Post-transfer editing mechanism of a D-aminoacyl-tRNA deacylase-like domain in threonyl-tRNA synthetase from archaea

To ensure a high fidelity during translation, threonyl-tRNA synthetases (ThrRSs) harbor an editing domain that removes noncognate L-serine attached to tRNAThr. Most archaeal ThrRSs possess a unique editing domain structurally similar to D-aminoacyl-tRNA deacylases (DTDs) found in eubacteria and eukaryotes that specifically removes D-amino acids attached to tRNA. Here, we provide mechanistic insights into the removal of noncognate L-serine from tRNAThr by a DTD-like editing module from Pyrococcus abyssi ThrRS (Pab-NTD). High-resolution crystal structures of Pab-NTD with pre- and post-transfer substrate analogs and with L-serine show mutually nonoverlapping binding sites for the seryl moiety. Although the pre-transfer editing is excluded, the analysis reveals the importance of main chain atoms in proper positioning of the post-transfer substrate for its hydrolysis. A single residue has been shown to play a pivotal role in the inversion of enantioselectivity both in Pab-NTD and DTD. The study identifies an enantioselectivity checkpoint that filters opposite chiral molecules and thus provides a fascinating example of how nature has subtly engineered this domain for the selection of chiral molecules during translation.     

Effect of temperature,cultivars, injury of root and inoculums load of Ralstonia solanacearum to cause bacterial wilt of tomato

Bacterial wilt, caused by Ralstonia solanacearum , is responsible for severe losses in tomato crops in the world. In the present study, the effect of temperature, cultivars of tomato, injury of root system and inoculums load of R. solanacearum to cause bacterial wilt disease under control conditions was undertaken. Three strains UTT-25, HPT-3 and JHT-5 of R. solanacearum were grown at 5–40?°C in vitro to study, the effect of temperature on the growth of bacteria and maximum growth was found at 30?°C after 72?h in all the strains. Twenty-one days old seedlings of two cultivars of tomato i.e. N-5 (moderately resistant) and Pusa Ruby (highly susceptible) were transplanted into the pots and inoculated with R. solanacearum strain UTT-25 (5 × 10⁸?cfu/ml), mechanically injured and uninjured roots of the plant. The plants were allowed to grow at 20, 25, 30 and 35?°C at National Phytotron Facility, IARI, New Delhi to study the effect of temperature on intensity of bacterial wilt disease. Maximum wilt disease intensity was found 98.73 and 95.9 % in injured roots of Pusa Ruby and N-5 cultivars of tomato at 35?°C on 11th days of inoculation, respectively. However, no wilt disease was observed in both the cultivars at 20?°C up to 60?days. For detection of R. solanacearum from asymptomatic tomato plants, hrpB-based sequence primers (Hrp_rs2F and Hrp_rs2R) amplified at 323?bp was used in bio-PCR to detect R. solanacearum from crown, mid part of stem and upper parts of the plant. Another experiment was conducted to find out the inoculum potential of R. solanacearum strain UTT-25 to cause bacterial wilt in susceptible cultivar Pusa Ruby. The bacteria were inoculated at concentration of bacterial suspension 10 to 10¹⁰?cfu/ml in injured and uninjured roots of the plants separately and injured root accelerated wilt incidence and able to cause wilt disease 63.3% by 100?cfu/ml of R. solanacearum, while no disease appeared at 10?cfu/ml on the 11th day of inoculation in injured and uninjured roots of the plant.     

Molecular modeling and spectroscopic studies of semustine binding with DNA and its comparison with lomustine–DNA adduct formation

Chloroethyl nitrosoureas constitute an important family of cancer chemotherapeutic agents, used in the treatment of various types of cancer. They exert antitumor activity by inducing DNA interstrand cross-links. Semustine, a chloroethyl nitrosourea, is a 4-methyl derivative of lomustine. There exist some interesting reports dealing with DNA-binding properties of chloroethyl nitrosoureas; however, underlying mechanism of cytotoxicity caused by semustine has not been precisely and completely delineated. The present work focuses on understanding semustine–DNA interaction to comprehend its anti-proliferative action at molecular level using various spectroscopic techniques. Attenuated total reflection–Fourier transform infrared (ATR-FTIR) spectroscopy is used to determine the binding site of semustine on DNA. Conformational transition in DNA after semustine complexation is investigated using circular dichroism (CD) spectroscopy. Stability of semustine–DNA complexes is determined using absorption spectroscopy. Results of the present study demonstrate that semustine performs major-groove-directed DNA alkylation at guanine residues in an incubation-time–drug-concentration-dependent manner. CD spectral outcomes suggest partial transition of DNA from native B-conformation to C-form. Calculated binding constants (K_a) for semustine and lomustine interactions with DNA are 1.53?×?10³ M^?1 and 8.12?×?10³ M^?1, respectively. Moreover, molecular modeling simulation is performed to predict preferential binding orientation of semustine with DNA that corroborates well with spectral outcomes. Results based on comparative study of DNA-binding properties of semustine and lomustine, presented here, may establish a correlation between molecular structure and cytotoxicity of chloroethyl nitrosoureas that may be instrumental in the designing and synthesis of new nitrosourea therapeutics possessing better efficacy and fewer side effects.     

Microglia and the control of autoreactive T cell responses

Microglial activation is one of the earliest and most prominent features of nearly all CNS neuropathologies often occurring prior to other indicators of overt neuropathology. Whether microglial activation in seemingly healthy CNS tissue during the early stages of several is a response to early stages of neuronal or glial distress or an early sign of microglial dysfunction causing subsequent neurodegeneration is unknown. Here we characterize and discuss how changes in the CNS microenvironment (neuronal activity/viability, glial activation) lead to specific forms of microglial activation. Specifically, we examine the potential role that TREM-2 expressing microglia may play in regulating the effector function of autoreactive T cell responses. Thus, we suggest that ubiquitous suppression of microglial activation during CNS inflammatory disorders rather than targeted manipulation of microglial activation, may in the end be maladaptive leading to incomplete remission of symptoms.     

