Mycobacterium tuberculosis groE promoter controls the expression of the bicistronic groESL1 operon and shows differential regulation under stress conditions

Heat shock promoters of mycobacteria are strong promoters that become rapidly upregulated during macrophage infection and thus serve as valuable candidates for expressing foreign antigens in recombinant BCG vaccine. In the present study, a new heat shock promoter controlling the expression of the groESL1 operon was identified and characterized. Mycobacterium tuberculosis groESL1 operon codes for the immunodominant 10 kDa (Rv3418c, GroES/Cpn10/Hsp10) and 60 kDa (Rv3417c, GroEL1/Cpn60.1/Hsp60) heat shock proteins. The basal promoter region was 115 bp, while enhanced activity was seen only with a 277-bp fragment. No promoter element was seen in the groES-groEL1 intergenic region. This operon codes for a bicistronic mRNA transcript as determined by reverse transcriptase-PCR and Northern blot analysis. Primer extension analysis identified two transcriptional start sites (TSSs) TSS1 (-236) and TSS2 (-171), out of which one (TSS2) was heat inducible. The groE promoter was more active than the groEL2 promoter in Mycobacterium smegmatis. Further, it was found to be differentially regulated under stress conditions, while the groEL2 promoter was constitutive.

Expression of the extracellular domain of the human heat-stable enterotoxin receptor in Escherichia coli and generation of neutralizing antibodies

The entire extracellular domain of the human heat-stable enterotoxin (ST) receptor as well as a truncated N-terminal domain were cloned as glutathione S-transferase fusion proteins and expressed in Escherichia coli. The recombinant fusion proteins were purified from both the cytosol and the inclusion body fractions by selective detergent extraction followed by glutathione-agarose affinity chromatography. The purified protein, corresponding to the entire extracellular domain, bound the stable toxin peptide with an affinity comparable to that of the native receptor characterized from the human colonic T84 cell line. No binding was observed with the N-terminal truncated fragment of the receptor under similar conditions, Polyclonal antibodies were raised to the entire extracellular domain fusion protein as well as the truncated extracellular domain fusion protein, and the antibodies were purified by affinity chromatography. Addition of the purified antibodies to T84 cells inhibited ST binding and abolished ST-mediated cGMP production, indicating that critical epitopes involved in ligand interaction are present in the N-terminal fragment of the receptor, Purified antibodies recognized a single protein of M(r) 160,000 Da on Western blotting with T84 membranes, corresponding to a size of the native glycosylated receptor in T84 cells. These studies are the first report of the expression, purification, and characterization of any member of the guanylyl cyclase family of receptors in E. coli and show that binding of the toxin to the extracellular domain of the receptor is possible in the absence of any posttranslational modifications such as glycosylation. The recombinant fusion proteins as well as the antibodies that we have generated could serve as useful tools in the identification of critical residues of the extracellular domain involved in ligand interaction.

Identification and expression of the 20 kd structural protein gene of colitis bacteriophage

The 2.3 kb BamHI fragment from the colitis bacteriophage DNA was transcribed and translated into a 20 kd structural protein P6, in a coupled transcription-translation system derived from Escherichia coli. This protein was expressed in vivo by the 2.3 kb DNA cloned in pBR322. The gene with the regulatory elements for this protein was located on the 680 bp AvaII fragment of the insert DNA. It hybridized with two RNAs of sizes 520 and 1630 nucleotides indicating that both are messengers for the 20 kd protein. Dot-blot hybridization showed that the transcripts for P6 reached a maximum level at 12 min after phage infection.

Expression of the Japanese encephalitis virus NS3 and NS2b proteins as glutathione S-transferase fusions

Flaviviruses generate their structural and nonstructural proteins by proteolytic processing of a single large polyprotein precursor. These proteolytic events are brought about both by host cell signalase and a virally encoded protease. The virally encoded proteolytic activity has been shown to reside within the nonstructural protein 3 (NS3) and requires the product of the nonstructural 2b (NS2b) gene. In order to obtain sufficient quantities of pure NS2b and NS3 proteins for kinetic analysis, we have expressed both these proteins in recombinant systems as fusions to glutathione S-transferase (GST). The fusion constructs were driven by the strong bacteriophage T7 promoter. Transfection of these constructs into the African green monkey kidney cell line CV-1 previously infected with a recombinant vaccinia virus expressing the T7 RNA polymerase resulted in synthesis of the fusion proteins. Both the fusion proteins could be purified to homogeneity in a single step using a glutathione agarose affinity matrix.

