RNomics of Thermus themophilus HB8 by DNA microarray and next-generation sequencing.

By using the data obtained by the DNA microarray analysis for the intergenic regions applied to RNA samples extracted from Thermus thermophilus HB8, seven small non-coding RNAs, TtR-1 to TtR-7, were found to be expressed in the cells growing in rich and/or minimal media. By analysing the time course of the expression for the cell growth in combination with the sequence comparison to the known RNAs, two RNAs, TtR-1 and TtR-2, are suggested to be riboswitches. The existence of the seven RNAs and the exact sequence and length, ranging 77-284 nt, were confirmed by the next-generation sequencing. By the combination of these two high-throughput techniques, our understanding of RNAs in the cell will be increased significantly.

Sanguiin H-6, a constituent of Rubus parvifolius L., inhibits receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis and bone resorption in vitro and prevents tumor necrosis factor-α-induced osteoclast formation in vivo.

BACKGROUND: Osteoclasts are multinucleated bone-resorbing cells that differentiate in response to receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL). Enhanced osteoclastogenesis contributes to bone diseases, such as osteoporosis and rheumatoid arthritis. Rubus parvifolius L. is traditionally used as an herbal medicine for rheumatism; however, its detailed chemical composition and the molecular mechanisms responsible for its biological action have not been elucidated. PURPOSE: To investigate the mechanisms by which R. parvifolius L. extract and its major constituent sanguiin H-6, inhibit osteoclastogenesis and bone resorption. METHODS: Cell proliferation, cell differentiation, and bone resorption were detected in vitro. Inhibition of signaling pathways, marker protein expression, and protein nuclear translocation were evaluated by western blot analysis. Tumor necrosis factor-α (TNF-α)-mediated osteoclastogenesis was examined in vivo. RESULTS: R. parvifolius L. extract inhibited the bone-resorption activity of osteoclasts. In addition, sanguiin H-6 markedly inhibited RANKL-induced osteoclast differentiation and bone resorption, reduced reactive oxygen species production, and inhibited the phosphorylation of inhibitor of NF-κB alpha (IκBα) and p38 mitogen-activated protein kinase. Sanguiin H-6 also decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), cathepsin K, and c-Src. Moreover, sanguiin H-6 inhibited the nuclear translocation of NFATc1, c-Fos, and NF-κB in vitro, as well as TNF-α-mediated osteoclastogenesis in vivo. CONCLUSIONS: Our data revealed that R. parvifolius L. has anti-bone resorption activity and suggest that its constituent, sanguiin H-6, can potentially be used for the prevention and treatment of bone diseases associated with excessive osteoclast formation and subsequent bone destruction.

Ultrastructural alterations of osteocyte morphology via loaded implants in rabbit tibiae.

Osteocytes are crucial cells that control bone responses to mechanical loading. However, the effects of mechanical loading on osteocytes around dental implants are unclear. The aim of this study was to investigate whether mechanical loading via bone-integrated implants influences osteocyte number and morphology in the surrounding bone. Fourteen anodized Ti-6Al-4V alloy dental implants were placed in seven Japanese white rabbits, and implants in each rabbit were subjected to mechanical loading (50N, 3Hz for 1800 cycles, 2 days/week) along the implant long axis. Eight weeks after the initiation of loading, histomorphometric analysis and microcomputed tomography were performed. Scanning electron microscopy (SEM) was also performed with an acid etching technique using longitudinal and cross-sectional specimens. More bone formation around loaded implants was noted. In the implant neck, osteocytes tended to be more spherical with increased dendrite processes around loaded implants, while spindle-shaped osteocytes without increased dendrite processes were observed around unloaded implants in both longitudinal and cross-sectional images. In the bottom area, morphological changes in osteocytes were observed around loaded implants; however, dendrite processes did not differ in longitudinal or cross-sectional images, regardless of mechanical loading. These findings indicate that increased osteocyte numbers and developed dendrite processes are associated with anabolic bone responses to mechanical loading. The combination of acid etching and SEM imaging is a useful technique to assess ultrastructural osteocyte morphology around dental implants.

