The O-polysaccharide of Pseudomonas syringae pv. mori NCPPB 1656 is a beta-(1-->2)-linked homopolymer of L-rhamnose.

The O-polysaccharide from the lipopolysaccharide of the phytopathogenic bacterium Pseudomonas syringae pv. mori NCPPB 1656 was studied by sugar analysis along with 1H and 13C NMR spectroscopy and found to be a new beta-(1-->2)-linked homopolymer of L-rhamnose.

Monoclonal antibodies against human Tudor-SN.

Tudor-SN is a multifunctional regulator of gene expression that has been shown to function as a transcriptional co-activator, regulator of miRNA processing, mRNA splicing, and stability. Tudor-SN has also been identified as a component in RNA-induced silencing complex. Here we have produced and characterized seven monoclonal antibody (MAb) clones against human Tudor-SN. Antibodies were generated against the fourth staphylococcal nuclease-like domain (SN4) and the Tudor domain of human Tudor-SN. The MAbs recognize the Tudor-SN protein in Western blot analysis and immunoprecipitation, and detect the specific antigen in immunohistochemistry assays. One of the antibody clones also recognizes the Drosophila melanogaster and Danio rerio Tudor-SN. Immunocytochemistry of HeLa cells revealed Tudor-SN localization in nucleolus, suggesting a possible new function for the protein in the compartment. An extensive expression analysis in human tissue arrays shows moderate to high expression of Tudor-SN in a wide range of organs and tissues, especially in epithelial cell types.

Extracellular HIV-1 Nef increases migration of monocytes.

Infiltration of human immunodeficiency virus type 1 (HIV-1)-infected and uninfected monocytes/macrophages in organs and tissues is a general phenomenon observed in progression of acquired immunodeficiency syndrome (AIDS). HIV-1 protein Nef is considered as a progression factor in AIDS, and is released from HIV-1-infected cells. Here, we show that extracellular Nef increases migration of monocytes. This effect is (i) concentration-dependent, (ii) reaches the order of magnitude of that induced by formyl-methyonyl-leucyl-proline (fMLP) or CC chemokine ligand 2 (CCL2)/monocyte chemotactic protein (MCP)-1, (iii) inhibited by anti-Nef monoclonal antibodies as well as by heating, and (iv) depends on a concentration gradient of Nef. Further, Nef does not elicit monocytic THP-1 cells to express chemokines such as CCL2, macrophage inhibitory protein-1alpha (CCL3) and macrophage inhibitory protein-1beta (CCL4). These data suggest that extracellular Nef may contribute to disease progression as well as HIV-1 spreading through affecting migration of monocytes.

Subcellular localization of pentachlorophenol 4-monooxygenase in Sphingobium chlorophenolicum ATCC 39723.

We have studied the subcellular localization of pentachlorophenol 4-monooxygenase (PCP4MO) in Sphingobium chlorophenolicum ATCC 39723 during induction by pentachlorophenol (PCP). Using a monoclonal antibody CL6 specific to the native and recombinant PCP4MO, the enzyme was primarily found soluble as determined by immunoblot and ELISA analyses of cellular fractions. However, the enzyme was observed both in the soluble and membrane-bound forms during induction for 2-4 h, suggesting its translocation out from the cytoplasm. Electron microscopy confirmed that PCP4MO was predominantly present in the cytoplasm at 1 h, whereas at 4 h significant amount was detected also in the membrane and periplasm. After 6 h, the majority of PCP4MO was in the periplasm and only small amount was bound to the inner membrane or present in the cytoplasm. The results indicate that after biosynthesis PCP4MO in S. chlorophenolicum is exported via the inner membrane to the final location in the periplasm.

Structure of the O polysaccharides and serological classification of Pseudomonas syringae pv. porri from genomospecies 4.

Strains of Pseudomonas syringae pv. porri are characterized by a number of pathovar-specific phenotypic and genomic characters and constitute a highly homogeneous group. Using monoclonal antibodies, they all were classified in a novel P. syringae serogroup O9. The O polysaccharides (OPS) isolated from the lipopolysaccharides (LPS) of P. syringae pv. porri NCPPB 3365 and NCPPB 3364T possess multiple oligosaccharide O repeats, some of which are linear and composed of l-rhamnose (l-Rha), whereas the major O repeats are branched with l-rhamnose in the main chain and GlcNAc in side chains (structures 1 and 2). Both branched O repeats, which differ in the position of substitution of one of the Rha residues and in the site of attachment of GlcNAc, were found in the two strains studied, O repeat 1 being major in strain NCPPB 3365 and 2 in strain NCPPB 3364T. [formula: see text]. The relationship between OPS chemotype and serotype on one hand and the genomic characters of P. syringae pv. porri and other pathovars delineated in genomospecies 4 on the other hand is discussed.

Chicken avidin-related proteins show altered biotin-binding and physico-chemical properties as compared with avidin.

Chicken avidin and bacterial streptavidin are proteins familiar from their use in various (strept)avidin-biotin technological applications. Avidin binds the vitamin biotin with the highest affinity known for non-covalent interactions found in nature. The gene encoding avidin (AVD) has homologues in chicken, named avidin-related genes (AVRs). In the present study we used the AVR genes to produce recombinant AVR proteins (AVRs 1, 2, 3, 4/5, 6 and 7) in insect cell cultures and characterized their biotin-binding affinity and biochemical properties. Amino acid sequence analysis and molecular modelling were also used to predict and explain the properties of the AVRs. We found that the AVR proteins are very similar to avidin, both structurally and functionally. Despite the numerous amino acid substitutions in the subunit interface regions, the AVRs form extremely stable tetramers similar to those of avidin. Differences were found in some physico-chemical properties of the AVRs as compared with avidin, including lowered pI, increased glycosylation and, most notably, reversible biotin binding for two AVRs (AVR1 and AVR2). Molecular modelling showed how the replacement Lys(111)-->isoleucine in AVR2 alters the shape of the biotin-binding pocket and thus results in reversible binding. Both modelling and biochemical analyses showed that disulphide bonds can form and link monomers in AVR4/5, a property not found in avidin. These, together with the other properties of the AVRs described in the present paper, may offer advantages over avidin and streptavidin, making the AVRs applicable for improved avidin-biotin technological applications.