Isolation of 3 beta,20 alpha-hydroxysteroid oxidoreductase from sheep fetal blood

3 beta,20 alpha-Hydroxysteroid oxidoreductase has been isolated from ovine fetal blood by a 2,370-fold purification scheme of ammonium sulfate fractionation, calcium phosphate gel adsorption, affinity chromatography, and fast performance liquid chromatography. A new high performance liquid chromatography-based assay for measuring 20 alpha-reductase activity is described. The enzyme is a monomer with a molecular weight of 35,000 and uses NADPH as a cofactor for reductase activity. It reduces progesterone to 4-pregnen-20 alpha-ol-3-one or 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol with kinetic characteristics of Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1 or Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1, respectively. 5 alpha-Dihydrotestosterone competitively inhibits 20 alpha-reductase activity with a Ki value of 102 microM.     

犬脑内动脉构筑的显微X线解剖学研究

犬脑11只,经生理盐水冲洗脑血管后,注入20％钡胶液,切成0.2～1.0厘米的厚片,用显微X线法研究犬脑内各级动脉的构筑,其结果:1.皮质动脉的管径平均为25.9±0.005微米,平均长度为888.0±0.241微米。其形态与发出部位有关,分别呈栅状和瓶刷状。2.髓质动脉的管径平均为49.9±0.007微米。呈直线或孤形向心走行。3.皮质下动脉的管径平均为38.7±0.009微米。呈新月形或蟹钳状分布。4.豆纹动脉和内囊动脉的平均管径为70.0±0.021微米。呈锐角、反血流方向发自母干,再呈“S”形上升。5.丘脑动脉的平均管径为63.7±0.019微米,主要从下方进丘脑,呈树枝状分支。     

Nitrogen fixation ability of exopolysaccharide synthesis mutants of Rhizobium sp. strain NGR234 and Rhizobium trifolii is restored by the addition of homologous exopolysaccharides.

Several transposon Tn5-induced mutants of the broad-host-range Rhizobium sp. strain NGR234 produce little or no detectable acidic exopolysaccharide (EPS) and are unable to induce nitrogen-fixing nodules on Leucaena leucocephala var. Peru or siratro plants. The ability of these Exo- mutants to induce functioning nodules on Leucaena plants was restored by coinoculation with a Sym plasmid-cured (Nod- Exo+) derivative of parent strain NGR234, purified EPS from the parent strain, or the oligosaccharide from the EPS. Coinoculation with EPS or related oligosaccharide also resulted in formation of nitrogen-fixing nodules on siratro plants. In addition, an Exo- mutant (ANU437) of Rhizobium trifolii ANU794 was able to form nitrogen-fixing nodules on white clover in the presence of added EPS or related oligosaccharide from R. trifolii ANU843. These results demonstrate that the absence of Rhizobium EPSs can result in failure of effective symbiosis with both temperate and subtropical legumes.     

Regulation of two nickel-requiring (inducible and constitutive) hydrogenases and their coupling to nitrogenase in Methylosinus trichosporium OB3b.

Two uptake hydrogenases were found in the obligate methanotroph Methylosinus trichosporium OB3b; one was constitutive, and a second was induced by H2. Both hydrogenases could be assayed by measuring methylene blue reduction anaerobically or by coupling their activity to nitrogenase acetylene reduction activity in vivo in an O2-dependent reaction. The H2 concentration for half-maximal activity of the inducible and constitutive hydrogenases in both assays was 0.01 and 0.5 bar (1 and 50 kPa), respectively, making it easy to distinguish these enzymes from one another both in vivo and in vitro. Hydrogen uptake was shown to be coupled to ATP synthesis in methane-starved cells. Methane, methanol, formate, succinate, and glucose all repressed the H2-mediated synthesis of the inducible hydrogenase. Furthermore, this enzyme was only expressed in N-starved cultures and was repressed by NH4+ and NO3-; synthesis of the constitutive hydrogenase was not affected by excess N in the growth medium. In nickel-free, EDTA-containing medium, the activities of these two enzymes were negligible; however, both enzyme activities appeared rapidly following the addition of nickel to the culture. Chloramphenicol, when added along with nickel, had no effect on the rapid appearance of either the constitutive or inducible activity, indicating that nickel is not required for synthesis of the hydrogenase apoproteins. These observations all suggest that these hydrogenases are nickel-containing enzymes. Finally, both hydrogenases were soluble and could be fractionated by 20% ammonium sulfate; the constitutive enzyme remained in the supernatant solution, while the inducible enzyme was precipitated under these conditions.     

NAD-linked aldehyde dehydrogenase for aerobic utilization of L-fucose and L-rhamnose by Escherichia coli.

