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Biomedical subjects

Y Liu

Publications and source records attributed to Y Liu.

At least 19 recordsLinked to original sources
PubMed1996

Two differentially expressed genes in normal human prostate tissue and in carcinoma.

Alterations in transcriptional control may contribute directly to carcinogenesis. Using the differential display technique in prostate cancer cells compared to normal prostate epithelial cells, we identified a down-regulated gene and an up-regulated gene in cancer cells. The down-regulated gene encodes human epithelial tropomyosin (TMe1), a member of the family of actin filament-binding proteins. The up-regulated gene encodes cytochrome c oxidase subunit VIc (COSVIc), a protein of the respiration chain in the mitochondrial inner membrane. The differential display pattern was confirmed by Northern hybridization in both prostate tissue and cell lines. In situ hybridization of malignant prostate epithelial tissue using a digoxigenin-labeled antisense riboprobe detected strong staining for mRNA of COSVIc, as opposed to very weak staining in normal prostate epithelium. The expression pattern of COSVIc may be a useful marker for studying the alteration of energy metabolism in cancer cells and for the diagnosis of prostate cancer.

Base Sequence↗
PubMed1996

Cannabinoid receptors are coupled to nitric oxide release in invertebrate immunocytes, microglia, and human monocytes.

The present study demonstrates that stereoselective binding sites for anandamide, a naturally occurring cannabinoid substance, can be found in invertebrate immunocytes and microglia. The anandamide-binding site is monophasic and of high affinity, exhibiting a Kd of 34.3 nM with a Bmax of 441 fmol/mg protein. These sites are highly selective, as demonstrated by the inability of other types of signaling molecules to displace [3H]anandamide. Furthermore, this binding site is coupled to nitric oxide release in the invertebrate tissues examined as well as in human monocytes. Interestingly, the cannabinoid-stimulated release of nitric oxide initiates cell rounding. Thus, these cannabinoid actions resemble those of opiate alkaloids. In this regard, we demonstrate that these signaling systems use the same effector system, i.e. nitric oxide release, but separate receptors. Last, the presence of a cannabinoid receptor in selected evolutionary diverse organisms indicates that this signaling system has been conserved for more than 500 million years.

Animals↗
PubMed1996

Vitamin K-dependent carboxylase: mRNA distribution and effects of vitamin K-deficiency and warfarin treatment.

A cDNA encoding vitamin K-dependent gamma-glutamyl carboxylase was cloned from a human Hep G2 cDNA library. The RNA transcript of the enzyme was found to be widely distributed in various human and rat tissues with liver showing the highest level. The carboxylase transcription in liver was not affected in rats treated with a single dose of warfarin (10 mg/kg) when measured up to 48 hours after the dose, though, at 12 hours, carboxylase activity measured in liver microsomes was elevated 5.4 fold over controls (p < 0.001). In rats fasted for 72 hours there was no affect on transcription in the liver while hepatic carboxylase activity increased 4.1 fold (p < 0.001). These data suggest that the increase in activity of the liver carboxylase in warfarin treated or fasted rats was not regulated by transcription but more likely was due to a posttranscriptional mechanism.

Actins↗
PubMed1996

The human FGF-8 gene localizes on chromosome 10q24 and is subjected to induction by androgen in breast cancer cells.

