A. Tietz

Molecular characterization and high expression during oocyte development of a shrimp ovarian cortical rod protein homologous to insect intestinal peritrophins

Abstract Penaeoid shrimp oocytes nearing the completion of oogenesis are enveloped in an acellular vitelline envelope and possess extracellular cortical rods (CRs) that extended into the cortical cytoplasm. These cortical specializations are precursors of the jelly layer (JL) of the egg. In searching for highly expressed mRNAs during oogenesis in the marine shrimp (Penaeus semisulcatus), two related cDNAs have been isolated that encode a mature protein of 250 amino acid residues. The deduced amino acid sequences revealed the presence of repeated cysteine-rich domains that are related to the chitin-binding domains of insect intestinal peritrophins. Similar cysteine-rich domains were reported in insect intestinal mucin, crustacean tachycitin, and invertebrate chitinases. The shrimp ovarian peritrophin (SOP) is glycosylated and can bind chitin when extracted from CRs. Its apparent molecular mass in SDS-PAGE is 29–35 kDa and 33–36 kDa, under nonreducing or reducing conditions, respectively. SOP is a major protein of CRs and the JL, and was immunodetected in ovaries; purified CRs; fertilized eggs that were surrounded by a JL matrix; and in the cloudy, whitish flocculent material appearing in sea water immediately after spawning. Immunolocalization in tissue sections determined that SOP was present in oocyte cytoplasm and in extraoocytic CRs. Shrimp expressed SOP mRNA in ovaries at all oocyte developmental stages, whereas expression in the hepatopancreas was restricted to vitellogenic stages. SOP mRNA was abundant in the shrimp ovary and was detected before the presence of the corresponding protein. This is the first demonstration that a protein with similar features to insect intestinal peritrophins is a component of CRs and is therefore a main precursor of the JL of spawned shrimp eggs.

Isolation and characterization of the high-density lipoproteins from the hemolymph and ovary of the penaeid shrimp Penaeus semisulcatus (de Haan): apoproteins and lipids.

The high-density lipoproteins (HDLs) found in the male and female hemolymph of Penaeus semisulcatus de Haan were isolated by NaBr (1.22 g/ml) followed by sucrose gradient (5-25%) ultracentrifugation. The male HDL contained one protein, lipoprotein 1 (LP1), composed of one 110-kDa peptide subunit. The female HDL contained two proteins: 1) the LP1 that was immunoidentical to the male LP1 and was similarly composed of one 110-kDa peptide subunit and 2) vitellogenin (Vg), reacting positively with the rabbit antiserum generated against vitellin (Vt) that was isolated from vitellogenic ovaries. Both Vg and Vt consisted mainly of three polypeptide subunits (200, 120, and 80 kDa) as revealed by denatured PAGE and Western blot. The LP1 from males or females did not react with the Vt rabbit antiserum. Similarly, Vg and Vt did not react with the rabbit antiserum prepared against LP1. Phospholipids (PL) constituted 71-76% of the total lipids in the hemolymph and HDLs of both male and female hemolymph. Cholesterol (Ch) amounted to 17-20%, and small amounts (5%) of diacylglycerols (DAG) were also carried by these HDLs. Both the PL and DAG contained highly unsaturated fatty acids (20:5 omega 3 and 22:6 omega 3) that are transported from the food or hepatopancreas to the tissues, including the vitellogenic ovaries in females. In the present study we show for the first time the separate lipid composition of female LP1 and Vg and compare them with the lipids attached to the Vt. Vg had a lower lipid content than LP1 (540 and 1089 mg/g protein, respectively). Differences were also found in the relative abundance of PL, Ch, and DAG classes in the LP1 in comparison with Vg. Furthermore, small amounts (approximately 3.8%) of triacylglycerols (TAG) were found only in the hemolymph of vitellogenic females, and they were associated with the Vg. Although Vg and Vt were composed of similar polypeptides, their lipid composition was different Vt, in contrast to Vg, carried considerable amounts of TAG (approximately 22%) and only trace amounts of DAG. The significance of the TAG in the hemolymph of vitellogenic females is not known, and the functional relationship between Vg and Vt requires future extensive studies. Lipids were not detected in hemocyanin that was purified from clotted hemolymph.

