using mitochondrial respiratory chain toxins, such as aminooxyacetic acid, 1-methyl-4-phenylpyridinium, malonic acid and 3-nitropropionic acid, suggest that such processes may indeed be involved in neurotoxicity. Striatal and nigral degeneration induced by mitochondrial toxins in rodents resembles the neuropathology seen in humans suffering from Huntington's or Parkinson's disease, and can be prevented either by decortication or by NMDA receptor antagonists. Such experimental observations suggest that glutamate may be involved in neuronal death leading to neurodegenerative disorders in humans. If so, glutamate antagonists may offer a therapeutic approach for retarding the progression of these disabling disorders.
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  • 12
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    Cellular and molecular life sciences 48 (1992), S. 516-519 
    ISSN: 1420-9071
    Keywords: Lipid ; neuroepithelial cells ; calcium ; neurulation ; neural tube ; chick embryo ; mouse embryo
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract In situ precipitation of calcium (Ca2+) with fluoride and antimonate shows that Ca2+-specific precipitate is localized almost exclusively within lipid droplets of neuroepithelial cells during neural tube formation in chick and mouse embryos. The density of Ca2+ precipitate within lipid droplets is generally greater in the apical ends of cells situated in regions of the neuroepithelium that are actively engaged in bending. These findings suggest that lipid droplets, in addition to providing a source of metabolic fuel for developing neuroepithelial cells, also serve as Ca2+-storage and-releasing sites during neurulation.
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  • 13
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    Cellular and molecular life sciences 52 (1996), S. 831-833 
    ISSN: 1420-9071
    Keywords: Paramecium caudatum ; thigmotaxis ; Ja-value ; CNR ; calcium ; ruthenium red ; LaCl3 ; caffeine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract To understand the role of calcium ions in thigmotaxis inParamecium caudatum, the effects of caffeine, ruthenium red and lanthanum (LaCl3) on thigmotaxis were examined. Thigmotaxis in the CNR mutant, which lacks voltage-dependent Ca2+-channels in the ciliary membrane, was also examined. Ruthenium red and LaCl3 suppressed thigmotaxis inP. caudatum, while caffeine enhanced it. The CNR mutant showed hardly any thigmotaxis. It can be thought that an increase in Ca2+ influx and the intraciliary concentration of Ca2+ ions induces thigmotaxis inParamecium.
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  • 14
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    European journal of clinical pharmacology 41 (1991), S. 11-16 
    ISSN: 1432-1041
    Keywords: Budesonide ; Prednisolone ; calcium ; phosphate ; healthy volunteers ; osteoporosis ; mineral metabolism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Medicine
    Notes: Summary In a randomized, double-blind crossover study, the effects of 0.8, 1.6 and 3.2 mg/day inhaled budesonide and 5, 10 and 20 mg/day oral prednisolone on mineral metabolism were compared. Twelve healthy subjects (4 m, 8 f) were treated for 1 week at each dosage level, the graduated dosages being given in ascending order. Budesonide and prednisolone were given twice daily and once daily, respectively, which reflects the schedules common in clinical practice. Serum calcium and the regulatory hormones of calcium metabolism (parathyroid hormone, vitamin D metabolites and calcitonin) were not changed either by prednisolone or budesonide. Prednisolone significantly increased 24 h and 08.00 h fasting urinary calcium excretion and decreased renal calcium reabsorption, while budesonide had little or no effect on urinary calcium loss and increased renal reabsorption at the highest dose level. Both drugs significantly increased renal phosphate reabsorption and serum phosphate levels, but prednisolone caused greater increases than budesonide. In conclusion, during short-term treatment with the dosages used, inhaled budesonide had less effect on calcium and phosphate metabolism than oral prednisolone, and so it may have a lesser action on the skeleton of the type contributing to osteoporosis during long-term treatment.
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  • 15
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    European journal of clinical pharmacology 16 (1979), S. 211-215 
    ISSN: 1432-1041
    Keywords: fluoride ; bioavailability ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Medicine
    Notes: Summary The effect of milk products on the gastrointestinal absorption of fluoride from sodium fluoride tablets was studied in five healthy subjects. Two different diets were tested: (1) 250 ml standardized milk (3% fat) and (2) 500 ml of milk, 3 pieces of white bread with cheese and 150 ml of yoghurt. The 100% bioavailability of sodium fluoride tablets during fasting was greatly decreased by coadministration of milk products: with Diet 1 the absolute bioavailability calculated from combined plasma and urine data was in the range 50–79% and with Diet 2 it ranged from 50–71%. It is suggested that the decreased bioavailability produced by dairy products should be taken into account when establishing fluoride dosage regimens for prophylaxis of caries.
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  • 16
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    European journal of clinical pharmacology 41 (1991), S. 521-523 
    ISSN: 1432-1041
    Keywords: Verapamil ; Bone metabolism ; calcium ; calcium metabolism ; PTH
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Medicine
    Notes: Summary The effect of the calcium antagonist verapamil on calcium homoeostasis and bone metabolism has been investigated in a double-blind randomized placebo-controlled study. Ten patients randomized to verapamil 120 mg t.i.d. and 9 patients randomized to placebo in The Danish Verapamil Infarction Trial II took part in the study. Bone formation, estimated by 24-h whole body retention of diphosphonate (WBR), osteocalcin, alkaline phosphatase and calcium metabolic indices, was recorded before the start of medication and after 1 and 6 months of treatment. Baseline calcium metabolic variables were not significantly different between the two study groups. There were no significant differences in WBR (0.38 vs 0.37), osteocalcin level (8.2 vs 8.0 μg/l) or alkaline phosphatase (218 vs 200 U/l) after treatment for 6 months with verapamil compared to placebo. Serum PTH, calcium and phosphate levels were also not affected by verapamil. The results suggest that prolonged treatment with clinical doses of verapamil does not affect indices of calcium and bone metabolism in humans.
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  • 17
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    European journal of clinical pharmacology 38 (1990), S. 189-193 
    ISSN: 1432-1041
    Keywords: estramustine phosphate ; prostatic cancer ; gastrointestinal absorption ; food intake ; calcium ; drug interaction ; pharmacokinetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Medicine
    Notes: Summary The effect of milk and food on the pharmacokinetics of estramustine phosphate was investigated in six patients with prostatic cancer. In a randomized three-way cross-over study, the patients were given single doses of the drug together with low calcium water, low calcium food and milk. The evaluation was based upon the plasma concentration of two metabolites, estromustine and estrone, as parent drug could not be detected in plasma. The tmax and lag time of estromustine were significantly increased by milk and food intake and Cmax and AUC were significantly decreased. In comparison with water, the AUC of estromustine was 41% when the drug was taken with milk and 67% after simultaneous intake of standardized food. Corresponding figures for the peak values were 32 and 57%, respectively. The effect of milk and food intake on the pharmacokinetics of estrone was similar. Studies in vitro demonstrated that the dissolution of estramustine phosphate disodium was markedly impaired in the presence of calcium. It was concluded that the rate and extent of absorption of estramustine phosphate were decreased when the drug was taken with milk or food due to the formation of a poorly absorbable calcium complex. To obtain high and reproducible absorption of Estracyt®, the drug should not be taken together with milk, milk products or other calcium-rich food or drugs.
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  • 18
    ISSN: 1573-4919
    Keywords: mucosa ; membranes ; smooth muscle ; calcium ; adenosine triphosphatase ; arteries ; myocardium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Heart tissue contains large amounts of the protein encoded by the Ca2+ pump gene SERCA2. The SERCA2 RNA can be spliced alternatively to produce mRNA encoding the proteins SERCA2a and SERCA2b which differ in their C-terminal sequences. In this study we report the tissue distribution of SERCA2a and SERCA2b isoforms byin situ hybridization to rabbit heart and stomach. The expression of SERCA2 mRNA was high in myocardial cells, being the highest in the atrial region. In contrast, there was more SERCA2 protein in Western blots in ventricles than in atria. Myocardial cells expressed predominantly the mRNA for the isoform SERCA2a. Whereas the stomach smooth muscle and the neuronal plexus expressed SERCA2 at levels much lower than myocardial cells, the expression was very high in the stomach mucosa. Mucosa contained mainly the mRNA for SERCA2b. From immunocytochemistry it was concluded that the anti-heart SR Ca2+ pump antibody IID8 reacted much better with heart and surface mucosal cells in the stomach than with the stomach smooth muscle, and that IID8 reactivity was intracellular. In contrast PM4A2B, an antibody against the plasma membrane Ca2+ pump, reacted well with heart and stomach smooth muscle, plexus and mucosa, and its localization appeared to be in the plasma membrane. Thus, stomach smooth muscle expressed SERCA2b mRNA and protein at low levels, mucosa expressed SERCA2b mRNA and protein at high levels, atria and ventricle expressed SERCA2a mRNA and protein at high levels, mRNA being more in atria, but protein being more in ventricles.
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  • 19
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    Molecular and cellular biochemistry 127-128 (1993), S. 229-237 
    ISSN: 1573-4919
    Keywords: calcium ; contraction ; smooth muscle ; myosin ; protein phosphorylation ; second messengers
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Phosphorylation of the regulatory light chain of myosin by the Ca2+/calmodulin-dependent myosin light chain kinase plays an important role in smooth muscle contraction, nonmuscle cell shape changes, platelet contraction, secretion, and other cellular processes. Smooth muscle myosin light chain kinase is also phosphorylated, and recent results from experiments designed to satisfy the criteria of Krebs and Beavo for establishing the physiological significance of enzyme phosphorylation have provided insights into the cellular regulation and function of this phosphorylation in smooth muscle. The multifunctional Ca2+/calmodulin-dependent protein kinase II phosphorylates myosin light chain kinase at a regulatory site near the calmodulin-binding domain. This phosphorylation increases the concentration of Ca2+/calmodulin required for activation and hence increases the Ca2+ concentrations required for myosin light chain kinase activity in cells. However, the concentration of cytosolic Ca2+ required to effect myosin light chain kinase phosphorylation is greater than that required for myosin light chain phosphorylation. Phosphorylation of myosin light chain kinase is only one of a number of mechanisms used by the cell to down regulate the Ca2+ signal in smooth muscle. Since both smooth and nonmuscle cells express the same form of myosin light chain kinase, this phosphorylation may play a regulatory role in cellular processes that are dependent on myosin light chain phosphorylation.
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  • 20
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    Molecular and cellular biochemistry 136 (1994), S. 11-22 
    ISSN: 1573-4919
    Keywords: magnesium ; calcium ; mag-fura-2 ; fura-2 ; magnesium regulation ; BC3H-1 cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The magnesium buffer coefficient (B Mg) was calculated for BC3H-1 cells from the rise in cytosolic Mg2+ activity observed when magnesium was released from ATP after iodoacetate (IAA) and NaCN treatment. The basal cytosolic Mg2+ activity (0.54±0.1 mM) measured with mag-fura-2 doubled when 4.54 mM magnesium was liberated from ATP:B Mg was 12.9 indicating that a 1 mM increase in Mg2+ activity requires an addition of about 13 mM magnesium. The accuracy of this value depends on these assumptions: (a) all of the magnesium released from ATP stayed in the cells; (b) the rise in Mg2+ was not secondary to pH-induced changes inB Mg; (c) mag-fura-2 measured Mg2+ and not Ca2+; and (d) the accuracy of the mag-fura-2 calibration. Total magnesium did not change in response to IAA/CN treatment, thus the change in Mg2+ activity reflected a redistribution of cell magnesium. pH changes induced by NH4Cl pulse and removal had little effect on Mg2+ activity and the changes were slower than and opposite to pH-induced changes in Ca2+ activity measured by fura-2. Ca2+ responses were temporally uncopled from Mg2+ responses when the cells were treated with IAA only and in no cases did Ca2+ levels rise above 1 μM, showing that the mag-fura-2 is responding to Mg2+. Additional studies demonstrated that ∼90% of the mag-fura-2 signal was cytosolic in origin. The remaining non-diffusible mag-fura-2 either was bound to cytosolic membranes or sequestered in organelles with the fluorescence characteristics of the Mg2+-complexed form, even when cytosolic free Mg2+ activity was approximately 0.5 mM. This bound mag-fura-2 would appear to increase the Kd and thus clearly limits the accuracy of our estimmate forB Mg. Despite this limitation, we demonstrate that Mg2+ is tightly regulated in face of large changes in extracellular Mg2+, and that the interplay observed between pH, Ca2+ and Mg2+ activities strongly supports the hypothesis that these factors interact through a shared buffer capacity of the cell.
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  • 21
    ISSN: 1573-4919
    Keywords: regucalcin ; calcium-binding protein ; insulin ; calcium ; gene expression ; rat liver
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of refeeding on the expression of Ca2+-binding protein regucalcin mRNA in the liver of fasted rats was investigated. When rats were fasted overnight, the hepatic regucalcin mRNA level was reduced about 70% of that in feeding rats. Refeeding produced a remarkable elevation of hepatic regucalcin mRNA level (about 150–170% of fasted rats). Liver regucalcin concentration was appreciably increased by refeeding, although it was not altered by fasting. The oral administration of glucose (2 g/kg body weight) to fasted rats caused a significant increase in hepatic regucalcin mRNA level. Moreover, hepatic regucalcin mRNA level was clearly elevated by a single subcutaneous administration of insulin (10 and 100 U/kg) to fasted rats. The hormonal effect was not further enhanced by the simultaneous administration of calcium chloride (250 mg Ca/kg) to fasted rats, although calcium administration stimulated regucalcin mRNA expression in the liver. The present study suggests that the expression of hepatic regucalcin mRNA stimulated by refeeding is significantly involved in the action of insulin and/or calcium as stimulating factors.
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  • 22
    ISSN: 1573-4919
    Keywords: calcium ; calcium channels ; smooth muscle ; sarcolemma ; coronary artery
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    Notes: Abstract Tension generation and Ca2+ flux in smooth muscle varies depending upon the diameter of a vessel and its location. The purpose of the present investigation was to determine if the biochemical characteristics of the Na+−Ca2+ exchanger and the Ca2+ channel differ in sarcolemmal membrane preparations isolated from a large conduit vessel (thoracic aorta) or from large and small coronary arteries. We also investigated the possibility of differences between sarcolemmal membranes isolated from coronary arteries dissected from the right and left ventricles. The purification of the sarcolemmal membranes was of a similar magnitude amongst the different groups. Contamination of the sarcolemmal membranes with other membranous organelles was negligible and similar amongst the groups. The Km and Vmax of Na+-dependent Ca2+ uptake in sarcolemmal vesicles was similar amongst the groups. Calcium channel characteristics were examined by measuring [3H]PN200-110 binding to sarcolemmal vesicles. The right coronary artery membranes from both large and small caliber vessels exhibited a higher Kd and the small right coronary artery sarcolemmal preparation had a lower maximal binding density for [3H] PN200-110. The results suggest that the right coronary artery, and in particular the small diameter right coronary artery, possesses altered Ca2+ channel characteristics in isolated sarcolemmal membranes.
