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The Pregnane and Xenobiotic Receptor (PXR; NR1I2) is a ligand-modulated transcription factor that belongs to the nuclear receptor superfamily. It is expressed at higher levels primarily in liver and intestine as compared to the levels in several other organs. It is activated by a broad spectrum of xenobiotics and endobiotics. The primary function of PXR is to regulate the expression of drug metabolizing enzymes and transporters and prevent the accumulation of toxic chemicals in the body, thereby maintaining body's homeostasis. In this study, we identified a C/T single nucleotide polymorphism at position −831 from the transcriptional start site of the PXR gene promoter and examined the functional significance of this variant using both the luciferase reporter gene assays and electrophoretic mobility shift assays (EMSA). Transient transfection experiments showed that the T-allele was associated with significantly greater transcriptional activity than the C-allele of SNP rs3814055. These results indicate that the −831C/T polymorphism has a direct effect on transcriptional regulation of PXR gene. This allelic variation may be a potential genetic marker that can help identify individuals at higher risk for Inflammatory Bowel Disease (IBD). [ABSTRACT FROM AUTHOR]

The role of nuclear receptor PXR in detoxification and clearance of xenobiotics and endobiotics is well-established. However, its projected role in hepatic cancer is rather illusive where its expression is reported altered in different cancers depending on the tissue-type and microenvironment. The expression of PXR, its target genes and their biological or clinical significance have not been examined in hepatic cancer. In the present study, by generating DEN-induced hepatic cancer in mice, we report that the expression of PXR and its target genes CYP3A11 and GSTa2 are down-regulated implying impairment of hepatic detoxification capacity. A higher state of inflammation was observed in liver cancer tissues as evident from upregulation of inflammatory cytokines IL-6 and TNF-α along with NF-κB and STAT3. Our data in mouse model suggested a negative correlation between down-regulation of PXR and its target genes with that of higher expression of inflammatory proteins (like IL-6, TNF-α, NF-κB). In conjunction, our findings with relevant cell culture based assays showed that higher expression of PXR is involved in reduction of tumorigenic potential in hepatic cancer. Overall, the findings suggest that inflammation influences the expression of hepatic proteins important in drug metabolism while higher PXR level reduces tumorigenic potential in hepatic cancer. [ABSTRACT FROM AUTHOR]

Besides its important role in embryonic development and homeostatic self-renewal in adult tissues, Wnt/β-catenin signaling exerts both anti-inflammatory and proinflammatory functions. This is, at least partially, due to either repressing or enhancing the NF-κB pathway. Similarly, the NF-κB pathway either positively or negatively regulates Wnt/β-catenin signaling. Different components of the two pathways are involved in this crosstalk, forming a complex regulatory network. This review summarizes our current understanding of the molecular mechanisms underlying the cross-regulation between the two pathways and discusses their involvement in inflammation and i nflammation-associated diseases such as cancer. [ABSTRACT FROM AUTHOR]

Cicer arietinum L. (chickpea) is the third most important food legume crop. We have generated the draft sequence of a desi-type chickpea genome using next-generation sequencing platforms, bacterial artificial chromosome end sequences and a genetic map. The 520-Mb assembly covers 70% of the predicted 740-Mb genome length, and more than 80% of the gene space. Genome analysis predicts the presence of 27 571 genes and 210 Mb as repeat elements. The gene expression analysis performed using 274 million RNA-Seq reads identified several tissue-specific and stress-responsive genes. Although segmental duplicated blocks are observed, the chickpea genome does not exhibit any indication of recent whole-genome duplication. Nucleotide diversity analysis provides an assessment of a narrow genetic base within the chickpea cultivars. We have developed a resource for genetic markers by comparing the genome sequences of one wild and three cultivated chickpea genotypes. The draft genome sequence is expected to facilitate genetic enhancement and breeding to develop improved chickpea varieties. [ABSTRACT FROM AUTHOR]

Pregnane X Receptor (PXR) is an important ligand-activated nuclear receptor functioning as a 'master regulator' of expression of phase I, phase II drug metabolizing enzymes, and members of the drug transporters. PXR is primarily expressed in hepatic tissues and to lesser extent in other non-hepatic tissues both in human and in mice. Although its expression profile is well studied but little is known about the regulatory mechanisms that govern PXR gene expression in these cells. In the present study, we have cloned and characterized over 5 kb (24963 to +54) region lying upstream of mouse PXR transcription start site. Promoter-reporter assays revealed that the proximal promoter region of up to 1 kb is sufficient to support the expression of PXR in the mouse liver cell lines. It was evident that the 500 bp proximal promoter region contains active binding sites for Ets, Tcf, Ikarose and nuclear factor families of transcription factors. Electrophoretic mobility shift assays demonstrated that the minimal region of 134 bp PXR promoter was able to bind Ets-1 and b-catenin proteins. This result was further confirmed by chromatin immunoprecipitation analysis. In summary, the present study identified a promoter region of mouse PXR gene and the transregulatory factors responsible for PXR promoter activity. The results presented herein are expected to provide important cues to gain further insight into the regulatory mechanisms of PXR function. [ABSTRACT FROM AUTHOR]

