The Mycobacterium tuberculosis Rv2745c Plays an Important Role in Responding to Redox Stress

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death from an infectious disease worldwide. Over the course of its life cycle in vivo, Mtb is exposed to a plethora of environmental stress conditions. Temporal regulation of genes involved in sensing and responding to such conditions is therefore crucial for Mtb to establish an infection. The Rv2745c (clgR) gene encodes a Clp protease gene regulator that is induced in response to a variety of stress conditions and potentially plays a role in Mtb pathogenesis. Our isogenic mutant, Mtb:ΔRv2745c, is significantly more sensitive to in vitro redox stress generated by diamide, relative to wild-type Mtb as well as to a complemented strain. Together with the fact that the expression of Rv2745c is strongly induced in response to redox stress, these results strongly implicate a role for ClgR in the management of intraphagosomal redox stress. Additionally, we observed that redox stress led to the dysregulation of the expression of the σ^H/σ^E regulon in the isogenic mutant, Mtb:ΔRv2745c. Furthermore, induction of clgR in Mtb and Mtb:ΔRv2745c (comp) did not lead to Clp protease induction, indicating that clgR has additional functions that need to be elucidated. Our data, when taken together with that obtained by other groups, indicates that ClgR plays diverse roles in multiple regulatory networks in response to different stress conditions. In addition to redox stress, the expression of Rv2745c correlates with the expression of genes involved in sulfate assimilation as well as in response to hypoxia and reaeration. Clearly, the Mtb Rv2745c-encoded ClgR performs different functions during stress response and is important for the pathogenicity of Mtb in-vivo, regardless of its induction of the Clp proteolytic pathway.     

Inhibition of Nitric Oxide Synthase Blocks N-Methyl-D-Aspartate-, Quisqualate-, Kainate-, Harmaline-, and Pentylenetetrazole-Dependent Increases in Cerebellar Cyclic GMP In Vivo

The synthesis of nitric oxide by brain slices has been demonstrated in several laboratories. In addition, in vitro studies have demonstrated stimulation of nitric oxide synthesis by excitatory amino acid receptor agonists. These data have led to the hypothesis that this readily diffusible "intercellular messenger molecule" acts to generate a cascade effect by activating guanylate cyclase in several cell types and thereby augment levels of the second messenger cyclic GMP (cGMP). Therefore, we evaluated this hypothesis in vivo, by testing the actions of the nitric oxide synthase inhibitor N-mono-methyl-L-arginine (NMMA) on elevations in level of mouse cerebellar cGMP generated by excitatory amino acid receptor agonists. The stimulatory effects of D-serine, quisqualate, and kainate were all found to be antagonized by this enzyme inhibitor. In addition, NMMA antagonized the increases in cerebellar cGMP level elicited by harmaline and pentylenetetrazole, pharmacological agents that augment endogenous excitatory amino acid transmission. Our data are, therefore, the first in vivo demonstration that nitric oxide is an important "messenger molecule" in the cerebellum, mediating the actions of kainate, quisqualate, and N-methyl-D-aspartate receptor agonists on guanylate cyclase. These data are consistent with previous in vitro findings with kainate and N-methyl-D-aspartate.     

Phosphatidylserine-stimulated production of N-acyl-phosphatidylethanolamines by Ca2+-dependent N-acyltransferase

N-acyl-phosphatidylethanolamine (NAPE) is known to be a precursor for various bioactive N-acylethanolamines including the endocannabinoid anandamide. NAPE is produced in mammals through the transfer of an acyl chain from certain glycerophospholipids to phosphatidylethanolamine (PE) by Ca²⁺-dependent or -independent N-acyltransferases. The ε isoform of mouse cytosolic phospholipase A₂ (cPLA₂ε) was recently identified as a Ca²⁺-dependent N-acyltransferase (Ca-NAT). In the present study, we first showed that two isoforms of human cPLA₂ε function as Ca-NAT. We next purified both mouse recombinant cPLA₂ε and its two human orthologues to examine their catalytic properties. The enzyme absolutely required Ca²⁺ for its activity and the activity was enhanced by phosphatidylserine (PS). PS enhanced the activity 25-fold in the presence of 1?mM CaCl₂ and lowered the EC₅₀ value of Ca²⁺ >8-fold. Using a PS probe, we showed that cPLA₂ε largely co-localizes with PS in plasma membrane and organelles involved in the endocytic pathway, further supporting the interaction of cPLA₂ε with PS in living cells. Finally, we found that the Ca²⁺-ionophore ionomycin increased [¹⁴C]NAPE levels >10-fold in [¹⁴C]ethanolamine-labeled cPLA₂ε-expressing cells while phospholipase A/acyltransferase-1, acting as a Ca²⁺-independent N-acyltransferase, was insensitive to ionomycin for full activity. In conclusion, PS potently stimulated the Ca²⁺-dependent activity and human cPLA₂ε isoforms also functioned as Ca-NAT.     

