Transcription of human resistin gene involves an interaction of Sp1 with peroxisome proliferator-activating receptor gamma (PPARγ)

Background: Resistin is a cysteine rich protein, mainly expressed and secreted by circulating human mononuclear cells. While several factors responsible for transcription of mouse resistin gene have been identified, not much is known about the factors responsible for the differential expression of human resistin. Methodology/Principal Finding: We show that the minimal promoter of human resistin lies within ~80 bp sequence upstream of the transcriptional start site (-240) whereas binding sites for cRel, CCAAT enhancer binding protein α (C/EBP-α), activating transcription factor 2 (ATF-2) and activator protein 1 (AP-1) transcription factors, important for induced expression, are present within sequences up to -619. Specificity Protein 1(Sp1) binding site (-276 to -295) is also present and an interaction of Sp1 with peroxisome proliferator activating receptor gamma (PPARγ) is necessary for constitutive expression in U937 cells. Indeed co-immunoprecipitation assay demonstrated a direct physical interaction of Sp1 with PPARγ in whole cell extracts of U937 cells. Phorbol myristate acetate (PMA) upregulated the expression of resistin mRNA in U937 cells by increasing the recruitment of Sp1, ATF-2 and PPARγ on the resistin gene promoter. Furthermore, PMA stimulation of U937 cells resulted in the disruption of Sp1 and PPARγ interaction. Chromatin immunoprecipitation (ChIP) assay confirmed the recruitment of transcription factors phospho ATF-2, Sp1, Sp3, PPARγ, chromatin modifier histone deacetylase 1 (HDAC1) and the acetylated form of histone H3 but not cRel, C/EBP-α and phospho c-Jun during resistin gene transcription. Conclusion: Our findings suggest a complex interplay of Sp1 and PPARγ along with other transcription factors that drives the expression of resistin in human monocytic U937 cells

The Co-Operonic PE25/PPE41 Protein Complex of Mycobacterium tuberculosis Elicits Increased Humoral and Cell Mediated Immune Response

BACKGROUND: Many of the PE/PPE proteins are either surface localized or secreted outside and are thought to be a source of antigenic variation in the host. The exact role of these proteins are still elusive. We previously reported that the PPE41 protein induces high B cell response in TB patients. The PE/PPE genes are not randomly distributed in the genome but are organized as operons and the operon containing PE25 and PPE41 genes co-transcribe and their products interact with each other. METHODOLOGY/PRINCIPAL FINDING: We now describe the antigenic properties of the PE25, PPE41 and PE25/PPE41 protein complex coded by a single operon. The PPE41 and PE25/PPE41 protein complex induces significant (p<0.0001) B cell response in sera derived from TB patients and in mouse model as compared to the PE25 protein. Further, mice immunized with the PE25/PPE41 complex and PPE41 proteins showed significant (p<0.00001) proliferation of splenocyte as compared to the mice immunized with the PE25 protein and saline. Flow cytometric analysis showed 15-22% enhancement of CD8+ and CD4+ T cell populations when immunized with the PPE41 or PE25/PPE41 complex as compared to a marginal increase (8-10%) in the mice immunized with the PE25 protein. The PPE41 and PE25/PPE41 complex can also induce higher levels of IFN-gamma, TNF-alpha and IL-2 cytokines. CONCLUSION: While this study documents the differential immunological response to the complex of PE and PPE vis-à-vis the individual proteins, it also highlights their potential as a candidate vaccine against tuberculosis

Mechanistic Insights into a Novel Exporter-Importer System of Mycobacterium tuberculosis Unravel Its Role in Trafficking of Iron

Elucidation of the basic mechanistic and biochemical principles underlying siderophore mediated iron uptake in mycobacteria is crucial for targeting this principal survival strategy vis-à-vis virulence determinants of the pathogen. Although, an understanding of siderophore biosynthesis is known, the mechanism of their secretion and uptake still remains elusive.Here, we demonstrate an interplay among three iron regulated Mycobacterium tuberculosis (M.tb) proteins, namely, Rv1348 (IrtA), Rv1349 (IrtB) and Rv2895c in export and import of M.tb siderophores across the membrane and the consequent iron uptake. IrtA, interestingly, has a fused N-terminal substrate binding domain (SBD), representing an atypical subset of ABC transporters, unlike IrtB that harbors only the permease and ATPase domain. SBD selectively binds to non-ferrated siderophores whereas Rv2895c exhibits relatively higher affinity towards ferrated siderophores. An interaction between the permease domain of IrtB and Rv2895c is evident from GST pull-down assay. In vitro liposome reconstitution experiments further demonstrate that IrtA is indeed a siderophore exporter and the two-component IrtB-Rv2895c system is an importer of ferrated siderophores. Knockout of msmeg_6554, the irtA homologue in Mycobacterium smegmatis, resulted in an impaired M.tb siderophore export that is restored upon complementation with M.tb irtA.Our data suggest the interplay of three proteins, namely IrtA, IrtB and Rv2895c in synergizing the balance of siderophores and thus iron inside the mycobacterial cell

