Oestradiol decreases rat apolipoprotein AI transcription via promoter site B.

Oestrogens protect against ischaemic heart disease in the post-menopausal female by increasing serum concentrations of apolipoprotein (apo) AI and the abundance of high-density lipoprotein particles. In men and experimental male animals, the administration of oestrogen has variable effects on apo AI expression. As the major mode of oestrogen action on target genes involves regulating promoter activity and hence transcription, oestrogen is expected to alter transcription of the apo AI gene. To test this hypothesis, the effect of 17beta-oestradiol (E(2)), on rat apo AI promoter activity in male hepatoma HuH-7 cells, was tested by co-transfecting a reporter template, pAI.474.CAT containing-474 to-7 of the rat apo AI promoter and an oestrogen receptor (ER) expression vector, pCMV-ER. Transfected cells exposed to E(2) showed a dose-dependent decrease in chloramphenicol acetyltransferase (CAT)-activity, with a maximum 91+/-1.5% reduction at 1 microM E(2). Deletional analysis of the promoter localized the inhibitory effect of ER and E(2) to site B (-170 to-144) with an adjacent 5' contiguous motif, site S (-186 to-171) acting as an amplifier. HuH-7 cell nuclear extracts showed binding activities with both sites S and B, but recombinant human ER did not. Furthermore, nuclear extracts from E(2)-treated HuH-7 cells showed weaker binding activity to site B, but not to site S. In summary, the inhibitory effect of ER and E(2) on rat apo AI gene activity is mediated by a promoter element, site B. This inhibitory effect arises from a mechanism that does not involve direct ER binding to the B-element. The conclusion that E(2) inhibits apo AI transcription was confirmed in vivo. Treatment of male adult Sprague-Dawley rats with up to 200 microg E(2) for 7 days decreased apo AI protein and hepatic mRNA by 72+/-21% and 68+/-1.4% respectively. Results of 'run-on' transcription of the apo AI gene in isolated hepatic nuclei showed a 55% decrease in hormone-treated male rats. These findings suggest that E(2) exerts primarily an inhibitory effect within male hepatic nuclei.

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.

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.

CD4+/CD8+ T-lymphocyte ratio: effects of rehydration before exercise in dehydrated men.

Effects of fluid ingestion on CD4+/CD8+ T-lymphocyte cell ratios were measured in four dehydrated men (ages 30-46 yr) before and after 70 min of supine submaximal (71% VO2max) lower extremity cycle exercise. Just before exercise, Evans blue dye was injected for measurement of plasma volume. The subjects then drank one of six fluid formulations (12 ml.kg-1) in 3-4 min. All six mean posthydration (pre-exercise) CD4+/CD8+ ratios (Becton-Dickinson Fluorescence Activated Cell Sorter and FACScan Consort-30 software program [San Jose, CA]) were below the normal range of 1.2-1.5; mean (+/- SE) and range were 0.77 +/- 0.12 and 0.39-1.15, respectively. The post-exercise ratios increased: mean = 1.36 +/- 0.15 (P < 0.05) and range = 0.98-1.98. Regression of mean CD4+/CD8+ ratios on mean plasma osmolality resulted in pre- and post-exercise correlation coefficients of -0.76 (P < 0.10) and -0.92 (P < 0.01), respectively. The decreased pre-exercise ratios (after drinking) were probably not caused by the Evans blue dye but appeared to be associated more with the stress (osmotic) of dehydration. The increased post-exercise ratios to normal levels accompanied the rehydration and were not due to the varied electrolyte and osmotic concentrations of the ingested fluids or to the varied vascular volume shifts during exercise. Thus, the level of subject hydration and plasma osmolality may be factors involved in the mechanism of immune system modulation induced by exercise.

Short-term strenuous exercise training: effects on blood pressure and hormonal levels in mild hypertension.

The effect of a six-week strenuous exercise training programme (modified Bruce protocol, treadmill, three times per week) on resting and exercising blood pressure, heart rate, plasma catecholamines, chromogranin A, renin activity and aldosterone levels was investigated in 15 patients with mild hypertension. An identical exercise test was conducted at baseline and study close (six weeks). At follow-up, seven to ten days after study close, patients completed an exercise test of equivalent intensity to that at baseline, achieving comparable heart rate levels at maximal exercise. On each occasion, blood pressure, heart rate and hormonal variables were measured at rest (supine), maximal exercise and ten minutes after stopping exercise. Resting and exercising blood pressure and heart rate were reduced by the six-week exercise regimen. There was a trend, although not statistically significant, for resting plasma noradrenaline levels to be lower at study close. The reduction in blood pressure and heart rate at maximal exercise was associated with a significant attenuation of the plasma renin response to exercise. Plasma catecholamines also appeared to be lower after exercise training, although this effect was not statistically significant. Plasma levels of chromogranin A and aldosterone measured at rest and maximal exercise were not influenced by the exercise regimen. Further controlled studies are required to corroborate the results of this preliminary study.

