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G Theofan

Publications and source records attributed to G Theofan.

29 records · Page 2Linked to original sources
PubMed1989

Bone Gla protein messenger ribonucleic acid is regulated by both 1,25-dihydroxyvitamin D3 and 3',5'-cyclic adenosine monophosphate in rat osteosarcoma cells.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] regulates the synthesis of bone gamma-carboxyglutamic acid (Gla) protein (BGP) by osteoblastic cells. In this study we examined the effect of cAMP, alone and in combination with 1,25-(OH)2D3, on the regulation of BGP mRNA levels in ROS 17/2 rat osteosarcoma cells. Elevation of intracellular cAMP levels by cAMP analogs or by isobutylmethylxanthine (IBMX), forskolin, or PTH, resulted in increased BGP mRNA levels and BGP secretion after 1 day of treatment. The effects of these agents were additive with 1,25-(OH)2D3 in stimulating BGP gene expression. After 4 days of treatment, pertussis toxin (PT) and 1,25-(OH)2D3 were synergistic in stimulating BGP mRNA, and the effect of PT could be mimicked by (Bu)2cAMP, IBMX, forskolin, cholera toxin, and to a lesser extent by PTH. The effect of 1-day treatment with cAMP alone and the synergistic effect with 1,25-(OH)2D3 on the stimulation of BGP mRNA were dependent on cell density, while basal and 1,25-(OH)2D3-stimulated synthesis were not. Cyclic AMP inhibited ROS 17/2 cell growth after 1 day of treatment, an effect that was also dependent on initial cell density. After 4 days of treatment, 1,25-(OH)2D3, cAMP, and PT all demonstrated inhibition of cell growth. When cells were treated with actinomycin D, both 1,25-(OH)2D3 and cAMP stimulation of BGP mRNA were blocked. In addition, neither agent was effective in enhancing BGP mRNA stability when prestimulated cells were exposed to actinomycin D.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1988

Molecular structure of the chicken vitamin D-induced calbindin-D28K gene reveals eleven exons, six Ca2+-binding domains, and numerous promoter regulatory elements.

The seco-steroid hormone 1,25-dihydroxyvitamin D3 is known to induce the expression of a calcium binding protein termed calbindin-D28K in a variety of target tissues. In order to comprehend the mechanism of induction we have cloned and sequenced the chicken calbindin-D28K gene. The gene spans some 18.5 kilobases (kb) of chromosomal DNA from the putative Cap site to the polyadenylation site of the 2.8 kb mRNA. It is split into 11 coding exons by 10 intervening sequences. The promoter region of this gene is markedly G + C-rich (60-80%) extending from -225 to +400. Within this region we find 70 CpG dinucleotides, four G-C boxes, and numerous known promoter regulatory signals. These putative regulatory signals include a TATA box (ATAAATA) at -30 and a CAT box (CCAAT) at -326. Ten additional variant CAT boxes are found in the upstream promoter region (-218 to -770) of this gene. Furthermore we have identified a glucocorticoid-like responsive element at -410 (TCTACACACTGTTCC) and this element overlaps a metal responsive element (TGCACTC) and a variant CAT box (CCAAAT) and juxtaposes an enhancer-like core element (AAATGGT) on its 3'-side. In addition, the calbindin-D28K promoter is composed of a variety of simple repeated sequences, some of which are components of putative regulatory signals. All splice junctions were found to conform to the GT-AG rule. A consensus sequence of the 5'-splice junction reads AG/GTAAG-TTATA. A consensus sequence of the 3'-splice site consists of two elements: a pyrimidine track (mainly T) followed by ACAG/G-T. A two-dimensional model of calbindin-D28K was constructed which projects the existence of 6 alpha-helix-loop-alpha-helix regions characteristic of calcium binding domains. The 3'-end of the gene consists of a single large (2039 base pair) uninterrupted exon, an organizational feature common to other members of the calcium binding protein gene family which include calmodulin, parvalbumin, Spec I, myosin light chains, etc. Another feature common to the gene family is the presence of the repeated sequence ATTT or TTTA located in the 3'-untranslated exons. These simple repeat sequences could be involved in regulating mRNA degradation by serving as a ribonuclease recognition signal.

Amino Acid Sequence↗
PubMed1987

1,25-Dihydroxyvitamin D3 regulates tubulin expression in chick intestine.

