Enantiospecific synthesis of heterocycles linked to purines: different apoptosis modulation of enantiomers in breast cancer cells.

The issue of chiral drug is now a major theme in the design, discovery and development of new drugs. It has been shown for many pharmaceuticals that only one enantiomer contains the desired activity, and the synthesis of such drug molecules in their optically pure form is becoming increasingly important. Mitsunobu reaction was carried out between (R)- and (S)-3,4-dihydro-2H-1,5-benzoxathiepin-3-ol and purines under microwave irradiation. A contraction into a six-membered ring takes place with concomitant inversion at the stereocentre with excellent enatiomeric excesses giving rise to the homochiral 9-(2,3-dihydro-1,4-benzoxathiin-3-ylmethyl)-9H-purines. The anti-tumour activity of all enantiomers is reported against the caspase-3-deficient MCF-7 and the wild type SKBR-3 human breast cancer cells. The most active homochiral compound displays an IC50 of 1.85 µM and induces inhibition of the translation initiation factor eIF2α. All homochiral compounds included in this study show different apoptotic effects between both enantiomers with levels up to 99%. We have analyzed caspase-mediated apoptotic pathways on enantiomers and racemates. We have found a homochiral derivative that activates the canonical intrinsic caspase-8/caspase-3 apoptotic pathway on the MCF-7 cells, and a racemic compound that induces caspase-2 activation. Moreover, we demonstrate the involvement of caspase activation during cell death induced by these compounds in SKBR-3 cells.

Melatonin synthetic analogs as nitric oxide synthase inhibitors.

Nitric oxide (NO), which is produced by oxidation of L-arginine to L-citrulline in a process catalyzed by different isoforms of nitric oxide synthase (NOS), exhibits diverse roles in several physiological processes, including neurotransmission, blood pressure regulation and immunological defense mechanisms. On the other hand, an overproduction of NO is related with several disorders as Alzheimer's disease, Huntington's disease and the amyotrophic lateral sclerosis. Taking melatonin as a model, our research group has designed and synthesized several families of compounds that act as NOS inhibitors, and their effects on the excitability of N-methyl-D-aspartate (NMDA)-dependent neurons in rat striatum, and on the activity on both nNOS and iNOS were evaluated. Structural comparison between the three most representative families of compounds (kynurenines, kynurenamines and 4,5-dihydro-1H-pyrazole derivatives) allows the establishment of structure-activity relationships for the inhibition of nNOS, and a pharmacophore model that fulfills all of the observed SARs were developed. This model could serve as a template for the design of other potential nNOS inhibitors. The last family of compounds, pyrrole derivatives, shows moderate in vitro NOS inhibition, but some of these compounds show good iNOS/nNOS selectivity. Two of these compounds, 5-(2-aminophenyl)-1H-pyrrole-2-carboxylic acid methylamide and cyclopentylamide, have been tested as regulators of the in vivo nNOS and iNOS activity. Both compounds prevented the increment of the inducible NOS activity in both cytosol (iNOS) and mitochondria (i-mtNOS) observed in a MPTP model of Parkinson's disease.

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).

Toxicological studies on 2,4,6-tribromoanisole.

