XbaI Restriction Site Research Articles

Enhancement in T1 lipase purification recovery using the novel construct pGEX4T1/His-T1

The Geobacillus zalihae strain T1 produces a thermostable T1 lipase that could be used for industrial purposes. Previously, the GST-T1 lipase was purified through two chromatographic steps: affinity and ion exchange (IEX) but the recovery yield was only 33%. To improve the recovery yield to over 80%, the GST tag from the pGEX system was replaced with a poly-histidine at the N-terminal of the T1 lipase sequence. The novel construct of pGEX/His-T1 lipase was developed by site-directed mutagenesis, where the XbaI restriction site was introduced upstream of the GST tag, allowing the removal of tag via double digestion using XbaI and EcoRI (existing cutting site in the pGEX system). Fragment of 6 × His-T1 lipase fusion was synthesized, cloned into the pGEX4T1 system, and expressed in Escherichia coli BL21 (DE3) pLysS, resulting in lipase-specific activity at 236 U/mg. The single purification step of His-T1 lipase was successfully achieved using nickel Sepharose 6FF with an optimized concentration of 5 mM imidazole for binding, yielding the recovery of 98%, 1,353 U/mg lipase activity, and a 5.7-fold increase in purification fold. His-T1 lipase was characterized and was found to be stable at pH 5–9, active at 70 °C, and optimal at pH 9.

The Effect of Cell Surface Expression and Linker Sequence on the Recruitment of Arrestin to the GIP Receptor

The glucose-dependent insulinotropic polypeptide (GIP) and the glucagon-like peptide-1 (GLP-1) receptor are important targets in the treatment of both type 2 diabetes mellitus (T2DM) and obesity. Originally identified for their role in desensitization, internalization and recycling of G protein-coupled receptors (GPCRs), arrestins have since been shown to act as scaffolding proteins that allow GPCRs to signal in a G protein-independent manner. While GLP-1R has been reported to interact with arrestins, this aspect of cell signaling remains controversial for GIPR. Using a (FRET)-based assay we have previously shown that yellow fluorescent protein (YFP)-labeled GIPR does not recruit arrestin. This GIPR-YFP construct contained a 10 amino acid linker between the receptor and a XbaI restriction site upstream of the YFP. This linker was not present in the modified GIPR-SYFP2 used in subsequent FRET and bioluminescence resonance energy transfer (BRET) assays. However, its removal results in the introduction of a serine residue adjacent to the end of GIPR’s C-terminal tail which could potentially be a phosphorylation site. The resulting receptor was indeed able to recruit arrestin. To find out whether the serine/arginine (SR) coded by the XbaI site was indeed the source of the problem, it was substituted with glycine/glycine (GG) by site-directed mutagenesis. This substitution abolished arrestin recruitment in the BRET assay but only significantly reduced it in the FRET assay. In addition, we show that the presence of a N-terminal FLAG epitope and influenza hemagglutinin signal peptide were also required to detect arrestin recruitment to the GIPR, most likely by increasing receptor cell surface expression. These results demonstrate how arrestin recruitment assay configuration can dramatically alter the result. This becomes relevant when drug discovery programs aim to identify ligands with “biased agonist” properties.

Cloning of sequence of homologous crt-cluster in Streptomyces globisporus 1912-бп

Aim. The aim was to set influence to the additional homologous crt-cluster on carotenogenesis of cells of streptomycete recipient. Methods. For this purpose transformation by the hybrid plasmid pWC 9,6 was conducted. This plasmid contained the fragment of the crt-cluster sequence (9576 bp) of Crt+-mutant S. globisporus Crt4 in a Crt--recipient S. globisporus 1912-бп. To construct this hybrid plasmid, a fragment of PLR-copies of sequence of the crt-cluster of mutant S. globisporus 1912 Crt4 was cloned in the shuttle vector pWHM4 (6.6 kb). Insertion was done into unique restriction sites for endonucleases XbaI and HindIII in a polylinker of this vector. These endonucleases have not restriction sites into the crt-cluster sequence. Results. The plasmid pWC 9,6 (16.2 kb) that contains the crt-cluster sequence (9576 bp) of the Crt+-variant Crt4 of the strain S. globisporus 1912 was constructed. The plasmid successfully functions in the cells of both recipients (E. coli XL1 Blue and S. globisporus 1912-бп). It provides to them resistance to the corresponding antibiotics. The plasmid pWC 9,6 stably keeps its molecular size (16.2 kb). However, indisputable proofs of expression of the crt-clusters in transformants were not got. Conclusions. The plasmid pWC 9,6, that is able to transform and stably function in the cells of both recipient microorganisms (Streptomyces and E. coli) was constructed.
 Keywords: crt-cluster, shuttle vector, cloning, resistance, PCR.

