Reversible photo-regulation of a hammerhead ribozyme using a diffusible effector

The potential utility of catalytic RNAs and DNAs (ribozymes and deoxyribozymes, respectively) as reagents in molecular biology as well as therapeutic agents for a variety of human diseases, has long been recognized. Although naturally occurring RNA-cleaving ribozymes are typically not subject to feedback control, rational methodologies for the creation of allosteric ribozymes, by functional combination of ribozyme and ligand-responsive aptamer RNA elements, have existed for some years. Here, we report the in vitro selection of RNA aptamers specific for binding one but not the other of two light-induced isomers of a dihydropyrene photo-switch compound, and the utilization of such an aptamer for the construction of the UG-dihydropyrene ribozyme, an allosteric hammerhead ribozyme whose catalysis is controllable by irradiation with visible versus ultraviolet light. In the presence of micromolar concentrations of the photo-switch compound, the ribozyme behaves as a two-state switch, exhibiting a >900-fold difference in catalytic rates between the two irradiation regimes. We anticipate that the UG-dihydropyrene, and other ribozymes like it, may find significant application in the developmental biology of model organisms such as Drosophila melanogaster and Caenorhabditis elegans, as well as in the biomedical sciences.     

The role of the omega subunit of RNA polymerase in expression of the relA gene in Escherichia coli

The rpoZ gene for the omega subunit of Escherichia coli RNA polymerase constitutes single operon with the spoT gene, which is responsible for the maintenance of stringent response under nutrient starvation conditions. To identify the physiological role of the omega subunit, we compared the gene expression profile of wild-type Escherichia coli with that of an rpoZ deleted strain by microarray analysis using an E. coli DNA chip. Here we report on a set of genes which show changes in expression profile following the removal of rpoZ. We have seen that relA, which is responsible for the synthesis of the stringent factor ppGpp and many ribosomal proteins, exhibited noticeable changes in mRNA levels and were therefore further analyzed for their expression using a GFP/RFP two-fluorescent protein promoter assay vector. In the absence of rpoZ, the promoter for the relA gene was severely impaired, but the promoters from the ribosomal protein genes were not affected as much. Taking these results together we propose that the omega subunit is involved in regulation of the relA gene, but induction of the stringently controlled genes in the absence of rpoZ is, at least in part, attributable to a decrease in ppGpp level.     

Microarray analysis of Mu transposition in Salmonella enterica, serovar Typhimurium: transposon exclusion by high-density DNA binding proteins

All organisms contain transposons with the potential to disrupt and rearrange genes. Despite the presence of these destabilizing sequences, some genomes show remarkable stability over evolutionary time. Do bacteria defend the genome against disruption by transposons? Phage Mu replicates by transposition and virtually all genes are potential insertion targets. To test whether bacteria limit Mu transposition to specific parts of the chromosome, DNA arrays of Salmonella enterica were used to quantitatively measure target site preference and compare the data with Escherichia coli. Essential genes were as susceptible to transposon disruption as non‐essential ones in both organisms, but the correlation of transposition hot spots among homologous genes was poor. Genes in highly transcribed operons were insulated from transposon mutagenesis in both organisms. A 10 kb cold spot on the pSLT plasmid was near parS, a site to which the ParB protein binds and spreads along DNA. Deleting ParB erased the plasmid cold spot, and an ectopic parS site placed in the Salmonella chromosome created a new cold spot in the presence of ParB. Our data show that competition between cellular proteins and transposition proteins on plasmids and the chromosome is a dominant factor controlling the genetic footprint of transposons in living cells.     

Characterization and role of Peptidase N from Salmonella enterica serovar Typhimurium

ATP-independent peptidases are important during the distal steps of cytosolic protein degradation. The contribution of a member of this group, Peptidase N (PepN) was studied in Salmonella enterica serovar Typhimurium (Salmonella typhimurium). The DeltapepN strain displays greatly reduced cleavage of 9 out of a total of 13 exopeptidase substrates, demonstrating a significant contribution of PepN to cytosolic aminopeptidase activity. The cleavage profile of purified S. typhimurium PepN is Arg>Ala>Thr, demonstrating broad specificity. Comparative biochemical studies with purified PepN from Escherichia coli and S. typhimurium revealed the latter to be distinct: S. typhimurium PepN cleaves Thr-AMC more efficiently and is less sensitive to inhibition by N-ethylmaleimide. Studies with DeltapepN and PepN overexpression demonstrated its importance for growth during nutritional downshift in combination with high temperature stress. In summary, S. typhimurium PepN contributes significantly to cytosolic aminopeptidase activity and its role is manifested under selected stress conditions.     

