The role of genetic variants in CRP in radiographic severity in African Americans with early and established rheumatoid arthritis.

This study investigates the association of CRP (C-reactive protein) single-nucleotide polymorphisms (SNPs) with plasma CRP levels and radiographic severity in African Americans with early and established rheumatoid arthritis (RA). Using a cross-sectional case-only design, CRP SNPs were genotyped in two independent sets of African Americans with RA: Consortium for the Longitudinal Evaluation of African Americans with RA (CLEAR 1) and CLEAR 2. Radiographic data and CRP measurements were available for 294 individuals from CLEAR 1 (median (interquartile range (IQR) 25-75) disease duration of 1 (0.6-1.6) year) and in 407 persons from CLEAR 2 (median (IQR 25-75) disease duration of 8.9 (3.5-17.7) years). In CLEAR 1, in adjusted models, the minor allele of rs2808630 was associated with total radiographic score (incident rate ratio 0.37 (95% confidence interval (CI) 0.19-0.74), P-value=0.0051). In CLEAR 2, the minor allele of rs3093062 was associated with increased plasma CRP levels (P-value=0.002). For each rs3093062 minor allele, the plasma CRP increased by 1.51 (95% CI 1.15-1.95) mg dl(-1) when all the other covariates remained constant. These findings have important implications for assessment of the risk of joint damage in African Americans with RA.

Pseudomonas moraviensis subsp. stanleyae, a bacterial endophyte of hyperaccumulator Stanleya pinnata, is capable of efficient selenite reduction to elemental selenium under aerobic conditions.

AIMS: To identify bacteria with high selenium tolerance and reduction capacity for bioremediation of wastewater and nanoselenium particle production. METHODS AND RESULTS: A bacterial endophyte was isolated from the selenium hyperaccumulator Stanleya pinnata (Brassicaceae) growing on seleniferous soils in Colorado, USA. Based on fatty acid methyl ester analysis and multi-locus sequence analysis (MLSA) using 16S rRNA, gyrB, rpoB and rpoD genes, the isolate was identified as a subspecies of Pseudomonas moraviensis (97.3% nucleotide identity) and named P. moraviensis stanleyae. The isolate exhibited extreme tolerance to SeO3(2-) (up to 120 mmol l(-1)) and SeO4(2-) (>150 mmol l(-1)). Selenium oxyanion removal from growth medium was measured by microchip capillary electrophoresis (detection limit 95 nmol l(-1) for SeO3(2-) and 13 nmol l(-1) for SeO4(2-)). Within 48 h, P. moraviensis stanleyae aerobically reduced SeO3(2-) to red Se(0) from 10 mmol l(-1) to below the detection limit (removal rate 0.27 mmol h(-1) at 30 °C); anaerobic SeO3(2-) removal was slower. No SeO4(2-) removal was observed. Pseudomonas moraviensis stanleyae stimulated the growth of crop species Brassica juncea by 70% with no significant effect on Se accumulation. CONCLUSIONS: Pseudomonas moraviensis stanleyae can tolerate extreme levels of selenate and selenite and can deplete high levels of selenite under aerobic and anaerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas moraviensis subsp. stanleyae may be useful for stimulating plant growth and for the treatment of Se-laden wastewater.

Genetic variants associated with methotrexate efficacy and toxicity in early rheumatoid arthritis: results from the treatment of early aggressive rheumatoid arthritis trial.

Methotrexate (MTX) has emerged as first-line therapy for early moderate-to-severe rheumatoid arthritis (RA), but individual variation in treatment response remains unexplained. We tested the associations between 863 known pharmacogenetic variants and MTX response in 471 Treatment of Early Aggressive Rheumatoid Arthritis Trial participants with early RA. Efficacy and toxicity were modeled using multiple regression, adjusted for demographic and clinical covariates. Penalized regression models were used to test joint associations of markers and/or covariates with the outcomes. The strongest genetic associations with efficacy were in CHST11 (five markers with P<0.003), encoding carbohydrate (chondroitin 4) sulfotransferase 11. Top markers associated with MTX toxicity were in the cytochrome p450 genes CYP20A1 and CYP39A1, solute carrier genes SLC22A2 and SLC7A7, and the mitochondrial aldehyde dehydrogenase gene ALDH2. The selected markers explained a consistently higher proportion of variation in toxicity than efficacy. These findings could inform future development of personalized therapeutic approaches.

