Pelvic haematoma following orchidectomy: a pitfall in the staging of non-seminomatous germ cell tumour.
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Adult↗
Biomedical subjects
S A Russell
Publications and source records attributed to S A Russell.
Selenium increases hydrogenase expression in autotrophically cultured Bradyrhizobium japonicum and is a constituent of the purified enzyme.
We have investigated the effect of added selenite on autotrophic growth and the time course of hydrogen oxidation derepression in Bradyrhizobium japonicum 122DES cultured in a medium purified to remove selenium compounds. In addition, hydrogenase was purified to near homogeneity and examined for the specific incorporation of Se into the enzyme. The addition of Se at 0.1 microM significantly increased total cell protein and hydrogenase specific activity of harvested cells. Also, the addition of SeO3(2-) enhanced the time course of hydrogenase derepression by 133%, whereas VO3, AsO2(2-), SO2(2-), and TeO3(2-) failed to substantially affect hydrogenase derepression. During the final chromatographic purification of hydrogenase, a striking coincidence in peaks of protein content, Se radioactivity, and hydrogenase activity of fractions was obtained. The total Se content expressed per milligram of protein increased manyfold during the purification procedure. The mean Se content of the purified hydrogenase was 0.56 +/- 0.13 mol of Se per mol of enzyme. These results indicate that Se is an important element in the H2 metabolism of B. japonicum and that hydrogenase from B. japonicum is a seleno protein.
Culture Media↗
Nickel as a micronutrient element for plants.
The detrimental effects of excessive Ni on plant growth have been well known for many years. More recent evidence indicates that Ni is required in small amounts for normal plant growth and development. Ni is an essential component of urease in plants and microorganisms. A deficiency of Ni in plants is reported to result in necrotic lesions in leaves in response to toxic accumulations of urea. Urease plays an essential role in mobilization of nitrogenous compounds in plants, a process that is especially important during seed germination and fruit formation when protein reserves are degraded into amino acids. Arginine, an abundant amino acid in plants, when degraded produces urea as a product and urease is needed for urea utilization. Theories of urea formation during allantoin degradation in Glycine max have been recently refuted. In G. max ureides apparently are metabolized via an amidohydrolase reaction with subsequent degradation of ureidoglycine, yielding glyoxylate, NH+4 and CO2. No evidence is available for the formation of urea in this pathway. Nitrogen-fixing symbionts, such as Rhizobium and Bradyrhizobium, contain two known Ni enzymes: urease and hydrogenase. Optimum growth of nodulated legumes and actinorhizal plants may depend on an adequate supply of Ni to meet the requirements of the Ni-requiring enzymes in host plants and endophytes. The seeds of severely Ni-deficient Hordeum are completely inviable, thus providing conclusive evidence for the essentiality of Ni for this species. The evidence indicates that Ni must be added to the list of micronutrient elements generally required by plants.
Nickel↗
Purification, properties, and distribution of ascorbate peroxidase in legume root nodules.
All aerobic biological systems, including N(2)-fixing root nodules, are subject to O(2) toxicity that results from the formation of reactive intermediates such as H(2)O(2) and free radicals of O(2). H(2)O(2) may be removed from root nodules in a series of enzymic reactions involving ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. We confirm here the presence of these enzymes in root nodules from nine species of legumes and from Alnus rubra. Ascorbate peroxidase from soybean nodules was purified to near homogeneity. This enzyme was found to be a hemeprotein with a molecular weight of 30,000 as determined by sodium dodecyl sulfate gel electrophoresis. KCN, NaN(3), CO, and C(2)H(2) were potent inhibitors of activity. Nonphysiological reductants such as guaiacol, o-dianisidine, and pyrogallol functioned as substrates for the enzyme. No activity was detected with NAD(P)H, reduced glutathione, or urate. Ascorbate peroxidation did not follow Michaelis-Menten kinetics. The substrate concentration which resulted in a reaction rate of (1/2) V(max) was 70 micromolar for ascorbate and 3 micromolar for H(2)O(2). The high affinity of ascorbate peroxidase for H(2)O(2) indicates that this enzyme, rather than catalase, is responsible for most H(2)O(2) removal outside of peroxisomes in root nodules.
Journal Article↗
Symbiotic Expression of Cosmid-Borne Bradyrhizobium japonicum Hydrogenase Genes.
