Anaerobic utilization of phosphite and hypophosphite by Bacillus sp.

A Bacillus sp. capable of utilizing phosphite and hypophosphite under anaerobic conditions was isolated from Cape Canerval soil samples. The organism was isolated on a glucose-mineral salts medium with phosphate deleted. Anaerobic cultivation of this isolate resulted in decreases in the hypophosphite or phosphite concentration, increases in turbidity, cell count, and dry-cell weight, and decreases in pH and glucose concentration. The optimum hypophosphite concentration for this isolate was 60 microgram/ml, whereas the optimum phosphate concentration was greater than 1,000 microgram/ml, suggesting that higher concentrations of hypophosphite may be toxic to this isolate. Hypophosphite or phosphite utilization was accompanied by little or no detectable accumulation of phosphate in the medium, and 32P-labeled hypophosphite was incorporated into the cell as organic phosphate. When phosphate was present in the medium, the isolate failed to metabolize phosphite. In the presence of phosphite and hypophosphite, the isolate first utilized phosphite and then hypophosphite.     

Culicoides, the vector of epizootic hemorrhagic disease in white-tailed deer in Kentucky in 1971.

The biting gnat, Culicoides variipennis (Coquillett), was shown to be a vector of epizootic hemorrhagic disease (EHD) in white-tailed deer, Odocoileus virginianus, in Kentucky because of virus isolations from parous females. Epidemiological evidence showed a close relationship of this vector to the animal host during an outbreak of EHD in penned deer. Larval breeding sites of C. variipennis were found and C. variipennis was the most abundant biting fly present during the outbreak. Females of C. variipennis were commonly observed biting deer, swine, cattle and, occasionally, man.     

Effects of polyamines on partial reactions of membrane (Na+ + K+)-ATPase.

Spermine and spermidine inhibit the (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) reaction so that the effect increases as the ionic content due to Na+ and K+ in the reaction is reduced. Several other amines inhibit (Na+ + K+)-ATPase to varying degress and methylglyoxal-bis-(guanylhydrazone) was the most potent inhibitor among those tested. The inhibition by polyamines of the ATPase is uncompetitive with respect to Mg2+ and ATP activation of the reaction. Various naturally occurring polyamines and other amines inhibited Na+ activation of (Na+ + K+)-ATPase as well as Na+-dependent phosphoenzyme formation in an apparently competitive manner with respect to Na+. Likewise, K+-activation of (Na+ + K+)-ATPase as well as K+-p-nitrophenyl phosphatase was inhibited in an apparently competitive manner with respect to K+. Both the cation charge and structure (e.g., aliphatic chain length) may contribute to the inhibitory effects of the amines; however, Na+ sites appear to be more sensitive to cation charge than the aliphatic chain length of the amine, whereas the opposite appears to be true for K+ sites. The results do not indicate a specific effect of polyamines on (Na+ + K+)-ATPase or its partial reactions.     

Localization by immunofluorescence and by light- and electron-microscopic immunoperoxidase techniques of Tamm–Horsfall glycoprotein in adult hamster kidney

1. Tamm-Horsfall glycoprotein was isolated from hamster urine and antiserum against it was produced in rabbits. Immunoglobulin G was isolated from the antiserum. 2. Indirect methods of immunofluorescence staining were applied to kidney sections previously fixed by both perfusion and immersion methods. Tamm-Horsfall glycoprotein was identified associated with only the cells of the ascending limb of the loop of Henle and the distal convoluted tubule. Maculae densae were free of the glycoprotein. 3. Indirect immunoperoxidase procedures with light microscopy were applied to kidney sections. The results extended those found by immunofluorescence by showing that the glycoprotein is largely associated with the plasma membrane of the cells. Macula densa cells were shown to be free of the glycoprotein, although the luminal surface of the remaining cells in the transverse section of the nephron at that region was shown to contain it. 4. A variety of immuno-electron-microscopic techniques were applied to sections previously fixed in a number of ways. Providing periodate/lysine/paraformaldehyde was used as the fixative, the glycoprotein was often seen to be present not only on the luminal surface of the cells of the thick ascending limb of the loop of Henle and of the distal convoluted tubule, but also on the basal plasma membrane, including the infoldings. 5. It is generally accepted that the hyperosmolarity in the medulla of the kidney results from passage of Cl(-) ions with their accompanying Na(+) ions across the single cell layer of the lumen of the thick ascending limb of the loop of Henle, a region of the nephron with relatively high impermeability to water. We suggest that Tamm-Horsfall glycoprotein operates as a barrier to decrease the passage of water molecules by trapping the latter at the membrane of the cells. Our hypothesis requires the glycoprotein on the basal plasma membrane also.     

