Carbon and nitrogen metabolism in legume root nodules

The literature concerning the metabolism of carbon compounds during the reduction, assimilation and translocation of nitrogen in root nodules of leguminous plants is reviewed. The reduction of dinitrogen requires an energy source (ATP) and a reluctant which are both supplied by respiratory catabolism of carbohydrates produced by the host plant. Photosynthates are also required to generate the carbon skeletons for amino acid or urcide synthesis during the assimilation of ammonia produced by the bacteria within the nodule tissue. Competition for photosynthates occurs between the bacteroids, nodule tissue and the various vegetative and reproductive sinks in the host plant. The nature of carbon compounds involved in these processes, their routes of metabolism, the mechanisms of control and the partitioning of metabolises between the various sites of utilization are only poorly understood. It is apparent that dinitrogen is reduced to ammonia in the bacteroids. Both fast- and slow-growing strains of Rhizobium possess the Entner-Doudoroff pathway of glucose catabolism, and some, if not all, enzymes of the Emden-Meyerhof pathway. Some bacterial cultures also metabolize carbon through the ketogluconate pathway but only the fast-growing strains of cultured rhizobia possess the key enzyme of the pentose phosphate pathway (6-phosphogluconate dehydrogenase). The host cells are thought to contain the complete Emden-Meyerhof pathway and tricarboxylic acid cycle, which provides the carbon skeletons for assimilation of the ammonia, formed by the bacteroids, into α-amino acids. A pathway of anapleurotic carbon conservation, operative in the host cells, synthesizes oxaloacetic acid through β-carboxylation of phosphoenol pyruvate. This process could be important in the recapture and assimilation of respired CO₂ in the rhizosphere. The main route of assimilation of ammonia produced by the bacteroids would appear to be via the glutamine synthetase-glutamate synthase pathway in the host cells. However, glutamate dehydrogenase may also be involved in ammonia assimilation. These enzymes also occur in in vitro cultures of Rhizobium and in bacteroids where they presumably participate in the synthesis of amino acids for growth of the bacteria or bacteroids. Nitrogen assimilated into glutamine or glutamate is exported from the nodules in a variety of forms, which include asparagine, glutamine, aspartate, homoserine and allantoates, in proportions which depend on the legume species. Studies on regulation of the overall process have focussed on expression of bacteroid genes and on the control of enzyme activity, at the level of nitrogenase and enzymes of nitrogen assimilation in particular. However, due to the wide range of experimental techniques, environmental conditions and plant species which have been used, no clear conclusions can yet be drawn. The pathways of carbon flow in nitrogen metabolism, particularly in relation to the synthesis of ureides and the regulation of carbon metabolism, remain key areas for future research in symbiotic nitrogen fixation.     

The effect of histone hyperacetylation on the nuclease sensitivity and the solubility of chromatin

We have examined the effects of histone hyperacetylation upon nuclease digestion of nuclei and subsequent fractionation of chromosomal material in the presence of MgCl2. DNase I shows a maximum sensitivity towards hyperacetylated nuclei at somewhat elevated ionic strengths (150-200 mM NaCl), whereas micrococcal nuclease exhibits no specificity for acetylated nuclei over a broad range of ionic strengths. Fractionation in the presence of MgCl2 of hyperacetylated nuclei digested with micrococcal nuclease results in a substantial increase in the amount of soluble chromatin relative to that obtained with control nuclei. This increased yield of Mg2+-soluble chromatin results from the recruitment into this fraction of oligonucleosomes containing extremely hyperacetylated histones. These results suggest that contiguous nucleosomes containing highly acetylated histones may be altered in their ability to interact with themselves and with other nucleosomes.     

Cytochalasin B blocks sperm incorporation but allows activation of the sea urchin egg

Cytochalasin B (CB) (2 × 10⁻⁶ M) prevents the incorporation of sperm into the eggs of Lytechinus pictus and Strongylocentrotus purpuratus as judged by light and transmission electron microscopy (TEM). At lower concentrations of CB (2 × 10⁻⁷ M), sperm are successfully incorporated into the egg, but their migration in the area of the egg cortex is impaired. The site of action of CB on the sperm may be on the initial rotation of the sperm nucleus in the cortex; the subsequent migration is not affected by CB. Although sperm incorporation is prevented at the higher CB concentrations, the eggs become activated—as judged by cortical reaction, increased protein synthesis and increased respiration. These findings raise the concept that egg activation by sperm could result from some pre-fusion event and hence that sperm-egg fusion would not be a prerequisite for the triggering of development. An alternative hypothesis is that fusion occurs between the acrosome process membrane and egg membrane, but since CB has destroyed the integrity of the cortex actin, the fusion bridge is so weak that it cannot be maintained without some contractile or cytoskeletal support by the cortex. The sperm may activate the CB-treated egg in the same manner as pricking with a microelectrode sometimes does.     

Transformation of Saccharomyces cerevisiae with yeast mitochondrial DNA linked to two micron circular yeast plasmid

Mitochondrial DNA from a petite mutant of yeast carrying an oligomycin resistance determinant has been ligated in vitro to 2 μm yeast plasmid DNA. The recombinant DNA so produced has been used to transform an oligomycin sensitive strain of Saccharomyces cerevisiae to oligomycin resistance at a frequency approaching 50 times the spontaneous mutation rate to oligomycin resistance. The majority of transformants showed genetic properties suggesting that recombination between the transforming DNA and the resident mtDNA has occurred. The properties of a subclass of oligomycin resistance transformants suggested that in these cells the transforming DNA has not become stably integrated into the mitochondrial genome of the recipient cell.     

