Acta biologica et medica Germanica最新文献

We describe an enzyme-immunoassay for the determination of factor VIII antigen. After representation of the isolation of proteins the enzyme-immunoassay is presented. The principle of the method is the following: Test plasma is mixed with rabbit antibody in excess and incubated at 37 degrees C. The incubation mixture is added to polystyrene tubes, which are coated with human factor VIII. The rabbit antibody is available to adhere to factor VIII coating the tube and can be detected with an enzyme-labeled antibody to rabbit IgG. This method is sensitive to 7.8 . 10(-3) U/ml factor VIII antigen; the variation coefficient is 10.9%.

Soluble guanylate cyclase activity from guinea pig heart is inhibited by increasing concentrations of sodium citrate. The Ki value was found to be 2.83 +/- 0.05 mM in the presence of 3 mM Mn2+ and 0.6 mM GTP. Citrate acts by lowering Vmax and increasing the apparent values of Km for GTP and K0.5 for Mn2+ and Mg2+. The soluble guanylate cyclase, activated by sodium nitroprusside, was also inhibited by citrate. This inhibitory action of citrate was not restricted to soluble guanylate cyclase activity of the heart and has been demonstrated also in the supernatant of lung, liver, diencephalon and in the homogenate of blood platelets. Since citrate is known to be an important intermediate of metabolism, its intracellular concentration may be also of relevance for guanylate cyclase activity.

Permanent diabetes was produced in 16 out of 55 dogs by partial pancreatectomy (77% of the calculated organ weight) and simultaneous infusion of 2 mg/kg streptozotocin into the superior pancreaticoduodenal artery. The animals exhibited hyperglycemia, absolute lack of endogenous B-cell function, and ketosis, but no exocrine pancreatic insufficiency. 21 animals needed up to 7 additional subsequent intravenous streptozotocin injections (15 mg/kg each at intervals of 3 days). In 18 animals the procedure failed to render them diabetic; they died mainly from toxic effects of the drug. There were severe pathohistological changes in all streptozotocin-treated animals. Besides the well known alterations of the islets of Langerhans, lymphocytic inflammations were found in numerous organs including the exocrine pancreas. In most cases they were combined with degenerative changes of the organ parenchyma, particularly in kidney and liver. These findings were not correlated to the sex of the animals, to the occurrence and severity if diabetes, to the time of survival, or to the streptozotocin dose applied. But they were obviously related to the clinical picture existing besides diabetes. It is concluded that the model of experimental diabetes presented might be useful in a carnivorous big animal species but that toxic streptozotocin effects are to be expected when the dose administered exceeds 2 mg/kg.

Labelled hemoglobin-haptoglobin (ham-hap), galactosylated serum albumin (gal-SA) and polymeric immunoglobulin A (p IgA) were injected intravenously to rats or mice. The labels disappeared from the plasma with a half-time of about 5 min and were almost entirely found associated with the liver where degradation products progressively appear. The uptake of hem-hap and gal-SA are partially saturable as a function of the plasmatic concentration and the uptake of gal-SA can be completely inhibited by the simultaneous injection of asialofetuin. About 45 min after injection to rats, labelled material appears in the bile in amounts corresponding to 3.9% of the injected dose (hem-hap), 2.8% (gal-SA) and 60.1% (p IgA). The molecular weight of the labelled material transferred into the bile has been characterized: it consists almost entirely of intact IgA and for about 60% of intact hem-hap and gal-SA. Cell fractionation experiments indicate that 4 min after injection, the label is associated with components which equilibrate around a density of 1.13 g/cm3 and which dissociate from marker enzymes of Golgi complex, plasma membrane and lysosomes. Longer times after injection (from 20 min for hem-hap and gal-SA to 1 h for p IgA) labelled material appears, within lysosomes. To explain all these data, we suggest that after binding to plasma membrane receptors, the ligands are rapidly interiorized into pinocytic vesicles which fuse with lysosomes. Most of the hem-hap and gal-SA molecules but only part of p IgA would be released and subsequently digested; these vesicles would dissociate from lysosomes and fuse with the biliary membrane where the molecules still bound to the membrane of the vesicles would be detached and excreted into the bile.

