Age at maturity in landlocked and anadromous Atlantic salmon parr from two Québec rivers

Synopsis Age at maturity in male Atlantic salmon parr from landlocked populations in the Watshishou and Musquaro Rivers is significantly greater than in anadromous populations from the same rivers. We conclude that high post-smolt mortality in anadromous stocks is conducive to male parr maturity at an early age. We also suggest that the lower proportion of maturing male parr in landlocked stocks may be related to competition among males for mates and the smaller ultimate size of spawning adult landlocked salmon.     

Selective inhibition by methoxyamine of the apurinic/apyrimidinic endonuclease activity associated with pyrimidine dimer-DNA glycosylases from Micrococcus luteus and bacteriophage T4

The UV endonucleases [endodeoxyribonuclease (pyrimidine dimer), EC 3.1.25.1] from Micrococcus luteus and bacteriophage T4 possess two catalytic activities specific for the site of cyclobutane pyrimidine dimers in UV-irradiated DNA: a DNA glycosylase that cleaves the 5'-glycosyl bond of the dimerized pyrimidines and an apurinic/apyrimidinic (AP) endonuclease that thereupon incises the phosphodiester bond 3' to the resulting apyrimidinic site. We have explored the potential use of methoxyamine, a chemical that reacts at neutral pH with AP sites in DNA, as a selective inhibitor of the AP endonuclease activities residing in the M. luteus and T4 enzymes. The presence of 50 mM methoxyamine during incubation of UV- (4 kJ/m2, 254 nm) treated, [3H]thymine-labeled poly(dA).poly(dT) with either enzyme preparation was found to protect completely the irradiated copolymer from endonucleolytic attack at dimer sites, as assayed by yield of acid-soluble radioactivity. In contrast, the dimer-DNA glycosylase activity of each enzyme remained fully functional, as monitored retrospectively by release of free thymine after either photochemical- (5 kJ/m2, 254 nm) or photoenzymic- (Escherichia coli photolyase plus visible light) induced reversal of pyrimidine dimers in the UV-damaged substrate. Our data demonstrate that the inhibition of the strand-incision reaction arises because of chemical modification of the AP sites and is not due to inactivation of the enzyme by methoxyamine. Our results, combined with earlier findings for 5'-acting AP endonucleases, strongly suggest that methoxyamine is a highly specific inhibitor of virtually all AP endonucleases, irrespective of their modes of action, and may therefore prove useful in a wide variety of DNA repair studies.     

Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains

Summary A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamB receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD ^- mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.     

A protein induced by NGF in PC12 cells is stored in secretory vesicles and released through the regulated pathway.

We have previously described the isolation of a cDNA clone corresponding to an mRNA rapidly induced to high levels in PC12 cells by treatment with NGF. We report here the complete amino acid sequence of the protein (named VGF8a) as deduced by nucleotide sequencing of overlapping cDNA clones. VGF8a is particularly rich in proline residues and has a conspicuous number of short stretches of basic amino acid residues which may represent potential targets for proteolytic cleavage. Antibodies directed against recombinant VGF8a-beta-galactosidase fusion proteins were used for immunofluorescent staining of the protein in PC12 cells as well as for its localization, by Western blot analysis, in subfractions of cell homogenates. We demonstrate that in PC12 cells, VGF8a protein is stored in secretory vesicles and is released in response to a variety of stimuli that are known to induce the regulated secretion of neurotransmitters.     

