Biomedical subjects

A P Reddy

Publications and source records attributed to A P Reddy.

At least 19 recordsLinked to original sources

PubMed2005

Serotonin-related gene expression in female monkeys with individual sensitivity to stress.

Female cynomolgus monkeys exhibit different degrees of reproductive dysfunction with moderate metabolic and psychosocial stress. In this study, the expression of four genes pivotal to serotonin neural function was assessed in monkeys previously categorized as highly stress resistant (n=3; normal menstrual cyclicity through two stress cycles), medium stress resistant (n=5; ovulatory in the first stress cycle but anovulatory in the second stress cycle), or low stress resistant (i.e. stress-sensitive; n=4; anovulatory as soon as stress is initiated). In situ hybridization and quantitative image analysis was used to measure mRNAs coding for SERT (serotonin transporter), 5HT1A autoreceptor, MAO-A and MAO-B (monoamine oxidases) at six levels of the dorsal raphe nucleus (DRN). Optical density (OD) and positive pixel area were measured with NIH Image software. In addition, serotonin neurons were immunostained and counted at three levels of the DRN. Finally, each animal was genotyped for the serotonin transporter long polymorphic region (5HTTLPR). Stress sensitive animals had lower expression of SERT mRNA in the caudal region of the DRN (P<0.04). SERT mRNA OD in the caudal DRN was positively correlated with serum progesterone during a pre-stress control cycle (P<0.0007). 5HT1A mRNA OD signal tended to decline in the stress-sensitive group, but statistical difference between averages was lacking in analysis of variance. However, 5HT1A mRNA signal was positively correlated with control cycle progesterone (P<0.009). There was significantly less MAO-A mRNA signal in the stress-sensitive group (P<0.007) and MAO-A OD was positively correlated with progesterone from a pre-stress control cycle (P<0.007). MAO-B mRNA exhibited a similar downward trend in the stress-sensitive group. MAO-B OD also correlated with control cycle progesterone (P<0.003). There were significantly fewer serotonin neurons in the stress-sensitive group. All animals contained only the long form of the 5HTTLPR. Thus, all serotonin-related mRNAs examined in the dorsal raphe to date were lower (SERT, MAO-A) or exhibited a lower trend (5HT1A, MAO-B) in the stress sensitive animals, which probably reflects the lower number of serotonin neurons present.

Amenorrhea↗

PubMed2001

Application of matrix solid-phase dispersion in the determination of dibenzo[a,l]pyrene content of experimental animal diets used in a large-scale tumor study.

A method utilizing matrix solid-phase dispersion (MSPD) was developed for isolation and determination of dibenzo[a,l]pyrene (DBP) in experimental rainbow-trout diets used in a large-scale carcinogenesis study. A 0.5 g sample of moist ration containing 0-225 ppm DBP (dry basis) was mixed with 2 g C18 sorbent and benzo[a]pyrene internal standard was added to the mixture. Extraction and clean-up were accomplished in a single step by extracting the sample mixture with hexane-benzene 4:1 from a cartridge containing 2 g Florisil. DBP was quantified by HPLC on a C5 bonded phase column with fluorescence detection. Mean analytical recovery of DBP from control diet spiked at three concentration levels was 101 to 107% with relative standard deviations of 1 to 7%. The limit of detection of DBP was equivalent to 0.014 ppm in the ration. Application of the method to verification of DBP levels in trout rations from the carcinogenesis study is described. Control ration (0 ppm DBP) was screened for possible DBP contamination and none was found. This is the first report on analysis of DBP in experimental animal diets.

Animal Feed↗

PubMed2001

Marker assisted selection of bacterial blight resistance genes in rice.

