Binding protein dependent transport of C4-dicarboxylates in Rhodobacter capsulatus

The characteristics of malate transport into aerobically grown cells of the purple photosynthetic bacterium Rhodobacter capsulatus were determined. A single transport system was distinguished kinetically which displayed a K_t value of 2.9 ± 1.2 μM and V_max of 43 ± 6 nmol · min^-1 · mg^-1 protein. Competition experiments indicated that the metabolically related C4-dicarboxylates succinate and fumarate are also transported by this system. Malate uptake was sensitive to osmotic shock and evidence from the binding of radiolabelled malate and succinate to periplasmic protein fractions indicated that transport is mediated by a dicarboxylate binding protein. The activity of the transport system was studied as a function of external and internal pH and it was found that a marked activation of uptake occurred at intracellular pH values greater than 7. The use of a high affinity binding protein dependent system to transport a major carbon and energy source suggests that Rhodobacter capsulatus would be capable of obtaining growth sustaining quantities of C4-dicarboxylates even if these were present at very low concentrations in the environment.     

Organization and expression of a second chromosome follicle cell gene cluster in Drosophila

Four genes expressed during the period of vitelline membrane formation are clustered within 8 kb of DNA in region 26A of the second chromosome. Temporal and quantitative difference in the profiles of accumulated RNA suggest that the genes are independently regulated although they are selectively expressed during the stages of vitelline membrane biosynthesis. In situ hybridization and S1 analyses of RNAs from fractionated eggchambers established that these genes are active only in the follicle cells. S1 mapping with in vitro synthesized RNA probes shows that three of the genes are tandemly oriented. All four appear to be intronless. In vitro translation products from hybrid-selected RNAs indicate that two of these genes code for major vitelline membrane proteins. Sequence analysis of these two genes support this conclusion. The cell- and stage-specific expression of the other two genes, encoding less abundant RNAs, suggests that they also play a role in early eggshell production.     

Stability and substructure of cardiac myosin subfragment 1 and isolation and properties of its heavy-chain subunit

The substructure and the thermal stability of the subunit interactions of bovine cardiac myosin subfragment 1 (SF1) have been examined. The results are in agreement with previous reports that the cardiac protein is cleaved in a very similar manner [Flink, I. L., & Morkin, E. (1982) Biophys. J. 37, 34; Korner, M., Thiem, N. V., Cardinaud, R., & Lacombe, G. (1983) Biochemistry 22, 5843-5847] but at a much faster rate [Applegate, D., Azarcon, A., & Reisler, E. (1984) Biochemistry 23, 6626-6630] than the skeletal protein. Additionally, it is found that the long-lived, steady-state intermediates formed by these proteins with MgATP at high ionic strength differ in their susceptibilities to tryptic attack especially at the 27K/50K junction of the associated heavy chains, suggesting a different conformation for these intermediates of the cardiac and skeletal SF1's. The thermal stability of the subunit interactions under conditions approaching the physiological state was examined by thermal ion-exchange chromatography of cardiac SF1 at 39.5 degrees C in the presence of MgATP. This results in the separation of part of the protein as the isolated heavy chain which is found to exhibit high levels of ATPase activity in the absence and presence of actin. Tryptic digestion of cardiac SF1 prior to thermal ion-exchange chromatography produces greater dissociation, with the heavy chain in this case being isolated as a complex of 27K, 50K, and 18-20K fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. IV. Recognition and activation by cloned glycoproteins gB and gD

Results of studies in mice and clinical observations in man indicate that T cell-mediated immunity is important in resistance to herpes simplex virus (HSV) infections. This study was undertaken to elucidate the viral antigen specificity of human HSV-immune T cells. Purified HSV-1 glycoproteins gB-1 and gD-1, cloned and expressed in mammalian cells, were found to stimulate proliferation of, and interleukin 2 (IL 2) production by, peripheral blood lymphocytes (PBL) of HSV seropositive individuals, indicating the presence of memory T cells to gB-1 and gD-1 in individuals with serologic evidence of immunity to HSV. Second, T cell clones, generated by stimulation of PBL with HSV-1, were found to recognize gB-1 or gD-1, as evidenced by the ability of the clones to proliferate in response to stimulation with gB-1 or gD-1 in the absence of exogenous IL 2. Third, HSV-specific T cell clones, lytic for HSV-1 or both HSV-1- and HSV-2-infected autologous target cells, were generated after stimulation of PBL with purified cloned gB-1 or gD-1. Our findings, that human HSV-specific T cells can recognize and be activated by HSV subunit antigens gB-1 or gD-1, imply that these glycoproteins play a role in human T cell-mediated immunity to HSV and support the contention that a gB-1 or gD-1 subunit vaccine may be protective in man.     

