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D Roy

Publications and source records attributed to D Roy.

At least 163 records · Page 9Linked to original sources
PubMed1996

[Acute torsion of an accessory spleen].

A case of acute torsion of an accessory spleen is reported. In a 15-year-old girl was admitted to hospital with acute abdominal pain. Ultrasonography showed a round, hypoechoic, solid mass. Computed tomography demonstrated a low-density mass with peripheral enhancement after intravenous contrast medium. These findings suggest the presence of a capsule. At laparotomy, the patient was found to have torsion and infarction of an accessory spleen. Sonographic and computed tomographic findings are discussed.

Acute Disease↗
PubMed1995

Perturbation of cell cycle kinetics in the mammary gland by stilbene estrogen, diethylstilbestrol (DES).

In the present study we have examined the effect of stilbene estrogen on the proliferative activity, cell cycle kinetics and the differentiation of the mammary gland of female Noble rats. The exposure of animals to stilbene estrogen (diethylstilbestrol) or natural estrogen (estrone) (10 micrograms/24 h) significantly increased proliferation in mammary epithelial cells. Both labeling index and growth fraction were increased by DES or estrone treatment. In addition, both stilbene and natural estrogen exposure altered cell cycle kinetics. Stilbene estrogen exposure to animals increased the conversion of mammary epithelial cells from G0 to G1 and G1 to S-phase cells by 8.3- and 8.5-fold, respectively, compared to that of control. Differentiation measured by the degree of lobular maturation revealed that the conversion of immature structures to mature structures was significantly increased by 2.4-fold in response to DES or estrone exposure compared to that of control. Perturbation of cell cycle is considered as a risk factor for the development of cancer. Changes in proliferation and cell cycle have been shown to lead to genetic instability, ultimately resulting in cell transformation. Our results indicated an increase in labeling index and growth fraction and a perturbation in cell cycle kinetics from DES or estrone exposure. Perturbation of cell cycle in response to stilbene estrogen exposure may produce adverse effects in the mammary glands of the Noble rats.

Animals↗
PubMed1995

In vitro conversion of environmental estrogenic chemical bisphenol A to DNA binding metabolite(s).

In the present study we have investigated the ability of bisphenol A to be converted to reactive metabolite(s) and its potential to bind to DNA. In this in vitro study we show that bisphenol A is oxidized by 70% to bisphenol o-quinone. The evidence for the formation of bisphenol o-quinone was shown by UV, IR and GC-MS. The new product, bisphenol o-quinone, had a maximum UV absorption at 386 nM, the appearance of an IR characteristic of unsaturated carbonyl (1690 cm-1) and a mass of 242. The chemical reaction of deoxyguanosine monophosphate (dGMP) or DNA with bisphenol o-quinone produced 6-8 adducts. The in vitro incubation of DNA with bisphenol A in the presence of peroxidase activation system also produced one major and seven minor adducts. The chromatographic mobilities of major DNA adducts four and six formed by bisphenol A in the presence of peroxidase activation system closely matched those of spots four and six obtained by chemical reaction between DNA or dGMP with bisphenol o-quinone. Based on these data it appears that bisphenol A is converted to DNA binding metabolites in vitro. Whether irreversible binding of bisphenol A to DNA through metabolic activation may be responsible for some of the toxic effects produced by bisphenol A is not clear.

Benzhydryl Compounds↗
PubMed1995

In vivo binding of diethylstilbestrol to nuclear proteins of kidneys of Syrian hamsters.

