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Biomedical subjects

S Wu

Publications and source records attributed to S Wu.

At least 235 records · Page 13Linked to original sources
PubMed1998

Critical role of both retinoid nuclear receptors and retinoid-X-receptors in mediating growth inhibition of ovarian cancer cells by all-trans retinoic acid.

Retinoids have been shown to inhibit the growth of a number of human tumor cells, including several ovarian adenocarcinoma cell lines. All-trans retinoic acid (RA) is an effective growth suppressor of CA-OV3 cells but not SK-OV3 cells. Since the effects of RA are known to be mediated via the nuclear receptors (RARs and RXRs), we initially compared levels of the various RARs and RXRs in the CA-OV3 and SK-OV3 cell lines. The RA resistant SK-OV3 cells expressed reduced levels of RAR-alpha and RXR-alpha. Furthermore, induction of RAR-alpha by RA was impaired in the RA resistant SK-OV3 cells as was RARE binding and RARE-dependent transcriptional activity. These results suggested that changes in the amounts and/or activity of RARs and/or RXR-alpha could determine the growth response of ovarian tumor cells to RA. This was confirmed by modulating the levels of RARs and RXR-alpha in the SK-OV3 cells using the LacSwitch inducible expression system. Stably transfected clones of RA resistant SK-OV3 cells exhibited a significant inhibition of growth by RA treatment when RAR-alpha was induced. Overexpression of both RAR-alpha and RXR-alpha resulted in a level of growth inhibition nearly equal to that exhibited by the RA sensitive CA-OV3 cell line. Similar results were obtained when a combination of RXR-alpha and either RAR-beta or RAR-gamma was overexpressed in SK-OV3 cells. Our results show that the nuclear receptors and RXR-alpha play a critical role in mediating growth suppression by RA in ovarian cancer cells.

Blotting, Western↗
PubMed1998

GRIFIN, a novel lens-specific protein related to the galectin family.

The vertebrate lens is a relatively simple cellular structure that has evolved to refract light. The ability of the lens to focus light on the retina derives from a number of properties including the expression at high levels of a selection of soluble proteins referred to as the crystallins. In the present study, we have used differential cDNA display techniques to identify a novel, highly abundant and soluble lens protein. Though related to the family of soluble lectins called galectins, it does not bind beta-galactoside sugars and has atypical sequences at normally conserved regions of the carbohydrate-binding domain. Like some galectin family members, it can form a stable dimer. It is expressed only in the lens and is located at the interface between lens fiber cells despite the apparent lack of any membrane-targeting motifs. This protein is designated GRIFIN (galectin-related inter-fiber protein) to reflect its exclusion from the galectin family given the lack of affinity for beta-galactosides. Although the abundance, solubility, and lens-specific expression of GRIFIN would argue that it represents a new crystallin, its location at the fiber cell interface might suggest that its primary function is executed at the membrane.

Amino Acid Sequence↗
PubMed1998

Identification and requirement of three ribosome binding domains in dsRNA-dependent protein kinase (PKR).

The interferon-inducible, double-stranded (ds) RNA-dependent protein kinase (PKR) regulates protein synthesis initiation by phosphorylating the alpha-subunit of eukaryotic translation initiation factor 2 (eIF-2). The amino-terminal half of PKR contains two dsRNA binding domains, and the kinase domain resides in the carboxy-terminal half of the protein. PKR is a ribosomal-associated protein. In this report, we provide evidence that PKR contains three ribosome interaction sites, two that are localized in each of the dsRNA binding domains and one that is localized in the kinase domain. All three domains can associate with polysomes independently. The ribosome association of the dsRNA binding domains requires dsRNA binding activity. Ribosome interaction of either the individual or the combined dsRNA binding domains was disrupted by 0.1 M KCl. In contrast, the ribosome interaction of intact PKR and the isolated kinase domain was largely resistant to 0.5 M KCl. These results indicate that all three domains of PKR contribute to the high-affinity ribosomal association. After dissociation of polysomes with EDTA, both intact PKR and the isolated kinase domain were primarily associated with the 60S ribosomal subunit. Coexpression of the adenovirus VAI RNA, an RNA polymerase III gene product that binds and inactivates PKR, disrupted ribosomal association of intact PKR, but not of the isolated PKR kinase domain. The results support a model where VAI RNA induces a major conformational change in PKR to prohibit ribosome association of all interaction sites. In contrast, other inhibitors of PKR including vaccinia virus E3L and K3L gene products, and the HIV trans-activating response (TAR) element binding protein TRBP, did not disrupt ribosome association of PKR. The results suggest a novel mechanism by which viral RNAs may inactivate PKR through disrupting ribosome association.

