Defects of immune cells in the senescence-accelerated mouse: a model for learning and memory deficits in the aged

AM1 and PM3 complete geometry optimizations were performed on 19 arotinoids congeneric with (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)-1-propenyl] benzoic acid (TTNPB), a very potent agent in carcinoprevention and carcinotherapy. Sixteen TTNPB conformations with close energy were obtained and characterized; four representative conformations were then studied for 14 derivative compounds, for which we found a substantial non-planarity of the two aromatic moieties. Large rotational flexibility of the arotinoid ring fragments was predicted by both methods. Very low barriers (0.4-3.9 kcal/mol) were found for the tetralenyl ring rotation. The two methods also agreed in predicting benzoic acid moiety rotation in a wide range of torsion angle values except those close to 0 or 180 degrees for which the PM3 rotational barriers were found to be considerably lower than the AM1 ones. This high conformational flexibility of arotinoid molecules may facilitate their favorable orientation in the process of fitting to the receptor sites.     

Use of trans-retinoic acid in the treatment of acute promyelocytic leukemia

OBJECTIVE: To discuss acute promyelocytic leukemia (APL) and review the literature concerning differentiation treatment of APL with trans-retinoic acid (t-RA). DATA SOURCES: English-language articles concerning APL or its treatment with t-RA were identified with a MEDLINE search. STUDY SELECTION: All studies available at the time of article preparation, which addressed t-RA treatment in APL, were selected. DATA EXTRACTION: Data extraction and assessment were performed subjectively by the authors. An extensive discussion of specific study details is included in the article. DATA SYNTHESIS: APL is a unique subset of acute myelogenous leukemia and is typified by an accumulation of malignant promyelocytes in the bone marrow. Within the granulocyte cell cycle of a patient with APL, differentiation has been halted at the level of the promyelocyte, preventing formation of mature granulocytes. Upon treatment with traditional cytotoxic chemotherapy, complete remission rates of approximately 70 percent, with a five-year survival ranging from 25 to 40 percent have been achieved. In most patients with APL, a characteristic chromosomal t(15q+;17q-) translocation has been found, which may be responsible for the production of an aberrant retinoic acid receptor-alpha. Therefore, t-RA induction therapy has been investigated and has produced promising results. Administration of t-RA in dosages of 45-100 mg/m2/d has induced complete remissions. The apparent mechanism of t-RA is the induction of promyelocyte differentiation and maturation. The most common adverse effects noted have been dry skin, cheilitis, and headaches. CONCLUSIONS: Upon consideration of the initial trials, t-RA appears to be a promising and unique treatment for APL.     

Synthesis and characterization of heteroarotinoids demonstrate structure specificity relationships

Heteroarotinoids are synthetic retinoids derived from trans-retinoic acid and the arotinoid structures and include a heteroatom in a five- or six-membered cyclic ring. This is the first systematic study of influences of the heteroatom, ring size, number of aryl groups, and terminal side chain on retinoid receptor specificity. Two new heteroarotinoids were synthesized and characterized. Although all heteroarotinoids activated RAR receptors, two dominant associations between structure and specificity were identified across all compounds. The six-membered ring conferred increased RARbeta specificity over the five-membered ring. The sulfur atom conferred greater specificity for RARgamma than the oxygen atom. RARalpha specificity was attenuated by a combination of influences from the heteroatom and aryl groups. In summary, the heteroatom and cyclic ring size exerted dominant effects, while the number of aryl rings and terminal side chain had attenuating effects on retinoid receptor specificity of heteroarotinoids.     

1-[Ethoxyamino)methyl]-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepins: a new class of antianaphylactic agents

The synthesis and biological activity in the rat passive cutaneous anaphylaxis (PCA) test of a new class of compounds, 1-[(ethoxyamino)methyl]-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepins, are reported. These compounds are synthesized from the adduct of 1-(bromomethyl)-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepins, are reported. These compounds are synthesized from the adduct of 1-(bromomethyl)-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepin and ethylene glycol or chloroethanol. The influence of the amine function on activity in the rat PCA is discussed. Aryl- or heteroarylpiperazines favor activity with this class of compounds.     