Cyclodextrins but not Compactin Inhibit the Lateral Diffusion of Membrane Proteins Independent of Cholesterol

Cholesterol and glycosphingolipid‐enriched membrane domains, termed lipid rafts, were proposed to play important roles in trafficking and signaling events. These functions are inhibited following putative disruption of rafts by cholesterol depletion, commonly induced by treatment with methyl‐β‐cyclodextrin (MβCD). However, several studies showed that the lateral diffusion of membrane proteins is inhibited by MβCD, suggesting that it may have additional effects on membrane organization unrelated to cholesterol removal. Here, we investigated this possibility by comparison of the effects of cholesterol depletion by MβCD and by metabolic inhibition (compactin), and of treatment with α‐CD, which does not bind cholesterol. The studies employed two series of proteins (Ras and influenza hemagglutinin), each containing as internal controls related mutants that differ in raft association. Mild MβCD treatment retarded the lateral diffusion of both raft and non‐raft mutants, whereas similar cholesterol reduction (30–33%) by metabolic inhibition enhanced selectively the diffusion of the raft‐associated mutants. Moreover, α‐CD also inhibited the diffusion of raft and non‐raft mutants, despite its lack of effect on cholesterol content. These findings suggest that the widely used treatment with CD to reduce cholesterol has additional, cholesterol‐independent effects on membrane protein mobility, which do not necessarily distinguish between raft and non‐raft proteins.

Effects of salts and ionophores on proline transport in a moderately halophilic halotolerant bacterium

The effect of salt on proline uptake in a moderately halophilic halotolerant bacterium was studied. Cells were grown either on low salt or high salt media. A correlation was found between the salt concentrations in the growth media and the optimal concentration for uptake. The uptake rate was stimulated 2--3-fold by NaCl, as compared to KCl. The Km, V and activation energies values for proline uptake, as well as the external pH effect, were similar in low-salt-grown cells and high-salt-grown cells. This suggests that the halotolerance of the transport system is not due to alterations of the system during growth at various conditions, but rather to its intrinsic ability to function under extreme environmental conditions. The uptake was inhibited by cyanide and carbonyl cyanide m-chlorophenylhydrazone, but not by arsenate, indicating that the electrochemical proton gradient (delta mu- H+), generated by respiration, is the main driving force for proline transport. In low-salt-grown cells, at pH 6.0, partial inhibition was exerted by nigericin or valinomycin, whereas at pH 8.0 the uptake was inhibited by valinomycin only. Similar, although less pronounced effects were found in high-salt-grown cells. The data suggest that at pH 6.0 proline transport is driven by delta mu- H+ (composed of electrical potential (delta psi) and pH gradient), whereas at pH 8.0 delta psi is the main driving force. Procedures of pretreatment with EDTA were developed to enable the penetration of the ionophores into the cells.

Response of Penaeus indicus females at two different stages of ovarian development to a lethal infection with Vibrio penaeicida

An association between vitellogenesis and the immune system was suggested in crustaceans from studies on plasma lipoproteins. The present research studies the effect of an experimentally induced bacterial infection on vitellogenesis in females of the shrimp Penaeus indicus, as a model for penaeid species. Pre-vitellogenic and vitellogenic P. indicus females were experimentally infected with an extremely pathogenic bacterium, Vibrio penaeicida. The peak in mortality occurred earlier in pre-vitellogenic animals than in vitellogenic ones, although the final mortality level ( approximately 64-74%) 52h post-infection was nearly the same for the two groups. Twenty hours after infection, the total number of haemocytes was significantly reduced in vitellogenic females while there was no change in the pre-vitellogenic group. Protein synthesis in ovaries was not significantly affected by infection, at the two stages of ovarian development. No differences were found in mRNA levels of shrimp ovarian peritrophin protein (SOP), but preliminary results showed that mRNA expression of vitellin (VT) was reduced in a heavily infected vitellogenic female. The total amount of lipids in the haemolymph of vitellogenic females was almost twice higher than that of pre-vitellogenic ones. However, there was no change in the total content of lipids, lipid classes and fatty acid distribution in haemolymph or hepatopancreas following infection. Although vitellogenic and pre-vitellogenic females probably respond differently to a lethal bacterial infection, physiological differences may be concealed by the rapid onset of mortality.

