Synthesis and biological properties of 7alpha-cyano derivatives of the (17alpha,20E/Z)-[125I]iodovinyl- and 16alpha-[125I]iodo-estradiols

The synthesis, receptor binding affinity, estrogenic potency and tissue distribution of the 7alpha-cyano derivatives of the (17alpha,20E/Z)-[125I]iodovinyl-(CIVE) and 16alpha-[125I]iodo-estradiols (CIE) are reported. The iodovinyl derivatives were prepared via the (17alpha,20E/Z)-tri-n-butylstannyl intermediates, derived from the addition of tri-n-butyl tin hydride to the 17alpha-ethynyl group of the 7alpha-cyano-17alpha-ethynylestradiol, using triethylborane as a catalyst. The no-carrier-added [125I]-CIVE isomers were prepared via the same stereospecific reaction. [125I]-CIE was prepared from 7alpha-cyano-16beta-bromoestradiol via halogen exchange with Na125I. Addition of the 7alpha-cyano group to 16alpha-iodoestradiol did not affect estrogen receptor binding affinity (RBA of CIE is 115). However the estrogenic potential of CIE, as measured by the capacity to stimulate the expression of the pS2 gene, was reduced to 1% as compared to that of estradiol. Addition of a 7alpha-cyano group to the (17alpha,20E/Z)-IVE isomers reduced the RBA to 21 and 36, respectively, while the estrogenic potential was reduced to 2-3% of that of estradiol. Uterus uptake in immature rats of the 125I-labeled CIVE 20E-isomer and the 16alpha-iodo CIE peaked at 0.5h post injection while the (17alpha,20Z)-CIVE isomer showed a maximum only past 5h post injection. Uptake of all three 125I-labeled 7alpha-cyanoestrogens was suppressed by the co-injection of non-radioactive estradiol confirming the role of estrogen receptors in the localization process. Uterus retention pattern differ substantially from those of the analogues 7alpha-methylestrogens, which were previously shown to give high maximum 125I-uptake values at 2h post injection. Overall our data indicate that addition of a 7alpha-cyano group to 123I-labeled estrogens does not improve their potential to serve as SPECT agents for the imaging of estrogen receptor densities in breast cancer.     

The 1.9 Å Structure of Human α-N-Acetylgalactosaminidase: The Molecular Basis of Schindler and Kanzaki Diseases

α-N-acetylgalactosaminidase (α-NAGAL; E.C. 3.2.1.49) is a lysosomal exoglycosidase that cleaves terminal α-N-acetylgalactosamine residues from glycopeptides and glycolipids. In humans, a deficiency of α-NAGAL activity results in the lysosomal storage disorders Schindler disease and Kanzaki disease. To better understand the molecular defects in the diseases, we determined the crystal structure of human α-NAGAL after expressing wild-type and glycosylation-deficient glycoproteins in recombinant insect cell expression systems. We measured the enzymatic parameters of our purified wild-type and mutant enzymes, establishing their enzymatic equivalence. To investigate the binding specificity and catalytic mechanism of the human α-NAGAL enzyme, we determined three crystallographic complexes with different catalytic products bound in the active site of the enzyme. To better understand how individual defects in the α-NAGAL glycoprotein lead to Schindler disease, we analyzed the effect of disease-causing mutations on the three-dimensional structure.     

A rapid, homogeneous, fluorescence polarization binding assay for peroxisome proliferator-activated receptors alpha and gamma using a fluorescein-tagged dual PPARalpha/gamma activator

Peroxisome proliferator-activated receptors (PPARs) and other members of the nuclear hormone receptor family are important drug targets for the treatment of metabolic diseases. PPARalpha and PPARgamma play crucial roles in lipid and glucose metabolism, respectively. Therefore, screening methods that help to rapidly identify activators of these receptors should be of considerable value. A homogeneous fluorescence polarization (FP) ligand binding assay capable of rapidly identifying ligands that bind to both PPARalpha and PPARgamma has been developed using purified PPARalpha or PPARgamma ligand binding domains and a fluorescein-labeled analog (FLA) of a potent dual PPARalpha/gamma activator. FLA activator showed good binding affinity toward both PPARalpha (K(i)=0.7microM) and PPARgamma (K(i)=0.4microM). The binding of FLA activator was rapid and reached a plateau within 10 min. The resulting FP signal was stable for at least 18h. The FP binding assay performed robustly in a 384-well format, and the average Z' value was 0.77. There was a good correlation between the binding potency (IC(50) values) and rank order of binding potency for a panel of standard PPAR ligands obtained in FP binding assay and scintillation proximity assay or gel filtration binding assays using (3)H-labeled PPARalpha (r(2)=0.99) and PPARgamma (r(2)=0.99) ligands. There was also a good correlation of IC(50) values obtained by FP binding assay and scintillation proximity assay for the clinically used PPAR activators. Thus, the FP binding assay with a single fluorescein-labeled PPARalpha/gamma dual activator offers a homogeneous nonradioactive, sensitive, robust, and less expensive high-throughput assay for detecting compounds that bind to both PPARgamma and PPARalpha. Using this FP binding assay, we have identified a large number of PPARalpha/gamma dual activators. A similar assay platform may be easily adapted to other members of the nuclear hormone receptor family.     

