Polychlorinated biphenyl congeners that increase the glucuronidation and biliary excretion of thyroxine are distinct from the congeners that enhance the serum disappearance of thyroxine.

Polychlorinated biphenyl (PCB) congeners differentially reduce serum thyroxine (T(4)) in rats, but little is known about their ability to affect biliary excretion of T(4). Thus, male Sprague-Dawley rats were orally administered Aroclor-1254, Aroclor-1242 (32 mg/kg per day), PCB-95, PCB-99, PCB-118 (16 mg/kg per day), PCB-126 (40 µg/kg per day), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (3.9 µg/kg per day), or corn oil for 7 days. Twenty-four hours after the last dose, [(125)I]T(4) was administered intravenously, and blood, bile, and urine samples were collected for quantifying [(125)I]T(4) and in bile [(125)I]T(4) metabolites. Serum T(4) concentrations were reduced by all treatments, but dramatic reductions occurred in response to Aroclor-1254, PCB-99 [phenobarbital (PB)-type congener], and PCB-118 (mixed-type congener). None of the treatments increased urinary excretion of [(125)I]T(4). Aroclor-1254, PCB-118, TCDD, and PCB-126 (TCDD-type congener) increased biliary excretion of T(4)-glucuronide by 850, 756, 710, and 573%, respectively, corresponding to marked induction of hepatic UDP-glucuronosyltransferase (UGT) activity toward T(4). PCB-95 and PCB-99 did not induce UGT activity; therefore, the increased biliary excretion of T(4)-glucuronide was related to the affinity of congeners for the aryl hydrocarbon receptor. The disappearance of [(125)I]T(4) from serum was rapid (within 15-min) and was increased by Aroclor-1254, PCB-99 and PCB-118. Thus, reductions in serum T(4) in response to PCBs did not always correspond with UGT activity toward T(4) or with increased biliary excretion of T(4)-glucuronide. The rapid disappearance of [(125)I]T(4) from the serum of rats treated with PB-like PCBs suggests that increased tissue uptake of T(4) is an additional mechanism by which PCBs may reduce serum T(4).

G2 as an emerging rotavirus strain in pediatric gastroenteritis in southern Italy.

BACKGROUND: Human rotaviruses (HRVs) represent a major cause of acute gastroenteritis in children worldwide. It is estimated that they are responsible for a large number of diarrhea-associated hospitalizations in childhood each year. In Italy, limited data are available on the patterns of distribution of HRV G and P types. We report here the results of 2 years of rotavirus strain surveillance among children with severe gastroenteritis diagnosed in the town of Portici, Campania, southern Italy. METHODS: A total of 421 stool specimens from children between 6 months and 5 years of age and presenting acute diarrhea were collected and tested by routine diagnostic tests for HRV, adenovirus, astrovirus, norovirus, and common bacterial pathogens. RESULTS: The laboratory results showed that 110 of the 225 (26.1%) virus-positive samples contained HRVs. The different G and P rotavirus genotypes were analyzed by polymerase chain reaction (PCR). Among the VP7 genotypes identified, G1 and G2 were predominant, with percentages of 48.2 and 30.9%, respectively. G4, G9, and G10 were detected in a minority of cases. Among the VP4 genotypes, P[8] occurred the most frequently (56.4%), followed by P[4] (31.8%), and only a few P[10] and P[11] at percentages of 1.8 and 0.9%, respectively. CONCLUSION: Our epidemiological data of HRV strains will contribute to assessing the magnitude of the problem of HRV in the south of Italy.

Bioavailability of dioxin in soil from a 2,4,5-T manufacturing site.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) is a highly toxic contaminant produced in the manufacture of phenoxy herbicides. Despite its high TCDD content, soil from a contaminated area associated with a 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) manufacturing site in Newark, New Jersey, did not induce acute toxicity when administered to guinea pigs (the most sensitive species) by gavage. Analysis of liver samples demonstrated low bioavailability of TCDD from this soil. A comparative analysis of soils showed that Soxhlet extraction was necessary for the determination of TCDD on Newark soil, whereas solvent extraction was sufficient for soil from Times Beach, Missouri. The difference in the bioavailability of TCDD from these soils is correlated with TCDD extractability and may be related to the different compositions of the soils.

(Q)SAR studies to design new human choline kinase inhibitors as antiproliferative drugs.

