[Electrically Evoked Auditory Nerve Compound Action Potentials in Nucleus CI24M Cochlear Implant Users]

Lin Chuang Er Bi Yan Hou Ke Za Zhi = Journal of Clinical Otorhinolaryngology. Jan, 2002  |  Pubmed ID: 11944479

To study the feasibility and clinical applicability of electrically evoked auditory nerve compound action potentials (ECAP) in young patients with Nucleus CI24M cochlear implants.

Heterogeneity of Normal Prion Protein in Two- Dimensional Immunoblot: Presence of Various Glycosylated and Truncated Forms

Journal of Neurochemistry. Jun, 2002  |  Pubmed ID: 12065622

The common use of one-dimensional (1-D) immunoblot with a single monoclonal antibody (Mab) engenders the notion that the normal or cellular prion protein (PrP(C) ) comprises few and simple forms. In this study we used two-dimensional (2-D) immunoblot with a panel Mabs to various regions of the prion protein to demonstrate the complexity of the PrP(C) present in human brain. We distinguished over 50 immunoblot spots, each representing a distinct PrP(C) species based on combinations of different molecular weights and isoelectric points (pIs). The PrP(C) heterogeneity is due to the presence of a full-length and two major truncated forms as well as to the diversity of the glycans linked to most of these forms. The two major truncated forms result from distinct cleavage sites located at the N-terminus. In addition, enzymatic removal of sialic acid and lectin binding studies indicate that the glycans linked to the full-length and truncated PrP(C) forms differ in their structure and ratios of the glycoforms. The truncation of PrP(C) and the heterogeneity of the linked glycans may play a role in regulating PrP(C) function. Furthermore, the presence of relatively large quantities of different PrP(C) species may provide additional mechanisms by which the diversity of prion strains could be generated.

[The Extensibility and Retractility of Surgical Margins in Digestive Tract Cancer]

Zhonghua Wai Ke Za Zhi [Chinese Journal of Surgery]. Apr, 2002  |  Pubmed ID: 12133357

To study the extensibility and retractility of the surgical margins in digestive system neoplasms.

Cell-surface Prion Protein Interacts with Glycosaminoglycans

The Biochemical Journal. Nov, 2002  |  Pubmed ID: 12186633

We used ELISA and flow cytometry to study the binding of prion protein PrP to glycosaminoglycans (GAGs). We found that recombinant human PrP (rPrP) binds GAGs including chondroitin sulphate A, chondroitin sulphate B, hyaluronic acid, and heparin. rPrP binding to GAGs occurs via the N-terminus, a region known to bind divalent cations. Additionally, rPrP binding to GAGs is enhanced in the presence of Cu2+ and Zn2+, but not Ca2+ and Mn2+. rPrP binds heparin strongest, and the binding is inhibited by certain heparin analogues, including heparin disaccharide and sulphate-containing monosaccharides, but not by acetylated heparin. Full-length normal cellular prion protein (PrPC), but not N-terminally truncated PrPC species, from human brain bind GAGs in a similar Cu2+/Zn2+-enhanced fashion. We found that GAGs specifically bind to a synthetic peptide corresponding to amino acid residues 23-35 in the N-terminus of rPrP. We further demonstrated that while both wild-type PrPC and an octapeptide-repeat-deleted mutant PrP produced by transfected cells bound heparin at the cell surface, the PrP N-terminal deletion mutant and non-transfectant control failed to bind heparin. Binding of heparin to wild-type PrPC on the cell surface results in a reduction of the level of cell-surface PrPC. These results provide strong evidence that PrPC is a surface receptor for GAGs.

Intercellular Transfer of the Cellular Prion Protein

The Journal of Biological Chemistry. Dec, 2002  |  Pubmed ID: 12359724

The cellular prion protein (PrP(C)) is a glycosylphosphatidylinositol (GPI)-anchored protein. We investigated whether PrP(C) can move from one cell to another cell in a cell model. Little PrP(C) transfer was detected when a PrP(C) expressing human neuroblastoma cell line was cultured with the human erythroleukemia cells IA lacking PrP(C). Efficient transfer of PrP(C) was detected with the presence of phorbol 12-myristate 13-acetate, an activator of protein kinase C. Maximum PrP(C) transfer was observed when both donor and recipient cells were activated. Furthermore, PrP(C) transfer required the GPI anchor and direct cell to cell contact. However, intercellular protein transfer is not limited to PrP(C), another GPI-anchored protein, CD90, also transfers from the donor cells to acceptor cells after cellular activation. Therefore, this transfer process is GPI-anchor and cellular activation dependent. These findings suggest that the intercellular transfer of GPI-anchored proteins is a regulated process, and may have implications for the pathogenesis of prion disease.

Dimeric and Monomeric Bacillus Subtilis RNase P Holoenzyme in the Absence and Presence of Pre-tRNA Substrates

Biochemistry. Oct, 2002  |  Pubmed ID: 12390025

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that catalyzes the 5' maturation of tRNA precursors. The bacterial RNase P holoenzyme is composed of a large, catalytic RNA and a small protein. Our previous work showed that Bacillus subtilis RNase P forms a specific "dimer" that contains two RNase P RNA and two RNase P protein subunits in the absence of substrate. We investigated the equilibrium and the structure of the dimeric and the monomeric holoenzyme in the absence and presence of substrates by synchrotron small-angle X-ray scattering, 3' autolytic processing, and hydroxyl radical protection. In the absence of substrate, the dimer-monomer equilibrium is sensitive to monovalent ions and the total holoenzyme concentration. At 0.1 M NH4Cl, formation of the dimer is strongly favored, whereas at 0.8 M NH4Cl, the holoenzyme is a monomer. Primary hydroxyl radical protection in the dimer is located in the specificity domain, or domain I, of the RNase P RNA. The ES complex with a substrate containing a single tRNA is always monomeric. In contrast, the dominant ES complex with a substrate containing two tRNAs is dimeric at 0.1 M NH4Cl and monomeric at 0.8 M NH4Cl. Our results show that the B. subtilis holoenzyme can be a dimer and a monomer, and the fraction of the dimer is very sensitive to the environment. Under a variety of conditions, both the holoenzyme dimer and monomer can be present in significant amounts. Because the majority of tRNA genes are organized in large operons and because of the lack of RNase E in B. subtilis, a dimeric holoenzyme may be necessary to facilitate the processing of large precursor tRNA transcripts. Alternatively, the presence of two forms of the RNase P holoenzyme may be required for other yet unknown functions.

Getting Hotter with RNA

Nature Structural Biology. Nov, 2002  |  Pubmed ID: 12402034

Crystal Structure of the Specificity Domain of Ribonuclease P

Nature. Feb, 2003  |  Pubmed ID: 12610630

RNase P is the only endonuclease responsible for processing the 5' end of transfer RNA by cleaving a precursor and leading to tRNA maturation. It contains an RNA component and a protein component and has been identified in all organisms. It was one of the first catalytic RNAs identified and the first that acts as a multiple-turnover enzyme in vivo. RNase P and the ribosome are so far the only two ribozymes known to be conserved in all kingdoms of life. The RNA component of bacterial RNase P can catalyse pre-tRNA cleavage in the absence of the RNase P protein in vitro and consists of two domains: a specificity domain and a catalytic domain. Here we report a 3.15-A resolution crystal structure of the 154-nucleotide specificity domain of Bacillus subtilis RNase P. The structure reveals the architecture of this domain, the interactions that maintain the overall fold of the molecule, a large non-helical but well-structured module that is conserved in all RNase P RNA, and the regions that are involved in interactions with the substrate.

Guanidine Hydrochloride Extraction and Detection of Prion Proteins in Mouse and Hamster Prion Diseases by ELISA

The Journal of Pathology. Apr, 2003  |  Pubmed ID: 12635145

Current detection of transmissible spongiform encephalopathy (TSE) relies on the proteolytic generation of a protease-resistant core from the scrapie isoform of prion protein (PrP(Sc)) followed by immunoblotting. This process is non-quantitative, time-consuming, and technically demanding. Recently, an alternative in vitro test for TSE based on the differential extraction of brain homogenates using guanidine hydrochloride followed by DELFIA (Dissociation Enhanced Lanthanide FluoroImmunoAssay) has been developed. In the present study, this approach was adopted using a panel of anti-PrP monoclonal antibodies (MAbs) in conventional sandwich enzyme-linked immunosorbent assay (ELISA) to investigate hamster and two distinct strains of mouse prion diseases. Although PrP species were present in both soluble and insoluble fractions from normal as well as TSE samples, only the PrP species in the insoluble fractions from the latter samples were protease-resistant. In addition, certain anti-PrP MAb pairs could distinguish the PrP species in infected brains from those in the normal samples. The ability to differentiate disease-associated PrP isoforms without proteinase K digestion could serve as a panacea for developing a reliable and rapid diagnostic test for prion diseases.

On the Same Cell Type GPI-anchored Normal Cellular Prion and DAF Protein Exhibit Different Biological Properties

Biochemical and Biophysical Research Communications. Apr, 2003  |  Pubmed ID: 12659837

Normal cellular prion protein (PrP(C)) and decay-accelerating factor (DAF) are glycoproteins linked to the cell surface by glycosylphosphatidylinositol (GPI) anchors. Both PrP(C) and DAF reside in detergent insoluble complex that can be isolated from human peripheral blood mononuclear cells. However, these two GPI-anchored proteins possess different cell biological properties. The GPI anchor of DAF is markedly more sensitive to cleavage by phosphatidylinositol-specific phospholipase C (PI-PLC) than that of PrP(C). Conversely, PrP(C) has a shorter cell surface half-life than DAF, possibly due to the fact that PrP(C) but not DAF is shed from the cell surface. This is the first demonstration that on the surface of the same cell type two GPI-anchored proteins differ in their cell biological properties.

RNA Folding: Models and Perspectives

Current Opinion in Structural Biology. Jun, 2003  |  Pubmed ID: 12831881

Intrinsic events during RNA folding include conformational search and metal ion binding. Several experimentally testable models have been proposed to explain how large ribozymes accomplish folding. Future challenges include the validation of these models, and the correlation of experimental results and theoretical simulations.

Mid-infrared Spectroscopic Measurement of Ionic Dissociative Materials in the Metabolic Pathway

Applied Spectroscopy. Dec, 2003  |  Pubmed ID: 14686773

We determine the pH dependency of the mid-infrared spectra in aqueous solution of the organic dissociative materials in the metabolic pathway: saccharide phosphates (G6P, F6P), adenosine, and its phosphates (ATP, ADP, AMP). The series of molar absorbance spectra for these reagents were obtained in a pH range of about 2 to 11 with a Fourier transform infrared (FT-IR) spectrometer equipped with a horizontal diamond attenuated total reflection (ATR) sampling accessory. We also provide a method of infrared spectral extraction of ionic dissociative materials by performing a linear least-square fitting utilizing the formulas of ionic dissociation equilibrium shift, and we obtain the infrared spectrum of each ionic species of the dissociative materials: G6P-, G6P2-; F6P-, F6P2-; ATP2-, ATP3-, ATP4-; ADP-, ADP2-, ADP3-; AMP, AMP-, AMP2-; and adenosine+, adenosine0. The infrared spectral structure of each ionic species of the dissociative materials in the metabolic pathway are discussed. Additionally, the possibility for a quantification system of the concentrations of the organic dissociative materials in varying pH is suggested.

[The Surgical Treatment of Cancer in the Base of Tongue]

Lin Chuang Er Bi Yan Hou Ke Za Zhi = Journal of Clinical Otorhinolaryngology. Dec, 2003  |  Pubmed ID: 15017714

To seek for a better approach for the resection of carcinoma of the base of tongue.

Prion Protein is Ubiquitinated After Developing Protease Resistance in the Brains of Scrapie-infected Mice

The Journal of Pathology. May, 2004  |  Pubmed ID: 15095484

Although the key event in the pathology of prion diseases is thought to be the conversion of cellular prion protein (PrP(C)) to the protease-resistant scrapie species termed PrP(Sc), the factors that contribute to neurodegeneration in scrapie-infected animals are poorly understood. One probable determinant could be when the accumulation of PrP(Sc) in infected brain overwhelms the ubiquitin-proteasome system and triggers the degenerative cascade. In the present study, it was found that in mouse brains infected with the ME7 scrapie strain, the level of ubiquitin protein conjugates increased significantly at approximately 144 days post-infection (pi) when clinical signs first become apparent. This elevation correlated with the detection of protease-resistant PrP(Sc) and a decline in two endopeptidase activities associated with proteasome function. However, ubiquitination of PrP was only detected at the terminal stage, 3 weeks after the development of clinical symptoms (approximately 165 days pi). These results suggest that ubiquitination of PrP is a late event phenomenon and this conjugation occurs after the formation of protease-resistant PrP(Sc). Whether this post-translational modification and the impairment of proteasome function are pivotal events in the pathogenesis of prion diseases remains to be determined.

Minute Gastric Carcinoid Tumor with Regional Lymph Node Metastasis: a Case Report and Review of Literature

World Journal of Gastroenterology : WJG. Aug, 2004  |  Pubmed ID: 15285046

We have encountered an unusual case of gastric carcinoid tumor. Gastroscopic examination of this 32-year-old male patient showed a smooth protrusion at the greater curvature of the gastric body with a central depression, identified by subsequent biopsy as carcinoma. The patient had a normal serum gastrin level and was negative for anti-parietal cell antibody. Histological examination of the resected gastric tissues showed that the tumor was a carcinoid, 0.3 cm x 0.3 cm in size with only one regional lymph node metastasis. We reviewed the pathogenesis, clinical presentation, diagnosis and treatment of gastric carcinoids and raise the possibility of being a lymph vessel-related metastasis even for a minute carcinoid tumor. Sentinel lymph node biopsy is recommended for surgery of minute carcinoid tumors.

