Distribution of Hydrogen-Metabolizing Bacteria in Alfalfa Field Soil

H₂ evolved by alfalfa root nodules during the process of N₂ fixation may be an important factor influencing the distribution of soil bacteria. To test this hypothesis under field conditions, over 700 bacterial isolates were obtained from fallow soil or from the 3-mm layer of soil surrounding alfalfa (Medicago sativa L.) root nodules, alfalfa roots, or bindweed (Convolvulus arvensis L.) roots. Bacteria were isolated under either aerobic or microaerophilic conditions and were tested for their capacity to metabolize H₂. Isolates showing net H₂ uptake and ³H₂ incorporation activity under laboratory conditions were assigned a Hup⁺ phenotype, whereas organisms with significant H₂ output capacity were designated as a Hout⁺ phenotype. Under aerobic isolation conditions two Hup⁺ isolates were obtained, whereas under microaerophilic conditions five Hup⁺ and two Hout⁺ isolates were found. The nine isolates differed on the basis of 24 standard bacteriological characteristics or fatty acid composition. Five of the nine organisms were isolated from soil around root nodules, whereas the other four were found distributed among the other three soil environments. On the basis of the microaerophilic isolations, 4.8% of the total procaryotic isolates from soil around root nodules were capable of oxidizing H₂, and 1.2% could produce H₂. Two of the Hup⁺ isolates were identified as Rhizobium meliloti by root nodulation tests, but the fact that none of the isolates reduced C₂H₂ under the assay conditions suggested that the H₂ metabolism traits were associated with various hydrogenase systems rather than with nitrogenase activity. Results from this study support the concept that H₂ evolution by alfalfa root nodules has a significant effect on the surrounding microenvironment and influences the number and diversity of bacteria occupying that region.     

Identification of 1H resonances from the bait region of human alpha 2-macroglobulin and effects of proteases and methylamine

The 1H NMR spectrum of human alpha 2-macroglobulin, Mr 716,000, consists of predominantly extremely broad unresolved resonances but also has nine relatively sharp (delta nu 1/2 less than 25 Hz) resonances from aromatic residues. By treatment of alpha 2-macroglobulin with methylamine, chymotrypsin, and subtilisin, it has been shown that eight of these resonances arise from bait region residues. More specifically, assignment has been made of resonances at 6.80 and 7.11 ppm to the ortho and meta protons, respectively, of tyrosine-685 and tentative assignment of a resonance at 7.29 ppm to the aromatic protons of phenylalanine-684. C2 proton resonances from five histidine residues are also visible. Four of these are attributed to residues in the bait region or immediately adjacent to this, at positions 675, 694, 699, and 704. The sharpness of resonances from bait region residues demonstrates the great flexibility of this region of the polypeptide. It is proposed that the flexible region extends from residue 675 to residue 710. These resonances are all affected by proteolytic cleavage in the bait region but are not influenced by the subsequent conformational rearrangement of the whole protein tetramer. The significance of these findings is discussed in relation to the current structural models of alpha 2-macroglobulin.     

A study of anti-group A streptococcal monoclonal antibodies cross-reactive with myosin

Anti-group A streptococcal monoclonal antibodies were obtained from BALB c/BYJ mice immunized with purified membranes from M type 5 Streptococcus pyogenes. Two of the anti-streptococcal monoclonal antibodies were previously shown to cross-react with muscle myosin. In this study the monoclonal antibodies were reacted with tissue sections of normal human heart and skeletal muscle. Antibody binding was estimated by indirect immunofluorescence and immunoperoxidase techniques. Both of the monoclonal antibodies (36.2.2 and 54.2.8) investigated in this report reacted with heart and/or skeletal muscle sections. When evaluated by immunofluorescence, monoclonal antibody 54.2.8 demarcated the periphery of cardiac striated muscle cells and reacted to a lesser degree with subsarcolemmal components. Monoclonal antibody 36.2.2 failed to react with heart sections, but both of the monoclonal antibodies reacted strongly with skeletal muscle sections. Results similar to those observed with indirect immunofluorescence were obtained with the immunoperoxidase technique. By Western immunoblotting and competitive inhibition assays, monoclonal antibodies 36.2.2 and 54.2.8 both were found to react with the heavy chain of skeletal muscle myosin. However, only 54.2.8 reacted with the heavy chain of cardiac myosin. The specificity of the monoclonal antibodies for subfragments of skeletal muscle myosin indicated that monoclonal antibody 36.2.2 was specific for light meromyosin fragments, whereas 54.2.8 reacted with both heavy and light meromyosin. The data demonstrated that two monoclonal antibodies against streptococci were specific for skeletal muscle and/or cardiac myosin and for subfragments of the myosin molecule. The reactions of the monoclonal antibodies with human tissue sections were consistent with the immunochemical reactions of the monoclonal antibodies with both denatured and native myosin.     

