Deficient transcription of mouse mast cell protease 4 gene in mutant mice of mi/mi genotype.
(33/16452)
The mi locus encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (hereafter called MITF). We reported that expression of the mouse mast cell protease 5 (MMCP-5) and MMCP-6 genes were deficient in cultured mast cells (CMC) derived from mutant mice of mi/mi genotype. Despite the reduced expression of both MMCP-5 and MMCP-6, their regulation mechanisms were different. Because MMCP-5 is a chymase and MMCP-6 a tryptase, there was a possibility that the difference in regulation mechanisms was associated with their different characteristics as proteases. We compared the regulation mechanisms of another chymase, MMCP-4, with those of MMCP-5 and MMCP-6. The expression of the MMCP-4 gene was also deficient in mi/mi CMC. The overexpression of the normal (+) MITF but not of mi-MITF normalized the poor expression of the MMCP-4 gene in mi/mi CMC, indicating the involvement of +-MITF in transactivation of the MMCP-4 gene. Although MMCP-4 is chymase as MMCP-5, the regulation of MMCP-4 expression was more similar to MMCP-6 than to MMCP-5. We also showed the deficient expression of granzyme B and cathepsin G genes in mi/mi CMC. Genes encoding granzyme B, cathepsin G, MMCP-4, and MMCP-5 are located on chromosome 14. Because all these genes showed deficient expression in mi/mi CMC, there is a possibility that MITF might regulate the expression of these genes through a locus control region. (+info)
Regulation of the human apolipoprotein AIV gene expression in transgenic mice.
(34/16452)
The apolipoprotein (Apo) AI-CIII-AIV gene cluster has a complex pattern of gene expression that is modulated by both gene- and cluster-specific cis-acting elements. In particular the regulation of Apo AIV expression has been previously studied in vivo and in vitro including several transgenic mouse lines but a complete, consistent picture of the tissue-specific controls is still missing. We have analysed the role of the Apo AIV 3' flanking sequences in the regulation of gene expression using both in vitro and in vivo systems including three lines of transgenic mice. The transgene consisted of a human fragment containing 7 kb of the 5' flanking region, the Apo AIV gene itself and 6 kb of the 3' flanking region (-7+6 Apo AIV). Accurate analysis of the Apo AIV mRNA levels using quantitative PCR and Northern blots showed that the 7+6 kb Apo AIV fragment confers liver-specific regulation in that the human Apo AIV transgene is expressed at approximately the same level as the endogenous mouse Apo AIV gene. In contrast, the intestinal regulation of the transgene did not follow, the pattern observed with the endogenous gene although it produced a much higher intestinal expression following the accepted human pattern. Therefore, this animal model provides an excellent substrate to design therapeutic protocols for those metabolic derangements that may benefit from variations in Apo AIV levels and its anti-atherogenic effect. (+info)
NADH-glutamate synthase in alfalfa root nodules. Genetic regulation and cellular expression.
(35/16452)
NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary nitrogen assimilation in alfalfa (Medicago sativa L.) root nodules. Here we report that in alfalfa, a single gene, probably with multiple alleles, encodes for NADH-GOGAT. In situ hybridizations were performed to assess the location of NADH-GOGAT transcript in alfalfa root nodules. In wild-type cv Saranac nodules the NADH-GOGAT gene is predominantly expressed in infected cells. Nodules devoid of bacteroids (empty) induced by Sinorhizobium meliloti 7154 had no NADH-GOGAT transcript detectable by in situ hybridization, suggesting that the presence of the bacteroid may be important for NADH-GOGAT expression. The pattern of expression of NADH-GOGAT shifted during root nodule development. Until d 9 after planting, all infected cells appeared to express NADH-GOGAT. By d 19, a gradient of expression from high in the early symbiotic zone to low in the late symbiotic zone was observed. In 33-d-old nodules expression was seen in only a few cell layers in the early symbiotic zone. This pattern of expression was also observed for the nifH transcript but not for leghemoglobin. The promoter of NADH-GOGAT was evaluated in transgenic alfalfa plants carrying chimeric beta-glucuronidase promoter fusions. The results suggest that there are at least four regulatory elements. The region responsible for expression in the infected cell zone contains an 88-bp direct repeat. (+info)
A single limit dextrinase gene is expressed both in the developing endosperm and in germinated grains of barley.