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae is one of the most important diseases affecting rice production in Asia. We were interested in surveying rice genotypes that are popularly used in the Indian breeding program for conferring resistance to bacterial blight, using 11 STMS and 6 STS markers. The basis of selection of these DNA markers was their close linkage to xa5, xa13, and Xa21 genes and their positions on the rice genetic map relative to bacterial blight resistance genes. Eight lines were found to contain the xa5 gene while two lines contained Xa21 gene and none of the lines contained the xa13 gene with the exception of its near-isogenic line. Using the polymorphic markers obtained in the initial survey, marker-assisted selection was performed in the F3 population of a cross between IR-64 and IET-14444 to detect lines containing multiple resistance genes. Of the 59 progeny lines analyzed, eight lines contained both the resistance genes, xa5 and Xa4.

Breeding↗

PubMed2001

On-screen vector-based ultrasound assessment of vesical neck movement.

OBJECTIVE: We sought to develop a vector-based assessment to determine the magnitude and direction of bladder neck movements, as well as to assess whether probe movement relative to the pubis needs to be taken into account. STUDY DESIGN: Ten nulliparous continent, 10 primiparous continent, and 10 primiparous stress-incontinent women were recruited. Perineal ultrasound scanning was performed in standing women while they were resting, performing the Valsalva maneuver, coughing, and performing Kegel exercises. A direct on-screen assessment of bladder neck displacement from rest to the peak of dynamic activity relative to the pubic axis was made. Transducer movement was assessed by measuring the displacement of the pubic bone. RESULTS: The method was feasible because measurements were possible in all 30 subjects. Vesical neck and pubic point movement in millimeters (+/- SD) and the percentage error if pubic point movement is not accounted for are as follow: strain, vesical neck 16.9 +/- 6.1 and pubic point 4.8 +/- 3.9, 28%; cough, vesical neck 10.2 +/- 5.4, pubic point 2.9 +/- 3.4, 33%; Kegel exercise, vesical neck 7.0 +/- 3.6 and pubic point 0.7 +/- 1.4, 37%. Similar discrepancies in angle were found and are presented. Uncorrected direction of vesical neck and pubic point movement in degrees and the percentage error if pubic point movement is not accounted for are as follow: strain, vesical neck 169.4 +/- 18.5 and pubic point 214.0 +/- 56.7, 18%; cough, vesical neck 162.0 +/- 12.8, pubic point 238.4 +/- 27.4, 22%; Kegel exercise, vesical neck -0.9 +/- 12.7 and pubic point -4.8 +/- 20.6, 87%. Test-retest reliability correlations were more than an r value of 0.7 in all measures and 86% of the measurements greater than 0.8. CONCLUSION: The vector-based system provides a simple method for quantifying distance and direction of vesical neck motion, as well as localizing the resting vesical neck position.

Adult↗

PubMed2000

Use of three different marker systems to estimate genetic diversity of Indian elite rice varieties.

Genetic diversity among 42 Indian elite rice varieties, which is important for selection of parents for conventional breeding and hybrid program, was evaluated using three different types of DNA markers and parentage analysis. Random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and sequence tagged microsatellite site (STMS) markers resulted in mean heterozygosity values of 0.429, 0.675 and 0.882 over all loci, respectively, and marker index values of 2.21, 4.05 and 5.49, respectively. The three molecular marker systems together provide wider genome coverage and, therefore, would be a better indicator of the genetic relationships among the 42 elite rice cultivars than those revealed using individual molecular markers. A total of 153 bands (91%) were polymorphic out of 168 bands amplified, considering all the markers together. The average genetic similarity coefficient across all the 861 cultivar pairs was 0.70 while the average coefficient of parentage was 0.10. Cluster analysis revealed that there was a very poor correlation (correlation coefficient <0.1) between dendrograms generated using coefficients of parentage and molecular marker generated genetic similarities, which can be attributed to selection pressure, genetic drift, sampling of loci and unknown relationships among supposedly unrelated ancestors.

DNA, Plant↗

PubMed2000

Influence of beta-naphthoflavone on 7,12-dimethylbenz(a)anthracene metabolism, DNA adduction, and tumorigenicity in rainbow trout.

