Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro

ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.     

Vanadium and diabetes

We demonstrated in 1985 that vanadium administered in the drinking water to streptozotocin (STZ) diabetic rats restored elevated blood glucose to normal. Subsequent studies have shown that vanadyl sulfate can lower elevated blood glucose, cholesterol and triglycerides in a variety of diabetic models including the STZ diabetic rat, the Zucker fatty rat and the Zucker diabetic fatty rat. Long-term studies of up to one year did not show toxicity in control or STZ rats administered vanadyl sulfate in doses that lowered elevated blood glucose. In the BB diabetic rat, a model of insulin-dependent diabetes, vanadyl sulfate lowered the insulin requirement by up to 75%. Vanadyl sulfate is effective orally when administered by either single dose or chronic doses. It is also effective by the intraperitoneal route. We have also been able to demonstrate marked long-terrn effects of vanadyl sulfate in diabetic animals following treatment and withdrawal of vanadyl sulfate. Because vanadyl sulfate is not well absorbed we have synthesized and tested a number of organic vanaditun compounds. One of these, bismaltolato-oxovanadiurn IV (BMOV), has shown promise as a therapeutic agent. BMOV is 2-3x more potent than vanadyl sulfate and has shown less toxicity. Recent studies from our laboratory have shown that the effects of vanadium are not due to a decrease in food intake and that while vanadium is deposited in bone it does not appear to affect bone strength or architecture. The mechanism of action of vanadium is currently under investigation. Several studies indicate that vanadiun is a phosphatase inhibitor and that vanadium can activate serine/threonine kineses distal to tbe insulin receptor presumably by preventing dephosphorylation due to inhibition of phosphatases Short-term clinical trials using inorganic vanadium compounds in diabetic patients have been promising.     

Hydrogen sulfide stimulates catecholamine secretion in rainbow trout (Oncorhynchus mykiss)

We tested the hypothesis that endogenously produced hydrogen sulfide (H(2)S) can potentially contribute to the adrenergic stress response in rainbow trout by initiating catecholamine secretion from chromaffin cells. During acute hypoxia (water Po(2) = 35 mmHg), plasma H(2)S levels were significantly elevated concurrently with a rise in circulating catecholamine concentrations. Tissues enriched with chromaffin cells (posterior cardinal vein and anterior kidney) produced H(2)S in vitro when incubated with l-cysteine. In both tissues, the production of H(2)S was eliminated by adding the cystathionine beta-synthase inhibitor, aminooxyacetate. Cystathionine beta-synthase and cystathionine gamma-lyase were cloned and sequenced and the results of real-time PCR demonstrated that with the exception of white muscle, mRNA for both enzymes was broadly distributed within the tissues that were examined. Electrical field stimulation of an in situ saline-perfused posterior cardinal vein preparation caused the appearance of H(2)S and catecholamines in the outflowing perfusate. Perfusion with the cholinergic receptor agonist carbachol (1 x 10(-6) M) or depolarizing levels of KCl (1 x 10(-2) M) caused secretion of catecholamines without altering H(2)S output, suggesting that neuronal excitation is required for H(2)S release. Addition of H(2)S (at concentrations exceeding 5 x 10(-7) M) to the perfusion fluid resulted in a marked stimulation of catecholamine secretion that was not observed when Ca(2+)-free perfusate was used. These data, together with the finding that H(2)S-induced catecholamine secretion was unaltered by the nicotinic receptor blocker hexamethonium, suggest that H(2)S is able to directly elicit catecholamine secretion via membrane depolarization followed by Ca(2+)-mediated exocytosis.     

Serratia marcescens as a rapid indicator of Microctonus hyperodae oviposition activity in Listronotus maculicollis and potential application of the technique to host-specificity testing

Listronotus maculicollis (Dietz) (Coleoptera: Curculionidae) is a potential novel host of the braconid parasitoid Microctonus hyperodae Loan, but initial studies have shown that levels of parasitism are lower than in the natural host L. bonariensis (Kuschel). A novel bacterial indicator test was used to determine whether the lower level of parasitism was due to behavioural factors, lack of oviposition, or host resistance. The incidence of ovipositor penetration by the parasitoid M. hyperodae into adult L. maculicollis was measured by immersing the ovipositor of the parasitoid in the facultative pathogen, Serratia marcescens Bizio. Adult weevils were then exposed to parasitoids for up to 72 h and rapid mortality used as an indicator of oviposition penetration. Survival was assessed after six days and surviving weevils were dissected and examined for parasitoid larvae. Mortality among L. maculicolis exposed to parasitoids treated with S. marcescens was significantly higher (P<0.001) than the controls but significantly lower (P<0.001) than in the natural host, L. bonariensis. Dissection of weevils exposed to uncontaminated parasitoids revealed that parasitism in L. maculicolis was significantly (P<0.001) less than parasitism in L. bonariensis. Serratia marcescens-induced mortality plus parasitism of surviving weevils in the parasitoid plus bacteria treatments produced a similar overall effect. Application of bacteria to the parasitoid ovipositor provided a rapid, simple test for ovipositor penetration, which shows potential for separation of behavioural and physiological defence mechanisms in parasitoid/host range studies.     

15N estimates of nitrogen fixation by white clover (Trifolium repens L.) growing in a mixture with ryegrass (Lolium perenne L.)

The ¹⁵N isotope dilution technique and the N difference method were used to estimate N₂ fixation by clover growing in a mixture with ryegrass, in a field experiment and a controlled environment experiment. Values obtained using N difference were approximately 25% lower than those estimated using ¹⁵N isotope dilution. In the field experiment there was a measured N benefit to grass growing with clover, equivalent to 42.7 kgN ha^-1. The grass in the mixture had a lower atom %¹⁵N content and a higher N content than grass in a monoculture; therefore values for N₂ fixation were different depending on choice of control plant i.e. monoculture or mixture grass. In the controlled environment experiment there were no significant differences between either the atom %¹⁵N contents or the N contents of monoculture grass and grass growing in a mixture with clover. It is concluded that there is a long term indirect transfer of N from clover to associated grass which can lead to errors in estimates of N₂ fixation.