Trypanosoma cruzi: in vitro activity of Epoxy-α-Lap, a derivative of α-lapachone, on trypomastigote and amastigote forms

Chagas disease is an endemic parasitic infection caused by Trypanosomacruzi that affects 18-20 million people in Central and South America. Recently we described the Epoxy-α-Lap, an oxyran derivative of α-lapachone, which presents a low toxicity profile and a high inhibitory activity against T.cruzi epimastigotes forms, the non-infective form of this parasite. In this work we described the trypanocidal effects of Epoxy-α-Lap on extracellular (trypomastigote) and intracellular (amastigote) infective forms of two T. cruzi strains (Y and Colombian) known by their different infective profile. Our results showed that Epoxy-α-Lap is lethal to trypomastigote Y and Colombian strains (97% and 84%, respectively). Interestingly, Epoxy-α-Lap also showed a trypanocidal effect in human macrophage infected with T. cruzi Y (85.6%) and Colombian (71.9%) strains amastigote forms. Similar effects were observed on T. cruzi amastigote infected Vero cells (96.4% and 95.0%, respectively). Our results pointed Epoxy-α-Lap as a potential candidate for Chagas disease chemotherapy since it presents trypanocidal activity on all T. cruzi forms with low) toxicity profile.     

Triumphalone, a diketone from the volatile oil of the leaves of Melaleuca triumphalis, and its spontaneous conversion into isotriumphalone

The major component (35-65%) of the volatile oil obtained by steam distillation of the leaves of Melaleuca triumphalis has been identified as (rel)-1beta-pentyl-1alpha,6alpha-dihydroxy-3,3,5,5-tetramethylcyclohexa-2,4-dione (trivial name triumphalone). Relative stereochemistry was established by nuclear Overhauser experiments and X-ray studies on the 2-(3,5-dinitrobenzoic acid) derivative. The remainder of the oil was composed of mono- and sesquiterpene hydrocarbons and alcohols. On prolonged standing the presence of a rearrangement product of triumphalone was observed which was characterized as (rel)-1beta-pentyl-1alpha,3alpha-dihydroxy-4,4,6,6-tetramethylcyclohexa-2,5-dione (trivial name isotriumphalone), presumably arising from an acid catalyzed shift of the pentyl group from C-1 to C-2.     

Regioselectivity of taxoid-O-acetyltransferases: heterologous expression and characterization of a new taxadien-5alpha-ol-O-acetyltransferase

In addition to the anticancer drug Taxol, yew (Taxus) species produce a large variety of other taxane diterpenoids which differ mainly in the type of acyl and aroyl groups appended to the many hydroxyl functions on the taxane core; acetate esters are particularly common. Taxol bears an acetate at C10 and another at C4 thought to originate by intramolecular migration of a C5 acetate function in the process of oxetane ring formation, but many other naturally occurring taxoids bear acetate groups at C1, C2, C7, C9, and C13, in addition to C5 and C10. cDNAs encoding a taxoid 5alpha-O-acetyltransferase (taxadien-5alpha-ol as substrate) and a taxoid 10beta-O-acetyltransferase (10-deacetylbaccatin III as substrate) have been acquired from a recently isolated family of Taxus acyl/aroyltransferase clones. To explore the origins of other acetylated taxoids, the group of recombinant Taxus acyltransferases was investigated with a range of polyhydroxylated taxoids as substrates. From this survey, a new acetyltransferase clone (denoted TAX19) was identified that was capable of acetylating taxadien-5alpha-ol with activity comparable to that of the previously identified 5alpha-O-acetyltransferase (clone TAX1). However, when these two recombinant enzymes were presented with taxadien-triol and tetraol substrates, they exhibited different regiospecificities. The TAX1 enzyme preferentially acetylates the "northern" hemisphere hydroxyls at C9 and C10, whereas the TAX19 enzyme preferentially acetylates the "east-west" pole positions at C5 and C13. The TAX1 enzyme possesses the lowest KM value with taxadien-5alpha-ol (an early pathway metabolite) as substrate, with much higher KM values for the polyhydroxylated taxoid substrates, whereas the TAX19 enzyme possesses lower KM values (than the TAX1 transferase) for all taxoid substrates tested. These results suggest that both TAX1 and TAX19 acyltransferases may function at the early C5 acetylation step of taxoid metabolism, and that the TAX19 acyltransferase, because of its broader specificity for polyhydroxylated taxoids, may also function later in metabolism and be responsible for the production of many other acetylated taxoids.     

