Erythrocyte membrane proteins reactive with human (warm-reacting) anti-red cell autoantibodies.

Immunoglobulin G (IgG) autoantibodies of 20 patients with autoimmune hemolytic anemia (AHA) were used in immunoaffinity assays with surface-radioiodinated human red blood cells (RBCs), and detergent-solubilized products were analyzed by SDS-PAGE/autoradiography. Four membrane proteins were identified as candidate autoantigens: a nonglycosylated polypeptide with an apparent molecular mass of 34 kD (p34) that was expressed in all available RBC phenotypes except Rhnull but differed consistently in apparent molecular mass from the 32-kD Rh(D) polypeptide co-isolated by IgG allo-anti-D; a heterogenous 37-55-kD glycoprotein, also deficient in Rhnull RBCs, which disappeared after deglycosylation by N-glycanase, with the appearance of a sharp, new approximately 31-kD band distinct from p34 and from Rh(D) polypeptide; a approximately 100-kD major membrane glycoprotein identified by immunoblotting as the band 3 anion transporter; and glycophorin A (GPA), also confirmed by immunoblotting. GP37-55 was not seen in the absence of p34, and both proteins are likely to be members of the Rh family. Indeed, a 34-kD polypeptide band and 37-55-kD poly-disperse "smear," isolated concurrently from the same labeled RBCs by IgG allo-anti-e, were indistinguishable from their autoantibody-isolated counterparts and may well be the same protein identified at different epitopes by the auto- and allo-antibodies. Individual AHA patients' autoantibodies isolated p34 and gp37-55, alone or in combination with band 3 (nine cases); strong band 3 alone (five cases); and combinations of band 3 with GPA (six cases). The autoantibodies of three additional patients whose AHA had been induced by alpha-methyldopa also isolated p34 and gp37-55.

Human cytosolic sulfotransferase 2B1: isoform expression, tissue specificity and subcellular localization.

Sulfation is an important Phase II conjugation reaction involved in the synthesis and metabolism of steroids in humans. Two different isoforms (2B1a and 2B1b) are encoded by the sulfotransferase (SULT) 2B1 gene utilizing different start sites of transcription resulting in the incorporation of different first exons. SULT2B1a and SULT2B1b are 350 and 365 amino acids in length, respectively, and the last 342 aa are identical. Message for both SULT2B1 isoforms is present in human tissues although SULT2B1b message is generally more abundant. However, to date only SULT2B1b protein has been detected in human tissues or cell lines. SULT2B1b is localized in the cytosol and/or nuclei of human cells. A unique 3'-extension of SULT2B1b is required for nuclear localization in human BeWo placental choriocarcinoma cells. Nuclear localization is stimulated by forskolin treatment in BeWo cells and serine phosphorylation has been identified in the 3'-extension. SULT2B1b is selective for the sulfation of 3beta-hydroxysteroids such as dehydroepiandrosterone and pregnenolone, and may also have a role in cholesterol sulfation in human skin. The substrate specificity, nuclear localization, and tissue localization of SULT2B1b suggest a role in regulating the responsiveness of cells to adrenal androgens via their direct inactivation or by preventing their conversion to more potent androgens and estrogens.

Expression of cytosolic sulfotransferases in normal mammary epithelial cells and breast cancer cell lines.