Summary [ABSTRACT FROM AUTHOR]

DNA methylation affects important developmental processes in both plants and animals. The process of methylation of cytosines at C-5 is catalysed by DNA methyltransferases (MTases), which are highly conserved, both structurally and functionally, in eukaryotes. In this study, we identified and characterized cytosine DNA MTase genes that are activated with the onset of reproductive development in rice. The rice genome ( Oryza sativa L. subsp. japonica) encodes a total of 10 genes that contain the highly conserved MTase catalytic domain. These genes have been categorized into subfamilies on the basis of phylogenetic relationships. A microarray-based gene expression profile of all 10 MTases during 22 stages/tissues that included 14 stages of reproductive development and five vegetative tissues together with three stresses, cold, salt and dehydration stress, revealed specific windows of MTase activity during panicle and seed development. The expression of six methylases was specifically/preferentially upregulated with the initiation of floral organs. Significantly, one of the MTases was also activated in young seedlings in response to cold and salt stress. The molecular studies presented here suggest a greater role for these proteins and the epigenetic process in affecting genome activity during reproductive development and stress than was previously anticipated. [ABSTRACT FROM AUTHOR]

The basic leucine (Leu) zipper (bZIP) proteins compose a family of transcriptional regulators present exclusively in eukaryotes. The bZIP proteins characteristically harbor a bZIP domain composed of two structural features: a DNA-binding basic region and the Leu zipper dimerization region. They have been shown to regulate diverse plant-specific phenomena, including seed maturation and germination, floral induction and development, and photomorphogenesis, and are also involved in stress and hormone signaling. We have identified 89 bZIP transcription factor-encoding genes in the rice (Oryza sativa) genome. Their chromosomal distribution and sequence analyses suggest that the bZIP transcription factor family has evolved via gene duplication. The phylogenetic relationship among rice bZIP domains as well as with bZIP domains from other plant bZIP factors suggests that homologous bZIP domains exist in plants. Similar intron/exon structural patterns were observed in the basic and hinge regions of their bZIP domains. Detailed sequence analysis has been done to identify additional conserved motifs outside the bZIP domain and to predict their DNA-binding site specificity as well as dimerization properties, which has helped classify them into different groups and subfamilies, respectively. Expression of bZIP transcription factor-encoding genes has been analyzed by full-length cDNA and expressed sequence tag-based expression profiling. This expression profiling was complemented by microarray analysis. The results indicate specific or coexpression patterns of rice bZIP transcription factors starting from floral transition to various stages of panicle and seed development. bZIP transcription factor-encoding genes in rice also displayed differential expression patterns in rice seedlings in response to abiotic stress and light irradiation. An effort has been made to link the structure and expression pattern of bZIP transcription factor-encoding genes in rice to their function, based on the information obtained from our analyses and earlier known results. This information will be important for functional characterization of bZIP transcription factors in rice. [ABSTRACT FROM AUTHOR]

F-box proteins constitute a large family in eukaryotes and are characterized by a conserved F-box motif (approximately 40 amino acids). As components of the Skp1p-cullin-F-box complex, F-box proteins are critical for the controlled degradation of cellular proteins. We have identified 687 potential F-box proteins in rice (Oryza sativa), the model monocotyledonous plant, by a reiterative database search. Computational analysis revealed the presence of several other functional domains, including leucine-rich repeats, kelch repeats, F-box associated domain, domain of unknown function, and tubby domain in F-box proteins. Based upon their domain composition, they have been classified into 10 subfamilies. Several putative novel conserved motifs have been identified in F-box proteins, which do not contain any other known functional domain. An analysis of a complete set of F-box proteins in rice is presented, including classification, chromosomal location, conserved motifs, and phylogenetic relationship. It appears that the expansion of F-box family in rice, in large part, might have occurred due to localized gene duplications. Furthermore, comprehensive digital expression analysis of F-box protein-encoding genes has been complemented with microarray analysis. The results reveal specific and/or overlapping expression of rice F-box protein- encoding genes during floral transition as well as panicle and seed development. At least 43 F-box protein-encoding genes have been found to be differentially expressed in rice seedlings subjected to different abiotic stress conditions. The expression of several F-box protein-encoding genes is also influenced by light. The structure and function of F-box proteins in plants is discussed in light of these results and the published information. These data will be useful for prioritization of F-box proteins for functional validation in rice. [ABSTRACT FROM AUTHOR]

