Acta biologica et medica Germanica最新文献

Modification of the serine and histidine residue in the active centre of thermitase with diisopropylfluorophosphate (DFP) or L-1-tosylamide-2-phenylethyl chloromethylketon (TPCK), and of the only SH-group of the enzyme, with Hg-compounds causes an activity loss against hydrolysis of 4-nitrophenylacetate. While the modification of cysteine prevents reaction of serine and histidine in the active centre of the enzyme with DFP and TPCK, respectively, the Hg2+- and CF3Hg+-binding to the SH-group after modification of essential amino acid residues in the active centre is retained. To elucidate the interaction of the SH-group with the active centre, the modified products of thermitase were investigated for their thermostability. Ca2+-ions were found to have a stabilizing effect on all the modified products of thermitase, as well as on the native enzyme. Simultaneous modification of the cysteine and serine leads to an increase in thermostability of thermitase, whilst double modification at the cysteine and histidine causes destabilization of the enzyme.

Dimethylnitrosamine (DMN)-N-demethylation by 10,000 x g liver supernatant follows a typical Michaelis-Menten kinetics with one apparent Km (4.0 . 10(-4) M) and V max (0.460 nmole/mg . min) of 30-day-old male rats. The investigation of hepatic DMN-N-demethylation in dependence on age reveals maximum activities in 30- to 60-day-old male rats. The demethylating enzyme activity is inducible by phenobarbital (PB), not by 3-methylcholanthrene (3-MC). Pretreatment of animals with PB plus 3-MC resulted in reduced inducibility compared with PB-pretreatment alone. Probably this is not due to an inhibition of PB-induction by 3-MC, but effected by PB residues in the liver tissue and in the incubation mixture. The elimination rate of PB in young rats seems to be retarded if high doses of the inducers PB and 3-MC are administered simultaneously. Additionally changes in inducible cytochrome P-450 subspecies have to be considered.

The rate constant of the Na+ efflux of human erythrocytes in isotonic solutions of various ionic strength varied over NaCl concentration was measured. The Na+ efflux remained constant over a wide range of ionic strength. Only under conditions where the transmembrane potential was near O mV, a local minimum could be detected. The rate constant of the ouabain-insensitive part of the Na+ efflux exhibited a strong increase at reduced exterior ionic strength. When reducing the extracellular NaCl concentration and at the same time equivalently increasing the extracellular KCl concentration in solutions of physiological ionic strength, a reduction of the rate constant of the Na+ efflux was found. It was established that an increase of the intracellular pH increases the rate constant of the Na+ efflux. A change of transmembrane potential from -7 to 52 mV at constant intracellular pH had no influence on the Na+ efflux. The change of the exterior surface potential of erythrocytes by preincubation with neuraminidase had no influence on the Na+ efflux in the range of the ionic strength studied.

Supernatants of human lymphocyte cultures incubated with cyclosporin A (CS-A) for 90 min reveal an inhibitory effect on the electrophoretic mobility of guinea-pig macrophages. This effect is distinctly demonstrable already at CS-A concentrations from 10-7 micrograms/ml on with a maximum at 10-3 micrograms/ml. An inhibitory action of CS-A on PHA-induced lymphocyte proliferation is detectable only beyond 10-2 micrograms/ml CS-A. The CS-A induced lymphokine is comparable to that stimulated by other ways (PHA, ConA).

Behavioral and neurochemical results refer to an impairment of the dopaminergic system produced by hypoxia. On that account we determined the chemically (K+, amphetamine) and electrically stimulated release of dopamine from rat striatum slices which were preloaded with labeled dopamine following the animals' exposure to hypobaric hypoxia. The effect of hypoxia consists in a marked inhibition of dopamine release depending both on the degree of hypoxia and on the kind of stimuli used. Dopamine release inhibition reflects not only the direct effect of hypoxia, which seems to be connected especially with the vesicular compartment, but also adaption and restitution processes. Regarding the effects on rats at the age of two weeks the resistance of young animals against hypoxia may be founded also upon missing vesicular mechanisms of distinct vulnerability.

The gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC) and the glycolytic enzyme pyruvate kinase (PK), regulating pyruvate metabolism, were determined in the livers of 71 fetuses, which were developed of 25 dams on the 80th, 100th, 106th, and 112th day of gestation (length: 115 days) by Caesarean section. For comparative purposes the same enzymes were estimated in 12 naturally born piglets immediately after delivery. During the period under examination (the last third of gestation) the total activity of PEPCK has its highest values at the 80th day, the PC and PK activity at the 100th day of gestation. The activities of all 3 enzymes decrease during the last 2 weeks of gestation until birth. The cytosolic part of PEPCK amounts to 10-15 per cent of total activity and develops in a parallel manner. In newborn piglets a further decline of the PC and total PEPCK activity can be observed, but the cytosolic PEPCK activity remains constant, and therefore its relative proportion increases to 25% of the total activity. The PK activity rises distinctly (1.5 times). The role of these enzymes in the carbohydrate metabolism of the fetus, especially in gluconeogenesis, is discussed in detail.

Using a radiochemical method the activity of the gluconeogenic key enzymes phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC) was determined in liver homogenates of 58 piglets (from birth to 10 days of life), 4 weaners and 18 fattening pigs. Simultaneously, the cytosolic PEPCK activity was estimated. In liver of newborn piglets (immediately after birth without suckling) total PEPCK activity is one half, cytosolic PEPCK activity one sixth and PC activity one half of that occurring in adult pig's liver. During regular suckling within the first 12 h of life, the total PEPCK activity of piglet liver rises slowly, however, cytosolic PEPCK activity increases 3 to 4 times and PC activity in the mitochondria twice. After the 2nd day of life the activity of both enzymes reaches a level which is kept during the whole suckling period: total PEPCK activity is 1.5 to 2-fold, cytosolic PEPCK activity is 2 to 3-fold higher, and PC activity is at the level existing at birth. In weaners cytosolic PEPCK activity further rises to the level existing in the liver of adult pigs. The PEPCK and PC activities in the liver at birth enables the piglet for gluconeogenesis, but due to the postnatal increase of enzyme activities, the prerequisites for an intensive synthesis of glucose are given only after the 2nd day of life.

There is presented a procedure for determining the phase fractions in a cell population (i.e. the fractions of the G1-, S- and G2 + M-cells) from the corresponding DNA histogram obtained by flow cytometry. The evaluation procedure can regard arbitrary apriori information about the model parameters. It is based on a mathematical model of the DNA distribution for a growing cell population which contains the phase fractions and some further values as parameters. The parameter estimations rest on the Maximum-Likelihood-Principle by which the parameters of the theoretical model must be determined in such a way that the measured histogram corresponds to a maximum probability. As a consequence of the model flexibility especially the S-phase fraction which can be determined independently by labeling techniques, can be regarded as a fixed predetermined value. Such apriori information enhances the preciseness of the parameter estimation. The applications have shown that the determination of the S-phase fraction by means of flow cytometry can be very unprecise especially under partial synchronization. For the application of the evaluation programs (programming language ALGOL) the histograms must be free of cell detritus and clumping.

Plasma membranes from tumor cells in enzootic bovine leukosis and from normal bovine lymphoid cells of different sources have been isolated and characterized. The glycoprotein composition of the different membranes has been studied by SDS-PAGE and by analysis of the carbohydrate composition as well as by lectin binding of glycoprotein fractions obtained by phenol treatment of the membranes. No differences in the electrophoretical glycoprotein pattern could be detected comparing (nonmalignant) cells from persistent lymphocytosis and tumour cells, suggesting that malignancy is not associated with the gain or loss of a major glycoprotein. A 151.5 kD glycoprotein, present in membranes from normal lymph node lymphocytes, seems to be absent in the membranes of peripheral blood lymphocytes as well as of tumour cells. The loss of this glycoprotein might thus be associated with a loss of sessility of bovine lymphoid cells. The carbohydrate analysis and lectin binding of extracted glycoproteins revealed a decreased fucosylation accompanied by an increased exposure of galactose residues as well as a loss or a decreased complexity of N-glycosidically bound oligosaccharides in the tumour cell glycoproteins compared with those of normal cells. These findings are discussed with regard to disturbed growth regulation of leukosis tumour cells.