Genetic association of htSNPs across the major histocompatibility complex with rheumatoid arthritis in an African-American population.

Notwithstanding the well-established association of HLA-DRB1 shared epitope alleles, interest remains in identifying additional major histocompatibility complex (MHC) region variants associated with rheumatoid arthritis (RA). We used a panel of 1201 haplotype-tagging single nucleotide polymorphisms (SNPs) designed for African Americans to find genetic variants associated with RA in a 3.8-Mb region encompassing the MHC. Conditioning on seven covariates, including HLA-DRB1 risk alleles and population structure, we identified an SNP in HLA-DOA (rs9276977) significantly associated with RA; minor allele frequency (MAF) 0.27 in cases versus 0.21 in controls, odds ratio (+/-95% confidence interval)=2.86 (1.61, 5.31). Genotyping of rs9276977 in an independent sample of African-American RA patients and controls did not replicate the association (MAF 0.28 in cases versus 0.27 in controls). This study points to the potential association of a SNP in the HLA-DOA gene with RA in African Americans, but also underscores the importance of replication of findings in larger patient cohorts.

Causes of death in remote symptomatic epilepsy.

OBJECTIVE: To determine the causes of death of individuals with developmental disabilities that occur more frequently among those with remote symptomatic epilepsy (i.e., epilepsy occurring in persons with developmental delay or identified brain lesions) than for those without. METHODS: The authors compared causes of mortality in persons with (n = 10,030) and without (n = 96,163) history of epilepsy in a California population of persons with mild developmental disabilities, 1988 to 2002. Subjects had traumatic brain injury, cerebral palsy, Down syndrome, autism, or a developmental disability with other or unknown etiology. There were 721,759 person-years of data, with 2,397 deaths. Underlying causes of death were determined from the State of California's official mortality records. Cause-specific death rates and standardized mortality ratios (SMRs) were computed for those with and without epilepsy relative to subjects in the California general population. Comparisons were then made between SMRs of those with and without epilepsy, and CIs on the ratios of SMRs were determined. RESULTS: Death rates for persons with epilepsy were elevated for several causes. The greatest excess was due to seizures (International Classification of Diseases-9 [ICD-9] 345; SMR 53.1, 95% CI 28.0 to 101.0) and convulsions (ICD-9 780.3; SMR 25.2, 95% CI 11.7 to 54.2). Other causes occurring more frequently in those with epilepsy included brain cancer (SMR 5.2, 95% CI 2.2 to 12.1), respiratory diseases (SMR 1.7, 95% CI 1.2 to 2.5), circulatory diseases (SMR 1.3, 95% CI 1.0 to 1.7), and accidents (SMR 2.7, 95% CI 1.9 to 3.7), especially accidental drowning (SMR 12.8, 95% CI 7.0 to 23.2). CONCLUSIONS: Remote symptomatic epilepsy is associated with an increased risk of death. Seizures, aspiration pneumonia, and accidental drowning are among the leading contributors.

Accretion of low-metallicity gas by the Milky Way.

Models of the chemical evolution of the Milky Way suggest that the observed abundances of elements heavier than helium ('metals') require a continuous infall of gas with metallicity (metal abundance) about 0.1 times the solar value. An infall rate integrated over the entire disk of the Milky Way of approximately 1 solar mass per year can solve the 'G-dwarf problem'--the observational fact that the metallicities of most long-lived stars near the Sun lie in a relatively narrow range. This infall dilutes the enrichment arising from the production of heavy elements in stars, and thereby prevents the metallicity of the interstellar medium from increasing steadily with time. However, in other spiral galaxies, the low-metallicity gas needed to provide this infall has been observed only in associated dwarf galaxies and in the extreme outer disk of the Milky Way. In the distant Universe, low-metallicity hydrogen clouds (known as 'damped Ly alpha absorbers') are sometimes seen near galaxies. Here we report a metallicity of 0.09 times solar for a massive cloud that is falling into the disk of the Milky Way. The mass flow associated with this cloud represents an infall per unit area of about the theoretically expected rate, and approximately 0.1-0.2 times the amount required for the whole Galaxy.

