Biosynthesis of a novel 3-deoxy-D-manno-oct-2-ulosonic acid-containing outer core oligosaccharide in the lipopolysaccharide of Klebsiella pneumoniae

The core oligosaccharide region of Klebsiella pneumoniae lipopolysaccharide contains some novel features that distinguish it from the corresponding lipopolysaccharide region in other members of the Enterobacteriaceae family, such as Escherichia coli and Salmonella. The conserved Klebsiella outer core contains the unusual trisaccharide 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-(2,6)-GlcN-(1,4)-GalUA. In general, Kdo residues are normally found in the inner core, but in K. pneumoniae, this Kdo residue provides the ligation site for O polysaccharide. The outer core Kdo residue can also be non-stoichiometrically substituted with an l-glycero-d-manno-heptopyranose (Hep) residue, another component more frequently found in the inner core. To understand the genetics and biosynthesis of core oligosaccharide synthesis in Klebsiella, the gene products involved in the addition of the outer core GlcN (WabH), Kdo (WabI), and Hep (WabJ) residues as well as the inner core HepIII residue (WaaQ) were identified. Non-polar mutations were created in each of the genes, and the resulting mutant lipopolysaccharide was analyzed by mass spectrometry. The in vitro glycosyltransferase activity of WabI and WabH was verified. WabI transferred a Kdo residue from CMP-Kdo onto the acceptor lipopolysaccharide. The activated precursor required for GlcN addition has not been identified. However, lysates overexpressing WabH were able to transfer a GlcNAc residue from UDP-GlcNAc onto the acceptor GalUA residue in the outer core.     

Isolation of three zebrafish dachshund homologues and their expression in sensory organs, the central nervous system and pectoral fin buds

Drosophila dachshund (dac) interacts with sine oculis (so), eyes absent (eya) and eyeless (ey) to control compound eye development. We have cloned three zebrafish dac homologues, dachA, dachB and dachC, which are expressed widely, in distinct but overlapping patterns. Expression of all three is found in sensory organs, the central nervous system and pectoral fin buds. dachA is also expressed strongly in the somites and dachC in the neural crest and pronephros. These expression domains overlap extensively with those of zebrafish pax, eya and six family members, the homologues of Drosophila ey, eya and so, respectively. This is consistent with the proposal that Dach, Eya, Six and Pax family members may form networks, similar to that found in the fly eye, in the development of many vertebrate organs.     

Systematics of the grey mullets (Teleostei: Mugiliformes: Mugilidae): molecular phylogenetic evidence challenges two centuries of morphology-based taxonomy

The family Mugilidae comprises mainly coastal marine species that are widely distributed in all tropical, subtropical and temperate seas. Mugilid species are generally considered to be ecologically important and they are a major food resource for human populations in certain parts of the world. The taxonomy and systematics of the Mugilidae are still much debated and based primarily on morphological characters. In this study, we provide the first comprehensive molecular systematic account of the Mugilidae using phylogenetic analyses of nucleotide sequence variation at three mitochondrial loci (16S rRNA, cytochrome oxidase I, and cytochrome b) for 257 individuals from 55 currently recognized species. The study covers all 20 mugilid genera currently recognized as being valid. The family comprises seven major lineages that radiated early on from the ancestor to all current forms. All genera that were represented by two species or more, except Cestraeus, turned out to be paraphyletic or polyphyletic. Thus, the present phylogenetic results generally disagree with the current taxonomy at the genus level and imply that the anatomical characters used for the systematics of the Mugilidae may be poorly informative phylogenetically. The present results should provide a sound basis for a taxonomic revision of the mugilid genera. A proportion of the species with large distribution ranges (including Moolgarda seheli, Mugil cephalus and M. curema) appear to consist of cryptic species, thus warranting further taxonomic and genetic work at the infra-generic level.     

The C-terminal domain of the nucleotide-binding domain protein Wzt determines substrate specificity in the ATP-binding cassette transporter for the lipopolysaccharide O-antigens in Escherichia coli serotypes O8 and O9a

The polymannan O-antigenic polysaccharides (O-PSs) of Escherichia coli O8 and O9a are synthesized via an ATP-binding cassette (ABC) transporter-dependent pathway. The group 2 capsular polysaccharides of E. coli serve as prototypes for polysaccharide synthesis and export via this pathway. Here, we show that there are some fundamental differences between the ABC transporter-dependent pathway for O-PS biosynthesis and the capsular polysaccharide paradigm. In the capsule system, mutants lacking the ABC transporter are viable, and membranes isolated from these strains are no longer able to synthesize polymer using an endogenous acceptor. In contrast, E. coli strains carrying mutations in the membrane component (Wzm) and/or the nucleotide-binding component (Wzt) of the O8 and O9a polymannan transporters are nonviable under conditions permissive to O-PS biosynthesis and take on an aberrant elongated cell morphology. Whereas the ABC transporters for capsular polysaccharides with different structures are functionally interchangeable, the O8 and O9a exporters are specific for their cognate polymannan substrates. The E. coli O8 and O9a Wzt proteins contain a C-terminal domain not present in the corresponding nucleotide-binding protein (KpsT) from the capsule exporter. Whereas the Wzm components are functionally interchangeable, albeit with reduced efficiency, the Wzt components are not, indicating a specific role for Wzt in substrate specificity. Chimeric Wzt proteins were constructed in order to localize the region involved in substrate specificity to the C-terminal domain.     

