Biomedical subjects

Liang Shi

Publications and source records attributed to Liang Shi.

30 records · Page 2Linked to original sources

PubMed2004

Comparative analysis of eukaryotic-type protein phosphatases in two streptomycete genomes.

Inspection of the genomes of Streptomyces coelicolor A3(2) and Streptomyces avermitilis reveals that each contains 55 putative eukaryotic-type protein phosphatases (PPs), the largest number ever identified from any single prokaryotic organism. Unlike most other prokaryotic genomes that have only one or two superfamilies of eukaryotic-type PPs, the streptomycete genomes possess the eukaryotic-type PPs that belong to four superfamilies: 2 phosphoprotein phosphatases and 2 low-molecular-mass protein tyrosine phosphatases in each species, 49 Mg(2+)- or Mn(2+)-dependent protein phosphatases (PPMs) and 2 conventional protein tyrosine phosphatases (CPTPs) in S. coelicolor A3(2), and 48 PPMs and 3 CPTPs in S. avermitilis. Sixty-four percent of the PPs found in S. coelicolor A3(2) have orthologues in S. avermitilis, indicating that they originated from a common ancestor and might be involved in the regulation of more conserved metabolic activities. The genes of eukaryotic-type PP unique to each surveyed streptomycete genome are mainly located in two arms of the linear chromosomes and their evolution might be involved in gene acquisition or duplication to adapt to the extremely variable soil environments where these organisms live. In addition, 56 % of the PPs from S. coelicolor A3(2) and 65 % of the PPs from S. avermitilis possess at least one additional domain having a putative biological function. These include the domains involved in the detection of redox potential, the binding of cyclic nucleotides, mRNA, DNA and ATP, and the catalysis of phosphorylation reactions. Because they contained multiple functional domains, most of them were assigned functions other than PPs in previous annotations. Although few studies have been conducted on the physiological functions of the PPs in streptomycetes, the existence of large numbers of putative PPs in these two streptomycete genomes strongly suggests that eukaryotic-type PPs play important regulatory roles in primary or secondary metabolic pathways. The identification and analysis of such a large number of putative eukaryotic-type PPs from S. coelicolor A3(2) and S. avermitilis constitute a basis for further exploration of the signal transduction pathways mediated by these phosphatases in industrially important strains of streptomycetes.

Computational Biology↗

PubMed2004

Evolution of the PPM-family protein phosphatases in Streptomyces: duplication of catalytic domain and lateral recruitment of additional sensory domains.

Originally identified from eukaryotes, the Mg2+- or Mn2+-dependent protein phosphatases (PPMs) are a diverse group of enzymes whose members include eukaryotic PP2C and some prokaryotic serine/threonine phosphatases. In a previous study, unexpectedly large numbers of PPMs were identified in two Streptomyces genomes. In this work, a phylogenetic analysis was performed with all the PPMs available from a wide variety of microbial sources to determine the evolutionary origin of the Streptomyces PPM proteins. Consistent with earlier hypotheses, the results suggested that the microbial PPMs were relatively recent additions from eukaryotic sources. Results also indicated that the Streptomyces PPMs were divided into two major subfamilies at an early stage of their emergence in Streptomyces genomes. The first subfamily, which contains only six Streptomyces PPMs, possesses a catalytic domain whose sequence and architecture are similar to that of eukaryotic PPMs; the second subfamily contains 89 Streptomyces PPMs that lack the 5a and 5b catalytic domain motifs, similar to the PPMs SpoIIE and RsbU of Bacillus subtilis. Significant gene duplication was observed for the PPMs in the second subfamily. In addition, more than half (54 %) of the Streptomyces PPMs from the second subfamily were found to have at least one additional sensory domain, most commonly the PAS or the GAF domain. Phylogenetic analysis showed that these domains tended to be clustered according to the putative physiological functions rather than taxonomic relationship, implying that they might have arisen as a result of domain recruitment in a late evolutionary stage. This study provides an insight into how Streptomyces spp. may have expanded their PPM-based signal transduction networks to enable them to respond to a greater range of environmental changes.

Catalytic Domain↗

PubMed2004

The CorA Mg2+ transporter is a homotetramer.

