Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family

Summary The Brassica napus rapeseed cultivar Topas contains an acetohydroxyacid synthase (AHAS) multigene family consisting of five members (AHAS 1–5). DNA sequence analysis indicate that AHAS1 and AHAS3 share extensive homology. They probably encode the AHAS enzymes essential for plant growth and development. AHAS2 has diverged significantly from AHAS1 and AHAS3 and has unique features in the coding region of the mature polypeptide, transit peptide and upstream non-coding DNA, which raises the possibility that it has a distinct function. AHAS4 and AHAS5 have interrupted coding regions and may be defective. The complexity of the AHAS multigene family in the allotetraploid species B. napus is much greater than reported for Arabidopsis thaliana and Nicotiana tabacum. Analysis of the presumptive progenitor diploid species B. campestris and B. oleracea indicated that AHAS2, AHAS3 and AHAS4 originate from the A genome, whereas AHAS1 and AHAS5 originate from the C genome. Further variation within each of the AHAS genes in these species was found.     

U-61,431F, a stable prostacyclin analogue, inhibits the proliferation of bovine vascular smooth muscle cells with little antiproliferative effect on endothelial cells.

The effects of U-61,431F, ciprostene, a stable prostacyclin analogue, were examined on the proliferation of cultured quiescent bovine aortic endothelial cells (EC) and smooth muscle cells (SMC). After stimulation with 5% fetal calf serum, U-61,431F suppressed both the DNA synthesis and proliferation of SMC dose-dependently at the concentration of 3-100 microM, but had no effect on either of them in EC at a concentration of up to 30 microM. The inhibitory effect on DNA synthesis was greater in SMC than in EC at 3-50 microM. When SMC were stimulated with platelet-derived growth factor (PDGF) for 2 hrs followed by a 22-hr incubation with insulin, U-61,431F (1-50 microM) administered at the time of PDGF stimulation did not inhibit DNA synthesis. SMC initiated and terminated DNA synthesis at about 15-18 h and 24 h after stimulation with serum, respectively. Inhibition of DNA synthesis in serum-stimulated SMC as a function of the addition time of U-61,431F reduced at 3-12 h after the stimulation. U-61,431F raised the cyclic AMP (cAMP) content in SMC. Moreover, a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and a more specific cAMP phosphodiesterase inhibitor, Ro 20-1724, augmented the inhibition of DNA synthesis in SMC concomitant with further elevation of cAMP level. These results suggest that U-61,431F inhibits DNA synthesis of SMC acting in the progression stage rather than in the competence stage, with little antiproliferative effect on EC. cAMP may play an important role in its antiproliferative action in SMC.     

Analysis of bacterial populations in a grassland soil according to rates of development on solid media

Abstract The process of colony formation by bacteria from grassland soil sampled in April, July and September was simulated by a colony-forming curve (CFC). The CFC was a super-imposition of several component curves (cCFC) given theoretically by the first order reaction (FOR) model [3,6]. The pattern of FOR model curves was not influenced by the time of sampling and four cCFCs were always recognized during an incubation period of 160 h. It was considered that the CFC describes an inherent property of the bacterial population of the field. Bacterial isolates were obtained from colonies produced in each of four cCFCs on agar plates. Isolates corresponding to one cCFC were classified as one group. The bacterial isolates were characterized by morphological and physiological tests and subsequently clustered. Few oligotrophic bacteria were obtained among bacteria which produced visible colonies within 63 h of incubation time. On the other hand, approx. 50% of bacteria which produced v colonies after 63 h were oligotrophic bacteria. The time required for the appearance of the first colony, t _r of the FOR model, was very similar in the isolates belonging to one group. A close linear relationship was observed between t _r value and doubling time of isolates.     

Purification and properties of 3 alpha-hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum from human intestine

3 alpha-Hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum, isolated from human intestinal bacteria, was capable of converting 3-ketoglycyrrhetic acid to 3 alpha-hydroxyglycyrrhetic acid. The enzyme was purified to homogeneity by means of butyl-Toyopearl 650M, Sephadex G-150, Matrex Red A, Toyopearl HW-55S, and isoelectric focusing column chromatographies. The purified enzyme showed a specific activity of 156 mumol/min.mg toward 3 alpha-hydroxyglycyrrhetic acid, and showed a single band on SDS-polyacrylamide gel electrophoresis. The apparent molecular weight was 53,000, as estimated by gel filtration, and 30,000, as judged by SDS-polyacrylamide gel electrophoresis. Its isoelectric point was 5.2. The enzyme showed absolute specificity for the 3 alpha-hydroxyl and 3-ketonic groups of 18 alpha- or 18 beta-glycyrrhetic acid and required NADP+ and NADPH as cosubstrates. The enzyme did not act on any 3 alpha-hydroxyl or 3-ketonic group of steroids or bile acids. The enzyme is a novel type of enzyme, defined as 3 alpha-hydroxy-glycyrrhetinate dehydrogenase, being quite different from 3 alpha-hydroxysteroid dehydrogenase [EC 1.1.1.50].     

