Growth hormone and seawater adaptation in coho salmon, Oncorhynchus kisutch

1. Effects of growth hormone (GH) were examined on short-term aspects of seawater adaptation in coho salmon smolts. 2. Injection of somatostatin (SRIF) immediately prior to seawater entry suppressed plasma GH levels, but did not have any significant effects at 6 or 12 hr on hematocrits, plasma glucose or plasma Na+ levels. 3. Plasma GH levels increased 250% within 36 hr after seawater exposure. 4. Plasma glucose levels, in contrast, were significantly lower in the seawater fish after 36 hr post-exposure. 5. Plasma Na+ levels increased to 190 mEq/1 by 24 hr but subsequently returned to freshwater levels while hematocrits showed no significant changes over the 72 hr of exposure. 6. The significance of these results is discussed in terms of successful seawater adaptation in coho salmon.     

The effects of low pH on egg and alevin survival of kokanee and sockeye salmon, Oncorhynchus nerka

1. The effects of low pH water on embryogenesis and vitellogenesis in kokanee and sockeye salmon (Oncorhynchus nerka) were investigated. Eggs were exposed to low pH from fertilization to 45 days post-median hatch or to an episodic exposure at pH 4.0. Adult kokanee were also exposed to low pH just prior to ovulation and spawning. 2. The most sensitive stages of development during chronic or episodic exposure to low pH were early embryonic development and newly-hatched alevins. 3. Incubation of eggs at low pH caused a lower median survival, delayed hatching, higher alevin mortality and reduced the efficiency of yolk conversion to tissue of yolk-sac alevins. Those effects were more pronounced when the eggs were fertilized at low pH. 4. Exposure of sexually mature kokanee salmon to acidified water reduced egg and alevin survival, delayed embryo hatching and decreased the percent hatch. Those effects were more pronounced when their eggs were incubated at low pH.     

Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers.

An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model.     

Oxidative bioconversion of toluene to 1,3-butadiene-1,4-dicarboxylic acid (cis,cis-muconic acid)

Pseudomonas sp. strains, isolated from soil, utilized toluene as their sole carbon source through ameta cleavage pathway. Strains metabolizing toluene through anortho cleavage pathway were selected from the wild typemeta strain. Theortho pathway strains were subjected to chemostat selection to obtain a fast-growing strain with doubling time reduced from 14 to 1.2 h. Benzoale and antibiotics enrichment selection procedures were utilized to select a blocked mutant. The blocked mutant grew on acetate as its sole carbon source and oxidatively converted toluene tocis, cis-muconic acid. Double-blocked and muconate-permeable mutants were also selected to reduce reversion frequency and to enhance muconic acid production. In shake-flask experiments, muconic acid at 3.5 g/l was obtained after 2 days of fermentation. In a 14 l fermenter, muconic acid was produced at 45 g/l in 4 days of controlled fed-batch fermentation. The oxidative bioconversion process was also demonstrated in a 1500 l fermenter.

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Résumé Des souches dePseudomonas sp., isolées du sol, ont utilisé le toluène comme seule source de carbone par la vole de la rupture de cycle enmeta. On a sélectionné des souches métabolisant le toluène par la voie de la rupture de cycle enortho, à partir de la souche sauvage de typemeta. Les souches de la voieortho ont été soumises à la sélection en chémostat pour obtenir des souches à croissance rapide dont le temps de doublement est rédult de 14 à 1.2 h. Les procédures de sélection par enrichissement sur benzoale et antibiotiques ont été utilisées pour sélectionner un mutant bloqué. Le mutant bloqué croît sur acétate comme seule source de carbone et convertit le toluène par voie oxydative en acidecis,cis-muconique. On a également sélectionné des mutants doublement bloqués et perméables au muconate pour réduire la lréquence de réversion et pour augmenter la production d'acide muconique. En expérimentation en flacons agités, on a obtenu 3.5 g/l d'acide muconique après 2 jours de fermentation. En fermentuer de 14 l, on a produit 45 g/l d'acide muconique en 4 jours de fermentation contrôlée en milieu non renouvelé à allmentation étagée. Le processus de bioconversion oxydative a également été démontré en fermenteur de 1500 l.

     

Expression of the chemically synthesized gene for ribonuclease T1 in Escherichia coli using a secretion cloning vector

The gene for ribonuclease T1 from Aspergillus oryzae has been chemically synthesized using the segmental support technique. An Escherichia coli clone producing the ribonuclease at high levels was constructed by linking the gene downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E. coli), using the secretion cloning vector pIN-III-ompA2. This strategy was employed in order to circumvent a possible toxic effect of the gene product on the host cell. Active ribonuclease containing four additional amino acids at the N-terminus could be isolated from the periplasmic fraction of the host. The final yield after purification was 20 mg enzyme/l liquid culture. With respect to immunological, catalytic and specific behaviour, no qualitative differences could be detected between the enzyme from the over-producing E. coli strain and ribonuclease T1 isolated from A. oryzae.     

Cyproheptadine metabolites inhibit proinsulin and insulin biosynthesis and insulin release in isolated rat pancreatic islets

The contribution of drug metabolites to cyproheptadine (CPH)-induced alterations in endocrine pancreatic -cells was investigated by examining the inhibitory activity of CPH and its biotransformation products, desmethylcyproheptadine (DMCPH), CPH-epoxide and DMCPH-epoxide, on hormone biosynthesis and secretion in pancreatic islets isolated from 50-day-old rats. Measurement of (pro)insulin (proinsulin and insulin) synthesis using incorporation of ³H-leucine showed that DMCPH-epoxide, DMCPH and CPH-epoxide were 22, 10 and 4 times, respectively, more potent than CPH in inhibiting hormone synthesis. The biosynthesis of (pro)insulin was also inhibited by CPH and DMCPH-epoxide in islets isolated from 21-day-old rat fetuses. The inhibitory action of CPH and its metabolites was apparently specific for (pro)insulin, and the synthesis of other islet proteins was not affected. Other experiments showed the metabolites of CPH were active in inhibiting glucose-stimulated insulin secretion but were less potent than the parent drug in producing this effect. CPH and its structurally related metabolites, therefore, have differential inhibitory activities on insulin synthesis and release. The observation that CPH metabolites have higher potency than CPH to inhibit (pro)insulin synthesis, when considered with published reports on the disposition of the drug in rats, indicate that CPH metabolites, particularly DMCPH-epoxide, are primarily responsible for the insulin depletion observed when the parent compound is given to fetal and adult animals.Abbreviations CPH cyproheptadine - CPH-epoxide cyproheptadine-10-11-epoxide - DMCPH desmethylcyproheptadine - DMCPH-epoxide desmethylcyproheptadine-10,11-epoxide - HPLC high-performance liquid chromatography - KBB Krebs biocarbonate buffer Recipient of a Society of Toxicology Predoctoral Research Fellowship.Present address: Department of Biochemistry, The University of Hong Kong, Hong Kong.