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Biomedical subjects

E Li

Publications and source records attributed to E Li.

At least 19 recordsLinked to original sources
PubMed2006

Fascin is a potential biomarker for early-stage oesophageal squamous cell carcinoma.

BACKGROUND: Fascin, an actin-binding protein, is usually expressed at a low level in normal epithelium, but is markedly up regulated in several types of carcinomas. Reports on fascin expression in oesophageal squamous cell carcinoma (ESCC) and precancerous lesions remain rare. AIM: To show the roles of fascin in the progression from normal epithelium to invasive ESCC. METHODS: Fascin expression in 102 sections embedded in paraffin wax, including samples of normal mucosa (n = 20), dysplasia (n = 10), ESCC (n = 62) and special sections (n = 10) of a full-length mucosa layer from the distant margin to the cancer focus of the excised oesophagus, and 49 fresh specimens of ESCC was analysed by immunohistochemistry, western blot and real-time reverse transcription-polymerase chain reaction. Fascin expression in ESCC cell lines was also investigated. RESULTS: In the immunohistochemical study, the positive rate of fascin was significantly higher in the tumour tissue than in the normal epithelium (p = 0.020), but no significant difference was shown between ESCC and dysplasia (p = 1.000). Immunostaining for fascin was only apparent in the basal layer of the normal epithelium. However, in the dysplasia, positive staining was observed in most of the heterogeneous cells from the basal layer to the granular layer of the epithelium. Fascin expression was seen to increase progressively from the normal epithelium to invasive ESCC. Up regulation of fascin was observed in 87.76% (43/49) and 77.55% (38/49) of the specimens, respectively, using western blot and real-time reverse transcription-polymerase chain reaction assays; 80% (4/5) of ESCC cell lines also expressed fascin at a high level. Furthermore, overexpression of fascin was markedly correlated with cell proliferation and lymph node metastasis. CONCLUSIONS: These findings suggested that fascin was associated with the transformation and development of ESCC and implicated the potential of fascin as a novel biomarker that would allow the tumour to be identified at an early stage in high-risk individuals.

Adult↗
PubMed2006

Establishment and maintenance of DNA methylation patterns in mammals.

In mammals, CpG methylation patterns are established and maintained during development by the Dnmt1 and Dnmt3 families of DNA methyltransferases. These enzymes share conserved catalytic motifs in their C-terminal regions, but have unique N-terminal regulatory domains. Studies over the past several years have shed light on the molecular mechanisms by which DNA methylation patterns are regulated. This review focuses on recent advances in defining the functional domains of DNA methyltransferases and identifying interacting proteins that may contribute to the functional specializations of these enzymes.

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

Effect of TSIX disruption on XIST expression in male ES cells.

XIST and its antisense partner, TSIX, encode non-coding RNAs and play key roles in X chromosome inactivation. Targeted disruption of TSIX causes ectopic expression of XIST in the extraembryonic tissues upon maternal transmission, which subsequently results in embryonic lethality due to inactivation of both X chromosomes in females and a single X chromosome in males. TSIX, therefore, plays a crucial role in maintaining the silenced state of XIST in CIS and regulates the imprinted X inactivation in the extraembryonic tissues. In this study, we examined the effect of TSIX disruption on XIST expression in the embryonic lineage using embryonic stem (ES) cells as a model system. Upon differentiation, XIST is ectopically activated in a subset of the nuclei of male ES cells harboring the TSIX-deficient X chromosome. Such ectopic expression, however, eventually ceased during prolonged culture. It is likely that surveillance by the X chromosome counting mechanism somehow shuts off the ectopic expression of XIST before inactivation of the X chromosome.

Animals↗
PubMed2001

p35 and p39 are essential for cyclin-dependent kinase 5 function during neurodevelopment.

Cyclin-dependent kinase 5 (Cdk5) plays a pivotal role in brain development and neuronal migration. Cdk5 is abundant in postmitotic, terminally differentiated neurons. The ability of Cdk5 to phosphorylate substrates is dependent on activation by its neuronal-specific activators p35 and p39. There exist striking differences in the phenotypic severity of Cdk5-deficient mice and p35-deficient mice. Cdk5-null mutants show a more severe disruption of lamination in the cerebral cortex, hippocampus, and cerebellum. In addition, Cdk5-null mice display perinatal lethality, whereas p35-null mice are viable. These discrepancies have been attributed to the function of other Cdk5 activators, such as p39. To understand the roles of p39 and p35, we created p39-null mice and p35/p39 compound-mutant mice. Interestingly, p39-null mice show no obvious detectable abnormalities, whereas p35(-/-)p39(-/-) double-null mutants are perinatal lethal. We show here that the p35(-/-)p39(-/-) mutants exhibit phenotypes identical to those of the Cdk5-null mutant mice. Other compound-mutant mice with intermediate phenotypes allow us to determine the distinct and redundant functions between p35 and p39. Our data strongly suggest that p35 and p39 are essential for Cdk5 activity during the development of the nervous system. Thus, p35 and p39 are likely to be the principal, if not the only, activators of Cdk5.