Breast cancers require the presence of estrogens for the maintenance of growth at some time in the course of their development, as does normal breast tissue. Sulfation is an important process in the metabolism and inactivation of steroids, including estrogens, because the addition of the charged sulfonate group prevents the binding of the steroid to its receptor. Also, many of the therapeutic and chemopreventive agents used in the treatment of breast cancer are substrates for the sulfotransferases (STs). The activity and expression of four cytosolic STs, which are the human phenol-sulfating and monoamine-sulfating forms of phenol ST (PST), dehydroepiandrosterone ST, and estrogen ST (hEST), were assayed in normal breast cells and in breast cancer cell lines. ST activities and immunoreactivities were assayed in the estrogen receptor-positive human breast cancer cell lines ZR-75-1, T-47D and MCF-7; in the estrogen receptor-negative breast cancer cell lines BT-20, MDA-MB-468, and MDA-MB-231; and in normal human mammary epithelial cells. The PSTs were the most highly expressed ST activities in the breast cancer cell lines, although the levels of activity varied significantly. ZR-75-1 and BT-20 cells possessed the highest levels of activity of the human phenol-sulfating form of PST. The breast cancer cell lines showed only trace levels of dehydroepiandrosterone ST and hEST activities. In contrast, hEST was the only ST detectable in human mammary epithelial cells. Understanding the regulation of ST activity in these breast cancer and normal breast cell lines will improve our knowledge of the role of sulfation in breast cancer and provide a model with which to study the mechanism of action of estrogens in mammary cells.

Regulation of estrogen sulfotransferase in human endometrial adenocarcinoma cells by progesterone.

During the secretory phase of the human menstrual cycle, the endometrium is minimally responsive to the estrogens secreted from the ovaries. Conjugation of beta-estradiol (E2) with sulfate is thought to be an important mechanism in the regulation of the levels of active E2 in endometrial tissue. Estrogen sulfation is reportedly increased during the secretory phase in response to the high levels of progesterone secreted by the ovaries. Estrogen sulfotransferase (hEST), a distinct form of human cytosolic sulfotransferase (ST) with an affinity for E2 and estrone at low nanomolar concentrations, has recently been cloned and expressed in mammalian cells and in bacteria (J Steroid Biochem Mol Biol 52:529, 1995). At least two other forms of human cytosolic ST, dehydroepiandrosterone ST (hDHEA-ST) and the phenol-sulfating form of phenol-ST (hP-PST), also conjugate estrogens but at micromolar concentrations. This report describes the specific induction of hEST in human Ishikawa endometrial adenocarcinoma cells by progesterone as a model for the increases in estrogen sulfation observed in women during the secretory phase of the menstrual cycle. Treatment of Ishikawa cells with 10 microns progesterone for 48 h resulted in a 7-fold increase in the sulfation of 20 nM E2. The sulfation of selective substrates for human dehydroepiandrosterone sulfotransferase (hDHEA-ST) and the two forms of phenol sulfotransferase (hP-PST, hM-PST) were not affected by treatment with progesterone. The levels of immunoreactive hEST and hEST mRNA in the Ishikawa cells were both increased by progesterone, whereas the levels of immunoreactive hDHEA-ST, hP-PST, and hM-PST were not altered. hEST activity was not induced by treatment of Ishikawa cells with varying concentrations of E2, testosterone, or cortisol. The induction of hEST by progesterone was inhibited by RU-486, indicating that progesterone is acting via the progesterone receptor. These results indicate that progesterone is capable of specifically inducing hEST and estrogen sulfation in human Ishikawa adenocarcinoma cells and suggest a mechanism for increasing estrogen sulfation in the endometrium during the secretory phase of the menstrual cycle.

Human fetal pancreas--a potential source for transplantation.

Human fetal pancreas (HFP) represents an ideal tissue source for transplantation into diabetic patients. Transplantation of HFP lacks many of the technical problems associated with whole organ transplantation and HFP is readily available. In order to proceed with clinical HFP transplantation it must be demonstrated that HFP can reverse experimentally induced diabetes, that HFP can respond to glucose challenge in a manner similar to adult tissue, and that the immunogenicity of HFP can be reduced. We transplanted HFP (13-17 weeks gestational age) beneath the kidney capsule of streptozotocin-induced diabetic BALB/c nu/nu mice. Within 6 to 8 weeks following transplantation, 6 out of 7 (88%) animals became normoglycemic. Oral glucose tolerance tests were performed to determine in vivo graft function. Results in cured animals were identical to those of normal BALB/c nu/nu mice. In vitro insulin response to glucose challenge demonstrated that grafted HFP was capable of insulin secretion in the presence of high glucose while fresh fetal tissue was not. Human passenger leukocytes, identified immunohistologically at various times after transplantation with monoclonal antibodies to HLA-DR and Leu 10, were greatly reduced by 32 weeks posttransplant. Our data demonstrate that HFP will differentiate and mature in the diabetic nude mouse and that human passenger leukocyte content can be reduced. These findings suggest that HFP is functionally suitable for transplantation into diabetic patients.