We demonstrate here that stilbene estrogen (diethylstilbestrol) is converted to nuclear protein binding metabolite(s) both in vitro and in vivo. In vitro reaction of DES with nuclei from hamster liver or kidney in the presence of cumene hydroperoxide or NADPH revealed binding of [3H]DES in nuclear proteins (histones; nonhistones precipitable by 2% TCA, NH2; nonhistones soluble in 2% TCA, NH30). The binding was significantly inhibited by cytochromes P450 inhibitors. In an in vitro system [3H]DES quinone, one of the metabolites of DES, was able to bind to pure nonhistone proteins RNA polymerase and DNA polymerase. The binding of [3H]DES quinone to nonhistones RNA polymerase and DNA polymerase was inhibited by low molecular weight thiols, i.e. glutathione and cysteine, or thiol modifiers, such as n-ethylmaleimide, dithionitrobenzoic acid and hydroxymercuric benzoate. DES and DES metabolites inhibited transcriptional activity. In vivo [3H]DES was able to bind to nuclear proteins of hamster liver, kidneys and testes. The level of in vivo [3H]DES binding to all three types of nuclear proteins (histones, NH2, NH30) in the kidney (target organ) was two or more fold higher than that observed in the liver or testis (nontarget organs). Four nuclear NH30 proteins (mol wts.: 56, 37, 33 and 28 kDa) were irreversibly bound to [3H]DES in vivo. The in vivo binding of [3H]DES to transcriptionally active chromatin NH30 proteins also was observed. The data reported here establish that DES was able to bind to liver or kidney nuclear proteins in vitro, which was catalyzed by nuclear enzymes when fortified with an appropriate cofactor. DES quinone may be one of the protein binding metabolites. DES and DES metabolites inhibited transcriptional activity. The level of in vivo binding of [3H] DES to nuclear proteins of kidney (target organ) was double in comparison with that observed in liver or testis (nontarget organs). In vivo modifications in the chromatin proteins may be a factor in the development of DES-induced renal carcinogenesis is not clear.

Animals↗
PubMed1995

Phosphorylation of tyrosine residues of RNA polymerase II and other nuclear proteins by active chromatin tyrosine kinase(s).

We demonstrate here for the first time that protein tyrosine kinases are present in the active chromatin of nucleus. The presence of tyrosine kinase activity in the active chromatin was initially determined using poly (Glu,Na-Tyr;4:1) (PGT) as a tyrosine phosphorylatable substrate. Active chromatin in the presence of cofactors phosphorylated PGT at a rate of 40 pmol/min. The phosphorylation of PGT by active chromatin was inhibited by 41, 47 and approximately 95% with genistein, n-ethylmaleimide and quercetin (known inhibitors of tyrosine kinases), respectively. A Lineweaver-Burk plot revealed an apparent Km of 50 micrograms/ml and Vmax of 45 pmol/min for active chromatin tyrosine kinase(s). Analyses of phosphorylation of endogenous substrates by immunoprecipitation, western blotting and phosphoamino acids revealed that active chromatin protein tyrosine kinase(s) are able to phosphorylate tyrosine residues of the large subunit of RNA pol II and several other active chromatin proteins. The ability of AC-PTKs to phosphorylate many proteins of active chromatin components argues strongly for its role(s) in regulating transcription.

Animals↗
PubMed1995

In vivo DNA adduct formation by bisphenol A.

We have previously shown that bisphenol A (BPA) is oxidized to bisphenol-o-quinone in the presence of activation system and that the chemical reaction of DNA or deoxyguanosine 3'-monophosphate (dGMP) with bisphenol-o-quinone produces adducts. In the present study, using the 32P-postlabeling technique, we have investigated the in vivo DNA adduct formation by BPA by examining covalent modification in DNA. Administration of a single or multiple dose of 200 mg/kg of BPA to CD1 male rats produced two major and several minor adducts in liver DNA. The two major in vivo adducts matched the adduct profile of DNA or dGMP-bisphenol-o-quinone. To determine how BPA may be converted to DNA-binding metabolites, adducts were examined after incubation of DNA with BPA in the presence of a microsomal activation system. The in vitro incubation of BPA with DNA in the presence of a microsomal activation system revealed one major adduct and several minor adducts. The formation of adducts in DNA by BPA in the presence of a microsomal activation system was drastically decreased by known inhibitors of cytochrome P450. Adduct formation in DNA when cumene hydroperoxide or NADPH was used as a cofactor showed adducts with similar chromatographic mobilities as those from the reaction of dGMP-bisphenol-o-quinone. These data demonstrate that BPA is capable of binding covalently to DNA. DNA binding can be inhibited by the inhibitors of cytochrome P450. One of the DNA-binding metabolite(s) both in vitro and in vivo may be bisphenol-o-quinone. Covalent modifications in DNA by in vivo exposure of BPA may be a factor in the induction of hepatotoxicity.