Epitope Scanning Reveals Gain and Loss of Strain Specific Antibody Binding Epitopes Associated with the Conversion of Normal Cellular Prion to Scrapie Prion

Journal of Neurochemistry. Sep, 2004  |  Pubmed ID: 15312175

We used anti-prion (PrP) monoclonal antibodies (Mabs) in different combinations to scan changes in the availability of antibody binding epitopes--using an epitope scanning assay--in brain homogenates from normal mice, and from mice infected with either ME7 or 139 A strains of infectious scrapie prion (PrPSc). In ME7-infected brains, the epitope detected by the Mab pair 8B4/8H4 is reduced, while the epitope detected by the Mab pair 8F9/11G5 is increased. Mab 8F9/11G5 detect a conformational epitope on PrPSc because the rise in Mab 8F9/11G5 binding is sensitive to a denaturing agent but resistant to proteinase K (PK). While the increase in Mab 8F9/11G5 binding correlates with the presence of PK-resistant PrP and clinical signs of infection, the reduction in Mab 8B4/8H4 binding is detected earlier. Fractionation of the ME7-infected brain homogenate in sucrose gradient revealed that the PrPSc species detected by the epitope scanning assay are heterogeneous in size, with a molecular mass of approximately > or = 2000-kDa. We also investigated whether these findings were applicable to two other strains of PrPSc, namely 87 V and 22 L. We found that the decrease in Mab 8B4/8H4 binding detected in ME7-infected brains was also detected in 87 V-infected brains but not in 22 L-infected brains. In contrast, the increase in Mab 8F9/11G5 binding detected in ME7- and 139 A-infected brains was also detected in 22 L-infected brains but not in 87 V-infected brains. Therefore, each prion strain has its unique conformation, and we can monitor the conversion of normal cellular prion (PrPC) to PrPSc based on the changes in the antibody binding patterns. The epitope can be decreased or increased, linear or conformational, detected late or early during infection, in a strain specific manner.

Basis for Structural Diversity in Homologous RNAs

Science (New York, N.Y.). Oct, 2004  |  Pubmed ID: 15459389

Large RNA molecules, such as ribozymes, fold with well-defined tertiary structures that are important for their activity. There are many instances of ribozymes with identical function but differences in their secondary structures, suggesting alternative tertiary folds. Here, we report a crystal structure of the 161-nucleotide specificity domain of an A-type ribonuclease P that differs in secondary and tertiary structure from the specificity domain of a B-type molecule. Despite the differences, the cores of the domains have similar three-dimensional structure. Remarkably, the similar geometry of the cores is stabilized by a different set of interactions involving distinct auxiliary elements.

Additional Bedtime H2-receptor Antagonist for the Control of Nocturnal Gastric Acid Breakthrough

Cochrane Database of Systematic Reviews (Online). 2004  |  Pubmed ID: 15495095

Nocturnal gastric acid breakthrough(NAB) is defined as intragastric pH<4 for more than one continuous hour overnight. Adding H2-receptor antagonists (H2RAs)at bedtime to high-dose proton pump inhibitors is likely to enhance nocturnal gastric pH control and decrease nocturnal gastric acid breakthrough.

Surface-modified Poly(methyl Methacrylate) Capillary Electrophoresis Microchips for Protein and Peptide Analysis

Analytical Chemistry. Dec, 2004  |  Pubmed ID: 15571346

Polymeric materials have emerged as appealing alternatives to conventional inorganic substrates for the fabrication of microscale analytical systems; however, native polymeric surfaces typically require covalent modification to ensure optimum biocompatibility. 2-Bromoisobutyryl bromide was immobilized on poly(methyl methacrylate) (PMMA) substrates activated using an oxygen plasma. Atom-transfer radical polymerization was then performed to graft poly(ethylene glycol) (PEG) on the PMMA surface. PMMA microcapillary electrophoresis (muCE) devices made with the covalently modified surfaces exhibited substantially reduced electroosmotic flow and nonspecific adsorption of proteins on microchannel surfaces. Experiments using fluorescein isothiocyanate-conjugated bovine serum albumin indicated that both column efficiency and migration time reproducibility were 1 order of magnitude better with derivatized compared to untreated PMMA muCE chips. Fast, reproducible, and efficient separations of proteins and peptides were demonstrated using the PEG-grafted PMMA muCE chips. All analyses were completed in less than 60 s, and separation efficiencies as high as 5.2 x10(4) plates for a 3.5-cm-long separation channel were obtained. These results demonstrate the general applicability of surface-grafted PMMA microdevices for a broad range of protein analyses.

Single-molecule Studies Highlight Conformational Heterogeneity in the Early Folding Steps of a Large Ribozyme

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2004  |  Pubmed ID: 14704266

The equilibrium folding of the catalytic domain of Bacillus subtilis RNase P RNA is investigated by single-molecule fluorescence resonance energy transfer (FRET). Previous ensemble studies of this 255-nucleotide ribozyme described the equilibrium folding with two transitions, U-to-I(eq)-to-N, and focused on the I(eq)-to-N transition. The present study focuses on the U-to-I(eq) transition. Comparative ensemble measurements of the ribozyme construct labeled with fluorescein at the 5' end and Cy3 at the 3' end show that modifications required for labeling do not interfere with folding and help to define the Mg(2+) concentration range for the U-to-I(eq) transition. Histogram analysis of the Mg(2+)-dependent single-molecule FRET efficiency reveals two previously undetermined folding intermediates. The single-molecule FRET trajectories exhibit non-two-state and nonergodic behaviors at intermediate Mg(2+) concentrations on the time scale of seconds. The trajectories at intermediate Mg(2+) concentrations are classified into five classes based on three FRET levels and their dynamics of interconversion within the measured time range. This heterogeneity, together with the observation of "nonsudden jump" FRET transitions, indicates that the early folding steps of this ribozyme involve a series of intermediates with different degrees of kinetic isolation and that folding occurs under kinetic control and involves many "local" conformational switches. A free energy contour is constructed to illustrate the complex folding surface.

Lymphatic Mapping and Sentinel Node Biopsy in Gastric Cancer

American Journal of Surgery. Feb, 2004  |  Pubmed ID: 14769318

To determine the feasibility and significance of lymphatic mapping and sentinel lymph node biopsy (SLNB) in patients with gastric cancer.

Interaction of the Bacillus Subtilis RNase P with the 30S Ribosomal Subunit

RNA (New York, N.Y.). Mar, 2004  |  Pubmed ID: 14970393

Ribonuclease P (RNase P) is a ribozyme required for the 5' maturation of all tRNA. RNase P and the ribosome are the only known ribozymes conserved in all organisms. We set out to determine whether this ribonucleoprotein enzyme interacts with other cellular components, which may imply other functions for this conserved ribozyme. Incubation of the Bacillus subtilis RNase P holoenzyme with fractionated B. subtilis cellular extracts and purified ribosomal subunits results in the formation of a gel-shifted complex with the 30S ribosomal subunit at a binding affinity of approximately 40 nM in 0.1 M NH(4)Cl and 10 mM MgCl(2). The complex does not form with the RNase P RNA alone and is disrupted by a mRNA mimic polyuridine, but is stable in the presence of high concentrations of mature tRNA. Endogenous RNase P can also be detected in the 30S ribosomal fraction. Cleavage of a pre-tRNA substrate by the RNase P holoenzyme remains the same in the presence of the 30S ribosome, but the cleavage of an artificial non-tRNA substrate is inhibited eightfold. Hydroxyl radical protection and chemical modification identify several protected residues located in a highly conserved region in the RNase P RNA. A single mutation within this region significantly reduces binding, providing strong support on the specificity of the RNase P-30S ribosome complex. Our results also suggest that the dimeric form of the RNase P is primarily involved in 30S ribosome binding. We discuss several models on a potential function of the RNase P-30S ribosome complex.

Fabrication of Calcium Fluoride Capillary Electrophoresis Microdevices for On-chip Infrared Detection

Journal of Chromatography. A. Feb, 2004  |  Pubmed ID: 14971507

In this paper, we demonstrate microfluidic capillary electrophoresis (CE) devices made in CaF2 , for optical detection in a broad spectral range. We have designed methods for micromachining and enclosing capillaries in CaF2. The utility of these microdevices has been shown through CE analysis of fluorescently labeled amino acids. We have also performed infrared spectroscopy for analyte identification in microfluidic CaF2 channels. These CaF2 microdevices open the door to microchip separations with optical detection in the ultraviolet, visible, and infrared spectral regions.

[Non-steroidal Anti-inflammatory Agents for Chemoprevention of Colorectal Polyps: a Meta-analysis]

Zhonghua Nei Ke Za Zhi [Chinese Journal of Internal Medicine]. Jan, 2004  |  Pubmed ID: 14990012

To assess whether or not non-steroidal anti-inflammatory agents (NSAIDs) might prevent colorectal polyps.

Exploring the Regulation of TRNA Distribution on the Genomic Scale

Journal of Molecular Biology. Mar, 2004  |  Pubmed ID: 15001350

Though up to 20% of the total RNA in bacterial cells is tRNA, the regulation of tRNA distribution on the genomic level remains unclear. tRNA distribution is governed by four processes: transcription, processing of precursor tRNA, degradation of precursor tRNA and degradation of mature tRNA. To elucidate the relationship between these processes in the regulation of tRNA production, the relative tRNA distribution was measured using a microarray specifically designed for tRNA. We developed a procedure that selectively labels 3'-CCA-containing RNAs with the fluorophores Cy3 or Cy5. The labeled tRNAs were then hybridized to microarrays printed with complementary DNA probes. The regulation of tRNA distribution in Bacillus subtilis was explored for a wild-type strain and a mutant strain with significantly decreased levels of RNase P, the enzyme required for the 5' maturation of all tRNA. The strains were either grown under a variety of conditions at doubling times ranging from 0.1 to 2.2 doublings per hour to investigate growth-related changes in the tRNA abundance or treated with the transcriptional inhibitor rifampicin to analyze mature tRNA degradation. Our results confirm that transcription and processing contribute significantly to the distribution of the 35 tRNA species in B.subtilis, and suggest a role for the degradation of precursor tRNA. Mature tRNA degradation occurs with little specificity for individual tRNA species and on the hour time-scale, indicating that degradation of mature tRNA plays only a minor role in the regulation of tRNA distribution. Aside from transcription, the final tRNA distribution appears to be derived from a balance between processing and precursor degradation activities.

A Study of the Lengthening and Contractility of the Surgical Margins in Digestive Tract Cancer

American Journal of Surgery. Mar, 2004  |  Pubmed ID: 15006583

The lengthening and the contractility of the digestive tract were studied.

Biochemical Fingerprints of Prion Diseases: Scrapie Prion Protein in Human Prion Diseases That Share Prion Genotype and Type

Journal of Neurochemistry. Jan, 2005  |  Pubmed ID: 15606903

The phenotype of human prion diseases is influenced by the prion protein (PrP) genotype as determined by the methionine (M)/valine (V) polymorphism at codon 129, the scrapie PrP (PrPSc) type and the etiology. To gain further insight into the mechanisms of phenotype determination, we compared two-dimensional immunoblot profiles of detergent insoluble and proteinase K-resistant PrP species in a type of sporadic Creutzfeldt-Jakob disease (sCJDMM2), variant CJD (vCJD) and sporadic fatal insomnia (sFI). Full-length and truncated PrP forms present in the insoluble fractions were also separately analyzed. These three diseases were selected because they have the same M/M PrP genotype at codon 129 and the same type 2 PrPSc, but different etiologies, also sCJDMM2 and sFI are sporadic, whereas vCJD is acquired by infection. We observed minor differences in the PrP detergent-insoluble fractions between sCJDMM2 and vCJD, although both differ in the corresponding fractions from sFI. We detected more substantial heterogeneity between sCJDMM2 and vCJD in the two-dimensional blots of the proteinase K-resistant PrP fraction suggesting that different PrP species are selected for conversion to proteinase K-resistant PrP in sCJDMM2 and vCJD. These differences are mostly, but not exclusively, due to variations in the type of the N-linked glycans. We also show that the over-representation of the highly glycosylated forms distinctive of the proteinase K-resistant PrPSc of vCJD in one-dimensional blots is due to differences in both the amount and the natures of the glycans. Overall, these findings underline the complexity of phenotypic determination in human prion diseases.

Biochemical Fingerprints of Prion Infection: Accumulations of Aberrant Full-length and N-terminally Truncated PrP Species Are Common Features in Mouse Prion Disease

Journal of Virology. Jan, 2005  |  Pubmed ID: 15613322

Infection with any one of three strains of mouse scrapie prion (PrPSc), 139A, ME7, or 22L, results in the accumulation of two underglycosylated, full-length PrP species and an N-terminally truncated PrP species that are not detectable in uninfected animals. The levels of the N-terminally truncated PrP species vary depending on PrPSc strain. Furthermore, 22L-infected brains consistently have the highest levels of proteinase K (PK)-resistant PrP species, followed by ME7- and 139A-infected brains. The three strains of PrPSc are equally susceptible to PK and proteases papain and chymotrypsin. Their protease resistance patterns are also similar. In sucrose gradient velocity sedimentation, the aberrant PrP species partition with PrPSc aggregates, indicating that they are physically associated with PrPSc. In ME7-infected animals, one of the underglycosylated, full-length PrP species is detected much earlier than the other, before both the onset of clinical disease and the detection of PK-resistant PrP species. In contrast, the appearance of the N-terminally truncated PrP species coincides with the presence of PK-resistant species and the manifestation of clinical symptoms. Therefore, accumulation of the underglycosylated, full-length PrP species is an early biochemical fingerprint of PrPSc infection. Accumulation of the underglycosylated, full-length PrP species and the aberrant N-terminally truncated PrP species may be important in the pathogenesis of prion disease.

