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  • Articles  (306)
  • Biochemistry and Biotechnology  (306)
  • 2015-2019
  • 1990-1994  (306)
  • 1950-1954
  • 1993  (306)
  • Chemistry and Pharmacology  (255)
  • Medicine  (51)
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  • Articles  (306)
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  • 2015-2019
  • 1990-1994  (306)
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  • 1
    Electronic Resource
    Electronic Resource
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 261-266 
    ISSN: 0884-3996
    Keywords: GroESL ; Lux-R-I complex ; V. fischeri ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: htpR- (rpoH, σ32 minus) strain of E. coli harbouring the whole lux system of Vibrio fischeri is very dim. We have recently shown that GroESL proteins fully recover the expression of the lux system in this strain. This work has been undertaken to study our assumption that the GroESL proteins stabilize the LuxR protein, thus enhancing the formation of LuxR-Inducer complex. E. coli htpR- cells harbouring the luxR gene were unable to bind extracellularly added inducer, while late logarithmically growing htpR+ strain bound small quantities of the inducer. Reduction in the nutrient content of the growth medium resulted in a large increase in the capability of these cells to bind the inducer. htpR+ or htpR- E. coli strains harbouring both the luxR and the groESL genes bound large quantities of the inducer. The molecular and ecological significance of these results is discussed.
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  • 2
    Electronic Resource
    Electronic Resource
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993) 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 3
    Electronic Resource
    Electronic Resource
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993) 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 4
    Electronic Resource
    Electronic Resource
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 1-1 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 5
    Electronic Resource
    Electronic Resource
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 3-14 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In the 13 years since it was first published the ‘Uniform requirements for manuscripts submitted to biomedical journals’ (the Vancouver style) has proved popular with both authors and editors; over 400 journals have stated that they will consider manuscripts that conform to its requirements and we know that many more do so. The fourth edition, reproduced here, incorporates recent amendments made by the group.
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  • 6
    Electronic Resource
    Electronic Resource
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 301-305 
    ISSN: 0884-3996
    Keywords: Bioluminescence ; Ca2+-activated photoprotein ; obelin ; singlet oxygen ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The luminescence of obelin is initiated by NaOCl in a reaction mixture containing no calcium. The addition of Mn2+ enhances the light emission 〉300-fold. Sodium azide and histidine, as singlet oxygen quenchers, inhibit NaOCl-activated obelin luminescence in the presence or absence of Mn2+. This suggests that the addition of NaOCI to the mixture causes singlet oxygen formation (stimulated by Mn2+ ions), and singlet oxygen initiates the light-emitting reaction.
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  • 7
    Electronic Resource
    Electronic Resource
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 315-323 
    ISSN: 0884-3996
    Keywords: Anhydride (norbornene) cured epoxy ; thermal oxidation ; chemiluminescence ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Networks were prepared by curing diglycidyl ether of bisphenol A (DGEBA) with variable concentrations of norbornene anhydride (NA). Almost completely cured samples with anhydride/epoxide (A/E) inolar ratios of respectively 0.8, 0.9, 1.0 and 1.1, and one incompletely cured sample with A/E = 1.0, were studied by chemiluminescence in the temperature range 135-220°C, using isothermal stationary or non-stationary (atmosphere change) exposures. The comparison of kinetic curves of intensity variation reveals: the importance of unreacted epoxide groups as sources of highly emissive radical species, the lowering effect of oxidation products, and the increasing effect of the decrease of macromolecular mobility due to crosslinking in the case of the incompletely cured sample. Most of the features of kinetic curves obtained in non-stationary experiments are explained in terms of radical formation mechanisms during exposure in inert atmosphere. The results show clearly that chemiluminescence is due to reactions of peroxy radicals rather than hydroperoxide groups.
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  • 8
    Electronic Resource
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    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 39-48 
    ISSN: 0884-3996
    Keywords: Microtox® ; nicotine ; cotinine ; biological monitoring ; urine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The aim was to investigate the factors influencing light emission from Photobacterium phosphoreum in the Microtox® test to interpret bioassay results for urine. Four reference urines were assessed as reference materials for the bioassay. Nicotine and cotinine were investigated as urinary markers for tobacco exposure. The optimum luminescence conditions were: 1.85%-3.25% NaCl, 0.33-0.58 mol/L ionic strength, and pH 5.8-6.7. Low pH values and high concentration of toxic trace metals were important factors in this study. Unexpacted toxicity for a Standard Reference Material was attributed to zinc contamination. Nicotine and cotinine together exhibited antagonistic effects in 2% saline but this could not be observed in the urines because of substantial urine toxicity. Thus practical urinary biological monitoring with the Microtox® test necessitates excretion of metabolites and compounds that are much more toxic than the urine components. Also, separation of the effects of physical factors like pH, ionic strength and dilution is essential before chemical toxicity effects can be assigned. This is the first report of Microtox® EC50 values for nicotine and cotinine. The results have application to environmental samples since analyses are often uncontrolled relative to pH, ionic strength and dilution.
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  • 9
    ISSN: 0884-3996
    Keywords: Kits ; reagents ; immunoassay ; probes ; labels ; nucleic acid hybridization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: This survey was compiled between January and November 1992 from public domain information requested by the author from companies specializing in these products. It includes individual sections giving the name and address of each company together with brief details of their (a) kits used for assay of certain analytes in the routine laboratory and (b) reagents used mainly as research tools. This survey is a companion to a previous survey about 90 commercially available luminometers (Stanley PE. J Biolumin Chemilum 1992; 7: 77-108 and 157-69).
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  • 10
    Electronic Resource
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    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 267-291 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The following references encompass a review of recent literature where the in vivo expression of eukaryotic and/or prokaryotic luciferases provide for sensitive reporters of cellular activity. The list is subdivided into prokaryotic and eukaryotic applications. We have included the uses of luciferases in elucidating the control of gene expression or for monitoring cell viability. We have not included papers cited by Stanley and Stewart (J Biolumin Chemilumin 1990; 5:141-52) nor have we included papers on the structure and regulation of luciferases as this now substantial literature will be the subject of a future review. References cited in both this review and previous ones are referred to by the number assigned to them in the earlier review.
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  • 11
    Electronic Resource
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    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 331-331 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 12
    Electronic Resource
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    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 153-158 
    ISSN: 0884-3996
    Keywords: Chemiluminescence ; bronchoalveolar lavage ; alveolar macrophages ; monocytes ; adherence ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The chemiluminescence of peripheral blood monocytes and alveolar macrophages was determined in the presence of luminol and lucigenin, either before or after the cell adherence to the luminometer curvettes. In the case of monocytes, cell adherence induces an increase of luminol-dependent chemiluminescence and has almost no effect on the lucigenin-dependent chemiluminescence. However, it shows a strong inhibition of the lucigenin-dependent chemiluminescence and almost no effect on luminol-dependent chemiluminescence, in the case of alveolar macrophages. These results show that adhesion to plastic alters the metabolic burst of both monocytes and alveolar macrophages. Although the mechanisms are poorly understood, they seem to be related to the modifications that take place during the differentiation of peripheral monocytes to alveolar macrophages.
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  • 13
    Electronic Resource
    Electronic Resource
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 159-167 
    ISSN: 0884-3996
    Keywords: Mononuclear cells ; chemiluminescence ; antigen stimulation ; interferon-gamma ; T lymphocytes ; Candida albicans ; Rabies (Lyssa) virus ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Stimulation of phagocytes by several cytokines causes superoxide generation and consequently chemiluminescence. Since antigen-activated lymphocytes generate cytokines, we investigated whether antigen recognition by mononuclear cells, which contain both lymphocytes and monocytes, is accompanied by changes in lucigenin-dependent chemiluminescence. Mononulcear cells which underwent antigen-induced proliferation showed a delayed rise in lucigenin-dependent chemiluminescence in the absence of other stimuli. The common recall antigen Candida albicans increased spontaneous chemiluminescence of mononuclear cells from unselected donors up to 20-fold over control values after 48-72h of culture. With Rabies virus vaccine as specific antigenic stimulus, only mononuclear cells from rabies immunized individuals responded with enhanced delayed chemiluminescence. In contrast to opsonized zymosan and phorbol myristate acetate, antigens induced no oxidative burst within one hour after addition. Delayed mononuclear cel chemiluminescence was inhibited by the superoxide scavenger superoxide dismutase and by di-phenylene iodonium, a selective inhibitor of the phagocyte NADPH oxidase. A neutralizing monoclonal antibody against interferon-gamma completely abrogated antigen-induced chemiluminescence. Recombinant interferon-gamma by itself induced delayed mononuclear cell chemiluminescence. Thus, antigen-induced delayed mononuclear cell chemiluminescence represents activation of phagocyte NADPH oxidase by interferon-gamma generated by activated lymphocytes.
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  • 14
    Electronic Resource
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    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 241-246 
    ISSN: 0884-3996
    Keywords: Chemiluminescent FIA ; bile acid ; glucose ; ATP ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We have developed a chemiluminescent flow injection method for analysis of bile acid, glucose and ATP using the chemiluminescent assay of NADH using 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(IL)/microperoxidase (m-POD) system and immobilized enzyme reactors such as 3α-hydroxysteroid dehydrogenase, glucosedehydrogenase, hexokinase and glucose-6-phosphate dehydrogenase. The standard curves were obtained in the range of 5 ∼ 100 pmol for bile acid, 0.5 ∼ 5.0 nmol for glucose and 10-7 ∼ 10-5 mol/L for ATP. The coefficient of variation for each assay was not more than 4.1% for bile acid, 2.3% for glucose and 5.3% for ATP, respectively.
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  • 15
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The abstracts of the talks and posters at this recent two day meeting are reproduced below. An exhibit of equipment and reagents was also held concurrently with the meeting.