A comparison of calcium phosphate coprecipitation and electroporation. Implications for studies on the genetic effects of DNA damage.

Plasmid-based transfection assays provide a rapid means to measure homologous and nonhomologous recombination in mammalian cells. Often it is of interest to examine the stimulation of recombination by DNA damage induced by radiation, genotoxic chemicals, or nucleases. Transfection is frequently performed by using calcium phosphate coprecipitation (CPP), because this method is well suited for handling large sample sets, and it does not require expensive reagents or equipment. Alternative transfection methods include lipofection, microinjection, and electroporation. Since DNA strand breaks are known to stimulate both homologous and nonhomologous recombination, the induction of nonspecific damage during transfection would increase background recombination levels and thereby reduce the sensitivity of assays designed to detect the stimulation of recombination by experimentally induced DNA damage. In this article, we compare the stimulatory effects of nuclease-induced double-strand breaks (DSBs) on homologous and nonhomologous recombination for molecules transfected by CPP and by electroporation. Although electroporation yielded fewer transfectants, both nonhomologous and homologous recombination were stimulated by nuclease-induced DSBs to a greater degree than with CPP. Ionizing radiation is an effective agent for inducing DNA strand breaks, but previous studies using CPP generally showed little or no stimulation of homologous recombination among plasmids damaged with ionizing radiation. By contrast, we found clear dose-dependent enhancement of recombination with irradiated plasmids transfected using electroporation. Thus, electroporation provides a higher signal-to-noise ratio for transfection-based studies of damage-induced recombination, possibly reflecting less nonspecific damage to plasmid DNA during transfection of mammalian cells.

Mycoplasma pneumoniae: a frequent cause of pneumonia among U.S. Marines in southern California.

From August 1993 through April 1994, U.S. Marines (98% male, median age 20 years) who were hospitalized with radiographically confirmed pneumonia were prospectively studied for evidence of acute Mycoplasma pneumoniae infection. Overall, 32 (36.4%) of the 88 patients with paired sera had evidence of acute infection by an elevated immunoglobulin M titer (22.7%), a 4-fold rise in immunoglobulin G titer (9.1%), a positive polymerase chain reaction result (11.1%), and/or a positive culture (5.8%). No specific symptoms or clinical findings were strong predictors of M. pneumoniae infection. Among patients with evidence of acute M. pneumoniae infection, admitting clinicians chose other pathogens as more likely etiologic agents 46.4% of the time, and over the course of the hospitalization, 10% of patients failed to receive appropriate antibiotics. These data indicate that M. pneumoniae may cause a high proportion of pneumonias among military personnel and should be considered in empiric treatment and prophylaxis.

Fabry-Perot CCD annular-summing spectroscopy: study and implementation for aeronomy applications.

The technique of Fabry-Perot CCD annular-summing spectroscopy, with particular emphasis on applications in aeronomy, is discussed. Parameter choices for optimizing performance by the use of a standard format CCD array are detailed. Spectral calibration methods, techniques for determining the ring pattern center, and effects imposed by limited radial resolution caused by superpixel size, variable by on-chip binning, are demonstrated. The technique is carefully evaluated experimentally relative to the conventional scanning Fabry-Perot that uses a photomultiplier detector. We evaluate three extreme examples typical of aeronomical spectroscopy using calculated signal-to-noise ratios. Predicted sensitivity gains of 10-30 are typical. Of the cases considered, the largest savings in integration time are estimated for the day sky thermospheric O(1)D case, in which the bright sky background dominates the CCD read noise. For profile measurements of faint night sky emission lines, such as exospheric hydrogen Balmer-α, long integration times are required to achieve useful signal-to-noise ratios. In such cases, CCD read noise is largely overcome. Predictions of a factor of 10-15 savings in integration time for night sky Balmer-α observations are supported by field tests. Bright, isolated night sky lines such as thermospheric O(1)D require shorter integration times, and more modest gains dependent on signal level are predicted. For such cases it appears from estimate results that the Fabry-Perot CCD annular-summing technique with a conventional rectangular format may be outperformed by a factor of 2-5 by special CCD formats or by unusual optical coupling configurations that reduce the importance of read noise, based on the ideal transmission for any additional optics used in these configurations.

