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  • 1
    ISSN: 1573-4919
    Keywords: 6-phosphofructo-1-kinase ; aging ; heart ; energy metabolism ; metabolic regulation ; isozymes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Relative to 2–3 month rats, total 6-phosphofructo-1-kinase (PFK) activity in heart atria from 12 month rats declined 31%; but, by 24 months it was decreased by only 13%. PFK activities from 12 and 24 month ventricles relative to the 2–3 month rat were decreased by 40% and 30%, respectively. This change in PFK activity in each heart region was associated with alterations of subunit composition. In heart atria from 12 and 24 month rats when compared to 3 month rats, the levels of L-type subunit were not significantly different; but the levels of the M-type subunit were decreased by 43% and 38%, respectively. With respect to levels in 2–3 month atria, the C-type subunit in 12 month atria decreased by 27%; and at 24 months it increased by 31%. Making the same comparison for the heart ventricle at 12 and 24 months, L-type subunit decreased by 30% and 24% respectively; M-type subunit decreased by approximately 47%; and the C-type subunit increased 1.9 and 4.7 fold, respectively. These age-related changes of subunit composition in atrial and ventricular PFK isozyme pools led to changes in their kinetic and regulatory properties suggesting that the aged rat could exhibit a diminished capacity to produce ATP from glucose.
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  • 2
    ISSN: 1573-4919
    Keywords: Rb and p53 genes ; gene expression ; colorectal cancers ; colon carcinoma cell lines ; cell cycle
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We have compared the expression of the retinoblastoma (Rb) and p53 genes in normal human fibroblasts, colon carcinoma cell lines, matched pairs of colorectal tumor tissues and adjacent normal mucosa and in synchronized human diploid fibroblast cell line W138. The increased expression of Rb and p53 RNA was observed in a majority of colorectal cancers in comparison to adjacent normal mucosa and is accompanied by proportional increase in the expression of histone H3 gene. The Rb and p53 RNA levels varied significantly between the various colon carcinoma cell lines. However, we found that the expression of Rb and p53 RNA is regulated differently in cell cycle synchronized normal human fibroblasts. The Rb mRNA level did not change with the position in the cell cycle and did not differ significantly whether the cells were serum deprived or in 10% serum. But p53 mRNA expression follows the same pattern as histone H3 mRNA.
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  • 3
    ISSN: 1573-4919
    Keywords: glutathione peroxidase ; catalase ; superoxide dismutase ; enzyme stability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Activities of the anti-oxidative enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase were studied in rat tissues to determine the ability of detergents both to solubilize the enzymes and also to stabilize enzyme activity. Rat brain, heart and liver were homogenized in 0.1M KCl, 0.1% sodium dodecyl sulfate, 0.1% lubrol, or 0.1% cetyl-trimethylammonium bromide. In general lubrol was more effective than the other solutions in solubilizing GPx and catalase. Lubrol and 0.1M KCl were equally effective in solubilizing SOD. The highest enzyme activities were (1) SOD: 2484 ng/mg (brain), 2501 ng/mg (heart), and 5586 ng/mg (liver); (2) GPx: 224 mU/mg (brain), 1870 mU/mg (heart), and 7332 mU/mg (liver); (3) catalase: 2.8 mU/mg (brain), 10.6 mU/mg (heart), and 309 mU/mg (liver). While cetyl trimethylammonium bromide is marginally better than sodium dodecyl sulfate in solubilizing active enzyme, neither ionic detergent has any advantage over lubrol or 0.1M KCl. For catalase and GPx, enzyme activity loss with time is biphasic. After initial, rapid activity loss (1–5 days for GPx and 7–10 days for catalase) the differences noted among the homogenizing solutions disappear and very little if any activity loss is noted over the next 2–3 weeks. For catalase and GPx, only baseline enzyme activity from t = 0 – 3 weeks is found in the most chaotropic solution, 0.1% sodium dodecyl sulfate while biphasic activity loss is most pronounced in 0.1% lubrol. These results may indicate active GPx and catalase species stabilized by a lipid-like environment. Correlatingin vitro catalase or GPx measurements within vivo anti-oxidative protection may underestimate tissue defences.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Molecular and cellular biochemistry 107 (1991), S. 33-44 
    ISSN: 1573-4919
