Myostatin induces cachexia by activating the ubiquitin proteolytic system through an NF-kappaB-independent, FoxO1-dependent mechanism

Myostatin, a transforming growth factor-beta (TGF-beta) super-family member, has been well characterized as a negative regulator of muscle growth and development. Myostatin has been implicated in several forms of muscle wasting including the severe cachexia observed as a result of conditions such as AIDS and liver cirrhosis. Here we show that Myostatin induces cachexia by a mechanism independent of NF-kappaB. Myostatin treatment resulted in a reduction in both myotube number and size in vitro, as well as a loss in body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 was reduced, while NF-kappaB (the p65 subunit) localization and expression remained unchanged. In addition, promoter analysis has confirmed Myostatin inhibition of myoD and pax3. An increase in the expression of genes involved in ubiquitin-mediated proteolysis is observed during many forms of muscle wasting. Hence we analyzed the effect of Myostatin treatment on proteolytic gene expression. The ubiquitin associated genes atrogin-1, MuRF-1, and E214k were upregulated following Myostatin treatment. We analyzed how Myostatin may be signaling to induce cachexia. Myostatin signaling reversed the IGF-1/PI3K/AKT hypertrophy pathway by inhibiting AKT phosphorylation thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. Therefore, our results suggest that Myostatin induces cachexia through an NF-kappaB-independent mechanism. Furthermore, increased Myostatin levels appear to antagonize hypertrophy signaling through regulation of the AKT-FoxO1 pathway.     

Influence of cytoplasmic male sterility on expression of different mechanisms of resistance in sorghum to Atherigona soccata (Diptera: Muscidae)

Atherigona soccata (Rondani) (Diptera: Muscidae) is one of the most important pests of sorghum, Sorghum bicolor (L.) Moench, in Asia, Africa, and the Mediterranean Europe. Exploitation of cytoplasmic male sterility (CMS) for hybrid production has resulted in considerable narrowing of the genetic base and may increase the vulnerability of this crop to insect pests. Therefore, we studied the expression of different mechanisms of resistance in sorghum to A. soccata in CMS (A) and maintainer (B) lines of 12 genotypes under field and greenhouse conditions. The CMS lines of A. soccata-resistant genotypes were preferred for oviposition (78.5 versus 71.5% plants with eggs) and suffered greater deadheart incidence (47.6 versus 41.6%) than the corresponding maintainer lines, whereas such differences were not apparent in CMS lines belonging to the susceptible genotypes (92.7 versus 92.3% plants with eggs and 75.6 versus 74.6% deadhearts) under multichoice field conditions. Similar differences also were observed under controlled conditions in the greenhouse. The larval period (9.0 versus 8.8 d) and pupal mortality (18.4 versus 13.4%) were greater on maintainer lines than that on the CMS lines in the resistant group. The male and female pupal weights, fecundity, and antibiosis index were greater on the CMS than on the maintainer lines. The maintainer lines showed better recovery resistance than the CMS lines, but no such differences were observed in tiller deadhearts. The differences in susceptibility to A. soccata were greater in the A. soccata resistant CMS and maintainer lines than in the CMS and maintainer lines belonging to susceptible genotypes. Conversion of A. soccata-resistant genotypes into alternate less susceptible cytoplasmic backgrounds may be undertaken for developing sorghum hybrids with stable resistance to A. soccata.     

Molecular iodine induces caspase-independent apoptosis in human breast carcinoma cells involving the mitochondria-mediated pathway

Molecular iodine (I2) is known to inhibit the induction and promotion of N-methyl-n-nitrosourea-induced mammary carcinogenesis, to regress 7,12-dimethylbenz(a)anthracene-induced breast tumors in rat, and has also been shown to have beneficial effects in fibrocystic human breast disease. Cytotoxicity of iodine on cultured human breast cancer cell lines, namely MCF-7, MDA-MB-231, MDA-MB-453, ZR-75-1, and T-47D, is reported in this communication. Iodine induced apoptosis in all of the cell lines tested, except MDA-MB-231, shown by sub-G1 peak analysis using flow cytometry. Iodine inhibited proliferation of normal human peripheral blood mononuclear cells; however, it did not induce apoptosis in these cells. The iodine-induced apoptotic mechanism was studied in MCF-7 cells. DNA fragmentation analysis confirmed internucleosomal DNA degradation. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling established that iodine induced apoptosis in a time- and dose-dependent manner in MCF-7 cells. Iodine-induced apoptosis was independent of caspases. Iodine dissipated mitochondrial membrane potential, exhibited antioxidant activity, and caused depletion in total cellular thiol content. Western blot results showed a decrease in Bcl-2 and up-regulation of Bax. Immunofluorescence studies confirmed the activation and mitochondrial membrane localization of Bax. Ectopic Bcl-2 overexpression did not rescue iodine-induced cell death. Iodine treatment induces the translocation of apoptosis-inducing factor from mitochondria to the nucleus, and treatment of N-acetyl-L-cysteine prior to iodine exposure restored basal thiol content, ROS levels, and completely inhibited nuclear translocation of apoptosis-inducing factor and subsequently cell death, indicating that thiol depletion may play an important role in iodine-induced cell death. These results demonstrate that iodine treatment activates a caspase-independent and mitochondria-mediated apoptotic pathway.     

