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Biomedical subjects

Q X Li

Publications and source records attributed to Q X Li.

At least 55 records · Page 3Linked to original sources
PubMed1998

Inhibition of platelet activation by the Alzheimer's disease amyloid precursor protein.

The amyloid precursor protein (APP) of Alzheimer's disease is abundantly expressed in the platelet alpha-granule where its role remains unclear. This study describes a novel function for APP in regulating human platelet activation. Preincubation of platelet-rich plasma with recombinant secreted APP (sAPP) isoforms dose-dependently inhibited platelet aggregation and secretion induced by ADP or adrenaline. Similarly, sAPP potently inhibited low-dose thrombin-induced activation in washed platelet suspensions, indicating that the activity does not require plasma cofactors. There were no functional differences between sAPP forms with or without the Kunitz protease inhibitor domain or derived from either alpha- or beta-secretase cleavage. In fact, the N-terminal cysteine-rich region of APP (residues 18-194) was as effective as the entire sAPP region in the inhibition of platelet activation. The inhibitory activity of sAPP correlated with a significant reduction in the agonist-induced production of the arachidonic acid (AA) metabolites thromboxane B2 and prostaglandin E2. However, sAPP did not affect AA-induced platelet aggregation or secretion, indicating the enzymatic conversion of AA was not inhibited. The addition of a threshold dose of AA reversed the sAPP-inhibition of agonist-induced platelet activation. This suggests that sAPP decreases the availability of free AA, although the mechanism is not yet known. These data provide evidence that the release of sAPP upon platelet degranulation may result in negative feedback regulation during platelet activation.

Adenosine Diphosphate↗
PubMed1998

Acetylcholinesterase is increased in the brains of transgenic mice expressing the C-terminal fragment (CT100) of the beta-amyloid protein precursor of Alzheimer's disease.

Acetylcholinesterase (AChE) expression is markedly affected in Alzheimer's disease (AD). AChE activity is lower in most regions of the AD brain, but it is increased within and around amyloid plaques. We have previously shown that AChE expression in P19 cells is increased by the amyloid beta protein (A beta). The aim of this study was to investigate AChE expression using a transgenic mouse model of A beta overproduction. The beta-actin promoter was used to drive expression of a transgene encoding the 100-amino acid C-terminal fragment of the human amyloid precursor protein (APP CT100). Analysis of extracts from transgenic mice revealed that the human sequences of full-length human APP CT100 and A beta were overexpressed in the brain. Levels of salt-extractable AChE isoforms were increased in the brains of APP CT100 mice. There was also an increase in amphiphilic monomeric form (G1A) of AChE in the APP CT100 mice, whereas other isoforms were not changed. An increase in the proportion of G1A AChE was also detected in samples of frontal cortex from AD patients. Analysis of AChE by lectin binding revealed differences in the glycosylation pattern in APP CT100 mice similar to those observed in frontal cortex samples from AD. The results are consistent with the possibility that changes in AChE isoform levels and glycosylation patterns in the AD brain may be a direct consequence of altered APP metabolism.

Acetylcholinesterase↗
PubMed1998

Secretion of Alzheimer's disease Abeta amyloid peptide by activated human platelets.

Alzheimer's disease (AD) is characterized by the deposition of Abeta (betaA4) peptides of 39 to 43 amino acid residues, which are normal cellular metabolic products derived by proteolysis of the amyloid precursor protein (APP). The physiologic function of Abeta/APP in vivo is poorly understood. We analyzed human platelets for Abeta production by immunoprecipitation coupled to immunoblotting. A 4-kd Abeta fragment that comigrates with an Abeta40 synthetic peptide and reacts with several antibodies specific for the N- and C-termini of Abeta is detected. The majority of platelet Abeta appears to end at residue 40, as determined by immunoreactivity with an Abeta40-specific antibody. Furthermore, Abeta is secreted upon platelet stimulation with the physiologic agonists thrombin and collagen, together with secretion of soluble APP (sAPP). A comparison between serum and plasma shows a 1.6-fold increase in Abeta levels and a 2.4-fold increase in sAPP levels in serum. This is consistent with the view that platelets are the primary source of circulating Abeta and APP. The release of platelet Abeta by physiologic stimuli suggests that it may play a role in platelet aggregation and coagulation or in the repair mechanisms associated with injury.

Amyloid beta-Peptides↗
PubMed1997

A novel metalloprotease in rat brain cleaves the amyloid precursor protein of Alzheimer's disease generating amyloidogenic fragments.