Adenoviridae↗
PubMed1998

Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells.

Mitochondrial membrane potential, in situ, is an important indicator of mitochondrial function and dysfunction. Because of recent interest in the role of mitochondria in signaling, cell injury and cell death, there is a need for a convenient, sensitive and accurate method for the measurement of the mitochondrial membrane potential, Deltapsim, in situ, in a heterogeneous cell population. We have adapted a flow cytometry method for the quantitative measurement of DeltaPsim which utilizes the lipophilic, cationic, fluorescent probe 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)). We developed a new protocol in which cells are equilibrated with very low dye concentrations (<1 nM). Only under these condition, the cell fluorescence appears to be correlated with the magnitude of DeltaPsim, as evident from the sensitivity of the fluorescence to low concentrations of uncouplers, ionophores and inhibitors of the mitochondrial proton pumps. The magnitude of the plasma membrane potential, DeltaPsip, also affects cell fluorescence, and a procedure that corrects for this effect is outlined. This method offers a distinct advantage over existing methods for estimation of Deltapsim by flow cytometry.

Animals↗
PubMed1998

Molecular cloning and characterization of a rabbit eIF2C protein.

Rabbit eIF2C (94kDa) has been shown to play important roles in the eukaryotic peptide chain initiation process. In this study, the primary structure of rabbit eIF2C is determined by cDNA cloning. Based on the partial amino acid sequences of Endolys C cleaved fragments, degenerate oligonucleotides were synthesized and used as primers for the polymerase chain reaction to amplify the corresponding cDNA fragment from a rabbit liver cDNA library. This fragment was subsequently used to screen for larger cDNAs. Marathon cDNA amplification and 5'-rapid amplification of cDNA ends were used to confirm the translation start site. Sequences from the overlapping clones were assembled into a 3599-bp composite sequence, which contains a single open reading frame that translates into a 813-deduced amino acid sequence. Northern blot analysis of rabbit liver ploy(A)+ RNA yielded a single message species at approximately 4.6kb. Western blot analysis of rabbit reticulocyte lysate using polyclonal antibody against the 94kDa eIF2C detected a higher-molecular-weight polypeptide (140kDa). No 94kDa polypeptide was detected. The cloned cDNA was further characterized by in-vitro transcription-coupled translation in reticulocyte lysate. The translated product was precipitated with antibodies against eIF2C. Genomic Southern blot analysis indicates that the rabbit eIF2C is a single copy gene. Sequence analysis reveals that rabbit eIF2C has strong homology with a hypothetical protein in Caenorhabditis elegans.

Amino Acid Sequence↗
PubMed1998

Glial fibrillary acidic protein-apolipoprotein E (apoE) transgenic mice: astrocyte-specific expression and differing biological effects of astrocyte-secreted apoE3 and apoE4 lipoproteins.

The epsilon4 allele of apolipoprotein E (apoE) is associated with increased risk for Alzheimer's disease (AD) and poor outcome after brain injury. In the CNS, apoE is expressed by glia, predominantly astrocytes. To define the potential biological functions of different human apoE isoforms produced within the brain, transgenic mice were generated in which human apoE3 and apoE4 expression is under control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. These animals were then bred back to apoE knock-out mice. Human apoE protein is found within astrocytes and the neuropil throughout development and into the adult period, as assessed by immunocytochemistry and immunoblot analysis in several GFAP-apoE3 and E4 lines. Cultured astrocytes from these mice secrete apoE3 and apoE4 in lipoproteins that are high-density lipoprotein-like in size. When primary hippocampal neurons are grown in the presence of astrocyte monolayers derived from these transgenic mice, there is significantly greater neurite outgrowth from neurons grown in the presence of apoE3-secreting astrocytes compared with apoE4-secreting or apoE knock-out astrocytes. These effects are not dependent on direct astrocyte-neuron contact and appear to require the low-density lipoprotein receptor-related protein. These data suggest that astrocyte-secreted, apoE3-containing lipoproteins have different biological effects than apoE4-containing lipoproteins. In addition to providing information regarding the role of astrocyte-secreted apoE lipoproteins in the normal brain, these animals will also be useful in models of both AD and CNS injury.