island biogeography of Day Geckos (Phelsuma) in the Indian Ocean

Summary Step-wise multiple regression was employed to probe the determinants of species diversity of day geckos (Phelsuma) in the Indian Ocean. Independent variables were area, elevation, and two measures of isolation. Distance from Madagascar and island height (an indicator of habitat diversity) were the two most important predictors of species richness. Similar studies on other taxa rarely find isolation to be a major factor. The relatively poor dispersal abilities of reptiles may explain why isolation, rather than attributes of the islands, are more important in this case. The regressions also indicate that habitat diversity (assumed to correlate with maximum island elevation) is more important than area per se in determining species diversity. These results agree with predictions of the equilibrium theory of island biogeography, but historical processes have also greatly influenced species richness.     

The functional domains of coagulation factor VIII:C

A lack of factor VIII:C, manifested as a bleeding disorder due to the absence of clot formation, is known as hemophilia A, an X chromosome-linked inherited disease afflicting 1-2 males/10,000. To determine the minimum functional domain(s) essential for factor VIII:C activity, we have expressed the amino-terminal (92-kDa) and carboxyl-terminal (80-kDa) proteolytic cleavage products as individual, secreted polypeptides in monkey cells without the 909-residue central region. We have found that neither terminal domain alone is able to promote coagulation in factor VIII:C-deficient plasma. However, when the 92- and 80-kDa peptides are co-expressed, clotting activity is readily detected. Thus, these two chains alone constitute an active or activatable complex. The central domain is required neither for activity nor for the assembly of an active complex from two chains expressed in trans. These results suggest that a truncated derivative of factor VIII:C may be useful in coagulation therapy.     

Calcium in lymphotoxin-mediated cytolysis: cellular pools, fluxes, and role of extracellular concentration

This study explores, by kinetic analysis, the movement of calcium among cellular pools of target cells in which cytotoxicity is induced by human lymphotoxin, and evaluates the requirement for calcium in this reaction. We employed the kinetic model of Borle to quantitate flux rates and pool sizes. It was found that the rate of flux between the surface (plasma membrane-glycocalyx) compartment and the intracellular compartment was greatly increased. The size of the total exchangeable intracellular calcium pool was not altered, but there was an apparent decrease in the size of the surface calcium pool. This latter phenomenon may be related to the blebbing and exfoliation of plasma membranes under the influence of the lymphokine. Lymphotoxin-induced cytotoxicity is observed in calcium-free medium and over a range of calcium concentrations. These results argue against cell death due to a massive in rush of calcium into the cell under these circumstances.     

Structural basis of anthracycline selectivity for unilamellar phosphatidylcholine vesicles: an equilibrium binding study

Fluorescence anisotropy titration was used to determine the equilibrium binding affinities of several anthracycline antitumor antibiotics for sonicated dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) vesicles at 27.5 degrees C. Eight daunomycin analogues, all differing from the parent by one structural change in the aglycon portion of the molecule, as well as four anthracycline congeners modified in the amino sugar were studied. Double-reciprocal plots were used to determine overall binding affinities (K). It was shown that structural changes in both the aglycon and amino sugar portions of the daunomycin molecule strongly modulated K values for DMPC and DPPC bilayers. For modifications in the aglycon portion of an anthracycline, a correlation between drug hydrophobicity and membrane affinity was observed. The number of binding sites per phospholipid molecule (n) and the apparent association constant (Kapp) where K = nKapp, were determined at several temperatures for adriamycin, daunomycin, and carminomycin. The n values were found to be independent of temperature for fluid-phase DMPC or solid-phase DPPC bilayers. The Kapp values (25 degrees C) ranged from (0.82-4.4) X 10(5) M-1 for DMPC vesicles to (4.4-7.3) X 10(5) M-1 for DPPC vesicles. Although the Kapp values for the three drugs were similar for a particular bilayer, major differences were noted in the values of n and, therefore, in the overall vesicle affinities (nKapp). van't Hoff plots showed that anthracycline binding was exothermic; in all cases but one binding was accompanied by a decrease in entropy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction potential of iron in transferrin

The reduction potential of Fe3+ in transferrin was measured spectrophotometrically by equilibration with methyl viologen in the presence of sodium dithionite. For an ionic strength near 0.1 M at 25 degrees C and pH 7.3 under 0.048 atm. CO2, half of the iron is reduced at a potential near -0.40 V (vs. standard hydrogen electrode). At least one disulfide bond of the protein is partially reduced at a potential of -0.44 V, as evidenced by reaction with [14C]iodoacetate.