Amino acid substitutions conferring resistance to quinolones in Mycobacterium tuberculosis have generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance in M. tuberculosis, we expressed recombinant DNA gyrases in Escherichia coli and characterized them in vitro. Wild-type and GyrB-E540V DNA gyrases were reconstituted in vitro by mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5-to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5-to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance in M. tuberculosis.A major human infectious disease, tuberculosis (TB) is estimated to affect approximately one-third of the world's population, and 95% of cases occur in developing countries (15,30,31). Current estimates show that approximately 9.4 million new cases and nearly 1.7 million deaths from TB occur each year, and TB remains a major cause of premature death (36).The increased incidence of multidrug-resistant (MDR) TB (TB resistant to more than two anti-TB drugs, including rifampin and isoniazid [35]) has hampered the treatment and control of TB and is associated with an increase in mortality rates in people with TB (3,37,40). Consequently, the required drug dosage for the treatment of TB has dramatically increased (38), and fluoroquinolones (FQs) are now considered to be important second-line anti-TB agents (13,20
Abstract:We examined the possibility that c-Jun N-terminal kinase (JNK) and nuclear factor KB (NF-KB) might be involved in intracellular signaling cascades that mediate NMDA-initiated neuronal events. Exposure of cortical neurons to 100 1iM NMDA induced activation of JNK within 1 mm. Activity of JNK was further increased over the next 5 mm and then declined by 30 mm. Similarly, ionomycin, a selective Ca 2 ionophore, induced activation of JNK. The NMDA-induced activation of JNK was abrogated in the absence of extracellular Ca2~,suggesting that Ca2~entry is necessary and sufficient for the JNK activation. Immunohistochemistry with anti-NF-KB antibody demonstrated nuclear translocation of NF-KB within 5mm following NMDA treatment. NMDA treatment also enhanced the DNA binding activity of nuclear NF-KB in a Ca2-dependent manner. Treatment with 3 mM aspirin blocked the NMDA-induced activation of JNK and NE-KB. Neuronal death following a brief exposure to 100.tM NMDA was Ca2dependent and attenuated by addition of aspirin or sodium salicylate. The present study suggests that Ca2~influx is required for NMDA-induced activation of JNK and NF-KB as well as NMDA neurotoxicity. This study also implies that aspirin may exert its neuroprotective action against NMDA through blocking the NMDA-induced activation of NF-KB and JNK. Key Words: Ca2-c-Jun N-terminal kinase-Nuclear factor KB-NMDA-Aspirin-Cortical neurons. J. Neurochem. 71, 1390Neurochem. 71, -1395Neurochem. 71, (1998Neurochem. 71, ). 1978Sloviter, 1983; Rothman, 1984;Choi et a!., 1988). Due to the large Ca2~conductance and voltage dependence, NMDA receptors are thought to play a major role in neurodegeneration as well as learning and memory Collingridge and Bliss, 1995). Extensive evidence indicates that downstream signals of NMDA neurotoxicity involve activation of several Ca2-dependent enzymes such as calpain (Siman and Noszek, 1988), phospholipase A 2 (Lazarewicz et a!., 1990), neuronal nitric oxide synthase (Dawson et al., 1991), and endonucleases (Kure et al., 1991).A number of reports suggest that nuclear factor KB (NF-KB) and mitogen-activated protein kinase (MAPK) may participate in the process of glutamate neurotoxicity. For example, glutamate treatment induces activation of NF-KB through NMDA receptors in cerebellar neurons (Guerrini et al., 1995;Kaltschmidt et al., 1995). The activation of NF-KB is necessary for NMDA-induced neurotoxicity in cerebellar and hippocampal neurons (Grilli et al., 1996). In addition, glutamate has been shown to activate MAPKs (Kawasaki et al., 1997;Schwarzschild et al., 1997).In light of the possibility that NF-KB and c-Jun Nterminal kinase (JNK) may play a role in the process of NMDA-mediated neuronal events, we conducted experiments to study whether NF-KB and JNK could be activated by NMDA in cortical neurons. In addition, Glutamate and aspartate, the major excitatory neurotransmitters in the brain, become toxic to central neurons via excess activation of the ionotropic receptors sensitive to NMDA, kainate, or cx-amino-3-hy...
Within the endoplasmic reticulum (ER), mannoses and glucoses, donated from dolichol-phosphate-mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositolmannosyltransferase I (GPI-MT-I), which transfers the first of the four mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was <10% in the absence of PIG-X, indicating that PIG-X stabilizes PIG-M. We found that Saccharomyces cerevisiae Pbn1p/YCL052Cp, which was previously reported to be involved in autoprocessing of proproteinase B, is the functional homologue of PIG-X; Pbn1p is critical for Gpi14p/YJR013Wp function, the yeast homologue of PIG-M. This is the first report of an essential subcomponent of glycosyltransferases using dolichol-phosphate-monosaccharide.
GPI mannosyltransferase I (GPI-MT-I) transfers the first mannose to a GPI-anchor precursor, glucosamine-(acyl)phosphatidylinositol [GlcN-(acyl)PI]. Mammalian GPI-MT-I consists of two components, PIG-M and PIG-X, which are homologous to Gpi14p and Pbn1p in Saccharomyces cerevisiae, respectively. In the present study, we disrupted yeast GPI14 and analysed the phenotype of gpi14 yeast. The gpi14 haploid cells were inviable and accumulated GlcN-(acyl)PI. We cloned PIG-M homologues from human, Plasmodium falciparum (PfPIG-M) and Trypanosoma brucei (TbGPI14), and tested whether they could complement gpi14-disrupted yeast. None of them restored GPI-MT-I activity and cell growth in gpi14-disrupted yeast. However, gpi14-disrupted yeast cells with human PIG-M, but not with PfPIG-M or TbGPI14, grew slowly but significantly when they were supplemented with rat PIG-X. This suggests that the association of PIG-X and PIG-M for GPI-MT-I activity is not interchangeable between mammals and the other lower eukaryotes.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired type hemolytic disorder. Hematopoietic cells of patients with PNH are deficient in glycosylphosphatidylinositol (GPI) anchored membrane proteins. Since some membrane-bound complement inhibitors, such as CD59 and decay accelerating factor (DAF), are GPI anchored proteins, abnormal cells from patients with PNH are sensitive to complement attack. Their myeloid and erythroid cells are affected more than their lymphoid cells. Patients whose B cells were severely deficient in GPI anchored proteins were chosen to establish cell lines by Epstein-Barr virus mediated transformation. The lines established (SS-1-, TK-1-, and TK-14- cell lines) had the following characteristics of PNH. First, GPI anchored proteins were completely absent from the surface of SS-1- and TK-14- cells, and were expressed at very low levels on TK-1- cells, whereas polypeptide anchored proteins were normally expressed on these three lines. Secondly, DAF mRNAs of the SS-1- cell line were qualitatively and quantitatively indistinguishable from those of a control, wild-type cell line. Third, pro-CD59 and pro-DAF molecules were detected intracellularly in these cell lines, their pro-CD59 being smaller and more hydrophilic than that from a wild-type cell line. These cell lines should be useful in further studies on the pathogenesis of PNH.
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