Structural analysis of three His32 mutants of DsbA: Support for an electrostatic role of His32 in DsbA stability

DsbA, a 21-kDa protein from Escherichia coli, is a potent oxidizing disulfide catalyst required for disulfide bond formation in secreted proteins. The active site of DsbA is similar to that of mammalian protein disulfide isomerases, and includes a reversible disulfide bond formed from cysteines separated by two residues (Cys3O-Pro31-His32-Cys33). Unlike most protein disulfides, the active-site disulfide of DsbA is highly reactive and the oxidized form of DsbA is much less stable than the reduced form at physiological pH. His32, one of the two residues between the active-site cysteines, is critical to the oxidizing power of DsbA and to the relative instability of the protein in the oxidized form. Mutation of this single residue to tyrosine, serine, or leucine results in a significant increase in stability (of similar to 5-7 kcal/mol) of the oxidized His32 variants relative to the oxidized wild-type protein. Despite the dramatic changes in stability, the structures of all three oxidized DsbA His32 Variants are very similar to the wild-type oxidized structure, including conservation of solvent atoms near the active-site residue, Cys3O. These results show that the His32 residue does not exert a conformational effect on the structure of DsbA. The destabilizing effect of His32 on oxidized DsbA is therefore most likely electrostatic in nature.

Candidate-Gene Approach in Posttraumatic Stress Disorder After Urban Violence: Association Analysis of the Genes Encoding Serotonin Transporter, Dopamine Transporter, and BDNF

Posttraumatic stress disorder (PTSD) is a prevalent, disabling anxiety disorder marked by behavioral and physiologic alterations which commonly follows a chronic course. Exposure to a traumatic event constitutes a necessary, but not sufficient, factor. There is evidence from twin studies supporting a significant genetic predisposition to PTSD. However, the precise genetic loci still remain unclear. The objective of the present study was to identify, in a case-control study, whether the brain-derived neurotrophic factor (BDNF) val66met polymorphism (rs6265), the dopamine transporter (DAT1) three prime untranslated region (3`UTR) variable number of tandem repeats (VNTR), and the serotonin transporter (5-HTTPRL) short/long variants are associated with the development of PTSD in a group of victims of urban violence. All polymorphisms were genotyped in 65 PTSD patients as well as in 34 victims of violence without PTSD and in a community control group (n = 335). We did not find a statistical significant difference between the BDNF val66met and 5-HTTPRL polymorphism and the traumatic phenotype. However, a statistical association was found between DAT1 3`UTR VNTR nine repeats and PTSD (OR = 1.82; 95% CI, 1.20-2.76). This preliminary result confirms previous reports supporting a susceptibility role for allele 9 and PTSD.

Mutation analysis of the CDKN2A promoter in Australian melanoma families

Approximately 50% of all melanoma families worldwide show linkage to 9p21-22, but only about half of these have been shown to contain germ line CDKN2A mutations. It has been hypothesized that a proportion of these families carry mutations in the noncoding regions of CDKN2A. Several Canadian families have been reported to carry a mutation in the 5' UTR, at position -34 relative to the start site, which gives rise to a novel AUG translation initiation codon that markedly decreases translation from the wild-type AUG (Liu et al., 1999). Haplotype sharing in these Canadian families suggested that this mutation is of British origin. We sequenced 1,327 base pairs (bp) of CDKN2A, making up 1,116 bp of the 5' UTR and promoter, all of exon 1, and 61 bp of intron 1, in at least one melanoma case from 110 Australian families with three or more affected members known not to carry mutations within the p16 coding region. In addition, 431 bp upstream of the start codon was sequenced in an additional 253 affected probands from two-case melanoma families for which the CDKN2A mutation status was unknown. Several known polymorphisms at positions -33, -191, -493, and -735 were detected, in addition to four novel variants at positions 120, -252, -347, and -981 relative to the start codon. One of the probands from a two-case family was found to have the previously reported Q50R mutation. No family member was found to carry the mutation at position -34 or any other disease-associated mutation. For further investigation of noncoding CDKN2A mutations that may affect transcription, allele-specific expression analysis was carried out in 31 of the families with at least three affected members who showed either complete or indeterminate 9p haplotype sharing without CDKN2A exonic mutations. Reverse transcription polymerase chain reaction and automated sequencing showed expression of both CDKN2A alleles in all family members tested. The lack of CDKN2A promoter mutations and the absence of transcriptional silencing in the germ line of this cohort of families suggest that mutations in the promoter and 5' UTR play a very limited role in melanoma predisposition. (C) 2001 Wiley-Liss, Inc.

