Jitendra N. Singh

Transcriptional analysis and functional characterization of a gene pair encoding iron regulated xenocin and immunity proteins of Xenorhabdus nematophila

We describe a two-gene cluster encoding a bacteriocin, xenocin, and the cognate immunity protein in the insect-pathogenic bacterium Xenorhabdus nematophila, which infects and kills larval stages of the common crop pest Helicoverpa armigera. The two genes, xcinA and ximB, are present in the genome as a single transcriptional unit, which is regulated under SOS conditions. The stress-inducible promoter was activated by mitomycin C, glucose, and Fe(3+) depletion and at an elevated temperature when it was tested in Escherichia coli cells. Expression of the xenocin protein alone in E. coli inhibited the growth of this organism. The growth inhibition was abolished when the immunity protein was also present. A recombinant xenocin-immunity protein complex inhibited the growth of E. coli indicator cells when it was added exogenously to a growing culture. Xenocin is an endoribonuclease with an enzymatically active C-terminal domain. Six resident bacterial species (i.e., Bacillus, Enterobacter, Enterococcus, Citrobacter, Serratia, and Stenotrophomonas species) from the H. armigera gut exhibited sensitivity to recombinant xenocin when the organisms were grown under iron-depleted conditions and at a high temperature. Xenocin also inhibited the growth of two Xenorhabdus isolates. This study demonstrates that Fe(3+) depletion acts as a common cue for synthesis of xenocin by X. nematophila and sensitization of the target strains to the bacteriocin.

The Cytotoxic Fimbrial Structural Subunit of Xenorhabdus nematophila Is a Pore-Forming Toxin

We have purified a fimbrial shaft protein (MrxA) of Xenorhabdus nematophila. The soluble monomeric protein lysed larval hemocytes of Helicoverpa armigera. Osmotic protection of the cells with polyethylene glycol suggested that the 17-kDa MrxA subunit makes pores in the target cell membrane. The internal diameter of the pores was estimated to be >2.9 nm. Electron microscopy confirmed the formation of pores by the fimbrial subunit. MrxA protein oligomerized in the presence of liposomes. Electrophysiological studies demonstrated that MrxA formed large, voltage-gated passive-diffusion channels in lipid bilayers.

Effects of 4-phenyl butyric acid on high glucose-induced alterations in dorsal root ganglion neurons

Mechanisms and pathways involving in diabetic neuropathy are still not fully understood but can be unified by the process of overproduction of reactive oxygen species (ROS) such as superoxide, endoplasmic reticulum (ER) stress, downstream intracellular signaling pathways and their modulation. Susceptibility of dorsal root ganglion (DRG) to internal/external hyperglycemic environment stress contributes to the pathogenesis and progression of diabetic neuropathy. ER stress leads to abnormal ion channel function, gene expression, transcriptional regulation, metabolism and protein folding. 4-phenyl butyric acid (4-PBA) is a potent and selective chemical chaperone; which may inhibit ER stress. It may be hypothesized that 4-PBA could attenuate via channels in DRG in diabetic neuropathy. Effects of 4-PBA were determined by applying different parameters of oxidative stress, cell viability, apoptosis assays and channel expression in cultured DRG neurons. Hyperglycemia-induced apoptosis in the DRG neuron was inhibited by 4-PBA. Cell viability of DRG neurons was not altered by 4-PBA. Oxidative stress was significantly blocked by the 4-PBA. Sodium channel expression was not altered by the 4-PBA. Our data provide evidence that the hyperglycemia-induced alteration may be reduced by the 4-PBA without altering the sodium channel expression.

Diabetic-induced increased sodium channel activity attenuated by tetracaine in sensory neurons in vitro.

