Expressed Sequence Tags for Bovine Muscle Satellite Cells,Myotube Formed-Cells and Adipocyte-Like Cells

Background

Muscle satellite cells (MSCs) represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs) and adipocyte-like cells (ALCs), we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs.

Results

A total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248) highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of dimethylarginine dimethylaminohydrolase2 (DDAH2) during myogenesis and hemoglobin subunit alpha2 (HBA2) during transdifferentiation in C2C12 were assayed as a case study. DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG) expression corresponding with the slight change in cell morphology. In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay.

Conclusion

In this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to enable better understanding of the mechanism of muscle differentiation and transdifferentiation.     

Production of Napiergrass as a Bioenergy Feedstock Under Organic Versus Inorganic Fertilization in the Southeast USA

Napiergrass (Pennisetum purpureum Schum.) is a high-yielding perennial biomass crop that is well adapted to the Southeast USA where poultry litter is readily available. This research was conducted to compare biomass production and nutrient utilization of napiergrass fertilized with either poultry litter or inorganic fertilizer. Each spring, approximately 100 kg ha^?1 of N, 40 kg ha^?1 P, and 90 kg ha^?1 K were applied as poultry litter or equivalent inorganic fertilizer. Biomass was harvested each winter after senescence. For the first 2 years, dry matter yield did not differ among treatments, but in the third and fourth years, yields declined in all treatments and were lowest in the unfertilized treatment. Biomass N concentration and N removal were greatest in the inorganic treatment. In general, N removal exceeded the amount applied, suggesting that higher application rates may be necessary to maintain yields. Biomass P concentration and total P uptake were greatest in the litter fertilized treatment, demonstrating that napiergrass can remove some of the excess P from applied litter. Soil cores were taken periodically to assess changes in soil properties. After 2 years of production, soil pH in the surface layer (0–15 cm) was lower in the inorganic treatment than in the other treatments. After 4 years, total soil C had increased by an average of 3,180 kg ha^?1 though fertilizer treatments did not differ. Yield declined in all treatments after 4 years and N supplementation is recommended for production in upland fields.     

Screening of Surface-associated Bacteria from the Mexican Red Alga Halymenia floresii for Quorum Sensing Activity

Microbiology - Macroalgae host a dense bacterial epibiome forming surface biofilms, which act as a biological defense by protecting the surface from macrofoulers. During experimental cultivation of...     

Preferential recognition of epitopes on peroxynitrite-modified alpha-2-macroglobulin by circulating autoantibodies in rheumatoid arthritis patients

Abstract

Autoimmune responses against post-translationally modified antigens are a hallmark of several autoimmune diseases. In this work, we have studied the changes in alpha-2-macroglobulin (α₂M) upon modification by peroxynitrite. Furthermore, we have evaluated the immunogenicity of modified α₂M in experimental rabbits and rheumatoid arthritis (RA) patients. Peroxynitrite-modified α₂M showed disturbed microenvironment and altered aromatic residues under UV and fluorescence studies. Aggregation, reduction in β-sheet content, production of nitrotyrosine and shift in amide I and II bands were observed in the modified α₂M by polyacrylamide gel electrophoresis besides CD and FTIR spectroscopic analysis. The exposure of hydrophobic clusters and changes in contact positions were observed in ANS and ThT binding assays. Immunological studies using ELISA showed peroxynitrite-modified α₂M as highly immunogenic producing high titre of specific antibodies in immunized rabbits. Cross-reactivity studies revealed the polyspecificity of the elicited antibodies. Direct binding ELISA and competitive inhibition studies confirmed the presence of circulating antibodies in the sera of RA patients having high specificity towards the peroxynitrite-modified α₂M as compared to the native α₂M. Sera from healthy (normal) human subjects showed lower binding with the native and modified protein. This study confirms that peroxynitrite induces structural modifications in α₂M and makes it immunogenic. The presence of neo-antigenic determinants on modified α₂M with enhanced binding for circulating autoantibodies in RA patients could offer new possibilities for diagnosis and etiopathology of the disease.