Rat hepatonuclear factor PS-1 regulates tissue-specific activity of the S14 promoter in vitro.

We have previously identified a rat hepatonuclear factor, PS-1 that binds to the thyroid hormone responsive gene, S14. To determine whether PS-1 is involved in regulating tissue-specific expression of the S14 gene, we have correlated the DNA binding activity of PS-1 with mRNA-S14 expression in a variety of tissues. Gel retardation analysis revealed a pattern of binding to the recognition site that was characteristic of tissues with high levels of mRNA-S14, a different pattern was found in tissues which do not express the gene. Competition studies using mutant oligonucleotides showed that the first 4 nucleotides and the CAAT motif contained within the PS-1 recognition sequence are essential for protein binding. C/EBP, a CCAAT-transcription factor binds to the PS-1 recognition site thus raising the possibility that both C/EBP and PS-1 may belong to the same family of proteins. Next we used a cell-free transcription assay to measure activity of a template, pS14-GFC(-72), that contained the PS-1 sequence. The pS14-GFC(-72) template was active in hepatonuclear extracts but deletion of or competition with the PS-1 binding sequence rendered the construct inactive. A template containing three PS-1 binding sequences increased S14 promoter activity by 12- to 13-fold. In nuclear extracts from spleen and testis, relative S14 promoter activity was only 2% of that in the liver, this observation mimicked closely in vivo expression of the gene. Mixing together extracts from liver and spleen in varying proportions, prior to incubation with S14 template, yielded a linear increase in S14 promoter activity that correlated with the amount of liver extract in the reaction. This finding is consistent with the absence of an essential factor or factors in spleen that is/are required for S14 promoter activity in vitro. In summary, PS-1 binds to a DNA sequence that contains a CAAT motif and appears to play a critical role in determining tissue-specific activity of the S14 promoter in vitro.

Secretion of vitamin A and retinol-binding protein into plasma is depressed in rats by N-(4-hydroxyphenyl)retinamide (fenretinide).

In clinical trials the cancer preventive drug N-(4-hydroxyphenyl)retinamide (HPR) markedly lowers plasma concentrations of retinol and retinol-binding protein (RBP). Five hours after injection of HPR (51 mumol/kg), serum concentrations of retinol and RBP were 33 and 42% lower, respectively, than control values in rats. Because the mean transit time for retinol disappearance from serum of HPR-treated rats (1.9 h) was similar to that for radiolabeled retinol in control rats in previous studies, plasma retinol turnover is apparently not accelerated by HPR treatment. To study the effects of HPR on the secretion of the retinol-RBP complex from liver, control or HPR-treated rats were injected with chylomicrons containing [3H]vitamin A and [14C]triglycerides. Both labels were rapidly cleared from plasma in the two groups. In control rats [3H]retinol concentrations began to increase in plasma after 30 min due to liver secretion of retinol bound to RBP. In HPR-treated rats, secretion was apparently inhibited because the amount of [3H]retinol bound to RBP at 4.66 h was only 2.6% of the control level. We conclude that HPR partially blocks the secretion of the retinol-RBP complex from liver and other tissues, and thus depresses plasma concentrations of vitamin A and RBP.

The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies.

Hybridoma fusions with hamster hosts were undertaken to generate mAbs to mouse spleen dendritic cells. Two mAb were obtained and used to uncover the distinct integrins of these APC. One, 2E6, bound a determinant common to all members of the CD11/CD18 family, most likely the shared 90 kD CD18 beta chain. 2E6 immunoprecipitated the characteristic beta 2 integrin heterodimers from lymphocytes (p180, 90; CD11a) and macrophages (p170,90; CD11b), but from dendritic cells, a p150,90 (presumably CD11c) integrin was the predominant species. 2E6 inhibited the binding function of the CD11a and CD11b integrins on B cells and macrophages in appropriate assays, but 2E6 exerted little or no inhibition on the clustering of dendritic cells to T cells early in primary MLR, suggesting a CD11/CD18-independent mechanism for this binding. The second mAb, N418, precipitated a 150, 90 kD heterodimer that shared the 2E6 CD18 epitope. This N418 epitope may be the murine homologue of the previously characterized human CD11c molecule, but the epitope was only detected on dendritic cells. N418 did not react with peritoneal macrophages, anti-Ig-induced spleen B blasts, or bulk lymph node cells. When used to stain sections of spleen, N418 stained dendritic cells in the T-dependent areas, much like anti-class II mAbs that were also generated in these fusions. In addition, N418 revealed nests of dendritic cells that punctuated the rim of marginal zone macrophages between red and white pulp. This localization positioned most dendritic cells at regions where arterial vessels and T cells enter the white pulp. We conclude that the p150, 90 heterodimer is the major beta 2 integrin of spleen dendritic cells, and we speculate that it may function to localize these APC at sites that permit access to the recirculating pool of resting T cells.

c-Ha-ras down regulates the alpha-fetoprotein gene but not the albumin gene in human hepatoma cells.