Characterization of the substrate binding site of polyenoic fatty acid isomerase, a novel enzyme from the marine alga Ptilota filicina

The substrate binding site of polyenoic fatty acid isomerase (PFI) has been investigated using a series of alternate substrates and by examination of the pH dependence on the kinetic parameters of PFI with selected substrates. The pH dependence profile of PFI with EPA [(5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid] shows the enzyme to be catalytically active over a wide pH range, with activity being optimal below pH 6.0. Analysis of the kinetic parameters of DHA [(4Z,7Z,10Z,13Z,16Z,19Z)-docosahexen oic acid]; adrenic acid [(7Z,10Z,13Z,16Z)-docosatetraenoic acid]; EPA; arachidonic acid [(5Z,8Z,11Z,14Z)-eicosatetraenoic acid]; anandamide (arachidonyl-N-ethanolamide); and eicosatrienoic acid [(5Z,8Z,11Z)-eicosatrienoic acid] demonstrates that substrates possessing omega-3 olefins (DHA and EPA) have the lowest K(m) values (1.9 and 9.6 microM, respectively). EPA and arachidonic acid showed the highest V(max) values (6.0 and 2.8 micromol min(-1) mg(-1), respectively). The twenty carbon omega-9 fatty acid eicosatrienoic acid showed a relatively large K(m) and had a V(max) approximately 20-fold less than EPA. Anandamide, a substrate analog lacking an ionizable carboxylate, showed a K(m) similar to the other omega-6 fatty acids (arachidonic acid and adrenic acid); however, the V(max) was approximately 5-fold lower than arachidonic acid and 8-fold lower than EPA. Moreover, anandamide demonstrated no pH dependency on its kinetic parameters over a range where EPA showed a 27-fold decrease in V/K(m). NMR spectroscopy was used to determine the structure of the product from reaction of PFI with DHA. These data showed the compound to be (4Z,7Z,9E,11E,16Z,19Z)-docosahexenoi c acid. Reaction of PFI with dihomo-gamma-linolenic acid resulted in the development of two products, one with the characteristic chromophore of a conjugated triene, the other with a chromophore characteristic of a conjugated diene. Analysis of the products from these reactions of PFI, in conjunction with the kinetic parameters from the alternate substrates, provides compelling evidence that the enzyme preferentially orients the substrate in the catalytic site with respect to the methyl terminus.     

alpha-Methylene-gamma-butyrolactones: synthesis and vasorelaxing activity assay of coumarin, naphthalene, and quinoline derivatives

Certain alpha-methylene-gamma-butyrolactone derivatives of coumarin, naphthalene, and quinoline were synthesized and evaluated for vasorelaxing effects on isolated rat thoracic aorta. The 7-[(2,3,4,5-tetrahydro-2-methyl-4-methylene-5-oxo-2-furanyl)methoxy]-2H- 1- benzopyran-2-ones, which have an aliphatic methyl substituent at the lactone C2, were more active than their C2-phenyl counterparts against high-K+ (80 mM) medium, Ca2+ (1.9 mM)-induced vasoconstriction and the norepinephrine (NE, 3 microM)-induced phasic and tonic constrictions (2a vs. 2b; 2c vs. 2d; 2e vs. 2f; 2g vs. 2h). Although 3-chloro-7-[(2,3,4,5-tetrahydro-2-methyl-4-methylene-5-oxo-2- furanyl)methoxy]-4-methyl-2H-1-benzopyran-2-one (2g) demonstrated the most potent inhibitory activities on the NE-induced phasic and tonic constrictions at concentrations of as low as 10 micrograms/ml, it possesses both affinity for NE-receptor and intrinsic activity to trigger the vasoconstriction. However, 8-[(2,3,4,5-tetrahydro-2-methyl-4-methylene-5-oxo-2- furanyl)methoxy]quinoline (10a) and other quinoline derivatives (11a, 12a) are pure irreversible non-competitive blockers of NE-receptor with no intrinsic activity. The aromatic ring played an important role in the vasorelaxing effects of alpha-methylene-gamma-butyrolactones; naphthalene was inactive, quinolines exhibited only affinity to the alpha-receptor, and coumarins possessed both affinity and intrinsic activity.     