Metabolism, DNA adduction, and tumor induction by 7, 12-dimethylbenz(a)anthracene (DMBA) were examined in cultured trout liver cells and in vivo in trout. Modulating CYP1A1 activity indicated this enzyme plays a significant role in metabolizing DMBA to water-soluble compounds in isolated trout liver cells. The major DMBA metabolites identified in trout liver cells were 10-, 11-, 8,9-, and 5,6-DMBA dihydrodiols, and DMBA, 2- or 3- or 4-phenol; 7-OH-methyl-12-methyl-benz(a)anthracene and 12-OH-methyl-7-methyl-benz(a)anthracene were minor metabolites. A very small amount of DMBA-3,4-dihydrodiol was detected, and polar metabolites, which did not migrate with any DMBA metabolite standards, were observed. Incubating trout hepatocytes with DMBA-3, 4-dihydrodiol produced three prominent, nonpolar adducts indistinguishable from those in mouse embryo cells. However, DMBA-DNA adducts, formed in trout in vivo or in trout liver cells exposed to DMBA, were predominantly more polar than those formed in mouse embryo fibroblasts, and levels of DMBA-DNA adducts formed in trout liver cells were not significantly altered by modulating CYP1A1 activity. No significant repair of DMBA-DNA adducts was detected in cultured trout liver cells over a 48-h period, supporting previous studies indicating that fish are less efficient than mammals in repairing polyaromatic hydrocarbon DNA adducts. Compared to animals receiving DMBA alone, beta-naphthoflavone pretreatment in vivo did not affect hepatic CYP1A1, DMBA-DNA adducts, nor hepatic tumor response; but did significantly reduce tumor response in two other target organs. These results collectively indicate that DMBA bioactivation to DNA-binding metabolites in trout liver cells and mouse embryo cells predominantly involve different metabolic pathways to form the DNA-binding intermediates.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1999

Inhibition of dibenzo[a,l]pyrene-induced multi-organ carcinogenesis by dietary chlorophyllin in rainbow trout.

Cancer chemoprevention by dietary chlorophyllin (CHL) was investigated in a rainbow trout multi-organ tumor model. In study 1, duplicate groups of 130 juvenile trout were treated for 2 weeks with control diet, 500 p.p.m. dibenzo[a,l]pyrene (DB[a,l]P) or 500 p.p.m. DB[a,l]P + 2052 p.p.m. CHL, then returned to control diet. DB[a,l]P alone proved somewhat toxic but induced high tumor incidences in liver (61%), stomach (91%) and swimbladder (53%) 11 months after initiation. CHL co-feeding abrogated DB[a,l]P acute toxicity and reduced tumor incidences to 18% in liver, 34% in stomach and 3% in swimbladder (P </= 0.01). A second tumor and DNA adduct study using a non-toxic initiation protocol (200 p.p.m. DB[a,l]P +/- 4000 p.p.m. CHL for 4 weeks) confirmed these results. Potential CHL inhibitory mechanisms were investigated. Dietary CHL inhibited hepatic DB[a, l]P-DNA adducts in the two tumor studies by 89 and 76%, respectively. CHL was shown to complex strongly with DB[a,l]P (K(d1,2) = 1.59 +/- 0.01 microM, stoichiometry 2CHL:DB[a,l]P) and strongly inhibited DB[a,l]P mutagenesis in the Salmonella assay. Significant inhibition occurred at CHL concentrations substantially less than stoichiometric with DB[a,l]P and thus not reflecting simple DB[a,l]P sequestration via complexation. These initial findings suggest that CHL chemoprevention reflects complexation that might limit DB[a,l]P uptake in vivo, antimutagenic mechanisms such as catalytic degradation of the proximate electrophile in target cells, or both. These results demonstrate that dietary CHL is a reproducibly effective chemopreventive agent for DB[a,l]P multi-organ tumorigenesis in trout and suggest that reduced DB[a,l]P-DNA adducts may be predictive biomarkers of CHL reduction of DB[a,l]P-initiated hepatic tumors.

Animals↗

PubMed1999

Experimental hepatic tumorigenicity by environmental hydrocarbon dibenzo[a,l]pyrene.

