Induction of apoptosis in sea bream fibroblasts by Vibrio harveyi haemolysin and evidence for an anti-apoptotic role of heat shock protein 70.

In this study, we exposed black sea bream, Mylio macrocephalus (Basilewsky), fibroblast (BSF) and silver sea bream, Sparus sarba Forsskål, fibroblast (SSF) cell lines to a recombinant Vibrio harveyi haemolysin (VHH) and investigated mechanisms involved in apoptosis. A decrease in mitochondrial membrane potential, followed by an increase in caspase 3 activity, occurred within 2-8 h of VHH exposure, in both cell lines; however, VHH did not alter cellular levels of reactive oxygen species. As heat shock protein 70 (HSP70) is known to prevent the onset of apoptosis in certain mammalian cells, we aimed to test whether such a protective effect is operative in VHH-exposed fibroblasts. The amounts of HSP70 were elevated in SSF and BSF via an acute heat shock or an acute heat shock followed by a 6 h recovery. It was found that the VHH-mediated reduction in mitochondrial membrane potential was suppressed in cells that had a 6 h post-heat shock recovery, and the protective effect of heat shock-induced HSP70 was attenuated following treatment of cells with the HSP70 inhibitor, quercetin. This study demonstrates how haemolysin causes cell death via induction of apoptosis and provides evidence as to the role of HSP70 as an anti-apoptotic factor.

The purification and characterization of a Trichoderma harzianum exochitinase.

A chitinolytic enzyme was purified from the culture filtrate of T. harzianum (T198) by precipitation with ammonium sulphate followed by affinity binding to swollen chitin and release with 10% (v/v) acetic acid. The molecular weight of the enzyme was calculated to be 28 and 27.5 kD by gel filtration chromatography and SDS-PAGE, respectively. The isoelectric point of the enzyme was 7.4. The pH optimum for activity was 3.5 and maximum activity was obtained at 50 degrees C. The enzyme displayed activity on a wide array of chitin substrates of more than two N-acetylglucosamine units in length. HPLC analysis of hydrolysis products demonstrated that the enzyme was an exochitinase releasing N-acetylglucosamine only.

Cloning and characterization of the gene encoding beta subunit of mitochondrial processing peptidase from the basidiomycete Lentinula edodes.

We have isolated the gene encoding the beta subunit of mitochondrial processing peptidase (beta-MPP) from the shiitake mushroom Lentinula edodes. It is a nuclear gene with two small introns. Comparison with known beta-MPP genes revealed that the L. edodes gene is most closely related with that from Neurospora crassa, with 60.8% identities and 87% similarity in the amino-acid sequences. The deduced L. edodes beta-MPP peptide sequence contains the inverse zinc-binding motif (H-X-E-H) that has been found in a large family of zinc-binding metalloproteinases including bacterial proteinases, insulin degrading enzymes and beta-MPPs. The two histidines are thought to contribute two of the three residues for zinc binding. The expression of L. edodes beta-MPP is higher during the development of the fruiting bodies, suggesting that higher mitochondrial activities may be required to meet the energy demand in the rapid growth of the fruiting bodies.

Effects of GH, prolactin and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba.

Heat shock protein 70 (HSP70) expression was assessed in hepatic tissue of a marine teleost Sparus sarba after exogenous hormone administration. Using a PCR-amplified, homologous HSP70 cDNA clone, as a probe in Northern analysis, we detected a 2.3 kb transcript which was elevated after exposure to a temperature 7 degrees C above the ambient. For our studies on hormonal effects on HSP70 expression, groups of fish were administered recombinant bream GH (rbGH), ovine prolactin (oPRL) or cortisol daily over a 7-day period. Quantification of hepatic HSP70 transcript revealed that the administration of GH and PRL significantly reduced HSP70 mRNA abundance by 42% and 54% from saline-injected fish respectively. Also hepatic HSP70 levels were reduced by 76% and 64% as determined by immunoblotting after rbGH and oPRL treatment respectively. The administration of exogenous cortisol did not alter hepatic HSP70 mRNA or protein levels in S. sarba. The results obtained in this study are the first evidence for hormonal modulation of heat shock protein expression in fish. The significance of these results is discussed within the context of current knowledge on the roles of these hormones in teleostean stress response.

