Crystal structure of isotibolone: a major degradation product of tibolone

Isotibolone is frequently found as an impurity in tibolone, a drug used for hormone reposition of post-menopause women, due to some inadequate tibolone synthesis or as a result of degradation during drug storage. The presence of isotibolone impurities should be detected and quantified in active pharmaceutical ingredient products of tibolone before its use in the manufacturing of medicaments. The X-ray powder diffraction technique offers the possibility of quantifying isotibolone amounts at different stages of drug production and storage, from the chemical synthesis to the final formulation. In the course of a study involving the quantitative analysis of isotibolone by X-ray powder diffraction, the authors determined the structure of the title compound using a recently developed approach (A. Gomez and S. Kycia, J. Appl. Crystallogr. 2011, 44, 708-713). The structure is monoclinic, space group P2(1) (4), with unit cell parameters a = 6.80704(7) angstrom, b = 20.73858(18) angstrom, c = 6.44900(6) angstrom, beta = 76.4302(5)degrees, V = 884.980(15) angstrom(3) and two molecules per unit cell (Z = 2). The molecules are hydrogen bonded in the ab plane forming layers that are held together in the crystal by van der Waals interactions along the c-axis.

In vitro efficacy of the essential oil of Piper cubeba L. (Piperaceae) against Schistosoma mansoni

In this paper, cercariae, schistosomula, and adult Schistosoma mansoni worms were incubated in vitro with the essential oil of Piper cubeba (PC-EO) at concentrations from 12.5 to 200 mu g/mL, and the viability was evaluated using an inverted microscopy. The effects of PC-EO at 100 and 200 mu g/mL on the stages of S. mansoni were similar to those of the positive control (PZQ at 12.5 mu g/mL), with total absence of mobility after 120 h. However, at concentrations from 12.5 to 50 mu g/mL, PC-EO caused a reduction in the viability of cercariae and schistosomula when compared with the negative control groups (RPMI 1640 or dechlorinated water) or (RPMI 1640 + 0.1% DMSO or dechlorinated water + 0.1% DMSO). On the other hand, adult S. mansoni worms remained normally active when incubated with PC-EO at concentrations of 12.5 and 25 mu g/mL, and their viabilities were similar to those of the negative control groups. In addition, at concentrations ranging from 50 to 200 mu g/mL, separation of all the coupled adult worms was observed after 24 h of incubation, which is related to the fact of the reduction in egg production at this concentration. The main chemical constituents of PC-EO were identified by gas chromatography-mass spectrometry as being sabinene (19.99%), eucalyptol (11.87%), 4-terpineol (6.36%), beta-pinene (5.81%), camphor (5.61%), and delta-3-carene (5.34%). The cytotoxicity of the PC-EO was determined, and a significant cytotoxicity was only obtained in the concentration of 200 mu g/mL after 24 h treatment. The results suggest that PC-EO possesses an effect against cercariae, schistosomula, and adult worms of the S. mansoni.

alpha,beta-Unsaturated Diazoketones as Platforms in the Asymmetric Synthesis of Hydroxylated Alkaloids. Total Synthesis of 1-Deoxy-8,8a-diepicastanospermine and 1,6-Dideoxyepicastanospermine and Formal Synthesis of Pumiliotoxin 251D

A versatile and concise approach for the stereoselective synthesis of mono-, di-, and trihydroxylated indolizidines is presented in four to six steps from Cbz-prolinal and a diazophosphonate. The key steps involved a Wolff rearrangement, followed by a stereoselective dihydroxylation/epoxidation reaction, from an alpha,beta-unsaturated diazoketone. The strategy also permits extension to the synthesis of many natural hydroxylated indolizidine alkaloids as demonstrated in the formal synthesis of pumiliotoxin 251D.

A novel xylan degrading beta-D-xylosidase: purification and biochemical characterization

Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular beta-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. beta-xylosidase is a glycoprotein (39 % carbohydrate content) and has a molecular mass of 137 kDa by SDS-PAGE, with optimal temperature and pH at 70 A degrees C and 3.0-5.5, respectively. beta-xylosidase was stable in acidic pH (3.0-6.0) and 70 A degrees C for 1 h. The enzyme was activated by 5 mM MnCl2 (28 %) and MgCl2 (20 %) salts. The beta-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-beta-d-xylopyranoside, exhibiting apparent K-m and V-max values of 0.66 mM and 39 U (mg protein)(-1) respectively, and to a lesser extent p-nitrophenyl-beta-d-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel beta-d-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by beta-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose.