The amyloid protein (A beta or beta A4) is the major constituent of amyloid plaques in the Alzheimer's disease brain. A beta is cleaved from the amyloid precursor protein (APP) by a mechanism which is poorly understood. Cell culture studies suggest that APP may be cleaved by secretases within the late Golgi compartment. Studies performed so far have mainly used exogenous APP and synthetic peptides as substrates. For this study, a Golgi and plasma membrane-enriched fraction was isolated from rat brain and incubated at 37 degrees C at pH 7.2 to study the degradation of endogenous APP. The breakdown of APP was accompanied by the concomitant generation of A beta-containing C-terminal fragments, in a time-dependent fashion. The metal ion chelators EDTA and 1,10-phenanthroline inhibited this degradation. The inhibition by EDTA was reversed by 50 microM Zn2+ but not by other metal ions. The protease activity was not inhibited by cysteine, serine or aspartic protease inhibitors nor was it inhibited by compounds which are inhibitors of known metalloendopeptidases and matrix metalloproteinases (cFP, phosphoramidon and TIMP-2). Our data suggest that a novel Zn(2+)-dependent metalloprotease activity associated with a Golgi and plasma membrane-enriched fraction can degrade endogenous APP to generate A beta containing C-terminal fragments. This protease may generate amyloidogenic fragments of APP which may serve as precursors for A beta.

Alzheimer Disease↗
PubMed1997

Collection and normal levels of the amyloid precursor protein in plasma.

The amyloid precursor protein is contained in platelet alpha granules and released with degranulation. Methods are described to control for amyloid precursor protein release from platelets during blood collection and processing. In normal subjects (n = 97; age range, 44-84 years), the average plasma level of amyloid precursor protein was 6.5 +/- 1.8 ng/ml.

Adult↗
PubMed1997

Determination of phloxine B and uranine in water by capillary zone electrophoresis.

Phloxine B and uranine are color additives in drugs and cosmetics as well as potential photoactive insecticides. A capillary zone electrophoretic (CZE) method is developed to determine phloxine B and uranine in water. A fused-silica capillary (67 cm, 75-micron i.d.) and borate buffer are used. Migration of phloxine B and uranine increases slightly as the pH of the running buffer increases between the range of 8-9. Although there are only slight effects of ionic strength on the analyte migration in the range of 0-20 mM NaCl in the running buffer, the migration of phloxine B and uranine increases as the percentage of methanol in the samples increases. Methanol shows little effect on the quantitation of phloxine B and uranine. The CZE procedure is applied to determine phloxine B and uranine fortified in tap and stream water samples. Solid-phase extraction is employed to recover the analytes spiked in the water samples. Recoveries range from 87-112% for phloxine B spiked at 10-200 ppb in the tap and stream water. Uranine recoveries are 86-91% at fortification levels of 10-50 ppb in the water samples.

Buffers↗
PubMed1996

Syncytium formation by recombinant HTLV-II envelope glycoprotein.

Infection by human T-cell leukemia viruses (HTLV-I and HTLV-II) induces syncytium formation in certain cell types in vitro. Recombinant vaccinia viruses expressing the HTLV-II envelope (Env) gp61-II were used to infect human cells, and syncytium formation was observed, demonstrating that HTLV-II Env expression was sufficient to mediate syncytium formation in appropriate cells. This syncytium formation could be blocked by sera from HTLV-II-infected individuals. Infection with these recombinant vaccinia viruses caused fusion in mouse/human hybrid cells containing only human chromosome 17, whereas there was no obvious syncytium formation in mouse cells. This fusogenic phenotype of recombinant gp61-II is likely a property of the specific interaction between HTLV-II envelope protein and the HTLV cellular receptor, encoded by a gene that was previously indicated to be on human chromosome 17. On this basis, we developed a sensitive syncytium formation assay, using lacZ gene activation, for detecting the presence of the HTLV receptor(s). We used this fusion assay to test for HTLV receptor activity on a variety of cells. Our findings indicate that the HTLV receptor is widely distributed among species and cell types, including, to a limited extent, murine cells.

Animals↗
PubMed1996

In vitro production of stable Epstein-Barr virus-positive epithelial cell clones which resemble the virus:cell interaction observed in nasopharyngeal carcinoma.

