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Major Bands (major + bands)
Selected AbstractsA novel T cell cytokine, secreted osteoclastogenic factor of activated T cells, induces osteoclast formation in a RANKL-independent mannerARTHRITIS & RHEUMATISM, Issue 11 2009Leonard Rifas Objective Chronic T cell activation is central to the etiology of rheumatoid arthritis (RA), an inflammatory autoimmune disease that leads to severe focal bone erosions and generalized systemic osteoporosis. Previous studies have shown novel cytokine-like activities in medium containing activated T cells, characterized by potent induction of the osteoblastic production of interleukin-6 (IL-6), an inflammatory cytokine and stimulator of osteoclastogenesis, as well as induction of an activity that directly stimulates osteoclast formation in a manner independent of the key osteoclastogenic cytokine RANKL. This study was undertaken to identify the factors secreted by T cells that are responsible for these activities. Methods Human T cells were activated using anti-human CD3 and anti-human CD28 antibodies for 72 hours in AIM V serum-free medium to obtain T cell,conditioned medium, followed by concentration and fractionation of the medium by fast-protein liquid chromatography. Biologically active fractions were resolved using sodium dodecyl sulfate,polyacrylamide gel electrophoresis. Major bands were analyzed by mass spectrometry, and a major candidate protein was identified. This novel cytokine was cloned, and its expression was analyzed using recombinant DNA technologies. Results A single novel cytokine that could induce both osteoblastic IL-6 production and functional osteoclast formation in the absence of osteoblasts or RANKL and that was insensitive to the effects of the RANKL inhibitor osteoprotegerin was identified in the activated T cell,conditioned medium; this cytokine was designated secreted osteoclastogenic factor of activated T cells (SOFAT). Further analysis of SOFAT revealed that it was derived from an unusual messenger RNA splice variant coded by the threonine synthase,like 2 gene homolog, which is a conserved gene remnant coding for threonine synthase, an enzyme that functions only in microorganisms and plants. Conclusion SOFAT may act to exacerbate inflammation and/or bone turnover under inflammatory conditions such as RA or periodontitis and in conditions of estrogen deficiency. [source] Quantitative measurement of collagen methylation by capillary electrophoresisELECTROPHORESIS, Issue 20 2004Jing Zhang Abstract Collagen methylation has been exploited in various applications involving living cells. We have observed correlation between the collagen methylation with the rate of cell proliferation in three-dimensional (3-D) microenvironment. To quantify the degree of collagen methylation, we have developed a capillary zone electrophoresis method. Using a polyvinyl alcohol-coated fused-silica capillary and UV detection at 200 nm, we have optimized pH and separated the native collagen into three major bands in phosphate buffer (50 mM, pH 2.5) with 0.05% hydroxypropylmethylcellulose. Under these conditions, the methylated collagens were separated into four major bands, which changed with different methylation reaction conditions. We propose an index to quantify the degree of collagen methylation that also correlates with their effects on cell proliferation. [source] Effect of humic material on the bacterioplankton community composition in boreal lakes and mesocosmsENVIRONMENTAL MICROBIOLOGY, Issue 5 2005Kaisa Haukka Summary The bacterioplankton community composition in two Finnish forest lakes with different content of humic substances was studied by denaturing gradient gel electrophoresis (DGGE) and sequencing of the major bands. The same dominant bacterial phylotypes were detected in the bacterioplankton communities of clear-water Lake Ahvenlammi and humic Lake Sammalisto. For 4 years, in every water layer, Actinobacteria was the dominant and Verrucomicrobia the second most common phylum. In the hypolimnion, other dominant phyla were also found. We set up a mesocosm experiment to assess the effect of a sudden load of allochthonous humus extract to the bacterioplankton community composition. Changes in the bacterial communities were followed in four control and four humus extract-added mesocosms for 50 days. In the humic mesocosms the phylotypes of allochthonous Proteobacteria arriving with the humus extract were initially prevalent but disappeared during the first weeks. After this the Actinobacteria -dominated communities