A novel and potent trypsin inhibitor peptide, AC-17, from the skin secretion of the edible frog, Rana esculenta

Protease inhibitors are found in many venoms and evidence suggests that they occur widely in amphibian skin secretions. Kunitz inhibitors have been found in the skin secretions of bombinid toads and ranid frogs, Kazal inhibitors in phyllomedusine frogs and Bowman–Birk inhibitors in ranid frogs. Selective protease inhibitors could have important applications as therapeutics in the treatment of diseases in which discrete proteases play an aetiologcal role. Here we have examined the skin secretion of the edible frog, Rana esculenta, for protease inhibitors using trypsin as a model. HPLC fractions of secretions were screened for inhibitory activity using a chromogenic substrate as reporter. Three major peptides were resolved with trypsin inhibitory activity in HPLC fractions — one was a Kunitz-type inhibitor, a second was a Bowman–Birk inhibitor but the third represented a novel class of trypsin inhibitor in European frog skin. Analysis of the peptide established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17. Peptide AC-17 resembled a typical “Rana box” antimicrobial peptide but while it was active against Escherichia coli (MIC 30 µM) it was devoid of activity against Staphylococcus aureus and of haemolytic activity. In contrast, the peptide was a potent inhibitor of trypsin with a Ki of 5.56 µM. AC-17 represents the prototype of a novel trypsin inhibitor from the skin secretion of a European ranid frog that may target a trypsin-like protease present on the surface of Gram-negative bacteria.

Avandia (TM) - From molecule to market for type 2 diabetes.

Type 2 diabetes is a disease fast approaching epidemic proportions throughout the world. From insulin sensitizers to PPARg agonists, the lecture will outline the successful medicinal chem. and biol. strategy underpinning the discovery and selection of Avandia, an innovative new medicine for this disease.

Up-regulation of GABA transporters and GABAA Receptor α1 subunit in tremor rat hippocampus

The loss of GABAergic neurotransmission has been closely linked with epileptogenesis. The modulation of the synaptic activity occurs both via the removal of GABA from the synaptic cleft and by GABA transporters (GATs) and by modulation of GABA receptors. The tremor rat (TRM; tm/tm) is the parent strain of the spontaneously epileptic rat (SER; zi/zi, tm/tm), which exhibits absence-like seizure after 8 weeks of age. However, there are no reports that can elucidate the effects of GATs and GABAA receptors (GABARs) on TRMs. The present study was conducted to detect GATs and GABAR a1 subunit in TRMs hippocampus at mRNA and protein levels. In this study, total synaptosomal GABA content was significantly decreased in TRMs hippocampus compared with control Wistar rats by high performance liquid chromatography (HPLC); mRNA and protein expressions of GAT-1, GAT-3 and GABAR a1 subunit were all significantly increased in TRMs hippocampus by real time PCR and western blot, respectively; GAT-1 and GABAR a1 subunit proteins were localized widely in TRMs and control rats hippocampus including CA1, CA3 and dentate gyrus (DG) regions whereas only a wide distribution of GAT-3 was observed in CA1 region by immunohistochemistry. These data demonstrate that excessive expressions of GAT-1 as well as GAT-3 and GABAR a1 subunit in TRMs hippocampus may provide the potential therapeutic targets for genetic epilepsy.

Chemotherapy-Induced Activation of ADAM-17: A Novel Mechanism of Drug Resistance in Colorectal Cancer

Purpose: We have shown previously that exposure to anticancer drugs can trigger the activation of human epidermal receptor survival pathways in colorectal cancer (CRC). In this study, we examined the role of ADAMs (a disintegrin and metalloproteinases) and soluble growth factors in this acute drug resistance mechanism.

Experimental Design: In vitro and in vivo models of CRC were assessed. ADAM-17 activity was measured using a fluorometric assay. Ligand shedding was assessed by ELISA or Western blotting. Apoptosis was assessed by flow cytometry and Western blotting.

Results: Chemotherapy (5-fluorouracil) treatment resulted in acute increases in transforming growth factor-a, amphiregulin, and heregulin ligand shedding in vitro and in vivo that correlated with significantly increased ADAM-17 activity. Small interfering RNA–mediated silencing and pharmacologic inhibition confirmed that ADAM-17 was the principal ADAM involved in this prosurvival response. Furthermore, overexpression of ADAM-17 significantly decreased the effect of chemotherapy on tumor growth and apoptosis. Mechanistically, we found that ADAM-17 not only regulated phosphorylation of human epidermal receptors but also increased the activity of a number of other growth factor receptors, such as insulin-like growth factor-I receptor and vascular endothelial growth factor receptor.

Conclusions: Chemotherapy acutely activates ADAM-17, which results in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. Thus, pharmacologic inhibition of ADAM-17 in conjunction with chemotherapy may have therapeutic potential for the treatment of CRC.