There is an evident need of low-cost vertebrates to be used in experimental carcinogenesis. Medaka (Oryzias latipes) provide a useful vertebrate model system for investigating tissue tropism of carcinogens and the action mechanisms of environmental contaminants posing a potential risk to human health. Juvenile medaka 2 months of age fed diets containing 100 ppm (dry weight basis) dibenzo[a,l]pyrene (DBP) for 28 days responded with hepatic neoplasia predominately of hepatocellular origin. When sampled 9 months following the termination of carcinogen exposure, medaka showed 26% incidence of neoplasia and 25% hepatic neoplasia, compared with 8% total neoplasia and 0% hepatic neoplasia in control fish. The predominant spontaneous neoplasms in this group of medaka were ovarian germ cell tumors. Hepatic neoplasia occurred at a higher incidence in female DBP-treated medaka than in males (11/29 vs 5/36). Nonneoplastic lesions observed in the livers of DBP-exposed fish included spongiosis hepatis, globular hyaline eosinophilic cytoplasmic inclusions in hepatocytes, foci of hepatocellular degeneration, extensive cytomegaly, and karyomegaly of hepatocytes. No activating exon I mutations in the one ras protooncogene examined were detected among six liver neoplasms. These results indicate that medaka are sensitive to the tumorigenic effects of the environmental carcinogen DBP, administered by dietary exposure.

Adenoma, Liver Cell↗

PubMed1997

In vivo aflatoxin B1 metabolism and hepatic DNA adduction in zebrafish (Danio rerio).

The zebrafish (Danio rerio) is assuming prominence in developmental genetics research. By comparison, little is known of tumorigenesis and nothing is known of carcinogen metabolism in this species. This study evaluated the ability of zebrafish to metabolize a well-characterized human carcinogen, aflatoxin B1 (AFB1), to phase I and phase II metabolites and assessed hepatic AFB1-DNA adduction in vivo. Fish i.p. injected with 50-400 micrograms [3H]AFB1/kg body wt displayed a linear dose response for hepatic DNA binding at 24 hr. AFB1-DNA adduct levels among treatments showed no statistical difference over the period from 1 to 21 days after injection, suggesting poor adduct repair in this species. DNA binding in female fish was 1-7-fold higher than that in males (p < 0.01). An in vitro AFB1 metabolism assay verified that zebrafish liver extracts oxidize AFB1 to the 8,9-epoxide proximate electrophile (Km = 79.0 +/- 16.4 microM, Vmax = 11.7 +/- 1.4 pmol/min/mg protein at 28 degrees C). The excretion of AFB1 and its metabolites was also examined by HPLC. As is typical of other fish studied, major metabolites excreted were aflatoxicol (AFL) and aflatoxicol-glucuronide (AFL-g), followed by unreacted AFB1. AFL appeared as early as 5 min after injection, whereas AFL-g was a significant metabolite after 18 hr. This study shows that in vivo administration of AFB1 to zebrafish results in moderate adduction of the carcinogen to liver DNA and that zebrafish have the capacity for both phase I and phase II metabolism of AFB1. The approximate fourfold difference between rainbow trout and zebrafish AFB1-DNA covalent binding index appears insufficient to explain the relative resistance of zebrafish to dietary AFB1 hepatocarcinogenicity.

Aflatoxin B1↗

PubMed1996

Identification of a human CD4-CDR3-like surface involved in CD4+ T cell function.

The CD4 molecule is expressed on the surface of helper T cells. This molecule contains four tandem external immunoglobulin-like domains (D1-D4), a transmembrane domain, and a cytoplasmic tail. Through the use of molecular modeling techniques, peptide analogs of the CDR3-like region of the human CD4 molecule, analog hPGP, a cyclized peptide 13 amino acids long, was synthesized and tested for its ability to inhibit proliferation in human mixed lymphocyte reactions. A conservative amino acid substitution was made at position 5 (D --> N) to increase its activity and designated hPGP(N). A series of alanine substitution peptides were synthesized based on the sequence of hPGP(N) to determine the importance of each residue to the peptide's function. The substitutions of amino acids in positions 3, 7, and 8 had essentially no effect on the inhibitory activity of hPGP(N), while substitutions of amino acids in positions 4 and 6 increased its inhibitory effect. Alanine substitutions of amino acids in positions 2, 5, and 9 dramatically decreased the inhibitory effect of analog hPGP(N). Molecular modeling of the native CD4-CDR3-like domain suggested that the residues corresponding to positions 2, 5, and 9 of the peptide formed a contiguous surface representing the active site.

