Biomedical subjects

Tao Pan

Publications and source records attributed to Tao Pan.

At least 19 recordsLinked to original sources

PubMed2006

Tissue-specific differences in human transfer RNA expression.

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.

Animals↗

PubMed2006

Diversity of tRNA genes in eukaryotes.

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.

Animals↗

PubMed2006

Test for detection of disease-associated prion aggregate in the blood of infected but asymptomatic animals.

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.

Animals↗

PubMed2006

A systematic, ligation-based approach to study RNA modifications.

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.

DNA Methylation↗

PubMed2006

Structure of ribonuclease P--a universal ribozyme.

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.

Bacterial Proteins↗

PubMed2006

Additional bedtime H2-receptor antagonist for the control of nocturnal gastric acid breakthrough: a Cochrane systematic review.

OBJECTIVES: 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. METHODS: Eligible trials were identified by searching the Cochrane Central Register of Controlled Trials (CENTRAL) (Cochrane Library Issue 2, 2004), MEDLINE (January 1966-June 2004), EMBASE (January 1980-June 2004) and CINAHL (January 1982-June 2004). Additional hand-searching was conducted on the proceedings of correlated conferences, eight important Chinese journals and references of all included trials. All randomized controlled trials evaluating H(2)RAs for the control of NAB were eligible for inclusion. The systematic review was conducted using methods recommended by The Cochrane Collaboration. RESULTS: Only two randomized crossover studies, comprising 32 participants, met the inclusion criteria. Because the design, dosage and duration of the treatments were different between the studies, it was not possible to conduct meta-analysis. There were no consistent conclusions found between the two included studies in evaluating H(2)RAs for the control of NAB. CONCLUSIONS: No implications for practice at this stage can be concluded. Appropriately designed large-scale randomized controlled trials with long-term follow up are needed to determine the effects of additional bedtime H(2)RAs in suppressing NAB.

Drug Administration Schedule↗

PubMed2006

RNA folding during transcription.

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.

Computer Simulation↗

PubMed2006

Structural basis for altering the stability of homologous RNAs from a mesophilic and a thermophilic bacterium.

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.

Base Sequence↗

PubMed2006

[A clinical analysis of intractable spontaneous epistaxis with 289 cases reviewed].

OBJECTIVE: To explore the clinical characteristics and the prevention strategy of intractable spontaneous epistaxis. METHOD: Two hundred and eighty-nine patients with intractable spontaneous epistaxis were retrospectively analyzed. RESULT: Intractable spontaneous epistaxis was characterized by the onset season, age, sex, and the associated disease. Nasal septums were the most common sites of bleeding. Recurrent epistaxis was mainly associated with the failure of locating the bleeding sites. CONCLUSION: Control of elevated blood pressure is essential for the prevention of intractable spontaneous epistaxis. Once epistaxis occurs, the importance of the first treatment for the haemostasis should be emphasized. For the uncontrollable epistaxis, multiple interventions are necessary.

Adolescent↗

PubMed2005

Structure of a folding intermediate reveals the interplay between core and peripheral elements in RNA folding.

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.

Bacillus subtilis↗

PubMed2005

Crystal structure of the RNA component of bacterial ribonuclease P.

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.

Catalytic Domain↗

PubMed2005

Calorie controlled diet for chronic asthma.

BACKGROUND: 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. OBJECTIVES: To observe the effect of dietary calorie reduction on chronic asthma in adults or children. SEARCH STRATEGY: We searched the Cochrane Airways Group trials register using prespecified terms. We assessed bibliographies from included studies, and contacted authors of known studies for additional information about published and unpublished trials. Date of most recent search: May 2004 SELECTION CRITERIA: Randomised-controlled trials of dietary calorie reduction were included. DATA COLLECTION AND ANALYSIS: Three authors assessed each study independently. Disagreement was resolved by consensus. Quality assessment was performed independently. MAIN RESULTS: One trial of fair methodological quality with a total of 38 patients suffering from chronic asthma was included. There were significant increases in FEV(1) and FVC in the active treatment group compared with control. No data pertaining serious adverse effects were reported from the interventions. AUTHORS' CONCLUSIONS: There is currently a very small amount of evidence assessing the effects of dietary interventions intended as part of a wide-ranging weight-loss programme. Whilst we are unable to recommend these strategies as concomitant interventions with drug-based therapy for the specific management of asthma, dietary interventions such as weight-loss programmes may provide benefits in specific patients. However, the impact of a calorie-controlled diet on the signs and symptoms of asthma in the general asthmatic population is yet to be established.

Adult↗

PubMed2005

Real-time RNA profiling within a single bacterium.

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.

Bacterial Proteins↗

PubMed2005

Phase-changing sacrificial materials for solvent bonding of high-performance polymeric capillary electrophoresis microchips.

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.

Acetonitriles↗

PubMed2005

Mechanistic insights on the folding of a large ribozyme during transcription.

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.

Base Sequence↗

PubMed2005

Selective charging of tRNA isoacceptors induced by amino-acid starvation.

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.

Amino Acids↗

PubMed2005

Biochemical fingerprints of prion diseases: scrapie prion protein in human prion diseases that share prion genotype and type.

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.

Biomarkers↗

PubMed2005

Novel antibody-lectin enzyme-linked immunosorbent assay that distinguishes prion proteins in sporadic and variant cases of Creutzfeldt-Jakob disease.

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.

Biotinylation↗