Animals↗
PubMed1995

Decreased cyclic inositol phosphohydrolase activity in hamster renal tumors and human renal cell carcinomas.

Cyclic inositol phosphohydrolase (cIPH) (EC 3.1.4.36) hydrolyzes the cyclic phosphodiester bond of cyclic inositol monophosphate to yield inositol 1-phosphate. Renal tissue in various species has been shown to contain the highest activity of this enzyme. Cyclic inositol monophosphate, the substrate for this enzyme, has been implicated in the regulation of cell density. Thus, we determined whether activity of this enzyme is modulated in renal tumor. Samples of hamster renal tumors induced by diethylstilbestrol (DES) demonstrated 87% less cIPH activity than control hamster kidney. After 8 days of DES treatment of hamsters, a 28% decrease in the cIPH activity in kidney was observed. Interestingly, comparison of the cIPH activity in normal human kidney cortex, medulla, and renal cell carcinoma gave the following values (mean +/- SD), 10.6 +/- 4.6 (9), 6.7 +/- 4.6 (5), and 1.9 +/- 2.3 (8), respectively. Our study demonstrates that cIPH activity is reduced in hamster and human renal tumors.

Animals↗
PubMed1995

Molecular cloning and expression of DNA encoding ovine interleukin 2.

We have generated DNA encoding the mature form of ovine interleukin 2 (IL-2) by polymerase chain reaction (PCR) using primers complementary to sequences at the 5' and 3' ends of human, murine and bovine IL-2 cDNA. The predicted PCR product of 400 bp was ligated into the yeast Ty-P1 galactose-inducible expression vector pOGS40 which was used to transform yeast spheroplasts. The fusion protein, with a Factor Xa proteolytic cleavage site between ovine IL-2 and the P1 fusion partner, was expressed from galactose-induced transformed yeast. P1:IL-2 fusion protein, which self-assembles into virus-like particles (VLPs) due to the interaction of the P1 protein, was purified from lysates of mechanically disrupted yeast by centrifugation on a discontinuous sucrose gradient. Fusion protein was detected in Western blot analysis with polyclonal antisera raised to recombinant bovine IL-2. Soluble recombinant ovine IL-2 was released from the P1 fusion protein by cleavage with Factor Xa enzyme. After purification recombinant ovine IL-2 was functionally active as shown by its ability to support the proliferation of Con A-activated T cells and was capable of generating maedi visna virus-specific cytotoxic T cells from primed precursor cells. The availability of recombinant ovine IL-2 will greatly help the analysis of the specificity of pathogen-specific cells in the sheep.

Amino Acid Sequence↗
PubMed1995

Histone nuclear proteins are irreversibly modified by reactive metabolites of diethylstilbestrol.

