Polyclonal antibodies mediated immobilization of a peroxidase from ammonium sulphate fractionated bitter gourd (Momordica charantia) proteins

Polyclonal antibody bound Sepharose 4B support has been exploited for the immobilization of bitter gourd peroxidase directly from ammonium sulphate precipitated proteins. Immunoaffinity immobilized bitter gourd peroxidase exhibited high yield of immobilization. IgG-Sepharose 4B bound bitter gourd peroxidase showed a higher stability against heat, chaotropic agents (urea and guanidinium chloride), detergents (cetyl trimethyl ammonium bromide and Surf Excel), proteolytic enzyme (trypsin) and water-miscible organic solvents (propanol, THF and dioxane). The activity of immobilized bitter gourd peroxidase was significantly enhanced in the presence of cetyl trimethyl ammonium bromide and after treatment with trypsin as compared to soluble enzyme.     

Implication of Nitric Oxide Under Salinity Stress: The Possible Interaction with Other Signaling Molecules

Journal of Plant Growth Regulation - It is a well-established fact that nitric oxide (NO) is a multifaceted signaling molecule, which plays diverse role in organisms. In the past two decades,...     

Doubly Spin-Labeled RNA as an EPR Reporter for Studying Multicomponent Supramolecular Assemblies

mRNAs are involved in complicated supramolecular complexes with human 40S and 80S ribosomes responsible for the protein synthesis. In this work, a derivative of nonaribonucleotide pUUCGUAAAA with nitroxide spin labels attached to the 5′-phosphate and to the C8 atom of the adenosine in sixth position (mRNA analog) was used for studying such complexes using double electron-electron resonance/pulsed electron-electron double resonance spectroscopy. The complexes were assembled with participation of tRNA^Phe, which targeted triplet UUC of the derivative to the ribosomal peptidyl site and predetermined location of the adjacent GUA triplet coding for Val at the aminoacyl (A) site. The interspin distances were measured between the two labels of mRNA analog attached to the first nucleotide of the peptidyl site bound codon and to the third nucleotide of the A site bound codon, in the absence/presence of second tRNA bound at the A site. The values of the obtained interspin distances agree with those calculated for available near-atomic structures of similar complexes of 40S and 80S ribosomes, showing that neither 60S subunit nor tRNA at the A site have a noticeable effect on arrangement of mRNA at the codon-anticodon interaction area. In addition, the shapes of distance distributions in four studied ribosomal complexes allowed conclusions on conformational flexibility of mRNA in these complexes. Overall, the results of this study are the first, to our knowledge, demonstration of double electron-electron resonance/pulsed electron-electron double resonance application for measurements of intramolecular distances in multicomponent supramolecular complexes involving intricate cellular machineries and for evaluating dynamic properties of ligands bound to these machineries.     

Determination of potential role of antioxidative status and circulating biochemical markers in the pathogenesis of ethambutol induced toxic optic neuropathy among diabetic and non-diabetic patients

The present study was designed to explore the antioxidative status and circulating biochemical markers having a potential role in the pathogenesis of ethambutol (EMB) induced toxic optic neuropathy (TON) among diabetic and non-diabetic patients.Fifty patients under complete therapy of EMB for tuberculosis were included in the present study. Inclusion criteria for patients were to receive EMB everyday during treatment, a dose of 25 mg/kg for initial 2 months and 15 mg/kg during the rest of therapy period. We conducted color vision and visual acuity test for all patients.Fifteen out of fifty EMB induced TON patients, were found to be diabetic. Color vision and visual acuity test results were evaluated for diabetic and non-diabetic as well as twenty age matched controls. The results demonstrated a significant pattern of circulating biochemical markers between the studied groups. Data regarding hematological (RBC, p value = 0.02; Hemoglobin, p value = 0.02), hepatic (total bilirubin, p value = 0.01), renal (urea, p value = 0.03; creatinine, p value = 0.007), lipid (total cholesterol, p value = 0.01; total triglycerides, p value = 0.03) and antioxidative (superoxide dismutase, p value = 0.005; glutathione, p value = 0.02; catalase, p value = 0.02) profile showed a highly significant difference among the studied groups specially patients with diabetes. Malondialdehyde (MDA) level had gone significantly up in diabetic TON patients (p value = 0.02), in comparison to other antioxidants and vitamins (Vit). Vit-A, E, B₁, B₁₂ and Zinc seem to be playing a major role in the pathogenesis of TON, specially Vit-E and B₁ surpassed all the antioxidants as having highly significant inverse relationships with MDA (MDA vs Vit-E, r = −0.676^** and MDA vs Vit-B₁, r = −0.724^** respectively).We conclude that during the ethambutol therapy the decreased levels of Vit-E and Vit-B₁ possibly play a role in the development of TON and may be used as therapeutic agents to lessen the deleterious effects of ethambutol.     