Alanine↗

PubMed1995

All-trans retinoic acid induces cellular retinol-binding protein in human skin in vivo.

We examined the regulation of cellular retinol-binding protein (CRBP) mRNA and protein expression in human skin in vivo by all-trans retinoic acid and all-trans retinol. Treatment of human skin for 24 h with all-trans retinoic acid (0.1%) or all-trans retinol (1.6%) induced CRBP mRNA 5.5-fold (p < 0.01, n = 10) and 5.7-fold (p < 0.01, n = 5), respectively, compared with skin treated with vehicle or sodium lauryl sulfate (used as an irritant control). In vitro translation of poly A+ RNA from all-trans retinoic acid, all-trans retinol, sodium lauryl sulfate, and vehicle-treated human skin demonstrated that the observed increased CRBP mRNA in all-trans retinoic acid- and all-trans retinol-treated skin was able to direct increased (2.3-2.9-fold) CRBP protein synthesis. Riboprobe in situ hybridization revealed that CRBP mRNA was uniformly elevated throughout the epidermis and in dermal cells after all-trans retinoic acid treatment of human skin. Western analysis revealed that CRBP protein was elevated 3.2-fold (p < 0.01, n = 6) and 3.0-fold (p < 0.01, n = 6) after all-trans retinoic acid treatment of human skin in vivo for 24 and 96 h, respectively, compared with vehicle- and sodium lauryl sulfate-treated skin. In addition, functional CRBP levels measured by [3H]all-trans retinol binding were elevated 1.9-fold (p < 0.01, n = 6) and 3.5-fold (p < 0.01, n = 6) at 24 and 94 h, respectively, after all-trans retinoic acid treatment, compared with vehicle- or sodium lauryl sulfate-treated skin. Gel mobility shift analysis revealed that retinoid receptors in nuclear extracts from human skin formed a specific complex with a DNA probe containing the retinoic acid response element in the mouse CRBP gene. Monoclonal antibodies to nuclear retinoid receptors demonstrated that predominantly retinoic acid receptor-alpha/retinoid X receptor-alpha heterodimers bound to the CRBP retinoic acid response element. These data demonstrate that CRBP expression in human skin in vivo is regulated by exogenous all-trans retinoic acid and all-trans retinol.

Base Sequence↗

PubMed1995

Application of retinol to human skin in vivo induces epidermal hyperplasia and cellular retinoid binding proteins characteristic of retinoic acid but without measurable retinoic acid levels or irritation.

