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Biomedical subjects

W Li

Publications and source records attributed to W Li.

At least 667 records · Page 37Linked to original sources
PubMed1998

C2-ceramide attenuates prostaglandin F2alpha-induced vasoconstriction and elevation of [Ca2+]i in canine cerebral vascular smooth muscle.

Sphingolipids have emerged as important components of signal transduction pathways involved in a variety of cellular processes. In the present study, we examined the effects of C2-ceramide, a cell-permeable sphingolipid, on contraction of canine cerebral vascular smooth muscle and intracellular free Ca2+ ([Ca2+]i). C2-ceramide (10(-8)-10(-4) M) alone did not elicit any significant changes in either basal tension or resting levels of [Ca2+]i in canine cerebrovascular muscle. However, C2-ceramide (10(-7)-10(-4) M) attenuated prostaglandin F2alpha (PGF2alpha)-induced contractions in isolated canine cerebrovascular smooth muscle rings. C2-ceramide (10(-5) M) inhibited the secondary phasic rise of [Ca2+]i evoked by PGF2alpha in cultured canine cerebral vascular smooth muscle cells, resulting in decreases in the elevation in [Ca2+]i. NO inhibitors (L-NNA, L-NMMA), an inhibitor of prostanoid synthesis (indomethacin), an inhibitor of opiate actions and several inhibitors of the pharmacologic actions of various vasoactive amines all failed to interfere with the vasorelaxant response of C2-ceramide. Our results suggest that the sphingomyelin signaling pathway may play an important regulatory role in cerebral arterial wall tone.

Animals↗
PubMed1998

Conformational and dynamic changes of Yersinia protein tyrosine phosphatase induced by ligand binding and active site mutation and revealed by H/D exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Protein tyrosine phosphatases (PTPase) play important roles in the intracellular signal transduction pathways that regulate cell transformation, growth, and proliferation. Here, solvent accessibility is determined for backbone amide protons from various segments of wild-type Yersinia PTPase in the presence or absence of 220 microM vanadate, a competitive inhibitor, as well as an active site mutant in which the essential cysteine 403 has been replaced by serine (C403S). The method consists of solution-phase H/D exchange, followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform ion cyclotron resonance mass spectrometry. Proteolytic segments spanning approximately 93.5% of the primary sequence are analyzed. Binding of vanadate reduces the H/D exchange rate throughout the protein, both for the WpD loop and for numerous other residues that are shielded when that loop is pulled down over the active site on binding of the inhibitor. The single active site C403S mutation reduces solvent access to the WpD loop itself, but opens up the structure in several other segments. Although the 3D structure of the ligand-bound C403S mutant is similar to that of the wild-type PTPase, and the C403S mutant and the wild-type enzyme display similar affinities for vanadate, the thermodynamics for binding of vanadate is different for the two proteins. Collectively, these results establish the flexibility of the WpD loop (previously inferred by comparing PTPase X-ray single-cyrstal diffraction structures in the presence and absence of a tungstate inhibitor), as well as several other signficant changes in segment exposure and/or flexibility that are not evident from X-ray structures.

Amino Acid Sequence↗
PubMed1998

Myelin gene expression after experimental contusive spinal cord injury.

After incomplete traumatic spinal cord injury (SCI), the spared tissue exhibits abnormal myelination that is associated with reduced or blocked axonal conductance. To examine the molecular basis of the abnormal myelination, we used a standardized rat model of incomplete SCI and compared normal uninjured tissue with that after contusion injury. We evaluated expression of mRNA for myelin proteins using in situ hybridization with oligonucleotide probes to proteolipid protein (PLP), the major protein in central myelin; myelin basic protein (MBP), a major component of central myelin and a minor component of peripheral myelin; and protein zero (P0), the major structural protein of peripheral myelin, as well as myelin transcription factor 1 (MYT1). We found reduced expression of PLP and MBP chronically after SCI in the dorsal, lateral, and ventral white matter both rostral and caudal to the injury epicenter. Detailed studies of PLP at 2 months after injury indicated that the density of expressing cells was normal but mRNA per cell was reduced. In addition, P0, normally restricted to the peripheral nervous system, was expressed both at the epicenter and in lesioned areas at least 4 mm rostral and caudal to it. Thus, after SCI, abnormal myelination of residual axons may be caused, at least in part, by changes in the transcriptional regulation of genes for myelin proteins and by altered distribution of myelin-producing cells. In addition, the expression of MYT1 mRNA and protein seemed to be upregulated after SCI in a pattern suggesting the presence of undifferentiated progenitor cells in the chronically injured cord.