Granulocyte and macrophage proliferation after injection of antitumor active and inactive strains of Corynebacterium parvum

Summary Corparvax, a strain of Corynebacterium parvum with strong antitumor activity, had a greater and more prolonged effect of increasing the production of granulocytes and macrophages than did a weak antitumor strain, CN5888. Following the injection of Coparvax to mice, there was a prompt and sustained increase in serum granulocyte/macrophage colony-stimulating activity, an increase in the number of spleen granulocyte/macrophage progenitor cells, an increased rate of proliferation of the bone marrow granulocyte/macrophage progenitor cells and an increase in the number of blood granulocytes and monocytes. The time courses of the increased rates of proliferation of granulocyte/macrophage progenitor cells following the injection of Coparvax were different in the bone marrow and the spleen, suggesting that local microenvironmental factors were also important.If immunostimulants such as C. parvum are to be used in chemoimmunotherapy programs, the kinetics of the increased proliferative rate of the granulocyte/macrophage progenitor cells may be important, since the more rapidly proliferating cells will be more affected by cell cycle-active chemotherapeutic agents.with the technical assistance of Beverly M. Dunne and L. Atherton     

Importance of experimental conditions in evaluating the malonyl-CoA sensitivity of liver carnitine acyltransferase. Studies with fed and starved rats.

The experiments reconfirm the powerful inhibitory effect of malonyl-CoA on carnitine acyltransferase I and fatty acid oxidation in rat liver mitochondria (Ki 1.5 microM). Sensitivity decreased with starvation (Ki after 18 h starvation 3.0 microM, and after 42 h 5.0 microM). Observations by Cook, Otto & Cornell [Biochem. J. (1980) 192, 955--958] and Ontko & Johns [Biochem. J. (1980) 192, 959--962] have cast doubt on the physiological role of malonyl-CoA in the regulation of hepatic fatty acid oxidation and ketogenesis. The high Ki values obtained in the cited studies are shown to be due to incubation conditions that cause substrate depletion, destruction of malonyl-CoA or generation of excessively high concentrations of unbound acyl-CoA (which offsets the competitive inhibition of malonyl-CoA towards carnitine acyltransferase I). The present results are entirely consistent with the postulated role of malonyl-CoA as the primary regulatory of fatty acid synthesis and oxidation in rat liver.     

Changes in the activities of the enzymes of hepatic fatty acid oxidation during development of the rat.

1. Changes in the activities of several enzymes involved in mitochondrial fatty acid oxidation were measured in livers of developing rats between late foetal life and maturity. The enzymes studied are medium- and long-chain ATP-dependent acyl-CoA synthetases of the outer mitochondrial membrane and matrix, GTP-dependent acyl-CoA synthetase, carnitine acyltransferase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, general 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase.     

Nuclear location of mammalian DNA polymerase activities.