Most of the signal transduction pathways are mediated by protein kinases regulating every aspect of cell function. Mutations which deregulate their expression or their function or both result in cancers. Therefore, protein kinase inhibitors have become the focus of development of new therapies for cancer. A comprehensive review of Choline kinase (ChoK) was published by us in 2003. Since then, molecular information of ChoK inhibitors has been accumulated. In this review, we intend to summarize the new lines of evidence that will include the design of the most active antiproliferative agents so far described against ChoK. Studies have been aimed at the establishment of structure-activity relationships and the structural parameters that define ChoK inhibitory and antiproliferative activities of a set of twenty-five acyclic biscationic pyridophane and forty acyclic biscationic quinolinephane compounds. The corresponding QSAR equation was obtained for the whole set of bisquinolinium compounds for the antiproliferative activity, taking into consideration the electronic parameter sigma(R) of R(4), the molar refractivity (MR) of R(8), and the lipophilic parameters clog P and pi(linker). The most potent antiproliferative agent shows an IC(50) = 0.45 microM, predicted by the QSAR equation, whilst its experimental value is IC(50) = 0.20 microM. Finally, toxicity assays were performed for the most promising compounds because of their interesting antiproliferative activities [IC(50 HT-29) = 0.70, 0.80, 1.50 and 1.90 microM] and low toxicity [LD(50) = 16.7, 12.5, > 25 and > 20 mg/kg of mouse]. These biological activities justify further analysis for antitumoral assays under in vivo conditions.

Characterization of latex-antineoplastic drug complexes by differential scanning calorimetry and microphotography.

Choline kinase inhibitors have recently been identified as potentially useful antitumoral agents. Here we determine the best conditions for obtaining drug-polymer complexes with 5-fluorouracil (5-FU), and JCR791B, a new drug representing a significant advance in the development of new molecules to inhibit tumour proliferation. As polymers we used the cellulose derivatives Aquacoat and Aquateric. The variables in the adsorption process measured were time to adsorbent-adsorbate equilibrium, pH and concentration. The drug-polymer complexes were characterized by differential scanning calorimetry and microphotography. Our results show that adsorption of 5-FU and JCR was similar with both polymers although slightly greater with Aquacoat. The chemical structure of the drug and its solubility in water and oil are fundamental characteristics that determine the performance of polymers as drug carriers able to provide controlled release.

Ionic and ligand-specific effects on the DNA binding of progesterone receptor bound to the synthetic progestin R5020 and the antiprogestin RU486.

Antiprogestin and other antihormones are valuable therapeutic agents in hormone-responsive cancers. A fundamental mechanism in the action of antiprogestin is its binding to PR,3 an intracellular protein that mediates the action of progesterone by direct interaction at the regulatory sites of responsive genes. To elucidate the mechanism of action of PR bound to agonistic and antagonistic ligands, we determined the binding affinity of rabbit uterine PR bound to R5020 and RU486, respectively, with different DNA sequences. We used 2 recombinant plasmid DNAs, pDHf14 and pDHf2, with 60- and 23-base pair inserts of potential Z-DNA-forming (dA-dC)n.(dG-dT)n sequences, respectively, parental plasmid pDPL6 with no insert, calf thymus DNA, and several synthetic polynucleotides in this study. The concentration of DNA required to elute 50% of the receptor bound to DNA-cellulose (EC50) was used as a measure of the relative binding affinity of the receptor for DNA. EC50 values of plasmids pDHf14 and pDHf2 were 1.2 +/- 0.5 (SD) and 2.5 +/- 0.6 micrograms/ml, respectively, for PR bound to R5020. In contrast, EC50 for control plasmid was 350 micrograms/ml. PR.R5020 had lower affinity for calf thymus DNA and polynucleotides compared with pDHf2 and pDHf14. Receptors complexed with the antiprogestin RU486 had lower affinity for the plasmids; EC50 values were 2.4 +/- 0.4 and 10 +/- 1 microgram/ml, respectively, for pDHf14 and pDHf2. This ligand-specific difference in DNA binding was amplified by the presence of 5 mM Mg2+ or Ca2+. The relative binding affinity of PR.R5020 to pDHf14 was 6- and 7-fold higher than that of PR.RU486, in the presence of 5 mM Mg2+ and Ca2+, respectively. These results show that PR.RU486 has lower binding affinity for specific DNA sequences than PR.R5020, but the binding affinity of both receptors is in a range that cannot preclude competitive interactions at the DNA recognition site. The effects of Mg2+ and Ca2+ on PR binding to DNA further suggest that these cations could affect PR recognition of DNA in a ligand-specific manner.