We investigated the clinical, histologic, and molecular responses of normal human skin to all-trans-retinol (ROL) application, compared to those induced by topical all-trans-retinoic acid (RA), and measured ROL-derived metabolites. Up to 1.6% ROL, 0.025% RA in vehicle (70% ethanol/30% propylene glycol), or vehicle alone were applied in a double-blind fashion to normal buttock skin and occluded for 4 d. ROL produced from none to only trace erythema, which was clinically and statistically insignificant, whereas RA induced a significant 3.7-fold increase in erythema score compared to vehicle (n = 10, p < 0.01). However, ROL induced significant epidermal thickening (1.5-fold at 1.6% ROL, p < 0.01), similar to RA (1.6-fold at 0.025% RA, p < 0.01), relative to the vehicle. ROL, compared with vehicle, also increased mRNA levels of cellular retinoic acid binding protein (CRABP-II) and cellular retinol binding protein (CRBP) genes as determined by Northern analysis (5-6-fold and 6-7-fold, respectively) and riboprobe in situ hybridization. CRABP-II and CRBP protein levels were also higher following ROL than vehicle treatment, as measured by ligand binding (3.2-fold, p < 0.001; n = 7) and Western analysis (3.6-fold, p < 0.003; n = 6), respectively. Epidermal retinyl ester (RE) content, measured after removal of stratum corneum, rose 240-fold (p < 0.005, n = 5) by 24 h of ROL occlusion. RA content, however, was undetectable or detectable only at trace amounts in all samples obtained at 0, 6, 24, and 96 h after ROL occlusion. Detectability of RA was not correlated with ROL treatment (compared to untreated normal skin, p = 0.86) or baseline skin ROL levels (average r = -0.1, p > 0.3). These data demonstrate that ROL application 1) produces trace erythema not significantly different from vehicle, whereas RA causes erythema; 2) induces epidermal thickening and enhances expression of CRABP-II and CRBP mRNAs and proteins as does RA; 3) causes marked accumulation of retinyl ester; and 4) does not significantly increase RA levels. Taken together, the data are compatible with the idea that ROL may be a prohormone of RA, because it produces changes in skin similar to those produced by RA but without measurable RA or irritation.

Administration, Cutaneous↗

PubMed1994

Immunological identification and functional quantitation of retinoic acid and retinoid X receptor proteins in human skin.

We have determined protein levels of total and individual nuclear retinoic acid (RAR-alpha, -beta, -gamma) and retinoid X (RXR-alpha, -beta, -gamma) receptors by ligand binding, Western analysis, and gel shift assays, in adult human skin, a major retinoid-responsive tissue. Total RARs and RXRs, measured by direct binding of specific ligands, were 0.24 +/- 0.01 fmol/micrograms (n = 13) and 1.26 +/- 0.08 fmol/micrograms (n = 7), respectively. These values calculated on an average per cell basis were 1790 RARs/cell and 9400 RXRs/cell. Similar results were obtained with competitive ligand binding assays. RAR-alpha, -beta, and -gamma were each specifically immunoprecipitated, and their levels determined by ligand binding assays of supernatants and Western analysis of precipitates. RAR-gamma was the most abundant, representing 87% of RAR protein. The remaining 12-14% of RAR protein was RAR-alpha. No RAR-beta was detected. Similar immunoprecipitation studies revealed that RXR-alpha represented 90% of RXR protein expressed in human skin. No RXR-beta or RXR-gamma proteins were detected by Western blot. Supershift gel retardation with antibodies to RARs detected probe-RAR-alpha and probe-RAR-gamma complexes in a 1 to 4 ratio. No probe-RAR-beta complex was detected. With antibodies to both RAR-gamma and RXR, a double supershifted complex was formed, indicating that RAR-gamma/RXR heterodimers bound to the probe. These data demonstrate 1) protein levels of RXRs are five times greater than RARs, 2) relative protein levels of RAR and RXR family members are compatible with their previously described relative mRNA levels, and 3) RXR-alpha/RAR-gamma heterodimers are the major retinoid receptors that have the potential to regulate transcription of target genes, in adult human skin.

Base Sequence↗

PubMed1994

Biophysical studies on fragments of the alpha-factor receptor protein.

The receptor for the alpha-factor mating pheromone of the yeast Saccharomyces cerevisiae consists of 431 amino acid residues and is a member of a family of membrane proteins predicted to have seven transmembrane helices. Fragments of the receptor corresponding to two of the transmembrane helices [residues 246-269 (M6) and 273-302 (M7)], two of the interhelical loops [residues 107-125 (E2) and 191-206 (E3)], and to a portion of the carboxyl terminus [residues 350-372 (CT)] were synthesized using solid-phase methodologies and purified to near homogeneity. CD was used to characterize the secondary structure of these peptides in trifluoroethanol (TFE), in TFE/water mixtures, in sodium dodecyl sulfate (SDS), and in the presence of dimyristoyl phosphatidylcholine (DMPC) liposomes. In TFE, M6 and M7 exhibited CD spectra consistent with highly helical peptides, whereas CT was partially helical. In contrast, E2 and E3 were either disordered or aggregated in this solvent. M6 did not partition well into DMPC vesicles whereas M7 remained helical. Both M6 and M7 assumed helical conformations in 25 mM SDS. The loop peptides and the carboxyl terminus peptide were either in a beta-structure or disordered in the presence of lipid. These findings represent the first biophysical evidence for conformations assumed by specific segments of the STE2 receptor protein.

