Biomedical subjects

Michael J Haas

Publications and source records attributed to Michael J Haas.

28 records · Page 2Linked to original sources

PubMed2003

Suppression of apolipoprotein AI gene expression in HepG2 cells by TNF alpha and IL-1beta.

Plasma inflammatory cytokines are elevated in obese subjects as well as in those with type 2 diabetes. This presumably results in systemic insulin resistance, characterized by a pro-atherogenic plasma lipid profile and reduced apolipoprotein AI (apoAI) protein levels. To determine how cytokine-mediated insulin resistance suppresses apoAI gene expression, we investigated the effect of tumor necrosis factor alpha (TNF alpha) and interleukin-1beta (IL-1beta) on apoAI protein, mRNA, and transcriptional activity in the human hepatoma cell line HepG2. ApoAI secretion was suppressed in a dose-dependent manner in HepG2 cells treated with both cytokines. ApoAI protein levels were 2892+/-22.0, 2263+/-117, 2458+/-25.0, 3401+/-152, 2333+/-248, 1520+/-41.5 and 956.0+/-11.0 arbitrary units (AU) in cells treated with 0, 0.3, 1.0, 3.0, 10, 30, and 100 ng/ml TNF alpha, achieving statistical significance in the 30 and 100 ng/ml range (P<0.0009). ApoAI protein levels were 4055+/-360, 3697+/-101, 3347+/-327, 1561+/-33.0, 1581+/-182, 810.0+/-59.5, and 1766+/-717 AU in cells treated with similar doses of IL-1beta, achieving statistical significance within the range of 3-100 ng/ml (P<0.02). ApoAI mRNA levels were suppressed 50.8% in HepG2 cells treated with 30 ng/ml TNF alpha for 24 h (P<0.05), and remained suppressed for up to 96 h. Similarly, treatment of cells with 30 ng/ml IL-1beta for 24 h, resulted in 42.9% reduction in apoAI mRNA levels (P<0.05) and remained suppressed for up to 96 h. In order to determine if the effect of TNF alpha and IL-1beta occurs at the transcriptional level, HepG2 cells were transfected with a chloramphenicol acetyltransferase (CAT) reporter gene plasmid containing the full-length apoAI promoter, and after 24 h, treated with TNF alpha (30 ng/ml), IL-1beta (30 ng/ml), or both cytokines. CAT activity was suppressed by both cytokines (24.0+/-1.9% acetylation in control cells vs. 5.6+/-1.2% (P<0.0004), 10.2+/-1.5% (P<0.0006), and 3.9+/-0.9% acetylation (P<0.0002) in cells treated with TNF alpha, IL-1beta, and the combination of both cytokines, respectively) suggesting that cytokine-mediated suppression occurs at the transcriptional level. Using a series of apoAI deletion constructs, the cytokine response element was mapped between nucleotides -325 and -186 (relative to the transcriptional start site). This region contains a previously identified and characterized cis-element, site A, which binds several different transcription factors. Finally, electrophoretic mobility shift assays (EMSA) showed that TNF alpha treatment of HepG2 cells is associated with reduced nuclear factor binding to site A. These studies suggest that inflammatory cytokines down-regulate apoAI expression at least partly through inhibition of binding of the nuclear factors to site A of the apoAI promoter.

Apolipoprotein A-I↗

PubMed2003

Effect of thyroid hormone responsive protein (THRP) expression on PC12 cell survival.