Hypercortisolemia does not affect the branchial osmoregulatory responses of the marine teleost Sparus sarba.

The effect of cortisol treatment on branchial Na(+)-K(+)-ATPase subunit mRNA abundance, enzyme activity, chloride cell number/morphometrics and serum electrolyte levels were investigated for the marine teleost Sparus sarba. Groups of fish received intraperitoneal injections of cortisol at a concentration of 4 micrograms/g body weight, daily, over a seven-day period. This dose of cortisol was sufficiently high enough to maintain a condition of hypercortisolemia as serum cortisol levels in treated fish were eleven fold higher than controls at time of sacrifice. By using branchial Na(+)-K(+)-ATPase alpha- and beta-subunit cDNA clones we were able to demonstrate that cortisol administration to S. sarba caused a significant elevation in the abundance of alpha-mRNA whereas the levels of beta-mRNA were unchanged. In addition Na(+)-K(+)-ATPase activity remained unaltered by cortisol administration. Branchial chloride cell number, exposure, apical area as well as serum Na+ and Cl- levels remained unchanged after cortisol administration. The results of this study suggest that elevated cortisol level may not necessarily translate into modulated branchial Na(+)-K(+)-ATPase activity and chloride cell function in hypo-osmoregulating marine fish.

Hormonal modulation of branchial Na+-K+-ATPase subunit mRNA in a marine teleost Sparus sarba.

The effect of hormone treatment on the abundance of Na+-K+-ATPase alpha- and beta-subunit mRNA in Sparus sarba branchial tissue was investigated. Groups of seawater (33/1000) and hypo-osmotic (6/1000) acclimated fish were injected daily, with either saline, cortisol, recombinant bream growth hormone (rbGH) or ovine prolactin (oPRL). Total RNA from branchial tissue was analyzed by Northern blotting using PCR amplified Na+-K+-ATPase alpha- and beta-subunit cDNA clones. Na+-K+-ATPase alpha- and beta- subunit transcripts of 3.3kb and 2.4kb respectively, were detected and their abundance, after hormone treatment was assessed using RNA dot blots. The abundance of subunit mRNAs increased 1.4-1.9 fold, relative to controls, after cortisol treatment. The alpha:beta mRNA ratio also increased in cortisol treated seawater acclimated fish. Growth hormone treatment did not cause any significant changes in Na+-K+-ATPase subunit mRNA, whereas prolactin significantly reduced alpha-subunit mRNA levels by approximately 0.5 fold in both seawater and hypo-osmotic conditions. The data from this study add further support to the generally accepted roles that cortisol and prolactin have in the modulation of Na+-K+-ATPase activity. It can be concluded from this study that S. sarba branchial Na+-K+-ATPase subunit expression is multihormonally regulated.

Larval development of silver sea bream (Sparus sarba): ontogeny of RNA-DNA ratio, GH, IGF-I, and Na(+)-K(+)-ATPase.

To ascertain some of the important biochemical and molecular events that take place during early larval development of silver sea bream (Sparus sarba), we undertook a study of changes in the morphology as well as the ontogeny of the RNA-DNA ratio, growth hormone (GH), insulin-like growth factor I (IGF-I) messenger RNA abundance, Na(+)-K(+)-ATPase subunit mRNA abundance, and Na(+)-K(+)-ATPase enzyme activity. Larvae samples were collected at 1 to 46 days posthatch (dph). At 7 dph the yolk sac was fully absorbed, and from 28 dph onward larvae underwent rapid developmental changes to the juvenile stage. The RNA-DNA ratio was highest at 1 dph, decreased to low levels between 7 and 21 dph, then increased by 28 dph, and then again by 46 dph. The ontogenetic profiles of GH, IGF-I, and Na(+)-K(+)-ATPase alpha1 and beta1 subunits were studied using reverse transcriptase polymerase chain reaction, coupled with radioisotope hybridization of immobilized DNA. Growth hormone abundance reached a constant and high level from 35 dph onward, whereas the IGF-I level reached a peak at 35 dph and then significantly decreased. Both Na(+)-K(+)-ATPase alpha1 and beta1 subunit mRNAs increased up to 35 dph, however, at 46 dph the alpha1 subunit remained high whereas the beta1 subunit decreased. Na(+)-K(+)-ATPase activity was low in 1-dph larvae but increased rapidly as development progressed. The importance of these findings is discussed within the context of larval development.

