The Dimerization Domain in DapE Enzymes Is Required for Catalysis

The emergence of antibiotic-resistant bacterial strains underscores the importance of identifying new drug targets and developing new antimicrobial compounds. Lysine and meso-diaminopimelic acid are essential for protein production and bacterial peptidoglycan cell wall remodeling and are synthesized in bacteria by enzymes encoded within dap operon. Therefore dap enzymes may serve as excellent targets for developing a new class of antimicrobial agents. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) converts N-succinyl-L,L-diaminopimelic acid to L,Ldiaminopimelic acid and succinate. The enzyme is composed of catalytic and dimerization domains, and belongs to the M20 peptidase family. To understand the specific role of each domain of the enzyme we engineered dimerization domain deletion mutants of DapEs from Haemophilus influenzae and Vibrio cholerae, and characterized these proteins structurally and biochemically. No activity was observed for all deletion mutants. Structural comparisons of wild-type, inactive monomeric DapE enzymes with other M20 peptidases suggest that the dimerization domain is essential for DapE enzymatic activity. Structural analysis and molecular dynamics simulations indicate that removal of the dimerization domain increased the flexibility of a conserved active site loop that may provide critical interactions with the substrate

Effect of triticale grain characteristics, scouring, and extrusion conditions on physico-chemical properties, antioxidant activity, and volatile compounds of flat bread

The objective of the study was to determine the effect of grain preparation technology, extrusion cooking conditions, and triticale cultivars on physico-chemical properties of crisp bread. Triticale cultivars used in the experiments differed in protein content, starch content, amylolytic activity, and viscosimetric properties. The type of the twinscrew extruder used has an effect on starch degradation, phenolic contents, and antioxidant activity of the product, but a greater effect on those characteristics is found for grain scouring prior to extrusion. All factors had an effect on volatile compound amounts in crisp bread, but the direction and range of these changes were random in character. Results of this study show that triticale grain can be useful raw material for the production of flat bread

Optimization of Benzoxazole-Based Inhibitors of Cryptosporidium parvum Inosine 5?-Monophosphate Dehydrogenase

Cryptosporidium parvum is an enteric protozoan parasite that has emerged as a major cause of diarrhea, malnutrition, and gastroenteritis and poses a potential bioterrorism threat. C. parvum synthesizes guanine nucleotides from host adenosine in a streamlined pathway that relies on inosine 5?-monophosphate dehydrogenase (IMPDH). We have previously identified several parasite-selective C. parvum IMPDH (CpIMPDH) inhibitors by high-throughput screening. In this paper, we report the structure–activity relationship (SAR) for a series of benzoxazole derivatives with many compounds demonstrating CpIMPDH IC50 values in the nanomolar range and >500-fold selectivity over human IMPDH (hIMPDH). Unlike previously reported CpIMPDH inhibitors, these compounds are competitive inhibitors versus NAD+. The SAR study reveals that pyridine and other small heteroaromatic substituents are required at the 2-position of the benzoxazole for potent inhibitory activity. In addition, several other SAR conclusions are highlighted with regard to the benzoxazole and the amide portion of the inhibitor, including preferred stereochemistry. An X-ray crystal structure of a representative E·IMP·inhibitor complex is also presented. Overall, the secondary amine derivative 15a demonstrated excellent CpIMPDH inhibitory activity (IC50 = 0.5 ± 0.1 nM) and moderate stability (t1/2 = 44 min) in mouse liver microsomes. Compound 73, the racemic version of 15a, also displayed superb antiparasitic activity in a Toxoplasma gondii strain that relies on CpIMPDH (EC50 = 20 ± 20 nM), and selectivity versus a wild-type T. gondii strain (200-fold). No toxicity was observed (LD50 > 50 ?M) against a panel of four mammalian cells lines

XRCC2 R188H (rs3218536), XRCC3 T241M (rs861539) and R243H (rs77381814) single nucleotide polymorphisms in cervical cancer risk

