Biomedica biochimica acta (Biomed Biochim Acta )

Publisher: Akademie der Wissenschaften der DDR. Presidium; Deutsche Gesellschaft für Experimentelle Medizin


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    Biomedica biochimica acta
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Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: In order to evaluate the role of cathepsin B (CB) as a proteinase involved in mammary tumor progression and its potential role as a tumor marker, we have measured the CB activity in cytosols of breast cancer tumor tissue using Z-Arg-Arg-AMC as the substrate and found a 23fold increase when compared to distantly located breast tissue from the same patient. In addition, urine of breast cancer patients under adjuvant chemotherapy was screened for CB immunoreactivity with a sandwich type enzyme immunoassay which revealed significant interindividual differences in concentrations with some urines containing immunoreactivity comparable to healthy controls. The urines were also investigated for CB activity but no differences between patients and controls were observed. However, urine contains thiol activatable proteinases causing substrate hydrolysis, which is to a varying extent inhibited by E-64 or Z-Phe-Phe-CHN2.
    Biomedica biochimica acta 02/1991; 50(4-6):555-60.
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    ABSTRACT: The known proportional increase in the amplitude of the pattern reversal visually evoked potential (VEP) with increasing stimulus area does not occur for the motion-onset VEP. When the stimulus area of the total field (6 degrees x 6 degrees) is compared to that of the half-field (6 degrees x 3 degrees), the N200 amplitude of the motion-onset VEP is not changed proportionally but remains almost constant. Reducing the pattern contrast beyond the saturation value for the motion-VEP yields essentially the same results. Contrary to the pattern reversal VEP, the amplitudes of the motion-onset VEP are found to be more pronounced for the nasal than for the temporal hemiretina. Our results support the notion of a genuine difference between the pattern- and the motion-analyzing visual system.
    Biomedica biochimica acta 02/1991; 50(9):1135-40.
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    ABSTRACT: The cDNAs for a number of multicatalytic proteinase (MCP) subunits have been cloned, characterized, and their primary structures have been determined. The mechanism for how MCP demonstrates its multicatalytic nature, especially protease activities, however, is still obscure, since no sequences similar to known protease sequences can be found in the sequences of MCP subunits thus far determined. To explain this fact, we propose a structural model for MCP: MCP consists of two classes of subunits, structural and catalytic, and the structural subunits constitute a "test-tube"-like container in which the other catalytic subunits sit and react with substrate. Most of the observations thus far obtained can be explained easily by this hypothesis, although various other possibilities are not excluded.
    Biomedica biochimica acta 02/1991; 50(4-6):459-64.
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    ABSTRACT: Some D-amino acid-substituted derivatives of bovine beta-casomorphin1-5 (CM) were found to share a particular affinity to central opioid receptors. As far as the antinociceptive potency of CM derivatives and their ability to produce tolerance is concerned, possible interactions of two selected CMs (Tyr-Pro-Phe-D-Pro-Gly, D-Pro4 and Tyr-Pro-D-Phe-Pro-Gly, D-Phe3) with morphine and, moreover, the doses necessary to induce tolerance were studied. Antinociception was tested by using the hot-plate test in experiments with mice. It was found that both CMs injected in doses up to 1 mumol/kg were not able to produce any sign of antinociception. It is all the more so interesting since such non-antinociceptive doses of D-Phe3 decreased analgesia induced by 15 mumol/kg morphine significantly. This could be regarded as a consequence of delta-receptor occupation by D-Phe3 and a resulting shift in the mu-receptor-mediated signal transduction. Finally, we found that repeated applications (once daily over a period of 20 days) induced a detectable tolerance to morphine. This suggests that opioid analgesia and tolerance must not be mediated by the same mechanisms and, moreover, that the mechanisms responsible for the development of tolerance are able to react more sensitive to an opioid.
    Biomedica biochimica acta 02/1991; 50(12):1255-60.
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    ABSTRACT: Prolyl endopeptidase, an enzyme exhibiting high specificity towards the Pro-Xaa bond, is thought to play an important role in the metabolism of biologically active peptides. We have purified the enzyme from pig muscle and observed significant differences in its kinetic behavior as compared to the extensively studied serine proteases, such as chymotrypsin. Thus, pH-dependence of, and kinetic deuterium isotope effects on, the rate constants indicated that the enzyme has two forms, which exhibit different activities and interconvert with changing pH. It can be concluded that a general base/acid-catalyzed acylation step is rate-limiting in the lower pH range, and an isotopically silent step, probably a conformational change preceding the acylation dominates the reaction in the physiological pH range.
    Biomedica biochimica acta 02/1991; 50(4-6):721-6.
