Identification of regions involved in enzymatic stability of peptide deformylase of Mycobacterium tuberculosis

Sequence analysis of peptide deformylase of Mycobacterium tuberculosis revealed the presence of insertions (residues 74 to 85) and an unusually long carboxy-terminal end (residues 182 to 197). Our results with deletion mutants indicated the contribution of these regions in maintaining enzymatic stability. Furthermore, we showed that the region spanning the insertions was responsible for maintaining resistance to oxidizing agents, like H(2)O(2).     

Variation in menstrual characters: a study between married and unmarried women of West Bengal

Menstrual cycle is an endocrine function of the ovary, controlled by the hypothalamus-pituitary-gonadal axis. Variation in menstrual characters across different ages, socioeconomic status, place of residence, ethnic groups and so on suggests that the function of hypothalamus-pituitary-gonadal axis is influenced by some internal and external factors. The objectives of the present study are to understand the variation in menstrual characters with respect to differential marital status of women and as well as to find out whether marital status can be a significant predictor of any menstrual character. Data have been collected on a total number of 200 participants (unmarried 100 and married 100) with the help of a well-tested questionnaire/schedule. Unmarried participants who are aged between 18 and 21 years and their closest blood related married kin members who are in the age group 25-35 years, have given birth to at least one child, presently not lactating or family way and are not using any hormonal contraceptives for the last one year period have been selected for the study. Bivariate analysis reveals that significant differences exist between married and unmarried women in menstrual characters like menstrual cycle length, nature of menstrual discharge and presence of premenstrual problems. Multivariate analysis also shows that marital status of a woman is one of the significant predictors of these menstrual characters (except nature of menstrual discharge). It may be concluded that probably a relationship exists between marital status of a woman and some of the menstrual characters.     

Selectable antibiotic resistance marker gene-free transgenic rice harbouring the garlic leaf lectin gene exhibits resistance to sap-sucking planthoppers

Rice, the major food crop of world is severely affected by homopteran sucking pests. We introduced coding sequence of Allium sativum leaf agglutinin, ASAL, in rice cultivar IR64 to develop sustainable resistance against sap-sucking planthoppers as well as eliminated the selectable antibiotic-resistant marker gene hygromycin phosphotransferase (hpt) exploiting cre/lox site-specific recombination system. An expression vector was constructed containing the coding sequence of ASAL, a potent controlling agent against green leafhoppers (GLH, Nephotettix virescens) and brown planthopper (BPH, Nilaparvata lugens). The selectable marker (hpt) gene cassette was cloned within two lox sites of the same vector. Alongside, another vector was developed with chimeric cre recombinase gene cassette. Reciprocal crosses were performed between three single-copy T₀ plants with ASAL- lox-hpt-lox T-DNA and three single-copy T₀ plants with cre-bar T-DNA. Marker gene excisions were detected in T₁ hybrids through hygromycin sensitivity assay. Molecular analysis of T₁ plants exhibited 27.4% recombination efficiency. T₂ progenies of L03C04(1) hybrid parent showed 25% cre negative ASAL-expressing plants. Northern blot, western blot and ELISA showed significant level of ASAL expression in five marker-free T₂ progeny plants. In planta bioassay of GLH and BPH performed on these T₂ progenies exhibited radical reduction in survivability and fecundity compared with the untransformed control plants.     

Solubilization,purification and reconstitution of Ca-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: Preservation of native structure and function of the enzyme by DHPC

The properties of Ca²⁺-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C₁₂E₈) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca²⁺-ATPase with a greater specific activity than solubilization with C₁₂E₈ or Triton X-100. DHPC was determined to be superior to C₁₂E₈; while that the C₁₂E₈ was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca²⁺-ATPase retained the E1Ca−E1*Ca conformational transition as that observed for native microsomes; whereas the C₁₂E₈ and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca²⁺ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C₁₂E₈ and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca²⁺-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C₁₂E₈ and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca²⁺ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca²⁺-ATPase retained more organized and native secondary conformation compared to C₁₂E₈ and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C₁₂E₈ and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca²⁺-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C₁₂E₈ and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme.     

High-frequency induction of multiple shoots and clonal propagation from rhizomatous nodal segments of Houttuynia cordata Thunb.—An ethnomedicinal herb of India

Summary This study reports an efficient and direct shoot bud differentiation and multiple shoot induction from nodal segments of underground stoloniferous rhizomes of Houttuynia cordata Thumb. The frequency of shoot bud regeneration was influenced by the type of cytokinin and concentrations. Among the various concentrations used, benzylaminopurine (BAP, 17.74 μM) or kinetin (Kn, 18.58 μM) was found to be most effective for rapid and maximum shoot but differentiation. The number of shoots per explant was higher (20.00±2.61) on Murashige and Skoog (MS) medium supplemented with Kn (18.58 μM) compared to BAP and 6-γ-γ-(dimethyl-allylamino)-purine (2iP) during initial 40-d-old culture. Subsequent shoot differentiation and multiplication were achieved in MS medium containing 9.29 μM Kn and 15% (v/v) coconut milk. Elongation and growth of multiple shoots were also obtained on MS medium containing either 2.32 μM Kn or 2.46 μM 2iP alone. The rate of shoot multiplication during subcultures declined with an increase in the size of proliferating shoot cluster. Reducing shoot cluster size to three to four shoots and subculturing together in shoot multiplication medium resulted in a better shoot multiplication and growth, which could be maintained for 2 yr. The elongated shoots (>20 mm) were successfully rooted on MS medium supplemented with 19.60 μM indole-3-butyric acid. Regenerated plants were successfully established in soil and were found to be healthy and uniform. The protocol reported in this study can be used for conservation and utilization of elite clone of H. cordata.     