Animals↗
PubMed1998

Integrin and cadherin synergy regulates contact inhibition of migration and motile activity.

Integrin receptors play a central role in cell migration through their roles as adhesive receptors for both other cells and extracellular matrix components. In this study, we demonstrate that integrin and cadherin receptors coordinately regulate contact-mediated inhibition of cell migration. In addition to promoting proliferation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A. Horwitz. 1996. J. Cell Biol. 133:169-184), ectopic expression of the alpha5 integrin in cultures of primary quail myoblasts promotes a striking contact-mediated inhibition of cell migration. Myoblasts ectopically expressing alpha5 integrin (alpha5 myoblasts) move normally when not in contact, but upon contact, they show inhibition of migration and motile activity (i.e., extension and retraction of membrane protrusions). As a consequence, these cells tend to grow in aggregates and do not migrate to close a wound. This phenotype is also seen with ectopic expression of beta1 integrin, paxillin, or activated FAK (CD2 FAK) and therefore appears to result from enhanced integrin-mediated signaling. The contact inhibition observed in the alpha5 myoblasts is mediated by N-cadherin, whose expression is upregulated more than fivefold. Perturbation studies using low calcium conditions, antibody inhibition, and ectopic expression of wild-type and mutant N-cadherins all implicate N-cadherin in the contact inhibition of migration. Ectopic expression of N-cadherin also produces cells that show inhibited migration upon contact; however, they do not show suppressed motile activity, suggesting that integrins and cadherins coordinately regulate motile activity. These observations have potential importance to normal and pathologic processes during embryonic development and tumor metastasis.

Animals↗
PubMed1998

[Detection of hemophilia A carriers by PCR analysis of hind III polymorphism in the factor VIII gene].

OBJECTIVE: To explore a scheme of using PCR analysis in the detection of carriers of Hind III polymorphism of factor VIII gene of hemophilia A. METHODS: Implicating intron 19 of the factor VIII gene of 6 patients with the hemophilia A and 207 unrelated X-chromosomes were amplified by PCR and were analysed by means of Amp-RFLPs of Hind III. RESULTS: The incidence of the polymorphic Hind III sites in the given population was found to be 0.29. The frequence of the Hind III heterozygotes in women calculated according to Hardy-Weinberg equation was 0.41, which proved to be informative enough for carrier detection and prenatal diagnosis of hemophilia A. 2 out of 6 families (33%)examined in this study were informative. CONCLUSION: The new scheme proved to be effective for hemophilia A carrier detection and prenatal diagnosis.

Deoxyribonuclease HindIII↗
PubMed1998

Effects of the M1 agonist xanomeline on processing of human beta-amyloid precursor protein (FAD, Swedish mutant) transfected into Chinese hamster ovary-m1 cells.

Complementary DNA (cDNA) encoding human beta-amyloid precursor protein familial Alzheimer's disease (FAD) Swedish mutant (beta APPSM) form was cloned into a mammalian expression vector (PK255) containing the CMV promoter. The vector was transfected into Chinese hamster ovary cells containing human muscarinic m1 receptors (CHO-m1), and clonal cells stably expressing beta APPSM were isolated. The effects of m1-receptor activation by the selective m1 agonist xanomeline and the non-selective muscarinic agonist carbachol on processing of beta APPSM to release soluble APP (APPs) and beta-amyloid peptide (A beta) were compared. Xanomeline stimulated APP release with a potency 1000-fold greater than that observed for carbachol. Concentrations of carbachol and xanomeline producing maximal effects on APPs release reduced the secretion of A beta by 28 and 46%, respectively. These results extend previous studies with xanomeline and suggest that cholinergic replacement therapy for Alzheimer's disease may reduce amyloid deposition.

Alzheimer Disease↗
PubMed1998

Effects of conformationally restricted synthetic retinoids on ovarian tumor cell growth.