Choline kinase inhibitors as a novel approach for antiproliferative drug design.

Recent progress in deciphering the molecular basis of carcinogenesis is of utmost importance to the development of new anticancer strategies. To this end, it is essential to understand the regulation of both normal cell proliferation and its alterations in cancer cells. We have previously demonstrated that in ras-transformed cells there is an increased level of phosphorylcholine (PCho) resulting from a constitutive activation on choiline kinase (ChoK). The importance of ChoK for the regulation of cell proliferation has also been proposed since an inhibitor for this enzyme, hemicholinium-3 (HC-3), drastically reduces entry into the S phase after stimulation with growth factors. Here we report the synthesis of several new compounds which are highly specific inhibitors for ChoK, with up to 1000-fold or 600-fold increased inhibitory activity, compared to HC-3 under ex vivo or in vitro conditions respectively. These novel compounds also drastically reduce entry into the S phase after stimulation with specific growth factors. A more profound inhibition of cell proliferation was observed in ras-, src- and mos-transformed cells in the presence of ChoK inhibitors, compared to their parental, untransformed NIH3T3 cells. By contrast, this effect was not observed in fos-transformed cells. While ras, src and mos transformation is associated with elevated levels of ChoK activity, fos-induced transformation does not affect ChoK activity. The inhibitory effect on proliferation of the new compounds correlates with their ability to inhibit the production of phosphorylcholine in whole cells, a proposed novel second messenger for cell proliferation. These results strongly support a critical role of choline kinase in the regulation of cell growth and makes this enzyme a novel target for the design of new antiproliferative and anticancer drugs.

New once-daily, highly effective rescue triple therapy after multiple Helicobacter pylori treatment failures: a pilot study.

BACKGROUND: Helicobacter pylori treatment failure is a growing problem in daily practice. AIM: To determine the efficacy of the combination of rabeprazole, levofloxacin and furazolidone as a rescue therapy. METHODS: Duodenal ulcer patients previously submitted, without success, to at least two H. pylori treatment regimens were included. Gastroscopy (urease test, histological examination and culture) and (13)C-urea breath test were performed. All patients received a combination of rabeprazole 20 mg, levofloxacin 500 mg and furazolidone 200 mg (two tablets) administered in a single dose in the morning for 10 days. Clinical examination and a new (13)C-urea breath test were performed 90 days after therapy. RESULTS: Twelve patients (eight females and four males), mean age 43 (30-58) years were included. Two patients failed to complete the treatment because of nausea and vomiting. Ten patients completed the study and took all the medications as advised. Culture was obtained in six patients: 100 and 83% of the samples were sensitive to furazolidone and levofloxacin, respectively. Per-protocol and intention-to-treat eradication rates were 100 and 83% (P = 0.019). CONCLUSIONS: the combination of rabeprazole, levofloxacin and furazolidone in a single daily dose for 10 days constitutes a highly-effective and low-cost alternative as a third-line therapy in patients infected with H. pylori.

[Acute urinary retention and hematuria secondary to metastatic renal clear-cell carcinoma to the penis]. / Retención urinaria aguda y hematuria secundarias a metástasis peneanas por carcinoma renal de células claras.

Renal cell carcinoma have a great capacity of dissemination and have a great variety of clinical presentation. We exposed a clinical note of a patient diagnosed of renal cell carcinoma who developed hematuria and acute urinary retention due to a penis metastasis. Next we review the literature about this topic.

QSAR-derived choline kinase inhibitors: how rational can antiproliferative drug design be?