Efficient Fluorescence Labeling of a Large RNA Through Oligonucleotide Hybridization

RNA (New York, N.Y.). Feb, 2005  |  Pubmed ID: 15613536

We present an efficient method of introducing fluorophore labels at selected locations in a large RNA. The method is based on specific and highly efficient hybridization between a fluorophore-containing DNA oligonucleotide and a modular hairpin loop replacing a functionally unimportant hairpin loop in the RNA. We demonstrate its feasibility using a 255-nucleotide RNA derived from the catalytic domain of RNase P from Bacillus subtilis. Hybridization of the DNA oligonucleotide to the modular hairpin loop minimally perturbs the structure and function of this RNA. This labeling scheme should be applicable in studies of RNA conformational dynamics by ensemble and single molecule fluorescence methods.

Selective Charging of TRNA Isoacceptors Induced by Amino-acid Starvation

EMBO Reports. Feb, 2005  |  Pubmed ID: 15678157

Aminoacylated (charged) transfer RNA isoacceptors read different messenger RNA codons for the same amino acid. The concentration of an isoacceptor and its charged fraction are principal determinants of the translation rate of its codons. A recent theoretical model predicts that amino-acid starvation results in 'selective charging' where the charging levels of some tRNA isoacceptors will be low and those of others will remain high. Here, we developed a microarray for the analysis of charged fractions of tRNAs and measured charging for all Escherichia coli tRNAs before and during leucine, threonine or arginine starvation. Before starvation, most tRNAs were fully charged. During starvation, the isoacceptors in the leucine, threonine or arginine families showed selective charging when cells were starved for their cognate amino acid, directly confirming the theoretical prediction. Codons read by isoacceptors that retain high charging can be used for efficient translation of genes that are essential during amino-acid starvation. Selective charging can explain anomalous patterns of codon usage in the genes for different families of proteins.

Novel Antibody-lectin Enzyme-linked Immunosorbent Assay That Distinguishes Prion Proteins in Sporadic and Variant Cases of Creutzfeldt-Jakob Disease

Journal of Clinical Microbiology. Mar, 2005  |  Pubmed ID: 15750071

We used different anti-prion protein (anti-PrP) monoclonal antibodies to capture either full-length or truncated PrP species and then used biotinylated lectin to compare the nature of the glycans on bound PrP species present in control, sporadic Creutzfeldt-Jakob disease (sCJD), or variant CJD (vCJD) brains. When full-length PrP species in these three groups were compared, no significant difference in the binding of concanavalin A or Aleuria aurantia lectin was detected. However, the binding of Ricinus communis agglutinin I (RCA) to sCJD and vCJD samples was significantly increased. In contrast, when only truncated PrP species were compared, only vCJD samples had more RCA binding activity. Therefore, while most of the RCA binding activity in sCJD is restricted to the full-length PrP species, the RCA binding activity in vCJD is associated with truncated and full-length PrP species. Furthermore, the RCA binding activity in sCJD and vCJD samples is mostly associated with proteinase K-resistant PrP species, a known signature of infectious prion. Therefore, PrP species in sCJD and vCJD have dissimilar lectin immunoreactivity, which reflects differences in their N-linked glycans. These differences may account for the distinct phenotypes of sCJD and vCJD.

Mechanistic Insights on the Folding of a Large Ribozyme During Transcription

Biochemistry. May, 2005  |  Pubmed ID: 15895996

RNA folding during transcription resembles folding in a cellular environment. We previously investigated the folding of a large ribozyme derived from a bacterial RNase P RNA during its transcription by the Escherichia coli RNA polymerase and the effect of the elongation factor NusA. We found that transcriptional pausing at a specific site induced by NusA significantly altered the folding pathway. In this work, we compare folding during transcription by the E. coli RNA polymerase of circularly permuted variants and site-specific mutants of the RNase P ribozyme to elucidate the molecular mechanism of transcriptional pausing and RNA folding. The effect of NusA-induced pausing depends on the order of RNA synthesis and only affects local folding of the RNA. Pausing likely prevents a misfolded structure between the 5' strand of a helix and its adjacent junction located in the specificity domain and a region known to bind single-stranded RNA located in the catalytic domain. These results lead to a structural model on how transcriptional pausing affects folding of RNase P RNA. Structural rearrangements of a nascent RNA transcript enhanced by transcriptional pausing may be a general feature of RNA folding during transcription.

Phase-changing Sacrificial Materials for Solvent Bonding of High-performance Polymeric Capillary Electrophoresis Microchips

Analytical Chemistry. Jun, 2005  |  Pubmed ID: 15924386

A new method for solvent bonding polymeric substrates to form microfluidic systems has been demonstrated. Prior to device sealing, channels in an embossed poly(methyl methacrylate) (PMMA) piece are filled with a heated liquid (paraffin wax) that forms a solid sacrificial layer at room temperature. The sacrificial material prevents the bonding solvent (acetonitrile) and softened PMMA from filling the channels. Once the sealing step is complete, the sacrificial layer is melted and removed, leaving enclosed microfluidic channels. We found that PMMA substrates welded together using this method could withstand internal pressures of >2250 psi, more than 1 order of magnitude higher than their thermally bonded counterparts. To demonstrate the usefulness of this method, microchip capillary electrophoresis (CE) devices in PMMA were created and tested. Amino acid and peptide mixtures were separated in <15 s, with >40,000 theoretical plates in a 2.5-cm separation distance. Electric fields as high as 1.5 kV/cm were applied in these microchips, and >300 CE runs were performed on a single device with no degradation of separation performance. The simplicity of the methods presented here and the improved robustness of the resulting devices should facilitate the broader implementation of polymer microchips in microfluidic analyses.

[The Changes of Fundamental Frequency and Formants of Vowel in Cochlear Implant Pre-lingual Children of Different Age]

Lin Chuang Er Bi Yan Hou Ke Za Zhi = Journal of Clinical Otorhinolaryngology. Feb, 2005  |  Pubmed ID: 15938202

To observe the changes of fundamental frequency and formants of vowel in cochlear implant pre-lingual children of different age and to provide the basis and direction for post-operative rehabilitation. To find the key period of language development from the aspect of phonetics and to illustrate the necessity of early implantation.

Real-time RNA Profiling Within a Single Bacterium

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2005  |  Pubmed ID: 15967986

Characterizing the dynamics of specific RNA levels requires real-time RNA profiling in a single cell. We show that the combination of a synthetic modular genetic system with fluorescence correlation spectroscopy allows us to directly measure in real time the activity of any specific promoter in prokaryotes. Using a simple inducible gene expression system, we found that induced RNA levels within a single bacterium of Escherichia coli exhibited a pulsating profile in response to a steady input of inducer. The genetic deletion of an efflux pump system, a key determinant of antibiotic resistance, altered the pulsating transcriptional dynamics and caused overexpression of induced RNA. In contrast with population measurements, real-time RNA profiling permits identifying relationships between genotypes and transcriptional dynamics that are accessible only at the level of the single cell.

[Voice Analysis in Pre-lingual Cochlear Implant Adults]

Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi = Chinese Journal of Otorhinolaryngology Head and Neck Surgery. Apr, 2005  |  Pubmed ID: 16008260

To observe voice characteristic of pre-lingual cochlear implant adults for cochlear implantation and phoniatrics.

Calorie Controlled Diet for Chronic Asthma

Cochrane Database of Systematic Reviews (Online). 2005  |  Pubmed ID: 16034941

The prevalence of asthma has increased in recent years. Epidemiological studies suggest a correlation between the onset of asthma and dietary nonallergic factors especially high calorie diet. These can be regarded as other potentially important risk factors.

Crystal Structure of the RNA Component of Bacterial Ribonuclease P

Nature. Sep, 2005  |  Pubmed ID: 16113684

Transfer RNA (tRNA) is produced as a precursor molecule that needs to be processed at its 3' and 5' ends. Ribonuclease P is the sole endonuclease responsible for processing the 5' end of tRNA by cleaving the precursor and leading to tRNA maturation. It was one of the first catalytic RNA molecules identified and consists of a single RNA component in all organisms and only one protein component in bacteria. It is a true multi-turnover ribozyme and one of only two ribozymes (the other being the ribosome) that are conserved in all kingdoms of life. Here we show the crystal structure at 3.85 A resolution of the RNA component of Thermotoga maritima ribonuclease P. The entire RNA catalytic component is revealed, as well as the arrangement of the two structural domains. The structure shows the general architecture of the RNA molecule, the inter- and intra-domain interactions, the location of the universally conserved regions, the regions involved in pre-tRNA recognition and the location of the active site. A model with bound tRNA is in agreement with all existing data and suggests the general basis for RNA-RNA recognition by this ribozyme.

Structure of a Folding Intermediate Reveals the Interplay Between Core and Peripheral Elements in RNA Folding

Journal of Molecular Biology. Sep, 2005  |  Pubmed ID: 16115647

Though the molecular architecture of many native RNA structures has been characterized, the structures of folding intermediates are poorly defined. Here, we present a nucleotide-level model of a highly structured equilibrium folding intermediate of the specificity domain of the Bacillus subtilis RNase P RNA, obtained using chemical and nuclease mapping, circular dichroism spectroscopy, small-angle X-ray scattering and molecular modeling. The crystal structure indicates that the 154 nucleotide specificity domain is composed of several secondary and tertiary structural modules. The structure of the intermediate contains modules composed of secondary structures and short-range tertiary interactions, implying a sequential order of tertiary structure formation during folding. The intermediate lacks the native core and several long-range interactions among peripheral regions, such as a GAAA tetraloop and its receptor. Folding to the native structure requires the local rearrangement of a T-loop in the core in concert with the formation of the GAAA tetraloop-receptor interaction. The interplay of core and peripheral structure formation rationalizes the high degree of cooperativity observed in the folding transition leading to the native structure.

[The Usage of the Whole Palate Flap in Maxillofacial Surgery]

Zhonghua Zheng Xing Wai Ke Za Zhi = Zhonghua Zhengxing Waike Zazhi = Chinese Journal of Plastic Surgery. May, 2005  |  Pubmed ID: 16128109

To study the clinical effects, the merits and shortcomings of the hard palate flap in repairing postoperative defects of oral soft tissue.

An Aggregation-specific Enzyme-linked Immunosorbent Assay: Detection of Conformational Differences Between Recombinant PrP Protein Dimers and PrP(Sc) Aggregates

Journal of Virology. Oct, 2005  |  Pubmed ID: 16160162

The conversion of the normal cellular prion protein, PrP(C), into the protease-resistant, scrapie PrP(Sc) aggregate is the cause of prion diseases. We developed a novel enzyme-linked immunosorbent assay (ELISA) that is specific for PrP aggregate by screening 30 anti-PrP monoclonal antibodies (MAbs) for their ability to react with recombinant mouse, ovine, bovine, or human PrP dimers. One MAb that reacts with all four recombinant PrP dimers also reacts with PrP(Sc) aggregates in ME7-, 139A-, or 22L-infected mouse brains. The PrP(Sc) aggregate is proteinase K resistant, has a mass of 2,000 kDa or more, and is present at a time when no protease-resistant PrP is detectable. This simple and sensitive assay provides the basis for the development of a diagnostic test for prion diseases in other species. Finally, the principle of the aggregate-specific ELISA we have developed may be applicable to other diseases caused by abnormal protein aggregation, such as Alzheimer's disease or Parkinson's disease.

[A Clinical Analysis of Intractable Spontaneous Epistaxis with 289 Cases Reviewed]

Lin Chuang Er Bi Yan Hou Ke Za Zhi = Journal of Clinical Otorhinolaryngology. Jan, 2006  |  Pubmed ID: 16570815

To explore the clinical characteristics and the prevention strategy of intractable spontaneous epistaxis.

Structural Basis for Altering the Stability of Homologous RNAs from a Mesophilic and a Thermophilic Bacterium

RNA (New York, N.Y.). Apr, 2006  |  Pubmed ID: 16581805

Tertiary RNA structures from thermophilic bacteria generally are more stable than their mesophilic homologs. To understand the structural basis of the increase in stability, we investigated equilibrium folding of the specificity domain (S-domain) of RNase P RNA from a mesophilic (Escherichia coli) and a thermophilic (Thermus thermophilus) bacterium. Equilibrium folding of both S-domains is described by a minimal, three-state folding scheme, U-to-I-to-N. In the I-to-N transition of the thermophilic S-domain, more structure forms and protections are stronger against T1 nuclease and hydroxyl radical reactions. Phylogenetic comparison in the context of the native structure reveals that among 39 nucleotide differences between these S-domains, 12 likely contribute to higher stability. These residues participate in extensive networks of hydrogen bonding, stacking, and metal ion coordination throughout the molecule. The thermophilic S-domain achieves higher stability by mutating strategic base pairs to G-C, decreasing surface accessibility of the native state, and increasing the amount of structure formation in the native folding transition. An E. coli S-domain mutant containing these 12 nt has the same stability and folding cooperativity as the T. thermophilus S-domain. E. coli S-domain mutants containing a subset of 4 or 6 nt have the same stability as the T. thermophilus S-domain but the same folding cooperativity as the E. coli S-domain. These results show that increasing stability can be accomplished by mutations within a local structure, but increasing folding cooperativity needs concerted changes among multiple structural units.

Structure of Ribonuclease P--a Universal Ribozyme

Current Opinion in Structural Biology. Jun, 2006  |  Pubmed ID: 16650980

Ribonuclease P (RNase P) is one of only two known universal ribozymes and was one of the first ribozymes to be discovered. It is involved in RNA processing, in particular the 5' maturation of tRNA. Unlike most other natural ribozymes, it recognizes and cleaves its substrate in trans. RNase P is a ribonucleoprotein complex containing one RNA subunit and as few as one protein subunit. It has been shown that, in bacteria and in some archaea, the RNA subunit alone can support catalysis. The structure and function of bacterial RNase P RNA have been studied extensively, but the detailed catalytic mechanism is not yet fully understood. Recently, structures of one of the structural domains and of the entire RNA component of RNase P from two different bacteria have been described. These structures provide the first atomic-level information on the structural assembly of the RNA component, and the regions involved in substrate recognition and catalysis. Comparison of these structures reveals a highly conserved core that comprises two universally conserved structural modules. Interestingly, the same structural core can be found in the context of different scaffolds.

