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Biomedical subjects

Yang Shi

Publications and source records attributed to Yang Shi.

At least 73 records · Page 4Linked to original sources
PubMed2003

Acetylation regulates subcellular localization of the Wnt signaling nuclear effector POP-1.

Lymphoid enhancer factor/T-cell factor (LEF/TCF) are transcription factors that mediate the Wnt signaling pathway, and have crucial roles during embryonic development in various organisms. Here we report that acetylation enhances nuclear retention of POP-1, the Caenorhabditis elegans LEF/TCF homolog, through increasing nuclear import and blocking nuclear export. We identify three lysines that are acetylated in vivo, and demonstrate their essential requirement for proper nuclear localization and biological activity of POP-1 during C. elegans embryogenesis. The conservation of these lysines among other LEF/TCF family members suggests that acetylation may be an important, evolutionarily conserved mechanism regulating subcellular distribution of LEF/TCF factors.

Acetylation↗
PubMed2003

Mammalian RNAi for the masses.

Just a couple of years ago, only biologists working with plants or Caenorhabditis elegans could use RNA-mediated interference (RNAi) technology to gain insight into gene function. However, the recent groundbreaking discovery that in vitro synthesized, 21- to 23-nucleotide, double-stranded RNAs can act as small interfering RNAs (siRNAs) to elicit gene-specific inhibition in mammalian cells has made RNAi possible in mammalian systems too. Reported only a year ago, mammalian RNAi is already changing our way of studying gene function in higher eukaryotes. And, a recent exciting advance allows delivery of siRNAs into mammalian cells by a DNA vector. In addition to providing a low-cost alternative to the chemically synthesized siRNAs, this DNA-vector-based strategy is capable of mediating stable target gene inhibition, thus allowing gene function analysis over an extended period of time.

Animals↗
PubMed2003

Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis.

RNA interference (RNAi) can achieve sequence-selective inactivation of gene expression in a wide variety of eukaryotes by introducing double-stranded RNA corresponding to the target gene. Here we explore the potential of RNAi as a therapy for amyotrophic lateral sclerosis (ALS) caused by mutations in the Cu, Zn superoxide dismutase (SOD1) gene. Although the mutant SOD1 is toxic, the wild-type SOD1 performs important functions. Therefore, the ideal therapeutic strategy should be to selectively inhibit the mutant, but not the wild-type SOD1 expression. Because most SOD1 mutations are single nucleotide changes, to selectively silence the mutant requires single-nucleotide specificity. By coupling rational design of small interfering RNAs (siRNAs) with their validation in RNAi reactions in vitro and in vivo, we have identified siRNA sequences with this specificity. A similarly designed sequence, when expressed as small hairpin RNA (shRNA) under the control of an RNA polymerase III (pol III) promoter, retains the single-nucleotide specificity. Thus, RNAi is a promising therapy for ALS and other disorders caused by dominant, gain-of-function gene mutations.

Alleles↗
PubMed2003

Smad6 recruits transcription corepressor CtBP to repress bone morphogenetic protein-induced transcription.

Smad6 and Smad7 are inhibitory Smads induced by transforming growth factor beta-Smad signal transduction pathways in a negative-feedback mechanism. Previously it has been thought that inhibitory Smads bind to the type I receptor and block the phosphorylation of receptor-activated Smads, thereby inhibiting the initiation of Smad signaling. Conversely, few studies have suggested the possible nuclear functions of inhibitory Smads. Here, we present compelling evidence demonstrating that Smad6 repressed bone morphogenetic protein-induced Id1 transcription through recruiting transcriptional corepressor C-terminal binding protein (CtBP). A consensus CtBP-binding motif, PLDLS, was identified in the linker region of Smad6. Our findings show that mutation in the motif abolished the Smad6 binding to CtBP and subsequently its repressor activity of transcription. We conclude that the nuclear functions and physical interaction of Smad6 and CtBP provide a novel mechanism for the transcriptional regulation by inhibitory Smads.

Alcohol Oxidoreductases↗
PubMed2003

Relationship between genetic polymorphism of cytochrome P450IIE1 and fatty liver.