The thyroid hormone responsive protein (THRP) is a novel gene product that remains responsive to thyroid hormone in the cerebral cortex of adult rats. The biological effects of THRP are currently unknown. Since thyroid hormones (TH) are known to cause cell death in primary neuronal cultures, the effect of exogenous THRP expression on PC12 cell viability was investigated. Co-transfection of the THRP expression plasmid with the selectable marker pSV2neo resulted in a lower number of surviving PC12 cells compared to transfection with pSV2neo and the empty vector, pSVL. Similar results were observed when PC12 cells were transfected with the plasmid pCMV. SPORT beta-gal with and without pSVL-THRP. However, expression of exogenous THRP in the colonic epithelial cell line Caco-2 and the glial cell line U251 had no effect on cell viability. Coexpression of THRP with either the wild-type (WT)-c-Abl or a kinase-defective mutant c-Abl (K290R) did not alter the cell viability changes induced by THRP alone. Under these experimental conditions the predominant form of cell death was necrosis as evidenced by in situ analyses, such as terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) and staining with membrane permeating and non-permeating nuclear dyes, Hoechst 33342 and propidium iodide respectively. In addition cell cycle arrest induced by THRP was demonstrated by reduced (3)H-thymidine incorporation into cellular DNA. The number of PC12 cells treated with 10(-7) M of l-3, 5, 3'-triiodothyronine (T(3)) was significantly reduced after the fourth day of culture. Treatment of the cells with T(3 )resulted in a dose dependent induction of THRP mRNA. It is concluded that: (1). THRP expression induces PC12 cell death; (2). under these experimental conditions the form of cell death is predominantly necrosis although cell cycle arrest may also occur; (3). the effect of THRP on cell viability is not modulated by c-Abl tyrosine kinase; and (4). the effect of T(3 )treatment on PC12 cell survival may be mediated by THRP.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Age-related changes in rat cerebral occludin and zonula occludens-1 (ZO-1).

The endothelial or epithelial tight junctions create a rate-limiting barrier to diffusion of solutes. A major determinant of the barrier function is the density of tight junction proteins. Since aging is associated with significant alterations in the blood-brain barrier (BBB) it is possible that specific tight junction proteins may be altered in the cerebrum of aging rats. To test this hypothesis, Western and Northern blot analysis were carried out to measure the steady-state level of occludin and zonula occludens-one (ZO-1) proteins and their mRNA in cerebral tissue of 3-, 12- and 24-month-old rats. The cerebral occludin content in 24-month-old rats (732.5+/-99.9 arbitrary units) was significantly reduced compared to 12-month-old rats (1043.4+/-131.8) or 3-month-old rats (1021.4+/-62.8), P<0.01. The cerebral ZO-1 protein content in 24-month-old rats (161.7+/-8.1 arbitrary units) and 12-month-old rats (144.3+/-35.9) were not significantly reduced compared to 4-month-old rats (189.0+/-27.2). The occludin mRNA content relative to G3PDH mRNA was 1.11+/-0.05, 1.11+/-0.07 and 1.00+/-0.05 in 3-, 12- and 24-month-old rats, respectively. The differences did not achieve statistical significance. The ZO-1 mRNA content of cerebral tissue relative to G3PDH mRNA was significantly increased in 24-month-old rats compared to 3-month-old rats (1.280+/-0.030 vs. 0.956+/-0.038), P<0.001. It is concluded that aging in rats may alter the molecular anatomy of the BBB by altering the content of select structural proteins of tight junctions.

Aging↗

PubMed2003

Effect of chromium on apolipoprotein A-I expression in HepG2 cells.

