Evaluation of a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the identification of Campylobacter strains isolated from poultry in Thailand

We have recently developed a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for identifying Campylobacter jejuni, C. coli and C. fetus. In the present study, the applicability of this assay was evaluated with 34 Campylobacter-like organisms isolated from poultry in Thailand for species identification and was compared with other assays including API Campy, 16S rRNA gene sequence, and hippuricase (hipO) gene detection. Of the 34 strains analyzed, 20, 10 and 1 were identified as C. jejuni, C. coli, and Arcobacter cryaerophilus, respectively, and 3 could not be identified by API Campy. However, 16S rRNA gene analysis, showed that all 34 strains are C. jejuni/coli. To discriminate between these 2 species, the hipO gene, which is specifically present in C. jejuni, was examined by PCR and was detected in 20 strains, which were identified as C. jejuni by API Campy but not in the remaining 14 strains. Collective results indicated that 20 strains were C. jejuni whereas the 14 strains were C. coli. When the cdt gene-based multiplex PCR was employed, however, 19, 20 and 19 strains were identified as C. jejuni while 13, 14 and 13 were identified as C. coli by the cdtA, cdtB and cdtC gene-based multiplex PCR, respectively. Pulsed-field gel electrophoresis revealed that C. jejuni and C. coli strains analyzed are genetically diverse. Taken together, these data suggest that the cdt gene-based multiplex PCR, particularly cdtB gene-based multiplex PCR, is a simple, rapid and reliable method for identifying the species of Campylobacter strains.     

Molecular analysis of RANKL-independent cell fusion of osteoclast-like cells induced by TNF-alpha, lipopolysaccharide, or peptidoglycan

Focusing on the final step of osteoclastogenesis, we studied cell fusion from tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells into multinuclear cells. TRAP-positive mononuclear cells before generation of multinuclear cells by cell fusion were differentiated from RAW264.7 cells by treatment with receptor activator of nuclear factor kappa B ligand (RANKL), and then the cells were treated with lipopolysaccharide (LPS), followed by culturing for further 12 h. LPS-induced cell fusion even in the absence of RANKL. Similarly, tumor necrosis factor (TNF)-alpha and peptidoglycan (PGN) induced cell fusion, but M-CSF did not. The cell fusion induced by RANKL, TNF-alpha, and LPS was specifically blocked by osteoprotegerin (OPG), anti-TNF-alpha antibody, and polymyxin B, respectively. LPS- and PGN-induced cell fusion was partly inhibited by anti-TNF-alpha antibody but not by OPG. When TRAP-positive mononuclear cells fused to yield multinuclear cells, phosphorylation of Akt, Src, extracellular signal-regulated kinase (ERK), p38MAPK (p38), and c-Jun NH2-terminal kinase (JNK) was observed. The specific chemical inhibitors LY294002 (PI3K), PP2 (Src), U0126 (MAPK-ERK kinase (MEK)/ERK), and SP600125 (JNK) effectively suppressed cell fusion, although SB203580 (p38) did not. mRNA of nuclear factor of activated T-cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP) during the cell fusion was quantified, however, there was no obvious difference among the TRAP-positive mononuclear cells treated with or without M-CSF, RANKL, TNF-alpha, LPS, or PGN. Collectively, RANKL, TNF-alpha, LPS, and PGN induced cell fusion of osteoclasts through their own receptors. Subsequent activation of signaling pathways involving PI3K, Src, ERK, and JNK molecules was required for the cell fusion. Although DC-STAMP is considered to be a requisite for cell fusion of osteoclasts, cell fusion-inducing factors other than DC-STAMP might be necessary for the cell fusion.     

Hyaline cartilage formation and enchondral ossification modeled with KUM5 and OP9 chondroblasts

What is it that defines a bone marrow‐derived chondrocyte? We attempted to identify marrow‐derived cells with chondrogenic nature and immortality without transformation, defining “immortality” simply as indefinite cell division. KUM5 mesenchymal cells, a marrow stromal cell line, generated hyaline cartilage in vivo and exhibited enchondral ossification at a later stage after implantation. Selection of KUM5 chondroblasts based on the activity of the chondrocyte‐specific cis‐regulatory element of the collagen α2(XI) gene resulted in enhancement of their chondrogenic nature. Gene chip analysis revealed that OP9 cells, another marrow stromal cell line, derived from macrophage colony‐stimulating factor‐deficient osteopetrotic mice and also known to be niche‐constituting cells for hematopoietic stem cells expressed chondrocyte‐specific or ‐associated genes such as type II collagen α1, Sox9, and cartilage oligomeric matrix protein at an extremely high level, as did KUM5 cells. After cultured OP9 micromasses exposed to TGF‐β3 and BMP2 were implanted in mice, they produced abundant metachromatic matrix with the toluidine blue stain and formed type II collagen‐positive hyaline cartilage within 2 weeks in vivo. Hierarchical clustering and principal component analysis based on microarray data of the expression of cell surface markers and cell‐type‐specific genes resulted in grouping of KUM5 and OP9 cells into the same subcategory of “chondroblast,” that is, a distinct cell type group. We here show that these two cell lines exhibit the unique characteristics of hyaline cartilage formation and enchondral ossification in vitro and in vivo. J. Cell. Biochem. 100: 1240–1254, 2007. © 2006 Wiley‐Liss, Inc.     