Expression of the receptor guanylyl cyclase C and its ligands in reproductive tissues of the rat: A potential role for a novel signaling pathway in the epididymis

Guanylyl cyclase C (GC-C) is a membrane-associated form of guanylyl cyclase and serves as the receptor for the heat-stable enterotoxin (ST) peptide and endogenous ligands guanylin, uroguanylin, and lymphoguanylin. The major site of expression of GC-C is the intestinal epithelial cell, although GC-C is also expressed in extraintestinal tissue such as the kidney, airway epithelium, perinatal liver, stomach, brain, and adrenal glands. Binding of ligands to GC-C leads to accumulation of intracellular cGMP, the activation of protein kinases G and A, and phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that regulates salt and water secretion. We examined the expression of GC-C and its ligands in various tissues of the reproductive tract of the rat. Using reverse transcriptase and the polymerase chain reaction, we demonstrated the presence of GC-C, uroguanylin, and guanylin mRNA in both male and female reproductive organs. Western blot analysis using a monoclonal antibody to GC-C revealed the presence of differentially glycosylated forms of GC-C in the caput and cauda epididymis. Exogenous addition of uroguanylin to minced epididymal tissue resulted in cGMP accumulation, suggesting an autocrine or endocrine activation of GC-C in this tissue. Immunohistochemical analyses demonstrated expression of GC-C in the tubular epithelial cells of both the caput epididymis and cauda epididymis. Our results suggest that the GC-C signaling pathway could converge on CFTR in the epididymis and perhaps control fluid and ion balance for optimal sperm maturation and storage in this tissue.

Roles for Transcription Factors Sp1, NF-kappa B, IRF3, and IRF7 in Expression of the Human IFNL4 Gene

The expression of a biologically active human IFN4 depends on the presence of a frameshift deletion polymorphism within the first exon of the interferon lambda 4 (IFNL4) gene. In this report, we use the lung carcinoma-derived cell line, A549, which is genetically viable to express a functional IFN4, to address transcriptional requirements of the IFNL4 gene. We show that the GC-rich DNA-binding transcription factor (TF) specificity protein 1 (Sp1) is recruited to the IFNL4 promoter and has a role in induction of gene expression upon stimulation with viral RNA mimic poly(I:C). By using RNAi and overexpression strategies, we also show key roles in IFNL4 gene expression for the virus-inducible TFs, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B), IFN regulatory factor 3 (IRF3), and IRF7. Interestingly, we also observe that overexpression of IFN4 influences IFNL4 promoter activity, which may further be dependent on the retinoic acid-inducible gene-I (RIG-I)-like receptor pathway. Together, our work for the first time reports on the functional characterization of the human IFNL4 promoter.

Determination of the orthotropic plate parameters of stiffened plates and grillages in free vibration

It is well known that the analysis of vibration of orthogonally stiffened rectangular plates and grillages may be simplified by replacing the actual structure by an orthotropic plate. This needs a suitable determination of the four elastic rigidity constants Dx, Dy, Dxy, D1 and the mass {Mathematical expression} of the orthotropic plate. A method is developed here for determining these parameters in terms of the sectional properties of the original plate-stiffener combination or the system of interconnected beams. Results of experimental work conducted on aluminium plates agree well with the results of the theory developed here.

Analysis of the beta-glucoside utilization (bgl) genes of Shigella sonnei: evolutionary implications for their maintenance in a cryptic state

The pattern of expression of the genes involved in the utilization of aryl beta-glucosides such as arbutin and salicin is different in the genus Shigella compared to Escherichia coli. The results presented here indicate that the homologue of the cryptic bgl operon of E. coli is conserved in Shigella sonnei and is the primary system involved in beta-glucoside utilization in the organism. The organization of the bgl genes in 5. sonnei is similar to that of E. coli; however there are three major differences in terms of their pattern of expression. (i) The bglB gene, encoding phospho-beta-glucosidase B, is insertionally inactivated in 5. sonnei. As a result, mutational activation of the silent bgl promoter confers an Arbutin-positive (Arb(+)) phenotype to the cells in a single step; however, acquiring a Salicin-positive (Sal(+)) phenotype requires the reversion or suppression of the bglB mutation in addition. (ii) Unlike in E. coli, a majority of the activating mutations (conferring the Arb(+) phenotype) map within the unlinked hns locus, whereas activation of the E. coli bgl operon under the same conditions is predominantly due to insertions within the bglR locus. (iii) Although the bgl promoter is silent in the wild-type strain of 5. sonnei (as in the case of E. coli), transcriptional and functional analyses indicated a higher basal level of transcription of the downstream genes. This was correlated with a 1 bp deletion within the putative Rho-independent terminator present in the leader sequence preceding the homologue of the bglG gene. The possible evolutionary implications of these differences for the maintenance of the genes in the cryptic state are discussed.