Characterization of G1 transit induced by the mitogenic-oncogenic viral Ki-ras gene product.

NRK rat kidney cells infected with a temperature-sensitive mutant of the Kirsten sarcoma virus (ts371) were transformed at 36 degrees C but were phenotypically nontransformed at 41 degrees C because of the abnormal thermolability of the oncogenic 21-kilodalton product of the viral Ki-ras gene. Thus tsK-NRK cells were rendered quiescent in a G0-G1 state by a 48-h incubation in serum-free medium at the nonpermissive, p21-inactivating temperature of 41 degrees C. The serum-starved cells could then be stimulated to transit G1 either as nontransformed cells by adding serum at 41 degrees C or as transformed cells by lowering the temperature to a p21-activating 36 degrees C. The viral p21 protein was as effective as serum in stimulating tsK-NRK cells to transit G1 and to start replicating DNA. While p21 effectively stimulated cells to transit G1 even in unconditioned, serum-free medium, they still needed cell-derived conditioning factors to subsequently divide. The p21 protein also enabled the cells to transit G1 in spite of an extracellular Ca2+ deficiency that inhibited the G1 transit of serum-stimulated cells. p21 activity was needed to stimulate both early and late G1 events. In contrast to serum, p21 did not stimulate total RNA or protein synthesis, but some RNA and protein synthesis must have been needed for the p21-driven G1 transit because it could be stopped by actinomycin D or cycloheximide.     

A monoclonal antibody identifies a 215 000-dalton nuclear envelope protein restricted to certain cell types

The monoclonal antibody, AGF2.3, was isolated from mice immunised with the human promyeloid cell line HL60. By immunofluorescence and immunoelectron microscopy the antibody was shown to bind to the nuclear envelope in uninduced HL60 cells. Immunofluorescent staining was reduced to very low levels in HL60 cells induced to mature to monocytes or neutrophils by addition of 12-0-tetradecanoylphorbol-13-acetate or dimethyl sulfoxide respectively. Blood neutrophils did not express the antigen. Weak immunofluorescent staining of cell nuclei was observed in peripheral blood lymphocytes and in sections of normal human kidney, tonsil and skin epithelium. The AGF2.3 antigen was strongly expressed on the nuclei of 21/21 haemopoietic cell lines and 21/25 permanent non-haemopoietic cell lines representing various cell types. In contrast, the antigen was not expressed by any of six primary (untransformed) cell cultures. These included fibroblasts, endothelial cells and keratinocytes. The antigen was expressed in the Q10 SV-40 transformed cell line derived from a non-expressing primary fibroblast culture. AGF2.3 antibody precipitated a protein with an apparent subunit molecular weight of approximately 215 kDa from Triton X-100 extracts of HL60 and HeLa cells labelled with 35S-methionine. This protein was not detectable in extracts of primary skin fibroblasts prepared in parallel. We conclude that AGF2.3 antibody recognises a previously undescribed protein associated with the nuclear envelope which is expressed at high levels in most transformed cell lines but which is weakly expressed or absent in normal tissues and primary cell cultures.     

Food and feeding ecology of piscivorous fishes at Lake St Lucia, Zululand

The food and feeding ecology of piscivorous fish in Lake St Lucia was monitored for two years. Piscivorous fishes feed predominantly on the planktivorous Gilchristella aestuarius and Thryssa vitrirostris but a wide range of prey species was recorded. Numbers of the predominant piscivores, Argyrosomus hololepidotus and Elops machnata , in an area appear to be related to the densities of their major prey, T. vitrirostris and G. aestuarius . Large piscivorous fishes are restricted to the deeper portions of the lake, whereas small piscivores such as Johnius belengerii and Terapon jarbua feed predominantly on small fishes in the littoral zone. The highly significant correlation between the composition of prey fish species in the lake and prey fish species in the diet of piscivorous fishes, indicates that piscivores are feeding in a density dependent manner. However, factors such as habitat, fish size and swimming speed of prey species are shown to be important in prey selection. Juvenile fish of species such as Sarotherodon mossambicus, Liza macrolepis and Acanthopagrus berda remain in shallow marginal areas, thus avoiding large piscivorous fishes. However by frequenting shallow areas these species become vulnerable to bird predators, especially egrets and herons.