Breast Tumors with Elevated Expression of 1q Candidate Genes Confer Poor Clinical Outcome and Sensitivity to Ras/PI3K Inhibition

Genomic aberrations are common in cancers and the long arm of chromosome 1 is known for its frequent amplifications in breast cancer. However, the key candidate genes of 1q, and their contribution in breast cancer pathogenesis remain unexplored. We have analyzed the gene expression profiles of 1635 breast tumor samples using meta-analysis based approach and identified clinically significant candidates from chromosome 1q. Seven candidate genes including exonuclease 1 (EXO1) are consistently over expressed in breast tumors, specifically in high grade and aggressive breast tumors with poor clinical outcome. We derived a EXO1 co-expression module from the mRNA profiles of breast tumors which comprises 1q candidate genes and their co-expressed genes. By integrative functional genomics investigation, we identified the involvement of EGFR, RAS, PI3K / AKT, MYC, E2F signaling in the regulation of these selected 1q genes in breast tumors and breast cancer cell lines. Expression of EXO1 module was found as indicative of elevated cell proliferation, genomic instability, activated RAS/AKT/MYC/E2F1 signaling pathways and loss of p53 activity in breast tumors. mRNA–drug connectivity analysis indicates inhibition of RAS/PI3K as a possible targeted therapeutic approach for the patients with activated EXO1 module in breast tumors. Thus, we identified seven 1q candidate genes strongly associated with the poor survival of breast cancer patients and identified the possibility of targeting them with EGFR/RAS/PI3K inhibitors.     

Enhanced Ca2+ transport and muscle relaxation in skeletal muscle from sarcolipin-null mice

Sarcolipin (SLN) inhibits sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pumps. To evaluate the physiological significance of SLN in skeletal muscle, we compared muscle contractility and SERCA activity between Sln-null and wild-type mice. SLN protein expression in wild-type mice was abundant in soleus and red gastrocnemius (RG), low in extensor digitorum longus (EDL), and absent from white gastrocnemius (WG). SERCA activity rates were increased in soleus and RG, but not in EDL or WG, from Sln-null muscles, compared with wild type. No differences were seen between wild-type and Sln-null EDL muscles in force-frequency curves or maximum rates of force development (+dF/dt). Maximum relaxation rates (-dF/dt) of EDL were higher in Sln-null than wild type across a range of submaximal stimulation frequencies, but not during a twitch or peak tetanic contraction. For soleus, no differences were seen between wild type and Sln-null in peak tetanic force or +dF/dt; however, force-frequency curves showed that peak force during a twitch and 10-Hz contraction was lower in Sln-null. Changes in the soleus force-frequency curve corresponded with faster rates of force relaxation at nearly all stimulation frequencies in Sln-null compared with wild type. Repeated tetanic stimulation of soleus caused increased (-dF/dt) in wild type, but not in Sln-null. No compensatory responses were detected in analysis of other Ca(2+) regulatory proteins using Western blotting and immunohistochemistry or myosin heavy chain expression using immunofluorescence. These results show that 1) SLN regulates Ca(2+)-ATPase activity thereby regulating contractile kinetics in at least some skeletal muscles, 2) the functional significance of SLN is graded to the endogenous SLN expression level, and 3) SLN inhibitory effects on SERCA function are relieved in response to repeated contractions thus enhancing relaxation rates.     

Attenuation of 4-nitroquinoline 1-oxide induced in vitro lipid peroxidation by green tea polyphenols

Lipid peroxidation is believed to play an important role in pathogenesis of diseases. 4-Nitroquiunoline 1-oxide (4-NQO) a potent oral carcinogen, widely used for induction of oral carcinogenesis, was found to induce lipid peroxidation in vivo and in vitro. Green tea contains high content of polyphenols, which are potent antioxidants. Thus green tea polyphenols (GP) can play a protective role in 4-NQO induced in vitro lipid peroxidation. 4-NQO at the concentration of 1.5 mM was found to induce lipid peroxidation in 5% liver homogenate in phosphate buffered saline and extent of lipid peroxidation at the different time intervals 0, 15, 30 and 45 min where studied by assessing parameters such as hydroxyl radical production (OH), thiobarbituric acid reactants (TBARS), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT). It was found that addition of 4-NQO caused an increase in OH and TBARS level and a decrease in activity of SOD, CAT and the levels of GSH. Simultaneous addition of GP 10 mg/ml significantly decreased lipid peroxidation and increased in antioxidant status. Thus, we conclude that GP, a potent antioxidant, was found to nullify 4-NQO induced lipid peroxidation in vitro and 4-NQO acts initially by causing oxidative stress and leads to carcinogenesis.     