Infectivity of Trypanosoma rhodesiense cultivated at 28 degrees C with various tsetse fly tissues

Metacyclic trypanosomes developed in populations of procyclic forms of four stocks of Trypanosoma brucei rhodesiense cultivated at 28 degrees C in a liquid medium containing explants of tsetse fly head-salivary glands, alimentary tract, abdominal body wall, or thoracic muscle. The cultures became infective for mice 7-16 days after they were prepared, and infective trypanosomes were present for prolonged periods. In the culture series of stock TRUM 545, infectivity persisted for 138 days when the cultures were terminated. Only one explant of thoracic muscle tissue was required for the production of metacyclic stages in stock TRUm 497 cultures. Infectivity titrations on trypanosome suspensions from cultures of stocks TRUM 497, TRUM 454, and TRUm 567 revealed that only a small proportion of the culture population was infective. Using stock TRUM 530, mice were infected consistently from inoculations of trypanosomes grown in the presence of explants; infectivity of the trypanosomes eased when the explants were removed from the flasks, but reappeared when they were returned to the cultures. Parasites grown in medium "conditioned" by explants produced sporadic infections in mice. The control cultures of trypanosomes grown in medium alone were generally not infective, but two of the stocks produced occasional parasitemias. Stained samples of infective inocula contained a few epimastigote-like and metacyclic-like trypanosomes.     

Development of metacyclic forms of Trypanosoma brucei sspp. in cultures containing explants of Phormia regina Meigen

When procyclic trypanosomes of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense were cultivated in Nunclon 25 cm2 flasks at 27 C in a liquid medium containing various tissue explants of Phormia regina Meigen, some of them developed into forms infective for mice. The infective stages were present at various periods of up to 29 days when the cultures were terminated. Larger numbers of explants of head-salivary glands than the other tissues used were required to produce infections. Infectivity titrations on trypanosome suspensions of T. b. brucei TRUM 252 and T. b. rhodesiense TRUM 497 indicated that only a small proportion of the populations was infective. Mice were rarely infected with trypanosomes grown in medium without explants. Only 1 mouse of the 11 inoculated developed a parasitemia from a control culture of T. b. rhodesiense TRUM 545. A few trypanosomes resembling epimastigotes and metacyclic forms were seen in stained samples of infective inocula.     

Reconstitution of mitochondrial F0.F1-ATPase with phosphatidylcholine using the nonionic detergent, octylglucoside

A reconstitution procedure has been developed for the incorporation of the mitochondrial F0.F1-ATPase into the bilayer of egg phosphatidylcholine vesicles. The nonionic detergent, octylglucoside, egg phosphatidylcholine, and the lipid-deficient, oligomycin-sensitive F0.F1-ATPase (Serrano, R., Kanner, B., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) were combined in a 4770:320:1 detergent/phospholipid/protein molar ratio and then centrifuged on a discontinuous sucrose gradient to isolate the F0.F1-phosphatidylcholine complex. The specific activity of the reconstituted F0.F1-ATPase was as high as 14.5 mumol/min/mg protein, whereas with no added lipid the activity ranged between 1.4 and 2.2 mumol/min/mg protein. This reconstituted preparation exhibited greater than 90% oligomycin sensitivity which demonstrated the intactness of the multisubunit enzyme complex. The phosphatidylcholine/protein molar ratio of the reconstituted F0.F1 was 250:1 with less than 0.4% of the added octylglucoside remaining. Titrations with both phosphatidylcholine and octylglucoside demonstrated that the specific activity and oligomycin sensitivity were highly dependent on the concentrations of both phospholipid and detergent in the original reconstitution mixture. Analysis of the reconstituted ATPase by electron microscopy demonstrated that the catalytic portion of the enzyme complex projected from the phospholipid bilayer with an orientation similar to that observed with submitochondrial particles. The F0.F1-phosphatidylcholine complex was able to trap inulin, which suggests a vesicular structure impermeable to macromolecules. The electrophoretic mobility of the complex was identical to that for liposomes of egg phosphatidylcholine alone. The reconstitution conditions utilized give rise to an enzyme-phospholipid complex with very low ionic charge that demonstrates high oligomycin-sensitive ATPase activity.     

