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PubMed2001

The alpha-subunit of the epithelial sodium channel is an aldosterone-induced transcript in mammalian collecting ducts, and this transcriptional response is mediated via distinct cis-elements in the 5'-flanking region of the gene.

Aldosterone stimulates Na(+) reabsorption in the collecting ducts by increasing the activity of the epithelial sodium channel, ENaC. Systemic administration of aldosterone increases alpha ENaC mRNA expression in mammalian kidney, suggesting that the alpha ENaC gene is a target for aldosterone action in the distal nephron. To determine whether aldosterone increases alpha ENaC gene transcription, a portion of the alpha ENaC 5'- flanking region coupled to luciferase was transfected into MDCK-C7 cells, a collecting duct cell line with aldosterone-stimulated Na(+) transport. Both dexamethasone and aldosterone stimulated alpha ENaC-coupled reporter gene activity via the glucocorticoid receptor (GR), and this response correlated with the effect of these hormones on endogenous alpha ENaC expression. The aldosterone-stimulated alpha ENaC expression was blocked by actinomycin D, and aldosterone had no effect on alpha ENaC mRNA decay, confirming a transcriptional effect. In HT-29 cells, a GR/mineralocorticoid receptor (MR)-deficient colonic cell line with constitutive alpha ENaC expression, cotransfection with GR or MR restored aldosterone-stimulated alpha ENaC gene transcription, although aldosterone had a functional preference for MR. Analysis of deletion constructs confirmed that a single imperfect glucocorticoid response element (GRE) is necessary and sufficient to confer the aldosterone responsiveness to the alpha ENaC gene promoter in MDCK-C7 and HT-29 cells. These results confirm that alpha ENaC is an aldosterone-induced transcript in the collecting duct and delineates the molecular mechanism for this effect.

Aldosterone↗
PubMed1999

Transcription from the P2 promoter of the growth hormone receptor gene involves members of the Sp transcription factor family.

The P2 promoter of the gene for growth hormone receptor is developmentally regulated and is differentially active in a number of tissues. Little is known about the identity of the transcription factors that participate to effect this pattern of transcription. Deletion analysis and transient transfection were used to localize a previously identified cis-acting element within the sheep P2 promoter to between positions -99 and -87. Gel mobility-shift assays with nuclear extracts from Chinese hamster ovary (CHO-K1) fibroblasts revealed that this sequence encompasses an atypical binding site for both Sp1 and two isoforms of Sp3. A gel mobility-shift scan of promoter sequences between -88 and +21 indicated the existence of three other binding sites for Sp1 and Sp3. One of these, designated site II and found by using a probe spanning -74 to -54, corresponds to a classical GC box consensus sequence. Site III (-63 to -41) and site IV (-27 to -5) harbour atypical Sp1/Sp3-binding sequences. Site-directed mutagenesis of site II or site IV decreased promoter activity by approx. 40%, whereas a promoter construct incorporating both mutations exhibited negligible (approx. 1%) activity. Co-transfection of expression plasmids encoding either Sp1 or Sp3 significantly transactivated reporter gene activity from a P2 promoter construct carrying all four Sp1/Sp3-binding sites (8-fold compared with 7.1-fold induction respectively). Sp1 is known to interact with a variety of other transcription factors to regulate the transcription of a number of differentially expressed genes. The identification of four binding sites for Sp1 and Sp3 within the P2 promoter of the gene for growth hormone receptor might point to other factors that interact to regulate the activity of this promoter in different tissues during foetal and post-natal development.

Animals↗
PubMed1992

Human gastric cathepsin E gene. Multiple transcripts result from alternative polyadenylation of the primary transcripts of a single gene locus at 1q31-q32.