Comparative Nucleotide and Amino Acid Sequence Analysis of the Sequence-Specific RNA-Binding Rotavirus Nonstructural Protein NSP3

NSP3, an acidic nonstructural protein, encoded by gene 7 has been implicated as the key player in the assembly of the 11 viral plus-strand RNAs into the early replication intermediates during rotavirus morphogenesis. To date, the sequence or NSP3 from only three animal rotaviruses (SA11, SA114F, and bovine UK) has been determined and that from a human strain has not been reported. To determine the genetic diversity among gene 7 alleles from group A rotaviruses, the nucleotide sequence of the NSP3 gene from 13 strains belonging to nine different G serotypes, from both humans and animals, has been determined. Based on the amino acid sequence identity as well as phylogenetic analysis, NSP3 from group A rotaviruses falls into three evolutionarily related groups, i.e., the SA11 group, the Wa group, and the S2 group. The SA 11/SA114F gene appears to have a distant ancestral origin from that of the others and codes for a polypeptide of 315 amino acids (aa) in length. NSP3 from all other group A rotaviruses is only 313 aa in length because of a 2-amino-acid deletion near the carboxy-terminus, While the SA114F gene has the longest 3' untranslated region (UTR) of 132 nucleotides, that from other strains suffered deletions of varying lengths at two positions downstream of the translational termination codon. In spite of the divergence of the nucleotide (nt) sequence in the protein coding region, a stretch of about 80 nt in the 3' UTR is highly conserved in the NSP3 gene from all the strains. This conserved sequence in the 3' UTR might play an important role in the regulation of expression of the NSP3 gene. (C) 1995 Academic Press, Inc.

Expression of VP1 protein of serotype A and O of foot-and-mouth disease virus in transgenic sunnhemp plants and its immunogenicity for guinea pigs

Recently, transgenic plants expressing immunogenic proteins of foot-and-mouth disease virus (FMDV) have been used as oral or parenteral vaccines against foot-and-mouth disease (FMD). They exhibit advantages like cost effectiveness, absence of processing, thermostability, and easy oral application. FMDV VP1 protein of single serotype has been mostly used as immunogen. Here we report the development of a bivalent vaccine with tandem-linked VP1 proteins of two serotypes, A and O, present in transgenic forage crop Crotalaria juncea. The expression of the bivalent protein in the transgenic plants was confirmed by Western blot analysis. Guinea pig reacted to orally or parenterally applied vaccine by humoral as well as cell-mediated immune responses including serum antibodies and stimulated lymphocytes, respectively. The vaccine protected the animals against a challenge with the virus of serotype A as well as O. This is the first report on the development of a bivalent FMD vaccine using a forage crop.

The chbG Gene of the Chitobiose (chb) Operon of Escherichia coli Encodes a Chitooligosaccharide Deacetylase

The chb operon of Escherichia coli is involved in the utilization of the beta-glucosides chitobiose and cellobiose. The function of chbG (ydjC), the sixth open reading frame of the operon that codes for an evolutionarily conserved protein is unknown. We show that chbG encodes a monodeacetylase that is essential for growth on the acetylated chitooligosaccharides chitobiose and chitotriose but is dispensable for growth on cellobiose and chitosan dimer, the deacetylated form of chitobiose. The predicted active site of the enzyme was validated by demonstrating loss of function upon substitution of its putative metal-binding residues that are conserved across the YdjC family of proteins. We show that activation of the chb promoter by the regulatory protein ChbR is dependent on ChbG, suggesting that deacetylation of chitobiose-6-P and chitotriose-6-P is necessary for their recognition by ChbR as inducers. Strains carrying mutations in chbR conferring the ability to grow on both cellobiose and chitobiose are independent of chbG function for induction, suggesting that gain of function mutations in ChbR allow it to recognize the acetylated form of the oligosaccharides. ChbR-independent expression of the permease and phospho-beta-glucosidase from a heterologous promoter did not support growth on both chitobiose and chitotriose in the absence of chbG, suggesting an additional role of chbG in the hydrolysis of chitooligosaccharides. The homologs of chbG in metazoans have been implicated in development and inflammatory diseases of the intestine, indicating that understanding the function of E. coli chbG has a broader significance.

Calcium Permeable AMPA Receptor-Dependent Long Lasting Plasticity of Intrinsic Excitability in Fast Spiking Interneurons of the Dentate Gyrus Decreases Inhibition in the Granule Cell Layer

