CC-chemokine receptor 2 required for bleomycin-induced pulmonary fibrosis

MCP-1, which signals via the CC chemokine receptor 2 (CCR2), is induced in lung fibrosis that is accompanied by mononuclear cell recruitment and activation of lung fibroblasts. To evaluate the role of CCR2 in lung fibrosis, CCR2 knockout (ko) mice were used in a model of bleomycin-induced lung fibrosis. Wild type (wt) and ko mice were injected endotracheally with bleomycin to induce lung injury and fibrosis, and then analyzed for degree of lung fibrosis and cytokine expression. The results showed significantly reduced fibrosis in ko mice as evidenced by decreased lung type I collagen gene expression and hydroxyproline content relative to those in wt mice. Lung TNF-alpha and TGF-beta1 expression was significantly lower in ko vs. wt mice, while MCP-1 expression was unaffected. Interestingly, lung alpha-smooth muscle actin (alpha-SMA) expression, a marker for myofibroblast differentiation, was also decreased in ko mice, which was confirmed by analysis of isolated lung fibroblasts. Fibroblasts from ko mice exhibited decreased responsiveness to TGF-beta1 induced alpha-SMA expression, which was associated with reduced expression of TGF-beta receptor II (TbetaRII) and Smad3. These findings suggest that CCR2 signaling plays a key role in bleomycin-induced pulmonary fibrosis by regulating fibrogenic cytokine expression and fibroblast responsiveness to TGF-beta.     

Effect of plasterboard composition on Stachybotrys chartarum growth and biological activity of spores

The effects of plasterboard composition on the growth and sporulation of Stachybotrys chartarum as well as on the inflammatory potential of the spores were studied. S. chartarum was grown on 13 modified plasterboards under saturated humidity conditions. The biomass was estimated by measuring the ergosterol content of the S. chartarum culture while the spore-induced cytotoxicity and production of nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), and interleukin-6 in mouse macrophages was used to illustrate the bioactivity of spores. The ergosterol content of S. chartarum correlated with the number of spores collected from plasterboards. The growth and sporulation decreased compared to that of the reference board in those cases where (i) the liner was treated with biocide, (ii) starch was removed from the plasterboard, or (iii) desulfurization gypsum was used in the core. Spores collected from all the plasterboards were toxic to the macrophages. The biocide added to the core did not reduce the growth; in fact, the spores collected from that board evoked the highest cytotoxicity. The conventional additives used in the core had inhibitory effects on growth. Recycled plasterboards used in the core and the board lacking the starch triggered spore-induced TNF-alpha production in macrophages. In summary, this study shows that the growth of a strain of S. chartarum on plasterboard and the subsequent bioactivity of spores were affected by minor changes to the composition of the core or liners, but it could not be totally prevented without resorting to the use of biocides. However, incomplete prevention of microbial growth by biocides even increased the cytotoxic potential of the spores.     

Isolation of antigens and antibodies by affinity chromatography

Antibody-antigen binding constants are commonly strong enough for an effective affinity purification of antibodies (by immobilized antigens) or antigens (by immobilized antibodies) to work out a straightforward purification method. A drawback is that antibodies are large protein molecules and subject to denaturation under conditions required for the elution from the complex. Structures of antigens can vary but usually antigens are also equally subject to similar problems. The lability of the components can sometimes make the procedure sophisticated, but usually in all cases it is possible to find a satisfactory approach. In certain cases, specific interactions of the Fc part of antibodies are more facile to exploit for their purification.     

A study of immunoglobulin G glycosylation in monoclonal and polyclonal species by electrospray and matrix-assisted laser desorption/ionization mass spectrometry

N-linked oligosaccharides were released from human and bovine polyclonal immunoglobulin G (IgG) obtained from commercial sources and also from a monoclonal IgG(1) secreted by murine B-lymphocyte hybridoma cells (CC9C10) grown under different serum-free conditions. These conditions differed according to their steady-state dissolved oxygen concentrations. This work is based on a previous quantitative study where released glycans were characterized by fluorophore-assisted carbohydrate electrophoresis (FACE) and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (J. P. Kunkel, D. C. H. Jan, J. C. Jamieson, and M. Butler, 1998, J. Biotechnol. 62, 55-71). In the present article, peptide-N-glycosidase F-released glycans from different species of polyclonal IgG and murine monoclonal IgG were characterized qualitatively by high-performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS). The glycans were also analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The MALDI mass spectrometer used allowed acquisition of MS and tandem MS data, which were useful in structural investigations at a more detailed level than allowed by FACE and HPAEC-PAD. Predominant N-linked structures, as determined by all techniques, were core-fucosyl asialyl biantennary chains with varying galactosylation. Minor amounts of afucosyl, bisected, and monosialyl oligosaccharides were also detected. In contrast to FACE and HPAEC-PAD, MALDI-double quadrupole/time-of-flight MS and HPLC/ESI-MS also detected low-abundance high-mannose and hybrid structures in some of the species under investigation.     