This review presents an overview of Choline Kinase (ChoK) inhibitors with antiproliferative activity. The consideration of ChoK as a novel target for the development of new anticancer drugs is justified. The synthesis of several derivatives based on structural modifications of hemicholinium-3 (HC-3) is not accompanied by potentiation of the neurological toxicity of HC-3. The increment of both ChoK inhibitory and antiproliferative activities was successfully obtained by the two following changes: a) substitution of the oxazonium moiety of HC-3 by several aromatic heterocycles, and b) using the 1,2-ethylene(bisbenzyl) moiety instead of the 4,4'-biphenyl fragment. In an attempt to understand the ChoK inhibitory activity, a quantitative structure-activity relationship was developed. The QSAR equations have described the forces involved in quantitative terms. The electron characteristic of the substituent at position 4 of the heterocycle and the lipophilic character of the whole molecule were found to significantly affect the antitumour activity in compounds 17-95. Trispyridinium compounds 91-95 are more potent than the bispyridinium ones 87-89 as ChoK inhibitors. Nevertheless, 91-95 are less active than 87-89 as antiproliferative agents because the latter show better lipophilicities to cross the cytosolic membranes. Inhibition of the growth of human tumours in nude mice has been demonstrated: Antitumour activity of compound 64 against human HT-29 produced a decrease of up to 70% in the size of the tumour in nude mice. These results indicate that ChoK can be used as a general target for anticancer drug design against Ras-dependent tumourigenesis.

From a classic approach in cancer chemotherapy towards differentiation therapy: acyclic and cyclic seven-membered 5-fluorouracil O,N-acetals.

Novel derivatives of 5-fluorouracil (5-FU) possessing a broader spectrum of antitumor activity and fewer toxic side effects than 5-FU have been sought. Herein, we report three different types of 5-FU O,N-acetals: a) a novel class of 5-fluorouracil-containing acyclonucleosides. The antitumor activities of such compounds were assessed against HEp human cells showing that (RS)-1-¿[3-(2-hydroxyethoxy)-1-cyclopentoxy]propyl¿-5-fluorouracil 3c is 4-fold more active than 5-FU. (RS)-1-¿[3-(2-hydroxyethoxy)-1-isopropoxy]propyl¿-5-fluorouracil 3b has important potential advantages over 5-FU because of its lower toxicity and its ability to induce myogenic differentiation in rhabdomyosarcoma cells. Our results suggest that this drug may be useful for differentiation therapy in this type of tumor; b) within the cyclic prodrugs of 5-FU, a series of new ring-expanded isosteres (1,4-oxaheteroepanes) of Ftorafur were synthesized. The level of diastereoselectivity in the preparation of cis and trans 1-(3-chloromethyl)-1,4-dioxepan-5-yl)-5-fluorouracil, although modest, suggests a potentially general approach for controlling the stereochemistry of this unexplored class of reactions involving the preparation of 5-FU seven-membered O,N-acetals; c) new 5-FU acyclic analogs containing two 5-FU moieties at both ends of the molecules with a linker having two amide bonds have been designed and synthesized. These bis(5-FU-O,N-acetals) show interesting antineoplastic activities against the HT-29 cell line.

Regulation of cell cycle and cyclins by 16alpha-hydroxyestrone in MCF-7 breast cancer cells.

It has been suggested that alterations in estradiol (E(2)) metabolism, resulting in increased production of 16alpha-hydroxyestrone (16alpha-OHE(1)), is associated with an increased risk of breast cancer. In the present study, we examined the effects of 16alpha-OHE(1)on DNA synthesis, cell cycle progression, and the expression of cell cycle regulatory genes in MCF-7 breast cancer cells. G(1) synchronized cells were treated with 1 to 25 nM 16alpha-OHE(1) for 24 and 48 h. [(3)H]Thymidine incorporation assay showed that 16alpha-OHE(1) caused an 8-fold increase in DNA synthesis compared with that of control cells, whereas E(2) caused a 4-fold increase. Flow cytometric analysis of cell cycle progression also demonstrated the potency of 16alpha-OHE(1) in stimulating cell growth. When G(1) synchronized cells were treated with 10 nM 16alpha-OHE(1) for 24 h, 62+/-3% of cells were in S phase compared with 14+/-3% and 52+/-2% of cells in the control and E(2)-treated groups respectively. In order to explore the role of 16alpha-OHE(1) in cell cycle regulation, we examined its effects on cyclins (D1, E, A, B1), cyclin dependent kinases (Cdk4, Cdk2), and retinoblastoma protein (pRB) using Western and Northern blot analysis. Treatment of cells with 10 nM 16alpha-OHE(1) resulted in 4- and 3-fold increases in cyclin D1 and cyclin A, respectively, at the protein level. There was also a significant increase in pRB phosphorylation and Cdk2 activation. In addition, transient transfection assay using an estrogen response element-driven luciferase reporter vector showed a 15-fold increase in estrogen receptor-mediated transactivation compared with control. These results show that 16alpha-OHE(1) is a potent estrogen capable of accelerating cell cycle kinetics and stimulating the expression of cell cycle regulatory proteins.

Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast cancer cells.

We investigated the effects of polyamine biosynthesis inhibition on the estrogenic signaling pathway of MCF-7 breast cancer cells using a protein-protein interaction system. Estrogen receptor (ER) linked to glutathione-S-transferase (GST) was used to examine the effects of two polyamine biosynthesis inhibitors, difluoromethylornithine (DFMO) and CGP 48664. ER was specifically associated with a 45 kDa protein in control cells. In cells treated with estradiol, nine proteins were associated with ER. Cells treated with polyamine biosynthesis inhibitors in the absence of estradiol retained the binding of their ER with a 45 kDa protein and the ER also showed low-affinity interactions with a number of cellular proteins; however, these associations were decreased by the presence of estradiol and the inhibitors. When samples from the estradiol+DFMO treatment group were incubated with spermidine prior to GST-ER pull down assay, an increased association of several proteins with ER was detected. The intensity of the ER-associated 45 kDa protein increased by 10-fold in the presence of 1000 microM spermidine. These results indicate a specific role for spermidine in ER association of proteins. Western blot analysis of samples eluted from GST-ER showed the presence of chicken ovalbumin upstream promoter-transcription factor, an orphan nuclear receptor, and the endogenous full-length ER. These results show that multiple proteins associate with ER and that the binding of some of these proteins is highly sensitive to intracellular polyamine concentrations. Overall, our results indicate the importance of the polyamine pathway in the gene regulatory function of estradiol in breast cancer cells.

Antagonistic and agonistic effects of tamoxifen: significance in human cancer.

Tamoxifen is a substituted triphenylethylene antiestrogen used in the adjuvant therapy and chemoprevention of breast cancer. The antiestrogenic activity of the compound has been attributed to its metabolism to an active 4-hydroxy derivative and the avid binding of the active metabolite to the estrogen receptor. Receptor binding of the antiestrogen alters the transcriptional activity normally attributed to the estradiol-bound estrogen receptor. Tamoxifen is both an antagonist and an agonist of the estrogen receptor. However, a molecular explanation exists for this apparent paradox. The dual action is a function of the estrogen receptor complex present in a particular cell or tissue. If a cell type requires activating factors 1 and 2 of the estrogen receptor to be functioning concurrently, tamoxifen is antagonistic. However, if a cell or tissue requires only activating factor 1 to interact with transcription factors at the promoter, tamoxifen is agonistic. The implication is that the investigators must understand the fundamental biology of the estrogen receptor complex in a tissue context before one can predict tissue activity of tamoxifen.

The effects of steroidal estrogens in ACI rat mammary carcinogenesis: 17beta-estradiol, 2-hydroxyestradiol, 4-hydroxyestradiol, 16alpha-hydroxyestradiol, and 4-hydroxyestrone.

Several investigators have suggested that certain hydroxylated metabolites of 17beta-estradiol (E2) are the proximate carcinogens that induce mammary carcinomas in estrogen-sensitive rodent models. The studies reported here were designed to examine the carcinogenic potential of different levels of E2 and the effects of genotoxic metabolites of E2 in an in vivo model sensitive to E2-induced mammary cancer. The potential induction of mammary tumors was determined in female ACI rats subcutaneously implanted with cholesterol pellets containing E2 (1, 2, or 3 mg), or 2-hydroxyestradiol (2-OH E2), 4-hydroxyestradiol (4-OH E2), 16alpha-hydroxyestradiol (16alpha-OH E2), or 4-hydoxyestrone (4-OH E1) (equimolar to 2 mg E2). Treatment with 1, 2, or 3 mg E2 resulted in the first appearance of a mammary tumor between 12 and 17 weeks, and a 50% incidence of mammary tumors was observed at 36, 19, and 18 weeks respectively. The final cumulative mammary tumor incidence in rats treated with 1, 2, or 3 mg E2 for 36 weeks was 50%, 73%, and 100% respectively. Treatment of rats with pellets containing 2-OH E2, 4-OH E2, 16alpha-OH E2, or 4-OH E1 did not induce any detectable mammary tumors. The serum levels of E2 in rats treated with a 1 or 3 mg E2 pellet for 12 weeks was increased 2- to 6-fold above control values (approximately 30 pg/ml). Treatment of rats with E2 enhanced the hepatic microsomal metabolism of E2 to E1, but did not influence the 2- or 4-hydroxylation of E2). In summary, we observed a dose-dependent induction of mammary tumors in female ACI rats treated continuously with E2; however, under these conditions 2-OH E2, 4-OH E2, 16alpha-OH E2, and 4-OH E1 were inactive in inducing mammary tumors.