Receptor complexes cotransported via polarized endocytic pathways form clusters with distinct organizations

Previously, FRET confocal microscopy has shown that polymeric IgA-receptor (pIgA-R) is distributed in a clustered manner in apical endosomes. To test whether different membrane-bound components form clusters during membrane trafficking, live-cell quantitative FRET was used to characterize the organization of pIgA-R and transferrin receptor (TFR) in endocytic membranes of polarized MDCK cells upon internalization of donor- and acceptor-labeled ligands. We show that pIgA-R and TFR complexes form increasingly organized clusters during cotransport from basolateral to perinuclear endosomes. The organization of these receptor clusters in basolateral versus perinuclear/apical endosomes is significantly different; the former showing a mixed random/clustered distribution while the latter highly organized clusters. Our results indicate that although both perinuclear and apical endosomes comprise pIgA-R and TFR clusters, their E% levels are significantly different suggesting that these receptors are packed into clusters in a distinct manner. The quantitative FRET-based assay presented here suggests that different receptor complexes form clusters, with diverse levels of organization, while being cotransported via the polarized endocytic pathways.     

Functional consequences of stably expressing a mutant calsequestrin (CASQ2D307H) in the CASQ2 null background

The role of calsequestrin (CASQ2) in cardiac sarcoplasmic reticulum (SR) calcium (Ca(2+)) transport has gained significant attention since point mutations in CASQ2 were reported to cause ventricular arrhythmia. In the present study, we have critically evaluated the functional consequences of expressing the CASQ2(D307H) mutant protein in the CASQ2 null mouse. We recently reported that the mutant CASQ2(D307H) protein can be stably expressed in CASQ2 null hearts, and it targets appropriately to the junctional SR (Kalyanasundaram A, Bal NC, Franzini-Armstrong C, Knollmann BC, Periasamy M. J Biol Chem 285: 3076-3083, 2010). In this study, we found that introduction of CASQ2(D307H) protein in the CASQ2 null background partially restored triadin 1 levels, which were decreased in the CASQ2 null mice. Despite twofold expression (relative to wild-type CASQ2), the mutant protein failed to increase SR Ca(2+) load. We also found that the Ca(2+) transient decays slower in the CASQ2 null and CASQ2(D307H) cells. CASQ2(D307H) myocytes, when rhythmically paced and challenged with isoproterenol, exhibit spontaneous Ca(2+) waves similar to CASQ2 null myocytes; however, the stability of Ca(2+) cycling was increased in the CASQ2(D307H) myocytes. In the presence of isoproterenol, Ca(2+)-transient amplitude in CASQ2(D307H) myocytes was significantly decreased, possibly indicating an inherent defect in Ca(2+) buffering capacity and release from the mutant CASQ2 at high Ca(2+) concentrations. We also observed polymorphic ventricular tachycardia in the CASQ2(D307H) mice, although lesser than in the CASQ2 null mice. These data suggest that CASQ2(D307H) point mutation may affect Ca(2+) buffering capacity and Ca(2+) release. We propose that poor interaction between CASQ2(D307H) and triadin 1 could affect ryanodine receptor 2 stability, thereby increasing susceptibility to delayed afterdepolarizations and triggered arrhythmic activity.     

Extracellular enzymatic activity profiles in fungi isolated from oil-rich environments

About 34 wild fungal species associated with edible oil mill wastes were isolated by the serial dilution technique. Methods for rapid screening of fungal species against production of extracellular enzymes such as amylase, protease, cellulase, and lipase are reported. Among all the species, Aspergillus versicolor exhibited high amlylolytic and gelatinolytic activity, whereas Penicillium citrinum showed only high amylolytic activity. Maximum cellulolytic activity was recorded for Absidia corymbifera, As. niger, Cunninghamella echinulata, Curvularia lunata, Fusarium solani, Mucor racemosus, Paecilomyces variotii, and Syncephalastrum racemosum. The fungal species Ab. corymbifera, As. fumigatus, As. japonicus, As. nidulans, As. terreus, Cun. verticillata, Cur. pallescens, F. oxysporum, Geotrichum candidum, M. racemosus, Pe. citrinum, Pe. frequentans, Rhizopus stolonifer, and Trichoderma viride exhibited maximum lipase activity. This study confirms the isolated fungi present on a wide range of substrates in the ambient environment, and these results could provide basic data for further investigations on fungal extracellular enzymes.First two authors equally contributed to this work     