Genetic characterization of Thrace honey bee populations of Turkey: restriction and sequencing of inter cytochrome C oxidase I-II (CoxI-CoxII) genes

In this study, the genetic characterization of Thrace honey bee populations of Turkey were identified using the mitochondrial gene region, 3′ end of cytochrome C oxidase I gene to 5′ end of cytochrome C oxidase II gene (inter CoxI-CoxII genes) by using XbaI PCR-RFLP and DNA sequencing. A total of 322 worker honey bees were collected from five different localities; 4 provinces (Tekirdağ, Kırklareli, Edirne and Çanakkale) and Gökçeada Island near Thrace. PCR RFLP and sequence data combined revealed three haplotypes. The most frequently observed haplotype (Type 1), revealed only one XbaI restriction site in inter CoxI-CoxII genes. Type 2 haplotype revealed additional XbaI restriction site and this digestion revealed three bands. The rarely seen third haplotype, which is first reported in this study, was only found in two of the samples that were collected from Gökçeada Island. According to Maximum Likelihood phylogenetic analysis which was constructed on the basis of the distance matrix, Type 1 haplotype was clustered in the same clade with A. m. caucasica and A. m. anatoliaca whereas Type 2 was clustered in the same clade with A. m. macedonica and A. m. carnica. These results may indicate that Carniolan or Macedonian honey bee subspecies or their ecotypes can be found in the Thrace region of Turkey, mainly in samples that were collected from Kırklareli and Edirne provinces near the borders with Greece and Bulgaria.

Genotypes and Haplotypes of the Estrogen Receptor α Gene (ESR1) Are Associated With Female-to-Male Gender Dysphoria

IntroductionGender dysphoria, a marked incongruence between one's experienced gender and biological sex, is commonly believed to arise from discrepant cerebral and genital sexual differentiation. With the discovery that estrogen receptor β is associated with female-to-male (FtM) but not with male-to-female (MtF) gender dysphoria, and given estrogen receptor α involvement in central nervous system masculinization, it was hypothesized that estrogen receptor α, encoded by the ESR1 gene, also might be implicated. AimTo investigate whether ESR1 polymorphisms (TA)n-rs3138774, PvuII-rs2234693, and XbaI-rs9340799 and their haplotypes are associated with gender dysphoria in adults. MethodsMolecular analysis was performed in peripheral blood samples from 183 FtM subjects, 184 MtF subjects, and 394 sex- and ethnically-matched controls. Main Outcome MeasuresGenotype and haplotype analyses of the (TA)n-rs3138774, PvuII-rs2234693, and XbaI-rs9340799 polymorphisms. ResultsAllele and genotype frequencies for the polymorphism XbaI were statistically significant only in FtM vs control XX subjects (P = .021 and P = .020). In XX individuals, the A/G genotype was associated with a low risk of gender dysphoria (odds ratio [OR] = 0.34; 95% CI = 0.16–0.74; P = .011); in XY individuals, the A/A genotype implied a low risk of gender dysphoria (OR = 0.39; 95% CI = 0.17–0.89; P = .008). Binary logistic regression showed partial effects for all three polymorphisms in FtM but not in MtF subjects. The three polymorphisms were in linkage disequilibrium: a small number of TA repeats was linked to the presence of PvuII and XbaI restriction sites (haplotype S-T-A), and a large number of TA repeats was linked to the absence of these restriction sites (haplotype L-C-G). In XX individuals, the presence of haplotype L-C-G carried a low risk of gender dysphoria (OR = 0.66; 95% CI = 0.44–0.99; P = .046), whereas the presence of haplotype L-C-A carried a high susceptibility to gender dysphoria (OR = 3.96; 95% CI = 1.04–15.02; P = .044). Global haplotype was associated with FtM gender dysphoria (P = .017) but not with MtF gender dysphoria. ConclusionsXbaI-rs9340799 is involved in FtM gender dysphoria in adults. Our findings suggest different genetic programs for gender dysphoria in men and women.Cortés-Cortés J, Fernández R, Teijeiro N, et al. Genotypes and Haplotypes of the Estrogen Receptor α Gene (ESR1) Are Associated With Female-to-Male Gender Dysphoria. J Sex Med 2017;14:464–472.