Receptor mechanisms and dose-response models for the effects of dioxins.

There is increasing evidence that receptor-mediated events impact one or more stages responsible for tumor development in experimental animals and humans. Although many chemicals and endogenous hormones require receptor interactions as a necessary event in their carcinogenic activity, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its structural analogs are the most visible examples of receptor-mediated carcinogens. TCDD, or dioxin as it is frequently called, interacts with the Ah receptor (AhR), which functions in a manner analogous to receptors for steroids. TCDD produces a wide spectrum of biochemical and toxic responses in in vitro and in vivo systems, and the Ah receptor is generally considered necessary for most if not all of these responses. Risk assessments for dioxin made by the United States and other countries throughout the world have been based on its carcinogenecity in experimental animals. Recently, epidemiology studies have indicated that TCDD is a human carcinogen at high doses. Because TCDD appears to be acting like a potent and persistent hormone agonist, it appears reasonable to incorporate mechanistic information on receptor-mediated events in risk assessments for TCDD. This information may be obtained from steroid receptor action and from molecular data on the Ah receptor. In this paper, we evaluate the scientific foundation on which mechanistic models for estimating dioxin's risks should be based. These models need to recognize the mechanisms possible for the diversity of biological responses that are initiated by a single receptor interacting with a single ligand. The U.S. EPA is currently reevaluating dioxin's risks by examining the possibility of developing biologically based models.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of nitric oxide synthase activity and neuronal response in rat striatum by melatonin and kynurenine derivatives.

Tryptophan is mainly metabolized in the brain through methoxyindole and kynurenine pathways. The methoxyindole pathway produces (among other compounds) melatonin, which displays inhibitory effects on human and animal central nervous systems, including a significant attenuation of excitatory, glutamate-mediated responses. The kynurenine pathway produces kynurenines that interact with brain glutamate-mediated responses. Nitric oxide (NO) increases glutamate release, and melatonin and kynurenines may act via modification of NO synthesis. In the present study, the effects of melatonin and four synthetic kynurenines were studied on the activity of rat striatal nitric oxide synthase (NOS) and on the response of rat striatal neurons to sensorimotor cortex (SMCx) stimulation, a glutamate-mediated response. Melatonin inhibited both NOS activity and the striatal glutamate response, and these effects were dose-related. Compound A (2-acetamide-4-(3-methoxyphenyl)-4-oxobutyric acid) did not inhibit NOS activity but inhibited the striatal response similarly to melatonin. Compound B (2-acetamide-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid) was more potent than melatonin in inhibiting both NOS activity and the striatal response. Compound C (2-butyramide-4-(3-methoxyphenyl)-4-oxobutyric acid) did not change NOS activity, but increased the striatal response. Compound D (2-butyramide-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid) showed potent inhibitory effects on both NOS activity and striatal glutamate-mediated response. A structure-related effect of the kynurenine derivatives was observed, and those with an amino group in position 2 of the benzenic ring had more potent effects than melatonin itself in inhibiting striatal NOS activity and the response of striatal neurons to SMCx.

Polyamine-mediated conformational perturbations in DNA alter the binding of estrogen receptor to poly(dG-m5dC).poly(dG-m5dC) and a plasmid containing the estrogen response element.