Increased vulnerability of hippocampal pyramidal neurons to the toxicity of kainic acid in OASIS-deficient mice.

The endoplasmic reticulum (ER) stress response is a defense system for dealing with the accumulation of unfolded proteins in the ER lumen. Old astrocyte specifically induced substance (OASIS) is known to be expressed in astrocytes and involved in the ER stress response; however the function of OASIS in the injured brain has remained unclear. In this study, we examined the roles of OASIS in neuronal degeneration in the hippocampi of mice intraperitoneally injected with kainic acid (KA). OASIS mRNA was strongly induced in response to KA injection, with a similar time course to the induction of ER molecular chaperone immunoglobulin heavy chain binding protein mRNA. In situ hybridization showed that KA injection causes induction of immunoglobulin heavy chain binding protein mRNA in glial fibrillary acidic protein-positive astrocytes as well as in pyramidal neurons, although up-regulation of OASIS mRNA was only detected in glial fibrillary acidic protein-positive astrocytes. Primary cultured astrocytes, but not the neurons of OASIS-/- mice, revealed reduced vulnerability to ER stress. Furthermore, pyramidal neurons in the hippocampi of OASIS-/- mice were more susceptible to the toxicity induced by KA than those of wild-type mice. Taken together, these data suggest that OASIS expressed in astrocytes plays important roles in protection against the neuronal damage induced by KA.

Two-dimensional phosphate-affinity gel electrophoresis for the analysis of phosphoprotein isotypes.

Herein, we describe three kinds of 2-DE using phosphate-affinity PAGE for the analysis of phosphoprotein isotypes. The first dimension is a urea-PAGE, IEF/NEPHGE, or SDS-PAGE, which are widely used. The second dimension is a phosphate-affinity SDS-PAGE using a phosphate-binding tag molecule, Phos-tag (Mn(2+)-Phos-tag SDS-PAGE). The first 2-D procedure coupling urea-PAGE and Mn(2+)-Phos-tag SDS-PAGE was applied to the separation of beta-casein phosphoisotypes. A typical protein sample containing multiple phosphoisotypes from beta-casein (with five phosphorylation sites) was prepared by partial dephosphorylation with alkaline phosphatase. The second procedure coupling IEF/NEPHGE and Mn(2+)-Phos-tag SDS-PAGE was applied to the separations of phosphoisotypes of caseins and in vitro kinase reaction products of Tau. The third procedure coupling normal SDS-PAGE and Mn(2+)-Phos-tag SDS-PAGE was applied to the separation of A431 cell lysates before and after stimulation with an epidermal growth factor. This procedure followed by immunoblotting with anti-mitogen-activated protein kinase(MAPK) and anti-Shc antibodies demonstrated the detection of phosphoisotypes in each protein isoform of MAPK1/2 (44 and 42 kDa) and Shc (66, 52, and 46 kDa) after the stimulation. By these novel 2-D procedures, the separations of phosphoprotein isotypes should be improved relative to those by current gel electrophoresis methods, including 1-D Mn(2+)-Phos-tag SDS-PAGE.

Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE.

Herein, we demonstrate the separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE. The phosphate-affinity site is a polyacrylamide-bound Phos-tag that enables the mobility shift detection of phosphoproteins from their nonphosphorylated counterparts. As the first practical example of the separation, we characterized the monophosphorylated Tau isotypes by each of three tyrosine kinases, c-Abl, MET, and Fyn. Each monophosphoisotype phosphorylated at the Tyr-394, Tyr-197, or Tyr-18 was detected as three distinct migration bands. As a further application, we extended this technique to the mobility shift analysis of His and Asp phosphoisotypes in the Sinorhizobium meliloti FixL/FixJ two-component system. FixL is autophosphorylated at the His-285 with ATP, and the phosphate group is transferred to the Asp-54 of FixJ and subsequently removed by the FixL phosphatase activity. Using this method, we first performed simultaneous detection of the phosphorylated and nonphosphorylated isotypes of FixL and FixJ generated in their phosphotransfer reaction in vitro. As a result, a monophosphoisotype of FixL containing the phosphorylated His residue was confirmed. As for FixJ, on the other hand, two monophosphoisotypes were detected as two distinct migration bands. One is a well-known isotype phosphorylated at the Asp-54. The other is a novel isotype phosphorylated at the His-84.