Animals↗
PubMed1998

Neuronal differentiation of precursors in the neocortical ventricular zone is triggered by BMP.

Neocortical neurons begin to differentiate soon after they are generated by mitoses at the surface of the ventricular zone (VZ). We provide evidence here that bone morphogenetic protein (BMP) triggers neuronal differentiation of neocortical precursors within the VZ. In cultures of dissociated neocortical neuroepithelial cells, BMPs increase the number of MAP-2- and TUJ1-positive cells within 24 hr of treatment. In explant cultures, BMP-4 treatment leads to an increase in the number of TUJ1-positive cells within the ventricular zone. Furthermore, truncated, dominant-negative, BMP type I receptor, introduced into neocortical precursors by retrovirus-mediated gene transfer, blocks neurite elaboration and migration out of the VZ. Finally, immunocytochemistry indicates that BMP protein is present at the VZ surface. Together, these results indicate that BMP protein is present within the VZ, that BMP is capable of promoting neuronal differentiation, and that signaling through BMP receptors triggers neuronal precursors to differentiate and migrate out of the VZ.

Animals↗
PubMed1998

Molecular staging of malignant melanoma: correlation with clinical outcome.

CONTEXT: For most solid tumors, the metastatic status of regional lymph nodes is the strongest predictor of relapse and survival. However, routine pathological examination of lymph nodes may underestimate the number of patients with melanoma who have nodal metastases. OBJECTIVE: To determine the clinical significance of a highly sensitive molecular assay for occult nodal metastases for the staging of patients with melanoma. DESIGN: A prospective cohort study of consecutive patients in which lymphatic mapping and sentinel lymph node (SLN) biopsy were performed on 114 melanoma patients with clinical stage I and stage II disease. The SLNs were bivalved, and half of each specimen was submitted for routine pathological examination. The other half was submitted for molecular detection of submicroscopic metastases using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for tyrosinase messenger RNA as a marker for the presence of melanoma cells. Patient follow-up averaged 28 months. SETTING: A major university-based melanoma referral center at a National Cancer Institute-designated cancer center. PATIENTS: A total of 114 patients with newly diagnosed cutaneous malignant melanoma who were at risk for regional nodal metastases. MAIN OUTCOME MEASURE: Melanoma recurrence and overall survival. RESULTS: Twenty-three patients (20%) had pathologically positive SLNs, and all of these patients were also RT-PCR positive. Of the 91 pathologically negative patients, 44 were RT-PCR negative and 47 were RT-PCR positive. There was a recurrence rate among 14 (61%) of the 23 patients who were both pathologically and RT-PCR positive and a recurrence rate among 1 (2%) of 44 patients who were both pathologically and RT-PCR negative. For patients who were upstaged by the molecular assay (pathologically negative, RT-PCR positive), there was a recurrence rate among 6 (13%) of 47 patients. The differences in recurrence rates and overall survival between the pathologically negative, RT-PCR-negative and pathologically negative, RT-PCR-positive patient groups were statistically significant (P= .02 for disease-free survival and for overall survival). In both univariate and multivariate regression analyses, the histological and RT-PCR status of the SLNs were the best predictors of disease-free survival. CONCLUSIONS: The use of an RT-PCR assay for detection of submicroscopic melanoma metastases in SLNs improved the prediction of melanoma recurrence and overall survival over routine pathological examination.

