Styrylheterocycles as a novel class inhibitor of cyclooxygenase-2-mediated prostaglandin E(2) production

The inhibitory effects of a series of styrylheterocycles on the production of cyclooxygenase-2-mediated prostaglandin E(2) (PGE(2)) were evaluated in lipopolysaccharide-stimulated RAW264.7 murine macrophages. A new series of potential inhibitors, including 3-[2-(4-methoxy-phenyl)-vinyl]-thiophene, have been identified, thus providing novel chemical leads for the further development of potential inhibitors in this capacity. The suppression of COX-2 mRNA expression by the active styrylheterocycles, in part, was involved in the inhibitory activity against the overproduction of PGE(2).     

Alpha,alpha-trehalose derivatives bearing guanidino groups as inhibitors to HIV-1 Tat-TAR RNA interaction in human cells

Replication of HIV-1 requires specific interactions of Tat protein with TAR RNA. Disruption of Tat-TAR RNA interaction could inhibit HIV-1 replication. Here four target compounds were designed and synthesized to bind to TAR RNA for blocking the interaction of Tat-TAR RNA. The core molecule 6,6'-diamino-6,6'-dideoxy-alpha,alpha-trehalose was obtained from selective bromination of, alpha,alpha-trehalose at C-6,6', followed by acetylation, azide displacement, deacetylation, and reduction. Coupling of the core molecule with the protected amino acid, then deprotection and guanidinylation generated the novel alpha,alpha-trehalose derivatives. Their abilities to inhibit Tat-TAR RNA interaction in human cells were determined by a Tat-dependent HIV-1 LTR-driven CAT assays.     

Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry

Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.     

Inhibiting apoptosis of CTLL-2 cells to enhance their GVL effects via anti-Fas ribozyme

Donor lymphocyte infusion (DLI) is an adoptiveimmunotherapy to achieve particular therapy aims forpatients accepting allogenetic hemopoietic stem celltransplantation [1–3]. Recently, many researches havetestified that the graft-versus-leukemia effect (GV…     

Identification and characteristics of a novel E1 like gene nUBE1L in human testis

A gene, presumably involved in spermatogenesis, was identified and characterized by using cDNA microarray. Hybridization intensity was 2.13 fold higher in adult testis than that in fetal testis.The full length of this gene was 4288bp and it encoded a 578 amino acid protein. Conserved structure and amino acid sequence analysis revealed that the protein contained 1 Thif-domain, 2 UBACT-domains,and a functional active site cysteine lay upstream of UBACT domain, all of them also existed in ubiquitin-activating enzyme E1 and E1 like proteins. So we named this gene as a novel ubiquitin-activating enzyme E1 like gene (nUBE1L). Expression profile showed that nUBE1L was predominantly expressed in testis.Comparison of the expression of nUBE1L in different developmental stages of testis indicated that it was highly expressed in adult testis. In conclusion, nUBE1L is a novel human E1 like gene highly expressed inadult testis, which plays key role in ubiquitin system, and accordingly influences spermatogenesis and male fertility.     

Flow cytometric application of helper adenovirus (HAd) containing GFP gene flanked by two parallel loxP sites to evaluation of 293 cre-complementing cell line and monitoring of HAd in Gutless Ad production

Gutless adenoviruses (GAds), namely, all gene-deleted adenoviruses, were developed to minimize their immune responses and toxic effects for a successful gene delivery tool in gene therapy. The Cre/loxP system has been widely used for GAd production. To produce GAd with a low amount of helper adenovirus (HAd) as byproduct, it is indispensable to use 293Cre cells expressing a high level of Cre for GAd production. In this study, we constructed the HAd containing enhanced green fluorescent protein gene flanked by two parallel loxP sites (HAd/GFP). The use of HAd/GFP with flow cytometry allows one to select 293Cre cells expressing a high level of Cre without using conventional Western blot analysis. Unlike conventional HAd titration methods such as plaque assay and end-point dilution assay, it also allows one to monitor rapidly the HAd as byproduct in earlier stages of GAd amplification. Taken together, the use of HAd/GFP with flow cytometry facilitates bioprocess development for efficient GAd production.     

