Mathurose Ponglikitmongkol

Genomic organization of the human oestrogen receptor gene.

The oestrogen receptor (ER) is a ligand‐activated transcription factor composed of several domains important for hormone binding, DNA binding and activation of transcription. We show here that the human ER gene is greater than 140 kb in length, split into eight exons and that the positions of these introns have been highly conserved when compared with the chicken progesterone receptor and are remarkably similar to those of one of the chicken thyroid hormone receptor genes. The N‐terminal A/B region, which is not conserved between the different members of the nuclear receptor family, is almost entirely encoded within a single exon. Notably each of the putative ‘zinc fingers’ of the receptor DNA‐binding domain is encoded separately, and the hormone‐binding domain is assembled from five exons. In addition, we find that the ER isolated from the human breast cancer cell line MCF‐7 contains a Gly‐400––Val mutation present in the hormone‐binding domain.

Synergistic activation of transcription by the human estrogen receptor bound to tandem responsive elements.

The synergistic action of the human estrogen receptor (hER) has been investigated using minimal promoters containing a TATA region and one or two estrogen responsive elements (EREs). We find that paired perfectly palindromic EREs act additively and independently of their relative spacing when positioned close to the TATA box. However, when moved 175 bp further upstream, perfectly palindromic EREs act synergistically and in a stereoalignment‐dependent manner. Less efficient imperfectly palindromic EREs display a stereoalignment‐dependent synergism even when positioned close to the TATA box. Analysis of binding in vitro of the hER to perfectly or imperfectly palindromic EREs by gel retardation and nitrocellulose filter binding assays does not provide any evidence for cooperative DNA binding. Conversely, the human progesterone receptor (hPR) clearly shows cooperative binding to paired responsive elements (PREs) under similar conditions in vitro. Thus, hER molecules bound to adjacent EREs must be properly stereoaligned to activate transcription synergistically in vivo. However, our data suggest that this synergism is not primarily due to cooperative binding to the EREs.

The transcriptional activation function located in the hormone-binding domain of the human oestrogen receptor is not encoded in a single exon.

Using GAL4 chimeric receptors, we have reported previously that the hormone‐binding domain (HBD) of the human oestrogen receptor (hER) contains an hormone‐inducible transcription activation function. We have extended that study here to show that this activation function represents the major activating domain in the hER in HeLa cells. In addition, we have expressed the various exons encoding the hER HBD as GAL4 fusion proteins and have shown that none contain a discrete activation function. Thus the activating domain of the hER HBD appears to be different from the recently characterized ‘simple’ activating domains, such as acidic ‘blob’ or amphipathic helix, and more likely corresponds to a protein surface created from dispersed elements and dependent upon the three‐dimensional folding of the HBD.

Diarylheptanoids, new phytoestrogens from the rhizomes of Curcuma comosa: Isolation, chemical modification and estrogenic activity evaluation.

Three new diarylheptanoids, a 1:2 mixture of (3S)- and (3R)-1-(4-methoxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol (13a and 13b) and 1-(4-hydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-one (15), together with two synthetically known diarylheptanoids 1,7-diphenyl-(1E,3E,5E)-1,3,5-triene (9) and 1-(4-hydroxyphenyl)-7-phenyl-(4E,6E)-4,6-heptadien-3-one (16), and nine known diarylheptanoids, 2, 8, 10-12, 14, a 3:1 mixture of 17a and 17b, and 18, were isolated from the rhizomes of Curcuma comosa Roxb. The absolute stereochemistry of the isolated compounds has also been determined using the modified Moshers method. The isolated compounds and the chemically modified analogues were evaluated for their estrogenic-like transcriptional activity using RT-PCR in HeLa cell line. Some of the isolated diarylheptanoids and their modified analogues exhibited estrogenic activity comparable to or higher than that of the phytoestrogen genistein. Based on the transcriptional activation of both estrogenic targets, Bcl-xL and ERbeta gene expression, the structural features for a diarylheptanoid to exhibit high estrogenic activity are the presence of an olefinic function conjugated with the aromatic ring at the 7-position, a keto group at the 3-position, and a phenolic hydroxyl group at the p-position of the aromatic ring attached to the 1-position of the heptyl chain.

A novel detection of a single plasmodium falciparum in infected blood

Detection of Plasmodium falciparum malaria by a specific DNA probe is a highly promising means for epidemiological surveillance of human malaria. However, none of presently available DNA probe methods could detect as little as a few parasites in infected blood. By amplification of a specific 206 base pairs P. falciparum DNA sequence using the polymerase chain reaction (PCR), as little as 0.01 picogram DNA or one-half of a parasite was sufficient for a specific detection. A PCR procedure for detection of P. falciparum in infected blood without prior DNA extraction was also developed which was sensitive for a single parasite. The procedure was simple and should be applicable for a large scale epidemiological study involving a very low parasitemia situation.

