PKC-regulated myogenesis is associated with increased tyrosine phosphorylation of FAK,Cas, and paxillin,formation of Cas-CRK complex,and JNK activation

Previous reports suggest that PKC plays an important role in regulating myogenesis. However, the regulatory signaling pathways are not fully understood. We examined the effects of PKC downregulation on signaling events during skeletal muscle differentiation. We found that downregulation of PKC results in increased myogenesis in C2C12 cells as measured by creatine kinase activity and myogenin expression. We showed that, during differentiation, downregulation of PKC expression results in increased tyrosine phosphorylation of FAK, Cas, and paxillin, concomitant with enhanced Cas-CrkII complex formation, which leads to activation of JNK2. But in proliferated muscle cells, PKC inhibition results in FAK and Cas tyrosine dephosphorylation. Further, disruption of actin cytoskeleton by cytochalasin D prevents the activation of FAK and Cas as well as the formation of Cas-CrkII complex stimulated by PKC downregulation during muscle cell differentiation. Finally, we observed that PKC downregulation increases the tyrosine phosphorylation of focal adhesion associated proteins. Based on the above data, we propose that PKC downregulation results in enhanced tyrosine phosphorylation of FAK, Cas, and paxillin, thus promoting the establishment of Cas-CrkII complex, leading to activation of JNK and that these interactions are dependent upon the integrity of actin cytoskeleton during muscle cell differentiation. Data presented here significantly contribute to elucidating the regulatory role of PKC in myogenesis possibly through integrin signaling pathway.     

The Comparative RNA Web (CRW) Site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs

Background

Comparative analysis of RNA sequences is the basis for the detailed and accurate predictions of RNA structure and the determination of phylogenetic relationships for organisms that span the entire phylogenetic tree. Underlying these accomplishments are very large, well-organized, and processed collections of RNA sequences. This data, starting with the sequences organized into a database management system and aligned to reveal their higher-order structure, and patterns of conservation and variation for organisms that span the phylogenetic tree, has been collected and analyzed. This type of information can be fundamental for and have an influence on the study of phylogenetic relationships, RNA structure, and the melding of these two fields.

Results

We have prepared a large web site that disseminates our comparative sequence and structure models and data. The four major types of comparative information and systems available for the three ribosomal RNAs (5S, 16S, and 23S rRNA), transfer RNA (tRNA), and two of the catalytic intron RNAs (group I and group II) are: (1) Current Comparative Structure Models; (2) Nucleotide Frequency and Conservation Information; (3) Sequence and Structure Data; and (4) Data Access Systems.

Conclusions

This online RNA sequence and structure information, the result of extensive analysis, interpretation, data collection, and computer program and web development, is accessible at our Comparative RNA Web (CRW) Site http://www.rna.icmb.utexas.edu. In the future, more data and information will be added to these existing categories, new categories will be developed, and additional RNAs will be studied and presented at the CRW Site.     

Role of S-adenosyl-L-methionine in potentiating cadmium mobilization by diethylenetriamine penta acetic acid in mice

The beneficial effects of S-adenosyl-L-methionine (SAM) in potentiating the mobilization of cadmium by cadmium trisodium diethylenetriamine penta acetic acid (DTPA) from the major target organs and restoration of depleted tissue glutathione (GSH), zinc and copper concentration, were determined in cadmium-exposed mice. The results indicated a significant depletion of cadmium concentration from the blood in DTPA plus SAM treated animals compared with DTPA or SAM alone treated groups. The treatment with SAM alone was also effective in correcting the zinc and GSH concentrations. The results indicated few beneficial effects of concomitant SAM administration during chelation of cadmium with DTPA.     

Human pS2/trefoil factor 1: production and characterization in Pichia pastoris

The recombinant protein human trefoil factor 1 (hTFF1), formerly called hpS2, has been produced for the first time in a yeast-based expression in Pichia pastoris. hTFF1 was secreted in large amounts in the extracellular medium of P. pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The fermentation broth containing hTFF1 was concentrated by tangential flow filtration prior to purification by anion- and cation-exchange chromatography, followed by preparative high-performance liquid chromatography. The resulting hTFF1 was found to be intact by Western blot analysis. Further analysis revealed mainly the presence of the monomeric form of the hTFF1 peptide. Finally, in vitro, the recombinant hTFF1 was shown to decrease proliferation of the HCT116 cancer cells.     

