Japanese Encephalitis Virus Utilizes the Canonical Pathway To Activate NF-kappa B but It Utilizes the Type I Interferon Pathway To Induce Major Histocompatibility Complex Class I Expression in Mouse Embryonic Fibroblasts

Flaviviruses have been shown to induce cell surface expression of major histocompatibility complex class I (MHC-I) through the activation of NF-kappa B. Using IKK1(-/-), IKK2(-/-), NEMO-/-, and IKK1-/- IKK2-/- double mutant as well as p50(-/-) RelA(-/-) cRel(-/-) triple mutant mouse embryonic fibroblasts infected with Japanese encephalitis virus (JEV), we show that this flavivirus utilizes the canonical pathway to activate NF-kappa B in an IKK2- and NEMO-, but not IKK1-, dependent manner. NF-kappa B DNA binding activity induced upon virus infection was shown to be composed of RelA: p50 dimers in these fibroblasts. Type I interferon (IFN) production was significantly decreased but not completely abolished upon virus infection in cells defective in NF-kappa B activation. In contrast, induction of classical MHC-I (class 1a) genes and their cell surface expression remained unaffected in these NF-kappa B-defective cells. However, MHC-I induction was impaired in IFNAR(-/-) cells that lack the alpha/beta IFN receptor, indicating a dominant role of type I IFNs but not NF-kappa B for the induction of MHC-I molecules by Japanese encephalitis virus. Our further analysis revealed that the residual type I IFN signaling in NF-kappa B-deficient cells is sufficient to drive MHC-I gene expression upon virus infection in mouse embryonic fibroblasts. However, NF-kappa B could indirectly regulate MHC-I expression, since JEV-induced type I IFN expression was found to be critically dependent on it.

Nitric oxide and KLF4 protein epigenetically modify class II transactivator to repress Major histocompatibility complex II expression during Mycobacterium bovis Bacillus Calmette-Guerin infection

Pathogenic mycobacteria employ several immune evasion strategies such as inhibition of class II transactivator (CIITA) and MHC-II expression, to survive and persist in host macrophages. However, precise roles for specific signaling components executing down-regulation of CIITA/MHC-II have not been adequately addressed. Here, we demonstrate that Mycobacterium bovis bacillus Calmette-Guerin (BCG)-mediated TLR2 signaling-induced iNOS/NO expression is obligatory for the suppression of IFN-gamma-induced CIITA/MHC-II functions. Significantly, NOTCH/PKC/MAPK-triggered signaling cross-talk was found critical for iNOS/NO production. NO responsive recruitment of a bifunctional transcription factor, KLF4, to the promoter of CIITA during M. bovis BCG infection of macrophages was essential to orchestrate the epigenetic modifications mediated by histone methyltransferase EZH2 or miR-150 and thus calibrate CIITA/MHC-II expression. NO-dependent KLF4 regulated the processing and presentation of ovalbumin by infected macrophages to reactive T cells. Altogether, our study delineates a novel role for iNOS/NO/KLF4 in dictating the mycobacterial capacity to inhibit CIITA/MHC-II-mediated antigen presentation by infected macrophages and thereby elude immune surveillance.

Processing of Mycobacterium tuberculosis bacilli by human monocytes for CD4+ alphabeta and gammadelta T cells: role of particulate antigen.

Mycobacterium tuberculosis readily activates both CD4+ and Vdelta2+ gammadelta T cells. Despite similarity in function, these T-cell subsets differ in the antigens they recognize and the manners in which these antigens are presented by M. tuberculosis-infected monocytes. We investigated mechanisms of antigen processing of M. tuberculosis antigens to human CD4 and gammadelta T cells by monocytes. Initial uptake of M. tuberculosis bacilli and subsequent processing were required for efficient presentation not only to CD4 T cells but also to Vdelta2+ gammadelta T cells. For gammadelta T cells, recognition of M. tuberculosis-infected monocytes was dependent on Vdelta2+ T-cell-receptor expression. Recognition of M. tuberculosis antigens by CD4+ T cells was restricted by the class II major histocompatibility complex molecule HLA-DR. Processing of M. tuberculosis bacilli for Vdelta2+ gammadelta T cells was inhibitable by Brefeldin A, whereas processing of soluble mycobacterial antigens for gammadelta T cells was not sensitive to Brefeldin A. Processing of M. tuberculosis bacilli for CD4+ T cells was unaffected by Brefeldin A. Lysosomotropic agents such as chloroquine and ammonium chloride did not affect the processing of M. tuberculosis bacilli for CD4+ and gammadelta T cells. In contrast, both inhibitors blocked processing of soluble mycobacterial antigens for CD4+ T cells. Chloroquine and ammonium chloride insensitivity of processing of M. tuberculosis bacilli was not dependent on the viability of the bacteria, since processing of both formaldehyde-fixed dead bacteria and mycobacterial antigens covalently coupled to latex beads was chloroquine insensitive. Thus, the manner in which mycobacterial antigens were taken up by monocytes (particulate versus soluble) influenced the antigen processing pathway for CD4+ and gammadelta T cells.