ANTI-TRYPANOSOMAL ACTIVITY OF 1,2,3,4,6-PENTA-O-GALLOYL-beta-D-GLUCOSE ISOLATED FROM Plectranthus barbatus Andrews (Lamiaceae)

MeOH extract from the leaves of Plectranthus barbatus Andrews (Lamiaceae), showed in vitro anti-trypanosomal activity. The bioassay-guided fractionation resulted in the isolation of a gallic acid derivative, identified as 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG), after thorough NMR and MS spectral analysis. Finally, this compound was tested against trypomastigote forms of T. cruzi and displayed an EC50 value of 67 mu M, at least 6.6-fold more effective than the standard drug benznidazole. This is the first occurrence of PGG in the Plectranthus genus and the first anti-parasitic activity described for PGG in the literature.

Lasiodiplodan, an exocellular (1 -> 6)-beta-d-glucan from Lasiodiplodia theobromae MMPI: production on glucose, fermentation kinetics, rheology and anti-proliferative activity

Lasiodiplodan, an exopolysaccharide of the (1 -> 6)-beta-d-glucan type, is produced by Lasiodiplodia theobromae MMPI when grown under submerged culture on glucose. The objective of this study was to evaluate lasiodiplodan production by examining the effects of carbon (glucose, fructose, maltose, sucrose) and nitrogen sources (KNO3, (NH4)(2)SO4, urea, yeast extract, peptone), its production in shake flasks compared to a stirred-tank bioreactor, and to study the rheology of lasiodiplodan, and lasiodiplodan's anti-proliferative effect on breast cancer MCF-7 cells. Although glucose (2.05 +/- A 0.05 g L-1), maltose (2.08 +/- A 0.04 g L-1) and yeast extract (2.46 +/- A 0.06 g L-1) produced the highest amounts of lasiodiplodan, urea as N source resulted in more lasiodiplodan per unit biomass than yeast extract (0.74 +/- A 0.006 vs. 0.22 +/- A 0.008 g g(-1)). A comparison of the fermentative parameters of L. theobromae MMPI in shake flasks and a stirred-tank bioreactor at 120 h on glucose as carbon source showed maximum lasiodiplodan production in agitated flasks (7.01 +/- A 0.07 g L-1) with a specific yield of 0.25 +/- A 0.57 g g(-1) and a volumetric productivity of 0.06 +/- A 0.001 g L-1 h(-1). A factorial 2(2) statistical design developed to evaluate the effect of glucose concentration (20-60 g L-1) and impeller speed (100-200 rpm) on lasiodiplodan production in the bioreactor showed the highest production (6.32 g L-1) at 72 h. Lasiodiplodan presented pseudoplastic behaviour, and the apparent viscosity increased at 60A degrees C in the presence of CaCl2. Anti-proliferative activity of lasiodiplodan was demonstrated in MCF-7 cells, which was time- and dose-dependent with an IC50 of 100 mu g lasiodiplodan mL(-1).

Faeces deposition on Amazonian Anthrosols as assessed from 5 beta-stanols

In the Amazon Basin, within a landscape of infertile soils, fertile Anthrosols of pre-Columbian origin occur (Amazonian Dark Earths or terra preta de Indio). These soils are characterized by high amounts of charred organic matter (black carbon, biochar) and high nutrient stocks. Frequently, they were considered as sign for intensive landscape domestication by way of sedentary agriculture and as sign for large settlements in pre-Columbian Amazonia. Beyond the archaeological interest in Amazonian Dark Earths, they increasingly receive attention because it is assumed that they could serve as a model for sustainable agriculture in the humid tropics (terra preta nova). Both questions lack information about the pre-Columbian practices which were responsible for the genesis of Amazonian Dark Earths. It has often been hypothesized that deposition of faeces could have contributed to the high nutrient stocks in these soils, but no study has focussed on this question yet. We analyzed the biomarkers for faeces 5 beta-stanols as well as their precursors and their 5 alpha-isomers in Amazonian Dark Earths and reference soils to investigate the input of faeces into Amazonian Dark Earths. Using Amazonian Dark Earths as example, we discuss the application of threshold values for specific stanols to evaluate faeces deposition in archaeological soils and demonstrate an alternative approach which is based on a comparison of the concentration patterns of 5 beta-stanols with the concentration patterns of their precursors and their 5 alpha-isomers as well as with local backgrounds. The concentration patterns of sterols show that faeces were deposited on Amazonian Dark Earths. (C) 2011 Elsevier Ltd. All rights reserved.

