Production of alkaline protease from Aspergillus oryzae isolated from seashore of Bay of Bengal

Aspergillus oryzae isolatedon  Potato dextrose agar  from soil samples of kottakoduru seashore of Bay of Bengal, Andhra Pradesh, India seashore of Bay of Bengal by spread plate method and was screened for alkaline protease production on Skim milk containing agar plates and identified by clear zones of protein hydrolysis around colonies. Different physical and chemical parameters such as pH, temperature, substrate concentration and incubation time were optimized for the better production of alkaline protease. The maximum protease activity was found at pH of 8 containing 10% wheat bran at 300C, after 72 hours of fermentation.ZnSO4was effective activator for protease activity and sodium dsulphate had shownmore than 50% inhibition of enzyme activity. Among the different oil cakes used for the production of enzyme the Sesame  oil cake proved to be suitable substrate after wheat bran for the production of protease by Aspergillus oryzae

Role of Nuclear Receptors in Lipid Dysfunction and Obesity-related Diseases

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 12 meeting in San Diego, CA. The presentations discussed the roles of a number of nuclear receptors in regulating glucose and lipid homeostasis, the pathophysiology of obesity-related disease states and the promise associated with targeting their activities to treat these diseases. While many of these receptors, in particular constitutive androstane receptor and pregnane X receptor and their target enzymes have been thought of as regulators of drug and xenobiotic metabolism, this symposium highlighted the advances made in our understanding of the endogenous functions of these receptors. Similarly, the advances made in our understanding of the mechanisms underlying bile acid signaling pathways in the regulation of body weight and glucose homeostasis illustrates the importance of using complementary approaches to elucidate this fascinating network of pathways. The observations that some receptors, like the farnesoid X receptor can function in a tissue specific manner via well defined mechanisms has important clinical implications particularly in the treatment of liver diseases. Finally, the novel findings that agents that selectively activate estrogen receptor β can effectively inhibit weight gain in a highfat diet model of obesity identifies a new role for this member of the steroid superfamily. Taken together, this symposium has revealed a number of significant findings that illustrate the promise associated with targeting a number of nuclear receptors for the development of new therapies to treat obesity and other metabolic disorders

Discovery and Preclinical Characterization of Novel Small Molecule TRK and ROS1 Tyrosine Kinase Inhibitors for the Treatment of Cancer and Inflammation

<div><p>Receptor tyrosine kinases (RTKs), in response to their growth factor ligands, phosphorylate and activate downstream signals important for physiological development and pathological transformation. Increased expression, activating mutations and rearrangement fusions of RTKs lead to cancer, inflammation, pain, neurodegenerative diseases, and other disorders. Activation or over-expression of ALK, ROS1, TRK (A, B, and C), and RET are associated with oncogenic phenotypes of their respective tissues, making them attractive therapeutic targets. Cancer cDNA array studies demonstrated over-expression of TRK-A and ROS1 in a variety of cancers, compared to their respective normal tissue controls. We synthesized a library of small molecules that inhibit the above indicated RTKs with picomolar to nanomolar potency. The lead molecule GTx-186 inhibited RTK-dependent cancer cell and tumor growth. <i>In vitro</i> and <i>in vivo</i> growth of TRK-A-dependent IMR-32 neuroblastoma cells and ROS1-overexpressing NIH3T3 cells were inhibited by GTx-186. GTx-186 also inhibited inflammatory signals mediated by NFκB, AP-1, and TRK-A and potently reduced atopic dermatitis and air-pouch inflammation in mice and rats. Moreover, GTx-186 effectively inhibited ALK phosphorylation and ALK-dependent cancer cell growth. Collectively, the RTK inhibitor GTx-186 has a unique kinase profile with potential to treat cancer, inflammation, and neuropathic pain.</p></div

GTx-186 inhibits inflammation <i>in vitro</i>.

