Effects of cardiac thin filament Ca2+: statistical mechanical analysis of a troponin C site II mutant.

Cardiac thin filaments contain many troponin C (TnC) molecules, each with one regulatory Ca2+ binding site. A statistical mechanical model for the effects of these sites is presented and investigated. The ternary troponin complex was reconstituted with either TnC or the TnC mutant CBMII, in which the regulatory site in cardiac TnC (site II) is inactivated. Regardless of whether Ca2+ was present, CBMII-troponin was inhibitory in a thin filament-myosin subfragment 1 MgATPase assay. The competitive binding of [3H]troponin and [14C]CBMII-troponin to actin.tropomyosin was measured. In the presence of Mg2+ and low free Ca2+ they had equal affinities for the thin filament. When Ca274+ was added, however, troponin's affinity for the thin filament was 2.2-fold larger for the mutant than for the wild type troponin. This quantitatively describes the effect of regulatory site Ca2+ on troponin's affinity for actin.tropomyosin; the decrease in troponin-thin filament binding energy is small. Application of the theoretical model to the competitive binding data indicated that troponin molecules bind to interdependent rather than independent sites on the thin filament. Ca2+ binding to the regulatory site of TnC has a long-range rather than a merely local effect. However, these indirect TnC-TnC interactions are weak, indicating that the cooperativity of muscle activation by Ca2+ requires other sources of cooperativity.     

M?ssbauer and electron nuclear double resonance study of oxidized bidirectional hydrogenase from Clostridium pasteurianum W5

The bidirectional hydrogenase from Clostridium pasteurianum W5 is an iron-sulfur protein containing approximately 12 Fe atoms and 12 labile sulfides. We have studied oxidized samples of the enzyme with M?ssbauer and electron nuclear double resonance (ENDOR) spectroscopy to elucidate the nature of the center that gives rise to the EPR signal with principal g-values at 2.10, 2.04, and 2.01. The g = 2.10 center exhibits two well-resolved 57Fe ENDOR resonances. One is isotropic with A1 = 9.5 MHz; the other is nearly isotropic with A2 = 17 MHz. These magnetic hyperfine coupling constants are substantially (approximately 50%) smaller than those observed for [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters. The M?ssbauer and ENDOR data, taken together, suggest that the g = 2.10 center contains at least two but not more than four iron atoms. Comparison of our data with recent results reported for Escherichia coli sulfite reductase and the ferricyanide-treated [4Fe-4S] cluster from Azotobacter vinelandii ferredoxin I suggests that the g = 2.10 center may possibly be formed, by oxidation, from a structure with a [4Fe-4S] core. The M?ssbauer spectra give evidence that at least 8 of the 12 Fe atoms of oxidized hydrogenase are organized in two ferredoxin-type [4Fe-4S] clusters, supporting conclusions derived previously from EPR studies of the reduced enzyme.     

5-Enolpyruvylshikimate-3-phosphate synthase from Escherichia coli--the substrate analogue bromopyruvate inactivates the enzyme by modifying Cys-408 and Lys-411

In order to identify the essential reactive amino acid residues of 5-enolpyruvylshikimate-3-phosphate synthase, the reaction of the enzyme with its substrate analogue bromopyruvate was investigated. Incubation of the enzyme with bromopyruvate resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first-order and saturation kinetics with a Kinact of 28 microM and a maximum rate constant of 0.31 min-1. The inactivation was prevented by preincubation of the enzyme with the substrates shikimate 3-phosphate, 5-enolpyruvylshikimate 3-phosphate or by the combination of shikimate 3-phosphate plus glyphosate (N-phosphonomethylglycine), an inhibitor of the enzyme. Addition of sodium [3H]borohydride to the reaction mixture had no effect on the rate of inactivation but resulted in the incorporation of 3H label to the modified enzyme. Upon 90% inactivation, approximately 1 mol of bromo[14C]pyruvate was incorporated per mole of enzyme modified in the absence or presence of sodium borohydride. When the enzyme was incubated with bromopyruvate in the presence of sodium [3H]borohydride, approximately 1 mol of 3H label was found to be associated per mole of the modified enzyme. Tryptic digestion of these labeled proteins followed by reverse phase chromatographic separation resulted in the isolation of three radioactive peptides. Analyses of these three peptides indicated that bromopyruvate inactivated the enzyme by modifying Cys-408 and Lys-411, which are conserved in all enzyme sequences studied to date.     

Metabolic utilization of linoleate and alpha-linolenate in cultured Sertoli cells.

1. The capacity of cultured Sertoli cells to synthesize long-chain polyunsatured fatty acids (PUFA) from the essential fatty acid (EFA) precursors 18:2 n-6 and 18:3 n-3 was tested, and the concentrations of each EFA required to obtain maximal incorporation into membrane lipids were determined. 2. The two EFA were added to the culture medium as free fatty acids complexed to albumin in a molar ratio of 12:1. 3. When the substrates were added individually, the maximal levels of biosynthesis were obtained with 0.7 micrograms/ml of 18:2 n-6 and 2 micrograms/ml of 18:3 n-3. 4. When the two EFA were added together, clear alterations in the behavior of the desaturases with regard to the n-6 and n-3 fatty acids were observed. 5. It was found that a concentration of 0.35 micrograms/ml of each EFA represented the "ideal" required level in order to ensure optimal incorporation of 22-carbon PUFA into the membrane lipids. 6. These results provide the first data on the definition of EFA requirements for Sertoli cells in culture.     