Human Papillomavirus (HPV) is the main cause of cervical cancer and its precursor lesions. Transformation may be induced by several mechanisms, including oncogene activation and genome instability. Individual differences in DNA damage recognition and repair have been hypothesized to influence cervical cancer risk. The aim of this study was to evaluate whether the double strand break gene polymorphisms XRCC2 R188H G>A (rs3218536), XRCC3 T241M C>T (rs861539) and R243H G>A (rs77381814) are associated to cervical cancer in Argentine women. A case control study consisting of 322 samples (205 cases and 117 controls) was carried out. HPV DNA detection was performed by PCR and genotyping of positive samples by EIA (enzyme immunoassay). XRCC2 and 3 polymorphisms were determined by pyrosequencing. The HPV-adjusted odds ratio (OR) of XRCC2 188 GG/AG genotypes was OR = 2.4 (CI = 1.1-4.9, p = 0.02) for cervical cancer. In contrast, there was no increased risk for cervical cancer with XRCC3 241 TT/CC genotypes (OR = 0.48; CI = 0.2-1; p = 0.1) or XRCC3 241 CT/CC (OR = 0.87; CI = 0.52-1.4; p = 0.6). Regarding XRCC3 R243H, the G allele was almost fixed in the population studied. In conclusion, although the sample size was modest, the present data indicate a statistical association between cervical cancer and XRCC2 R188H polymorphism. Future studies are needed to confirm these findings.Fil: Perez, Luis Orlando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Crivaro, Andrea Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Barbisan, Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Poleri, Lucía Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Golijow, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentin

Apoptotic cell clearance in chronic inflammation of lateral neck cysts

The mechanism driving accumulation of large numbers of apoptotic and necrotic neutrophils in inflamed lateral neck cysts (LNC), in the absence of infection, remains obscure. The cellular content of cysts obtained from 17 patients was co-cultured with human macrophages. Phagocytosis levels of cyst-derived neutrophils were determined and compared to the uptake of spontaneously apoptotic neutrophils. Simultaneously, the expression of cytokines in macrophages exposed to cyst contents was measured. In comparison to spontaneously apoptotic neutrophils, the phagocytosis of LNC-derived neutrophils by macrophages was inefficient. An inverse correlation between neutrophil content in LNC and their uptake was observed. Macrophages co-cultured with cyst contents responded with variable expression of IL-6, TNF-α and IL-10. The hindered clearance of apoptotic neutrophils in LNC may lead to secondary necrosis of these cells and stimulation of the inflammatory reaction. Together with local production of anti-inflammatory cytokines, this may fuel chronic inflammation in the cysts

A novel myelin P0–specific T cell receptor transgenic mouse develops a fulminant autoimmune peripheral neuropathy

Autoimmune-prone nonobese diabetic mice deficient for B7-2 spontaneously develop an autoimmune peripheral neuropathy mediated by inflammatory CD4+ T cells that is reminiscent of Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy. To determine the etiology of this disease, CD4+ T cell hybridomas were generated from inflamed tissue–derived CD4+ T cells. A majority of T cell hybridomas were specific for myelin protein 0 (P0), which was the principal target of autoantibody responses targeting nerve proteins. To determine whether P0-specific T cell responses were sufficient to mediate disease, we generated a novel myelin P0–specific T cell receptor transgenic (POT) mouse. POT T cells were not tolerized or deleted during thymic development and proliferated in response to P0 in vitro. Importantly, when bred onto a recombination activating gene knockout background, POT mice developed a fulminant form of peripheral neuropathy that affected all mice by weaning age and led to their premature death by 3–5 wk of age. This abrupt disease was associated with the production of interferon γ by P0-specific T cells and a lack of CD4+ Foxp3+ regulatory T cells. Collectively, our data suggest that myelin P0 is a major autoantigen in autoimmune peripheral neuropathy

The relation between paracetamol use and asthma:a GA2LEN European case-control study

Studies from the UK and USA suggest that frequent use of paracetamol (acetaminophen) may increase the risk of asthma, but data across Europe are lacking. As part of a multicentric case-control study organised by the Global Allergy and Asthma European Network (GA(2)LEN), it was examined whether or not frequent paracetamol use is associated with adult asthma across Europe. The network compared 521 cases with a diagnosis of asthma and reporting of asthma symptoms within the last 12 months with 507 controls with no diagnosis of asthma and no asthmatic symptoms within the last 12 months across 12 European centres. All cases and controls were selected from the same population, defined by age (2045 yrs) and place of residence. In a random effects meta-analysis, weekly use of paracetamol, compared with less frequent use, was strongly positively associated with asthma after controlling for confounders. There was no evidence for heterogeneity across centres. No association was seen between use of other analgesics and asthma. These data add to the increasing and consistent epidemiological evidence implicating frequent paracetamol use in asthma in diverse populations