Identification and characterization of cytosolic sulfotransferase activities in MCF-7 human breast carcinoma cells.

MCF-7 human mammary carcinoma cells have been reported to possess beta-estradiol and dehydroepiandrosterone sulfotransferase activities. These steroid sulfotransferase activities may be important in the metabolism and activity of different steroids in these cells. This report describes and characterizes both the enzymatic activity of three cytosolic sulfotransferases found in MCF-7 cells and the corresponding immunoblot analysis of these enzymes with specific anti-sulfotransferase antibodies. Two cytosolic sulfotransferases have been purified and characterized from human tissues which are capable of sulfating estrogens. These are the phenol-sulfating form of phenol sulfotransferase (P-PST) and the hydroxysteroid sulfotransferase, dehydroepiandrosterone sulfotransferase (DHEA-ST). The results of this study show that P-PST is the major cytosolic sulfotransferase found in MCF-7 cytosol and is responsible for most of the beta-estradiol sulfation in these cells. Although DHEA-ST activity was found in MCF-7 cytosol, this activity was only about 3% of the P-PST activity. Immunoblot analysis of MCF-7 cytosol detected both P-PST and lower levels of the monoamine-sulfating form of PST; however DHEA-ST could not be detected apparently because of low levels of expression. Human liver P-PST was expressed in Cos-7 Green monkey kidney fibroblasts and the ability of the cloned enzyme to sulfate beta-estradiol was investigated. This study indicates that P-PST is the prevalent cytosolic sulfotransferase in MCF-7 cytosol and is responsible for the majority of beta-estradiol sulfation in these cells.

Bacterial expression and characterization of a cDNA for human liver estrogen sulfotransferase.

A distinct human estrogen sulfotransferase (hEST-1) cDNA has been isolated from a human liver lambda Zap cDNA library using a PCR procedure. The enzymatically active protein has been expressed in two bacterial expression systems and the kinetic and immunologic properties of the enzyme have been characterized. The full-length cDNA for hEST-1 is 994 base pairs in length and encodes a 294 amino acid protein with a calculated molecular mass of 35,123 Da. Purified hEST-1 migrated with an apparent molecular mass of 35,000 Da during SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of hEST-1 expressed in E. coli with a rabbit anti-hEST-1 antibody yields a band of approximately 35,000 Da. The anti-hEST-1 antibody also detects a single band in human liver and jejunum cytosol which migrates with the same molecular mass as expressed hEST-1. There was also no cross-reactivity of hEST-1 with rabbit anti-hP-PST or rabbit anti-hDHEA-ST antibodies upon immunoblot analysis. hEST-1 was expressed in bacteria and purified to homogeneity. Expressed hEST-1 activity has a significantly greater affinity for estrogen sulfation than that found for the other human STs which conjugate estrogens. hEST-1 maximally sulfates beta-estradiol and estrone at concentrations of 20 nM. hEST-1 also sulfates dehydroepiandrosterone, pregnenolone, ethinylestradiol, and 1-naphthol, at significantly higher concentrations; however, cortisol, testosterone and dopamine are not sulfated. The results presented in this paper describe the expression and characterization of a human EST distinct from other human STs which sulfate estrogens. The high affinity of hEST-1 for estrogens indicates that this ST may be important in both the metabolism of estrogens and in the regulation of their activities.