<p>Cells were serum starved for 2 days, pre-treated with indicated concentrations of GTx-186 for 30 min and treated with growth factors or cytokines for 12–16 hrs. RNA was extracted, cDNA synthesized, and expression of inflammatory genes were measured by realtime PCR using Taqman primer and probes. All experiments were performed in triplicate and repeated at least three times. Values are expressed as Avg ± S.E. Dex-Dexamethasone; PMA-Phorbol Myristate Acetate; NGF-Nerve Growth Factor; TNF-α-Tumor Necrosis Factor; LPS-Lipopolysaccharide; MMP3-Matrix Metalloproeinase 3; IL-8-Interleukin 8; PC-12-Pheochromocytoma cells; IMR-32-Neuroblastoma cells; NHEK-Normal Human Epidermal Keratinocytes; LADMAC-Macrophage/Monocytes; RAW-264.7-Mouse macrophage cells.</p

Kinase assays to determine the effect of GTx-186 and staurosporine on the activity of the indicated kinases.

<p>Values are expressed as IC₅₀.</p

GTx-186 inhibits TRK-A-dependent neuroblastoma cell and tumor xenograft growth.

<p><b>A</b>. GTx-186 inhibits NGF-induced ERK (p42/44 MAPK) phosphorylation. IMR-32 neuroblastoma cells were serum starved for 2 days, pre-treated for 2 hrs with the indicated concentrations of GTx-186 and treated with 200 ng/ml NGF for 30 min. Cells were harvested and Western blot performed for phospho-ERK and total-ERK. <b>B</b>. GTx-186 inhibits proliferation of IMR-32 cells. IMR-32 cells were plated in growth medium and treated with indicated concentration of GTx-186 for 3 days. Cells were fixed and stained with sulforhodamine B (SRB) and optical density (OD) was measured at 535 nm. <b>C</b>. GTx-186 inhibits migration of IMR-32 cells. IMR-32 cells were plated in a platypus migration assay plate and were treated with vehicle, NGF, or combination of NGF and GTx-186. Images were captured under light microscope 12 hrs after treatment. <b>D–G</b>. GTx-186 inhibits IMR-32 neuroblastoma xenograft growth. IMR-32 cells were subcutaneously implanted in nude mice (10 million cells/mouse). Once tumors reached 100–200 mm³, animals (n = 8) were randomized and treated daily with vehicle or 20 mg/kg/day i.v. GTx-186. Tumor volume (D) and body weight (F) were measured every day for 4 days. The animals were sacrificed, tumors weighed (E), stored in formalin, and processed for TUNEL immunohistochemistry (G-Number of TUNEL positive cells (left panel) and intensity of TUNEL staining (right panel)). Values are expressed as Avg ± S.E. NGF-Nerve Growth Factor; Open bars are vehicle-treated and filled bars are GTx-186-treated samples. *-statistically significant at p<0.05; **-statistically significant at p<0.01.</p

GTx-186 inhibits growth of NIH3T3-FIG-ROS1 xenograft.

<p>NIH3T3 cells stably over-expressing FIG-ROS1 were implanted subcutaneously in nude mice (2 million cells/mouse). Once tumors reached 100–200 mm³, the animals were randomized and treated daily with vehicle, 20 mg/kg/day GTx-186 i.v., or 75 mg/kg p.o. crizotinib. Tumor volumes (A) and body weight (C) were measured biweekly. After sacrifice, tumor weights (B) were recorded and the amount of drug in serum and tumors were quantified using LC-MS/MS (D). *-significant at p<0.01; **-significant at p<0.001. Values are expressed as Avg ± S.E. of n = 8.</p

TRK-A and ROS1 are over-expressed in multiple cancers.

<p>Expression of TRK-A and ROS1 were quantified by realtime PCR in cDNAs from 381 samples from 22 different cancers and corresponding normal tissues. TRK-A and ROS1 expression were normalized to actin and represented as fold difference from normal non-cancerous samples using ddCt method. Average of normal samples was taken for the ddCt calculation. Ad.C-Adrenal cancer; Normal-cDNA from Non-cancerous tissues. Numbers under samples indicate normal samples.</p