Investigation of a suspected nosocomial transmission of blaKPC3-mediated carbapenem-resistant Klebsiella pneumoniae by whole genome sequencing

Whole genome sequencing (WGS) was compared to pulse-field gel electrophoresis (PFGE) of XbaI-digested genomic DNA, as methods by which to evaluate a potential transmission of carbapenem-resistant Klebsiella pneumoniae between 2 hospital inpatients. PFGE result demonstrated only 1-band difference between the isolates, suggesting probable relatedness. In contrast, while WGS data demonstrated the same sequence type and very similar chromosomal sequences, over 20 single nucleotide variants were identified between the isolates, bringing into question whether there was a transmission event. WGS also identified an additional plasmid, with an XbaI restriction site in the isolates of the second patient that was not identified by PFGE. While WGS provided additional information that was not available by PFGE, in this study, neither method could definitively conclude the relatedness between the isolates.

Precise cloning and tandem integration of large polyketide biosynthetic gene cluster using Streptomyces artificial chromosome system

BackgroundDirect cloning combined with heterologous expression of a secondary metabolite biosynthetic gene cluster has become a useful strategy for production improvement and pathway modification of potentially valuable natural products present at minute quantities in original isolates of actinomycetes. However, precise cloning and efficient overexpression of an entire biosynthetic gene cluster remains challenging due to the ineffectiveness of current genetic systems in manipulating large-sized gene clusters for heterologous as well as homologous expression.ResultsA versatile Escherichia coli-Streptomyces shuttle bacterial artificial chromosomal (BAC) conjugation vector, pSBAC, was used along with a cluster tandem integration approach to carry out homologous and heterologous overexpression of a large 80-kb polyketide biosynthetic pathway gene cluster of tautomycetin (TMC), which is a protein phosphatase PP1/PP2A inhibitor and T cell-specific immunosuppressant. Unique XbaI restriction sites were precisely inserted at both border regions of the TMC biosynthetic gene cluster within the chromosome of TMC-producing Streptomyces sp. CK4412, followed by site-specific recombination of pSBAC into the flanking region of the TMC gene cluster. The entire TMC gene cluster was then rescued as a single giant recombinant pSBAC by XbaI digestion of the chromosomal DNA as well as subsequent self-ligation. Next, the recombinant pSBAC construct containing the entire TMC cluster in E. coli was directly conjugated into model Streptomyces strains, resulting in rapid and enhanced TMC production. Moreover, introduction of the TMC cluster-containing pSBAC into wild-type Streptomyces sp. CK4412 as well as a recombinant S. coelicolor strain resulted in a chromosomal tandem repeat of the entire TMC cluster with 14-fold and 5.4-fold enhanced TMC productivities, respectively.ConclusionsThe 80-kb TMC biosynthetic gene cluster was isolated in a single integration vector, pSBAC. Introduction of TMC biosynthetic gene cluster in TMC non-producing strains has resulted in similar amount of TMC production yield. Moreover, over-expression of TMC biosynthetic gene cluster in original producing strain and recombinant S. coelicolor dramatically increased TMC production. Thus, this strategy can be employed to develop a custom overexpression scheme of entire metabolite pathway clusters present in actinomycetes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0325-2) contains supplementary material, which is available to authorized users.