We demonstrate for the first time that diethylstilbestrol (DES), a synthetic estrogen, is converted by nuclei to histone-binding metabolite(s). Reaction of [3H]DES with nuclei in the presence of cumene hydroperoxide or NADPH revealed binding of [3H]DES to histone nuclear proteins. Gel electrophoresis experiments revealed that all five histones, 1, 2A, 2B, 3, and 4, were irreversibly bound to [3H]DES. Histones 1 and 3 were more susceptible to the attack by [3H]DES quinone, a metabolite of DES, than histones 2A, 2B, or 4. The kinetic constants, Km and Vmax, of this binding reaction in the presence of cumene hydroperoxide were 10 microM and 750 pmol/mg protein/30 min, respectively. This binding was significantly inhibited by cytochromes P-450 inhibitors. Low-molecular-weight thiols, such as glutathione and cysteine, or thiol modifiers, such as n-ethylmaleimide, dithionitrobenzoic acid, and hydroxymercuric benzoate, drastically inhibited binding of [3H]DES quinone to histone 3. The binding of [3H]DES metabolites to both transcriptionally active and inactive chromatin histone proteins was observed. We conclude that DES is metabolized to histone-binding metabolites, presumably by nuclear cytochrome P-450. DES quinone may be one of the histone-binding DES metabolites. These data suggest that an analogous in vivo modification in the transcriptionally active chromatin histones by DES metabolites may influence gene function.

Animals↗
PubMed1995

Mitochondrial enzyme-catalyzed oxidation and reduction reactions of stilbene estrogen.

We have demonstrated for the first time that mitoplasts (i.e. mitochondria without outer membrane) were able to convert stilbene estrogen (diethylstilbestrol, DES) to reactive metabolites, which covalently bind to mitochondrial (mt)DNA. Depending on the cofactor used, mitochondrial enzymes catalyzed the oxidation and/or reduction of DES. DES was oxidized to DES quinone by peroxide-supported mitochondrial enzyme. A Lineweaver-Burk plot of rate of formation of DES quinone at various substrate concentrations yielded a Km of 33 microM and Vmax of 39 nmol/mg protein/min. The oxidation of DES to DES quinone by mitochondria was drastically decreased by known inhibitors of cytochrome P450. DES quinone was reduced to DES by mitoplasts in the presence of NADH. The Km and Vmax for the DES quinone reduction in the absence of mitoplasts and NADH were 3.2 microM and 5.6 nmol respectively. The reduction of DES quinone to DES by mitoplasts was significantly inhibited by inhibitors of cytochrome b5 reductase and diaphorase. DES quinone was also reduced to DES by pure diaphorase, a mitochondrial reducing enzyme, in the presence of NADH. The Km and Vmax for the DES quinone reduction by diaphorase were 9.0 microM and 4.3 nmol respectively. Under reaction conditions similar to oxidation of DES to DES quinone by mitoplasts, it was observed that mitochondrial metabolic products of DES were able to covalently bind to mtDNA. These data provide direct evidence of mitochondrial enzyme-catalyzed oxidation and reduction reactions of DES. In the cell, activation of DES in the mitochondria (the organelle in which mtDNA synthesis, mtDNA repair and transcription systems are localized) is of utmost importance, because an analogous in vivo mitochondrial metabolism of DES through covalent modifications in mitochondrial genome may produce instability in the mitochondrial genome of the cells. These modifications may in turn play a role in the development of DES-induced hepatocarcinogenicity.

Animals↗
PubMed1995

Organ-specific inhibition of types I, II and III transcriptional activity in hamsters exposed to stilbene estrogen.