TBA, or 2,4,6-tribromoanisole, is a musty-smelling metabolite of 2,4,6-tribromophenol that is used as a flame retardant and an antifungal agent for wooden pallets and packaging materials. The compound can impart its peculiar, often offensive, odor on product packaging to the concern of consumers for the safety of the package contents. These studies were conducted to evaluate the safety of TBA to humans ingesting products tainted with TBA. In addition to the 28-day oral study, a bacterial reverse mutation study was conducted, and confirmed that TBA was not mutagenic. To evaluate oral safety, TBA was evaluated in single dose and 5-day and 4-week repeated dose oral toxicity studies in rats. The test article, administered in single gavage doses of 2000, 5000 and 7500 mg/kg body weight (bw), in 5 daily repeated doses of 1000, 2000 or 3000 mg/kg bw/day or in 28 daily oral gavage doses of 0 001, 0.01, 100, and 1000 mg/kg bw/day did not result in any deaths. Also, the single and repeat dose studies resulted in no significant differences between control and treated groups on body weight gain, food consumption, clinical observations, blood biochemical values, and hematology findings. Treatment-related adverse findings were only detected in male rats during repeated dose studies and were associated with high plasma concentrations of TBA. The test article-related finding of hyaline droplets in the cortical tubular epithelium of kidneys was associated with increases in α(2 µ)-globulin content in the kidneys as indicated by the intensity of immunohistochemical staining. These findings were correlated with an increased weight of kidneys in males administered 1000mg/kgbw/day for 28days. Chemical induction of hyaline droplets containing α(2µ)-globulin in the renal proximal tubule is a process unique to the male rat and is not relevant for human risk assessment. Findings of increased liver weight with minimal centrilobular to diffuse hepatocellular hypertrophy in males treated with TBA at 1000mg/kg bw/day for 28days were considered to be an adaptive metabolic response to xenobiotic administration. The increased volume of urine, noted in both males and females treated with 1000mg/kg bw/day was considered adaptive and necessary to excrete the high xenobiotic burden resulting from TBA administration. TBA appeared to be highly bioavailable since high concentrations of TBA were detected in plasma, at 1, 4 and 8h after administration of TBA at 100 and 1000mg/kg bw for 1 and 28days. Levels were dose-related but did not clarify the course of TBA elimination with time after administration. These studies indicate that TBA, administered orally to rats, produced male rat-specific, treatment-related toxicity at the highest orally administered dose in repeated dose (5-day at 3000mg/kg bw and 28-day at 1000mg/kg bw) studies. Therefore, the NOAEL for the 28-day oral study was determined to be 1000mg/kg bw/day for the rat.

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.

Derivados de 4, 5-DIHIDRO-1H-PIRAZOL como inhibidores selectivos de nNOS: Síntesis y relaciones estructura-actividad / 4, 5-DIHYDRO-1H-PYRAZOLE as nNOS selective inhibitors: Synthesis and structure-activity relationship

INTRODUCCIÓN: La oxido nítrico sintasa (NOS) es la enzima que cataliza la biosíntesis de óxido nítrico (NO) a partir de L-arginina. 1 Hasta el momento, se han descubierto cuatro isoformas:2 nNOS, iNOS, eNOS y mtNOS. El NO es un biomensajero relacionado con importantes funciones fisiológicas. 3Sin embargo, se ha demostrado que una sobreproducción de NO por la nNOS está implicada en procesos neurodegenerativos. 4 Este hecho justifica la necesidad terapéutica de encontrar inhibidores selectivos de la nNOS que permitan luchar contra enfermedades tales como Alzheimer, Parkinson, esclerosis lateral amiotrófica y corea de Huntington. Nuestro grupo de investigación ha sintetizado y evaluado una serie de derivados kinurenínicos 5 1 y kinurenamínicos 6 2 como agentes neuroprotectores que resultaron desprovistos de actividad inhibitoria frente a la enzima kinurenina-3-hidroxilasa (KYN3OH), lo que demuestra que su actividad neuroprotectora se debe a la inhibición de la nNOS. OBJETIVO: Basándonos en estos antecedentes, hemos sintetizado y realizado la evaluación biológica in vitrofrente a las isoformas nNOS e iNOS de una serie de derivados de 4,5-dihidro-1H-pirazol con estructura general 3, con objeto de encontrar inhibidores selectivos de alguna de estas isoformas. METODOLOGÍA: Tomando como referencia los derivados kinurenínicos y kinurenamínicos, hemos sintetizado los análogos rígidos con un resto de 4,5-dihidro-1H-pirazol. Además de la restricción conformacional, se han llevado a cabo otras modificaciones, como la introducción de distintos sustituyentes en el anillo aromático y la modificación del grupo acilo en el anillo de pirazolina(AU)

CONCLUSIÓN /DISCUSIÓN: Todos los compuestos ensayados inhiben nNOS. La inhibición de iNOS es ínfima en la mayoría de los casos, por lo que se pueden considerar selectivos, y no hay inhibición de KYN3OH. Por consiguiente, el potencial neuroprotector de estos derivados se debe únicamente a la inhibición de nNOS(AU)

