The identification of 14 alpha,17 beta-dihydroxyandrost-4-en-3-one monohydrate and 14 alpha,17 beta-dihydroxyandrosta-1,4-dien-3-one monohydrate, metabolites of androstenedione in Mucor piriformis

The microorganism Mucor piriformis transforms androst-4-ene-3,17-dione into a major and several minor metabolites. X-ray crystallographic analysis of two of these metabolites was undertaken to determine unambiguously their composition and chirality. Crystals belong to the orthorhombic space-group P2(1)2(1)2(1), with a = 7.199(4) angstrom and a = 6.023(3) angstrom, b = 11.719(3) angstrom and b = 13.455(4) angstrom, c = 20.409(3) angstrom and c = 20.702(4) angstrom for the two title compounds, respectively. The structures have been refined to final R values of 0.060 and 0.040, respectively.

A large conductance Ca2+-activated K+ channel in alpha T3-1 pituitary gonadotrophs

The Ca2+-activated K+ channel in endocrine cells is responsible for membrane hyperpolarization and rhythmic firing of action potentials. The probability of opening of this channel is sensitive to intracellular-free Ca2+ concentration. In this study we have identified one such large conductance Ca2+-activated K+ channel in alpha T3-1 pituitary gonadotroph cell. This channel is ohmic with a unit conductance of 170 pS in symmetrical KCl (135 mM) and its current reverses near zero millivolts. When more than one channel is present in the patch membrane they open and close independent of each other, exhibiting no cooperativity between them as expected of a binomial distribution. The regulatory mechanism of this channel in modulating hormone secretion from alpha T3-1 gonadotroph cells is indicated.

Intramolecular Reactivity - Keto Participation in the Hydrolysis of N-Methyl-N-(9-Oxobicyclo[3.3.1]Nonan-2-Alpha-YL) Morpholinium Iodide, and A Cannizzaro-Type Reaction of 3-(2-Oxocyclohexyl)Propanal

Mechanistic studies of two intramolecular processes, nucleophilic displacement of N-methylmorpholinium in N-methyl-N-{9-oxobicyclo[3,3,1]nonan-2 alpha-yl}morpholinium iodide, anchimerically assisted by keto carbonyl, and a Cannizzaro-type reaction of 3-(2-oxocyclohexyl)propanal, occurring via axial hydride transfer onto the cyclohexanone, are reported.

Synthesis of acicular hydrogoethite (alpha-FeOOH center dot xH(2)O; 0.1 < x < 0.22) particles using morphology controlling cationic additives and magnetic properties of maghemite derived from hydrogoethite

A method for the preparation of acicular hydrogoethite (alpha -FeOOH.xH(2)O, 0.1 < x < 0.22) particles of 0.3-1 mm length has been optimized by air oxidation of Fe( II) hydroxide gel precipitated from aqueous (NH4)(2)Fe(SO4)(2) solutions containing 0.005-0.02 atom% of cationic Pt, Pd or Rh additives as morphology controlling agents. Hydrogoethite particles are evolved from the amorphous ferrous hydroxide gel by heterogeneous nucleation and growth. Preferential adsorption of additives on certain crystallographic planes thereby retarding the growth in the perpendicular direction, allows the particles to acquire acicular shapes with high aspect ratios of 8-15. Synthetic hydrogoethite showed a mass loss of about 14% at similar to 280 degreesC, revealing the presence of strongly coordinated water of hydration in the interior of the goethite crystallites. As evident from IR spectra, excess H2O molecules (0.1- 0.22 per formula unit) are located in the strands of channels formed in between the double ribbons of FeO6 octahedra running parallel to the c- axis. Hydrogoethite particles constituted of multicrystallites are formed with Pt as additive, whereas single crystallite particles are obtained with Pd (or Rh). For both dehydroxylation as well as H-2 reduction, a lower reaction temperature (similar to 220 degreesC) was observed for the former (Pt treated) compared to the latter (Pd or Rh) (similar to 260 degreesC). Acicular magnetite (Fe3O4) was prepared either by reducing hydrogoethite (magnetite route) or dehydroxylating hydrogoethite to hematite and then reducing it to magnetite (hematite- magnetite route). According to TEM studies, preferential dehydroxylation of hydrogoethite along < 010 > leads to microporous hematite. Maghemite (gamma -Fe2O3 (-) (delta), 0 < < 0.25) was obtained by reoxidation of magnetite. The micropores are retained during the topotactic transformation to magnetite and finally to maghemite, whereas cylindrical mesopores are formed due to rearrangement of the oxygen sublattice from hexagonal to cubic close packing during the conversion of hydrogoethite to magnetite and then to maghemite. Accordingly, three different types of maghemite particles are realized: strongly oriented multicrystalline particles, single crystalline acicular particles with micropores or crystallites having mesopores. Higher values of saturation magnetization ((s) = 74 emu g(-1)) and coercivity (H-c = 320 Oe) are obtained for single crystalline mesoporous particles. In the other cases, the smaller size of particles and larger distribution of micropores decreases sigma (s) considerably ( < 60 emu g(-1)) due to relaxation effects of spins on the surface atoms as revealed by Mossbauer spectroscopy.

