Purification of the NADPH:5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase from female rat pituitary cytosol

The NADPH:5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) [EC 1.1.1.50] which catalyzes the reversible conversion of 5 alpha-pregnane-3,20-dione (5 alpha-dihydroprogesterone; 5 alpha-DHP) to 3 alpha-hydroxy-5 alpha-pregnan- 20-one (3 alpha-,5 alpha-tetrahydroprogesterone; 3 alpha,5 alpha-THP) was purified to apparent homogeneity from female rat anterior pituitary cytosol by a three step micro-purification procedure. Specific activity of purified 3 alpha-HSOR was enriched 438-fold from that in pituitary cytosol using successive ion exchange, chromatofocusing and affinity column chromatography purification steps. 3 alpha-HSOR appears to be a monomer with an approximate molecular weight of 36 kDa and an isoelectric point of about 5.75. The purified enzyme appears as a single protein staining band (36 kDa) when examined by polyacrylamide gel electrophoresis and with both silver or Coomassie blue staining. Under non-dissociating electrophoretic conditions, all of the 3 alpha-HSOR activity co-migrated with the 36 kDa protein staining band. The purified enzyme in the presence of the preferred cofactor, NADPH, has an apparent Km for 5 alpha-DHP of 82 nM and a Vmax of 1.2 mumol of 3 alpha,5 alpha-THP formed per mg protein/30 min. The Km for NADPH was 0.71 microM. In the oxidative direction, the enzyme in the presence of NADP+ has a Km for 3 alpha,5 alpha-THP of 1.4 microM and a Vmax of 9.7 mumol of 5 alpha-DHP formed per mg protein/30 min. The Km for NADP+ was 1.6 microM.     

Seed size variation: magnitude,distribution, and ecological correlates

Summary We examined seed-mass variation in 39 species (46 populations) of plants in eastern-central Illinois, USA. The coefficient of variation of seed mass commonly exceeded 20%. Significant variation in mean seed mass occurred among conspecific plants in most species sampled (by hierarchical ANOVA), averaging 38% of total variance. For most species, within-plant variation was the larger component of total variance, averaging 62% of total variance. Variation in seed mass among fruits within crops was significant in most species tested.We conclude that variation in seed mass among and within plants is widespread and common. There was little evidence of trade-offs between number of seeds and mean or variance of seed mass, and little correlational evidence of local competition for maternal resources. No consistent ecological (dispersal mode and growth form) correlates of variance of seed mass were evident.     

Catabolic instability,plasmid gene deletion and recombination in Alcaligenes sp. BR60

An Alcaligenes sp. BR60, isolated from surface runoff waters of the Hyde Park industrial landfill, contained a novel 85 kb catabolic plasmid (pBR60) functional in 3-chlorobenzoate (3Cba) degradation. The plasmid exhibited a spontaneous 3.2% frequency of deletion of a 14 kb fragment specifying 3Cba degradation. The deletion mutant BR40 and mitomycin C cured strains were not able to grow on 3Cba and had reversion frequencies of less than 10^-10 cell^-1 generation^-1. Transformation or conjugation of pBR60 into cured strains restored catabolic activity. An EcoRI, BgIII, HindIII and SaII restriction map of the deletion region was constructed, and EcoRI and HindIII fragments spanning the deletion region of the plasmid were cloned in pUC18. Conjugation of resistance plasmid R 68.45 into Alcaligenes sp. BR60, with selection on antibiotics, resulted in the elimination of pBR60 and maintenance of unaltered R68.45. In 30% of the exconjugants, 3Cba degradative capacity was retained, although variation in the regulation of 3Cba degradation was observed in these strains. Hybridization of deletion region fragments to BgIII digested total DNA of BR60 and the R68.45 cured exconjugants revealed the presence of pBR60 deletion region sequences in the chromosome of exconjugants. Hybridization also revealed a repeated sequence flanking the deletion region of pBR60. Selection on 4-chlorobenzoate as a sole source of carbon and energy resulted in the isolation of 4Cba⁺ mutants of Alcaligenes sp. BR60.Abbreviations 3 and 4 Cba chlorobenzoic acid isomers and growth phenotypes - HPLC high pressure liquid chromatography - ATCC American Type Culture Collection     

Agroinfection and nucleotide sequence of cloned wheat dwarf virus DNA

Cloned DNA of the geminivirus wheat dwarf virus (WDV) was successfully used to infect seedling wheat plants. The clone was derived from circular double-stranded viral DNA isolated from naturally infected tissue. The initiation of infection was mediated by Agrobacterium tumefaciens using cloned dimeric WDV genomes in a binary Agrobacterium vector. The WDV DNA which comprised the infectious clone was sequenced and is compared with the published sequence of a Swedish isolate of the same virus. The results confirm that the single WDV genome component of 2.75 kb carries all the information necessary for production of viral symptoms, virus particles and viral double- and single-stranded DNA forms.     