We studied the effects of transfection of the normal c-Ha-ras gene, rasGly-12, and its oncogenic mutant, rasVal-12, on expression of the alpha-fetoprotein (AFP) and albumin genes in a human hepatoma cell line, HuH-7. The mutant and, to a lesser extent, the normal ras gene caused reduction of the AFP mRNA but not the albumin mRNA level in transfected HuH-7 cells. Cotransfection experiments with a rasVal-12 expression plasmid and a chloramphenicol acetyltransferase reporter gene fused to AFP regulatory sequences showed that rasVal-12 suppressed the activity of enhancer and promoter regions containing A + T-rich sequences (AT motif). In contrast, rasVal-12 did not affect the promoter activity of the albumin and human hepatitis B virus pre-S1 genes even though these promoters contain homologous A + T-rich elements. ras transfection appeared to induce phosphorylation of nuclear proteins that interact with the AFP AT motif, since gel mobility analysis revealed the formation of slow-moving complexes which was reversed by phosphatase treatment. However, similar changes in complex formation were observed with the albumin and hepatitis B surface antigen pre-S1 promoters. Therefore, this effect alone cannot explain the specific down regulation of the AFP promoter and enhancer activity. ras-mediated suppression of the AFP gene may reflect the process of developmental gene regulation in which AFP gene transcription is controlled by a G-protein-linked signal transduction cascade triggered by external growth stimuli.

Strenuous short-term dynamic exercise: effects on heart rate, blood pressure, potassium homeostasis, and packed cell volume in mild hypertension.

This study examined the effect of strenuous short-term dynamic exercise on the heart rate, blood pressure, plasma potassium and packed cell volume in mildly hypertensive subjects. At baseline a symptom-limited Bruce Exercise Protocol was carried out with blood pressure, heart rate, plasma potassium and packed cell volume measurements at fixed time points at rest and during and after the exercise. After 6 weeks of taking exercise sessions (Bruce protocol) to fatigue three times a week, the subjects were restudied. Blood pressure and the heart rate fell significantly at rest and during and after exercise. The packed cell volume was higher at all study points and plasma potassium was higher in the postexercise period after the exercise conditioning. Strenuous short-term exercise has a beneficial antihypertensive effect, raises packed cell volume and has a favourable effect on plasma potassium homeostasis.

Analysis of T lymphocyte subsets in tamarins with colitis and colon cancer.

The cotton-top tamarin, Saguinus oedipus, serves as an animal model for the study of human colon cancer. This New World monkey has a high incidence of colitis and colon cancer that develops spontaneously. Evidence suggests that these diseases may be the result of a virally induced immunodeficiency. We have shown that T4+/T8+ cell ratios are significantly altered in tamarins with acute colitis and colon cancers. The T4+/T8+ ratios were 1.50 +/- 0.09, 0.70 +/- 0.05, and 0.48 +/- 0.05 for negative controls, acute colitis, and cancer positive tamarins, respectively. Statistical analysis showed a significant difference (p less than or equal to .0005) between negative controls vs. acute colitis and cancer positive groups.

Isolation and partial chemical characterization of the IgG Fc receptor of human T lymphocytes and production of an antiserum.

We report here the isolation of an IgG Fc receptor from normal human T lymphocytes. The purified receptor has a nonreduced and a reduced component of molecular weights 120,000 and 60,000, respectively, and it was functionally active in the in vitro blocking of rosette formation between T lymphocytes and IgG-coated ox erythrocytes. An antiserum raised to the Fc receptor and an isolated F(ab')2 fragment of this antiserum, also blocked rosette formation between T cells and IgG-coated ox erythrocytes. In contrast, rosette formation between T lymphocytes and IgM-coated ox or sheep erythrocytes was not blocked by the F(ab')2 fragment, demonstrating the marked specificity of this antiserum for the IgG Fc receptor. In addition, this antiserum did not block the Fc receptors of non-T cells, indicating that the T-cell IgG Fc receptor has unique antigenic determinants not shared with B cells.

Isolation and partial characterization of a 67Ga-binding glycoprotein from Morris 5123C rat hepatoma.

A Glycoprotein, particularly high in tumors, has been extracted from Morris 5123C rat hepatomas and purified. The compound constitutes a major binding component for 67Ga in this hepatoma. It has a molecular weight of approximately 45,000. Its molecular weight was determined by sodium dodecyl sulfate:polyacrylamide gel electrophoresis and by Sephadex G-200 superfine gel filtration. The steps involved in its extraction and purification include ultrafiltration, gel filtration through Sephadex G-200 superfine, ion-exchange chromatography on diethylaminoethyl Sephadex A-50, and hydroxylapatite chromatography. The homogeneity of the compound was established by gel electrophoresis. The NH2-terminal residue, the percentage of nitrogen, the nonamino carbohydrate content, and the amino acid composition are reported.