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  • 16
    ISSN: 0884-3996
    Keywords: Prostaglandin E1 ; endothelium ; polymorphonuclear leukocyte ; cell interaction ; adherence ; oxygen-derived metabolites ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We investigated the effect of prostaglandin E1 on human polymorphonuclear leukocytes, in vivo. Polymorphonuclear leukocytes of a prostaglandin E1 and placebo study group were harvested and their function, as production of oxygen-derived metabolites and adherence to human cultured endothelial cells, was compared. Additionally, data obtained from polymorphonuclear leukocytes of a prostaglandin E1 and placebo group were compared with data obtained from polymorphonuclear leukocytes from 28 blood donors, who served as a control group.Production of oxygen-derived metabolites by polymorphonuclear leukocytes during contact with endothelial cells was measured by chemiluminescence. Chemiluminescence was significantly (p 〈 0.01) increased in the placebo group in comparison to the control group decreasing to values of control group after 6 d (post-trauma). Chemiluminescence response was not significantly suppressed in patients treated with prostaglandin E1 in comparison to the placebo group. Adherence of polymorphonuclear leukocytes (placebo group) to endothelial cells was significantly increased (p 〈 0.01) within the first 6 d post-trauma Following day 6, values were in the same range as values for the control group. Adherence was not significantly suppressed in patients treated with prostaglandin E1 in comparison to the placebo group. In conclusion, prostaglandin E1 at a dose of 20 ng/kg bw/min does not influence production of oxygenderived metabolites and adherence in polytraumatized patients in comparison to a placebo group. Additionally, production of oxygen-derived metabolites by polymorphonuclear leukocytes in response to endothelial cells is shown and it is evident that endothelial cells might influence production of oxygen derived metabolites by polymorphonuclear leukocytes.
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  • 17
    Electronic Resource
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    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 307-313 
    ISSN: 0884-3996
    Keywords: Luminol ; sodium hypochlorite ; hydrogen peroxide ; inhibition of chemiluminescence ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Two different mechanisms of inhibition of chemiluminescence in the oxidation of luminol by sodium hypochlorite were found. Most substances investigated in these experiments acted by scavenging NaOCI. This mechanism was independent of the concentration of hydrogen peroxide and the incubation time between luminol and inhibitors. The most potent inhibitors were substances containing SH groups. Compounds with amino groups as a target for HOCI/OCI- to yield chloramines were much less effective inhibitors. Another mechanism of inhibition was found for catalase. It depended on the presence of hydrogen peroxide in the incubation medium and the incubation time between luminol and catalase. The enzyme inhibited the luminescence by removing H2O2 at molar concentrations much smaller than those found for all other inhibitors. Our results confirm the present models of the mechanism of generation of luminescence in luminol oxidation.
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  • 18
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    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 332-332 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 19
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 21-24 
    ISSN: 0884-3996
    Keywords: Chemiluminescence ; haemodialysis ; oxalate determination ; urolithiasis ; peroxyoxalate chemiluminescence ; ascorbic acid ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We describe a new sensitive and specific method for determination of oxalate in human serum. By using the chemiluminescence decay of monoperoxyoxalic acid very low concentrations of oxalate (200 nmol/L) can be determined. The mean serum oxalate level in apparently healthy controls was 14.5 ± 8.5 m̈mol/L. Supplementation of ascorbic acid leads to an increase in serum oxalate level. While serum oxalate concentrations of calcium oxalate stone formers (x = 16.4 ± 9.8 m̈mol/L) are not significantly different from the control group, an extreme increase of serum oxalate is evident in haemodialysis patients. The serum oxalate concentration decreased during dialysis treatment from 141.4 ± 32.1 m̈mol/L to 36.4 ± 12.7 m̈mol/L.
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  • 20
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 33-37 
    ISSN: 0884-3996
    Keywords: Autoantibodies ; myeloperoxidase ; chemiluminescence ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We investigated interaction between antibodies directed against myeloperoxidase (anti-MPO) and myeloperoxidase (MPO) with chemiluminescence. Whole serum diluted 1/10 containing circulating anti-MPO antibodies as well as serum from normal blood donors inhibited myeloperoxidase (MPO) enzyme activity when incubated with 1.42 m̈g MPO. When further diluted the inhibitory effect was abolished. Incubation with purified human lgG fraction of anti-MPO, did not cause any inhibition when diluted 1/10 and incubated with 1.42 m̈g MPO. When adding MPO to normal sera a rapid increase of the enzyme activity was seen above 5 m̈g, and the inhibitory effect was completely abolished when 10 m̈g was added. Both sera from healthy individuals, as well as sera from patients with circulating anti-MPO inhibited MPO activity. The inability of pure lgG fractions from anti-MPO sera to inhibit MPO enzyme activity, clearly indicates the presence of an inhibitory factor, unspecific or specific, in serum.
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  • 21
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    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 141-145 
    ISSN: 0884-3996
    Keywords: Bioluminescence ; Lactococcus ; lux ; pH ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: High levels of constitutive aldehyde-dependent light emission were obtained from non-growing cells of Lactococcus lactis subsp. diacetylactis F712 transformed with luxA/B when they were suspended in buffered solutions. Inductions of light emission was time-dependent and was not due to growth, synthesis of luciferase or stimulation of metabolism by fermentable carbohydrate. The major factor controlling light emission in such cells appears to be the intracellular pH value. Experiments with ionophores indicated that a transmembrane pH gradient was not essential for light emission.
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  • 22
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 135-139 
    ISSN: 0884-3996
    Keywords: Alpha-fetoprotein ; chemiluminescence ; enzyme immunoassay ; b̃-D-galactosidase ; 5-bromo-4-chloro-3-indolyl-b̃-D-galactopyranoside ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We developed a sensitive chemiluminescent sandwich-type enzyme immunoassay (CLEIA) of alpha-fetoprotein (AFP) using b̃-D-galactosidase (b̃-gal) as a label and 5-bromo-4-chloro-3-indolyl-b̃-D-galactopyranoside as a substrate. The CL-EIA for AFP was performed using two monoclonal antibodies, one antibody is labelled with b̃-gal, the other is coated onto the inside surface of a polystyrene tube. The detection limit for AFP was 0.5 ng/mL, equivalent to 10 pg/assay tube. The coefficient of variation for within and between assay imprecision were 2.0%-4.9% (n = 10) and 4.4%-9.8% (n = 5), respectively. AFP values in serum determined by this method correlated well with those obtained by radioimmunoassay (n = 26, r = 0.99). This sensitive AFP assay can be performed within 4 h and can be used as a routine assay in clinical diagnosis.
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  • 23
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 169-182 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 24
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    Journal of Bioluminescence and Chemiluminescence 8 (1993) 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 25
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 215-235 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 26
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 247-252 
    ISSN: 0884-3996
    Keywords: Salmeterol ; beta-adrenergic agonist ; chemiluminescence ; lucigenin ; neutrophil ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Although beta-agonists remain an important aspect of the treatment of asthma, their role has recently been questioned. Salmeterol has recently been developed as a betaagonist with prolonged bronchodilator action. Using lucigenin-enhanced chemiluminescence, we have shown that salmeterol inhibits this aspect of phagocyte function in vitro in a concentration-dependent manner. However, salmeterol differs from classical beta2-agonists in that at concentrations between 10-5 and 10-3 mol/L, its effects on phagocytes cannot be completely reversed by washing the cells or by propanolol. The effects on phagocytes may not therefore be explicable on the basis of betaadrenergic mechanisms alone.
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  • 27
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 293-299 
    ISSN: 0884-3996
    Keywords: Growth ; luminescence intensity ; luminous bacteria ; biostimulator ; hydrolysate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The examination of four species of luminous bacteria Photobacterium leiognathi, Photobacterium phosphoreum, Vibrio fischeri and Vibrio harveyi has enabled us to reveal some nutrient medium components effecting growth, luminescence intensity and luciferase synthesis. These agents are nucleic components (nucleotides, nucleosides and amine bases), amino acids and vitamins, which are part of hydrolysates from the biomass of various lithotrophic microorganisms, hydrogen-oxidizing, ironoxidizing and carboxydobacteria. The effect of promoting agents essentially alters the physiological state and ultrastructure of the cells of luminous bacteria and increases luciferase biosynthesis two- to three-fold compared to a control.
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  • 28
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 25-31 
    ISSN: 0884-3996
    Keywords: Acridinium ester ; chemiluminescence ; flow injection ; stability ; decomposition ; kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Decomposition of phenyl acridinium-9-carboxylate is monitored using electrogenerated chemiluminescence in a flow system. The formation of the pseudobase from the acridinium ester [AE] is described by rate = k′1[AE] + k″1[AE][OH-]0.5, where k′1 = 0.020 ± 0.006 s-1 and k″1 = 2.1 ± 0.8 (L/mol)-0.5 s-1. Irreversible decomposition of the pseudobase is described by rate = k′2[AE][OH-], where k′2 = 20.1 ± 3.8 (L/mol s). These kinetic equations, plus measurement of variation in emission intensity for constant acridinium ester concentration, are used to predict the resulting emission intensity v. pH behaviour given various contact times (in the 0.25 to 25 s range) for the acridinium ester to be in an alkaline solution prior to initiation of the chemiluminescence reaction.
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  • 29
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 49-49 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 30
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 65-132 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: An abstract index is given immediately after the abstracts, on page 128.
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  • 31
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    Journal of Bioluminescence and Chemiluminescence 8 (1993) 
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    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 32
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    Journal of Bioluminescence and Chemiluminescence 8 (1993) 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 33
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 201-205 
    ISSN: 0884-3996
    Keywords: Bioluminescence ; chemiluminescence ; luciferin ; luminous fungus ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The chemical structure of two luciferin precursors PS-A and PS-B, isolated from the luminous mushroom Panellus stipticus, were determined as 1-O-decanoylpanal (2) and 1-O-dodecanoylpanal (3), respectively. Both PS-A and PS-B were converted into chemiluminescent luciferins by treatment with 50 mmol/l methylamine in a pH 3.5 buffer solution containing an anionic surfactant Tergitol 4 at 25-35ºC. The luciferins emitted chemiluminescence in a pH 7-8 buffer solution containing a cationic surfactant in the presence of O2 and O2-.