Isolation and expression of an isoform of rat estrogen sulfotransferase.

A new isoform of rat liver estrogen sulfotransferase (EST), rEST-6, which is distinct from the previously reported rat EST [Demyan et al., Molec. Endocrinol. 6 (1992) 589], has been cloned, expressed, purified and characterized. A PCR procedure using oligonucleotide primers synthesized to the 5'-nontranslated and 3'-nontranslated regions of the published rEST sequence was used to isolate rEST-6 cDNA. The cloned DNA is 1000 bp in length and encodes a protein of 295 amino acids with a calculated molecular mass of 35,300 Da. rEST-6 is selectively expressed in male rats, as confirmed by Northern blot and immunoblot analyses. Northern blot analysis of male and female rat liver RNA with the rEST-6 cDNA as a probe shows a band with male RNA but not with female RNA. Similarly, immunoblot analysis of male and female rat liver cytosols with an antibody to rat EST yields a strong immunoreactive band in rat liver cytosol from male rats but not from females. Subsequent to bacterial expression and purification of rEST-6, the enzyme was analyzed kinetically and shown to sulfate estrogens but not dehydroepiandrosterone, pregnenolone, cortisol or testosterone. Maximal sulfation activity towards both beta-estradiol and estrone occurred at a concentration of 1 microM with substrate inhibition at higher concentrations. These results indicate that multiple, closely related forms of EST are present in rat liver. Analysis of the activity and regulation of these different EST enzymes is important in understanding estrogen metabolism in rats.

Regulation of estrogen activity by sulfation in human Ishikawa endometrial adenocarcinoma cells.

Sulfation is an important conjugation reaction in the metabolism of steroids. Steroids sulfates do not interact with the appropriate hormone receptors; additionally, the presence of the charged sulfate moiety increases the aqueous solubility and excretion of most steroids. Estrogen sulfotransferase (EST) is the major form of human cytosolic ST involved in the conjugation of estrogens. EST is important in the inactivation of beta-estradiol (E2) during the luteal phase of the menstrual cycle. EST has a significantly higher affinity for the sulfation of E2 and 17alpha-ethinylestradiol (EE2) than for other potent estrogens such as diethylstilbestrol (DES) and equine estrogens. The ability of EST to sulfate these estrogenic compounds at physiologic concentrations is important in regulating their activation of the ER in estrogen responsive cells. Human Ishikawa endometrial adenocarcinoma (ISH) cells possess an estrogen receptor (ER)-regulated alkaline phosphatase (AlkPhos) which is used to assay ER activation. To study the effects of EST activity on the ER activation of different estrogenic compounds, ISH cells were stably transformed with an EST expression vector. Dose-response curves for the induction of AlkPhos activity by the different estrogenic compounds were generated with EST/ISH and control pcDNA/ISH cells. EST/ISH cells were 200-fold less sensitive to E2 and EE2 than were control cells. No differences were observed in the dose response curves for DES between EST/ISH and pcDNA/ISH cells. EST/ISH cells were approximately 3-10-fold less sensitive to the equine estrogens equilin and 17-equilin as compared to control cells. The ability of EST to decrease the ER activation of an estrogen correlates with the sulfation of these compounds at nanomolar concentrations by EST/ISH and pcDNA/ISH ISH cells. These results indicate that EST is capable of efficiently inactivating E2 and EE2 but is significantly less effective in inhibiting the ER binding of other potent estrogenic compounds.

Steroid sulfation by expressed human cytosolic sulfotransferases.

