Clarithromycin resistance prevalence and Icea gene status in Helicobacter Pylori clinical isolates in Turkish patients with duodenal ulcer and functional dyspepsia

Clarithromycin resistance in Helicobacter pylori is a principal cause of failure of eradication therapies, and its prevalence varies geographically. The IceA gene is a virulence factor associated with clinical outcomes. The objective of this study was to determine the current state of clarithromycin resistance prevalence, and to investigate the role of iceA genotypes in 87 Turkish adult patients (65 with functional dyspepsia and 22 with duodenal ulcer). A2143G and A2144G point mutations were tested by PCR-RFLP for clarithromycin resistance. Among the patients in the study, 28 patients were tested by agar dilution as well. Allelic variants of the iceA gene were identified by PCR. A total of 24 (27.6%) strains evidenced one of the mutations, either A2143G or A2144G. IceA1 was found to be positive in 28 of the strains (32.2%), iceA2 was positive in 12 (13.8%) and, both iceA1 and iceA2 were positive in 22 (25.3%) strains. In conclusion, we discovered no relationships between iceA genotypes and functional dyspepsia or duodenal ulcer, nor between clarithromycin resistance and iceA genotypes. Clarithromycin resistance appears to be more prevalent in Turkish patients.     

Purification and characterization of Tet(M), a protein that renders ribosomes resistant to tetracycline

The tet(M) tetracycline resistance gene has been found in a wide variety of clinically important bacteria. It has been shown previously (Burdett, V. (1986) J. Bacteriol. 165, 564-569) that the tet(M) gene product mediates resistance at the level of protein synthesis as judged by in vitro assay. Using this assay, large amounts of protein were purified from an Escherichia coli overproducer expressing the gene under control of a T7 promoter. The purified activity consists of a single polypeptide of molecular weight 68,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was confirmed to be the tet(M) gene product by amino-terminal sequence analysis. Purified Tet(M) has an associated ribosome-dependent GTPase with the specific activity being similar to that of the corresponding activity associated with elongation factor G. Since Tet(M) also displays substantial homology to elongation factor G throughout its length, Tet(M) may function as an analog of this elongation factor.     

Overexpression of heme oxygenase-1 in microenvironment mediates vincristine resistance of B-cell acute lymphoblastic leukemia by promoting vascular endothelial growth factor secretion

Chemoresistance often causes treatment failure of B-cell acute lymphoblastic leukemia (B-ALL). However, the mechanism remains unclear at present. Herein, overexpression of heme oxygenase-1 (HO-1) was found in the bone marrow stromal cells (BMSCs) from B-ALL patients developing resistance to vincristine (VCR), a chemotherapeutic agent. Two B-ALL cell lines Super B15 and CCRF-SB were cocultured with BMSCs transfected with lentivirus to regulate the expression of HO-1. Silencing HO-1 expression in BMSCs increased the apoptotic rates of B-ALL cell lines induced by VCR, whereas upregulating HO-1 expression reduced the rate. Cell cycle can be arrested in the G2/M phase by VCR. In contrast, B-ALL cells were arrested in the G0/G1 phase due to HO-1 overexpression in BMSCs, which avoided damage from the G2/M phase. Vascular endothelial growth factor (VEGF) in BMSCs, as a key factor in the microenvironment-associated chemoresistance, was also positively coexpressed with HO-1. VEGF secretion was markedly increased in BMSCs with HO-1 upregulation but decreased in BMSCs with HO-1 silencing. B-ALL cell lines became resistant to VCR when cultured with VEGF recombinant protein, so VEGF secretion induced by HO-1 expression may promote the VCR resistance of B-ALL cells. As to the molecular mechanism, the PI3K/AKT pathway mediated regulation of VEGF by HO-1. In conclusion, this study clarifies a mechanism by which B-ALL is induced to resist VCR through HO-1 overexpression in BMSCs, and provides a novel strategy for overcoming VCR resistance in clinical practice.     

Chloramphenicol acetyltransferase may confer resistance to fusidic acid by sequestering the drug.