Total synthesis of (+/-)-1,13-herbertenediol, (+/-)-alpha-herbertenol and (+/-)-beta-herbertenol

Total synthesis of alpha-herbertenol, beta-herbertenol and 1,13-herbertenediol, employing a Claisen rearrangement and ring-closing metathesis as key reactions for the generation of the cyclopentane containing vicinal quaternary carbons, has been described. (C) 2003 Elsevier Science Ltd. All rights reserved.

Luteinizing hormone regulates inhibin-alpha subunit expression through multiple signaling pathways involving steroidogenic factor-1 and beta-catenin in the macaque corpus luteum

We employed different experimental model systems to define the role of GATA4, beta-catenin, and steroidogenic factor (SF-1) transcriptional factors in the regulation of monkey luteal inhibin secretion. Reverse transcription polymerase chain reactions and western blotting analyses show high expression of inhibin-alpha, GATA4, and beta-catenin in corpus luteum (CL) of the mid-luteal phase. Gonadotropin-releasing hormone receptor antagonist-induced luteolysis model suggested the significance of luteinizing hormone (LH) in regulating these transcriptional factors. Inducible cyclic AMP early repressor mRNA expression was detected in the CL and no change was observed in different stages of CL. Following amino acid sequence analysis, interaction between SF-1 and beta-catenin in mid-stage CL was verified by reciprocal co-immunoprecipitation experiments coupled to immunoblot analysis. Electrophoretic mobility shift analysis support the role of SF-1 in regulating luteal inhibin-alpha expression. Our results suggest a possible multiple crosstalk of Wnt, cAMP, and SF-1 in the regulation of luteal inhibin secretion.

An approach to chiral amino acids via reductive amination of ketones: hydride reduction of 1-(S)-phenethyl amine derived Schiff bases of C-protected alpha-keto acids

Various 1-acyl-2,4,10-trioxaadamantanes were prepared from the corresponding 1-methoxycarbonyl derivatives, via conversion to the N-acylpiperidine derivatives followed by reaction with a Grignard reagent in refluxing THF. These alpha-keto orthoformates were converted to the corresponding imines with 1-(S)-phenethyl amine (TiCl4/Et3N/toluene/reflux), with the Schiff bases being reduced further with NaBH4 (MeOH/0 degrees C) into the corresponding 1-(S)-phenethyl amines (diastereomeric excess 91:9 by NMR). Hydrogenolysis of the phenethyl group (Pd-C/MeOH) finally led to the 1-(aminoalkyl)trioxaadamantanes, which are chiral C-protected alpha-amino acids, in excellent overall yields. (C) 2012 Elsevier Ltd. All rights reserved.