Engineered and Native Coenzyme B12-dependent Isovaleryl-CoA/Pivalyl-CoA Mutase

Adenosylcobalamin-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently demonstrated that an isobutyryl-CoA mutase variant, IcmF, a member of this enzyme family that catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation. Given the biotechnological potential of the isovaleryl-CoA/pivalyl-CoA mutase (PCM) reaction, we initially attempted to engineer IcmF to be a more proficient PCM by targeting two active site residues predicted based on sequence alignments and crystal structures, to be key to substrate selectivity. Of the eight mutants tested, the F598A mutation was the most robust, resulting in an ∼17-fold increase in the catalytic efficiency of the PCM activity and a concomitant ∼240-fold decrease in the isobutyryl-CoA mutase activity compared with wild-type IcmF. Hence, mutation of a single residue in IcmF tuned substrate specificity yielding an ∼4000-fold increase in the specificity for an unnatural substrate. However, the F598A mutant was even more susceptible to inactivation than wild-type IcmF. To circumvent this limitation, we used bioinformatics analysis to identify an authentic PCM in genomic databases. Cloning and expression of the putative AdoCbl-dependent PCM with an α2β2 heterotetrameric organization similar to that of isobutyryl-CoA mutase and a recently characterized archaeal methylmalonyl-CoA mutase, allowed demonstration of its robust PCM activity. To simplify kinetic analysis and handling, a variant PCM-F was generated in which the αβ subunits were fused into a single polypeptide via a short 11-amino acid linker. The fusion protein, PCM-F, retained high PCM activity and like PCM, was resistant to inactivation. Neither PCM nor PCM-F displayed detectable isobutyryl-CoA mutase activity, demonstrating that PCM represents a novel 5'-deoxyadenosylcobalamin-dependent acyl-CoA mutase. The newly discovered PCM and the derivative PCM-F, have potential applications in bioremediation of pivalic acid found in sludge, in stereospecific synthesis of C5 carboxylic acids and alcohols, and in the production of potential commodity and specialty chemicals.

Molecular Epidemiology of Mycobacterium tuberculosis Isolates in 100 Patients With Tuberculosis Using Pulsed Field Gel Electrophoresis.

Background:Tuberculosis (TB) is a widespread infectious disease. Today, TB has created a public health crisis in the world. Genotyping of Mycobacterium tuberculosis isolates is useful for surveying the dynamics of TB infection, identifying new outbreaks, and preventing the disease. Different molecular methods for clustering of M. tuberculosis isolates have been used.Objectives:During a one year study of genotyping, 100 M. tuberculosis isolates from patients referred to Pasteur Institute of Iran were collected and their genotyping was accomplished using pulsed field gel electrophoresis (PFGE) method.Materials and Methods:Identification of all M. tuberculosis isolates was accomplished using standard biochemical and species-specific polymerase chain reaction (PCR) methods. Antibiotic susceptibility tests were performed using proportional method. After preparing PFGE plaques for each isolate of M. tuberculosis, XbaI restriction enzyme was applied for genome digestion. Finally, the digested DNA fragments were separated on 1% agarose gel and analyzed with GelCompar II software.Results:Genotyping of the studied isolates in comparison with the molecular weight marker revealed two common types; pulsotype A with 71 isolates and one multidrug resistant mycobacterium (MDR) case, and pulsotype B including 29 isolates and three MDR cases. No correlation between the antibiotypes and pulsotypes was observed.Conclusions:Molecular epidemiology studies of infectious diseases have been useful when bacterial isolates have been clustered in a period of time and in different geographical regions with variable antibiotic resistance patterns. In spite of high geographical differences and different antibiotic resistant patterns, low genetic diversity among the studied TB isolates may refer to the low rate of mutations in XbaI restriction sites in the mycobacterial genome. We also identified three MDR isolates in low-incidence pulsotype B, which could be disseminated and is highly important to consider in TB surveillance programs to prevent the spread of MDR-TB isolates in the population.

Identification of five sea cucumber species through PCR-RFLP analysis

Sea cucumbers are traditional marine food and Chinese medicine in Asia. The rapid expansion of sea cucumber market has resulted in various problems, such as commercial fraud and mislabeling. Conventionally, sea cucumber species could be distinguished by their morphological and anatomical characteristics; however, their identification becomes difficult when they are processed. The aim of this study was to develop a new convenient method of identifying and distinguishing sea cucumber species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of mitochondrial cytochrome oxidase I gene (COI) was used to identifing five sea cucumber species (Apostichopus japonicus, Cucumaria frondosa, Thelenota ananas, Parastichopus californicus and Actinopyga lecanora). A 692 bp fragment of COI was searched for BamHI, KpnI, PstI, XbaI and Eco31I restriction sites with DNAMAN 6.0, which were then used to PCR-RFLP analysis. These five sea cucumber species can be discriminated from mixed sea cucumbers. The developed PCR-RFLP assay will facilitate the identification of sea cucumbers, making their source tracing and quality controlling feasible.