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  • 34
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 237-240 
    ISSN: 0884-3996
    Keywords: Luminometers ; radiometers ; low-light level imaging ; review ; survey ; immunoassay ; rapid microbiology ; hplc ; glc. kits ; reagents ; immunoassay ; probes ; labels ; nucleic acid hybridization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: This survey update was compiled in June 1993 and includes products not covered in the luminometer survey (Jan 1992; Stanley, PE. J Biolumin Chemilumin 1992; 7:77-108 and 7:157-69) and kit and reagent survey (Nov 1992; Stanley, PE. J Biolumin Chemilumin 1993; 8:51-63). Some products are new whilst for others details have only recently become available to the author. Technical details are provided together with company address and contact information.
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  • 35
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 15-20 
    ISSN: 0884-3996
    Keywords: Ascorbic acid ; endothelial cell ; neutrophil ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The effect of different concentrations (0.06, 0.6 and 6.0 mmol/L) of ascorbic acid on neutrophil-endothelial interaction was studied using an in vitro model of human umbilical cord vein endothelial cells and human neutrophils. The aim of the study was to determine changes in chemiluminescence response of neutrophils during adherence to endothelial cells. Because adherence of neutrophils to endothelial cells is an essential component in inflammatory processes leading to endothelial cell injury, the influence of ascorbic acid on adherence and endothelial cell injury have been investigated. Production of oxygen-derived metabolites, measured by chemiluminescence response of neutrophils, decreased significantly in the presence of 6 mmol/L ascorbic acid during coincubation of neutrophils and endothelial cells (p 〈 0.025). The adherence of neutrophils to endothelial cells was significantly decreased at a concentration of 6 mmol/L (p 〈 0.0005). The inhibition of neutrophil adherence to endothelial cells was correlated with a diminished neutrophil-mediated endothelial cell injury during incubation with 6 mmol/L ascorbic acid (p 〈 0.0005). The present results indicate that ascorbic acid might exert a protective effect on neutrophil-mediated endothelial cell injury by decreasing adherence of neutrophils to endothelial cells and by scavenging reactive oxygen metabolites. Moreover, the current investigation points to probable protective effect of ascorbic acid on oxidant-mediated cell damage in diseases (e.g., Adult Respiratory Distress Syndrome).
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  • 36
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    Journal of Bioluminescence and Chemiluminescence 8 (1993) 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 37
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 133-134 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 38
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 147-152 
    ISSN: 0884-3996
    Keywords: Bioluminescence ; hop ; ionophore ; Lactococcus ; lux ; pH ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Ionophores (carbonyl cyanide m-chlorophenylhydrazone; valinomycin; and the hopderived compounds colupulone, trans-isohumulone and trans-humulinic acid) reduced the rate of dodecanal-dependent light emission from luxA/B-transformed cells of Lactococcus lactis subsp. diacetylactis F712 when the cells were suspended in a buffered medium (pH 6.4) containing glucose. This allowed an assay for ionophores to be devised and permitted measurement of the effects of such compounds on the test organism by the use of a non-destructive technique in real time.
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  • 39
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 183-199 
    ISSN: 0884-3996
    Keywords: Flow injection ; chemiluminescence ; bioluminescence ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: This paper reviews publicaions that combine the technique of flow injection (FI) with chemiluminescence (CL) and bioluminescence (BL) detection, from the earliest papers in 1979/80 to mid-1992, and refers exclusively to reactions occurring in solution. Airsegmented systems and liquid chromatography with CL detection are not considered unless FI has been used to pre-optimize the system. The applications have been categorized in terms of the type of CL reaction; there are separate entries for luminol, peroxyoxalate, other CL reactions and BL reactions. Each of the four sections includes a table of applications that lists the analyte, the nature of the reaction, the sample matrix and the limit of detection.
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  • 40
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    Journal of Bioluminescence and Chemiluminescence 8 (1993), S. 207-213 
    ISSN: 0884-3996
    Keywords: Phagocytes ; respiratory burst ; oxygen species ; MCLA ; Dextran-T70 ; chemiluminescence ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Leukocyte oxidative function was investigated in a more physiological milieu than currently used in the chemiluminescence (CL) technique. Heparinized blood was mixed with 6% dextran-T70 (9:1) and the leukocyte-rich plasma obtained without centrifugation was used for the CL experiments (phagocyte count was adjusted to 0.7 × 106/mL with Hanks' buffer) (method A). In this medium, phagocytes responded to stimulation by opsonized zymosan, producing strong luminescence in the presence of 0.5 m̈ mol/L MCLA. CL was inhibited by superoxide dismutase, suggesting that the luminescence reaction was attributable to O2-. Granulocytes were also prepared by the usual method involving centrifugation and were then suspended in plasma (method B). Oxidative function of phagocytes prepared by the two methods was studied together with whole blood as aliquots diluted with Hanks' buffer up to a factor of 1000. Luminescence reached a peak value at a dilution factor of 16, but at very high dilutions luminescence decreased sharply. Significantly higher luminescence values were obtained with samples from method A. Luminescence of whole blood peaked at a dilution factor of 248 but it was less than the value obtained using samples prepared by method A or B. As samples prepared by method A contain all the leukocyte populations, platelets, residual red cells and plasma proteins, the assay of leukocyte-generated reactive oxygens using CL is attained in more physiological conditions than method B in which leukocytes may be damaged owing to repeated centrifugation and hypotonic shock.
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  • 41
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    Cell Biochemistry and Function 11 (1993), S. 257-261 
    ISSN: 0263-6484
    Keywords: PTH ; adenylate cyclase ; Ca2+ ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We studied the effect of PTH (10-100 nM) on transductive mechanisms (adenylate cyclase activity, Ca2+ metabolism, IP3 levels) in cell cultures derived from normal and otosclerotic human bone fragments. The cultured cells were osteoblast-like but with calcitonin-receptors still present and with PTH receptors coupled with the adenylate cyclase system.The results showed that PTH activated adenylate cyclase and increased the intracellular Ca2+ levels with qualitative and quantitative differences between the two cellular populations. In particular, otosclerotic cells responded less to hormone stimulation, which is in accord with the current hypothesis of a desensitization of the receptor/enzyme complex associated with the pathological status.
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  • 42
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    Cell Biochemistry and Function 11 (1993), S. 263-269 
    ISSN: 0263-6484
    Keywords: Proteoglycans ; senescence ; human skin fibroblasts ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The properties of proteoglycans (PGs) produced by normal human skin fibroblast were investigated with increasing passage. The increase of subculture number was associated with a constant increase in PG molecular size, which was particularly evident in cell layer extracts. In the cell layer, the ratio of DS-PGs/HS-PGs was markedly higher in early passage cultures. Moreover, the cell layer from young cells contained lower amounts of radioactivity incorporated into the most hydrophobic PG populations, suggesting that the PG core protein might also undergo significant modification with increasing subcultures. There was no significant difference in energy charge value between early and late passage cultures, whereas the NAD/NADH ratio was found to decrease markedly in senescent cells.
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  • 43
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    Cell Biochemistry and Function 11 (1993), S. 287-290 
    ISSN: 0263-6484
    Keywords: DNA primase ; nuclear matrix ; nuclear isolation ; heat exposure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have reinvestigated the association of DNA primase activity with the nuclear matrix prepared from exponentially growing HeLa S3 cells. We have found that 25-30 per cent of the nuclear primase activity resists extraction with 2 M NaCl and digestion with Dnase I. Unlike previous investigations, done with the same cell line, the results showed that nuclear matrix-bound DNA primase activity represented less than 10 per cent of the total cell activity. Association of high levels of primase activity with the nuclear matrix was strictly dependent on a 37°C incubation of isolated nuclei prior to subfractionation. Evidence was obtained that the method used for preparing nuclei can have a dramatic effect on the amount of primase activity which is recovered both in the postnuclear supernatant and in isolated nuclei, thus seriously affecting the interpretation of the results about the quantity of DNA primase activity bound to the nuclear matrix.
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  • 44
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    Cell Biochemistry and Function 11 (1993), S. 271-277 
    ISSN: 0263-6484
    Keywords: Neurofibromatosis ; human fibroblasts ; alkaline phosphodiesterase I ; tumourigenicity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Alkaline phosphodiesterase I from cultured fibroblasts from patients with neurofibromatosis was partially purified and characterized following extraction with Triton X-100, and fractionation with high-performance liquid chromatography. Some properties were compared with the enzyme extracted from normal-appearing fibroblasts. The isoelectric points of both the tumour and normal-appearing cell enzymes were 6·0. The enzyme required Zn2+ for its activity, was heat labile, and nicked superhelical covalently closed circular φX174 DNA. The activity was inhibited by GTP, DTT and EDTA. The native molecular weight of alkaline phosphodiesterase I was determined to be 430 000. No differences were found in properties of the tumour-derived and normal cell enzymes. On purification it was observed that the peak pattern of enzyme activity corresponded to that of 125 kDa protein, which was more abundant upon SDS-PAGE analysis in tumour cells than in normal cells. The most active fraction of isoelectric focusing, which was performed using disulfide cross-linked polyacrylamide gel, was used to produce an antibody. The bands of 125, 60 and 40 kDa were immuno-stained in tumour cell preparation. These results indicate that alkaline phosphodiesterase I, of which the molecular weight is probably 125 kDa, is over-expressed in tumour-derived fibroblasts from neurofibromatosis patients.
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  • 45
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    Cell Biochemistry and Function 11 (1993), S. 291-291 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 46
    ISSN: 0263-6484
    Keywords: 4-Hydroxynonenal (HNE) ; lipid peroxidation ; cell proliferation ; viability ; human carcinoma (HeLa) ; protein synthesis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The aim of this study was to analyze the growth response of HeLa cells over a prolonged period of time to a single exposure of physiological and supraphysiological concentrations of 4-hydroxynonenal (HNE), a peroxidation product of omega-6-polyunsaturated fatty acids. Furthermore, the growth modulating effect of serum factors, particularly albumin, on the growth pattern was examined. The effects of HNE on the growth rate and viability of the cells, as well as on the incorporation of labelled amino acids were monitored daily over a period of four days. Fetal calf serum not only had a growth stimualting effect but also modulated the action of HNE. In neither respect was albumin able to substitute for serum indicating that the influence of serum was not exerted via an albumin-HNE conjugate.HNE had a clear dose-dependent effect and a distinction could be made between a supraphysiological concentration (100 μM), which was primarily cytotoxic and a physiological range (below 10 μM) which showed growth modulatory effects. These effects consisted of a transient inhibition in the initial phase of the cell growth, which under optimal conditions (in presence of serum) was followed by a period of increased proliferation, compared to untreated control cultures, until confluence was attained. It is suggested that HNE is not only a toxic product of lipid peroxidation, but a physiological growth regulating factor as well.