resembled the bacterioplankton communities of the control mesocosms and Lake Ahvenlammi. Towards the end of the experiment the community patterns in all the mesocosms started to change slightly because of erratic occurrence of new proteobacterial phylotypes. Thus the effects of a sudden load of allochthonous humic material and bacteria to the bacterioplankton community composition were transient. [source] Bacillus pumilus SG2 isolated from saline conditions produces and secretes two chitinasesJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2007G. Ahmadian Abstract Aims: Isolation and characterization of chitinases from a halotolerant Bacillus pumilus. Methods and Results: Bacillus pumilus strain SG2 was isolated from saline conditions. It is able to produce chitinase activity at high salt concentration. SDS-PAGE analysis of the B. pumilus SG2 culture supernatant showed two major bands that were induced by chitin. The amino acid sequence of the two proteins, designated ChiS and ChiL, showed a high homology with the chitinase of B. subtilis CHU26, and chitinase A of B. licheniformis, respectively. N -terminal signal peptide of both proteins was also determined. The molecular weight and isoelectric point of the chitinases were determined to be 63 and 74 kDa, and 4·5 and 5·1, for ChiS and ChiL respectively. The genes encoding for both chitinases were isolated and their sequence determined. The regulation of the chitinase genes is under the control of the catabolite repression system. Conclusions: Secreted chitinase genes and their flanking region on the genome of B. pumilus SG2 have been identified and sequenced. Significance and Impact of the Study: This is the first report of a multiple chitinases-producing B. pumilus halotolerant strain. We have identified two chitinases by using a reverse genetics approach. The chitinases show resistance to salt. [source] Storage of biodegradable polymers by an enriched microbial community in a sequencing batch reactor operated at high organic load rateJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 11 2005Davide Dionisi Abstract The production of polyhydroxyalkanoates (PHAs) from organic acids by mixed bacterial cultures using a process based on aerobic enrichment of activated sludge, that selects for mixed microbial cultures able to store PHAs at high rates and yields, is described. Enrichment resulted from the selective pressure established by periodic feeding the carbon source in a sequencing batch reactor (SBR); a mixture of acetic, lactic and propionic acids was fed at high frequency (2 hourly), high dilution rate (1 d,1), and at high organic load rate (12.75 g chemical oxygen demand (COD) L,1 d,1). The performance of the SBR was assessed by microbial biomass and PHA production as well as the composition and polymer content of the biomass. A final batch stage was used to increase the polymer concentration of the excess sludge produced in the SBR and in which the behaviour of the biomass was investigated by determining PHA production rates and yields. The microbial biomass selected in the SBR produced PHAs at high rate [278 mg PHAs (as COD) g biomass (as COD),1 h,1, with a yield of 0.39 mg PHAs (as COD) mg removed substrates (as COD),1], reaching a polymer content higher than 50% (on a COD basis). The stored polymer was the copolymer poly(3-hydroxybutyrate/3-hydroxyvalerate) [P(HB/HV)], with an HV fraction of 18% mol mol,1. The microbial community selected in the SBR was analysed by DGGE (denaturing gradient gel electrophoresis). The operating conditions of the SBR were shown to select for a restricted microbial population which appeared quite different in terms of composition with respect to the initial microbial cenosis in the activated sludge used as inoculum. On the basis of the sequencing of the major bands in the DGGE profiles, four main genera were identified: a Methylobacteriaceae bacterium, Flavobacterium sp, Candidatus Meganema perideroedes, and Thauera sp. The effects of nitrogen depletion (ie absence of growth) and pH variation were also investigated in the batch stage and compared with the SBR operative mode. Absence of growth did not stimulate higher PHA production, so indicating that the periodic feed regime fully exploited the storage potential of the enriched culture. Polymer production rates remained high between pH 6.5 and 9.5, whereas the HV content in the stored polymer strongly increased as the pH value increased. This study shows that polymer composition in the final batch stage can readily be controlled independently from the feed composition in the