As in many other cell types, autoregulation of tubulin synthesis is evident in the intestinal epithelium of normal (vitamin D-replete) chicks: Suppression of protein (tubulin) synthesis by cycloheximide administration in vivo resulted within 30 min in a two-fold increase in RNA hybridizing with an alpha-tubulin probe. Vitamin D status revealed an additional regulatory component. alpha-Tubulin mRNA was elevated in vitamin D-deficient (-D) chicks and those treated with 1,25(OH)2D3 for 1-10 h prior to sacrifice, but declined precipitously 15-20 h after hormone, and in normal birds. These results suggested hormonally increased tubulin levels which in turn suppressed cellular alpha-tubulin mRNA. Analyses of total tubulin levels by [3H]-colchicine binding revealed low levels of the protein(s) in -D chicks, increased levels at 1-15 h after 1,25(OH)2D3, and maximum binding at 20 h after hormone and in normal birds.

Animals↗
PubMed1987

Expression of calbindin-D28K mRNA as a function of altered serum calcium and phosphorus levels in vitamin D-replete chick intestine.

The availability of specific cDNA probes to the chick intestinal calbindin-D28K (CaBP) mRNA has allowed us to assess the regulation of this mRNA in response to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) administration. It has previously been demonstrated that dietary calcium and phosphorus can effect alterations in the steady-state intestinal levels of chick CaBP. We have examined whether or not perturbations in dietary calcium and phosphorus have an effect on the expression of the intestinal mRNA coding for CaBP in the vitamin D-replete chick. We found altered protein levels of CaBP as expected; however there was surprisingly no difference in steady-state CaBP mRNA levels between the different dietary groups. These data suggest that calcium and phosphorus regulation of CaBP occurs at a post-transcriptional level. In addition, we have examined what effect dietary manipulations of calcium and phosphorus levels have on the response of the vitamin D-replete intestine to 1,25(OH)2D3 administration as assessed by CaBP mRNA changes. Administration of 1,25(OH)2D3 to vitamin D-replete chicks maintained on normal calcium and phosphorus levels resulted in a less than 2-fold increase in CaBP mRNA levels. Previous studies have demonstrated that receptor occupancy goes up 6-fold under these conditions; therefore there is apparently a very tight regulation of CaBP gene activity. 1,25(OH)2D3 administration to chicks raised on either low calcium, high calcium, or low phosphorus vitamin D-replete diets similarly showed only small changes in the intestinal CaBP mRNA levels; however there seemed to be qualitative differences in response attributable to the dietary alterations.

Animals↗
PubMed1986

Regulation of calbindin-D28K gene expression by 1,25-dihydroxyvitamin D3 is correlated to receptor occupancy.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) induces the synthesis of calcium binding protein (calbindin-D28K) and calbindin-D28K-mRNA in the chick intestine. We have examined the relationship between nuclear uptake of 1,25-(OH)2D3, 1,25-(OH)2D3 receptor occupancy levels, and transcription of the calbindin-D28K gene and found all three parameters to be highly correlated. All three events occur rapidly (within 15-30 min) following a single dose of 6.5 nmol of 1,25-(OH)2D3 to vitamin D-deficient chicks, reaching peak values by 1-2 h; by 4 h, values of all three parameters start to decline. Calbindin-D28K-mRNA begins to accumulate by 3-5 h but does not peak until 12 h following hormone administration. The levels of calbindin-D28K start to increase by 5-8 h and do not peak until 48 h after the 1,25-(OH)2D3 dose. These observations suggest that post-transcriptional regulatory mechanisms may be involved. Measurements of basal levels of calbindin-D28K gene transcription show that there is virtually no transcription in the vitamin D-deficient chick intestine, and a 12-fold induction in the intestine of vitamin D-replete chicks. This basal level of transcription in vitamin D-replete chick intestine is not inhibited by cycloheximide pretreatment. These results confirm the thesis that a major component of the mechanism of action of 1,25-(OH)2D3 is functioning as a steroid hormone, effected through the direct action of the seco-steroid-receptor complex on the initiation of transcription of specific genes.

Animals↗
PubMed1986

Effects of alpha-amanitin and cycloheximide on 1,25-dihydroxyvitamin D3-dependent calbindin-D28K and its mRNA in vitamin D3-replete chick intestine.