Identification of a zinc finger domain in the human NEIL2 (Nei-like-2) protein

The recently identified human NEIL2 (Nei-like-2) protein, a DNA glycosylase/AP lyase specific for oxidatively damaged bases, shares structural features and reaction mechanism with the Escherichia coli DNA glycosylases, Nei and Fpg. Amino acid sequence analysis of NEIL2 suggested it to have a zinc finger-like Nei/Fpg. However, the Cys-X2-His-X16-Cys-X2-Cys (CHCC) motif present near the C terminus of NEIL2 is distinct from the zinc finger motifs of Nei/Fpg, which are of the C4 type. Here we show the presence of an equimolar amount of zinc in NEIL2 by inductively coupled plasma mass spectrometry. Individual mutations of Cys-291, His-295, Cys-315, and Cys-318, candidate residues for coordinating zinc, inactivated the enzyme by abolishing its DNA binding activity. H295A and C318S mutants were also shown to lack bound zinc, and a significant change in their secondary structure was revealed by CD spectra analysis. Molecular modeling revealed Arg-310 of NEIL2 to be a critical residue in its zinc binding pocket, which is highly conserved throughout the Fpg/Nei family. A R310Q mutation significantly reduced the activity of NEIL2. We thereby conclude that the zinc finger motif in NEIL2 is essential for its structural integrity and enzyme activity.     

Recent developments in electrolytic devices for ion chromatography

Recent developments in new electrolytic devices that utilize the electrolysis of water and charge-selective electromigration of ions through ion-exchange media have significantly changed the routine operation of ion chromatographic methods. Examples of these new electrolytic devices include on-line eluent generators that produce high-purity electrolyte eluents using deionized water as the carrier stream, continuously regenerated trap columns that remove ionic contaminants in the eluents, and continuously regenerated suppressors that reduce eluent background conductance prior to conductivity detection. The combined use of these electrolytic devices has made it possible to perform various ion chromatographic separations using only deionized water as the mobile phase. This paper reviews the operation principles of these electrolytic devices and their applications in the ion chromatographic determination of anionic and cationic analytes.     

Effect of chromium and zinc on insulin signaling in skeletal muscle cells

Patients on total parenteral nutrition without Cr supplementation develop symptoms similar to those of diabetes. Zn has been implicated in diabetes because of its antioxidant properties and interaction with insulin. To study the effect of these metal ions on insulin signaling proteins, cultured mouse skeletal muscle cells was used as an in vitro model, as the tissue accounts for more than 80% of insulin-stimulated glucose disposal in the body. In the present study, it has been observed that both Cr and Zn, upon prolonged exposure, could stimulate tyrosine phosphorylation of insulin receptor (IR) even in the absence of insulin. Insulin-mediated IR tyrosine phosphorylation was enhanced by the treatment with both of the metal ions. Both Cr and Zn could phosphorylate insulin receptor substrate-1 (IRS-1). Phosphorylation of IRS-1 induced by metal ions was higher than that induced by insulin. Hence, both Cr and Zn were found to have insulin mimetic activity. Both of the metal ions were also found to potentiate insulin-mediated activation of IRS-1. The basal level of glucose uptake was also increased by prolonged treatment of the cells with the metal ions. The ions could also enhance the insulin-stimulated glucose uptake into the cells. Therefore, both Zn and Cr seem to have a positive effect on insulin signaling leading to glucose uptake.     

Evidence that CTR1-Mediated Ethylene Signal Transduction in Tomato is Encoded by a Multigene Family Whose Members Display Distinct Regulatory Features

Ethylene governs a range of developmental and response processes in plants. In Arabidopsis thaliana, the Raf-like kinase CTR1 acts as a key negative regulator of ethylene responses. While only one gene with CTR1 function apparently exists in Arabidopsis, we have isolated a family of CTR1- like genes in tomato ( Lycopersicon esculentum ). Based on amino acid alignments and phylogenetic analysis, these tomato CTR1- like genes are more similar to Arabidopsis CTR1 than any other sequences in the Arabidopsis genome. Structural analysis reveals considerable conservation in the size and position of the exons between Arabidopsis and tomato CTR1 genomic sequences. Complementation of the Arabidopsis ctr1-8 mutant with each of the tomato CTR genes indicates that they are all capable of functioning as negative regulators of the ethylene pathway. We previously reported that LeCTR1 expression is up-regulated in response to ethylene. Here, quantitative real-time PCR was carried out to detail expression for LeCTR1 and the additional CTR1 -like genes of tomato. Our results indicate that the tomato CTR1 gene family is differentially regulated at the mRNA level by ethylene and during stages of development marked by increased ethylene biosynthesis, including fruit ripening. The possibility of a multi-gene family of CTR1 -like genes in other species besides tomato was examined through mining of EST and genomic sequence databases.