Adult↗
PubMed1998

[Loss of heterozygosity microsatellite DNA on chromosome loci 3, 5, 7, 9 and 18 in human pancreatic cancer].

OBJECTIVE: Detecting the loss of heterozygosity in paraffin-embedded pancreatic cancer tissues. METHODS: Analysing loss of heterzygosity (LOH) of microsatellite DNA on chromosome loci 3,5,7,9 and 18 with PCR-SSLP-silver stain method in pancreatic cancer. RESULTS: In 45 sporadic pancreatic cancer samples and their paired control tissue, LOH was detected on site for D18S46( 18q21.1,31.0%), D18S474 (18q21.1, 20.0%), D9S176 (9q22-31,20.0%), D3S1234 (3p14.2,17.5%),D 3S1289 (3p21.1, 15.7%), D3S1481 (3p14.2,4.9%), D7S486 (7q22,3.6%), D5S365 (5q32, 3.2%) and D3S587 (3p24-26, 2.6%). CONCLUSION: Different percentages of loss of heterzygosity on specific chromosomal regions were found, and the meaning of the results was discussed. Some key genes may play a role in the pathogenesis of pancreatic cancer.

Adult↗
PubMed1998

Coexpression of IL-6 and soluble IL-6R causes nodular regenerative hyperplasia and adenomas of the liver.

Studies with tumor necrosis factor p55 receptor- and interleukin-6 (IL-6)-deficient mice have shown that IL-6 is required for hepatocyte proliferation and reconstitution of the liver mass after partial hepatectomy. The biological activities of IL-6 are potentiated when this cytokine binds soluble forms of its specific receptor subunit (sIL-6R) and the resulting complex interacts with the transmembrane signaling chain gp130. We show here that double transgenic mice expressing high levels of both human IL-6 and sIL-6R under the control of liver-specific promoters spontaneously develop nodules of hepatocellular hyperplasia around periportal spaces and present signs of sustained hepatocyte proliferation. The resulting picture is identical to that of human nodular regenerative hyperplasia, a condition frequently associated with immunological and myeloproliferative disorders. In high expressors, hyperplastic lesions progress with time into discrete liver adenomas. These data strongly suggest that the IL-6/sIL-6R complex is both a primary stimulus to hepatocyte proliferation and a pathogenic factor of hepatocellular transformation.

Adenoma↗
PubMed1998

[Effects of taurine and extraction of cristata L on serum Zn, Cu and Ca in rats].

Forty rats fed with basic diets were randomly divided into 4 groups. NG group were fed with basic diets. The other three groups were fed with high fat diets. The rats in TA group and EC group were given TA 100 mg/kg or EC 100 mg/kg each day respectively in addition to high fat diet for 8 weeks. The results showed that taurine and extraction of cristata L not only increased the level of red cell SOD and the content of serum Zn (P < 0.05), but also decreased the contents of TC, MDA in the wall of artery and decreased the level of serum LDH significantly (P < 0.05 or P < 0.01). TA and EC increased significantly the content of serum Cu and decreased the ratio of serum Cu to Zn in the high fat diet rats (P < 0.01), and decreased the contents of serum Ca also. The results indicate that TA and EC may play some role in lipid metabolism and inhibit atherosclerosis by regulating the levels of Zn, Cu and Ca in rats.

Animals↗
PubMed1998

Identification of Nck family genes, chromosomal localization, expression, and signaling specificity.