Mutant analysis revealed that complete utilization of L-fucose and L-rhamnose by Escherichia coli requires the activity of a common NAD-linked aldehyde dehydrogenase which converts L-lactaldehyde to L-lactate. Mutations affecting this activity mapped to the ald locus at min 31, well apart from the fuc genes (min 60) encoding the trunk pathway for L-fucose dissimilation (as well as L-1,2-propanediol oxidoreductase) and the rha genes (min 88) encoding the trunk pathway for L-rhamnose dissimilation. Mutants that grow on L-1,2-propanediol as a carbon and energy source also depend on the ald gene product for the conversion of L-lactaldehyde to L-lactate.     

The role of protein structure in the mitochondrial import pathway. Unfolding of mitochondrially bound precursors is required for membrane translocation

In order to examine the influence of protein structure on the post-translational import of a protein into mitochondria, the carboxyl-terminal 129 residues of F1-ATPase beta-subunit precursor (511aa) have been replaced with 61 residues of yeast copper metallothionein. Import of the F1 beta-copper metallothionein (beta CuMT) hybrid into mitochondria was as efficient as that of the F1 beta precursor in the absence of copper. Addition of copper to mitochondrial import reactions, which had no significant effect on import of the F1 beta-subunit precursor, blocked import of the beta CuMT protein. This copper-dependent transport block for the beta CuMT precursor occurred after the precursor was bound to mitochondria. Expression of the beta CuMT protein in vivo revealed that beta CuMT would bind copper and allow growth of a copper-sensitive yeast host on an otherwise inhibitory level of the cation as long as it was localized in the cytoplasm. These data indicate that the binding of copper by beta CuMT renders it refractile for partial unfolding which is necessary for its translocation into mitochondria. These observations provide an alternative scheme for the selection of mutants defective in mitochondrial import.     

Protein translocation into Escherichia coli membrane vesicles is inhibited by functional synthetic signal peptides

A synthetic peptide corresponding to the signal sequence of wild type Escherichia coli lambda-receptor protein (LamB) inhibits in vitro translocation of precursors of both alkaline phosphatase and outer membrane protein A into E. coli membrane vesicles (half-maximal inhibition at 1-2 microM). By contrast, the inhibitory effect was nearly absent in a synthetic peptide corresponding to the signal sequence from a mutant strain that harbors a deletion mutation in the LamB signal region and displays an export-defective phenotype for this protein in vivo. Two peptides derived from pseudorevertant strains that arose from the deletion mutant and exported LamB in vivo were found to inhibit in vitro translocation with effectiveness that correlated with their in vivo export ability. Controls indicated that these synthetic signal peptides did not disrupt the E. coli membrane vesicles. These results can be interpreted to indicate that the presequences of exported proteins interact specifically with a receptor either in the E. coli inner membrane or in the cytoplasmic fraction. However, biophysical data for the family of signal peptides studied here reveal that they will spontaneously insert into a lipid membrane at concentrations comparable to those that cause inhibition. Hence, an indirect effect mediated by the lipid bilayer of the membrane must be considered.     

Identification of a possible nucleotide binding site in CheW, a protein required for sensory transduction in bacterial chemotaxis

CheW is an essential component of the system which mediates chemotaxis in Salmonella typhimurium and Escherichia coli. Here we report the nucleotide sequence of the cheW gene as well as the purification and characterization of the CheW protein. The DNA sequence predicts a protein of 18,000 molecular weight. The pure protein exhibits an apparent molecular weight of 18,000 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Molecular sieve chromatography under nondenaturing conditions indicates a molecular weight of approximately 35,000, however. This result suggests that CheW is a homodimer. The predicted amino acid sequence between Thr-128 and Asp-160 fits a consensus exhibited by many proteins which bind purine nucleotides.     

Genetic analysis of human B cell hybridomas expressing a cross-reactive idiotype

We have examined the genetic basis for the expression of a human cross-reactive idiotype (CRI) commonly found on monoclonal IgM rheumatoid factors. The CRI was identified with a monoclonal antibody (17.109) and has been localized previously to the kappa-variable region. By using the human lymphoblastoid cell line WI-L2-729-HF2, and mononuclear cells from several sources, a panel of hybridomas was generated that produced 17.109 CRI-positive Ig. A recently cloned human germ-line V kappa III gene, Humkv305, served as a probe to identify genes which were rearranged and expressed in 17.109 CRI-positive and -negative hybridomas. This probe, when hybridized to human genomic DNA under stringent conditions, identified only two to five germ-line bands. In 10 separate 17.109 CRI-positive hybridoma clones, an additional rearranged V kappa band was identified. The probe did not anneal to rearranged V kappa bands in hybridoma clones that produced kappa-chains lacking the CRI. RNA dot-blot studies provided evidence for expression of genes hybridizing to the Humkv305 probe. The results indicate that the 17.109 CRI is a serologic marker for a single V kappa gene, or a small family of closely related V kappa genes, which is identified by the Humkv305 probe.