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    Cell Biochemistry and Function 11 (1993), S. 291-291 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
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    Cell Biochemistry and Function 11 (1993), S. 291-291 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 49
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    Cell Biochemistry and Function 11 (1993), S. 292-292 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 50
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    Cell Biochemistry and Function 11 (1993), S. 292-292 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 51
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    Electrophoresis 14 (1993), S. 27-35 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: DNA digest analysis with polymer-filled capillaries in capillary electrophoresis is described. The samples analyzed consisted of commercially available standards including a 100 base pair ladder with repeating units up to 2000 base pairs. Three DNA digests covered the most common small fragment ranges: up to 600 base pairs, up to 1500 base pairs and from 100 to 2500 base pairs. All samples were separated by traditional slab gel electrophoresis at various agarose concentrations and by automated capillary electrophoresis. The capillary electrophoretic separations were achieved with noncross-linked polyacrylamide from 6% monomer solutions. The acrylamide was polymerized inside the capillary, which was coated with a methacryloxysilane to insure binding of the polyacrylamide to the capillary wall. With 6% columns, excellent separations were observed up to 600 base pairs with base line resolution for fragments differing in less than 10 base pairs. Resolution power for fragments between 600 and 2200 decreased to about 300 base pairs. Compared to slab gels, capillary electrophoresis achieved better resolution in the low fragment range, whereas with the reported column composition, slab gels were superior above 600 base pairs. Fast access to the analysis of a specific sample, automation, and a larger dynamic range for sample load are further benefits of a DNA digest analysis by capillary electrophoresis.
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    Electrophoresis 14 (1993), S. 36-39 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A new method of capillary electrophoresis was established to separate the three cresol isomers. Cresols are of importance due to their use as insecticides, precursors of various dye intermediates, resins, fire-resistant fluids and other purposes. Reversion of the electroosmotic flow was carried out by a polycationic ammonium compound. The separations were carried out at high pH values (pH 12) to obtain complete dissociation of the cresols and short times of analysis (less than 5 min) through fast migration of the cresol towards the anode. Various aliphatic alcohols were added to improve peak shape and resolution.
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  • 53
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    Electrophoresis 14 (1993), S. 51-55 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In most silver staining methods the first step in the staining of proteins separated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis is a rather protracted fixation of the gels. Optimum fixation should be short, cause no background staining and effectively immobilize the proteins in the gel without masking the proteins for reaction with the staining solution. Further, the concentration of the fixing compounds should be as low as possible due to the potentional toxicity of fixatives. Fixation for only 5 min with mixtures of very low concentrations of formaldehyde and glutaraldehyde in ethanol, or a solution of formaldehyde or glutaraldehyde in picric acid and ethanol, fulfill these demands, provided that the gels were prefixed in ethanol-acetic acid for 10 min. As a consequence of these results a fast and sensitive silver staining procedure is proposed.
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  • 54
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    Electrophoresis 14 (1993), S. 40-50 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Photopolymerization of polyacrylamide gels in the presence of methylene blue (100 μM) and a redox couple (1 mM sodium toluenesulfinate, a reducer, and 50 μM dipheriyliodonium chloride, an oxidizer) has been investigated. The gel point, i.e. the time needed for onset of gelation upon illumination, has been found to lengthen progressively at lower temperatures and at lower light intensities. If the three catalysts are progressively diluted, the gel point does not vary for a threefold dilution, but gelation is greatly hampered below a 1:5 dilution of the three effectors. Photobleaching has been assessed as a function of liquid layer thickness (from 0.5 to 2 mm), of a progressive dye dilution (down to a fourfold dilution) and as a function of temperature. A maximum of elastic modulus is located in correspondence to a minimum of permeability (both situated at 5% cross-linker). It is found that methylene blue-activated polymerization produces polyacrylamide gels with elastic properties which are higher than in persulfate-activated gels, so far the most popular matrices for electrokinetic separations. Due to the ease of preparation, the full control of all experimental parameters, and the lack of oxidizing power of this catalyst system (as opposed to the strong oxidation power of persulfate catalysis), methylene blue catalysis is advocated as a valid alternative to other redox systems.
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  • 55
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    Electrophoresis 14 (1993), S. 65-68 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The use of Nile Red to track and stain low density lipoprotein (LDL) and modified LDL in agarose gels was investigated. Lipoproteins were prestained with Nile Red, a fluorescent dye, prior to electrophoresis. After 2 h of electrophoresis, the LDL and modified LDL were visualized using a UV transilluminator with an excitation wavelength of 302 nm. Spectrofluorometric analysis revealed that the Nile Red fluorescence of the stained LDL had an emission maximum of 609 nm. This rapid staining method of LDL and modified LDL can detect as little as 2.5 μg of LDL protein and permits the immediate visualization of these lipoproteins in agarose gels.
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  • 56
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    Electrophoresis 14 (1993), S. 56-64 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Giant proteins in the megadalton range (〉 0.5 MDa) appear to play important structural and functional roles in striated muscle. Titin (∼ 3 MDa) is involved in the generation of resting tension and the assembly and stability of the sarcomere in skeletal and cardiac muscle tissues, while nebulin (∼ 0.7 MDa) is thought to regulate thin filament length in skeletal muscle. Sodium dodecyl sulfate (SDS)-gel electrophoresis is an important tool in revealing the size, quantity and integrity of these giant proteins in muscle tissues. We report here a method for solubilizing, detecting and quantifying titin and nebulin from short segments of single fibers of the rabbit psoas muscle. Muscle proteins ranging from 15 kDa to 3MDa were resolved on 3.3-12% gradient polyacrylamide gels that were silver-stained and quantitated by densitometry. Presoaking fiber segments in a low ionic strength pH 8.4 buffer enhances the amount of solubilized titin and nebulin. Solubilizing the presoaked fiber segments with SDS at 60°C for 60 s maximizes the amount of intact titin; solubilizing at higher temperatures causes extensive degradation of titin. Detection sensitivity is sufficient to study titin and nebulin in fiber segments as short as 120 μm.
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  • 57
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    Electrophoresis 14 (1993), S. 69-72 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A method is described for electroblotting of proteins, separated by gradient polyacrylamide gel electrophoresis, onto an agarose gel matrix containing specific antibodies. Three proteins of different molecular weight, including human albumin, isolated and in plasma, human plasma transferrin and C3 complement were tested. Immunoblotting on agarose, compared with nitrocellulose, was quantitative and highly sensitive, with small amounts of protein (i.e., 100 pg) being detected. Moreover, albumin aggregates (i.e., dimer, trimer and tetramer) were blotted quantitatively in addition to the monomer, and their percentages were calculated. This method is sensitive, quantitative, reproducible, and includes fewer manipulations; furthermore, it is less expensive and does not require the use of toxic or carcinogenic agents.
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    Electrophoresis 14 (1993), S. 73-77 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The PhastTransfer system is a semi-dry electrophoretic unit designed to optimize the transfer of small amounts of protein. Because of its efficiency, we adapted the PhastTransfer system for the detection of labeled membrane molecules. Biotin was used as the membrane molecule label because it permitted the long-term storage of labeled lysates as well as the flexibility of derivatizing several different functional groups. After immunoaffinity separation using magnetic microspheres, the protein was electrophoretically separated with the PhastSystem and transferred with the PhastTransfer unit. Using an avidin-linked enzyme amplification system, less than 10 ng of loaded protein could be detected on the transfer membrane. Based on these findings, the PhastTransfer system is a fast, reproducible, and convenient method for the transfer of small quantities of labeled cell surface protein.
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  • 59
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A silver-staining procedure for enhancing the sensitivity of protein detection on nitrocellulose membranes in immunoblotting is described. After completing any peroxidase-Ni-diaminobenzidine immunostaining, nitrocellulose sheets are placed in a physical developer, containing sodium tungstate and ascorbic acid, until the desired grade of silver-intensification has been reached. In this way a 16- to 64-fold amplification of intensity of the initial immunostaining can be achieved. False positive silver staining of protein bands and of background are suppressed by replacing bovine serum albumin, the blocking agent most frequently used in immunoblotting, with skimmed milk.
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  • 60
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Immune response to a hapten of fluorescein isothiocyanate (FITC) in a single BALB/c strain mouse was analyzed by two-dimensional affinity electrophoresis (2D-AEP). Anti-FITC antibodies were induced by immunization with FITC-conjugated bovine serum albumin. The antibodies were separated into a large number of spots of IgG due to differences in their isoelectric points(pI) and binding affinities to the FITC ligand. These spots consisted of IgG families which were composed of several spots having an identical affinity to the ligand but a different pI. The spots were not clearly detected in the antiserum taken on day 7 after the primary immunization, but on day 21 the spots of IgG were clearly detected, with a high diversity and specificity for the ligand. The size and number of IgG spots were markedly increased by the secondary immunization; however, the third immunization did not increase the size and number of IgG spots. The IgG spots of each family were specifically stained with an antimouse IgG subclass antibody. Furthermore, a monoclonal antibody (FL-D6) was separated by 2D-AEP into a single family which consisted of seven IgG1 spots having an identical affinity to FITC but different pIs. Therefore, each of the IgG families of anti-FITC antibodies in the antiserum can be generated by a single clone of anti-FITC antibody-producing cells. The substitution of dextran T 2000 or lipopolysaccharide for bovine serum albumin as a carrier for FITC induced much smaller amounts of anti-FITC antibodies with a low diversity but high specificity to FITC.