Effect of growth-promoting 17ß estradiol, 19 nortestosterone and dexamethasone on circulating levels of nine potential biomarker candidates in veal calves

The use of screening methods based on the detection of biological effects of growth promoters is a promising approach to assist residue monitoring. To reveal useful effects on protein metabolism, male and female veal calves at 10 weeks of age were treated thrice with a combination of 25 mg 17ß-estradiol 3-benzoate and 150 mg 19-nortestosterone decanoate with 2 weeks intervals and finally once with 4 mg dexamethasone. Hormone-treated calves showed a significant accelerated growth rate over 6 weeks. Plasma samples of treated and control calves were analysed for immunoreactive inhibin (ir-inhibin), osteocalcin, insulin-like growth factor 1 (IGF-1), insulin-like growth factor-binding protein 2 (IGFBP-2), IGFBP-3, luteinzing hormone (LH), follicle-stimulating hormone (FSH) and prolactin using immunoaffinity assays. Hormone treatment did not affect levels of IGF-1, IGFBP-2, IGFBP-3, LH, FSH and prolactin. The concentration of circulating ir-inhibin decreased, however, significantly (P < 0.05) in bull calves upon administration of the sex steroids, whereas it remained unchanged in the female animals. Dexamethasone treatment decreased significantly (P < 0.05) circulating levels of osteocalcin in both female and male animals. Ir-inhibin and osteocalcin were, therefore, considered as candidates for a protein biomarker-based screening assay for detection of abuse of estrogens, androgens and/or glucocorticoids in cattle fattening, which is being developed in the framework of EU research project BioCop

Langerhans Cells Prime IL-17-Producing T Cells and Dampen Genital Cytotoxic Responses following Mucosal Immunization

Langerhans cells (LCs) are dendritic cells (DCs) localized in stratified epithelia, such as those overlaying skin, buccal mucosa, and vagina. The contribution of LCs to the promotion or control of immunity initiated at epithelial sites remains debated. We report in this paper that an immunogen comprising OVA linked to the B subunit of cholera toxin, used as delivery vector, was efficient to generate CTLs after vaginal immunization. Using Lang-EGFP mice, we evaluated the contribution of distinct DC subsets to the generation of CD4 and CD8 T cell responses. We demonstrate that the vaginal epithelium, unlike the skin epidermis, includes a minor population of LCs and a major subset of langerin(-) DCs. Intravaginally administered Ag is taken up by LCs and langerin(-) DCs and carried up to draining lymph nodes, where both subsets prime CD8 T cells, unlike blood-derived DCs, although with distinct capabilities. LCs prime CD8 T cells with a cytokine profile dominated by IL-17, whereas Lang(-) DCs induce IFN-gamma-producing T cells. Using Lang-DTR-EGFP mice to ensure a transient ablation of LCs, we found that these cells not only are dispensable for the generation of genital CTL responses but also downregulate these responses, by a mechanism that may involve IL-10 and IL-17 cytokines. This finding has implications for the development of mucosal vaccines and immunotherapeutic strategies designed for the targeting of DCs.

In vitro testing of Advanced JAX (TM) Bone Void Filler System: species differences in the response of bone marrow stromal cells to beta tri-calcium phosphate and carboxymethylcellulose gel

The Advanced JAX (TM) Bone Void Filler System (AJBVFS) is a novel bone graft material manufactured by Smith and Nephew Orthopaedics Ltd. and comprises beta tri-calcium phosphate granules with carboxymethylcellulose (CMC) gel as a handling agent. This study investigated the potential, in vitro, of the AJBVFS to function as a delivery system for cell therapy to enhance healing of bone defects. The attachment of rabbit bone marrow stromal cells (rbBMSCs), human BMSCs (hBMSCs) and human bone-derived cells (hBDCs) to JAX (TM) granules and the effect of CMC gel on cell proliferation and differentiation were investigated. There were slight species differences in the number and morphology of cells attached on the JAX (TM) granules with less rbBMSC attachment than human. All cells tolerated the presence of CMC gel and a reduction in cell number was only seen after longer exposure to higher gel concentrations. Low concentrations of CMC gel enhanced proliferation, alkaline phosphatase (ALP) expression and ALP activity in human cells but had no effect on rbBMSC. This study suggests that AJBVFS is an appropriate scaffold for the delivery of osteogenic cells and the addition of CMC gel as a handling agent promotes osteogenic proliferation and differentiation and is therefore likely to encourage bone healing.

A phase I study of the Heat Shock Protein 90 inhibitor alvespimycin (17-DMAG) given intravenously to patients with advanced, solid tumours.