Ethics of care and HIV: a case for rural women in India

Recent literature shows that ethics of care can be used as a theoretical basis to add a new, important dimension to social issues. This paper argues for a similar extension of the theoretical support from ethics of care to an area in bioethics. Specifically, it contends that a justification based ethics of care can be constructed to argue for a moral obligation to give some priority in the HIV-related initiatives to one of most vulnerable groups; namely, the rural women in India. In an epidemic situation this care-based approach has certain advantages as a moral justification over the usual traditional approaches.     

Complement activation in heart diseases. Role of oxidants

Increasing evidence demonstrated that atherosclerosis is an immunologically mediated disease. Myocardial ischemia/reperfusion injury is accompanied by an inflammatory response contributing to reversible and irreversible changes in tissue viability and organ function. Three major components are recognized as the major contributing factors in reperfusion injury. These are: (1) molecular oxygen; (2) cellular blood elements (especially the neutrophils); and (3) components of the activated complement system. The latter two often act in concert. Endothelial and leukocyte responses are involved in tissue injury, orchestrated primarily by the complement cascade. Anaphylatoxins and assembly of the membrane attack complex contribute directly and indirectly to further tissue damage. Tissue damage mediated by neutrophils can be initiated by complement fragments, notably C5a, which are potent stimulators of neutrophil superoxide production and adherence to coronary artery endothelium. The complement cascade, particularly the alternative pathway, is activated during myocardial ischemia/reperfusion. Complement fragments such as the anaphylatoxins C3a and C5a, are produced both locally and systematically, and the membrane attack complex is deposited on cell membranes and subsequent release of mediators such as histamine and platelet activating factor (PAF), thereby causing an increase in vascular permeability with concomitant manifestation of cellular edema. Complement increases the expression of CD18 on the neutrophils and increases P-selectin expression on the surface of the endothelium. Mitochondria may be a source of molecules that activate complements during ischemia/reperfusion injury to myocardium, providing therewith a stimulus for infiltration of polymorphonuclear leukocytes. Tissue salvage can be achieved by depletion of complement components, thus making evident a contributory role for the complement cascade in ischemia/reperfusion injury. The complexities of the complement cascade provide numerous sites as potential targets for therapeutic interventions designed to modulate the complement response to injury. The latter is exemplified by the ability of soluble form of complement receptor 1 (sCR1) to decrease infarct size in in vitro models of ischemia/reperfusion injury. The mechanism(s) that initiates complement activation is not clearly known, although loss of CD59 (protectin) from cells compromised by ischemia/reperfusion may contribute to direct damage of the coronary vascular bed by the terminal complement complex. Therapeutic approaches to ischemia/reperfusion injury in general, and especially those involving complements, are at the very beginning and their potential benefits have still to be adequately evaluated. It may be noted that complement activation has both positive and negative effects and, therefore, might be modulated rather than abruptly blunted.     

Role of protein kinase C in phospholemman mediated regulation of α₂β₁ isozyme of Na⁺/K⁺-ATPase in caveolae of pulmonary artery smooth muscle cells

We have recently reported that α(2)β(1) and α(1)β(1) isozymes of Na(+)/K(+)-ATPase (NKA) are localized in the caveolae whereas only the α(1)β(1) isozyme of NKA is localized in the non-caveolae fraction of pulmonary artery smooth muscle cell membrane. It is well known that different isoforms of NKA are regulated differentially by PKA and PKC, but the mechanism is not known in the caveolae of pulmonary artery smooth muscle cells. Herein, we examined whether this regulation occurs through phospholemman (PLM) in the caveolae. Our results suggest that PKC mediated phosphorylation of PLM occurs only when it is associated with the α(2) isoform of NKA, whereas phosphorylation of PLM by PKA occurs when it is associated with the α(1) isoform of NKA. To investigate the mechanism of regulation of α(2) isoform of NKA by PKC-mediated phosphorylation of PLM, we have purified PLM from the caveolae and reconstituted into the liposomes. Our result revealed that (i) in the reconstituted liposomes phosphorylated PLM (PKC mediated) stimulate NKA activity, which appears to be due to an increase in the turnover number of the enzyme; (ii) phosphorylated PLM did not change the affinity of the pump for Na(+); and (iii) even after phosphorylation by PKC, PLM still remains associated with the α(2) isoform of NKA.     

Detection and phylogenetic profiling of nodavirus associated with white tail disease in Malaysian <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis> de Man

White tail disease (WTD) is a serious viral disease in the hatcheries and nursery ponds of Macrobrachium rosenbergii in many parts of the world. A new disease similar to WTD was observed in larvae and post larvae of M. rosenbergii cultured in Malaysia. In the present study, RT-PCR assay was used to detect the causative agents of WTD, M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) using specific primers for MrNV RNA2 and XSV. The results showed the presence of MrNV in the samples with or without signs of WTD. However, XSV was only detected in some of the MrNV-positive samples. Phylogenetic analysis showed that the RNA2 of our Malaysian isolates were significantly different from the other isolates. Histopathological studies revealed myofiber degeneration of the tail muscles and liquefactive myopathy in the infected prawns. This was the first report on the occurrence of MrNV in the Malaysian freshwater prawn.