Regulation of human CD4(+) alphabeta T-cell-receptor-positive (TCR(+)) and gammadelta TCR(+) T-cell responses to Mycobacterium tuberculosis by interleukin-10 and transforming growth factor beta.

Mycobacterium tuberculosis is the etiologic agent of human tuberculosis and is estimated to infect one-third of the world's population. Control of M. tuberculosis requires T cells and macrophages. T-cell function is modulated by the cytokine environment, which in mycobacterial infection is a balance of proinflammatory (interleukin-1 [IL-1], IL-6, IL-8, IL-12, and tumor necrosis factor alpha) and inhibitory (IL-10 and transforming growth factor beta [TGF-beta]) cytokines. IL-10 and TGF-beta are produced by M. tuberculosis-infected macrophages. The effect of IL-10 and TGF-beta on M. tuberculosis-reactive human CD4(+) and gammadelta T cells, the two major human T-cell subsets activated by M. tuberculosis, was investigated. Both IL-10 and TGF-beta inhibited proliferation and gamma interferon production by CD4(+) and gammadelta T cells. IL-10 was a more potent inhibitor than TGF-beta for both T-cell subsets. Combinations of IL-10 and TGF-beta did not result in additive or synergistic inhibition. IL-10 inhibited gammadelta and CD4(+) T cells directly and inhibited monocyte antigen-presenting cell (APC) function for CD4(+) T cells and, to a lesser extent, for gammadelta T cells. TGF-beta inhibited both CD4(+) and gammadelta T cells directly and had little effect on APC function for gammadelta and CD4(+) T cells. IL-10 down-regulated major histocompatibility complex (MHC) class I, MHC class II, CD40, B7-1, and B7-2 expression on M. tuberculosis-infected monocytes to a greater extent than TGF-beta. Neither cytokine affected the uptake of M. tuberculosis by monocytes. Thus, IL-10 and TGF-beta both inhibited CD4(+) and gammadelta T cells but differed in the mechanism used to inhibit T-cell responses to M. tuberculosis.

Sex Determination - A Hypothesis Based On Noncoding Dna

Certain recent models of sex determination in mammals, Drosophila melanogaster, Caenorhabditis elegans, and snakes are examined in the light of the hypothesis that the relevant genetic regulatory mechanisms are similar and interrelated. The proposed key element in each of these instances is a noncoding DNA sequence, which serves as a high-affinity binding site for a repressor-like molecule regulating the activity of a major "sex-determining" gene. On this basis it is argued that, in several eukaryotes, (i) certain DNA sequences that are sex-determining are noncoding, in the sense that they are not the structural genes of a sex-determining protein; (ii) in some species these noncoding sequences are present in one sex and absent in the other, while in others their copy number or accessibility to regulatory molecules is significantly unequal between the two sexes; and (iii) this inequality determines whether the embryo develops into a male or a female.

Phenobarbitone-mediated translocation of the cytosolic proteins interacting with the 5 '-proximal region of rat liver CYP2B1/B2 gene into the nucleus

The positive element (PE) (-69 to -98 bp) within the 5'-proximal region of the CYP2B1B2 gene (+1 to -179 bp) of rat liver is essential for phenobarbitone (PB) response and gives a single major complex with the rat liver cytosol in gel shift analysis. This complex corresponds to complex I (top) of the three complexes given by the nuclear extracts. PB treatment of rats leads to a decrease in complex I formation with the cytosol and PE and an increase in the same with the nuclear extract in gel shift analysis. Both the changes are counteracted by simultaneous okadaic acid administration. The nuclear protein giving rise to complex I has been isolated and has an M-r of 26 kDa. The cytosolic counterpart consists of two species, 26 and 28 kDa, as revealed by Southwestern blot analysis using labeled PE. It is concluded that PB treatment leads to the translocation accompanied by processing of the cytosolic protein species into the nucleus that requires protein dephosphorylation. It is suggested that PB may exert a global regulation on the transcription of many genes by modulating the phosphorylation status of different protein factors involved in transcriptional regulation. (C) 2002 Elsevier Science (USA).

