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N C Wong

Publications and source records attributed to N C Wong.

At least 37 records · Page 2Linked to original sources
PubMed1999

Transcription factors and age-related decline in apolipoprotein A-I expression.

Apolipoprotein (apo)A-I alone or as a component of high density lipoprotein particles has antiatherogenic properties. The age-dependent decline in abundance of this protein may underlie the higher risk for developing occlusive coronary artery disease (CAD) in older individuals. Similar to humans, expression of rat apoA-I also declines with age. Results in rats showed that levels of serum apoA-I protein, hepatic mRNA, and transcription of the gene were decreased to 39%, 18%, and 38%, respectively, in 180-day-old animals compared to those of newborn rats. These findings suggest that a nuclear mechanism(s) may account for the decline in apoA-I expression. Accordingly, we examined hepatic nuclear binding activity to four specific cis-acting elements of the rat apoA-I promoter. There were age-dependent changes of binding activity to two proximal sites, B and C, but not to the more distal elements, IRCE and A. Decreased B-site binding activity correlated with lower mRNA levels encoding the activator, HNF-3beta. The age-dependent change in the pattern of binding to site C was due to a switch from the activator, HNF-4, to the repressor, ARP-1. In summary, the age-related decline in apoA-I expression may arise from a reduction in the activity of both cis-acting elements, B and C.

Aging↗
PubMed1998

Effects of glucose and insulin on rat apolipoprotein A-I gene expression.

We have examined the regulation of apolipoprotein A-I (apoA-I) gene expression in response to glucose and insulin. In Hep G2 cells, endogenous apoA-I mRNA was suppressed by one-half or induced 2-fold following 48 h of exposure to high concentrations of glucose (22.4 mM) or insulin (100 microunits/ml), respectively, compared with control. Transcriptional activity of the rat apoA-I promoter (-474 to -7) in Hep G2 cells paralleled endogenous mRNA expression, and this activity was dependent on the dose of glucose or insulin. Deletional analysis showed that a 50-base pair fragment spanning -425 to -376 of the promoter mediated the effects of both insulin and glucose. Within this DNA fragment there is a motif (-411 to -404) that is homologous to a previously identified insulin response core element (IRCE). Mutation of this motif abolished not only the induction of the promoter by insulin but also abrogated its suppression by glucose. Electrophoretic mobility shift assay analysis of nuclear extracts from Hep G2 cells revealed IRCE binding activity that formed a duplex with radiolabeled probe. The IRCE binding activity correlated with insulin induction of apoA-I expression. In summary, our data show that glucose decreases and insulin increases apoA-I promoter activity. This effect appears to be mediated by a single cis-acting element.

Animals↗
PubMed1998

Alphav integrins mediate adhesion and migration of breast carcinoma cell lines.

Integrins with the alphav subunit are involved in cell adhesion and cellular signaling. Some alphav integrins have been associated with tumor progression and dissemination. The objective of this study was to assess the contribution of alphav integrins to the adhesive and migratory behavior of cells derived from breast carcinoma (BCA). The expression and function of alphav integrins was characterized in three BCA cell lines which exhibit different metastatic potentials. These include MCF-7 cells which metastasize inefficiently, MDA-MB-231 cells, which have a moderate metastatic potential, and MDA-MB-435 cells, which metastasize extensively. Each cell type displays a different repertoire of alphav integrins on the cell surface. The complement of alphav integrins on each cell type influences their ability to adhere and migrate. The most striking difference among these cell lines was the expression of the alphavbeta3 integrin. The highly metastatic MDA-MB-435 cells express substantial levels of this receptor, whereas MDA-MB-231 and MCF-7 cells do not. The MDA-MB-435 cells showed a greater ability to adhere and to migrate and this functional difference can largely be attributed to the expression of alphavbeta3 integrin. This characterization is a first step toward determining the role of alphav integrins in animal models of BCA metastasis, and lends support to the hypothesis that the alphavbeta3 integrin can be a contributing factor in metastatic disease.

Antigens, CD↗
PubMed1997

Hepatocyte nuclear factor 4 inhibits the activity of site A from the rat apolipoprotein AI gene.