Acceleration of R5 HIV replication by polymorphonuclear neutrophils in cultures of macrophages

Cell-to-cell contact between monocyte-derived macrophages (MDM) and endothelial cells has resulted in the increased proliferation of CC chemokine receptor 5/M-tropic (R5) human immunodeficiency virus (HIV) in MDM. In the present study, R5 HIV replication was shown to be increased by polymorphonuclear neutrophils (PMN) in MDM cultures through the soluble factors released from these PMN. The replication of R5 HIV in MDM was greatly enhanced when PMN were added to cultures. An increase in the replication of R5 HIV was also demonstrated when the virus was replicated in MDM cultured in a double chamber transwell with PMN. Chemokine ligand (CCL) 2 and interleukin (IL)-10 were detected in culture fluids of PMN exposed to R5 HIV. The replication of R5 HIV was not accelerated in cultures of MDM and PMN in a double chamber transwell supplemented with a mixture of monoclonal antibodies directed against CCL2 and IL-10. Similarly, the replication of R5 HIV was accelerated in MDM cultures supplemented with a mixture of recombinant CCL2 and IL-10. These results indicated that, in response to the viral stimulation, PMN produce CCL2 and IL-10 and enhance the replication of R5 HIV in MDM cultures.     

Adenylate kinase in sea urchin embryonic cilia

Sea urchin embryos swim by ciliary movement. Hypertonic shock causes deciliation and loss of motility. Within 2-4 h, cilia regenerate and the embryos swim again. Regeneration of cilia occurs multiple times. The adenylate kinase (AK) activity of isolated cilia was studied. A 130-kDa Sp-AK isozyme, present in sperm flagella, is also present in embryonic cilia. AK activity is responsible for approximately 93% of nonmitochondrial ATP regeneration from ADP in embryonic cilia. This is unlike sea urchin sperm flagella, where approximately 31% of the nonmitochondrial ATP regeneration is from the 130-kDa Sp-AK isozyme and approximately 69% from the flagellar creatine kinase (Sp-CK). Embryos were deciliated 1-3 times and after a 2-h period of regeneration the major ciliary axonemal proteins such as the tubulins appeared constant in amount. However, a moderate decrease in ATPase activity, and a large decrease of total AK activity, were measured. The decrease in AK activity paralleled the decrease in embryo swimming velocity. Embryos were deciliated once and cilia regeneration followed for 4 h. ATPase activity recovered to control levels by 3 h, but AK activity and swimming velocity remained lower than in controls. Detergent solubility data and kinetic experiments indicate that, in addition to the 130-kDa Sp-AK, there is at least one additional AK isozyme in embryonic cilia. Analysis of the S. purpuratus genome indicates five AK isozymes in addition to the 130-kDa Sp-AK isozyme. Decreased swimming velocity of embryos with regenerated cilia suggests that regenerated cilia are not as functionally perfect as naturally grown cilia.     

Behavioral phenotypes of Disc1 missense mutations in mice

To support the role of DISC1 in human psychiatric disorders, we identified and analyzed two independently derived ENU-induced mutations in Exon 2 of mouse Disc1. Mice with mutation Q31L showed depressive-like behavior with deficits in the forced swim test and other measures that were reversed by the antidepressant bupropion, but not by rolipram, a phosphodiesterase-4 (PDE4) inhibitor. In contrast, L100P mutant mice exhibited schizophrenic-like behavior, with profound deficits in prepulse inhibition and latent inhibition that were reversed by antipsychotic treatment. Both mutant DISC1 proteins exhibited reduced binding to the known DISC1 binding partner PDE4B. Q31L mutants had lower PDE4B activity, consistent with their resistance to rolipram, suggesting decreased PDE4 activity as a contributory factor in depression. This study demonstrates that Disc1 missense mutations in mice give rise to phenotypes related to depression and schizophrenia, thus supporting the role of DISC1 in major mental illness.     

In-situ observation of tryptophan in larval cocoon silk of the hornet Vespa simillima xanthoptera cameron by ultraviolet resonance raman spectroscopy

We performed in-situ ultraviolet resonance Raman (UVRR) spectroscopy of the larval cocoon silk of the hornet, Vespa simillima xanthoptera Cameron, and compared the result with that of the silkworm, Bombix mori. The UVRR spectrum of the hornet cocoon differed markedly from that of the B. mori cocoon: peaks attributable to tyrosine (Tyr) were observed strongly, and tryptophan (Trp) peaks weakly, in the spectrum of the B. mori cocoon, whereas peaks attributable to Trp exclusively appeared in the spectrum of the hornet cocoon.     

Role of the ompT mutation in stimulated decrease in colony-forming ability due to intracellular protein aggregate formation in Escherichia coli strain BL21

Recently we found that the cells of Escherichia coli strain BL21 producing a fusion protein, GST-Sup35NM, show a much more rapid decrease in colony-forming ability in the stationary phase than control cells. In this study, it was found that an extract of the cells producing GST-Sup35NM forms fibrous protein polymers containing GST-Sup35NM. In the course of the study, we realized that strain BL21 carried the ompT mutation. We suspected that the deficiency in OmpT protease was responsible for the observed phenotype. To test this, we introduced the wild-type ompT gene into strain BL21, and found that the transformed cells recovered the wild-type phenotype. We concluded that OmpT protease, though known to localize on the cell surface, is involved in protein quality control within the cell.