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Y Liu

Publications and source records attributed to Y Liu.

At least 757 records · Page 42Linked to original sources
PubMed2000

Electrophoretic separation of proteins on a microchip with noncovalent, postcolumn labeling.

Proteins were separated by microchip capillary electrophoresis and labeled on-chip by postcolumn addition of a fluorogenic dye, NanoOrange, for detection by laser-induced fluorescence. NanoOrange binds noncovalently with hydrophobic protein regions to form highly fluorescent complexes. Kinetic measurements of complex formation on the microchips suggest that the reaction rate is near the diffusion limit under the conditions used for protein separation. Little or no band broadening is caused by the postcolumn labeling step. Lower limits of detection for model proteins, alpha-lactalbumin, beta-lactoglobulin A, and beta-lactoglobulin B, were <0.5 pg (approximately 30 amol) of injected sample. The relative fluorescence and reaction rates are compared with those of a number of other fluorogenic dyes used for protein labeling.

Electrophoresis, Capillary↗
PubMed2000

Effect of human neuronal tau on denaturation and reactivation of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase.

Human neuronal tau-40 (htau-40) has been used to study denaturation and renaturation of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). Inactivation of GAPDH incubated with tau was more distinguishably detected than that of control GAPDH during thermal and guanidine hydrochloride (GdnHCl) denaturation. However, tau did not influence the activity of GAPDH at room temperature or in solution without GdnHCl. A marked change in both the emission intensity and emission maximum of the intrinsic fluorescence at 335 nm of GAPDH with tau was observed when GdnHCl concentration was 0.8 M, but that of the control without tau occurred in 1.2 M GdnHCl. The first-order rate of the decrease in the fluorescence intensity of the enzyme with tau was approximately twice as great as that of GAPDH without tau. Kinetics of inactivation of GAPDH with tau in 0.2 M GdnHCl was a monophasic procedure, instead of the biphasic procedure followed by the control, as described before [He, Zhao, Yan and Li (1993) Biochim. Biophys. Acta 1163, 315-320]. Similar results were obtained when the enzyme was thermally denatured at 45 degrees C. It revealed that tau bound to the denatured GAPDH but not the native molecule. On the other hand, tau suppressed refolding and reactivation of GAPDH when this enzyme was reactivated by dilution of GdnHCl solution. Furthermore, tau improved the aggregation of the non-native GAPDH in solutions. It suggested that tau acted in an anti-chaperone-like manner towards GAPDH in vitro. However, tau lost that function when it was aggregated or phosphorylated by neuronal cdc2-like protein kinase. It showed that tau's anti-chaperone-like function depended on its native conformation.

Animals↗
PubMed2000

Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size.

While the transporters that accumulate classical neurotransmitters in synaptic vesicles have been identified, little is known about how their expression regulates synaptic transmission. We have used adenoviral-mediated transfection to increase expression of the brain vesicular monoamine transporter VMAT2 and presynaptic amperometric recordings to characterize the effects on quantal release. In presynaptic axonal varicosities of ventral midbrain neurons in postnatal culture, VMAT2 overexpression in small synaptic vesicles increased both quantal size and frequency, consistent with the recruitment of synaptic vesicles that do not normally release dopamine. This was confirmed using noncatecholaminergic AtT-20 cells, in which VMAT2 expression induced the quantal release of dopamine. The ability to increase quantal size in vesicles that were already competent for dopamine release was shown in PC12 cells, in which VMAT2 expression increased the quantal size but not the number of release events. These results demonstrate that vesicle transporters limit the rate of transmitter accumulation and can alter synaptic strength through two distinct mechanisms.

Adenoviridae↗
PubMed2000

Characterization of the human cysteinyl leukotriene 2 receptor.