The local fast-spiking interneurons (FSINs) are considered to be crucial for the generation, maintenance, and modulation of neuronal network oscillations especially in the gamma frequency band. Gamma frequency oscillations have been associated with different aspects of behavior. But the prolonged effects of gamma frequency synaptic activity on the FSINs remain elusive. Using whole cell current clamp patch recordings, we observed a sustained decrease of intrinsic excitability in the FSINs of the dentate gyrus (DG) following repetitive stimulations of the mossy fibers at 30 Hz (gamma bursts). Surprisingly, the granule cells (GCs) did not express intrinsic plastic changes upon similar synaptic excitation of their apical dendritic inputs. Interestingly, pairing the gamma bursts with membrane hyperpolarization accentuated the plasticity in FSINs following the induction protocol, while the plasticity attenuated following gamma bursts paired with membrane depolarization. Paired pulse ratio measurement of the synaptic responses did not show significant changes during the experiments. However, the induction protocols were accompanied with postsynaptic calcium rise in FSINs. Interestingly, the maximum and the minimum increase occurred during gamma bursts with membrane hyperpolarization and depolarization respectively. Including a selective blocker of calcium-permeable AMPA receptors (CP-AMPARs) in the bath; significantly attenuated the calcium rise and blocked the membrane potential dependence of the calcium rise in the FSINs, suggesting their involvement in the observed phenomenon. Chelation of intracellular calcium, blocking HCN channel conductance or blocking CP-AMPARs during the experiment forbade the long lasting expression of the plasticity. Simultaneous dual patch recordings from FSINs and synaptically connected putative GCs confirmed the decreased inhibition in the GCs accompanying the decreased intrinsic excitability in the FSINs. Experimentally constrained network simulations using NEURON predicted increased spiking in the GC owing to decreased input resistance in the FSIN. We hypothesize that the selective plasticity in the FSINs induced by local network activity may serve to increase information throughput into the downstream hippocampal subfields besides providing neuroprotection to the FSINs. (c) 2014 Wiley Periodicals, Inc.

Oestrogenic regulation and differential expression of WNT4 in the bonnet monkey and rodent epididymis

A role for oestrogen in regulating fluid reabsorption in the monkey epididymis was recently demonstrated. Here, these Studies are extended to identify potential oestrogen-regulated proteins in the cauda region of monkey epididymis treated with vehicle and oestrogen receptor antagonist (ICI 182780). Two-dimensional electrophoretic analysis was used to identify the proteins. The results indicated down-regulation of WNT4 in the ICI-182780-treated monkey cauda. In addition. the Wnt4f mRNA concentration was also reduced in the caput regions of ICI-182780-treated rats and oestrogen receptor knockout mice. WNT4 is a key regulator of gonadal differentiation in humans and mice and plays a pivotal role in early mouse embryogenesis. The results of the present Study establish the presence of WNT4 in the monkey epididymis and its regulation by oestrogen, and Suggest a role for WNT4 in maintaining epididymal homeostasis.

Comparative sequence analysis and expression in E-coli of the subgroup I-specific antigen VP6 from a G2 serotype human rotavirus IS2

VP6, the intermediate capsid protein of the virion, specifies subgroup specificity of rotavirus, It is also the most conserved, both at nucleotide and amino acid levels, among group A rotaviruses and is the target of choice for rotavirus detection, In this study we report the sequence of the subgroup I (SGI)-specific VP6 from the serotype G2 strain IS2 isolated from a child suffering from acute diarrhoea in Bangalore ana its comparison with the published VP6 sequences. Interestingly, IS2 gene 6 shared highest homology with that from bovine UK strain and the protein contained substitutions by lysine at amino acid positions 97 and 134, In contrast, the amino acids Met and Glu/Asp at these respective positions are highly conserved in all the other group A rotaviruses sequenced so far, These observations have obvious implications for the evolution of serotype G2 and G2-like strains circulating in India, The SGI VP6, of a human rotavirus, possessing epitopes that are conformationally similar to those found in the native protein in the virion, was successfully expressed in E. coli and purified for the first time by single-step affinity chromatography.

Expression of glucose transporter isoforms and the insulin receptor during hamster preimplantation embryo development

During preimplantation development, embryos of many species are known to express up to five isoforms of the facilitative glucose transporter proteins (GLUT). Development of hamster blastocysts is inhibited by glucose. We therefore investigated GLUT isoform and insulin receptor (IR) expression in hamster preimplantation embryos cultured in glucose-free medium from the 8-cell stage onwards. We show that GLUT1, 3 and 8 mRNA are constitutively expressed from the 8-cell to the blastocyst stage. The IR is expressed from the morula stage onwards. Messenger RNA of the insulin-responsive GLUT4 was not detected at any stage. GLUT1 and 3 were localised by immunocytochemistry. GLUT1 was expressed in both embryoblast and trophoblast, in the latter, mainly in basal and lateral membranes directed towards the blastocoel. and embryoblast. GLUT3 was exclusively localised in the apical. membrane of trophoblast cells. We show that hamster preimplantation embryos express several GLUT isoforms thus closely resembling embryos of other mammalian species. Despite endogenous IR expression, the insulin-sensitive isoform GLUT4 was not expressed, indicating that the insulin-mediated glucose uptake known from classical insulin target cells may not be relevant for hamster blastocysts.