Regulation of the immune response to tumor antigen. VIII. The effects of host specific anti-I-J antibodies on the immune response to tumors of different origin

The efficiency of in vivo therapy using alloantisera produced to interact specifically with I-J subregion encoded determinants has been investigated in two etiologically distinct syngeneic tumor systems, both of which have been shown to evoke suppressor T-cell host responses. Administration of 2 μl/day of anti-I-J^k alloantisera caused a significant reduction in the growth of the P815 methylcholanthrene (MCA)-induced mastocytoma or the 1316 ultraviolet (uv) radiation-induced fibrosarcoma in the syngeneic host. Inhibition of tumor growth with anti-I-J antibody treatment occurred in normal as well as in subcarcinogenically uv-treated hosts given the uv-induced 1316 fibrosarcoma, even though the normal host is capable of spontaneously rejecting the tumor graft in the absence of external manipulation. Evidence is also provided that the effects of anti-I-J antibody treatment are not due to direct interactions with the tumor cells, or to contaminating antiviral antibody activity within the antiserum. We have previously demonstrated the reduction of tumor growth in two antigenically distinct MCA-induced tumor systems (S1509a, SAI) using similar treatments. The data presented herein thus reinforce the possibility that such means of therapy may be beneficial to the treatment of a wide variety to tumor types where suppression represents a detrimental component of the host response, and may also provide some insight into the mechanisms underlying the effects of uv radiation on the immune response to tumor antigen.     

Chemically defined media for growth and sporulation of Entomophthora virulenta

Amino acids, salts, and vitamins were combined with dextrose to test their effect on growth and sporulation of Entomophthora virulenta in liquid shake culture. The addition of a vitamin solution to the tested media did not enhance growth or sporulation. MgSO₄·7H₂O was the only salt individually tested that allowed for good growth and sporulation. MgSO₄·7H₂O concentrations exceeding 250 mg/liter in media lacking other salts inhibited sporulation. A simple medium of l-arginine, l-leucine, glycine, and mineral salts allowed high growth and sporulation.     

The role of calmodulin in the structure and regulation of phosphorylase kinase from rabbit skeletal muscle.

A purification procedure is described for the initiation factors of protein synthesis from rabbit reticulocytes: (a) from the ribosomal wash and (b) from the postribosomal supernantant. A comparison is made between these preparations with respect to yield and specific activity. eIF-4A and eIF-4D occur mainly in the postribosomal supernatant; eIF-2, eIF-4C and eIF-5 are more evenly divided over both fractions, whereas eIF-1, eIF-3 and eIF-4B are found almost exclusively in the ribosomal wash. No significant difference in specific activity could be detected when factors from both sources were compared, with a possible exception of eIF-4A and eIF-4D.     

Critical problems with extraction of ATP for bioluminescence assay of plankton biomass

Recovery of ATP by boiling tris extraction was 90–95 percent greater in 1 liter grab samples than in concentrated net samples. ATP losses were attributed to insulating effects promoted by accumulation of detritus on filters. A series of extractions over a concentration range of whole or size-segregated plankters and cultured algae was made to determine volume of water to be filtered for optimum extraction efficiency. Accuracy of ATP assays was optimized by: (i) using large diameter (i.e. 47 mm) acetate filters; (2) limiting sample volume filtered to 50 ml when particulate organic carbon (POC) exceeded 0.4 mg l^–1; and (3) performing extractions in boiling tris maintained initially on a laboratory hot plate at 400°C as opposed to hot water bath at 100°C.Additional problems were encountered in using published cellular carbon: ATP ratios for conversion of ATP data to biomass as carbon. Ratios of POC: ATP in cultures of sheathed blue-green algae reached 550 : i, while non-sheathed forms yielded ratios near values previously reported for plankton communities. Difficulties in applying a uniform conversion factor may be expected in plankton communities containing significant volumes of sheathed blue-greens.     

Accumulation of iron by yersiniae.

Escherichia coli, Bacillus megaterium, and three species of yersiniae grew rapidly without significant production of soluble siderophores in a defined iron-sufficient medium (20 microM Fe3+). In iron-deficient medium (0.1 to 0.3 microM Fe3+) all organisms showed reduced growth, and there was extensive production of siderophores by E. coli and B. megaterium. Release of soluble siderophores by Yersinia pestis, Y. pseudotuberculosis, or Y. enterocolitica in this medium was not detected. Citrate (1 mM) inhibited growth of yersiniae in iron-deficient medium, indicating that the organisms lack an inducible Fe3+-citrate transport mechanism. Uptake of 59Fe3+ by all yersiniae was an energy-dependent saturable process, showing increased accumulation after adaptation to iron-deficient medium. Growth of Y. pseudotuberculosis and Y. enterocolitica but not Y. pestis on iron-limited solid medium was enhanced to varying degrees by exogenous siderophores (desferal, schizokinen, aerobactin, and enterochelin). Only hemin (0.1 pmol) or a combination of inorganic iron plus protoporphyrin IX promoted growth of Y. pestis on agar rendered highly iron deficient with egg white conalbumin (10 microM). Growth of Y. pseudotuberculosis and Y. enterocolitica was stimulated on this medium by Fe3+ or hemin. These results indicate that hemin can serve as a sole source of iron for yersiniae and that the organisms possess an efficient cell-bound transport system for Fe3+.