The human cytosolic sulfotransferases (STs), dehydroepiandrosterone sulfotransferase (DHEA-ST) and the phenol-sulfating form of phenol sulfotransferase, (P-PST), have been expressed in bacteria and used to investigate the ability of the cloned enzymes to conjugate steroids and related compounds. DHEA-ST was capable of sulfating all of the 3-hydroxysteroids, testosterone and estrogens tested as substrates. The 3-hydroxysteroids, androsterone, epiandrosterone and androstenediol, were conjugated at 50-60% of the rate of DHEA. Of the steroids tested, P-PST was capable of conjugating only the estrogens. The catechol estrogens, 2-hydroxyestradiol, 4-hydroxyestradiol and 4-hydroxyestrone, and compounds with estrogenic activity such as 17 alpha-ethynyl-estradiol and trans-4-hydroxytamoxifen, were also tested as substrates. DHEA-ST showed little or no sulfation activity with these compounds; however, all of these compounds were sulfated by P-PST. These results indicate that the expressed human STs are valuable in analyzing the overlapping substrate specificities of these enzymes and that P-PST may have an important role in the metabolism of estrogens and estrogenic compounds in human tissues.

Activation of benzylic alcohols to mutagens by rat and human sulfotransferases expressed in Escherichia coli.

Human hydroxysteroid sulfotransferase, human phenol-sulfating form of phenol sulfotransferase, rat hydroxysteroid sulfotransferase a and rat phenol sulfotransferase IV were expressed in Escherichia coli. Cytosol preparations of transformed bacteria were used as activating systems in mutagenicity tests with Salmonella typhimurium TA98. All test compounds, 1-hydroxymethylpyrene, 2-hydroxymethylpyrene, 1-(1-pyrenyl)ethanol, 9-hydroxymethylanthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene and 4H-cyclopenta[def]chrysen-4-ol, were activated by both hydroxysteroid sulfotransferases investigated. However, 1-(1-pyrenyl)ethanol was 67-fold more efficiently activated by the human enzyme, whereas 7-hydroxymethyl-12-methylbenz[a]anthracene was 27-fold more efficiently activated by the rat enzyme. The phenol sulfotransferases showed relatively low activities with the benzylic alcohols investigated. The only exception was 4H-cyclopenta[def]chrysen-4-ol, which was activated efficiently by rat phenol sulfotransferase IV. We had previously tested the ability of rat and human hepatic cytosol preparations to activate the same compounds. The results of a statistical analysis suggest that the activities of human hydroxysteroid sulfotransferase, rat hydroxysteroid sulfotransferase a and phenol sulfotransferase IV can account for a substantial portion of the activation of benzylic alcohols in human, female rat and male rat liver, respectively.

Enhancing the virulence of Streptococcus sobrinus in rats.

The purpose of this study was to explore the virulence of strains of Streptococcus sobrinus that had been re-isolated from a desalivated rat. Furthermore, we wished to determine the influence of desalivation on the acidogenicity of fasting and sucrose-pulsed dental plaque in rats infected by strains of S. sorbrinus. Experimental groups were formed and infected as follows: Group 1, desalivated animals, S. sorbrinus ATCC27352; Group 2, intact animals, infected as in Group 1; Group 3, desalivated animals, S. sorbrinus re-isolated from a desalivated animal that had been infected with the ATCC 27352 strain for five weeks; Group 4, intact animals, infected as in Group 3; Group 5, desalivated animals, S. sobrinus re-isolated from a desalivated animal that had been infected with the ATCC27352 strain for ten weeks; Group 6, intact animals, infected as in Group 5; Group 7, intact animals, no infection. All animals were fed cariogenic diet 2000 and sucrose-sweetened drinking water ad libitum. After five weeks, desalivated and intact animals that had been infected with isolates obtained from desalivated animals had significantly higher (p less than 0.05) smooth-surface-caries scores than did intact and desalivated animals infected with cultures of the original stock ATCC strain. Moreover, fasting plaque pH values were statistically lower in desalivated animals (Groups 1 and 3) than in intact animals (Groups 2, 6, and 7) (Turkey analysis, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Caries incidence in intact rats infected with Streptococcus sobrinus via transmission from desalivated cagemates.