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    Molecular and cellular biochemistry 154 (1996), S. 123-132 
    ISSN: 1573-4919
    Keywords: rat pancreas ; cholecystokinin-octapeptide ; magnesium ; calcium ; secretion ; cyclic AMP
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    Notes: Abstract This study investigates the effect of magnesium (Mg2+) on the secretory responses and the mobilization of calcium (Ca2+) and Mg2+ evoked by cholecystokinin-octapeptide (CCK-8) in the exocrine rat pancreas. In the isolated intact perfused pancreas CCK-8 (10−10 M) produced marked increases in juice flow and total protein output in zero and normal (1.1 mM) extracellular Mg2+ [Mg2+]o compared to a much reduced secretory response in elevated (5 mM and 10 mM) [Mg2+]o Similar effects of perturbation of [Mg2+]o on amylase secretion and 45Ca2+ uptake (influx) were obtained in isolated pancreatic segments. In pancreatic acinar cells loaded with the fluorescent bioprobe fura-2 acetomethylester (AM), CCK-8 evoked marked increases in cytosolic free Ca2+ concentration [Ca2+]i in zero and normal [Mg2+]o compared to a much reduced response in elevated [Mg2+]o Pretreatment of acinar cells with either dibutyryl cyclic AMP (DB2 cAMP) or forskolin had no effect on the CCK-8 induced changes in [Ca2+]i. In magfura-2-loaded acinar cells CCK-8 (10−8 M) stimulated an initial transient rise in intracellular free Mg2+ concentration [Mg2+]i followed by a more prolonged and sustained decrease. This response was abolished when sodium Na+ was replaced with N-methyl-D-glucamine (NMDG). Incubation of acinar cells with 10 mM Mg2+ resulted in an elevation in [Mg2+]i. Upon stimulation with CCK-8, [Mg2+]i. decreased only slightly compared with the response obtained in normal [Mg2+]o. CCK-8 caused a net efflux of Mg2+ in pancreatic segments; this effect was abolished when extracellular sodium [Na+]o was replaced with either NMDG or choline. The results indicate that Mg2+ can regulate CCK-8-evoked secretory responses in the exocrine pancreas possibly via Ca2+ mobilization. Moreover, the movement of Mg2+ in pancreatic acinar cells is dependent upon extracellular Na+.
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    Molecular and cellular biochemistry 146 (1995), S. 179-186 
    ISSN: 1573-4919
    Keywords: Ca2+-ATPase ; calcium ; nuclear DNA ; DNA fragmentation ; regucalcin ; regenerating rat liver
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    Notes: Abstract The alteration of calcium content, Ca2+-ATPase activity, DNA content and DNA fragmentation in the nuclei of regenerating rat liver was investigated. Liver was surgically removed about 70% of that of sham-operated rats. the reduced liver weight by partial hepatectomy was completely restored at 3 days after the surgery. Regenerating liver significantly increased Ca2+-ATPase activity and DNA content in the nuclei between 1 and 5 days after hepatectomy. The nuclear calcium content was clearly increased from 2 days after hepatectomy. The increase of Ca2+-ATPase activity in regenerating liver was clearly inhibited by the presence of trifluoperazine (10 μM), staurosporine (2.5 μM) and dibucaine (10 μM), which are inhibitors of calmodulin and protein kinase, in the enzyme reaction mixture. However, the nuclear enzyme activity in normal rat liver was not significantly altered by these inhibitors. Meanwhile, the increase of nuclear DNA content in regenerating liver was completely blocked by the administration of trifluoperazine (2.5 mg/100 g body weight), suggesting an involvement of calmodulin. Now, the nuclear DNA fragmentation was significantly decreased in regenerating liver, suggesting that this decrease is partly contributed to the increase in nuclear DNA content. The present study clearly demonstrates that regenerating liver enhances nuclear Ca2+-ATPase activity and induces a corresponding elevation of nuclear calcium content. This Ca2+-signaling system may be involved in the regulation of nuclear DNA functions in regenerating rat liver.
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  • 25
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    Molecular and cellular biochemistry 148 (1995), S. 67-72 
    ISSN: 1573-4919
    Keywords: regucalcin ; calcium ; protease ; calpain ; rat liver cytosol
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The increasing effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was characterized. The proteolytic activity was markedly elevated by the addition of regucalcin (0.1–0.5 μM) in the absence of Ca2+. This increase was not significantly altered by the presence of diisopropylfluorophsophate (DPF;2.5 mM)—although DFP caused a significant decrease in the proteolytic activity. Regucalcin (0.25 μM) additively enhanced the dithiothreitol (DTT; 1.0 mM)—increased proteolytic activity, while the regucalcin or DTT effect was completely abolished by NEM (5 mM), indicating that regucalcin may act on the SH group in proteases. Also, regucalcin (0.25 μM) enhanced the effect of Ca2+ (10 μM) increasing liver proteolytic activity, suggesting that regucalcin does not influence on the active sites for Ca2+ in proteases. Moreover, the proteolytic activity of regucalcin (0.25 μM) was significantly decreased by the presence of calpastatin (24 μg/ml), an inhibitor of Ca2+-activated neutral protease (calpain). Now, regucalcin (0.25 μM) increased about 7-fold the activity ofm-calpain isolated from rabbit skeletal muscle. These observations demonstrate that regucalcin directly activates cysteinyl-proteases. Regucalcin may have a role as a potent proteolytic activator in the cytoplasm of liver cells.
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  • 26
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    Molecular and cellular biochemistry 149-150 (1995), S. 203-212 
    ISSN: 1573-4919
    Keywords: calcium ; mitochondria ; FAD-glycerol 3-phosphate dehydrogenase ; pyruvate dehydrogenase ; oxoglutarate dehydrogenase ; isocitrate dehydrogenase
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    Notes: Abstract In mammalian cells, increases in calcium concentration cause increases in oxidative phosphorylation. This effect is mediated by the activation of four mitochondrial dehydrogenases by calcium ions; FAD-glycerol 3-phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol 3-phosphate dehydrogenase, being located on the outer surface of the inner mitochondrial membrane, is exposed to fluctuations in cytoplasmic calcium concentration. The other three enzymes are located within the mitochondrial matrix. While the kinetic properties of all of these enzymes are well characterised, the molecular basis for their regulation by calcium is not. This review uses information derived from calcium binding studies, analysis of conserved calcium binding motifs and comparison of amino acid sequences from calcium sensitive and non-sensitive enzymes to discuss how the recent cloning of several subunits from the four dehydrogenases enhances our understanding of the ways in which these enzymes bind calcium. FAD-glycerol 3-phosphate dehydrogenase binds calcium ions through a domain which is part of the polypeptide chain of the enzyme. In contrast, it is possible that the calcium sensitivity of the other dehydrogenases may involve separate calcium binding subunits.
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    Molecular and cellular biochemistry 158 (1979), S. 91-95 
    ISSN: 1573-4919
    Keywords: smooth muscle ; vascular ; ATPase ; calcium
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    Notes: Abstract Densities of sarcoplasmic reticulum (SR) Ca2+-pump were compared in proximal and distal segments of pig left coronary artery using two biochemical methods: acylphosphate formation and immunoreactivity in Western blots, and a functional assay based on contraction to SR Ca2+-pump inhibitors. In the microsomes prepared from smooth muscle, the level of the 115 kDa SR Ca2+-pump acylphosphate was 7.1 ± 0.3 -fold greater in distal than in proximal segments. Similarly in Western blots using these microsomes, the reactivity of the 115 kDa band to an anti-SR Ca2+-pump antibody was 5.3 ± 0.8 -fold greater in distal than in proximal segments. Endothelium free coronary artery rings contracted to the SR Ca2+-pump inhibitors Cyclopiazonic acid (CPA, EC50 = 0.19 ± 0.06 μM) and thapsigargin (EC50 = 0.0095 ± 0.0035 μM). With 10 μM CPA, the force of contraction per tissue wet weight was 4.2 ± 0.5-fold greater in distal than in proximal rings, and with 1 μM thapsigargin it was 4.0 ± 1.0 -fold greater. The contractions produced by 60 mM KCl were used as a control. In contrast to the CPA and thapsigargin, the force per mg tissue weight produced by 60 mM KCl did not differ significantly between the proximal and distal segments. Thus, the results from the two biochemical methods and those from the contractility data were all consistent with the smooth muscle in the distal segments of the coronary artery containing a higher density of the SR Ca2+-pump than the proximal segments.
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    Molecular and cellular biochemistry 179 (1998), S. 99-110 
    ISSN: 1573-4919
    Keywords: diaphragm ; oxygen-derived free radicals ; respiratory muscle fatigue ; nitric oxide ; sarcoplasmic reticulum ; calcium
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    Notes: Abstract It is now recognized that respiratory muscle fatigue contributes to the development of respiratory failure in some patients with lung disease. This observation has prompted an examination into the mechanisms of development of muscle fatigue, with the understanding that an elucidation of these processes may lead to new therapeutic approaches to the treatment of these patients. A series of recent studies examining this issue have, moreover, discovered that oxygen-derived free radicals generated during strenuous contraction may modulate respiratory muscle contractile function and contribute to the development of muscle fatigue. The data supporting this concept include: (a) direct (e.g. EPR, ESR studies) and indirect (evidence of lipid peroxidation, protein carbonyl formation, glutathione oxidation) evidence that there is heightened free radical production in contracting muscle, (b) evidence that pharmacologic depletion of muscle antioxidant stores increases degree of muscle fatigue present after a period of exercise, and (c) evidence that administration of agents that act as free radical scavengers retard the development muscle fatigue. Free radicals may produce these changes in muscle force generating capacity by interacting with and altering the function of a number of intracellular-biophysical processes (i.e. sarcolemmal action potential propagation, sarcoplasmic reticulum calcium handling, mitochondrial function, contractile protein interactions).
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  • 29
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    Keywords: heart cells ; taurine ; β-alanine ; taurine-Na+ cotransport ; CBDMB ; Na+-Ca2+ exchanger ; calcium ; nucleus ; confocal
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    Notes: Abstract Recent studies in heart cells have shown taurine to induce a sustained increase of both intracellular Ca2+ and Na+. These results led us to believe that the increase in Na+ by taurine could be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through Na+-Ca2+ exchange. Therefore, we investigated the effect of β-alanine, a blocker of the taurine-Na+ cotransporter and low concentrations of CBDMB (a pyrazine derivative, 5-(N-4chlorobenzyl)-2′,4′-dimethylbenzamil), a Na+-Ca2+ exchanger blocker on taurine-induced [Ca]i increase in embryonic chick heart cells. Using Fura-2 Ca2+ imaging and Fluo-3 Ca2+ confocal microscopy techniques, taurine (20 mM) as expected, induced a sustained increase in [Ca]i at both the cytosolic and the nuclear levels. Preexposure to 500 μM of the blocker of the taurine-Na+ cotransporter, β-alanine, prevented the amino acid-induced increase of total [Ca]i. On the other hand, application of β-alanine did not reverse the action of taurine on total [Ca]i. However, low concentrations of the Na+-Ca2+ exchanger blocker, CBDMB, reversed the taurine-induced sustained increase of cytosolic and nuclear free calcium (in presence or absence of β-alanine). Thus, the effect of taurine on [Ca]i in heart cells appears to be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through the Na+-Ca2+ exchanger.
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    Molecular and cellular biochemistry 190 (1999), S. 185-190 
    ISSN: 1573-4919
    Keywords: calcium ; calcium wave ; calcium oscillation ; inositol 1,4,5-trisphosphate receptor ; ryanodine receptor ; excitation-concentration coupling
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    Notes: Abstract After the seminal work of Ebashi and coworkers which established the essential role of the intracellular Ca2+ concentration ([Ca2+]i) in the regulation of skeletal muscle contraction, we have witnessed an explosive elongation of the list of cell functions that are controlled by the [Ca2+]i. In numerous instances, release of intracellular Ca2+ stores plays important roles in Ca2+ signalling which displays significant variation in spatio-temporal pattern. There are two families of Ca2+ release channels, ryanodine receptors and inositol 1,4,5-trisphosphate (IP3) receptors. These Ca2+ release channels are structurally and functionally similar. In particular, the activity of both types of channels is regulated by the [Ca2+]i. The [Ca2+]i dependence of the Ca2+ release channel activity provides both types of channels with properties of a Ca2+ signal amplifier. This function of the ryanodine receptor is important in striated muscle excitation-contraction coupling, whereas that of the IP3 receptor seems to be the basis of the generation of Ca2+ waves. Thus the wide variety of Ca2+ signalling patterns seem to be critically dependent on the [Ca2+]i dependence of the Ca2+ release channels.
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    Molecular and cellular biochemistry 190 (1999), S. 39-45 
    ISSN: 1573-4919
    Keywords: microcalorimetry ; calcium ; troponin C ; calmodulin ; parvalbumin
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    Notes: Abstract Results of microcalorimetric titrations of calcium-binding proteins with calcium or magnesium have been reviewed and evaluated. Results were analyzed mostly in terms of heat capacity changes, which is most closely related to the structural changes of the molecule on metal binding. Two high-affinity sites of rabbit skeletal troponin C are distinguishable in terms of their affinity to calcium and associated enthalpy changes. Heat capacity changes on calcium binding to one of the two high-affinity sites is negative and is in the range ascribed to the ligand binding. In contrast, that to the other of the high-affinity sites is large and positive, indicating that a substantial area of hydrophobic groups become exposed to the solvent. In frog skeletal troponin C, the anomalous positive heat capacity changes occur in one of the low-affinity calcium-specific sites, so that this may be involved in the regulation of contraction. Unlike skeletal troponin C, both of the two high-affinity sites of cardiac troponin C show negative heat capacity changes. In calmodulin, heat capacity changes are positive but small, indicating that calcium binding may induce clustering of the hydrophobic residues on the surface of the molecule. In parvalbumins, heat capacity changes are negative, characteristic of most ligand binding.
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    Molecular and cellular biochemistry 99 (1990), S. 25-32 
    ISSN: 1573-4919
    Keywords: calcium ; regucalcin ; protein synthesis ; aminoacyl-tRNA synthetase ; rat liver
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, onin vitro protein synthesis in the 5500g supernatant fraction of rat liver homogenate was investigated. Addition of Ca2+ up to 5.0 μM in the reaction mixture caused a significant decrease in protein synthesis. This decrease was saturated at 10 μM Ca2+. The Ca2+ effect was not reversed by the presence of regucalcin (2.0 μM); the protein caused a remarkable decrease in hepatic protein synthesis, and it enhanced significantly the Ca2− effect. Meanwhile, calmodulin (2.5-20 μg/ml), a calcium-binding protein, did not have an appreciable effect on the Ca2+ (10 μM)-induced decrease in hepatic protein synthesis. [3H]Leucyl-tRNA synthetase activity in the 105000g supernatant fraction (cytosol) of liver homogenate was markedly decreased by addition of Ca2+ (1.0–50 μM). This decrease was not reversed by the presence of regucalcin (2.0 μM); the protein (1.0–2.0 μM) caused a remarkable decrease in the enzyme activity. The present results suggest that regucalcin can regulate protein synthesis in liver cells.
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    Molecular and cellular biochemistry 95 (1990), S. 133-137 
    ISSN: 1573-4919
    Keywords: nifedipine ; neutrophil ; calcium
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Administration of Ca2+ channel blockers in cardiac disorders and the central role of Ca 2+ in modulating neutrophil functions, prompted us to investigate whether administration of nifedipine to mice would alter their natural resistance to infectious agents like Salmonella typhimurium. Neutrophil chemiluminescence (CL) in response to S. typhimurium was significantly (p 〈 0.01) decreased in mice fed with nifedipine (0.015 mg/kg body weight) over a period of six months. Intracellular killing of S. typhimurium by isolated neutrophils also decreased significantly (p 〈 0.01) and exponentially with nifedipine administration, representing a 42% fall at six months. In addition the drug administration lowered the survival rate of animals following challenge by a lethal dose of S. typhimurium (LD50 = 1 × 104 bacteria/animal). Our data suggest that long term administration of nifedipine lowers the natural resistance of mice to S. typhimurium owing to impaired neutrophil functions.