The pivotal role of apolipoprotein AI (Apo AI) in mediating reverse cholesterol transport has lead us to the study of transcription factors that influence the expression of this gene. Previous studies show that rat HNF-4 enhances the activity of a cis-acting site C in the rat Apo AI promoter. Since sites C and A share 80% homology, we have examined whether HNF-4 binds to and modulates the transcriptional activity of the A-motif. Results show that HNF-4 binds to site A. The transcriptional activity of site A in a human hepatoma cell line, HuH-7, increases 2-2.5-fold in the presence of antisense HNF-4, but the sense construct has no effect on the activity of the reporter template. The lack of an effect of HNF-4 on site A activity may be due to high endogenous levels of the factor in HuH-7 cells. However, in BHK cells HNF-4 clearly inhibits the transcriptional activity of site A. Together these findings suggest that in contrast to the enhancing effects of HNF-4 on site C, the same factor inhibits site A activity. Since hepatocytes normally contain the T3 receptor and this nuclear factor increases site A action, cotransfection of T3 receptor along with antisense HNF-4 further augments the activity of p5'A.CAT. In summary, rat HNF-4 binds to site A from rat Apo AI DNA, and this factor suppresses site A activity. HNF-4 interferes with the enhancer role of the T3 receptor and thus contributes negatively to the net expression of the Apo AI gene.

Animals↗
PubMed1997

Apolipoprotein A1 expression in young and aged rats is modulated by dietary carbohydrates.

To determine if dietary carbohydrates modulate apolipoprotein A1 (ApoA1) expression, plasma ApoA1 protein and hepatic ApoA1 mRNA levels were measured in young and aged rats maintained on a high-fructose (60% of diet weight consisting of fructose), or high-glucose (60% glucose) diet or fed regular rat chow for 10 days. Aged rats on regular chow had significantly higher plasma ApoA1 concentrations and hepatic ApoA1 mRNA than young rats maintained on this diet. Plasma ApoA1 and hepatic ApoA1 mRNA levels in young rats or aged rats maintained on the 60% fructose diet were significantly higher than in rats within the same age group maintained on regular rat chow (P < .01). Similar induction of ApoA1 protein and mRNA was found in rats maintained on the 60% glucose diet (P < .01). It is concluded that ApoA1 expression in rats is modulated by factors related to the nature of dietary carbohydrates rather than insulin resistance associated with high-fructose feeding.

Aging↗
PubMed1997

Beneficial effects of a novel thyromimetic on lipoprotein metabolism.

Although L-triiodothyronine (L-T3) lowers cholesterol, this hormone is not used to treat hypercholesterolemia because of its cardiotoxic effects. Thyromimetics, such as the novel compound CGS 23425, that mimic the beneficial but lack the detrimental effects of T3, may be useful in the treatment of hypercholesterolemia. To show that CGS 23425 has no cardiotoxicity, atrial contractility and force were both measured and found to be unchanged in rats treated with up to 10 mg/kg drug. The lipid lowering actions of this drug resulted in a 44% decrease in low-density lipoprotein (LDL) cholesterol in hypercholesterolemic rats treated with 10 microg/kg of the compound. Normal rats required a higher dose of 1000 microg/kg to elicit a similar 50% reduction in LDL cholesterol. Both CGS 23425 or T3 (10 nM) increased the specific binding of 125I-labeled LDL to Hep G2 cells and increased LDL receptor number by 44 and 49%, respectively. These data indicate that CGS 23425 enhances hepatic clearance of serum LDL cholesterol. Normal and fat-fed animals treated with the drug showed a dose-dependent increase in apolipoprotein AI, a protein that promotes the efflux of cholesterol from peripheral tissues. Transient transfection of a rat apolipoprotein AI promoter-chloramphenicol acetyltransferase construct, in human hepatoma cells, showed a dose-dependent increase in chloramphenicol acetyltransferase activity with EC50 values of 2 x 10(-12) M and 10(-10) M for thyroid hormone receptors beta1 and alpha1, respectively, with maximal responses at 10(-7) M. These data indicate that CGS 23425 is a thyromimetic that increases apolipoprotein AI expression via thyroid hormone receptor. In summary, CGS 23425 ameliorates hypercholesterolemia by increasing apolipoprotein A1 and the clearance of LDL cholesterol. Therefore, a compound like CGS 23425 may be useful for the prevention and reversal of atherosclerosis.

Animals↗
PubMed1996

Identification of functional positive and negative thyroid hormone-responsive elements in the rat apolipoprotein AI promoter.

