Expression, purification and characterization of a biologically active and thermally stable human lysyl oxidase

Lysyl oxidase (LOX), a promising therapeutic target for the progression of cancer and fibrosis, has not been well characterized yet. A major difficulty faced in LOX characterization is its lack of solubility in common buffers. In this study, mature LOX (mLOX) was cloned, purified and its purity was ascertained by mass spectroscopy. Through screening various buffers, 0.2 M glycine-NaOH buffer with 10% glycerol pH 8.0 was identified to maintain mLOX in its soluble state. About 67% of the refolded mLOX was found to be in copper bound state after His-tag removal. Catalytic properties Km and kcat were found to be 3.72 × 10−4 M and 7.29 ×103s−1. In addition, collagen cross-linking in ARPE-19 cells was augmented on exposure to mLOX, endorsing its biological activity. Circular Dichroism revealed that mLOX comprises 8.43% of α-helix and 22% of β-strand and it was thermally stable up to 90°C. Disulfide linkage imparts the structural stability in LOX which was experimentally ascertained with intrinsic and extrinsic fluorescence studies

Accelerating Drug Discovery Efforts for Trypanosomatidic Infections Using an Integrated Transnational Academic Drug Discovery Platform

According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion\u2013toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 (TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain

Prevalence of Sleep Abnormalities and Their Association with Metabolic Syndrome among Asian Indians: Chennai Urban Rural Epidemiology Study (CURES – 67)

Objective: To estimate the prevalence of sleep abnormalities and their association with glucose intolerance and metabolic syndrome (MS) in the normal-weight urban South Indian population. Methods: This population-based, cross-sectional study was carried out in 358 subjects aged 20-76 years randomly selected from the Chennai Urban Rural Epidemiology Study in South India. A validated questionnaire assessing various sleep abnormalities (snoring, daytime sleepiness, lack of refreshing sleep, and number of hours of sleep) was administered. All subjects underwent an oral glucose tolerance test, and anthropometric biochemical measurements were obtained to assess cardiometabolic risk factors including glucose intolerance. Diabetes risk was assessed using a previously validated Indian Diabetes Risk Score (IDRS). Results: The overall prevalence of snoring and daytime sleepiness was 40% and 59%, respectively. Snorers were more male, older, smokers, and had higher levels of cardiometabolic risk factors. Subjects with daytime sleepiness had higher body mass index (BMI) and abdominal obesity. Both snoring (50.9% vs 30.2%, p < 0.001) and daytime sleepiness (68% vs 49.7%, p < 0.001) were more prevalent among subjects with impaired glucose metabolism compared to those with normal glucose metabolism. Both sleep measures were associated with higher diabetes risk scores, as assessed by the IDRS (snoring: trend Χ2, 11.14, p = 0.001; daytime sleepiness: trend Χ2, 5.12, p = 0.024). Metabolic syndrome was significantly associated with snoring even after adjusting for age, sex, family history of diabetes, physical activity, smoking, and alcohol. Conclusion: The prevalence of snoring and daytime sleepiness is high among urban South Indians and these two sleep measures are associated with glucose intolerance, MS, and higher diabetes risk scores

New pregaliellalactonoids from Galiella rufa

An extract of a culture of the fungus Galiella rufa yielded several new derivatives of pregaliellalactone (1) that were characterised by NMR and MS experiments. The tetraene 4 has been reported previously, but was in this investigation isolated in sufficient amounts for the determination of the configuration of the C-4/C-5 double bond which is Z, not E. The possibility that any of the new compounds are involved in the biosynthesis of 1 is discussed. (C) 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved

Expression, purification and characterization of a biologically active and thermally stable human lysyl oxidase

105-116Lysyl oxidase (LOX), a promising therapeutic target for the progression of cancer and fibrosis, has not been well characterized yet. A major difficulty faced in LOX characterization is its lack of solubility in common buffers. In this study, mature LOX (mLOX) was cloned, purified and its purity was ascertained by mass spectroscopy. Through screening various buffers, 0.2 M glycine-NaOH buffer with 10% glycerol pH 8.0 was identified to maintain mLOX in its soluble state. About 67% of the refolded mLOX was found to be in copper bound state after His-tag removal. Catalytic properties Km and kcat were found to be 3.72 × 10−4 M and 7.29 ×103s−1. In addition, collagen cross-linking in ARPE-19 cells was augmented on exposure to mLOX, endorsing its biological activity. Circular Dichroism revealed that mLOX comprises 8.43% of α-helix and 22% of β-strand and it was thermally stable up to 90°C. Disulfide linkage imparts the structural stability in LOX which was experimentally ascertained with intrinsic and extrinsic fluorescence studies