The enzymes of phospholipid synthesis in Clostridium butyricum

We have examined extracts of Clostridium butyricum for several enzymes of phospholipid synthesis. Membrane particles were shown to catalyze the formation of CDP-diglyceride from [3H]CTP and phosphatidic acid. The reaction was dependent on Mg2+ and stimulated by monovalent cations. CDP-diglyceride formed in vitro was found to be a substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. The formation of phosphatidylglycerophosphate from added CDP-diglyceride and [U-14C]sn-glycerol-3-phosphate was dependent on Mg2+ and Triton X-100. The dephosphorylation of endogenously-generated phosphatidylglycerophosphate to yield phosphatidylglycerol was observed to be pH-dependent. The formation of phosphatidylserine from CDP-diglyceride and L-[3-14C]serine was stimulated by Mg2+ and Triton X-100. dCDP-diglyceride was a suitable substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. Phosphatidylserine decarboxylase activity was barely detectable in membrane particles from C. butyricum. The addition of E. coli membrane particles provided efficient phosphatidylserine decarboxylase activity in this system. Although plasmalogens are the principal lipids of C. butyricum, none of the products of phospholipid synthesis formed in vitro contained measurable amounts of plasmalogens. The subcellular distribution of both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase in C. butyricum was also studied. Both were found to be membrane-associated.     

Inhibition of cell wall synthesis and acylation of the penicillin binding proteins during prolonged exposure of growing Streptococcus pneumoniae to benzylpenicillin

Growing cultures of an autolysis-defective pneumococcal mutant were exposed to [3H]benzylpenicillin at various multiples of the minimal inhibitory concentration and incubated until the growth of the cultures was halted. During the process of growth inhibition, we determined the rates and degree of acylation of the five penicillin-binding proteins (PBPs) and the rates of peptidoglycan incorporation, protein synthesis, and turbidity increase. The time required for the onset of the inhibitory effects of benzylpenicillin was inversely related to the concentration of the antibiotic, and inhibition of peptidoglycan incorporation always preceded inhibition of protein synthesis and growth. When cultures first started to show the onset of growth inhibition, the same characteristic fraction of each PBP was in the acylated form in all cases, irrespective of the antibiotic concentration. Apparently, saturation of one or more PBPs with the antibiotic beyond these threshold levels is needed to bring about interference with normal peptidoglycan production and cellular growth. Although it was not possible to correlate the inhibition of cell wall synthesis or cell growth with the degree of acylation (percentage saturation) of any single PBP, there was a correlation between the amount of peptidoglycan synthesized and the actual amount of PBP 2b that was not acylated. In cultures exposed to benzylpenicillin concentrations greater than eight times the minimal inhibitory concentration, the rates of peptidoglycan incorporation underwent a rapid decline when bacterial growth stopped. However, in cultures exposed to lower concentrations of benzylpenicillin (one to six times the minimal inhibitory concentration) peptidoglycan synthesis continued at constant rate for prolonged periods, after the turbidity had ceased to increase. We conclude that inhibition of bacterial growth does not require a complete inhibition or even a major decline in the rate of peptidoglycan incorporation. Rather, inhibition of growth must be caused by an as yet undefined process that stops cell division when the rate of incorporation of peptidoglycan (or synthesis of protein) falls below a critical value.     

Changes in the vulnerable period of the rat myocardium during hypoxia, hyperventilation and heart failure

Changes in the duration and size of the vulnerable period of the myocardium in the presence of respiratory changes were studied in acute experiments on rats. The limits of the vulnerable period were determined by directly stimulating the heart during ventilation via the enlarged respiratory dead space, during hyperventilation and during heart failure. In the control group (normal ventilation without enlargement of the dead space), the vulnerable period lasted 5.7 +/- 0.76 ms. During ventilation via the enlarged dead space, hypercapnic hypoxaemia developed and the vulnerable period was markedly prolonged (18.55 +/- 5.29 ms) by a shift of its inner limit to the left. Hyperventilation caused normoxic to hyperoxic hypocapnia and markedly reduced the duration of the vulnerable period (8.17 +/- 2.21 and 9.31 +/- 2.38 ms respectively). The vulnerable period lengthened the most in heart failure (25.46 +/- 3.93), mainly as a result of a shift of its outer limit. In all the experimental groups there was a shift of the vulnerable period to the right, which was fastest in hypercapnic hypoxaemia and slowest in hyperoxic hypocapnia. The administration of Inderal (3 mg/kg i.p.) or Arfonad (50 mg/kg i.p.) markedly shortened the vulnerable period during hypercapnic hypoxaemia (9.87 +/- 2.78 and 9.32 +/- 2.16 ms respectively), but did not block the shift. Lengthening of the vulnerable period during hypercapnic hypoxaemia was probably due to activation of sympathetic nerves via beta-adrenergic receptors.     