Production, purification and characterization of two α-amylase isoforms from a newly isolated Aspergillus Oryzae strain S2

A new fungal strain that was isolated from old sweet soy sauce was identified, based on subsequent microscopic studies and analyses of rRNA18S gene sequence, intergenic region rRNA 18S-23S, and aflatoxins production tests, as an Aspergillus oryzae strain. The latter was noted to produce two extracellular α-amylases, namely AmyA and AmyB. The monitoring of alpha-amylase production in the presence and absence of various protease inhibitors indicated that AmyB could be formed from the proteolysis of AmyA. The enzymes were purified to homogeneity through fractional acetone precipitation, size exclusion, and anion exchange chromatography. The molecular masses estimated for AmyA and AmyB by SDS-PAGE were 50 and 42 kDa, respectively. The NH₂-terminal of the purified proteins showed the same amino acid sequences. Further biochemical characterization assays revealed that both enzymes attained maximal activity at pH 5.6 and 50 °C. They were activated and stabilized by Ca²⁺ and were noted to produce maltose and maltotriose as major starch hydrolysis end products. Overall, the findings of the present study indicate that both AmyA and AmyB exhibit a number of promising properties that make them potential strong candidates for application as additives in the bread making industry.     

Ent-pimarane type diterpenes from Gnaphalium gaudichaudianum

Fractionation of the methanol extract of Gnaphalium gaudichaudianum DC afforded one new and six known ent-pimarane diterpenes together with velutin, squalene and stigmasterol. The structure of the new compound was established on the basis of extensive 1D and 2D NMR spectroscopic data interpretation. Two of the isolated compounds exhibited moderate toxicity in the Artemia salina toxicity test.     

Isolation and characterization of two positional isomers of novel heterogeneous branched cyclomaltohexaoses (alpha-cyclodextrins) having a D-galactobiosyl residue on the side chain

Transgalactosylated products, 2-O-alpha-D-galactobiosyl-cyclomaltohexaoses (alpha-cyclodextrins, alphaCDs), were synthesized by alpha-galactosidase from coffee bean using melibiose and alphaCD as a donor substrate and an acceptor, respectively. Two positional isomers of 2-O-alpha-galactobiosyl-alphaCDs were isolated and purified by HPLC, and their structures were elucidated by FABMS and NMR spectroscopies, as well as by an enzymatic degradation method. The chromatographic behavior of these novel galactosylated alphaCDs was compared on three HPLC columns with different separation modes.     

5α,8α-Epidioxysterols from the gorgonian Eunicella cavolini and the ascidian Trididemnum inarmatum: Isolation and evaluation of their antiproliferative activity

Three new (1, 4, 9) and nine previously reported (2, 3, 5-8, 10-12) 5α,8α-epidioxysterols were isolated from the organic extracts of the gorgonian Eunicella cavolini and the ascidian Trididemnum inarmatum. The structures and relative configurations of 1-12 were established on the basis of detailed NMR spectroscopic analyses and comparison with the literature. The growth inhibitory effects of 1-12 were evaluated against MCF-7 human breast cancer cells. Compound 1, bearing a cyclopropyl moiety in the side chain, exhibited the highest antiproliferative activity.     

Diterpenes from the brown algae Dictyota dichotoma and Dictyota linearis

Nineteen secondary metabolites of the brown alga Dictyota dichotoma (Huds.) Lam. and fifteen metabolites of the brown alga D. linearis (Ag.) Grev. were isolated and their chemical structures were elucidated on the basis of their NMR and mass spectral data. The diterpenes isopachydictyolal (1) from D. dichotoma and 4alpha-acetyldictyodial (2) from D. linearis are new natural products. The antiviral activity of metabolites isolated in adequate amounts was evaluated in laboratory assays against Herpes simplex virus I (HSV I) and Poliomyelitis Virus I, using Vero cells as hosts.     