Genomic clones containing portions of the human cathepsin E (CTSE) gene were isolated from cosmid and lambda recombinant libraries. The regions corresponding to coding, the 5'- and 3'-untranslated, and the exon-intron boundaries of the CTSE gene were identified by sequence and hybridization analysis. The size and placement of the nine exons found in the 17.5-kilobase CTSE gene was highly conserved relative to other aspartic proteinases and provided additional evidence that these proteinases are derived from a common ancestral gene. Segregation and linkage analysis of two informative restriction fragment length polymorphisms (MspI and DraI) indicated that there is a single human CTSE locus located at chromosome 1q31-q32 which is closely linked to the renin gene. Three CTSE transcripts (3.6, 2.6, and 2.1 kilobases) were identified in gastric fundic and antral mucosa poly (A+) RNA, and these appeared identical in size and relative abundance to those contained in poly(A+) RNA from cultured gastric adenocarcinoma cell lines containing CTSE. Sequence analysis of cDNA clones and comparison with the 3'-flanking untranslated region in genomic clones provided evidence that alternative polyadenylation of the primary transcript resulted in the 2.6- and 2.1-kilobase transcripts which constituted greater than 95% of CTSE transcripts found in the stomach.

Amino Acid Sequence↗
PubMed2003

[Correlation between transcriptional activity of vascular endothelial growth factor (VEFG) and transcriptional activity of its receptors (FLT-1 and FLK-1) in low-grade squamous intraepithelial lesions (LSIL) of uterine cervix].

OBJECTIVES: In this report we focus on angiogenesis, as one component of a complex molecular relations in the process of neovascularisation in the low-grade intraepithelial changes (LSIL). Increasing number of publications indicates that the interrelation between isoforms of VEGF (VEGF121, VEGF145, VEGF165, VEGF183, VEGF189, VEGF206) but not total VEGF is responsible for angiogenesis, both in physiological and pathological processes. The molecular co-operation of the said isoforms and their receptors results in morphological presentation of "de novo" created vascular network that dynamically involves the entire connective tissue stroma of the uterine cervix. METHOD: The selection of intraepithelial pathology 38 cases to assess molecular activity of the given genes was based on colposcopy examination. Tissue specimens were taken for pathological investigation and to analyse transcriptional activity and mRNA alternative splicing of the angiogenesis genes. VEGF, Flt-1, Flk-1. To analyse quantitative gene expression RT-PCR TaqMan was performed. To estimate the dependence transcriptional activity of Flt-1 and Flk-1 on VEGF gene expression Spearman's correlation rank was performed. Differences with p < 0.05 were considered significant. RESULTS: The comparison of transcriptional activity of VEGF and its receptors revealed significant correlation between increase in the number of Flt-1 mRNA copies and enhanced mRNA expression of VEGF121 (p < 0.05), VEGF145 (p < 0.05), VEGF165 (p < 0.05), VEGF183 (p < 0.05), VEGF189 (p < 0.05), and VEGF206 (p < 0.05). Significant increase in the number of Flk-1 mRNA copies was observed in case of enhanced mRNA expression of VEGF121 (p < 0.05), VEGF145 (p < 0.05), VEGF183 (p < 0.05), VEGF189 (p < 0.05) and VEGF206 (p < 0.05). The number of sFlt-1 mRNA copies significantly correlated only with enhanced VEGF145 mRNA expression (p < 0.05). CONCLUSION: Changing intensification of transcriptional activity of VEGF gene and its receptors indicates on autocrine mechanism regulation of angiogenic genes activity in the first steep of carcinogenesis--LSIL.

Carcinoma, Squamous Cell↗
PubMed2005

[Clinical significance of detection of AML1/ETO fusion transcripts in childhood AML using real-time quantitative reverse transcription polymerase chain reaction].

