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Biomedical subjects

W Li

Publications and source records attributed to W Li.

At least 955 records · Page 53Linked to original sources
PubMed1996

Defective transcription of the IL-2 gene is associated with impaired expression of c-Fos, FosB, and JunB in anergic T helper 1 cells.

Anergic CD4+ Th cells do not produce IL-2 when challenged with Ag-pulsed accessory cells because of a transcriptional defect. In this work, we report that these anergic T cells are defective in their ability to up-regulate protein binding and transactivation at two critical IL-2 DNA enhancer elements: NF-AT (nuclear factor of activated T cells; a sequence that binds a heterotrimeric NFATp, Fos, and Jun protein complex) and Activator Protein-1 (AP-1) (that binds Fos and Jun heterodimers). Western blot analysis of nuclear extracts showed that the impaired DNA-protein interactions in anergic T cells were associated with poor expression of the inducible AP-1 family members c-Fos, FosB, and JunB. However, the reduced expression of these proteins was not the result of a global TCR/CD3-signaling defect because CD3 cross-linking induced an equivalent increase in intracellular-free calcium ions, as well as NFATp dephosphorylation, translocation to the nucleus, and DNA binding in both normal and anergic T cells. Thus, defective IL-2 gene transcription appears to be due, at least in part, to a selective block in the expression of the AP-1 Fos and Jun family members in anergic T cells.

Animals↗
PubMed1996

Expression of an ATP binding mutant of PKC-delta inhibits Sis-induced transformation of NIH3T3 cells.

In an effort to determine the role of protein kinase C-delta (PKC-delta) in cellular transformation mediated by the sis proto-oncogene, we cotransfected expression vectors containing cDNAs that encode for c-sis with an ATP binding mutant of PKC-delta (PKC-delta K376R) or wild type PKC-delta (PKC-delta WT) into NIH3T3 cells. Our results showed that expression of PKC-delta K376R severely impaired Sis-induced focus formation, whereas cotransfection of PKC-delta WT cDNA had no effect on Sis-mediated transformation. Consistent with this result, PKC-delta K376R expression also inhibited PDGF-BB-mediated anchorage-independent colony formation. While cotransfection of a vector containing a dominant negative mutant of ras (N17 ras) cDNA potently inhibited Sis-induced transformation, the expression of PKC-delta K376R did not block transformation mediated by v-H-Ras or v-Raf. In addition, PDGF-BB-induced Raf and mitogen-activated protein kinase activation, which are known to be downstream molecules in the Ras cascade, were not affected by the expression of PKC-delta K376R, indicating that PKC-delta and Ras are segregated in mediating Sis-induced transformation. Interestingly, expression of PKC-delta K376R strongly reduced TPA responsive element (TRE) transactivation induced by PDGF stimulation, suggesting that activation of TRE-containing genes, which may be involved in Sis-mediated transformation, are negatively regulated by expression of PKC-delta K376R.

3T3 Cells↗
PubMed1996

Two-dimensional crystallization of rabbit C-reactive protein on lipid monolayers.

Two-dimensional (2D) crystals of rabbit C-reactive protein (CRP) have been obtained by protein binding on lipid monolayers at the air/water interface. Two different types of crystalline arrays of CRP were obtained, by specific binding and non-specific adsorption to the lipids. Electron crystallographic analysis of the negatively stained specimens showed that the unit cell parameters of the CRP 2D crystals formed by specific binding were a=81 angstroms, b=78 angstroms, gamma=118.35 degrees, and those formed by nonspecific adsorption were a=74 angstroms, b=67 angstroms, gamma=95.5 degrees, both with the layer group p1. Projection maps were obtained at a resolution of 26 angstroms and 22 angstroms respectively. They showed that only the monomers of the CRP were packed in the 2D arrays and the orientations of the monomers on the lipid monolayers were different in the two types of crystals. By comparing the two projection maps, a preliminary shape of the CRP monomer has been derived. A model of the pentameric structure of the oligomeric CRP has been proposed.

Adsorption↗
PubMed1996

Nck inhibits NGF and basic FGF induced PC12 cell differentiation via mitogen-activated protein kinase-independent pathway.