Enterobacterial chloramphenicol acetyltransferase bound fusidic acid with high affinity, but did not acetylate the drug at an experimentally detectable rate. The enzyme may therefore confer resistance to fusidic acid by sequestering the drug and thereby preventing the drug from binding to translational elongation factor G.     

Association of soluble epoxide hydrolase gene polymorphism with insulin resistance in type 2 diabetic patients

The insulin resistance found in diabetes is influenced by vascular tone and local blood flow. Endothelial-derived hyperpolarizing factor (EDHF) functions as a potent vasodilator to regulate vascular tone, and its production is regulated by soluble epoxide hydrolase (sEH). In this study, we examined the genotype distribution and allele frequency of sEH gene G860A (Arg287Gln) polymorphism in Japanese subjects (n=499) (non-diabetic subjects, n=205; type 2 diabetic patients, n=294). Also, to accurately evaluate insulin resistance, we performed the euglycemic hyperinsulinemic clamp test for each type 2 diabetic patient (n=86) from whom agreement was obtained, and then examined a possible association of sEH gene G860A polymorphism with insulin resistance status. There was no significant difference in genotype distribution and allele frequency between non-diabetic subjects and type 2 diabetic patients. Interestingly, however, there was close association of sEH gene G860A (Arg287Gln) polymorphism with insulin resistance in type 2 diabetic patients, which was not observed in non-diabetic subjects. These results suggest that sEH and EDHF play some important role in the pathogenesis of insulin resistance found in type 2 diabetes.     

Cell cycle dependence of Hoechst 33342 dye cytotoxicity on sorted living cells

The effect of staining cellular DNA with the bisbenzimidazole dye Hoechst 33342 on the colony forming efficiency of Chinese Hamster Ovary Cells in different cell cycle phases has been studied. Exposures of 90 and 120 min to 5 microM Hoechst 33342 provided a considerable loss of clonogenicity depending on the cycle phase at staining procedure. The G2+M cells reveal to be the most sensitive fraction followed by the G1 cells. The highest resistance was found on S-phase cells with a colony forming efficiency exceeding that of the G2+M fraction by a factor of two.     

The forespore line of gene expression in Bacillus subtilis

Endospore formation by Bacillus subtilis involves three differentiating cell types, the predivisional cell, the mother cell, and the forespore. Here we report the program of gene expression in the forespore, which is governed by the RNA polymerase sigma factors sigma(F) and sigma(G) and the DNA-binding proteins RsfA and SpoVT. The sigma(F) factor turns on about 48 genes, including the gene for RsfA, which represses a gene in the sigma(F) regulon, and the gene for sigma(G). The sigma(G) factor newly activates 81 genes, including the gene for SpoVT, which turns on (in nine cases) or stimulates (in 11 cases) the expression of 20 genes that had been turned on by sigma(G) and represses the expression of 27 others. The forespore line of gene expression consists of many genes that contribute to morphogenesis and to the resistance and germination properties of the spore but few that have metabolic functions. Comparative genomics reveals a core of genes in the sigma(F) and sigma(G) regulons that are widely conserved among endospore-forming species but are absent from closely related, but non-spore-forming Listeria spp. Two such partially conserved genes (ykoU and ykoV), which are members of the sigma(G) regulon, are shown to confer dry-heat resistance to dormant spores. The ykoV gene product, a homolog of the non-homologous end-joining protein Ku, is shown to associate with the nucleoid during germination. Extending earlier work on gene expression in the predivisional cell and the mother cell, we present an integrated overview of the entire program of sporulation gene expression.     

G1/S cell cycle arrest provides anoikis resistance through Erk-mediated Bim suppression