The human melanocortin-1 receptor locus: analysis of transcription unit, locus polymorphism and haplotype evolution

The complete sequence of the MCIR locus has been assembled, the coding region of the gene is intronless and placed within a 12 kb region flanked by the NULP1 and TUBB4 genes. The immediate promoter region has an E-box site with homology to the M-box consensus known to bind the microphthalmia transcription factor (MITF), however, promoter deletion analysis and transactivation studies have failed to show activation through this element by MITF. Polymorphism within the coding region, immediate 5' promoter region and a variable number tandem repeat (VNTR) minisatellite within the locus have been examined in a collection of Caucasian families and African individuals. Haplotype analysis shows linkage disequilibrium between the VNTR and MCIR coding region red hair variant alleles which can be used to estimate the age of these missense changes. Assuming a mean VNTR mutation rate of 1% and a star phylogeny, we estimate the Arg151Cys variant arose 7500 years before the present day, suggesting these variants may have arisen in the Caucasian population more recently than previously thought. (C) 2001 Published by Elsevier Science B.V.

A longitudinal analysis of sex differences in bone mineral accrual in healthy 8-19-year-old boys and girls

Background: Although early in life there is little discernible difference in bone mass between boys and girls, at puberty sex differences are observed. It is uncertain if these differences represent differences in bone mass or just differences in anthropometric dimensions. Aim: The study aimed to identify whether sex independently affects bone mineral content (BMC) accrual in growing boys and girls. Three sites are investigated: total body (TB), femoral neck (FN) and lumbar spine (LS). Subjects and methods: 85 boys and 67 girls were assessed annually for seven consecutive years. BMC was assessed by dual energy X-ray absorptiometry (DXA). Biological age was defined as years from age at peak height velocity (PHV). Data were analysed using a hierarchical (random effects) modelling approach. Results: When biological age, body size and body composition were controlled, boys had statistically significantly higher TB and FN BMC at all maturity levels (p < 0.05). No independent sex differences were found at the LS (p > 0.05). Conclusion: Although a statistical significant sex effect is observed, it is less than the error of the measurement, and thus sex difference are debatable. In general, sex difference are explained by anthropometric difference

Penicillium glabrum cork colonising isolates – preliminary analysis of their genomic similari

Cork stopper manufacturing process includes an operation, known as stabilisation, by which humid cork slabs are extensively colonised by fungi. The effects of fungal growth on cork are yet to be completely understood and are considered to be involved in the so called “cork taint” of bottled wine. It is essential to identify environmental constraints which define the appearance of the colonising fungal species and to trace their origin to the forest and/or as residents in the manufacturing space. The present article correlates two sets of data, from consecutive years and the same season, of systematic biologic sampling of two manufacturing units, located in the North and South of Portugal. Chrysonilia sitophila dominance was identified, followed by a high diversity of Penicillium species. Penicillium glabrum, found in all samples, was the most frequent isolated species. P. glabrum intra-species variability was investigated using DNA fingerprinting techniques revealing highly discriminative polymorphic markers in the genome. Cluster analysis of P. glabrum data was discussed in relation to the geographical location of strains, and results suggest that P. glabrum arise from predominantly the manufacturing space, although cork resident fungi can also contrib

Morphometric Analysis of the Extensor Tendons of the Hallux and Potential Implications for Tendon Grafting