Mechanism of Fragility at BCL2 Gene Minor Breakpoint Cluster Region during t(14;18) Chromosomal Translocation

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Breaks in chromosome 18 are localized at the 3'-UTR of BCL2 gene or downstream and are mainly clustered in either the major breakpoint region or the minor breakpoint cluster region (mcr). The recombination activating gene (RAG) complex induces breaks at IgH locus of chromosome 14, whereas the mechanism of fragility at BCL2 mcr remains unclear. Here, for the first time, we show that RAGs can nick mcr; however, the mechanism is unique. Three independent nicks of equal efficiency are generated, when both Mg2+ and Mn2+ are present, unlike a single nick during V(D)J recombination. Further, we demonstrate that RAG binding and nicking at the mcr are independent of nonamer, whereas a CCACCTCT motif plays a critical role in its fragility, as shown by sequential mutagenesis. More importantly, we recapitulate the BCL2 mcr translocation and find that mcr can undergo synapsis with a standard recombination signal sequence within the cells, in a RAG-dependent manner. Further, mutation to the CCACCTCT motif abolishes recombination within the cells, indicating its vital role. Hence, our data suggest a novel, physiologically relevant, nonamer-independent mechanism of RAG nicking at mcr, which may be important for generation of chromosomal translocations in humans.

Efficient Dendrimer-DNA Complexation and Gene Delivery Vector Properties of Nitrogen-Core Poly(propyl ether imine) Dendrimer in Mammalian Cells

Dendrimers as vectors for gene delivery were established, primarily by utilizing few prominent dendrimer types so far. We report herein studies of DNA complexation efficacies and gene delivery vector properties of a nitrogen-core poly(propyl ether imine) (PETIM) dendrimer, constituted with 22 tertiary amine internal branches and 24 primary amines at the periphery. The interaction of the dendrimer with pEGFPDNA was evaluated through UV-vis, circular dichroism (CD) spectral studies, ethidium bromide fluorescence emission quenching, thermal melting, and gel retardation assays, from which most changes to DNA structure during complexation was found to occur at a weight ratio of dendrimer:DNA similar to 2:1. The zeta potential measurements further confirmed this stoichiometry at electroneutrality. The structure of a DNA oligomer upon dendrimer complexation was simulated through molecular modeling and the simulation showed that the dendrimer enfolded DNA oligomer along both major and minor grooves, without causing DNA deformation, in 1:1 and 2:1 dendrimer-to-DNA complexes. Atomic force microscopy (AFM) studies on dendrimer-pEGFP DNA complex showed an increase in the average z-height as a result of dendrimers decorating the DNA, without causing a distortion of the DNA structure. Cytotoxicity studies involving five different mammalian cell lines, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (MTT) assay, reveal the dendrimer toxicity profile (IC50) values of similar to 400-1000 mu g mL(-1), depending on the cell line tested. Quantitative estimation, using luciferase assay, showed that the gene transfection was at least 100 times higher when compared to poly(ethylene imine) branched polymer, having similar number of cationic sites as the dendrimer. The present study establishes the physicochemical behavior of new nitrogen-core PETIM dendrimer-DNA complexes, their lower toxicities, and efficient gene delivery vector properties.

The polycomb group gene EMF2B is essential for maintenance of floral meristem determinacy in rice

Polycomb Repressive Complex 2 (PRC2) represses the transcriptional activity of target genes through trimethylation of lysine 27 of histone H3. The functions of plant PRC2 have been chiefly described in Arabidopsis, but specific functions in other plant species, especially cereals, are still largely unknown. Here we characterize mutants in the rice EMF2B gene, an ortholog of the Arabidopsis EMBRYONIC FLOWER2 (EMF2) gene. Loss of EMF2B in rice results in complete sterility, and mutant flowers have severe floral organ defects and indeterminacy that resemble loss-of-function mutants in E-function floral organ specification genes. Transcriptome analysis identified the E-function genes OsMADS1, OsMADS6 and OsMADS34 as differentially expressed in the emf2b mutant compared with wild type. OsMADS1 and OsMADS6, known to be required for meristem determinacy in rice, have reduced expression in the emf2b mutant, whereas OsMADS34 which interacts genetically with OsMADS1 was ectopically expressed. Chromatin immunoprecipitation for H3K27me3 followed by quantitative (q)RT-PCR showed that all three genes are presumptive targets of PRC2 in the meristem. Therefore, in rice, and possibly other cereals, PRC2 appears to play a major role in floral meristem determinacy through modulation of the expression of E-function genes.