We have examined the effects of the transcriptional inhibitor alpha-amanitin and the translational inhibitor cycloheximide on levels of calbindin-D28K (28-kDa calcium binding protein, CaBP) and CaBP-mRNA in the vitamin D-replete chick intestine. Chicks were raised on one of four diets: "normal" (1% calcium, 0.6% phosphorus); high calcium (3.3% calcium, 0.5% phosphorus); low calcium (0.3% calcium, 0.6% phosphorus); or low phosphorus (1% calcium, 0.09% phosphorus). Chicks were then treated either with alpha-amanitin (20 micrograms/chick) or cycloheximide (600 micrograms/chick) 2 h prior to a dose of 6.5 nmol of 1,25-dihydroxyvitamin D3. Duodenal mucosa was collected from 0 to 120 min afterward and assayed for CaBP-mRNA by dot blot hybridization and for CaBP using an enzyme-linked immunosorbent assay. In the absence of inhibitor, CaBP levels were depressed by high calcium and elevated by low calcium or low phosphorus, as expected. These changes occurred, however, without a change in CaBP-mRNA levels. alpha-Amanitin had no effect on CaBP or on CaBP-mRNA levels in chicks raised on any of the diets. Cycloheximide inhibited CaBP levels, and surprisingly also inhibited CaBP-mRNA levels in all four dietary groups. These results indicate that continual protein synthesis is necessary for the expression of CaBP-mRNA, suggesting the existence of a rapidly turned over protein that may be required for stabilization or for processing of the chick intestinal CaBP messenger RNA.

Amanitins↗
PubMed1984

Characterization of the calf uterine progesterone receptor and its stabilization by nucleic acids.

The molecular and steroid hormone-binding properties of the calf uterine progesterone receptor and its interaction with nucleic acids were investigated. A positive cooperative binding interaction of [3H]progesterone with the receptor was evident from a nonlinear Scatchard plot and a Hill coefficient of 1.22 +/- 0.02. The range of progesterone receptor concentrations was 0.73-1.04 pmol/mg protein, approximately twice that of the estrogen receptor. Competitive binding assays revealed a high specificity for progesterone: R5020 greater than or equal to progesterone greater than deoxycorticosterone greater than 5 alpha-pregnane-3,20-dione much greater than 17 alpha-hydroxyprogesterone greater than or equal to 20 alpha-dihydroprogesterone greater than or equal to testosterone greater than or equal to estradiol greater than cortisol. Thus, a progesterone-specific receptor of high affinity and concentration is obtainable from calf uterus in large quantities without estrogen pretreatment. Thermal inactivation of the unoccupied progesterone receptor is inhibited by 10 mM sodium molybdate, whereas thermal inactivation of the ammonium sulfate-purified progesterone receptor is not. Thermal inactivation of the ammonium sulfate-purified receptor is inhibited by nucleic acids and polynucleotides; polyguanylate (poly G) is the most effective. DNA and poly G also effectively restore the progesterone-binding ability of the ammonium sulfate-purified receptor which had been lost due to heat inactivation. After incubation of the unoccupied receptor from 5-30 min at 25 C, the addition of poly G restored the receptor's [3H]progesterone-binding ability to control levels. These data suggest that the progesterone receptor's steroid-binding site is more readily inactivated by heat than is the DNA-binding site, and that nucleic acid binding induces a conformational change, which consequently restores the receptor's progesterone-binding site to functional activity.

Animals↗
PubMed1983

The in vitro synthesis of final maturational steroids by ovaries of brook trout (Salvelinus fontinalis) and yellow perch (Perca flavescens).