OBJECTIVE: Chromium is a key micronutrient required for lipid and carbohydrate metabolism. Some but not all clinical trials have associated use of chromium supplements with improved insulin sensitivity and lipid profile including increased high-density lipoprotein cholesterol levels. METHODS: Because apolipoprotein A-I (apoA-I) is the principal protein of high-density lipoprotein, the molecular pathways underlying chromium-related changes in apoA-I expression were studied in a human hepatoma cell line (HepG2) transfected with full-length apoA-I promoter attached to the reporter chloramphenicol acetyl transferase gene. RESULTS: Exposure of these cells to different concentrations of chromium chloride (0, 0.5, 1.0, and 3.0 mM) resulted in a dose-dependent decrease in apoA-I promoter activity (chloramphenicol acetyl transferase activity expressed as a percentage of an internal control was 99.4 +/- 7.2% in control cells versus 87.6 +/- 5.0%, 73.4 +/- 2.3%, and 36.6 +/- 3.9%, respectively, P < 0.01). Chromium chloride at 10 mM concentration was toxic and caused death in a large number of cells. Treating HepG2 cells with other minerals known to have insulin-sensitizing effects such as magnesium (1 mM), zinc (0.2 mM), and vanadyl sulfate (0.1 mM) significantly reduced apoA-I promoter activity in the presence and absence of 100 microU/mL of insulin. Northern blot analyses showed that the apoA-I mRNA content of cells treated with 0.2 mM of chromium chloride relative to G3PDH mRNA was not significantly increased compared with controls (0.652 +/- 0.122 versus 0.745 +/- 0.143, the ratio of apoA-I to glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA in control and chromium-treated cells, respectively). Western blot analyses of proteins secreted in culture media indicated that neither chromium treatment of the HepG2 cells (858.0 +/- 151.4 arbitrary units) nor treatment with magnesium (1323.3 +/- 175.7) or vanadium (1102 +/- 78.7) significantly altered apoA-I concentrations compared with controls (1061.7 +/- 114.7). However treatment of HepG2 cells with 0.2 mM of zinc significantly reduced apoA-I concentrations (291.0 +/- 29.2 versus 1061.7 +/- 114.7; P < 0.001). CONCLUSIONS: Supraphysiologic concentrations of chromium and other minerals with known insulin-sensitizing activity may reduce apoA-I promoter activity in cultured cells. Whether similar changes may occur in vivo remains to be shown. However, these observations do not support the use of pharmacologic amounts of chromium supplementation to enhance the cardioprotective lipid profile.

Antioxidants↗

PubMed2002

Partial characterization of a cerebral thyroid hormone-responsive protein.

The thyroid hormone-responsive protein (THRP) is expressed in rat cerebral tissue and has 83% overall sequence homology with c-Abl interactor protein, Abi-2, which is a substrate for the tyrosine kinase activity of c-Abl. Within the core region of the two proteins, the sequence similarity approaches 99%. To determine whether THRP is a rat homologue of Abi-2 or is a distinct protein with unique properties, the tissue distribution of THRP and Abi-2 mRNA's was examined using a sensitive ribonuclease protection assay and probes specific for THRP and Abi-2, respectively. The THRP mRNA content of cerebral tissue (1340.0 +/- 126.5 arbitrary units) was 2.3-fold higher than Abi-2 mRNA (581.3 +/- 73.7), while the ratio of hepatic content of THRP mRNA (209.0 +/- 49.1) to hepatic Abi-2 mRNA (2923.0 +/- 378.7) was only 0.07 (P < 0.004). Very low levels of Abi-2 mRNA, but not THRP mRNA, were also found in the heart and small intestine. Experiments with PC12 cells transfected with the full-length THRP cDNA and grown in the presence or absence of a tyrosine kinase inhibitor, along with experiments where PC12 cells were cotransfected with the THRP cDNA with or without the wild-type or mutant (tyrosine kinase deficient) c-Abl cDNA, showed that THRP is tyrosine phosphorylated; however, it is not a substrate for c-Abl. These studies demonstrate that THRP and Abi-2 have distinct tissue distribution and distinct biological properties.

Adaptor Proteins, Signal Transducing↗

PubMed2002

The isolation and identification of a toxic impurity in XP315 drug substance.

In early safety assessment studies with the experimental anti-neoplastic drug XP315, a toxic reaction was observed in dogs immediately after intravenous (iv) infusion. The reaction was characterized by severe erythema around the ears, eyes, face and body; ocular hyperemia; head shaking; swelling around the eyes, face, paws, head, neck and legs; scratching; and reddened gums, which lasted several hours after dosing. By fractionating the drug substance using preparative HPLC and then infusing the residues into dogs by iv, this reaction was traced to an impurity in the drug substance. Following the preparative isolation of the toxic impurity, characterization was performed using a combination of NMR and mass spectral methods. The proposed impurity was found to be structurally related and nearly twice the molecular weight of XP315, resulting from a dimerization by ring fusion of two 3-aminonaphthalene fragments during the synthetic process. This paper details the steps taken to isolate the toxic impurity and characterize its structure using off-line methods.