This study was aimed to investigate the clinical value of quantification of AML1/ETO fusion transcripts using real-time reverse transcription PCR. Fourteen AML1/ETO positive children out of 52 AML children were selected. A serial dilution of AML1/ETO plasmid was used as a template for the AML1/ETO real-time PCR. AML1/ETO was quantified according to the expression of the GAPDH housekeeping gene at new diagnosis and during/after chemotherapy and transplantation. SPSS statistics was used to analyze the data. The results showed that the ratio of AML1/ETO: GAPDH expression level at new diagnosis varied in the range 0.219-2.080 (median 0.648) among the patients, without relevance with percentage of blasts. The detection sensitivity was up to the dilution of 1:10(5). Six patients showed a slight decline of AML1/ETO (higher than 5 x 10(-2)) at 1 month, three of whom relapsed in the early stage and one later. Five patients had a higher level than 5 x 10(-3) at 3 months, three of whom relapsed. Four patients with always a higher level than 5 x 10(-3) all relapsed in early stage. After six months, four out of them with constant low-level expression (10(-4) - 10(-6)) were in continuous complete hematological remission (CCR). In another patient, a rapid rise of AML1/ETO transcripts could be detected at CR stage and he relapsed 5 months later. The AML1/ETO gene expression leveling off by 10(-5) - 10(-6) could be detected in 3 patients at their complete remission after 9 months. It is concluded that real-time RT-PCR is a suitable approach for quantifying AML1/ETO transcripts in monitoring of AML patients with t(8;21) during/after chemotherapy and provides data of diagnostic relevance.

Acute Disease↗
PubMedDate not supplied

[RNA polymerase III transcription apparatus: structure and transcription regulation].

RNA polymerase III is a complex multi-subunit enzyme directing transcription of small stable non-translated RNA genes: tRNAs, 5S rRNA, Alu-RNA, U6 snRNA genes and some others (class III genes). Because of its complexity the enzyme is the worst studied among three forms of eukariotic RNA polymerases, but it draws more attention of the researchers in recent years. The reason is that new data appeared about an essential role of RNA polymerase III RNA products in such important cell processes as growth, proliferation and differentiation. It was shown that the RNA product levels are changed depending on cell growth rate and cell cycle stage, during cancer transformation, virus infection and heat shock, and either depend on physiological state of the cell (slow and active proliferation and apoptosis). In this review we consider the structure and function of RNA polymerase III, its general transcription factors and holoenzyme, the structure of different class III gene promoters, the preinitiation complex assembly and the transcription cycle. The second part of the review is devoted to the regulation of class III gene transcription in dependence on cell cycle stage, growth factor influence and cell growth rate, during cell transformation and apoptosis.

Cell Cycle↗
PubMed1991

Alternative processing of androgen-binding protein RNA transcripts in fetal rat liver. Identification of a transcript formed by trans splicing.

Androgens and their nuclear receptor regulate genes necessary for development of the male phenotype, a process that is thought to be modulated by extracellular androgen carrier proteins. Two of these carrier proteins, testicular androgen-binding protein (ABP) and plasma sex hormone-binding globulin (SHBG), are encoded by the same gene, but differ in glycosylation and possibly amino acid sequence. To investigate ABP-SHBG gene expression in fetal rat liver, we analyzed RNA transcripts and expressed protein. These studies demonstrated a transient expression of ABP in hepatocytes during the time of testosterone-dependent differentiation of the Wolffian duct. Analysis of cDNA clones derived from fetal rat liver cDNA libraries identified two cDNAs encoded by the ABP-SHBG gene that represented alternatively spliced RNAs. One cDNA had an alternate exon 1, suggesting the function of another promoter in fetal liver. This cDNA also lacked testicular exon 6 DNA, an alteration that implicates the encoded protein in regulatory functions. The other cDNA represented a fused transcript of the ABP-SHBG gene (exons 1-5) and the histidine decarboxylase (HDC) gene, encoding a Mr 98,000 precursor protein. The two domains were joined at splice junctions of the ABP-SHBG and HDC genes, which were localized to rat chromosomes 10 and 3, respectively. Our results indicate that the joining of the two domains was by a trans (donor and acceptor)-splicing mechanism. Data from Northern hybridization experiments suggest the fusion transcript is present in fetal liver RNA. Polymerase chain reaction experiments with fetal liver cDNA further support the existence of an ABP-HDC fusion transcript, as well as the other alternate mRNA. Moreover, a Mr 93,000 immunoreactive protein was transiently expressed in fetal liver during the time of ABP and HDC gene expression. Expression of the fusion cDNA in COS cells yielded HDC activity and the predicted size protein (Mr = 93,000) on Western immunoblots.

