Mutagenesis of the Tra1 core region of RK2 by using Tn5: identification of plasmid-specific transfer genes.

The conjugation system of the IncP alpha plasmid RK2/RP4 is encoded by transfer regions designated Tra1, Tra2, and Tra3. The Tra1 core region, cloned on plasmid pDG4 delta 22, consists of the origin of transfer (oriT) and 2.6 kilobases of flanking DNA providing IncP alpha plasmid-specific functions that allow pDG4 delta 22 to be mobilized by the heterologous IncP beta plasmid R751. Tn5 insertions in pDG4 delta 22 define a minimal 2.2-kilobase region required for plasmid-specific transfer of oriT. The Tra1 core contains the traJ and traK genes as well as an 18-kilodalton open reading frame downstream of traJ. The traJ and traK genes were shown to be required for transfer by complementation of inserts within these genes. Genetic evidence for the role of the 18-kilodalton open reading frame in transfer was obtained, although this protein has not been detected in cell lysates. These studies indicate that at least three transfer proteins are involved in plasmid-specific interactions at oriT.     

Effects of dl-2-Amino-5-Phosphonovalerate on Metabolism of Catecholamines in Synaptosomes from Rat Brain

Incubation of synaptosomes from rat brain with DL-2-amino-5-phosphonovalerate (APV) stimulated an increased release of dopamine, and this effect was strictly dependent on the extrasynaptosomal calcium level. APV increased biosynthesis of dopamine from tyrosine by 30%, whereas monoamine oxidase activity was inhibited by 30%. When synaptosomes were incubated with radioactive dopamine, APV caused a large decrease in incorporation of label into 3,4-dihydroxyphenylacetic acid but greatly increased incorporation into norepinephrine and its N-methyl derivatives. Quantification of dopamine and its metabolites in synaptosomes, using electrochemical detection, indicated that the presence of APV resulted in changes in the absolute levels of the aforementioned dopamine metabolites similar to the changes in radiolabel incorporation. Omission of Ca2+ from the extrasynaptosomal medium greatly diminished the APV-induced changes in catecholamine metabolism. The metabolic changes appear to largely result from an increased intrasynaptosomal Ca2+ level due to the APV-induced increase in calcium permeability of the plasma membrane.     

Uniform 15N labeling of a fungal peptide: the structure and dynamics of an alamethicin by 15N and 1H NMR spectroscopy.

An alamethicin, secreted by the fungus Trichoderma viride and containing a glutamine at position 18 instead of the usual glutamic acid, has been uniformly labeled with 15N and purified by HPLC. The extent of 15N incorporation at individual backbone and side-chain sites was found to vary from 85% to 92%, as measured by spin-echo difference spectroscopy. The proton NMR spectrum of the peptide dissolved in methanol was assigned using correlation spectroscopies and nuclear Overhauser enhancements (NOE) measured in the rotating frame. The 15N resonances were assigned by the 2D 1H-15N correlation via heteronuclear multiple-quantum coherence experiment. NOEs and 3JNHC alpha H coupling constants strongly suggest that, in methanol, from Aib-3 to Gly-11, the peptide adopts a predominantly helical conformation, in agreement with previous 1H NMR studies [Esposito, G., Carver, J.A, Boyd, J., & Campbell, I.D. (1987) Biochemistry 26, 1043-1050; Banerjee, U., Tsui, F.-P., Balasubramanian, T.N., Marshall, G.R., & Chan, S I. (1983) J. Mol. Biol. 165, 757-775]. The conformation of the carboxyl terminus (12-20) is less well determined, partly because the amino acid composition reduces the number of NOEs and coupling constants which can be determined by 1H NMR spectroscopy. The 3JNHC alpha H in the C-terminus suggest the possibility of conformational averaging at Leu-12, Val-15, and Gln-19, an interpretation which is supported by a recent molecular dynamics simulation of the peptide [Fraternalli, F. (1990) Biopolymers 30, 1083-1099].(ABSTRACT TRUNCATED AT 250 WORDS)     

Restriction site mapping for three or more enzymes

Restriction site mapping requires a generator to put forwardpossible maps and a constraint checker to reject false maps.Ideally these combine to give an algorithm which calculatesa sound and complete solution set. Three algorithms for generationare presented and compared. Two decompose a multi-enzyme problem(3) into subproblems. The constraint checker is based on separationtheory. Some insights into the extent of constraint checkinginvolved in and the feasibility of more checking for three ormore enzymes are discussed. The trade-off between computationtime and the soundness of the solution set is examined. Received on July 30, 1989; accepted on April 4, 1990     

Bioavailability of selenium accumulated by selenite-reducing bacteria

The bioavailability of selenium (Se) was determined in bacterial strains that reduce selenite to red elemental Se (Se^o). A laboratory strain ofBacillus subtilis and a bacterial rod isolated from soil in the vicinity of the Kesterson Reservoir, San Joaquin Valley, CA, (Microbacterium arborescens) were cultured in the presence of 1 mM sodium selenite (Na₂SeO₃). After harvest, the washed, lyophilizedB. subtilis andM. arborescens samples contained 2.62 and 4.23% total Se, respectively, which was shown to consist, within error, entirely of Se^o. These preparations were fed to chicks as supplements to a low-Se, vitamin E-free diet. Three experiments showed that the Se in both bacteria had bioavailabilities of approx 2% that of selenite. A fourth experiment revealed that gray Se^o had a bioavailability of 2% of selenite, but that the bioavailability of red Se^o depended on the way it was prepared (by reduction of selenite). When glutathione was the reductant, bioavailability resembled that of gray Se^o and bacterial Se; when ascorbate was the reductant, bioavailability was twice that level (3–4%). These findings suggest that aerobic bacteria such asB. subtilis andM. arborescens may be useful for the bioremediation of Se-contaminated sites, i.e., by converting selenite to a form of Se with very low bioavailability.     