Enhancing effect of low culture temperature on specific antibody productivity of recombinant Chinese hamster ovary cells: clonal variation

To understand the different responses of recombinant Chinese hamster ovary (rCHO) cells to low culture temperature regarding specific productivity (q), 12 parental clones and their corresponding amplified clones producing a humanized antibody were cultivated at 32 and 37 degrees C. The specific growth rate of all clones, including both parental and amplified clones, decreased by 30-63% at 32 degrees C, compared to rates at 37 degrees C. In contrast, their specific antibody productivity (qAb) was significantly enhanced at 32 degrees C. Furthermore, the degree of qAb enhancement at 32 degrees C varied a lot from 4- to 25-fold among the parental clones. At 32 degrees C, most of the amplified clones, regardless of methotrexate (MTX) levels, also showed enhanced qAb but to a lesser extent than their parental clones. However, clone 14 amplified at 0.32 microM MTX (clone 14-0.32) and clone 20 amplified at 1 microM MTX (clone 20-1.00), unlike their parental clones, did not show enhanced qAb at 32 degrees C. Thus, it was found that the enhancing effect of low culture temperature on q of rCHO cells depends on clones. Taken together, the results obtained here emphasize the importance of clonal selection for the successful application of low culture temperature to the enhanced foreign protein production in rCHO cells.     

Biochemical characterization of protein complexes from the Helicobacter pylori protein interaction map: strategies for complex formation and evidence for novel interactions within type IV secretion systems

We have investigated a large set of interactions from the Helicobacter pylori protein interaction map previously identified by high-throughput yeast two-hybrid (htY2H)-based methods. This study had two aims: i) to validate htY2H as a source of protein-protein interaction complexes for high-throughput biochemical and structural studies of the H. pylori interactome; and ii) to validate biochemically interactions shown by htY2H to involve components of the H. pylori type IV secretion systems. Thus, 17 interactions involving 31 proteins and protein fragments were studied, and a general strategy was designed to produce protein-interacting partners for biochemical and structural characterization. We show that htY2H is a valid source of protein-protein complexes for high-throughput proteome-scale characterization of the H. pylori interactome, because 76% of the interactions tested were confirmed biochemically. Of the interactions involving type IV secretion proteins, three could be confirmed. One interaction is between two components of the type IV secretion apparatus, ComB10 and ComB4, which are VirB10 and VirB4 homologs, respectively. Another interaction is between a type IV component (HP0525, a VirB11 homolog) and a non-type IV secretion protein (HP01451), indicating that proteins other than the core VirB (1-11)-VirD4 proteins may play a role in type IV secretion. Finally, a third interaction was biochemically confirmed between CagA, a virulence factor secreted by the type IV secretion system encoded by the Cag pathogenicity island, and a non-type IV secretion protein, HP0496.     

Intermediate fertile Triticum aestivum （＋）Agropyron elongatum somatic hybrids are generated by low doses of UV irradiation

We report the production and characterization of somatic hybrids between Triticum aestivum L. and Agropyron elongatum (Host) Nevishi (the synonym is Thinopyrum ponticum). Asymmetric protoplast fusion was performed between Agropyron elongatum protoplasts irradiated with a low UV dose and protoplasts of wheat taken from nonregenerable suspension cultures. More than 40 green plantlets were obtained from 15 regenerated clones and one of them produced seeds. The phenotypes of the hybrid plants and seeds were intermediate between wheat and Agropyron elongatum. All of the regenerated calli and plants were verified as intergeneric hybrids on the basis of morphological observation and analysis of isozyme, cytological, 5SrDNA spacer sequences and random amplified polymorphic DNA (RAPD). RFLP analysis of the mitochondrial genome revealed evidence of random segregation and recombination of mtDNA.