Amino Acid Sequence↗
PubMed1987

Insulin negatively regulates albumin mRNA at the transcriptional and post-transcriptional level in rat hepatoma cells.

Treatment of cultured H4-II-E rat hepatoma cells with insulin causes a large decrease in cytoplasmic serum albumin mRNA. This effect is observed at low doses of insulin (ED50 = 2 pM), consistent with the effect being mediated by interaction of insulin with high affinity insulin receptors. The reduction in cytoplasmic albumin mRNA is first observed 8-12 h following insulin addition, and albumin mRNA continues to decrease up to 28 h following hormone addition. Northern blot analysis of purified poly(A)+ RNA has indicated that insulin causes a decrease in albumin mRNA relative to total cytoplasmic poly(A)+ RNA. In addition, one other specific mRNA, that encoded by the alpha-tubulin gene, is not decreased following insulin treatment. These results indicate that insulin induces a specific decrease in albumin mRNA. This effect is largely reversed if essential amino acids are added along with the insulin, suggesting that the insulin effect is related to limitation of the cells for essential amino acids. Insulin reduces transcription of the albumin gene 4.7-fold, as measured by nuclear transcription assays. However, this inhibition of albumin gene transcription does not fully account for the 57-fold decrease in albumin mRNA, indicating that insulin also exerts a negative effect on albumin mRNA at a post-transcriptional step.

Animals↗
PubMed1988

Sequences in the 5' proximal segment of the paused transcript affect NusA-mediated enhancement of transcriptional pausing.

NusA protein is a transcription elongation and termination factor that acts to enhance pausing of RNA chain growth by RNA polymerase at specific sites on DNA templates. We demonstrate that this enhancement of pausing in tR1, the transcription termination site between genes cro and cII of phage lambda, is inhibited by DNA oligonucleotides complementary to a segment of the nascent RNA just preceding the sequence that is thought to be a part of the stem of an RNA hairpin that is responsible for pausing, but not by oligonucleotides complementary to segments containing the boxA sequence or the sequences up to 40 nucleotides upstream from boxA. The results are consistent with a model proposed by Landick and Yanofsky (Landick, R., and Yanofsky, C. (1987) J. Mol. Biol. 196, 363-377) in which the NusA protein acting as a subunit of RNA polymerase in the elongation complex contacts a structure near the 3' end of the nascent transcript and uses the binding energy to shift the equilibrium between elongation and paused conformational isomers of the transcription complex in favor of the paused isomer.

Bacterial Proteins↗
PubMed1983

Transcription analyses with heteroduplex trp attenuator templates indicate that the transcript stem and loop structure serves as the termination signal.

DNA sequences that control transcriptional termination by prokaryotic RNA polymerases normally contain an inverted repeat, or self-complementary sequence, about 10 base pairs upstream from the site of RNA chain termination. Point mutations that interrupt this self-complementarity can reduce or eliminate RNA chain termination. We have constructed heteroduplex DNA templates using wild type and mutant attenuators for the Escherichia coli trp operon to probe the relative contributions of the two DNA strands in the termination process. Transcription analyses show that only the sequences in the transcribed DNA strand determine whether or not a heteroduplex terminator can function. This result strongly supports a model in which the formation of a stem and loop structure in the nascent RNA transcript is the signal for transcriptional termination.

DNA, Bacterial↗
PubMed1983

Hormonal regulation of transcription of rDNA. Inhibition of transcription during glucocorticoid-mediated inhibition of proliferation of lymphosarcoma P1798 cells in culture.