Hormonal status and phagocytic activity in sea bream infected with vibriosis.

Serum taken from female, sexually mature, silver sea bream (Sparus sarba) displaying either no symptoms of vibriosis, mild infection, severe infection or moribundity were assayed for a number of key hormones. Serum cortisol levels were not significantly different among symptomless, mildly- and severely-infected groups, whereas moribund fish displayed hypercortisolemia with a 14-fold increase in serum cortisol in comparison to symptomless fish. Serum estradiol levels were significantly reduced 19-fold in mildly-infected fish and remained at a low level as infection progressed, whereas serum testosterone increased gradually during vibriosis with a 1.8-fold increase in moribund groups in comparison to symptomless groups. Both serum thyroxine (T(4)) and triiodothyronine (T(3)) gradually decreased during vibriosis being 26- and 2.8-fold lower, respectively, in moribund fish in comparison to symptomless fish. The non-specific immune response, as determined by phagocytic activity, was also assessed using macrophages isolated from the pronephros and spleen of infected fish. Phagocytic indices significantly increased in mildly- and severely-infected fish and then decreased from these stimulated levels in moribund fish.

Modulation of beta-actin, insulin-like growth factor 1 and glucose-6-phosphate dehydrogenase gene expression during vibriosis of sea bream, Sparus (=Rhabdosargus) sarba Forsskål.

Gene expression changes, related to growth regulation, were investigated in silver sea bream, Sparus (=Rhabdosargus) sarba, subjected to vibriosis. Using reverse transcriptase polymerase chain reaction assays, transcript levels of beta-actin, insulin-like growth factor 1 (IGF-1) and glucose-6-phosphate dehydrogenase (G6PDH) were analysed. A tissue-specific response for beta-actin was found, as transcript levels in the kidney increased during early- and mid-stage vibriosis, but remained unchanged in liver tissue. The gene transcript levels of IGF-1 were found to decrease in both liver and kidney from an early stage of vibriosis. The gene transcript levels of G6PDH increased at early and mid stages of vibriosis in liver and kidney before decreasing during the late stage of vibriosis. Further studies on G6PDH demonstrated that chemical inhibition of G6PDH in silver sea bream fibroblasts resulted in increased cell death upon exposure to hydrogen peroxide. The results from the present study demonstrate the complexity of growth-related gene expression events that occur in fish tissues during disease.

Evidence for disruption of Na(+)-K(+)-ATPase and hsp70 during vibriosis of sea bream, Sparus (=Rhabdosargus) sarba Forsskål.

Osmoregulation via the sodium pump (Na(+)-K(+)-ATPase) and cytoprotection via expression of different families of heat shock protein (hsp) were studied at transcriptional/translational levels during progressive vibriosis in silver sea bream, Sparus (=Rhabdosargus) sarba Measurements of Na(+)-K(+)-ATPase activity showed that an early and drastic decline occurred in the kidney of infected fish. This reduction in Na(+)-K(+)-ATPase activity was not caused by transcriptional downregulation of genes coding for either the Na(+)-K(+)-ATPase alpha or beta subunits. Using specific antibodies, data from immunoassays showed that the decreased sodium pump activity was caused by the specific loss of the translated glycosylated Na(+)-K(+)-ATPase beta subunit. Data from immunoassays of different hsp families demonstrated that the expression of hsp90 and hsp60 remained unchanged throughout vibriosis whereas expression of the hsp70 family decreased in kidney and liver tissues. As hsp70 is a multigene family, the expression of the constitutive (hsc70) and inducible (hsp70) members of the hsp70 family were studied and it was found that hsp70 and hsc70 expression decreased from an early stage of infection in the kidney and the liver respectively. Reverse transcriptase-polymerase chain reaction analysis of the hsp70 transcription-inducing factor, hsf1, demonstrated that loss of cytoprotective function during vibriosis was mediated by a downregulation of hsf1 transcription.