Effect of crude sulphated polysaccharide from brown algae against acetaminophen-induced toxicity in rats

This study was conducted to examine the protective role of crude polysaccharide from brown seaweed Sargassum polycystum against acetaminophen-induced abnormality in blood glucose, serum albumin/globulin ratio, and liver glycogen, lactate, and pyruvate. Liver and renal tissue histology was performed to confirm the efficacy of Sargassum polysaccharide. A toxic dose of acetaminophen (800 mg/kg body weight intraperitoneally) induced severe abnormality in all basic parameters with apparent toxicity in liver (enlargement of hepatocytes, loss of cytoplasmic content with disruption in the hepatic plates and sinusoidal dilation) and renal tissue (glomerular damage with congestion of tubules). The isolated liver cells were stained with acridine orange and examined under fluorescence microscope, which revealed that the acetaminophen induced significant damage. In contrast, the rats pretreated with Sargassum polysaccharide (200 mg/kg body weight) daily for 3 weeks did not show liver and renal tissue with these severe aberrations induced by acetaminophen. Histology results were also consistent with analyzed basic biochemical parameters, which confirmed the effectiveness of the crude polysaccharide against acetaminophen-induced abnormality in rats.     

Is reduced SERCA2a expression detrimental or beneficial to postischemic cardiac function and injury? Evidence from heterozygous SERCA2a knockout mice

Recent studies have demonstrated that increased expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a improves myocardial contractility and Ca2+ handling at baseline and in disease conditions, including myocardial ischemia-reperfusion (I/R). Conversely, it has also been reported that pharmacological inhibition of SERCA might improve postischemic function in stunned hearts or in isolated myocardium following I/R. The goal of this study was to test how decreases in SERCA pump level/activity affect cardiac function following I/R. To address this question, we used a heterozygous SERCA2a knockout (SERCA2a+/-) mouse model with decreased SERCA pump levels and studied the effect of myocardial stunning (20-min ischemia followed by reperfusion) and infarction (30-min ischemia followed by reperfusion) following 60-min reperfusion. Our results demonstrate that postischemic myocardial relaxation was significantly impaired in SERCA2a+/- hearts with both stunning and infarction protocols. Interestingly, postischemic recovery of contractile function was comparable in SERCA2a+/- and wild-type hearts subjected to stunning. In contrast, following 30-min ischemia, postischemic contractile function was reduced in SERCA2a+/- hearts with significantly larger infarction. Rhod-2 spectrofluorometry revealed significantly higher diastolic intracellular Ca2+ in SERCA2a+/- hearts compared with wild-type hearts. Both at 30-min ischemia and 2-min reperfusion, intracellular Ca2+ levels were significantly higher in SERCA2a+/- hearts. Electron paramagnetic resonance spin trapping showed a similar extent of postischemic free-radical generation in both strains. These data provide direct evidence that functional SERCA2a level, independent of oxidative stress, is crucial for postischemic myocardial function and salvage during I/R.     

Isolation, purification and characterization of beta-1,3-glucan binding protein from the plasma of marine mussel Perna viridis

A beta-1,3-glucan binding protein (betaGBP) specific for laminarin (a beta-1,3-glucan) was detected for the first time in a mollusc, Perna viridis. betaGBP was isolated and purified from the plasma using laminarin precipitation and affinity chromatography on laminarin-Sepharose 6B, respectively. It agglutinated bakers yeast, bacteria, and erythrocytes and enhanced prophenoloxidase (proPO) activity of the plasma in a dose-dependent manner. The purified betaGBP appeared as a single band in native-PAGE and the purity was conformed by HPLC. The protein has a molecular weight estimate of 510kDa as determined by SDS-PAGE and in isoelectric focusing the purified betaGBP was focused as a single band at pI 5.3. betaGBP was found to possess inherent serine protease activity but lacked beta-1,3-glucanase activity and all these results suggest that plasma betaGBP of P. viridis functions as a recognition molecule for beta-1,3-glucan on the surface of microbial cell walls. This recognition and binding lead to the activation of the prophenoloxidase cascade mediated by the inherent serine protease activity of betaGBP. Presence of agglutinating activity and serine protease activity shows that betaGBP is a bifunctional protein. The findings are discussed in light of the importance of this protein in the innate immune response of P. viridis, and they implicate evolutionary link with similar proteins found in other invertebrates.