Androgen-induced growth factor (AIGF or FGF-8) was originally isolated from the conditioned medium of an androgen-dependent Shionogi carcinoma, SC-3, cell line. It shares structural similarity with other members of the FGF family. The temporal and spatial expression patterns of the FGF-8 gene suggest its involvement in gastrulation, regionalization of the brain, and organogenesis of the limb and face as an embryonic epithelial factor. In the adult, expression of FGF-8 is restricted to gonads including testes and ovaries. Since FGF-8 is identified as a corroborating gene in MMTV-induced mammary tumors in Wnt-1 transgenic mice and because FGF-8 manifested its autocrine mitogenic activity in SC-3 cells, it is possible that aberrant expression of FGF-8 may be present in human cancers which are hormone dependent. However, very little is known about human FGF-8. To determine whether FGF-8 plays a role in human breast cancer, we have isolated the full-length cDNA from SK-BR-3 breast cancer cells. We have also isolated the corresponding genomic DNA in a P1 cloning vector. The FGF-8 gene has been mapped to chromosome 1Oq24 using both somatic cell hybrid genetic analysis and fluorescence in situ hybridization. Finally, we show that FGF-8 gene expression in a human breast cancer cell line, MDA-MB-231, is inducible by androgen. The findings presented here will facilitate our understanding of the molecular mechanism underlying hormone-responsive breast and prostate cancers.

Adenocarcinoma↗
PubMed1996

Oxidized low density lipoprotein inhibits lipopolysaccharide-induced binding of nuclear factor-kappaB to DNA and the subsequent expression of tumor necrosis factor-alpha and interleukin-1beta in macrophages.

A large body of evidence suggests that oxidized LDL (oxLDL) has a role in atherogenesis. One effect is the impact on macrophage function. We have studied the effects of oxLDL and oxysterols on the binding of the transcription factors nuclear factor (NF)-kappaB and AP-1 to DNA. These transcription factors are involved in the regulation of several genes and expressed during activation of macrophages, for example by endotoxin (LPS). OxLDL did not induce binding of NF-kappaB. However, the LPS-induced response to NF-kappaB was substantially reduced after preincubation with oxLDL. Medium and highly oxidized LDL also decreased the constitutive DNA-binding of AP-1. Similar effects on AP-1-binding were seen with the oxysterols, 7beta-hydroxycholesterol, 24- hydroxy-, 25-hydroxy-, and 27-hydroxy-cholesterol. Our data therefore suggest an effect of oxLDL on the DNA-binding of AP-1, which might be mediated by the oxysterol content of oxLDL. A decreased LPS-induced TNF-alpha and IL-1beta mRNA and protein expression were found in macrophages incubated with oxLDL before LPS-exposure. These observations suggest that macrophages that internalize extensively oxidized LDL are suppressed in their response to inflammatory stimulation.

Base Sequence↗
PubMed1996

Morphine-induced conformational changes in human monocytes, granulocytes, and endothelial cells and in invertebrate immunocytes and microglia are mediated by nitric oxide.

We evaluated the contribution of nitric oxide (NO) to morphine-induced rounding of spontaneously activated (mobile) ameboid human monocytes, granulocytes, or arterial endothelial cells and invertebrate immunocytes and microglia. Morphine induced significant rounding and inactivation of ameboid cells within 20 min except for arterial endothelial cells, which became rounded 24 h after morphine exposure. The effects of morphine on cell conformation were blocked in the presence of N-nitro-L-arginine, a nitric oxide synthase inhibitor. Treatment of cells with the NO donor, sodium nitroprusside, induced cell rounding similar to that observed following morphine exposure, suggesting that NO release may mediate morphine-induced changes in cell conformation. The contribution of NO release to morphine-induced cell rounding was determined by direct evaluation of NO concentration in real-time using a NO-specific amperometric probe. Significant increases in NO concentration were observed 2 min after morphine stimulation, whereas morphine-induced NO release was markedly impaired by pretreatment with N-nitro-L-arginine or the opiate alkaloid antagonist, naloxone. In contrast, opioid peptides failed to induce NO release, consistent with our previous observations that demonstrated the failure of opioid peptides to promote cell rounding. Taken together, these data suggest that morphine-induced NO release may be mediated by activation of the opiate alkaloid-selective, opioid peptide-insensitive micro3 receptor, and that functional coupling of morphine to NO production has been conserved during evolution and may modulate cellular activation.

Animals↗
PubMed1996

An unusual gene arrangement for the putative chromosome replication origin and circadian expression of dnaN in Synechococcus sp. strain PCC 7942.