We have previously shown that stilbene estrogen (diethyl-stilbestrol, DES) covalently binds to nonhistone nuclear proteins both in vivo and in vitro. In this study, we demonstrate the differential effects of DES exposure on in organelle transcriptional activity in nuclei isolated from kidney (target organ of cancer) and liver (non target organ) of hamsters. Kidney RNA polymerase (RNA pol) I and III activities were significantly inhibited by 50% at days 8 and 15 of DES exposure compared to that of controls. Liver RNA pol I and III activities were only modestly inhibited (17 and 22%, respectively) by 2 and 8 days of DES exposure, respectively. However, longer exposure of DES to animals did not produce any significant effects on RNA pol I activity. The activity of RNA pol II was affected by DES exposure in both liver and kidney. DES treatment for two days resulted in an increase in RNA pol II activity in kidney. The enhanced enzyme activity was decreased to 50% of that of the control at 15 days of DES treatment. Unlike RNA pol I and III, RNA pol II activity in the liver was inhibited in a time-dependent fashion in response to DES exposure. To understand the mechanism of transcriptional inhibition by DES, we analyzed the effect of DES exposure on the expression of hepatic RNA pol II at both mRNA and protein levels and also phosphorylation of hepatic RNA pol II. The total amount of transcripts or protein contents of hepatic RNA pol II was not altered in response to DES exposure to hamster for 15 days. Total phosphorylation of hepatic RNA pol II was also not affected by 15 days DES exposure. However tyrosine phosphorylation of hepatic RNA pol II was lowered by 2.8-fold compared to that of control enzyme in response to DES exposure for 15 days. An inhibitory effect of DES on the total RNA polymerase activity in both kidney and liver nuclei in the presence of endogenous template was observed in vitro. No inhibitory effect of DES was observed in vitro on transcriptional activity in the presence of exogenously added DNA template. Based on these data it appears that the in vivo inhibition of transcription by DES may be due to alterations in chromatin template or the level of transcription regulating proteins and not due to decreased availability of the chromatin template and/or RNA polymerase. Whether DES related inhibition of transcriptional activity plays a role in the development of kidney cancer is not clear.

Animals↗
PubMed1995

Activation of phosphorylation of plasma membrane insulin-like growth factor-I receptors in the kidney of Syrian hamsters by diethylstilbestrol.

In the present work we have investigated activation of phosphorylation of plasma membrane insulin-like growth factor-I receptors (IGF-IR) by diethylstilbestrol (DES). Insulin-like growth factor-I (IGF-I) stimulated the activity of membrane protein tyrosine kinase(s) (PTK) in both normal and DES-treated hamster kidneys. The level of IGF-I-stimulated PTK(s) almost doubled after 15 days of DES treatment. Autophosphorylation experiments revealed that phosphorylation of a 95 kDa band (presumably the beta subunit of IGF-IR) was 2-fold higher in the membranes of kidney from DES-treated animals compared with controls. To understand the mechanism of activation of IGF-I-dependent PTK by DES, we investigated the relationship between the binding capacity of IGF-I to membrane proteins and the level of IGF-IR. The binding of [125I]IGF-I to membranes from the DES-treated group was 30% higher than that of age-matched normal kidney (P < 0.001). Scatchard analysis of the binding data for both normal and DES-treated hamster kidney revealed a single class binding site for IGF-I with a dissociation constant (Kd) of 4.1 and 4.6 nM and a maximum binding capacity (Bmax) of 1786 and 2086 fmol/200 micrograms protein respectively. Therefore, the difference observed in [125I]IGF-I binding between DES-treated and normal kidney membranes may be partially due to an increase in the number of IGF-I binding sites, with no change in the affinity of the receptors for IGF-I. An enhanced level of IGF-IRs in membranes from DES-treated animals was visualized by autoradiography following affinity labeling of membrane proteins subjected to SDS-PAGE. Under reducing conditions a molecular band of 132 kDa was evident. The 132 kDa band represents the alpha-subunit of IGF-IRs. Northern blot analyses revealed that DES treatment increased the level of IGF-IR mRNA 2-fold compared with that of controls. These findings suggest that an enhanced level of IGF-IR coupled with qualitative changes may be responsible for the activation of IGF-I-dependent PTK on DES exposure. Whether the stimulation of IGF-IR phosphorylation by exposure to a carcinogenic dose of DES may be a factor in the induction of renal cancer in Syrian hamsters is not clear.

Animals↗
PubMed1995

Estrone-induced cell proliferation and differentiation in the mammary gland of the female Noble rat.