Animals↗

PubMed2002

Diabetes-related changes in rat cerebral occludin and zonula occludens-1 (ZO-1) expression.

The endothelial or epithelial tight junctions create a barrier to diffusion of solutes. Since experimental diabetes mellitus is associated with considerable alterations in the blood-brain barrier (BBB), it is possible that specific tight junction proteins may be altered in diabetes. To test this hypothesis, Western and Northern blot analysis were carried out to measure the steady-state level of occludin and zonula occludens-one (ZO-1) proteins and mRNA levels in cerebral tissue of streptozotocin-induced diabetic rats and the results were compared to insulin treated diabetic rats and vehicle injected control rats. The cerebral occludin content in diabetic rats (115.4 +/- 18.6 arbitrary units) was significantly reduced compared to insulin-treated diabetic rats (649.1 +/- 141.2) or control rats (552.9 +/- 82.9), p < 0.001. The ZO-1 content of cerebral tissue from diabetic rats (1,240.6 +/- 199.7 arbitrary units) was not significantly altered compared to controls (1,310.8 +/- 256.9). The cerebral occludin mRNA content relative to G3PDH mRNA was 1.35 +/- 0.07 and 1.34 +/- 0.19 in control and diabetic rats respectively. The cerebral ZO-1 mRNA content relative to G3PDH mRNA in diabetic and control rats was 1.135 +/- 0.123 and 0.956 +/- 0.038 respectively. These differences did not achieve statistical significance. It is concluded that diabetes alters the molecular anatomy of the tight junctions in cerebral tissue by altering the content of select structural proteins.

Animals↗

PubMed2002

Effects of dehydroepiandrosterone on rat apolipoprotein AI gene expression in the human hepatoma cell line, HepG2.

Serum apolipoprotein AI (apoAI) levels correlate with the risk of developing atherosclerosis. Previous studies have suggested that dehydroepiandrosterone (DHEA) lowers high-density lipoprotein (HDL)-cholesterol levels. We investigated whether or not DHEA may lower HDL-cholesterol levels by suppressing apoAI gene transcription in hepatocytes. ApoAI mRNA levels, assessed by Northern blotting, were suppressed in HepG2 cells treated with DHEA (34%) (10 microg/mL) or testosterone (36%) (T, 1 microg/mL). Estradiol alone (E2, 1 microg/mL) had relatively little effect on apoAI mRNA levels, while E2 in combination with DHEA prevented a decrease in apoAI mRNA levels compared to DHEA alone. To determine whether these effects were due to changes in apoAI gene transcription, HepG2 cells were transfected with a plasmid carrying the full-length promoter of the rat apoAI gene ligated into a chloramphenicol acetyltransferase (CAT) reporter construct. The plasmid pCMV.SPORT-beta-gal was included in each transfection to normalize the data to transfection efficiency. Cells were then cultured in the presence or absence of DHEA (10 microg/mL), T (1 microg/mL), 17alpha-methyltestosterone (MTT, 1 microg/mL), 5alpha-dihydrotestosterone (DHT, 1 microg/mL), E2 (1 microg/mL), or a combination of DHEA plus E2, T plus E2, MTT plus E2, and DHT plus E2, for 24 hours. CAT activity, relative to beta-galactosidase activity, was reduced by 19.6%, 57.6%, 38.6%, and 54.6% with DHEA, T, DHT, and MTT addition, respectively. E2 increased CAT activity by 43.8%. When the androgens (ie, DHEA, T, DHT, or MTT) were combined with E2, apoAI promoter activity was suppressed. We conclude, therefore, that androgens downregulate apoAI promoter activity in the presence or absence of E2. However, the changes in mRNA levels do not always reflect changes in promoter activity, suggesting that these steroids may have additional post-transcriptional effects on steady-state apoAI mRNA levels. It remains to be established if the transcriptional effects we observed are mediated through an androgen response element.