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    Molecular and cellular biochemistry 99 (1990), S. 67-74 
    ISSN: 1573-4919
    Keywords: calcium ; lanthanide ; Europium ; sarcoplasmic reticulum ; ATP ; calcium occlusion ; tryptic digestion ; calcium-uptake
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Europium luminescence from europium bound to sarcoplasmic reticulum (Ca2+ Mg2+)-ATPase indicates that there are two high affinity calcium binding sites. Furthermore, the two calcium ions at the binding sites are highly coordinated by the protein as the number of H2O molecules surrounding the Ca2+ ions are 3 and 0.5. In the presence of ATP, calcium ions are occluded even further down to 2 and zero H2O molecules, respectively. The Ca2+ - Ca2+ intersite distance is estimated to be 8–9 Å and the average distance from the Ca2+ sites to CrATP is about 18 Å. Digestion of the (Ca2+ + Mg2+)-ATPase at the T2 site (Arg 198) causes uncoupling of Ca2+-transport from ATPase activity while calcium occlusion due to E1-P formation remains unchanged. Further tryptic digestion beyond T2 and in the presence of ATP diminishes Ca2+ occlusion to zero while 50% of the ATPase hydrolytic activity remains. Tryptic digestion beyond T2 and in the absence of ATP diminishes ATPase hydrolytic activity to 50% of normal while Ca2+ occlusion remains intact. These data are consistent with a mechanism in which the functional enzyme must be in the dimeric form for occlusion and calcium uptake to occur, but each monomer can hydrolyze ATP.
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    Molecular and cellular biochemistry 114 (1992), S. 97-103 
    ISSN: 1573-4919
    Keywords: magnesium ; calcium ; hydrogen ion ; fluorescent dyes ; heart cells
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Several aspects of Mg2+ homeostasis were investigated in cultured chicken heart cells using the fluorescent Mg2+ indicator, FURAPTRA. The concentration of cytosolic Mg2+ ([Mg2+]i) is 0.48 ± 0.03 mM (n = 31). To test whether a putative Na/Mg exchange mechanism controls [Mg2+]i below electrochemical equilibrium, we manipulated the Na+ gradient and assessed the effects on [Mg2+]i. When extracellular Na+ was removed, [Mg2+]i increased; this increase was not altered in Mg-free solutions, but was attenuated in Ca-free solutions. A similar increase in [Mg2+]i, which was dependent upon extracellular Ca2+, was observed when intracellular Na+ was raised by inhibiting the Na/K pump with ouabain. These results do not provide evidence for Na/Mg exchange in heart cells, but they suggest that Ca2+ can modulate [Mg2+]i. In addition, removing extracellular Na+ caused a decrease in intracellular pH (pHi), as measured by pH-sensitive microelectrodes, and this acidification was attenuated when Cat+ was also removed from the solution. These results suggest that Ca2+ and H+ interact intracellularly. Since changes in the Na+ gradient can also alter pHi, we questioned whether pH can modulate [Mg2+]i. pHi was manipulated by the NH4Cl prepulse method. NH4 +-evoked changes in pHi, as measured by the fluorescent indicator BCECF, were accompanied by opposite changes in [Mg2+]i; [Mg2+]i changed by −0.16 mM/unit pH. These NH4 +-evoked changes in [Mg2+]i were not caused by movements of Mg2+ or Ca2+ across the sarcolemma or by changes in cytosolic Ca2+. Additionally, pHi was manipulated by changing extracellular pH (pHo). When pHo was decreased from 7.4 to 6.3, pHi decreased by 0.64 units and [Mg2+]i increased by 0.12 mM; in contrast, when pHo was raised from 7.4 to 8.3, pHi increased by 0.6 units and [Mg2+]i did not change significantly. The results of our investigations suggest that Ca 2+ and H+ can modulate [Mg2+]i, probably by affecting cytosolic Mg2+ binding and/or subcellular Mg2+ transport and that such redistribution of intracellular Mg2+ may play an important role in Mg2+ homeostasis in cardiac cells.
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    Molecular and cellular biochemistry 135 (1994), S. 79-88 
    ISSN: 1573-4919
    Keywords: calcium ; nucleus ; calpain ; calmodulin ; cell division ; gene expression
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Calcium has long been known to play a role as a key cytoplasmic second messenger, but until relatively recently its possible involvement in nuclear signal transduction and the regulation of nuclear events has not been extensively studied. Evidence revealing the presence of transmembrane nuclear Ca2+ gradients and a variety of intranuclear Ca2+ binding proteins has fueled renewed interest in this key ion and its involvement in cell-cycle timing and division, gene expression, and protein activation. This review will offer an overview of the current state of knowledge and theory regarding calcium orchestration of nuclear functions and events and discuss possible future directions in this field of study.
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    Molecular and cellular biochemistry 135 (1994), S. 1-9 
    ISSN: 1573-4919
    Keywords: calcium ; contraction ; artery ; smooth muscle ; actin ; myosin ; phosphorylation ; calmodulin ; myosin light chain kinase
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The fact that smooth muscle exists in almost every hollow organ and is involved in a large number of disease states has led to a vast increase in smooth muscle research, covering areas from testing response to antagonists and agonists to measuring the molecular force generated by a single actin filament. Yet, the exact mechanisms regulating contractile response of smooth muscle remain unsolved. Calcium has been a central player in mediating smooth muscle contraction through binding with calmodulin, although there is evidence showing that under special circumstances smooth muscle can contract without change in intracellular Ca2+. In addition to the major regulatory pathway of Ca2+-calmodulin-mysoin light chain kinase, there are other thin filament linked regulatory mechanisms in which Ca2+-calmodulin dependent phosphorylation of calponin and caldesmon may be involved. Ca2+ sensitivity of smooth muscle contraction may vary under different situations and this has recently been recognized as an important regulatory mechanism. Examples are protein kinase C (PKC) dependent phosphorylation of myosin light chain kinase which results in partial inhibition of contraction, and activation of myosin light chain phosphatase. There is new evidence howing that not only does Ca2+ regulate contraction by regulating the interaction of contractile proteins in smooth muscle, but also that shortening of smooth muscle itself reduces intracellular Ca2+ concentration, via a negative feedback.
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    Molecular and cellular biochemistry 149-150 (1995), S. 263-265 
    ISSN: 1573-4919
    Keywords: NO synthase ; toxic metals ; signal transduction ; calcium
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This study was designed to evaluate thein vitro effects of transition heavy metal cations on activity of constitutive isoform of nitric oxide synthase (cNOS) in rat brain. NOS activity was determined in the cytosolic fractions of rat cerebral hemispheres by conversion of3H-L-arginine to3H-L-citrulline. Different concentrations of mercury (Hg2+), nickel (Ni2+), manganese (Mn2+), zinc (Zn2+), cadmium (Cd2+), lead (Pb2+) and calcium (Ca2+) were tested on NOS activity. While all the cations caused inhibition, there were differences in the apparent inhibition constants (Ki) among the cations. With the exception of calcium ion no other cation required preincubation with the enzyme preparation. These results indicate that while calcium ion modulate cNOS activity at regulatory site(s), inhibitory influence of toxic heavy metal cations may be exerted on the catalytic site(s) either by direct binding to it or by interfering with the electron transfer during catalysis.
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    Molecular and cellular biochemistry 168 (1997), S. 95-100 
    ISSN: 1573-4919
    Keywords: aluminium ; calcium ; brain ; neurodegenerative disease
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    Notes: Abstract The present study investigates the possible effects of chronic aluminium exposure on the various aspects of calcium homeostasis in the primate central nervous system. Aluminium administration caused a marked decline in the activity of Ca2+ ATPase in the monkey brain. The total calcium content was also significantly raised following aluminium exposure. Concomittant to the increase in the calcium content, the levels of lipid peroxidation were also augmented in the aluminium treated animals, thereby further accentuating the aluminium induced neuronal damage. In addition, aluminium had an inhibitory effect on the depolarization induced 45Ca2+ uptake via the voltage operated channels. The results presented herein, indicate that the toxic effects of aluminium could be mediated through modifications in the intracellular calcium homeostasis with resultant altered neuronal function.
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    ISSN: 1573-4919
    Keywords: nitric oxide ; endotoxin ; cardiomyocytes ; guanosine 3′, 5′-cyclic monophosphate ; calcium ; ADP-ribosylation ; phosphorylation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract To evaluate the effects of the in vivo endotoxin treatment of the rat on (1) the contractile responses in the subsequently isolated papillary muscle to adrenergic and cholinergic agonists and (2) the biochemical parameters (cyclic GMP, nitric oxide synthesis, protein phosphorylation and ADP-ribosyslation) in the subsequently isolated cardiomyocytes. Following the in vivo endotoxin treatment (4 mg/kg i.p., 18 h), contractile responses to increasing amounts of isoprenaline or to increasing amounts of oxotremorine in the presence of a fixed amount of isoprenaline were determined in isolated papillary strips. Activities of nitric oxide synthase, guanylyl cyclase, as well as phosphorylation of phospholamban and troponin-inhibitory subunit, and pertussis toxin-catalyzed and endogenous ADP-ribosylations were determined in the intact cardiomyocytes and subcellular fractions. The increase in the force of contraction by isoprenaline was reduced, while its inhibition by oxotremorine was greater in the endotoxin-treated papillary strips. The activities of both nitric oxide synthase, primarily of the inducible form of the enzyme, and cytosolic guanylyl cyclase were higher while the phosphorylations of both phospholamban and troponin-inhibitory subunit were of lesser magnitude in the cardiomyocytes following the in vivo endotoxin treatment. Pertussis toxin-catalyzed ADP-ribosylation of the 41 kDa polypeptide, which is the alpha subunit of Gi, was also decreased. The results of the present study support the postulate that alterations in both the cyclic AMP and cyclic GMP signalling cascade contribute to the myocardial dysfunction caused by endotoxin and cytokines.
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  • 41
    ISSN: 1573-4919
    Keywords: taurine ; heart cells ; calcium ; sodium ; confocal microscopy ; nucleus ; fluo-3 ; sodium green ; Ca2+ overload
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of taurine on the different types of ionic currents appears to depend on [Ca]o and [Ca]i and may also vary accordingly to tissue or cell type studied. Using microfluorometry and Ca2+ imaging techniques, short-term exposure (5–10 min) of single heart cells to taurine was found to increase total intracellular free Ca2+ in a concentration-dependent manner. However, long-term exposure of heart myocytes to taurine was found to decrease both nuclear and cytosolic Ca2+ without significantly changing either nuclear or cytosolic Na+ levels, as measured by 3-dimensional Ca2+ and Na+ confocal imaging techniques. Long- term exposure to taurine was found to prevent cytosolic and nuclear increases of Ca2+ induced by permanent depolarization of heart cells with high [K]o. This preventive effect of taurine on nuclear Ca2+ overload was associated with an increase of both cytosolic and nuclear free Na+. Thus, the effect of long-term exposure to taurine on intranuclear Ca2+ overload in heart cells seems to be mediated via stimulation of sarcolemmal and nuclear Ca2+ outflow through the Na+-Ca2+ exchanger.
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  • 42
    ISSN: 1573-4919
    Keywords: mitochondria ; calcium ; permeability transition ; vasopressin ; glucagon ; thapsigargin ; protein kineses and phosphatases ; rat hepatocytes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Ca2+ functions as an intracellular signal to transfer hormonal messages to different cellular compartments, including mitochondria, where it activates intramitochondrial Ca2+-dependent enzymes. However, excessive mitochondrial Ca2+ uptake can promote the mitochondrial permeability transition (MPT), a process known to be associated with cell injury. The factors controlling mitochondrial Ca2+ uptake and release in intact cells are poorly understood. In this paper, we investigate mitochondrial Ca2+ accumulation in intact hepatocytes in response to the elevation of cytosolic Ca2+ levels ([Ca2+]c) induced either by a hormonal stimulus (vasopressin), or by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump. After stimulation, cells were rapidly permeabilized for the determination of the mitochondrial Ca2+ content (Ca2+_m) and to analyze the susceptibility of the mitochondria to undergo the MPT. Despite very similar levels of [Ca2+]c elevation, vasopressin and thapsigargin had markedly different effects on mitochondrial Ca2+ accumulation. Vasopressin caused a rapid (〈 90 sec), but modest (〈 2 fold) increase in Ca2+m that was not further increased during prolonged incubations, despite a sustained [Ca2+]c elevation. By contrast, thapsigargin induced a net Ca2+ accumulation in mitochondria that continued for up to 30 min and reached Ca2+_m levels 10–20 fold over basal. Accumulation of mitochondrial Ca2+ was accompanied by a markedly increased susceptibility to undergo the MPT. Both mitochondrial Ca2+ accumulation and MPT activation were modulated by treatment of the cells with inhibitors of protein kineses and phosphatases. The results indicate that net mitochondrial Ca2+ uptake in response to hormonal stimulation is regulated by processes that depend on protein kinase activation. These controls are inoperative when the cytosol is flooded by Ca2+ through artificial means, enabling mitochondria to function as a Ca2+ sink under these conditions. (Mol Cell Biochem 174: 173–179, 1997)
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  • 43
    ISSN: 1573-4919
    Keywords: heart ; calcium ; magnesium ; contractility ; dietary ; L-type calcium current
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This study employs both dietary and physiological studies to investigate the relationship between calcium (Ca2+) and magnesium (Mg2+) signalling in the mammalian myocardium. Rats maintained on a low Mg2+ diet (LMD; 39 mg Kg-1 Mg2+ in food) consumed less food and grew more slowly than control rats fed on a control Mg2+ diet (CMD; 500 mg Kg-1 Mg2+ in food). The Mg2+ contents of the heart and plasma were 85 ± 3% and 34 ± 6.5%, respectively relative to the control group. In contrast, Ca2+ contents in the heart and plasma were 177 ± 5% and 95 ± 3%. The levels of potassium (K+) was raised in the plasma (129 ± 16%) and slightly decreased in the heart (88 ± 6%) compared to CMD. Similarly, sodium (Na+) contents were slightly higher in the heart and lowered in the plasma of low Mg2+ diet rats compared to control Mg2+ diet rat. Perfusion of the isolated Langendorff's rat heart with a physiological salt solution containing low concentrations (0-0.6 mM) of extracellular magnesium [Mg2+]0 resulted in a small transient increase in the amplitude of contraction compared to control [Mg2+]0 (1.2 mM). In contrast, elevated [Mg2+]0 (2-7.2 mM) caused a marked and progressive decrease in contractile force compared to control. In isolated ventricular myocytes the L-type Ca2+ current (ICa,L was significantly (p 〈 0.001) attenuated in cells dialysed with 7.1 mM Mg2+ compared to cells dialysed with 2.9 µM Mg2+. The results indicate that hypomagnesemia is associated with decrease levels of Mg2+ and elevated levels of Ca2+ in the heart and moreover, internal Mg2+ is able to modulate the Ca2+ current through the L-type Ca2+ channel which in turn may be involved with the regulation of contractile force in the heart.
