Fish larvae distribution among different habitats in coastal East Africa

Fish larvae abundances, diversity and trophic position across shallow seagrass, coral reef and open water habitats were examined to characterize their distribution in coastal East Africa. Larvae were identified to family and analysed for abundance differences between sites and habitats, trophic level using stable-isotope analysis and parental spawning mode. Abundances differed greatly between sites with the highest numbers of larvae occurring in the open-water and seagrass habitats. Larval fish diversity was high across habitats with 51 families identified with small differences between sites and among habitats. Notably, larvae of abundant large herbivorous fishes present in reef and seagrass habitats were almost completely absent at all sampling locations. In the seagrass, demersal spawned larvae were more abundant compared with the reef and open-water habitats. Stable-isotope analysis revealed that fish larvae have a varied diet, occupying trophic level two to three and utilizing planktonic prey. This study offers new insights into distributional aspects of fish larvae along the East African coast where such information is sparse.     

Tat1, a novel sulfate transporter specifically expressed in human male germ cells and potentially linked to rhogtpase signaling

RhoGTPases (Rho, Rac, and Cdc42) are known to regulate multiple functions, including cell motility, adhesion, and proliferation; however, the signaling pathways underlying these pleiotropic effects are far from fully understood. We have recently described a new RhoGAP (GTPase activating protein for RhoGTPases) gene, MgcRacGAP, primarily expressed in male germ cells, at the spermatocyte stage. We report here the isolation, through two-hybrid cloning, of a new partner of MgcRacGAP, very specifically expressed in the male germ line and showing structural features of anion transporters. This large protein (970 amino acids and a predicted size of 109 kDa), we provisionally designated Tat1 (for testis anion transporter 1), is closely related to a sulfate permease family comprising three proteins in human (DRA, Pendrin, and DTD); it is predicted to be an integral membrane protein with 14 transmembrane helices and intracytoplasmic NH(2) and COOH termini. In situ hybridization studies demonstrate that Tat1 and MgcRacGAP genes are coexpressed in male germ cells at the spermatocyte stage. On testis sections, Tat1 protein can be immunodetected in spermatocytes and spermatids associated with plasma membrane. Two-hybrid and in vitro binding assays demonstrate that MgcRacGAP stably interacts through its NH(2)-terminal domain with the Tat1 COOH-terminal region. Expression of Tat1 protein in COS7 cells generates a 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene and chloride-sensitive sulfate transport. Therefore we conclude that Tat1 is a novel sulfate transporter specifically expressed in spermatocytes and spermatids and interacts with MgcRacGAP in these cells. These observations raise the possibility of a new regulatory pathway linking sulfate transport to Rho signaling in male germ cells.     

Solid-supported enzymatic synthesis of pectic oligogalacturonides and their analysis by MALDI-TOF mass spectrometry

Solid-phase biosynthetic reactions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF), was used to gain insight into the biosynthesis of pectin oligomers. Sepharose supports bearing long pectic oligogalacturonides (OGAs) anchored through a disulfide-containing cleavable linker, were prepared. The OGAs (degrees of polymerization of 13 and 14) were efficiently immobilized through the reducing end via formation of an oxime linkage. These OGA-derivatized matrices were subsequently employed in novel solid-phase enzymatic reactions, with the pectin biosynthetic enzyme, alpha-1,4-galacturonosyltransferase, GalAT (solubilized from Arabidopsis thaliana) and the glycosyl donor, uridine diphosphate-galacturonic acid (UDP-GalA). Solid-supported biosynthesis was followed by cleavage of the immobilized OGAs and direct analysis of the products released into the liquid phases by MALDI-TOF mass spectrometry. In time course studies conducted with an immobilized (alpha-D-GalA)14 and limiting amounts of the glycosyl donor, the predominant product was an OGA extended by one GalA residue at the non-reducing end (i.e., (GalA)15). When UDP-GalA was added in approximately excess compared to immobilized (GalA)13, OGAs up to the 16-mer were synthesized, confirming the non-processivity of the GalAT in vitro.     

Platelet activating factor-induced apoptosis is inhibited by ectopic expression of the platelet activating factor G-protein coupled receptor

The pro-inflammatory lipid mediator platelet activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) accumulates in ischemia, epilepsy, and human immunodeficiency virus-1-associated dementia and is implicated in neuronal loss. The present study was undertaken to establish a role for its G-protein coupled receptor in regulating neurotoxicity. PC12 cells do not express PAF receptor mRNA as demonstrated by northern analysis and RT-PCR. In the absence of the G-protein coupled receptor, PAF (0.1-1 micro m) triggered chromatin condensation, DNA strand breaks, oligonucleosomal fragmentation, and nuclear disintegration characteristic of apoptosis. Lyso-PAF (0.001-1 micro m), the immediate metabolite of PAF, did not elicit apoptotic death. Concentrations of PAF or lyso-PAF that exceeded critical micelle concentration had physicochemical effects on plasma membrane resulting in necrosis. Apoptosis but not necrosis was inhibited by the PAF antagonist BN52021 (1-100 micro m) but not CV3988 (0.2-20 micro m). Ectopic PAF receptor expression protected PC12 transfectants from ligand-induced apoptosis. PAF receptor-mediated protection was inhibited by CV3988 (1 micro m). These data provide empirical evidence that: (i) PAF can initiate apoptosis independently of its G-protein coupled receptor; (ii) PAF signaling initiated by its G-protein coupled receptor is cytoprotective to PC12 cells; (iii) the pro- and anti-apoptotic effects of PAF on PC12 cells can be pharmacologically distinguished using two different PAF antagonists.     

