Importance of IL28B gene polymorphisms in hepatitis C virus genotype 2 and 3 infected patients.

BACKGROUND & AIMS: Genetic variation in the interleukin 28B (IL28B) gene has been associated with the response to interferon-alfa/ribavirin therapy in hepatitis C virus (HCV) genotype 1-infected patients. The importance of three IL28B single nucleotide polymorphisms (rs8099917, rs12980275 and rs12979860) for HCV genotype 2/3-infected patients is unknown. METHODS: In patients with chronic hepatitis C genotype 2/3 (n=267), IL28B host genotypes (rs8099917, rs12980275 and rs12979860) were analyzed for associations with sustained virologic response (SVR) to antiviral therapy with (pegylated) interferon-alfa and ribavirin and with respect to epidemiological, biochemical, and virological parameters. For comparison, hepatitis C genotype 1 patients (n=378) and healthy controls (n=200) were included. RESULTS: The rs12979860 CC genotype, lower age, and genotype 2 were significantly associated with SVR in HCV genotype 2/3-infected patients (p=0.01, p=0.03 and p=0.03, respectively). No association was observed for rs8099917 and rs12980275. In addition, an SVR in patients with rapid virologic response (RVR) was associated with the rs12979860 CC genotype (p=0.05), while for non-RVR no association was found. Furthermore, a significant association with a higher baseline viral load was observed for all three IL28B genotypes in genotype 1/2/3-infected patients. Finally, increasing frequencies of the rs12979860 CC genotypes were observed in genotype 1- (33.9%), genotype 3- (38.9%), and genotype 2-infected (51.9%) patients in comparison with healthy controls (49.0%) (p<0.01). CONCLUSIONS: In genotype 2/3-infected patients, rs12979860 was significantly associated with SVR. The frequency of the rs12979860 CC genotype is lower in HCV genotype 1 vs. genotype 2/3 patients. All major IL28B genotypes are associated with HCV-RNA concentration.

Autosomal-dominant distal myopathy associated with a recurrent missense mutation in the gene encoding the nuclear matrix protein, matrin 3.

Distal myopathies represent a heterogeneous group of inherited skeletal muscle disorders. One type of adult-onset, progressive autosomal-dominant distal myopathy, frequently associated with dysphagia and dysphonia (vocal cord and pharyngeal weakness with distal myopathy [VCPDM]), has been mapped to chromosome 5q31 in a North American pedigree. Here, we report the identification of a second large VCPDM family of Bulgarian descent and fine mapping of the critical interval. Sequencing of positional candidate genes revealed precisely the same nonconservative S85C missense mutation affecting an interspecies conserved residue in the MATR3 gene in both families. MATR3 is expressed in skeletal muscle and encodes matrin 3, a component of the nuclear matrix, which is a proteinaceous network that extends throughout the nucleus. Different disease related haplotype signatures in the two families provided evidence that two independent mutational events at the same position in MATR3 cause VCPDM. Our data establish proof of principle that the nuclear matrix is crucial for normal skeletal muscle structure and function and put VCPDM on the growing list of monogenic disorders associated with the nuclear proteome.

The promoter of the gene encoding 3',5'-cyclic adenosine monophosphate (cAMP) response element binding protein contains cAMP response elements: evidence for positive autoregulation of gene transcription.

The transcriptional transactivational activities of the phosphoprotein cAMP-response element-binding protein (CREB) are activated by the cAMP-dependent protein kinase A signaling pathway. Dimers of CREB bind to the palindromic DNA element 5'-TGACGTCA-3' (or similar motifs) called cAMP-responsive enhancers (CREs) found in the control regions of many genes, and activate transcription in response to phosphorylation of CREB by protein kinase A. Earlier we reported on the cyclical expression of the CREB gene in the Sertoli cells of the rat testis that occurred concomitant with the FSH-induced rise in cellular cAMP levels and suggested that transcription of the CREB gene may be autoregulated by cAMP-dependent transcriptional proteins. We now report the structure of the 5'-flanking sequence of the human CREB gene containing promoter activity. The promoter has a high content of guanosines and cytosines and lacks canonical TATA and CCAAT boxes typically found in the promoters of genes in eukaryotes. Notably, the promoter contains three CREs and transcriptional activities of a promoter-luciferase reporter plasmid transfected to placental JEG-3 cells are increased 3- to 5-fold over basal activities in response to either cAMP or 12-O-tetradecanoyl phorbol-14-acetate, and give 6- to 7-fold responses when both agents are added. The CREs bind recombinant CREB and endogenous CREB or CREB-like proteins contained in placental JEG-3 cells and also confer cAMP-inducible transcriptional activation to a heterologous minimal promoter. Our studies suggest that the expression of the CREB gene is positively autoregulated in trans.