Transcription of the antiatherogenic protein apolipoprotein AI is regulated by the thyroid hormone, L-triiodothyronine. Transient transfection and electrophoretic mobility shift assays were used to identify the cis-acting elements involved. In transient transfection assays, hormone bound to either thyroid hormone receptor alpha or beta exerts a positive effect through a thyroid hormone response element, site A (-208 to -193). In the absence of site A, liganded receptor alpha or beta have a negative effect on promoter activity. This negative effect is mediated by a 40 bp fragment spanning nucleotides -46 to -7. Closer examination of this region of the gene shows there to be a negative thyroid hormone response element at position -25 to -20 which is fused to the 3' end of the TATA element. Electrophoretic mobility shift assays show that bacterially expressed chicken or rat thyroid hormone receptor alpha 1 binds to site A, either as a homodimer or as a heterodimer with the human 9-cis-retinoic acid receptor alpha. In contrast, the negative thyroid hormone responsive element binds chicken thyroid hormone receptor alpha exclusively as a monomer. Site-directed mutagenesis of the negative thyroid hormone response element abolished the inhibitory effects of the hormone and increased basal promoter activity by up to 40-fold. These data suggest that functional positive and negative thyroid hormone response elements coexist within the rat apolipoprotein AI promoter and both elements contribute to the control of apolipoprotein AI gene expression.

Animals↗
PubMed1996

Effects of diabetes mellitus on hepatocyte nuclear factor 1 decrease albumin gene transcription.

We have previously reported that albumin gene transcription is reduced in diabetes mellitus (DM). The present study explored the mechanism by which albumin gene transcription is down-regulated in DM. Deletional studies and displacement of factors binding to site B of the albumin promoter indicated that the repressive effects of DM are mediated by nuclear factors binding to this site. Since hepatocyte nuclear factor 1 (HNF1) activates albumin promoter activity and is the predominant factor binding to site B, we examined HNF1. The abundance and binding activity of HNF1 were reduced in hepatonuclear extracts from diabetic compared to control rats. However, HNF1 mRNA levels were unchanged, suggesting that the effect of DM on HNF1 is at the post-transcriptional level. Extracts from diabetic animals also contained another protein, distinct from HNF1 and vHNF1, which bound to site B in gel retardation studies. In summary, our studies demonstrate that the reduced abundance and binding activity of HNF1 correlates with decreased albumin gene transcription in DM.

Animals↗
PubMed1996

Identification of A2-restricted hepatitis C virus-specific cytotoxic T lymphocyte epitopes from conserved regions of the viral genome.

We have focused on conserved regions of the hepatitis C Virus (HCV) genome to identify viral peptides that contain HLA class I binding motifs and bind with high affinity to the corresponding purified HLA molecules. Accordingly, we have identified 31 candidate epitopes in the HCV that have the potential to be recognized by either HLA-A1, A2.1-, A3, A11- or A24-restricted cytotoxic T lymphocytes (CTL). Twelve conserved peptides that bind HLA-A2.1 with high or intermediate affinity were tested for immunogenicity in vitro in human primary CTL cultures and in vivo by direct immunization of HLA-A2.1/Kb transgenic mice. Six HLA-A2.1-restricted CTL epitopes were immunogenic in both systems. At least three of these peptide epitopes were endogenously processed and presented for CTL recognition. Overall, these data illustrate the value of this approach for the development of virus-specific, peptide-based vaccines.

Amino Acid Sequence↗
PubMed1996

Age-related changes in the responsiveness of apolipoprotein A1 to thyroid hormone.

To determine the age-related changes in the effect of thyroid hormone on apolipoprotein A1 (ApoA1) gene expression, male Fischer 344 rats at 4 (young) and 26 (aged) mo of age were studied. Hyperthyroidism was induced with daily intraperitoneal injections of 3,5,3'-triiodothyronine (15 micrograms/100 g body wt) for 10 days. Hypothyroidism was induced with 0.025% methimazole in drinking water for 4 wk. Hyperthyroidism was associated with increased serum ApoA1 levels in young rats [7.52 +/- 0.41 vs. 3.67 +/- 0.30 optical density (OD); P < 0.01]. The increase in aged rats (6.5 +/- 0.87 vs. 5.14 +/- 0.09 OD) did not reach statistical significance. Hypothyroidism was not associated with significant changes in serum ApoA1 levels in either young or aged rats. Hyperthyroidism was associated with a 2.5-fold increase in ApoA1 mRNA in young rats and a 1.7-fold increase in aged rats. Hypothyroidism was associated with a 3.6-fold reduction in ApoA1 mRNA in young rats, but there was no significant change in aged hypothyroid rats. Mobility shift assays indicated that the binding of transacting factors to ApoA1 promoter increased in hyperthyroid young rats but not in hyperthyroid aged rats. It is concluded that aging in rats is associated with reduced ApoA1 responsiveness to thyroid hormones. This altered responsiveness could partly be the result of changes in the binding activity of nuclear factors to the ApoA1 promoter.