RNA Folding During Transcription

Annual Review of Biophysics and Biomolecular Structure. 2006  |  Pubmed ID: 16689632

The evolution of RNA sequence needs to satisfy three requirements: folding, structure, and function. Studies on folding during transcription are related directly to folding in the cell. Understanding RNA folding during transcription requires the elucidation of structure formation and structural changes of the RNA, and the consideration of intrinsic properties of the RNA polymerase and other proteins that interact with the RNA. This review summarizes the research progress in this area and outlines the enormous challenges facing this field. Significant advancement requires the development of new experimental methods and theoretical considerations in all aspects of transcription and RNA folding.

Additional Bedtime H2-receptor Antagonist for the Control of Nocturnal Gastric Acid Breakthrough: a Cochrane Systematic Review

Chinese Journal of Digestive Diseases. 2006  |  Pubmed ID: 16808794

To assess the effectiveness and safety of additional bedtime H(2)-receptor antagonists (H(2)RAs) in suppressing nocturnal gastric acid breakthrough (NAB) via a systematic review.

A Systematic, Ligation-based Approach to Study RNA Modifications

RNA (New York, N.Y.). Nov, 2006  |  Pubmed ID: 16963711

Over 100 different chemical types of modifications have been identified in thousands of sites in tRNAs, rRNAs, mRNAs, small nuclear RNAs, and other RNAs. Some modifications are highly conserved, while others are more specialized. They include methylation of bases and the ribose backbone, rotation, and reduction of uridine, base deamination, elaborate addition of ring structures, carbohydrate moieties, and more. We have developed a systematic approach to detect and quantify the extent of known RNA modifications. The method is based on the enzymatic ligation of oligonucleotides using the modified or unmodified RNA as the template. The efficiency of ligation is very sensitive to the presence and the type of modifications. First, two oligo pairs for each type of modification are identified. One pair greatly prefers ligation using the unmodified RNA template over the modified RNA template or vice versa. The other pair has equal reactivity with unmodified and modified RNA. Second, separate ligations with each of the two oligo pairs and the total RNA mixture are performed to detect the presence or absence of modifications. Multiple modification sites can be examined in the same ligation reaction. The feasibility of this method is demonstrated for three 2'O-methyl modification sites in yeast rRNA.

Diversity of TRNA Genes in Eukaryotes

Nucleic Acids Research. 2006  |  Pubmed ID: 17088292

We compare the diversity of chromosomal-encoded transfer RNA (tRNA) genes from 11 eukaryotes as identified by tRNAScan-SE of their respective genomes. They include the budding and fission yeast, worm, fruit fly, fugu, chicken, dog, rat, mouse, chimp and human. The number of tRNA genes are between 170 and 570 and the number of tRNA isoacceptors range from 41 to 55. Unexpectedly, the number of tRNA genes having the same anticodon but different sequences elsewhere in the tRNA body (defined here as tRNA isodecoder genes) varies significantly (10-246). tRNA isodecoder genes allow up to 274 different tRNA species to be produced from 446 genes in humans, but only up to 51 from 275 genes in the budding yeast. The fraction of tRNA isodecoder genes among all tRNA genes increases across the phylogenetic spectrum. A large number of sequence differences in human tRNA isodecoder genes occurs in the internal promoter regions for RNA polymerase III. We also describe a systematic, ligation-based method to detect and quantify tRNA isodecoder molecules in human samples, and show differential expression of three tRNA isodecoders in six human tissues. The large number of tRNA isodecoder genes in eukaryotes suggests that tRNA function may be more diverse than previously appreciated.

Tissue-specific Differences in Human Transfer RNA Expression

PLoS Genetics. Dec, 2006  |  Pubmed ID: 17194224

Over 450 transfer RNA (tRNA) genes have been annotated in the human genome. Reliable quantitation of tRNA levels in human samples using microarray methods presents a technical challenge. We have developed a microarray method to quantify tRNAs based on a fluorescent dye-labeling technique. The first-generation tRNA microarray consists of 42 probes for nuclear encoded tRNAs and 21 probes for mitochondrial encoded tRNAs. These probes cover tRNAs for all 20 amino acids and 11 isoacceptor families. Using this array, we report that the amounts of tRNA within the total cellular RNA vary widely among eight different human tissues. The brain expresses higher overall levels of nuclear encoded tRNAs than every tissue examined but one and higher levels of mitochondrial encoded tRNAs than every tissue examined. We found tissue-specific differences in the expression of individual tRNA species, and tRNAs decoding amino acids with similar chemical properties exhibited coordinated expression in distinct tissue types. Relative tRNA abundance exhibits a statistically significant correlation to the codon usage of a collection of highly expressed, tissue-specific genes in a subset of tissues or tRNA isoacceptors. Our findings demonstrate the existence of tissue-specific expression of tRNA species that strongly implicates a role for tRNA heterogeneity in regulating translation and possibly additional processes in vertebrate organisms.

Different Criteria for Radioactive Sentinel Lymph Nodes Has Different Impact on Sentinel Node Biopsy in Breast Cancer Patients

Journal of Surgical Oncology. Jun, 2007  |  Pubmed ID: 17252554

This study set out to determine the impact of different criteria for radioactive sentinel lymph nodes (SLNs) on sentinel lymph node biopsy (SLNB), and the optimal criteria for radioactive SLNs.

Normal Cellular Prion Protein is a Ligand of Selectins: Binding Requires Le(X) but is Inhibited by SLe(X)

The Biochemical Journal. Sep, 2007  |  Pubmed ID: 17497959

The normal PrP(C) (cellular prion protein) contains sLe(X) [sialyl-Le(X) (Lewis X)] and Le(X). sLe(X) is a ligand of selectins. To examine whether PrP(C) is a ligand of selectins, we generated three human PrP(C)-Ig fusion proteins: one with Le(X), one with sLe(X), and the other with neither Le(X) nor sLe(X). Only Le(X)-PrP(C)-Ig binds E-, L- and P-selectins. Binding is Ca(2+)-dependent and occurs with nanomolar affinity. Removal of sialic acid on sLe(X)-PrP(C)-Ig enables the fusion protein to bind all selectins. These findings were confirmed with brain-derived PrP(C). The selectins precipitated PrP(C) in human brain in a Ca(2+)-dependent manner. Treatment of brain homogenates with neuraminidase increased the amounts of PrP(C) precipitated. Therefore the presence of sialic acid prevents the binding of PrP(C) in human brain to selectins. Hence, human brain PrP(C) interacts with selectins in a manner that is distinct from interactions in peripheral tissues. Alternations in these interactions may have pathological consequences.

In-channel Atom-transfer Radical Polymerization of Thermoset Polyester Microfluidic Devices for Bioanalytical Applications

Electrophoresis. Aug, 2007  |  Pubmed ID: 17640094

A new technique for polymer microchannel surface modification, called in-channel atom-transfer radical polymerization, has been developed and applied in the surface derivatization of thermoset polyester (TPE) microdevices with poly(ethylene glycol) (PEG). X-ray photoelectron spectroscopy, electroosmotic flow (EOF), and contact angle measurements indicate that PEG has been grafted on the TPE surface. Moreover, PEG-modified microchannels have much lower and more pH-stable EOF, more hydrophilic surfaces and reduced nonspecific protein adsorption. Capillary electrophoresis separation of amino acid and peptide mixtures in these PEG-modified TPE microchips had good reproducibility. Phosducin-like protein and phosphorylated phosducin-like protein were also separated to measure the phosphorylation efficiency. Our results indicate that PEG-grafted TPE microchips have broad potential application in biomolecular analysis.

Detection of Misfolded Prion Protein in Blood with Conformationally Sensitive Peptides

Transfusion. Aug, 2007  |  Pubmed ID: 17655586

The long-standing goal of a preclinical diagnostic test for transmissible spongiform encephalopathy (TSE) has recently become urgent because of the discovery that humans with variant Creutzfeldt-Jakob disease can transmit disease via blood transfusions.

Recombinant Anti-HBsAg Fab Blocks Hepatitis B Virus Infection After Orthotopic Liver Transplantation

Hepatobiliary & Pancreatic Diseases International : HBPD INT. Aug, 2007  |  Pubmed ID: 17690031

Recurrence of hepatitis B virus (HBV) after orthotopic liver transplantation is very common in the absence of adequate prophylaxis and is often associated with poor prognosis because of the development of cirrhosis, fibrosing cholestatic hepatitis, or fulminant hepatitis. Therefore it is important to study the prevention of HBV reinfection after liver transplantation.

Identification of Recognition Residues for Ligation-based Detection and Quantitation of Pseudouridine and N6-methyladenosine

Nucleic Acids Research. 2007  |  Pubmed ID: 17881375

Over 100 chemical types of RNA modifications have been identified in thousands of sites in all three domains of life. Recent data suggest that modifications function synergistically to mediate biological function, and that cells may coordinately modulate modification levels for regulatory purposes. However, this area of RNA biology remains largely unexplored due to the lack of robust, high-throughput methods to quantify the extent of modification at specific sites. Recently, we developed a facile enzymatic ligation-based method for detection and quantitation of methylated 2'-hydroxyl groups within RNA. Here we exploit the principles of molecular recognition and nucleic acid chemistry to establish the experimental parameters for ligation-based detection and quantitation of pseudouridine (Psi) and N6-methyladenosine (m6A), two abundant modifications in eukaryotic rRNA/tRNA and mRNA, respectively. Detection of pseudouridylation at several sites in the large subunit rRNA derived from yeast demonstrates the feasibility of the approach for analysis of pseudouridylation in biological RNA samples.

Folding of a Universal Ribozyme: the Ribonuclease P RNA

Quarterly Reviews of Biophysics. May, 2007  |  Pubmed ID: 17931443

Ribonuclease P is among the first ribozymes discovered, and is the only ubiquitously occurring ribozyme besides the ribosome. The bacterial RNase P RNA is catalytically active without its protein subunit and has been studied for over two decades as a model system for RNA catalysis, structure and folding. This review focuses on the thermodynamic, kinetic and structural frameworks derived from the folding studies of bacterial RNase P RNA.

Folding of Noncoding RNAs During Transcription Facilitated by Pausing-induced Nonnative Structures

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2007  |  Pubmed ID: 17986617

RNA folding in the cell occurs during transcription. Expedient RNA folding must avoid the formation of undesirable structures as the nascent RNA emerges from the RNA polymerase. We show that efficient folding during transcription of three conserved noncoding RNAs from Escherichia coli, RNase P RNA, signal-recognition particle RNA, and tmRNA is facilitated by their cognate polymerase pausing at specific locations. These pause sites are located between the upstream and downstream portions of all of the native long-range helices in these noncoding RNAs. In the paused complexes, the nascent RNAs form labile structures that sequester these upstream portions in a manner to possibly guide folding. Both the pause sites and the secondary structure of the nonnative portions of the paused complexes are phylogenetically conserved among gamma-proteobacteria. We propose that specific pausing-induced structural formation is a general strategy to facilitate the folding of long-range helices. This polymerase-based mechanism may result in portions of noncoding RNA sequences being evolutionarily conserved for efficient folding during transcription.

[Development and Clinical Application of an Intramedullary Controlled Dynamic Nailing]

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi = Zhongguo Xiufu Chongjian Waike Zazhi = Chinese Journal of Reparative and Reconstructive Surgery. Dec, 2007  |  Pubmed ID: 18277685

To describe the design and application of a new intramedullary controlled dynamic nailing (ICDN).

[A Patient with Secretory Otitis Media Treated with Cochlear Implant and Canal Wall Removal and Reconstruction]

Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi = Chinese Journal of Otorhinolaryngology Head and Neck Surgery. Dec, 2007  |  Pubmed ID: 18335762

Test for Detection of Disease-associated Prion Aggregate in the Blood of Infected but Asymptomatic Animals

Clinical and Vaccine Immunology : CVI. Jan, 2007  |  Pubmed ID: 17079434

We have developed a sensitive in vitro assay for detecting disease-associated prion aggregates by combining an aggregation-specific enzyme-linked immunosorbent assay (AS-ELISA) with the fluorescent amplification catalyzed by T7 RNA polymerase technique (FACTT). The new assay, named aggregation-specific FACTT (AS-FACTT), is much more sensitive than AS-ELISA and could detect prion aggregates in the brain of mice as early as 7 days after an intraperitoneal inoculation of PrP(Sc). However, AS-FACTT was still unable to detect prion aggregates in blood of infected mice. To further improve the detection limit of AS-FACTT, we added an additional prion amplification step (Am) and developed a third-generation assay, termed Am-A-FACTT. Am-A-FACTT has 100% sensitivity and specificity in detecting disease-associated prion aggregates in blood of infected mice at late but still asymptomatic stages of disease. At a very early stage, Am-A-FACTT had a sensitivity of 50% and a specificity of 100%. Most importantly, Am-A-FACTT also detects prion aggregates in blood of mule deer infected with the agent causing a naturally occurring prion disease, chronic wasting disease. Application of this assay to cattle, sheep, and humans could safeguard food supplies and prevent human contagion.

Identifying Tinnitus Subgroups with Cluster Analysis

American Journal of Audiology. Dec, 2008  |  Pubmed ID: 19056922

We believe it is important to uncover tinnitus subgroups to identify subsets of patients most likely to benefit from different treatments. We review strategies for subgrouping based on etiology, subjective reports, the audiogram, psychoacoustics, imaging, and cluster analysis.