Concurrent Proinflammatory and Apoptotic Activity of a Helicobacter pylori Protein (HP986) Points to Its Role in Chronic Persistence

Helicobacter pylori induces cytokine mediated changes in gastroduodenal pathophysiology, wherein, the activated macrophages at the sub-mucosal space play a central role in mounting innate immune response against the antigens. The bacterium gains niche through persistent inflammation and local immune-suppression causing peptic ulcer disease or chronic gastritis; the latter being a significant risk factor for the development of gastric adenocarcinoma. What favors persistence of H. pylori in the gastric niches is not clearly understood. We report detailed characterization of a functionally unknown gene (HP986), which was detected in patient isolates associated with peptic ulcer and gastric carcinoma. Expression and purification of recombinant HP986 (rHP986) revealed a novel, ∼29 kDa protein in biologically active form which associates with significant levels of humoral immune responses in diseased individuals (p<0.001). Also, it induced significant levels of TNF-α and Interleukin-8 in cultured human macrophages concurrent to the translocation of nuclear transcription factor-κB (NF-κB). Further, the rHP986 induced apoptosis of cultured macrophages through a Fas mediated pathway. Dissection of the underlying signaling mechanism revealed that rHP986 induces both TNFR1 and Fas expression to lead to apoptosis. We further demonstrated interaction of HP986 with TNFR1 through computational and experimental approaches. Independent proinflammatory and apoptotic responses triggered by rHP986 as shown in this study point to its role, possibly as a survival strategy to gain niche through inflammation and to counter the activated macrophages to avoid clearance

Direct in-gel hybridization, without blotting, using nick-translated cloned DNA probe

Hybridization of DNA and RNA to labeled probes is central to molecular biology. A method is described here for hybridization of cloned DNA probes directly to DNA in agarose gels. This in-gel hybridization method has several advantages over conventional techniques using transfer to membranes. It is extremely rapid, highly sensitive, less expensive and particularly suited for high molecular weight genomic DNA analysis

Ancestral Mycobacterium tuberculosis genotypes in India: implications for TB control programmes

It has been a decade since the genome sequence of Mycobacterium tuberculosis was unraveled. The fruits of genomic technologies are yet to reach high burden countries such as India, where tuberculosis (TB) kills a huge number of patients. Paradoxically, despite increased cases of human immunodeficiency virus (HIV) infection and diabetes mellitus, TB cure rates in India have been consistently improving during the DOTS program. Does this mean that the underlying TB bacilli are somehow 'co-operating' with the TB control program implementers ? Genotypic analyses of the tubercle bacilli have identified a predominance of ancestral strains of M. tuberculosis in major parts of India in addition to various other lineages of modern evolutionary descent. Virulence and dissemination potentials of these ancestral strains are speculated to be 'low' as compared to the other 'aggressive' strains such as Beijing and LAM, which are expected to be more widespread in future, also in synergy with HIV and diabetes epidemics. We discuss the implications of the high prevalence of ancestral strains on TB control in India. It appears that despite a hypothetical 'ancestral advantage', future dynamics of tubercle bacilli in the back drop of surging HIV and diabetes incidences may pose a major healthcare problem in India in the years to come

Nucleotide sequence of a human satellite DNA

The nucleotide sequence of a 342-bp human alphoid satellite DNA is described

A novel probe for human DNA fingerprinting based on chi-like sequences

A synthetic oligodeoxyribonucleotide (oligo) containing crossover initiating hotspot-like sequences was designed on the assumption that hypervariability is partly due to the presence of molecular signals which promote recombination. This oligo, when used as a probe for human DNA fingerprinting, generated individual-specific DNA band patterns. The probability of two unrelated individuals having the same DNA band pattern, using this probe, was estimated to be 1.9 × 10<SUP>-13</SUP>

The translation initiation factor, PeIF5B, from Pisum sativum displays chaperone activity

We earlier documented the structural and functional characterization of PeIF5B factor from Pisum sativum that shows strong homology to the universal translation initiation factor eIF5B (Rasheedi et al., 2007, 2010 and ). We now show that PeIF5B is an unusually thermo-stable protein resisting temperatures up to 95 °C. PeIF5B prevents thermal aggregation of heat labile proteins, such as citrate synthase (CS) and NdeI, under heat stress or chemical denaturation conditions and promotes their functional folding. It also prevents the aggregation of DTT induced insulin reduction. GTP appears to stimulate PeIF5B-mediated chaperone activity. In-vivo, PeIF5B over expression significantly enhances, the viability of Escherichia coli cells after heat stress (50 °C). These observations lead us to conclude that PeIF5B, in addition to its role in protein translation, has chaperone like activity and could be likely involved in protein folding and protection from stress