Animals↗
PubMed2001

Local vs systemic corticosteroids in the treatment of carpal tunnel syndrome.

The authors compared the effectiveness of low-dose, short-term oral prednisolone vs local methylprednisolone injection in a prospective, double-blinded, parallel treatment study of carpal tunnel syndrome (CTS). A single injection of 15 mg methylprednisolone resulted in significant improvement in global symptom scores over a 12-week period. This study demonstrated the superiority of local steroid injection to oral steroid in the treatment of CTS.

Administration, Oral↗
PubMed2001

Characterization of a novel proapoptotic caspase-2- and caspase-9-binding protein.

Caspases play important roles in regulating apoptotic signaling pathways. Here we report the cloning, by the yeast two hybrid system with dominant negative caspase-2 as "bait," of a proapoptotic molecule named proapoptotic caspase adaptor protein (PACAP), encoded by a 372-base pair open reading frame. Binding of this novel protein to caspase-2 (casp-2) was confirmed in yeast two hybrid, in vitro, and in vivo assays. The deduced amino acid sequence revealed homology to functional motifs, including ATP and cytochrome c binding sites. PACAP mRNA was widely expressed in most human tissues; in transfected cells, PACAP was diffusely expressed in the cytoplasm. Bindings studies with the PACAP recombinant protein demonstrated specific binding to casp-2 and casp-9 but not to casp-3, -4, -7, or -8 in cell extracts. Cotransfection experiments showed that PACAP binds to casp-2 and -9 in 293T cells. In addition, studies with truncated PACAP demonstrated a requirement for residues 39-72 of PACAP for specific binding to casp-2 and -9. Transient transfection of PACAP into 293T human kidney cells and rat-1 fibroblasts triggered apoptosis at 24 h, which was at least in part prevented by an inhibitor of casp-3-like enzymes. Transfection of PACAP into human B cell lines using a retroviral system also triggered apoptotic cell death. In addition, transcription of PACAP in primary human B cells was dramatically down-regulated early after cellular activation by CD40L and Staphylococcus aureus and markedly up-regulated as the cells apoptose. These findings identify a novel proapoptotic caspase adaptor protein.

Adaptor Proteins, Signal Transducing↗
PubMed2001

The bifunctional Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2) protein is necessary for amebic growth and survival and requires an intact C-terminal domain for both alcohol dahydrogenase and acetaldehyde dehydrogenase activity.

The intestinal protozoan pathogen Entamoeba histolytica lacks mitochondria and derives energy from the fermentation of glucose to ethanol with pyruvate, acetyl enzyme Co-A, and acetaldehyde as intermediates. A key enzyme in this pathway may be the 97-kDa bifunctional E. histolytica alcohol dehydrogenase 2 (EhADH2), which possesses both alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase activity (ALDH). EhADH2 appears to be a fusion protein, with separate N-terminal ALDH and C-terminal ADH domains. Here, we demonstrate that EhADH2 expression is required for E. histolytica growth and survival. We find that a mutant EhADH2 enzyme containing the C-terminal 453 amino acids of EhADH2 has ADH activity but lacks ALDH activity. However, a mutant consisting of the N-terminal half of EhADH2 possessed no ADH or ALDH activity. Alteration of a single histidine to arginine in the putative active site of the ADH domain eliminates both ADH and ALDH activity, and this mutant EhADH2 can serve as a dominant negative, eliminating both ADH and ALDH activity when co-expressed with wild-type EhADH2 in Escherichia coli. These data indicate that EhADH2 enzyme is required for E. histolytica growth and survival and that the C-terminal ADH domain of the enzyme functions as a separate entity. However, ALDH activity requires residues in both the N- and C-terminal halves of the molecule.

Alcohol Dehydrogenase↗
PubMed2001

Production of functional antibodies generated in a nonlytic insect cell expression system.

A monoclonal antibody directed against the type 2 adenovirus (Ad2) penton base protein was cloned and expressed in Spodoptera frugiperda (Sf9) cells using a nonlytic vector system. The coding sequences for the immunoglobulin light and heavy chains were placed under the control of the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus immediate-early 2 (OpIE2) promoter. Transfected Sf9 cells continuously secreted the antibody which retained the ability to recognize both native and recombinant Ad2 penton base proteins. Bifunctional penton base antibodies were also generated by fusing a gene for a growth factor or a cytokine at the 3' end of the Ig constant heavy chain domain. The quantity and activity of recombinant antibodies generated in the nonlytic insect cell system could be determined relatively quickly compared to other expression systems. Moreover, these recombinant proteins were not subjected to proteolytic degradation as frequently occurs during baculovirus-mediated cell lysis and the levels of recombinant antibodies produced in the nonlytic system were comparable to those reported for cytolytic baculovirus vectors.