Optimized enzymatic synthesis of geranyl butyrate with lipase AY from Candida rugosa

Response surface methodology (RSM) and five-level, five-variable central composite rotatable design (CCRD) were used to evaluate the effects of synthetic variables, such as reaction time (1-9 h), temperature (25-65 degrees C), enzyme amount (10-50%), substrate molar ratio of geraniol to tributyrin (1:0.33-1:1), and added water amount (0-20%) on molar percent yield of geranyl butyrate, using lipase AY from Candida rugosa. Reaction time and temperature were the most important variables and substrate molar ratio had no effect on percent molar conversion. Based on contour plots, optimum conditions were: reaction time 9 h, temperature 35 degrees C, enzyme amount 50%, substrate molar ratio 1:0.33, and added water 10%. The predicted value was 100% and actual experimental value was 96.8% molar conversion. (c) 1996 John Wiley & Sons, Inc.     

Cloning and Expression of a 5-Hydroxytryptamine7 Receptor Positively Coupled to Adenylyl Cyclase

Abstract: In a number of different cell types, phosphorylation of a 63-kDa protein has been shown to increase rapidly in response to stimuli that lead to an increase in intracellular calcium. Here, a stimulus-sensitive protein at this molecular weight is identified in PC12 cells and rat cortical synaptosomes as phosphoglucomutase. In addition, the added phosphate is shown to be in an oligosaccharide terminating in phosphodiester-linked glucose. In synaptosomes, incorporated radioactivity, following incubation with [¹⁴C]glucose or the [β-³⁵S]phosphorothioate analogue of UDP-glucose, was found to increase within 5 s of stimulation and return to baseline within 25 s. Despite the many pathways utilizing glucose, this was the only detectable protein glycosylation observed in synaptosomes. These results indicate that cytoplasmic glycosylation is reversible and rapidly regulated, and suggest that phosphoglucomutase undergoes an alteration in function and/or topography in response to increases in intracellular calcium.     

Superoxide dismutase,catalase, and U78517F attenuate neuronal damage in gerbils with repeated brief ischemic insults

Repeated ischemic insults at one hour intervals result in more severe neuronal damage than a single similar duration insult. The mechanism for the more severe damage with repetitive ischemia is not fully understood. We hypothesized that the prolonged reperfusion periods between the relatively short ischemic insults may result in a pronounced generation of oxygen free radicals (OFRs). In this study, we tested the protective effects of superoxide dismutase (SOD) and catalase (alone or in combination), and U78517F in a gerbil model of repetitive ischemia. Three episodes (two min each) of bilateral carotid occlusion were used at one hour intervals to produce repetitive ischemia. Superoxide dismutase and catalase were infused via osmotic pumps into the lateral ventricles. Two doses of U78517F were given three times per animal, one half hour prior to each occlusion. Neuronal damage was assessed 7 days later in several brain regions using the silver staining technique. The Mann-Whitney U test was used for statistical comparison. Superoxide dismutase showed significant protection in the hippocampus (CA4), striatum, thalamus and the medial geniculate nucleus (MGN). Catalase showed significant protection in the striatum, hippocampus, thalamus, and MGN and the substantia nigra reticulata. Combination of the two resulted in additional protection in the cerebral cortex. Compared to the controls, there was little protection with a dose of 3 mg/kg of U78517F. There was significant protection with a dose of 10 mg/kg in the hippocampus (CA4), striatum, thalamus, medial geniculate nucleus and the substantia nigra reticulata. The significant protection noted with SOD, catalase or U78517F with repeated ischemia supports, the hypothesis that OFRs may play a role in neuronal damage in repeated cerebral ischemia.     

Distinct morphological and mito-inhibitory effects induced by TGF-β1, HGF and EGF on mouse,rat and human hepatocytes

TGF-1 is known as a potent inhibitor of proliferation of rat and human hepatocytes. In this study we show that the effects of TGF-1 are quite different on mouse hepatocytes. In rat and human hepatocytes, TGF-1 inhibited DNA synthesis and also inhibited the morphological changes induced by growth factors in rat and human hepatocytes. In contrast, addition of TGF-1 to mouse hepatocytes resulted in pronounced alterations in morphology of these cells. These changes were similar to those induced by HGF and EGF. The induction of structural changes by TGF-1 was noted only in mouse hepatocytes. Mouse hepatocytes were also much more resistant to the mito-inhibitory effect of TGF-1. These findings suggest profound differences in hepatocyte growth regulation between these species and may relate to observed differences in susceptibility to carcinogenesis.Abbreviations EGF epidermal growth factor - HGF hepatocyte growth factor - SF scatter factor - TGF-1 transforming growth factor beta type one