Amino Acid Sequence↗

PubMed1992

Characterization and purification of human retinoic acid receptor-gamma 1 overexpressed in the baculovirus-insect cell system.

The full-length cDNA for the human retinoic acid receptor-gamma 1 (RAR-gamma 1) has been expressed to high levels in Spodoptera frugiferda (Sf9) cells using the baculovirus expression system. Western blot analysis revealed that RAR-gamma 1 expression increased between 32 and 60 h post-infection. The recombinant receptor was expressed primarily as a nuclear protein and displayed a molecular mass of 50 kDa as determined by SDS/PAGE and gel-filtration chromatography, consistent with its cDNA-deduced size. Based on ligand binding, 2 x 10(6) RAR-gamma 1 molecules were expressed per Sf9 cell, a level approx. 2000 times greater than in mammalian cells. The receptor was partially purified 300-fold by sequential anion-exchange, gel-filtration and DNA affinity chromatographies. The overexpressed receptor specifically bound all-trans-retinoic acid (RA) and the synthetic retinoid CD367 with high affinity (Kd 0.15 nM and 0.23 nM respectively). The RA metabolites 4-hydroxy-RA and 4-oxo-RA were poor competitors for [3H]CD367 binding to recombinant RAR-gamma 1 (K(i) > 1 microM), indicating that 4-oxidation of RA greatly reduces its affinity for RAR-gamma 1. Gel-retardation analysis demonstrated that RAR-gamma 1 specifically bound the RA response element of the mouse RAR-beta gene. RAR-gamma 1 species expressed from recombinant baculovirus (in Sf9 cells) and vaccinia virus (in HeLa cells) exhibited similar affinities for RA and CD367 and had comparable DNA-binding properties in gel-retardation experiments. Moreover, a similar requirement for additional DNA-binding stimulatory factor(s) was observed in both cases. These results provide a basis for the use of baculovirus-expressed RAR-gamma 1 in further functional and structural studies.

Amino Acid Sequence↗

PubMed1992

Short-term retinoic acid treatment increases in vivo, but decreases in vitro, epidermal transglutaminase-K enzyme activity and immunoreactivity.

Epidermal transglutaminase-K is believed to catalyze the covalent linking of loricrin and involucrin to form cross-linked (CE) envelopes. In normal skin, transglutaminase-K is expressed as a band immediately below the stratum corneum, whereas in psoriasis and healing skin its expression is considerably expanded throughout the suprabasal layers. We have investigated whether the hyperproliferative state induced by short-term application of topical retinoic acid is similarly characterized by an increase in transglutaminase-K enzyme activity and immunoreactivity. Retinoic acid (0.1% cream) or vehicle were applied to human skin and occluded for 4 d. Skin biopsies were obtained for measurement of transglutaminase-K and transglutaminase-C activity and immunoreactivity. For comparison, cultured normal human keratinocytes were incubated for 4 d in the presence of 1 microM retinoic acid and the subsequent transglutaminase-K activity and immunoreactivity measured. Transglutaminase-K activity was increased 2.8 times in retinoic acid compared to vehicle-treated skin (p less than 0.005, n = 12) whereas there was no significant difference in transglutaminase-C activity. However, transglutaminase-K mRNA levels were not significantly different between retinoic acid- and vehicle-treated skin. In vehicle-treated skin, transglutaminase-K immunoreactivity was limited to a narrow, substratum corneal band, but was considerably expanded in a diffuse suprabasal pattern in retinoic acid-treated epidermis. In contrast, transglutaminase-K immunostaining was decreased and its enzymatic activity reduced sixfold in retinoic acid-treated keratinocytes (p less than 0.01, n = 4). These results demonstrate that retinoic acid treatment in vivo, in contrast to in vitro, leads to not only increased transglutaminase-K protein expression but also increased enzymatic activity in the absence of detectable increases in mRNA levels. These data, taken with the previously reported lack of in vivo modulation of the differentiation markers keratins 1 and 10 by retinoic acid, indicate that certain aspects of keratinocyte terminal differentiation that are altered in vitro by retinoic acid do not occur in vivo in human skin.