Estrogen receptor alpha gene polymorphisms and risk of prostate cancer: a meta-analysis involving 18 studies

Genetic and epigenetic changes in the estrogen receptor alpha (ER-α) gene, according to multiple lines of evidence, might alter its expression and its downstream signaling thereby increasing the risk of developing prostate cancer. The purpose of this meta-analysis was to determine whether polymorphisms in two intronic restriction sites in the ER-α (PvuII and XbaI) gene contribute to prostate cancer. A literature search for eligible studies published before November 5, 2013 was conducted in the PubMed, Embase, China Biology Medicine (CBM), and CNKI databases. Pooled crude odds ratios (ORs) and their corresponding 95 % confidence intervals (CIs) were calculated. Eighteen case-control and cohort studies were included in this meta-analysis with a total 4,884 prostate cancer cases and 10,134 healthy controls. Two common ER-α polymorphisms were examined: IVS1-397 C>T (a.k.a. the PvuII restriction site, rs2234693) and IVS1-351 A>G (a.k.a. the XbaI restriction site, rs9340799). Results from this meta-analysis showed that the PvuII polymorphism was not significantly associated with prostate cancer risk in any of the racial subgroups, either by allelic or genotypic frequencies. However, this meta-analysis revealed that the G allele in the XbaI polymorphism was associated with a statistically significant increase in the risk of prostate cancer. In a stratified analysis based on ethnicity, the XbaI G allele remained significantly correlated with an increased risk of prostate cancer in Africans; this correlation, however, was not found in Caucasians or Asians. In summary, a positive association correlation was observed between frequencies of the XbaI (A>G) polymorphism and prostate cancer, especially in Africans, but not such correlation was found with regard to the frequency of the PvuII (C>T) polymorphism.

Remodeling of Marrow Hematopoietic Stem and Progenitor Cells by Non-self ST6Gal-1 Sialyltransferase

Glycans occupy the critical cell surface interface between hematopoietic cells and their marrow niches. Typically, glycosyltransferases reside within the intracellular secretory apparatus, and each cell autonomously generates its own cell surface glycans. In this study, we report an alternate pathway to generate cell surface glycans where remotely produced glycosyltransferases remodel surfaces of target cells and for which endogenous expression of the cognate enzymes is not required. Our data show that extracellular ST6Gal-1 sialyltransferase, originating mostly from the liver and released into circulation, targets marrow hematopoietic stem and progenitor cells (HSPCs) and mediates the formation of cell surface α2,6-linked sialic acids on HSPCs as assessed by binding to the specific lectins Sambucus nigra agglutinin and Polysporus squamosus lectin and confirmed by mass spectrometry. Marrow HSPCs, operationally defined as the Lin-c-Kit+ and Lin-Sca-1+c-Kit+ populations, express negligible endogenous ST6Gal-1. Animals with reduced circulatory ST6Gal-1 have marrow Lin-Sca-1+c-Kit+ cells with reduced S. nigra agglutinin reactivity. Bone marrow chimeras demonstrated that α2,6-sialylation of HSPCs is profoundly dependent on circulatory ST6Gal-1 status of the recipients and independent of the ability of HSPCs to express endogenous ST6Gal-1. Biologically, HSPC abundance in the marrow is inversely related to circulatory ST6Gal-1 status, and this relationship is recapitulated in the bone marrow chimeras. We propose that remotely produced, rather than the endogenously expressed, ST6Gal-1 is the principal modifier of HSPC glycans for α2,6-sialic acids. In so doing, liver-produced ST6Gal-1 may be a potent systemic regulator of hematopoiesis.

RESTRICTION FRAGMENT LENGTH POLYMORPHISM-BASED IDENTIFICATION OF 'DICKEYA SOLANI ', A NEW GENETIC CLADE OF DICKEYA spp.