Background: MADS-box transcription factors, besides being involved in floral organ specification, have also been implicated in several aspects of plant growth and development. In recent years, there have been reports on genomic localization, protein motif structure, phylogenetic relationships, gene structure and expression of the entire MADS-box family in the model plant system, Arabidopsis. Though there have been some studies in rice as well, an analysis of the complete MADS-box family along with a comprehensive expression profiling was still awaited after the completion of rice genome sequencing. Furthermore, owing to the role of MADS-box family in flower development, an analysis involving structure, expression and functional aspects of MADS-box genes in rice and Arabidopsis was required to understand the role of this gene family in reproductive development. Results: A genome-wide molecular characterization and microarray-based expression profiling of the genes encoding MADS-box transcription factor family in rice is presented. Using a thorough annotation exercise, 75 MADS-box genes have been identified in rice and categorized into MIKCc, MIKC*, Mα, Mβ and Mγ groups based on phylogeny. Chromosomal localization of these genes reveals that 16 MADS-box genes, mostly MIKCc-type, are located within the duplicated segments of the rice genome, whereas most of the M-type genes, 20 in all, seem to have resulted from tandem duplications. Nine members belonging to the Mβ group, which was considered absent in monocots, have also been identified. The expression profiles of all the MADS-box genes have been analyzed under 11 temporal stages of panicle and seed development, three abiotic stress conditions, along with three stages of vegetative development. Transcripts for 31 genes accumulate preferentially in the reproductive phase, of which, 12 genes are specifically expressed in seeds, and six genes show expression specific to panicle development. Differential expression of seven genes under stress conditions is also evident. An attempt has been made to gain insight into plausible functions of rice MADS-box genes by collating the expression data of functionally validated genes in rice and Arabidopsis. Conclusion: Only a limited number of MADS genes have been functionally validated in rice. A comprehensive annotation and transcriptome profiling undertaken in this investigation adds to our understanding of the involvement of MADS-box family genes during reproductive development and stress in rice and also provides the basis for selection of candidate genes for functional validation studies. [ABSTRACT FROM AUTHOR]

DNA topoisomerase 6 (TOP6) belongs to a novel family of type II DNA topoisomerases present, other than in archaebacteria, only in plants. Here we report the isolation of full-length cDNAs encoding putative TOP6 subunits A and B from rice ( Oryza sativa ssp. indica), preserving all the structural domains conserved among archaebacterial TOP6 homologs and eukaryotic meiotic recombination factor SPO11. OsTOP6A1 was predominantly expressed in prepollinated flowers. The transcript abundance of OsTOP6A2, OsTOP6A3 and OsTOP6B was also higher in prepollinated flowers and callus. The expression of OsTOP6A2, OsTOP6A3 and OsTOP6B was differentially regulated by the plant hormones, auxin, cytokinin, and abscisic acid. Yeast two-hybrid analysis revealed that the full-length OsTOP6B protein interacts with both OsTOP6A2 and OsTOP6A3, but not with OsTOP6A1. The nuclear localization of OsTOP6A3 and OsTOP6B was established by the transient expression of their β-glucuronidase fusion proteins in onion epidermal cells. Overexpression of OsTOP6A3 and OsTOP6B in transgenic Arabidopsis plants conferred reduced sensitivity to the stress hormone, abscisic acid, and tolerance to high salinity and dehydration. Moreover, the stress tolerance coincided with enhanced induction of many stress-responsive genes in transgenic Arabidopsis plants. In addition, microarray analysis revealed that a large number of genes are expressed differentially in transgenic plants. Taken together, our results demonstrate that TOP6 genes play a crucial role in stress adaptation of plants by altering gene expression. [ABSTRACT FROM AUTHOR]

Abstract: Small auxin-up RNAs (SAURs) are the early auxin-responsive genes represented by a large multigene family in plants. Here, we report the identification of 58 OsSAUR gene family members from rice (Oryza sativa japonica cv Nipponbare), the model monocot plant, by a reiterative database search and manual reannotation; 2 of these are pseudogenes. The coding sequences of OsSAURs do not possess any intron. Most of the predicted OsSAUR protein sequences harbor a putative nuclear localization signal at their N-terminus. Localized gene duplications appear to be the primary genetic event responsible for SAUR gene family expansion in rice. Interestingly, the duplication of OsSAURs was found to be associated with the chromosomal block duplication as well. The phylogenetic analysis revealed that the SAUR gene family expanded in rice and Arabidopsis due to species-specific expansion of the family in monocots and dicots. The auxin-responsive elements and downstream element are conserved in the upstream and downstream sequences, respectively, of OsSAURs. In addition to the 21 OsSAURs with full-length cDNA sequences and 20 with expressed sequence tags, gene expression analyses of at least 7 OsSAURs by RT-qPCR indicated that the majority of identified OsSAURs most likely are expressed in rice. The transcript abundance of the OsSAURs examined increased within a few minutes of exogenous auxin application with varying kinetics. The present study provides basic genomic information for the rice SAUR gene family and will pave the way for deciphering the precise role of SAURs in plant growth and development. [Copyright &y& Elsevier]