Biotransformations of 20-hydroxyecdysone and analogues by Curvularia lunata NRRL 2178

20-Hydroxyecdysone, the arthropod moulting hormone, was biotransformed by the fungus Curvularia lunata NRRL 2178 to the rare ecdysteroid, 2-dehydro-3-epi-20-hydroxyecdysone, and the novel 3alpha,9alpha-cyclo ecdysteroid analogue, (20R,22R)-3beta,14alpha,20,22,25-pentahydroxy-3alpha,9alpha-cyclo-5beta-cholest-7-en-2,6-dione in 14 and 44% yields, respectively. Ponasterone A and pterosterone were similarly biotransformed to the corresponding 2-dehydro-3-epi- and 3alpha,9alpha-cyclo-analogues.     

Topo II alpha reporter mice reveal proliferative regions in the epidermis and small intestine

We investigated topoisomerase II alpha promoter activity in epithelia. In confluent human keratinocytes the promoter (-557 to+90 bp) was upregulated by oncogenic Ras. In transgenic mice expressing GFP via the promoter, GFP expression in undamaged epidermis was confined to growing hair follicles. GFP was also expressed in intestinal crypts. GFP expression was upregulated in epidermis following wounding or phorbol ester treatment. In each situation GFP expression correlated with high endogenous topoisomerase II alpha expression and high proliferation. The mice therefore provide a useful model for non-invasive imaging of regions of high proliferation in normal or damaged epidermis.     

Hydroxylation of the sesterterpene leucosceptrine by the fungus Rhizopus stolonifer

The microbial transformation of leucosceptrine (1), the first member of class leucosesterterpenes, by Rhizopus stolonifer afforded two metabolites, 1alpha-hydroxyleucosceptrine (2), and 8alpha-hydroxyleucosceptrine (3).     

Estrogen regulation of c-fos gene expression through phosphatidylinositol-3-kinase-dependent activation of serum response factor in MCF-7 breast cancer cells

17Beta-estradiol (E2) induces proliferation and c-fos gene expression in MCF-7 cells and both responses are partially blocked by wortmannin and LY294002 which are inhibitors of phosphatidylinositol-3-kinase (PI3-K). Analysis of the c-fos gene promoter shows that the effects of wortmannin and LY294002 are associated with inhibition of E2-induced activation through the serum response factor (SRF) motif within the proximal serum response element at -325 and -296. E2 activates constructs containing multiple copies of the SRF (pSRF) and a GAL4-SRF fusion protein; these responses are accompanied by PI3-K-dependent phosphorylation of Akt and inhibited by wortmannin/LY294002, the antiestrogen ICI 182780, but not by the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD98059. Using a series of kinase inhibitors and dominant negative kinase expression plasmids, it was shown that the non-genomic activation of SRF by E2 was associated with src-ras-PI3-K pathway, thus, demonstrating hormonal activation of the SRE through src-ras activation of both PI3-K- and MAPK-dependent signaling pathways.     

Enhanced pro-inflammatory cytokine production in Galphai2-deficient mice on colitis prone and colitis resistant 129Sv genetic backgrounds

Mice deficient in G-protein subunit alphai2 develop colitis closely resembling human ulcerative colitis when raised on 129SvEv background. When backcrossing the Galphai2-deficiency into a 129SvJBom genetic background, surprisingly, mice did not develop colitis. In vitro stimulation of splenocytes with formalin-killed Staphylococcus aureus resulted in significantly increased production of interleukin-1beta, tumor necrosis factor, and interleukin-12p40 in Galphai2(-/-) as compared to control mice. The enhanced production of pro-inflammatory cytokines was seen in colitis prone as well as in colitis resistant genetic background. A similar outcome was seen upon stimulation with toxic shock syndrome toxin-1, a T cell superantigen, except that Galphai2(-/-) colitis resistant 129SvJBom splenocytes did not show increased production of IL-12p40 as compared to their controls.     

Neuroprotection by methanol extract of Uncaria rhynchophylla against global cerebral ischemia in rats

In traditional Oriental medicine, Uncaria rhynchophylla has been used to lower blood pressure and to relieve various neurological symptoms. However, scientific evidence related to its effectiveness or precise modes of action has not been available. Thus, in the current study, we evaluated neuroprotective effects of U. rhynchophylla after transient global ischemia using 4-vessel occlusion model in rats. Methanol extract of U. rhynchophylla administered intraperitoneally (100-1000 mg/kg at 0 and 90 min after reperfusion) significantly protected hippocampal CA1 neurons against 10 min transient forebrain ischemia. Measurement of neuronal cell density in CA1 region at 7 days after ischemia by Nissl staining revealed more than 70% protection in U. rhynchophylla-treated rats compared to saline-treated animals. In U. rhynchophylla-treated animals, induction of cyclooxygenase-2 in hippocampus at 24 hr after ischemia was significantly inhibited at both mRNA and protein levels. Furthermore, U. rhynchophylla extract inhibited TNF-alpha and nitric oxide production in BV-2 mouse microglial cells in vitro. These anti-inflammatory actions of U. rhynchophylla extract may contribute to its neuroprotective effects.     

