Comparative organization of nitrogen fixation-specific genes from Azotobacter vinelandii and Klebsiella pneumoniae: DNA sequence of the nifUSV genes.

In the facultative anaerobe Klebsiella pneumoniae 17 nitrogen fixation-specific genes (nif genes) have been identified. Homologs to 12 of these genes have now been isolated from the aerobic diazotroph Azotobacter vinelandii. Comparative studies have indicated that these diverse microorganisms share striking similarities in the genetic organization of their nif genes and in the primary structure of their individual nif gene products. In this study the complete nucleotide sequence of the nifUSV gene clusters from both K. pneumoniae and A. vinelandii were determined. These genes are identically organized on their respective genomes, and the individual genes and their products exhibit a high degree of interspecies sequence homology.     

The effect of analogues of chymostatin on lysosomal and non-lysosomal components of protein degradation in isolated hepatocytes

Of the proteinase inhibitors derived from Streptomyces spp., chymostatin is the most effective inhibitor of non-lysosomal proteolysis. As part of a systematic study of the structural features of the chymostatin molecule that are responsible for this inhibitory activity, a series of fifteen di- and tripeptide analogues of chymostatin were tested for their ability to suppress protein degradation in isolated primary hepatocytes. Protein degradation was assessed in two ways: by the release of radiolabel from proteins prelabelled in vivo (to which both lysosomal and non-lysosomal processes contribute) and by the rate of inactivation of tyrosine aminotransferase, a process that is exclusively non-lysosomal. All inhibitors were relatively non-toxic and did not affect the intracellular ATP levels, although some suppression of gluconeogenesis was observed in the presence of leupeptin, chymostatin or the analogues. Tripeptide phenylalanine aldehydes or semicarbazones were at least as effective as chymostatin in reducing protein degradation, whereas peptide alcohols were relatively ineffective. Replacement of the basic capreomycidine moiety in chymostatin with an arginine residue improved the inhibitory activity but equally, substitution of the arginine residue with an uncharged norleucine residue was without significant effect. The structural features that are optimal for inhibition of chymotrypsin or other serine proteinases (previously defined) are not as critical for inhibition of protein degradation in vivo.     

Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii.

Determination of a 28,793-base-pair DNA sequence of a region from the Azotobacter vinelandii genome that includes and flanks the nitrogenase structural gene region was completed. This information was used to revise the previously proposed organization of the major nif cluster. The major nif cluster from A. vinelandii encodes 15 nif-specific genes whose products bear significant structural identity to the corresponding nif-specific gene products from Klebsiella pneumoniae. These genes include nifH, nifD, nifK, nifT, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifW, nifZ, nifM, and nifF. Although there are significant spatial differences, the identified A. vinelandii nif-specific genes have the same sequential arrangement as the corresponding nif-specific genes from K. pneumoniae. Twelve other potential genes whose expression could be subject to nif-specific regulation were also found interspersed among the identified nif-specific genes. These potential genes do not encode products that are structurally related to the identified nif-specific gene products. Eleven potential nif-specific promoters were identified within the major nif cluster, and nine of these are preceded by an appropriate upstream activator sequence. A + T-rich regions were identified between 8 of the 11 proposed nif promoter sequences and their upstream activator sequences. Site-directed deletion-and-insertion mutagenesis was used to establish a genetic map of the major nif cluster.     

Effect of glycerol on thermolysin-catalyzed peptide bond synthesis

The effect of glycerol on the hydrolytic activity of thermolysin (EC 3.4.24.4) has been compared with the effect on the condensation of N-benzyloxycarbonyl-L-aspartic acid with L-phenylalanine methyl ester to form N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (Z X Asp X Phe X OMe), the precursor to the sweet-tasting compound L-aspartyl-L-phenylalanine methyl ester. Hydrolytic activity was measured by the degradation of azocasein and furylacryloyl-L-glycyl-L-leucinamide. Increasing concentrations of glycerol reversibly inhibited the hydrolytic activity of the enzyme toward both substrates. The inclusion of glycerol in the synthetic medium facilitated the production of Z X Asp X Phe X OMe in a water-soluble system but reduced the initial rate of peptide synthesis. Glycerol stabilized thermolysin against thermal denaturation.     

Lack of effect of oral magnesium on high blood pressure: a double blind study.

Seventeen unselected patients with mild to moderate essential hypertension and whose average supine blood pressure after two months'' observation with no treatment was 154/100 mm Hg were entered into a double blind randomised crossover study of one month''s treatment with magnesium aspartate (15 mmol magnesium/day) and treatment with placebo for a further month. This preparation of magnesium was well tolerated and did not cause diarrhoea. Despite a significant increase in plasma magnesium concentration and a significant increase in urinary excretion of magnesium while taking magnesium aspartate there was no fall in blood pressure compared with either treatment with placebo or values before treatment. The results provide no evidence for a role of dietary magnesium in the regulation of high blood pressure and are contrary to recent speculations.     

Sex-Limited Mitochondrial DNA Transmission in the Marine Mussel Mytilus Edulis

Mitochondrial DNA (mtDNA) was thought to be inherited maternally in animals, although paternal leakage has been reported in mice and Drosophila. Recently, direct evidence of extensive paternal inheritance of mtDNA has been found in the marine mussel Mytilus. We give evidence that whereas female mussels are homoplasmic for a genome that is transmitted to eggs, male mussels are heteroplasmic for this genome and for a second genome that is transmitted preferentially to sperm. The results provide support for the existence of separate male and female routes of mtDNA inheritance in mussels. The two genomes show a base sequence divergence exceeding 20% at three protein coding genes, consistent with long term maintenance of the heteroplasmic state. We propose that the two genomes differ in fitness in males and females, possibly as a result of interaction with nuclear genes.     

Unmasking and Abolition of Sudanophilia of the Mineralizing Front

The effect of various treatments on the Sudanophilia of the mineralizing fronts of hard tissues has been examined. We have shown that a variety of organic solvents. but not all lipid unmasking protocols. expose Sudanophilic lipids at the mineralizing fronts of dentine, enamel matrix, bone and cartilage by the extraction of a substance which is not Sudanophilic. A variety of organic solvents, but not all extraction protocols. abolish Sudanophilia at the mineralizing fronts of bone and cartilage. The present study indicates that only solvent mixtures containing methanol abolished Sudanophilia at the mineralizing fronts of dentine and enamel matrix.     

Diversity of Rhizobia Nodulating Phaseolus vulgaris L. in Two Kenyan Soils with Contrasting pHs

Rhizobia were isolated from two Kenyan soils with pHs of 4.5 and 6.8 and characterized on the basis of their host ranges for nodulation and nitrogen fixation, colony morphologies, restriction fragment fingerprints, and hybridization with a nifH probe. The populations of rhizobia nodulating Phaseolus vulgaris in the two soils were similar in numbers and in effectiveness of N(inf2) fixation but were markedly different in composition. The population in the Naivasha soil (pH 6.8) was dominated by isolates specific in host range for nodulation to P. vulgaris; these all had multiple copies, in most cases four, of the structural nitrogenase gene nifH. Only one of the isolates from this soil formed effective nodules on Leucaena leucocephala, and this isolate had only a single copy of nifH. By contrast, the population in the acid Daka-ini soil (pH 4.5) was composed largely of broad-host-range isolates which had single copies of nifH. The isolates from the Daka-ini soil which were specific to P. vulgaris generally had three copies of nifH, although one isolate had only two copies. These rhizobial isolates are indigenous to Kenyan soils and yet have marked similarities to previously described Rhizobium species from other continents.