Degradation of humic acids by the litter-decomposing basidiomycete Collybia dryophila

The basidiomycete Collybia dryophila K209, which colonizes forest soil, was found to decompose a natural humic acid isolated from pine-forest litter (LHA) and a synthetic (14)C-labeled humic acid ((14)C-HA) prepared from [U-(14)C]catechol in liquid culture. Degradation resulted in the formation of polar, lower-molecular-mass fulvic acid (FA) and carbon dioxide. HA decomposition was considerably enhanced in the presence of Mn(2+) (200 microM), leading to 75% conversion of LHA and 50% mineralization of (14)C-HA (compared to 60% and 20%, respectively, in the absence of Mn(2+)). There was a strong indication that manganese peroxidase (MnP), the production of which was noticeably increased in Mn(2+)-supplemented cultures, was responsible for this effect. The enzyme was produced as a single protein with a pI of 4.7 and a molecular mass of 44 kDa. During solid-state cultivation, C. dryophila released substantial amounts of water-soluble FA (predominantly of 0.9 kDa molecular mass) from insoluble litter material. The results indicate that basidiomycetes such as C. dryophila which colonize forest litter and soil are involved in humus turnover by their recycling of high-molecular-mass humic substances. Extracellular MnP seems to be a key enzyme in the conversion process.     

Evidence for sequential action of two ATPase active sites in yeast Msh2-Msh6

Bacterial MutS homodimers contain two ATPase active sites that have non-equivalent functions in DNA mismatch repair. The homologous Msh2-Msh6 complex in eukaryotes also has intrinsic ATPase activity that is essential for mismatch repair. Here, we investigate differences in the two putative ATPase active sites by examining the properties of heterodimers containing alanine substituted for an invariant glutamic acid in the active site of either Msh2, Msh6 or both. Mutation rates in wild type versus Glu-->Ala mutant haploid yeast strains indicate that both ATPase active sites are essential for mismatch repair activity in vivo. The properties of purified heterodimers suggest that the ATPase active site in Msh6 binds ATP with higher affinity and hydrolyzes ATP faster and with higher efficiency than does the ATPase active site in Msh2. This suggests sequential action of the two ATPase active sites, in which ATP binds to Msh6 first to trigger downstream events in mismatch repair.     

Quantitation of latex allergens

Minimizing allergen concentration in latex goods to prevent sensitization to natural rubber latex (NRL) and thereby the development of clinical allergy is acknowledged as of mutual interest for rubber manufacturers and regulatory health authorities. However, measuring total protein, the principal currently available method, cannot be deemed a satisfactory regulatory measure to control allergen content. Specific methods based on human IgE-containing reagents, such as radioallergosorbent test (RAST) inhibition, have been available in certain laboratories for demonstrating NRL allergens in rubber products but the methods lack standardization. Currently, one commercial test has become available for measuring individual NRL allergens by capture ELISA-based assays using monoclonal antibodies and purified or recombinant allergens. Such methods are specific, they can be properly standardized, and they are of sufficient sensitivity and reproducibility. Results from medical gloves collected in two national market surveys in Finland in 1995 and 1999, respectively, show that Hev b 6.02 and Hev b 5, the two major allergens for NRL-allergic adults, are the most abundant allergens regularly detectable in high- and moderate-allergen gloves. In addition, Hev b 3 and Hev b 1, the two major allergens for children with spina bifida, are also commonly found. In general, when the sum of the four allergens exceeded 1 microg/g, most NRL-allergic patients showed positive skin prick test reactions against them. Using these new methods assessment of threshold levels that could in due course become guidelines for the rubber industry and regulatory health authorities is becoming possible. Eventually, this progress is expected to lead to a declining incidence of latex allergy.     

Low fidelity DNA synthesis by a y family DNA polymerase due to misalignment in the active site

Sulfolobus solfataricus DNA polymerase IV (Dpo4) is a member of the Y family of DNA polymerases whose crystal structure has recently been solved. As a model for other evolutionarily conserved Y family members that perform translesion DNA synthesis and have low fidelity, we describe here the base substitution and frameshift fidelity of DNA synthesis by Dpo4. Dpo4 generates all 12 base-base mismatches at high rates, 11 of which are similar to those of its human homolog, DNA polymerase kappa. This result is consistent with the Dpo4 structure, implying lower geometric selection for correct base pairs. Surprisingly, Dpo4 generates C.dCMP mismatches at an unusually high average rate and preferentially at cytosine flanked by 5'-template guanine. Dpo4 also has very low frameshift fidelity and frequently generates deletions of even noniterated nucleotides, especially cytosine flanked by a 5'-template guanine. Both unusual features of error specificity suggest that Dpo4 can incorporate dNTP precursors when two template nucleotides are present in the active site binding pocket. These results have implications for mutagenesis resulting from DNA synthesis by Y family polymerases.