Alpha-glucosidase inhibitory activity of Syzygium cumini (Linn.) Skeels seed kernel in vitro and in Goto-Kakizaki (GK) rats

Syzygium cumini seed kernel extracts were evaluated for the inhibition of alpha-glucosidase from mammalian (rat intestine), bacterial (Bacillus stearothermophilus), and yeast (Saccharomyces cerevisiae, baker's yeast). In vitro studies using the mammalian alpha-glucosidase from rat intestine showed the extracts to be more effective in inhibiting maltase when compared to the acarbose control. Since acarbose is inactive against both the bacterial and the yeast enzymes, the extracts were compared to 1-deoxynojirimycin. We found all extracts to be more potent against alpha-glucosidase derived from B. stearothermophilus than that against the enzymes from either baker's yeast or rat intestine. In an in vivo study using Goto-Kakizaki (GK) rats, the acetone extract was found to be a potent inhibitor of alpha-glucosidase hydrolysis of maltose when compared to untreated control animals. Therefore, these results point to the inhibition of alpha-glucosidase as a possible mechanism by which this herb acts as an anti-diabetic agent.     

Effect of a novel steroid (PM-9) on the inhibition of 5alpha-reductase present in Penicillium crustosum broths

The conversion of testosterone (T) to 5alpha-dihydrotestosterone (DHT) has been demonstrated in Penicillium crustosum broth obtained from fermented pistachios, lemons and corn tortillas. Furthermore, the presence of 5alpha-reductase enzyme, which is responsible for this conversion, has been established by electrophoretical techniques in these cultures.5alpha-Reductase enzyme is also present in animal and human androgen-dependent tissues as well as in prostate and seminal vesicles. The increase of the conversion of T to DHT in prostate gland, has been related to some illnesses such as benign prostate hyperplasia and prostate cancer. Furthermore, treatment with 5alpha-reductase inhibitors such as finasteride reduces the prostate growth. These data have stimulated research for the synthesis of new molecules with antiandrogenic activity, whose biological effect needs to be demonstrated.The purpose of this study is to determine the inhibition pattern of 5alpha-reductase in P. crustosum by finasteride and the new steroidal compound PM-9. K(m) and V(max) values for T, were determined in the broths by Lineweaver-Burk plots using different testosterone concentrations. The inhibition pattern of finasteride and PM-9 was also determined by Lineweaver-Burk using different concentrations of T and inhibitors. Results show that finasteride and PM-9 inhibit 5alpha-reductase present in the broth in a competitive manner.     

Constituents of the stem bark of Discopodium penninervium and their LTB4 and COX-1 and -2 inhibitory activities

The stem bark of Discopodium penninervium afforded a withanolide, 6alpha,7alpha-epoxy-1-oxo-5alpha,12alpha,17alpha-trihydroxywitha-2,24-dienolide (1) and a coloratane sesquiterpene, 7alpha,11alpha-dihydroxy-4(13),8-coloratadien-12,11-olide (4) along with five known compounds, withanone (2), 5alpha,17beta-dihydroxy-6alpha,7alpha-epoxy-1-oxowitha-2,24-dienolide (3), 7alpha,11alpha-dihydroxy-8-drimen-12,11-olide (5), withasomnine (6), and (E,Z)-9-hydroxyoctadeca-10,12-dienoic acid (7). The identity of the compounds was established on the basis of spectroscopic data analysis. All compounds were assessed for inhibition of leukotriene metabolism in an in vitro bioassay using activated human neutrophile granulocytes, and for in vitro cycloxygenase-1 and -2 inhibition from sheep cotyledons and seminal vesicles, respectively. In the leukotriene biosynthesis assay all compounds tested at a concentration of 50 microM exhibited activity with percentage inhibitions ranging from 11.5 to 36.6. The withanolide, 1, displayed a 46.4% inhibition of COX-2 and a 22.9% inhibition of LTB(4) formation at 50 microM concentration. Compounds 4 and 6 inhibited LTB(4) biosynthesis but showed minor inhibition of COX-1 and COX-2. The remaining compounds, on the other hand, were found to be inactive on COX enzymes.