Bacteria belonging to Pectobacterium and Dickeya spp. (previously known as pectinolytic Erwinia spp.) cause soft rot diseases in a great variety of crops worldwide, particularly in potato, tomato, maize, and ornamental plants and other hosts. Infections by these pathogens may lead to severe economic losses to the crops, partially due to the lack of effective detection/identification tools and incomplete knowledge of their behavior in the environment. Since 2005, an increase in blackleg and soft rot diseases of potato has been observed in Europe due to the appearance of the new genetic clade of Dickeya spp. provisionally named ‘Dickeya solani’. This pathogen is more aggressive in most cases than bacteria of the Pectobacterium species and cause plant tissue maceration under a wide range of conditions. The present study provides a simple and reliable three-step PCR-based method for rapid differentiation of ‘D. solani’ isolates from other Dickeya spp. and Pectobacterium spp. Bacterial isolates are classified as Dickeya spp. on the basis of the PCR reaction with Dickeya spp. specific primers. Isolates identified positively as Dickeya spp. are tested by PCR for the presence of recA with the primers universal for bacteria of the former genus Erwinia. The PCR product is then digested with XbaI restriction endonuclease. The differentiation of ‘D. solani’ is based on the presence of a unique XbaI restriction site in the ‘D. solani’ recA gene that is absent in the recA sequences of other Dickeya species.

KHARON1 Mediates Flagellar Targeting of a Glucose Transporter in Leishmania mexicana and Is Critical for Viability of Infectious Intracellular Amastigotes

The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle.

Estrogen Receptor Alpha (Esr) Gene Polymorphism As Risk Factor For Type 2 Diabetes Mellitus (T2dm) In Javenese Menopause Women of Indonesia

Background: The number of menopausal women who suffer from low level estrogen-associated type-2 diabetes has been increasing recently. The role of estrogen in metabolism of glucose depends on estrogen receptor alpha expression that is regulated by estrogen receptor alpha gene (ESR ?). PvuII and XbaI polymorphisms in the ESR ? receptor may decrease the expression of ESR ? protein and receptor activity, thereby increasing the risk of developing type 2 diabetes mellitus in menopausal women. Purpose: to determine the ESR ? polymorphism as a risk factor for type 2 diabetes mellitus (DM) in menopause women of Javanese in Indonesia. Methods: Sixty five menopausal women were recruited for the study consisted of 40 women with T2DM and 25 women as control. PvuII and XbaI polymorphisms were determined by polymerase chain reacton-restriction fragment length polymorphism (PCR-RFLP). The absence of PvuII and XbaI restriction sites were indicated by “P1” and “X1” and presence by “P2” and “X2”, respectively. Chai Square test were used in statistical analyisis to measure Hardy Weinberg Equilibrium (HWE) and the risk of P1/P2 and X1/X2 allele for suffering T2DM. Results: PvuII genotype was distributed as; 22.5% (P1P1), 45% (P1P2), 32.5% (P2P2) while XbaI genotype was distributed as 10 % (X1X1), 62.5% (X1X2) and 27.5% (X2X2) in diabetics respectively. There was no difference in distribution of P1 and P2 between diabetics and non diabetics but difference for X1 and X2 existed between groups. The frequency of P2 allele was 55 % while P1 allele frequency is 45% in diabetics. X2 allele frequency was 58.8% while X1 allele is 41.2%. X2 allele had an impact on the 3.6 times higher risk of getting type 2 diabetes in Javanese menopausal women (OR = 3.662, CI = 1.711 to 7.840). Conclusions: PvuII and XbaI polymorphism was found in Javanese menopause women of Indonesia in patients with type 2 diabetes mellitus. The allele frequency of P2 and X2 are 55% and 58.8% respectively. X2 allele was found as a risk factor for type 2 diabetes mellitus in Javanese menopause women of Indonesia. Bangladesh Journal of Medical Science Vol. 12 No. 02 April’13 Page 171-179 DOI: http://dx.doi.org/10.3329/bjms.v12i2.14946

Molecular and Functional Analysis of the Large 5′ Promoter Region of CFTR Gene Revealed Pathogenic Mutations in CF and CFTR-Related Disorders