Activation of PPAR delta Regulates Ywhae (Encoding 14-3-3 epsilon Protein), a Gene Required for Ventricular Morphogenesis

Cardiac ventricular morphogenesis is a key developmental stage during which the ventricles grow considerably in size, but the transcriptional pathways controlling this process remains poorly understood. 14-3-3_ is a member of a conserved protein family that regulates a wide range of processes such as transcription, apoptosis and proliferation by binding to the phospho-serine/threonine residues of its target proteins. We found that deletion of the Ywhae gene (encoding 14-3-3_) in mice leads to abnormal ventricular morphogenesis and an embryonic cardiomyopathy (Cieslik KA et al, Circ. Res. 2008, abstract). Interestingly, we recently showed in cultured cells that the Ywhae gene is regulated directly by peroxisome proliferator-activated receptor _ (PPAR_) (Brunelli L et al, Circ. Res. 2007), a ligand-inducible nuclear receptor that controls energy metabolism and development. Postnatal cardiac-specific deletion of the Ppard gene in mice causes a lethal dilated cardiomyopathy, but it is still unknown whether PPAR_ regulates genes involved in heart development. We hypothesized that the expression of the Ywhae gene is responsive to PPAR_ during heart development. We confirmed that PPAR_ is expressed in the heart during development, and found higher expression at E10.5 compared to later gestational ages. We showed by immunofluorescence that a PPAR_ agonist (50 _M L-165,041 for 24 hr) upregulates 14-3-3_ in primary cardiomyocytes. We showed that when P19CL6 cells are driven towards cardiomyocyte lineage by dimethyl sulfoxide (DMSO), 14-3-3_ levels increase 4-fold, while L-165,041 treatment increases levels by an additional 50%. Based on previous work in mice (Leibowitz MD et al, FEBS Lett. 2000; Letavernier E et al, J. Am. Soc. Nephrol. 2005), we tested the response of Ywhae to PPAR_ in vivo . We fed 30 mg/kg/day L-165,041 to 14-3-3__/_ adult pregnant mice for 3 days starting at E9.5 and assessed Ywhae mRNA levels in embryonic hearts at E12.5. Baseline mRNA levels in Ywhae_/_ hearts were double that of Ywhae_/ hearts, while L-165,041 upregulated Ywhae mRNA levels in both Ywhae_/_ and Ywhae_/ hearts by 65%. These results indicate that Ywhae responds to PPAR_ in vivo, and suggest that PPAR_ regulates Ywhae during ventricular morphogenesis.

Calpastatin-mediated inhibition of calpains in the mouse brain prevents mutant ataxin 3 proteolysis, nuclear localization and aggregation, relieving Machado-Joseph disease.

Machado-Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-repetition of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within the ataxin 3 protein, which upon proteolysis may trigger Machado-Joseph disease. We investigated the role of calpains in the generation of toxic ataxin 3 fragments and pathogenesis of Machado-Joseph disease. For this purpose, we inhibited calpain activity in mouse models of Machado-Joseph disease by overexpressing the endogenous calpain-inhibitor calpastatin. Calpain blockage reduced the size and number of mutant ataxin 3 inclusions, neuronal dysfunction and neurodegeneration. By reducing fragmentation of ataxin 3, calpastatin overexpression modified the subcellular localization of mutant ataxin 3 restraining the protein in the cytoplasm, reducing aggregation and nuclear toxicity and overcoming calpastatin depletion observed upon mutant ataxin 3 expression. Our findings are the first in vivo proof that mutant ataxin 3 proteolysis by calpains mediates its translocation to the nucleus, aggregation and toxicity and that inhibition of calpains may provide an effective therapy for Machado-Joseph disease.

The 3' half of the mouse mammary tumor virus orf gene is not sufficient for its superantigen function in transgenic mice.