In eubacteria, the clustering of DnaA boxes around the dnaN (beta subunit of DNA polymerase III) and dnaA genes usually defines the chromosome replication origin (oriC). In this study, the dnaN locus from the cyanobacterium Synechococcus sp. strain PCC 7942 was sequenced. The gene order in this region is cbbZp-dnaN-orf288-purL-purF which contrasts with other eubacteria. A cluster of eleven DnaA boxes (consensus sequence: TTTTCCACA) was found in the intergenic region between dnaN and cbbZp. We also found a 41-bp sequence within this region that is 80% identical to the proposed oriC of Streptomyces coelicolor. Therefore, we propose that this intergenic region may serve as an oriC in Synechococcus. Using bacterial luciferase as a reporter, we also showed that dnaN is rhythmically expressed, suggesting that DNA replication could be under circadian control in this organism.

Bacterial Proteins↗
PubMed1996

UDP-galactose 4-epimerase: NAD+ content and a charge-transfer band associated with the substrate-induced conformational transition.

UDP-galactose 4-epimerase from Escherichia coli contains tightly bound NAD+, which participates in catalyzing the interconversion of UDP-galactose and UDP-glucose through its redox properties. The purified enzyme is a dimer of identical subunits that consists of a mixture of catalytically active subunits designated E.NAD+ and inactive, abortive complexes designated E.NADH.uridine nucleotide, in which the uridine nucleotide may be UDP-glucose, UDP-galactose, or UDP [Vanhooke, J. L., & Frey, P. A. (1994) J. Biol. Chem. 269, 31496-31404]. The abortive complexes are transformed into active E.NAD+ by denaturation of the purified enzyme at 4 degrees C in 6 M guanidine hydrochloride buffered at pH 7.0 in the presence of 0.126 mM NAD+ for 3 h, followed by dilution of guanidine hydrochloride to 0.18 M and of NAD+ to 0.076 mM for 2 h. The renatured enzyme is fully active and contains negligible amounts of NADH and uridine nucleotides. The extinction coefficent of the epimerase at 280 nm is 1.81 +/- 0.15 mL mg-1 cm-1 (epsilon 280 = 137 +/- 11 mM-1 cm-1), as determined by quantitative amino acid analysis and spectrophotometric measurements. This value allows the value of the extinction coefficient for the reduced enzyme (E.NADH)to be calculated as epsilon 344 = 5.7 mM-1 cm-1. On the basis of the new value of epsilon 280, analytical measurements of the nAD+ content of epimerase show that there are two molecules of NAD+ per dimer, which confirms conclusions from X-ray crystallography and revises the earlier bioanalytical determinations. The ultraviolet/visible absorption spectrum of E.NAD+ from denaturation-renaturation experiments reveals the presence of a broad absorption band extending from 300 nm to beyond 360 nm that cannot be attributed to NADH and appears to be a charge-transfer band. This band is partially bleached by UMP and almost totally abolished by UDP, indicating that the interactions leading to the charge-transfer band are altered by the uridine nucleotide-induced conformational change in this enzyme. This conformational change is associated with control of the chemical reactivity of NAD+ in the reaction mechanism.

Escherichia coli↗
PubMed1996

A role for Shc, Grb2, and Raf-1 in FcgammaRI signal relay.