Genetic diversity studies of Kherigarh cattle based on microsatellite markers

We report a genetic diversity study of Kherigarh cattle, a utility draught-purpose breed of India, currently declining at a startling rate, by use of microsatellite markers recommended by the Food and Agriculture Organization. Microsatellite genotypes were derived, and allelic and genotypic frequencies, heterozygosities and gene diversity were estimated. A total of 131 alleles were distinguished by the 21 microsatellite markers used. All the microsatellites were highly polymorphic, with mean (±s.e.) allelic number of 6.24 ±1.7, ranging 4–10 per locus. The observed heterozygosity in the population ranged between 0.261 and 0.809, with mean (±s.e.) of 0.574 ±0.131, indicating considerable genetic variation in this population. Genetic bottleneck hypotheses were also explored. Our data suggest that the Kherigarh breed has not experienced a genetic bottleneck in the recent past.     

Granzyme B, a new player in activation-induced cell death, is down-regulated by vasoactive intestinal peptide in Th2 but not Th1 effectors

Following antigenic stimulation and differentiation, Th1 and Th2 effector cells contribute differently to cellular and humoral immunity. Vasoactive intestinal peptide (VIP) induces Th2 responses by promoting Th2 differentiation and survival. In this study, we investigate the mechanisms for the protective effect of VIP against activation-induced cell death (AICD) of Th2 effectors. Surprisingly, microarray and protein data indicate that VIP prevents the up-regulation of granzyme B (GrB) in Th2 but not Th1 effectors. This is the first report of GrB expression in Th cells and of its involvement in activation-induced apoptosis. The enhanced responsiveness of Th2 cells to VIP is probably due to the higher expression of VIP receptors. The effect of VIP on Th2 survival and GrB expression is mediated through the VIP receptors 1 and 2 and cAMP signaling through exchange protein activated by cAMP and, to a lesser degree, protein kinase A. In addition to effects on GrB, VIP also down-regulates Fas ligand (FasL) and perforin (Pfr) expression. The extrinsic Fas/FasL pathway and the intrinsic GrB-dependent pathway act independently in inducing AICD. The mechanisms by which GrB induces cell death in Th1/Th2 effectors include both fratricide and suicide. Fratricide killing, prevalent in wild-type cells, is calcium and Pfr dependent, whereas the cell death of Pfr-deficient Th cells involves Fas and GrB but is calcium independent. This study identifies GrB as a new significant player in Th1/Th2 AICD and characterizes two mechanisms for the protective effect of VIP on Th2 survival, i.e., the down-regulation of GrB and FasL expression.     

Preventive effect of tannic acid on 2-acetylaminofluorene induced antioxidant level, tumor promotion and hepatotoxicity: a chemopreventive study

Tannic acid, present in almost every food derived from plants, has been widely investigated as a chemopreventive agent because, apart from its use as a food additive, pharmacological studies have demonstrated its many health-promoting properties. In this study, we show the modulatory effect of tannic acid on 2-acetylaminofluorene (2-AAF)-mediated hepatic oxidative stress and cell proliferation in rats. 2-AAF (50 mg/kg body weight) caused reduction in hepatic glutathione content and the activities of hepatic anti-oxidant enzymes and phase-II metabolizing enzymes with an enhancement of xanthine oxidase activity, lipid peroxidation and hydrogen peroxide content. 2-AAF treatment also induced serum oxaloacetate and pyruvate transaminase, lactate dehydrogenase and gamma-glutamyl transpeptidase. Treatment of rats orally with tannic acid (125 and 250 mg/kg body weight) resulted in significant recovery of hepatic glutathione content, antioxidant and phase-II metabolizing enzymes. Also, significant decreases in lipid peroxidation, xanthine oxidase, hydrogen peroxide generation and liver damage marker enzymes were observed. The antiproliferative efficacy of the tannic acid was also evaluated. The promotion parameters induced (ornithine decarboxylase activity and DNA synthesis) by 2-AAF administration in the diet with partial hepatectomy (PH) were also significantly suppressed, dose dependently, by tannic acid. Hence, we propose that tannic acid might suppress the promotion stage via inhibition of oxidative stress and polyamine biosynthetic pathway.     

