Biomedical subjects

Liang Shi

Publications and source records attributed to Liang Shi.

5 recordsLinked to original sources

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↗