Studies on the structure-function relationship of transporters require the availability of sufficient amounts of the protein in a functional state. In this paper, we report the functional expression, purification, and reconstitution of the human sodium/D-glucose cotransporterl (hSGLT1) in Pichia pastoris and ligand-induced conformational changes of hSGLT1 in solution as studied by intrinsic tryptophan fluorescence. hSGLT1 gene containing FLAG tag at position 574 was cloned into pPICZB plasmid, and the resulting expression vector pPICZB-hSGLT1 was introduced into P. pastoris strain GS115 by electroporation. Purification of recombinant hSGLT1 by nickel-affinity chromatography yields about 3 mg of purified recombinant hSGLT1 per 1-liter of cultured Pichia cells. Purified hSGLT1 migrates on SDS-PAGE with an apparent mass of 55 kDa. Kinetic analysis of hSGLT 1 in proteoliposomes revealed sodium- dependent, secondary active, phlorizin-sensitive, and stereospecific α-methyl-D-glucopyranoside transport, demonstrating its full catalytic activity. The position of the maximum intrinsic tryptophan fluorescence and titration with hydrophilic collisional quenchers KI, acrylamide, and trichloroethanol suggested that most of Trps in hSGLT1 in solution are in a hydrophobic environment. In the presence of sodium, sugars that have been identified earlier as substrate for the transporter increase intrinsic fluorescence in a saturable manner by a maximum of 15%. α-Methyl-D-glucopyranoside had the highest affinity (Kd = 0.71 mM), followed by D-glucose, D-galactose, D-mannose, and D-allose which showed a much lower affinity. L-Glucose was without effect. D-Glucose also increased the accessibility of the Tips to hydrophilic collisional quenchers. On the contrary phlorizin, the well-established inhibitor of SGLT1, decreased intrinsic fluorescence by a maximum of 50%, and induced a blue shift of maximum (5 nm). Again, the effects were sodium-dependent and saturable and a high affinity Kd of 5 μM was observed. In addition the surface of hSGLT1 was labeled with 1-anilinonaphthalene-8-sulfonic acid, a reporter molecule for the surface hydrophobicity. In the presence of sodium, addition of D-glucose decreased ANS fluorescence whereas phlorizin increased ANS fluorescence. Thus three conformational states of SGLT1 could be defined which differ in their packing density and hydrophobicity of their surface. They reflect properties of the empty carrier, the D-glucose loaded carrier facing the outside of membrane and the complex of the outside- orientated carrier with phiorizin. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis faces various stressful conditions inside the host and responds to them through a coordinated regulation of gene expression. We had previously reported identification of the virS gene of M. tuberculosis (Rv3082c) belonging to the AraC family of transcriptional regulators. In the current study, we show that the seven genes (Rv3083–Rv3089) which are present divergently to virS (Rv3082c) constitute an operon designated the mymA operon. Further investigation on the regulation of this operon showed that transcription of the mymA operon is dependent on the presence of VirS protein. A four-fold induction of the mymA operon promoter occurs specifically in wild-type M. tuberculosis and not in the virS mutant of M. tuberculosis (MtbΔvirS) when exposed to acidic pH. Expression of the mymA operon was also induced in infected macrophages by 10-fold over a 6-day period. To gain an insight into the function of the proteins encoded by this operon, we carried out a bioinformatic analysis, which suggested the involvement of these proteins in the modification of fatty acids required for cell envelope. This was supported by altered colony morphology and cell envelope structure displayed by the virS mutant of M. tuberculosis (MtbΔvirS). [Copyright &y& Elsevier]

Light can modulate development and differentiation of the vital photosynthetic organelle, chloroplast, by photomorphogenetic mechanisms involved in regulating transcription of various photosynthetic genes encoded by nuclear genome. The nuclear genes encode majority of proteins involved in photosynthesis. After perception, light signal is transduced via signaling intermediates, which have been identified using various approaches. These signaling components can either directly influence the binding of light regulatory trans-acting factors to cis-acting elements present in a photosynthetic gene promoter or modulate their activity by various means to facilitate transcription in response to light. Some cis-acting elements show a high degree of conservation among photo-responsive nuclear genes in plants and have been designated as light regulatory elements (LREs). The gene regulation seems to involve interplay of several cis-acting elements and regulatory factors. Additional information available about the post-transcriptional mechanisms responsible for light-regulated expression of photosynthetic genes suggests the importance of these levels in regulation biology. [ABSTRACT FROM AUTHOR]