Cells, Cultured↗

PubMed1991

DNA polymorphisms of the apolipoprotein AI/CIII/AIV gene cluster influence plasma cholesterol and triglyceride levels in the Mayans of the Yucatán Peninsula, Mexico.

The purpose of this work was to examine the influence of apolipoprotein gene variation on plasma lipid levels in a population of Mayan Indians of the Yucatán Peninsula, Mexico. Four restriction enzymes: XmnI, PstI, SstI, and PvuII, were used to detect restriction fragment length polymorphisms (RFLP) within the region of the apolipoprotein AI/CIII/AIV gene cluster. The frequencies of these polymorphisms in this Mayan population were similar to those reported for other Amerindian populations, but differed widely from those reported for Caucasian populations. The XmnI and SstI RFLPs were informative for association studies in this population, and we analyzed their influence on the quantitative variation of plasma cholesterol and triglycerides. Using a nonparametric analysis of variance, it is shown that the presence of the XmnI restriction site had a significant effect in lowering plasma cholesterol, whereas the presence of the restriction site for SstI had a significant effect in raising plasma triglycerides. Consequently, genetic indicators of both low and high risk for lipid-related diseases, such as atherosclerosis and coronary heart disease, seem to be present within the same gene region in this Mayan population.

Adult↗

PubMed1990

Elevation of rat liver mRNA for selenium-dependent glutathione peroxidase by selenium deficiency.

Selenium-dependent glutathione peroxidase (Se-GSH-Px, GSH-H2O2 oxidoreductase EC 1.11.1.9) is the best characterized selenoprotein in higher animals, but the mechanism whereby selenium becomes incorporated into the enzyme protein remains under investigation. To elucidate the mechanism of insertion of selenium into Ge-GSH-Px further, we have systematically analyzed and compared the results of Western blot, in vitro translation immunoprecipitation, and Northern blot experiments conducted with liver proteins and RNAs obtained from rats fed on selenium-deficient and selenium-supplemented diets. The anti-serum employed in this study was raised against an electrophoretically pure Se-GSH-Px preparation obtained from rat livers by a simplified purification procedure involving separation by high performance liquid chromatography on a hydrophobic interaction column. Different forms of Se-GSH-Px, including apo-protein, cross-reacted with this antiserum and Western blot analysis found no Se-GSH-Px protein present in livers from rats fed on selenium-deficient diets. By contrast, a distinct protein band corresponding to purified Se-GSH-Px was detected in livers from selenium-supplemented animals, a result consistent with the finding that the Se-GSH-Px activity was reduced to undetectable levels in livers of selenium-deficient rats. The in vitro translation experiments, however, indicated not only that mRNA for Se-GSH-Px was present during selenium deficiency but also that its translation products contained 2-3-fold as much immunoprecipitable protein as the products of poly(A) RNA from livers of selenium-supplemented rats. This result suggests that the Se-GSH-Px mRNA may be increased in the selenium-deficient state. Elevated levels of Se-GSH-Px mRNA were directly demonstrated in Northern blot experiments employing cDNA clone pGPX1211 as a probe. A similar increase in Se-GSH-Px mRNA was observed in such other tissues as kidney, testis, brain, and lung tissue, in selenium-deficient states. The present data support the co-translational mechanism for the incorporation of selenium into Se-GSH-Px in rat liver.

Animals↗