Limited DNA damage in human endothelial cells after hyperbaric oxygen treatment and protection from subsequent hydrogen peroxide exposure.

BACKGROUND: In vitro studies on hyperbaric oxygen (HBO) therapy suggest that HBO may cause DNA damage, but this has not been evaluated using endothelial cells. METHODS: Human umbilical cord endothelial cells (HUVECs) were exposed either to H(2)O(2) or to HBO for 90 min, with or without subsequent H(2)O(2) exposure. Measurements included the comet assay for DNA damage, and reduced and oxidised glutathione levels. RESULTS: HUVECs showed sensitivity to H(2)O(2) (EC(50) of 0.2mM for DNA migration). A single 90 min HBO treatment at 2.2 ATA caused a statistically significant (ANOVA, P<0.05) increase of DNA migration in HUVECs to 6.8 ± 0.3% (mean ± SEM, n=8), which returned to normal levels (4.9 ± 0.1%, n=6) after 24h. Further exposure to 0.2mM H(2)O(2) after HBO treatment significantly increased the DNA migration in HBO-treated cells immediately post-treatment; but 24h later the cells showed 22% less DNA damage and higher glutathione than controls. CONCLUSION: A single HBO exposure causes limited DNA damage to HUVECs, which repairs quickly. HBO treatment protects against H(2)O(2)-induced DNA damage and involves cellular glutathione. SIGNIFICANCE: Endothelial cells are unlikely to be compromised during HBO therapy.

The combined oral contraceptive pill and the assumed 28-day cycle.

Some studies involving women taking the combined oral contraceptive pill (COCP) have on occasion assumed the COCP group to have a rigid 28-day pharmaceutically driven cycle. Anecdotal evidence suggests otherwise, with many women adjusting their COCP usage to alter the time between break-through bleeds for sporting and social reasons. A prospective field study involving 533 scuba diving females allowed all menstrual cycle lengths (COCP and non-COCP) to be observed for up to three consecutive years (St Leger Dowse et al. 2006). A total of 29% of women were COCP users who reported 3,241 cycles. Of these cycles, only 42% had a rigid 28-day cycle, with the remainder varying in length from 21 to 60 days. When performing studies involving the menstrual cycle, it should not be assumed that COCP users have a rigid confirmed 28-day cycle and careful consideration should be given to data collection and analysis. The effects of differing data interpretations are shown.

Quantification of gene transcription and enzyme activity for functionally important proteolytic enzymes during early development in the Pacific oyster Crassostrea gigas.

Gene transcripts and enzyme activities were quantified for a selection of functionally important aminopeptidases at 2-day intervals throughout the first 72 days of development in the Pacific oyster Crassostrea gigas. Leucine aminopeptidase (LAP) and cathepsin B (CathB) gene transcripts were quantified using fluorogenic ('real time') PCR. LAP and CathB gene transcripts were detected at all time points. The proportion of CathB transcripts remained essentially constant and low throughout development (Ct<35). The proportion of LAP transcripts was often similar (Ct<30), but with a distinct peak in transcript abundance at day 19 (Ct approximately 23). CathB and cathepsin D (CathD) enzyme activities were measured biochemically. Whilst CathD activity peaked at day 19, LAP and CathB activities both peaked at day 24. The closely coupled increase in transcript and enzyme activity for LAP indicates regulation at the transcriptional level. Alternatively, the peak in enzyme activity for CathB without enhanced transcriptional activity suggests post-transcriptional regulation. Similar mechanisms of regulation for LAP and CathB have been observed in both plants and mammals, indicating widespread conservation.

Transcript analysis of the genes encoding aminopeptidase N and alanine aminotransferase, two enzymes involved in protein turnover, in the Pacific oyster, Crassostrea gigas.

Molecular probes have been developed to detect aminopeptidase N (ApN) and alanine aminotransferase (ALAT) transcripts in the Pacific oyster Crassostrea gigas. Degenerate primers were designed using ApN and ALAT sequences stored in the EMBL database. Amplification of C. gigas genomic DNA using these primers resulted in amplification of a 344-bp ApN fragment and a 530-bp alanine aminotransferase fragment. The deduced amino acid sequence of the ApN fragment displayed 75 and 73% identities with sequences of ApN from human and mouse, respectively. The deduced amino acid sequence of the ALAT fragment displayed 57% identity both with human and rat ALAT. An ApN transcript of approximately 3.1 kb was detected by northern blotting in larvae and in adult digestive gland and gonadal tissue. No transcript was detected in adult adductor muscle. An ALAT transcript of approximately 2 kb was similarly detected in larvae and in adult gonadal tissue, but was undetectable in adult digestive gland and adductor muscle. Transcript detection employing RT-PCR demonstrated low-level expression of both ApN and ALAT in all studied tissues, in both larvae and adults.

Stable production of human gastric lipase by chromosomal integration in the fission yeast Schizosaccharomyces pombe.

Strains of the fission yeast Schizosaccharomyces pombe have been constructed containing single or multiple chromosomally integrated copies of an expression cassette for production of human gastric lipase. Integrant strains of S. pombe secrete active lipase and are stable for lipase production over a minimum of 50 generations in non-selective media. Lipase activity levels for integrant strains containing up to three tandem copies of the expression cassette are strongly correlated with copy number of the cassette in both complete and minimal media. Lipase activity is higher in complete medium than in minimal medium. Strains carrying three chromosomally integrated expression cassette copies can be grown without selection in complete medium and are capable of significantly higher lipase activities than strains containing the expression cassette on a multicopy plasmid.

Production of human gastric lipase in the fission yeast Schizosaccharomyces pombe.

A cDNA encoding human gastric lipase (hGL) has been expressed on multicopy plasmids in the fission yeast Schizosaccharomyces pombe (Sp). Active lipase is secreted from transformants containing the hGL cDNA under the control of either the Sp adh1 promoter (Padh1) or the plant cauliflower mosaic virus (CaMV) 35S promoter. Cell-wall-associated lipase activities are greatest in the early logarithmic growth phase and with Padh1. Western blot analysis indicates that a protein of identical molecular mass to natural hGL is secreted by Sp, although the major secreted product is of a higher molecular mass than either native hGL or recombinant hGL produced in the budding yeast Saccharomyces cerevisiae (Sc). Several distinct hGL are present within cells at all growth phases. Treatment of these proteins with endoglycosidase H gives rise to a single species equivalent in size to deglycosylated natural hGL, indicating that most of these are glycosylation intermediates. An hGL of similar molecular mass accumulates intracellularly in Sp when a modified version of cDNA is used which lacks the sequence encoding the natural secretory signal peptide. Production of hGL markedly slows the growth rate of Sp. The average copy number per cell of the plasmid expressing the hGL cDNA from the recombinant Padh1 is 2-3, as compared with 11-12 for the control plasmid.