AIM: To study the correlation between genetic polymorphism of cytochrome P450IIE1 (CYPIIE1) and fatty liver. METHODS: Peripheral blood mononuclear cells were collected in 56 patients with fatty liver, 26 patients without fatty liver and 20 normal controls. Then PCR-RFLP was used to analyze genetic polymorphism of CYPIIE1 in monocytes on the region of Pst I and Rsa I. RESULTS: The frequency of homozygotic C1 gene in patients with alcoholic fatty liver (28.6%), obese fatty liver (38.5%), or diabetic fatty liver (33.3%) was significantly lower than that of the corresponding patients without fatty liver (100%, 100% and 80% respectively), while the frequency of C2 genes, including C1/C2 and C2/C2, was significantly higher (71.4%/0%, 61.5%/0%, and 66.7%/20%) (P<0.01). The frequency distribution of the above genes of non-fatty liver patients (100%/0, 100%/0, and 80%/20%) was not significantly different from that of the normal controls (85%/15%) (P>0.05). CONCLUSION: The genetic polymorphism of CYPIIE1 on the position of Pst I and Rsa I is related to the susceptibility of fatty liver. Besides, C2 gene may play a key role in the pathogenesis of fatty liver.

Cytochrome P-450 CYP2E1↗
PubMed2003

Studies on specific interaction of beta-2-glycoprotein I with HBsAg.

AIM: To observe the binding activity of beta-2-glycoprotein I(beta(2)GPI) to hepatitis B surface antigen (HBsAg) and the possible roles of beta(2)GPI in hepatitis B virus (HBV) infection. METHODS: The rationale of ELISA methods and ELISA-based research method and ligand-blotting technique were used to detect the specific interaction of beta(2)GPI with HBsAg. RESULTS: With the increase of rHBsAg, the binding of beta(2)GPI to rHBsAg elevated, and these changes had statistic significance. When we added non- biotinlyated beta(2)GPI, the OD value significantly decreased though they still were positively relevant to rHBsAg, suggesting non- biotinlyated beta(2)GPI competed with biotinlyated beta(2)GPI to saturate the binding sites on rHBsAg. Meanwhile BSA was used as negative control to substitute for rHBsAg coating the plates. The results indicated no interaction between beta(2)GPI and BSA, suggesting the affinity of beta(2)GPI to rHBsAg was specific. The ligand blotling indicated that beta(2)GPI might bind to rHBsAg no matter whether it was under reduced condition or not. CONCLUSION: The binding of beta(2)GPI to HBsAg suggests that beta(2)GPI may be a carrier of HBV and that beta(2)GPI may play important roles in HBV infection.

Binding Sites↗
PubMed2003

Inhibitory effect of all-trans retinoic acid on human hepatocellular carcinoma cell proliferation.

AIM: To study the inhibitory effect of all-trans retinoic acid on human hepatocellular carcinoma cell line SMMC-7721 and to explore the mechanism of its effect. METHODS: SMMC-7721 cells were divided into two groups, one treated with all-trans retinoic acid (ATRA) for 5 days and the other as a control group. Light microscope and electron microscope were used to observe the morphological changes. Telomerase activity was analyzed with silver-stained telomere repeated assay protocal (TRAP). Expression of Caspase-3 was demonstrated with western blot. RESULTS: ATRA-treated cells showed differentiation features including small and pyknotic nuclei, densely stained chromatin and fewer microvilli. Besides, ATRA could inhibit the activity of telomerase, promote the expression of Caspase-3 and its activation. CONCLUSION: Telomerase activity and Caspase-3 expression are changed in human hepatocellular carcinoma cell line SMMC-7721 treated with all-trans retinioc acid. The inhibition of telomerase activity and the activation of Caspase-3 may be the key steps through which ATRA inhibits the proliferation of SMMC-7721 cell line.

Antineoplastic Agents↗
PubMed2003

A possible receptor for beta 2 glycoprotein I on the membrane of hepatoma cell line smmc7721.