The expression of cosmid-borne Bradyrhizobium japonicum hydrogenase genes in alfalfa, clover, and soybean nodules harboring Rhizobium transconjugants was studied. Cosmid pHU52 conferred hydrogen uptake (Hup) activity in both free-living bacteria and in nodules on the different plant hosts, although in nodules the instability of the cosmid resulted in low levels of Hup activity. In contrast, cosmid pHU1, which does not confer Hup activity on free-living bacteria, gave a Hup phenotype in nodules on alfalfa and soybean. Nodules formed by B. japonicum USDA 123Spc(pHU1) recycled about 90% of nitrogenase-mediated hydrogen evolution. Both subunits of hydrogenase (30- and 60-kilodalton polypeptides) were detected in enzyme-linked immunosorbent assays of bacteroid preparations from nodules harboring B. japonicum strains with pHU1 or pHU52. Neither pHU53 nor pLAFR1 conferred detectable Hup activity in either nodules or free-living bacteria. Based on the physical maps of pHU1 and pHU52, it is suggested that a 5.5-kilobase EcoRI fragment unique to pHU52 contains a gene or part of a gene required for Hup activity in free-living bacteria but not in nodules. This conclusion is supported by the observation that two Tn5 insertions in the chromosome of B. japonicum USDA 122DES obtained by marker exchange with Tn5-mutagenized pHU1 abolished Hup activity in free-living bacteria but not in nodules.
Journal Article↗
Physiology, biochemistry, and genetics of the uptake hydrogenase in rhizobia.
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Genes, Bacterial↗
Enzymatic reactions of ascorbate and glutathione that prevent peroxide damage in soybean root nodules.
The critical problem of oxygen toxicity for nitrogen-fixing organisms may be related to damage caused by oxygen radicals and peroxides. An enzymatic mechanism is described for removal of peroxides in root nodules of soybean (Glycine max). The system utilizes ascorbate as an antioxidant and glutathione as a reductant to regenerate ascorbate. The enzymes involved are ascorbate peroxidase (ascorbate:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1), and glutathione reductase (NADPH:oxidized-glutathione oxidoreductase, EC 1.6.4.2). The reactions are essentially the same as those involving scavenging of H(2)O(2) in chloroplasts. Glutathione peroxidase (glutathione:hydrogenperoxide oxidoreductase, EC 1.11.1.9) was not detected. During the course of early nodule development, ascorbate peroxidase and dehydroascorbate reductase activities and total glutathione contents of nodule extracts increased strikingly and were positively correlated with acetylene reduction rates and nodule hemoglobin contents. The evidence indicates an important role of glutathione, ascorbate, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase as components of a peroxide-scavenging mechanism in soybean root nodules.
Journal Article↗
Intra- and interspecies transfer and expression of Rhizobium japonicum hydrogen uptake genes and autotrophic growth capability.
Cosmids containing hydrogen uptake genes have previously been isolated in this laboratory. Four new cosmids that contain additional hup gene(s) have now been identified by conjugal transfer of a Rhizobium japonicum 122DES gene bank into a Tn5-generated Hup(-) mutant and screening for the acquisition of Hup activity. The newly isolated cosmids, pHU50-pHU53, contain part of the previously isolated pHU1 but extend as far as 20 kilobases beyond its border. pHU52 complements five of six Hup(-) mutants and confers activity on several Hup(-) wild-type R. japonicum strains in the free-living state and where tested in nodules. Transconjugants obtained from interspecies transfer of pHU52 to Rhizobium meliloti 102F28, 102F32, and 102F51 and Rhizobium leguminosarum 128C53 showed hydrogen-dependent methyleneblue reduction, performed the oxyhydrogen reaction, and showed hydrogen-dependent autotrophic growth by virtue of the introduced genes. The identity of the presumptive transconjugants was confirmed by antibiotic-resistance profiles and by plant nodulation tests.
Journal Article↗
Determination of the hydrogenase status of individual legume nodules by a methylene blue reduction assay.
We adapted a method for the rapid screening of colonies of free-living Rhizobium japonicum for hydrogenase activity to determine the hydrogenase status of individual soybean nodules. Crude bacteroid suspensions from nodules containing strains known to be hydrogen uptake positive (Hup) caused a localized decolorization of filter paper disks, whereas suspensions from nodules arising from inoculation with hydrogen uptake-negative (Hup) mutants or strains did not decolorize the disks. The reliability of the method was demonstrated by its successful application to 29 slow-growing rhizobia. The Hup phenotype on methylene blue filters agreed with that determined amperometrically with either methylene blue or oxygen as the electron acceptor.