Proto-oncogene Nck, an adapter molecule containing three SH3 and one SH2 domains, binds to cell surface receptors and mediates mitogenic effects in the cells. Overexpression of Nck caused cell transformation in vitro and tumor formation in the nude mice. The mechanism of this action by Nck, however, remained unclear. Rat adrenal pheochromocytoma cell line PC12 provides a useful system for studying growth factor-regulated cell proliferation and differentiation. Serum and epidermal growth factor (EGF) stimulate proliferation, whereas nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) cause growth arrest and sympathetic neurite outgrowth in these cells. To study the function of Nck, we generated stable clones of PC12 cells overexpressing the human Nck. We report here that the overexpressed Nck caused continued proliferation of PC12 cells even in the presence of NGF and blocked both the NGF- and bFGF-induced neurite outgrowth. Anti-sense but not sense oligonucleotides to the human Nck resumed the NGF-induced differentiation, indicating the specific inhibitory effect of Nck. Interestingly, Nck did not interfere with the kinetics of NGF- and EGF-stimulated protein tyrosine phosphorylation and the mitogen-activated protein kinase (MAPK) activation, suggesting that Nck inhibited the induced PC12 cell differentiation via a MAPK-independent mechanism. This study has provided a useful system for further understanding the function of Nck.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Heregulin is rapidly translocated to the nucleus and its transport is correlated with c-myc induction in breast cancer cells.

Heregulins (neuregulins) are a family of proteins known to interact and activate the receptor tyrosine kinases ErbB2 in association with ErbB3 or ErbB4. Using immunofluorescence microscopy, electron microscopy autoradiography, and SDS-PAGE analysis of nuclear fractions, we show that the heregulin-beta1(1-244) isoform is rapidly internalized and translocated to the nucleus of SK-BR-3 breast cancer cells as an intact molecule. Heregulin-beta 1(1-244) treatment up-regulated expression of c-myc mRNA and protein, which was also observed to undergo its own translocation from the cytosol to the nucleus. c-myc thus appears to be a cellular target gene of HRGbeta 1(1-244), and its induction may be related to the nuclear translocation of heregulin.

Breast Neoplasms↗
PubMed1996

Identification of a domain within the carboxyl-terminal region of the beta platelet-derived growth factor (PDGF) receptor that mediates the high transforming activity of PDGF.

We have reported previously that a chimeric platelet-derived growth factor receptor (PDGFR) possessing the ligand binding domain of the alpha PDGFR and the intracellular domain of the beta PDGFR (alpha 340 beta 342 R) was markedly more efficient than the wild type alpha PDGFR (alpha RWT) in its ability to enhance PDGF-A transforming activity in NIH/3T3 fibroblasts. To determine the region within the cytoplasmic domain of beta PDGFR that confers this higher transforming activity, we generated several additional alpha/beta PDGFR chimerae. When a chimeric PDGFR possessing the first 933 amino-terminal amino acids from the alpha PDGFR and the final 165 amino acids from the carboxyl-terminal of the beta PDGFR (alpha 933 beta 942 R) was cotransfected with the PDGF-A gene into NIH/3T3 cells, it showed a similar high efficiency to enhance PDGF-A chain transforming activity as alpha 340 beta 342 R. However, when chimeric PDGFRs in which either the kinase insert domain (alpha beta RKI) or the last 79 amino acids from the carboxyl-terminal end of the beta PDGFR (alpha 1024 beta 1028 R) were substituted into alpha PDGFR sequences were cotransfected with PDGF-A, they showed similar low efficiencies in enhancing transforming activity as the alpha RWT. These results predicted that the 86 amino acids following the tyrosine kinase 2 domain of beta PDGFR (amino acid residues 942-1027) were responsible for the higher transforming activity of beta PDGFR. To confirm this finding, we next constructed a chimera in which amino acid residues 942-1028 of the beta PDGFR (alpha beta 942-1028R) were substituted for those in the alpha PDGFR. Cotransfection experiments indicated that alpha beta 942-1028R increased transforming activity of PDGF-A to similar extent as the alpha 933 beta 942R, or alpha 340 beta 342R. Therefore, our findings define a critical domain within the noncatalytic region of beta PDGFR intracellular domain that confers the higher focus forming activity mediated by the beta PDGFR.

3T3 Cells↗
PubMed1996

Elevated connexin43 immunoreactivity at sites of amyloid plaques in Alzheimer's disease.

The distribution of the astrocytic gap junctional protein, connexin43 (Cx43) was compared immunohistochemically with that of amyloid plaques in Alzheimer's Disease (AD) brain. By light microscopy, cortical areas containing numerous beta/A4 amyloid plaques exhibited increased immunostaining density for Cx43 and some plaques corresponded exactly to sites of intensified Cx43 immunoreactivity. By electron microscopy, Cx43 was localized to astrocytic gap junctions in AD brain. Increased Cx43 expression in AD may represent an attempt to maintain tissue homeostasis by augmented intercellular communication via gap junction formation between astrocytic processes that invest senile plaques, or alternatively, an aberrant induction of astrocytic Cx43 expression which may further compromise homeostasis and exacerbate pathological conditions in the microenvironment of amyloid plaques.

Alzheimer Disease↗
PubMed1996

Different conformations and site selectivity of HO-2-Co(III)-bleomycin A2 and Co (III)-bleomycin A2 bound to DNA oligomers.