Estrogenic activity of a dieldrin/toxaphene mixture in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based estrogen receptor assays: no apparent synergism

The estrogenic activity of dieldrin, toxaphene, and an equimolar mixture of both compounds (dieldrin/toxaphene) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 human breast cancer cells, and in yeast-based reporter gene assays. Treatment of the animals with 17beta-estradiol (E2) (0.0053 kg/day x3) resulted in a 3.1-, 4.8-, and 7.8-fold increase in uterine wet weight, peroxidase activity, and progesterone receptor binding, respectively. In contrast, treatment with 2.5, 15 and 60 micromol/kg (x3) doses of toxaphene, dieldrin, or dieldrin/toxaphene (equimolar) did not significantly induce a dose-dependent increase in any of the E2-induced responses. The organochlorine pesticides alone and the binary mixture did not bind to the mouse uterine estrogen receptor (ER) in a competitive binding assay using [3H]E2 as the radioligand. In parallel studies, estrogenic activities were determined in MCF-7 cells by using a cell proliferation assay and by determining induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with plasmids containing estrogen-responsive 5'-promoter regions from the rat creatine kinase B and human cathepsin D genes. E2 caused a 24-fold increase in CAT activity in MCF-7 cells transiently transfected with creatine kinase B and a 3.8-fold increase in cells transiently transfected with the human cathepsin D construct. Treatment of MCF-7 cells with dieldrin, toxaphene, or an equimolar mixture of dieldrin plus toxaphene (10(-8)-10(-5) M) did not significantly induce cell proliferation or CAT activity in the transient transfection experiment with both plasmids. The relative competitive binding of the organochlorine pesticides was determined by incubating MCF-7 cells with 10(-9) M [3H]E2 in the presence or absence of 2 x 10(-7) M unlabeled E2 (to determine nonspecific binding), toxaphene (10(-5) M), dieldrin (10(-5) M), and equimolar concentrations of the dieldrin plus toxaphene mixture (10(-5) M). The binding observed for [3H]E2 in the whole cell extracts was displaced by unlabeled E2, whereas the organochlorine pesticides and binary mixture exhibited minimal to nondetectable competitive binding activity. E2 caused a 5000-fold induction of beta-galactosidase (beta-gal) activity in yeast transformed with the human ER and a double estrogen responsive element upstream of the beta-gal reporter gene. Treatment with 10(-6)-10(-4) M chlordane, dieldrin, toxaphene, or an equimolar mixture of dieldrin/toxaphene did not induce activity, whereas 10(-4) M endosulfan caused a 2000-fold increase in beta-gal activity. Diethylstilbestrol caused a 20-fold increase in activity in yeast transformed with the mouse ER and a single estrogen responsive element upstream of the beta-gal reporter gene. Dieldrin, chlordane, toxaphene, and endosulfan induced a 1.5- to 4-fold increase in activity at a concentration of 2.5 x 10(-5) M. Synergistic transactivation was not observed for any equimolar binary mixture of the pesticides at concentrations of either 2.5 x 10(-5) M or 2.5 x 10(-4) M. The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based reporter gene assays, the activities of both dieldrin and toxaphene were minimal, and no synergistic interactions were observed with a binary mixture of the two compounds.     