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    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The methodology for the simultaneous analysis of protein synthesis concomitant with protein phosphorylation/dephosphorylation is described. The technique consists of metabolic labeling of rat liver epithelial (RLE) cells with [32P]orthophosphate and [35S]methionine, performing two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of the mixed samples, followed by silver staining and subsequent autoradiography of the dried silver stained 2-D PAGE electrophoretograms using two films placed back-to-back. The first film, which is positioned in direct contact with the dried silver-stained gel, visualized both exposure to 35S and 32P while the second film recorded exposure to only 32P due to the differential energy levels of the two isotopes. The juxtapositioning of the silver-stained images with the two autoradiographic film images permits the unambiguous mapping of the phosphorylated polypeptides back to their corresponding silver-stained and methionine-labeled counterparts. This strategy provides quantitative information utilizing both silver staining (measure of constitutive levels of protein expression) and metabolic labeling to measure rates of protein synthesis and/or degradation and phosphorylation and/or dephosphorylation using [35S]methionine and [32P]orthophosphate, respectively. We have utilized this methodology for the in vitro analysis of transforming growth factor type β1 (TGF-β1)-mediated signal transduction in RLE cells and have identified three nuclear polypeptides, 1 (pI4.95/Mr 97 kDa), 2 (5.00/85 kDa) and 3 (4.90/84 kDa) whose phosphorylation status is rapidly and transiently modulated by TGF-β1. The methodology described should have wide applications in studies where it is desirous to measure protein synthesis and/or degradation concomitant with signal transduction pathways involving protein phosphorylation.
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    Electrophoresis 14 (1993), S. 168-168 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 63
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    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Apolipoprotein B-100 is the principal protein component of lipoproteins with very low, intermediate, and low density. The interaction of apoB-100 with low density lipoprotein (LDL) receptors is responsible for the uptake of LDL into cells. An AT-rich hypervariable region is located adjacent to the 3′ end of the apoB gene. It consists of a variable number of tandemly repeated sequences (VNTR). Two approaches were used to analyze this polymorphism. In both, the region harboring the VNTR was amplified with the polymerase chain reaction (PCR). In the first method, fluorescently labeled primers were used in the PCR reactions and products were separated in agarose gels by means of an automated fluorescent fragment analyzer. In the second method, PCR products were analyzed in denaturing polyacrylamide gels and detected with silver staining. Even in the highly sophisticated automated system, agarose gel electrophoresis did not always enable unequivocal assignment of VNTR alleles. In contrast, denaturing polyacrylamide gel electrophoresis made it possible to distinguish the 15 bp differences between the VNTR alleles in a precise and simple manner. The VNTR polymorphism was typed in 234 individuals. Among these were 136 patients with coronary artery disease and 74 healthy controls. Thirteen alleles could be distinguished. The allele containing 49 repeats (VNTR-49) was found in 9.2% of the coronary artery disease patients and in 4.7% of the controls. Thus, the VNTR-49 allele increases relative coronary risk by about twofold. It is concluded that the apoB VNTR polymorphism is a potentially useful genetic marker. Since agarose gel electrophoresis may lead to ambiguous results, we prefer typing by denaturing polyacrylamide gel electrophoresis. This has to be accounted for, especially if the apoB VNTR polymorphism is applied to forensic studies.
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  • 64
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    Electrophoresis 14 (1993), S. 191-201 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In dye-sensitized polymerization, some paradoxical phenomena have been found, such as an anomalous decrease of reaction rate with decrementing thicknesses of the gelling layers. By mathematical modelling, and experimental verification, it has been found that high incorporation efficiencies (〉 95% conversion of monomers into the growing polymer) can only be obtained at the correct levels of dye in the gelling solution and by using the proper light intensity. Paradoxically, if levels of sensitizer or incident light intensities are too high, the rate of photosen-sitizer consumption is too high, as compared with the rate of monomer incorporation, so that dye depletion occurs prior to chain elongation, and the reaction suddenly comes to a stop. In methylene-blue catalysis, the curve correlating the monomer incorporation with the catalyst concentration exhibits a maximum, indicating poor conversions both below and above a critical catalyst level. When correctly used, photopolymerization still offers unique advantages over chemical (persulfate) polymerization, such as absence of oxidizing conditions and control at the onset of reaction, while guaranteeing high conversion efficiencies (〉 95%).
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  • 65
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    Electrophoresis 14 (1993), S. 235-237 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Isoelectric focusing of human orosomucoid (ORM) was studied following different sample treatment. It is shown that: (i) alkylation with iodoacetamide leads to a drastic change in the isoelectric point (pI) of both ORM1 F2 and ORM2 A gene products and greatly improves the discrimination between ORM1 F1 and ORM1 F2; (ii) previous reduction of the molecule with dithiothreitol partially inhibits the pI transitions with resultant artifactual ORM1 F1F2S patterns that correspond in most cases to F2S phenotypes. With the technique now described, the persistence of three ORM1 gene products was found in only one individual and the segregation analysis is consistent with the existence of a rare ORM1*F2S haplotype.
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  • 66
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    Keywords: Chemistry ; Biochemistry and Biotechnology
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    Topics: Biology , Chemistry and Pharmacology
    Notes: Blue dextran was incorporated into the stacking layer of one-dimensional polyacrylamide gels to prevent the migration of albumin into the resolving gel during the electrophoresis of native plasma samples. Human and horse plasma proteins normally masked by albumin were revealed by this method.
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  • 67
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    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: To study the clonal events occurring during ontogeny of the humoral immune system, we evaluated plasma immunoglobulin (Ig) production in term newborns and young children by high-resolution two-dimensional gel electrophoresis. The clonality pattern of Ig light (L) chains from healthy newborns (n = 19) was similar to that observed on protein maps of their mothers or of normal adults (n 〉 100), that is, rare distinguishable small spots in a cloud-like large band of indiscrete Ig L chain spots (polyclonal pattern; maternal Igs). Analysis of plasma samples obtained from infants between 1 month and 5 years of age (n = 55) revealed discrete but evident alterations of the clonality of Ig production. Between the 2nd and 4th months of life, transient attenuation of the “polyclonal background” was observed in association with the appearance of an increasing number of well-resolved Ig L chain spots (corresponding to plasma Ig concentrations between 0.5 and 1 g/L per spot). This “restricted” clonal pattern was progressivly less apparent on protein maps of infants older than 2 year and evolved towards a “normal” adult polyclonal pattern at the age of 5. These results suggest that the development of the B-cell clones is heterogeneous, either through limited outgrowth of precursor cells or through selective antigenic pressures.
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  • 68
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: To understand how comparatively simple macromolecular components become biological systems, studies are made of the morphogenesis of bacteriophages. Pulsed field agarose gel electrophoresis (PFGE) has contributed to these studies by: (i) improving the length resolution of both mature, linear, double-stranded bacteriophage DNAs and the concatemers formed both in vivo and in vitro by the end-to-end joining of these mature bacteriophage DNAs, (ii) improving the resolution of circular conformers of bacteriophage DNAs, (iii) improving the resolution of linear single-stranded bacteriophage DNAs, (iv) providing a comparatively simple technique for analyzing protein-DNA complexes, and (v) providing a solid-phase quantitative assay for all forms of bacteriophage DNA; solid-phase assays are both less complex and more efficient than liquid-phase assays such as rate zonal centrifugation. Conversely, studies of bacteriophages have contributed to PFGE the DNA standards used for determining the length of nonbacteriophage DNAs. Among the solid-phase assays based on PFGE is an assay for excluded volume effects.
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  • 69
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    Electrophoresis 14 (1993), S. 296-303 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The position and velocity of a band of double-stranded, linear DNA from bacteriophage G were measured during 120° pulsed-field gel electrophoresis, using a video micrometer. Both the x and y coordinates were determined simultaneously in the plane of a 1% agarose gel; x is the mean drift direction. For pulse durations T greater than the tube renewal time T*, the path traced by the band of 670 kb DNA in the xy plane was in remarkably good accord with that predicted by Southern's ratchet model. However, the measured instantaneous velocity vx showed a sharp backward spike each time the field changed direction, with amplitude about twice the mean drift velocity. This spike is not consistent with models which assume a constant curvilinear velocity of DNA in a tube, nor with the biased reptation model without fluctuations. The corresponding measurements of vy showed a sharp positive spike with amplitude more than 3 times the plateau velocity in the y direction; neither model predicted this. The sharp velocity spikes are consistent with the idea that, for T〉 T*, a large fraction of the DNA chains are stretched into U-shaped or herniated configurations. When the field changes direction, the arms of the U's and the hernias recoil rapidly in response to intramolecular DNA chain tension. Because the base of a U or hernia is fixed by gel fibers, the center of mass of the chain recoils backward every time the field changes direction.
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    Electrophoresis 14 (1993), S. 313-321 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In agarose gel electrophoresis, in a steady, continuous field, it is well known that the mobility μs versus size M relation for linear DNAs (L-DNAs) can be divided into three regimes: Ogston regime I for small DNAs, where M dependence of μs is weak; entangled but unstretched regime II for intermediate-size L-DNAs (of M 〈 20 kbp), where μs σ M-1 so that efficient fractionation is possible; and entangled and stretched regime III for large L-DNAs, where M dependence of μs is again weak. Although μs and the regime boundaries can be altered by adjusting the gel concentration Cgel and/or the field strength E, the features of the M dependence of μs are essentially unchanged. As to the effect of DNA topology on μs, we found that in dilute gels (Cgel 〈 1.0 wt%) coiled, circular DNAs (C-DNAs) of 2-7 kbp size migrate faster than L-DNAs of comparable size, while in concentrated gels (Cgel 〉 1.5 wt%) C-DNAs migrate much slower than L-DNAs. To facilitate separation of large DNAs in the regime III range, we proposed biased sinusoidal field gel electrophoresis (BSFGE), which utilizes a sinusoidal field of strength Es and frequency f superposed on a steady bias field of strength Eb. Striking results in BSFGE of low bias (Eb 〈 Es) conditions were that (i) the effective mobility μ at low f (μo) is higher than that of μ∞ at high f, which is equal to the steady field value μs, and (ii) for large DNAs of M 〉 20 kbp the μ exhibits a minimum μp (pin-down mobility) at a frequency fp (pin-down frequency) specific to M, Cgel, and the field strengths in such a way that fp σ M-1 Cgel -1Eb. Esα with α changing from 0 to 2 ∼ 3 at a value of Es dependent on Eb. The μp values appear to fall on the extrapolated portion of the regime II log(μs) versus log M curve. These results are interpreted in terms of the current dynamical models of DNA gel electrophoresis and also with the results of direct observation by fluorescence microscopy on migrating T4dC DNA of 166 kbp in a steady field and under several BSFGE conditions.