Transforming growth factor beta and apoptosis in leprosy skin lesions: possible relationship with the control of the tissue immune response in the Mycobacterium leprae infection

The course of leprosy depends of the host immune response which ranges from the lepromatous pole (LL) to the tuberculoid pole (TT). A comparative study was conducted in 60 patients with the LL and TT The results showed a mean expression of TGF-beta of 339 +/- 99.4 cells/field for TT and of 519.2 +/- 68.2 cells/field for LL. Frequency of apoptosis was 6.3 +/- 1.8 in TT and 14.0 +/- 6.1 in LL. A correlation (p = 0.0251) between TGF-beta and caspase-3 in the LL was found. This finding indicates a role of TGF-beta and apoptosis in the immune response in leprosy. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Lack of beta(2)-AR improves exercise capacity and skeletal muscle oxidative phenotype in mice

beta(2)-adrenergic receptor (beta(2)-AR) agonists have been used as ergogenics by athletes involved in training for strength and power in order to increase the muscle mass. Even though anabolic effects of beta(2)-AR activation are highly recognized, less is known about the impact of beta(2)-AR in endurance capacity. We presently used mice lacking beta(2)-AR [beta(2)-knockout (beta(2) KO)] to investigate the role of beta(2)-AR on exercise capacity and skeletal muscle metabolism and phenotype. beta(2) KO mice and their wild-type controls (WT) were studied. Exercise tolerance, skeletal muscle fiber typing, capillary-to-fiber ratio, citrate synthase activity and glycogen content were evaluated. When compared with WT, beta 2KO mice displayed increased exercise capacity (61%) associated with higher percentage of oxidative fibers (21% and 129% of increase in soleus and plantaris muscles, respectively) and capillarity (31% and 20% of increase in soleus and plantaris muscles, respectively). In addition, beta 2KO mice presented increased skeletal muscle citrate synthase activity (10%) and succinate dehydrogenase staining. Likewise, glycogen content (53%) and periodic acid-Schiff staining (glycogen staining) were also increased in beta 2KO skeletal muscle. Altogether, these data provide evidence that disruption of beta(2)AR improves oxidative metabolism in skeletal muscle of beta 2KO mice and this is associated with increased exercise capacity.

Novel aryl beta-aminocarbonyl derivatives as inhibitors of Trypanosoma cruzi trypanothione reductase: binding mode revised by docking and GRIND2-based 3D-QSAR procedures

Trypanothione reductase has long been investigated as a promising target for chemotherapeutic intervention in Chagas disease, since it is an enzyme of a unique metabolic pathway that is exclusively present in the pathogen but not in the human host, which has the analog Glutathione reductase. In spite of the present data-set includes a small number of compounds, a combined use of flexible docking, pharmacophore perception, ligand binding site prediction, and Grid-Independent Descriptors GRIND2-based 3D-Quantitative Structure-Activity Relationships (QSAR) procedures allowed us to rationalize the different biological activities of a series of 11 aryl beta-aminocarbonyl derivatives, which are inhibitors of Trypanosoma cruzi trypanothione reductase (TcTR). Three QSAR models were built and validated using different alignments, which are based on docking with the TcTR crystal structure, pharmacophore, and molecular interaction fields. The high statistical significance of the models thus obtained assures the robustness of this second generation of GRIND descriptors here used, which were able to detect the most important residues of such enzyme for binding the aryl beta-aminocarbonyl derivatives, besides to rationalize distances among them. Finally, a revised binding mode has been proposed for our inhibitors and independently supported by the different methodologies here used, allowing further optimization of the lead compounds with such combined structure- and ligand-based approaches in the fight against the Chagas disease.