The steroidogenic profile produced by ovaries of brook trout (Salvelinus fontinalis) and yellow perch (Perca flavescens) was examined using radioactive incorporation incubations. The radioactive metabolites produced were identified and were tested for their ability to induce germinal vesicle breakdown (GVBD) in oocytes in in vitro bioassays. Brook trout ovarian tissue converted [14C]progesterone to five metabolites that were effective in inducing GVBD in the bioassays: 5 beta-pregnanedione, 17 alpha-hydroxyprogesterone, deoxycorticosterone, and two more polar metabolites that were not identified. One of these two metabolites comigrated with 11-deoxycortisol and 17 alpha-hydroxy,20 beta-dihydroprogesterone (17 alpha, 20 beta prog) in thin layer chromatography. Androstenedione, testosterone, and estradiol-17 beta were also identified as metabolites of progesterone. Brook trout ovarian tissue did not extensively metabolize [14C]pregnenolone. Dehydroepiandrosterone and 17 alpha-hydroxypregnenolone were tentatively identified as metabolites of pregnenolone. Yellow perch ovarian tissue converted [14C]progesterone or [14C]pregnenolone to six metabolites that were effective in inducing GVBD in the bioassays: 5 alpha-pregnanedione, 17 alpha-hydroxyprogesterone, 20 alpha-dihydroprogesterone, 17 alpha-hydroxy, 20 alpha-dihydroprogesterone (tentatively identified), and two other unidentified metabolites. One of these two metabolites comigrated with 11-deoxycortisol and 17 alpha, 20 beta prog in thin layer chromatography. The only metabolite that induced ovulation of yellow perch oocytes in vitro was the metabolite comigrating with 11-deoxycortisol and 17 alpha, 20 beta prog. Androstenedione and testosterone were also identified as metabolites produced by yellow perch ovarian tissue. The results show that the ovaries of both species produce steroids that are active in the induction of oocyte final maturation.

Animals↗
PubMedDate not supplied

Safety and immunogenicity of REMUNE in HIV-infected Thai subjects.

The safety and immunogenicity of REMUNE, an HIV-specific immune based therapy for HIV infection, was evaluated in a cohort of 30 HIV infected subjects in Thailand. This therapy utilizes a gp120 depleted inactivated virus (HZ321), which exhibits a high degree of conservation with the core antigens of both type B' and E strains of HIV, the predominant Thailand isolates. The treatment was well tolerated, with no serious adverse events reported over the course of the 4-month trial. Treatment in which four doses were administered with REMUNE appeared to boost HIV-specific immune responses, with approximately 75% of the treated subjects demonstrating an increase in either the repertoire or the intensity of the serological response to HIV as measured by Western blot. CD4%, viral load, and weight remained stable over the course of the 4-month study relative to baseline values. Viral subtyping of this cohort revealed a predominance of type 'E'. These data suggest that REMUNE is safe and immunogenic in seropositive Thai subjects and supports further study of the therapeutic potential of REMUNE to treat HIV-1 infection.

AIDS Vaccines↗
PubMedDate not supplied

Tumor necrosis factor alpha and human immunodeficiency virus-specific functional immune responses after immunization with Gp120-depleted, inactivated HIV-1 in incomplete Freund's adjuvant (REMUNE) in HIV-1-seropositive subjects.

OBJECTIVE: We examined the relation between tumor necrosis factor-alpha (TNF-alpha) levels and human immunodeficiency virus type 1 (HIV-1)-specific functional immune responses, as measured by HIV-1 antigen-stimulated lymphocyte proliferation and beta-chemokine production after immunization with gp120-depleted, inactivated HIV-1 in incomplete Freund's adjuvant (i.e., HIV-1 Immunogen; REMUNE, The Immune Response Corporation, Carlsbad, CA, U.S.A.). STUDY DESIGN/METHODS: HIV-1-seropositive subjects who enrolled in an open-label study were immunized with REMUNE every 12 weeks and monitored for 60 weeks. HIV-1 antigen-stimulated lymphocyte proliferation and RANTES production were measured in peripheral blood mononuclear cells (PBMCs). TNF-alpha levels were measured in serum. RESULTS: TNF-alpha (P = 0.0003) significantly decreased and HIV-1 antigen-stimulated RANTES production (P = 0.002) and lymphocyte proliferation (P = 0.07) increased after immunization with REMUNE. TNF-alpha levels negatively correlated with HIV-1 antigen-stimulated RANTES production (r = -0.71; P = 0.0002) and lymphocyte proliferation (r = -0.37; P = 0.09). CONCLUSIONS: This study demonstrated decreased TNF-alpha levels with a concomitant augmentation of HIV-specific functional immunity in subjects immunized with REMUNE. Because TNF-alpha has been implicated in the induction of anergy in HIV-1 infection, the ability to decrease TNF-alpha may allow the immune system to respond to HIV and non-HIV antigens. Larger studies are being conducted to confirm the clinical utility of REMUNE in combination with potent antiviral drugs.

AIDS Vaccines↗