Patients with cystic fibrosis (CF) manifest a multisystemic disease due to mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR); despite extensive testing of coding regions, a proportion of CF alleles remains unidentified. We studied 118 patients with CF and CFTR-related disorders, most with one or both unknown mutations after the scanning of CFTR coding regions, and a non-CF control group (n = 75) by sequencing the 6000-bp region at the 5' of the CFTR gene. We identified 23 mutations, of which 9 were novel. We expressed such mutations in vitro using four cell systems to explore their functional effect, relating the data to the clinical expression of each patient. Some mutations reduced expression of the gene reporter firefly luciferase in various cell lines and may act as disease-causing mutations. Other mutations caused an increase in luciferase expression in some cell lines. One mutation had a different effect in different cells. For other mutations, the expression assay excluded a functional role. Gene variants in the large 5' region may cause altered regulation of CFTR gene expression, acting as disease-causing mutations or modifiers of its clinical phenotype. Studies of in vitro expression in different cell systems may help reveal the effect of such mutations.

The Polysialyltransferases Interact with Sequences in Two Domains of the Neural Cell Adhesion Molecule to Allow Its Polysialylation

The neural cell adhesion molecule (NCAM) is the major substrate for the polysialyltransferases (polySTs), ST8SiaII/STX and ST8SiaIV/PST. The polysialylation of NCAM N-glycans decreases cell adhesion and alters signaling. Previous work demonstrated that the first fibronectin type III repeat (FN1) of NCAM is required for polyST recognition and the polysialylation of the N-glycans on the adjacent Ig5 domain. In this work, we highlight the importance of an FN1 acidic patch in polyST recognition and also reveal that the polySTs are required to interact with sequences in the Ig5 domain for polysialylation to occur. We find that features of the Ig5 domain of the olfactory cell adhesion molecule (OCAM) are responsible for its lack of polysialylation. Specifically, two basic OCAM Ig5 residues (Lys and Arg) found near asparagines equivalent to those carrying the polysialylated N-glycans in NCAM substantially decrease or eliminate polysialylation when used to replace the smaller and more neutral residues (Ser and Asn) in analogous positions in NCAM Ig5. This decrease in polysialylation does not reflect altered glycosylation but instead is correlated with a decrease in polyST-NCAM binding. In addition, inserting non-conserved OCAM sequences into NCAM Ig5, including an "extra" N-glycosylation site, decreases or completely blocks NCAM polysialylation. Taken together, these results indicate that the polySTs not only recognize an acidic patch in the FN1 domain of NCAM but also must contact sequences in the Ig5 domain for polysialylation of Ig5 N-glycans to occur.

238: Lack of association between estrogen receptor α gene polymorphisms and myocardial infarction in Tunisian population

Coronary artery disease (CAD) is a multifactorial disease, its expression probably being influenced by the interaction of genetic and environmental risk factors. Genetic variation in the estrogen receptor-alpha gene (ERS1) has been associated with CAD in several populations worldwide, but results are still controversial. In the present study, we examined the association between the c.454-397T>C: PvuII restriction site (rs2234693) and c.454-351A>G: XbaI restriction site (rs9340799) polymorphisms (SNPs) and myocardial infarction (MI) in a sample of the Tunisian population. The case group included 294 patients with MI, and the control group comprised 275 individuals with angiographically normal coronary arteries and without signs or symptoms of MI. ESR1 PvuII and XbaI polymorphism was determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. Genotype distributions of the SNPs in the control and patient groups were consistent with those expected for samples in Hardy-Weinberg equilibrium (p> 0.05). The Genotype distribution and the relative allelic frequencies of the -397T>C and - 351A>G polymorphisms were not significantly different between the control and patient groups (p=NS). Moreover, the odds ratio for MI associated with the c.454-397C (OR=1.02, 95% CI 0.81-1.30), and c.454-351G (OR=1.24, 95% CI 0.98-1.58) variants failed to reach statistical significance. In the current study, we were unable to demonstrate a significant association of the ESR1 c.454-397T>C and c.454-351A>G polymorphisms with MI. These results do not support the extent of risk associated with developing MI previously reported for the ESR1 c.454-397T>C and c.454-351A>G polymorphisms.

GRK2 Protein-mediated Transphosphorylation Contributes to Loss of Function of μ-Opioid Receptors Induced by Neuropeptide FF (NPFF2) Receptors

Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of μ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit β-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex.

Histone Deacetylase 6 (HDAC6) Deacetylates Survivin for Its Nuclear Export in Breast Cancer

Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.