The present study was aimed to explore correlation between the altered pain perception and Na(+) channel activity in diabetic animals as well as the effect of tetracaine on sensory neurons of diabetic rat. In streptozotocin-induced diabetic rats behavioral nociceptive parameters were assessed. The Na(+) current (INa) was obtained using whole-cell voltage-clamp configuration in dorsal root ganglion (DRG) neurons isolated from diabetic rat (in vitro). In addition, the effects of tetracaine on altered Na(+) channel activity associated with diabetes in small DRG neurons were evaluated. After induction of diabetes mechanical allodynia, thermal hyperalgesia and Na(+) channel activity were altered significantly in 4th and 6th week in relation to the control. Altered pain parameters were in correlation with increased INa in time-dependent manner. In comparison to age-matched control (-1.10±0.20nA) the INa was found to be -2.49±0.21nA at 4th week and -3.71±0.28nA at 6th week. The increased activity of Na(+) channels was blocked by tetracaine even in diabetic condition. The depression of the INa on tetracaine exposure was not sensitive to the voltage or time. The conductance curve shifted towards right around -8.0mV. The alterations in neuropathic pain associated with diabetes and Na(+) channel activity has been clearly correlated in time-dependent manner. The INa density was increased significantly with the progression of neuropathic pain. Local anesthetic, tetracaine potentially blocked the Na(+) channel activity in diabetic sensory neurons.

hERG Potassium Channels in Drug Discovery and Development

Potassium (K+) channels play a central role in the electrical activity of excitable cells. Although there are variety of potassium channels, scientists have developed immense interest in human ether-a-go-go-related gene (hERG) potassium channels due to their involvement in life-threatening cardiac arrhythmia. hERG is a gene that encodes the pore-forming α-subunit of a voltage-gated potassium channel expressed in nervous and cardiac tissue including atrium, ventricles, purkinje fiber, SA node and AV node. Potassium flow through hERG channel plays an important role in action potential repolarization, particularly in ventricular muscle. Blockade of hERG potassium channel via pharmacological interventions or hereditary mutations of genes encoding the channel is associated with a prolongation of cardiac ventricular repolarization, that is long QT syndrome (LQTS), a disorder that predisposesindividuals to life-threatening arrhythmias and substantial risk of sudden death. Inherited or drug-induced mutations in hERG channel lead to disruption of delayed rectifier potassium current (IKr), increase in cardiac excitability subsequently torsades de pointes and sudden death. A large number of putative disease-causing mutations in hERG have been identified in affected families so far, yet mechanism behind these mutations is unspecified and undistinguished. Therefore, entire paradigm of drug discovery has shifted towards the safety of the new molecules to screen for potential cardiac arrhythmogenic effects. Non-clinical assays are not sensitive enough to accurately predict QT prolongation liabilities in humans. For this reason, International Conference on Harmonization (ICH) safety pharmacology S7B guidelines were proposed for new chemical entities. According to these guidelines, thorough studies (in vitro and in vivo) on QT are required for virtually all newly developed pharmaceutical agents. In this article, an overview on hERG channels, their functions and dysfunctions, therapeutic agents modulating these channels and associated QT prolongation, and assay have been discussed.

Hyper-insulinemia increases the glutamate-excitotoxicity in cortical neurons: A mechanistic study

Abstract Insulin resistance in type‐2 diabetic condition increases the risk of stroke and cognitive deficits in which involvement of glutamate has been postulated. It has been hypothesized that hyper‐insulinemia in cortical neurons increases the vulnerability towards glutamate‐induced excitotoxicity. To mimic insulin resistance, cortical neurons were incubated with high insulin (1 &mgr;M) and high glucose (50 mM final concentration) in in‐vitro condition for 24 h. Pre‐treatment of cortical neurons with high insulin blocked acute insulin‐induced activation of Akt and GSK‐3&bgr; but not in the case of high glucose. Our results demonstrate that chronic high insulin exposure increases glutamate‐induced excitotoxity, which was blocked by insulin receptor antagonist (S961) and GSK‐3&bgr; inhibitor (SB216763). These inhibitors also ameliorated pAkt (Ser473) and pGSK‐3&bgr;(Ser9) levels after chronic insulin exposure. Increase in glutamate‐excitotoxicity in insulin‐resistant cortical neurons was found to be associated with increased expression of PICK1. However, GluR2 did not get altered in hyper‐insulinemia condition. This study demonstrates that hyper‐insulinemia increases glutamate excitotoxicity which could be attributed to activation of GSK‐3&bgr; and increased expression of PICK1.