The activation of the serine/threonine kinase, Raf-1, serves to connect upstream protein tyrosine kinases to downstream signaling events. We previously reported that FcgammaRI stimulation of interferon gamma-differentiated U937 cells (termed U937IF cells) induces a mobility shift in Erk2. Herein, we report that cross-linking of FcgammaRI receptor in U937IF cells induces a marked tyrosine phosphorylation of Raf-1 (10-fold increase). Tyrosine phosphorylation of Raf-1 is induced by FcgammaRI activation and not by PMA (1 microg/ml), N-formyl-Met-Leu-Phe (1 microM), calcium ionophore (1 microM), thrombin (0.05 unit/ml), FcgammaRII, or FcgammaRIII stimulation. The kinetics of Raf-1 tyrosine phosphorylation is rapid, reaching peak levels 1-2 min after FcgammaRI activation, and the tyrosine phosphorylation of Raf-1 precedes the activation of the respiratory burst. FcgammaRI cross-linking induces the tyrosine phosphorylation of Shc; tyrosine-phosphorylated Shc binds to Grb2 forming a Shc-Grb2 complex. The data provide evidence that the FcgammaRI receptor signals via the upstream activation of nonreceptor protein tyrosine kinases, which leads to the subsequent activation of Ras family GTPases and serine/threonine kinases, Raf-1 and mitogen-activated protein kinase.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Lineage-specific regulators couple cell lineage asymmetry to the transcription of the Caenorhabditis elegans POU gene unc-86 during neurogenesis.

The POU homeo box gene unc-86 specifies neuroblast and neural identities in the developing Caenorhabditis elegans nervous system. After an asymmetric neuroblast division, unc-86 is expressed in one of two daughter cells in 27 lineage classes that are not obviously related by function or position. We show here that unc-86 transcriptional regulatory regions detect cell lineage asymmetry to activate unc-86 expression in one of two neuroblast daughter cells. Distinct regulatory regions activate unc-86 expression in particular sets of sublineages. Therefore the unc-86 regulatory region integrates distinct cell lineage asymmetry cues to activate unc-86 expression in the many classes of neuroblast cell lineages. In agreement with such lineage-specific regulation of unc-86 asymmetric activation, mutations in lin-11 (LIM homeo box), ham-1, and lin-17 affect the asymmetry of unc-86 expression in particular cell lineages, and mutations in lin-32 (achaete/scute family), vab-3 (Pax-6 homolog) and egl-5 (Abd-B homolog) affect the establishment of unc-86 expression in other cell lineages. Homologs of unc-86 and many of these unc-86 regulators have been implicated in control of neurogenesis in vertebrates and invertebrates. These data suggest that unc-86 acts in a phylogenetically conserved pathway that couples neuroblast cell lineage asymmetry to the generation of diverse neural types.

Animals↗
PubMed1996

Vitamin D receptors repress basal transcription and exert dominant negative activity on triiodothyronine-mediated transcriptional activity.

We have examined vitamin D receptor (VDR), thyroid hormone receptor (TR), and retinoid X receptor beta (RXR beta) binding to vitamin D response elements (VDREs), two thyroid hormone response elements (TREs) (DR4 and F2), and a retinoic acid response element (DR5). VDR/RXR bound well to the VDREs and to DR4 and DR5 using the electrophoretic mobility shift assay. Surprisingly, VDR/RXR also bound well to F2, which contains half-sites arranged as an inverted palindrome. In co-transfection experiments using CV-1 cells, we observed that VDR repressed basal transcription in the absence of ligand on DR3 and osteopontin VDREs and F2, but had no effect on DR4 or DR5. VDR selectively mediated ligand-dependent transcription on only VDREs. VDR also exhibited dominant negative activity as it blocked triiodothyronine (T3)-mediated transcriptional activity on DR4 and F2. These results demonstrate that VDR/RXR heterodimers can bind promiscuously to a wide range of hormone response elements, including inverted palindromes. Moreover, they show that unliganded VDRs, similar to TRs and retinoic acid receptors, can repress basal transcription. Last, they also suggest a novel repressor function of VDR on T3-mediated transcription which may be significant in tissues where VDR and TR are co-expressed.

Animals↗
PubMed1996

An amperometric new methylene blue N-mediating sensor for hydrogen peroxide based on regenerated silk fibroin as an immobilization matrix for peroxidase.