Communicated by Ramaswamy H. Sarma     

A Comparison of Effects of Broad-Spectrum Antibiotics and Biosurfactants on Established Bacterial Biofilms

Current antibiofilm solutions based on planktonic bacterial physiology have limited efficacy in clinical and occasionally environmental settings. This has prompted a search for suitable alternatives to conventional therapies. This study compares the inhibitory properties of two biological surfactants (rhamnolipids and a plant-derived surfactant) against a selection of broad-spectrum antibiotics (ampicillin, chloramphenicol and kanamycin). Testing was carried out on a range of bacterial physiologies from planktonic and mixed bacterial biofilms. Rhamnolipids (Rhs) have been extensively characterised for their role in the development of biofilms and inhibition of planktonic bacteria. However, there are limited direct comparisons with antimicrobial substances on established biofilms comprising single or mixed bacterial strains. Baseline measurements of inhibitory activity using planktonic bacterial assays established that broad-spectrum antibiotics were 500 times more effective at inhibiting bacterial growth than either Rhs or plant surfactants. Conversely, Rhs and plant biosurfactants reduced biofilm biomass of established single bacterial biofilms by 74–88 and 74–98 %, respectively. Only kanamycin showed activity against biofilms of Bacillus subtilis and Staphylococcus aureus. Broad-spectrum antibiotics were also ineffective against a complex biofilm of marine bacteria; however, Rhs and plant biosurfactants reduced biofilm biomass by 69 and 42 %, respectively. These data suggest that Rhs and plant-derived surfactants may have an important role in the inhibition of complex biofilms.     

Slow Regulated Release of H2S Inhibits Oxidative Stress Induced Cell Death by Influencing Certain Key Signaling Molecules

Hydrogen sulphide (H₂S) is one of three gaseous signaling molecules after nitric oxide and carbon monoxide. Various H₂S donor compounds have been synthesized to study its physiological function. Among these compounds sodium hydrosulphide (NaHS), a donor of releasing H₂S rapidly have shown to be protective in certain neuronal cell line but several in vivo studies have generated conflicting data. Furthermore several slow releasing H₂S donors have been shown to have positive effects on cells in culture. The intracellular concentration of H₂S and hence its rate of production may be a factor in keeping the balance between its neuroprotective and toxic effects. The present study was undertaken to deduce how a rapid releasing H₂S donor (NaHS) as opposed to a slow releasing donor (ADTOH), affect oxidative stress related intracellular components and survival of RGC-5 cells. It was concluded that when RGC-5 cells are exposed to the toxic effects of glutamate in combination with buthionine sulfoxime (Glu/BSO), ADTOH was more efficacious in inhibiting apoptosis, scavenging reactive oxygen species (ROS), stimulation of glutathione (GSH) and gluthathione-S-transferase (GST). Western blot and qPCR analysis showed ADTOH increased the levels of Nrf2, HO-1, PKCα, p-Akt, Bcl-2 and XIAP but caused a decrease of Nfκβ and xCT greater than NaHS. This study is first to compare the efficacy of two H₂S donor drugs as potential neuroprotectants and demonstrate that slow regulated release of H₂S to cell culture can be more beneficial in inhibiting oxidative stress induced cell death.     

Rat model of fractionated (2 Gy/day) 60 Gy irradiation of the liver: long-term effects

The liver is considered a radiosensitive organ. However, in rats, high single-dose irradiation (HDI) showed only mild effects. Consequences of fractionated irradiation (FI) in such an animal model have not been studied so far. Rats were exposed to selective liver FI (total dose 60 Gy, 2 Gy/day) or HDI (25 Gy) and were killed three months after the end of irradiation. To study acute effects, HDI-treated rats were additionally killed at several time points between 1 and 48 h. Three months after irradiation, no differences between FI and HDI treatment were found for macroscopically detectable small “scars” on the liver surface and for an increased number of neutrophil granulocytes distributed in the portal fields and through the liver parenchyma. As well, no changes in HE-stained tissues or clear signs of fibrosis were found around the portal vessels. Differences were seen for the number of bile ducts being increased in FI- but not in HDI-treated livers. Serum levels indicative of liver damage were determined for alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (γGT) and lactate dehydrogenase (LDH). A significant increase of AP was detected only after FI while HDI led to the significant increases of AST and LDH serum levels. By performing RT-PCR, we detected up-regulation of matrix metalloproteinases, MMP-2, MMP-9, MMP-14, and of their inhibitors, TIMP-1, TIMP-2 and TIMP-3, shortly after HDI, but not at 3 month after FI or HDI. Overall, we saw punctual differences after FI and HDI, and a diffuse formation of small scars at the liver surface. Lack of “provisional clot”-formation and absence of recruitment of mononuclear phagocytes could be one explanation for scar formation as incomplete repair response to irradiation.