Journal Article↗
Characterization of Rhizobium japonicum hydrogen uptake genes.
Recombinant cosmids from a gene library of the DNA from Hup+ Rhizobium japonicum 122DES previously have been shown to restore hydrogenase activity when transferred by conjugation into certain Hup- mutants of R. japonicum. We generated a restriction map covering 32.2 kilobases of this cosmid DNA. At least 25.3 kilobases of the cosmid pHU1 were shown to have the same arrangement as those in the genome of strain 122DES. Analysis of Tn5 insertions into the 122DES genome indicates that hup-specific sequences occur in a region spanning about 15 kilobases of insert DNA within pHU1. Introduction of pHU1 into five out of six R. japonicum Hup- mutants resulted in a Hup+ phenotype in some transconjugants. Three of the mutations appear to be in transcriptional units completely contained within pHU1, whereas the other two must be in genes that are at least partially contained within pHU1. pBR235 derivatives containing fragments of hup DNA can be transferred into the R. japonicum Hup- mutant PJ18nal if the derivatives contain a region of homology with the R. japonicum genome. The hup mutation in strain PJ18nal appears to be dominant. The hup genes in R. japonicum strain 122DES appear to be organized in at least two, and probably three, transcriptional units.
Base Sequence↗
Nickel: A micronutrient element for hydrogen-dependent growth of Rhizobium japonicum and for expression of urease activity in soybean leaves.
Soybean plants and Rhizobium japonicum 122 DES, a hydrogen uptake-positive strain, were cultured in media purified to remove Ni. Supplemental Ni had no significant effect on the dry matter or total N content of plants. However, the addition of Ni to both nitrate-grown and symbiotically grown plants resulted in a 7- to 10-fold increase in urease activity (urea amidohydrolase, EC 3.5.1.5) in leaves and significantly increased the hydrogenase activity (EC 1.18.3.1) in isolated nodule bacteroids. When cultured under chemolithotrophic conditions, free-living R. japonicum required Ni for growth and for the expression of hydrogenase activity. Hydrogenase activity was minimal or not detectable in cells incubated either without Ni or with Ni and chloramphenicol. Ni is required for derepression of hydrogenase activity and apparently protein synthesis is necessary for the participation of Ni in hydrogenase expression. The addition of Cr, V, Sn, and Pb in place of Ni failed to stimulate the activity of hydrogenase in R. japonicum and urease in soybean leaves. The evidence indicates that Ni is an important micronutrient element in the biology of the soybean plant and R. japonicum.
Journal Article↗
Respiratory and Nitrogenase Activities of Soybean Nodules Formed by Hydrogen Uptake Negative (Hup) Mutant and Revertant Strains of Rhizobium japonicum Characterized by Protein Patterns.
Rates of respiratory CO(2) loss and nitrogenase activities of H(2) uptake-negative mutant strains and H(2) uptake-positive revertant strains of Rhizobium japonicum have been investigated. Two-dimensional gel protein patterns of bacteroids formed by inoculation of soybeans (Glycine max L.) with these two strains show that they are closely related and revealed only one obvious difference between them. On the basis of molecular weight standards, it was concluded that the missing protein spot in the H(2) uptake-negative mutant strain could be caused by a failure of the mutant to synthesize hydrogenase. Nodules formed by the H(2) uptake-negative mutant strain evolved respiratory CO(2) at a rate of about 10% higher than that of nodules formed by the H(2) uptake-positive revertant strain. During short-term experiments employed, rates of both C(2)H(2) reduction and (15)N(2) fixation varied considerably among replicate samples and no statistically significant differences between mutant and revertant strains were observed. It was observed that increasing the partial pressure of O(2) over nodules significantly decreased the proportion of nitrogenase electrons allocated to H(+).
Journal Article↗
Revertible hydrogen uptake-deficient mutants of Rhizobium japonicum.