The contractile and inflammatory actions of the cysteinyl leukotrienes (CysLTs), LTC(4), LTD(4), and LTE(4), are thought to be mediated through at least two distinct but related CysLT G protein-coupled receptors. The human CysLT(1) receptor has been recently cloned and characterized. We describe here the cloning and characterization of the second cysteinyl leukotriene receptor, CysLT(2), a 346-amino acid protein with 38% amino acid identity to the CysLT(1) receptor. The recombinant human CysLT(2) receptor was expressed in Xenopus oocytes and HEK293T cells and shown to couple to elevation of intracellular calcium when activated by LTC(4), LTD(4), or LTE(4). Analyses of radiolabeled LTD(4) binding to the recombinant CysLT(2) receptor demonstrated high affinity binding and a rank order of potency for competition of LTC(4) = LTD(4) LTE(4). In contrast to the dual CysLT(1)/CysLT(2) antagonist, BAY u9773, the CysLT(1) receptor-selective antagonists MK-571, montelukast (Singulair(TM)), zafirlukast (Accolate(TM)), and pranlukast (Onon(TM)) exhibited low potency in competition for LTD(4) binding and as antagonists of CysLT(2) receptor signaling. CysLT(2) receptor mRNA was detected in lung macrophages and airway smooth muscle, cardiac Purkinje cells, adrenal medulla cells, peripheral blood leukocytes, and brain, and the receptor gene was mapped to chromosome 13q14, a region linked to atopic asthma.

Adrenal Medulla↗
PubMed2000

Non-replicating Epstein-Barr virus-based plasmids extend gene expression and can improve gene therapy in vivo.

To date, no gene transfer vector has produced prolonged gene expression following a single intravenous injection and then efficiently re-expressed the delivered gene following repeated systemic injection into immunocompetent hosts. To overcome these limitations, a gene therapy regimen using non-replicating Epstein-Barr virus (EBV)-based expression plasmids was developed. One plasmid contains the FR (EBV family of repeats) sequence and the expressed gene. The other encodes Epstein-Barr nuclear antigen 1 (EBNA-1), but lacks FR. Although unable to replicate in mice, intravenous co-injection of EBV-based plasmids in cationic liposome-DNA complexes (CLDCs) substantially prolonged luciferase gene expression. The use of a two-vector system limited host exposure to the EBNA-1 gene product. Furthermore, this EBV-based vector system could be intravenously re-injected multiple times into immunocompetent mice without loss of transfection efficiency. Use of this vector system significantly improved the therapeutic efficacy of the biologically important human granulocyte colony-stimulating factor gene. Delivery of the human granulocyte colony-stimulating factor gene in EBV-based plasmids increased circulating white blood counts for at least 2 months following a single CLDC-based intravenous co-injection. Conversely, white blood counts were never elevated following injection of CLDCs lacking EBV-derived elements. Thus, this EBV-based plasmid vector system both markedly prolongs gene expression at therapeutic levels and efficiently and repeatedly re-transfects immunocompetent hosts. These properties of EBV-based plasmid vectors appear to be due, at least in part, to the documented abilities of the EBNA-1 protein both to retain FR-containing DNA intracellularly and within the nucleus and to block anti-EBNA-1 cytotoxic T cell responses.

Animals↗
PubMed2000

SNAP-25 functional domains in SNARE core complex assembly and glutamate release of cerebellar granule cells.

Synaptosomal associated protein of 25 kDa (SNAP-25) is a member of the SNARE protein complex that has been implicated in synaptic vesicle docking and fusion. In this report, we have generated SNAP-25 mutants and assayed their functions in SNARE complex formation and glutamate release from cultured rat cerebellar granule cells. In vitro binding studies show that a deletion mutant lacking the C-terminal 181-206 amino acid sequence inhibits the formation of the SNARE core complex. Additional deletion of an N-terminal 1-31 amino acid sequence abolished this inhibitory activity. Adenovirus-mediated gene transfer is used to overexpress wild-type and mutant SNAP-25 in cerebellar granule cells. Neurons overexpressing the wild-type protein show slight reductions in glutamate release, ranging from 10 to 15% in both the developing and mature granule cells. A 30-35% inhibition is obtained with the C-terminal deletion mutant, and the inhibitory effect is abolished in the N- and C-terminal double deletion mutant. These results demonstrate that the SNARE core complex exists in a dynamic and reversible state, and the formation of the core complex is necessary for neurotransmitter release in neurons.