Proper attachment to the extracellular matrix is essential for cell survival. Detachment from the extracellular matrix results in an apoptotic process termed anoikis. Anoikis induction in MCF-10A mammary epithelial cells is due not only to loss of survival signals following integrin disengagement, but also to consequent downregulation of epidermal growth factor (EGFR) and loss of EGFR-induced survival signals. Here we demonstrate that G(1)/S arrest by overexpression of the cyclin-dependent kinase inhibitors p16(INK4a), p21(Cip1), or p27(Kip1) or by treatment with mimosine or aphidicolin confers anoikis resistance in MCF-10A cells. G(1)/S arrest-mediated anoikis resistance involves suppression of the BH3-only protein Bim. Furthermore, in G(1)/S-arrested cells, Erk phosphorylation is maintained in suspension and is necessary for Bim suppression. Following G(1)/S arrest, known proteins upstream of Erk, including Raf and Mek, are not activated. However, retained Erk activation under conditions in which Raf and Mek activation is lost is observed, suggesting that G(1)/S arrest acts at the level of Erk dephosphorylation. Thus, anoikis resistance by G(1)/S arrest is mediated by a mechanism involving Bim suppression through maintenance of Erk activation. These results provide a novel link between cell cycle arrest and survival, and this mechanism could contribute to the survival of nonreplicating, dormant tumor cells that avert apoptosis during early stages of metastasis.     

Association analysis of the plasminogen activator inhibitor-1 4G/5G polymorphism in Hispanics and African Americans: the IRAS family study

OBJECTIVE: Plasminogen activator inhibitor type-1 (PAI-1) plays a central role in fibrolysis and has recently been hypothesized to influence components of the insulin resistance syndrome. We consider whether the 4G/5G polymorphism influences components of insulin resistance and obesity solely through PAI-1 protein levels or also though a secondary pathway. In addition, we explore whether transforming growth factor (TGF-beta1), a key regulator of PAI-1 expression, modifies the influence of the PAI-1 4G/5G polymorphism on these traits. METHODS AND RESULTS: The Insulin Resistance and Atherosclerosis (IRAS) Family Study genotyped 287 African American (18 pedigrees) and 811 Hispanic American (45 pedigrees) individuals for the 4G/5G PAI-1 and two TGF-beta1 polymorphisms (R25P, C-509T). Individuals were recruited from three clinical centers located in San Antonio (urban Hispanic), San Luis Valley (rural Hispanic) and Los Angeles (African American). The presence of the 4G PAI-1 allele was positively associated with PAI-1 protein level (combined sample p < 0.0001). Hispanic Americans average 65% higher PAI-1 protein levels than African Americans (p < 0.0001). Consistently across ethnic groups, increased PAI-1 protein levels were associated with increased insulin resistance and overall and central obesity (p value < 0.0001, combined sample). Adjusting for PAI-1 protein levels, there was evidence of an association of PAI-1 genotype (4G) with insulin sensitivity (p < 0.002) and subcutaneous fat (p < 0.01). These associations were not influenced by TGF-beta1 genotypes. CONCLUSIONS: PAI-1 protein is a strong correlate of insulin resistance (IR) and obesity in Hispanics and African Americans. However, PAI-1 4G/5G polymorphism appears to influence insulin resistance and obesity beyond its direct influence on serum PAI-1 protein levels.     

HIV-1 Vif can directly inhibit apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G-mediated cytidine deamination by using a single amino acid interaction and without protein degradation

The human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G), also known as CEM-15, is a host-cell factor involved in innate resistance to retroviral infection. HIV-1 viral infectivity factor (Vif) protein was shown to protect the virus from APOBEC3G-mediated viral cDNA hypermutation. The mechanism proposed for protection of the virus by HIV-1 Vif is mediated by APOBEC3G degradation through ubiquitination and the proteasomal pathway. Here we show that in Escherichia coli the APOBEC3G-induced cytidine deamination is inhibited by expression of Vif without depletion of deaminase. Moreover, inhibition of deaminase-mediated bacterial hypermutation is dependent on a single amino acid substitution D128K that renders APOBEC3G resistant to Vif inhibition. This single amino acid was elegantly proven by other authors to determine species-specific sensitivity. Our results show that in bacteria this single amino acid substitution controls Vif-dependent blocking of APOBEC3G that is dependent on a strong protein interaction. The C-terminal region of Vif is responsible for this strong protein-protein interaction. In conclusion, our experiments suggest a complement to the model of Vif-induced degradation of APOBEC3G by bringing to relevance that deaminase inhibition can also result from a direct interaction with Vif protein.     