[A New Type Water Supplement Mode of Urban Wetland Park and Its Effects in Purification and Ecology]

Ying Yong Sheng Tai Xue Bao = The Journal of Applied Ecology / Zhongguo Sheng Tai Xue Xue Hui, Zhongguo Ke Xue Yuan Shenyang Ying Yong Sheng Tai Yan Jiu Suo Zhu Ban. Dec, 2008  |  Pubmed ID: 19288726

With the Rosebush Wetland Park in Changzhou as a case, a new type water supplement mode for urban wetland park, i.e., "vertical-flow plus horizontal-flow", was constructed, and its effects in water purification, ecology, and economic advantages were evaluated. The results showed that this water supplement mode could not only improve the landscape of the water bodies in urban wetland park, but also enhance their quality, making it satisfy the requirement for human full-body exposure. Furthermore, the operation cost of the mode was as lower as 5%-25% of direct municipal pipe-water supply and other routine technique solutions, suggesting that this water supplement mode had potential positive ecological effects and economic advantages.

A New Intramedullary Nailing Device for the Treatment of Femoral Shaft Fractures: a Biomechanical Study

Clinical Biomechanics (Bristol, Avon). Mar, 2008  |  Pubmed ID: 18079030

The treatment of choice for early mobilization of femoral fractures is surgery, which traditionally employs plates and screws or intramedullary nails. We examined the biomechanical properties of a new femoral nail system. The new intramedullary propping nailing system, made of a stainless-steel alloy, consists of a nail shaft, inner rod, tensile screw, end cape and two interlocked screws.

A Large Collapsed-state RNA Can Exhibit Simple Exponential Single-molecule Dynamics

Journal of Molecular Biology. May, 2008  |  Pubmed ID: 18402978

The process of large RNA folding is believed to proceed from many collapsed structures to a unique functional structure requiring precise organization of nucleotides. The diversity of possible structures and stabilities of large RNAs could result in non-exponential folding kinetics (e.g. stretched exponential) under conditions where the molecules have not achieved their native state. We describe a single-molecule fluorescence resonance energy transfer (FRET) study of the collapsed-state region of the free energy landscape of the catalytic domain of RNase P RNA from Bacillus stearothermophilus (C(thermo)). Ensemble measurements have shown that this 260 residue RNA folds cooperatively to its native state at >or=1 mM Mg(2+), but little is known about the conformational dynamics at lower ionic strength. Our measurements of equilibrium conformational fluctuations reveal simple exponential kinetics that reflect a small number of discrete states instead of the expected inhomogeneous dynamics. The distribution of discrete dwell times, collected from an "ensemble" of 300 single molecules at each of a series of Mg(2+) concentrations, fit well to a double exponential, which indicates that the RNA conformational changes can be described as a four-state system. This finding is somewhat unexpected under [Mg(2+)] conditions in which this RNA does not achieve its native state. Observation of discrete well-defined conformations in this large RNA that are stable on the seconds timescale at low [Mg(2+)] (<0.1 mM) suggests that even at low ionic strength, with a tremendous number of possible (weak) interactions, a few critical interactions may produce deep energy wells that allow for rapid averaging of motions within each well, and yield kinetics that are relatively simple.

Single-molecule Nonequilibrium Periodic Mg2+-concentration Jump Experiments Reveal Details of the Early Folding Pathways of a Large RNA

Proceedings of the National Academy of Sciences of the United States of America. May, 2008  |  Pubmed ID: 18448679

The evolution of RNA conformation with Mg(2+) concentration ([Mg(2+)]) is typically determined from equilibrium titration measurements or nonequilibrium single [Mg(2+)]-jump measurements. We study the folding of single RNA molecules in response to a series of periodic [Mg(2+)] jumps. The 260-residue catalytic domain of RNase P RNA from Bacillus stearothermophilus is immobilized in a microfluidic flow chamber, and the RNA conformational changes are probed by fluorescence resonance energy transfer (FRET). The kinetics of population redistribution after a [Mg(2+)] jump and the observed connectivity of FRET states reveal details of the folding pathway that complement and transcend information from equilibrium or single-jump measurements. FRET trajectories for jumps from [Mg(2+)] = 0.01 to 0.1 mM exhibit two-state behavior whereas jumps from 0.01 mM to 0.4 mM exhibit two-state unfolding but multistate folding behavior. RNA molecules in the low and high FRET states before the [Mg(2+)] increase are observed to undergo dynamics in two distinct regions of the free energy landscape separated by a high barrier. We describe the RNA structural changes involved in crossing this barrier as a "hidden" degree of freedom because the changes do not alter the detected FRET value but do alter the observed dynamics. The associated memory prevents the populations from achieving their equilibrium values at the end of the 5- to 10-sec [Mg(2+)] interval, thereby creating a nonequilibrium steady-state condition. The capability of interrogating nonequilibrium steady-state RNA conformations and the adjustable period of [Mg(2+)]-jump cycles makes it possible to probe regions of the free energy landscape that are infrequently sampled in equilibrium or single-jump measurements.

Affinity Monolith-integrated Poly(methyl Methacrylate) Microchips for On-line Protein Extraction and Capillary Electrophoresis

Analytical Chemistry. Jul, 2008  |  Pubmed ID: 18479142

Immunoaffinity monolith pretreatment columns have been coupled with capillary electrophoresis separation in poly(methyl methacrylate) (PMMA) microchips. Microdevices were designed with eight reservoirs to enable the electrically controlled transport of selected analytes and solutions to carry out integrated immunoaffinity extraction and electrophoretic separation. The PMMA microdevices were fabricated reproducibly and with high fidelity by solvent imprinting and thermal bonding methods. Monoliths with epoxy groups for antibody immobilization were prepared by direct in situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in a porogenic solvent consisting of 70% 1-dodecanol and 30% cyclohexanol. Antifluorescein isothiocyanate was utilized as a model affinity group in the monoliths, and the immobilization process was optimized. A mean elution efficiency of 92% was achieved for the monolith-based extraction of fluorescein isothiocyanate (FITC)-tagged human serum albumin. FITC-tagged proteins were purified from a contaminant protein and then separated electrophoretically using these devices. The developed immunoaffinity column/capillary electrophoresis microdevices show great promise for combining sample pretreatment and separation in biomolecular analysis.

[Biomechanical Test of Intramedullary Controlled Dynamic Nailing]

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi = Zhongguo Xiufu Chongjian Waike Zazhi = Chinese Journal of Reparative and Reconstructive Surgery. Jun, 2008  |  Pubmed ID: 18630568

To explore the biomechanical properties of a new intramedullary controlled dynamic nailing (ICDN).

Affinity Monolith Preconcentrators for Polymer Microchip Capillary Electrophoresis

Electrophoresis. Aug, 2008  |  Pubmed ID: 18702050

Developments in biology are increasing demands for rapid, inexpensive, and sensitive biomolecular analysis. In this study, polymer microdevices with monolithic columns and electrophoretic channels were used for biological separations. Glycidyl methacrylate-co-ethylene dimethacrylate monolithic columns were formed within poly(methyl methacrylate) microchannels by in situ photopolymerization. Flow experiments in these columns demonstrated retention and then elution of amino acids under conditions optimized for sample preconcentration. To enhance analyte selectivity, antibodies were immobilized on monoliths, and subsequent lysozyme treatment blocked nonspecific adsorption. The enrichment capability and selectivity of these affinity monoliths were evaluated by purifying fluorescently tagged amino acids from a mixture containing green fluorescent protein (GFP). Twenty-fold enrichment and 91% recovery were achieved for the labeled amino acids, with a >25 000-fold reduction in GFP concentration, as indicated by microchip electrophoresis analysis. These devices should provide a simple, inexpensive, and effective platform for trace analysis in complex biological samples.

Immunization with Plasmid DNA Encoding Influenza A Virus Nucleoprotein Fused to a Tissue Plasminogen Activator Signal Sequence Elicits Strong Immune Responses and Protection Against H5N1 Challenge in Mice

Journal of Virological Methods. Dec, 2008  |  Pubmed ID: 18789973

DNA vaccination is an effective means of eliciting both humoral and cellular immunity. Most of influenza vaccines targeted at hemagglutinin (HA) show efficient immunogenicity for protecting subjects against influenza virus infection. However, major antigenic variations of HA may facilitate the virus in developing resistance against such vaccines. DNA vaccines encoding conserved antigens protect animals against diverse viral subtypes, but their potency requires further improvement. In the present study, a DNA vaccine encoding the conserved nucleoprotein (NP) with a tissue plasminogen activator (tPA) signal sequence (ptPAs/NP) was generated, and immune responses were examined in vaccinated mice. A higher level of NP expression and secretion was observed in lysates and supernatants of the cells transfected with ptPAs/NP when compared to a plasmid encoding the wild-type full-length NP (pflNP). Immunofluorescence studies showed the cytoplasmic localization of the NP protein expressed from ptPAs/NP, but not from pflNP. In mice, the ptPAs/NP vaccine elicited higher levels of the NP-specific IgG and CD8(+) T cell-stimulating responses than that of pflNP. Vaccination with ptPAs/NP efficiently cleared the homologous H5N1 influenza virus in the infected lungs and induced partial cross-protection against heterologous, highly pathogenic H5N1 strains in mice. Our results may contribute to the development of protective immunity against diverse, highly pathogenic H5N1 virus subtypes.

A Novel Approach of Prophylaxis to HBV Recurrence After Liver Transplantation

Virology. Dec, 2008  |  Pubmed ID: 18945464

Liver transplantation (LT) in patients with hepatitis B virus (HBV) infection is associated with a high rate of graft loss and poor survival, unless re-infection can be prevented. Human hepatitis B immune globulin (HBIG) and nucleoside analogues (NA) have long been utilized to prevent re-infection. Previously, we generated a human monoclonal antibody (mAb), HB that recognizes the surface antigen of hepatitis B virus (HBV). We have constructed a secreted version of HB and cloned its genes into recombinant adeno-associated virus (AAV). We compared the efficiency of AAV vector after a single injection via intramuscular or intravenous routes without additional intervention. Then, we evaluated the activity of antibody HB in tree shrews treated with rAAV-HB and in vitro experiments. So, intramuscular injection of rAAV-HB was a suitable method for the immunoprophylaxis of HBV infection. This human antibody will be useful for the immunoprophylaxis of HBV infection.

Electrical Stimulation of the Cochlea to Reduce Tinnitus

Seminars in Hearing. Nov, 2008  |  Pubmed ID: 20333263

This article reviews possible neural correlates of tinnitus, including an increase in rate, a decrease in rate, periodic activity, synchronous activity across neurons, and an edge between active and inactive neurons. We make some suggestions regarding how electrical current might alter these patterns of neural activity. For example, if tinnitus were represented with periodic neural activity, then electrical stimulation would need to disrupt this periodicity. Some cases of cochlear electrical stimulation are reviewed that show the tinnitus can be reduced or eliminated with cochlear electrical stimulation although it varies across individuals. Finally, after summarizing some key observations, we suggest some next steps to bring this into a clinical application.

Aqua-bis(2,2'-bipyridine-κN,N')(1H-indole-2-carboxyl-ato-κO)nickel(II) 1H-indole-2-carboxyl-ate Dihydrate

Acta Crystallographica. Section E, Structure Reports Online. 2008  |  Pubmed ID: 21581519

The hydro-thermal reaction of Ni(2)(OH)(2)CO(3) with 2,2'-bipyridine and 2-indolyl-formic acid in CH(3)OH/H(2)O at 423 K for 7 d produced the novel Ni(II) complex [Ni(C(9)H(6)NO(2))(C(10)H(8)N(2))(2)(H(2)O)](C(9)H(6)NO(2))·2H(2)O. The asymmetric unit of the title compound consists of a monovalent [Ni(L)(bpy)(2)(H(2)O)](+) cation (bpy is 2,2'-bipyridine and L is 1H-indole-2-carboxyl-ate), an L anion and two solvent water mol-ecules. In the [Ni(L)(bpy)(2)(H(2)O)](+) cations, the Ni atom coordinates to four N atoms from the two bpy ligands and two O atoms, one from a L anion and the other from a water mol-ecule to complete an significantly distorted NiN(4)O(2) octa-hedron. The coordinated and solvate water mol-ecules form an extensive series of O-H⋯O hydrogen bonds. N-H⋯O and C-H⋯O hydrogen bonds are also present and the mol-ecules are inter-linked, forming a three-dimensional network.

RNA Folding During Transcription: Protocols and Studies

Methods in Enzymology. 2009  |  Pubmed ID: 20946770

RNA folds during transcription in the cell. Compared to most in vitro studies where the focus is generally on Mg(2+)-initiated refolding of fully synthesized transcripts, cotranscriptional RNA folding studies better replicate how RNA folds in a cellular environment. Unique aspects of cotranscriptional folding include the 5'- to 3'-polarity of RNA, the transcriptional speed, pausing properties of the RNA polymerase, the effect of the transcriptional complex and associated factors, and the effect of RNA-binding proteins. Identifying strategic pause sites can reveal insights on the folding pathway of the nascent transcript. Structural mapping of the paused transcription complexes identifies important folding intermediates along these pathways. Oligohybridization assays and the appearance of the catalytic activity of a ribozyme either in trans or in cis can be used to monitor cotranscriptional folding under a wide range of conditions. In our laboratory, these methodologies have been applied to study the folding of three highly conserved RNAs (RNase P, SRP, and tmRNA), several circularly permuted forms of a bacterial RNase P RNA, a riboswitch (thiM), and an aptamer-activated ribozyme (glmS).