Animals↗
PubMed2000

BmK AS: new scorpion neurotoxin binds to distinct receptor sites of mammal and insect voltage-gated sodium channels.

This study was undertaken to assess the binding properties of BmK AS on both mammal and insect excitable cell membranes. It was found that BmK AS bound specifically to a single class of non-interacting binding sites on both rat brain and cockroach nerve cord synaptosomes with high affinity (K(d) = 1.49 +/- 0.14 and 0.79 +/- 0.29 nM) and low capacity (B(max) = 1.39 +/- 0.09 and 6.60 +/- 1.25 pmol/mg protein), respectively. Binding kinetics showed that BmK AS could bind and reach equilibrium quickly, and dissociate partially from its binding sites on both kinds of synaptosomes. The binding of BmK AS was independent of membrane potential. Veratridine could not modify the binding of BmK AS. The competitive binding assay showed that specific binding of (125)I-BmK AS could be significantly inhibited by native BmK AS, BmK AS-1, BmK IT2 and BmK IT on both synaptosomes. Unexpectedly, only about 20-30% binding of BmK AS on mammal synaptosomes was inhibited by BmK I at 10(-5)-10(-9) M, but not on insect synaptosomes. It thus suggests that BmK AS type neurotoxins might bind to a distinct receptor site of sodium channels on mammal and insect excitable cell membranes with a manner similar to beta-scorpion toxins.

Animals↗
PubMed2000

Intermediates Produced in the Electron-Transfer Processes of Phenothiazine/Semiconductor Systems.

Phenothiazine radical cation PTH(+*) and phenothiazine dication PTH(2+) produced by photoinduced electron transfer in phenothiazine/semiconductor systems have been studied by using ESR, UV-visible absorption, and fluorescence spectroscopic methods. It is found that the PTH(+*) was generated by electron transfer from (1)PTH* and (3)PTH* to the conduction band of the semiconductors besides PTH(+*) resulted in the photoionization of PTH as well as PTH(2+) produced by electron transfer from PTH(+*) to the conduction band of the semiconductors successively. Very significant supplementary information provided by UV-visible absorption, ESR, and resonance Raman spectra of PTH(+*) and PTH(2+), which were obtained by oxidation of PTH with lead tetraacetate, not only confirmed the two-step mono-electron-transfer mechanism proposed for interpretation of electron-transfer processes in PTH/semiconductor systems but also demonstrated that the two successive electron-transfer steps were corresponding to the removal of a p-electron at the nitrogen atom and a p-electron at the sulfur atom in the PTH molecule, respectively. Consequently, the nonplanar configuration of the PTH molecule changed into a planar configuration of the PTH(2+). Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

Characterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from embryonic rat spinal cord.

Recent advances in the isolation and characterization of neural precursor cells suggest that they have properties that would make them useful transplants for the treatment of central nervous system disorders. We demonstrate here that spinal cord cells isolated from embryonic day 14 Sprague-Dawley and Fischer 344 rats possess characteristics of precursor cells. They proliferate as undifferentiated neurospheres in the presence of EGF and bFGF and can be maintained in vitro or frozen, expanded and induced to differentiate into both neurons and glia. Exposure of these cells to serum in the absence of EGF and bFGF promotes differentiation into astrocytes; treatment with retinoic acid promotes differentiation into neurons. Spinal cord cells labeled with a nuclear dye or a recombinant adenovirus vector carrying the lacZ gene survive grafting into the injured spinal cord of immunosuppressed Sprague-Dawley rats and non-immunosuppressed Fischer 344 rats for up to 4 months following transplantation. In the presence of exogenously supplied BDNF, the grafted cells differentiate into both neurons and glia. These spinal cord cell grafts are permissive for growth by several populations of host axons, especially when combined with exogenous BDNF administration, as demonstrated by penetration into the graft of axons immunopositive for 5-HT and CGRP. Thus, precursor cells isolated from the embryonic spinal cord of rats, expanded in culture and genetically modified, are a promising type of transplant for repair of the injured spinal cord.

