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.     

Alterations in osteoclast morphology following osteoprotegerin administration in the magnesium-deficient mouse

In the present study, we used osteoprotegerin (OPG), which blocks osteoclastogenesis, to correct and thus explain the hypercalcemia that is seen during dietary Mg deficiency in the mouse. Control and Mg-deficient mice received injections for 12 days of either OPG or vehicle only. Serum Ca was similar in Mg-deficient mice treated with OPG and in control mice receiving OPG (9.2±0.3 mg/dl vs. 9.2±0.5). Both groups had significantly higher serum Ca than controls or Mg-deficient animals receiving vehicle alone. Surprisingly, Mg-depleted mice that received OPG in doses that inhibit osteoclastic bone resorption remained hypercalcemic. Because mature osteoclasts still present in the marrow might be hyperactive, we examined osteoclast morphology at the light microscopic and ultrastructural level. Light microscopic examination of trabecular bone showed few osteoclasts in OPG-treated mice. Ultrastructural examination revealed that osteoclasts in OPG-treated mice have decreased contact with the endosteal bone surface and absence of a ruffled border. Because the morphology of the existing pool of mature osteoclasts did not enhance resorption, another mechanism, such as increased intestinal absorption of Ca in Mg-deficient mice, likely contributes to the hypercalcemia observed during Mg deficiency.     

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.     

Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [γ-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [γ-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.     

Structure of rapidly frozen gap junctions

The structure of gap junctions in the rabbit ciliary epithelium, corneal endothelium, and mouse stomach and liver was studied with the freeze-fracturing technique after rapid freezing to near 4 degrees K from the living state. In the ciliary epithelium, the connexons were randomly distributed, separated by smooth membrane matrix. In the corneal endothelium, both random and crystalline arrangements of the connexons were observed. In the stomach and liver, the connexons were packed but not crystalline. Experimental anoxia or lowered pH caused crystallization of the connexons within 20-30 min. In the ciliary epithelium, the effects of prolonged anoxia or low pH could not be reversed . In addition, invaginated or annular gap junctions increased in number, but their connexons were usually distributed at random. Rapid freezing thus demonstrates that gap junctions of different tissues are highly pleiomorphic in the living state, and this may explain their variations in structure after chemical fixation. The slow time-course and irreversibility of the morphological changes induced by prolonged anoxia or low pH suggest that connexon crystallization may be a long-term consequence rather than the morphological correlate of the switch to high resistance.     

Fungal infections of ear with special reference to chronic suppurative otitis media

Fungus were found to take important role in ear infections of the 344 patients (CSOM 286, Otomycosis 44, Otitis externa 14), significant fungal infections (with positive smear and culture) were detected on 49%, 79.5%, 66.6% patients respectively. 84.8% patients were detected both by smear and culture, 14.1% patients by culture and 0.1% patients in smear preparation only. In CSOM patients, age predominated in 20–27 yrs group, sex in male below 30 yrs, and Aspergillus flavus, A. niger, Penicillium, A. fumigatus in mycelial fungus, Candida albicans, C. parapsillosis in yeast. But in 18 post antibiotic fungus infected patients Penicillium and A. niger were the important isolates. In otomycosis and otitis externa patients A. niger took the main role.     

Calmodulin and rat vitamin D-dependent calcium-binding proteins: Biochemical and immunochemical comparison

Purified vitamin D-dependent rat intestinal (M_r 10,000) and rat renal (M_r 28,000) calcium-binding proteins (CaBPs) have been compared to vertebrate calmodulin, and the vitamin D-dependent CaBPs have been found to be distinct from calmodulin by biochemical and immunochemical criteria. Rat renal and rat intestinal CaBPs do not stimulate 3′,5′-cyclic nucleotide phosphodiesterase, do not compete with iodinated calmodulin for binding to phenothiazine-Sepharose conjugates, do not cross-react immunochemically, and do not contain N^?-trimethyllysine. In addition, although calmodulin exhibits a characteristic calcium-dependent mobility shift on polyacrylamide gels in the presence of sodium dodecyl sulfate, a similar mobility shift is not observed for the vitamin D-dependent CaBPs. Immunocytochemically, calmodulin has a widespread localization in the kidney, whereas CaBP is present specifically in the distal tubules of the kidney. These localizations suggest a specialized role for CaBP in the kidney. Thus, although the vitamin D-dependent CaBPs and calmodulin are similar in that they are small, acidic, calcium-binding proteins, these two classes of proteins are biochemically and immunochemically distinct.