We have used conformationally restricted retinoids to investigate the role of individual RAR subtypes and RXR in mediating the growth response of ovarian tumor cells to retinoids. Our results show that treatment of all-trans-RA-sensitive CAOV-3 cells with retinoids that bind and activate a single RAR or RXR led to a partial inhibition of growth. Treatment of all-trans-RA- resistant SKOV-3 cells did not alter growth. Maximum inhibition of growth, comparable to that observed following treatment with natural retinoids such as all-trans-RA and 9-cis-RA, was obtained only following treatment with a combination of an RAR-selective compound and an RXR-selective one. These results suggest that activation of both RAR and RXR classes is required in order to obtain maximum inhibition of ovarian tumor cell growth by retinoids. In addition, one compound, AHPN, was found to inhibit both RA-sensitive CAOV-3 and RA-resistant SKOV-3 cells. Further study of the effects of this retinoid showed that AHPN acts through an apoptotic pathway. Taken together, our results suggest that retinoids may serve as effective anti-proliferative agents in the treatment of ovarian cancer.

Apoptosis↗
PubMed1998

Orientation of the heme vinyl groups in the hydrogen sulfide-binding hemoglobin I from Lucina pectinata.

Hemoglobin I (HbI) from the claim Lucina pectinata is a unique heme protein that binds and transfers hydrogen sulfide (H2S) to a symbiotic bacteria. The metcyano, metaquo, carbon monoxy, oxy, and deoxy complexes of HbI were studies by resonance Raman (RR) spectroscopy, and the metacyano and carbon monoxy complexes were also studied by 1H-NMR. The results indicate a unique orientation of the heme 2-vinyl group relative to other heme proteins. The RR spectra of the HbICO, metHbICN, metHbIH2O, HbIO2 and deoxyHbI heme derivatives show a band at 1621 cm-1 and a shoulder at 1626 cm-1, indicative of an out-of-plane position for one of the vinyls relative to the other one. Spin-lattice relaxation properties of protons in the metHbICN complex also suggest a unique orientation for the heme 2-vinyl group of HbI. The longitudinal relaxation time (T1) for the 2-H alpha, 2-H beta c, and H beta t protons are 120 ms, 115 ms, and 135 ms, respectively. The data from both techniques suggest an out-of-plane and trans-oriented 2-vinyl group, and an in-plane and cis-oriented 4-vinyl group for the low-spin complexes of HbI. These results imply that the electron withdrawing character of the out-of-plane vinyl group contributes to the stability of the heme Fe+3 oxidation state, facilitates the binding of the H2S ligand, and promotes the stability of this ferric H2S complex.

Animals↗
PubMed1998

Treatment of hepatocellular carcinoma with the cellular tumor vaccines generated by in vitro modification of tumor cells with non gene transfer approaches.

Anti-tumor immune responses are mediated primarily by T cells. Down regulation of major histocompatibility complex (MHC) and the molecules that costimulate the immune responses is associated with defective signaling of tumor cells for T cell activation. In vitro fusion of autologous tumor cells with antigen presenting cells (APCs) or treatment of tumor cells with a combination of cytokines significantly increased the expression of MHC class I and adhesion molecules on tumor cell surfaces that costimulate host immune responses. The hybrid cells generated by fusion of tumor cells with APCs and the tumor cells treated in vitro with a combination of cytokines and pre-incubated with a bispecific monoclonal antibody (bi-Mab) cross-linking antigen on tumor cells to CD28 on T cells, become immunogenic and able to stimulate naive T cells with generation of tumor specific cytotoxic T cells both in vitro and in vivo. Immunization with the modified tumor cells elicits an immune response mediated by both CD4+ and CD8+ T cells. This response protected against a parental tumor cell challenge and cured established tumors. The approach was effective in both low immunogenic and non-immunogenic tumor systems. Modification of tumor cells with tumor:APC fusion or the two-step procedure may provide a strategy for development of tumor vaccines that is effective for cancer immunotherapy.

Animals↗
PubMed1998

LY341495 is a nanomolar potent and selective antagonist of group II metabotropic glutamate receptors.