Influence of platelet-activating factor on leukotriene D4-induced contractions of the guinea pig parenchymal strip

Platelet-activating factor (PAF) and sulphidopeptide leukotrienes, such as leukotriene D4 (LTD4), are potent constrictors that are probably released simultaneously in a variety of inflammatory respiratory events. The purpose of the present study was to determine whether LTD4-induced contractions of guinea pig parenchymal lung strips (GPPS) are modified in the presence of PAF. The contractile responses of isolated GPPS to cumulative doses of LTD4, acetylcholine, histamine, and potassium chloride in the presence of PAF (0.1 nM, 0.1 microM) were compared with parallel controls. There was no significant alteration of the response to acetylcholine and potassium chloride and the PAF-induced inhibition of the response to histamine, although significant, was not concentration dependent. In contrast, PAF in a concentration range from 0.1 nM to 1.0 microM caused a marked, concentration-dependent reduction of LTD4-induced contractions. Pretreatment with the PAF receptor antagonist, BN52021, prevented the attenuation of LTD4-induced contraction by PAF. The attenuation of LTD4-induced contraction by PAF was also prevented by pretreatment with indomethacin or with the thromboxane synthase inhibitor U63,557A, but not by pretreatment with the lipoxygenase inhibitors BW755c or nordihydroguaiaretic acid. Thus inhibition of LTD4-induced GPPS contraction by PAF is receptor dependent and probably secondary to thromboxane generation. The respiratory smooth muscle response to leukotrienes may be modified significantly by concomitant PAF release.     

Yeast myosin heavy chain mutant: maintenance of the cell type specific budding pattern and the normal deposition of chitin and cell wall components requires an intact myosin heavy chain gene

Recent studies with myosin heavy chain mutants in the slime mold Dictyostelium discoideum and the yeast Saccharomyces cerevisiae indicate that the myosin heavy chain gene is not essential for cell survival under laboratory growth conditions. However, cells lacking a normal myosin heavy chain gene demonstrate substantial alterations in growth and cell division. In this study, we report that a disruption mutant in the rod portion of the yeast myosin heavy chain gene, MYO1, produces abnormal chitin distribution and cell wall organization at the mother-bud neck in a high proportion of dividing cells. It is suggested that this phenotype is the cause of the cell division defect and the osmotic sensitivity of yeast MYO1 mutants. In the absence of a normal MYO1 polypeptide, yeast cells alter their cell type specific budding pattern. It is concluded that an intact myosin heavy chain gene is required to maintain the cell type specific budding pattern and the correct localization and deposition of chitin and cell wall components during cell growth and division.     

Ras-mediated cell cycle arrest is altered by nuclear oncogenes to induce Schwann cell transformation.

The cellular responses to ras and nuclear oncogenes were investigated in purified populations of rat Schwann cells. v-Ha-ras and SV40 large T cooperate to transform Schwann cells, inducing growth in soft agar and allowing proliferation in the absence of added mitogens. Expression of large T alone reduces their growth factor requirements but is insufficient to induce full transformation. In contrast, expression of v-Ha-ras leads to proliferation arrest in Schwann cells expressing a temperature-sensitive mutant of large T at the restrictive temperature. Cells arrest in either the G1 or G2/M phases of the cell cycle, and can re-enter cell division at the permissive temperature even after prolonged periods at the restrictive conditions. Oncogenic ras proteins also inhibit DNA synthesis when microinjected into Schwann cells. Adenovirus E1a and c-myc oncogenes behave similarly to SV40 large T. They cooperate with Ha-ras oncogenes to transform Schwann cells, and prevent ras-induced growth arrest. Thus nuclear oncogenes fundamentally alter the response of Schwann cells to a ras oncogene from cell cycle arrest to transformation.     

Diffusion gradient plates for herbicide toxicity tests on micro-algae and cyanobacteria

Single dimension agar gel diffusion gradient plates were used to test the tolerance of several micro-algae and cyanobacteria to five herbicides. Growth of most of the test organisms was prevented by terbutryn and diuron at concentrations between 1·1 and 2·8 μg/ml (minimum inhibitory concentration, MIC). Chlorpropham was intermediate in toxicity whilst monuron and atrazine generally gave higher MIC values (up to 17·1 μg/ml). The cyanobacteria were mostly more tolerant than the algae, but susceptibility to the herbicides was variable even between strains of an individual algal species. The gradient plate technique offers the advantages of simplicity and low cost in toxicity screening studies with herbicides or other biologically active chemicals and xenobiotics.