Analysis of Synonymous Codon Usage in Classical Swine Fever Virus

Virus Genes. Feb, 2009  |  Pubmed ID: 18958611

Using the complete genome sequences of 35 classical swine fever viruses (CSFV) representing all three genotypes and all three kinds of virulence, we analyzed synonymous codon usage and the relative dinucleotide abundance in CSFV. The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in CSFV. Furthermore, we observed that the relative abundance of dinucleotides in CSFV is independent of the overall base composition but is still the result of differential mutational pressure, which also shapes codon usage. In addition, other factors, such as the subgenotypes and aromaticity, also influence the codon usage variation among the genomes of CSFV. This study represents the most comprehensive analysis to date of CSFV codon usage patterns and provides a basic understanding of the mechanisms for codon usage bias.

High Levels of TRNA Abundance and Alteration of TRNA Charging by Bortezomib in Multiple Myeloma

Biochemical and Biophysical Research Communications. Jul, 2009  |  Pubmed ID: 19450555

In multiple myeloma (MM), malignant plasma cells produce large amounts of antibodies and have highly active protein translational machinery. It is not known whether regulation of the abundance and aminoacylation (charging) of transfer RNA (tRNA) takes place in myeloma cells to accommodate for the increased amount of protein translation. Using tRNA-specific microarrays, we demonstrate that tRNA levels are significantly elevated in MM cell lines compared to normal bone marrow cells. We furthermore show that the addition of the proteasome inhibitor, bortezomib (Velcade, PS-341) results in decreased charging levels of tRNAs, in particular those coding for hydrophobic amino acids. These results suggest that tRNA properties are altered in MM to accommodate for its increased need for protein translation, and that proteasome inhibition directly impacts protein synthesis in MM through effects on tRNA charging.

Effect of Mn-Zn Ferrite on Apatite-wollastonite Glass-ceramic (A-W GC)

Biomedical Materials (Bristol, England). Aug, 2009  |  Pubmed ID: 19525575

Magnetic bioactive glass-ceramics (M GC) were prepared by doping apatite-wollastonite glass-ceramic (A-W GC) with Mn-Zn ferrite. The effect of different contents of Mn-Zn ferrite on the phase structure, magnetic property and bioactivity of A-W GC was investigated. X-ray powder diffraction results showed that A-W GC exhibited apatite, fluorapatite and wollastonite as the main phases. The doping of Mn-Zn ferrite caused the formation of a new phase Zn(0.75)Mn(0.75)Fe(1.5)O(4) in M GC. The amount of this new phase increased with increasing content of Mn-Zn ferrite. Under a magnetic field of 7.96 x 10(5) A m(-1), the saturation magnetization of M GC increased from 4.63 to 9.7 A m(2) kg(-1), but the coercive forces of M GC decreased from 2.39 x 10(4) to 7.56 x 10(3) A m(-1) as the Mn-Zn ferrite content increased from 5% to 20% in the material. The bioactivity of samples was evaluated by soaking in simulated body fluid (SBF). The results showed that the doping of Mn-Zn ferrite decreased the bioactivity of A-W GC dramatically. It took 7 days for an apatite layer to form on the surface of A-W GC, while at least 30 days was needed for an apatite layer forming on the surface of M GC.

Genome-wide Analysis of TRNA Charging and Activation of the EIF2 Kinase Gcn2p

The Journal of Biological Chemistry. Sep, 2009  |  Pubmed ID: 19546227

When cells are subjected to nutritional stress, uncharged tRNAs accumulate and activate Gcn2p phosphorylation of eukaryotic initiation factor-2 (eIF2) and the general amino acid control pathway. The Gcn2p regulatory domain homologous to histidyl-tRNA synthetases is proposed to bind to uncharged tRNA, directly contributing to activation of Gcn2p. Here we apply a microarray technology to analyze genome-wide changes in tRNA charging in yeast upon activation of Gcn2p in response to amino acid starvation and high salinity, a stress not directly linked to nutritional deficiency. This microarray technology is applicable for all eukaryotic cells. Strains were starved for histidine, leucine, or tryptophan and shown to rapidly induce Gcn2p phosphorylation of eIF2. The relative charging level of all tRNAs was measured before and after starvation, and Gcn2p activation and the intracellular levels of the starved amino acid correlate with the observed decrease in tRNA charging. Interestingly, in some cases, tRNAs not charged with the starved amino acid became deacylated more rapidly than tRNAs charged with the starved amino acid. This increase in uncharged tRNA levels occurred although the intracellular levels for these non-starved amino acids remained unchanged. Additionally, treatment of a wild-type strain with high salinity stress showed transient changes in the charging of several different tRNAs. These results suggest that Gcn2p can be activated by many different tRNA species in the cell. These results also depict a complex cellular relationship between tRNA charging, amino acid availability, and non-nutrient stress. These relationships are best revealed by simultaneous monitoring of the charging level of all tRNAs.

Virus-like Particle Vaccine Comprised of the HA, NA, and M1 Proteins of an Avian Isolated H5N1 Influenza Virus Induces Protective Immunity Against Homologous and Heterologous Strains in Mice

Viral Immunology. Jul, 2009  |  Pubmed ID: 19594398

Highly pathogenic avian influenza H5N1 virus represents a growing threat for an influenza pandemic. Development of effective vaccines for H5N1 is a priority for pandemic preparedness. Focusing on influenza virus-like particles (VLPs) has been suggested as a promising vaccine approach. Recent VLP vaccination efforts have been concentrated on the H5N1 strains isolated from humans. Because all confirmed cases of human H5N1 infection were directly transmitted from infected poultry, it is of interest to develop VLP vaccines comprised of antigenic proteins of avian H5N1 strains in order to compare their efficacy in fighting diverse H5N1 strains with vaccines developed using human isolates. In this study, we generated a VLP vaccine composed of the HA, NA, and M1 proteins of the avian H5N1 influenza virus isolate A/chicken/Hubei/489/2004, which seems to occupy a unique phylogenetic position; it belongs to neither clade 1 nor clade 2. Upon infection of Sf9 insect cells using recombinant baculoviruses, the co-expressed HA, NA, and M1 proteins self-assembled and released into the culture medium as VLPs. In a mouse model, purified VLPs elicited an effective antibody response and conferred complete protection against heterologous human H5N1 influenza virus, as well as a homologous avian H5N1 influenza virus isolate. Our work provides further evidence that vaccination with influenza VLPs may be a productive approach to achieve protection against diverse H5N1 strains.

TRNA Over-expression in Breast Cancer and Functional Consequences

Nucleic Acids Research. Nov, 2009  |  Pubmed ID: 19783824

Increased proliferation and elevated levels of protein synthesis are characteristics of transformed and tumor cells. Though components of the translation machinery are often misregulated in cancers, what role tRNA plays in cancer cells has not been explored. We compare genome-wide tRNA expression in cancer-derived versus non-cancer-derived breast cell lines, as well as tRNA expression in breast tumors versus normal breast tissues. In cancer-derived versus non-cancer-derived cell lines, nuclear-encoded tRNAs increase by up to 3-fold and mitochondrial-encoded tRNAs increase by up to 5-fold. In tumors versus normal breast tissues, both nuclear- and mitochondrial-encoded tRNAs increase up to 10-fold. This tRNA over-expression is selective and coordinates with the properties of cognate amino acids. Nuclear- and mitochondrial-encoded tRNAs exhibit distinct expression patterns, indicating that tRNAs can be used as biomarkers for breast cancer. We also performed association analysis for codon usage-tRNA expression for the cell lines. tRNA isoacceptor expression levels are not geared towards optimal translation of house-keeping or cell line specific genes. Instead, tRNA isoacceptor expression levels may favor the translation of cancer-related genes having regulatory roles. Our results suggest a functional consequence of tRNA over-expression in tumor cells. tRNA isoacceptor over-expression may increase the translational efficiency of genes relevant to cancer development and progression.

Enhanced Protective Immunity Against H5N1 Influenza Virus Challenge by Vaccination with DNA Expressing a Chimeric Hemagglutinin in Combination with an MHC Class I-restricted Epitope of Nucleoprotein in Mice

Antiviral Research. Mar, 2009  |  Pubmed ID: 19135483

DNA vaccination is an effective means of eliciting both humoral and cellular immune responses. The hemagglutinin (HA) surface protein of influenza A virus is a major target of protective antibody responses induced by virus infection or by vaccination and is widely considered to be the antigen of choice for an influenza vaccine. Cytotoxic T lymphocyte (CTL) responses directed against the conserved nucleoprotein (NP) are thought to play an important role in clearing virus and promoting survival and recovery from influenza. In this study, we developed a novel DNA vaccine approach using a chimeric plasmid consisting of the HA of H5N1 influenza virus in which an MHC class I-restricted NP-specific CTL epitope (NP147-155) was inserted. Immunogenicity and antiviral efficacy of this vaccine was assessed in mouse models. A similar level of HA expression was achieved in 293T cells transfected with pHA/NP(147-155) compared to that with pHA. Besides eliciting the specific anti-HA antibody responses, vaccination using pHA/NP(147-155) in mice induced NP epitope-specific CD8(+) T cell responses, which are generally not inducible by vaccination with pHA alone. After H5N1 influenza virus challenge, BALB/c mice vaccinated with pHA/NP(147-155) exhibited reduced inflammation severity and lung viral titers compared to those vaccinated with pHA. Our work may contribute to improvement of HA-based influenza DNA vaccines.

Efficient Chemical Synthesis of AppDNA by Adenylation of Immobilized DNA-5'-monophosphate

Organic Letters. Mar, 2009  |  Pubmed ID: 19191584

AppDNA is an intermediate in enzyme-catalyzed DNA ligation reactions, and its efficient enzymatic synthesis requires a donor-template duplex of at least 11 base pairs in length. An efficient chemical synthesis of AppDNA with the coupling of an adenosine 5'-phosphorimidazolidate to an immobilized DNA-5'-monophosphate as the key step is described. The adenylation efficiencies of DNA-5'-monophosphate were excellent for oligonucleotides containing less than 11 nucleotides and at least 50% for oligonucleotides containing 15-25 nucleotides.

Langerhans' Cell Histiocytosis of the Spine in Children with Soft Tissue Extension and Chemotherapy

International Orthopaedics. Jun, 2009  |  Pubmed ID: 18338168

The objectives of this paper were to look into the possible incidence of obvious soft tissue extension from Langerhans' cell histiocytosis (LCH) of the spine in children and to evaluate the effects of chemotherapy for those patients. Eighteen patients with histopathological diagnosis of LCH were reviewed and nine with obvious paravertebral soft tissue extension were included in this study. Soft tissue extension was involved in the spinal canal and/or around the vertebral body in eight cases and posterior involvement was prevalent in one case. Eight patients experienced neurological symptoms. All received chemotherapy and one had surgical treatment. The mean follow-up time was 30.3 months. Soft tissue extension disappeared completely in all patients. No clinical evidence of disease was observed at the most recent follow-up. The incidence of LCH of the spine in children with obvious soft tissue extension was up to 50%. Chemotherapy is safe and effective, and surgical decompression was probably not necessary for most patients.

Development of a Solid-phase Extraction-enzyme-linked Immunosorbent Assay for the Determination of 17beta-19-nortestosterone Levels in Antifatigue Functional Foods

Journal of Food Science. Oct, 2009  |  Pubmed ID: 19799684

17beta-19-nortestosterone (17beta-NT) has been illegally used in antifatigue functional foods to promote muscle growth and improve endurance. A rapid and sensitive solid-phase extraction-enzyme-linked immunosorbent assay (SPE-ELISA) method was developed and successfully applied to analyze the levels of 17beta-NT in antifatigue functional foods. A polyclonal antibody against 17beta-NT was produced from rabbits immunized with the 17beta-NT-BSA conjugate, and a competitive direct enzyme-linked immunosorbent assay was developed for the rapid detection of 17beta-NT. The concentration causing 50% inhibition (IC(50)) and the limit of detection (LOD) were found to be 0.08 and 0.0055 ng/mL, respectively; this was better than methods previously reported that had a LOD of 2.4 ng/mL. C(18) cartridges were investigated for use in removing the effects of matrix in foods, and the sample purification protocol was optimized. Using the developed SPE-ELISA method, recoveries of functional food samples were obtained in the range of 71% to 91.5%. Moreover, 2 kinds of antifatigue functional foods were analyzed using the established ELISA and HPLC methods. The correlation coefficient of the results obtained using the 2 methods was greater than 0.98. Thus, the preliminary evaluation of the SPE-ELISA method proved that it is a specific, sensitive, and precise tool that can be used for the practical detection of 17beta-NT in various antifatigue functional food samples.

Additional Bedtime H2-receptor Antagonist for the Control of Nocturnal Gastric Acid Breakthrough

Cochrane Database of Systematic Reviews (Online). 2009  |  Pubmed ID: 19821323

Nocturnal gastric acid breakthrough (NAB) is defined as intragastric pH<4 for more than one continuous hour overnight. Adding H(2)-receptor antagonists (H2RAs) at bedtime to high-dose proton pump inhibitors is likely to enhance nocturnal gastric pH control and decrease nocturnal gastric acid breakthrough.

Endoscopic Mucosal Resection for Early Gastric Cancer

Cochrane Database of Systematic Reviews (Online). 2009  |  Pubmed ID: 19821324

The treatment of early gastric cancer (EGC) using endoscopy, namely endoscopic mucosal resection (EMR), has been adopted for about 20 years, but the effectiveness and safety of the modality are still controversial. Furthermore, the risk of bias of trials of this technique has not been assessed systematically.