In a systematic approach to understand the transcriptional machinery of mycobacteria, we had previously isolated and characterized mycobacterial promoter regions. In this study, we have investigated molecular interactions between mycobacterial RNA polymerase holoenzyme, reconstituted with different sigma subunits and the promoter element of the Mycobacterium tuberculosis gene pknH (Rv1266c), a representative of promoters belonging to the ‘extended −10’ class. In vitro transcription assays using the pknH promoter and reconstituted RNA polymerase holoenzyme demonstrated that transcription from the pknH promoter is specifically initiated by σA, the principal sigma factor of mycobacteria. DNase I protection assay and deletion studies with the pknH promoter revealed that the minimal region required for optimal transcription carries the sequence from position −37 to position +6. Moreover, mutation in the TGN motif of the pknH promoter resulted in the loss of >75% of its activity. Binding of RNA polymerase with wild-type promoter as well as its TG− mutant revealed that the TGN motif is required for the transition from a close complex into an open complex. Further, it was observed that the presence of the TGN motif reduces the thermal energy required for the conversion of a close complex into an open complex, necessary for initiation of transcription. [Copyright &y& Elsevier]

The psbQ gene encoding a 16-kDa polypeptide of the oxygen-evolving complex of photosystem II has been isolated from Arabidopsis thaliana and characterized. The gene consists of a 28 nucleotide long leader sequence, two introns and three exons encoding a 223-amino-acid precursor polypeptide. The first 75 amino acids act as a transit peptide for the translocation of the polypeptide into the thylakoid lumen. Expression studies show that the gene is light-inducible and expresses only in green tissues with high steady-state mRNA levels in leaves. Using this gene as a probe, restriction fragment length polymorphism between two ecotypes, Columbia and Estland, has also been detected. [ABSTRACT FROM PUBLISHER]

The 33-kDa polypeptide of the oxygen-evolving complex of photosystem II is nuclear-encoded. The single psbO gene of Arabidopsis thaliana, as suggested by Southern hybridization, has been isolated from the genomic library and sequenced. The sequence analysis has revealed that the psbO gene harbors two introns and encodes a precursor polypeptide of 332 amino acid residues; the first 85 amino acid residues represent the transit peptide and the following 247 amino acids constitute the mature polypeptide. The hydrophilic nature of the 33-kDa protein is confirmed by the presence of 27% charged residues. Northern analysis of the total RNA from Arabidopsis indicates that a 1.2-kb transcript represents the psbO gene. It is expressed in a tissue-specific manner — the steady-state transcript levels being highest in the leaves and virtually undetectable in the roots. Also, expression of the psbO gene is development-dependent and regulated by light in young Arabidopsis seedlings. In a constitutively photomorphogenic mutant of Arabidopsis, pho2 (plumular hook open 2), the psbO gene is de-repressed in young, dark-grown seedlings, resulting in increased transcript abundance compared to the wild-type. These studies, thus, define the influence of at least one regulatory component for psbO expression. [ABSTRACT FROM PUBLISHER]

The psbP gene encoding the precursor of 23-kDa polypeptide of the oxygen-evolving complex of photosystem II has been isolated from Arabidopsis thaliana genomic library and sequenced. The gene harbors three introns and encodes a mature polypeptide of 186 amino acid residues and a transit peptide of 77 amino acid residues. The deduced molecular mass of the mature polypeptide is 23.5 kDa and it contains 22.6% charged amino acid residues which may contribute to the hydrophilic nature of the protein. The transcript encoded by psbP gene of Arabidopsis is approximately 1.3 kb long. In wild-type Arabidopsis seedlings, its expresssion is organ-specific and is regulated by endogenous developmental cues, light and sucrose. In a constitutively photomorphogenic mutant of Arabidopsis, designated as pho1, the psbP gene is partly derepressed in young, dark-grown seedlings, resulting in a slightly higher level of the transcript. Additionally, the pho1 mutant shows slow accumulation of psbP transcript upon illumination of young, dark-grown seedlings. However, the derepression is not markedly displayed on dark-adaptation of pho1 plants grown in continuous light. These studies, therefore, define the activity of at least one cellular effector involved in regulation of psbP expression. [ABSTRACT FROM PUBLISHER]

The ability of Mycobacterium tuberculosis to respond and adapt to various stresses such as oxygen/nitrogen radicals and low pH inside macrophages is critical for the persistence of this human pathogen inside its host. We have previously shown that an AraC/XylS-type transcriptional regulator, VirS, which is induced in low pH, is involved in remodeling the architecture of the bacterial cell envelope. However, how VirS influences gene expression to coordinate these pH responses remains unclear. Here, using a genetic biosensor of cytoplasmic pH, we demonstrate that VirS is required for the intracellular pH maintenance in response to acidic stress and inside acidified macrophages. Furthermore, we observed that VirS plays an important role in blocking phagosomal-lysosomal fusions. Transcriptomics experiments revealed that VirS affects the expression of genes encoding metabolic enzymes, cell-wall envelope proteins, efflux pumps, ion transporters, detoxification enzymes, and transcriptional regulators expressed under low-pH stress. Employing electrophoretic mobility-shift assays, DNA footprinting, and in silico analysis, we identified a DNA sequence to which VirS binds and key residues in VirS required for its interaction with DNA. A significant role of VirS in M. tuberculosis survival in adverse conditions suggested it as a potential anti-mycobacterial drug target. To that end, we identified VirS inhibitors in a virtual screen; the top hit compounds inhibited its DNA-binding activity and also M. tuberculosis growth in vitro and inside macrophages. Our findings establish that VirS mediates M. tuberculosis responses to acidic stress and identify VirS-inhibiting compounds that may form the basis for developing more effective anti-mycobacterial agents. [ABSTRACT FROM AUTHOR]

