Esterase Gene Expression Research Articles

Identification of 5' flanking sequences that affect human pancreatic cholesterol esterase gene expression

Pancreatic cholesterol esterase (CEL) is shown to play a significant role in cholesterol metabolism. As the hydrolytic property of CEL is important for transport of lipid esters, the extent of its expression is an important factor in the metabolism of lipids. Therefore, to identify the elements that modulate the transcription of its mRNA, we obtained several cosmid clones carrying the CEL gene. From one of these cosmid clones a 6.5-kb SmaI fragment that hybridizes to the 5' untranslated region of CEL cDNA was subcloned. Primer extension and S1 protection assays revealed that the 5' untranslated region is relatively short (only 20 nucleotides long). An analysis of the 5' flanking sequence revealed typical TATA and CCAAT boxes that impart tissue specificity. Further, consensus sequences of several cis elements described earlier could also be detected in this region. To identify the promoter sequences, various deletion constructs of the 5' region were made using polymerase chain reaction. These constructs were subcloned into a bacterial plasmid vector carrying chloramphenicol acetyltransferase (CAT) as the reporter gene and transfected into HepG-2 cells. CAT activity in the cell homogenate of the transfected cells was measured 48 h after transfection. Results showed that the promoter activity of human pancreatic CEL mRNA is in a large segment of 5' flanking sequences spanning the -10 and -930 nucleotides of its gene.

Identification of 5' flanking sequences that affect human pancreatic cholesterol esterase gene expression

Identification of 5' flanking sequences that affect human pancreatic cholesterol esterase gene expression

Transcriptional regulation of dog prostate arginine esterase gene by androgens

These studies were designed to define the molecular events involved in the modulation of dog prostate arginine esterase gene expression following short castration intervals and androgen treatment. Arginine esterase enzymatic activity and protein levels decreased about 50% 24 h after castration. Thereafter, a more progressive decrease was observed, resulting in 2-4-fold lower levels in 12-day castrates than in the intact controls. Total prostatic arginine esterase mRNA levels slowly decreased during the first five days after castration but more abruptly thereafter and were about 150-fold lower in 12-day castrated animals. By contrast, in isolated prostatic nuclei, levels of arginine esterase RNA precursors and mature transcripts rapidly fell following orchiectomy, with a 50–70% decrease 24 h after castration. Nuclear run-on experiments confirmed that the latter effects were the result of decreased arginine esterase gene transcription. All these changes could be at least partially reversed by administration of testosterone cypionate. Furthermore, no striking modifications in the proportion of epithelial/stromal cells in the prostatic tissue were observed following orchiectomy. These results show that castration and androgens exert very rapid effects on the gene expression of arginine esterase, and that the regulation occurs at the transcriptional level.

IL-2 and IL-5 gene expression in response to alloantigen in liver allograft recipients and in vitro.

IL-2 and IL-5 gene expression in response to alloantigen was studied in liver allograft recipients and in an in vitro system. Seventy-seven sequential liver allograft biopsies from 22 patients were analyzed for IL-2 and IL-5 mRNA by polymerase chain reaction and Southern blot hybridization. Message for IL-5 was present in 74% of allografts with rejection, 46% of allografts with resolving rejection, and 33% of allografts with no evidence of rejection. The frequency of IL-5 transcripts in rejecting allografts was significantly different than the frequency of IL-5 transcripts in grafts without evidence of rejection (P = 0.003). Message for IL-2 was detected in 29% of rejecting allografts, 18% of allografts without evidence of rejection, and 43% of allografts with resolving rejection. There was no significant association between IL-2 gene expression and the histopathological status of the allograft. Interestingly, 9 of 15 biopsies that contained IL-2 message in the no rejection and resolving rejection categories went on to display rejection shortly thereafter. IL-2 and IL-5 gene expression rarely occurred simultaneously within allografts. An in vitro system consisting of irradiated, allogeneic stimulator cells and normal peripheral blood mononuclear cells as responders was established to further investigate alloantigen-driven IL-2 and IL-5 production. Both IL-2 and IL-5 were produced in response to alloantigen as determined by specific bioassays. Maximal levels of IL-5 activity in culture supernatants generally followed maximal IL-2 levels by 24 hr, but both IL-2 and IL-5 production were dramatically inhibited by CsA. Analysis of cytokine gene expression revealed that IL-2 transcription peaked within the initial 24 hr of culture, whereas IL-5 transcription was maximal at 120 hr of culture. The expression of a CTL-specific serine esterase gene was similar to IL-5 in that it was maximal during the latter phases of the culture period. Thus, both human IL-2 and IL-5 are produced in response to alloantigen and are inhibitable by CsA. These data suggest that IL-2 and IL-5 may participate in cellular pathways of tissue damage within the rejecting allograft.