Although several tendon sources are available for reconstructive surgical procedures, all have one or more shortcomings. The aim of this work was to evaluate if the extensor tendons of the hallux showed anatomical characteristics that could make them an additional source for tendon grafting procedures.The authors performed a detailed morphometric analysis of the extensor tendons of the hallux in 26 lower limbs in order to evaluate the putative association of anatomical variants with hallux valgus, and to attempt to assess the feasibility of using part of the extensor apparatus of the hallux as a source of tendon for grafting procedures.An accessory extensor hallucis longus ten-don was found in 92.3% of cases. The extensor hallucis brevis tendon length was 10.5 ± 0.6 cm; its width was 0.5 ± 0.1 cm, and its thickness varied between 1-2 mm, making it a potentially good candidate as a source of ten-don grafts. Several anatomical variations were observed, namely the fusion of the tendons of the extensor hallucis brevis and the accessory extensor hallucis longus muscles in the distal part of the foot.This new therapeutic option, if implemented, would possibly increase the supply of autogenous donor tissue for reconstructive procedures, thereby enhancing the reconstructive surgeon’s armamentarium.

Analysis of Highly Conserved Regions of the 3'UTR of MECP2 Gene in Patients with Clinical Diagnosis of Rett Syndrome and Other Disorders Associated with Mental Retardation

In this work we explored the role of the 3'UTR of the MECP2 gene in patients with clinical diagnosis of RTT and mental retardation; focusing on regions of the 3'UTR with almost 100% conservation at the nucleotide level among mouse and human. By mutation scanning (DOVAM-S technique) the MECP2 3'UTR of a total of 66 affected females were studied. Five3'UTR variants in the MECP2 were found (c.1461+9G>A, c.1461+98insA, c.2595G>A, c.9961C>G and c.9964delC) in our group of patients. None of the variants found is located in putative protein-binding sites nor predicted to have a pathogenic role. Our data suggest that mutations in this region do not account for a large proportion of the RTT cases without a genetic explanation.

Does Ad Hoc Coronary Intervention Reduce Radiation Exposure? – Analysis of 568 Patients

Background:Advantages and disadvantages of ad hoc percutaneous coronary intervention have been described. However little is known about the radiation exposure of that procedure as compared with the staged intervention.Objective:To compare the radiation dose of the ad hoc percutaneous coronary intervention with that of the staged procedureMethods:The dose-area product and total Kerma were measured, and the doses of the diagnostic and therapeutic procedures were added. In addition, total fluoroscopic time and number of acquisitions were evaluated.Results:A total of 568 consecutive patients were treated with ad hoc percutaneous coronary intervention (n = 320) or staged percutaneous coronary intervention (n = 248). On admission, the ad hoc group had less hypertension (74.1% vs 81.9%; p = 0.035), dyslipidemia (57.8% vs. 67.7%; p = 0.02) and three-vessel disease (38.8% vs. 50.4%; p = 0.015). The ad hoc group was exposed to significantly lower radiation doses, even after baseline characteristic adjustment between both groups. The ad hoc group was exposed to a total dose-area product of 119.7 ± 70.7 Gycm2, while the staged group, to 139.2 ± 75.3 Gycm2 (p < 0.001).Conclusion:Ad hoc percutaneous coronary intervention reduced radiation exposure as compared with diagnostic and therapeutic procedures performed at two separate times.

β-Glucan antigenemia assay for the diagnosis of invasive fungal infections in patients with hematological malignancies: a systematic review and meta-analysis of cohort studies from the Third European Conference on Infections in Leukemia (ECIL-3).