The in vitro pharmacology of a structurally novel compound, LY341495, was investigated at human recombinant metabotropic glutamate (mGlu) receptor subtypes expressed in non-neuronal (RGT, rat glutamate transporter) cells. LY341495 was a nanomolar potent antagonist of 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD)-induced inhibition of forskolin-stimulated cAMP formation at mGlu2 and mGlu3 receptors (respective IC50S of 0.021 and 0.014 microM). At group I mGlu receptor expressing cells, LY341495 was micromolar potent in antagonizing quisqualate-induced phosphoinositide (PI) hydrolysis, with IC50 values of 7.8 and 8.2 microM for mGlu1a and mGlu5a receptors, respectively. Among the human group III mGlu receptors, the most potent inhibition of L-2-amino-4-phosphonobutyric acid (L-AP4) responses was seen for LY341495 at mGlu8, with an IC50 of 0.17 microM. LY341495 was less potent at mGlu7 (IC50 = 0.99 microM) and least potent at mGlu4 (IC50 = 22 microM). Binding studies in rat brain membranes also demonstrated nanomolar potent group II mGlu receptor affinity for LY341495, with no appreciable displacement of ionotropic glutamate receptor ligand binding. Thus, LY341495 has a unique range of selectivity across the mGlu receptor subtypes with a potency order of mGlu3 > or = mGlu2 > mGlu8 > mGlu7 >> mGlu1a = mGlu5a > mGlu4. In particular, LY341495 is the most potent antagonist yet reported at mGlu2, 3 and 8 receptors. Thus, it represents a novel pharmacological agent for elucidating the function of mGlu receptors in experimental systems.

Amino Acids↗
PubMed1998

The effect of photon noise on the detection of white flashes.

Thresholds for detecting brief, white, foveal test flashes drop abruptly within 0.2 sec of the offset of a white adapting field. The magnitude of the abrupt drop is proportional to the square root of field intensity (square root of I) correct for bleaching and dark light. Thresholds are then stable out to 1.6 sec for 200 msec tests, or recover only slightly for 20 msec tests. These results exclude some simple deterministic models in which Weber-like gain controls in the luminance pathway are assumed to recover exponentially in the dark, but can be explained parsimoniously if turning off the field abolishes photon-driven noise, improving the S/N ratio while leaving visual responsivity virtually unaltered. This theory was first put forward by Krauskopf and Reeves [(1980) Vision Research, 20, 193-196] for S-cone thresholds; it implies that the Weber law for increment thresholds is not due to a single gain control, but rather expresses the product of two distinct square root of I factors, adjustment of responsivity and photon-driven noise. Removal of the noise, not recovery of gain, permits thresholds to fall in early dark adaptation.

Adaptation, Ocular↗
PubMed1998

Group III human metabotropic glutamate receptors 4, 7 and 8: molecular cloning, functional expression, and comparison of pharmacological properties in RGT cells.

Cloning and expression in a stable mammalian cell line co-transfected with a glutamate transporter (RGT cells) were used as tools for studying the functions and pharmacological properties of group III metabotropic glutamate receptors (mGluRs). Complementary DNAs (cDNAs) encoding the human mGluR4, human mGluR7, and human mGluR8 were isolated from human cerebellum, fetal brain or retinal cDNA libraries. The human mGluR4, mGluR7 and mGluR8 receptors were 912, 915 and 908 amino acid residues long and share 67-70% amino acid similarity with each other and 42-45% similarity with the members of mGluR subgroups I and II. The human mGluR4 and mGluR7 had amino acid identity of 96% and 99.5% with rat mGluR4 and 7, respectively, whereas the human mGluR8 has 98.8% amino acid identity with the mouse mGluR8. The nucleotide and amino acid sequences in the coding region of human mGluR4 and mGluR7 were found to be identical to the previously published sequences by Flor et al. and Makoff et al. Following stable expression in RGT cells, highly significant inhibitions of forskolin stimulation of cAMP production by group III agonists were found for each receptor. The relative potencies of the group III agonist L-AP4 varied greatly between the group III clones, being mGluR8>mGluR4 >> mGluR7. The reported group II mGluR agonist L-CCG-I was a highly potent mGluR8 agonist (EC50=0.35 microM), with significant agonist activities at both mGluR4 (EC50=3.7 microM) and mGluR7 (EC50=47 microM). The antagonist potency of the purported group III mGluR antagonist MPPG also varied among the receptors being human mGluR8 >> mGluR4 = mGluR7. The expression and second messenger coupling of human group III mGluRs expressed in the RGT cell line are useful to clearly define the subtype selectivities of mGluR ligands.