A lymphosarcoma cell line designated P1798.S6M has been characterized with respect to glucocorticoid responsiveness in culture. Cells ceased to proliferate in the presence of 10(-7) M dexamethasone. Cell viability remained high and glucocorticoid-sensitive cells could be rescued from cultures treated with dexamethasone for 24 or 48 h. These data indicate that P1798.S6M undergoes reversible arrest in the presence of dexamethasone. The system was used to study the effects of mitotic arrest upon transcription of rDNA. Incorporation of [methyl-3H]methionine into rRNA was rapidly inhibited and pulse-chase experiments indicated that 28 S RNA was not synthesized after 24 h of exposure to dexamethasone. Hybridization studies indicated that the amount of pre-rRNA was reduced by 90 to 95% in cells treated for 24 h. Transcription studies were carried out in isolated nuclei. Twenty-four hours after addition of dexamethasone, template-bound RNA polymerase I activity decreased by 89 to 96%. Total RNA polymerase I activity did not decrease, whereas disengaged nuclear polymerase I activity increased dramatically. Filter hybridization studies indicated that labeling of nascent pre-rRNA chains in vitro was inhibited 93%. These data are interpreted as follows: Dexamethasone reversibly inhibits proliferation of P1798.S6M cells and transcription of rDNA. Total RNA polymerase I activity does not decrease, but the amount of template-bound enzyme is reduced with a concomitant increase in the amount of disengaged polymerase I. This indicates that initiation of transcription is inhibited in cells undergoing mitotic arrest in the presence of dexamethasone.

Animals↗
PubMed1980

Regulation of gene transcription by estrogen and progesterone. Lack of hormonal effects on transcription by Escherichia coli RNA polymerase.

Estrogen and progesterone markedly stimulate transcription of ovalbumin and conalbumin (transferrin) genes in chick oviduct as measured by hybridization of labeled RNA synthesized in isolated nuclei to immobilized plasmid DNA containing these gene sequences. Using this direct assay for specific gene transcription, we explored the basis of previous reports indicating that steroid hormones also cause changes in oviduct chromatin structure that can be detected by Escherichia coli RNA polymerase. We observed no effect of these hormones on the ability of E. coli RNA polymerase to transcribe specifically the conalbumin and ovalbumin genes 8 1/2 h after hormone administration when transcription of these genes by endogenous RNA polymerase was elevated 5- and 30-fold, respectively. Furthermore, we were unable to detect any significant effect of either of these hormones on the total number of E. coli RNA polymerase binding sites in oviduct nuclei or chromatin. In contrast, after several days of hormone administration, we detected an apparent preferential ovalbumin RNA synthesis by E. coli RNA polymerase and this effect could be transferred to unstimulated nuclei by a 0.35 M salt extract of active nuclei. However, further experiments revealed that this preferential ovalbumin RNA synthesis is an artifact produced by transcription from contaminating ovalbumin mRNA. We conclude that E. coli RNA polymerase does not recognize steroid hormone-induced changes in oviduct chromatin.

Amanitins↗
PubMed1995

Angiotensin II down-regulates the vascular smooth muscle AT1 receptor by transcriptional and post-transcriptional mechanisms: evidence for homologous and heterologous regulation.

The vascular angiotensin II (ANG II) receptor (AT1) is a central component of the renin-angiotensin system; thus, regulation of its expression is likely to be important in cardiovascular responsiveness. We demonstrate that ANG II down-regulates its receptor in rat aortic vascular smooth muscle cells. Incubation for 4 hr with 100 nM ANG II decreased AT1 mRNA and protein by 70% and 35%, respectively. This homologous down-regulation was concentration and time dependent and was blocked by the AT1 antagonist losartan. It did not appear to be mediated by protein kinase C or other protein kinases but was dependent on the sustained signaling pathway sensitive to phenylarsine oxide. Heterologous down-regulation was observed with the agonists alpha-thrombin and ATP and the cAMP-increasing agent forskolin. ANG II inhibited transcription by 50% and destabilized the AT1 mRNA. Down-regulation of AT1 mRNA was blocked by transcription and translation inhibitors, suggesting that it required expression of a protein factor or factors. These results indicate that ANG II down-regulates its vascular receptor by both transcriptional and post-transcriptional mechanisms. Homologous and heterologous down-regulation of the AT1 receptor may participate in the coordinated physiological adaptation of vascular tone to vasoactive hormones.