The purpose of this study was to determine the influence of caging desalivated S. sobrinus-infected rats on the caries incidence in intact, previously uninfected cagemates. Weanlings were divided as follows: Stage I: Twenty desalivated animals paired with 20 intact animals (Group I); 20 intact animals paired with 20 intact animals (Group II). Animals were infected with S. sobrinus and fed diet 2000 and 10% sucrose water. After five weeks, ten paired animals from each group were killed. Desalivated animals had statistically higher (p less than 0.05) caries scores than did other animals. Stage II: The remaining Stage I infected animals from Groups I and II were paired with 40 new uninfected animals 15 days of age and fed cariogenic diets. After five weeks, new intact animals paired with Stage I desalivated animals had significantly higher smooth-surface enamel caries scores (38.0; SE 7.84) than new intact animals paired with Stage I intact animals (11.15; SE 3.69). Moreover, new intact animals caged for only five weeks with Stage I desalivated animals developed smooth-surface caries (38.0; SE 7.84) to a degree comparable with that in all Stage I intact animals in the experiment for ten weeks (36.6, SE 9.5; and 30.05, SE 4.1). The data suggest that desalivation may select a readily transmissible strain of S. sobrinus, which demonstrates enhanced virulence.

The effect of chronic atropine treatment on salivary composition and caries in rats.

Many instances of salivary dysfunction in humans can be traced to the use of medications that have hyposalivary side-effects. In this study, atropine, a cholinergic antagonist, was administered chronically to rats by use of osmotic mini-pumps. Steady-state blood levels, similar to levels obtained in human multiple oral dosing, were thus maintained. Atropine delivered in this manner for 24 days was found to decrease protein concentration of parotid saliva (p less than 0.05) elicited by pilocarpine, and to increase smooth-surface caries scores (p less than 0.05) in rats fed a cariogenic diet. Parotid saliva collected via ductal cannulation from rats subjected to chronic atropine administration (and stimulated to secrete by pilocarpine) exhibited increased levels of two basic proline-rich proteins (Peak A and SP-3), as evaluated by SDS-PAGE, compared with those observed in saliva from controls. Cannulation of sublingual glands in animals receiving high doses of atropine produced no measurable secretion upon pilocarpine stimulation. Carbachol stimulation of dispersed cell aggregates of sublingual glands from sham-operated and high-dose atropine groups indicated that the glands responded similarly once the antagonist was washed from the system, implying that the lack of secretion in vivo was caused by antagonism of the cholinergic receptor by atropine. Our observations suggest that this model system can be exploited for determination of the effects of chronic administration of hyposalivary drugs on salivary composition and caries rates.

Regulation of estrogen activity by sulfation in human MCF-7 breast cancer cells.

Estrogen metabolism is closely associated with the growth, progression, and treatment of breast cancer because many breast cancers are dependent upon estrogens for both growth and progression. Factors that affect the intracellular metabolism of estrogens may be critical in altering the effects of estrogens on breast cancer cells. MCF-7 cells have been used as a model system to study the effects of estrogens on breast cancer cellular growth. Because normal human mammary epithelial (HME) cells contain estrogen sulfotransferase (EST), which is involved in the inactivation of estrogens via sulfation, and MCF-7 cells do not possess this enzyme, the absence of EST may be critical to the growth of MCF-7 cells in the presence of estrogens. To study the effects of EST on cellular growth, MCF-7 cells stably transformed with an EST expression vector were compared to control cells transformed with vector only. Sulfation of 20 nM E2 occurs more rapidly with MCF-7 cells transformed with EST than with the control cells, thereby rendering E2 physiologically inactive. Additionally, these EST/MCF-7 cells sulfate 20 nM 17 alpha-ethinylestradiol (EE2) at a rate similar to that for E2 but sulfate 20 nM diethylstibestrol (DES) much more slowly; these results correlate with the kinetic characteristics of EST for these steroids. EST/MCF-7 cells require higher concentrations of E2 to stimulate growth than do control MCF-7 cells, hypothetically because EST is inactivating E2 via sulfation, rendering it incapable of binding to the estrogen receptor (ER). The effects of EE2 are similar to those of E2 whereas DES is effective at lower concentrations because it is not inactivated by EST. Neither control nor EST/MCF-7 cells grow well in the complete absence of estrogens, as would be expected because MCF-7 cells are estrogen dependent. However, in medium that has not been treated to remove endogenous estrogens, EST/MCF-7 cells grow more slowly than control cells, most likely because EST is inactivating the estrogens in the medium, making them ineffective in stimulating growth. EST/MCF-7 cells possess EST at levels similar to HME cells and are less responsive to estrogens than are MCF-7 cells lacking EST. The loss of EST may be a factor in oncogenesis, which leads to altered estrogen metabolism in breast carcinoma cells.