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Summary A discontinuous preparative polyacrylamide gel electrophoresis system has been developed and used to purify both the nicked and unnicked forms of tetanus toxin. The system was also used to prepare purified H and L chain peptides from the nicked toxin. The results show that the endogenous protease(s), which convert unnicked toxin to the nicked form, produce multiple species of nicked toxin, and heterogeneity in the H and L chains. The major amino termini of the toxins and their peptide components are: extract toxin, proline; filtrate toxin, proline, serine and asparagine; L chain, proline; and H chain, serine and asparagine. The L chain is located in the amino terminal position of the toxin molecule and the H chain the carboxy terminal end. A model is proposed to explain these results. Using the analytical ultracentrifuge, we have determined the molecular weights of extract and filtrate toxins to be 140 000 ± 5 000 and 128 000 ± 3 000, respectively. Using S DS-polyacrylamide gel electrophoresis we estimate the molecular weights of the H and L chains to be 87 000 and 48 000 daltons, respectively. Circular dichroic spectra of the toxins and their peptide components indicate that: the major tryptophanyl band in the toxin is contributed almost entirely by the H chain, the microenvironments of all the aromatics and disulfides in the two toxins appear to have small if any differences, the two toxins show little difference in their ordered secondary structure, and the two peptides when separated from one another still retain 80% of the helical structure that is present in the intact toxin but show a considerable loss of β-structure. The crystalline form of the nicked toxin has a hexagonal symmetry with two dimensional reciprocal lattice constants of 1/150 Å−1 and 1/150 Å−1. The crystals appear to belong to the two dimensional plane group P6 suggesting that each unit cell contains 6 or a multiple of 6 toxin molecules.
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  • 5
    ISSN: 1573-4919
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Summary Nuclei isolated from ischemic liver show a consistent reduction of their RNA synthesis. The reduction persists at high ionic strength and in the presence of heparin, when RNA synthesis in vitro is fully activated and occurs in the presence as well as in the absence of a-amanitin. Both the number of transcribing polymerase molecules and the rate of elongation of initiated polynucleotide chains seem to be equally affected.
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  • 6
    ISSN: 1573-4919
    Keywords: ribosomal protein ; hepatectomy ; cycloheximide ; insulin ; phosphopeptide mapping ; phosphoprotein phosphatases
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Summary Spontaneous S6 phosphatase activities dephosphorylating Ser(P)-235 and Ser(P)-236 of the ribosomal protein S6 were measured and compared in microsomes and cytosol of rat liver. The substrate used, small (40S) ribosomal subunits 32P-labelled in vitro by protein kinase A, contained phosphorylated S6 (mainly in the dephosphorylated form) and some minor phosphorylated species. The microsomal and cytosolic S6 phosphatase activities displayed a number of distinct properties. The microsomal activity, representing ca 20% of the S6 phosphatase activity in the post-mitochondrial supernatant, was mainly due to a type-1 phosphatase and dephosphorylated only S6. The remaining post-mitochondrial S6 phosphatase activity, which was fully recovered in the cytosol, and appeared to result from a combination of type-1 (43%) and type 2 (57%) phosphatases, acted on S6 as well as on the minor phosphorylated species. The microsomal activity was 50% inhibited by MgCl2 (l0 mM) and was stimulated at least 4.3 fold by MnCl2 (1 mM), while the cytosolic activity was inhibited only 18% by Mg2+ (10 mM) and was increased 2.2 fold by Mn2+ (1 mM). The microsomal activity was increased 10% (P 〈 0.06) by lower doses of insulin (25 U/Kg) and 14% (P 〈 0.05) by vanadate, but was not significantly (P 〉 0.10) affected by larger doses of insulin (100 U/kg), hepatectomy or cycloheximide. By comparison the cytosolic S6 phosphatase activity was unresponsive to insulin and vanadate, but was decreased 14% and 17% (P 〈 0.05) by hepatectomy and cycloheximide. It is concluded that (i) there. are clear differences between the microsomal and cytosolic S6 phosphatase activities, which may be relevant to their specific functions in the cell, and (ii) the inhibition of cytosolic S6 phosphatase activity by hepatectomy and cycloheximide may contribute to the increase in hepatic S6 phosphorylation induced by these treatments.