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  • 44
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    Molecular and cellular biochemistry 177 (1997), S. 209-214 
    ISSN: 1573-4919
    Keywords: regucalcin ; calmodulin ; calcium ; cyclic AMP phosphodiesterase ; rat kidney cytosol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of regucalcin, a novel Ca2+-binding protein, on Ca2+/ calmodulin-dependent cyclic adenosine monophosphate (AMP) phosphodiesterase activity in the cytosol of rat renal cortex was investigated. Regucalcin with physiologic concentration (10-7 M) in rat kidney had no effect on cyclic AMP phosphodiesterase activity in the absence of CaCl2 and calmodulin. However, the activatory effect of both CaCl2 (10 µM) and calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was markedly inhibited by the addition of regucalcin (10-8 to 10-6 M) in the enzyme reaction mixture. The inhibitory effect of regucalcin on the enzyme activity was also seen in the presence of CaCl2 (5-50 µM) or calmodulin (5-50 U/ml) with increasing concentrations. The presence of trifluoperazine (10 µM), an antagonist of calmodulin, caused a partial inhibition of Ca2+ /calmodulin-dependent cyclic AMP phosphodiesterase activity. This inhibition was further enhanced by the addition of regucalcin (10-7 M). The inhibitory effect of regucalcin (10-7 M) was not seen in the presence of 20 µM trifluoperazine. Moreover, the activatory effect of calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was not entirely seen, when calmodulin was added 10 min after incubation in the presence of CaCl2 (10 µM) and regucalcin (10-7 M). The present results demonstrates that regucalcin has an inhibitory effect on Ca2+ /calmodulin-dependent cyclic AMP phosphodiesterase activation in the cytosol of rat renal cortex.
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  • 45
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    Molecular and cellular biochemistry 180 (1998), S. 53-57 
    ISSN: 1573-4919
    Keywords: diabetes ; cardiomyopathy ; lipids ; lipoprotein lipase ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract It has been established that diabetes results in a cardiomyopathy, and increasing evidence suggests that an altered substrate supply and utilization by cardiac myocytes could be the primary injury in the pathogenesis of this specific heart muscle disease. For example, in diabetes, glucose utilization is insignificant, and energy production is shifted almost exclusively towards β-oxidation of free fatty acids (FFA). FFA's are supplied to cardiac cells from two sources: lipolysis of endogenous cardiac triglyceride (TG) stores, or from exogenous sources in the blood (as free acid bound to albumin or as TG in lipoproteins). The approximate contribution of FFA from exogenous or endogenous sources towards β-oxidation in the diabetic heart is unknown. In an insulin-deficient state, adipose tissue lipolysis is enhanced, resulting in an elevated circulating FFA. In addition, hydrolysis of the augmented myocardial TG stores could also lead to high tissue FFA. Whatever the source of FFA, their increased utilization may have deleterious effects on myocardial function and includes the abnormally high oxygen requirement during FFA metabolism, the intracellular accumulation of potentially toxic intermediates of FFA, a FFA-induced inhibition of glucose oxidation, and severe morphological changes. Therapies that target these metabolic aberrations in the heart during the early stages of diabetes could potentially delay or impede the progression of more permanent sequelae that could ensue from otherwise uncontrolled derangements in cardiac metabolism.
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  • 46
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    Molecular and cellular biochemistry 179 (1998), S. 135-145 
    ISSN: 1573-4919
    Keywords: calcium ; non-lysosomal proteases ; muscle damage ; neutrophils ; muscle regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract It is well established that periods of increased contractile activity result in significant changes in muscle structure and function. Such morphological changes as sarcomeric Z-line disruption and sarcoplasmic reticulum vacuolization are characteristic of exercise-induced muscle injury. While the precise mechanism(s) underlying the perturbations to muscle following exercise remains to be elucidated, it is clear that disturbances in Ca2+ homeostasis and changes in the rate of protein degradation occur. The resulting elevation in intracellular [Ca2+] activates the non-lysosomal cysteine protease, calpain. Because calpain cleaves a variety of protein substrates including cytoskeletal and myofibrillar proteins, calpain-mediated degradation is thought to contribute to the changes in muscle structure and function that occur immediately following exercise. In addition, calpain activation may trigger the adaptation response to muscle injury. The purpose of this paper is to: (i) review the chemistry of the calpain-calpastatin system; (ii) provide evidence for the involvement of the non-lysosomal, calcium-activated neutral protease (calpain) in the response of skeletal muscle protein breakdown to exercise (calpain hypothesis); and (iii) describe the possible involvement of calpain in the inflammatory and regeneration response to exercise.
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  • 47
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    Keywords: aortic cells ; steady state R-type Ca2+ channel ; ET-1 ; insulin ; calcium ; G-protein
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    Notes: Abstract In single rabbit aortic smooth muscle cells, and at a concentration known to induce a maximum sustained increase of intracellular Ca2+ via activation of the steady-state voltage dependent R-type Ca2+ channels, endothelin-1 (10-7 M) and insulin (80 μU/ml) were found to induce a sustained increase in cytosolic free Ca2+ ([Ca]i) levels that was significantly attenuated by pre-treatment with either pertussis toxin (PTX), cholera toxin (CTX) or removal of extracellular Ca2+. However, both PTX and CTX failed to inhibit the sustained depolarization-evoked sustained Ca2+ influx and [Ca]i elevation via activation of the R-type Ca2+ channels. Moreover, ET-1 and insulin-evoked sustained increases in Ca2+ influx were not attenuated by the selective PKC inhibitor, bisindolylmaleimide (BIS), or the specific L-type Ca2+ channel blocker, nifedipine, but were completely reversed by the R-type Ca2+ channel blocker, (-) PN 200-110 (isradipine). These data suggest that both insulin and ET-1 activate the nifedipine-insensitive but isradipine-sensitive steady state voltage dependent R-type Ca2+ channels present on rabbit VSMCs and these channels are directly coupled to PTX and CTX sensitive G protein(s).
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  • 48
    ISSN: 1573-4919
    Keywords: hyperosmolality ; hyperglycemia ; calcium ; smooth muscle cells ; diabetes
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Abstract Hyperglycemia and/or hyperosmolality may disturb calcium homeostasis in vascular smooth muscle cells (SMCs), leading to altered vascular contractility in diabetes. To test this hypothesis, the KCl induced increases in [Ca2+]i in primarily cultured vascular SMCs exposed to different concentrations of glucose were examined. With glucose concentration in solutions kept at 5.5 mM, KCl induced a fast increase in [Ca2+]i which then slowly declined (type 1 response) in 83% of SMCs from non-diabetic rats. In 9% of non-diabetic SMCs KCl induced a slow increase in [Ca2+]i (type 2 response). Interestingly, under the same culture conditions KCl induced type 1 and type 2 responses in 47 and 35% of SMCs from diabetic rats. When SMCs from non-diabetic or diabetic rats were cultured in 36 mM glucose, KCl induced a fast increase in [Ca2+]i which, however, maintained at a high level (type 3 response). The sustained level of [Ca2+]i in the presence of KCl was significantly higher in cells cultured with 36 mM glucose than that in non-diabetic cells cultured with 5.5 mM glucose. Furthermore, the hyperglycemia-induced alterations in calcium mobilization were similarly observed in cells cultured in high concentration of mannitol (30.5 mM) or L-glucose, indicating that hyperosmolality was mainly responsible for the abnormal calcium mobilization in diabetic SMCs.
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  • 49
    ISSN: 1573-4919
    Keywords: CGRP-1 receptor ; HEK-293 cells ; calcium ; cholera toxin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Calcitonin gene-related peptide (CGRP) is a neuropeptide with diverse biological properties including potent vasodilating activity. Recently, we reported the cloning of complementary DNAs (cDNAs) encoding the human and porcine CGRP receptors which share significant amino acid sequence homology with the human calcitonin receptor, a member of the recently described novel subfamily of G-protein-coupled 7TM receptors. Activation of this family of receptors has been shown to result in an increase in intracellular cAMP accumulation and calcium release. In this study, we demonstrate that HEK-293 cells expressing recombinant CGRP receptors (HEK-293HR or PR) respond to CGRP with increased intracellular calcium release (EC50 = 1.6 nM) in addition to the activation of adenylyl cyclase (EC50 = 1.4 nM). The effect of CGRP on adenylyl cyclase activation and calcium release was inhibited by CGRP (8-37), a CGRP receptor antagonist. Both effects were mediated by cholera toxin-sensitive G-proteins, but these two signal transduction pathways were independent of each other. While cholera toxin pretreatment of HEK-293PR cells resulted in permanent activation of adenylyl cyclase, the same pretreatment resulted in an inhibition of CGRP-mediated [Ca2+]i release. Pertussis toxin was without effect on CGRP-mediated responses. In addition, CGRP-mediated calcium release appears to be due to release from a thapsigargin-sensitive intracellular calcium pool. These results show that the recombinant human as well as porcine CGRP receptor can independently increase both cAMP production and intracellular calcium release when stably expressed in the HEK-293 cell line.
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  • 50
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    Molecular and cellular biochemistry 93 (1990), S. 27-34 
    ISSN: 1573-4919
    Keywords: neutrophil ; superoxide ; calcium ; nifedipine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Summary Superoxide production by mice neutrophils was inhibited by nifedipine exposure in a dose dependent manner. The inhibition of Ca2+ uptake elicited by nifedipine did not appear to account for the observed effect as the extracellular Ca2+ enrichment and depletion did not produce a significant reversal of the inhibition. Cytosolic free Ca2+ as measured by Quin 2AM fluorescence did not show any significant change, indicating that the effect was independent of the inhibition of Ca2+ influx. In addition nifedipine caused a significant inhibition (p 〈 0.01) in NADPH oxidase activity. Our data indicates that nifedipine inhibits superoxide production independent of inhibiting Ca2+ inflow and supports the hypothesis that Ca2+ antagonists affect cellular functions by non Ca2+ mediated process as well.
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  • 51
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    Keywords: myosin ; calcium ; calmodulin ; type II kinase
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Brain type II Ca2+/calmodulin-dependent protein kinase was found to phoshorylate smooth muscle myosin, incorporating maximally ∼ 2 mol of phosphoryl per mol of myosin, exclusively on the 20,000 dalton light chain subunit. After maximal phosphorylation of myosin or the isolated 20,000 dalton light chain subunit by myosin light chain kinase, the addition of type II Ca2+/calmodulin-dependent protein kinase led to no further incorporation indicating the two kinases phosphorylated a common site. This conclusion was supported by two dimensional mapping of tryptic digests of myosin phosphorylated by the two kinases. By phosphoamino acid analysis the phosphorylated residue was identified as a serine. The phosphorylation by type II Ca 2+/calmodulin-dependent protein kinase of myosin resulted in enhancement of its actin-activated Mg2+-ATPase activity. Taken together, these data strongly support the conclusion that type II Ca2+/calmodulin-dependent protein kinase phosphorylates the same amino acid residue on the 20,000 dalton light chain subunit of smooth muscle myosin as is phosphorylated by myosin light chain kinase and suggest an alternative mechanism for the regulation of actin-myosin interaction.
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  • 52
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    Keywords: calcium ; regucalcin ; deoxyuridine 5′-triphosphatase ; rat liver cytosol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on deoxyuridine 5′-triphosphatase (dUTPase) in the cytosol of rat liver was investigated. Addition of Ca2+ up to 5.0 µM to the enzyme reaction mixture caused a significant decrease of dUTPase activity, while Zn2+, Cd2+, Co2+, Al3+, Mn2+ and Ni2+ (10 µM) did not have an appreciable effect. The Ca2+-induced decrease of dUTPase activity was reversed by the presence of regucalcin; the effect was complete at 1.0 µM of the protein. Regucalcin had no effect on the basal activity of the enzyme. Meanwhile, the reversible effect of regucalcin on the Ca2+ (10 µM)-induced decrease of dUTPase activity was not altered by the coexistence of Cd2+ or Zn2+ (10 µM). The present data suggest that liver cytosolic dUTPase is uniquely regulated by Ca2+ of various metals, and that the Ca2+ effect is reversed by regucalcin.
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  • 53
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    Molecular and cellular biochemistry 111 (1992), S. 71-76 
    ISSN: 1573-4919
    Keywords: neutrophil ; free oxygen radicals ; calcium ; endothelial cell growth factors ; prostaglandins ; indomethacin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The exposure of cardiac cells to OFR generated artificially, showed a marked decrease (p 〈 0.01) in cellular utilization of glucose alongwith a significant decrease in calcium uptake (p 〈 0.05). We have also provided evidence for a direct relationship of neutrophil OFR production with the extent of myocardial ischemia in patients of myocardial infarction. Our data provides evidence for implication of OFR in myocardial injury and the pivotal role played by modulators like calcium, ECGF and prostaglandins in potentiating damage to the myocardium.
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  • 54
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    Molecular and cellular biochemistry 111 (1992), S. 17-24 
    ISSN: 1573-4919
    Keywords: ischemia/reperfusion injury ; oxygen radicals ; sarcoplasmic reticulum ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The purpose of this study was to explore the role of singlet oxygen in cardiovascular injury. To accomplish this objective, we investigated the effect of singlet oxygen [generated from photoactivation of rose-bengal] on the calcium transport and Ca2+-ATPase activity of cardiac sarcoplasmic reticulum and compared these results with those obtained by superoxide radical, hydrogen peroxide and hydroxyl radical. Isolated cardiac SR exposed to rose bengal (10 nM) irradiated at (560 nm) produced a significant inhibition of Ca 2+ uptake; from 2.27 ± 0.05 to 0.62 ± 0.05 µmol Ca+/mg.min (mean ± SE) (P 〈 0.01) and Ca2+-ATPase activity from 2.08 ± 0.05 µmol Pi/min. mg to 0.28 ± 0.04 µmol Pi/min. mg (mean ± SE) (P 〈 0.01). The inhibition of calcium uptake and Ca2+-ATPase activity by rose bengal derived activatedoxygen (singlet oxygen) was dependent on the duration of exposure and intensity of light. The singlet oxygen scavengers ascorbic acid and histidine significantly protected SR Ca2+-ATPase against rose bengal derived activated oxygen species but superoxide dismutase and catalase did not attenuate the inhibition. SDS-polyacrylamide gel electrophoresis of SR exposed to photoactivated rose bengal up to 14 min, demonstrated complete loss of Ca2+-ATPase monomer band which was significantly protected by histidine. Irradiation of rose bengal also caused an 18% loss of total sulfhydryl groups of SR. On the other hand, superoxide (generated from xanthine oxidase action on xanthine) and hydroxyl radical (0.5 mM H2O2 + Fe2+ -EDTA) as well as H2O2 (12 mM) were without any effect on the 97,000 dalton Ca2+-ATPase band ofsarcoplasmic reticulum. The results suggest that oxidative damage of cardiac sarcoplasmic reticulum may be mediated by singlet oxygen. This may represent an important mechanism by which the oxidative injury to the myocardium induces both a loss of tension development and arrhythmogenesis.