Privileged structure-based ligands for melanocortin receptors-tetrahydroquinolines, indoles, and aminotetralines

Substitution of the aryl sulfonamide moiety contained in MC4 agonist 1 with bicyclic heterocycles and aminotetralines produced compounds with MC4 activity. The heterocycles represent alternative privileged structures to that contained in 1. Compounds in which the polar group of the privileged structure was displayed in an endocyclic fashion were not as active as the parent agonist 1, while those with an exocyclic polar group afforded activity competitive with 1.     

The role of endothelin-1 in myocarditis and inflammatory cardiomyopathy: old lessons and new insights

Endothelin-1 has emerged as an important participant in the pathophysiology of a variety of cardiovascular diseases, where it may act on endocrine, paracrine and autocrine bases. Here we review its regulated biosynthesis, receptor-mediated signaling, and functional consequences in the heart, with particular emphasis on cardiac development and disease. Exploring published data employing molecular genetic mouse models of endothelin dysregulation, we highlight its heretofore underappreciated role as a pro-inflammatory cytokine. We also present novel micro-array data from one such mouse model, which implicate the specific downstream pathways that may mediate endothelin-1's effects.     

Immunochemical identification of coenzyme Q0-dihydrolipoamide adducts in the E2 components of the alpha-ketoglutarate and pyruvate dehydrogenase complexes partially explains the cellular toxicity of coenzyme Q0

Coenzyme Q(0) (Q(0)), a strong electrophile, is toxic to insulin-producing cells. Q(0) was incubated with rat and human pancreatic islets and INS-1 insulinoma cells, and its attachment to cellular proteins was studied with Western analysis using antiserum raised against the benzoquinone ring structure of ubiquinone (anti-Q). Q(0) covalently bonded to two proteins, one of 50 kDa and another of 70 kDa. Both proteins were found to be mitochondrial in human and rat islet cells and in many rat organs. Mitochondria were incubated with Q(0), and affinity-purified anti-Q was used to immunoprecipitate the 50-kDa protein. Amino acid sequencing identified it as dihydrolipoamide succinyltransferase, the E2 component of the alpha-ketoglutarate dehydrogenase complex (KDC). Western analysis also showed that Q bonds to the E2 components of the purified KDC and (0)the pyruvate dehydrogenase complex (PDC). Dihydrolipoamide acetyltransferase, the E2 of the PDC, has a molecular mass of 70 kDa, and the 70-kDa protein was inferred to be this enzyme. Q(0) was found to bond only to proteins containing dihydrolipoate, and in preparations of mitochondria, thiol reducing agents facilitated the attachment of Q(0), but oxidizing agents prevented it, suggesting that Q(0) bonds to thiols of dihydrolipoamide. Incubation of human or pig PDC with Q(0) followed by matrix-assisted laser desorption ionization time-of-flight and liquid chromatography/electrospray ionization mass spectrometry analyses of chymotrypsin-digested peptides of PDC E2 confirmed that Q(0) bonds to the dihydrolipoamide in these proteins. In mitochondria, coenzymes Q(1) and Q(2) did not bond to the 50-kDa protein but competed with the bonding of Q(0) to this protein. The prevention by Q(1) of characteristics the bonding of Q(0) to KDC E2, as well as other of the Q(0) effect, are reminiscent of the action of Q(0) on the mitochondrial permeability transition pore described previously (Fontaine, E., Ichas, F., and Bernardi, P. (1998) J. Biol. Chem. 273, 25734-25740).     

EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival,Cell Invasion,Focal Adhesions,and Mammalian Target of Rapamycin Activation

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130^Cas. Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130^Cas. WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.     

Acorus calamus extracts and nickel chloride: prevention of oxidative damage and hyperproliferation response in rat kidney

Nickel, a major environmental pollutant, is known for its clastogenic, toxic, and carcinogenic potential. In this article, we report the effect of Acorus calamus on nickel chloride (NiCl2)-induced renal oxidative stress, toxicity, and cell proliferation response in male Wistar rats. NiCl2 (250 micromol/kg body weight/mL) enhanced reduced renal glutathione content (GSH), glutathione- S-transferase (GST), glutathione reductase (GR), lipid peroxidation (LPO), H2O2 generation, blood urea nitrogen (BUN), and serum creatinine with a concomitant decrease in the activity of glutathione peroxidase (GPx) (p < 0.001). NiCl2 administration also dose-dependently induced the renal ornithine decarboxylase (ODC) activity several-fold as compared to salinetreated control rats. Similarly, renal DNA synthesis, which is measured in terms of [3H] thymidine incorporation in DNA, was elevated following NiCl2 treatment. Prophylactic treatment of rats with A. calamus (100 and 200 mg/kg body weight po) daily for 1 wk resulted in the diminution of NiCl2- mediated damage, as evident from the downregulation of glutathione content, GST, GR, LPO, H2O2 generation, BUN, serum creatinine, DNA synthesis (p < 0.001), and ODC activity (p < 0.01) with concomitant restoration of GPx activity. These results clearly demonstrate the role of oxidative stress and its relation to renal disfunctioning and suggest a protective effect of A. calamus on NiCl2-induced nephrotoxicity in a rat experimental model.