ATP-Binding Cassette Transporters↗
PubMed1998

Structure, function, and molecular modeling approaches to the study of the intestinal dipeptide transporter PepT1.

The proton-coupled intestinal dipeptide transporter, PepT1, has 707 amino acids, 12 putative transmembrane domains (TMD), and is of importance in the transport of nutritional di- and tripeptides and structurally related drugs, such as penicillins and cephalosporins. By using a combination of molecular modeling and site-directed mutagenesis, we have identified several key amino acid residues that effect catalytic transport properties of PepT1. Our molecular model of the transporter was examined by dividing it into four sections, parallel to the membrane, starting from the extracellular side. The molecular model revealed a putative transport channel and the approximate locations of several aromatic and charged amino acid residues that were selected as targets for mutagenesis. Wild type or mutagenized human PepT1 cDNA was transfected into human embryonic kidney (HEK293) cells, and the uptake of tritiated glycylsarcosine [3H]-(Gly-Sar) was measured. Michaelis-Menton analysis of the wild-type and mutated transporters revealed the following results for site-directed mutagenesis. Mutation of Tyr-12 or Arg-282 into alanine has only a very modest effect on Gly-Sar uptake. By contrast, mutation of Trp-294 or Glu-595 into alanine reduced Gly-Sar uptake by 80 and 95%, respectively, and mutation of Tyr-167 reduced Gly-Sar uptake to the level of mock-transfected cells. In addition, preliminary data from fluorescence microscopy following the expression of N-terminal-GFP-labeled PepT1Y167A in HEK cells indicates that the Y167A mutation was properly inserted into the plasma membrane but has a greatly reduced Vmax.

Amino Acid Sequence↗
PubMed1998

The integrated activities of IRF-2 (HiNF-M), CDP/cut (HiNF-D) and H4TF-2 (HiNF-P) regulate transcription of a cell cycle controlled human histone H4 gene: mechanistic differences between distinct H4 genes.

Maximal transcription of a prototypical cell cycle controlled histone H4 gene requires a proliferation-specific in vivo genomic protein/DNA interaction element, Site II. Three sequence-specific transcription factors interact with overlapping recognition motifs within Site II: interferon regulatory factor IRF-2 (HiNF-M), the putative H4 subtype-specific protein H4TF-2 (HiNF-P), and HiNF-D which represents a complex of the homeodomain protein CDP/cut, CDC2, cyclin A and pRB. However, natural sequence variation in the Site II sequences of different human H4 genes abolishes binding of specific trans-acting factors; the functional consequences of these variations have not been investigated. To address the precise contribution of H4 promoter factors to the level of H4 gene transcription, we performed a systematic mutational analysis of Site II transcriptional motifs. These mutants were tested for ability to bind each of the Site II cognate proteins, and subsequently evaluated for ability to confer H4 transcriptional activity using chimeric H4 promoter/CAT fusion constructs in different cell types. We also analyzed the effect of over-expressing IRF-2 on CAT reporter gene expression driven by mutant H4 promoters and assessed H4 transcriptional control in cells nullizygous for IRF-1 and IRF-2. Our results show that the recognition sequence for IRF-2 (HiNF-M) is the dominant component of Site II and modulates H4 gene transcription levels by 3 fold. However, the overlapping recognition sequences for IRF-2 (HiNF-M), H4TF-2 (HiNF-P) and CDP/cut (HiNF-D) together modulate H4 gene transcription levels by at least an order of magnitude. Thus, maximal activation of H4 gene transcription during the cell cycle in vivo requires the integrated activities of multiple transcription factors at Site II. We postulate that the composite organization of Site II supports responsiveness to multiple signalling pathways modulating the activities of H4 gene transcription factors during the cell cycle. Variations in Site II sequences among different H4 genes may accommodate differential regulation of H4 gene expression in cells and tissues with unique phenotypic properties.

Animals↗