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  • 55
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    Molecular and cellular biochemistry 130 (1994), S. 137-147 
    ISSN: 1573-4919
    Keywords: microtubules ; calcium ; colchicine ; posttranslational modifications ; fish ; cow
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Microtubule proteins were isolated by a temperature-dependent assembly-disassembly method from brain tissue of for cold-temperate fish; one fresh water fish (Oncorhynchus mykiss), and three marine fish (Labrus berggylta, Zoarces viviparus andGadus morhua). The α-tubulins from all four fish species were acetylated. The α-tubulins from the marine fish were composed of a mixture of tyrosinated and detyrosinated tubulin, while the fresh water fish tubulin only reacted with an antibody against detyrosinated tubulin. The isolated microtubules had a similar MAP composition. A 400 kD protein and a MAP2-like protein were found, but MAP1 was missing. All microtubules disassembled upon cooling to 0°C. In spite of these common characteristics, the assembly of microtubules fromLabrus berggylta was inhibited by colchicine and calcium, in contrast to the assembly of microtubules fromOncorhynchus mykiss andZoarces viviparus. For the latter, colchicine was not completely inhibitory even at a concentration as high as 1 mM, and calcium induced the formation of both loosely and densely coiled ribbons. The effects of calcium and colchicine on microtubules fromOncorhynchus mykiss andZoarces viviparus were modulated by either fish or cow MAPs, indicating that the effects are due to intrinsic properties of the fish tubulins and not the MAPs. In view of these findings, our results suggest that there is not correlation between colchicine sensitivity, inability of calcium to inhibit microtubule assembly, and acetylation and detyrosination.
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  • 56
    ISSN: 1573-4919
    Keywords: lymphocyte ; calcium ; glucose transporter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Summary Generalised metabolic and electrolyte disturbances are known to accompany both plasma and surface virus infections. We have investigated whether these infections could impair the transport of Ca2+ from cells under conditions of controlled concentrations of the energy substrate glucose. Thus, cytosolic calcium levels ([Ca2+]i) were measured in single isolated lymphocytes obtained from healthy volunteers or those suffering from coryza. Before making measurements using a Ca2+-sensitive fluorescent dye indo 1, we incubated lymphocytes in buffers containing 0 mM-, 5.6 mM- or 11.2 mM-[glucose]. We found that [Ca2+]i of lymphocytes obtained from the sick were significantly higher than those from healthy controls both at 0 mM and 5.6 mM-[glucose], and that [Ca2+]i was inversely related to the media glucose concentration for both groups. These results suggest a diminished capacity of cation pumping in viral infections, such as coryza, in relationship to the available glucose as energy substrate.
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  • 57
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    Molecular and cellular biochemistry 136 (1994), S. 105-111 
    ISSN: 1573-4919
    Keywords: domoic acid ; excitotoxicity ; adenylate cyclase ; cyclic AMP ; calcium ; calmodulin ; forskolin ; Gpp(NH)p ; brain (rat)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Adenylate cyclase activity measured by the formation of cyclic AMP in rat brain membranes was inhibited by a shellfish toxin, domoic acid (DOM). The inhibition of enzyme was dependent on DOM concentration, but about 50% of enzyme activity was resistant to DOM-induced inhibition. Rat brain supernatant resulting from 105,000×g centrifugation for 60 min, stimulated adenylate cyclase activity in membranes. Domoic acid abolished the supernatant-stimulated adenylate cyclase activity. The brain supernatant contains factors which modulate adenylate cyclase activity in membranes. The stimulatory factors include calcium, calmodulin, and GTP. In view of these findings, we examined the role of calcium and calmodulin in DOM-induced inhibition of adenylate cyclase in brain membranes. Calcium stimulated adenylate cyclase activity in membranes, and further addition of calmodulin potentiated calcium-stimulated enzyme activity in a concentration dependent manner. Calmodulin also stimulated adenylate cyclase activity, but further addition of calcium did not potentiate calmodulin-stimulated enzyme activity. These results show that the rat brain membranes contain endogenous calcium and calmodulin which stimulate adenylate cyclase activity. However, calmodulin appears to be present in membranes in sub-optimal concentration for adenylate cyclase activation, whereas calcium is present at saturating concentration. Adenylate cyclase activity diminished as DOM concentration was increased, reaching a nadir at about 1 mM. Addition of calcium restored DOM-inhibited adenylate cyclase activity to the control level. Similarly, EGTA also inhibited adenylate cyclase activity in brain membranes in a concentration dependent manner, and addition of calcium restored EGTA-inhibited enzyme activity to above control level. The fact that EGTA is a specific chelator of calcium, and that DOM mimicked adenylate cyclase inhibition by EGTA, indicate that calcium mediates DOM-induced inhibition of adenylate cyclase activity in brain membranes. While DOM completely abolished the supernatant-, and Gpp (NH)p-stimulated adenylate cyclase activity, it partly blocked calmodulin-, and forskolin-stimulated adenylate cyclase activity in brain membranes. These results indicate that DOM may interact with guanine nucleotide-binding (G) protein and/or the catalytic subunit of adenylate cyclase to produce inhibition of enzyme in rat brain membranes.
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  • 58
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    Molecular and cellular biochemistry 135 (1994), S. 89-98 
    ISSN: 1573-4919
    Keywords: calcium ; cell death ; nuclei ; apoptosis ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The last decade has seen the rapid development of research investigating the molecular mechanisms whereby hormones, peptide growth factors and cytokines regulate cell metabolism, differentiation and proliferation. One general signalling mechanism used to transfer the information delivered by agonists into appropriate intracellular compartments involves the rapid Ca2+ redistribution throughout the cell, which results in transient elevations of the cytosolic free Ca2+ concentration. Ca2+ signals are required for a number of cellular processes including the activation of nuclear processes such as gene transcription and cell cycle events. The latter require that appropriate Ca2+ signals elicited in response to agonists be transduced across the nuclear envelope. It has generally been assumed that small molecules, metabolites and ions could freely diffuse across the nuclear envelope. Nevertheless several findings during the past few years have suggested that nuclear pore permeability can be regulated and that ion transport systems and ion-selective channels may exist on the nuclear membranes and regulate intranuclear processes. Intranuclear Ca2+ fluctuations can affect chromatin organization, induce gene expression and also activate cleavage of nuclear DNA by nucleases during programmed cell death or apoptosis. The possible mechanisms involved in nuclear Ca2+ transport and the control of nuclear Ca2+-dependent enzymes in apoptosis is discussed below.
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  • 59
    ISSN: 1573-4919
    Keywords: endothelin-1 ; ventricular cardiomyocytes ; contraction ; calcium ; heart failure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Endothelin (ET-1) is found at elevated concentrations in the plasma of patients with heart failure and in animal models of cardiomyopathy. The peptide is a potent positive inotropic agent, the effects of which are mediated by increases in cytosolic Ca2+ in cardiomyocytes. The object of this study was to investigate at the cellular level, the actions of ET-1 on contractile function and on Ca2+ currents in heart-failed ventricular myocardium. Male New Zealand White rabbits (8 wks) were treated with twice weekly injections of epirubicin (4 mg/kg/wk, n=7) or with saline (n=7) for 6 wks, followed by a washout period of 2 wks. Ventricular cardiomyocytes were isolated from rabbit hearts using Langendorff perfusion with collagenase; contractile function was examined using a video microscopy method, and L-type Ca2+ currents were recorded using a whole-cell patch-clamp technique. ET-1 produced a concentration-dependent increase in contractile response (% increase from basal value) to a maximum at 1 nM ET-1 of 69 ± 11% (mean ± S.D.) in control cardiomyocytes and 33 ± 6% in heart-failed cells. However, there was no significant change in the EC50 obtained with ET-1 for healthy (0.31 ± 0.1 nM) and for failed cardiomyocytes (0.24 ± 0.1 nM). The effects of ET-1 on L-type Ca2+ channels were similar with a peak amplitude at 1 nM ET-1 of −3.26 ± 0.8 ⋬ in control cardiomyocytes and −3.32 ± 0.9 nA in heart-failed cells. The attenuation of the contractile response to ET-1 in heart-failed cells may reflect a desensitization of ET receptors as a consequence of elevated circulating levels of ET and was not reflected by alteration of transmembrane Ca2+ conductance. It is probable, therefore, that multiple signalling pathways are involved in the actions of ET on ventricular myocardium.
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  • 60
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    Molecular and cellular biochemistry 163-164 (1996), S. 125-130 
    ISSN: 1573-4919
    Keywords: cardiac myocytes ; early after depolarisations ; delayed after depolarisations ; calcium ; sarcoplasmic reticulum ; noradrenaline
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We investigated the effect of 10−8 M noradrenaline (NA) on [Ca2+], and electrical activity of single myocytes of guinea-pig ventricular myocardium loaded with Indo 1-AM. Membrane potential was recorded by means of the patch electrode and patch amplifier set to the current clamp mode. Cells were stimulated at a rate of 30/min by 3 ms pulses of the current injected through the recording electrode. Superfusion of NA resulted in slight shortening of action potentials (APs), increase in rate of rise and amplitude of the respective Ca2+ transients, and appearance of secondary Ca2+ transients of two kinds: 1. appearing before repolarisation of AP and decay of the preceding Ca2+ transient were completed and 2. appearing between the APs. We named them early after-transients (EAT) and delayed after-transients (DAT), respectively. Without any additional intervention EATS caused some prolongation of APs duration and DATs resulted in subthreshold delayed after-depolarisations (DADS). When sarcolemmal K+ conductance was decreased by tetraethylammonium (TEA) in the patch electrode or 20 μM BaCl2 in the Tyrode solution, EATs initiated early after depolarizations (EADs) and DATs initiated suprathreshold DADs triggering full-sized APs. Superfusion of 30.0 mM Na+ (replaced with LiCl) resulted in reduction of AP duration by -70% and appearance of DATs. Also, the frequent multiple oscillations of Ca 2+ concentration were often observed. Neither DATs nor the oscillations had any affect on electrical activity of the cells. Their electrogenicity could not be increased by TEA or 20.0 μM Ba2+. EATs and DATs and their respective EADs and DADs could not be initiated by NA or low Na+ superfusion in the cells pretreated with 2 × 10−7 M thapsigargin, a selective blocker of Ca2+-ATPase of sarcoplasmic reticulum (SR). We conclude that in contrast to the current hypothesis, EADs can be initiated by Ca2+ released early in the cardiac cycle from the overloaded SR, and that electrogenicity of both types of Ca2+ oscillations critically depends on the sarcolemmal K+ conductance.
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  • 61
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    Molecular and cellular biochemistry 187 (1998), S. 47-55 
    ISSN: 1573-4919
    Keywords: Mimosa pudica ; apyrase ; arabinogalactan ; calcium ; circular dichroism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Mimosa pudica Linn leaves with pulvini contain unique isoforms (I and II) of apyrase enzyme (EC 3.6.1.5). The activity of isoform I depends on divalent cation Mn2+. This isoform is associated noncovalently with the polysaccharide, containing mainly of galactose and arabinose sugars. The apparent molecular mass of these 2 isoforms are 36 and 34 Kd respectively. The association of the polysaccharide with the isoform I has been found to be Ca2+ dependent which is endogenously present in this isoform. Removal of Ca2+ and polysaccharide from the enzyme (isoform I) leads to an inactivation. The enzyme activity can be restored when both Ca2+ and endogenous polysaccharide fraction were added at an optimal molar ratio of Ca2+:protein of 7:1. The endogenous polysaccharide can be replaced by the standard arabinogalactan. No other sugar or polysaccharide except the arabinogalactan can restore the apyrase activity. Calcium mediates a conformational change in the protein which helps in association of polysaccharide as evidenced from fluorometric and far UV-CD studies to restore the enzymic activity. Neither any interaction of the polysaccharide with the protein is detected in absence of Ca2+ nor the enzyme activity could be recovered under such condition.
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  • 62
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    Molecular and cellular biochemistry 187 (1998), S. 1-10 
    ISSN: 1573-4919
    Keywords: oxidant ; cardiovascular system ; signal transduction ; calcium ; mitogen activated protein kinases ; nuclear transcription factors ; tyrosine kinase ; protein kinase C ; superoxide ; hydrogen peroxide ; ischemia-reperfusion ; atherosclerosis ; phospholipases ; apoptosis ; antioxidant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Although oxidants such as superoxide (O2.-) and hydrogen peroxide (H2O2) play a role in host-mediated destruction of foreign pathogens yet excessive generation of oxidants may lead to a variety of pathological complications in the cardiovascular system. An important mechanism by which oxidants cause dysfunction of the cardiovascular system appears to be due to the increase in intracellular free Ca2+ concentration. Oxidants cause cellular Ca2+ mobilization by modulating activities of a variety of regulators such as Na+/H+ and Na+/Ca2+ exchangers, Na+/K+ ATPase and Ca2+ ATPase and Ca2+ channels that are associated with Ca2+ transport in the plasma membrane and the sarco(endo)plasmic reticular membrane of myocardial cells. Recent research have suggested that the increase in Ca2+ level by oxidants plays a pivotal role in indicing several protein kinases such as protein kinase C, tyrosine kinase and mitogen activated protein kinases. Oxindant-mediated alteration of different signal transduction systems and their interations eventually regulate a variety of pathological conditoins such as atherosclerosis, apoptosis and necrosis in the myocardium
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  • 63
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    Molecular and cellular biochemistry 104 (1991), S. 81-86 
    ISSN: 1573-4919
    Keywords: phosphoinositides ; phosphatidylcholine ; phospholipases ; diacylglycerol ; phosphatidate ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract There is much evidence that G-proteins transduce the signal from receptors for Ca2+-mobilizing agonists to the phospholipase C that catalyzes the hydrolysis of phosphoinositides. However, the specific G-proteins involved have not been identified. We have recently purified a 42 kDa protein from liver that activates phosphoinositide phospholipase C and cross-reacts with antisera to a peptide common to G-protein α-subunits. It is proposed that this protein is the a-subunit of the G-protein that regulates the phospholipase in this tissue. Ca2+-mobilizing agonists and certain growth factors also promote the hydrolysis of phosphatidylcholine through the activation of phospholipases C and D in many cell types. This yields a larger amount of diacylglycerol for a longer time than does the hydrolysis of inositol phospholipids. Consequently phosphatidylcholine breakdown is probably a major factor in long-term regulation of protein kinase C. The functions of phosphatidic acid produced by phospholipase D are speculative, but there is evidence that it is a major source of diacylglycerol in many cell types. The regulation of phosphatidylcholine phospholipases is multiple and involves direct activation by G-proteins, and regulation by Ca2+ protein kinase C and perhaps growth factor receptor tyrosine kinases.
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  • 64
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    Keywords: heart ; vascular endothelium ; vascular smooth muscle ; confocal microscopy ; pH ; calcium ; sodium ; voltage probe ; heart ; endothelin-1 ; Angiotensin II ; PAF ; nucleus ; mitochondria ; SR ; cardiomyopathy ; cells interaction ; R-type Ca2+ channel ; excitation-contraction coupling ; dystrophic mouse
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Abstract In recent years, fluorescence microscopy imaging has become an important tool for studying cell structure and function. This non invasive technique permits characterization, localisation and qualitative quantification of free ions, messengers, pH, voltage and a pleiad of other molecules constituting living cells. In this paper, we present results using various commercially available fluorescent probes as well as some developed in our laboratory and discuss the advantages and limitations of these probes in confocal microscopy studies of the cardiovascular system.
