The mechanism of aconitase: 1.8 A resolution crystal structure of the S642a:citrate complex

The crystal structure of the S642A mutant of mitochondrial aconitase (mAc) with citrate bound has been determined at 1.8 A resolution and 100 K to capture this binding mode of substrates to the native enzyme. The 2.0 A resolution, 100 K crystal structure of the S642A mutant with isocitrate binding provides a control, showing that the Ser --> Ala replacement does not alter the binding of substrates in the active site. The aconitase mechanism requires that the intermediate product, cis-aconitate, flip over by 180 degrees about the C alpha-C beta double bond. Only one of these two alternative modes of binding, that of the isocitrate mode, has been previously visualized. Now, however, the structure revealing the citrate mode of binding provides direct support for the proposed enzyme mechanism.     

Effect of cisplatin on Allium cepa root meristem cells

Allium cepa root growth was retarded by cisplatin treatment in a dose-dependent manner. A decrease in the mitotic index (MI) and an increase in the number of interphase cells was seen in cisplatin treated root tips. An increase in the frequency of abnormal mitoses and chromosomal aberrations was also observed in cisplatin treated groups which indicates its genotoxic effect on plant cells. The endogenous glutathione (GSH) level in the root tips decreased significantly after cisplatin treatment which may favour its increased interaction with cellular DNA thereby developing enhanced chromosomal aberrations and affecting cell divisions and root growth. It is suggested that the decrease in endogenous GSH may be related to the development of cisplatin-mediated genotoxic effects in plants.     

Effect of cigarette smoke on lipid peroxidation and antioxidant enzymes in albino rat

Effect of cigarette smoke on lipid peroxidation (LPX) and antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione-S-transferase (GST) in various organs like brain, heart, lung, liver and kidney of the albino rats exposed to cigarette smoke for 30 min/day for a period of 30 days were assayed. It was observed that the lipid peroxide levels in liver, lung and kidney were enhanced in case of animals exposed to cigarette smoke, whereas brain and heart did not show any change as compared to control animals. The activity of the antioxidant enzymes was also elevated in liver, lung and kidney of the test animals whereas, brain and heart did not show any change in the activities of all of these antioxidant enzymes except glutathione-s-transferase which was increased in brain also. The level of reduced glutathione (GSH) was lowered in liver, lung and kidney of the tested animals when compared with the control animals but there was no significant change in brain and heart. The results of our study suggest that cigarette smoke induces lipid peroxidation in liver, lung and kidney, and the antioxidant enzymes levels were enhanced in order to protect these tissues against the deleterious effect of the oxygen derived free radicals. The depletion of reduced glutathione in these organs could be due to it's utilization by the tissues to mop off the free radicals.     

Activation of Glut1 glucose transporter in response to inhibition of oxidative phosphorylation.

We have previously shown that exposure of Clone 9 cells to hypoxia, cyanide, or azide results in an acute stimulation of glucose transport that is largely mediated by "activation" of glucose transporter (Glut1) sites preexisting in the plasma membrane. However, it is not known whether inhibition of oxidative phosphorylation only at its terminal step, or at any of its steps, leads to the glucose transport response. Hence, the effect of azide (5 mM), rotenone (1 microM), rotenone (1 microM) plus thenoyltrifluoroacetone (TTFA) (5 microM), antimycin A (0.3 microM), dinitrophenol (0.25 mM), carbonyl cyanide m-chlorophenylhydrazone (CCCP) (2.5 microM), and oligomycin B (0.15 microM) on glucose transport was determined. All of the above agents elicited a similar approximately 4-fold stimulation of cytochalasin B (CB)-inhibitable 3-O-methyl glucose (3-OMG) uptake in Clone 9 cells. The stimulatory effect of azide on 3-OMG uptake was not inhibited by antioxidants 2-mercaptopropionyl glycine (1.2 mM) and 1,10-phenanthroline (40 microM), while, in contrast, the antioxidants attenuated the stimulation of glucose transport in response to 250 microM H(2)O(2) by approximately 50%. To differentiate between an increase in the number of functional Glut1 sites in the plasma membrane (in the absence of "translocation") versus an increase in the "intrinsic activity" of Glut1, the effect of azide on the energy of activation (E(a)) of glucose transport was measured. The E(a) was determined by measuring the rate of CB-inhibitable 3-OMG uptake at 24.0, 28.0, 35. 0, and 40 degrees C. The E(a) of control Clone 9 cells and of cells exposed to 10 mM azide for 2 h was 32,530 +/- 1830 and 31,220 +/- 600 J/mol, respectively (P > 0.1), while the rate of CB-inhibitable 3-OMG uptake was 9.3 +/- 0.7-fold higher in azide-treated cells. It is concluded that (i) inhibition of oxidative phosphorylation, at any of its steps, leads to a stimulation of glucose transport, and (ii) the mechanism of stimulation of glucose transport in response to azide appears to be predominately mediated by an apparent increase in the number of functional Glut1 sites in the plasma membrane (instead of an increase in their "intrinsic activity"), suggesting an "unmasking" mechanism.     

Human LAT1, a subunit of system L amino acid transporter: molecular cloning and transport function

We report here on the cloning and functional characterization of human LAT1, a subunit of the amino acid transport system L. The hLAT1 cDNA, obtained from a human placental cDNA library, codes for a protein of 507 amino acids. When functionally expressed in mammalian cells together with the heavy chain of the rat 4F2 antigen (r4F2hc), hLAT1 induces the transport of neutral amino acids. When expressed independently, neither hLAT1 nor r4F2hc was capable of amino acid transport to any significant extent. Thus, the hLAT1-r4F2hc heterodimeric complex is responsible for the observed amino acid transport. The transport process induced by the heterodimer is Na+ independent and is not influenced by pH. It recognizes exclusively neutral amino acids with high affinity. LAT1-specific mRNA is expressed in most human tissues with the notable exception of the intestine.     