In pig fetuses (19 of 8 dams) developed by Caesarean section the dry matter and protein content of the kidneys and their PEPCK activity remain constant during the last third (from 80th to 112th day) of gestation. After birth the dry matter content of the kidneys rises slowly, but their protein content remarkably. In the kidneys of suckling piglets (17 animals of 3 offsprings) the FDPase activity remains at the same level from birth to the 9th day of life, while in the same time the G6Pase activity rises 1.5-2 times. In the kidneys of newborn piglets the total PEPCK activity increases 3-4 times and the activity of the cytosolic enzyme 2-3 times during the first 12 hours of life. At the end of the first week of life the total PEPCK activity decreases by one-third, while the activity of the cytosolic enzyme remains stable. In the kidneys of slaughter pigs (n = 7) the dry matter content and the FDPase activity are significantly higher, the protein content and the G6Pase activity are the same as in the kidneys of piglets. The total PEPCK activity is one-half, the activity of the cytosolic enzyme one-third lower than in the kidneys of piglets. In the kidneys of adult pigs the PEPCK activity is localized to equal parts in the cytosol and in the mitochondria, but in some development stages the mitochondrial part exceeds that of the cytosol. In adult pigs the PEPCK activity of the renal cortex is 2.5-3 times higher than that of the renal medulla.

Thermitase has been investigated as a means for obtaining single cells from tissue material, for enzymatic detachment of cultivated cells from the substrate and rarification of cells with subsequent passaging of mouse embryonic fibroblasts. The action of this enzyme was compared with that of "trypsin for cell cultivation". Tissue digestion showed that thermitase at 1/50 of the enzyme concentration needed with trypsin, was 1.5 fold more effective in yielding single cells. In cell cultivation thermitase is able to detach cells from the substrate at the same low concentration of 0.05 mg enzyme/ml and to give sufficient rarification, without the need of adding complexing agents. Rate of attachment, cell form and multiplication in subcultures corresponded to those after application of trypsin. The best results in cell detachment and rarification and the most uniform cell morphology were obtained with thermitase at a concentration of 0.025 mg enzyme/ml under addition of 4 mM of complexing agent. At that, thermitase proved 50 fold more effective than trypsin. Another advantage of thermitase is its better storage quality at 4 degrees C in dissolved form.

Female A/J mice were immunized with sheep red blood cells (SRBC) before mating and boosted a few days before delivery. The progeny of these mothers was immunologically tolerant against SRBC at the level of plaque forming cells (PFC). The state of unresponsiveness was antigen specific. Exchange of the newborn mice between control mothers and immunized ones shows clearly that the tolerance is induced by factors present in the milk or the colostrum, respectively. Some others findings suggest that antibodies of the mothers and not small amounts of the injected antigen are responsible for the nearly complete suppression of the immune response of the offspring.

Leucocytes and spleen contain four different types of protein proteinase inhibitors. Two of them can be inactivated by cathepsin D. In this work biochemical and immunological studies of the inactivation of I-2 by cathepsin D are presented. Polyacrylamide gel electrophoretic examinations indicate that cathepsin D inactivates I-2 by hydrolysis of the inhibitor molecule. The conversion of the active inhibitor into inactive protein proceeds catalytically. The studies on the inhibitor mechanism of the isoinhibitors of I-1 type explain the unusual inhibitor property of this type of inhibitor to inhibit two different types of proteinases, cysteine and serine. The evidence suggests that the inhibitory mechanism is based on an active sulfhydryl group of the inhibitor which may interact with the disulfide bridge of the inhibited proteinase.

Out of nine compounds known to induce accumulation of autophagosomes, seven were found to inhibit degradation of endogenous protein and all of them to inhibit degradation of an exogenous protein (asialofetuin) in isolated rat hepatocytes. On the basis of these findings we propose two possible common mechanisms by which the drugs may cause autophagosome accumulation: 1) The inhibition of protein degradation may result in a decrease in the intracellular amino acid levels, a change which in turn serves as a stimulus for increased autophagic sequestration. 2) Disturbance of the function of the lysosomes may reduce their ability to fuse with newly formed autophagosomes, thereby causing accumulation of the latter.

The relative proportions of alpha-, beta-, and pre-beta-lipoproteins in the fetal serum of mouse, rat, rabbit, guinea pig, mini-pig, and man were determined by agarose gel electrophoresis and subsequent scan evaluation. No distinct species-independent, fetal-specific common features could be detected. In mouse, rabbit and rat an increase in alpha-lipoproteins and a decrease of the proportion of beta-lipoproteins can be observed during prenatal development. In human cord serum of the 30th week alpha-LP are prevalent; at delivery their proportion is still 50%, like in the serum of adults. Five of the species investigated reveal low, if any, pre-beta-lipoprotein levels in fetal serum, while those in the adult organism amount to 19-33%. The lipoprotein spectrum of the mini-pig at the end of the gestational period is identical with that of the adult animal. In the serum of the fetal and adult guinea pig no alpha-lipoprotein band is detectable. On the other hand, a pre-albumin fraction appeared in the prenatal period, amounting to 25% of total lipoproteins. In total, the findings reported here reflect a largely species-specific development of the fetal lipoprotein patterns.