Avaliação intra-operatória da viabilidade instestinal por imagem infravermelha / Perioperative assessment of the intestinal viability by infrared imaging

A isquemia mesentérica aguda é uma emergência vascular com alto índice de mortalidade e sua incidência vem aumentando nas últimas décadas. Determinar a necessidade de ressecar a alça intestinal, qual segmento preservar e avaliar a viabilidade do segmento preservado continua sendo um desafio para o cirurgião, o qual se vale, quase que exclusivamente, de critérios clínicos intra-operatórios para decidir a conduta. Diversos métodos experimentais estão sendo estudados para avaliar a viabilidade intestinal. Realizou-se revisão da literatura do período de 1975 a 2007 sobre a utilização da imagem infravermelha na avaliação intra-operatória da viabilidade intestinal. Concluiu-se que a imagem infravermelha poderá ser um método sensível, rápido e confiável para a avaliação intra-operatória da viabilidade intestinal.

Molecular mechanisms responsible for aberrant splicing of SERCA1 in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disorder associated with an expansion of CTG trinucleotide repeats in the 3'-untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. The RNA gain-of-function hypothesis proposes that mutant DMPK mRNA alters the function and localization of alternative splicing regulators, which are critical for normal RNA processing. Previously, we found alternative splicing variants of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1), which excluded exon 22, in skeletal muscle of DM1 patients. In the present study, we analyzed the molecular mechanisms responsible for the splicing dysregulation of SERCA1. Five 'YGCU(U/G)Y' motifs that could potentially serve as Muscleblind-like 1, (MBNL1)-binding motifs, are included downstream from the SERCA1 exon 22. Exon trapping experiments showed that MBNL1 acts on the 'YGCU(U/G)Y' motif, and positively regulates exon 22 splicing. Of the five MBNL1 motifs in intron 22, the second and third sites were important for regulation of exon 22 splicing, but the other three binding sites were not required. Overexpression of the CUG repeat expansion of DMPK mRNA resulted in exclusion of exon 22 of SERCA1. These results suggest that sequestration of MBNL1 into the CUG repeat expansion of DMPK mRNA could cause the exclusion of SERCA1 exon 22, and the expression of this aberrant splicing form of SERCA1 could affect the regulation of Ca(2+) concentration of sarcoplasmic reticulum in DM patients.

Label-free kinase profiling using phosphate affinity polyacrylamide gel electrophoresis.

Herein we describe three applications of label-free kinase profiling using a novel type of phosphate affinity polyacrylamide gel electrophoresis. The phosphate affinity site is a polyacrylamide-bound dinuclear Mn2+ complex that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart. The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in phosphorylation status. The activity profiles of six kinds of kinases, glycogen synthase kinase-3beta, cyclin-dependent kinase 5/p35, protein kinase A, mitogen-activated protein kinase (MAPK), casein kinase II, and calmodulin-dependent protein kinase II, were determined using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple electrophoresis migration bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes in the epidermal growth factor-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting with anti-Shc and anti-MAPK antibodies. The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was determined using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (an ATP competitor). In the kinase assay, the slower migration band, monophosphorylated Abltide-GST, increased time-dependently, whereas the faster migration band, nonphosphorylated Abltide-GST, decreased. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the faster and slower migration bands, which showed an IC50 value of 1.6 microM in the presence of 0.10 mM ATP.