Novel enzyme-linked immunosorbent assay systems for the quantitative analysis of connective tissue growth factor (CTGF/Hcs24/CCN2): detection of HTLV-I tax-induced CTGF from a human carcinoma cell line

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24/CCN2) is known as a multifunctional growth factor. It stimulates proliferation, migration, and extracellular matrix production of mesenchymal cells, and is highly expressed in hypertrophic chondrocytes. In this study, we constructed useful ELISA systems for the analysis of CTGF and its modular fragments. For this objective we prepared four different antihuman CTGF monoclonal antibodies. One, specific for the VWC module, was utilized as the detecting antibody, and the other three, recognizing CT, IGFBP, and VWC modules, respectively, were employed as capture antibodies. Then we established three novel quantitative analysis systems for CTGF. The first system recognizing CT and VWC modules was useful to measure full-length CTGF with improved sensitivity. Utilizing this system, we found significant enhancement of CTGF production from a human carcinoma cell line transduced by HTLV-I tax gene, where the finding indicates the possible involvement of Tax in carcinogenesis. The second system, seeing IGFBP and VWC modules, could quantify not only CTGF, but also may be useful to analyze processed N-terminal fragments. The third system, utilizing capture and detection antibodies against the VWC module, was able to quantify the VWC module only, while it did not recognize full-length CTGF. Since CTGF is actually processed into subfragments, and functional assignment of each module is of interest, these systems are expected to contribute to the progress of CTGF investigations.     

Comparable response of ccn1 with ccn2 genes upon arthritis: An in vitro evaluation with a human chondrocytic cell line stimulated by a set of cytokines

BACKGROUND: The chondrosarcoma-derived HCS-2/8 has been known to be an excellent model of human articular chondrocytes. By mimicking the arthritic conditions through the treatment of HCS-2/8 cells with cytokines, we estimated the gene expression response of ccn1 and ccn2 during the course of joint inflammation in vitro. RESULTS: In order to mimic the initiation of inflammation, HCS-2/8 cells were treated with tumor necrosis factor (TNF)-alpha. To induce pro-inflammatory or reparative responses, TGF-beta was employed. Effects of an anti-inflammatory glucocorticoid were also evaluated. After stimulation, expression levels of ccn1 and ccn2 were quantitatively analyzed. Surprisingly, not only ccn2, but also ccn1 expression was repressed upon TNF-alpha stimulation, whereas both mRNAs were uniformly induced by transforming growth factor (TGF)-beta and a glucocorticoid. CONCLUSION: These results describing the same response during the course of inflammation suggest similar and co-operative roles of these 2 ccn family members in the course of arthritis.     

AIP1/WDR1 supports mitotic cell rounding

The actin cytoskeleton plays a fundamental role in configuring cell shapes and movements. Actin interacting protein 1 (AIP1)/tryptophan-aspartate-repeat protein 1 (WDR1) induces actin severing and disassembly cooperating with ADF/cofilin. We found that mitotic cell flattening but not rounding was manifested by suppression of AIP1/WDR1 in cells. This mitotic cell flattening was not due to any changes in phosphorylation and distribution of cofilin in cells. We carried out a direct observation of actin filament severing/disassembly assay and found that phosphorylated cofilin still somewhat severs/disassembles actin filaments and that AIP1/WDR1 effaces this in vitro. We suggest that the phosphorylation of ADF/cofilin will be insufficient to completely inhibit actin turnover during mitosis, and that AIP1/WDR1 could abort the severing/disassembly activity somewhat still carried out due to phosphorylated ADF/cofilin. This mechanism could be required to induce cell morphologic changes, especially mitotic cell rounding.     

Control of the redox potential of Pseudomonas aeruginosa cytochrome c551 through the Fe-Met coordination bond strength and pKa of a buried heme propionic acid side chain

Pseudomonas aeruginosa cytochrome c(551) and a series of its mutants exhibiting various thermostabilities have been studied by paramagnetic (1)H NMR and cyclic voltammetry in an effort to elucidate the molecular mechanisms responsible for control of the redox potentials (E degrees ') of the proteins. The study revealed that the E degrees ' value of the protein is regulated by two molecular mechanisms operating independently of each other. One is based on the Fe-Met coordination bond strength in the protein, which is determined by the amino acid side chain packing in the protein, and the other on the pK(a) of the heme 17-propionic acid side chain, which is affected by the electrostatic environment. The former mechanism alters the magnitude of the E degrees ' value throughout the entire pH range, and the latter regulates the pK values reflected by the pH profile of the E degrees ' value. These findings provide novel insights into functional regulation of the protein, which could be utilized for tuning the E degrees ' value of the protein by means of protein engineering.     