Comparison of glucokinase in C3H/He and C58 mice that differ in their hepatic activity

Partially purified preparations of the hepatic glucokinase from C3H/He and C58 inbred mice have been used to explore the molecular basis for the observed twofold difference in activity between the strains. The single codominant gene that appears to regulate activity, the alleles of which are designated Gk^a and Gk^b, respectively, for the two strains, could represent a structural gene change. This now seems unlikely because the mouse enzyme, although showing small differences from rat glucokinase, appeared to be identical in the two strains with respect to thermal stability, electrophoretic mobility in agarose gels, and kinetic properties such as the apparent K _m values for MgATP^2– and glucose and the unique cooperative interaction with the latter substrate. The enzymes also reacted identically in a range of immunological tests (double-diffusion, immunoelectrophoresis, immune precipitation and immune inhibition assays) and ELISA immune inhibition assays indicated that the twofold difference in activity was due to a similar difference in antigenically active enzyme. Genetic control over the physiologically significant regulation of enzyme amount is therefore probable.This work has been supported in part by a grant from the British Diabetic Association and a Training Studentship to PAJ from the Medical Research Council (U.K.).     

Uptake Hydrogenase Activity Determined by Plasmid pRL6JI in Rhizobium leguminosarum Does Not Increase Symbiotic Nitrogen Fixation

We examined three groups of wild baboons (Papio cynocephalus) in Amboseli National Park, Kenya, to determine the prevalence of aerobic antibiotic-resistant fecal bacteria in nonhuman primates with and without contact with human refuse. Using standard isolation and replica plating techniques, we found only low numbers of antibiotic-resistant gram-negative enteric bacteria in two groups of baboons leading an undisturbed existence in their natural habitat and having limited or no contact with humans. However, resistance was significantly higher among enteric bacteria from the third group of baboons living in close proximity to a tourist lodge and having daily contact with unprocessed human refuse. Conjugation studies and analysis of the cell DNA by gel electrophoresis showed that in many cases resistance was plasmid-borne and transferable. These data suggest that wild nonhuman primates in frequent contact with human debris have a higher proportion of antibiotic-resistant enteric bacteria than do conspecifics without this contact. The findings further suggest that such groups of wild animals may constitute a heretofore overlooked source of antibiotic resistance in the natural environment.     

Early stimulation of ATP turnover induced by growth factors : Synergistic effect of EGF and insulin and correlation with DNA synthesis

Addition of a mixture of EGF + insulin to quiescent cell cultures synergistically stimulates the cells to reinitiate DNA synthesis and cell division. We have previously demonstrated that this mixture rapidly increases ATP turnover in quiescent cells. The present work shows that each of the two growth factors, EGF and insulin, when added separately to quiescent cells was able to stimulate the phosphorylation of the organic acid-soluble compounds (Po) pool and ATP turnover. The stimulation of ATP turnover was closely correlated with the increase in phosphorylation of the Po pool which suggests that Po labelling reflects the ATP turnover. In many experiments, the synergy between the two growth factors on the early increase in phosphorylation of the Po pool was clearly shown. Doubling the concentration of EGF (12-24 ng/ml) or insulin (50-100 ng/ml) did not increase early stimulation of phosphorylation of the Po pool, whereas simultaneous addition of the two growth factors induced a greater stimulation than that of each growth factor separately added. The augmentation in Po labelling after addition of EGF or insulin alone was transient. The synergistic effect of the two growth factors was more significant when determined 150 or 300 min after growth-factor addition. In our experimental conditions, each of the two growth factors, EGF and insulin, was able to induce a stimulation of DNA synthesis. However, the best stimulatory effect was observed with the mixture of the two which synergistically increased DNA synthesis determined between 6 and 24 h after growth-factor addition. The comparison between DNA replication and Po labelling suggests a correlation between the increase in DNA replication and in the total ATP synthesized in the first 5 h after cell stimulation by growth factors added separately or in combination.