Animals↗

PubMed2002

Apolipoprotein A-I expression in rats is not altered by troglitazone.

Insulin is known to upregulate apolipoprotein A-I (apoA-I) promoter activity and to increase apoA1 gene expression in vivo. To determine if enhancement of insulin action with insulin sensitizers can also increase the apoA-I expression, we studied the in vivo effect of troglitazone, a potent insulin sensitizer, on the expression of rat hepatic and intestinal apoA-I mRNA using Northern blot analysis. The plasma, hepatic, and intestinal apoA-I content was also measured with immunoblot analysis using a specific anti-rat apoA-I antiserum. Troglitazone, given mixed with rat chow (0.2%) for 18 days, did not increase either plasma or tissue apoA-I mRNA or protein content. Intestinal apoA-I mRNA content relative to glyceraldehyde-3 phosphate dehydrogenase (G(3)PDH) mRNA was significantly lower compared with hepatic tissue content in both control and troglitazone-treated rats. The effect of troglitazone on the rat apoA-I promoter was examined using transient transfection analysis in HepG2 cells transfected with the apoA-I-chloramphenicol acetyl transferase (CAT) reporter plasmid (pAI.474.CAT). CAT activity (percentage acetylation of chloramphenicol as means +/- SEM) was not significantly different in ethanol (vehicle)-treated cells compared with cells treated with troglitazone (50.5% +/- 2.5% in control cells vs 57.7% +/- 8.2% and 53.5% +/- 4.2% in cells treated with 10 and 100 mM troglitazone, respectively). It is concluded that troglitazone doses known to achieve insulin sensitization did not enhance rat apoA-I promoter activity sufficiently to result in an increased apoA-I mRNA or protein expression in the intact rat. However, peroxisome proliferator activator receptor (PPAR) agonists that have significant PPAR alpha activity in addition to their PPAR gamma effects, may well be able to induce apoA-I expression.

Animals↗

PubMedDate not supplied

Suppression of hyperglycemia-induced superoxide formation and endothelin-1 gene expression by carvedilol.

Carvedilol (CV) is a beta-blocker with favorable effects on cardiovascular disease. To determine whether CV can prevent increases in superoxide (SO) production due to hyperglycemia, human umbilical vein endothelial cells (HUVEC) were treated with either 100 or 500 mg/dL dextrose in the presence or absence of 0.1, 1.0, and 10 micromol/L CV. Superoxide levels were measured using the hydroethidine (HE) fluorescence method. Generation of SO was linear from time 0 to 60 minutes. At 60 minutes, the HE fluorescence in cells treated with 500 mg/dL dextrose (123.3+/-4.9 units) was significantly higher than that in control cells treated with 100 mg/dL dextrose (84.0+/-3.5 units) (P<0.002). Addition of 0.1, 1.0, and 10 micromol/L CV to cells treated with 500 mg/dL dextrose decreased SO generated to 113.3+/-1.8, 98.7+/-8.3, and 70.0+/-1.0 units, respectively (P<0.13, P<0.05, and P<0.004, respectively). Cellular endothelin-1 mRNA and endothelin-1 protein secreted in culture media were significantly increased in the presence of 500 mg/dL dextrose. The addition of 10 micromol/L CV significantly decreased both endothelin-1 (1-21) mRNA and protein levels. Measurements of media lactate dehydrogenase activity indicated that CV inhibited cytotoxicity caused by 500 mg/dL dextrose. These findings suggest that CV not only prevents dextrose-induced SO generation in endothelial cells but may also have favorable effects on gene expression and cell survival.

Antihypertensive Agents↗