The Relationship Between Tinnitus Pitch and the Audiogram

International Journal of Audiology. May, 2009  |  Pubmed ID: 19842803

We studied the relationship between tinnitus pitch and the audiogram in 195 patients. Patients with tone-like tinnitus reported a higher pitch (mean = 5385 Hz) compared to those with a noise-like quality (mean = 3266 Hz). Those with a flat audiogram were more likely to report: a noise-like tinnitus, a unilateral tinnitus, and have a pitch < 2000 Hz. The average duration of bilateral tinnitus (12 years) was longer than that of unilateral tinnitus (5 years). Older subjects reported a less severe tinnitus handicap questionnaire score. Patients with a notched audiogram often reported a pitch or=8000 Hz. We failed to find a relationship between the pitch and the edge of a high frequency hearing loss. Some individuals did exhibit a pitch at the low frequency edge of a hearing loss, but we could find no similar characteristics among these subjects. It is possible that a relationship between pitch and audiogram is present only in certain subgroups.

Innate Immune and Chemically Triggered Oxidative Stress Modifies Translational Fidelity

Nature. Nov, 2009  |  Pubmed ID: 19940929

Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 to 100,000 couplings. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.

Changes in the Tinnitus Handicap Questionnaire After Cochlear Implantation

American Journal of Audiology. Dec, 2009  |  Pubmed ID: 19949236

To determine (a) changes in the Tinnitus Handicap Questionnaire (THQ) for patients using cochlear implants, (b) differences between patients who receive total or partial relief, and (c) identifiable characteristics of those who report tinnitus after implantation.

Profiling Non-lysyl TRNAs in HIV-1

RNA (New York, N.Y.). Feb, 2010  |  Pubmed ID: 20007329

During its assembly, human HIV-1 selectively packages the tRNA(Lys) isoacceptors, including tRNA(Lys3), the primer for the reverse transcriptase. However, other low molecular weight RNA species are also seen in the virus. We profiled the tRNAs packaged into HIV-1 using microarray analysis and validated our results by two-dimensional gel electrophoresis and RT-PCR. In addition to tRNA(Lys) isoacceptors, tRNA(Asn) and the rare isoacceptor of tRNA(Ile) are also selectively packaged. In Gag viral-like particles missing the GagPol protein, overall tRNA incorporation is reduced by >80%. This reduction is significantly greater than can be accounted for by the reduction in tRNA(Lys) isoacceptors, tRNA(Asn) and tRNA(Ile), suggesting that incorporation of other tRNAs may also require the GagPol protein. These results demonstrate selective incorporation of non-lysyl tRNAs into HIV-1 and highlight the application of microarrays as a novel method to study tRNA incorporation into viruses.

Functional Analysis of Human TRNA Isodecoders

Journal of Molecular Biology. Feb, 2010  |  Pubmed ID: 20026070

tRNA isodecoders share the same anticodon but have differences in their body sequence. An unexpected result from genome sequencing projects is the identification of a large number of tRNA isodecoder genes in mammalian genomes. In the reference human genome, more than 270 isodecoder genes are present among the approximately 450 tRNA genes distributed among 49 isoacceptor families. Whether sequence diversity among isodecoder tRNA genes reflects functional variability is an open question. To address this, we developed a method to quantify the efficiency of tRNA isodecoders in stop-codon suppression in human cell lines. First, a green fluorescent protein (GFP) gene that contains a single UAG stop codon at two distinct locations is introduced. GFP is only produced when a tRNA suppressor containing CUA anticodon is co-transfected with the GFP gene. The suppression efficiency is examined for 31 tRNA isodecoders (all contain CUA anticodon), 21 derived from four isoacceptor families of tRNASer genes, 7 from five families of tRNALeu genes, and 3 from three families of tRNAAla genes. We found that isodecoder tRNAs display a large difference in their suppression efficiency. Among those with above background suppression activity, differences of up to 20-fold were observed. We were able to tune tRNA suppression efficiency by subtly adjusting the tRNA sequence and inter-convert poor suppressors into potent ones. We also demonstrate that isodecoder tRNAs with varying suppression efficiencies have similar stability and exhibit similar levels of aminoacylation in vivo. Our results indicate that naturally occurring tRNA isodecoders can have large functional variations and suggest that some tRNA isodecoders may perform a function distinct from translation.

Extended Structures in RNA Folding Intermediates Are Due to Nonnative Interactions Rather Than Electrostatic Repulsion

Journal of Molecular Biology. Apr, 2010  |  Pubmed ID: 20188108

RNA folding occurs via a series of transitions between metastable intermediate states for Mg(2+) concentrations below those needed to fold the native structure. In general, these folding intermediates are considerably less compact than their respective native states. Our previous work demonstrates that the major equilibrium intermediate of the 154-residue specificity domain (S-domain) of the Bacillus subtilis RNase P RNA is more extended than its native structure. We now investigate two models with falsifiable predictions regarding the origins of the extended intermediate structures in the S-domains of the B. subtilis and the Escherichia coli RNase P RNA that belong to different classes of P RNA and have distinct native structures. The first model explores the contribution of electrostatic repulsion, while the second model probes specific interactions in the core of the folding intermediate. Using small-angle X-ray scattering and Langevin dynamics simulations, we show that electrostatics plays only a minor role, whereas specific interactions largely account for the extended nature of the intermediate. Structural contacts in the core, including a nonnative base pair, help to stabilize the intermediate conformation. We conclude that RNA folding intermediates adopt extended conformations due to short-range, nonnative interactions rather than generic electrostatic repulsion of helical domains. These principles apply to other ribozymes and riboswitches that undergo functionally relevant conformational changes.

Integration of General Amino Acid Control and Target of Rapamycin (TOR) Regulatory Pathways in Nitrogen Assimilation in Yeast

The Journal of Biological Chemistry. May, 2010  |  Pubmed ID: 20233714

Two important nutrient-sensing and regulatory pathways, the general amino acid control (GAAC) and the target of rapamycin (TOR), participate in the control of yeast growth and metabolism during changes in nutrient availability. Amino acid starvation activates the GAAC through Gcn2p phosphorylation of translation factor eIF2 and preferential translation of GCN4, a transcription activator. TOR senses nitrogen availability and regulates transcription factors such as Gln3p. We used microarray analyses to address the integration of the GAAC and TOR pathways in directing the yeast transcriptome during amino acid starvation and rapamycin treatment. We found that GAAC is a major effector of the TOR pathway, with Gcn4p and Gln3p each inducing a similar number of genes during rapamycin treatment. Although Gcn4p activates a common core of 57 genes, the GAAC directs significant variations in the transcriptome during different stresses. In addition to inducing amino acid biosynthetic genes, Gcn4p in conjunction with Gln3p activates genes required for the assimilation of secondary nitrogen sources such as gamma-aminobutyric acid (GABA). Gcn2p activation upon shifting to secondary nitrogen sources is suggested to occur by means of a dual mechanism. First, Gcn2p is induced by the release of TOR repression through a mechanism involving Sit4p protein phosphatase. Second, this eIF2 kinase is activated by select uncharged tRNAs, which were shown to accumulate during the shift to the GABA medium. This study highlights the mechanisms by which the GAAC and TOR pathways are integrated to recognize changing nitrogen availability and direct the transcriptome for optimal growth adaptation.

The Efficacy of a Voice Training Program: a Case-control Study in China

European Archives of Oto-rhino-laryngology : Official Journal of the European Federation of Oto-Rhino-Laryngological Societies (EUFOS) : Affiliated with the German Society for Oto-Rhino-Laryngology - Head and Neck Surgery. Jan, 2010  |  Pubmed ID: 19826827

The objective of this study was to design a voice training program for Chinese speakers, and to evaluate its efficacy. It was a prospective, randomized, case-control study practiced in three middle schools in Beijing, China. Teachers in the treatment group received voice training for 4 weeks, whereas the control group subjects received no treatment. The voice training program, which was adapted for Chinese, contained vocal hygiene education and group voice training. The outcome was assessed by voice handicap index (VHI), maximum phonation time (MPT) and acoustic analysis parameters including, noise to harmonic ratio (NHR), jitter and shimmer. The results showed that at the onset of the study, no significant differences were found between the subjects in two groups for VHI, MPT and NHR. VHI of treatment group subjects who received voice training decreased significantly, whereas VHI of control group subjects showed no significant change. Treatment group MPT was significantly increased after training, whereas the control group one presented no significant change during the same period. NHR in treatment group decreased significantly after training, whereas the one in control group showed no significant change. There were no significant changes for jitter and shimmer in both groups before and after the study. So we conclude that the voice training program is suitable to treat voice diseases, particularly for middle school teachers. This result provided reliable evidence for carrying out further voice training in China.

Extractive Fermentation in Cloud Point System for Lipase Production by Serratia Marcescens ECU1010

Applied Microbiology and Biotechnology. Feb, 2010  |  Pubmed ID: 19798498

Extractive microbial fermentation for production of lipase by Serratia marcescens ECU1010 has been carried out in cloud point system. The cloud point system is composed of mixture nonionic surfactants with a ratio of Triton X-114 to Triton X-45 4:1 in aqueous solution. The lipase prefers to partition into the surfactant rich phase (coacervate phase) whereas the cells and other hydrophilic proteins retain in the dilute phase of cloud point system. Thus, a concentration factor 4.2-fold and a purification factor 1.3-fold of the lipase have been achieved in the extractive fermentation process. This is the first report about extractive fermentation of proteins in cloud point system.

Efficacy of Concurrent Chemoradiotherapy Plus Adjuvant Chemotherapy on Advanced Cervical Cancer

Chinese Journal of Cancer. Nov, 2010  |  Pubmed ID: 20979696

Concurrent chemoradiotherapy for cervical carcinoma develops rapidly and has become a common and standard therapy in recent years. Both the local control rate and survival rate of patients were increased and the risk of death fell by 30%-50%. This study aimed to explore the efficacy of concurrent chemoradiotherapy plus adjuvant chemotherapy on and the treatment compliance of the patients with advanced cervical squamous cell carcinoma.

Discrete Structure of an RNA Folding Intermediate Revealed by Cryo-electron Microscopy

Journal of the American Chemical Society. Nov, 2010  |  Pubmed ID: 21038867

RNA folding occurs via a series of transitions between metastable intermediate states. It is unknown whether folding intermediates are discrete structures folding along defined pathways or heterogeneous ensembles folding along broad landscapes. We use cryo-electron microscopy and single-particle image reconstruction to determine the structure of the major folding intermediate of the specificity domain of a ribonuclease P ribozyme. Our results support the existence of a discrete conformation for this folding intermediate.

Structure of a Bacterial Ribonuclease P Holoenzyme in Complex with TRNA

Nature. Dec, 2010  |  Pubmed ID: 21076397

Ribonuclease (RNase) P is the universal ribozyme responsible for 5'-end tRNA processing. We report the crystal structure of the Thermotoga maritima RNase P holoenzyme in complex with tRNA(Phe). The 154 kDa complex consists of a large catalytic RNA (P RNA), a small protein cofactor and a mature tRNA. The structure shows that RNA-RNA recognition occurs through shape complementarity, specific intermolecular contacts and base-pairing interactions. Soaks with a pre-tRNA 5' leader sequence with and without metal help to identify the 5' substrate path and potential catalytic metal ions. The protein binds on top of a universally conserved structural module in P RNA and interacts with the leader, but not with the mature tRNA. The active site is composed of phosphate backbone moieties, a universally conserved uridine nucleobase, and at least two catalytically important metal ions. The active site structure and conserved RNase P-tRNA contacts suggest a universal mechanism of catalysis by RNase P.

[Cochlear Implant in Patient with Chronic Otitis Media After Subtotal Petrosectomy]

Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi = Chinese Journal of Otorhinolaryngology Head and Neck Surgery. Dec, 2010  |  Pubmed ID: 21215057

Goos-Hänchen Shifts of the Reflected Waves from a Cold, Inhomogeneous, and Magnetized Plasma Slab

Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics. Jan, 2010  |  Pubmed ID: 20365487

We discuss theoretically the Goos-Hänchen (GH) shifts of the reflected waves from a cold, inhomogeneous, and magnetized plasma slab by using the invariant imbedding approach. Aiming at the linear and parabolic electron-density profiles, we demonstrate numerically the dependences of the co- and cross-polarized GH shifts on the angle of incidence, external static magnetic field, and the thickness of the plasma slab. The results show that the different electron-density profiles of plasma can result in the very different dependences of the GH shifts on the angle of incidence, external magnetic field, and the slab's thickness; the GH shifts can be switched between the considerably large positive and negative values under certain conditions. Particularly, without altering the structure of the plasma slab, the GH shifts can be manipulated by modifying the angle of incident or the external static magnetic field.

An Evolutionarily Conserved Mechanism for Controlling the Efficiency of Protein Translation

Cell. Apr, 2010  |  Pubmed ID: 20403328

Recent years have seen intensive progress in measuring protein translation. However, the contributions of coding sequences to the efficiency of the process remain unclear. Here, we identify a universally conserved profile of translation efficiency along mRNAs computed based on adaptation between coding sequences and the tRNA pool. In this profile, the first approximately 30-50 codons are, on average, translated with a low efficiency. Additionally, in eukaryotes, the last approximately 50 codons show the highest efficiency over the full coding sequence. The profile accurately predicts position-dependent ribosomal density along yeast genes. These data suggest that translation speed and, as a consequence, ribosomal density are encoded by coding sequences and the tRNA pool. We suggest that the slow "ramp" at the beginning of mRNAs serves as a late stage of translation initiation, forming an optimal and robust means to reduce ribosomal traffic jams, thus minimizing the cost of protein expression.

[Endoscopic Surgery Using the Low-temperature Plasma Radiofrequency for Nasal Hemangioma]

Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi = Chinese Journal of Otorhinolaryngology Head and Neck Surgery. Mar, 2010  |  Pubmed ID: 20450697

To evaluate the effect of endoscopic surgery using the low-temperature plasma radiofrequency for nasal hemangioma.