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  • 65
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    Keywords: cyclosporin A ; mitochondrial permeability transition ; reperfusion injury ; cyclophilin ; oxidative stress ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract When loaded with high (pathological) levels of Ca2+, mitochondria become swollen and uncoupled as the result of a large non-specific increase in membrane permeability. This process, known as the mitochondrial permeability transition (MPT), is exacerbated by oxidative stress and adenine nucleotide depletion. These conditions match those that a heart experiences during reperfusion following a period of ischaemia. The MPT is caused by the opening of a non-specific pore that can be prevented by sub-micromolar concentrations of cyclosporin A (CsA). A variety of conditions that increase the sensitivity of pore opening to [Ca2+], such as thiol modification, oxidative stress, increased matrix volume and chaotropic agents, all enhance the binding of matrix cyclophilin (CyP) to the inner mitochondrial membrane in a CsA-sensitive manner. In contrast, ADP, membrane potential and low pH decrease the sensitivity of pore opening to [Ca2+] without affecting CyP binding. We present a model of pore opening involving CyP binding to a membrane target protein followed by Ca2+-dependent triggering of a conformational change to induce channel opening. Using the ischaemic/reperfused rat heart we have shown that the mitochondrial pore does not open during ischaemia, but does do so during reperfusion. Recovery of heart during reperfusion is improved in the presence of 0.2 µM CsA, suggesting that the MPT may be critical in the transition from reversible to irreversible reperfusion injury. (Mol Cell Biochem 174: 167–172, 1997)
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  • 66
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    Keywords: diabetes ; Ca2+-Mg2+-ATPase ; calcium ; liver plasma membrane
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The alteration in calcium transport in the liver of rats with streptozocin(STZ)-diabetic state was investigated. STZ (6 mg/100 g body weight) was subcutaneously administered in rats, and 1 or 2 weeks later they were sacrificed by bleeding. STZ administration caused a remarkable elevation of serum glucose concentration. Liver calcium content was significantly increased by STZ administration. Hepatic plasma membrane (Ca2+-Mg2+)-ATPase activity was markedly elevated by STZ administration. This increase was completely abolished by the presence of staurosporine (10-7-10-5 M), an inhibitor of protein kinase C, in the enzyme reaction mixture, suggesting an involvement of protein kinase C signalling. Moreover, the STZ-induced increase in liver plasma membrane (Ca2+-Mg2+)-ATPase activity was significantly raised by the presence of okadaic acid (10-5 and 10-4 M). Meanwhile, the STZ-increased (Ca2+-Mg2+)-ATPase activity was not appreciably altered by the presence of anti-regucalcin IgG in the reaction mixture, indicating that the activatory protein regucalcin does not participate in the elevation of the enzyme activity. The present study demonstrates that STZ-induced diabetes causes the increase in hepatic plasma membrane (Ca2+-Mg2+)-ATPase activity of rats.
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  • 67
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    Molecular and cellular biochemistry 184 (1998), S. 393-400 
    ISSN: 1573-4919
    Keywords: ATP synthase ; phosphorylation potential ; cytosolic pH ; reperfusion damage ; calcium ; free radicals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract A short period of ischemia followed by reperfusion produces a state of affairs in which the cells' potential for surviving longer ischemia is enhanced. This is called ischemic preconditioning. The effects of preconditioning are also related to the reperfusion damage which ensues upon tissue oxygenation. The role of the cellular energy state in reperfusion damage remains an enigma, although ischemic preconditioning is known to trigger mechanisms which contribute to the prevention of unnecessary ATP waste. In some species up to 80% of ATP hydrolysis in ischemia can be attributed to mitochondrial F1-F0-ATPase (ATP synthase), and a role for its inhibitor protein (IF1) in ATP preservation has been proposed. Although originally regarded as limited to large animals with a slow heart beat, inhibition by IF1 is probably a universal phenomenon. Coincidentally with ATPase inhibition, the decline in cellular ATP slows down, but even so the difference in ATP concentration between preconditioned and non-conditioned hearts is still small at the final stages of a long ischemia, when the beneficial effect of preconditioning is observable, although the energy state during reperfusion remains low in hearts which do not recover.
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  • 68
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    Keywords: calcium ; Ca2+-ATPase ; DNA fragmentation ; liver nuclei ; liver injury ; carbon tetrachloride
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    Notes: Abstract The alteration in calcium transport in the liver nuclei of rats orally administered carbon tetrachloride (CCl4) was investigated. Rats received a single oral administration of CCl4(5, 10, and 25%, 1.0ml/100 g body weight), and 5, 24 and 48 h later the animals were sacrificed. The administration of CCl4 (25%) caused a remarkable elevetion of calcium content in the liver tissues and the nuclei of rats. Liver nuclear Ca2+-ATPase activity was markedly decreased by CCl4 (25%) administration. The presence of dibutyryl cyclic AMP(10-4 and 10-3 M) or inositol 1,4,5-trisphosphate (10-6 and 10-5 M) in the enzyme reaction mixture caused a significant decrease in Ca2+-ATPase activity in the liver nuclei obtained from normal rat, while the enzyme activity was significantly increased by calmodulin (1.0 and 2.0 μg/ml). These signaling factor's effects were completely impaired in the liver nuclei obtained from CCl4 (25%)-administered rats. DNA fragmentation in the liver nuclei obtained from CCl4 -administered rats was significantly decreased by the presence of EGTA (2 mM) in the reaction mixture, suggesting that the endogenous calcium activates nuclear DNA fragmentation. The present study demonstrates that calcium transport system in the liver nuclei is impaired by liver injury with CCl4 administration in rats.
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  • 69
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    Molecular and cellular biochemistry 194 (1999), S. 159-164 
    ISSN: 1573-4919
    Keywords: free radicals ; ischemia-reperfusion ; sarcoplasmic reticulum ; Ca2+-Mg2+-ATPase ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Reactive oxygen species (ROS, free radicals) produced during cardiac ischemia and reperfusion can damage the contractile functions of arteries. The sarcoplasmic reticulum (SR) Ca2+ pump in coronary artery smooth muscle is very sensitive to ROS. Here we show that contractions of de-endothelialized rings from porcine left coronary artery produced by the hormone Angiotensin II and by the SR Ca2+ pump inhibitors cyclopiazonic acid and thapsigargin correlate negatively with the tissue weight. In contrast, the contractions due to membrane depolarization by high KCl correlate positively. Peroxide also produces a small contraction which correlates negatively with the tissue weight. When artery rings are treated with peroxide and washed, their ability to contract with Angiotensin II, cyclopiazonic acid and thapsigargin decreases. Thus, the SR Ca2+ pump may play a more important role in the contractility of the smaller segments of the coronary artery than in the larger segments. These results are consistent with the hypothesis that ROS which damage the SR Ca2+ pump affect the contractile function of the distal segments more adversely than of the proximal segments.
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  • 70
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    Molecular and cellular biochemistry 194 (1999), S. 173-177 
    ISSN: 1573-4919
    Keywords: calcium ; ATPase ; central nervous system ; phencyclidine ; inhibition ; in vitro ; in vivo
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Phencyclidine (PCP) is a potent psychotomimetic drug of abuse and has profound effect on the functioning of the central nervous system (CNS). Many of the CNS functions are known to be mediated by calcium (Ca2+). In the present study we have investigated the effects of PCP on Ca2+ ATPase activity in rat brain both in vitro and in vivo. For in vitro studies, synaptic membrane fractions prepared from normal rat brain were incubated with PCP at different concentrations (25-100 μM) before the addition of substrate. For n vivo studies, rats were treated with a single moderate dose of PCP (10 mg/kg, IP) and animals were sacrificed at 1,2, 6 and 12 h after treatment. Ca2+ ATPase activity in synaptic membrane fractions was assayed by estimation of inorganic phosphate. PCP inhibited the Ca2+ ATPase in vitro in a concentration dependent manner with significant effect at 50 and 100 μM. A significant time-dependent reduction of the Ca2+ ATPase activity was evident in vivo. As early as 2 h after the treatment of rats with PCP the ATPase activity was significantly reduced. The reduction of Ca2+ ATPase observed even at 12 h after treatment suggesting a prolonged presence of the drug in the brain tissue. Further, kinetic studies in vitro indicated PCP to be a competitive inhibitor of Ca2+ ATPase with respect to the substrate, ATP. The present findings indicate that PCP inhibits synaptic membrane Ca2+ ATPase thus altering cellular Ca2+ homeostasis in CNS which may partially explain the pharmacological effects of the drug and/or its neurotoxicity.
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  • 71
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    Keywords: ultraviolet radiation ; oxidative stress ; calcium ; phospholipase A2 ; thrombin ; V79 fibroblast
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract V79 fibroblasts were treated with ultraviolet (UV) C radiation alone as well as in conjunction with chronic oxidative stress. The effects of these treatments on calcium signaling were observed at 30 min post-irradiation. In the absence of extracellular calcium, thrombin released calcium from internal stores of UVC-irradiated V79 fibroblasts even after exposure to neomycin. In neomycin-treated control and chronic oxidative stress cells, no calcium release by thrombin was observed after chelation of external calcium. Calcium release by thrombin from internal stores of UV-irradiated and neomycin-treated cells was completely abolished by pretreatment with N-acetyl cysteine and dexamethasone. Cellular total soluble thiol content which is a good indicator of cellular reduced glutathione (GSH) level was significantly elevated 30 min after ultraviolet radiation, indicating an adaptive response after oxidative stress. Chronic oxidative stress alone resulted in a much smaller increase in GSH but chronic oxidative stress in conjunction with UVC produced a very prominent elevation in GSH levels. Our data suggest that thrombin can cause calcium release from internal stores of ultraviolet-irradiated fibroblasts which is independent of phosphatidylinositol bisphosphate hydrolysis and is directly related to the level of oxidative stress. Involvement of phopholipase A2 and a role for its products as possible mediators of calcium release from intracellular stores, is strongly indicated.
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  • 72
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    Molecular and cellular biochemistry 197 (1999), S. 25-29 
    ISSN: 1573-4919
    Keywords: regucalcin ; anti-regucalcin antibody ; protein phosphatase ; calcium ; rat liver cytosol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of anti-regucalcin monoclonal antibody on neutral phoshatase activity in rat liver cytosol was investigated. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate toward phosphatase asssy. Liver cytosolic phosphatase activity with three phosphoaminoacids was significantly increased in the presence of anti-regucalcin antibody (100 and 200 ng/ml) in the enzyme reaction mixture with calcium chloride (0.1 mM) or EGTA (1.0 mM). The effect of anti-regucalcin antibody was completely abolished in the presence of exogenous regucalcin (1.0 μM), indicating the involvement of endogenous regucalcin. The anti-regucalcin anti body- increased phosphatase activity was not significantly altered in the presence of trifluoperazine (20 μM), an antagonist of calmodulin, or akadaic acid (10 μM), an inhibitor of protein phosphatase, although these inihibitors caused a slight decrease in liver cytosolic phosphatase activity. The effect of endogenous regucalcin might be not related to calmodulin, and it was insensitive to okadaic acid. The present findings suggest that endogenous regucalcin is involved in the regulation of protein phasphatase in rat liver cytoplasm.
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  • 73
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    Keywords: isolated cardiac mitochondria ; cyclosporin A ; calcium ; magnesium ; oxidative phosphorylation ; high energy phosphate production ; Krebs cycle intermediates
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This study was designed to determine the effect of calcium and ADP-Mg on the oxidative phosphorylation in isolated cardiac mitochondria. The influence of cyclosporin A was also evaluated. The mitochondria were extracted from rat ventricles. Their oxidative phosphorylations were determined in two respiration media with different free Ca2+ concentrations. Respiration was determined with palmitoylcarnitine and either ADP- or ADP-Mg. With elevated free Ca2+concentrations and ADP-Mg, the transition state III to state IV respiration did not occurred. The ADP:O ratio was reduced. The phenomenon was not observed in the other experimental conditions (low free Ca2+ concentration with either ADP- or ADP-Mg or elevated free Ca2+ concentration with ADP-). Uncoupling was allied with a constant AMP production, which maintained an elevated ADP level in the respiration medium and prevented the return to state IV respiration. It was also observed in a respiration medium devoid of free Ca2+ when the mitochondria were pre-loaded with Ca2+. Uncoupling was inhibited by cyclosporin A. Furthermore, the Krebs cycle intermediates released from14C-palmitoylcarnitine oxidation revealed that succinate was increased by elevated free Ca2+ and ADP-Mg. Succinate is a FAD-linked substrate with low respiration efficiency. Its accumulation could account for the decreased ADP:O ratio. The Ca2+- and ADP-Mg-induced uncoupling might be partly responsible for the mechanical abnormalities observed during low-flow ischemia. (Mol Cell Biochem 000: 000-000, 1999)
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  • 74
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    Keywords: Salmonella typhimurium ; diarrhoea ; porins ; calcium ; protein kinase C ; free radicals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Attachment of Salmonella typhimurium to epithelial surfaces elicit significant alterations in different cell signalling events which lead to the development of disease. The present investigation was conducted to evaluate the effect of immunization of rats with porins, on gut physiologic markers following challenge with S. typhimurium. Male albino Wistar rats were immunized with purified porins and challenged by intragastric infection with S. typhimurium. Electrolyte transport, levels of different second messengers and inflammatory mediators were studied. A net absorption of transepithelial fluxes of Na+ and Cl- in immunized-challenged group and secretion in infected group was found. Ca2+ and 3-O-methyl-D-glucose fluxes did not show any change. Significant increase in the levels of [Ca+]i, cAMP, membrane form of protein kinase C, prostaglandins, NADPH oxidase, Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, total oxygen free radicals, reactive nitrogen intermediates, citrulline and lipid peroxidation was found in the infected group. However, in the immunized-challenged group, the values of all the parameters were found to be almost the same as that of control as well as immunized groups. Na+, K+-ATPase and calmodulin levels were unaltered in all the groups of animals. The results of this study thus suggest that immunization of rats with purified Salmonella porins followed by subsequent challenge with the organism might be helpful for the prevention of multiple physiologic derangements in isolated ideal cells.
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  • 75
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    Molecular and cellular biochemistry 151 (1995), S. 39-47 
    ISSN: 1573-4919
    Keywords: 7B2 ; calcium ; protein aggregation ; secretogranins ; protein sorting
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract To study the behavior of the neuroendocrine polypeptide 7B2 in the presence of calcium, various fragments of this molecule were produced inEscherichia coli as fusion proteins to glutathione S-transferase (GST). Addition of millimolar concentrations of Ca2+ to purified preparations of hybrid molecules carrying the N-terminal segment of 7B2 induced precipitation in a manner dependent on protein and cation concentrations. This precipitation occurred at pH 7.5 but not at pH 5.2. It was augmented by 4 and 8 mM ATP, and reduced by 12 and 24 mM ATP. ADP had a similar but weaker effect. Calcium failed to cause precipitation of GST alone or of GST fused to the C-terminal peptide 7B2156–186. However, when the latter protein was mixed with a GST protein carrying a short fragment of the N-terminal region of 7B2, both proteins were precipitated by calcium. Except for the pH dependence, the behavior of 7B2 fusion proteins in the presence of calcium and adenosine nucleotides are reminiscent of those exhibited by chromogranins and secretogranins, which, like 7B2, are acidic proteins found in the secretory granules of a variety of neuroendocrine cells. As suggested for other granins, this property may underlie the segregation of 7B2 fragments into secretory granules.