Methods for induction and utilization of variability in the improvement of an apomictic grass,Dichanthium annulatum complex

Summary Many problems and difficulties are encountered in making genetic improvements in plants where both apomixis and polyploidy occur together. From biosystematic studies on an agamic species complex, Dichanthium annulatum, information is presented on: (A) Mechanisms which create variability in apomicts — (i) genome building and reduction, (ii) hybridization between ecotypes of facultative apomicts, (iii) fertilization of unreduced gametes, (iv) introgressive hybridization, (v) preferential pairing and genotypic control of bivalent formation and (vi) induced mutation; (B) Embryo-sac variations, vis-a-vis sexual/apomictic sacs — (i) production of sexual embryo-sac in apomicts, (ii) balance between apomixis and sexual process, (iii) effect of environment and experimental manipulation of the type of embryo-sac; and (C) Heterosis and fixation of apomixis.The utilization and exploitation of these mechanisms and phenomena for accelerating the genetic improvement of apomictic plants is discussed.Mating systems impose certain restrictions on the breeding methodology to be used in the genetic improvement of crop plants. Allogamous species have built-in mechanisms for self-improvement and, for them, the breeding techniques are well worked out. Little information is, however, available on the procedures to be followed for the genetic improvement of apomicts. Recently gathered information on the causal mechanisms of apomixis and its mode of inheritance, the genetic systems which regulate the balance between apomixis and sexuality, the physical and chemical agents for artificial induction of sexuality in apomicts, and the processes which promote variability and adaptive polymorphism in apomicts show a way for the creation, exploitation and fixation of superior genotypes. Such information, based on biosystematic studies on an agamic species complex, Dichanthium annulatum, at the Oklahoma State University, Stillwater, Oklahoma, U.S.A., is presented here.Breeding procedures commonly followed for the genetic improvement of apomicts are outlined below:1. Collection of varieties, strains or ecotypes from diverse sources; 2. Evaluation of the germ plasm for the presence of desirable characters; 3. Building up of selection indices and estimation of genetic parameters; 4. Determination of mode of reproduction and isolation of sexual types or clones; 5. Hybridization using the sexual types; 6. Progeny testing, comparisons, multiplication and release of superior types.Thus, the success of the breeding programme would depend on the range of variability already present in the germ plasm collections, the relative proportion of sexual/apomictic seed produced and the exploitation of variability from the crossbred progenies. Since large collections of plants with different genotypes are not often available, one would like to look for the mechanisms which can create variability in the apomicts. Such mechanisms are as follows.     

In situ and in vitro characterization of the cellular immune response in erythema nodosum leprosum

We sought to evaluate cell-mediated immune responses in erythema nodosum leprosum (ENL), a reactional state occurring in lepromatous leprosy. Skin biopsies from patients with leprosy were studied with monoclonal antibodies against T lymphocyte antigenic determinants, interleukin 2 (IL 2), and IL 2 receptors (Tac) by using immunoperoxidase staining of frozen sections. Peripheral blood lymphocytes from 18 ENL patients were tested in vitro for lepromin-induced suppression of Con A stimulation. Serial studies of seven lepromatous patients who developed ENL during the course of the study showed increases in both the Leu-3a:Leu-2a ratio and the number of IL 2-positive cells. IL 2-positive cells comprised 0.3% of the cells in all of the ENL lesions studied as compared with the 0.03% found in nonreactional lepromatous lesions (P less than 0.001). Lepromin-induced suppression of the Con A response, present in nonreactional lepromatous patients, significantly decreased in patients developing the ENL reaction, but returned after recovery from ENL. These changes in tissues and peripheral blood suggest that the pathogenesis of ENL is related to cell-mediated immune processes. Despite these immunologic changes, however, ENL patients do not recover antigen-specific skin tests or eliminate Mycobacterium leprae.