(36/16452)
The single gene encoding limit dextrinase (pullulan 6-glucanohydrolase; EC 3.2.1.41) in barley (Hordeum vulgare) has 26 introns that range in size from 93 to 822 base pairs. The mature polypeptide encoded by the gene has 884 amino acid residues and a calculated molecular mass of 97,417 D. Limit dextrinase mRNA is abundant in gibberellic acid-treated aleurone layers and in germinated grain. Gibberellic acid response elements were found in the promoter region of the gene. These observations suggest that the enzyme participates in starch hydrolysis during endosperm mobilization in germinated grain. The mRNA encoding the enzyme is present at lower levels in the developing endosperm of immature grain, a location consistent with a role for limit dextrinase in starch synthesis. Enzyme activity was also detected in developing grain. The limit dextrinase has a presequence typical of transit peptides that target nascent polypeptides to amyloplasts, but this would not be expected to direct secretion of the mature enzyme from aleurone cells in germinated grain. It remains to be discovered how the enzyme is released from the aleurone and whether another enzyme, possibly of the isoamylase group, might be equally important for starch hydrolysis in germinated grain. (+info)
Antisense expression of the CK2 alpha-subunit gene in Arabidopsis. Effects on light-regulated gene expression and plant growth.
(37/16452)
The protein kinase CK2 (formerly casein kinase II) is thought to be involved in light-regulated gene expression in plants because of its ability to phosphorylate transcription factors that bind to the promoter regions of light-regulated genes in vitro. To address this possibility in vivo and to learn more about the potential physiological roles of CK2 in plants, we transformed Arabidopsis with an antisense construct of the CK2 alpha-subunit gene and investigated both morphological and molecular phenotypes. Antisense transformants had a smaller adult leaf size and showed increased expression of chs in darkness and of cab and rbcS after red-light treatment. The latter molecular phenotype implied that CK2 might serve as one of several negative and quantitative effectors in light-regulated gene expression. The possible mechanism of CK2 action and its involvement in the phytochrome signal transduction pathway are discussed. (+info)
Adventitial delivery minimizes the proinflammatory effects of adenoviral vectors.
(38/16452)
PURPOSE: Adenovirus-mediated arterial gene transfer is a promising tool in the study of vascular biology and the development of vascular gene therapy. However, intraluminal delivery of adenoviral vectors causes vascular inflammation and neointimal formation. Whether these complications could be avoided and gene transfer efficiency maintained by means of delivering adenoviral vectors via the adventitia was studied. METHODS: Replication-defective adenoviral vectors encoding a beta-galactosidase (beta-gal) gene (AdRSVnLacZ) or without a recombinant gene (AdNull) were infused into the lumen or the adventitia of rabbit carotid arteries. Two days after infusion of either AdRSVnLacZ (n = 8 adventitial, n = 8 luminal) or AdNull (n = 4 luminal), recombinant gene expression was quantitated by histochemistry (performed on tissue sections) and with a beta-gal activity assay (performed on vessel extracts). Inflammation caused by adenovirus infusion was assessed 14 days after infusion of either AdNull (n = 6) or vehicle (n = 6) into the carotid adventitia. Inflammation was assessed by means of examination of histologic sections for the presence of neointimal formation and infiltrating T cells and for the expression of markers of vascular cell activation (ICAM-1 and VCAM-1). To measure the systemic immune response to adventitial infusion of adenovirus, plasma samples (n = 3) were drawn 14 days after infusion of AdNull and assayed for neutralizing antibodies. RESULTS: Two days after luminal infusion of AdRSVnLacZ, approximately 30% of luminal endothelial cells expressed beta-gal. Similarly, 2 days after infusion of AdRSVnLacZ to the adventitia, approximately 30% of adventitial cells expressed beta-gal. beta-gal expression was present in the carotid adventitia, the internal jugular vein adventitia, and the vagus nerve perineurium. Elevated beta-gal activity (50- to 80-fold more than background; P <.05) was detected in extracts made from all AdRSVnLacZ-transduced arteries. The amount of recombinant protein expression per vessel did not differ significantly between vessels transduced via the adventitia (17.1 mU/mg total protein [range, 8.1 to 71.5]) and those transduced via a luminal approach (10.0 mU/mg total protein [range, 3.9 to 42.6]). Notably, adventitial delivery of AdNull did not cause neointimal formation. In addition, vascular inflammation in arteries transduced via the adventitia (ie, T-cell infiltrates and ICAM-1 expression) was confined to the adventitia, sparing both the intima and media. Antiadenoviral neutralizing antibodies were present in all rabbits after adventitial delivery of AdNull. CONCLUSION: Infusion of adenoviral vectors into the carotid artery adventitia achieves recombinant gene expression at a level equivalent to that achieved by means of intraluminal vector infusion. Because adventitial gene transfer can be performed by means of direct application during open surgical procedures, this technically simple procedure may be more clinically applicable than intraluminal delivery. Moreover, despite the generation of a systemic immune response, adventitial infusion had no detectable pathologic effects on the vascular intima or media. For these reasons, adventitial gene delivery may be a particularly useful experimental and clinical tool. (+info)
DNA topoisomerase IIalpha and -beta expression in human ovarian cancer.