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  • 76
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    Molecular and cellular biochemistry 151 (1995), S. 55-60 
    ISSN: 1573-4919
    Keywords: regucalcin ; calcium ; gene expression ; kidney damage ; rat kidney cortex
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The alteration of Ca2+-binding protein regucalcin mRNA expression in the kidney cortex of rats administered cisplatin and cephaloridine, which can induce kidney damage, was investigated. Cisplatin (0.25, 0.5 and 1.0 mg/100 g body weight) or cephaloridine (25, 50 and 100 mg/100 g) was intraperitoneally administered in rats, and 1, 2 and 3 days later they were sacrificed. The alteration in serum findings after the administration of cisplatin (1.0 mg/100 g) or cephaloridine (50 and 100 mg/100 g) demonstrated chemically induced kidney damage; blood urea nitrogen (BUN) concentration increased markedly and serum inorganic phosphorus or calcium concentration decreased significantly. Moreover, the administration of cisplatin (1.0 mg/100 g) or cephaloridine (100 mg/100 g) caused a remarkable increase of calcium content in the kidney cortex of rats, indicating kidney damage. The expression of regucalcin mRNA in the kidney cortex was markedly reduced by the administration of cisplatin or cephaloridine in rats, when the mRNA levels were analyzed by Northern blotting using rat liver regucalcin cDNA (0.9 kb). The mRNA decreases were seen with the used lowest dose of cisplatin or cephaloridine. The present study clearly demonstrates that the mRNA expression of Ca2+-binding protein regucalcin in the kidney cortex of rats is decreased by chemically induced kidney damage.
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  • 77
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    Keywords: hyperthyroid heart ; high energy phosphates ; oxidative metabolism ; cardiac work ; calcium ; 31P-NMR spectroscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of calcium activation on energy production was investigated in isolated perfused hearts from rats treated with triiodothyronine (T3) during 15 days (0.2 mg/kg/day) and in hearts of rats allowed to recover after T3-treatment during 15 days. Changes in phosphorylated compound concentrations were followed in the isolated hearts perfused with a glucose-pyruvate medium by 31P-NMR spectroscopy, when the external calcium concentration was increased from 0.5–1, 1.5 and 2 mM. As expected, T3-treatment resulted in the hypertrophy of the heart (50% increase in HW/BW) that was nearly reversible 15 days after discontinuation of the treatment. When compared to controls, creatine, phosphocreatine (PCr) and glycogen contents were lower (58, 24 and 17% decrease respectively) in the hypertrophied hearts and higher (10, 14 and 18% respectively) after regression of hypertrophy. Intracellular pH, ATP, inorganic phosphate concentrations and the phosphorylation potential were not altered under T3-treatment and after regression of hypertrophy, while calculated free ADP concentration was lower in hypertrophied hearts (control: 40±2 μM, T3-treatment: 21±1 μM, regression: 37±1 μM). Increasing the calcium concentration induced a similar increase in left ventricular developed pressure in the three groups of hearts, with inorganic phosphate concentration increasing with cardiac work. The PCr concentration slightly decreased while the ATP concentration did not change. In spite of different initial PCr concentrations, the evolutions of PCr and Pi concentrations for each stepwise increase in external calcium were similar in the three groups. It is concluded that, in spite of the well-known decrease in efficiency induced by the drug, the mechanisms of PCr (ATP) production ramain able to respond to an acute moderate increase in energy demand provoked by a physiological stimulus. This adaptation also persists after the treatment when the energy metabolism balance is apparently improved.
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    Molecular and cellular biochemistry 151 (1995), S. 149-155 
    ISSN: 1573-4919
    Keywords: SERCA2 ; ATPase ; calcium ; transport ; vascular
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Pig coronary artery cultured smooth muscle cells were skinned using saponin. In the presence of an ATP-regenerating system and oxalate, the skinned cells showed an ATP-dependent azide insensitive Ca2+-uptake which increased linearly with time for 〉1 h. The Ca2+-uptake occurred with Km values of 0.20±0.03 μM for Ca2+ and 400±34 μM for MgATP2−. Thapsigargin and cyclopiazonic acid inhibited this uptake with IC50 values of 0.13±0.02 and 0.56±0.04 μM, respectively. These properties of SR Ca2+-pump are similar to those reported for membrane fractions isolated from fresh smooth muscle of coronary artery and other arteries. However, optimum pH of the uptake in the skinned cells (6.2) was lower than that reported previously using isolated membranes (6.4–6.8).
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  • 79
    ISSN: 1573-4919
    Keywords: calcium ; regucalcin ; calmodulin ; cyclic nucleotide phosphodiesterase ; rat liver
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    Notes: Abstract The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on Ca2+/calmodulin-dependent cyclic nucleotide (AMP) phosphodiesterase activity in rat liver cytosol was investigated. The addition of Ca2+ (50 µM) and calmodulin 160 U/ml in the enzyme reaction mixture caused a significant increase in cyclic AMP phosphodiesterase activity. This increase was inhibited by the presence of regucalcin (0.5-3.0 µM); the inhibitory effect was complete at 1.0 µM. Regucalcin (1.0 µM) did not have an appreciable effect on basal activity without Ca2+ and calmodulin. The inhibitory effect of regucalcin was still evident even at several fold higher concentrations of calmodulin (160–480 U/ml). However, regucalcin (1.0 µM) did not inhibit Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity in the presence of 100 and 200 µM Ca2+ added. Meanwhile, Cd2 (25–100 µM)-induced decrease in Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity was not reversed by the presence of regucalcin (1.0 µM). The present results suggest that regucalcin can regulate Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity due to binding Ca2+ in liver cells.
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  • 80
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    Molecular and cellular biochemistry 154 (1996), S. 113-121 
    ISSN: 1573-4919
    Keywords: heart cells ; nucleus ; calcium ; R-type channel ; excitation-contraction coupling ; pacemaker activity ; Fura-2 ; Fluo-3 ; confocal microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In the present study, Fluo-3 Ca2+ measurement and confocal microscopy techniques were used in order to localize cytosolic [ ]c and nuclear [ ]n free Ca2+ distribution in resting and spontaneously contracting single heart cells from 10-day-old chick embryos. In resting single cells, the concentration of Ca2+ in the cytoplasm was lower than that in the nucleus. Increasing cytosolic free Ca2+ from 100–1600 nM gradually increased [Ca2+]n with a maximum capacity near 1200 nM. Results from Fura-2 microfluorometry and Fluo-3 confocal microscopy suggest a potential cross talk between the increase of cytosolic free Ca2+ and the uptake and release of Ca2+ by the nucleus during spontaneous contraction of single myocytes. Calcium waves in spontaneously contracting cells were found to spread from one cell to the next with the nucleus acting as a fluorescent beacon in which Ca2+ levels remained elevated for several milliseconds even after cytosolic Ca2+ had returned to near basal values. These results strongly suggest that the nucleus plays a negative and positive feedback role in controlling cytosolic free Ca2+ concentration during excitation-contraction coupling in heart cells.
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  • 81
    ISSN: 1573-4919
    Keywords: calcium ; calcium transport ; brain ; calcium-regulating hormone ; calcium-antagonist ; energy dependency
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The characterization of calcium accumulation in the brain of rats administered orally calcium chloride solution was investigated. Rats received a single oral administration of calcium (15–50 mg/100 g body weight), and they were sacrificed by bleeding-between 15 and 120 min after the administration. The administration of calcium (50 mg/100 g) produced a significant increase in serum calcium concentration and a corresponding elevation of brain calcium content, indicating that the transport of calcium into the brain is associated with the elevation of serum calcium levels. The increase in brain calcium content by calcium administration was not appreciably altered by the pretreatment with Ca2+ channel blockers (verapamil or diltiazem with the doses of 1.5 and 3.0 mg/100 g). In thyroparathyroidectomized rats, the administration of calcium (50 mg/100 g) caused a significant increase in brain calcium content, indicating that calcium-regulating hormones do not participate in the brain calcium transport. Now, brain calcium content was clearly elevated by fasting (overnight), although serum calcium level was not significantly altered. Calcium administration to fasted rats induced a further elevation of brain calcium content as compared with that of control (fasted) rats. The fasting-induced increase in brain calcium content was appreciably restored by refeeding. This restoration was also seen by the oral administration of glucose (0.4 g/100 g) to fasted rats. The present study demonstrates that serum calcium is transported to brain, and that the increased brain calcium is released promptly. The release of calcium from brain may be involved in energy metabolism, and this release may be weakened by the reduction of glucose supply into brain. The finding suggests a physiological significance of energy-dependent mechanism in the regulation of brain calcium.
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  • 82
    ISSN: 1573-4919
    Keywords: hydroxyl radical ; oxidant ; hydrogen peroxide ; smooth muscle tissue ; mitochondria ; calcium
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We sought to investigate the mechanism(s) by which the oxidant H2O2 stimulates Ca2+ release from mitochondria of bovine pulmonary vascular smooth muscle tissue and to test the hypothesis that hydroxyl radical is involved in this phenomenon. Treatment of the smooth muscle tissue with 1 mM H2O2 dramatically stimulated hydroxyl radical generation as measured by methane (CH4) production by GLC using dimethylsulfoxide (DMSO) as the substrate. Pretreatment of the mitochondria with the hydroxyl radical scavanger dimethylthiourea (DMTU) prevented the increase in CH4 production caused by H2O2. In the absence of EGTA, H2O2 caused stimulation of Ca2+ release from mitochondria occurred with a lag time of about 4 min. Addition of EGTA to Ca2+ loaded mitochondria resulted an immediate loss of Ca2+ and that has been found to be augmented by H2O2. The release of Ca2+ by H2O2 did not appear to occur with concommitant increase in sucrose entry into, K+ release from, and swelling of mitochondria when the Ca2+ cycling was prevented by EGTA. These observations suggested that H2O2-mediated Ca2+ release from bovine pulmonary vascular smooth muscle tissue mitochondria occurred (i) through the involvement of hydroxyl radical; (ii) via specific pathway(s); and (iii) did not appear to happen primarily via nonspecific ‘pore’ formation.
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  • 83
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    Molecular and cellular biochemistry 173 (1997), S. 169-175 
    ISSN: 1573-4919
    Keywords: regucalcin ; calcium-binding protein ; calcium ; nuclear RNA synthesis ; regenerating ratliver
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of regucalcin, a Ca2+-bindingf protein isolated from rat livercytosol, on ribonucleic acid (RNA) synthesis in the nuclei of normal ratliver and of regenerating rat liver was investigated. The liver weight at 1day after partial hepatectomy was increased about 50% of that ofsham-operated (control) rats. Calcium chloride (1.0-20 µM Ca2+ asfinal concentration) was added into the reaction mixture of nuclear RNAsynthesis. RNA synthesis was established by incorporation of [3H]-uridine5'-triphosphate (UTP) into the nuclear RNA. Addition of Ca2+ (5 and 10µM) caused a significant increase of RNA synthesis in the nuclei fromcontrol rat liver. Such effect of Ca2+ was potentiated in the nuclei ofregenerating liver; nuclear RNA synthesis was increased about 2 fold by the1.0 and 2.5 µM Ca2+ addition. The stimulatory effect of Ca2+ wassignificantly inhibited by the presence of a-amanitin (10-8 M), an inhibitorof RNA polymerase II. The presence of regucalcin (0.25 and 0.5 µM)significantly inhibited RNA synthesis in the nuclei from control rat liverand from regenerating rat liver. The inhibitory effect of regucalcin wasremarkable in the presence of EGTA (0.5 mM), and it was weakened by theaddition of Ca2+ (5 µM). Such regucalcin effect was not seen in thepresence of a-amanitin. The presence of anti-regucalcin IgG in the reactionmixture significantly increased RNA synthesis in the nuclei from control ratliver, indicating that the endogenous regucalcin may be involved in nuclearRNA synthesis. The present resuits demonstrate that regucalcin can inhibitnuclear RNA synthesis in rat liver. Regucalcin may have an inhibitory rolein the regulation of liver nuclear RNA synthesis.
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  • 84
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    Molecular and cellular biochemistry 149-150 (1995), S. 175-182 
    ISSN: 1573-4919
    Keywords: rat pancreas ; cholecystokinin ; magnesium ; calcium ; acetylcholine ; amylase secretion
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    Notes: Abstract Application of either acetylcholine (ACh, 10−5 M) or cholecystokininoctapeptide (CCK-8, 10−8 M) to the isolated rat pancreas elicited large increases in amylase secretion, radiolabelled45Ca2+ influx and cytosolic free calcium [Ca2+]i levels in zero and normal (1.1 mM) extracellular magnesium [Mg2+]o. Elevated [Mg2+]o significantly (p〈0.001) reduced the secretagogueevoked secretory responses and Ca2+ mobilisation. Stimulation of pancreatic segments with either ACh (10−5 and 10−6 M) or CCK-8 (10−8 and 10−10 M) resulted in marked elevation in Mg2+ concentration in effluent samples (net efflux). On removal of either ACh or CCK-8, Mg2+ concentration returned to resting level. In pancreatic acinar cells loaded the flourescent dye magfura, ACh and CCK-8 evoked marked reduction in cytosolic free Mg2+ concentration [Mg2+]i compared to the resting value of 0.82±0.03 mM (n=50) in normal medium in the absence of secretagogues. In elevated [Mg2+]o (10 mM) medium, [Mg2+]i rises to 0.98±0.04 mM (n=6). Addition of CCK-8 led to only a small reduction in [Mg2+ i in elevated [Mg2+]o. In Mg2+ loaded pancreatic acinar cells, Mg2+ is released in a time dependent manner and this efflux of Mg2+ was sensitive to sodium, extracellular amiloride (1 mM), dinitrophenol (10 mM) and lidocaine (1 mM). The results indicate that Mg2+ is acting as an intracellular messenger to regulate the mobilisation of Ca2+ which in turn mediates enzyme secretion.
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  • 85
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    Molecular and cellular biochemistry 176 (1997), S. 317-326 
    ISSN: 1573-4919
    Keywords: calcium ; metabolism ; glucose ; hypoxia ; rat brain
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In a previous communication we reported that glucose deprivation from KHRB medium resulted in a marked stimulation of Ca2+ uptake by brain tissue, suggesting a relationship between glucose and Ca2+ homeostasis in brain tissue [17]. Experiments were carried out to investigate the significance of glucose in Ca2+ transport in brain cells. The replacement of glucose with either D-methylglucoside or 2-deoxyglucose, non-metabolizable analogues of glucose, resulted in stimulation of Ca2+ uptake just as by glucose deprivation. These data show that glucose metabolism rather than glucose transfer was necessary to stimulate Ca2+ uptake in brain tissue. Inhibition of glucose metabolism with either NaF, NaCN, or iodoacetate resulted in stimulation of Ca2+ uptake similar to that produced by glucose deprivation. These results lend further support for the concept that glucose metabolism is essential for Ca2+ homeostasis in brain. Anoxia promotes glucose metabolism through glycolytic pathway to keep up with the demand for ATP by cellular processes (the Pasteur effect). Incubation of brain slices under nitrogen gas did not alter Ca2+ uptake by brain tissue, as did glucose deprivation and the inhibitors of glucose metabolism. We conclude that glucose metabolism resulting in the synthesis of ATP is essential for Ca2+ homeostasis in brain. Verapamil and nifedipine which block voltage-gated Ca2+ channels, did not alter Ca2+ uptake stimulated by glucose deprivation, indicating that glucose deprivation-enhanced Ca2+ uptake was not mediated by Ca2+ channels. Tetrodotoxin which specifically blocks Na+ channels, abolished Ca2+ uptake enhanced by glucose deprivation, but had no effect on Ca2+ uptake in presence of glucose (controls). These results suggest that stimulation of Ca2+ uptake by glucose deprivation may be related to Na+ transfer via Na-Ca exchange in brain.