Evaluation of recombinant outer membrane protein based vaccine against Salmonella Typhimurium in birds

Food-borne diseases caused by Salmonella enterica from poultry sources represent an important public health problem and no reliable control by vaccination has proved effective despite research. The aim of the present study was to evaluate the use of recombinant OmpC protein for immunization of birds to elucidate its protection against virulent Salmonella Typhimurium. The recombinant OmpC protein was prepared after cloning and expressing ompC gene and was characterized by SDS-PAGE and Western blot analyses. The protein preparations were tested as vaccine candidate in layer birds by comparing the immune response, protection and organ clearance against crude lysate and control. The biologically functional recombinant 43 kDa truncated OmpC protein proved to be a good immunogen which induced a significantly high humoral immune response than control. At the same time, it primed a stable cell-mediated immune response. A protective index (based on faecal shedding of organism) of rOmpC based preparations ranged between 50 and 75% as observed for 3 weeks after challenge. Therefore, the protein preparations conferred satisfactory protection against challenge infections with virulent strains of S. Typhimurium as evidenced by limited faecal shedding and minimal detection of Salmonella from edible tissues and eggs. These findings suggest the possibility to explore the use of S. enterica OMP protein for the production of novel vaccine.     

Electrochemical characterization of Geobacter sulfurreducens cells immobilized on graphite paper electrodes

Bacteria able to transfer electrons to conductive surfaces are of interest as catalysts in microbial fuel cells, as well as in bioprocessing, bioremediation, and corrosion. New procedures for immobilization of Geobacter sulfurreducens on graphite electrodes are described that allow routine, repeatable electrochemical analysis of cell-electrode interactions. Immediately after immobilizing G. sulfurreducens on electrodes, electrical current was obtained without addition of exogenous electron shuttles or electroactive polymers. Voltammetry and impedance analysis of pectin-immobilized bacteria transferring electrons to electrode surfaces could also be performed. Cyclic voltammetry of immobilized cells revealed voltage-dependent catalytic current similar to what is commonly observed with adsorbed enzymes, with catalytic waves centered at -0.15 V (vs. SHE). Electrodes maintained at +0.25 V (vs. SHE) initially produced 0.52 A/m(2) in the presence of acetate as the electron donor. Electrical Impedance Spectroscopy of coatings was also consistent with a catalytic mechanism, controlled by charge transfer rate. When electrodes were maintained at an oxidizing potential for 24 h, electron transfer to electrodes increased to 1.75 A/m(2). These observations of electron transfer by pectin-entrapped G. sulfurreducens appear to reflect native mechanisms used for respiration. The ability of washed G. sulfurreducens cells to immediately produce electrical current was consistent with the external surface of this bacterium possessing a pathway linking oxidative metabolism to extracellular electron transfer. This electrochemical activity of pectin-immobilized bacteria illustrates a strategy for preparation of catalytic electrodes and study of Geobacter under defined conditions.     

Chemical modification of alpha-chymotrypsin for obtaining high transesterification activity in low water organic media

Alpha-chymotrypsin was made more hydrophilic by modifying 11 (out of 16) ε-amino groups with pyromellitic dianhydride. The hydrophilic preparation was precipitated with n-propanol. This preparation gave significantly higher initial rates at the optimum a_w (127.51 nmol mg^?1 min^?1 in n-octane and 21.30 nmol mg^?1 min^?1 in acetonitrile at a_w=0.33) compared with the lyophilized preparation (53.50 nmol mg^?1 min^?1 in n-octane and 0.26 nmol mg^?1 min^?1 in acetonitrile at a_w=0.97). FT-IR showed that the precipitate of modified alpha-chymotrypsin has a higher content of alpha-helices and beta-sheets compared to the lyophilized powder.     

In vitro propagation,clonal fidelity and phytochemical analysis of <Emphasis Type="Italic">Rhodiola imbricata</Emphasis> Edgew: a rare trans-Himalayan medicinal plant

The present study focuses on development of a micropropagation protocol for true to type plants of Rhodiola imbricata, an endangered medicinal plant found in trans-Himalayan Leh-Ladakh region of India. It also aims at analyzing the pharmaceutically important secondary metabolites and antioxidant potential of in vitro and in vivo plants. Various cytokinins and auxins were tested for shoot proliferation and in vitro rooting of the microshoots, respectively. Random primers were used for checking genetic uniformity at different stages of micropropagation. Pharmaceutically important secondary metabolites of R. imbricata such as Rosavin, total polyphenols and free radical scavenging activity were analyzed by HPLC. Among different cytokinins used, BAP (5 µM) and TDZ (1 µM) were found to perform better in terms of shoot proliferation, shoot length and number of leaves as compared to other concentrations. For rooting of microshoots, a lower concentration of NAA (0.5 µM) yielded more efficient rooting of micro shoots (17.33 roots per micro shoot). In vitro rooted microshoots were hardened and showed 60% survival rate. The content of gallic acid, chlorogenic acid and 4-hydroxybenzoic acid was higher in the in vivo plant. The amount of ferulic acid was higher in the in vitro raised plant when compared to field grown plant. Furthermore, caffeic acid and p-coumaric acid were higher in the in vitro raised plants as compared to field grown plants. This work will facilitate in conservation of this endangered herb and provide necessary plant materials for various biotechnological and pharmaceutical applications.