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    ABSTRACT: Calcium-phosphate induced fusion of human erythrocytes was investigated using an assay based on the relief of the fluorescence selfquenching of a fluorescent amphiphile incorporated into ghost membranes as it occurs when labeled membranes are fused with unlabeled membranes. Measuring ghost fusion up to Ca(2+)-concentrations of 4 mM in the presence of 10 mM phosphate, both an acceleration of the fusion process and an enhancement of the fusion extent with increasing amounts of calcium were observed. Fusion takes place even in the case when calcium-phosphate complexes were formed in the suspension medium prior to the addition of ghosts. These results are in contrast to previous observations, obtained by an internal aqueous content mixing assay, where fusion of ghosts was inhibited at Ca(2+)-concentrations greater than 1.75 mM, and preformed calcium-phosphate complexes were not able to induce fusion. The results point to the necessity of utilizing content mixing assays in conjunction with the lipid dilution assay to get a more detailed insight in membrane fusion mechanism(s).
    Biomedica biochimica acta 02/1991; 50(3):251-6.
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    ABSTRACT: Hereditary spherocytosis (HS) is characterised with many deviations of red blood cells properties. We investigated a group of 14 patients with mild HS, 7 of whom were splenectomised. We found changes in the content and/or turnover of polyphosphoinositides and phosphatidic acid accompanied by the higher generation of inositol 1,4,5-trisphosphate. We suggest that the activation of the phosphoinositide signalling system may be crucial for the manifestation of HS.
    Biomedica biochimica acta 02/1991; 50(1):93-100.
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    ABSTRACT: We have examined the effects of salicylate on fluxes of lactate metabolism in rat hepatocytes using a steady state model. Salicylate produces an uncoupling effect, an inhibition of gluconeogenesis, a marked activation of pyruvate dehydrogenase flux, and an inhibition of endogenous fatty acid oxidation. Agents with known functions such as dinitrophenol and dichloroacetate were also compared in this system. The in vitro inhibition of gluconeogenesis caused by salicylate is not primarily related to the uncoupling effect. The fact that octanoate, but not palmitate, overcomes the salicylate inhibition of gluconeogenesis suggests that salicylyl CoA is involved in the inhibition. To relate in vitro studies to Reye's syndrome in vivo, in which medium chain dicarboxylic acids accumulate, we have also examined the effects of monomethyl suberate on liver lactate metabolism. This half ester is taken up by the hepatocytes, and causes inhibition of lactate gluconeogenesis, and uncoupling. Both salicylate and monomethyl suberate inhibit the oxidation of 0.2 mM octanoate by hepatocytes.
    Biomedica biochimica acta 02/1991; 50(7):921-30.
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    ABSTRACT: L-ornithine has growth potential for parenteral nutrition and as a component for biologically active peptides. A process for enzymatic conversion of L-arginine to L-ornithine with arginase has been developed and tested on a pilot scale. With activation of arginase by Mn2+ and stabilization by ascorbic acid, the enzyme is sufficiently active and stable for application in large-scale L-ornithine synthesis.
    Biomedica biochimica acta 02/1991; 50(10-11):S249-55.
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    ABSTRACT: Administration of stobadine, a cardioprotective substance in investigation prevents a decrease in the content of protein SH groups and glutathione in hearts of rats treated with high doses of isoproterenol (ISO) (30 mg/kg). Moreover, stobadine also attenuated the increase in the content of malondialdehyde and activities of catalase and glutathione reductase as well as a diminution in the GSH/GSSG ratio observed in heart mitochondria isolated from ISO-treated animals. Since stobadine may be considered as a scavenger of reactive oxygen species (ROS), the above effects of the latter substance support the assumption about a possible involvement of reactive oxygen species (ROS) in some processes initiated by administration of ISO in doses inducing cardiac hypertrophy. However our results also indicate that ROS-mediated processes are not necessarily involved in the mechanism of induction of cardiac hypertrophy itself.
    Biomedica biochimica acta 02/1991; 50(12):1251-4.
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    ABSTRACT: Synthesis of dipeptides benzoyl Arginine leucinamide and kyotorphin catalyzed by highly stabilized derivatives of trypsin and chymotrypsin have been performed. Extreme experimental conditions could be tested and parameters of industrial interest could be improved provided the high activity and stability of the derivatives in these unfavourable environments. Thermodynamically controlled synthesis catalyzed by trypsin could be optimized and 97% conversion was obtained in 90% organic cosolvents. 100% yields were achieved in kinetically controlled synthesis catalyzed by trypsin in aqueous medium in the presence of IM Ammonium Sulphate. Higher starting concentrations of poorly soluble substrates of chymotrypsin could be used in a reaction medium containing 50% DMF and 95% yield were obtained.
    Biomedica biochimica acta 02/1991; 50(10-11):S110-3.
  • Biomedica biochimica acta 02/1991; 50(10-11):S193-6.
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    ABSTRACT: Freezing of the aqueous reaction mixtures can cause dramatically increased peptide yields in protease-catalyzed kinetically controlled peptide synthesis reactions. The "Freeze-concentration model" which implies that the enzymic reaction within a macroscopically frozen system takes place in an unfrozen liquid phase is presented as a reasonable basis for the understanding of the effect of freezing. It is shown that L-amino acid esters are effective nucleophiles in alpha-chymotrypsin-catalyzed acyl transfer reactions when these are performed in frozen state.
    Biomedica biochimica acta 02/1991; 50(10-11):S84-9.