The binding estrogen receptor (ER) to the upstream regions of estrogen-responsive genes, the estrogen response elements (ERE), is of fundamental importance in the regulation of gene expression by estradiol. Multiple cell-specific factors affect ER-ERE binding and modulate the responses of estradiol. We studied the role of polyamines in the recognition of ER, a ligand-activated transcription factor, with a left-handed Z-DNA forming polynucleotide as well as with a plasmid containing ERE. Polyamines are cellular organic cations with multiple functions in cell growth and differentiation. Polyamines induce Z-DNA conformation in alternating purine-pyrimidine sequences. To understand the role of polyamine-induced DNA conformational transition in ER-DNA interaction, we studied the binding of partially purified rabbit uterine ER to poly(dG-m5dC).poly(dG-m5dC). The induction of Z-DNA in the polynucleotide was monitored by circular dichroism and ultraviolet spectroscopic measurements. Binding of ER to poly(dG-m5dC).poly(dG-m5dC) increased from 15% to approx. 50-60% in the presence of 7.5 mM putrescine, 0.5 mM spermidine or 0.25 mM spermine. Maximal binding of ER to the polynucleotide was observed near the midpoint of the B-DNA to Z-DNA transition of the polynucleotide. N1-acetyl spermidine and N1-acetyl spermine facilitated the B-DNA to Z-DNA transition and the binding of ER although they were less effective than the unacetylated analog. Co(NH3)6(3+), a trivalent inorganic cation, also provoked the B-DNA to Z-DNA transition of the polynucleotide and increased its binding to ER. At higher polyamine concentrations, there was an inhibition of ER binding to the polynucleotide. In the presence of polyamines, the binding of ER to a plasmid containing ERE was 2-3-fold higher than that to a control plasmid devoid of ERE. Polyamine-induced facilitation of ER-ERE binding was also confirmed by gel mobility shift assay. Our data indicate that conformational perturbations, similar to that of the early stages of B-DNA to Z-DNA transition, are important in the recognition of ER and ERE.

Transcriptional suppression of estrogen receptor gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

TCDD, the most potent congener of the polychlorinated dioxins, has been shown to be an antiestrogen. The mechanisms of TCDD-induced antiestrogenicity are still under investigation. In this study, we investigated the effects of TCDD on the expression of the estrogen receptor (ER) gene. We studied the levels of un-spliced ER transcript (hnRNA) as well as the ER mRNA in ovary, uterus and liver of TCDD-treated mice with different genetic backgrounds. To quantitate the ER hnRNA levels, the intron and exon boundary of ER hnRNA was amplified by competitive RT-PCR. The ER mRNA from these mice was quantitated by competitive RT-PCR amplifying exons separated by an intron. ER hnRNA and ER mRNA levels were quantitated 4 days after a single i.p. dose of TCDD (5 microg/kg) in female C57BL/6J (B6) mice, which carry the responsive allele to TCDD. TCDD treatment significantly (p < 0.05) suppressed the levels of ER hnRNA in the ovary (27.4%) and uterus (21.9%). The decreases in ER hnRNA were coordinated with significant (p < 0.01) decreases in ER mRNA in ovary (57.7%) and uterus (37.6%). There was a significant decrease (20.3%, p < 0.05) in liver ER mRNA, however, the changes of ER hnRNA in liver were not significant. The coordinated decreases in ER hnRNA and mRNA in TCDD-treated mice suggest a suppression of transcription of the ER gene. We performed the same study on DBA/2J (D2) mice, which possess the "non-responsive" allele of the aryl hydrocarbon receptor (AhR). These mice demonstrated no significant decrease in either the ER mRNA or hnRNA after TCDD treatment. Overall, these results suggest that TCDD suppresses the gene expression of the ER receptor by decreasing its transcription, and the AhR plays an important role in mediating this response.

Release of chemotactic factors by veins during preparation for arterial bypass.

With the use of arterial bypass release neutrophil chemotactic factors, saphenous veins were removed from dogs by means of standard surgical technique and were distended to a pressure of 200 mm Hg for 15 minutes with cold (4 degrees C) plasmalyte solution. Veins cannulated in situ and flushed with cold (4 degrees C) plasmalyte solution served as negative controls. Experimental and control flush solutions were assayed for the presence of neutrophil chemotactic factors with the use of modified Boyden chambers. Chemotactic activity from the distended vein grafts was 2.2 to 7.2 times the control value. Biologic chemotactic activity was demonstrated as well. These results suggest a role for the vein graft itself in the recruitment of neutrophils to implanted veins by releasing chemoattractants.