Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes

ANK is a multipass transmembrane protein transporter thought to play a role in the export of intracellular inorganic pyrophosphate and so to contribute to the pathophysiology of chondrocalcinosis. As transforming growth factor-beta-1 (TGF-β1) was shown to favor calcium pyrophosphate dihydrate deposition, we investigated the contribution of ANK to the production of extracellular inorganic pyrophosphate (ePPi) by chondrocytes and the signaling pathways involved in the regulation of Ank expression by TGF-β1. Chondrocytes were exposed to 10 ng/mL of TGF-β1, and Ank expression was measured by quantitative polymerase chain reaction and Western blot. ePPi was quantified in cell supernatants. RNA silencing was used to define the respective roles of Ank and PC-1 in TGF-β1-induced ePPi generation. Finally, selective kinase inhibitors and dominant-negative/overexpression plasmid strategies were used to explore the contribution of several signaling pathways to Ank induction by TGF-β1. TGF-β1 strongly increased Ank expression at the mRNA and protein levels, as well as ePPi production. Using small interfering RNA technology, we showed that Ank contributed approximately 60% and PC-1 nearly 20% to TGF-β1-induced ePPi generation. Induction of Ank by TGF-β1 required activation of the extracellular signal-regulated kinase (ERK) pathway but not of p38-mitogen-activated protein kinase or of protein kinase A. In line with the general protein kinase C (PKC) inhibitor calphostin C, Gö6976 (a Ca²⁺-dependent PKC inhibitor) diminished TGF-β1-induced Ank expression by 60%, whereas a 10% inhibition was observed with rottlerin (a PKCδ inhibitor). These data suggest a regulatory role for calcium in TGF-β1-induced Ank expression. Finally, we demonstrated that the stimulatory effect of TGF-β1 on Ank expression was inhibited by the suppression of the Ras/Raf-1 pathway, while being enhanced by their constitutive activation. Transient overexpression of Smad 7, an inhibitory Smad, failed to affect the inducing effect of TGF-β1 on Ank mRNA level. These data show that TGF-β1 increases ePPi levels, mainly by the induction of the Ank gene, which requires activation of Ras, Raf-1, ERK, and Ca²⁺-dependent PKC pathways in chondrocytes.     

Role of <Emphasis Type="Italic">Serratia marcescens</Emphasis> ACE2 on diesel degradation and its influence on corrosion

A facultative anaerobic species Serratia marcescens ACE2 isolated from the corrosion products of diesel transporting pipeline in North West, India was identified by 16S rDNA sequence analysis. The role of Serratia marcesens ACE2 on biodegradation of diesel and its influence on the corrosion of API 5LX steel has been elucidated. The degrading strain ACE2 is involved in the process of corrosion of steel API 5LX and also utilizes the diesel as an organic source. The quantitative biodegradation efficiency (BE) of diesel was 58%, calculated by gas-chromatography–mass spectrum analysis. On the basis of gas-chromatography–mass spectrum (GC–MS), Fourier Transform infrared spectroscopy (FTIR) and X-ray diffractometer (XRD), the involvement of Serratia marcescens on degradation and corrosion has been investigated. This basic study will be useful for the development of new approaches for detection, monitoring and control of microbial corrosion.     

Transducin-like enhancer of Split-1 (TLE1) combines with Forkhead box protein G1 (FoxG1) to promote neuronal survival

Transducin-like enhancer of split-1 (TLE1) plays a critical role in the regulation of neurogenesis by inhibiting the differentiation of neural progenitor cells into neurons. Although TLE1 is also expressed highly in the postnatal brain and through adulthood, its role in postmitotic neurons is not clear. Using cultures of cerebellar granule neurons, we show that expression of TLE1 is reduced in neurons primed to die. Reestablishment of elevated TLE1 levels by ectopic expression protects neurons from death, whereas suppression of TLE1 expression in otherwise healthy neurons induces cell death. These results show that TLE1 is necessary for the maintenance of neuronal survival. Experiments using pharmacological inhibitors as well as expression of point mutants indicate that phosphorylation of TLE1 by casein kinase-2 (CK2) at Ser-239 and Ser-253 is necessary for its survival-promoting activity. TLE1-mediated survival is also inhibited by pharmacological inhibition of PI3K-Akt signaling but not by inhibitors of Raf-MEK-ERK signaling or other molecules, including histone deacetylases, calcium calmodulin kinase, or CK1. The survival-promoting activity of TLE1 depends critically on interaction with FoxG1, another protein involved in the regulation of neurogenesis and shown previously to promote survival of postmitotic neurons. Likewise, the ability of FoxG1 to promote neuronal survival depends on TLE1. Taken together, our study demonstrates that TLE1 cooperates with FoxG1 to promote neuronal survival in a CK2- and PI3K-Akt-dependent manner.     