Animals↗
PubMed2000

Glutathione depletion in PC12 results in selective inhibition of mitochondrial complex I activity. Implications for Parkinson's disease.

Oxidative stress appears to play an important role in degeneration of dopaminergic neurons of the substantia nigra (SN) associated with Parkinson's disease (PD). The SN of early PD patients have dramatically decreased levels of the thiol tripeptide glutathione (GSH). GSH plays multiple roles in the nervous system both as an antioxidant and a redox modulator. We have generated dopaminergic PC12 cell lines in which levels of GSH can be inducibly down-regulated via doxycycline induction of antisense messages against both the heavy and light subunits of gamma-glutamyl-cysteine synthetase, the rate-limiting enzyme in glutathione synthesis. Down-regulation of glutamyl-cysteine synthetase results in reduction in mitochondrial GSH levels, increased oxidative stress, and decreased mitochondrial function. Interestingly, decreases in mitochondrial activities in GSH-depleted PC12 cells appears to be because of a selective inhibition of complex I activity as a result of thiol oxidation. These results suggest that the early observed GSH losses in the SN may be directly responsible for the noted decreases in complex I activity and the subsequent mitochondrial dysfunction, which ultimately leads to dopaminergic cell death associated with PD.

Animals↗
PubMed2000

B7-1, IFN gamma and anti-CTLA-4 co-operate to prevent T-cell tolerization during immunotherapy against a murine T-lymphoma.

We previously reported on a murine T lymphoma cell line, BW-Sp3, with inherent immunogenicity. BW-Sp3 tumors can elicit an anti-tumor CD8(+) CTL response capable of mediating a regression of subcutaneous tumors. However, this immune response is inadequate to eliminate cancer cells completely in a significant percentage of the recipients, resulting in progressing tumors. In this tumor model, tumor progression correlated with a tolerization of tumor-reactive T cells and cellular immunotherapy of tumor bearing animals, with or without B7-mediated costimulation, even increased tumor progression (Raes et al, 1998). In the present study, we investigated whether the co-expression of IFN gamma, together with B7-1, could have beneficial effects on immunotherapy. Although immunotherapy with IFN gamma and B7-1 single transfectants tended to tolerize anti-tumor T-cells and consequently increased tumor growth, the B7-1/IFN gamma double transfectants resulted in a more beneficial outcome. This phenomenon correlated with an increased CTL-inducing potential of the double transfectants. Secondly, we wondered whether CTLA-4 signalling was involved in the down-regulation of the anti-tumor response. Indeed, when immunotherapy was provided along with anti-CTLA-4, the protection by B71/IFN gamma double transfectants was further improved and the tumor-promoting effect of BW-Sp3(B7-1) was compensated for. Our results indicate that B7-1, IFN gamma and the blockade of CTLA-4 cooperate to tilt the balance in favour of tumor elimination, while either factor alone fails to do so or even promotes tumor growth.

Abatacept↗
PubMed2000

Breast-cancer diagnosis with nipple fluid bFGF.

Early diagnosis of breast cancer is the key to extending survival of breast-cancer patients. We found that the concentrations of nipple fluid bFGF (basic fibroblast growth factor) was significantly increased in breast-cancer patients compared with concentrations in controls (1717 ng/L [SD 706] vs 19 ng/L [19]; Student's t test p=0.027). Measurement of bFGF in nipple fluid could be a useful diagnostic tool for breast cancer, and deserves further study.

Biomarkers, Tumor↗
PubMed2000

Growth factors inactivate the cell death promoter BAD by phosphorylation of its BH3 domain on Ser155.