Adenosine Triphosphate↗
PubMed1995

CD28 costimulation up-regulates long-term IL-2R beta expression in human T cells through combined transcriptional and post-transcriptional regulation.

Costimulatory molecules such as CD28 are required for induction of T cell clonal expansion and for prevention of T cell unresponsiveness. In combination with either CD3 or CD2 triggering, CD28 was shown to enhance T cell proliferation, cytolytic activity, production of cytokines and especially of IL-2, and expression of the IL-2R alpha-chain (IL-2R alpha). We and others have demonstrated that the costimulatory effect of CD28 on both IL-2 and IL-2R alpha expression results from a coordinated transcriptional activation of their genes and transcript stabilization. We show here that the CD28 stimulation, together with CD2, leads to a prolonged up-regulation of the constitutive expression of the IL-2R beta-chain in human peripheral T cells. As for IL-2R alpha, the increase in IL-2R beta gene expression seems to result from both transcriptional activation and transcript stabilization. In addition, IL-2 differentially regulates its own receptors, as only expression of IL-2R alpha, but not of IL-2R beta, is largely inhibited, at both the mRNA and protein levels, by blocking IL-2R mAbs. We propose that the long lasting T cell proliferation mediated by the CD2 and CD28 costimulation is mainly the consequence of the high and prolonged expression of both the IL-2R alpha- and beta-chains.

Blotting, Northern↗
PubMed1993

Angiotensin II regulates parathyroid hormone-related protein expression in cultured rat aortic smooth muscle cells through transcriptional and post-transcriptional mechanisms.

Parathyroid hormone-related protein (PTHrP), a tumor product responsible for malignancy-associated hypercalcemia, is also produced in many normal tissues, including vascular smooth muscle cells (SMC). As PTHrP exhibits vasodilatory properties, we postulated that other vasoactive agents may control PTHrP gene expression in SMC. Addition of angiotensin II to serum-deprived SMC resulted in a marked induction of PTHrP mRNA by 2 h, with a peak (6-10-fold) at 4-6 h. Angiotensin II effects on PTHrP gene expression were inhibited by saralasin, an angiotensin II receptor antagonist, and blocked by actinomycin D and cycloheximide, suggesting a requirement for gene transcription and protein synthesis. Nuclear run-off assays revealed a 3-fold increase in PTHrP gene transcription 1 h after angiotensin II treatment. Angiotensin II also prolonged PTHrP mRNA half-life by 2-3-fold. Angiotensin-induced PTHrP mRNA is partially dependent on cyclooxygenase products and protein kinase C activation. Other vasoconstrictor substances, including serotonin and bradykinin, also stimulated PTHrP expression, whereas the vasodilator atrial natriuretic peptide did not. Addition of recombinant PTHrP-(1-141) significantly inhibited angiotensin II-induced SMC DNA synthesis. PTHrP expression is increased by angiotensin II through transcriptional and post-transcriptional mechanisms. In addition, PTHrP modulates the effect of angiotensin II on SMC proliferation. This suggests that PTHrP acts locally in SMC, possibly to oppose the vasoactive and/or growth-promoting effects of vasoconstrictor agents such as angiotensin II.

Angiotensin II↗
PubMed1993

Transcriptional activation of the macrophage colony-stimulating factor gene by IL-2 is associated with secretion of bioactive macrophage colony-stimulating factor protein by monocytes and involves activation of the transcription factor NF-kappa B.