Already a dozen molecules share binding to the Src homology (SH) 3 domains of human Nck, an SH3-SH3-SH3-SH2 adapter protein. We reason that there may be multiple gene members of Nck to accommodate the large binding repertoires. Here we report identification of novel human and mouse Nck genes and rename them as the Nckalpha and Nckbeta genes (including the human Nckalpha, human Nckbeta, mouse Nckalpha, and mouse Nckbeta genes). Nckalpha and Nckbeta share 68% amino acid identity, whereas the two Nckalpha and two Nckbeta across the species show 96% identity to each other. The human Nckbeta gene is mapped to 2q12, whereas the human Nckalpha gene has previously been mapped at 3q21. Antibodies specifically against Nckalpha and Nckbeta detect Nckalpha and Nckbeta with an identical molecular mass in the same cells of various origins. Ectopically expressed Nckbeta, but not its SH2 domain mutant, strongly inhibits epidermal growth factor- and platelet-derived growth factor-stimulated DNA synthesis. Consistently, epidermal growth factor receptor and platelet-derived growth factor receptor preferentially interact with Nckbeta over Nckalpha in vitro. This study indicates that Nck is a multiple gene family and that each gene may have its own signaling specificity. Because previous anti-Nck (human Nckalpha) antibodies cross-react with Nckbeta, reassessment of those studies with specific Nck genes would be necessary.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Induction of T cell anergy by high concentrations of immunodominant native peptide is accompanied by IL-10 production and a block in JNK activity.

The ability to induce anergy in antigen-specific T cells has potential therapeutic value for altering pathologic immune responses. This study was undertaken to further analyze changes in cytokine production and intracellular signaling during anergy induction using high concentrations of native peptide ligand of tetanus toxoid (TT)- and myelin basic protein (MBP)-specific human T cell lines. The TT-selected T cell line could be rendered unresponsive to its dominant epitope in a dose-dependent manner (IC50 = 0.03 microg/ml). The TT-selected line, as well as three T cell clones established from this line, continued to produce IFN-gamma and significantly increased IL-4 and IL-10 production when anergy was induced with high concentrations of the immunodominant epitope. JNK enzymatic activity was blocked in anergized T cells. The MBP-selected line could likewise be rendered unresponsive by incubation with supraoptimal concentrations of immunodominant peptide and anergy induction was accompanied by IL-10 release. Both T cell lines could be anergized by the autopresentation of native peptide since anergy was induced in cultures lacking fresh antigen-presenting cells. This study shows that the mitogen-activated protein kinase cascade is blocked when anergy is induced to high concentrations of soluble peptide.

Aged↗
PubMed1998

Organophosphorus neuropathy target esterase inhibitors selectively block outgrowth of neurite-like and cell processes in cultured cells.

This study compares two direct-acting neuropathy target esterase (NTE) inhibitors (mipafox and 2-octyl-4H-1,3,2-benzodioxophosphorin 2-oxide (OBDPO)), a metabolic precursor to an NTE inhibitor (tri-o-cresyl phosphate or TOCP) and a potent acetylcholinesterase inhibitor (chlorpyrifos oxon or CPO) for their effects on outgrowth of neurite-like and cell processes and on viability in differentiated cultured cells (rat adrenal pheochromocytoma (PC-12) and brain glial tumor (C6)). The direct-acting NTE inhibitors block process outgrowth by 50% or more at 50-100 microM for OBDPO and 100-200 microM for mipafox, well below their cytotoxic levels (EC50 values, 445-474 microM for OBDPO and 1021-1613 microM for mipafox). In contrast, the effects on process development for TOCP and CPO parallel their cytotoxicity. These findings suggest that inhibition of neurite-like and cell process outgrowth by OBDPO and mipafox may be associated with NTE inhibition.

Acetylcholinesterase↗
PubMed1998

Genetic deletion of AT2 receptor antagonizes angiotensin II-induced apoptosis in fibroblasts of the mouse embryo.

To examine whether angiotensin II (Ang II) can trigger apoptosis via Ang II type 2 (AT2) receptor, two genotypes of skin fibroblasts cultured from the AT2 receptor gene targeting homozygous (AT2-/-) and wild-type (AT2+/+) mouse embryos, respectively, were studied when exposed to Ang II. In the AT2+/+ fibroblasts, mRNA expression of the AT2 receptor was modulated by Ang II in a dose-dependent manner and apoptosis appeared with the convincing features of internucleosomal DNA fragmentation and DNA content decrease after stimulation with Ang II, whereas Ang II had no significant impact on the AT2-/- fibroblasts due to the AT2 receptor gene deletion. This is the first report using a gene targeting study to demonstrate that Ang II induces apoptosis through the AT2 receptor in the fibroblasts of the mouse embryo.