Thyroid hormone receptor β (THRβ) is a member of the nuclear receptor superfamily of ligand-modulated transcription factors. Upon ligand binding, THRβ sequentially recruits the components of transcriptional machinery to modulate target gene expression. In addition to regulating diverse physiological processes, THRβ plays a crucial role in hypothalamus-pituitary-thyroid axis feedback regulation. Anomalies in THRβ gene/protein structure are associated with onset of diverse disease states. In this study, we investigated disease-inflicting truncated variants of THRβ using in-silico analysis and cell-based assays. We examined the THRβ truncated variants on multiple test parameters, including subcellular localization, ligand-receptor interactions, transcriptional functions, interaction with heterodimeric partner RXR, and receptor-chromatin interactions. Moreover, molecular dynamic simulation approaches predicted that shortened THRβ-LBD due to point mutations contributes proportionally to the loss of structural integrity and receptor stability. Deviant subcellular localization and compromised transcriptional function were apparent with these truncated variants. Present study shows that ' mitotic bookmarking' property of some THRβ variants is also affected. The study highlights that structural and conformational attributes of THRβ are necessary for normal receptor functioning, and any deviations may contribute to the underlying cause of the inflicted diseases. We anticipate that insights derived herein may contribute to improved mechanistic understanding to assess disease predisposition. • Cellular and physiological functions of T 3 are mediated via THRβ. • Genetic alterations in the THRB gene cause several thyroid-related disorders. • Introduction of premature stop codon by a critical SNP/mutation truncates THRβ. • Truncated THRβ variants exhibit aberrant cellular functions to inflict diseases. • Understanding disease-inflicting THRβ variants may aid novel therapeutic avenues. [ABSTRACT FROM AUTHOR]

Abstract: The real-time polymerase chain reaction (PCR) data requires normalization with an internal control gene expressed at constant levels under all the experimental conditions being analyzed for accurate and reliable gene expression results. In this study, the expression of 12 candidate internal control genes, including ACT1, EF1α, GAPDH, IF4a, TUB6, UBC, UBQ5, UBQ10, 18SrRNA, 25SrRNA, GRX and HSP90, in a diverse set of 18 tissue samples representing different organs/developmental stages and stress conditions in chickpea (Cicer arietinum L.) has been validated. Their expression levels vary considerably in various tissue samples analyzed. The expression levels of EF1α and HSP90 are most constant across various organs/developmental stages analyzed. Similarly, the expression levels of IF4a and GAPDH are most constant across various stress conditions. A set of two most stable genes is found sufficient for accurate and reliable normalization of real-time PCR data in the given set of tissue samples of chickpea. The genes with most constant expression identified in this study should be useful for normalization of gene expression data in a wide variety of tissue samples in chickpea. [Copyright &y& Elsevier]

For accurate and reliable gene expression results, normalization of real-time PCR data is required against a control gene, which displays highly uniform expression in living organisms during various phases of development and under different environmental conditions. We assessed the gene expression of 10 frequently used housekeeping genes, including 18S rRNA, 25S rRNA, UBC, UBQ5, UBQ10, ACT11, GAPDH, eEF-1α, eIF-4a, and β-TUB, in a diverse set of 25 rice samples. Their expression varied considerably in different tissue samples analyzed. The expression of UBQ5 and eEF-1α was most stable across all the tissue samples examined. However, 18S and 25S rRNA exhibited most stable expression in plants grown under various environmental conditions. Also, a set of two genes was found to be better as control for normalization of the data. The expression of these genes (with more uniform expression) can be used for normalization of real-time PCR results for gene expression studies in a wide variety of samples in rice. [Copyright &y& Elsevier]

The regulation of chloroplast gene expression by light involves multiple signaling components. In an earlier study, we demonstrated the role of calcium and phosphorylation in regulating the expression of photosynthesis-related plastid genes, psbA, psaA and rbcL, using rice as a model monocot system. This work has been extended further to examine the possible involvement of heterotrimeric GTP-binding proteins and calmodulin. Vacuum infiltration of 5-day-old etiolated rice seedlings with G-protein agonists, cholera toxin and GTPγS, increased the steady-state transcript levels of the plastid genes. The antagonists/inhibitors of calmodulin action, trifluoperazine and W7, inhibited the light-induced increase in steady-state transcript levels of these genes. The light-regulated expression of photosynthetic genes was also adversely affected by tagetitoxin, a specific inhibitor of plastid-encoded RNA polymerase. These results indicate the involvement of various signaling components in transduction of light signal that probably also recruits PEP to regulate plastid gene expression. [Copyright &y& Elsevier]