SBR. Copyright © 2005 Society of Chemical Industry [source] Alteration of argyrophilic nucleolar organizer region associated (Ag-NOR) proteins in apoptosis-induced human salivary gland cells and human oral squamous carcinoma cellsJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 4 2001Yasuhiro Morimoto Abstract: The level of argyrophilic nucleolar organizer regions (AgNORs) and AgNOR-associated proteins (Ag-NOR proteins) varies with cell activity, including ribosomal biogenesis occurring in proliferating cells. Proteins associated with some AgNORs are detected by a specific silver staining. To investigate a possible relationship between apoptosis and the AgNORs or Ag-NOR proteins, we examined the changes of AgNORs and Ag-NOR proteins during apoptosis in a human salivary gland cell line, HSG cells, and a human oral squamous carcinoma cell line, SCC-25 cells. Apoptosis was induced by treatment of HSG and SCC-25 cells with okadaic acid. Proteins prepared from HSG and SCC-25 cells treated with varying concentrations of okadaic acid (OA) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to transfer membranes and staining for Ag-NOR proteins by modified Western blot analysis. Four major bands (110 kDa, 43 kDa, 39kDa, and 37 kDa) were detected in the proteins obtained from the control cells. The level of the 110-kDa protein decreased in the proteins prepared from OA-induced apoptotic cells; however, the reaction intensity of the other three bands was changed in apoptotic cells. An additional band of an 80-kDa Ag-NOR protein appeared and increased in the apoptotic cells. Cellular fractionation of HSG cells and SCC-25 cells was done with or without apoptotic induction. An 80-kDa Ag-NOR protein was detected in the nuclear fraction prepared from the apoptotic cells, while the 110-kDa protein decreased in the nuclear fraction of these cells. The 110-kDa Ag-NOR protein may be nucleolin (C23) as deduced from its AgNOR staining features, including molecular weight. The 80-kDa protein may be the cleavage product of the 110-kDa protein. In the cell-free apoptotic system, in which intact nuclei of HSG cells were incubated with the cytosol fraction of apoptotic HSG and SCC-25 cells, the 80-kDa Ag-NOR protein was detected in nuclei incubated with the cytosol fraction of apoptotic cells, while the level of the 110-kDa protein decreased. The changes of Ag-NOR proteins in nuclei prepared from SCC-25 cells incubated with cytosol fractions prepared from HSG and SCC-25 cells were identical to those of the HSG cells. The alternation of AgNORs in apoptosis-induced HSG cells was also examined using double staining with Hoechst 33342 and silver nitrate. Hoechst staining revealed typical apoptotic nuclei, which exhibited highly fluorescent condensed chromatin in OA-treated HSG cells. Silver grains representing AgNORs were not detected in the cells undergoing apoptosis. The dual-imposition view confirmed that AgNORs, which are visible as dots in nucleoli in the control cells, disappeared from the apoptotic nuclei of HSG cells. Our results indicate that the 110-kDa nucleolar Ag-NOR protein is associated with apoptosis and is cleaved during apoptosis. [source] Immunoreactivity of peptides generated by limited proteolysis of 71-kDa cell wall protein of Mycobacterium tuberculosis H37Ra using PLG-microparticlesLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2000N. Dhiman Peptide mapping by limited proteolysis of a highly protective 71-kDa cell wall-associated protein of Mycobacterium tuberculosis H37Ra was carried out in order to identify key protective determinants within the native protein. The 71-kDa protein, which had an isoelectric point of 4·25, was digested into eight major bands at 48 h using trypsin and pepsin at equal enzyme to protein ratios (pH 5·5). The in vitro lymphocyte reactivity of individual peptides suggested P1, P2 and P5 to be significantly immunoreactive in mice immunized with native 71-kDa-polylactide-coglyeolide (PLG); however, the reactivity was significantly lower than that of the native 71-kDa protein. Immunization of mice with a pooled fraction (upper fraction-71 kDa) of more immunoreactive peptides (consisting of P1 and P2) did not further boost their immunoreactivity. However, P1 and P2 exhibited comparable or even higher lymphocyte proliferation in human tuberculous and control subjects. These data suggest distinct antigenic specificities in humans and mice and further substantiate the use of the 71-kDa protein or its peptides P1 and P2 as potential vaccine candidates for tuberculosis. [source] | |||||||||||||