G12-G13-LARG-mediated signaling in vascular smooth muscle is required for salt-induced hypertension

The tone of vascular smooth muscle cells is a primary determinant of the total peripheral vascular resistance and hence the arterial blood pressure. Most forms of hypertension ultimately result from an increased vascular tone that leads to an elevated total peripheral resistance. Regulation of vascular resistance under normotensive and hypertensive conditions involves multiple mediators, many of which act through G protein-coupled receptors on vascular smooth muscle cells. Receptors that mediate vasoconstriction couple with the G-proteins G(q)-G11 and G12-G13 to stimulate phosphorylation of myosin light chain (MLC) via the Ca2+/MLC kinase- and Rho/Rho kinase-mediated signaling pathways, respectively. Using genetically altered mouse models that allow for the acute abrogation of both signaling pathways by inducible Cre/loxP-mediated mutagenesis in smooth muscle cells, we show that G(q)-G11-mediated signaling in smooth muscle cells is required for maintenance of basal blood pressure and for the development of salt-induced hypertension. In contrast, lack of G12-G13, as well as of their major effector, the leukemia-associated Rho guanine nucleotide exchange factor (LARG), did not alter normal blood pressure regulation but did block the development of salt-induced hypertension. This identifies the G12-G13-LARG-mediated signaling pathway as a new target for antihypertensive therapies that would be expected to leave normal blood pressure regulation unaffected.     

Separation of penicillin G from phenylacetic acid in a supported liquid membrane system

The separation of penicillin G (Pen G) from phenylacetic acid (PAA) by use of a supported liquid membrane (SLM) system with Amberlite LA-2 dissolved in 1-decanol, supported on a microporous polypropylene membrane, was studied. The results show that the individual permeability of each component in mixture was lower than that in a single compartment system and, it suggests a strong transport competition between Pen G and PAA. The SLM system in this study proved to be a promising process for the selective separation of Pen G from PAA. The maximum separation factor was found to be 1.8 under a liquid membrane resistance controlled mechanism. (c) 1994 John Wiley & Sons, Inc.     

Heterotrimeric Gα protein Pga1 from Penicillium chrysogenum triggers germination in response to carbon sources and affects negatively resistance to different stress conditions

Heterotrimeric Gα protein Pga1 of Penicillium chrysogenum controls vegetative growth, conidiation and secondary metabolite production. In this work we studied the role of Pga1 in spore germination and resistance to different stress conditions. Strains G203R-T (expressing the dominant inactivating pga1(G203R) allele) and Δpga1 (deleted pga1) showed a delayed and asynchronic germination pattern, and a decrease in the percentage of germination, which occurred in only 70-80% of the total conidia. In contrast, in strains expressing the dominant activating pga1(G42R) allele, germination occurred at earlier times and in 100% of conidia. In addition, strains with the pga1(G42R) allele were able to bypass the carbon source (glucose or sucrose) requirement for germination in about 64% of conidia. Thus Pga1 plays an important, but not essential, role in germination, mediating carbon source sensing. Regulation of germination by Pga1 is probably mediated by cAMP, as intracellular levels of this secondary messenger undergo a peak before the onset of germination only in strains with an active Pga1. Pga1 activity is also a determinant factor in the resistance to different stress conditions. Absence or inactivation of Pga1 allow growth on SDS-containing minimal medium, increase resistance of conidia to thermal and oxidative stress, and increase resistance of vegetative mycelium to thermal and osmotic stress. In contrast, constitutive activation of Pga1 causes a decrease in the resistance of conidia to thermal stress and of vegetative mycelium to thermal and osmotic stress. Together with our previously reported results, we show in this work that Pga1 plays a central role in the regulation of the whole growth-developmental program of this biotechnologically important fungus.     