The Mouse Mammary Tumor Virus (MMTV) long terminal repeat contains an open reading frame (orf) of 960 nucleotides encoding a 36 kDa polypeptide with a putative transmembrane domain and five N-glycosylation sites in the N-terminal part of the protein. Transgenic mice bearing either the complete or the 3' terminal half of the orf sequence of MMTV-GR under the control of the SV40 promoter were raised. As shown previously by FACS analysis transgenic mice which express the complete orf gene have a significant deletion of V beta 14 expressing T cells at 6 weeks of age. Here we show that no clonal deletion of V beta 14 bearing T cells takes place in transgenic mice that contain orf sequences from the fifth ATG to the termination codon. The pattern of tissues expressing the truncated transgene was studied by the Polymerase Chain Reaction (PCR) and was very similar to the one obtained in the V beta 14 deleting animals. These data suggest that the amino-terminal portion of the ORF protein (pORF) is required for a superantigen function, while our previous data indicated that determinants from the carboxy-terminus play an important role for TCR V beta specificity.

Gene transfer of the Ly-3 chain gene of the mouse CD8 molecular complex: co-transfer with the Ly-2 polypeptide gene results in detectable cell surface expression of the Ly-3 antigenic determinants.

The CD8 molecule is a glycoprotein expressed on a subset of mature T lymphocytes. It has been postulated to be a receptor for class I major histocompatibility complex molecules. In the mouse, CD8 is a heterodimer composed of Ly-2 and Ly-3 chains. We have isolated and analyzed cDNA and cosmid clones corresponding to the Ly-3 subunit. One of the isolated, cosmid clones was subsequently transfected, alone or in combination with the Ly-2 gene, into mouse Ltk- cells. Analysis of the Ly-2,3 molecules expressed at the surface of the double transfectants indicated that they are serologically and biochemically indistinguishable from their normal counterparts expressed on lymphoid cells. Ltk- cells transfected with the Ly-2 gene alone were shown to react with a subset of anti-CD8 monoclonal antibodies whereas Ly-3 transfectants did not stain with any of the anti-Ly-3 antibodies employed in this study. Since at least one of these antibodies (53-5.8) has been previously shown to recognize an epitope which is retained on the Ly-3 subunit after dissociation of the heterodimeric Ly-2,3 complex, these observations suggest that the expression of the Ly-2 polypeptide is required to permit the detectable cell surface expression of the antigenic determinants carried by the Ly-3 subunit.

Transcriptional regulation by triiodothyronine of the UDP-glucuronosyltransferase family 1 gene complex in rat liver. Comparison with induction by 3-methylcholanthrene.

This study demonstrates that the expression of the phenol UDP-glucuronosyltransferase 1 gene (UGT1A1) is regulated at the transcriptional level by thyroid hormone in rat liver. Following 3,5, 3'-triiodo-L-thyronine (T3) stimulation in vivo, there is a gradual increase in the amount of UGT1A1 mRNA with maximum levels reached 24 h after treatment. In comparison, induction with the specific inducer, 3-methylcholanthrene (3-MC), results in maximal levels of UGT1A1 mRNA after 8 h of treatment. In primary hepatocyte cultures, the stimulatory effect of both T3 and 3-MC is also observed. This induction is suppressed by the RNA synthesis inhibitor actinomycin D, indicating that neither inducer acts at the level of mRNA stabilization. Indeed, nuclear run-on assays show a 3-fold increase in UGT1A1 transcription after T3 treatment and a 6-fold increase after 3-MC stimulation. This transcriptional induction by T3 is prevented by cycloheximide in primary hepatocyte cultures, while 3-MC stimulation is only partially affected after prolonged treatment with the protein synthesis inhibitor. Together, these data provide evidence for a transcriptional control of UGT1A1 synthesis and indicate that T3 and 3-MC use different activation mechanisms. Stimulation of the UGT1A1 gene by T3 requires de novo protein synthesis, while 3-MC-dependent activation is the result of a direct action of the compound, most likely via the aromatic hydrocarbon receptor complex.

The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of a factor (Doa10) and human TEB4, components of the endoplasmic reticulum-associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.