Human peripheral blood monocytes (Mo) constitutively display the beta-chain of the receptor for IL-2, whereas expression of the IL-2R alpha-chain is not constitutive but inducible with IL-2. Here we report that binding of human IL-2 to its binding site leads to transcriptional activation of the macrophage CSF (M-CSF) gene in Mo resulting in accumulation of M-CSF mRNA and subsequent release of bioactive M-CSF protein as demonstrated by ELISA and inhibition of IL-2 induced release of an activity-stimulating growth of monocyte-type colonies by a neutralizing anti-M-CSF antibody. Transcriptional activation of the M-CSF gene by IL-2 is preceded by enhanced binding activity of the transcription factor NF-kappa B to its recognition sequence in the 5' regulatory enhancer region of the M-CSF gene. Moreover, using a heterologous promoter (herpes thymidine kinase) construct containing the NF-kappa B consensus sequence, it is shown that NF-kappa B binding by an IL-2-induced monocyte-derived nuclear protein confers reporter gene (human growth hormone) activity. Taken together, our findings indicate that IL-2 induces gene expression of M-CSF in human blood-derived Mo and provide evidence for involvement of NF-kappa B in transcriptional regulation of this gene.

Animals↗
PubMed1996

Cholesterol homeostasis is modulated by amphiphiles at transcriptional and post-transcriptional loci.

A variety of amphiphiles inhibit plasma membrane cholesterol esterification and induce 3-hydroxy-3-methylglutaryl-coenzyme A reductase accumulation in cultured cells; among these are steroids, hydrophobic amines, phenothiazines, ionophores, colchicine, and lysophosphatides. It has been proposed that these amphiphiles signal a sterol deficiency to regulatory sites by blocking the movement of plasma membrane cholesterol into the cell (Lange, Y., and Steck, T. L. 1994. J. Biol. Chem. 269: 29371-29374). If this were the case, these agents also should enhance transcription of sterol responsive genes and stabilize 3-hydroxy-3-methylglutaryl-coenzyme A reductase. As a test of this hypothesis, the effect of the amphiphiles on such transcriptional and post-transcriptional events was assessed. A mouse embryo cell line was transfected with a construct containing the promoter for the human low density lipoprotein receptor upstream of the DNA sequence coding for chloramphenicol acyltransferase (CAT). Incubation of these cells for 7-18 h with the aforementioned agents caused the level of expression of the promoter/CAT construct to increase 2- to 9-fold. We showed further that the amphiphiles stimulated 3-hydroxy-3 methylglutaryl-coenzyme A reductase activity by increasing gene transcription as well as by decreasing degradation of the enzyme. These are the predicted homeostatic responses to cell cholesterol deficiency. These findings support the hypothesis that certain amphiphiles falsely signal a cholesterol deficiency to the intracellular sites regulating cholesterol homeostasis.

3T3 Cells↗
PubMed1998

Significance of reverse transcription polymerase chain reaction in the detection of human cytomegalovirus gene transcripts in thoracic organ transplant recipients.

BACKGROUND: Cytomegalovirus disease is a major cause of morbidity in transplant recipients. We have evaluated the clinical value of detecting viral mRNA transcripts for the diagnosis of active infection leading to disease in recipients of thoracic organ transplants. METHODS: Blood samples from 10 transplant recipients were analyzed before transplantation and weekly after transplantation for 12 weeks. The profile of viral immediate-early, early, and late gene expression was determined by the reverse transcription polymerase chain reaction and compared with cytomegalovirus (pp65) antigenemia and host antibody status (serologic study). RESULTS: Two patients showed no active cytomegalovirus infection, one had asymptomatic infection detected serologically and seven patients had development of symptomatic infection with a significant serologic change. Viral immediate-early mRNA transcript was detectable in all 10 patients, including the two with no active infection. Early and late gene expression occurred in seven patients who were all antigenemia positive and in whom disease developed. Of the seven patients with development of antigenemia, six showed viral early and late gene expression before pp65 antigenemia, whereas one patient showed antigenemia before early and late gene expression. CONCLUSION: We have shown that the detection of viral early and late gene expression by reverse transcription polymerase chain reaction can act as diagnostic markers of cytomegalovirus disease with expression of early gene preceding the detection of antigenemia in most cases. In contrast, viral immediate early gene expression did not correlate with clinical infection. This diagnostic approach could be useful in the treatment of thoracic organ transplant recipients.

Antibodies, Viral↗