OBJECTIVES: To study the interaction of beta-2-glycoprotein I (beta 2GPI) with the membrane of hepatocytes and determine whether beta 2GPI participates in HBV infection. METHODS: Ligand blotting, fluorescence microscopy, and fluorescence activated cell sorter (FACS) analysis were used to detect the specific interaction of beta 2GPI with the hepatoma cell line smmc7721, the gastric carcinoma cell line SGC7901, and the lymphoma cell line HL-60. RESULTS: A specific 40 kDa beta 2GPI band was observed by ligand blotting in the case of smmc7721 cells. No such band was observed in SGC7901 or HL-60 cells. Fluorescence microscopy also revealed specific binding of FITC-beta 2GPI to smmc7721 cells, but neither to SGC7901 nor HL-60 cells. FACS analysis demonstrated that the binding rate of FITC-beta 2GPI to smmc7721 cells was significantly higher than these in SGC7901 and HL-60 cells (P < 0.01). The binding rate to smmc7721 cells did not increase with increasing amounts of FITC-beta 2GPI. CONCLUSIONS: There is a specific beta 2GPI-binding protein on the membrane of hepatoma cells in cell line smmc7721 which as the beta 2GPI receptor may participate in HBV infection of hepatocytes.

Animals↗
PubMed2002

RNA interference reveals a requirement for myocyte enhancer factor 2A in activity-dependent neuronal survival.

RNA interference (RNAi) provides a powerful method of gene silencing in eukaryotic cells, including proliferating mammalian cells. However, the utility of RNAi as a method of gene knock-down in primary postmitotic mammalian neurons remained unknown. Here, we asked if RNAi might be utilized to allow the assessment of the biological function of a specific gene in the nervous system. We employed a U6 promoter-driven DNA template approach to induce hairpin RNA-triggered RNAi to characterize the role of the transcription factor myocyte enhancer factor 2A (MEF2A) in the neuronal activity-dependent survival of granule neurons of the developing rat cerebellum. We found that the expression of MEF2A hairpin RNAs leads to the efficient and specific inhibition of endogenous MEF2A protein expression in primary cerebellar granule neurons. We also found that RNAi of MEF2A reduces significantly MEF2 response element-mediated transcription in granule neurons and inhibits activity-dependent granule neuron survival. Taken together, our RNAi experiments have revealed that MEF2A plays a critical role in activity-dependent neuronal survival. In addition, our findings indicate that RNAi does operate in postmitotic mammalian neurons and thus offers a rapid genetic method of studying gene function in the development and function of the mammalian nervous system.

Animals↗
PubMed2002

Chronic hypoxia increases endothelial nitric oxide synthase generation of nitric oxide by increasing heat shock protein 90 association and serine phosphorylation.

Chronic hypoxia increases endothelial nitric oxide synthase (eNOS) production of nitric oxide (*NO) and cardioprotection in neonatal rabbit hearts. However, the mechanism by which this occurs remains unclear. Recent studies suggest that heat shock protein 90 (hsp90) alters eNOS function. In the present study, we examined the role of hsp90 in eNOS-dependent cardioprotection in neonatal rabbit hearts. Chronic hypoxia increased recovery of postischemic left ventricular developed pressure (LVDP). Geldanamycin (GA), which inhibits hsp90 and increases oxidative stress, decreased functional recovery in normoxic and hypoxic hearts. To determine if a loss in *NO, afforded by GA, decreased recovery, GA-treated hearts were perfused with S-nitrosoglutathione (GSNO) as a source of *NO. GSNO increased recovery of postischemic LVDP in GA-treated normoxic and hypoxic hearts to baseline levels. Although chronic hypoxia decreased phosphorylated eNOS (S1177) levels by approximately 4- to 5-fold and total Akt and phosphorylated Akt by 4- and 5-fold, it also increased hsp90 association with eNOS by more than 3-fold. Using hydroethidine (HEt), a fluorescent probe for superoxide, we found that hypoxic hearts contained less ethidine (Et) staining than normoxic hearts. Normoxic hearts generated 3 times more superoxide by an N(omega)-nitro-L-arginine methyl ester (L-NAME)-inhibitable mechanism than hypoxic hearts. Taken together, these data indicate that the association of hsp90 with eNOS is important for increasing *NO production and limiting eNOS-dependent superoxide anion generation. Such changes in eNOS function appear to play a critical role in protecting the myocardium against ischemic injury.

Animals↗
PubMed2002

CDK-9/cyclin T (P-TEFb) is required in two postinitiation pathways for transcription in the C. elegans embryo.