Structural and molecular basis of interaction of HCV non-structural protein 5A with human casein kinase 1 alpha and PKR

Background: Interaction of non-structural protein 5A (NS5A) of Hepatitis C virus (HCV) with human kinases namely, casein kinase 1 alpha (ck1 alpha) and protein kinase R (PKR) have different functional implications such as regulation of viral replication and evasion of interferon induced immune response respectively. Understanding the structural and molecular basis of interactions of the viral protein with two different human kinases can be useful in developing strategies for treatment against HCV. Results: Serine 232 of NS5A is known to be phosphorylated by human ck1 alpha. A structural model of NS5A peptide containing phosphoacceptor residue Serine 232 bound to ck1 alpha has been generated using the known 3-D structures of kinase-peptide complexes. The substrate interacting residues in ck1 alpha has been identified from the model and these are found to be conserved well in the ck1 family. ck1 alpha - substrate peptide complex has also been used to understand the structural basis of association between ck1 alpha and its other viral stress induced substrate, tumour suppressor p53 transactivation domain which has a crystal structure available. Interaction of NS5A with another human kinase PKR is primarily genotype specific. NS5A from genotype 1b has been shown to interact and inhibit PKR whereas NS5A from genotype 2a/3a are unable to bind and inhibit PKR efficiently. This is one of the main reasons for the varied response to interferon therapy in HCV patients across different genotypes. Using PKR crystal structure, sequence alignment and evolutionary trace analysis some of the critical residues responsible for the interaction of NS5A 1b with PKR have been identified. Conclusions: The substrate interacting residues in ck1 alpha have been identified using the structural model of kinase substrate peptide. The PKR interacting NS5A 1b residues have also been predicted using PKR crystal structure, NS5A sequence analysis along with known experimental results. Functional significance and nature of interaction of interferon sensitivity determining region and variable region 3 of NS5A in different genotypes with PKR which was experimentally shown are also supported by the findings of evolutionary trace analysis. Designing inhibitors to prevent this interaction could enable the HCV genotype 1 infected patients respond well to interferon therapy.

HIF-1 alpha (1772C > T) polymorphism as marker for breast cancer development

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) is an important transcription factor that regulates different cellular responses to hypoxia. HIF-1 alpha is rapidly degraded by von Hippel-Lindau (VHL) protein under normoxic conditions and stabilized under hypoxia. A common variant of HIF-1 alpha (1772C > T) (rs 11549465) polymorphism, corresponding to an amino acid change from proline to serine at 582 position within the oxygen-dependent degradation domain, results in increased stability of the protein and altered transactivation of its target genes. The present study was aimed to find the association between HIF-1 alpha (1772C > T) (rs 11549465) polymorphism and breast cancer development. For this purpose, 348 primary breast cancer patients and 320 healthy and age-matched controls were genotyped through PCR-RFLP method. The genotype frequencies were compared between patients and controls, and their influence on clinical characteristics of breast cancer patients was analyzed. Our study revealed a significant increase of TT genotype in breast cancer patients compared to controls (p = 0.038). Further, TT genotype and T allele were found to be associated with progesterone receptor (PR)-negative status (p < 0.09). None of the clinical variables revealed significant association with HIF-1 alpha (1772C > T) (rs 11549465) polymorphism.

The reduced-alpha-width of the lowest 1¯ state of ^(16)O

Cross sections for the reaction ¹²C(α,γ)¹⁶O have been measured for a range of center-of-mass alpha particle energies extending from 1.72 MeV to 2.94 MeV. Two 8"x5" NaI (Tℓ) crystals were used to detect gamma rays; time-of-flight technique was employed to suppress cosmic ray background and background due to neutrons arising mainly from the ¹³C(α,n)¹⁶O reaction. Angular distributions were measured at center-of-mass alpha energies of 2.18, 2.42, 2.56 and 2.83 MeV. Upper limits were placed on the amount of radiation cascading through the 6.92 or 7.12-MeV states in ¹⁶O. By means of theoretical fits to the measured electric dipole component of the total cross section, in which interference between the 1¯ states in ¹⁶O at 7.12 MeV and at 9.60 MeV is taken into account, it is possible to extract the dimensionless, reduced-alpha-width of the 7.12-MeV state in ¹⁶O. A three-level R-matrix parameterization of the data yields the width Θ_α,F² = 0.14^+0.10_-0.08. A "hybrid" R-matrix-optical-model parameterization yields Θ_α,F² = 0.11^+0.11_-0.07. This quantity is of crucial importance in determining the abundances of ¹²C and ¹⁶O at the end of helium burning in stars.