In this study we examined the influence of estrone on proliferation and differentiation in the mammary gland of female Noble rats. Estrone treatment increased proliferation in mammary epithelial cells at days 3 and 7, and peaked by day 11 of estrone exposure. In addition, estrone exposure altered cell cycle kinetics. Animals exposed to estrone for 11 days demonstrated a 9-fold increase in the cells in G1 phase and a 48-fold increase in the cells in S phase, compared to those of controls. Differentiation, measured by the degree of lobular maturation, was significantly increased in the treatment group by day 7 and continued to mature through day 21 post estrone implant. The appearance of the mammary glands in these animals approached the morphology normally found during pregnancy and lactation. In a subset of animals we examined the effects of a concurrent treatment with luteolin, a bioflavonoid which has been shown to inhibit estrone binding to type II estrogen receptors. Luteolin significantly reversed the effects of estrone treatment on both proliferation and differentiation in these animals. Changes in proliferation and cell cycle kinetics have been shown to lead to genetic instability, ultimately resulting in cell transformation. Our results indicated an increase in proliferative cells by > 2-fold and a perturbation in cell cycle kinetics from estrone exposure. These changes may play a role in the induction of estrone-induced mammary cancer in the Noble rat model. In addition, the anti-proliferative action of luteolin suggests that it may play a protective role in estrone-induced mammary carcinogenesis.

Animals↗
PubMed1995

Transforming growth factor alpha gene expression in cultured human keratinocytes is unaffected by cellular aging.

BACKGROUND: Cultured human keratinocyte grafts have been shown to stimulate endogenous reepithelialization of both chronic nonhealing and acute partial-thickness wounds. This effect is most likely mediated by cytokines that stimulate keratinocyte growth, such as transforming growth factor alpha. The effect of cellular age on cytokine expression by cultured grafts used for this purpose is presently undefined. In this study, transforming growth factor alpha gene expression in cultured foreskin keratinocytes from donors varying in age from 2 to 82 years was analyzed semiquantitatively by two separate methods, ie, Northern hybridization and competitive polymerase chain reaction. RESULTS: No pattern of decline in transforming growth factor alpha messenger RNA expression with increasing cellular age was observed by either analysis. CONCLUSION: The results indicate that expression of transforming growth factor alpha by cultured grafts may not be significantly affected by increasing cellular age and suggest that, even in the elderly, cultured autografts may be effective as pharmacologic agents for wound treatment.

Adult↗
PubMed1995

Risk factors and angiographic profile of coronary artery disease in young.

A total of 124 patients of ischaemic heart disease under 40 years of age (96 with myocardial infarction and 28 with angina) were studied for risk factors of coronary artery disease. Electrocardiogram, treadmill test, lipid profile and coronary arteriography were done in all cases. Smoking (56.4%) and hyperlipidaemia (30.6%) emerged as the major risk factors. Further stratification of lipid profile revealed that predictive value of hypercholesterolaemia could be enhanced by considering the different ratios of lipoproteins and indices of atherogenicity. Coronary arteriography revealed a preponderance of single vessel disease (48.4%)-left anterior descending being most commonly involved (71.8%). Increase in low density lipoprotein fraction was related to multivessel involvement.

Adult↗
PubMed1995

[Spontaneous dissection of the cervical arteries].

The spontaneous cervical artery dissection (SCAD) is one of the main causes of cerebro-vascular accident in the young adult. It may results from minor cervical trauma. The pathophysiological basis of the neurological complications remain unclear. For that reason, the treatment of SCAD is often empirical. In a retrospective study (1984-1994), we have reviewed 20 cases admitted to Notre-Dame Hospital for a SCAD confirmed by angiography. The initial clinical diagnosis was missed in 65% of the cases despite a suggestive symptomatology (headache 80%, hemiparesis 65% and Horner 45%). We have studied the clinical evolution as related to blood pressure, volemia, haematocrit and the administered treatment (aspirin, Coumadin). 60% of the patients (mean age 39) deteriorated in the hours following their admission. Their was one death (5%) and 85% had neurological sequelae. This study suggest that SCAD is a potentially serious disease. A delayed diagnosis may lead to a poor prognosis. Early aggressive treatment aiming at an optimal cerebral perfusion could improve the patients outcome.

Adolescent↗