Thyrotropin releasing hormone stimulation of GABA-gated but not basal chloride ion influx in rat cerebellum

Potential interaction between gamma-amino butyric acid (GABA) and thyrotropin-releasing hormone (TRH) in eliciting a variety of central nervous system (CNS)-related biologic activities is well known; however, the mechanism underlying this interaction is not clearly defined. To gain further insight into this interaction, we examined the effects of TRH and two of its central nervous system selective analogs, DN 1417 and TA 0910, on basal and GABA-mediated chloride ion influx into rat cerebellar neurosynaptosomes. The results of these studies show that TRH may facilitate GABA action by augmenting chloride ion influx and hyperpolarization of cerebellar neurons.     

Primary subcutaneous Nocardia asteroides infection in a renal allograft recipient

Nocardia asteroides is an important opportunistic pathogen in immunocompromised hosts. The primary infection is usually in the lungs and is followed by dissemination to other parts of the body. Primary subcutaneous infection with Nocardia asteroides has been reported rarely (three reports) and no such case has been reported in a renal transplant recipient. We describe here a case of renal transplant recipient who developed primary subcutaneous infection with Nocardia asteroides within one and half years of the transplantation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Altering Cellular Signaling Pathways Enhance Gene Silencing Activity of shRNA,shRNA.Ribozyme,and shRNA.Antisense in Neuroblastoma Cells

1. RNA interference (RNAi) is a multicomponent machinery that operates in a sequence-specific manner to repress the expression of genes in most eukaryotic cells.2. Here we wanted to investigate in a murine neuroblastoma cell line (NBP2) (a) if replacement of the loop of the short hairpin RNA (shRNA) with a hammerhead ribozyme (shRNA.RZ) or an antisense oligonucleotide (shRNA.AS) would affect the efficacy of gene suppression, and (b) if activation or inhibition of signaling pathways would enhance the efficacy of shRNA, shRNA.RZ, and shRNA.AS complex in gene silencing.3. We used U6-driven expression of these shRNAs to target either a short-lived green fluorescent protein (d2EGFP) or an endogenous cyclophilin A (CyP-A) gene in a d2EGFP expressing NBP2 cell line (NBP2-PN25).4. Activation of the cAMP signaling pathway or inhibition of phosphatidylinositol 3-kinase (PI3K) enhanced the efficacy of shRNA and shRNA.RZ complex in reducing the expression of d2EGFP shRNA.RZ complex was as efficacious as shRNA in reducing the expression of d2EGFP and CyP-A shRNA.AS complex showed a slightly lower efficacy than shRNA alone in decreasing d2EGFP expression. In contrast, the U6-driven hammerhead ribozyme targeted to d2EGFP showed no gene silencing activity.5. This report describes novel strategies of modifying shRNA and altering signaling pathways to affect siRNA-mediated gene silencing in a neuronal cell line.     

Epidemiology and molecular typing of Candida isolates from burn patients

This study, spread over a span of 2 years describes Candida infections in burn patients of an Indian hospital. A total of 220 burn patients were monitored and Candida could be isolated from 138 patients. A total of 228 different Candida species were obtained from various body locations of these patients. Species identification revealed that Candida albicans was the most predominant (45) followed by Candida tropicalis(33), Candida glabrata (13.5), C. parapsilosis (4), C. krusei (2.75) and C. kefyr (1.75). DNA fingerprinting of all C. albicans isolates was done by using CARE-2 probe. Fingerprinting analyses of all the C. albicans strains revealed that strains collected from different patients were different. It is noteworthy that patients with disseminated candidiasis had a similar, but unique strain isolated from all body locations, suggesting a possibility that commensal isolates might be turning pathogenic. Taken together, this is probably the first ever detailed survey of Candidainfections in burn patients in India and is expected to lead to better clinical management of this group of patients.     

Antioxidant effect of zinc in humans

Oxidative stress is known to be an important contributing factor in many chronic diseases. We tested the hypothesis that in healthy normal volunteers zinc acts as an effective anti-inflammatory and antioxidant agent. Ten normal volunteers were administered daily oral zinc supplementation (45 mg zinc as gluconate) and 10 volunteers received placebo for 8 weeks. Plasma zinc, MDA, HAE, and 8-OHdG levels; LPS-induced TNF-alpha and IL-1beta mRNA; and ex vivo TNF-alpha-induced NF-kappaB activity in mononuclear cells (MNC) were determined before and after supplementation. In subjects receiving zinc, plasma levels of lipid peroxidation products and DNA adducts were decreased, whereas no change was observed in the placebo group. LPS-stimulated MNC isolated from zinc-supplemented subjects showed reduced mRNA for TNF-alpha and IL-1beta compared to placebo. Ex vivo, zinc protected MNC from TNF-alpha-induced NF-kappaB activation. In parallel studies using HL-60, a promyelocytic cell line, we observed that zinc enhances the upregulation of mRNA and DNA-specific binding for A20, a transactivating factor which inhibits the activation of NF-kappaB. Our results suggest that zinc supplementation may lead to downregulation of the inflammatory cytokines through upregulation of the negative feedback loop A20 to inhibit induced NF-kappaB activation. Zinc administration to human subjects with conditions associated with increased oxidative stress should be explored.