INTRODUCTION: Nitric Oxide Synthase (NOS) is the enzyme which catalyses the biosynthesis of Nitric Oxide (NO)from L-arginine 1. Four NOS isoforms have been described: 2 nNOS, iNOS, eNOS and mtNOS. NO is a biological messenger involved in several physiologic processes. 3 However, an over production of NO by nNOS produces neurotoxicity which has been associated with various neurological disorders 4.Therefore, it is necessary to found nNOS inhibitors to fight pathologies such as Alzheimer’s disease, Parkinson, amyotrophic lateral sclerosis and Huntington’s disease. In previous papers, our research group have described the synthesis of a series of kynurenine 5 1 and kynurenamine 6 2 derivatives as neuroprotective agents which are not active versus kynurenine-3-hydroxylase (KYN3OH). This fact demonstrates that their neuroprotective activity is only due to then NOS inhibition. 2 3 OBJECTIVE: Basing on these precedents, we have developed and evaluated in vivo, versus nNOS and iNOS, a series of 4,5-dihydro-1H-pyrazole derivatives with general structure 3 in order to find new selective compounds. METHODOLOGY: Taking kynurenine and kynurenamine derivatives as reference, we have synthesized rigid analogous with a ring of 4,5-dihydro-1H-pyrazole . Besides the conformacional restriction, other modification shave been carried out, as the introduction of different substituents in the aromatic ring and the modification of the acyl group in the pyrazoline ring . CONCLUSION /DISCUSION: All compounds inhibit nNOS. In most of cases, the inhibition of iNOS is negligible. Thus, they can be considered selective. On the other hand, there is no KYN3OH inhibition. Consequently, the neuroprotective potential of these derivatives is due only to the inhibition of nNOS(AU)

(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.

Structural elucidation of a new delta2-pyrazoline derivatives using 1H and 13C NMR spectroscopy.

This paper describes the unequivocal structural elucidation of a new kind of Delta2-pyrazoline derivatives carried out by means of monodimensional 1H and 13C NMR spectroscopies, bidimensional ones such as HMBC and HMQC experiments, and NOEDIFF effects. Conformational analysis of this molecule agrees very well with the experimentally NOEDIFF effects found.

[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.

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.

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.

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.

El papel de los radicales libres en la etiología de la ateroesclerosis / Atherosclerosis and the rol of ROS and RNS

Los mecanismos moleculares que originan la aterosclerosis no están claros, sin embargo se conoce que anormalidades en las señales mediadas por las especies reactivas de oxígeno (ERO) y de nitrógeno (ERN) pueden desencadenar la patogénesis de la enfermedad cuando se asocian con factores de riesgo como hiperlipidemia, hiperglicemia y cambios en las fuerzas hemodinámicas. En esta revisión nos proponemos resaltar los diferentes mecanismos desencadenados por las ERO y ERN que llevan al desarrollo de la aterogénesis incluyendo la regulación de la transcripción de varios genes

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.

Mechanism of suppression of cytochrome P-450 1A1 expression by tumor necrosis factor-alpha and lipopolysaccharide.

Proinflammatory cytokines, such as tumor necrosis factor (TNF)-alpha, interleukin-1beta, and lipopolysaccharides (LPS), suppress the gene expression of cytochrome P-450 1A1 (cyp1a1). The mechanism of the suppression is not well understood. In present study, we show that activation of nuclear factor-kappaB (NF-kappaB) is a critical event leading to the suppression of cyp1a1 gene expression, thus providing an underlying mechanism for the TNF-alpha- and LPS-induced cyp1a1 suppression. We demonstrated that: (i) inducible RelA expression down-regulated aryl hydrocarbon receptor (AhR) activated reporter gene; (ii) the suppressive effects of LPS and TNF-alpha on the AhR-activated reporter gene could be blocked by pyrrolidine dithiocarbamate, which is known to inhibit NF-kappaB action; and (iii) TNF-alpha and LPS-imposed repression could be reversed by the NF-kappaB super repressor (SRIkappaBalpha), thus demonstrating the specific involvement of NF-kappaB. Furthermore, nuclear receptor coactivators p300/CBP and steroid receptor coactivator-1 act individually as well as cooperatively to reverse the suppressive effects by NF-kappaB on the AhR-activated reporter gene, suggesting that these transcriptional coactivators serve as the common integrators for the two pathways, thereby mediating the cross-interactions between AhR and NF-kappaB. Finally, using the chromatin immunoprecipitation assay, we demonstrated that AhR ligand induces histone H4 acetylation at the cyp1a1 promoter region containing the TATA box, whereas TNF-alpha inhibits this acetylation, suggesting that AhR/NF-kappaB interaction converges at level of transcription involving chromatin remodeling.