CorA is a primary Mg2+ transporter for Bacteria and Archaea. The C-terminal domain of approximately 80 amino acids forms three transmembrane (TM) segments, which suggests that CorA is a homo-oligomer. A Cys residue was added to the cytoplasmic C terminus (C317) of Salmonella enterica serovar Typhimurium CorA with or without mutation of the single periplasmic Cys191 to Ser; each mutant retained function. Oxidation of the Cys191Ser Cys317 CorA gave a dimer. Oxidation of Cys317 CorA showed a dimer plus an additional band, apparently cross-linked via both Cys317 and C191. To determine oligomer order, intact cells or purified membranes were treated with formaldehyde or carbon disulfide. Higher-molecular-mass bands formed, consistent with the presence of a tetramer. Cross-linking of the Bacillus subtilis CorA expressed in Salmonella serovar Typhimurium similarly indicated a tetramer. CorA periplasmic soluble domains from both Salmonella serovar Typhimurium and the archaeon Methanococcus jannaschii were purified and shown to retain structure. Formaldehyde treatment showed formation of a tetramer. Finally, previous mutagenesis of the CorA membrane domain identified six intramembrane residues forming an apparent pore that interacts with Mg2+ during transport. Each was mutated to Cys. In mutants carrying a single intramembrane Cys residue, spontaneous disulfide bond formation that was enhanced by oxidation with Cu(II)-1,10-phenanthroline was observed between monomers, indicating that these Mg2+-interacting residues within the membrane are very close to their cognate residue on another monomer. Thus, CorA appears to be a homotetramer with a TM segment of one monomer physically close to the same TM segment of another monomer.

Amino Acid Substitution↗

PubMed2004

A room-temperature synthesis of titanium nitride hollow spheres.

Titanium nitride hollow spheres were synthesized by the reaction of TiCl4 and NaNH2 at room temperature. X-ray powder diffraction (XRD) pattern could be indexed as cubic TiN with the lattice constants of a = 4.236 A. Transmission electron microscopy (TEM) images showed hollow spheres with diameter about 200 nm. A possible formation mechanism of TiN hollow spheres was discussed.

Coated Materials, Biocompatible↗

PubMed2003

Differential gene expression analysis by micro-preparative capillary gel electrophoresis.

Differential display analysis by cDNA fractionation, collection of differentially expressed fractions of interests and their downstream characterization is demonstrated. cDNA pools from two strains of Cochliobolus heterostrophus fungus were generated by specific restriction digestion and selective ligation. Micropreparative separation and isolation of differentially expressed transcript representatives were accomplished by high-performance capillary gel electrophoresis. The collected individual DNA molecules were polymerase chain reaction amplified and sequenced to create expressed sequence tags for the genes of interests. High resolving power and sensitivity of capillary gel electrophoresis enabled fast and automated processing of minute amounts of cDNA samples with high precision.

Base Sequence↗

PubMed2003

Micropreparative capillary gel electrophoresis of DNA: rapid expressed sequence tag library construction.

A capillary gel electrophoresis based automated DNA fraction collection technique was developed to support a novel DNA fragment-pooling strategy for expressed sequence tag (EST) library construction. The cDNA population is first cleaved by BsaJ I and EcoR I restriction enzymes, and then subpooled by selective ligation with specific adapters followed by polymerase chain reaction (PCR) amplification and labeling. Combination of this cDNA fingerprinting method with high-resolution capillary gel electrophoresis separation and precise fractionation of individual cDNA transcript representatives avoids redundant fragment selection and concomitant repetitive sequencing of abundant transcripts. Using a computer-controlled capillary electrophoresis device the transcript representatives were separated by their size and fractions were automatically collected in every 30 s into 96-well plates. The high resolving power of the sieving matrix ensured sequencing grade separation of the DNA fragments (i.e., single-base resolution) and successful fraction collection. Performance and precision of the fraction collection procedure was validated by PCR amplification of the collected DNA fragments followed by capillary electrophoresis analysis for size and purity verification. The collected and PCR-amplified transcript representatives, ranging up to several hundred base pairs, were then sequenced to create an EST library.

Animals↗

PubMed2002

Chronic ethanol increases lipopolysaccharide-stimulated Egr-1 expression in RAW 264.7 macrophages: contribution to enhanced tumor necrosis factor alpha production.

Increased production of tumor necrosis factor alpha (TNFalpha) is associated with the development of alcoholic liver disease. Culture of RAW264.7 macrophages with 25 mm ethanol for 48 h increased lipopolysaccharide (LPS)-stimulated accumulation of tumor necrosis factor alpha (TNFalpha) peptide and mRNA by 2-fold. We investigated whether chronic ethanol-induced increases in the DNA binding and/or promoter activity of the key transcription factors regulating LPS-stimulated TNFalpha promoter activity contribute to increased TNFalpha expression. Binding of Egr-1 to the TNFalpha promoter was increased by 2.5-fold after ethanol exposure, whereas NFkappaB binding was decreased to 30% of control. AP-1 binding was not affected. Changes in binding activity were paralleled by an increased contribution of the Egr-1 binding site and a decreased contribution of the NFkappaB site to LPS-stimulated TNFalpha promoter activity. Overexpression of dominant negative Egr-1 prevented the ethanol-induced increase in LPS-stimulated TNFalpha mRNA accumulation. Chronic ethanol exposure enhanced LPS-stimulated Egr-1 promoter-driven CAT expression and transcription of Egr-1. Induction of Egr-1 is dependent on ERK1/2 activation in other systems. Therefore, we investigated whether the ERK1/2 pathway mediated the chronic ethanol-induced increases in Egr-1 and TNFalpha. Increased Egr-1 promoter activity and TNFalpha mRNA accumulation after chronic ethanol were both prevented by overexpression of dominant negative ERK1/2. LPS-stimulated ERK1/2 phosphorylation was increased 2-fold in cells cultured with ethanol compared with controls. These results demonstrate that enhanced LPS-dependent activation of Egr-1 contributes to increased TNFalpha production after chronic ethanol exposure.