Patterns of morphological variation within myelin internodes of normal peripheral nerve: quantitative analysis by confocal microscopy.

Knowledge of variations in the morphology of normal myelinated peripheral nerve fibres is fundamental to subsequent interpretation of neuropathology. It would be advantageous for structural analysis of normal variations to be based on entire myelin internodes, but acquisition of such data via the classic approach of nerve fibre teasing has been hindered by limitations in optical resolution and specimen preparation. This study addressed these limitations through a new confocal imaging method which permits detailed visualisation of individual myelinated fibres in intact peripheral nerve trunks, and quantitated previously unrecognised patterns of morphological variation within normal internodes. The study focused particularly on Schmidt-Lanterman incisures, the narrow cytoplasmic channels which traverse normal compact myelin and provide foci for disruption of the compact sheath in a number of peripheral neuropathies. Analysis was based on confocal fluorescence images of multiple sequential internodes, traced within posterior tibial nerve trunks of adult male Sprague-Dawley rats. The strength of relationships between internodal size variables (length, fibre diameter, myelin sheath thickness) and total number of incisures per internode were documented. Each internode was divided into 4 regions of equivalent length (regions 1-4), and variations in the distribution of incisures and Schwann cell nuclear location were evaluated. Regional variations were consistent, irrespective of differences in fibre diameter, myelin sheath thickness, and internodal length. Expressed in terms of proximodistal orientation, there was a unimodal distribution of incisures within internodes of this fibre population (diameter range 5-9 microns), with region 3 containing the highest number of incisures and region 4 the lowest (P < 0.05). The Schwann cell nucleus was located more frequently in region 3 than in region 2 (P < 0.01). Contrary to previous reports, an incisure was found in close association with the nucleus in at least 50% of internodes. Documentation of frequent incisure-nuclear association and consistent patterns of variation within internodes extends knowledge of the microanatomy of normal peripheral nerve, and may provide insight into the functional role of incisures. Demonstration of such patterns in normal nerve may contribute to the understanding of pathological change, for example progression of ovoid formation from midinternodal regions during wallerian degeneration.

Characterization of a mammalian cell line that exhibits spontaneous and ultraviolet light-induced hypermutability while retaining resistance to cell killing by ultraviolet light.

Chronic exposure of V79 cells to 80 daily doses of 150 J/M2, 290-330-nm ultraviolet light (UVB) produced a mixed cell population that was found to be generally more resistant to cell killing by both UVB and UVC (254 nm) than the wild-type cells. Several subclones from this population were studied for their survival and mutation responses and then one was chosen for further characterization based on this data. The studies carried out on this subclone, designated N806, show that its spontaneous HPRT mutation rate is approximately 10 times higher than that of wild-type V79 cells and it is almost three times more mutable than the wild-type cells when both are induced by UVB or UVC. The mutation responses of N806 and MI2G cells to 50-kVp X-rays are different, but the N806 cells do not appear to be hypermutable as they are with UV. N806 cells are also moderately more resistant to the cytotoxic effects of UV radiation but are more sensitive than MI2G cells when exposed to X-rays. Assays to measure the removal of cyclobutane pyrimidine dimers (CPDs) and the incision step of nucleotide excision repair have revealed no detectable difference in the repair capacities of N806 and parental V79 cells. These results suggest that chronic, protracted UV irradiation may be able to induce a 'mutator phenotype' in a subpopulation of the progenitor cells.

Imaging myelinated nerve fibres by confocal fluorescence microscopy: individual fibres in whole nerve trunks traced through multiple consecutive internodes.