Typhoid fever survey in two localities in Vietnam

As a part of multipurpose health survey of the population in Vietnam the antibodies against S. typhi were determined by the micromethod using haemagglutination test (O-antigen 9, 12) and agglutination test using standard H-diagnostic antigen (d). Totally 292 sera were examined, 139 from Duyen Thai village and 154 from Mai Chau. The data on vaccination against typhoid fever are recorded only in 102 persons. The positivity on Vi antibodies is very high--70% in Duyen Thai and 47% in Mai Chau. This finding is significant according to the high titres in the carriers of S. typhi. The titres of all antibodies are lower in Mai Chau area situated in mountains then in crowded lowlands of Duen Thai. The level of antibodies is decreasing with age. The frequency distribution of antibodies by age proves endemicity of the disease in area, where a large part of population is infected already before reaching 20 years of age. The effectivities of vaccination is discussed.     

Diversity ofFrankia strains isolated from a single alder stand

One hundredFrankia strains isolated from variousAlnus species in a single alder stand were tested for plasmid presence. Plasmid DNA was observed in five of the frankiae strains and was analyzed. We found that plasmids with a similar molecular weight exhibited in fact minor divergences in restriction patterns. The genetic diversity among the five isolates which contained plasmids and seven isolates which contained no plasmid DNA were examined by using restriction endonucleas digestions, Southern hybridization ofnifHDK,nifAB genes, andFrankia cryptic DNA fragments determined at random. Results indicate that genomic DNA digestion patterns and Southern hybridizations to anifHDK probe were not able to discriminate between closely related frankiae. On the other hand, plasmid presence, Southern hybridization to anifAB proble or to a crypticFrankia probe allowed us to delineate groupings of these isolates.     

Postnatal development of the cholecystokinin-gastrin family of peptides in the brain and gut of rat

The average levels of CCK-8- and CCK-like peptides present in aqueous and acidic extracts of the brain of the newborn rat were, respectively, 5.3 and 2.9 pmol/g wet weight. These low levels increased 20- and 10-fold, respectively, so as to attain, at 25–30 days post partum, values comparable to those found in adult brain. There was also a rapid deposition of CCK-gastrin-like peptides in the whole gut between day 20 post coitum and day 10 post partum, when maximal concentrations were attained. Later, less CCK-gastrin peptides than other gut components were accumulated during the progressive weaning of the young rats, so that the average concentrations of CCK-gastrin-like peptides were, at day 30, similar to those observed in adult gut.     

Viscidic acid A and B,two ent-labdane derivatives from Chrysothamnus viscidiflorus

The chemical investigation of Chrysothamnus viscidiflorus afforded, in addition to known bisabolene derivatives, elemicin and p-hydroxyacetophenone, two new diterpene acids. Their structures were determined, by spectroscopic methods and some chemical transformations, as ent-labd-8(17),13E-dien-15-ol-18-oic acid (viscidic acid A) and ent-labd-8(17),13E-dien-15-acetoxy-18-oic acid (visidic acid B).     

Covalent binding to glutathione of the DNA-alkylating antitumor agent, S23906-1.

The benzoacronycine derivative, S23906-1, was characterized recently as a novel potent antitumor agent through alkylation of the N2 position of guanines in DNA. We show here that its reactivity towards DNA can be modulated by glutathione (GSH). The formation of covalent adducts between GSH and S23906-1 was evidenced by EI-MS, and the use of different GSH derivatives, amino acids and dipeptides revealed that the cysteine thiol group is absolutely required for complex formation because glutathione disulfide (GSSG) and other S-blocked derivatives failed to react covalently with S23906-1. Gel shift assays and fluorescence measurements indicated that the binding of S23906-1 to DNA and to GSH are mutually exclusive. Binding of S23906-1 to an excess of GSH prevents DNA alkylation. Additional EI-MS measurements performed with the mixed diester, S28053-1, showed that the acetate leaving group at the C1 position is the main reactive site in the drug: a reaction scheme common to GSH and guanines is presented. At the cellular level, the presence of GSH slightly reduces the cytotoxic potential of S23906-1 towards KB-3-1 epidermoid carcinoma cells. The GSH-induced threefold reduction of the cytotoxicity of S23906-1 is attributed to the reduced formation of lethal drug-DNA covalent complexes in cells. Treatment of the cells with buthionine sulfoximine, an inhibitor of GSH biosynthesis, facilitates the formation of drug-DNA adducts and promotes the cytotoxic activity. This study identifies GSH as a reactant for the antitumor drug, S23906-1, and illustrates a pathway by which GSH may modulate the cellular sensitivity to this DNA alkylating agent. The results presented here, using GSH as a biological nucleophile, fully support our initial hypothesis that DNA alkylation is the major mechanism of action of the promising anticancer drug S23906-1.