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  • 7
    ISSN: 1573-4919
    Keywords: heart sarcolemmal membrane ; sarcolemmal Ca2+/Mg2+ ATPase ; concanaval in A binding ; phospholipid content ; cholesterol content ; polysaccharide content
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The Ca2+/Mg2+ ATPase, which is activated by millimolar concentrations of Ca2+ or Mg2+, was solubilized from rat heart plasma membrane by employing lysophosphatidylcholine, CHAPS, Nal, EDTA and Tris-HCI at pH 7.4. The enzyme was purified by sucrose density gradient, Affi-Gel Blue column and Sepharose 6B column chromatography. The purified enzyme was seen as a single peptide band in the sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of about 90,000. The apparent molecular weight of the holoenzyme as determined under non-dissociating conditions by gel filtration on Sepharose 6B column was about 180,000 indicating two subunits. The enzyme was insensitive to ouabain, verapamil, vanadate, oligomycin, N,N-dicyclohexylcarbodiimide and NaN3, but was markedly inhibited by 20 µM gramacidin S and 50 µM trifluoperazine. Analysis of the purified Ca2+/Mg2+ ATPase revealed the presence of 17 amino acids where leucine, glutamic acid and aspartic acid were the major components and histidine, cysteine and methionine were the minor components. The purified enzyme was associated with 19.7 µmol phospholipid/mg protein which was 60 times higher than the phospholipid content in plasma membrane. The cholesterol content in the purified enzyme preparation was 0.75 µmol/mg protein and this represented an 8-fold enrichment over plasma membrane. The glycoprotein nature of the enzyme was evident from the positive periodic acid-Schiff staining of the purified Cau2+/MgATPase in the sodium dodecyl sulfate polyacrylamide gel. The polysaccharide content of the enzyme was enriched 8-fold over plasma membrane; neurominidase treatment decreased the polysaccharide content. Concanavalin A prevented the ATP-dependent inactivation of the purified Ca2+/Mg2+ ATPase and was found to bind to the purified enzyme with a KD of 576 nM and Bmax of 4.52 nmol/mg protein. The results indicate that Ca2+/Mg2+ ATPase is a glycoprotein and contains a large amount of lipids.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Molecular and cellular biochemistry 107 (1991), S. 161-168 
    ISSN: 1573-4919
    Keywords: antigenic determinants ; histone Hl° ; immunoblotting ; peptides
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In order to study the antigenic structure of histone Hl° the purified protein from mouse liver was subjected to different chemical and enzymatic treatments (CNBr, acetic acid, trypsin, chymotrypsin). The resulting peptides were fractionated in SDS-containing or acid-urea polyacrylamide gels, transferred by electroblotting onto nitrocellulose paper and probed with specific rabbit anti-H1° antiserum. The C-terminal fragments 99–193 (obtained following acetic acid hydrolysis) and 107–193 (obtained by chymotrypsin digestion) also exhibited strong immunoreactivity. Fragment 1–30 (CNBr cleavage) contained antigenic determinants while the shorter fragments 1–22 and 1–28 (acetic acid hydrolysis) failed to show any detectable reactivity. It was concluded that, in contrast to histone H5 whose reactivity is mainly concentrated to the globular domain of the molecule, the antigenic determinants in histone H1° are more or less evenly distributed along the polypeptide chain with the possible exception of the short unstructured N-nose.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    ISSN: 1573-4919
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 10
    ISSN: 1573-4919
    Keywords: HIV-1 gp-160 synthetic peptide ; anti-HIV-1 xenogeneic immune RNA ; interspecies transfer of immunity ; anti-HIV-1 cellular immunological memory
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract A sheep was immunized with a synthetic peptide corresponding to amino acid residues 586–606 of the precursor envelope protein GP-160 of the HIV-1 including a conserved epitope region of the GP-41 transmembrane protein in the mature viral particles, referred to as SM 284 HIV-1 [1]. It is demonstrated that immune RNA extracted from the lymphoid organs of the immunized animal (SM 284 HIV-1 I-RNA) was able to transfer immune cellular reactivity to SM 284 HIV-1 in vitro to human and rabbit lymphocytes and in vivo to Cebus apella monkeys. The transfer was detected by the leukocyte adherence inhibition test (LAI) as an indicator of cellular reactivity. One of the most relevant results was the demonstration that SM 284 HIV-1 I-RNA was able to induce cellular immunological memory in vivo in monkeys. These results may be relevant to delineate a new alternative for immunomodulation against HIV infection.
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