Two-dimensional gel analysis of swine histocompatibility antigens

Miniature swine MHC antigens from three inbred herds were examined by two-dimensional gel electrophoresis. These antigens were found to constitute a series of complex glycoproteins displaying haplotype-specific patterns that allowed the distinction of both class I and class II molecules among the three haplotypes. Selected outbred pig antisera reacted with a subset of class I antigens, suggesting the presence of at least two distinct molecular species among these antigens. Similarly, alloantisera reacting with mouse Ia antigens and a monoclonal anti-human DR were shown to immunoprecipitate a subset of class II molecules. Examination of the cells from two recombinant haplotypes demonstrated that both independent recombinational events took place between the class I and class II genes.     

Use of fibre-optic endoscopes in studies of gastric digestion in carnivorous vertebrates

1. Two methods of assessing gastric digestion rates of three prey types fed to Sooty albatrosses Phoebetria fusca were compared: removal of stomach contents, using a water-flushing stomach pump (a technique used commonly in diet studies), and inspection using a fibre-optic gastroscope (a previously unused technique). 2. The stomach pump yielded quantitative information, but proved stressful and resulted in incomplete recovery of meals ingested 3-6 hr before pumping. Gastric morphology of the animals studied and digestion state of their stomach contents may influence the effectiveness of this technique. 3. Inspection using the gastroscope yielded qualitative information only but permitted serial inspection of the same animal, and was less stressful than the stomach pump. Times for total evacuation of the stomach were 6-12 hr less when estimated using the gastroscope than when using the stomach pump. 4. The specifications of endoscopes relevant to their use by biologists are given. 5. Previous non-medical biological uses of endoscopes are given. Potential uses include underwater observations, sampling of digestive juices and stomach linings for enzyme analyses, observations of ingested prey, and assessment of parasite infestation.     

Identification of the 190 kD microtubule-associated protein in cultured fibroblasts and its association with interphase and mitotic microtubules

We previously investigated the biochemical characteristics of microtubule-associated proteins (MAPs) of the adrenal medulla and adrenal cortex and found that they contain a new kind of MAP with a molecular weight of 190,000 (190 kD MAP) as a major species (Kotani, S., H. Murofushi, S. Maekawa, C. Sato, and H. Sakai. Eur. J. Biochem. 156, 23-29, 1986). We now have used an affinity purified anti-(190 kD MAP) antibody and show by indirect immunofluorescent microscopy the association of this MAP with microtubules in situ in TIG-3 cells (human embryonic lung fibroblasts). The 190 kD MAP was present along the interphase and mitotic microtubules, and there was no marked difference between the staining pattern with anti-tubulin and that with anti-(190 kD MAP) antibodies, evidence that the localization of 190 kD MAP is not restricted to the subset of microtubules. We also isolated MAPs from TIG-3 cells and identified their 190 kD MAP as a major heat-stable component. Several other unidentified polypeptides were recovered in the MAP fraction specifically.     

Correlation between bilayer destabilization and activity enhancement by diacylglycerols in reconstituted Ca-ATPase vesicles

Using the reconstituted Ca-ATPase vesicles as a model system, we demonstrated that the presence of 1,2-dioleoyl-sn-glycerol (diolein) in the membrane introduces a pronounced enhancement in the Ca-transport function of Ca-ATPase, while the 1,2-dipalmitoyl-sn-glycerol (dipalmitin) does not. We also found by both 31P NMR and freeze-fraction electron microscopy that diolein destabilized lipid bilayers to a greater extent than did dipalmitin. We conclude that the tendency of diacylglycerols to destabilize the phospholipid bilayer is related to their capacity to enhance the activity of the membrane calcium pump.     

Modulation of murine hemopoiesis in vivo by a synthetic hemoregulatory pentapeptide (HP 5b)

Abstract. Degeneration of the archenteron in middle gastrulae occurred in the presence of α,α'-dipyridyl or Zn²⁺, inhibitors of prolyl hydroxylase. In the presence of these substances the archenteron degenerated and was eventually destroyed. Adding Fe²⁺ to the embryo culture containing α,α'-dipyridyl protected the archenteron from further degeneration, but the collapsed archenteron was not restored to the upright position. At the late gastrula stage, α,α'-dipyridyl did not cause the degeneration of the archenteron. Treatment of the embryos by α,α'-dipyridyl, starting at the swimming blastula stage, resulted in the production of many mesenchyme-like cells but archenteron was not produced in the embryos. Addition of Fe²⁺ to α,α'-dipyridyl culture, just before the beginning of gastrulation of normal embryos, resulted in the formation of normal archenteron. α,α'-Dipyridyl inhibited hydroxylation of proline residues of collagen in sea urchin embryos and Fe²⁺ prevented the inhibition by α,α'-dipyridyl. Respiration was not inhibited by α,α'-dipyridyl.