Current methods of morphological analysis do not permit detailed imaging of individual myelinated fibres over substantial lengths without disruption of neighbouring, potentially significant, cellular and extracellular relationships. We report a new method which overcomes this limitation by combining aldehyde-induced fluorescence with confocal microscopy. Myelin fluorescence was intense relative to that from other tissue components, enabling individual myelinated nerve fibres to be traced for distances of many millimeters in whole PNS nerve trunks. Image obtained with a Bio-Rad MRC-600 confocal laser scanning microscope clearly displayed features of PNS and CNS myelinated fibres including nodes of Ranvier; fibre diameter; sheath thickness and contour; branch points at nodes; as well as (in the PNS) Schmidt-Lanterman incisures and the position of Schwann cell nuclei. Direct comparisons using the same specimens (whole nerve trunks; also teased fibres) showed confocal imaging to be markedly superior to conventional fluorescence microscopy in terms of contrast, apparent resolution and resistance to photobleaching. Development of the fluorophore was examined systemically in sciatic nerves of young adult rats. In separate experiments, animals were perfused systemically using (1) 5% glutaraldehyde; (2) Karnovsky's solution; (3) 4% paraformaldehyde; buffered with either 0.1 M sodium phosphate or sodium cacodylate (pH 7.4). The concentration of glutaraldehyde in the fixative solution was the principal determinant of fluorescence intensity. Confocal imaging was achieved immediately following perfusion with 5% glutaraldehyde or Karnovsky's. Fluorescence intensity increased markedly during overnight storage in these fixatives and continued to increase during subsequent storage in buffer alone. The fluorophore was stable and resistant to fading during storage (15 months at least), enabling data collection over extended periods. To demonstrate application of the method in neuropathology, individual fibres in transected sciatic nerve trunks were traced through multiple successive internodes: Classical features of Wallerian degeneration (axonal swelling and debris; ovoid formation and incisure changes; variation among fibres in the extent of degeneration) were displayed. The method is compatible with subsequent ultrastructural examination and will complement existing methods of investigation of myelinated fibre anatomy and pathology, particularly where preservation of 3-dimensional relationships or elucidation of spatial gradients are required.

Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Several human genes related to DNA excision repair (ER) have been isolated via ER cross-species complementation (ERCC) of UV-sensitive CHO cells. We have now isolated and characterized cDNAs for the human ERCC5 gene that complement CHO UV135 cells. The ERCC5 mRNA size is about 4.6 kb. Our available cDNA clones are partial length, and no single clone was active for UV135 complementation. When cDNAs were mixed pairwise with a cosmid clone containing an overlapping 5'-end segment of the ERCC5 gene, DNA transfer produced UV-resistant colonies with 60 to 95% correction of UV resistance relative to either a genomic ERCC5 DNA transformant or the CHO AA8 progenitor cells. cDNA-cosmid transformants regained intermediate levels (20 to 45%) of ER-dependent reactivation of a UV-damaged pSVCATgpt reporter plasmid. Our evidence strongly implicates an in situ recombination mechanism in cDNA-cosmid complementation for ER. The complete deduced amino acid sequence of ERCC5 was reconstructed from several cDNA clones encoding a predicted protein of 1,186 amino acids. The ERCC5 protein has extensive sequence similarities, in bipartite domains A and B, to products of RAD repair genes of two yeasts, Saccharomyces cerevisiae RAD2 and Schizosaccharomyces pombe rad13. Sequence, structural, and functional data taken together indicate that ERCC5 and its relatives are probable functional homologs. A second locus represented by S. cerevisiae YKL510 and S. pombe rad2 genes is structurally distinct from the ERCC5 locus but retains vestigial A and B domain similarities. Our analyses suggest that ERCC5 is a nuclear-localized protein with one or more highly conserved helix-loop-helix segments within domains A and B.

Electroporation-mediated gene transfer efficiency is reduced by linear plasmid carrier DNAs.