Characterization of expressed human phenol-sulfating phenol sulfotransferase: effect of mutating cys70 on activity and thermostability.

The cDNA for human liver phenol-sulfating phenol sulfotransferase (P-PST) has been cloned and the active enzyme expressed in Cos cells and bacteria. Analysis of the sequence identified two cysteine residues, one of which is highly conserved in the phenol sulfotransferase gene family. Previous studies with the pure human liver enzyme suggested that the conserved cysteine may be involved in binding substrates. Bacterial expression of P-PST with the cysteine converted to a serine indicates that the cysteine is not essential for activity or substrate binding, however, the mutant enzyme is significantly more sensitive to thermal inactivation.

Identification and characterization of cytosolic sulfotransferases in normal human endometrium.

Understanding the factors which alter estrogen metabolism and activity in endometrial tissue is important because unopposed estrogen stimulation is an important risk factor in the development of endometrial carcinoma. The cyclic progression of the endometrium through proliferative and secretory phases is normally under the control of the ovarian hormones beta-estradiol (E2) and progesterone. One mechanism by which progesterone inhibits the activity of E2 in secretory endometrium is by elevating the degree of E2 sulfation, thereby reducing its ability to bind to the estrogen receptor and elicit a cellular response. Our laboratories have investigated the cytosolic sulfotransferases (STs) found in biopsies of both proliferative and secretory endometrium obtained from five normal pre-menopausal women who were not taking any drugs or steroids. Two of the human cytosolic STs were detected in human endometrial tissues. The phenol-sulfating form of phenol ST (P-PST) was found at varying levels in cytosol from both proliferative and secretory endometrium in all of the women studied but with no consistent correlation to the phase of the menstrual cycle. In contrast, estrogen ST (EST) was not detected in the proliferative endometrial cytosol of any of the women studied but was consistently found in all of the secretory endometrial cytosols. The presence and levels of these STs was confirmed by ST activity studies, immunoblot analysis and Northern blot analysis. These results indicate that the expression of EST in human endometrial tissues varies with the phase of the menstrual cycle and is most likely regulated by progesterone secreted from the ovaries.

Induction of rat hepatic drug metabolizing enzymes by dimethylcyclosiloxanes.