Aging↗
PubMed1996

Phlorizin or vanadate treatment reverses impaired expression of albumin and hepatocyte nuclear factor 1 in diabetic rats.

Diabetes decreases transcription of the albumin gene. The role of hyperglycemia in mediating this suppression of albumin gene activity is unclear. To study the effect of glucose in vivo, we treated diabetic rats with phlorizin or vanadate, two agents that ameliorate hyperglycemia without increasing the levels of circulating insulin. When glucose was normalized in diabetic rats with either agent, the hepatic levels of albumin mRNA became indistinguishable from those in nondiabetic animals. In light of our previous observation that diabetes decreases the abundance of hepatocyte nuclear factor 1 (HNF1), the predominant factor increasing albumin gene transcription, we wondered whether glucose normalization in diabetes would alter HNF1. Both the levels and DNA binding activity of HNF1 were restored to control values when phlorizin or vanadate was administered to diabetic rats. These findings suggest that hyperglycemia is integrally involved in mediating the suppression of albumin gene expression in diabetes. The effect of hyperglycemia on HNF1 suggests that glucose affects albumin expression at the level of transcription.

Animals↗
PubMed1996

Regulation of protamine gene expression in an in vitro homologous system.

An in vitro transcription system from the trout testis nuclei was developed to study trout protamine gene expression. The protamine promoter contains, among others, two regulatory elements: 1) a cAMP-responsive element or CRE element (TGACGTCA) which is present in position 5' to TATA box, and 2) GC box (CCGCCC) which is present in position 3' to TATA box. The removal of the CRE-binding protein by titration (by the addition of appropriate oligonucleotides to the incubation mixture) resulted in a decrease in transcription of the protamine gene. These results were confirmed by experiments in which the pure CRE-binding factor (TPBP1) was used, as well as by those where a stimulatory effect of cAMP on protamine promoter transcription was observed. On the other hand, addition of oligonucleotides containing the GC-box sequence enhanced the protamine gene transcription indicating that the protein (Sp1 like) which binds to this sequence acts as a repressor of protamine gene expression. These results confirm the previously proposed model which suggested that the GC box played a role in negative regulation of the protamine gene expression. Involvement of some other factors in this process was also discussed.

Animals↗
PubMed1996

Glucocorticoid increases rat apolipoprotein A-I promoter activity.

The observation that glucocorticoids increase the abundance of apolipoprotein A-I led us to a search for potential underlying mechanism(s). In this report, we show that the synthetic glucocorticoid, dexamethasone, injected into rats increases serum levels of apoA-I protein, hepatic mRNA and "run-on' transcription of the gene by 3-, 5-, and 2-fold, respectively. Results of transient transfection studies of the rat apoA-I promoter reveal that effects of dexamethasone are mediated by a cis-acting site B (-170 to -145). Dexamethasone treatment of hepatoma cells enhances the DNA binding activity of nuclear factors that bind this site. Unexpectedly, site B does not contain a consensus glucocorticoid receptor recognition motif nor binds to bacterially expressed glucocorticoid receptor. These results indicate that the actions of glucocorticoids on site B involve indirect mechanisms. Site B is comprised of a direct repeat of a nonanucleotide and mutation of either one abolishes the effect of glucocorticoid. Additionally, the transcriptional activity of site B in response to dexamethasone is amplified by a 5' sequence called site S (-186 to -171). Dexamethasone has no effect on site S in the absence of site B. In summary, our data show that dexamethasone increases rat apoA-I gene expression by an indirect mechanism.

Animals↗
PubMed1995

Age-related changes in apolipoprotein A-I expression.