Antisense inhibition of pectin esterase gene expression in transgenic tomatoes

SummaryTomato plants (Lycopersicon esculentum Mill. cv. Ailsa Craig) were transformed with chimaeric pectin esterase (PE; EC 3.1.1.11) antisense RNA genes, based on a cDNA isolated from a tomato fruit library. PE enzyme activity in ripe fruit of these plants was reduced by up to 93%, as a result of inhibition of PE mRNA accumulation from an endogenous gene, or genes. No major differences in fruit development and ripening were apparent, although pectin remained more heavily esterified (methylated) at all stages of fruit development, thus confirming that PE has a role in pectin de‐esterification in vivo. PE enzyme activity was unaffected in leaves or roots of the transformants, although antisense RNA transcripts were detected. This observation, and immunological differences between PE isoenzymes in leaves and roots compared to fruit, indicate differential expression of PE isoenzymes in these organs.

Induction of perforin and serine esterases in a murine cytotoxic T lymphocyte clone.

The expression of perforin and serine esterase (SE) activities and genes was examined in a murine cytotoxic T lymphocyte line (R8i) that does not require exogenous IL-2 for proliferation. Although perforin (hemolytic) activity was detected in unstimulated R8i, it was induced 2- to 14-fold in the presence of IL-2, IL-3, IL-4, and IL-6, and to a lesser degree (less than 4-fold) by TNF and IFN-gamma. A transient induction was also observed at the mRNA level. Peak perforin protein and mRNA levels were reached within 24 h and started to decline 48 h after stimulation. A trypsinlike SE activity which cleaves the chromogenic substrate N, alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester was also induced 2- to 4-fold in the presence of the various IL tested. At the mRNA level, the message for SE SE1/granzyme A/Hanukah factor was absent from R8i whereas SE2/granzyme B/CTLA-1 increased by greater than 3-fold in the presence of IL-2, IL-3, IL-4, and IL-6 and occurred with the same kinetics and pattern as perforin. The induction response occurred without any enhancement of cell proliferation, suggesting that the cytokines tested may provide a direct differentiation signal to CTL. The induction response was abrogated effectively by inhibitors of protein (cycloheximide or emetine) and RNA (actinomycin D) syntheses. These findings suggest that the various IL may provide both a growth signal and a differentiation signal to CTL, resulting in the direct activation of perforin and SE genes.

Perforin and serine esterase gene expression in stimulated human T cells. Kinetics, mitogen requirements, and effects of cyclosporin A.

A pore-forming protein (PFP; perforin) and various serine esterases (SE) have been identified in the cytoplasmic granules of CTL and NK cells. Perforin and several SE have recently been cloned. Northern blotting analysis was performed here using cDNA probes specific for human perforin and two SE (SE 1/HS and SE 2/GB) to monitor the levels of specific mRNAs in mitogen-stimulated primary human T cells. These mRNAs were rapidly induced by IL-2 with optimal responses at 300 U/ml. After IL-2 treatment, mRNAs for perforin, SE 1, and SE 2 peaked at 12-24 h and decreased after 48 h. The three mRNAs were also induced in T cells treated with a combination of PMA plus lectin, OKT3 mAb, or plastic-adherent accessory cells. However, the induction induced by PMA/mitogen followed a slower kinetics, peaking at 48 h. In general, we found that SE 1 mRNA was more readily induced by IL-2, while SE 2 responded better to PMA/mitogen. Similar patterns of mRNA expression were observed for both unprimed T cells and PHA-primed T blasts. After stimulation with IL-2 and PMA/mitogen, the T8+ subset was shown to be the main producer of perforin, SE 1, and SE 2. Low levels of all three mRNAs, however, were also detected in the T4+ subset. The induction of all three mRNAs by either IL-2 or PMA/mitogen was partially blocked by the immunosuppressive drug cyclosporin A (CsA), but not by the biologically inactive analogue cyclosporin H. Together, these results point to some similarities and differences with upregulation of granule mediator mRNAs relative to lymphokine mRNAs. Both sets of genes require two signals for their induction by mitogens. In contrast to lymphokines, there is a strong response of granule mRNAs to IL-2, and the induction of these transcripts is only partially blocked by CsA.

Analysis of naturally occurring delayed-type hypersensitivity reactions in leprosy by in situ hybridization.