Low molecular weight dimethylcyclosiloxanes (DMCS) are important precursors in the synthesis of polydimethysiloxane polymers widely used in industry, and in medical and personal care products. The objective of this study was to characterize the ability of two DMCS, octamethylcyclosiloxane (D4) and decamethylcyclopentasiloxane (D5) to induce drug metabolizing enzymes in rats. Male and female Sprague-Dawley rats were administered 1, 5, 20, or 100 mg/kg D4 or D5 in corn oil daily by gavage for 4 days. Changes in the levels of activity and/or immunoreactivity of CYP1A1/2, CYP2B1/2, CYP3A1/2 and NADPH cytochrome P450 reductase in liver microsomes were examined. Significant increases were observed in the liver to body weight ratio in female rats administered either D4 or D5 at doses > or = 20 mg/kg. Increases in the liver to body weight ratio were observed in male rats treated with > or = 100 mg/kg D5 but not with D4. Relatively large increases in CYP2B1/2 enzymatic activity and immunoreactive protein were observed with increasing concentrations of both D4 and D5. Significant increases in 7-pentoxyresorufin O-depentylase (PROD) activity were also detected in male and female rats given D4 at doses > or = 5 mg/kg. D5 increased PROD activity in male rats at doses > or = 20 mg/kg and in female rats at doses > or = 5 mg/kg. 7-Ethoxyresorufin O-deethylase (EROD) activity was increased in both male and female rats receiving > or = 20 mg/kg D4 or > or = 5 mg/kg D5; however, no changes were detected in CYP1A1/2 immunoreactive protein in rats of either sex. D4 and D5 caused significant increases in CYP3A1/2 immunoreactive protein in only male rats treated with 100 mg/kg of either compound. However, significant increases were detected in CYP3A1/2 immunoreactive protein in female rats at D4 doses > or = 20 mg/kg and D5 doses > or = 5 mg/kg. Induction of NADPH cytochrome P-450 reductase immunoreactive protein was observed with D4 in female rats and in both male and female rats with D5. Induction of CYP2B/1/2, CYP3A1/2 and NADPH cytochrome P450 reductase was observed in rats treated with 50 mg/kg phenobarbital by intraperitoneal injection. Maximal CYP2B induction detected with D4 was approximately 50% of the increase observed with phenobarbital. In summary, D4 and D5 induced CYP2B1/2 in adult rat liver in a manner similar to that observed with phenobarbital; however, differences were observed between D4 and D5 in their ability to induce CYP3A1/2 and NADPH cytochrome P450 reductase. Female rats were more sensitive to the inductive properties of low doses of both DMCS than male rats whereas male rats were more responsive to phenobarbital induction.

Influence of sweetening agents in solution on dental caries in desalivated rats.

Sucralose (trichlorogalactosucrose), sorbitol and aspartame in drinking water induced little or no caries in desalivated rats infected with Streptococcus sobrinus and Actinomyces viscosus and receiving their essential nutrition by gastric gavage. In contrast, sucrose and fructose induced extensive decay. Only sucrose could sustain implantation of Strep. sobrinus in these animals. The populations of A. viscosus were sparse (0.3-0.5%) in the animals given fructose and sucrose. Large populations of A. viscosus occurred in the controls and in those given sucralose, sorbitol and aspartame. In a second experiment, where animals were also desalivated and receive diet 2000 ad libitum, sucrose in solution promoted caries whereas sucralose, aspartame and saccharin were without effect. Addition of 10 parts/10(6) F overcame the caries-promoting effect of sucrose in solution. There was no interaction between fluoride and other sweetening agents that affected the incidence of caries.

The effect of chronic propranolol treatment on salivary composition and caries in the rat.

Many drugs are known to affect salivary secretion. The purpose of this study was to explore the chronic effects of a commonly used beta-adrenergic blocker, propranolol. Adult rats were desalivated or treated for 28 days with propranolol HCl (10 or 20 mg/kg, daily) or sterile buffer (sham-operated control) using osmotic pumps for delivery. The parotid and submandibular glands of each rat were cannulated and secretion elicited by pilocarpine (10 mg/kg, intravenous). There were no statistical differences in salivary protein content (Lowry) or output among the groups (ANOVA, p greater than 0.05). Analysis of salivary proteins by SDS-PAGE revealed a constant profile for submandibular secretions, but peak A and SP-3 proline-rich proteins were not detectable in parotid saliva of animals treated with propranolol for the entire experiment. Significantly increased smooth-surface (p = 0.0003) and sulcal (p = 0.0011) caries scores were found within these propranolol groups (ANOVA). The findings provide further evidence that chronic administration of propranolol alters salivary composition by decreasing proline-rich proteins and concurrently enhances susceptibility to caries.