A simple and effective procedure was described for the immobilization of peroxidase in regenerated silk fibroin membrane prepared from waste silk. The membranes of regenerated silk fibroin with or without peroxidase, before or after the ethanol treatment, were characterized by ir spectra. An amperometric H202 sensor, based on the immobilized peroxidase in regenerated silk fibroin membrane, in the use of new methylene blue N as an electron transfer mediator, was fabricated. The characteristics of the sensor with respect to linearity, response time, effect of pH and temperature, stability, and reproducibility were investigated. Dependences of Michaelis-Menten constant KMapp on the concentration of the mediator, and the applied potential were also studied and the results were presented. The sensor was highly sensitive to H2O2 with a detection limit of 1.0 x 10(-7)M and with response time of less than 40 s.

Biosensing Techniques↗
PubMed1996

Visualizing a specific contact in the HIV-1 Tat protein fragment and trans-activation responsive region RNA complex by photocross-linking.

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a stem-loop structure containing two helical stem regions separated by a trinucleotide bulge. The Tat protein contains a basic RNA-binding region (amino acids 49-57) located in the carboxyl-terminal half of the protein, and peptides containing this basic domain of Tat protein can bind TAR RNA with high affinities. We synthesized a 31-amino acid Tat fragment (amino acids 42-72) containing the basic region and part of flanking regulatory core domain that formed a specific complex with TAR RNA. Upon UV irradiation (254 nm), this Tat fragment cross-linked covalently with TAR RNA. Sites of cross-links were determined on both the TAR RNA and Tat protein fragment by RNA and protein sequencing, respectively. These results revealed that guanosine 26 of TAR RNA was cross-linked with tyrosine 47 of the Tat peptide. Our results provide the first physical evidence for a direct amino acid-base contact in Tat-TAR complex. Recently, orientation of the Tat-(42-72) was determined in our laboratory by psoralen.Tat-(42-72) conjugate (Wang, Z., and Rana, T. M. (1995) J. Am. Chem. Soc. 117, 5438-5444). On the basis of our findings, we suggest a model in which Tat binds to TAR RNA by inserting the basic recognition sequence into the major groove with an orientation where lysine 41 in the core domain of Tat contacts the lower stem and Tyr47 is close to G26 of TAR RNA. The knowledge of the orientation of Tat and details of other interactions with TAR RNA in Tat-TAR complex has significant implications for understanding gene regulation in HIV-1.

Amino Acid Sequence↗
PubMed1996

Facile liquid chromatographic enantioresolution of native amino acids and peptides using a teicoplanin chiral stationary phase.

The glycopeptide antibiotic teicoplanin is shown to be a highly effective stationary phase chiral selector for the resolution of underivatized amino-acid and imino-acid enantiomers. Fifty four of these compounds (including all chiral protein amino acids) as well as a number of dipeptides were resolved. Hydro-organic mobile phases are used and no buffers or added salts are needed in most cases. Hence the purified analytes are easily isolated in pure form, if needed, by evaporating of the solvent. The effect of pH, organic modifier type and amount are discussed. The enantioselective separation mechanism is examined using both molecular modeling and retention data. The strongest stereoselective interaction is for carboxy-terminated D-amino-acids. In case of peptides, it is not necessary for these to be a D-, D-, terminal sequence for strong interactions. In some cases, including Ala-Ala, the L-, D-, terminal sequence showed greater interaction with the teicoplanin chiral stationary phase.

Amino Acid Sequence↗
PubMed1996

Toxic effects of ethylene oxide residues on bovine embryos in vitro.

The potential of ethylene oxide (EtO) residues in exposed plastic tissue culture dishes to adversely affect bovine oocyte maturation, fertilization and subsequent embryonic development was monitored. In experiment 1, the effects of aeration time and aeration combined with washing of EtO-gassed culture dishes on the extent of residual toxicity were investigated. There was no cleavage in any treatment in which oocytes were matured and fertilized in dishes exposed to EtO. EtO residues caused functional degeneration of oocytes even when culture dishes were aerated for more than 12 days post EtO-exposure and repeatedly washed. In experiment 2, the residual toxicity of EtO gas on in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) were evaluated. Cleavage rate significantly decreased and post-cleavage development was retarded in ova maintained in dishes treated with EtO either during IVM or IVF. EtO residues may be more detrimental to spermatozoa than to oocytes which may have been the primary cause of fertilization failure during IVF.