Differential expression of the Ebola virus GP(1,2) protein and its fragments in E. coli

Bacterial expression platforms are frequently used for the expression and production of different recombinant proteins. The full length Ebola virus (EBOV) GP(1,2) gene and subfragments of the GP(1) gene were cloned in a bacterial expression vector as a C-terminal His(6) fusion protein. Surprisingly, the full length EBOV GP(1,2) gene could not be expressed in Escherichia coli. The subfragments of GP(1) were only expressed in small amounts with the exception of one small fragment (subfragment D) which was expressed at very high levels as inclusion bodies. This was seen even in the in vitro translation system with no expression of full length GP(1,2), GP(1) subfragments A and C and low level expression of subfragment B. Only the subfragment D showed high level of expression. In E. coli (Top10), the recombinant GP(1) subfragment D protein was expressed exclusively as an insoluble approximately 25 kDa His(6) fusion protein, which is the expected size for a non-glycosylated recombinant protein. The IMAC purified and refolded non-glycosylated protein was used to immunize mice for the development of monoclonal anti-EBOV antibodies which successfully yielded several monoclonal antibodies with different specificities. The monoclonal and polyclonal antiserum derived from the animals immunized with this recombinant GP(1) subfragment D protein was found to specifically recognize the full length glycosylated EBOV GP(1,2) protein expressed in mammalian 293T cells, thus, demonstrating the immunogenicity of the recombinant subfragment.     

Visceral Dracunculiasis: A Case Report from East Medinipore District,West Bengal,India

A case of visceral dracunculiasis in a female patient is reported from east Medinipur district, West Bengal, India. It is the first report from the eastern India. The patient from rural West Bengal underwent laparoscopic cholecystectomy on 13th March 2002 and after 3 days the patient was released with a drain in the upper abdomen because of persistence of fluid of about 50 ml per day. The patient was to readmit after 8 days with a history of expulsion of one worm through the drain tube and next day another four living worms were expelled through the tube. Subsequently the drain dried up and the patient was released after removing the tube. The worms are identified as Dracunculus medinensis, possibly remaining within the abscess developing adjacent to the gall bladder and it is a case of visceral dracunculiasis reported first time from eastern India.     

Nardostachys jatamansi Protects Against Cold Restraint Stress Induced Central Monoaminergic and Oxidative Changes in Rats

Cold restraint stress (CRS) model exerts similar effect as physiological stress because it combines emotional stress (escape reaction) and physical stress (muscle work). It is well established that various responses to stress are regulated by sympathoadrenal system, brain monoaminergic systems and oxidative processes. Nardostachys jatamansi (NJE) is known to possess soothing and sedative action on the central nervous system. The present investigation was performed to explore the anti-stress activity of NJE on CRS model, through its effect on biochemical and neurochemical alterations. The rats were restrained in metallic chambers for 3?h at 4?°C was followed by sacrifice and assessment of stress related alterations. Hydro-ethanolic (30:70) extract of NJE was administrated orally at the doses of 200 and 500?mg/kg for 14?days and compared with vehicle control and Panax ginseng (100?mg/kg). Effects of NJE on CRS induced oxidative stress including reduced glutathione, glutathione peroxidase, glutathione reductase, glutathione-s-transferase were estimated. Dopamine, norepinephrine, serotonin and 5-hydroxy indole acetic acid were measured in the cerebral cortex, hippocampus and hypothalamus by HPLC electrochemical detector. NJE at both doses significantly inhibited CRS induced oxidative stress. It significantly mitigated CRS induced altered level of neurotransmitters in different brain regions. The study implied that NJE has the ability to provide protection against CRS induced oxidative stress and neurochemical alterations. Findings indicated that NJE revealed potent anti-stress effect implicating its therapeutic importance in stress-related disorders.     

Synergistic effect of salt mixture on the gelation temperature and morphology of methylcellulose hydrogel

Gelation temperature of methylcellulose (MC) can be altered by adding different additives. Pure MC showed sol-gel transition at 60°C. Sodium citrate and sodium tartrate were used alone and in combination to see the effect of individual salt and combination of salts on the gelation temperature of MC. The gelation temperature of all the binary and ternary combinations of MC and salts were measured with different methods such as test tube tilting method (TTM), UV-vis spectroscopy, viscometry, and by rheometer and also the morphology of gels were characterized with the help of environmental scanning electron microscopy (ESEM). It was observed that when 0.1M sodium citrate (NaC) and 0.1M sodium tartrate (NaT) were used separately, the gelation temperature of MC was reduced up to 44°C and 47°C respectively but when mixture of NaC and NaT (0.1(M) NaC and 0.1(M) (NaT)) were used the gelation temperature was further reduced to 36°C. It was clear from ESEM images that when NaC and NaT were used separately the formation of network was not distinguishable. But, well-connected network structure was observed when a mixture 0.1M NaC and 0.1M NaT was used.