The metazoan transcription elongation factor P-TEFb (CDK-9/cyclin T) is essential for HIV transcription, and is recruited by some cellular activators. P-TEFb promotes elongation in vitro by overcoming pausing that requires the SPT-4/SPT-5 complex, but considerable evidence indicates that SPT-4/SPT-5 facilitates elongation in vivo. Here we used RNA interference to investigate P-TEFb functions in vivo, in the Caenorhabditis elegans embryo. We found that P-TEFb is broadly essential for expression of early embryonic genes. P-TEFb is required for phosphorylation of Ser 2 of the RNA Polymerase II C-terminal domain (CTD) repeat, but not for most CTD Ser 5 phosphorylation, supporting the model that P-TEFb phosphorylates CTD Ser 2 during elongation. Remarkably, although heat shock genes are cdk-9-dependent, they can be activated when spt-4 and spt-5 expression is inhibited along with cdk-9. This observation suggests that SPT-4/SPT-5 has an inhibitory function in vivo, and that mutually opposing influences of P-TEFb and SPT-4/SPT-5 may combine to facilitate elongation, or insure fidelity of mRNA production. Other genes are not expressed when cdk-9, spt-4, and spt-5 are inhibited simultaneously, suggesting that these genes require P-TEFb in an additional mechanism, and that they and heat shock genes are regulated through different P-TEFb-dependent elongation pathways.

Animals↗
PubMed2002

Activation of protein kinases in chronically hypoxic infant human and rabbit hearts: role in cardioprotection.

BACKGROUND: Many infants who undergo heart surgery have a congenital cyanotic defect in which the heart is chronically perfused with hypoxic blood. However, the signaling pathways by which infant hearts adapt to chronic hypoxia and resist subsequent surgical ischemia is unknown. METHODS AND RESULTS: We determined the activation and translocation of protein kinase C (PKC) isoforms and mitogen activated protein kinases (MAP kinases) in 15 infants with cyanotic (SaO2<85%) or acyanotic (SaO2>95%) heart defects undergoing surgical repair and in 80 rabbits raised from birth in a hypoxic (SaO2<85%) or normoxic (SaO2>95%) environment. Tissues from infant human and rabbit hearts were processed for Western and in vitro kinase analysis. In human infants with cyanotic heart defects, PKCepsilon, p38 MAP kinase, and JUN kinase but not p42/44 MAP kinase were activated and translocated from the cytosolic to the particulate fraction compared with acyanotic heart defects. In rabbit infants there was a parallel response for PKCepsilon, p38 MAP kinase, and JUN kinase similar to humans. In infant rabbit hearts inhibition of PKCepsilon with chelerythrine, p38 MAP kinase, with SB203580 and JUN kinase with curcumin abolished the cardioprotective effects of chronic hypoxia but had no effects on normoxic hearts. CONCLUSIONS: Infant human and rabbit hearts adapt to chronic hypoxia through activation of PKCepsilon, p38 MAP kinase, and JUN kinase signal transduction pathways. These pathways may be responsible for cardioprotection in the chronically hypoxic infant rabbit heart.

Activating Transcription Factor 2↗
PubMed2002

A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms including plants, Caenorhabditis elegans, and Drosophila. The discovery that synthetic double-stranded, 21-nt small interfering RNA triggers gene-specific silencing in mammalian cells has further expanded the utility of RNAi into mammalian systems. Here we report a technology that allows synthesis of small interfering RNAs from DNA templates in vivo to efficiently inhibit endogenous gene expression. Significantly, we were able to use this approach to demonstrate, in multiple cell lines, robust inhibition of several endogenous genes of diverse functions. These findings highlight the general utility of this DNA vector-based RNAi technology in suppressing gene expression in mammalian cells.

Animals↗
PubMed2002

Functional requirement for histone deacetylase 1 in Caenorhabditis elegans gonadogenesis.