Increase in the stability and helical content of estrogen receptor alpha in the presence of the estrogen response element: analysis by circular dichroism spectroscopy.

Ligand-dependent stabilization of the estrogen receptor (ER) is often postulated, with limited support from experimental data. We studied the thermal unfolding of recombinant ERalpha by circular dichroism (CD) spectroscopy. The T(M) of unfolding of ERalpha was 38 +/- 2.4 degrees C, and the van't Hoff enthalpy of unfolding was 31.7 +/- 3.4 kcal/mol in the absence of ligands. Addition of estradiol (E(2)) increased the T(M) to 43.6 +/- 2.3 degrees C, while addition of E(2) and an oligonucleotide harboring the estrogen response element (ERE) increased the T(M) to 47.9 +/- 1.6 degrees C. Addition of the antiestrogen 4-hydroxytamoxifen (HT) alone did not increase the T(M); however, a combination of HT and the ERE increased the T(M) to 48.9 +/- 1.0 degrees C. The ERE alone increased the T(M) to 46.1 +/- 0.9 degrees C. Addition of E(2) alone had no effect on the apparent enthalpy of unfolding; however, the ERE alone increased the apparent enthalpy from 31.7 to 36.1 kcal/mol. ERalpha samples containing the ERE also exhibited an increase in the negative ellipticity at 208 and 222 nm, relative to that of ligand-free ERalpha, suggesting a stabilization of the alpha-helix. CD data analysis further showed that the presence of the ERE caused a large increase in alpha-helical content of ERalpha in both the presence and absence of the ligands. This increase in alpha-helical content of ERalpha was not observed in the presence of a nonspecific oligonucleotide. These results show that the ERE can increase the thermal stability of ERalpha, enhance its alpha-helical content, and facilitate the cooperativity of the folding transition.

LUMO energy of model compounds of bispyridinium compounds as an index for the inhibition of choline kinase.

Eleven derivatives of 1,1'-[1,2-ethylenebis(benzene-1,4-diylmethylene)]bis(4-pyridinium) dibromides bearing various groups at C-4 of the pyridinium moiety were synthesized and examined for their inhibition of choline kinase (ChoK) and antiproliferative activities. The C-4 substituents include electron-releasing, neutral or electron-withdrawing groups. A one-parameter regression equation has been derived which satisfactorily describes the ex vivo inhibitory potency of ChoK of the title compounds. The electronic effect plays a critical function in the ex vivo inhibition of ChoK although the role of electrostatic interactions could be altered due to a solvation process of both ChoK and ligands.

Oral bioavailability of lead and arsenic from a NIST standard reference soil material.

The oral bioavailability of soil contaminants is measured using in vitro or in vivo techniques. Current efforts in our laboratory are focused on the comparisons of in vitro methods for bioavailability estimation with the presently employed in vivo techniques, such as animal models. We present a comparison of two techniques for oral bioavailability estimation: in vitro dissolution and in vivo rat feeding using a standard reference soil. Lead (Pb) and arsenic (As) were chosen because of the range of concentration in this soil as well as the large historical database of bioavailability values for these metals. Metal solubility was measured using a sequential soil extraction in synthetic analogues of human saliva, gastric and intestinal fluids. The soluble metal was defined as the bioaccessible fraction. Oral bioavailability of Pb and As was measured in Sprague Dawley rats by determining metal levels in the major organs and urine, feces, and blood at 1-, 2-, and 3-day time points. Extractions to determine bioaccessibility yielded a gastric component of 76.1% and 69.4% for Pb and As, respectively, and intestinal components were 10.7% and 65.9%. The oral bioavailability of the standard reference soil was 0.7% and 37.8% for Pb and As, respectively. Bioaccessibility was greater than bioavailability for both metals in both gastrointestinal compartments. Although Pb had the highest soil concentration of the selected metals, it was the least bioavailable, while As was highly available in both the in vitro and in vivo method. These types of data allow for an in vitro-in vivo comparison of a soil whose metal concentrations have been certified and validated.