BACKGROUND: Invasive fungal infections (IFIs) are life-threatening complications in patients with hemato-oncological malignancies, and early diagnosis is crucial for outcome. The compound 1,3-β-D-glucan (BG), a cell wall component of most fungal species, can be detected in blood during IFI. Four commercial BG antigenemia assays are available (Fungitell, Fungitec-G, Wako, and Maruha). This meta-analysis from the Third European Conference on Infections in Leukemia (ECIL-3) assessed the performance of BG assays for the diagnosis of IFI in hemato-oncological patients. METHODS: Studies reporting the performance of BG antigenemia assays for the diagnosis of IFI (European Organization for Research and Treatment of Cancer and Mycoses Study Group criteria) in hemato-oncological patients were identified. The analysis was focused on high-quality cohort studies with exclusion of case-control studies. Meta-analysis was performed by conventional meta-analytical pooling and bivariate analysis. RESULTS: Six cohort studies were included (1771 adult patients with 414 IFIs of which 215 were proven or probable). Similar performance was observed among the different BG assays. For the cutoff recommended by the manufacturer, the diagnostic performance of the BG assay in proven or probable IFI was better with 2 consecutive positive test results (diagnostic odds ratio for 2 consecutive vs one single positive results, 111.8 [95% confidence interval {CI}, 38.6-324.1] vs 16.3 [95% CI, 6.5-40.8], respectively; heterogeneity index for 2 consecutive vs one single positive results, 0% vs 72.6%, respectively). For 2 consecutive tests, sensitivity and specificity were 49.6% (95% CI, 34.0%-65.3%) and 98.9% (95% CI, 97.4%-99.5%), respectively. Estimated positive and negative predictive values for an IFI prevalence of 10% were 83.5% and 94.6%, respectively. CONCLUSIONS: Different BG assays have similar accuracy for the diagnosis of IFI in hemato-oncological patients. Two consecutive positive antigenemia assays have very high specificity, positive predictive value, and negative predictive value. Because sensitivity is low, the test needs to be combined with clinical, radiological, and microbiological findings.

Cytological and isoenzyme analysis of the Bucay and Quevedo cytotypes of the onchocerciasis vector Simulium exiguum (Diptera: Simuliidae) in Ecuador

Four cytotypes of Simulium exiguum occur in Ecuador, where this morphospecies is the primary vector of onchocerciasis. In this paper, we give the first full description of the banding pattern of the larval polytene chromosomes of the Quevedo cytotypes differ from the chromosomal standard sequence (of the Cayapa cytotype) by the fixed inversions IIL-5 and IIL-6. The Quevedo cytotype additionally differs from the standard and Bucay cytotypes by processing a differentiated X chromosome, wich is indicated by the inversion IIS-A. As the degree of reproductive isolation between the Bucay and Quevedo cytotypes has not yet been estabilished, they must be regarded as intraspecific variants of the same species. In fact, isoenzyme characterizations showed that the Bucay and Quevedo cytotypes are differentiated only to the extent expected of incipient species or geographical populations. Moreover, the sibiling species status previously given to the Bucay cytotype needs be reassessed, there being inadequate analysis from areas in Ecuador where Bucay occurs in sympatry with the standard Cayapa cytotype. No isoenzyme electromorphs were discovered that identified all or mostadult females of any one (cytotype-pure) collection.

Genome-wide meta-analysis of common variant differences between men and women.

The male-to-female sex ratio at birth is constant across world populations with an average of 1.06 (106 male to 100 female live births) for populations of European descent. The sex ratio is considered to be affected by numerous biological and environmental factors and to have a heritable component. The aim of this study was to investigate the presence of common allele modest effects at autosomal and chromosome X variants that could explain the observed sex ratio at birth. We conducted a large-scale genome-wide association scan (GWAS) meta-analysis across 51 studies, comprising overall 114 863 individuals (61 094 women and 53 769 men) of European ancestry and 2 623 828 common (minor allele frequency >0.05) single-nucleotide polymorphisms (SNPs). Allele frequencies were compared between men and women for directly-typed and imputed variants within each study. Forward-time simulations for unlinked, neutral, autosomal, common loci were performed under the demographic model for European populations with a fixed sex ratio and a random mating scheme to assess the probability of detecting significant allele frequency differences. We do not detect any genome-wide significant (P < 5 × 10(-8)) common SNP differences between men and women in this well-powered meta-analysis. The simulated data provided results entirely consistent with these findings. This large-scale investigation across ~115 000 individuals shows no detectable contribution from common genetic variants to the observed skew in the sex ratio. The absence of sex-specific differences is useful in guiding genetic association study design, for example when using mixed controls for sex-biased traits.