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    Electrophoresis 14 (1993), S. 349-354 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Properties of agarose potentially relevant to PFGE (pulsed-field gel electrophoresis) are reviewed, and some new information is presented. Agarose polymers appear to have molecular weights in the range of 100 000 to 200 000 Da, but this is not tightly related to the effective gel strenght. Agarose has some residual charge, and hence exhibits electroendosmosis (EEO). It is possible to markedly increase the speed of separation of DNA molecules by using agarose of low EEO, especially in low ionic strenght, non-borate buffers. This increase is especially noticeable in the relatively long experiments required for separation of large DNAs. It is also possible to increase the range of separation in a single run by use of step gradients of agarose concentration, which allows visualization of yeast chromosomes and lambda-phage restriction fragments in the same lane. Because of the strong influence of concentration on separation, it may be useful for investigators to control water content and related variables. Our lack of knowledge of the detailed microstructure of gels may be barrier to complete understanding of PFGE.
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    Electrophoresis 14 (1993), S. 371-372 
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    Keywords: Chemistry ; Biochemistry and Biotechnology
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    Electrophoresis 14 (1993), S. 492-501 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The electrophoretic mobility of single-stranded DNA in denaturing polyacrylamide gel-filled capillaries is analyzed as a function of the applied electric field. The resultant mobility plots are complex functions of the fragment size and electric field. Traditionally, these plots are separated into three mobility regimes corresponding to Ogston sieving, reptation without stretching and reptation with stretching theories. However, none of these theories accurately models the variations in mobility that we observe with electric field strength. As a result, we propose a modification of the Ogston sieving theory which accounts for the stretching of migrating DNA molecules in the direction of the electric field. This theory assumes that the applied electric field in conjunction with the gel matrix distorts the DNA, altering the effective size of the migrating molecule. The stretched DNA offers a smaller cross-section to the gel pore and thus sieves as though it were a smaller molecule. In this modified Ogston theory, the electrophoretic mobility depends only on the applied electric field, the size of the fragment, and constants which are independent of size and field strength. The modified Ogston equation accurately predicts the mobilities of DNA fragments in all three mobility regimes9 providing a single, simple model to account for all of the observed behavior.
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    Electrophoresis 14 (1993), S. 531-539 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The determination of inorganic cations and anions by capillary electrophoresis/mass spectrometry (CE/MS) is reported using an ion spray-sheath flow interface coupling. A twelve-component synthetic mixture of cations which included the positive ions of K, Ba, Ca, Mn, Cd, Co, Pb, Cr, Ni, Zn, Ag, and Cu was loaded into the capillary column at levels ranging from 30 to 300 pg, separated by CE, and detected by indirect UV and in the full-scan (m/z 35-450) positive ion CE/MS mode using an aqueous buffer containing 30 mM creatinine and 8 mM α-hydroxyisobutyric acid, pH 4.8. Creatinine forms adducts with the cations which are observed in the gas phase and requires rather high (120 electron volts) declustering energy to dissociate. This produces a reduction in charge state to form the free, singly charged, inorganic cations which are observed in the mass spectra. CE/MS analysis of an aqueous acidic extract of used aircraft engine oil revealed high levels of lead as well as lower levels of chromium and nickel. CE-indirect UV analysis of a synthetic mixture containing 300 pg each of 11 inorganic ions, which included the anions of Br, Cl, NO2, NO3, S2O3, N3, SCN, SO4, SeO4, oxalate, and MoO4, is shown. The running buffer which affected this separation contained 5 mM ammonium dichromate, 10 mM ammonium acetate, and 20 mM diethylenetriamine at pH 9.3. Although indirect UV detection revealed good separation of these anions, CE/MS analysis of this mixture was complicated by interfering ion current signals from the cluster ions formed by the interaction between the additives and the analytes. Thus only three of these singly charged anions, e.g. Br, SCN, and HSeO4, could be satisfactorily detected in this mixture by CE/MS.
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    Electrophoresis 14 (1993), S. 559-559 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 76
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    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Temperature gradient gel electrophoresis (TGGE) and related methods are widely employed to detect mutations in DNA fragments. DNA melting map calculations and GC clamps have been used to enhance the detection of mutations. While generally successful, these methods have not always revealed base changes within a DNA fragment. Previous work suggested that mutations are detected if they are in a DNA's first melting domain, and the melting domain is well separated from final strand dissociation. Two criteria from the DNA melting theory were established to determine when both of these conditions are met. The criteria involve calculating the derivative melting curve as well as the melting map of a DNA sequence. The approach was applied to the cDNA sequence of the human p53 gene. Mutations in the p53 gene are common in human cancers and are generally located in four ‘hot spot’ regions. Calculations indicated that three DNA fragments are needed to detect base substitutions in the four hot-spot regions. Predicted melting behavior was experimentally tested with eight single base substitutions distributed among the four hot-spot regions. All mutations tested behaved as predicted and were detected by vertical TGGE. Heteroduplex DNAs formed by melting and reannealing various ratios of wild type and mutant DNA fragments were also examined. Results indicated that point mutations can be detected by ethidium bromide staining from samples containing 10% mutant and 90% wild-type sequences.
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    Electrophoresis 14 (1993), S. 597-600 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In agarose gel electrophoresis, periodically inverting or interrupting the applied field may greatly accelerate the migration of polystyrene microspheres, in a manner varying with pulse times, and the observed zones are made sharper. The particles concerned are just large enough that under constant field they appear not to enter the gel at all or to migrate very slowly: and merely lowering the applied field may also enhance their electrophoretic migration, though to a lesser extent than with field pulsing. These effects may be accounted for by gel mesh flexibility which, varying with the nature of the migrating species, may either help or hinder migration.
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    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Standard protocols for sample preparation for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) typically involve the combined use of heat and a reductant to fully disrupt protein-protein interactions and allow for constant ratios of SDS-binding to individual polypeptides. However, 14C-labeled forms of the membrane-bound, active-site-containing 27 kDa polypeptide of ammonia monooxygenase from Nitrosomonas europaea undergo an aggregation reaction when cells or membranes are heated in the presence of SDS-PAGE sample buffer. The aggregate produced after heating at 100°C is a soluble complex which fails to enter the stacking gel in discontinuous SDS-PAGE gels. The extent of the aggregation reaction is dependent on the temperature of sample preparation, and the reaction exhibits first-order kinetics at 65°C and 100°C (rates constants = 0.07 and 0.35 min-1, respectively). The rate of the aggregation reaction is further dependent on the concentration of reductant used in the sample buffer. However, the concentration of SDS does not significantly affect the rate of aggregation. The aggregated form of the 27 kDA polypeptide can be isolated by gel-permeation chromatography in the presence of SDS. The aggregated protein can also be returned to the monomeric state by incubation at high pH in the presence of SDS. The aggregation reaction also occurs with 14C2H2-labeled polypeptides in other species of autotrophic nitrifiers and a methanotrophic bacterium which expresses the particulate form of methane monooxygenase. We conclude that strongly hydrophobic amino acid sequences present in ammonia monooxygenase are responsible for the aggregation phenomenon.
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    Electrophoresis 14 (1993), S. 638-643 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Mouse liver proteins were classified into metal-binding and non-binding proteins by combining immobilized metal ion affinity chromatography (IMAC) and twodimensional electrophoresis (2-DE). The proteins were fractionated by three metal ions, Zn2+, Ni2+ and Cu2+, immobilized on iminodiacetic acid and then separated by 2-DE. The total number of protein spots resolved by 2-DE increased approximately twofold when the proteins were prefractionated by IMAC. By establishing 2-DE standard patterns, 371 proteins were selected and then characterized according to their specificity in binding the three different metal ions. Only 48 proteins did not bind to any of the three metal ions investigated. Cu2+ was the most efficient ion in binding different proteins (310) compared to the other metals. Cu2+ bound to 42 proteins specifically and to 268 proteins unspecifically. Both Zn2+ and Ni2+ showed specific affinity only to four proteins.
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    Cell Biochemistry and Function 11 (1993) 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 81
    ISSN: 0263-6484
    Keywords: Cellular inorganic phosphate ; hormone action ; intracellular metabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Uptake of orthophosphate (Pi) by osteoblast-like cells is known to be stimulated by parathyroid hormone (PTH), but effects on intracellular [Pi] have not been investigated. Here we show in rat osteoblast-like cells (UMR 106-06) that PTH (10-11 to 10-7 M) increases both 32Pi uptake and cellular [Pi] by up to 50 per cent. 1,25 Dihydroxyvitamin D3 (1,25D) (10-12 to 10-6 M) and salmon calcitonin (CT) (10-12 to 10-6 g ml-1) also increased cellular [Pi] (by up to 60 per cent), but the percentage increases in total cellular 32Pi uptake were smaller. The effects of 1,25D were transient (observable at 80 min and 6 h but not 24 h), and were also observed with 24,25 dihydroxy- and 25 hydroxyvitamin D3. Transient degradation of organic phosphorus pools to Pi might contribute to this increased [Pi]. These pools remain to be identified but were not shown to be phospholipids. Foetal bovine serum also affected cellular [Pi]. Care is therefore needed in distinguishing direct hormonal effects on cellular [Pi] from indirect effects arising from changes in the rate of cell growth.