Nuclei were isolated from monolayer cultures of mouse and human cells using a nonaqueous procedure of cell fractionation in which lyophilized cells were homogenized and centrifuged in 100% glycerol. In previous work we have shown that the nuclear pellet and cytoplasmic supernatant fraction contained 10% or less of the nucleic acids characteristic of the other cell fraction. Aqueous extracts made from fresh cultures and from nonaqueous material at each step of the fractionation procedure were assayed fro DNA polymerase activity. Activities were normalized to DNA contents of extracted material. Specific activity was preserved quantitatively through freezing and drying the cells, but was found to be unstable in glycerol suspensions with approximate half-lives and 1 h at 23 degrees and 4 h at 0-4 degrees. Activities were relatively stable at -25 degrees, however, so that by homogenizing only 15 min at 4 degrees and centrifuging at -25 degrees we preserved approximately 85% of the specific activity of fresh cultures in the nonaqueous nuclear fraction. Sedimentation analyses showed that the nuclear fraction contained both DNA polymerase-alpha and-beta in approximately the proportions expected if all polymerase activities were confined to the nucleus in living cells. DNA polymerase-alpha was found to be more unstable in glycerol suspensions than DNA polymerase-beta. Nuclear location of both activities was found in exponential cultures and in 3T3 mouse cultures synchronized in the G1 and S phases of the cell division cycle. We found no evidence for cytoplasmic factors affecting nuclear polymerase activities. We have concluded that the two major DNA polymerases are nuclear although one, DNA polymerase-alpha, frequently is present as a weakly bound nuclear protein.     

Hepatic pyruvate kinase. Regulation by glucagon, cyclic adenosine 3'-5'-monophosphate, and insulin in the perfused rat liver.

A reversible interconversion of two kinetically distinct forms of hepatic pyruvate kinase regulated by glucagon and insulin is demonstrated in the perfused rat liver. The regulation does not involve the total enzyme content of the liver, but rather results in a modulation of the substrate dependence. The forms of pyruvate kinase in liver homogenates are distinguished by measurements of the ratio of the enzyme activity at a subsaturating concentration of P-enolpyruvate (1.3 mM) to the activity at a saturating concentration of this substrate (6.6 mM). A low ratio form of pyruvate kinase (ratio between 0.1 and 0.2) is obtained from livers perfused with 10(-7) M glucagon or 0.1 mM adenosine 3':5'-monophosphate (cyclic AMP). A high ratio form of the enzyme is obtained from livers perfused with no hormone (ratio = 0.35 to 0.45). The regulation of pyruvate kinase by glucagon and cyclic AMP occurs within 2 min following the hormone addition to the liver. Insulin (22 milliunits/ml) counteracts the inhibition of pyruvate kinase caused by 5 X 10(-11) M glucagon, but has only a slight influence on the enzyme properties in the absence of the hyperglycemic hormone. The low ratio form of pyruvate kinase obtained from livers perfused with glucagon or cyclic AMP is unstable in liver extracts and will revert to a high ratio form within 10 min at 37 degrees or within a few hours at 0 degrees. Pyruvate kinase is quantitatively precipitated from liver supernatants with 2.5 M ammonium sulfate. This precipitation stabilizes the enzyme and preserves the kinetically distinguishable forms. The kinetic properties of the two forms of rat hepatic pyruvate kinase are examined using ammonium sulfate precipitates from the perfused rat liver. At pH 7.5 the high ratio form of the enzyme has [S]0.5 = 1.6 +/- 0.2 mM P-enolpyruvate (n = 8). The low ratio form of enzyme from livers perfused with glucagon or cyclic AMP has [S]0.5 = 2.5 +/- 0.4 mM P-enolpyruvate (n = 8). The modification of pyruvate kinase induced by glucagon does not alter the dependence of the enzyme activity on ADP (Km is approximately 0.5 mM ADP for both forms of the enzyme). Both forms are allosterically modulated by fructose 1,6-bisphosphate, L-alanine, and ATP. The changes in the kinetic properties of hepatic pyruvate kinase which follow treating the perfused rat liver with glucagon or cyclic AMP are consistent with the changes observed in the enzyme properties upon phosphorylation in vitro by a clyclic AMP-stimulated protein kinase (Ljungstr?m, O., Hjelmquist, G. and Engstr?m, L. (1974) Biochim. Biophys. Acta 358, 289--298). However, other factors also influence the enzyme activity in a similar manner and it remains to be demonstrated that the regulation of hepatic pyruvate kinase by glucagon and cyclic AMP in vivo involes a phosphorylation.