Comprehensive analysis of RET common and rare variant patientsin a series of Spanish Hirschsprung patients confirms a synergistic effect of both kinds of events

Background. RET is the major gene associated to Hirschsprung disease (HSCR) with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. In the present study, we have performed a comprehensive study of our HSCR series evaluating the involvement of both RET rare variants (RVs) and common variants (CVs) in the context of the disease. Methods. RET mutational screening was performed by dHPLC and direct sequencing for the identification of RVs. In addition Taqman technology was applied for the genotyping of 3 RET CVs previously associated to HSCR, including a variant lying in an enhancer domain within RET intron 1 (rs2435357). Statistical analyses were performed using the SPSS v.17.0 to analyze the distribution of the variants. Results. Our results confirm the strongest association to HSCR for the "enhancer" variant, and demonstrate a significantly higher impact of it in male versus female patients. Integration of the RET RVs and CVs analysis showed that in 91.66% of cases with both kinds of mutational events, the enhancer allele is in trans with the allele bearing the RET RV. Conclusions. A gender effect exists on both the transmission and distribution of rare coding and common HSCR causing mutations. In addition, these RET CVs and RVs seem to act in a synergistic way leading to HSCR phenotype.

Recurrence of keratoconus characteristics: a clinical and histologic follow-up analysis of donor grafts.

PURPOSE: To report on clinical corneal topography, histopathologic analysis, and fine structure findings in failed grafts after penetrating keratoplasty (PK) for keratoconus (KC). DESIGN: Retrospective, consecutive, interventional case series with histologic and clinical correlation. PARTICIPANTS: Twelve corneal buttons were obtained from consecutive patients undergoing repeated PK 10 to 28 years after the initial PK for KC. The indication for regrafting was endothelial deficiency in seven cases, irreversible immune graft rejection in two cases, and corneal ectasia in three cases. METHODS: Removed corneal buttons were examined by light and transmission electron microscopy. A potential correlation between the clinical and videokeratoscopic findings and the microscopic structural observations was analyzed. RESULTS: Preoperative simulated keratometry measured by TMS-1 (Tomey, New York, NY) or EyeSys CAS (EyeSys Technology, Houston, TX) ranged from 49.8 to 66.1 diopters. A pattern compatible with KC characteristics was observed in all cases. Fine structure analysis revealed Bowman's layer disruption or folds and stromal deposits in all corneal buttons. However, central corneal thinning was not present in any of the removed buttons. CONCLUSIONS: Structure changes compatible with the diagnosis of KC were observed in all donor buttons many years after PK on KC recipients. Recurrence of the KC characteristics may result from graft repopulation by recipients' keratocytes, aging of the grafted tissue, or both.

Analysis of the cytolytic T lymphocyte response of melanoma patients to the naturally HLA-A*0201-associated tyrosinase peptide 368-376.

The human tyrosinase gene codes for two distinct antigens that are recognized by HLA-A*0201-restricted CTLs. For one of them, tyrosinase peptide 368-376, the sequence identified by mass spectrometry in melanoma cell eluates differs from the gene-encoded sequence as a result of posttranslational modification of amino acid residue 370 (asparagine to aspartic acid). Here, we used fluorescent tetrameric complexes ("tetramers") of HLA-A*0201 and tyrosinase peptide 368-376 (YMDGTMSQV) to characterize the CD8+ T-cell response to this antigen in lymphoid cell populations from HLA-A2 melanoma patients. Taking advantage of the presence of significant numbers of tetramer-positive CD8+ T cells in tumor-infiltrated lymph node cells from a melanoma patient, we derived polyclonal and monoclonal tyrosinase peptide 368-376-specific CTLs by tetramer-guided flow cytometric sorting. These CTLs efficiently and specifically lysed HLA-A*0201- and tyrosinase-positive melanoma cells. As assessed with tyrosinase peptide variants, the fine antigen specificity of the CTLs was quite diverse at the clonal level. Flow cytometric analysis of PBMCs stained with tetramers showed that tyrosinase peptide 368-376-specific CD8+ T cells were hardly detectable in peripheral blood of melanoma patients. However, significant numbers of such cells were detected after short-term stimulation of CD8+ lymphocytes with tyrosinase peptide 368-376 in 6 of 10 HLA-A2 melanoma patients. Taken together, these findings emphasize the significant contribution of the natural tyrosinase peptide 368-376 to the antigenic specificities recognized by the tumor-reactive CTLs that may develop in HLA-A2 melanoma patients.