Starvation-induced changes in rat brain corticotropin-releasing factor (CRF) and pituitary-adrenocortical response

Starvation-induced changes in CRF concentration in major brain regions and abnormalities in the pituitary-adrenal axis were examined in rats using rat CRF radioimmunoassay. The CRF concentrations in the hypothalamus and cerebellum were significantly reduced in the completely starved rats, while those in the midbrain, thalamus and neurointermediate lobe of the pituitary were significantly increased in the semi-starved or completely starved rats. No significant changes in the CRF concentrations were found in the pons, medulla oblongata and cerebral cortex. In the completely starved rats, the serum ACTH level was significantly reduced, whereas the serum corticosterone level was markedly elevated. These observations suggest that starvation may stimulate the CRF-ACTH-corticosterone system and that not only hypothalamic CRF but also extrahypothalamic CRF may be discretely related to feeding behavior or starvation. The reduced serum ACTH level in starved rats may be ascribed to the negative feedback effect of the elevated serum corticosterone.     

Reactivity of a sulfhydryl group of the ras oncogene product p21 modulated by GTP binding

We have studied the sensitivity of sulfhydryl groups of a highly purified p21 protein of the v-rasH oncogene to a thiol-specific reagent, N-ethylmaleimide (NEM). Approximately 70% of GTP binding and autokinase activities of p21 were inactivated by NEM, and excessive amounts of GTP or GDP protected p21 activities. Thiol titration revealed the presence of one fast reactive cysteine residue, the susceptibility of which is modulated by GTP binding. A total of 4 and 6 residues, respectively, became titratable upon denaturation and reduction, suggesting the presence of a disulfide bond. This GTP-modulated sulfhydryl group was identified as Cys-80 in the following tryptic peptide sequence: NH2-Thr-Gly-Glu-Gly-Phe-Leu-Cys-Val-Phe-Ala-Ile-Asn-Asn-Thr-Lys-COOH. This is based on the comparative tryptic peptide mapping of [14C]NEM-modified p21 in the presence and absence of GTP. The GTP-modulated peptide co-chromatographed with a synthetic peptide of the predicted sequence. Amino acid analysis of the purified [14C]NEM-modified peptide from tryptic digests of p21 also confirmed its identity. This region of p21 shares an extensive sequence homology with various G-proteins and appears to be in the vicinity of the GTP-binding domain of these proteins.     

Sialic acid moiety is responsible for the charge heterogeneity and the biological potency of rat lutropin

To elucidate a possible role of sialic acid moiety in the electrical heterogeneity of rat pituitary lutropin, seven components separated were individually treated with neuraminidase. Some intermediates with isoelectric points corresponding to the native components were concomitantly seen at the serial stages of the enzyme treatment. All the treated components showed an isoelectric point of about 10.0 which was the same to the isoelectric point of one of the seven components. Desialylation of the components with less biological activity caused enhancement of the in vitro cyclic AMP producing- and testosterone producing-activities as well as the binding activity to the receptor. It is concluded that the number of sialic acid moiety in lutropin is responsible for the charge heterogeneity and the biological potency of the hormone.     

The base-exchange enzyme activities of sarcolemma and sarcoplasmic reticulum from rat heart

The Ca2+ dependent incorporation of [14C]ethanolamine, L-[14C]serine and [14C]choline into phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine, respectively, were investigated in membrane preparations from rat heart. The ethanolamine and serine base-exchange enzyme-catalyzed reactions were associated with the sarcolemma and sarcoplasmic reticulum. There was a 17.2-fold and 6.8-fold enrichment, respectively, of the serine and the ethanolamine base-exchange enzyme activities in the sarcolemma compared to the starting whole homogenate. The sarcoplasmic reticulum was enriched in the ethanolamine and serine base-exchange enzyme activities. The choline base-exchange enzyme activity of all membranes fractions was negligible compared to the ethanolamine or serine base-exchange enzyme activities. The apparent Km for the ethanolamine and serine base-exchange enzyme in sarcolemma was 14 microM and 25 microM, respectively. The pH optimum for these base-exchange activities was 7.5-8.0. There was a dependence upon Ca2+ for these reactions with a 1 or 4 mM concentration required for maximal activity. The properties of the sarcoplasmic reticulum base-exchange enzymes were similar to the sarcolemmal base-exchange enzymes.     

Reduction in brain immunoreactive corticotropin-releasing factor (CRF) in spontaneously hypertensive rats

The brain CRF concentration of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) was examined by rat CRF radioimmunoassay. Anti-CRF serum was developed by immunizing rabbits with synthetic rat CRF. Synthetic rat CRF was also used as tracer and standard. The displacement of 125I-rat CRF by serially diluted extracts of male Wistar rats hypothalamus, thalamus, midbrain, pons, medulla oblongata, cerebral cortex, cerebellum and neurointermediate lobe was parallel to the displacement of synthetic rat CRF. In both WKY and SHR the highest levels of CRF immunoreactivity were shown by the hypothalamus and neuro-intermediate lobe, and considerable CRF immunoreactivity was also detected in other brain regions. The CRF immunoreactivity in the hypothalamus, neurointermediate lobe, midbrain, medulla oblongata and cerebral cortex was significantly reduced in SHR and it may suggest that CRF abnormality may be implicated in the reported abnormalities in the pituitary-adrenal axis, autonomic response and behavior of SHR.