Animals↗

PubMed2002

Analysis of differential gene expression by ligation specificity-based transcript profiling.

An open architecture mRNA profiling technology, LEAD (Ligation specificity-based Expression Analysis Display), was developed for studying differential gene expression. This method utilizes restriction enzymes with N(m) degeneracy in their recognition/cleavage sequences to fractionate cDNA population (N represents any one of the four possible bases; while m > or = 1, represents the number of degenerate bases). The fractionated cDNAs are subpooled by selective ligation with specific adapters, and then amplified and labeled by PCR. Fluorescent-labeled cDNA fingerprints are separated by electrophoresis as distinct bands with unique size and sequence, and quantified electronically by using the LEAD Finder program. The specificity of ligation and the uniform efficiency of PCR reaction allow precise quantification of differential gene expression among samples. Transcripts of low abundance (1/100,000 copies) can be detected, allowing the status of nearly all mRNA to be monitored. Because of its sequence independence, this technology can be used to monitor gene expression in both model and nonmodel systems lacking whole genome information. It can also be applied to separate and collect different cDNA species fingerprints to build a nonredundant EST library for microarray and other applications.

Animals↗

PubMed2002

Lipopolysaccharide stimulation of ERK1/2 increases TNF-alpha production via Egr-1.

Lipopolysaccharide (LPS) is a potent activator of tumor necrosis factor-alpha (TNF-alpha) production by macrophages. LPS stimulates the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and increases TNF-alpha mRNA and protein accumulation in RAW 264.7 murine macrophages. However, the role of ERK1/2 activation in mediating LPS-stimulated TNF-alpha production is not well understood. Inhibition of ERK1/2 activation with PD-98059 or overexpression of dominant negative ERK1/2 decreased LPS-induced TNF-alpha mRNA quantity. LPS rapidly increased early growth response factor (Egr)-1 binding to the TNF-alpha promoter; this response was blunted in cells treated with PD-98059 or transfected with dominant-negative ERK1/2. Using a chloramphenicol acetyltransferase reporter gene linked to the Egr-1 promoter, we show that LPS increased Egr-1 promoter activity via an ERK1/2-dependent mechanism. These results delineate the role of ERK1/2 activation of Egr-1 activity in mediating LPS-induced increases in TNF-alpha mRNA expression in macrophages.

Animals↗

PubMed2002

[Study on the effects of disposing of pig manure with EM and OL].

The effects of EM and OL on the color, pH value, stink and the various kinds of harmful microbes of pig manure were analyzed. The results show that EM and OL could significantly reduce the stink and restrain the number of harmful microbes. At the same time, the rotting process of pig manure was accelerated, and the number of flies around pig manure reduced.

Animals↗

PubMedDate not supplied

Characterization of purified c-type heme-containing peptides and identification of c-type heme-attachment sites in Shewanella oneidenis cytochromes using mass spectrometry.

We describe methods for mass spectrometric identification of heme-containing peptides from c-type cytochromes that contain the CXXCH (X=any amino acid) sequence motif. The heme fragment ion yielded the most abundant MS/MS peak for standard heme-containing peptides with one amino acid difference for both 2+ and 3+ peptide charge states; both sequence and charge affect the extent of heme loss. Application to Shewanella oneidenis demonstrated the utility of this approach for identifying c-type heme-containing peptides from complex proteome samples.

Amino Acid Motifs↗

PubMedDate not supplied

Simple protein complex purification and identification method for high-throughput mapping of protein interaction networks.

Most current methods for purification and identification of protein complexes use endogenous expression of affinity-tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, and gel separation and in-gel digestion prior to mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pull-down assay with denaturing elution, trypsin digestion in organic solvent, and LC-ESI MS/MS protein identification using SEQUEST analysis. Our method is simple and easy to scale-up and automate, making it suitable for high-throughput mapping of protein interaction networks and functional proteomics.

Chromatography, Liquid↗