Carrier DNA has generally been found to stimulate DNA-mediated gene transfer of Chinese hamster ovary (CHO) cells by calcium phosphate coprecipitation. In studies employing electroporation, however, we observed that linear plasmid DNA was inhibitory to the transfection of CHO cells. This unexpected result prompted us to explore the effects of various types and forms of plasmid, cosmid, and chromosomal DNAs on transfection efficiencies. Both carrier DNA form and type were found to influence transfection efficiencies. Circular and linear forms of plasmid carrier DNA had opposite effects: circular plasmids increased and linear plasmids decreased transfection efficiencies. These effects were independent of homology with the selected plasmid and are probably independent of homologous recombination mechanisms. Bacterial genomic DNA failed to stimulate transfection, while calf thymus and cosmid DNA consisting primarily of human sequences stimulated transfection significantly. Our results have importance for plasmid-based experiments in mammalian cells such as those involving the induction of interplasmid homologous recombination.

Transcription stimulates homologous recombination in mammalian cells.

Transcription stimulates homologous recombination in Saccharomyces cerevisiae and has been implicated in the control of recombinational events during the development of mammalian immune systems. Here, we describe a plasmid-based system in which an inducible promoter from the mouse mammary tumor virus is located upstream of heteroallelic neomycin genes carried on two plasmids. Pairs of plasmids are introduced into Chinese hamster ovary cells by electroporation, and recombination is monitored by scoring colonies resistant to the aminoglycoside G418. When transcription is induced with dexamethasone, a synthetic glucocorticoid hormone, and double-strand breaks are introduced at mutation sites, recombination is stimulated sixfold over noninduced levels. Inducing transcription in circular substrates or in substrates cleaved at sites distant from the mutations has no detectable effect on recombination between neomycin genes. Results are presented that are consistent with the observed stimulation of recombination occurring before plasmids integrate into the cellular DNA. Our results are discussed in relation to molecular models for extrachromosomal recombination in mammalian cells.

Effect of duration of exposure to benzo(a)pyrene diol-epoxide on neoplastic transformation, mutagenesis, cytotoxicity, and total covalent binding to DNA of rodent cells.

We examined the effect of different durations of exposure (20 sec to 24 hr) to (+/-) 7-beta,8 alpha-dihydroxy-9 alpha, 10 alpha -epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE I) on the induction of transformation in C3H/10T 1/2 cells and of mutations to 6-thioguanine resistance in Chinese hamster ovary cells (CHO), as well as on BPDE I-DNA binding in these two cell lines. A 20-sec exposure of the cells to BPDE I was sufficient to induce mutations and morphological transformation in vitro. However, the transformation frequency in CH3 mouse-embryo-derived 10T 1/2 cells increased twofold and the frequency of mutations in CHO cells sixfold when the exposure time to BPDE I was increased from 20 sec to 8 h. Cytotoxicity increased under similar conditions. A large number of BPDE I-DNA adducts were formed in both cell lines within the first 15-min of exposure of the cells to this ultimate carcinogen. The total covalent binding did not increase with longer than 15-min incubation times. These results suggest that in addition to its covalent binding to DNA, BPDE I may influence other cellular mechanism(s) that are responsible for the initiation of transformation and mutagenesis.

Induction and repair of closely opposed pyrimidine dimers in Saccharomyces cerevisiae.

Pyrimidine dimer-DNA glycosylase activity prepared from Micrococcus luteus has been used to develop an enzyme-sensitive site assay for the detection and quantification of closely opposed pyrimidine dimers in the nuclear DNA of UV-irradiated yeast. With this assay, closely opposed dimers were found to be induced as a linear function of dose from 0 to 200 J/m2 (254 nm). Closely opposed dimer frequencies decreased during the incubation of UV-irradiated, excision repair-proficient cells under liquid-holding conditions in the dark and during post-irradiation exposure of excision-deficient cells to photoreactivating light. Incubation of excision-deficient cells in the dark had no effect on the frequency of closely opposed dimers for up to 16 h. These results indicate that closely opposed dimers in UV-irradiated yeast are subject to repair by enzymatic photoreactivation and/or by dark-repair processes dependent, at least in part, upon functions necessary for normal excision repair. The genetic and biochemical implications of these results are discussed.