Angiotensin II↗
PubMed1998

Regulation of cytochrome P450 enzymes by aryl hydrocarbon receptor in human cells: CYP1A2 expression in the LS180 colon carcinoma cell line after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin or 3-methylcholanthrene.

It has been difficult to study the regulation of cytochrome P4501A2 (CYP1A2) because expression of this enzyme is reported to be limited or absent in cell culture. We found that CYP1A2 can be induced significantly by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (MC), or benz[a]anthracene in the human colon carcinoma cell line LS180. TCDD and MC each caused a dramatic elevation of CYP1A2 mRNA, as assessed by reverse transcription-polymerase chain reaction or by northern blot analysis. TCDD also increased immunoreactive CYP1A2 protein and the activity of phenacetin-O-deethylase, a diagnostic catalytic marker for CYP1A2. The induction of CYP1A2 at all levels (mRNA, protein, catalytic activity) was concentration- and time-dependent: the EC50 for mRNA induction by TCDD = 0.5 nM, and by MC = 1.4 microM. Inducible CYP1A2 mRNA also was detected at lower levels in two other human cell lines, the hepatoma cell line HepG2 and the breast carcinoma cell line MCF-7. CYP1A1 and CYP1B1, additional CYP1 enzymes regulated by the aryl hydrocarbon receptor (AHR), also were inducible by TCDD and MC in LS180 cells; their concentration-dependent induction was highly correlated with induction of CYP1A2 at mRNA, protein, and catalytic levels. CYP1B1 was constitutively expressed and inducible in the LS180, MCF-7, and HepG2 cell lines as well as in the human choriocarcinoma cell line JEG-3 and the squamous cell carcinoma line A431. CYP1A2 was neither constitutively expressed nor inducible in A431 or JEG-3 cells. The expression of mRNAs encoding the regulators of CYP1 enzymes-the AHR and its heterodimerization partner, the ARNT (AH receptor nuclear translocator) protein-was not altered by treatment with TCDD or MC. However, the cytosolic content of AHR protein and ARNT protein was depleted substantially following treatment with TCDD. The LS180 cell line should constitute a good model for further mechanistic studies on AHR-regulated CYP1A2 expression.

Aryl Hydrocarbon Receptor Nuclear Translocator↗
PubMed1998

CCAAT enhancer- binding protein beta is required for normal hepatocyte proliferation in mice after partial hepatectomy.

After two-thirds hepatectomy, normally quiescent liver cells are stimulated to reenter the cell cycle and proliferate to restore the original liver mass. The level of bZIP transcription factor CCAAT enhancer-binding protein beta (C/EBPbeta) increases in the liver during the period of cell proliferation. The significance of this change in C/EBP expression is not understood. To determine the role of C/EBPbeta in the regenerating liver, we examined the regenerative response after partial hepatectomy in mice that contain a targeted disruption of the C/EBPbeta gene. Posthepatectomy, hepatocyte DNA synthesis was decreased to 25% of normal in C/EBPbeta -/- mice. The reduced regenerative response was associated with a prolonged period of hypoglycemia that was independent of expression of C/EBPalpha protein and gluconeogenic genes. C/EBPbeta -/- livers showed reduced expression of immediate-early growth-control genes including the Egr-1 transcription factor, mitogen-activated protein kinase protein tyrosine phosphatase (MKP-1), and HRS, a delayed-early gene that encodes an mRNA splicing protein. Cyclin B and E gene expression were dramatically reduced in C/EBPbeta -/- livers whereas cyclin D1 expression was normal. The abnormalities in immediate-early gene expression in C/EBPbeta -/- livers were distinct from those seen in IL-6 -/- livers. These data link C/EBPbeta to the activation of metabolic and growth response pathways in the regenerating liver and demonstrate that C/EBPbeta is required for a normal proliferative response.

Animals↗
PubMed1998

Dual recognition and the role of specificity-determining residues in colicin E9 DNase-immunity protein interactions.