Diarylheptanoid Phytoestrogens Isolated from the Medicinal Plant Curcuma comosa: Biologic Actions in Vitro and in Vivo Indicate Estrogen Receptor–Dependent Mechanisms

Background Diarylheptanoids isolated from Curcuma comosa Roxb. have been recently identified as phyto estrogens. However, the mechanism underlying their actions has not yet been identified. Objectives We characterized the estrogenic activity of three active naturally occurring diarylheptanoids both in vitro and in vivo. Methods We characterized mechanisms of estrogenic action of the diarylheptanoids (3S)-1,7-diphenyl-(6E)-6-hepten-3-ol (D1), 1,7-diphenyl-(6E)-6-hepten-3-one (D2), and (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (D3) by using a real-time polymerase chain reaction assay, a mammalian transfection model, and a uterotrophic assay in mice. Results All diarylheptanoids up-regulated estrogen-responsive genes in estrogen-responsive breast cancer cells (MCF-7). In HepG2 cells transfected with estrogen receptor (ER) β or different ERα functional receptor mutants and the Vit-ERE-TATA-Luc reporter gene, all diarylheptanoids induced transcription through a ligand-dependent human ERα-ERE–driven pathway, which was abolished with ICI 182,780 (ER antagonist), whereas only D2 was active with ERβ. An ERα mutant lacking the functional AF2 (activation function 2) region was not responsive to 17β-estradiol (E2) or to any of the diarylheptanoids, whereas ERα lacking the AF1 domain exhibited wild-type–like activity. D3 markedly increased uterine weight and proliferation of the uterine epithelium in ovariectomized mice, whereas D1 and D2 were inactive. D3, like E2, up-regulated lactoferrin (Ltf) gene expression. The responses to D3 in the uterus were inhibited by ICI 182,780. In addition, D3 stimulated both classical (Aqp5) and nonclassical (Cdkn1a) ER-mediated gene regulation. Conclusions The results suggest that the D3 diarylheptanoid is an agonist for ER both in vitro and in vivo, and its biological action is ERα selective, specifically requiring AF2 function, and involves direct binding via ER as well as ERE-independent gene regulation.

Stage specificity of Plasmodium falciparum telomerase and its inhibition by berberine.

Telomerase activity in synchronized Plasmodium falciparum during its erythrocytic cycle was examined using the TRAP assay. Telomerase activity was detected at all stages of the parasite intraerythrocyte development, with higher activity in trophozoite and schizont stages compared with ring form. Berberine, extracted from Arcangelisia flava (L.) Merr., inhibited telomerase activity in a dose-dependent manner over a range of 30-300 microM, indicating that P. falciparum telomerase might be a potential target for future malaria chemotherapy.

Estrogenic Activity of Diarylheptanoids from Curcuma comosa Roxb. Requires Metabolic Activation

Curcuma comosa Roxb. has traditionally been used as a dietary supplement for health promotion in peri- and postmenopausal women in Thailand. We investigated the estrogenic activity of 7 naturally occurring diarylheptanoids from the extracts of C. comosa both in vitro and in vivo. A yeast recombinant system containing human estrogen receptor alpha, coactivator TIF2 and a beta-galactosidase reporter gene was used to determine estrogenic activity of diarylheptanoids metabolically activated with rat liver S9-fraction prior to the assay. The most potent compound was (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol, with a relative potency of 4% compared to 17beta-estradiol. The metabolic activation of diarylheptanoids markedly enhanced their efficiency. The chemical structure required for estrogenic activity of diarylheptanoids was the presence of a keto group at C3 and absence of hydroxyl moiety in ring B. Only diarylheptanoids showing full estrogenic efficiency in vitro were able to elicit uterotrophic activity of in immature ovariectomized rat. This is the first evidence for in vivo estrogenic activity of diarylheptanoids from C. comosa. This novel class of natural phytoestrogens has the potential to be developed for use as dietary supplement in the treatment of menopausal symptoms.

Alterations of organ histopathology and metallolhionein mRNA expression in silver barb, Puntius gonionotus during subchronic cadmium exposure

Common silver barb, Puntius gonionotus, exposed to the nominal concentration of 0.06 mg/L Cd for 60 d, were assessed for histopathological alterations (gills, liver and kidney), metal accumulation, and metallothionein (MT) mRNA expression. Fish exhibited pathological symptoms such as hypertrophy and hyperplasia of primary and secondary gill lamellae, vacuolization in hepatocytes, and prominent tubular and glomerular damage in the kidney. In addition, kidney accumulated the highest content of cadmium, more than gills and liver. Expression of MT mRNA was increased in both liver and kidney of treated fish. Hepatic MT levels remained high after fish were removed to Cd-free water. In contrast, MT expression in kidney was peaked after 28 d of treatment and drastically dropped when fish were removed to Cd-free water. The high concentrations of Cd in hepatic tissues indicated an accumulation site or permanent damage on this tissue.

Differential localization of HPV16 E6 splice products with E6-associated protein

High-risk Human Papillomavirus (HPV) is the etiological agent associated with the majority of anogenital cancers. The primary HPV oncogenes, E6 and E7, undergo a complex splicing program resulting in protein products whose purpose is not fully understood. Previous mouse studies have confirmed the existence of a translated product corresponding to the E6*I splice product. In terms of function, the translated E6*I protein has been shown to bind to E6 protein and to E6 associated protein (E6AP). E6*I has an inhibitory effect on E6-mediated p53 degradation in E6 expressing cells. In order to analyze the relationship between E6*I and full-length E6 in relation to localization, we created a series of green fluorescent protein (GFP) fusion products. The localization of these proteins with reference to E6AP in vivo remains unclear. Therefore, we investigated the cellular distribution of different forms of E6 with reference to E6AP. E6 and E6*I proteins, expressed from a wild type E6 gene cassette, were dispersed in the nucleus and the cytoplasm. Whereas, the E6 splice donor mutant (E6MT) was primarily localized to the nucleus. E6*I protein and E6AP were found to co-localize mainly to the cytoplasm, whereas the co-localization of full-length E6 protein and E6AP, if at all, was found mainly at the perinuclear region. These results suggest a functional relationship between the E6*I and full-length E6 protein which correlates with their localization and likely is important in regulation of the E6-E6AP complex.