Cytotoxicity and apoptosis produced by arachidonic acid in HepG2 cells overexpressing human cytochrome P-4502E1

The goal of the current study was to evaluate the effects of arachidonic acid, as a representative polyunsaturated fatty acid, on the viability of a HepG2 cell line, which has been transduced to express human cytochrome P4502E1 (CYP2E1). Arachidonic acid produced a concentration- and time-dependent toxicity to HepG2-MV2E1-9 cells, which express CYP2E1, but little or no toxicity was found with control cells. In contrast to arachidonic acid, oleic acid was not toxic to the HepG2-MV2E1-9 cells. The cytotoxicity of arachidonic acid involved a lipid peroxidation type of mechanism since toxicity was enhanced after depletion of cellular glutathione; formation of malondialdehyde and 4-hydroxy-2-nonenal was markedly elevated in the cells expressing CYP2E1, and toxicity was prevented by antioxidants and the iron chelator desferrioxamine. The CYP2E1-dependent arachidonic acid toxicity appeared to involve apoptosis, as demonstrated by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and DNA laddering experiments. Trolox, which prevented toxicity of arachidonic acid, also prevented the apoptosis. Transfection with a plasmid containing bcl-2 resulted in complete protection against the CYP2E1-dependent arachidonic acid toxicity. It is proposed that elevated production of reactive oxygen intermediates by cells expressing CYP2E1 can cause lipid peroxidation, which subsequently promotes apoptosis and cell toxicity when the cells are enriched with polyunsaturated fatty acids such as arachidonic acid.     

Inducibility of cytochromes P-4502E1 and P-4501A1 in the rat pancreas

Cytochrome P-450 (CYP) isoenzymes have been incriminated in the toxicity and carcinogenicity of various xenobiotics in different tissues, but prior measurements of their activity in pancreatic microsomes have been disappointing. We now applied new isolation methods and a highly sensitive procedure to assay for the metabolism of p-nitrophenol and 7-ethoxyresorufin, specific substrates for CYP2E1 (2E1) and CYP1A1 (1A1), respectively. 2E1 and 1A1 content was estimated with high-resolution chemiluminescent Western blots using recombinant 2E1 and 1A1 as standards. We found that p-nitrophenol hydroxylase activity was 5.07 +/- 0.66 and 1.50 +/- 0.26 pmol/ min/mg of protein in pancreatic microsomes of ethanol-fed and control rats, respectively. Chronic ethanol treatment increased 2E1 content in pancreatic microsomes 3.6-fold. Activity and content of 2E1 were also assessed in hepatic microsomes: specific activity (expressed per 2E1 content) was similar in pancreatic and hepatic microsomes. There was also an inductive effect of 3-methylcholanthrene (MC) on 1A1 in pancreatic microsomes. Pancreatic microsomal 7-ethoxyresorufin-O-dealkylation activity in MC-treated rats was 19.6 +/- 1.7 pmol/min/mg of protein, 61-fold higher than in controls. MC treatment increased the 1A1 content in pancreatic microsomes 42-fold. These results demonstrate that, in pancreatic microsomes, ethanol and MC exert striking inductive effects on 2E1 and 1A1 activities, which could play a role in the pathogenesis of pancreatitis and/or pancreatic cancer.     

Encapsulation of the topoisomerase I inhibitor GL147211C in pegylated (STEALTH) liposomes: pharmacokinetics and antitumor activity in HT29 colon tumor xenografts