Cloning Of Rice Dna And Identification Of Transfer-Rna Gene Clones

THE rapid development of recombinant DNA technology has brought forth a revolution in biology'>", it aids us to have a closer look at the 'way genes are organized, eS11 ecially in the complex eucaryotic genornes'<", Although many animal and yeast genes have been studied in detail using recombinant DNA technology, plant genes have seldom been targets for such studie., Germination is an ideal process to study gene expression .because it effects a . shift in the metabolic status of seeds from a state of 'dormancy to an active one. AJ;l understanding of gene organization and regulation darin.g germination can be accomplblted by molecular cloning of DNA from seeds lik.e rice. To study the status of histone, rRNA tRNA and other genes in the rice genome, a general method was developed to clone eucarvotic DNA in a' plasmid vector pBR 322. This essentially ~ involves the following steps. The rice embryo and plasmid pBR 322 DNAs were cut witll restriction endonuclease Bam Hi to generate stick.Y ends, The plasmid DNA was puosphatased, the DNA~ ware a~·tnealed and joined 'by T4 phage DNA ligase. The recombinant DNA molecules thus produced were transjerred into E. coli and colonies containing them Were selected by their sensitivity to tetracycline and resistance to ampicillin, Two clones were identified . 2S haVing tRNA genes by hybridization of the DNA in the clones \vitl1 32P-la.belled rice tRNAs.

Sodium santalbate-dimethyl sulfate inclusion complex

A molecular inclusion complex has been obtained from the major acetylenic acid, santalbic acid (octadec-11-en-9-ynoic acid ortrans-11-octadecen-9-ynoic acid) of the seed oil ofSantalum album L. by a simple treatment of its sodium salt with dimethyl sulfate. Aqueous solutions (0.5–1%) of the complex produce good lather and have efficient cleansing (detergent) action on grease and dirt particles.

Identification of a novel nucleolin related protein (NRP) gene expressed during rat spermatogenesis

Nucleolin is a major nucleolar phosphoprotein involved in various steps of ribosome biogenesis in eukaryotic cells. As nucleolin plays a significant role in ribosomal RNA transcription we were interested in examining in detail the expression of nucleolin across different stages of spermatogenesis and correlate with the transcription status of ribosomal DNA in germ cells.

Sodium santalbate-dimethyl sulfate inclusion complex

A molecular inclusion complex has been obtained from the major acetylenic acid, santalbic acid (octadec-11-en-9-ynoic acid ortrans-11-octadecen-9-ynoic acid) of the seed oil ofSantalum album L. by a simple treatment of its sodium salt with dimethyl sulfate. Aqueous solutions (0.5–1%) of the complex produce good lather and have efficient cleansing (detergent) action on grease and dirt particles.

Identification and functional characterization of a cis-acting positive DNA element regulating CYP 2B1/B2 gene transcription in rat liver

A positive cis-acting DNA element in the near 5'-upstream region of the CYP2B1/B2 genes in rat liver was found to play an important role in the transcription of these genes. An oligonucleotide covering -69 to -98 nt mimicked the gel mobility shift pattern given by the fragment -179 to +29 nt, which was earlier found adequate to confer the regulatory features of this gene. Two major complexes were seen, of which the slower and faster moving complexes became intense under uninduced and Phenobarbitone-induced conditions respectively. Minigene cloned DNA plasmid covering -179 to +181 nt in pUC 19 and Bal 31 mutants derived from this parent were transcribed in whole nuclei and cell free transcription extracts and mutants containing only upto -75 nt of the upstream were poorly transcribed. Transcription extracts from phenobarbitone-injected rat liver nuclei were significantly more active than extracts from uninduced rats in transcribing the minigene constructs. Addition of the oligonucleotide (-69 to -98nt) specifically inhibited the transcription of the minigene construct (-179 to +181 nt) in the cell free transcription system. It is therefore, concluded that the region -69 to -98 nt acts as a positive cis-acting element in the transcription of the CYP2B1/B2 genes and in mediating the inductive effects of phenobarbitone.

Use of protein A gene fusions for the analysis of structure-function relationship of the transactivator protein C of bacteriophage Mu

A sensitive dimerization assay for DNA binding proteins has been developed using gene fusion technology. For this purpose, we have engineered a gene fusion using protein A gene of Staphylococcus aureus and C gene, the late gene transactivator of bacteriophage Mu. The C gene was fused to the 3' end of the gene for protein A to generate an A- C fusion. The overexpressed fusion protein was purified in a single step using immunoglobulin affinity chromatography. Purified fusion protein exhibits DNA binding activity as demonstrated by electrophoretic mobility shift assays. When the fusion protein A-C was mixed with C and analyzed for DNA binding, in addition to C and A-C specific complexes, a single intermediate complex comprising of a heterodimer of C and A-C fusion proteins was observed. Further, the protein A moiety in the fusion protein A-C does not contribute to DNA binding as demonstrated by proteolytic cleavage and circular dichroism (CD) analysis. The assay has also been applied to analyze the DNA binding domain of C protein by generating fusions between protein A and N- and C-terminal deletion mutants of C. The results indicate a role for the region towards the carboxy terminal of the protein in DNA binding. The general applicability of this method is discussed.