Valproic Acid Induced Differentiation and Potentiated Efficacy of Taxol and Nanotaxol for Controlling Growth of Human Glioblastoma LN18 and T98G Cells

Glioblastoma shows poor response to current therapies and warrants new therapeutic strategies. We examined the efficacy of combination of valproic acid (VPA) and taxol (TX) or nanotaxol (NTX) in human glioblastoma LN18 and T98G cell lines. Cell differentiation was manifested in changes in morphological features and biochemical markers. Cell growth was controlled with down regulation of vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), nuclear factor-kappa B (NF-κB), phospho-Akt (p-Akt), and multi-drug resistance (MDR) marker, indicating suppression of angiogenic, survival, and multi-drug resistance pathways. Cell cycle analysis showed that combination therapy (VPA and TX or NTX) increased the apoptotic sub G1 population and apoptosis was further confirmed by Annexin V-FITC/PI binding assay and scanning electron microscopy. Combination therapy caused activation of caspase-8 and cleavage of Bid to tBid and increased Bax:Bcl-2 ratio and mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF). Upregulation of calpain and caspases (caspase-9 and caspase-3) and substrate degradation were also detected in course of apoptosis. The combination of VPA and NTX most effectively controlled the growth of LN18 and T98G cells. Therefore, this combination of drugs can be used as an effective treatment for controlling growth of human glioblastoma cells.     

Cytidine 5'-monophospho-N-acetylneuraminic acid or a related compound is the low Mr factor from human red blood cells which induces gonococcal resistance to killing by human serum

A low-Mr factor which induces gonococcal resistance to complement-mediated serum killing has been partially purified from lysates of mixed red and buffy coat cells from human blood. The lysates were dialysed against Tris buffer for 24 h at 25 degrees C with the diffusate being continuously recycled through a column of QAE-Sephadex A25. After elution in an NaCl gradient, the active fractions were both desalted and further purified on Sephadex G10. A second fractionation on QAE-Sephadex A25 and desalting with Sephadex G10 preceded further purification by repeated high-pressure liquid chromatography (HPLC) using a DEAE anion exchange column and desalting with Sephadex G10. Less than 500 micrograms of material showing one peak in HPLC was obtained from 1 litre of blood. After NMR had indicated the possible presence of pyrimidine nucleotide, carbohydrate and N-acetyl groups, nanogram quantities of a commercial preparation of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) were shown to induce gonococci to serum resistance. The synthetic CMP-NANA also co-eluted with the preparation from blood cells in HPLC, and the two materials were indistinguishable in their patterns of acid and heat lability. Furthermore, the resistance-inducing activity of both materials was inhibited by cytidine monophosphate, which is known to inhibit sialylation reactions by CMP-NANA. It appears therefore that the resistance-inducing factor is CMP-NANA or a closely related compound. If the factor is CMP-NANA, biological activities indicated that the cell lysate from 1 litre of blood contained about 40 micrograms, and the most purified preparation contained only about 1%. With this minute amount in a mixture, the presence of CMP-NANA or a closely related analogue could not be established unequivocally by NMR.     

Mutations in the eIF(iso)4G translation initiation factor confer high resistance of rice to Rice yellow mottle virus

We report here evidence of the role that the isoform of the eukaryotic translation initiation factor 4G (eIF(iso)4G) plays in naturally occurring resistance in plant/virus interactions. A genetic and physical mapping approach was developed to isolate the Rymv1 locus controlling the high recessive resistance to Rice yellow mottle virus (RYMV) in the rice (Oryza sativa) variety Gigante. The locus was mapped to a 160-kb interval containing a gene from the eIF(iso)4G family. The stable transformation of a resistant line with the cDNA of this gene, derived from a susceptible variety, resulted in the loss of resistance in transgenic plants. The allelic variability of this gene was analysed in three resistant and 17 susceptible varieties from different cultivated rice species or subspecies. Compared with susceptible varieties, resistant varieties present specific alleles, characterized by either amino acid substitutions or short amino-acid deletions in the middle domain of the protein. The structure of this domain was modelled and showed that the substitutions were clustered on a small surface patch. This suggests that this domain may be involved in an interaction with the virus.     

Chromosomal Localization of the Structural Genes of the Polypeptide Chain Elongation Factors

A survey of the polypeptide chain elongation factors in potentially sexually compatible genera was carried out. Factors from Escherichia coli and Proteus mirabilis were found to be clearly distinguishable by immunochemical and electrophoretic techniques. Mapping of the structural genes of these factors was undertaken by a study of the gene products in genetically defined E. coli-P. mirabilis hybrid diploid strains. It was found that the EF G factor mapped within 5 min of the streptomycin resistance locus, but the EF Ts factor did not map in this region.