Bipolar localization of putative photoreceptor protein for phototaxis in thermophilic cyanobacterium Synechococcus elongatus

We identified an open reading frame from a database of the entire genome of Synechococcus elongatus, the product of which was very similar to pixJ1, which was proposed as photoreceptor gene for phototaxis in Synechocystis sp. PCC6803 [Yoshihara et al. (2000) Plant Cell Physiol. 41: 1299]. The mRNA of S. elongatus pixJ (SepixJ) was expressed in vivo as a part of the product of an operon. SePixJ was detected exclusively in the membrane fraction after cell fractionation. Immunogold labeling of SePixJ in ultra-thin sections indicated that it existed only in both ends of the rod-shaped cell; probably bound with the cytoplasmic membrane.     

Identification of an antifreeze lipoprotein from Moraxella sp. of Antarctic origin

We found six bacteria capable of producing antifreeze protein (AFP) from Ross Island, Antarctica. Among these AFP-producing bacteria, strain No. 82 had the highest antifreeze activity and was identified as Moraxella sp. The optimum temperature and pH for the production of AFP were 5 degrees C and 7.0, respectively. After partially purifying the AFP from the culture supernatant using 60% saturation of ammonium sulfate, only the 52-kDa protein band (100 microg/ml) which eluted from SDS-PAGE indicated antifreeze activity by the formation of hexagonal crystals. Furthermore, we confirmed that this AFP was a lipoprotein by the lipid stain test and treatment with some enzymes and that it had no ice-nucleating activity. Also, the N-terminal amino acid sequence of this AFP had high similarity with that of outer membrane proteins from Moraxella (Branhamella) catarrhalis. This is the first report of AFP-producing bacteria in Antarctica and an antifreeze lipoprotein (AFLP) from Moraxella sp.     

Comparison of insulin-like growth factor I receptor and insulin receptor purified from human placental membranes

Insulin-like growth factor (IGF)-I receptor purified from human placental membranes as previously described (LeBon, T. R., Jacobs, S., Cuatrecasas, P., Kathuria, S., and Fujita-Yamaguchi, Y. (1986) J. Biol. Chem. 261, 7685-7689) was characterized. The IGF-I receptor was similar to the insulin receptor with respect to subunit structure (beta-alpha-alpha-beta), apparent sizes of deglycosylated alpha (Mr = approximately 88,000) and beta (Mr = approximately 67,000) subunits, and amino acid composition of the subunits. Monoclonal antibody specific to each receptor recognized its own receptor whereas polyclonal anti-human insulin receptor antibody cross-reacted with the IGF-I receptor, indicating that the receptors share one or more antigenic sites. Further characterization of the purified IGF-I receptor tyrosine-protein kinase activity indicated that by analogy with the insulin receptor the monomeric alpha beta form of the IGF-I receptor appears to have higher kinase activity than the intact receptor in the alpha 2 beta 2 form. The most significant difference between the two receptors was found in the N-terminal amino acid sequences of their alpha subunits, which apparently show 60% identity. The IGF-I receptor alpha subunit lacks residues corresponding to the N-terminal 4 amino acids of the insulin receptor alpha subunit. These results provide the first direct proof that the IGF-I receptor is a molecule distinct from the insulin receptor despite numerous similarities.     

1,4-Dioxane degradation potential of members of the genera <Emphasis Type="Italic">Pseudonocardia</Emphasis> and <Emphasis Type="Italic">Rhodococcus</Emphasis>

In recent years, several strains capable of degrading 1,4-dioxane have been isolated from the genera Pseudonocardia and Rhodococcus. This study was conducted to evaluate the 1,4-dioxane degradation potential of phylogenetically diverse strains in these genera. The abilities to degrade 1,4-dioxane as a sole carbon and energy source and co-metabolically with tetrahydrofuran (THF) were evaluated for 13 Pseudonocardia and 12 Rhodococcus species. Pseudonocardia dioxanivorans JCM 13855^T, which is a 1,4-dioxane degrading bacterium also known as P. dioxanivorans CB1190, and Rhodococcus aetherivorans JCM 14343^T could degrade 1,4-dioxane as the sole carbon and energy source. In addition to these two strains, ten Pseudonocardia strains could degrade THF, but no Rhodococcus strains could degrade THF. Of the ten Pseudonocardia strains, Pseudonocardia acacia JCM 16707^T and Pseudonocardia asaccharolytica JCM 10410^T degraded 1,4-dioxane co-metabolically with THF. These results indicated that 1,4-dioxane degradation potential, including degradation for growth and by co-metabolism with THF, is possessed by selected strains of Pseudonocardia and Rhodococcus, although THF degradation potential appeared to be widely distributed in Pseudonocardia. Analysis of soluble di-iron monooxygenase (SDIMO) α-subunit genes in THF and/or 1,4-dioxane degrading strains revealed that not only THF and 1,4-dioxane monooxygenases but also propane monooxygenase-like SDIMOs can be involved in 1,4-dioxane degradation.