Graphical abstract Highlights • Characterization of hydrophilic fraction of Clematis flammula aerial parts with Gas Chromatography and Mass Spectrometry/ cytotoxicity of Clematis flammula hydrolate towards human colon cancer cells (HCT-116)/Radical oxygen species production after treatment with hydrolate of Clematis flammula / Clematis hydrolate synergizes the effect of Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Gene expression. Abstract This work focused on characterizing hydrophilic fractions of Clematis flammula (CFl). The data here clearly demonstrated that hydrolate fractions act as a free radical scavengers and inhibited proliferation of different cell lines in a time- and concentration-dependent manner, transwell, and with a significant cytotoxic effect. Treating cells with CFl had the effect of suppressing cell growth attenuated by ROS generation in colonic carcinoma. Moreover, CFl in HCT116 cells suppressed survival, proliferation, invasion, angiogenesis and metastasis in vitro by inhibiting gene expression. Following CFl treatment, caspases and PARP cleavage were detected. The up- and down-regulated genes obtained from the WBA of the effect of CFl showed that several biological processes were associated with apoptosis and induction of G1 cell cycle arrest. CFl synergizes the effect of TRAIL by down-regulating the expression of cell survival proteins involved in apoptosis compared to cells treated with CFl or TRAIL alone. Our findings showed that CFl sensitizes apoptosis in TRAIL-resistant cells by activating MAPKs, SP1, and CHOP, that induced DR5 expression. Overall, our data showed that CFl is a promising antitumor agent through kinases and transcription factor induction, both of which are required to activate TRAIL receptors. Colon inflammation induced by LPS was inhibited by CFl hydrolate. [ABSTRACT FROM AUTHOR]

Nuclear receptor PXR is predominantly expressed in liver and intestine. Expression of PXR is observed to be dysregulated in various metabolic disorders indicating its involvement in disease development. However, information available on mechanisms of PXR self-regulation is fragmentary. The present investigation identifies some of the regulatory elements responsible for its tight regulation and low cellular expression. Here, we report that the PXR-promoter is a target for some key transcription factors like PU.1/Ets-1, Pax5, LEF-1 and c-Jun. Interestingly, we observed that PXR-promoter responsiveness to Pax5, LEF-1 and c-Jun, is considerably enhanced by Ets transcription factors (PU.1 and Ets-1). Co-transfection of cells with Ets-1, LEF-1 and c-Jun increased PXR-promoter activity by 5-fold and also induced expression of endogenous human PXR. Site-directed mutagenesis and transfection studies revealed that two Ets binding sites and two of the three LEF binding sites in the PXR-promoter are functional and have a positive effect on PXR transcription. Results suggest that expression of Ets family members, in conjunction with Pax5, LEF-1 and c-Jun, lead to coordinated up-regulation of PXR gene transcription. Insights obtained on the regulation of PXR gene have relevance in offering important cues towards normal functioning as well as development of several metabolic disorders via PXR signaling. [ABSTRACT FROM AUTHOR]

Abstract: Many studies have supported a role for inflammation in prostate tumour growth. However, the contribution of inflammation to the development of castration-resistant prostate cancer remains largely unknown. Based on observations that aberrant expression of the proinflammatory protein tissue transglutaminase (TG2) is associated with development of drug resistance and metastatic phenotype in multiple cancer types, we determined TG2 expression in prostate cancer cells. Herein we report that human prostate cancer cell lines with low expression of androgen receptor (AR) had high basal levels of TG2 expression. Also, overexpression of TG2 negatively regulated AR mRNA and protein expression and attenuated androgen sensitivity of prostate cancer cells. TG2 expression in prostate cancer cells was associated with increased invasion and resistance to chemotherapy. Mechanistically, TG2 activated nuclear factor (NF)-κB and induced epithelial–mesenchymal transition. TG2/NF-κB-mediated decrease in AR expression resulted from transcriptional repression involving cis-interaction of NF-κB in a complex with TG2 with the 5′-untranslated region of AR. Negative regulation of AR could be partially abrogated by repression of TG2 or NF-κB (p65/RelA) by gene-specific small interfering RNA. These results suggested that a novel pathway links androgen dependence with TG2-regulated inflammatory signalling and hence may make TG2 a novel therapeutic target for the prevention and treatment of castration-resistant prostate cancer. [Copyright &y& Elsevier]