Schistosomicidal evaluation of flavonoids from two species of Styrax against Schistosoma mansoni adult worms

Context: Schistosomiasis is a major health problem worldwide. Thus, the search for new schistosomicidal agents from natural sources can provide prototypes for drug discovery. Objective: The present study investigated the chemical composition of the EtOAc fractions of Styrax pohlii Pohl (Styracaceae) (EF-SP) aerial parts and S. camporum A. DC. leaves (EF-SC), as well as schistosomicidal activities against Schistosoma mansoni adult worms, which have not yet been studied. Materials and methods: The crude ethanol extracts of S. camporum leaves and S. pohlii aerial parts (EE-SC and EE-SP) were partitioned with n-hexane, EtOAc, and n-BuOH. The EtOAc fractions were purified by preparative HPLC. The crude extracts, EtOAc fractions and pure compounds were tested against S. mansoni adult worms in vitro. Results: The purification procedure resulted in the isolation of kaempferol-3-O-(2 '',4 ''-di-O-(E)-p-coumaroyl)-beta-D-glucopyranoside (1), kaempferol-3-O-(2 '',6 ''-di-O-(E)-p-coumaroyl)-beta-D-glucopyranoside (2), quercetin (3), and kaempferol (4). The bioassay results indicated that EE-SC, EF-SC, EF-SP, and compounds 2 and 4 are able to separate coupled S. mansoni adult worms. Additionally, EE-SC, EF-SP, and compound 4 killed the adult schistosomes in vitro at 100 mu g/mL and 100 mu M. Discussion and conclusion: This is the first time that the presence of compounds 1-2 in S. pohlii and 3-4 in S. camporum has been reported. Additionally, biological results indicated that S. pohlii and S. camporum have great potential as a source of active compounds.

The inactive form of glycogen synthase kinase-3 beta is associated with the development of carcinomas in galectin-3 wild-type mice, but not in galectin-3-deficient mice

Galectin-3 has been implicated in the tumor development via its mediation of the Wnt signaling pathway. Likewise, glycogen synthase kinase-3beta (GSK3 beta) also plays a role in the Wnt signaling pathway by controlling the levels of cytoplasmic beta-catenin. Altered GSK3 beta expression has been described in various tumors, but to date, there are no studies evaluating its expression in models of oral carcinogenesis. Additionally, it is unknown whether the absence of galectin-3 regulates the expression of GSK3 beta. To this end, Gal3-deficient (Gal3(-/-)) and wild-type (Gal3(+/+)) male mice were treated with 4NQO for 16 weeks and sacrificed at week 16 and 32. The tongues were removed, processed, and stained with H&E to detect dysplasias and carcinomas. An immunohistochemical assay was performed to determine the level of P-GSK3 beta-Ser9 expression in both groups. Carcinomas were more prevalent in Gal3(+/+) than Gal3(-/-) mice (55.5% vs. 28.5%), but no statistical difference was reached. In the dysplasias, the proportion of cells positive for P-GSK3 beta-Ser9 was slightly higher in Gal3(+/+) than Gal3(-/-) mice (63% vs. 61%). In the carcinomas, a significant difference between Gal3(+/+) and Gal3(-/-) mice was found (74% vs. 59%; p=0.02). P-GSK3 beta-Ser9-positive cells slightly decreased from the progression of dysplasias to carcinomas in Gal3(-/-) mice (61% vs. 59%; p>0.05). However, a significant increase in P-GSK3 beta-Ser9 expression was observed from dysplasias to carcinomas in Gal3(+/+) mice (63% vs. 74%; p=0.01). In conclusion, these findings suggest that fully malignant transformation of the tongue epithelium is associated with increased P-GSK3 beta-Ser9 expression in Gal3(+/+) mice, but not in Gal3(-/-) mice.