Variability of the 3' terminal of the polymerase gene of Grapevine leafroll-associated virus 3 isolates from Vale do São Francisco, Brazil

Many viral diseases, including leafroll, which is of great economic importance, affect grapevines (Vitis spp.). A complex of eight viruses [Grapevine leafroll-associated virus (GLRaV) -1 to 8] is associated with this disease. The objective of this study was to compare the variability of the 3' terminal region of the polymerase gene of three isolates of GLRaV-3 (Grapevine leafroll-associated virus-3), from Submédio do Vale do Rio São Francisco (Petrolina-PE) with that of other isolates available at the GenBank, including an isolate from North America and another from Southern Brazil. The viral RNA was extracted from three infected ELISA reactive plants and a fragment of 340 bp was amplified, by RT-PCR, using primers that recognize that portion of the polymerase gene found between nucleotides 8267 and 8606. The three isolates from Vale do Rio São Francisco named Pet-1, Pet-2 and Pet-3, showed similarities ranging from 98% and 94%, respectively to the isolates from North America (AF037268) and Southern Brazilian (AF438411). Considering the whole genome, the main variation found was one amino acid change at position 2766 (F2766Y). These preliminary data indicate the existence of a natural variation among GLRaV-3 isolates from grapevines. This could be due to the vegetative propagation and long cycle of the plant, associated with the error-prone nature of RNA-dependent RNA polymerase.

Variability of the coat protein gene of Grapevine leafroll-associated virus 3 in Brazil

Leafroll is an economically important disease affecting grapevines (Vitis spp.). Nine serologically distinct viruses, Grapevine leafroll-associated virus-1 through 9, are associated with this disease. The present study describes the coat protein gene sequence of four GLRaV-3 isolates occurring in the São Francisco River basin, Northeastern Brazil. The viral RNA was extracted from GLRaV-3 ELISA-positive plants and the complete coat protein gene was amplified by RT-PCR. Sequences were generated automatically and compared to the complete coat protein sequence from North American (NY1) and Chinese (Dawanhong Nº2 and SL10) GLRaV-3 isolates. The four studied isolates, named Pet-1 through 4, showed deduced amino acid identities of 98-100% (Pet-1 through 3) and 95% (Pet-4) with North American and Chinese isolates. A total of seventeen amino acid substitutions was detected among the four characterized isolates in comparison to the NY1, Dawanhong No.2 and SL10 sequences. The results indicated the existence of natural variation among GLRaV-3 isolates from grapevines, also demonstrating a lack of correlation between sequence data and geographic origin. This variability should be considered when selecting regions of the viral genome targeted for reliable and consistent virus molecular detection.

Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies

Grapevine leafroll-associated virus 3 (GLRaV-3), the main viral species of the grapevine leafroll complex, causes yield and quality reduction in grapes (Vitis spp.). The coat protein gene was RT-PCR-amplified from total RNA extracted from infected grapevine leaves and the amplified fragment was cloned and completely sequenced. The fragment was subsequently subcloned into the pRSET-C expression vector. The recombinant plasmid was used to transform Escherichia coli BL21:DE3 and express the capsid protein. The coat protein, fused to a 6 His-tag, was purified by affinity chromatography using an Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE and Western blot. The in vitro-expressed protein was quantified and used for rabbit immunizations. The antiserum was shown to be sensitive and specific for the detection of GLRaV-3 in grapevine extracts in Western blot and DAS-ELISA assays, with no unspecific or heterologous reactions against other non-serologically related viruses being observed.

Gene transfer of Chlorella vulgaris n-3 fatty acid desaturase optimizes the fatty acid composition of human breast cancer cells

Chlorella vulgaris has the gene of n-3 fatty acid desaturase (CvFad3), which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs) or convert n-6 to n-3 PUFAs. The objective of the present study was to examine whether the CvFad3 gene from C. vulgaris can be functionally and efficiently expressed in human breast cancer cells and whether its expression can exert a significant effect on cell fatty acid composition. We inserted the CvFad3 gene into the plasmid pEGFP-C3 to construct the eukaryotic expression vector pEGFP-C3-n-3 and to express the n-3 Fad gene in human breast cancer cells (MCF-7 cells). Transfection of MCF-7 cells with the recombinant vector resulted in a high expression of n-3 fatty acid desaturase. Lipid analysis indicated that the ratio of n-6/n-3 PUFAs was decreased from 6:1 in the control cells to about 1:1 in the cells expressing the n-3 fatty acid desaturase. Accordingly, the CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of the MCF-7 cell membrane. The expression of the CvFad3 gene can decrease cell proliferation and promote cell apoptosis. This study demonstrates that the CvFad3 gene can dramatically balance the ratio of n-6/n-3 PUFAs and may provide an effective approach to the modification of the fatty acid composition of mammalian cells, also providing a basis for potential applications of its transfer in experimental and clinical settings.

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.