Adaptive evolution of primate TRIM5 alpha, a gene restricting HIV-1 infection

Recent studies showed that nonhuman primate TRIM5 alpha can efficiently block HIV-1 infection in human cell lines. It can also restrict other retroviruses, therefore, suggested as a general defender against retrovirus infection. Here, we present an evolutionary analysis of TRIM5 alpha in primates. Our results demonstrated that TRIM5a has been evolving rapidly in primates, which is likely caused by Darwinian positive selection. The SPRY domain of TRM5 alpha, which may be responsible for recognition of incoming viral capsids showed higher nonsynonymous/synonymous substitution ratios than the non-SPRY domain, indicating that the adaptive evolution of TRIM5a ill primates might be an innate strategy developed in defending retrovirus infection during primate evolution. In addition, the comparative protein sequence analysis suggested that the amino acid substitution pattern at a single site (344R/Q/P) located in the SPRY domain may explain the differences in Susceptibilities of HIV-1 infection in diverse primate species. (c) 2005 Elsevier B.V. All rights reserved.

Alpha-momorcharin inhibits HIV-1 replication in acutely but not chronically infected T-lymphocytes

AIM: To identify the anti-human immunodeficiency virus type 1 (HIV-1) activities of alpha-momorcharin ( alpha-MMC) from Momordica charantia in acutely and chronically infected lymphocytes. METHODS: The anti-HIV activities of alpha-MMC were examined by 1) the inhibition of syncytia formation induced by HIV-1 III B; 2) reduction of p24 core antigen expression level and decrease in numbers of HIV antigen positive cells in acutely and chronically infected cultures. The cytotoxic effects of alpha-MMC was tested by trypan blue dye exclusion or colorimetric MTT assay. RESULTS: alpha-MMC was found to obviously inhibit HIV-1 III B-inducing C8166 syncytia formation and markedly reduced both expression of p24 core antigen and the numbers of HIV antigen positive cells in acutely but not chronically HTV-1-infected culture. The median effective concentration (EC50) in these assays were 0.016, 0.07, and 0.32 mg.L-1, respectively. CONCLUSION: alpha-MMC is a unique component of momorcharin with anti-HIV activity, and markedly inhibited HIV-1 replication in acutely but not chronically HIV-1-infected T-lymphocytes.

Measurement of the Interaction Between Recombinant I-domain from Integrin alpha 2 beta 1 and a Triple Helical Collagen Peptide with the GFOGER Binding Motif Using Molecular Force Spectroscopy.

The role of the collagen-platelet interaction is of crucial importance to the haemostatic response during both injury and pathogenesis of the blood vessel wall. Of particular interest is the high affinity interaction of the platelet transmembrane receptor, alpha 2 beta 1, responsible for firm attachment of platelets to collagen at and around injury sites. We employ single molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) to study the interaction of the I-domain from integrin alpha 2 beta 1 with a synthetic collagen related triple-helical peptide containing the high-affinity integrin-binding GFOGER motif, and a control peptide lacking this sequence, referred to as GPP. By utilising synthetic peptides in this manner we are able to study at the molecular level subtleties that would otherwise be lost when considering cell-to-collagen matrix interactions using ensemble techniques. We demonstrate for the first time the complexity of this interaction as illustrated by the complex multi-peaked force spectra and confirm specificity using control blocking experiments. In addition we observe specific interaction of the GPP peptide sequence with the I-domain. We propose a model to explain these observations.