The Bcl-2 family protein BAD promotes apoptosis by binding through its BH3 domain to Bcl-x(L) and related cell death suppressors. When BAD is phosphorylated on either Ser(112) or Ser(136), it forms a complex with 14-3-3 in the cytosol and no longer interacts with Bcl-x(L) at the mitochondria. Here we show that phosphorylation of a distinct site Ser(155), which is at the center of the BAD BH3 domain, directly suppressed the pro-apoptotic function of BAD by eliminating its affinity for Bcl-x(L). Protein kinase A functioned as a BAD Ser(155) kinase both in vitro and in cells. BAD Ser(155) was found to be a major site of phosphorylation induced following stimulation by growth factors and prevented by protein kinase A inhibitors but not by inhibitors of the phosphatidylinositol 3-kinase/Akt pathway. Growth factors inhibited BAD-induced apoptosis in both a Ser(112)/Ser(136)- and a Ser(155)-dependent fashion. Thus, growth factors engage an anti-apoptotic signaling pathway that inactivates BAD by direct modification of its BH3 cell death effector domain.

Amino Acid Sequence↗
PubMed2000

Processing by endoplasmic reticulum mannosidases partitions a secretion-impaired glycoprotein into distinct disposal pathways.

In the early secretory pathway, a distinct set of processing enzymes and family of lectins facilitate the folding and quality control of newly synthesized glycoproteins. In this regard, we recently identified a mechanism in which processing by endoplasmic reticulum mannosidase I, which attenuates the removal of glucose from asparagine-linked oligosaccharides, sorts terminally misfolded alpha(1)-antitrypsin for proteasome-mediated degradation in response to its abrogated physical dissociation from calnexin (Liu, Y., Choudhury, P., Cabral, C., and Sifers, R. N. (1999) J. Biol. Chem. 274, 5861-5867). In the present study, we examined the quality control of genetic variant PI Z, which undergoes inappropriate polymerization following biosynthesis. Here we show that in stably transfected hepatoma cells the additional processing of asparagine-linked oligosaccharides by endoplasmic reticulum mannosidase II partitions variant PI Z away from the conventional disposal mechanism in response to an arrested posttranslational interaction with calnexin. Intracellular disposal is accomplished by a nonproteasomal system that functions independently of cytosolic components but is sensitive to tyrosine phosphatase inhibition. The functional role of ER mannosidase II in glycoprotein quality control is discussed.

Animals↗
PubMed2000

Participation of syntaxin 1A in membrane trafficking involving neurite elongation and membrane expansion.

Syntaxin 1A has been implicated to play an important role in neurotransmitter release by regulating synaptic vesicle fusion. The protein is also suggested to be required for other types of membrane fusion such as cellularization during embryonic development. In the current study, we overexpressed syntaxin 1A, SNAP-25b, and VAMP-2 in PC12 cells using recombinant adenoviruses, and determined their effects on membrane trafficking involving neurite outgrowth. It was found that overexpression of syntaxin 1A inhibited NGF-induced neurite extension, and the expressed syntaxin was localized to the plasma membrane, intracellular membranes, and the neurite tips. SNAP-25 overexpression slightly enhanced neurite elongation, whereas no significant changes in neurite growth was observed in VAMP-overexpressing cells. The effect of syntaxin 1A in general membrane trafficking was further studied by transient transfection of non-neuronal cells. Syntaxin 1A expression in HEK 239 and NIH3T3-L1 caused the cells to lose their normal morphology, leading to round and smaller cells. Deletion of the C-terminal sequence containing the H3 helix and the membrane anchoring domains of syntaxin abolished its ability to induce cell morphology changes, whereas removal of the N-terminal 1-170 amino acid sequence did not affect this activity. These findings suggest that in addition to its well documented role in synaptic vesicle fusion, syntaxin may function in other non-synaptic membrane trafficking such as neurite outgrowth and membrane expansion.

3T3 Cells↗