A new method for fish leucocyte counting and partial differentiation by flow cytometry

A simple and rapid method for analysis of fish blood cells is presented. Carp (Cyprinus carpio) blood was diluted 200 times with Hanks' solution containing 1 microg/ml of DiOC6(3) which is a fluorescent, lipophilic dye. After staining for 10 min, the blood cells were measured by a flow cytometer (FACS). Several blood cell populations were identified by different FL-1 (green fluorescence), FSC (forward scatter), and SSC (side scatter) properties. FL-1 v. SSC or FSC v. SSC dot-plot of stained blood cells displayed five separate cell populations: erythrocytes: a mixture of thrombocytes plus lymphocytes; monocytes; neutrophils; and basophils. The number of each type of blood cell counted by the FACS was in good agreement with those counted microscopically.     

The structure of jack bean urease. The complete amino acid sequence, limited proteolysis and reactive cysteine residues

The amino acid sequence of jack bean urease has been determined. The protein consists of a single kind of polypeptide chain containing 840 amino acid residues. The subunit relative molecular mass calculated from the sequence is 90,770, indicating that urease is composed of six subunits. Out of 25 histidine residues in urease, 13 were crowded in the region between residues 479 and 607, suggesting that this region may contain the nickel-binding site. Limited tryptic digestion cleaved urease at two sites, Lys-128 and Lys-662. Proteolytic products were not dissociated and retained full enzymatic activity. Five tryptic peptides containing the reactive cysteine residues were isolated and characterized with the aid of sulfhydryl-specific reagents, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine and N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide. The reactive cysteine residues were located at positions 59, 207, 592, 663, and 824. The possibility that Cys-59, Cys-207, Cys-663, and Cys-824 are involved in the urease activity of the enzyme has been eliminated. Cys-592, which is essential for enzymatic activity, is located in the above-mentioned histidine-rich region.     

The lactosylceramide binding specificity of Helicobacter pylori

The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay. Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere. By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding. A selective binding of H. pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay. Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers. An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin. By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases. Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.      

cDNAs and deduced amino acid sequences of subunits in the binding component of mouse bactericidal factor, Ra-reactive factor: similarity to mannose-binding proteins.

The complement-dependent bactericidal factor, Ra-reactive factor, binds specifically to Ra polysaccharide, which is common to some strains of Gram-negative enterobacteria, and its is a complex of proteins composed of a polysaccharide-binding component and a component that is presumably responsible for the complement activation. The former component consists of two different 28-kDa polypeptides, P28a and P28b. We determined the partial amino acid sequences of P28a and P28b, and the results indicated that these polypeptides were similar to two species of mannose-binding protein, MBP-C and MBP-A (alternative names, liver and serum mannan-binding proteins, respectively), which have been isolated from rat liver and/or serum [Drickamer, K., Dordal, M. S., & Reynolds, L. (1986) J. Biol. Chem. 261, 6878-6887; Oka, S., Itoh, N., Kawasaki, T., & Yamashina, I. (1987) J. Biochem. 101, 135-144]. Thus, we cloned the respective cDNAs, using as probes synthetic oligonucleotides for which the sequences had been deduced from the amino acid sequences of P28a and P28b and of rat MBP cDNAs. The primary structures of P28a and P28b deduced from the cloned cDNAs are homologous to one another. They have three domains, a short NH2-terminal domain, a collagen-like domain, and a domain homologous to regions of some carbohydrate-binding proteins, as has been reported for rat MBPs. Southern and Northern blotting analyses using these cDNAs indicated that the P28a and P28b polypeptides are the products of two unique mouse genes which are expressed in hepatic cells.     

Molecular evolution of a Y-chromosomal repetitive sequence family in the genus Mus

A 522-base-long Y-chromosomal sequence was isolated from a BALB/c genomic library and was designated "BF046." It is repeated about 200 times in the male genome, and a difference was detected between the Mus musculus musculus and the M. m. domesticus type Y chromosomes. BF046- related sequences were present over the entire length of the Y chromosome as visualized by in situ hybridization. Southern blot analysis against DNAs isolated from eight species in the genus Mus showed that BF046-related sequences were amplified in the Y chromosomes of three closely related species: M. musculus, M. spicilegus, and M. spretus. To gain insight into the stability of the BF046 sequence family, we isolated 18 additional clones from these three mouse species and compared their sequences. The M. musculus sequences differed from the M. spicilegus and M. spretus sequences by two indels. The remaining parts of the sequences were very similar, but both parsimony and distance-based analytical methods divided the sequences into the same four subgroups, with each species having its own subgroup(s). Thus, the Y chromosomes of M. musculus, M. spicilegus, and M. spretus can be distinguished from one another.      

Hepatitis C virus core protein binds to a DEAD box RNA helicase.

Approximately 4 million Americans are infected with the hepatitis C virus (HCV), making it a major cause of chronic liver disease. Because of the lack of an efficient cell culture system, little is known about the interaction between HCV and host cells. We performed a yeast two-hybrid screen of a human liver cell cDNA library with HCV core protein as bait and isolated the DEAD box protein DBX. DBX has significant amino acid sequence identity to mouse PL10, an ATP-dependent RNA helicase. The binding of DBX to HCV core protein occurred in an in vitro binding assay in the presence of 1 M NaCl or detergent. When expressed in mammalian cells, HCV core protein and DBX were co-localized at the endoplasmic reticulum. In a mutant strain of Saccharomyces cerevisiae, DBX complemented the function of Ded1p, an essential DEAD box RNA helicase. HCV core protein inhibited the growth of DBX-complemented mutant yeast but not Ded1p-expressing yeast. HCV core protein also inhibited the in vitro translation of capped but not uncapped RNA. These findings demonstrate an interaction between HCV core protein and a host cell protein involved in RNA translation and suggest a mechanism by which HCV may inhibit host cell mRNA translation.     

Geographical and temporal variation in the diet of Bank Cormorants Phalacrocorax neglectus in South Africa

The Bank Cormorant Phalacrocorax neglectus is endemic to the Benguela upwelling ecosystem off southwest Africa and is classified as Endangered owing to a recent large reduction in its number. It is thought that food scarcity, including a decreased abundance of West Coast rock lobster Jasus lalandii, has been a major driver of the decrease, yet its diet in South Africa is poorly known. We collected 941 pellets regurgitated by Bank Cormorants, at 18 South African breeding colonies during 1975–1985, and 1 523 pellets at 17 colonies during 1995–2002. The species composition of the diet (% numbers) was significantly different between the two periods, with widespread decreases in proportions of rock lobster in the west and of octopus and cuttlefish Sepia spp. at most localities. These taxa were replaced in the diet by fish, including Gobiidae and Clinidae. The pelagic goby Sufflogobius bibarbatus, an important prey of Bank Cormorants in Namibia, was absent from pellets collected in 1975–1985 but common at northern localities from 1995–2002. Composition of the diet by frequency of occurrence was only determined for 1995–2002, when rock lobster was present in 67% of all samples collected, cuttlefish in 39%, and Clinidae in 32%. Data for 1975–1985 and 1995–2002 showed that carapace lengths of rock lobsters eaten by Bank Cormorants averaged 56 mm (range 22–82 mm) and 50 mm (range 22–75 mm), respectively, which compares to the minimum legal size of 75 mm for fisheries in South Africa. This energy- rich prey item was an important constituent of the diet in the winter breeding period.