The immunity protein Im2 can bind and inhibit the noncognate endonuclease domain of the bacterial toxin colicin E9 with a Kd of 19 nM, 6 orders of magnitude weaker than that of the cognate immunity protein Im9 with which it shares 68% sequence identity. Previous work from our laboratory has shown that the specificity differences of these four-helix immunity proteins is due almost entirely to helix II which is largely variable in sequence in the immunity protein family. From alanine scanning mutagenesis of Im9 in conjunction with high-field NMR data, a dual recognition model for colicin-immunity protein specificity has been proposed whereby the conserved residues of helix III of the immunity protein act as the anchor of the endonuclease binding site while the variable residues of helix II control the specificity of the protein-protein interaction. In this work, we identify three residues (at positions 33, 34, and 38) in helix II which define the specificity differences of Im2 and Im9 for colicin E9 and, using alanine mutagenesis of the putative endonuclease binding surface of Im2, compare the distribution of binding energies for conserved and nonconserved sites in both immunity proteins. This comparison highlights the conserved residues of both Im2 and Im9 as the major determinants of E9 DNase binding energy. Conversely, the nonconserved, specificity-determining residues only contribute to the E9 DNase binding energy in the cognate Im9 protein, while in the noncognate immunity protein Im2, they either destabilize the complex or do not contribute to the binding energy. This comparative alanine scan of two immunity proteins therefore supports the dual recognition mechanism of selectivity in colicin-immunity protein interactions and provides a basis for understanding specificity in other protein-protein interaction systems involving structurally conserved protein families.

Alanine↗
PubMed1998

Donor pretreatment with Flt-3 ligand augments antidonor cytotoxic T lymphocyte, natural killer, and lymphokine-activated killer cell activities within liver allografts and alters the pattern of intragraft apoptotic activity.

BACKGROUND: Liver allografts are accepted across major histocompatibility complex (MHC) barriers in mice and induce donor-specific tolerance without requirement for immunosuppressive therapy. There is evidence that passenger leukocytes may play a key role in tolerance induction. Flt-3 ligand (FL) is a recently cloned hematopoietic cytokine that strikingly augments functional dendritic cells (DCs) within lymphoid and nonlymphoid tissue. METHODS: The expression of costimulatory molecules and MHC class II antigen on DCs isolated from livers of FL-treated B10 (H2b) mice (10 microg/day; 10 days) was examined by flow cytometric analysis, and their allostimulatory activity assessed in primary mixed leukocyte cultures. B10 livers from FL-treated donors were transplanted orthotopically into naive C3H (H2k) recipients. Donor cells (MHC class II+) in recipient spleens were identified by immunohistochemistry. Antidonor cytotoxic T lymphocyte activity, and both natural killer and lymphokine-activated killer cell activities of graft nonparenchymal cells and host splenocytes were determined using isotope release assays. Apoptotic activity within liver grafts was determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling. RESULTS: DCs isolated from livers of FL-treated donor mice exhibited increased cell surface expression of CD40, CD80, CD86, and IAb, and augmented T cell allostimulatory activity compared with controls. Within 24 hr of organ transplantation, the numbers of donor IAb+ cells within recipient spleens was augmented substantially compared with normal liver recipients. Livers from FL-treated donors were rejected acutely (median survival time, 5 days), whereas control B10 liver allografts survived >100 days. Nonparenchymal cells from rejecting grafts 4 days after transplantation exhibited increased antidonor cytotoxic T lymphocyte, natural killer, and lymphokine-activated killer cell activities compared with cells from spontaneously accepted grafts. This augmented cytotoxic reactivity was associated with histologic evidence of injury to bile duct epithelium and vascular endothelium that was not readily evident in controls. CONCLUSION: Thus, although normal livers provide allostimulatory signals sufficient to elicit an antidonor immune response, regulatory mechanisms that may include apoptosis of graft-infiltrating T cells, and that are overcome by augmenting the number of functional donor DCs, may account for inherent liver tolerogenicity.

Animals↗