Animals↗
PubMed1996

Role of NAD(P)H:quinone oxidoreductase (DT-diaphorase) in cytotoxicity and induction of DNA damage by streptonigrin.

The metabolism, cytotoxicity, and genotoxicity of streptonigrin (SN) w ere determined in two human colon carcinoma cell lines: HT-29 with high NAD(P)H:quinone oxidoreductase (EC 1.6.99.2, DTD) activity and BE with undetectable DTD activity. Dicumarol-sensitive oxidation of NADH was observed with HT-29 cytosol, but not with BE cytosol. Oxygen consumption was also observed using HT-29 cytosol, but was absent with BE cytosol. Dicumarol inhibited oxygen consumption with HT-29 cytosol, but deferoxamine had no effect, suggesting that divalent metal cations were not necessary for efficient auto-oxidation of SN hydroquinone. In cytotoxicity studies, SN was much more toxic to the DTD-rich HT-29 cells than to the DTD-deficient BE cells. Deferoxamine decreased toxicity in both cell lines, implicating hydroxyl radicals produced during Fenton-type reactions as the toxic species. In the genotoxicity assay, SN induced a much higher incidence of DNA strand breaks in HT-29 cells than in BE cells, and deferoxamine protected against DNA strand breaks in both cell lines. Some evidence of DNA repair was also observed in the two cell lines. These results support an important role for DTD in the cytotoxicity of SN in the high DTD HT-29 colon carcinoma cell line.

Antibiotics, Antineoplastic↗
PubMed1996

Tyrosine phosphorylation at the membrane-microfilament interface: a p185neu-associated signal transduction particle containing Src, Abl and phosphorylated p58, a membrane- and microfilament-associated retroviral gag-like protein.

Microfilaments are associated with the microvillar membrane in the 13762 ascites rat mammary carcinoma cells by stable interaction with a large, multimeric signal transduction particle (STP) containing the (proto)oncogene receptor p185(neu). In vitro kinase assays on isolated microvilli and microvillar fractions enriched in the putative signal transduction particle showed a high specific activity of tyrosine kinase activity compared to that of membranes from EGF receptor-overexpressing A431 cells maximally activated by EGF. Assays of velocity sedimentation fractions from microvillar lysates in the presence and absence of the exogenous tyrosine kinase substrate poly-glu-tyr polypeptide (poly-E(4)Y) suggested association of the tyrosine kinase activity with STP-enriched microvillar fractions. The particulate fractions also contained discrete endogenous tyrosine-phosphorylated proteins, including prominent bands of approximately 42 and 58 kDa. Addition of ATP to these fractions resulted in a rapid increase in tyrosine phosphorylation of these and several other proteins, as detected by anti-phosphotyrosine blots. Immunoprecipitation and immunoblotting with anti-phosphotyrosine antibody of SDS-solubilized ascites cells and microfilament core fractions showed nine major bands; the electrophoretic mobilities of most of these in the cell immunoprecipitate and microfilament core were the same. In vivo and in situ phosphorylation, phosphoamino acid analysis, immunoprecipitation, 2-dimensional isoelectric focusing/SDS PAGE and immunoblot analysis showed that one of the prominent substrates is p58(gag), a retroviral Gag-like cytoplasmic STP component implicated in stabilizing microfilament-membrane interactions. Immunoblotting identified two peripheral membrane tyrosine kinases, p6O(src) and p120(abl), stably associated with the p185(neu)-containing signal transduction particle. These results provide further evidence for the constitutive activation of this aggressive mammary tumor and suggest a rote for phosphorylation of p58(gag) in organization of the STP at the membrane-microfilament interface in these cells.

Adenosine Triphosphate↗