Pericentrin forms a complex with intraflagellar transport proteins and polycystin-2 and is required for primary cilia assembly

Primary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases. Here, we show that the centrosome protein pericentrin (Pcnt) colocalizes with IFT proteins to the base of primary and motile cilia. Immunogold electron microscopy demonstrates that Pcnt is on or near basal bodies at the base of cilia. Pcnt depletion by RNA interference disrupts basal body localization of IFT proteins and the cation channel polycystin-2 (PC2), and inhibits primary cilia assembly in human epithelial cells. Conversely, silencing of IFT20 mislocalizes Pcnt from basal bodies and inhibits primary cilia assembly. Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions. Pcnt antibodies coimmunoprecipitate IFT proteins and PC2 from several cell lines and tissues. We conclude that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella.     

Exopolysaccharide production by a new Halomonas strain CRSS isolated from saline lake Cape Russell in Antarctica growing on complex and defined media

A haloalkalophilic Halomonas strain CRSS, isolated from salt sediments in Antarctica, produced exocellular polysaccharides (EPS) up to 2.9 g g(-1) dry cells. Acetate was the most efficient carbon source for EPS production. The composition of media strongly affected the nature of the polymers; a mannan and a xylo-mannan, were obtained when cells were grown on complex media. Acetate was the most efficient carbon source for EPS production and in presence of this substrate, a new polysaccharide, a fructo-glucan, was produced. The EPS fraction was composed by glucose, fructose, glucosamine and galactosamine in relative proportions of 1:0.7:0.3:trace.     

The <Emphasis Type="Italic">vaa</Emphasis> locus of <Emphasis Type="Italic">Mycoplasma hominis</Emphasis> contains a divergent genetic islet encoding a putative membrane protein

Background  

The Mycoplasma hominis vaa gene encodes a highly variable, surface antigen involved in the adhesion to host cells. We have analysed the structure of the vaa locus to elucidate the genetic basis for variation of vaa.     

Adamantylsulfanyl- and N-adamantylcarboxamido-derivatives of heterocycles and phenols: synthesis, crystal structure, tumor necrosis factor-alpha production-enhancing properties, and theoretical considerations

The synthesis of several adamantylthio heterocycles and S-adamantylated thiocresols is reported. The attack of the adamantyl cation formed from 1-adamantan-1-ol in refluxing trifluoroacetic acid provides the corresponding adamantylsulfanyl compounds. The use of the adamantyl cation in the Ritter reaction gave the number of N-adamantylcarboxamide derivatives of heterocyclic and phenolic compounds. Adamantylsulfanyl heterocycles, contrary to N-adamantylcarboxamido compounds, enhanced the production of tumor necrosis factor alpha (TNF-alpha) in genetically modified murine melanoma cells transduced with the gene for human TNF-alpha. The highest activity, comparable to that of the most-active previously described for 6-methyl-2-[(adamantyl)amino]pyridine, showed 2-thioadamantyl derivatives of pyridine and 6-methylpyridine. The crystal structures of carboxamido and sulfanyl analogues of 2-(1-adamantylamino)pyridine have been studied, and consecutive quantum-chemical calculations have been performed. The low TNF-alpha production stimulatory activity of N-adamantylcarboxamido-pyridine compared to the sulfanyl and amino analogues is postulated to be linked to the conformational rigidity of the former one enhanced by the formation of the intramolecular H-bond. The comparison of the activity of 2-(1-adamantylsulfanyl)-6-methylpyridine and (1-adamantylsulfanyl)-2-methylbenzene proved the importance of the ring N-atom, whereas the differences in activity between bromo and methyl analogues pointed at electronic rather than steric influence of ortho-substituents on the biological activity.     

Ascorbate restores lifespan of superoxide-dismutase deficient yeast

Yeast (Saccharomyces cerevisiae) mutants lacking CuZn-superoxide dismutase (CuZnSOD) are hypersensitive to oxygen and have significantly decreased replicative life span. Both these defects can be ameliorated by exogenous ascorbate. The effect of ascorbate on life span is complicated by auto-oxidation of its compound in the medium. If negative effects of auto-oxidation are prevented by exchange of the medium, ascorbate prolongs not only mean but also maximal replicative life span of the yeast in the atmosphere of air and of pure oxygen. These results demonstrate that life span shortening due to the lack of a vital antioxidant enzyme can be ameliorated by a low-molecular weight antioxidant.     

Construction and Use of a Broad-Host-Range Plasmid Expressing the lamB Gene for Utilization of Bacteriophage λ Vectors in the Marine Bacterium Vibrio harveyi

The remarkable success of Escherichia coli as a model organism in molecular genetics was dependent, among other things, on its susceptibility to genetic manipulation. Many versatile and sophisticated genetic tools for molecular biology studies are derived from bacteriophage λ. However, this bacteriophage is specific for E. coli, and thus λ-based techniques have been restricted to this bacterium. Plasmids expressing the E. coli gene coding for bacteriophage λ receptor were reported previously, and introduction of such plasmids into cells of some other bacteria made them sensitive to phage λ infection. However, we found that these systems were not efficient for Vibrio harveyi, one of the most frequently investigated species of marine bacteria. Here we describe construction of a broad-host-range plasmid expressing the lamB gene. Introduction of this plasmid to V. harveyi cells and expression of lamB made this strain susceptible to bacteriophage λ adsorption and λ DNA injection. Foreign genetic material could be introduced into cells of this strain using a cosmid vector. Received August 30, 2000; accepted January 30, 2001.     

Planaria FoxA (HNF3) homologue is specifically expressed in the pharynx-forming cells

We have isolated a planarian Forkhead box A (FoxA, a new name for a gene group containing HNF3 alpha,beta,gamma)-related gene, DjFoxA, and examined its spatial and temporal distribution in both intact and regenerating planarians by in situ hybridization. In intact worms, DjFoxA is specifically expressed in the cells participating in pharynx development in the region surrounding the pharynx, which is located in the central portion of the body. During regeneration, DjFoxA-positive cells appear in the pharynx-forming region and migrate to the midline to form a pharynx rudiment. These results suggest that DjFoxA is specifically expressed in the cells participating in pharynx formation and has an evolutionarily conserved function in digestive tract formation.     

Synthesis of NF-kappaB activation inhibitors derived from epoxyquinomicin C

In order to develop new inhibitors of NF-kappaB activation, we designed and synthesized dehydroxymethyl derivatives of epoxyquinomicin C, namely, DHM2EQ and its regioisomer DHM3EQ. These derivatives were synthesized from 2,5-dimethoxyaniline in 5 steps. Since DHM2EQ was more active and less toxic than DHM3EQ, its stereochemical configuration was determined by X-ray crystallographic analysis. Each enantiomer of the protected DHM2EQ was separated by a chiral column and deprotected. DHM2EQ inhibited TNF-alpha-induced activation of NF-kappaB in human T cell leukemia cells, and also inhibited collagen-induced arthritis in a rheumatoid model in mice.