On the mechanism of palmitic acid-induced apoptosis: the role of a pore induced by palmitic acid and Ca2+ in mitochondria

Palmitic acid (Pal) is known to promote apoptosis (Sparagna G et al (2000) Am J Physiol Heart Circ Physiol 279: H2124–H2132) and its amount in blood and mitochondria increases under some pathological conditions. Yet, the mechanism of the proapoptotic action of Pal has not been elucidated. We present evidence for the involvement of the mitochondrial cyclosporin A-insensitive pore induced by Pal/Ca²⁺ complexes in the apoptotic process. Opening of this pore led to a fall of the mitochondrial membrane potential and the release of the proapoptotic signal cytochrome c. The addition of cytochrome c prevented these effects and recovered membrane potential, which is in contrast to the cyclosporin A-sensitive mitochondrial permeability transition pore. Oleic and linoleic acids prevented the Pal/Ca²⁺-induced pore opening in the intact mitochondria, this directly and significantly correlating with the effect of these fatty acids on Pal-induced apoptosis in cells (Hardy S et al (2003) J Biol Chem 278: 31861–31870). The specific probe for cardiolipin, 10-N-nonyl acridine orange, inhibited formation of this pore.     

Role of proteins in resistance mechanism of Pseudomonas fluorescens against heavy metal induced stress with proteomics approach

The genus Pseudomonas is a group of gram-negative, motile, rod-shaped bacteria known for their metabolic versatility. One of the species is Pseudomonas fluorescens, which has an efficient system for detoxification of industrial waste. Other aspects include, catabolic versatility, excellent root colonizing abilities and capacity to produce a wide range of antifungal metabolites. They are also known for their resistance and survival in the presence of several organic and inorganic pollutants. P. fluorescens has also been isolated from metal polluted water and soils but the elucidation of proteins responsible for its survival is still not clear. The aim of the study was to elucidate the differential protein expression of this bacterium when exposed to heavy metal stress, using two-dimensional electrophoresis. The proteins spo VG and enolase showed upregulation during the bacterial exposure to lead and copper. Hypothetical protein showed downregulation when bacterium was exposed to cobalt. Some proteins like xylosyltransferase, ORF 18 phage phi KZ, OMP H1 and translational elongation factor EF-Tu appeared only during their exposure to cobalt. These were absent in the control condition. Analysis of the differentially expressed proteins as well as the newly synthesized proteins along with the results obtained growth and enzyme activity indicate the involvement of all these factors in the survival of this organism in the presence of heavy metals.     

TspMI, a thermostable isoschizomer of XmaI (5′C/CCGGG3′): characterization and single molecule imaging with DNA

TspMI, a thermostable isoschizomer of XmaI from a Thermus sp., has been characterized. The enzyme was purified to homogeneity using Cibacron-Blue 3GA agarose, Heparin agarose, SP sephadex C50, and Mono-Q fast protein liquid chromatography and was found to be a homodimer of 40 kDa. Restriction mapping and run-off sequencing of TspMI-cleaved DNA ends depicted that it cleaved at 5′C/CCGGG3′ to generate a four-base, 5′-CCGG overhang. The enzyme was sensitive to methylation of second and third cytosines in its recognition sequence. TspMI worked optimally at 60°C with 6 mM Mg²⁺, no Na⁺/K⁺, and showed no star activity in the presence of 25% glycerol. The enzyme could efficiently digest the DNA labeled with a higher concentration of YOYO-I (one dye molecule to one nucleotide), making it a useful candidate for real-time imaging experiments. Single molecule interaction between TspMI and λ DNA was studied using total internal reflection fluorescence microscopy. The enzyme survived 30 polymerase chain reaction (PCR) cycles in the presence of 10% glycerol and 0.5 M trehalose without any activity loss and, hence, is suitable for incorporation in restriction-endonuclease-mediated selective-PCR for various applications.Electronic Supplementary Material Supplementary material for this article is available at