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    Cell Biochemistry and Function 11 (1993), S. 35-44 
    ISSN: 0263-6484
    Keywords: Tunicamycin ; acetylated low density lipoprotein ; macrophages ; endocytosis ; proteolysis and chloroquine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effect of tunicamycin (TM) on the metabolism of acetylated low-density lipoprotein (AcLDL) was examined to determine whether N-linked glycosylation is required for the proper function of the AcLDL pathway. Proteolytic degradation of [125I]-AcLDL was increased twofold in the presence of TM. This did not occur via an increase in total lysosomal enzyme activity or extracellular proteolysis; rather, the rate of uptake of [125I]-AcLDL was increased. The enhanced degradation of AcLDL did not lead to a commensurate increase in the rate of synthesis of cholesteryl oleate. Conversely, the rate of cholesterol esterification was reduced in the presence of TM. The uptake of [125I]-AcLDL was more sensitive to inhibition by chloroquine in TM-treated cells. However, the presence of TM did not affect the ability of chloroquine to inhibit constitutive recycling of AcLDL binding sites. These results suggest that N-linked glycosylation may be involved in the regulation of AcLDL metabolism in J774 cells.
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    Cell Biochemistry and Function 11 (1993), S. 63-69 
    ISSN: 0263-6484
    Keywords: DNA ; osteoarthritis ; osteoporosis ; Feulgen ; microdensitometry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The normal amount of DNA in human diploid nuclei was determined by the use of the Feulgen reaction measured by microdensitometry. The DNA-content of nuclei in normal human articular cartilage was determined in nuclei of zones 3 and 4 of cartilage of the femoral head removed from osteoporotic fractured necks of femur. Analysis of the results indicated that a degree of synthesis of DNA occurred even in these zones of very elderly persons. Results on these zones in the articular cartilage of osteoarthritic joints indicated that different populations occurred. In some there was DNA-synthesis related to tetraploidy; in others, the DNA was very stable to acid hydrolysis with no sign of biosynthetic activity; in the last group, which contained erosions of the superficial zones, the DNA was unstable to hydrolysis.
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    Cell Biochemistry and Function 11 (1993), S. 119-124 
    ISSN: 0263-6484
    Keywords: UMR-106-01 cells ; phosphate transport ; alanine transport ; Na+/H+ exchange ; BCECF ; cycloheximide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The rat osteosarcoma cell line UMR-106-01 has an osteoblast-like phenotype. When grown in monolyer culture these cells transport inroganic phosphate and L-alanine via Na+-dependent transport systems. Exposure of these cells to a low phosphate medium for 4 h produced a 60-70 per cent increase in Na+-dependent phosphate uptake compared to control cells maintained in medium with a normal phosphate concentration. In contrast, Na+-dependent alanine uptake and Na+-independent phosphate uptake were not changed during phosphate deprivation. The increased phosphate uptake was due, in part, to an increased Vmax and was blocked completely by pretreatment with cycloheximide (70 μM). In these cells recovery of intracellular pH after acidification with NH4Cl is due primarily to the Na+/H+ exchange system. The rate of this recovery process, monitored with a pH sensitive indicator (BCECF), was decreased by more than 50 per cent in phosphate-deprived cells compared to controls indicating that Na+/H+ exchange was inhibited during phosphate deprivation.
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    Cell Biochemistry and Function 11 (1993), S. 131-135 
    ISSN: 0263-6484
    Keywords: Cytotoxicity ; benzo(c)fluorene derivative ; cell culture ; cell proliferation ; unbalanced growth ; cell metabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The highest concentration of 9-hydroxybenfluron (HBF) tested, namely 4·05 μmol1-1, induced immediate cytotoxic effects which were manifested by total inhibition of cell proliferation after only 24 h of culture. In a certain proportion of the cells cytolytic effects were observed at longer times of culture. Lower concentrations of HBF induced toxicity that was concentration- and time-dependent.The toxic effect appeared to occur in two phases. Cells, which had lost their ability to divide, did not stop their metabolism in which glutamine was the main source of energy. The results suggest that HBF primarily interferes with one of the phases of the cell cycle and only secondarily influences energy processes. Because benfluron (BF) and HBF have similar effects, it is suggested that the cytotoxicity of BF can be ascribed to the influence of its metabolite, HBF.
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  • 86
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    Cell Biochemistry and Function 11 (1993), S. 153-153 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 87
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    Cell Biochemistry and Function 11 (1993), S. 145-151 
    ISSN: 0263-6484
    Keywords: Hypothyroidism ; pancreatic islets ; FAD-glycerophosphate dehydrogenase ; insulin secretion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In thyroidectomized rats, the activity of FAD-linked glycerophosphate dehydrogenase was severely diminished in liver homogenates but not affected significantly in pancreatic islet homogenates, whilst the activity of 2-ketoglutarate dehydrogenase was decreased modestly in both liver and islet homogenates. Likewise, in intact islets of thyroidectomized rats, the generation of3HOH from [2-3H]glycerol was not decreased, and the ratio between oxidative and total glycolysis not significantly lower than in islets from sham-operated rats, at least in the presence of a high concentration of D-glucose. Nevertheless impaired oxidation of both D-[3,4-14C]glucose and D-[6-14C]glucose was observed in islets of thyroidectomized rats, the relative magnitude of such a decrease being more pronounced at a low than at a high D-glucose concentration. Such metabolic anomalies coincided with a lower level of plasma insulin and a decreased output of insulin by islets incubated at low (2·8 mM), but not higher, concentrations of D-glucose. It is concluded that hypothyroidism does not mimic the deficiency in islet FAD-linked glycerophosphate dehydrogenase activity found in rats with inherited or acquired non-insulin-dependent diabetes.
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  • 88
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 89
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    Cell Biochemistry and Function 11 (1993), S. 155-158 
    ISSN: 0263-6484
    Keywords: Pancreatic islets ; succinate dehydrogenase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Succinate dehydrogenase activity was measured in rat pancreatic islet homogenates incubated in the presence of [1,4-14C]succinate, the reaction velocity being judged through the generation of 14CO2 in the auxiliary reactions catalysed by pig heart fumarase and chicken liver NADP-malate dehydrogenase. In the presence of 1·0 mM succinate, the reaction velocity averaged 5·53 ± 0·44 pmol min-1 μg-1 islet protein. The Km for succinate was close to 0·4 mM and the enzymic activity was restricted to mitochondria. These kinetic results indicate that, under the present experimental conditions, the activity of succinate dehydrogenase does not vastly exceed that of either NAD-isocitrate dehydrogenase or the 2-ketoglutarate dehydrogenase complex, at least when the latter enzymes are activated by ADP and/or Ca2+. Nevertheless, the activity of succinate dehydrogenase is sufficient to account for the increase in O2 uptake evoked in intact islets by the monomethyl ester of succinic acid. It could become a rate-limiting step of the Krebs cycle in models of B-cell dysfunction.
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  • 90
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    Cell Biochemistry and Function 11 (1993), S. 159-165 
    ISSN: 0263-6484
    Keywords: Propranolol ; iodide ; thyroid cell ; antithyroid drug ; thyroid hormone ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effect of propranolol on the process of thyroid hormone formation was studied in a physiological culture system. Porcine thyroid follicles were preincubated with propranolol for 24 h. Iodide transport, iodine organification, and de novo thyroid hormone formation were measured by incubating these follicles with the mixture of carrier-free 0·1 μCi Na 125I and 50 nM NaI for 2 to 6 h at 37°C. A concentration of propranolol greater than 100 μM inhibited iodide transport in a dose-dependent manner; this inhibition was non-competitive with iodide and independent of thyrotropin (TSH). Reduced iodine organification and thyroid hormone formation was seen with 150 μM propranolol or greater. The inhibitory action of propranolol was not caused by beta-blocking activity, since D-propranolol (devoid of beta-blocking activity) inhibited iodide transport, and other beta-blockers (metoprolol, atenolol, and labetalol) did not inhibit iodide transport. The inhibition of iodide transport was most likely caused by membrane stabilizing activity since quinidine, which possess the same membrane stabilizing activity as propranolol, also inhibited iodide transport. TSH-mediated cAMP generation and Na +K+ ATPase activity, membrane functions for iodide transport, were unaffected by propranolol.Our study has shown, for the first time, that propranolol has a direct antithyroid action, namely inhibition of iodide transport in the intact thyroid follicle.
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  • 91
    ISSN: 0263-6484
    Keywords: β-glucosidase ; fibroblasts ; Gaucher's disease ; fluorogenic probes ; microspectrofluorimetry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Beta-glucosidase activity was evaluated in situ by means of fluorogenic probes in normal human fibroblasts and fibroblasts from homozygous carriers of the Gaucher trait. Probe internalization, targeting to lysosomes and post-cleavage probe retention were the primary concerns. Internalization and targeting were attempted by in situ photosensitized labilization of lysosomal membranes, lysosomotropic detergents and the use of low density lipid (LDL) or the receptor ligand apolipoprotein E (ApoE). Post-cleavage increase of fluorescence with fluoresceinyl (bis) betaglucopyranoside was appreciably above the rather large pre-cleavage emission. In cells incubated overnight with nonylumbelliferylbetaglucoside (UG9) in the presence of bovine serum albumin and in the absence of ApoE, the probe was dealt with as a cytotoxic agent, accumulating in a paranuclear cap, most likely comprising elements of the endoplasmic reticulum (ER) and Golgi apparatus. Targeting of UG9 to lysosomes occurred within 1 to 3 h of preincubation in the presence of ApoE. There was some evidence of specificity, as Gaucher fibroblasts exhibited weaker cleavage of UG9 (by 50 per cent or more) compared to normal fibroblasts, but in the Gaucher cells there was some residual beta-glucosidase activity. Cleavage of UG9 was nearly totally suppressed in Gaucher cells treated with the beta-glucosidase inhibitor, conduritol B epoxide, for 24 h to 7 days. Suppression in the control fibroblasts was evident but to a lesser degree. The in situ method of fluorogenic assay established for beta-glucosidase deficiency, is in principle applicable to enzyme deficiencies in other lysosomal storage diseases, or to evaluate enhanced enzyme activity following gene therapy.