We have developed mutants of Rhizobium japonicum which are deficient in H2 uptake capacity (Hup-) and which spontaneously revert to the parent type at a frequency consistent with that of a single-point mutation (ca. 1.0 x 10(-09)). The mutagenesis by nitrous acid and the selection of the Hup- phenotype by using penicillin and chemolithotrophy as enrichment for chemolithotrophy-deficient strains are described. Two mutants retain low but reproducible levels of ribulose bisphosphate-dependent CO2 fixation when grown on a low-carbon medium under an atmosphere of 1% O2, 4% H2, 5% CO2, and 90% N2. Neither O2 nor the artificial electron acceptors phenazine methosulfate or methylene blue supported detectable H2 uptake by the free-living Hup- mutants or by their bacteroids. Plant growth experiments under bacteriologically controlled conditions were conducted to assess the mutants' performance as inocula for soybean plants. Plants inoculated with Hup- strains had lower dry weights and contained less total N than did plants inoculated with the parent Hup+ strain. Use of either the Hup- mutants or the Hup+ parent strain as inocula, however, did not significantly affect the acetylene-reducing activity or the fresh weight of nodules. These results, obtained with apparently isogenic lines of H2 uptake-deficient R. japonicum, provide strong support for a beneficial role of the H2 uptake phenotype in legume symbiosis.
Carbon Dioxide↗
Oxyleghemoglobin-mediated Hydrogen Oxidation by Rhizobium japonicum USDA 122 DES Bacteroids.
Oxyleghemoglobin was used to supply low concentrations of O(2) to H(2)-oxidizing bacteroids from Rhizobium japonicum USDA 122 DES. The H(2) oxidation system of these bacteroids was capable of effectively utilizing O(2) at the low concentrations of O(2) expected to be found in soybean nodules. Apparent K(m) values of approximately 10 nanomolar O(2) have been calculated for the oxyhydrogen reaction. These values include the K(m) values for both H(2) oxidation and endogenous substrate oxidation. Even in the presence of oxyleghemoglobin, H(2) additions stimulated C(2)H(2) reduction, reduced the rate of endogenous respiration and maintained the ATP contents of bacteroids. In our reconstituted oxyleghemoglobin and bacteriod system, we estimate that the H(2) oxidation system is capable of recycling all of the H(2) evolved during the N(2) fixation process.
Journal Article↗
Investigation of the H(2) Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids.
The H(2)-oxidizing complex in Rhizobium japonicum 122 DES bacteroids failed to catalyze, at a measurable rate, (2)H(1)H exchange from a mixture of (2)H(2) and (1)H(2) in presence of (2)H(2)O and (1)H(2)O, providing no evidence for reversibility of the hydrogenase reaction in vivo. In the H(2) oxidation reaction, there was no significant discrimination between (2)H(2) and (1)H(2), indicating that the initial H(2)-activation step in the over-all H(2) oxidation reaction is not rate-limiting. By use of improved methods, an apparent K(m) for H(2) of 0.05 micromolar was determined. The H(2) oxidation reaction in bacteroids was strongly inhibited by cyanide (88% at 0.05 millimolar), theonyltrifluoroacetone, and other metal-complexing agents. Carbonyl cyanide m-chlorophenylhydrazone at 0.005 millimolar and 2,4-dinitrophenol at 0.5 millimolar inhibited H(2) oxidation and stimulated O(2) uptake. This and other evidence suggest the involvement of cytochromes and nonheme iron proteins in the pathway of electron transport from H(2) to O(2). Partial pressures of H(2) at 0.03 atmosphere and below had a pronounced inhibitory effect on endogenous respiration by bacteroid suspensions. The inhibition of CO(2) evolution by low partial pressures of H(2) suggests that H(2) utilization may result in conservation of oxidizable substrates and benefits the symbiosis under physiological conditions. Succinate, acetate, and formate at concentrations of 50 millimolar inhibited rates of H(2) uptake by 8, 29, and 25%, respectively. The inhibition by succinate was noncompetitive and that by acetate and formate was uncompetitive. A concentration of 11.6 millimolar CO(2) (initial concentration) in solution inhibited H(2) uptake by bacteroid suspensions by 18%. Further research is necessary to establish the significance of the inhibition of H(2) uptake by succinate, acetate, formate, and CO(2) in the metabolism of the H(2)-uptake-positive strains of Rhizobium.
Journal Article↗
Expression of hydrogenase activity in free-living Rhizobium japonicum.