To determine the age-related changes in apolipoprotein A-I (ApoA1) expression, male Fischer 344 rats at 4 (young), 12 (intermediate age), and 24-26 (aged) months of age were studied. Immunoblot analysis of plasma proteins indicated that 26-month-old rats (1.79 +/- 0.16 mg/ml) and 12-month-old rats (2.23 +/- 0.11 mg/ml) have significantly higher plasma ApoA1 concentrations compared to 4-month-old rats (1.14 +/- 0.15 mg/ml) P < 0.001. Hepatic ApoA1 mRNA was approx. 2-fold higher in aged rats compared to 12-month-old and 4-month-old rats. This increase in hepatic ApoA1 mRNA in aged rats was also reflected in the increased translation of ApoA1 mRNA in vitro. Reduced mRNA turnover may account for the increased hepatic ApoA1 mRNA content in 26-month-old rats, since the rate of ApoA1 gene transcription as measured with nuclear run off assays was significantly reduced with age. The ApoA1 synthesis in vivo, as measured by [14C]leucine incorporation at 30 min, was reduced in aged rats compared to young rats (170.5 +/- 10.2 vs. 253.9 +/- 7.7 cpm per liver) P < 0.001 probably as a result of changes related to cellular metabolism rather than an alteration inherent to the ApoA1 mRNA translatability. The age-related increase in plasma ApoA1 protein is probably secondary to reduced metabolic clearance rate of ApoA1 protein or is the result of increased intestinal synthesis of ApoA1.

Aging↗
PubMed1994

Bedside assessment of skin-fold thickness. A useful measurement for distinguishing Cushing's disease from other causes of hirsutism and oligomenorrhea.

BACKGROUND: The known catabolic effects of glucocorticoid excess on protein metabolism prompted us to devise a method to assess this measure in reproductive-aged females with Cushing's disease. Since collagen protein is a major component of skin, decreased abundance of this protein should cause a reduction in skin-fold thickness. To determine whether skin-fold thickness is useful as an added tool in the diagnosis of Cushing's disease, we compared this value in female patients with Cushing's disease with those who presented with a similar set of symptoms. METHODS: This open prospective study was conducted in an endocrinology clinic at a tertiary care center. The study population consisted of 88 females in the reproductive age group who presented to the clinic with hirsutism, oligomenorrhea, and/or obesity. Measurement of skin-fold thickness, body mass index, Ferriman-Gallwey index, and serum testosterone were performed in all patients. RESULTS: Skin-fold thickness in the patients with Cushing's disease was 1.5 +/- 0.2 mm (range, 1.0 to 1.8 mm). This value was significantly (P < .01) lower than that in controls or subjects with other disorders that have a similar set of presenting symptoms. CONCLUSIONS: Bedside assessment of skin-fold thickness is an easy, low-cost, and noninvasive test for distinguishing Cushing's disease from disorders with similar presenting symptoms in females of reproductive age. Assessment of skin-fold thickness should be used as an adjunct to current physical and biochemical study of patients with symptoms suggestive of Cushing's disease.

Adolescent↗
PubMed1994

Characteristics of a negative thyroid hormone response element.

Thyroid hormones may stimulate or repress transcriptional activity depending upon the specific gene. Whereas, a palindromic DNA sequence, TREpal, mediates positive regulation by thyroid hormone, the negative response element (nT3RE) remains undefined. Therefore, we have examined the DNA sequences that mediate the inhibitory effects of thyroid hormone on the transcription of the beta-subunit gene of rat thyrotropin (rTSH beta). In rat pituitary tumor cells (GH3), transient expression of plasmid constructs containing the putative nT3RE of rTSH beta mediated negative regulation by L-triiodothyronine (T3). Since this nT3RE contained sequences which resembled a half-site motif of the consensus T3RE and the idealized palindrome (TREpal), we tested a construct containing this half-site motif in the same cells. T3 decreased the activity of this plasmid. Cotransfection studies in T3-receptor (T3R)-deficient cells indicated that either alpha or beta isoforms of T3R were required for the inhibitory effects of the hormone. Both T3R isomers bind to DNA sequences containing the nT3RE from rTSH beta DNA or the half-site motif of TREpal. In summary, our results show that the repressive properties of T3 are mediated by a nT3RE from rTSH beta. Unexpectedly, this motif resembles a half-site component of TREpal which enhances promoter activity in response to T3.

Animals↗