PURPOSE: A phase I study to define toxicity and recommend a phase II dose of the HSP90 inhibitor alvespimycin (17-DMAG; 17-dimethylaminoethylamino-17-demethoxygeldanamycin). Secondary endpoints included evaluation of pharmacokinetic profile, tumor response, and definition of a biologically effective dose (BED). PATIENTS AND METHODS: Patients with advanced solid cancers were treated with weekly, intravenous (i.v.) 17-DMAG. An accelerated titration dose escalation design was used. The maximum tolerated dose (MTD) was the highest dose at which = 1/6 patients experienced dose limiting toxicity (DLT). Dose de-escalation from the MTD was planned with mandatory, sequential tumor biopsies to determine a BED. Pharmacokinetic and pharmacodynamic assays were validated prior to patient accrual. RESULTS: Twenty-five patients received 17-DMAG (range 2.5-106 mg/m(2)). At 106 mg/m(2) of 17-DMAG 2/4 patients experienced DLT, including one treatment-related death. No DLT occurred at 80 mg/m(2). Common adverse events were gastrointestinal, liver function changes, and ocular. Area under the curve and mean peak concentration increased proportionally with 17-DMAG doses 80 mg/m(2) or less. In peripheral blood mononuclear cells significant (P

High-performance liquid chromatographic determination of 17 beta-estradiol and 17 beta-estradiol-3-acetate solubilities and diffusion coefficients in silicone elastomeric intravaginal rings

A rapid, sensitive reversed-phase high-performance liquid chromatographic method has been developed for the determination of in vitro release of 17 beta-estradiol and its ester prodrug, 17 beta-estradiol-3-acetate, from silicone intravaginal rings. Partial hydrolysis of the acetate under the aqueous conditions provided by the 1% benzalkonium chloride release medium necessitates its conversion to 17 beta-estradiol prior to HPLC analysis. Both steroid peaks have been fully resolved from the benzalkonium chloride peaks by the reported chromatographic method,which employs a C-18 bonded reversed-phase column, an acetonitrile-water (50:50, v/v) mobile phase and a UV detection wavelength of 281 nm. The peak area versus 17 beta-estradiol concentration was found to be linear over the range of 0.0137-1347 mu g ml(-1) The HPLC method has also been used to determine the silicone solubilities and diffusion coefficients of the two related steroids. The almost 100-fold increase in 17 beta-estradiol-3-acetate release from the silicone core-type intravaginal rings compared to 17 beta-estradiol is shown to be due to a 60-fold increase in silicone solubility and a one and a half-fold increase in diffusitivity. The results demonstrate that an effective estrogen replacement therapy dose of 17 beta-estradiol may be administered from a silicone intravaginal reservoir device containing the labile 17 beta-estradiol-3-acetate prodrug. (C) 2000 Elsevier Science B.V. All rights reserved.

A comprehensive aerosol spray method for the rapid photocatalytic grid area analysis of semiconductor photocatalyst thin films

Indicator inks, previously shown to be capable of rapidly assessing photocatalytic activity via a novel photo-reductive mechanism, were simply applied via an aerosol spray onto commercially available pieces of Activ (TM) self-cleaning glass. Ink layers could be applied with high evenness of spread, with as little deviation as 5% upon UV-visible spectroscopic assessment of 25 equally distributed positions over a 10 cm x 10 cm glass cut. The inks were comprised of either a resazurin (Rz) or dichloroindophenol (DCIP) redox dye with a glycerol sacrificial electron donor in an aqueous hydroxyethyl cellulose (HEC) polymer media. The photo-reduction reaction under UVA light of a single spot was monitored by UV-vis spectroscopy and digital images attained from a flat-bed scanner in tandem for both inks. The photo-reduction of Rz ink underwent a two-step kinetic process, whereby the blue redox dye was initially reduced to a pink intermediate resorufin (Rf) and subsequently reduced to a bleached form of the dye. In contrast, a simple one-step kinetic process was observed for the reduction of the light blue redox dye DCIP to its bleached intermediates. Changes in red-green-blue colour extracted from digital images of the inks were inversely proportional to the changes seen at corresponding wavelengths via UV-visible absorption spectroscopy and wholly indicative of the reaction kinetics. The photocatalytic activity areas of cuts of Activ (TM) glass, 10 cm x 10 cm in size, were assessed using both Rz and DCIP indicator inks evenly sprayed over the films: firstly using UVA lamp light to activate the underlying Activ (TM) film (1.75 mW cm(-2)) and secondly under solar conditions (2.06 +/- 0.14 mW cm(-2)). The photo-reduction reactions were monitored solely by flat-bed digital scanning. Red-green-blue values of a generated 14 x 14 grid (196 positions) that covered the entire area of each film image were extracted using a Custom-built program entitled RGB Extractor(C). A homogenous degradation over the 196 positions analysed for both Rz (Red colour deviation = 19% UVA, 8% Solar: Green colour deviation = 17% UVA, 12% Solar) and DCIP (Red colour deviation = 22% UVA, 16% Solar) inks was seen in both UVA and solar experiments, demonstrating the consistency of the self-cleaning titania layer on Activ (TM). The method presented provides a good solution for the high-throughput photocatalytic screening of a number of homogenous photocatalytically active materials simultaneously or numerous positions on a single film; both useful in assessing the homogeneity of a film or determining the best combination of reaction components to produce the optimum performance photocatalytic film. (C) 2010 Elsevier B.V. All rights reserved.