A medium is described on which selected Rhizobium japonicum strains express hydrogenase (H(2) uptake) activity under free-living conditions. Low concentrations of carbon substrates, decreased oxygen tension, and the quantity of combined nitrogen in the medium were major factors influencing hydrogenase expression. Hydrogenase activity was dependent upon a preincubation period in the presence of H(2) under conditions such that the cells did not exhibit nitrogenase activity. H(2) uptake rates were easily measured amperometrically in aerobically or anaerobically prepared suspensions from free-living cultures. Six R. japonicum strains that formed nodules with the ability to utilize H(2) oxidized this gas when grown in free-living cultures. In comparison six randomly chosen strains forming nodules that lost H(2) in air either showed no or low capacity to take up H(2) under free-living conditions. The reduction of triphenyltetrazolium chloride in an agar medium was used to detect strains capable of oxidizing H(2). This method has enabled us to isolate a spontaneous R. japonicum mutant strain that has lost the ability to utilize H(2). This mutant strain forms nodules that evolve H(2) but other symbiotic characteristics appear normal. This strain will be useful in evaluating the importance of the hydrogenase system in the nitrogen-fixing process of legumes.
Journal Article↗
Hydrogen reactions of nodulated leguminous plants: I. Effect of rhizobial strain and plant age.
The ATP-dependent evolution of H(2) catalyzed by nitrogenase and the hydrogenase-catalyzed oxidation of H(2) have been implicated as factors influencing the efficiency of energy utilization in the N(2) fixation process. The effects of rhizobial strain and plant age on the H(2)-evolving and H(2)-utilizing activity of leguminous root nodules are described in this manuscript. Two classes of legume-Rhizobium combinations were observed in studies with soybeans (Glycine max L. Merr.) and cowpeas (Vigna unguiculata L. Walp.). One group evolved H(2) in air; the other group did not exhibit net evolution of H(2). The latter group metabolized H(2) formed within the nodule through the action of a hydrogenase. The capacity to oxidize H(2) was strongly linked to the strain of Rhizobium used to inoculate cowpeas and soybeans. Although the magnitude of H(2) evolution in air changed during vegetative growth of a given symbiont, the ratio of H(2) evolved in air to total nitrogenase activity was not appreciably altered during this period. No consistent difference in nitrogenase activity as measured by the C(2)H(2) reduction assay was observed between symbionts with an active hydrogenase and those that apparently lack the enzyme and evolve H(2). The effects of the two reactions of H(2) on total N(2) fixation and yield must now be established.
Journal Article↗
Acetylene reduction (nitrogen fixation) associated with corn inoculated with Spirillum.
Sorghum and corn breeding lines were grown in soil in field and greenhouse experiments with and without an inoculum of N2-fixing in Spirillum strains from Brazil. Estimated rates of N2 fixation associated with field-grown corn and sorghum plants were less than 4 g of N2/ha per day. The mean estimated N2-fixation rates determined on segments of roots from corn inoculated with Spirillum and grown in the greenhouse at 24 to 27 degrees C were 15 g of N2/ha per day (16 inbreds), 25 g of N2/ha per day (six hybrids), and 165 g of N2/ha per day for one hybird which was heavily inoculated. The corresponding mean rates determined from measurements of in situ cultures of the same series of corn plants (i.e., 16 inbreds, six hybrids, and one heavily inoculated hybrid) were 0.4, 2.3, and 1.1 g of N2/ha per day, respectively. Lower rates of C2H2 reduction were associated with control corn cultures which had been treated with autoclaved Spirillum than with cultures inoculated with live Spirillum. No C2H2 reduction was detected in plant cultures treated with ammonium nitrate. Numbers of nitrogen-fixing bacteria on excised roots of corn plants increased an average of about 30-fold during an overnight preincubation period, and as a result acetylene reduction assays of root samples after preincubation failed to serve as a valid basis for estimating N2 fixation by corn in pot cultures. Plants grown without added nitrogen either with or without inoculum exhibited severe symptoms of nitrogen deficiency and in most cases produced significantly less dry weight than those supplied with fixed nitrogen. Although substantial rates of C2H2 reduction by excised corn roots were observed after preincubation under limited oxygen, the yield and nitrogen content of inoculated plants and the C2H2-reduction rates by inoculated pot cultures of corn, in situ, provided no evidence of appreciable N2 fixation.
Acetylene↗