Influence of tail versus cardiac sampling on blood glucose and lipid profiles in mice

Blood is collected during animal experimentation to measure haematological and metabolic parameters. It cannot be assumed that circulating blood has the same composition irrespective of its location, and indeed, differences in the composition of blood sampled from the arterial and venous compartments have been reported. Here we investigated whether blood collected by cardiac puncture (CP) versus that collected following removal of the distal 1 mm of the tail tip (TT) differs with respect to glucose and lipid profiles in male C57BL/6J mice at 4, 7, 20 and 28 weeks of age. Blood was first collected from the TT of unanaesthetized mice, which were then immediately anaesthetized using ketamine/xylazine, and a second blood sample was collected by CP. The CP glucose concentration was significantly higher than TT glucose by a positive bias averaging +80% (P < 0.01), irrespective of the age of the mice. Conversely, the concentrations of the CP lipids, including total cholesterol, high-density lipoprotein cholesterol and triglyceride were lower than TT lipids by a negative bias averaging -25% (P < 0.05). These observations highlight the difficulty in measuring and comparing metabolic parameters such as glucose and lipid between one blood compartment and another. They illustrate the need to standardize sampling sites, especially when repeated blood sampling is required.     

Acute effects of prostaglandin E(1) and E(2) on vascular reactivity and blood flow in situ in the chick chorioallantoic membrane

The chick chorioallantoic membrane (CAM) subserves gas exchange in the developing embryo and shell-less culture affords a unique opportunity for direct observations over time of individual blood vessels to pharmacologic interventions. We tested a number of lipids including prostaglandins PGE(1&2) for vascular effects and signaling in the CAM. Application of PGE(1&2) induced a decrease in the diameter of large blood vessels and a concentration-dependent, localized, reversible loss of blood flow through small vessels. The loss of flow was also mimicked by misoprostol, an agonist for 3 of 4 known PGE receptors, EP(2-4), and by U46619, a thromboxane mimetic. Selective receptor antagonists for EP(3) and thromboxane each partially blocked the response. This is a first report of the effects of prostaglandins on vasoreactivity in the CAM. Our model allows the unique ability to examine simultaneous responses of large and small vessels in real time and in vivo.     

Acaricidal activity of Cassia alata against Rhipicephalus (Boophilus) annulatus

Using adult immersion test, the acaricidal activity of ethanolic extracts of leaves of Cassia alata L. was studied against Rhipicephalus (Boophilus) annulatus. The efficacy was assessed by measuring per cent adult mortality, inhibition of fecundity and hatching rate. The ethanolic extract of C. alata produced a concentration dependant increase in the adult tick mortality. The highest mortality (45.8%) and inhibition of fecundity (10.9%) were observed at the highest concentration tested (100 mg/ml). The plant extract did not affect egg hatchability.     

Protective role of Cop in Rip2/caspase-1/caspase-4-mediated HeLa cell death

CARD only protein (Cop) was recently identified as a protein with significant homology with the CARD of caspase-1. We have conducted functional studies on Cop and report on its role as an inhibitor of cell death in a broad range of cell death paradigms. A notable exception in the ability of Cop to inhibit cell death pertains to its inability to inhibit ER stress-mediated cell death. Furthermore, in addition to the known interaction of Cop and caspase-1, we demonstrated a novel interaction of Cop with caspase-4. We propose that Cop's action to prevent TNF-alpha-induced cell death may operate independently of the mitochondrial death pathway. Furthermore, Cop overexpression inhibits Bid cleavage. In summary, Cop inhibition of cell death, at least to a certain extent, results from its interference with the activation of caspase-1 and caspase-4. Understanding the mechanistic details modulating caspase cell death pathways should provide important information for the development of therapies for diseases featuring aberrant caspase activation. Cop, as an inhibitor of an important apical caspase cell death axis, may provide a tool for modulating pathological cell death.     