Adenoviridae↗
PubMed2001

Integrin alpha(v)beta1 is an adenovirus coreceptor.

The human embryonic kidney (HEK293) cell line, commonly used for recombinant adenovirus (Ad) propagation, does not express the Ad coreceptor alpha(v)beta3 or alpha(v)beta5 integrins, yet these cells are efficiently infected by Ad vectors. Here we demonstrate that Ad binds to HEK293 cells via the fiber receptor CAR and is subsequently internalized via interaction with integrin alpha(v)beta1. Function-blocking antibodies directed against alpha(v) or beta1, but not beta3, beta5, or alpha5, integrin subunits block Ad infection and viral endocytosis. Therefore, alpha(v)beta1 serves as a coreceptor for Ad infection, and the lack of beta3 and/or beta5 but the relatively high expression of alpha(v)beta1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.

Adenoviridae↗
PubMed2001

The role of effectors of the activin signalling pathway, activin receptors IIA and IIB, and Smad2, in patterning of tooth development.

The gene for activin betaA is expressed in the early odontogenic mesenchyme of all murine teeth but mutant mice show a patterning defect where incisors and mandibular molars fail to develop but maxillary molars develop normally. In order to understand why maxillary molar tooth development can proceed in the absence of activin, we have explored the role of mediators of activin signalling in tooth development. Analysis of tooth development in activin receptor II and Smad2 mutants shows that a similar tooth phenotype to activin betaA mutants can be observed. In addition, we identify a novel downstream target of activin signalling, the Iroquois-related homeobox gene, Irx1, and show that its expression in activin betaA mutant embryos is lost in all tooth germs, including the maxillary molars. These results strongly suggest that other transforming growth factor beta molecules are not stimulating the activin signalling pathway in the absence of activin. This was confirmed by a non-genetic approach using exogenous soluble receptors to inhibit all activin signalling in tooth development, which reproduced the genetic phenotypes. Activin, thus, has an essential role in early development of incisor and mandibular molar teeth but this pathway is not required for development of maxillary molars.

Activin Receptors, Type II↗
PubMed2001

Regulation of imprinted X-chromosome inactivation in mice by Tsix.

In mammals, X-chromosome inactivation is imprinted in the extra-embryonic lineages with paternal X chromosome being preferentially inactivated. In this study, we investigate the role of Tsix, the antisense transcript from the Xist locus, in regulation of Xist expression and X-inactivation. We show that Tsix is transcribed from two putative promoters and its transcripts are processed. Expression of Tsix is first detected in blastocysts and is imprinted with only the maternal allele transcribed. The imprinted expression of Tsix persists in the extra-embryonic tissues after implantation, but is erased in embryonic tissues. To investigate the function of Tsix in X-inactivation, we disrupted Tsix by insertion of an IRESbetageo cassette in the second exon, which blocked transcripts from both promoters. While disruption of the paternal Tsix allele has no adverse effects on embryonic development, inheritance of a disrupted maternal allele results in ectopic Xist expression and early embryonic lethality, owing to inactivation of both X chromosomes in females and single X chromosome in males. Further, early developmental defects of female embryos with maternal transmission of Tsix mutation can be rescued by paternal inheritance of the Xist deletion. These results provide genetic evidence that Tsix plays a crucial role in maintaining Xist silencing in cis and in regulation of imprinted X-inactivation in the extra-embryonic tissues.

Animals↗
PubMed2001

Solution structure of cytochrome b(5) mutant (E44/48/56A/D60A) and its interaction with cytochrome c.

Using 1617 meaningful NOEs with 188 pseudocontact shifts, a family of 35 conformers of oxidized bovine microsomal cytochrome b5 mutant (E44/48/56A/D60A) has been obtained and is characterized by good resolution (rmsd to the mean structure are 0.047 +/- 0.007 nm and 0.095 +/- 0.008 nm for backbone and heavy atoms, respectively). The solution structure of the mutant, when compared with the X-ray structure of wild-type cytochrome b(5), has no significant changes in the whole folding and secondary structure. The binding between cytochrome b(5) and cytochrome c shows that the association constant of the mutant-cytochrome c complex is much lower than the one for wild-type complex (2.2 x 10(4) M(-1) vs. 5.1 x 10(3) M(-1)). The result suggests the four acidic residues have substantial effects on the formation of the complex between cytochrome b(5) and cytochrome c, and therefore it is concluded reasonably that the electrostatic interaction plays an important role in maintaining the stability and specificity of the complex formed. The competition between the ferricytochrome b(5) mutant and [Cr(oxalate)(3)](3-) for ferricytochrome c shows that site III of cytochrome c, which is a strong binding site to wild-type cytochrome b(5), still binds to the mutant with relatively weaker strength. Our results indicate that certain bonding geometries do occur in the interaction between the present mutant and cytochrome c and these geometries, which should be quite different from the ones of the Salemme and Northrup models.

Amino Acid Sequence↗