The topoisomerase I inhibitor GL147211C [7-[(4-methylpiperazino)methyl]-10,11-(ethylenedioxy)-(20S)-campto thecin trifluoroacetate], a camptothecin analogue, has significant activity in tumor cell cytotoxicity assays in vitro and antitumor activity in both animal tumor models and human patients. Its toxicity is significant, however, effectively limiting the amount of drug that can be administered and its clinical utility. To determine whether the therapeutic index of GL147211C could be improved, the drug was encapsulated in long-circulating, pegylated (STEALTH) liposomes (SPI-355). The pharmacokinetics and antitumor activity of SPI-355 were compared to those of nonliposomal GL147211C. The plasma pharmacokinetics of SPI-355 in rats were typical of those of other pegylated liposomal formulations, with significantly increased blood circulation time; the dose-corrected area under the curve and Cmax of SPI-355 (10 mg/kg) were 1250- and 35-fold higher, respectively, than those of nonliposomal GL14711C (8.72 mg/kg). The comparative antitumor activity of SPI-355 and nonliposomal GL1472211C was evaluated in nude mice implanted with HT29 colon carcinoma xenografts. SPI-355 was 20-fold more effective than GL147211C in inhibiting tumor growth (1 mg/kg SPI-355 and 20 mg/kg GL147211C) and produced durable complete remissions of tumors at well-tolerated dose levels that were >5-fold lower than the maximally tolerated dose of GL147211C, which induced no durable complete responses. Signs of toxicity were similar between the two drugs, but liposome encapsulation increased the toxicity of drug approximately 4-fold, with increased weight loss and several deaths with SPI-355 (5 mg/kg SPI-355 versus 20 mg/kg GL147211C). Despite the increased toxicity seen with SPI-355, the therapeutic index of the liposomal formulation was increased approximately 5-fold over that of nonliposomal GL147211C, suggesting that such a pegylated liposomal formulation could demonstrate increased therapeutic index in human patients.     

Ferulic acid dehydrodimers from wheat bran: isolation, purification and antioxidant properties of 8-O-4-diferulic acid

Wheat bran contains several ester-linked dehydrodimers of ferulic acid, which were detected and quantified after sequential alkaline hydrolysis. The major dimers released were: trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl)-7-methoxy-2, 3- dihydrobenzofuran-3-carboxylic acid (5-8-BendiFA), (Z)-beta-[4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy]-4-hydroxy-3-methox ycinnamic acid (8-O-4-diFA) and (E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid (5-5-diFA). trans-7-hydroxy-1-(4-hydroxy-3methoxyphenyl)-6-methoxy-1,2-dihydro - naphthalene-2,3-dicarboxylic acid (8-8-diFA cyclic form) and 4,4'-dihydroxy-3,3'-dimethoxy-beta,beta'-bicinnamic acid (8-8-diFA non cyclic form) were not detected. One of the most abundant dimers, 8-O-4-diFA, was purified from de-starched wheat bran after alkaline hydrolysis and preparative HPLC. The resultant product was identical to the chemically synthesised 8-O-4-dimer by TLC and HPLC as confirmed by 1H-NMR and mass spectrometry. The absorption maxima and absorption coefficients for the synthetic compound in ethanol were: lambda max: 323 nm, lambda min: 258 nm, epsilon lambda max (M-1 cm-1): 24,800 +/- 2100 and epsilon 280 (M-1 cm-1): 19,700 +/- 1100. The antioxidant properties of 8-O-4-diFA were assessed using: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes and; (b) scavenging of the radical cation of 2,2'-azinobis (3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue, Trolox C. The 8-O-4-diFA was a better antioxidant than ferulic acid in both lipid and aqueous phases. This is the first report of the antioxidant activity of a natural diferulate obtained from a plant.     

Alterations in cholesterol sulfate and its biosynthetic enzyme during multistage carcinogenesis in mouse skin

A novel series of nonpeptide angiotensin II antagonists containing the acrylamide group at the 4-position of the imidazole ring was synthesized and their antagonistic activity was examined by functional assay in rabbit aorta. The acrylamide group was selected as a large lipophilic surrogate for the chloro group of EXP3174. A structure-activity relationship study of the acrylamide moiety has shown that substitution at the 4-position with the N-methyl-3,3-dimethylacrylamide group resulted in the optimal compound, 2-butyl-4-[(3,3-dimethylacryloyl)methyl-amino]-1-[[2'-(1H-tetra zol-5- yl)biphenyl-4-yl]methyl]-1H-imidazole-5-carboxylic acid (1), which was superior to EXP3174 in vitro. Since 1 showed only poor activity against angiotensin II-induced pressor response in rats after oral administration, the carboxylic acid function of 1 was converted into prodrug esters (13). Among these, the 1-[(ethoxycarbonyl)oxy]ethyl ester (13a) showed the most potent and longest-lasting activity when given orally to rats. When administered orally to conscious furosemide-treated dogs, 13a showed an approximately 3-fold increased hypotensive activity in comparison with DuP 753. These data suggest that 13a may be an useful agent for the treatment of angiotensin II-dependent diseases, such as hypertension.     