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  • 92
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    Cell Biochemistry and Function 11 (1993), S. 211-219 
    ISSN: 0263-6484
    Keywords: Lectins ; cocaine ; morphine ; human peripheral blood mononuclear cells (PBMNC) ; common acute lymphoblastic leukemia antigen (CALLA) ; neutral endopeptidase 3.4.24.11 (NEP) ; phospholipase C; 5′-nucleotidase ; subcellular localization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have tested the effect of alkaloids (cocaine, morphine) and enkephalins on neutral endopeptidase of peripheral blood mononuclear cells activated by lectins. When treated with concanavalin A and cocaine, peripheral blood mononuclear cells showed an enhanced activity (+110 per cent) of the membrane neutral endopeptidase, which was not related to the expression of the common acute lymphoblastic leukemia antigen at the cell surface, although both molecules have the identical amino acid sequence. Phytohemagglutinin-P, morphine and synthetic enkephalins did not induce the activity of neutral endopeptidase nor the expression of common acute lymphoblastic leukemia antigen. Our findings suggested that the drugs of abuse, cocaine and morphine, affected specific membrane constituents without altering proliferation, subcellular localization of membrane enzymes or the surface immune phenotype of peripheral blood mononuclear cells.
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  • 93
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    Cell Biochemistry and Function 11 (1993), S. 201-209 
    ISSN: 0263-6484
    Keywords: Unsaturated fatty acid ; phosphatidylcholine ; phosphatidylethanolamine ; monocyte (U937 cells) ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in monocyte-like leukemia U937 cells was monitored by adding [3H]choline, [14C]ethanolamine or [14C]glycerol to the culture media; incorporation into phospholipid (PL) increased with time. The effect of unsaturated fatty acids (UFA) on PC and PE synthesis was investigated by pretreating U937 cells for 72h with 10 μM 18:1 (n -9), 18:2 (n -6), 18:3 (n -3), 20:4 (n -6) and 20:5 (n -3). The UFA caused no alteration in cell growth, as evidenced by light microscopy and the incorporation of [3H]thymidine and [3H]leucine. Total cellular uptake of radioactive precursors remained unaffected by all the treatments. Pretreatment with 20:5 resulted in approximately 25 per cent reduction in the incorporation of [3H]choline into PL, while no significant effect was detected with the other UFAs. 18:3, 20:4 and 20:5 depressed the incorporation of [14C]ethanolamine into PL by 34 per cent, 28 per cent and 49 per cent respectively. However, there was no redistribution of label with any of the treatments. 18:3, 20:4 and 20:5 also antagonized the stimulatory effect of endotoxin (LPS) on PC and PE synthesis. In addition, the incorporation from [14C]glycerol into PC and PE was reduced by 18:3, 20:4 and 20:5.Although the PL composition of the cells remained essentially unaffected, our study shows that chronic treatment of U937 cells with n -3 PUFA (20:5) depressed PC and PE synthesis, and 18:3 and 20:4 also caused inhibition of PE synthesis.
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  • 94
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    Cell Biochemistry and Function 11 (1993), S. ii 
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    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 95
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    Cell Biochemistry and Function 11 (1993), S. 225-230 
    ISSN: 0263-6484
    Keywords: Electrophoresis ; endothelia ; glycoproteins ; lectins ; smooth muscle cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Endothelial and smooth muscle cells were isolated from porcine aorta and kept in short-term culture. To determine the terminal carbohydate composition of the plasma membranes from both cell populations, the cells were incubated with a panel of fluorescein-labelled lectins. Both cell populations shared a number of terminal carbohydrates, but the N-galactosamine specific lectin Wistaria floribunda agglutinin labelled only the endothelial cells. A lectin which selectively labelled smooth muscle cells was not found. Western blot analysis of isolated endothelial cell membrane glycoproteins indicated that most membrane glycoproteins are labelled by Wistaria floribunda agglutinin.
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  • 96
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    Cell Biochemistry and Function 11 (1993), S. 231-239 
    ISSN: 0263-6484
    Keywords: Priming ; neutrophil modulation ; adhesion ; endotoxin ; chemotaxis ; cyclic AMP ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Neutrophils, treated with sequential additions of bacterial products such as endotoxin (E. Coli lipopolysaccharide, LPS) and the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), undergo to metabolic activation and express membrane-anchoring proteins that promote adhesion to serum-coated culture wells. By investigating the dose-response relationships of these phenomena, we have found that: (a) resting neutrophils do not produce a significant amount of superoxide (O2-) and show only minimal adhesion to serum-coated plastic surfaces; (b) fully activatory doeses (〉 5 × 10-8 M) of fMLP induce the release of O2- and a significant increase of the cell adhesion; (c) pretreatment of the cells for 1 h with LPS augments cell adhesion to serum-coated culture wells in the absence of further stimulation and primes the neutrophils to enhanced fMLP-dependent O2- release; (d) addition of low, substimulatory doses of fMLP (from 10-10 M to 5 × 10-9 M) inhibits and reverses the adhesion of LPS-treated cells, (e) high fMLP doses (〉 10-7 M) are additive to LPS in promoting adhesion. Phorbol-myristate acetate (〉 10-9M) increased adhesion in both normal and LPS-treated neutrophils, but low doses of this stimulant did not inhibit adhesion. Low doses (10-9 M) of fMLP increased intracellular cyclic AMP in both normal and LPS-treated neutrophils, suggesting that stimulus-induced rises in cAMP may be the negative signal responsible for down-modulation of adhesion. Low (5 × 10-9 M) and high (5 × 10-7 M) fMLP doses induced the same increase of expression of CD11/CD18 integrins, indicating that the inhibition of adhesion caused by low doses is not due to quantitative down-regulation of integrins. These findings may provide an in vitro model of the complex biological events involved in the regulation of neutrophil adhesion.
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  • 97
    ISSN: 0263-6484
    Keywords: Inflammatory stimulus ; lymphocyte ; glycolysis ; glutaminolysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Key enzyme activities of glycolysis, the pentose-phosphate pathway, the Krebs' cycle and glutaminolysis were measured in lymphocytes obtained from the control (CC), thioglycollate-injected (TG) and Walker 256 tumour-implanted (WT) groups, non-immune and immune inflammatory stimuli, respectively. The rates of incorporation of [2-14C]-thymidine and [5-3H]-uridine into cultured lymphocytes were also determined. The results indicated that the rates of both [2-14C]-thymidine and [5-3H]-uridine incorporation were enhanced in lymphocytes obtained from thioglycollate-injected (by an average of 80 per cent) and tumour-implanted animals (by 2·4-fold) as compared to control rats. Lymphocyte hexokinase activity diminished both in the TG (23 per cent) and WT (61 per cent) groups, whereas glucose 6-phosphate dehydrogenase activity was not altered due to the non-immune inflammatory stimulus, being reduced (23 per cent) in WT rats as compared to CC. The activity of lymphocyte citrate synthase was lowered by thioglycollate (39 per cent) and tumour-implantation (46 per cent). In contrast, glutaminase activity was augmented in lymphocytes from the TG (41 per cent) and was not modified in the WT groups. Taken as a whole, the presence of the Walker 256 tumour did not affect the capacity for glutamine utilization but depressed glucose metabolism in these cells. On the other hand, the non-immune inflammatory stimulus suppressed the activities of glycolysis and the Krebs' cycle and enhanced that of glutaminolysis in lymphocytes.
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  • 98
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    Cell Biochemistry and Function 11 (1993), S. 13-23 
    ISSN: 0263-6484
    Keywords: Cellular inorganic phosphate ; cellular orthophosphate ; intracellular metabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Osteoblast-like cells possess Na-dependent transporters which accumulate orthophosphate (Pi) from the extracellular medium. This may be important in bone formation. Here we describe parallel measurements of Pi uptake and cellular [Pi] in such cells from the rat (UMR 106-01 and UMR 106-06) and human (OB), and in non-osteoblastic human fibroblasts (Detroit 532 (DET)). In UMR 106-01, cellular [Pi] was weakly dependent on extracellular [Pi] and higher than expected from passive transport alone. [32Pi]-uptake was inhibited by Na deprivation, but paradoxically increased on K deprivation. With Na, 87 per cent of cellular 32P was found in organic phosphorus pools after only 5 min. Na deprivation also decreased cellular [Pi], in both UMR 106-01 and DET, but the decrease was smaller than that in [32Pi]-uptake. Ouabain decreased [32Pi]-uptake and cellular [Pi] in DET, but not in UMR 106-01. Regulation of cellular [Pi] is therefore at least partly dependent on Na/Pi co-transport, but this does not seem to be an exclusive property of osteoblasts.
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  • 99
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    Cell Biochemistry and Function 11 (1993), S. 1-11 
    ISSN: 0263-6484
    Keywords: Prion protein ; prion gene expression ; scrapie ; N2a cells ; mouse neuroblastoma cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The scrapie prion protein, PrPSc, is formed from its isoform, the cellular PrPc. There is evidence available indicating that PrPSc is necessary component of the infectious prion particle to cause a series of transmissible spongiform encephalopathies. We have used immunocytochemistry and RNA blotting techniques to investigate if infection with prions results in an increased PrP gene expression. For the experiments we used N2a cells which had been infected with prions (ScN2a cells). We demonstrated by confocal laser scanning microscopy that PrP-protein was present in the nucleus (predominantly in the nucleoli) of ScN2a cells. Analysis of the PrP-mRNA levels both in N2a- and in ScN2a cells using cDNA encoding PrPc revealed no marked alteration of the mRNA steady state level between the two cell strains. Likewise, in run-off experiments no changes in either PrP-specific transcription or in general transcriptional activity were found. The half-life of PrP-mRNA was found to be identical in both cell strains (7 h). Taken together, these results show that PrPSc and /or PrPc is present in the nucleus (nucleoli) of ScN2a cells but does not display and effect on the expression of the PrP gene.
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  • 100
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    Cell Biochemistry and Function 11 (1993), S. 107-117 
    ISSN: 0263-6484
    Keywords: Ethanol ; phosphatidylcholine ; phosphatidylethanolamine ; U937 cells (monocyte) ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effect of ethanol (ETOH) on the incorporation of [14C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [14C] 18 : 1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (≤ 1·0 per cent; v/v) had no effect. At concentrations greater than 1·5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased.The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [3H]choline or [14C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (≥ 1·5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40-50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [14C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18 : 1 and PL-base, the ratios of incorporation (base/18 : 1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [14C] ethanolamine into [14C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway.
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