Conformational properties of HO2(-)-Co(III)-bleomycin A2 (Form I) and Co(III)-bleomycin (Form II) bound to DNA oligomers offering either principal cleavage site for the drug, d(GGAAGCTTCC)2 or d(AAACGTIT)2, have been studied by NMR methods. Form I binds in slow exchange to these oligomers. It retains most of its solution nuclear Overhauser effects (NOEs) upon binding to either oligomer. Pyrimidinyl methyl protons from the metal domain of the drug make an NOE connection with a G5 2-amino proton on DNA. The bithiazole intercalates between base pairs involving either C6 and T7 or T6 and T7 of the two DNA molecules, according to NOE connections between the bithiazole protons and protons from these bases and changes in the positions of their chemical shifts. Form II also retains most of its solution NOEs upon association with the first oligomer. However, in contrast to Form I it binds to DNA in fast exchange on the NMR time scale over the temperature range of 5-35 degrees C and does not break the degeneracy of the DNA proton chemical shifts. No intermolecular NOEs between Form II and the 10-mer have been detected. Likewise, the major perturbation in chemical shift of the histidine H2 and guanine G5 protons seen in Form I-DNA adducts is absent in Form II-DNA. The association constant of Form II with d(GGAAGCTTCC)2 in 20 mM HEPES buffer at pH 7.4 and 25 degrees C is 1.7 x 10(5) M(-1), and 1.0 mol of Form II bind per mol of 10-mer.

Antibiotics, Antineoplastic↗
PubMed1996

Selection of trypsin inhibitors in phage peptide library.

The newly developed techniques of peptide libraries have become a conventional and efficient method in screening ligands of proteins of interest. We present here the successful results of selection of trypsin inhibitors in a phage hexapeptide library. After affinity selection and activity assay, peptide sequences, deduced from DNA sequencing of the phage peptides with the most striking trypsin activity, share some common features with trypsin inhibitors reported. All of the phage peptides selected out and those native and synthetic trypsin inhibitors reported are composed of three parts: (a) positively charged part (Arg, Lys or their analogs); (b) polar part that may form hydrogen bonds with Ser in the active site of trypsin; (c) hydrophobic part that interacts with the nonpolar region of trypsin active site.

Amino Acid Sequence↗
PubMed1996

Properties of chicken erythrocyte histone deacetylase associated with the nuclear matrix.

Histone H2B is deacetylated more rapidly than H3 and H4 in chicken immature erythrocytes. Histone deacetylase from chicken immature erythrocytes was partially purified, and the histone specificities of the multiple histone deacetylase forms were determined. Ion-exchange (Q-Sepharose) and gel-exclusion (Superdex 200) chromatography of extracts from erythrocyte nuclei showed two forms (HD1 and HD2) of histone deacetylase. HD1, with a molecular mass of about 55 kDa, preferred free H3-H4 relative to H2A-H2B, while HD2, with a molecular mass of approx. 220 kDa, had a slight preference for H3-H4. HD1 and HD2 differed in pH- and ion-strength-dependence. HD2 dissociated into HD1 when treated with 1.6 M NaCl or when applied to a Q-Sepharose column. The enzymic properties of nuclear-matrix-bound histone deacetylase showed a striking difference from that of HD1 and HD2, particularly in its strong preference for H2A-H2B. Treatment of the nuclear matrix with 1.6 M NaCl and 1% 2-mercaptoethanol solubilized histone deacetylase which chromatographed as 400 and 220 kDa forms on a Superdex 200 column. The solubilized enzyme retained its histone preference for H2A-H2B. Chromatography of the nuclear-matrix-derived enzyme on Q-Sepharose yielded one peak of enzyme activity with chromatographic properties and histone specificities similar to those of HD1. These results provide support for the active form of the enzyme in situ being a high-molecular mass complex associated with proteins that are components of the nuclear matrix. Substrate preference of the enzyme is governed by the proteins associated with the histone deacetylase.

Animals↗
PubMed1996

Blocked signal transduction to the ERK and JNK protein kinases in anergic CD4+ T cells.

T cells activated by antigen receptor stimulation in the absence of accessory cell-derived costimulatory signals lose the capacity to synthesize the growth factor interleukin-2 (IL-2), a state called clonal anergy. An analysis of CD3- and CD28-induced signal transduction revealed reduced ERK and JNK enzyme activities in murine anergic T cells. The amounts of ERK and JNK proteins were unchanged, and the kinases could be fully activated in the presence of phorbol 12-myristate 13-acetate. Dephosphorylation of the calcineurin substrate NFATp (preexisting nuclear factor of activated T cells) also remained inducible. These results suggest that a specific block in the activation of ERK and JNK contributes to defective IL-2 production in clonal anergy.

Animals↗