Histone acetylation and deacetylation have been implicated in the regulation of gene expression. Molecular studies have shown that histone deacetylases (HDACs) function as transcriptional repressors. However, very little is known about their roles during development in multicellular organisms. We previously demonstrated that inhibition of maternal and zygotic expression of histone deacetylase 1 (HDA-1) causes embryonic lethality in Caenorhabditis elegans. Here, we report the identification of an hda-1 genetic mutant which has also been called a gon-10 mutant (for gonadogenesis defective 10) and show that loss of HDA-1 zygotic expression results in specific postembryonic defects in gonadogenesis and vulval development. We provide evidence that the lag-2 gene, which plays a role in gonadogenesis and vulval development and encodes a Notch ligand, is derepressed in gon-10 animals, suggesting that lag-2 may be a target of HDA-1. Our findings reveal a novel and specific function for the ubiquitously expressed HDA-1 in C. elegans gonadogenesis and place hda-1 in the Notch signaling pathway.

Amino Acid Sequence↗
PubMed2001

HAT activity is essential for CBP-1-dependent transcription and differentiation in Caenorhabditis elegans.

The p300/CBP family of transcriptional coactivators possesses multiple functional domains, including a histone acetyltransferase (HAT) and several activation domains. A number of models have been proposed to account for their roles in transcriptional activation, including interactions with basal transcription machinery and chromatin remodeling. However, individual contributions of these domains to transcriptional activation and their significance in living organisms remain unclear. We addressed the importance of the HAT activity of CBP-1, the worm ortholog of p300/CBP, in Caenorhabditis elegans with three different and complementary approaches. These include allele-specific RNA-mediated interference (RNAi), genetic rescue and the use of a specific chemical inhibitor of the p300/CBP HAT. Our findings demonstrate that HAT activity is of primary importance for CBP-1 to regulate transcription and to promote differentiation during C. elegans embryogenesis.

Acetyltransferases↗
PubMedDate not supplied

A simple solid phase mass tagging approach for quantitative proteomics.

New mass-tagging reagents for quantitative proteomics measurements have been designed using solid phase peptide synthesis technology. The solid phase mass tags have been used to accurately measure the relative amounts of cysteine-containing peptides in model peptide mixtures as well as in mixtures of tryptic digests in the femtomol range. Measurements were made using both matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and online reversed-phase capillary liquid chromatography coupled through a nanoelectrospray interface to an ion trap mass spectrometer (capillary LC/ESI-MS). Results of mass-tagging experiments obtained from these two mass spectrometry techniques and their relative advantages and disadvantages for identification and quantitation of mass tagged peptides are compared. These reagents provide a simple, rapid and cost-effective alternative to currently available mass tagging technologies.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

2D LC/MS analysis of membrane proteins from breast cancer cell lines MCF7 and BT474.

Membrane proteins play a central role in the interaction of the cell with its environment and in the function of subcellular organelles. The current study focused on developing a better understanding of the membrane proteome of two well-characterized breast cancer cell lines. Membranes from osmotically lysed BT474 and MCF7 cells were treated with cyanogen bromide followed by a combination of trypsin and Staphylococcus V8 protease to obtain hydrophilic peptides from membrane proteins. The complex peptide mixtures obtained were separated by 2-dimensional liquid chromatography coupled online with a nano-electrospray ionization ion trap mass spectrometer (2D LC/nanoESI-MS). The strong cation exchange column used in the first dimension of the separation was eluted in an automated fashion using a series of salt steps of increasing concentration. Peptides eluted from each of the salt steps were separated using a capillary reversed-phase HPLC column, the output of which was directed through a nano-electrospray fused silica tip into the mass spectrometer. Peptides were fragmented by collision-induced dissociation (CID) and analyzed by data-dependent MS/MS followed by database searching using the Sequest algorithm. Analysis of the data revealed both similarities and expected differences between proteins identified from these cell lines. As demonstrated by others, mRNA and the HER2/neu protein tyrosine kinase-linked receptor in BT474 cells is up regulated compared to its level in MCF7, while the expression of the estrogen receptor alpha is known to be up regulated in MCF7 cells. As expected, our studies showed identification of peptides from HER2 in BT474 while estrogen receptor peptides were detected in the MCF7 line. A total of 604 proteins were identified from BT474 membranes while 313 proteins were found from MCF7. The results are discussed in terms of the known differences in both protein and mRNA expression between these two breast cancer cell lines and also in the context of other known phenotypic differences between these cells.

Breast Neoplasms↗