Differential retinoic acid radiosensitization of cervical carcinoma cell lines

The potential of retinoic acid as a radiosensitizer was investigated using SiHa and CC-1 human uterine cervical carcinoma cell lines, representative of high- and low-grade lesions, respectively. SiHa was significantly (P < 0.05) radiosensitized, whereas CC-1 was not. Although 48 h of treatment with 5 microM 13-cis-retinoic acid prior to irradiation was sufficient to induce radiosensitization, continuation of treatment after irradiation significantly increased the effect (P < 0.05). Three hypotheses were tested to explain the different responses of the two lines. One hypothesis was that SiHa is more sensitive to retinoic acid than CC-1. Measurement of growth revealed that SiHa was more sensitive to growth inhibition by retinoic acid than CC-1. The second hypothesis was that retinoic acid increases the proportion of G1-phase cells in SiHa but not in CC-1. This was found not to be true, because a retinoic acid treatment schedule that induced radiosensitization did not alter cell cycle distribution profiles in the absence of radiation. The third hypothesis was that retinoic acid alters the cell cycle response of SiHa but not CC-1 to radiation. Postirradiation cell cycle profiles revealed that retinoic acid increased G1 delay in SiHa, whereas CC-1 exhibited no significant G1 delay. Both lines exhibited G2 delays that were unaffected by retinoic acid. In conclusion, radiosensitization of SiHa but not CC-1 may be explained by different sensitivities to retinoic acid and differences in postirradiation cell cycle responses. Radiosensitization at radiation doses used clinically was observed when retinoic acid was administered both before and after irradiation.     

Regulation of tyrosine hydroxylase and aromatic L-amino acid decarboxylase by dopaminergic drugs

We provide evidence that dopamine receptors differentially modulate tyrosine hydroxylase and aromatic L-amino acid decarboxylase in the mouse striatum. The dopamine D1 receptor family (D1-like) antagonist, R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benazepine (SCH 23390), elevated aromatic L-amino acid decarboxylase activity and protein content in striatum, as well as the mRNA for the enzyme in midbrain. The dopamine D1-like receptor agonist, (+/-)-1-phenyl-2,3,4,5-tetrahydro-(1 H)-3-benzazepine-7,8-diol (SKF 38393), had no effect on aromatic L-amino acid decarboxylase. The dopamine D1-like drugs had no effect on tyrosine hydroxylase. In contrast, the dopamine D2 receptor family (D2-like) antagonists haloperidol and spiperone elevated both tyrosine hydroxylase and aromatic L-amino acid decarboxylase activities. The increase in aromatic L-amino acid decarboxylase activity was accompanied by elevated enzyme protein content but not mRNA. The dopamine D2-like receptor agonists, bromocriptine, quinpirole and (+/-)-7-hydroxydipropylaminotetralin (7-OH-DPAT), all decreased striatal tyrosine hydroxylase. Under the conditions used, bromocriptine and 7-OH-DPAT, but not quinpirole, decreased aromatic L-amino acid decarboxylase activity of striatum. Both the dopamine D1- and D2-like receptor antagonists enhanced the turnover of striatal dopamine to differing degrees, as judged by the ratio of acid metabolites of dopamine to dopamine. Taken together our results indicate that aromatic L-amino acid decarboxylase can be modulated independently of tyrosine hydroxylase.     

