Chemical modification of lysine and arginine residues in the myosin regulatory light chain inhibits phosphorylation

The contribution of lysine and arginine residues to the substrate specificity of the myosin light-chain kinase has been studied using chemically modified myosin light chains. Succinylation or maleylation of the myosin light chains caused complete inhibition of their phosphorylation. Modification of 50% of the lysine residues resulted in 90% inhibition of phosphorylation and this was accompanied by a 25-fold increase in the apparent Km. In contrast, phosphorylation of the myosin light chains by the cAMP-dependent protein kinase was relatively insensitive to lysine modification, with only a 15% reduction in phosphorylation following succinylation of 50% of the lysine residues. Treatment with either cyclohexane-1,2-dione or camphorquinone-10-sulfonic acid resulted in between 90 and 98% inhibition of myosin light-chain phosphorylation. These reagents caused modification of both lysine and arginine residues, and accordingly only part of the inhibition can be attributed to arginine modification. Modification of all of the cysteine and methionine residues caused only a 40% inhibition of phosphorylation. The results of this study support the concept that lysine and arginine residues act as essential specificity determinants for the myosin light-chain kinase in protein substrates.     

Peritoneal macrophages exposed to purified macrophage colony-stimulating factor (M-CSF) suppress mitogen- and antigen-stimulated lymphocyte proliferation

The effect of M-CSF-exposed macrophages on murine splenic lymphocyte responses was determined. Resident peritoneal macrophages incubated with purified M-CSF for 48 hr inhibited lymphocyte proliferation to Con A, PHA, and listerial antigen as determined by [3H]TdR uptake, and inhibited Con A-stimulated lymphocyte IL 2 production. The inhibition was similar to that observed with macrophages from BCG-infected mice. Maximal suppression occurred at M-CSF concentrations of 500 U/ml or greater and when the incubation time with M-CSF was 48 hr or more. M-CSF effect was specific because rabbit anti-M-CSF IgG blocked the suppression whereas control rabbit IgG did not. Secretory products of macrophages could not be implicated in this interaction. Catalase and indomethacin, alone or together, did not reverse the inhibition. In addition, putative suppressive factors were not detected in supernatants of M-CSF-stimulated macrophages. Lymphocytes that were removed from macrophage monolayers and were recultured in medium plus Con A were able to proliferate. Macrophages stimulated by M-CSF therefore appear to have inhibitory activity for proliferating lymphocytes, and may play a role in immunoregulatory mechanisms.     

The hydrolysis of extracellular adenine nucleotides by cultured endothelial cells from pig aorta. Feed-forward inhibition of adenosine production at the cell surface

The time course of the extracellular reaction sequence ATP----ADP----AMP----adenosine has been examined during recirculation of substrate solutions over cultured pig aortic endothelial cells attached to polystyrene beads. This permits the study of reactions at volume to cell surface ratios approaching those of small blood vessels. When endothelial cells were presented with an initial bolus of ATP, high concentrations of the intermediates ADP and AMP developed before significant conversion of AMP to adenosine occurred. Further, the higher the initial ATP concentration, the slower the conversion of AMP to adenosine. Kinetic constants for each reaction were estimated by fitting simulated reaction curves to observed time courses. Apparent Km values estimated in this way agreed well with those reported for initial velocity measurements (ATPase = 300 microM; ADPase = 240 microM; and 5'-nucleotidase = 26 microM). The ratio of maximum velocities was ATPase:ADPase:AMPase = 6:1.5:1, with absolute values varying among cell batches. The data could only be fitted if the model incorporated inhibition of 5'-nucleotidase by ATP or ADP, and satisfactory fitting was achieved with a Ki value for ADP of 5 microM. These kinetic properties maximize the time separation of the intermediate pools. In vivo, at sites of platelet degranulation, they would create a time gap proportional to the size of the initial release between release of ADP (a proaggregatory milieu) and the appearance of adenosine (an anti-aggregatory milieu).     

Glycosaminoglycan synthesis is depressed during mitosis and elevated during early G1

[35S]Sulfate incorporation was measured in populations of Chinese hamster ovary cells enriched for mitotics, early G1 cells, and interphase monolayers or suspensions. Incorporation was determined by biochemical analysis of extracts and quantitative autoradiography of thick sections. 90% of [35S]sulfate was incorporated into glycosaminoglycan (GAG). Incorporation was depressed fourfold in mitotics and stimulated by from two- to three-fold in early G1 cells relative to mixed interphase cells. GAG synthesis was maintained into late G2. Thus, the rate of GAG biosynthesis was correlated temporally with the detachment and reattachment of cells to substrate. Inhibitors of protein synthesis brought about the rapid arrest of GAG biosynthesis. However, xylosides, which bypass the requirement for core protein, did not bring oligosaccharide sulfation in mitotics to interphase levels. These observations indicate an inhibition of Golgi processing and are consistent with a generalized defect of membrane vesicle-mediated transport during mitosis.     