Effects of chlorocyclizine on pulmonary lipid metabolism in rats

Pulmonary lipidosis was induced in rats by including 0.36 and 0.54% chlorocyclizine in their diet. Chemical analyses of the lung tissue revealed a very marked increase in phosphatidylcholine concentration. Phosphatidylglycerol and phosphatidylinositol concentrations were also markedly increased. An increase in the phosphatidylcholine content was also observed in lavage fluid and macrophages. Microscopic examination of the cell fraction showed that almost all the cells of the lavage fluid were macrophages and that histochemically demonstrable acid esterase activity was mostly inversely related to storage of lipids in the cells. Sonication of macrophages isolated from normal or chlorocyclizine-treated rats yielded a soluble acid phospholipase (pH optimum, 4.0) and a neutral (pH optimum, 8.2) membrane-bound, CaCl2-dependent enzyme. An inhibitory effect of chlorocyclizine in vitro on the activity of the soluble phospholipase was shown.

THE EFFECTS OF DIETARY PHOSPHOLIPIDS ENRICHED WITH PHOSPHATIDYLETHANOLAMINE ON BILE AND RED CELL MEMBRANE LIPIDS IN HUMANS

The role of phospholipids in biliary cholesterol solubilization and crystallization has only recently begun to be appreciated. Phospholipid vesicles are believed to be the metastable carrier from which cholesterol nucleates. Cholesterol crystallization is influenced by the phospholipid species in bile. Feeding rats and hamsters with diets enriched in phospholipids or their precursors, especially ethanolamine, resulted in reduced cholesterol saturation of bile. Although whole phospholipids are normal dietary constituents, the effects and safety of phospholipid components have not been tested in humans. In the present study, we have evaluated the effects of a dietary phospholipid mixture, enriched with phosphatidylethanolamine, on human bile and red blood cell membrane lipid composition. Five ambulatory volunteers having a chronic indwelling T-tube, with an intact enterohepatic circulation, were investigated. Thirty-six grams of phospholipids (54% phosphatidylethanolamine, 54% linoleyl acyl chains) were added to their daily diet for fourteen days. Biliary nucleation time, cholesterol carriers, as well as plasma, red blood cell membrane, and bile lipid compositions, were monitored. Following phospholipid supplementation, the proportion of linoleyl chains (18:2) in biliary phospholipids increased significantly from 31.1±1.2 to 37.7±5.3%, while that of oleyl chains (18:1) decreased from 11.4±1.6 to 9.6±1.1%. These changes were accompanied by an increase of linoleate and its metabolite, arachidonate, in red cell membranes. Phospholipid feeding did not cause any side effects, and no significant changes in biliary nucleation time, cholesterol, phospholipid, or bile salt concentrations, or in the distribution of cholesterol within micelles or vesicles. We conclude that phospholipid feeding is safe, and can be effective as a vehicle for lecithin fatty acyl chain modulation of bile and lipid membranes. These findings may provide a basis for a controlled modulation of biliary phospholipids to increase cholesterol solubility in bile.

Lipid composition during sexual development of the noble crayfish Asyacus astacus and effect of a fungal infection

Summary Previous published results (Hall et al., 1995) showed that two plasma proteins involved in the immune response in the crayfish (Pacifastacus leniusculus) are lipoproteins, suggesting an apparent functional association between lipid transport and the immune response system in crustaceans. The crayfish Astacus astacus is sensitive to a disease caused by the fungus Aphanomyces astaci, which is responsible for high mortalities in the natural environment. Since vitellogenesis is accompanied by a massive accumulation and transport of lipids to ovaries, the present study examines the effect of the crayfish plague fungus A. astaci on lipid transport during sexual development in the crayfish A. astacus. Comparison of the plasma lipid composition between animals sampled before and at the end of the ovarian development cycle revealed that mature females contained more lipids than males or immature females. The high-density lipo-protein (HDL) hemolymph fractions contained four to seven times more lipids than the very high-density lipoprotein (VHDL) fractions; triacylglycerols (TAG) were detected only in the HDL of females. There were no significant changes in hemolymph lipid levels in vitellogenic females subjected to an experimental fungal infection. However, in males exposed to the same experimental conditions, there was a significant decrease in phospholipids (PL) concomitant with an increase in diacylglycerols (DAG) and cholesterol (CH). No differences were observed in the lipid content of either the hepatopancreas (of males and females) or the ovaries due to infection, except a slight decrease in the ovarian PL level. Clotting protein was found relatively more abundant in plasma samples obtained from infected animals after subjecting them to polyacrylamide gel electrophoresis under native conditions. The results of the present study suggest that long-term exposure to a sub lethal dose of the fungus may allow in future studies, a better evaluation of the effect of an infection on lipid composition in shrimp plasma and tissue samples.