Amplification of gene expression using both 5'- and 3'-untranslated regions of GLUT1 glucose transporter mRNA

Cis-regulatory elements located at either the 5'- or 3'-untranslated region (UTR) of the GLUT1 glucose transporter mRNA increase the expression of luciferase reporter genes. The aim of the present study was to investigate the possible cooperative effects of 5'- and 3'-UTRs of the GLUT1 mRNA on the expression of a luciferase reporter gene in cultured brain endothelial cells. Luciferase reporter genes containing control elements in nucleotides (nt) 1-171 of GLUT1 5'-UTR, or nt 2100-2300 of GLUT1 3'-UTR produced a 10- and 6-fold increase in the expression of the luciferase reporter gene compared to the control vector containing no GLUT1 regulatory sequences, respectively. The insertion of both GLUT1 mRNA cis-regulatory elements increased 59-fold the activity of luciferase compared to controls. Data presented here demonstrate that cis-regulatory elements located at both the 5'- and 3'-UTR of GLUT1 mRNA increase expression of a reporter gene in an independent manner.     

Effect of ramosetron on short-circuit current response in rat colonic mucosa

We investigated the effects of ramosetron (YM060, (-)-(R)-5-[(1-methyl-1H-indol-3-yl)carbonyl]-4,5,6,7-tetrahydro-1 H-benzimidazole monohydrochloride) on the short-circuit current (Isc) responses to 5-HT receptor agonists in the rat distal colon, and compared its potency to that of other 5-HT3 receptor antagonists. 5-Hydroxytryptamine (5-HT) concentration-dependently increased Isc. The Isc response to 5-HT was partially reduced by tetrodotoxin and ramosetron, and strongly inhibited by GR113808 ([[1-[(2-methyl-sulphonyl) amino]ethyl]-4-piperidin-yl]methyl 1-methyl-1 H-indole-3-carboxylate). 2-Methyl-5-HT and 5-methoxytryptamine also increased Isc. The former response was inhibited by ramosetron, and the latter was abolished by GR113808. Ramosetron, YM114 (KAE-393, (-)-(R)-5-[(1-indolinyl)carbonyl]-4,5,6,7-tetrahydro-1 H-benzimidazole monohydrochloride) and granisetron concentration-dependently antagonized the Isc responses to 2-methyl-5-HT with reduction in the maximal response at higher concentrations. Apparent pA2 values for these antagonists were 10.40, 10.37 and 8.99, respectively. Ondansetron produced clear rightward shifts of the concentration-response curves to 2-methyl-5-HT, with a pA2 value of 8.53. These results suggest that 5-HT increases Isc through the 5-HT3 and 5-HT4 receptors, and that ramosetron is a potent and selective 5-HT3 receptor antagonist in rat colonic mucosa.     

A model to describe how a point mutation of the estrogen receptor alters the structure-function relationship of antiestrogens

The antiestrogen tamoxifen [(Z)-1(p-beta-dimethylamino-ethoxyphenyl)-1,2- diphenylbut-1-ene] is an effective anticancer agent for the treatment of hormone responsive breast cancer. Previous studies have demonstrated that a point mutation in the estrogen receptor (ER) resulted in an alteration of the pharmacology of 4-hydroxytamoxifen, the active metabolite of tamoxifen (Jiang et al, Mol Endocrinol 6:2167-2174, 1992). We have extended our studies to evaluate the effect of a point mutation, a Val substitution for Gly at amino acid 400 in the ligand binding domain of ER, on the pharmacology of other antiestrogens in ER stable transfectants derived from the ER-negative breast cancer cell line MDA-MB-231 CL10A. The compounds were tested with or without estradiol-17 beta (E2) for their effects on cell growth in cells expressing the wild type ER (S30) or the mutant ER (ML alpha 2H) or in control antisense ER transfectant AS23 which does not express ER protein. MCF-7 cells, which express the wild type ER, were also used as a control. The growth of AS23 cells was not affected by any of the compounds at a concentration of 1 microM. E2 stimulated the growth of MCF-7 cells but inhibited the growth of ER transfectants S30 and ML alpha 2H. The ML alpha 2H cells were about 10 to 100-fold less sensitive to E2 and antiestrogens than S30 and MCF-7 cells. Keoxifene, an antiestrogen with a high affinity for the ER, maintained antiestrogenic activities in both ER transfectants and MCF-7 cells.(ABSTRACT TRUNCATED AT 250 WORDS)