Cationic amino acid transport by two renal epithelial cell lines: LLC-PK1 and MDCK cells

LLC-PK1 and MDCK cells take up cationic amino acids (lysine and arginine) by a specific sodium independent transport system. Uptake is inhibited by ornithine in LLC-PK1 and MDCK cells either in the presence or absence of sodium and by glutamine or homoserine in MDCK cells in the presence of sodium. Trans-stimulation of uptake occurs in the presence of intracellular cationic amino acids. Experiments with valinomycin or with different extracellular potassium concentrations suggest that uptake is dependent on the membrane potential of these cells. These transport features are similar to those previously ascribed to a transport system denominated y+ in other cells. Further experiments suggested that this carrier system is localised to the basolateral membrane in each cell type.     

Differential susceptibility of cultured neural cells to the human coronavirus OC43.

By using cell-type-specific markers and neural cultures derived from various areas of the nervous system, it has been possible to identify various interactions between OC43 virus and mouse oligodendrocytes, neurons, astrocytes, and fibroblasts. Neurons derived from dorsal root ganglia produced viral antigen and infectious virus. Astrocytes and fibroblasts both produced viral antigen but not infectious virus. Oligodendrocytes produced neither infectious virus nor viral antigen. Human embryo brain cells, including astrocytes, were susceptible to OC43 infection but did not produce infectious virus.     

Characterization of virulent and avirulent A/chicken/Pennsylvania/83 influenza A viruses: potential role of defective interfering RNAs in nature.

In April 1983, an influenza virus of low virulence appeared in chickens in Pennsylvania. Subsequently, in October 1983, the virus became virulent and caused high mortality in poultry. The causative agent has been identified as an influenza virus of the H5N2 serotype. The hemagglutinin is antigenically closely related to tern/South Africa/61 (H5N3) and the neuraminidase is similar to that from human H2N2 strains (e.g., A/Japan/305/57) and from some avian influenza virus strains (e.g., A/turkey/Mass/66 [H6N2]). Comparison of the genome RNAs of chicken/Penn with other influenza virus isolates by RNA-RNA hybridization indicated that all of the genes of this virus were closely related to those of various other influenza virus isolates from wild birds. Chickens infected with the virulent strain shed high concentrations of virus in their feces (10(7) 50% egg infective dose per g), and the virus was isolated from the albumin and yolk of eggs layed just before death. Virus was also isolated from house flies in chicken houses. Serological and virological studies showed that humans are not susceptible to infection with the virus, but can serve as short-term mechanical carriers. Analysis of the RNA of the viruses isolated in April and October by gel migration and RNA-RNA hybridization suggested that these strains were very closely related. Oligonucleotide mapping of the individual genes of virulent and avirulent strains showed a limited number of changes in the genome RNAs, but no consistent differences between the virulent and avirulent strains that could be correlated with pathogenicity were found. Polyacrylamide gel analysis of the early (avirulent) isolates demonstrated the presence of low-molecular-weight RNA bands which is indicative of defective-interfering particles. These RNAs were not present in the virulent isolates. Experimental infection of chickens with mixtures of the avirulent and virulent strains demonstrated that the avirulent virus interferes with the pathogenicity of the virulent virus. The results suggest that the original avirulent virus was probably derived from influenza viruses from wild birds and that the virulent strain was derived from the avirulent strain by selective adaptation rather than by recombination or the introduction of a new virus into the population. This adaptation may have involved the loss of defective RNAs, as well as mutations, and thus provides a possible model for a role of defective-interfering particles in nature.     

Effect of Unilateral Decortication on Choline Acetyltransferase Activity in the Nucleus Basalis and Other Areas of the Rat Brain

Acetyl-coenzyme A: choline O-acetyltransferase (EC 2.3.1.6) (ChAT) enzyme activity was measured in the nucleus basalis and other microscopically identified brain areas at various times after unilateral cortical lesions were made in the rat. Initially, a significant decrease in ChAT activity was detected in the nucleus basalis ipsilateral to the lesion. However, after 120 days ChAT activity had apparently recovered, as levels of the enzyme at that time were not significantly different from control values. No changes in ChAT activity could be detected in any of the other brain areas similarly studied. The significance of these findings and their relationship to the morphological changes seen in neurones of the nucleus basalis after cortical lesions are discussed.     

Inhibition of chlorophyll synthesis inHordeum vulgare by 3-amino 2,3-dihydrobenzoic acid (gabaculin)

Gabaculin (3-amino 2,3-dihydrobenzoic acid) is shown to be a very potent inhibitor of chlorophyll formation inHordeum vulgate. Exposure of leaf segments to 30/M gabaculin results in an 80% inhibition of chlorophyll synthesis, and this is paralleled by a decrease in carotenoid. Dual-inhibitor studies with dioxoheptanoic acid, which is an inhibitor of inolaevulinic acid dehydratase, show that gabaculin inhibits an earlier step than dioxoheptanoic acid and affects -aminolaevulinic acid synthesis rather than its subsequent metabolism.