Induction of embryogenicTriticum aestivum L. calli. I. Quantification of genotype and culture medium effects

Somatic embryo (embryoid) formation from immature-embryo-derived calli was quantified in replicated experiments involving 10Triticum aestivum L. genotypes. Several published media formulations, which had previously been optimized for wheat tissue culture, were tested for each genotype. Embryos from each plant were randomly assigned to each medium. Percentage precocious germination of immature embryos and mean percentage scutellar callus per explant were recorded. Embryoids per callus were determined by microscopic examination at 28 and 56 days. There were highly significant differences among genotypes, media, and individual plants from which explants were taken. A medium based on double the Murashige and Skoog (MS) inorganic salt concentration was significantly better than other media. Inclusion of all MS vitamins appeared essential for optimal response. Two genotypes were tested in a second experiment where both 3,6-dichloro-o-anisic acid (9.05 M) and 6-furfurylaminopurine (0.46 M) were substituted for 2,4-dichlorophenoxyacetic acid (4.52 M) in either double or normal MS medium. This substitution significantly increased embryoid formation at 28 days. Additions of either 6-furfurylaminopurine or coconut water increased precocious germination of both embryo explants and embryoids.This study was supported in part by NASA-Ames Cooperative Agreement No. NCC2-139. Contribution of the Utah Agricultural Experiment Station, Utah State University, Logan, UT, Journal Paper No. 3358.     

Nitrate reductase and its role in nitrate assimilation in plants

Nitrate reductase (EC 1.6.6.1) is an enzyme found in most higher plants and appears to be a key regulator of nitrate assimilation as a result of enzyme induction by nitrate. The biochemistry of nitrate reductase has been elucidated to a great extent and the role that nitrate reductase plays in regulation of nitrate assimilation is becoming understood.     

Phytochrome-mediated light regulation of nitrate reductase expression in squash cotyledons

In etiolated squash (Cucurbita maxima L.) cotyledons, nitrate-inducible NADH:nitrate reductase activity and protein were increased in darkness by red light pulses with red/far-red photoreversibility. Continuous far-red light also led to increased levels of nitrate reductase activity and protein. Poly(A)+RNA, which hybridizes to squash nitrate reductase cDNA, was also increased by light treatments. Thus, we found that after nitrate triggering, nitrate reductase expression appears to be regulated by light via phytochrome.     

Biochemical and ultrastructural characterization of the 1,4-dihydropyridine receptor from rabbit skeletal muscle. Evidence for a 52,000 Da subunit

The 1,4-dihydropyridine receptor purified from rabbit skeletal muscle contains four polypeptide components of 175,000 Da (nonreduced)/150,000 Da (reduced), 170,000, 52,000, and 32,000 Da (Leung, A. T., Imagawa, T., and Campbell, K. P. (1987) J. Biol. Chem. 262, 7943-7946). A monoclonal antibody specific to the 52,000-Da polypeptide component of the dihydropyridine receptor has been produced and used in immunoprecipitation and immunoblotting experiments to demonstrate that the 52,000-Da polypeptide is an integral subunit of the purified dihydropyridine receptor. Peptide mapping experiments with 32P-labeled dihydropyridine receptor have also demonstrated that the 52,000-Da polypeptide is distinct from and not a proteolytic fragment of the 170,000-Da subunit. Densitometric scanning of Coomassie Blue-stained sodium dodecyl sulfate-polyacrylamide gels of the purified dihydropyridine receptor has demonstrated that the 52,000-Da polypeptide exists in a 1:1 stoichiometric ratio with the 170,000-, 175,000/150,000-, and 32,000-Da subunits of the dihydropyridine receptor. Electron microscopy of the freeze-dried, rotary-shadowed dihydropyridine receptor has shown that the preparation contains a homogeneous population of 16 x 22-nm ovoidal particles large enough to contain all four polypeptides of the dihydropyridine receptor. The particles have two distinct components of similar size which may represent the location in the molecule of the two larger subunits.     

Aortic velocity contours at abdominal branches in anesthetized dogs

Pulsed Doppler velocimetry was used to record a grid of velocity waveforms 0.5 cm downstream of the cephalic mesenteric and left renal branches of the aorta in anesthetized dogs. Aortic velocity contours at different phases of the pulse cycle were developed from the grid of waveforms. Transient flow recirculation occurred in the post-systolic phase of the pulse cycle at the aortic wall opposite the branch artery. There was no recirculation at the systolic peak but there was asymmetry of the velocity profile. In contrast, in the in vivo post-stenotic velocity field recirculation persists throughout most of the cycle. These results compare well with similar results from pulsatile model studies. Spectral width of the Doppler signal was not increased in any of the velocity waveforms immediately downstream of the branches, this suggests that flow is relatively undisturbed in these locations.