The extreme C terminus of rat liver carnitine palmitoyltransferase I is not involved in malonyl-CoA sensitivity but in initial protein folding

We previously reported that the N-terminal domain (1-147 residues) of rat liver carnitine palmitoyltransferase I (L-CPTI) was essential for import into the outer mitochondrial membrane and for maintenance of a malonyl-CoA-sensitive conformation. Malonyl-CoA binding experiments using mitochondria of Saccharomyces cerevisiae strains expressing wild-type L-CPTI or previously constructed chimeric CPTs (Cohen, I., Kohl, C., McGarry, J.D., Girard, J., and Prip-Buus, C. (1998) J. Biol. Chem. 273, 29896-29904) indicated that the N-terminal domain was unable, independently of the C-terminal domain, to bind malonyl-CoA with a high affinity, suggesting that the modulation of malonyl-CoA sensitivity occurred through N/C intramolecular interactions. To assess the role of the C terminus in malonyl-CoA sensitivity, a series of C-terminal deletion mutants was generated. The kinetic properties of Delta772-773 and Delta767-773 deletion mutants were similar to those of L-CPTI, indicating that the last two highly conserved Lys residues in all known L-CPTI species were not functionally essential. By contrast, Delta743-773 deletion mutant was totally inactive and unfolded, as shown by its sensitivity to trypsin proteolysis. Because the C terminus of the native folded L-CPTI could be cleaved by trypsin without inducing protein unfolding, we concluded that the last 31 C-terminal residues constitute a secondary structural determinant essential for the initial protein folding of L-CPTI.     

Aspartic proteinase in Dugesia tigrina (Girard) planaria

A proteolytic activity was identified in Dugesia tigrina planaria using the chromogenic substrate Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu-O4MP. The activity of the enzyme increased four times during the regeneration and presented a maximum at 120 hr being higher in tail than head regenerating segments. The protease that displays this activity was purified from worms by a single step on pepstatin-agarose followed by gel-filtration high performance liquid chromatography. The purification resulted in a 34-fold increase in specific activity and the final yield was 10%. The active D. tigrina hydrolase appears to be a dimeric protein composed of identical subunits with 34 kDa associated by disulphide bridges similar to vertebrate cathepsin D. By SDS-PAGE several bands were detected but upon gel filtration HPLC one proteolytically active component, termed Asp-68, was detected and isolated. The maximal activity was observed in a range between pH 3.5-5.0 and the enzyme became inactivated at a pH value above 7.2. The purified enzyme was not inhibited by inhibitors from serine (aprotinin, TPCK, PMSF and TLCK), metallo (EDTA) and cysteine proteinase (E-64) classes. In contrast, inhibitors such as pepstatin, EPNP, and 4-beta-PMA efficiently inhibited the activity of the 68-kDa protease.     

Absent effect of zinc deficiency on the oxidative stress of erythrocytes in chronic uremic rats

Both anemia and zinc deficiency are commonly observed in patients with chronic uremia. Oxidative stress of red blood cells (RBC) has been suggested to participate in the development of anemia in these patients with chronic uremia due to reduced life span of RBC. Whether zinc deficiency aggravates the effect of oxidative stress on RBC of chronic uremia is still not understood. We thus performed the study to determine the influence of zinc deficiency on the oxidative stress of RBC in uremic rats. Zinc deficiency was induced by long-term dietary zinc deficiency. Five-sixth nephrectomy (5/6 Nx) was used to produce chronic uremia. Experiment was carried out in the following five groups: normal control (NL), chronic uremia (Nx), chronic uremia + dietary zinc deficiency (Nx-D), Nx-D + zinc supplement (Nx-DZ) and Chronic uremia + pair-fed (Nx-PF). Osmotic fragility and lipid peroxidation of RBC were used to evaluate the oxidative stress of RBC. Five weeks after 5/6 nephrectomy (Nx), 5/6 Nx rats present a syndrome of uremia to elevate the levels of plasma creatinine and urea, and reduce the level of plasma zinc (1.12 +/- 0.08 vs 1.35 +/- 0.05 ug/ml). But they does not find to produce anemia and to increase osmotic fragility and lipid peroxidation in RBC. Dietary zinc deficiency in Nx-D group produced severe anorexia and reduced plasma zinc and selenium levels and the activity of RBC-GPX. Yet in Nx-D rats, osmotic fragility and susceptibility of lipid peroxidation in red cells did not increase, because of the increase of plasma copper level (1.85 +/- 0.3 vs 1.41 +/- 0.05 microg/ml) and RBC-SOD activity (1.95 +/- 0.27 vs 0.78 +/- 0.05 unit/g Hb). Zinc supplement in Nx-D rats (Nx-DZ group) recovered the appetite and normalized the levels of plasma zinc, copper and selenium. Food restriction in 5/6 Nx rats (Nx-PF group) decreased plasma copper level and increased osmotic fragility of RBC and elevated the susceptibility of lipid peroxidation after stressing RBC with H2O2 Because Nx-PF rats presented a lower RBC-SOD activity (0.44 +/- 0.11 vs 0.78 +/- 0.05 unit/g Hb) and a lower plasma copper level. We further found a positive relationship (r=0. 802,p<0.01) between plasma copper level and RBC-SOD activity in normal and uremic rats. This study suggests that RBC-SOD activity may play an important role in preventing RBC oxidative stress. Plasma copper level may be a marker of RBC-SOD activity. We conclude, in chronic uremia, zinc deficiency doses not result in RBC oxidative stress as plasma copper level is normal, but may affect the absorption of intestinal nutrition.