Genome-wide Analysis of N1-methyl-adenosine Modification in Human TRNAs

RNA (New York, N.Y.). Jul, 2010  |  Pubmed ID: 20484468

The N(1)-methyl-Adenosine (m(1)A58) modification at the conserved nucleotide 58 in the TPsiC loop is present in most eukaryotic tRNAs. In yeast, m(1)A58 modification is essential for viability because it is required for the stability of the initiator-tRNA(Met). However, m(1)A58 modification is not required for the stability of several other tRNAs in yeast. This differential m(1)A58 response for different tRNA species raises the question of whether some tRNAs are hypomodified at A58 in normal cells, and how hypomodification at A58 may affect the stability and function of tRNA. Here, we apply a genomic approach to determine the presence of m(1)A58 hypomodified tRNAs in human cell lines and show how A58 hypomodification affects stability and involvement of tRNAs in translation. Our microarray-based method detects the presence of m(1)A58 hypomodified tRNA species on the basis of their permissiveness in primer extension. Among five human cell lines examined, approximately one-quarter of all tRNA species are hypomodified in varying amounts, and the pattern of the hypomodified tRNAs is quite similar. In all cases, no hypomodified initiator-tRNA(Met) is detected, consistent with the requirement of this modification in stabilizing this tRNA in human cells. siRNA knockdown of either subunit of the m(1)A58-methyltransferase results in a slow-growth phenotype, and a marked increase in the amount of m(1)A58 hypomodified tRNAs. Most m(1)A58 hypomodified tRNAs can associate with polysomes in varying extents. Our results show a distinct pattern for m(1)A58 hypomodification in human tRNAs, and are consistent with the notion that this modification fine tunes tRNA functions in different contexts.

The AlkB Domain of Mammalian ABH8 Catalyzes Hydroxylation of 5-methoxycarbonylmethyluridine at the Wobble Position of TRNA

Angewandte Chemie (International Ed. in English). Nov, 2010  |  Pubmed ID: 20583019

Selective Control of Amino Acid Metabolism by the GCN2 EIF2 Kinase Pathway in Saccharomyces Cerevisiae

BMC Biochemistry. 2010  |  Pubmed ID: 20684782

When eukaryotic cells are deprived of amino acids, uncharged tRNAs accumulate and activate the conserved GCN2 protein kinase. Activated Gcn2p up-regulates the general amino acid control pathway through phosphorylation of the translational initiation factor eIF2. In Saccharomyces cerevisiae, Gcn2p is the only kinase that phosphorylates eIF2 to regulate translation through this mechanism. We addressed changes in yeast growth and tRNA aminoacylation, or charging, during amino acid depletion in the presence and absence of GCN2. tRNA charging was measured using a microarray technique which simultaneously measures all cytosolic tRNAs. A fully prototrophic strain, and its isogenic gcn2 Delta counterpart, were used to study depletion for each of the 20 amino acids, with a focus on Trp, Arg, His and Leu, which are metabolically distinct and together provide a good overview on amino acid metabolism.

[Spatial Pattern of Vegetation Landscape Diversity in Longitudinal Range-Gorge Region, Southwestern China]

Ying Yong Sheng Tai Xue Bao = The Journal of Applied Ecology / Zhongguo Sheng Tai Xue Xue Hui, Zhongguo Ke Xue Yuan Shenyang Ying Yong Sheng Tai Yan Jiu Suo Zhu Ban. Dec, 2010  |  Pubmed ID: 21442994

Based on the China 1:1000000 vegetation type map, and by using GIS spatial analysis, the spatial pattern of major vegetation landscape diversity indices and its relationships with environmental factors in Longitudinal Range-Gorge Region (LRGR) were analyzed. The proper scale for studying the vegetation landscape diversity in LRGR was 2000 m. In the study region, an obvious regional difference was observed in the vegetation landscape diversity indices, exhibiting typical longitudinal "corridor" and latitudinal "barrier" characteristics. The correlations between the vegetation landscape diversity indices and environmental elements were significant, and the regional difference in the environmental elements was the main factor controlling the spatial pattern of vegetation landscape diversity indices. The "corridor-barrier" function of the longitudinal range-gorge terrain made a spatial redistribution of hydro-thermal conditions, being the main cause of the special pattern of the vegetation landscape diversity in LRGR.

Design and Analysis of MEMS Based PVDF Ultrasonic Transducers for Vascular Imaging

Sensors (Basel, Switzerland). 2010  |  Pubmed ID: 22163683

Polyvinilidene fluoride (PVDF) single-element transducers for high-frequency (>30 MHz) ultrasound imaging applications have been developed using MEMS (Micro-electro-Mechanical Systems) compatible techniques. Performance of these transducers has been investigated by analyzing the sources and effects of on-chip parasitic capacitances on the insertion-loss of the transducers. Modeling and experimental studies showed that on-chip parasitic capacitances degraded the performance of the transducers and an improved method of fabrication was suggested and new devices were built. New devices developed with minimal parasitic effects were shown to improve the performance significantly. A 1-mm aperture PVDF device developed with minimal parasitic effects has resulted in a reduction of insertion loss of 21 dB compared with devices fabricated using a previous method.

Syntheses of Two 5-hydroxymethyl-2'-deoxycytidine Phosphoramidites with TBDMS As the 5-hydroxymethyl Protecting Group and Their Incorporation into DNA

The Journal of Organic Chemistry. May, 2011  |  Pubmed ID: 21462947

5-Hydroxymethylcytosine (5-hmC) is a newly discovered DNA base modification in mammalian genomic DNA that is proposed to be a major epigenetic mark. We report here the syntheses of two new versions of phosphoramidites III and IV from 5-iodo-2'-deoxyuridine in 18% and 32% overall yields, respectively, with TBDMS as the 5-hydroxyl protecting group. Phosphoramidites III and IV allow efficient incorporation of 5-hmC into DNA and a "one-step" deprotection procedure to cleanly remove all the protecting groups. A "two-step" deprotection strategy is compatible with ultramild DNA synthesis, which enables the synthesis of 5hmC-containing DNA with additional modifications.

Misacylation of Specific Nonmethionyl TRNAs by a Bacterial Methionyl-tRNA Synthetase

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2011  |  Pubmed ID: 21482813

Aminoacyl-tRNA synthetases perform a critical step in translation by aminoacylating tRNAs with their cognate amino acids. Although high fidelity of aminoacyl-tRNA synthetases is often thought to be essential for cell biology, recent studies indicate that cells tolerate and may even benefit from tRNA misacylation under certain conditions. For example, mammalian cells selectively induce mismethionylation of nonmethionyl tRNAs, and this type of misacylation contributes to a cell's response to oxidative stress. However, the enzyme responsible for tRNA mismethionylation and the mechanism by which specific tRNAs are mismethionylated have not been elucidated. Here we show by tRNA microarrays and filter retention that the methionyl-tRNA synthetase enzyme from Escherichia coli (EcMRS) is sufficient to mismethionylate two tRNA species, and , indicating that tRNA mismethionylation is also present in the bacterial domain of life. We demonstrate that the anticodon nucleotides of these misacylated tRNAs play a critical role in conferring mismethionylation identity. We also show that a certain low level of mismethionylation is maintained for these tRNAs, suggesting that mismethionylation levels may have evolved to confer benefits to the cell while still preserving sufficient translational fidelity to ensure cell viability. EcMRS mutants show distinct effects on mismethionylation, indicating that many regions in this synthetase enzyme influence mismethionylation. Our results show that tRNA mismethionylation can be carried out by a single protein enzyme, mismethionylation also requires identity elements in the tRNA, and EcMRS has a defined structure-function relationship for tRNA mismethionylation.

Cellular Dynamics of RNA Modification

Accounts of Chemical Research. Dec, 2011  |  Pubmed ID: 21615108

Five decades of research have identified more than 100 ribonucleosides that are post-transcriptionally modified. Many modified nucleosides are conserved throughout bacteria, archaea, and eukaryotes, while some are unique to each branch of life. However, the cellular and functional dynamics of RNA modification remain largely unexplored, mostly because of the lack of functional hypotheses and experimental methods for quantification and large-scale analysis. Many RNA modifications are not essential for life, which parallels the observation that many well-characterized protein and DNA modifications are not essential for life. Instead, increasing evidence indicates that RNA modifications can play regulatory roles in cells, especially in response to stress conditions. In this Account, we review some examples of RNA modification that are dynamically controlled in cells. We also discuss some recently developed methods that have enhanced the ability to study the cellular dynamics of RNA modification. We discuss four specific examples of RNA modification in detail here. We begin with 4-thio uridine (s(4)U), which can act as a cellular sensor of near-UV light. Then we consider queuosine (Q), which is a potential biomarker for malignancy. Next we examine N(6)-methyl adenine (m(6)A), which is the prevalent modification in eukaryotic messenger RNAs (mRNAs). Finally, we discuss pseudouridine (ψ), which is inducible by nutrient deprivation. We then consider two recent technical advances that have stimulated the study of the cellular dynamics in modified ribonucleosides. The first is a genome-wide method that combines primer extension with a microarray. It was used to study the N(1)-methyl adenine (m(1)A) hypomodification in human transfer RNA (tRNA). The second is a quantitative mass spectrometric method used to investigate dynamic changes in a wide range of tRNA modifications under stress conditions in yeast. In addition, we discuss potential mechanisms that control dynamic regulation of RNA modifications as well as hypotheses for discovering potential RNA demodification enzymes. We conclude by highlighting the need to develop new tools and to generate additional hypotheses for how these modifications function in cells. The study of the cellular dynamics of modified RNA remains a largely open area for new development, which underscores the rich potential for important advances as researchers drive this emerging field to the next level.

TRNA: Vast Reservoir of RNA Molecules with Unexpected Regulatory Function

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2011  |  Pubmed ID: 21933958

N6-methyladenosine in Nuclear RNA is a Major Substrate of the Obesity-associated FTO

Nature Chemical Biology. Dec, 2011  |  Pubmed ID: 22002720

We report here that fat mass and obesity-associated protein (FTO) has efficient oxidative demethylation activity targeting the abundant N6-methyladenosine (m(6)A) residues in RNA in vitro. FTO knockdown with siRNA led to increased amounts of m(6)A in mRNA, whereas overexpression of FTO resulted in decreased amounts of m(6)A in human cells. We further show the partial colocalization of FTO with nuclear speckles, which supports the notion that m(6)A in nuclear RNA is a major physiological substrate of FTO.

[Clinical and Pathologic Features of 33 Children with Thin Basement Membrane Nephropathy]

Zhongguo Dang Dai Er Ke Za Zhi = Chinese Journal of Contemporary Pediatrics. Mar, 2011  |  Pubmed ID: 21426649

[Diagnostic Significance of Spinal Cord Conduction Velocity in the Diagnosis of Central Lesions in Subacute Combined Degeneration]

Zhonghua Yi Xue Za Zhi. Oct, 2011  |  Pubmed ID: 22321932

To study explore the roles of spinal cord conduction velocity (SCCV) test in the diagnosis of spinal lesions in patients with subacute combined degeneration (SCD) of spinal cord and examine the values of SCCV in the localization of spinal lesions.

Transcriptional Pausing Coordinates Folding of the Aptamer Domain and the Expression Platform of a Riboswitch

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2012  |  Pubmed ID: 22331895

Riboswitches are cis-acting elements that regulate gene expression by affecting transcriptional termination or translational initiation in response to binding of a metabolite. A typical riboswitch is made of an upstream aptamer domain and a downstream expression platform. Both domains participate in the folding and structural rearrangement in the absence or presence of its cognate metabolite. RNA polymerase pausing is a fundamental property of transcription that can influence RNA folding. Here we show that pausing plays an important role in the folding and conformational rearrangement of the Escherichia coli btuB riboswitch during transcription by the E. coli RNA polymerase. This riboswitch consists of an approximately 200 nucleotide, coenzyme B12 binding aptamer domain and an approximately 40 nucleotide expression platform that controls the ribosome access for translational initiation. We found that transcriptional pauses at strategic locations facilitate folding and structural rearrangement of the full-length riboswitch, but have minimal effect on the folding of the isolated aptamer domain. Pausing at these regulatory sites blocks the formation of alternate structures and plays a chaperoning role that couples folding of the aptamer domain and the expression platform. Pausing at strategic locations may be a general mechanism for coordinated folding and conformational rearrangements of riboswitch structures that underlie their response to environmental cues.

Rationalization and Prediction of Selective Decoding of Pseudouridine-modified Nonsense and Sense Codons

RNA (New York, N.Y.). Mar, 2012  |  Pubmed ID: 22282339

A stop or nonsense codon is an in-frame triplet within a messenger RNA that signals the termination of translation. One common feature shared among all three nonsense codons (UAA, UAG, and UGA) is a uridine present at the first codon position. It has been recently shown that the conversion of this uridine into pseudouridine (Ψ) suppresses translation termination, both in vitro and in vivo. Furthermore, decoding of the pseudouridylated nonsense codons is accompanied by the incorporation of two specific amino acids in a nonsense codon-dependent fashion. Ψ differs from uridine by a single N(1)H group at the C5 position; how Ψ suppresses termination and, more importantly, enables selective decoding is poorly understood. Here, we provide molecular rationales for how pseudouridylated stop codons are selectively decoded. Our analysis applies crystal structures of ribosomes in varying states of translation to consider weakened interaction of Ψ with release factor; thermodynamic and geometric considerations of the codon-anticodon base pairs to rank and to eliminate mRNA-tRNA pairs; the mechanism of fidelity check of the codon-anticodon pairing by the ribosome to evaluate noncanonical codon-anticodon base pairs and the role of water. We also consider certain tRNA modifications that interfere with the Ψ-coordinated water in the major groove of the codon-anticodon mini-helix. Our analysis of nonsense codons enables prediction of potential decoding properties for Ψ-modified sense codons, such as decoding ΨUU potentially as Cys and Tyr. Our results provide molecular rationale for the remarkable dynamics of ribosome decoding and insights on possible reprogramming of the genetic code using mRNA modifications.