Analysis of tissue lesions of the major reactional states of leprosy was undertaken to study the immune mechanisms underlying regulation of cell-mediated immunity and delayed-type hypersensitivity (DTH) in man. In situ hybridization hybridization of reversal reaction biopsy specimens for INF-gamma mRNA expression revealed a 10-fold increase in specific mRNA-containing cells over that observed in unresponsive lepromatous patients. Expression of huHF serine esterase, a marker for T cytotoxic cells, were fourfold increased in reversal reaction and tuberculoid lesions above that detected in unresponsive lepromatous individuals. Immunohistology of reversal reactions confirmed a selective increase of Th and T cytotoxic cells in the cellular immune response. Of interest, the microanatomic location of these serine esterase mRNA-containing cells was identical to the distribution of CD4+ cells. Analysis of erythema nodosum leprosum (ENL) lesions revealed differences in the underlying immune processes in comparison with reversal reaction lesions. Although phenotypic Th cells predominated in ENL lesions, IFN-gamma and serine esterase gene expression were markedly reduced. We suggest that reversal reactions represent a hyperimmune DTH response characterized by a selective increase of CD4+ IFN-gamma producing cells and T cytotoxic cells, which result in the clearing of bacilli and concomitant tissue damage. In contrast, ENL reactions may be viewed as a transient diminution of Ts cells and activity leading to a partial and transient augmentation in cell-mediated immunity, perhaps sufficient to result in antibody and immune complex formation, but insufficient to clear bacilli from lesions.

The genetics and expression of an esterase locus in Anopheles gambiae.

The main polymorphic system of esterase isoenzymes in adults of the G3 laboratory strain of Anopheles gambiae consists of two to five major bands of activity per individual. The bands are designated 5S, 5F, 13, 14, and 15. In genetic crosses, the genes which coded for the bands assorted as three codominant alleles, Est A, Est B, and Est C, at a single autosomal locus. Homozygotes for the Est C allele were significantly underrepresented among backcross progeny. The developmental pattern of esterase expression was examined. Esterase gene expression in embryos was first detectable between 2 and 12 hr after oviposition. The initiation or termination of expression of some of the bands corresponded to boundaries between developmental stages. Most of the esterase fractions were not specifically localized within the tissues tested, with the exception of a series of bands which were restricted largely to adult male testes.

Identification and sequence determination of a cDNA clone for tomato pectin esterase

Cell wall softening during tomato fruit ripening is brought about through the action of a number of pectolytic enzymes. We have reported previously the cloning and characterisation of the cDNA for the major cell wall softening (degrading) enzyme, polygalacturonase [Grierson, D., Tucker, G. A., Keen, J., Ray, J., Bird, C. R. and Schuch, W. (1986) Nucleic Acids Res. 14, 8595-8603]. We have now isolated a cDNA clone for tomato pectin esterase, an enzyme also implicated in cell wall softening. Here we report the structure of this cDNA and compare it with the structure of pectin esterase derived from amino acid sequence experiments [Markovic, O. and Jornvall, H. (1986) Eur. J. Biochem. 158, 455-462]. We have used the pectin esterase cDNA clone to analyse pectin esterase gene expression during development and ripening of normal and mutant fruit.

Genetics of Somatic Mammalian Cells: Demonstration of a Human Esterase Activator Gene Linked to the AdeB Gene

Prototrophic hybrids formed from an adenine-requiring Chinese hamster cell and human fibroblasts uniformly display new esterase activity that differs from that of either parental cell in electrophoretic mobility and substrate specificity. The hybrids that grew in the selective medium and possessed the new esterase activity had a single extra chromosome that resembled a B-group human chromosome. When clones of such hybrid cells were cultured in nonselective medium, they rapidly reverted to inability to synthesize adenine, disappearance of the new esterase activity, and simultaneous loss of the extra human chromosome. Esterase activity like that of the hybrid is present in cells of various Chinese hamster, but not human, tissues. It is postulated that particular Chinese hamster esterase genes became inactive after longterm cultivation, and that, in the hybrid cell, a human activator gene linked to the adeB gene and located on a human B-group chromosome reactivated expression of these Chinese hamster esterase genes.

Tissue Specific Esterase Expression In Indigenous Chicken Of Bangladesh

The present investigation was done in the esterase gene expression analysis of the Red Jungle fowl Gallus gallus of Bangladesh utilizing the polyacrylamide gel electrophoresis (PAGE) technique. Each allele produces a biochemical message that helps to produce various protein phenotypes those were expressed in the form of bands on the polyacrylamide gel bed. Altogether four different esterase bands were recorded in both Red Jungle (RJ) fowl and Naked Neck (NN) fowl of Bangladesh. These were denoted as Est-1 (2), Est-2(1), Est-3 (21) and Est-4 (13) based on the relative mobility on the gel. Among them Est-2 is very common in almost all tissues. Comparing the esterase in both genetic groups, their expression intensity even the relative mobility provided a unique feature that they were similar. The investigation indicates that esterase gene between the Red Jungle fowl and Naked Neck fowl of Bangladesh has a close relationship. DOI: http://dx.doi.org/10.3329/bjas.v38i1-2.9908 BJAS 2009; 38(1-2): 15-21