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  • 86
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    Molecular and cellular biochemistry 201 (1999), S. 159-167 
    ISSN: 1573-4919
    Keywords: phospholipases A1, A2 and C ; Ureaplasma urealyticum ; calcium ; plasma membrane ; phospholipids ; pH ; detergents
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The presence of endogenous phospholipase A (PL-A) activity of U. urealyticum hydrolyzing the acyl ester bond and phospholipase C (PL-C) activity hydrolyzing the phosphodiester bond is primarily localized in the membranes of ureaplasmas. Characterization of the membrane PL-A and PL-C activity in exponential growing cells of serovars 3, 4, and 8 was investigated. The pH optimum was about 8.5-9 for phospholipase A1 (PL-A1) in the three serovars. A more acidic pH optimum of 6 was observed for phospholipase A2 (PL-A2) enzymes in serovars 3 and 4. However, a very significant stimulation of PL-A2 activity in serovar 8 occurred around pH 7. The specific activity of PL-A2 was always 50-100 fold higher than PL-A1 activity in the pH range studied. Ca2+ ions only slightly stimulated PL-A1 activity in all 3 serovars. PL-A2 activity was stimulated about 6-fold from 0.5-0.8 mM Ca2+ ion concentrations for serovar 3 and 12-fold for serovar 8. Only lower concentrations (0.2-0.4 mM) of calcium stimulated PL-A2 activity in serovar 4. EDTA inhibition corresponded to Ca2+ stimulation for PL-A2 activity for serovars 3 and 8. A general stimulation of PL-A2 activity by diethyl ether was evident but the degree of stimulation varied with the serovar. Sodium deoxycholate enhanced PL-A activity of serovars 4 and 3, but partially inhibited that of serovar 8. PL-A activity in the three serovars were not significantly affected by p-hydroxymercuribenzoate, a marker of -SH groups in the enzyme. All 3 serovars were inactivated by heat. A broad pH optimum for PL-C activity was evident around 7-8. Diethyl ether enhanced PL-C activity of serovar 8. Sodium deoxycholate and heat were inhibitory to PL-C activity. The results demonstrate that the major characteristics of ureaplasma membrane bound PL-A and PL-C are basically similar to those of other mollicutes and bacteria. However, the major differences in the specific characteristics of specially PL-A1 and PL-A2 suggest that the ureaplasma phospholipases are unique enzymes different from the phospholipases of bacteria. Both the PL-A and PL-C enzymes function over the broad range at which ureaplasma can grow, pH 5-9 essential for survival. The ureaplasma PL-As are also markedly different from one serovar to another. This variation in specific activity could contribute significantly to differences in virulence among serovars in specific host milieus. There is significant variation from acidic pH of the vagina and alveolar surface of the lung to a more neutral pH of the endometrium and placenta. There are marked differences in calcium concentrations under specific circumstances in various host tissues. Thus the differences in specific activity among the phospholipases of the serovars of U. urealyticum may be of physiological importance in interactions with host tissues and pathogenesis of disease.
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  • 87
    ISSN: 1573-0646
    Keywords: somatostatin ; angiogenesis ; somatostatin receptors ; signal transduction ; xanthines ; calcium ; proteins
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  • 88
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    Bulletin of experimental biology and medicine 83 (1977), S. 155-158 
    ISSN: 1573-8221
    Keywords: ouabain ; cyclic AMP ; calcium ; mitochondria
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    Topics: Biology , Medicine
    Notes: Abstract The action of ouabain and cyclic AMP on the Ca-accumulating capacity and outflow of Ca2+ ions from loaded rat heart mitochondria was studied by the tetracycline probe method. In the course of the investigations no effect of ouabain on these processes was found. Cyclic AMP did not act on Ca binding by the mitochondrial membrane but it induced rapid liberation of Ca2+ from organelles loaded with these ions.
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  • 89
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    Bulletin of experimental biology and medicine 81 (1976), S. 639-641 
    ISSN: 1573-8221
    Keywords: blood plasma ; magnesium ; calcium ; strontium ; cation-exchange method
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    Topics: Biology , Medicine
    Notes: Abstract Adsorption of Mg++, Ca++, and Sr++ ions from physiological solutions and from solutions containing canine blood plasma and physiological saline in different proportions was studied by a cation-exchange method on KU-2×8 resin in the balanced salt form. Equilibrium constants were calculated from the experimental results and used to determine the molar fractions (or percentage content) of “free” and “bound” ions of the corresponding metals.
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  • 90
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    Bulletin of experimental biology and medicine 86 (1978), S. 1582-1585 
    ISSN: 1573-8221
    Keywords: hypoparathyroidism ; calcium ; mono- and polysynaptic reflex ; posttetanic potentiation ; efferent output
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    Notes: Abstract Experiments on parathyroidectomized cats with motor disorders of different degrees of severity showed disturbance of spinal reflex activity, in the form of activation of motoneurons and the interneuron system, weakening of posttetanic potentiation, and facilitation of conduction in the reflex arc. If severe tetany was present monosynaptic responses were reduced or, in some cases, abolished completely and polysynaptic reflex discharges were potentiated. After intravenous injection of calcium chloride into the experimental animals a tendency was found for recordable reflexes to return. It is concluded that disturbance of the functional state of the spinal segmental apparatus was largely due to hypocalcemia and it may be one of the causes of tetany in hypoparathyroidism.
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  • 91
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    Bulletin of experimental biology and medicine 88 (1979), S. 696-699 
    ISSN: 1573-8221
    Keywords: hypertension ; ACTH ; lipolysis ; calcium
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    Topics: Biology , Medicine
    Notes: Abstract The action of ACTH on lipolysis was studied in the adipose tissue of rats with spontaneous and renal hypertension and also in normotensive rats of corresponding control groups. The sensitivity of the adipose tissue of SHR rats to ACTH was shown to be higher than in the normotensive control. Evidence was obtained that this increase in sensitivity is due to the state or quantity of intracellular calcium. In rats with renal hypertension no such increase in sensitivity of their adipose tissue to ACTH was found.
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  • 92
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    Bulletin of experimental biology and medicine 111 (1991), S. 151-154 
    ISSN: 1573-8221
    Keywords: zinc ; calcium ; diabetes ; pancreatic islets
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  • 93
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    Bulletin of experimental biology and medicine 109 (1990), S. 50-52 
    ISSN: 1573-8221
    Keywords: HeLa ; histamine ; calcium ; phorbol esters
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  • 94
    ISSN: 1573-675X
    Keywords: Activation markers ; apoptosis ; calcium ; cytotoxicity ; mistletoe lectins ; Viscum album.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract This review focuses on the cytotoxic properties of Viscum album L. (VAL). Apart from well-established results of protein synthesis inhibition by the mistletoe lectins (MLs), namely their catalytic A chain, there is now convincing evidence that the VAL-mediated cytotoxicity is mainly due to an induction of apoptosis. Among the more than 1,000 proteins detected in VAL, the MLs and the viscotoxins (VTs) are the predominant toxic proteins. Using purified components, such as the D-galactose-specific ML I, the N-acetyl-D-galactosamine-specific ML II and ML III, crude VTs and oligosaccharides, only the MLs induced apoptosis. The in vitro studies suggest that interaction of lectin B chains with appropriate receptors on the cell surface activates distinct signalling pathways that ultimately leads to apoptosis in a large fraction of cells, while others survive, however, with a conservation of their DNA. Inhibition of protein synthesis by the A chain of the hololectin probably accelerates the B chain-induced course of events.
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  • 95
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    Cell biology and toxicology 9 (1993), S. 141-148 
    ISSN: 1573-6822
    Keywords: genistein ; xenobiotic receptor ; calcium ; inositol ; zinc ; nickel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Cd2+ provokes inositol trisphosphateproduction and releases stored Ca2+, apparently by binding to a zinc site in the external domain of an orphan receptor. One pM Cd2+ evokes an immediate spike in cytosolic free Ca2+, which is similar to that evoked by bradykinin. Platelet-derived growth factor (PDGF) also increases free Ca2+ in human dermalfibroblasts, but there is a distinct lag before free Ca2+ rises in response to PDGF. Genistein, which selectively inhibits tyrosine kinases, markedly inhibited Ca2+ mobilization evoked by PDGF. Calcium mobilization triggered by cadmium or bradykinin was relatively insensitive to genistein. The PDGF receptor is known to be a tyrosine kinase, whichphosphorylates and thereby activatesphospholipase Cψ, whereas a G protein couples the bradykinin receptor to anotherphospholipase C isoform. These findings support the hypothesis that the orphan receptor triggered by cadmium is coupled to phospholipase C via a G protein.
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  • 96
    ISSN: 1573-6822
    Keywords: calcium ; dihydropyridine ; lactotroph ; TRH
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    Topics: Biology , Medicine
    Notes: Abstract Prolactin (PRL) release and intracellular free calcium concentration [Ca2+]i were measured in two populations of normal rat lactotrophs (light and heavy fractions) in culture. Spontaneous PRL release of heavy fraction cells was more sensitive to dihydropyridines (DHPs; Bay K 8644 and nifedipine) when compared to the light fraction lactotrophs. The stimulatory effect of thyrotropin-releasing hormone (TRH) on PRL release from heavy fraction cells was inhibited by Cd2+ and mimicked by Bay K 8644. Indo-1 experiments revealed that TRH-increased [Ca2+]i was reversibly inhibited by Cd2+. In a Ca2+-free EGTA-containing medium, TRH did not modify [Ca2+]i.
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  • 97
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    Cell biology and toxicology 10 (1994), S. 317-322 
    ISSN: 1573-6822
    Keywords: bradykinin ; calcium ; ionomycin ; scanning cytometry
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    Topics: Biology , Medicine
    Notes: Abstract Ligand-receptor affinity is classically demonstrated by measuring ligand binding density to a specific site on membrane preparations, and receptor function is studied by measuring calcium flux, cell by cell, using microspectrofluorimetry. In order to study these phenomena in a larger cell population, calcium flux was measured in MRC-5 cell line expressing the B2 receptor for bradykinin using an ACAS 570 scanning cytometer. Following incorporation of fluo3/AM, different ligands were studied, singly or in association with bradykinin. This study confirmed that only the B2 receptor is present on the plasma membrane of MRC-5 cells. Bradykinin binding to the B2 receptor was not modified by a B1 agonist (Des-Arg9-bradykinin) or by a B1 antagonist (Des-Arg9-[Leu8]-bradykinin) but was inhibited by a B2 agonist ([Hyp3]-bradykinin) and a B2 antagonist (HOE 140). The source of free calcium was also studied in comparison with ionomycin. The intensity of the calcium peak after binding of bradykinin is independent of the concentration of extracellular calcium. Preincubation with diltiazem or TMB-8 did not modify calcium flux indicating that transduction of the signal after bradykinin binding in this cell line is independent of voltage-dependent channels and does not require mobilization of intracellular calcium blocked by TMB-8. In conclusion, scanning cytometry can be used to study ligand-receptor binding and to obtain results rapidly from multiple cells. Recording of individual cell variations and kinetics enables identification of active agonists or antagonists and consequently the selection of new compounds.
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  • 98
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    Cell biology and toxicology 11 (1995), S. 1-10 
    ISSN: 1573-6822
    Keywords: calcium ; hippocampal neurons ; neuronal differentiation ; organic lead ; triethyl lead
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Triethyl lead is the major metabolite of tetraethyl lead, which is used in industrial processes and as an antiknock additive to gasoline. We tested the hypothesis that low levels of triethyl lead (0.1 nmol/L to 5μmol/L) interfere with the normal development of cultured E18 rat hippocampal neurons, possibly through increases in intracellular free calcium ion concentration, [Ca2+]in. The study assessed survival and differentiation using morphometric analysis of individual neurons. We also looked at short-term (up to 3.75-h) changes in intracellular calcium using the calcium-sensitive dye fura-2. Survival of neurons was significantly reduced at 5 μmol/L, and overall production of neurites was reduced at ≥2 μmol/L. The length of axons and the number of axons and dendrites were reduced at ≥1 μmol/L. Neurite branching was inhibited at 10 nmol/L for dendrites and 100 nmol/L for axons. Increases in intracellular calcium were observed during a 3.75-h exposure of newly plated neurons to 5 μmol/L triethyl lead. These increases were prevented by BAPTA-AM; which clamps [Ca2+]in at about 100 nmol/L. Culturing neurons with BAPTA-AM and 5 μmol/L triethyl lead did not reverse the effects of triethyl lead, suggesting that elevation of [Ca2+]in is not responsible for decreases in survival and neurite production. Triethyl lead has been shown to disrupt cytoskeletal elements, particularly neurofilaments, at very low levels, suggesting a possible mechanism for its inhibition of neurite branching at nanomolar concentrations.
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  • 99
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    Cell biology and toxicology 12 (1996), S. 245-249 
    ISSN: 1573-6822
    Keywords: acrolein ; bronchial hyperresponsiveness ; calcium ; ozone ; excitation-contraction coupling
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The aims of this work were (1) to determine the dose-response relationship between ex vivo exposure to oxidizing pollutants such as nitrogen dioxide (NO2), the aldehyde acrolein, and ozone (O3), and the reactivity to agonists in isolated human bronchial smooth muscle; and (2) to investigate the alterations in the cellular mechanisms of human airway smooth muscle contraction induced by such exposures. Experiments were performed in isolated human bronchi obtained at thoracotomy. Isometric contraction in response to a variety of agonists was compared between pollutant-exposed preparations and paired controls. Short exposures to NO2, acrolein, or O3 altered the subsequent airway smooth muscle responsiveness in a dose-dependent manner. The cellular mechanisms producing the airway hyperresponsiveness observed in vitro are shared by the three pollutants and include alterations in airway smooth muscle excitation-contraction coupling as well as indirect effects on neutral endopeptidase activity.
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  • 100
    ISSN: 1573-6822
    Keywords: ATP ; energy metabolism ; calcium ; diltiazem ; rat liver perfusion ; ATP
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    Notes: Abstract Diltiazem causes vasoconstriction in the liver when present at high concentrations, an action that is strictly Ca2+-dependent. Diltiazem is also active on energy metabolism. This toxic action could be partly a consequence of hemodynamic effects. In the absence of Ca2+, the hemodynamic effects are no longer present and, consequently, Ca2+-free experiments are useful for distinguishing between hemodynamics-dependent and hemodynamics-independent effects. The experimental system used was the hemoglobin-free perfused rat liver from fed and fasted rats. Diltiazem was infused at various concentrations in the presence and absence of Ca2+. Several metabolic parameters were measured: lactate and pyruvate production (glycolysis), glycogenolysis, oxygen uptake, gluconeogenesis, and the cellular levels of lactate, pyruvate, glucose, AMP, ADP, and ATP. The effects of diltiazem can be divided into three groups: (1) Effects that are strictly dependent on the Ca2+-mediated hemodynamic action. This group comprises inhibition of oxygen uptake at all concentrations (50–500 μmol/L) inhibition of lactate, pyruvate, and glucose release at high concentrations; the decrease in cellular ATP; the increase in cellular AMP; and the cellular accumulation of glucose and lactate. (2) Effects that are independent of the hemodynamic action. The most relevant effect of this type is inhibition of gluconeogenesis. (3) Effects that are influenced by Ca2+ but are independent of the hemodynamic effects. This is the typical case of lactate and glucose release from endogenous glycogen, whose stimulation by low diltiazem concentrations is more pronounced in the presence of Ca2+ than in its absence.
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