(39/16452)
To study DNA topoisomerase IIalpha (Topo-IIalpha) and -beta expression and regulation in human ovarian cancer, 15 ovarian tumour samples were investigated. To compare different levels of expression, the samples were screened for topo IIalpha and -beta mRNA with Northern blotting and a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay for Topo-IIalpha mRNA. Additionally, protein levels were determined with Western blotting and topoisomerase II activity levels with the decatenation assay. The results obtained were compared with each other and with the tumour volume index of the samples. In tumours with a tumour volume index > or = 50%, the mRNA levels (as determined by Northern blotting) and protein levels for each isozyme were in accordance. Additionally, correlations were found between Topo-IIalpha RT-PCR data and Topo-IIalpha Northern blot results, and between Topo-IIalpha RT-PCR data and Topo-IIalpha protein levels. Interestingly, Topo-IIbeta protein levels correlated better with Topo-II activity than Topo-IIalpha protein levels. In eight ovarian cystadenoma samples, no Topo-IIalpha protein could be found. In only three out of eight of these cystadenomas, Topo-IIbeta protein could be detected. These findings suggest that Topo-IIalpha and Topo-IIbeta protein levels are up-regulated in ovarian cancer and may indicate that Topo-IIbeta is an interesting target for chemotherapy in ovarian tumours. (+info)
Increased expression of the RIalpha subunit of the cAMP-dependent protein kinase A is associated with advanced stage ovarian cancer.
(40/16452)
The primary element in the cAMP signal transduction pathway is the cAMP-dependent protein kinase (PKA). Expression of the RIalpha subunit of type I PKA is elevated in a variety of human tumours and cancer cell lines. The purpose of this study was to assess the prognostic importance of RIalpha expression in patients with ovarian cancer. We have evaluated the expression of RIalpha in a panel of human ovarian tumours (n = 40) and five human ovarian cancer cell lines using quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The human ovarian cell lines OAW42 and OTN14 express high endogenous levels of RIalpha mRNA and protein (at significantly higher mRNA levels than high tissue expressors, P < 0.05). The ovarian cell line A2780 expresses low endogenous levels of RIalpha mRNA and protein (also at higher mRNA levels than low tissue expressors, P < 0.05). Quantitative RT-PCR revealed no significant difference in RIalpha mRNA expression between different ovarian histological subtypes in this study. No associations were found between RIalpha mRNA expression and differentiation state. RIalpha mRNA expression was significantly associated with tumour stage (P = 0.0036), and this remained significant in univariate analysis (P = 0.0002). A trend emerged between RIalpha mRNA expression levels and overall survival in univariate analysis (P = 0.051), however, by multivariate analysis, stage remained the major determinant of overall survival (P = 0.0001). This study indicates that in ovarian epithelial tumours high RIalpha mRNA expression is associated with advanced stage disease. RIalpha expression may be of predictive value in ovarian cancer and may be associated with dysfunctional signalling pathways in this cancer type. (+info)