Evaluation of active versus passive uptake of metabolites by Rhizobium japonicum bacteroids.

Rhizobium japonicum bacteroids were isolated anaerobically from soybean [Glycine max (L.) Merr] nodules. The bacteroids, which were capable of acetylene reduction and respiration, were used to study the uptake of metabolites by a method which permits correction for nonspecific adsorption of metabolites and estimation of total cell volume. These determinations permit active uptake to be assessed from metabolite accumulation against a concentration gradient. Succinate, malate, alpha-ketoglutarate, and glutamate were absorbed via an active mechanism. Plots of 1/V versus 1/[S] for succinate and malate indicated the presence of two uptake components: a saturable and presumably active or carrier-mediated component and a nonsaturable and presumably passive component. The uptake of glucose, malonate, D-pinitol, myo-inositol, and glucose 6-phosphate was slow and not active.     

Expression in Bacillus subtilis of the gene for human urogastrone using synthetic ribosome binding sites

Summary A chemically synthesised gene coding for human urogastrone which was earlier cloned in E. coli (Smith et al. 1982) has now been cloned into expression vectors for Bacillus subtilis Two types of constructs have been made, one giving production of methionylurogastrone and the other giving rise to a methionyl-urogastrone- galactosidase fusion polypeptide facilitating quantification of expression levels.The ribosome binding sites used in the expression plasmids are synthetically made oligonucleotides residing on short restriction fragments to allow easy replacement by other ribosome binding sites.Using shuttle vectors and constitutive promoters from Bacillus phages 105 and SPP1, we were able to detect levels of expression amounting to a few thousand molecules per cell during logarithmic growth in both E. coli and B. subtilis.     

Ionophore A23187, Verapamil, Protonophores, and Veratridine Influence the Release of γ-Aminobutyric Acid from Synaptosomes by Modulation of the Plasma Membrane Potential Rather than the Cytosolic Calcium

The release of GABA induced by veratridine shows no correlation with the synaptosomal Ca content and is therefore not mediated by the release of mitochondrial Ca. Instead, with both Ca-repleted and -depleted synaptosomes, the extent of GABA efflux is correlated with the decrease in plasma membrane potential. The slow release of GABA induced by protonophores and the Ca-dependent release induced by ionophore A23187 are also consequences of the depolarization of the plasma membrane, rather than of elevated cytosolic Ca. Finally, the ability of verapamil to inhibit the release of GABA induced by low veratridine concentrations is due to the ability of the Ca channel inhibitor to antagonize the action of veratridine, rather than to inhibit Ca entry into the synaptosome. It is concluded that it is essential to monitor plasma membrane potentials in experiments in which amino acid efflux from synaptosomes is induced.     

Characterization of Cholecystokinin from the Human Brain

Human forms of cholecystokinin have not previously been characterized chemically. In this study, we have extracted and purified the predominant molecular form of cholecystokinin present in human cerebral cortex. The peptide was characterized by amino acid analysis, automated peptide sequencing, and fast atom bombardment mass spectrometry. It appears to be identical to porcine cholecystokinin-octapeptide, with the sequence of Asp-Tyr(SO3)-Met-Gly-Trp-Met-Asp-Phe(NH2). This structural identity is consistent with the observations that the peptide in human brain and porcine cholecystokinin-octapeptide are recognized similarly by a battery of antisera to porcine cholecystokinin; that they coelute from several chromatographic systems, including gel filtration, ion exchange, and reversed-phase; and that they possess similar biological activities.     

Inhibition of exogenous NADH oxidation in plant mitochondria by chlorotetracycline in the presence of calcium ions

Chlorotetracycline inhibits the uncoupled oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L.) mitochondria extensively (over 80%) and rapidly (inhibition complete in 10 s) in the presence of added Ca²⁺. Half-maximal inhibition is observed at 15 μM chlorotetracycline in the presence of 2 mM Ca²⁺. The oxidation of succinate is only affected marginally by chlorotetracycline plus Ca²⁺. The inhibition of NADH oxidation and the fluorescence of CTC are well correlated. Mn²⁺ is the only other cation which shows an (increased) inhibition in the presence of chlorotetracycline. The inhibition by Ca²⁺ and chlorotetracycline disappears at acid pH, and the pH optimum in their presence is 6.4. The inhibition caused by other lipid-soluble Ca²⁺-chelators is not reversible or is enhanced by the addition of excess Ca²⁺. In contrast, inhibition caused by relatively water-soluble chelators is completely reversed by added Ca²⁺. It is suggested that a neutral 1:2 complex is formed between Ca²⁺ and chlorotetracycline which can substitute for Ca²⁺ bound at sites in the lipophilic phase of the inner mitochondrial membrane, which are essential for the activity of the external NADH dehydrogenase.     

Natural immunity to murine gonococcal bacteremia: roles of complement, leucocytes, and sex

The roles of the serum bactericidal system, inflammatory cells, and sex in resisting gonococcal infection were studied in a murine model of gonococcal bacteremia. The role of serum killing in defense was investigated with complement component 5 deficient (C5-deficient) (B1O.D2/OSN) and normal (B1O.D2/NSN) mice. No significant differences were found between LD50's with either murine serum-sensitive or serum-resistant gonococci in those two mouse strains. However, in vitro experiments revealed a heat-stable factor in mouse serum which killed gonococci. Thus it appeared that the C5-deficient mouse is not a good model for the study of the role of C-mediated killing in resistance to gonococcal infection. Mice with Chediak-Higashi disease were used to study the role of phagocytes and natural killer cells. The difference in LD50's between affected mice (C57B1/6J beige J) and controls (C57B1/6J) was significant. The CBA/N mice, which have a B-cell maturation defect, were no more resistant to infection than control mice, which was taken as further evidence that B cells were less important than other leucocytes in innate immunity to gonococcal infection. Finally, male mice were significantly more resistant than female mice to gonococcal bacteremia. Thus, in this study the two most important determinants of resistance to gonococcal infection were inflammatory cells and sex.     

Effects of Nitrogen Sources on Cellulose and Synthetic Lignin Degradation by Phanerochaete chrysosporium

Phanerochaete chrysosporium degraded cellulose faster with organic nitrogen sources than with NH₄Cl. Simple and complex nitrogen sources added at the time of inoculation to N-limited cultures of P. chrysosporium, with glucose as carbon/energy source, transiently stimulated degradation of synthetic [¹⁴C]lignin to ¹⁴CO₂. The same nitrogen sources added 5 days after inoculation, when the cultures were entering secondary metabolism, delayed ¹⁴CO₂ production. The various N sources affected synthetic lignin degradation in defined medium differently than lignin degradation in aspen wood.