Abstract: An efficient global control of tuberculosis requires development of alternative vaccination strategies that can enhance the efficacy of existing BCG vaccine. In this study, we evaluated the protective efficacy of a recombinant BCG (rBCG) vaccine over-expressing iron-cofactored superoxide dismutase (SOD-A), one of the prominent oxidative stress response proteins of Mycobacterium tuberculosis. Contrary to our expectations, over-expression of SOD-A resulted in the abrogation of BCG''s ability to confer protection in guinea pig as well as in murine model. Analysis of immune responses revealed that over-expression of SOD-A by rBCG has pleiotropic effects on innate and adaptive immune responses. Macrophages infected in vitro with rBCG exhibited a marked reduction in apoptosis and microbicidal potential. In addition, rBCG vaccination of mice resulted in a reduced IFNγ and increased IL10 production when compared with the BCG vaccination. Further, we show that rBCG vaccination failed to generate an effective multi-functional CD4 T cell response. Altogether, our findings suggest that over-expression of SOD-A in BCG enhances the immuno-suppressive properties of BCG, characterized by skewing of immune responses towards Th2 type, an inefficient multi-functional T cell response and reduced apoptosis and microbicidal potential of macrophages leading to abolishment of BCG''s protective efficacy. [Copyright &y& Elsevier]

Abstract: Owing to its highly immunodominant nature and ability to induce long-lived memory immunity, ESAT-6, a prominent antigen of Mycobacterium tuberculosis, has been employed in several approaches to develop tuberculosis vaccines. Here, for the first time, we combined ESAT-6 based recombinant BCG (rBCG) and DNA vaccine (DNAE6) in a prime boost approach. Interestingly, in spite of inducing an enhanced antigen specific IFN-γ response in mice, a DNAE6 booster completely obliterated the protection imparted by rBCG against tuberculosis in guinea pigs. Analysis of immunopathology and cytokine responses suggests involvement of an exaggerated immunity behind the lack of protection imparted by this regimen. [Copyright &y& Elsevier]

Abstract: Complement receptor 1 (CR1/CD35) levels on erythrocytes and related CR1 polymorphisms have been associated with response to falciparum malaria in populations inhabiting malaria-endemic regions. Differences in disease association profiles of its low expression alleles have been observed in populations from different regions of the world. We analyzed the influence of CR1 levels and associated SNPs on susceptibility/resistance to falciparum malaria in Indian populations. Two CR1 SNPs [exon 22 (A/G) and intron 27 (A/T)] define the low expression (L) CR1 allele in populations inhabiting a Plasmodium falciparum–endemic and a nonendemic region of India. Populations of the endemic region have very low red blood cell surface CR1 levels and higher frequencies of the exon 22 and intron 27 mutant L alleles. Whereas low CR1 levels correlated with susceptibility to severe malaria in the nonendemic region, high CR1 levels were associated with manifestation of disease in the endemic region. In addition, the exon 22 L allele was a risk factor for severe malaria in the nonendemic region. Absence of correlation between levels of tumor necrosis factor– α, interferon-γ, and interleukin-6 with CR1 levels in patients with severe disease indicated that RBC CR1 levels in individuals are not the major determinants of pro-inflammatory cytokine release during infection. Our results are interpreted in the context of differences in the pathogenesis of severe malaria in the malaria-endemic and nonendemic region. [Copyright &y& Elsevier]

The SET domains are conserved amino acid sequences present in chromosomal proteins that contribute to the epigenetic control of gene expression by altering regional organization of the chromatin structure. The SET domain proteins are divided into four subgroups as categorized by their Drosophila members; enhancer of zeste (E(Z)), trithorax (TRX), absent small or homeotic 1 (ASH1) and supressor of variegation (SU(VAR)3–9). Homologs of all four classes have been characterized in yeast, mammals and plants. We report here the isolation and characterization of rice (Oryza sativa L. subspecies indica) cDNA, OsiEZ1, as a monocot member of this family. The OsiEZ1 cDNA is 3133 bp long with an ORF of 2799 bp, and the predicted amino acid sequence (895 residues) corresponds to a protein of ca. 98 kDa. All the characteristic domains known to be conserved in E(Z) homologs (subgroup I) of SET domain containing proteins are present in OsiEZ1. In the rice genome, a 7499 bp long OsiEZ1 sequence is split into 17 exons interrupted by 16 introns. Southern analysis indicates that OsiEZ1 is represented as single copy in the rice genome. Expression studies revealed that the OsiEZ1 transcript level was highest in rice flowers, almost undetectable in developing seeds of 1–2 days post-fertilization but increased significantly in young seeds of 3–5 days post-fertilization. The OsiEZ1 transcript was barely detectable in mature zygotic embryos, but its levels were significantly higher in callus derived from rice scutellum, somatic embryos and young seedlings. The OsiEZ1/GUS recombinant protein was confined to the nucleus in living cells of particle-bombarded onion peels. The expression of OsiEZ1 complemented a set1Δ Saccharomyces cerevisiae mutant that is impaired in telomeric silencing. We suggest that the nuclear-localized OsiEZ1 has a role in regulating various aspects of plant development, and this control is most likely brought about by repressing the activity of downstream regulatory genes. [Copyright &y& Elsevier]