Chronic cold stress in mice induces a regulatory phenotype in macrophages: Correlation with increased 11 beta-hydroxysteroid dehydrogenase expression

Susceptibility to infections, autoimmune disorders and tumor progression is strongly influenced by the activity of the endocrine and nervous systems in response to a stressful stimulus. When the adaptive system is switched on and off efficiently, the body is able to recover from the stress imposed. However, when the system is activated repeatedly or the activity is sustained, as during chronic or excessive stress, an allostatic load is generated, which can lead to disease over long periods of time. We investigated the effects of chronic cold stress in BALB/c mice (4 degrees C/4 h daily for 7 days) on functions of macrophages. We found that chronic cold stress induced a regulatory phenotype in macrophages, characterized by diminished phagocytic ability, decreased TNF-alpha and IL-6 and increased IL-10 production. In addition, resting macrophages from mice exposed to cold stress stimulated spleen cells to produce regulatory cytokines, and an immunosuppressive state that impaired stressed mice to control Trypanosoma cruzi proliferation. These regulatory effects correlated with an increase in macrophage expression of 11 beta-hydroxysteroid dehydrogenase, an enzyme that converts inactive glucocorticoid into its active form. As stress is a common aspect of modern life and plays a role in the etiology of many diseases, the results of this study are important for improving knowledge regarding the neuro-immune-endocrine interactions that occur during stress and to highlight the role of macrophages in the immunosuppression induced by chronic stress. (C) 2011 Elsevier Inc. All rights reserved.

Progression to Adrenocortical Tumorigenesis in Mice and Humans through Insulin-Like Growth Factor 2 and beta-Catenin

Dysregulation of the WNT and insulin-like growth factor 2 (IGF2) signaling pathways has been implicated in sporadic and syndromic forms of adrenocortical carcinoma (ACC). Abnormal beta-catenin staining and CTNNB1 mutations are reported to be common in both adrenocortical adenoma and ACC, whereas elevated IGF2 expression is associated primarily with ACC. To better understand the contribution of these pathways in the tumorigenesis of ACC, we examined clinicopathological and molecular data and used mouse models. Evaluation of adrenal tumors from 118 adult patients demonstrated an increase in CTNNB1 mutations and abnormal beta-catenin accumulation in both adrenocortical adenoma and ACC. In ACC, these features were adversely associated with survival. Mice with stabilized beta-catenin exhibited a temporal progression of increased adrenocortical hyperplasia, with subsequent microscopic and macroscopic adenoma formation. Elevated Igf2 expression alone did not cause hyperplasia. With the combination of stabilized beta-catenin and elevated Igf2 expression, adrenal glands were larger, displayed earlier onset of hyperplasia, and developed more frequent macroscopic adenomas (as well as one carcinoma). Our results are consistent with a model in which dysregulation of one pathway may result in adrenal hyperplasia, but accumulation of a second or multiple alterations is necessary for tumorigenesis. (Ant J Pathol 2012, 181:1017-1033; http://dx.doi.org/10.1016/j.ajpath.2012.05.026)

Beta-alanine (Carnosyn (TM)) supplementation in elderly subjects (60-80 years): effects on muscle carnosine content and physical capacity

The aim of this study was to investigate the effects of beta-alanine supplementation on exercise capacity and the muscle carnosine content in elderly subjects. Eighteen healthy elderly subjects (60-80 years, 10 female and 4 male) were randomly assigned to receive either beta-alanine (BA, n = 12) or placebo (PL, n = 6) for 12 weeks. The BA group received 3.2 g of beta-alanine per day (2 x 800 mg sustained-release Carnosyn (TM) tablets, given 2 times per day). The PL group received 2 x (2 x 800 mg) of a matched placebo. At baseline (PRE) and after 12 weeks (POST-12) of supplementation, assessments were made of the muscle carnosine content, anaerobic exercise capacity, muscle function, quality of life, physical activity and food intake. A significant increase in the muscle carnosine content of the gastrocnemius muscle was shown in the BA group (+85.4%) when compared with the PL group (+7.2%) (p = 0.004; ES: 1.21). The time-to-exhaustion in the constant-load submaximal test (i.e., TLIM) was significantly improved (p = 0.05; ES: 1.71) in the BA group (+36.5%) versus the PL group (+8.6%). Similarly, time-to-exhaustion in the incremental test was also significantly increased (p = 0.04; ES 1.03) following beta-alanine supplementation (+12.2%) when compared with placebo (+0.1%). Significant positive correlations were also shown between the relative change in the muscle carnosine content and the relative change in the time-to-exhaustion in the TLIM test (r = 0.62; p = 0.01) and in the incremental test (r = 0.48; p = 0.02). In summary, the current data indicate for the first time, that beta-alanine supplementation is effective in increasing the muscle carnosine content in healthy elderly subjects, with subsequent improvement in their exercise capacity.