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    ABSTRACT: In medical research and clinical practice, the increasing application of filtration measurements, to characterize the erythrocyte deformability has aroused an intensive discussion about possible erroneous interpretations of measured values (filterability indices). In the present paper the influence of white blood cells (WBC) on these indices is described and discussed in relation to a mathematical model of the filtration process. We found for two gravity filtration systems, using cellulose filters and concentrated erythrocytes suspensions sensitivities of their filterability indices of (1 +/- 0.1)% per 100 WBC/microliter (initial filtration rate measurement) and (3 +/- 0.3)% per 100 WBC/microliter (hemofiltrometer). In comparison to Nuclepore filtration systems with sensitivities of 5-12% per 100 WBC/microliters, cellulose filters were much less sensitive to WBC. The necessity, advantages and disadvantages of additional procedures for WBC removal are discussed. A standardization of residual WBC-count in limits of 500 +/- 300 WBC/microliters at a hematocrit of 60% is proposed. Fitting of the filtration curves of the hemofiltrometer to a model function may either improve the accuracy of estimation of filterability indices or divide them into two model parameters with different WBC-sensitivities: the initial filtration rate parameter having a low WBC-sensitivity ((0.9 +/- 0.2)% per 100 WBC/microliter) and the occlusion related parameter having a 12 times higher sensitivity ((11 +/- 1)% per 100 WBC/microliter). Interpretation, advantages and disadvantages of the model parameters are discussed also in comparison to the filterability index.
    Biomedica biochimica acta 02/1991; 50(2):159-68.
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    ABSTRACT: Synthesis of LHRH N-terminal pentapeptide Glp1-His2-Trp3-Ser4-Tyr5-OMe (6) involving 3 + 2 enzymatic coupling has been developed. Synthetic strategy features the formation of one peptide bond (Glp-His) by chemical coupling and three peptide bonds by means of papain (Trp-Ser, Ser-Tyr) or alpha-chymotrypsin (His-Trp). High efficiency of this six-step synthesis is demonstrated by 44% overall yield. Its advantages are the use of inexpensive enzymes, simple isolation of intermediates and final pentapeptide, and easy recovery of substrates.
    Biomedica biochimica acta 02/1991; 50(10-11):S205-8.
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    ABSTRACT: The transglycosylation from raffinose and lactose to Aloc-Ser-OMe is catalyzed respectively by alpha and beta galactosidases. Transglycosylation from cellobiose has been achieved with beta-glucosidase. The simplicity of the enzymatic synthesis, the stereospecificity of the condensations in one-pot reactions and the ease of purification give the method value for large scale preparation of beta-linked derivatives. The protective groups of the serine residue can be cleaved under mild conditions: the ester group has been removed quantitatively by papain catalyzed hydrolysis and the Aloc group by a Pd (0) hydrostannolytic cleavage.
    Biomedica biochimica acta 02/1991; 50(10-11):S231-6.
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    ABSTRACT: Long-Evans hooded rats were not able neither to reach criterion of active avoidance tasks in a Y-maze and in a jump test, nor in brightness discrimination after lesions of the NRTP without preoperative experience in these tests. After preoperative consolidation of active avoidance, the retention of Y-maze avoidance performance was zero and of jump test avoidance at 25%. The Y-maze avoidance was again relearned except in the task with 2:2 alternation of goals whereas retention of brightness discrimination was not affected. In the jump test, avoidance relearning was evidently retarded. Ambulatory and exploratory behaviour in the open field were significantly reduced after NRTP lesions. The NRTP is evidently involved in the establishment and control of goal-directed behaviour.
    Biomedica biochimica acta 02/1991; 50(2):169-74.
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    ABSTRACT: In human, goat and chick amniotic fluid the contents of lecithin, lysolecithin and phosphatidyl ethanolamine increase during embryonic development. The sphingomyelin content however, increased during the early period only and declined during the latter part of development.
    Biomedica biochimica acta 02/1991; 50(8):955-8.
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    ABSTRACT: The interaction of delta (Ser50)-hirudin with alpha-thrombin has been investigated. Deletion of Ser50 of r-hirudin caused a 2.7 fold increase of the Ki for its complex with alpha-thrombin. Determination of the rate constants kon and koff for complex formation showed that this effect was mainly due to a change in koff.
    Biomedica biochimica acta 02/1991; 50(4-6):707-10.
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    ABSTRACT: The ratio of hydrolysis to aminolysis product in papain-catalyzed acyl transfer reactions using various nucleophiles was determined. The data are interpreted in terms of binding specificity. The acyl transfer reactions were performed using the acyl donor Mal-Phe-Ala-OEtCl. The analysis of the structure-activity relationships of the hydrophobic S1'-P1' contact indicates that the S1' subsite can accommodate maximally three methyl(ene) groups. Hydrophilic amino acid side chains are better bound to S1' than can be explained by their hydrophobicities. The S2' as well as the S3' binding subsite exhibits a preference for space-filling hydrophobic amino acid residues.
    Biomedica biochimica acta 02/1991; 50(10-11):S122-6.