Decolorisation, biodegradation and detoxification of benzidine based azo dye

The present study deals with the decolorisation, biodegradation and detoxification of Direct Black-38, a benzidine based azo dye, by a mixed microbial culture isolated from an aerobic bioreactor treating textile wastewater. The studies revealed a biotransformation of Direct Black-38 into benzidine and 4-aminobiphenyl followed by complete decolorisation and biodegradation of these toxic intermediates. From cytotoxicity studies, it was concluded that detoxification of the dye took place after degradation of the toxic intermediates by the culture.     

Epigallocatechin-3-gallate exhibits anti-fibrotic effect by attenuating bleomycin-induced glycoconjugates, lysosomal hydrolases and ultrastructural changes in rat model pulmonary fibrosis

Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix components in the alveolar space, which hampers normal respiration process. Pathophysiological enzymes, glycoprotein moieties and matrix degrading lysosomal hydrolases are key markers and play a crucial role in the progression of fibrosis. Bleomycin is an anti-neoplastic drug, used for the treatment of various types of cancers and induces pulmonary fibrosis due its deleterious side effect. Tea catechin epigallocatechin-3-gallate (EGCG) is known for its wide array of beneficial effects. The present study was intended to evaluate the beneficial efficacy of EGCG against bleomycin-induced glycoconjugates, lysosomal hydrolases and ultrastructural changes in the lungs of Wistar rats. Intratracheal instillation of bleomycin (6.5 U/kg body weight) to rats increased the activities of pathophysiological enzymes such as aspartate transaminase, alanine transaminase, lactate dehydrogenase and alkaline phosphatase, which were attenuated upon EGCG treatment. The increased level of hydroxyproline and histopathological parameters in bleomycin-induced rats were decreased upon EGCG treatment. Bleomycin-induced increase in the level of glycoconjugates was restored closer to normal levels on EGCG treatment. Furthermore, the increased activities of matrix degrading lysosomal enzymes in bleomycin-induced rats were reduced upon EGCG supplementation. Treatment with EGCG also attenuated bleomycin-induced ultrastructural changes as observed from transmission electron microscopy studies. The results of the present study put-forward EGCG as a potential anti-fibrotic agent due to its attenuating effect on potential fibrotic markers.     

Fusobacterium necrophorum: a ruminal bacterium that invades liver to cause abscesses in cattle

Fusobacterium necrophorum, a Gram-negative, rod-shaped, and an aerotolerant anaerobe, is a normal inhabitant of the rumen of cattle. The organism is in ruminal contents and adherent to the ruminal wall. Its role in ruminal fermentation is to metabolize lactic acid and degrade feed and epithelial proteins. The ruminal concentration is higher in grain-fed than forage-fed cattle. From the rumen, the organism gains entry into the portal circulation and is trapped in the liver to cause abscesses. The organism is an opportunistic pathogen and a primary causative agent of liver abscesses, an economically important disease of grain-fed cattle. Liver abscesses are often secondary to ruminal acidosis and rumenitis in grain-fed cattle. Two subspecies of F. necrophorum, subsp. necrophorum (biotype A) and subsp. funduliforme (biotype B), are recognized that can be differentiated based on morphological, biochemical, biological and molecular characteristics. The subsp. necrophorum is more virulent and is isolated more frequently from infections than the subsp. funduliforme. Several toxins or secreted products have been implicated as virulence factors. The major factors contributing to ruminal colonization and invasion into the liver are hemagglutinin, endotoxin and leukotoxin, of which leukotoxin is the protective antigen. In some conditions, the organism synergistically interacts with Arcanobacterium pyogenes, a facultative anaerobic organism and a secondary etiologic agent, to cause liver abscesses.