The interaction of Epstein-Barr virus (EBV) with epithelial cells and the consequent role of the virus in the aetiology of undifferentiated nasopharyngeal carcinoma (NPC) is poorly understood. One important obstacle to work in this area has been the lack of an epithelial cell culture system in which EBV is stably maintained. Using a previously described approach in which CR2-transfected epithelial cells (SVK-CR2) are rendered susceptible to transient EBV infection (Li et al., Nature 356, 347, 1992), we now demonstrate that the pattern of EBV latent gene transcription in these acutely infected epithelial cells differs from that observed in virus-infected primary B cells. In addition, some of these epithelial cells spontaneously entered the EBV lytic cycle. By cloning Akata virus-infected SVK-CR2 cells we generated two stable lines which remained EBV positive for more than 1.5 years at which time further subclones were isolated. These cloned lines carry the EBV genome as an episome and exclusively use the FQp promoter for driving EBNA1 transcription, display no Cp/Wp promoter activity, and express low levels of the LMP mRNAs. Unlike acutely infected SVK-CR2 cells, the cloned lines responded poorly to suspension-induced terminal differentiation and were impaired in their ability to enter the virus lytic cycle. These results, showing similarities between the cloned EBV-positive SVK-CR2 lines and NPC tumour cells, suggest that stable maintenance of EBV in epithelial cells may require an undifferentiated cellular environment.

Animals↗
PubMed1996

Structure-activity relationships for substrates and inhibitors of mammalian liver microsomal carboxylesterases.

PURPOSE: Carboxylesterases are important in the detoxification of drugs, pesticides and other xenobiotics. This study was to evaluate a series of substrates and inhibitors for characterizing these enzymes. METHODS: A series of novel aliphatic esters and thioesters were used in spectral assays to monitor human, murine and porcine esterases. A series of transition state mimics were evaluated as selective esterase inhibitors. RESULTS: Several alpha-alkyl thioacetothioates were found to be approximately 2 to 11-fold superior to commonly used substrates for monitoring carboxylesterase activity. Insertion of a heteroatom in the acid portion of these esters in the beta or gamma position relative to the carbonyl had a dramatic effect on enzyme activity with S or O substituents often improving the kCAT/K(M) ratio of the substrate and N decreasing it. Several alpha,alpha'-bis (2-oxo-3,3,3-trifluoropropylthio)alkanes proved to be potent selective transition state mimics of the esterase activity with IC50's from 10(-5) to 10(-9)M. CONCLUSIONS: This library of substrates and inhibitors are useful research tools for characterizing the numerous isozymes of carboxylesterases present in mammalian tissues.

Animals↗
PubMed1996

Vpr-induced cell cycle arrest is conserved among primate lentiviruses.

We previously reported that expression of human immunodeficiency virus type 1 strain NL4-3 (HIV-1(NL4-3))vpr causes cells to arrest in the G2 phase of the cell cycle. We examined the induction of cell cycle arrest by other HIV-1 isolates and by primary lentiviruses other than HIV-1. We demonstrate that the vpr genes from tissue culture-adapted or primary isolates of HIV-1 are capable of inducing G2 arrest. In addition, we demonstrate that induction of cell cycle arrest is a conserved function of members of two other groups of primate lentiviruses, HIV-2/simian immunodeficiency virus strain sm (SIVsm)/SIVmac and SIVagm. vpr from HIV-1, HIV-2, and SIVmac induced cell cycle arrest when transfected in human (HeLa) and monkey (CV-1) cells. vpx from HIV-2 and SIVmac did not induce detectable cell cycle arrest in either cell type, and SIVagm vpx was capable of inducing arrest in CV-1 but not HeLa cells. These results indicate that induction of cell cycle perturbation is a general property of lentiviruses that infect primates. The conservation of this viral function throughout evolution suggests that it plays a key role in virus-host relationships, and elucidation of its mechanism may reveal important clues about pathology induced by primary lentiviruses.

Alleles↗
PubMed1996

High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G.

Currently, amphotropic retroviral vectors are widely used for gene transfer into CD34+ hematopoietic progenitor cells. The relatively low levels of transduction efficiency associated with these vectors in human cells is due to low viral titers and limitations in concentrating the virus because of the inherent fragility of retroviral envelopes. Here we show that a human immunodeficiency virus type 1 (HIV-1)-based retroviral vector containing the firefly luciferase reporter gene can be pseudotyped with a broad-host-range vesicular stomatitis virus envelope glycoprotein G (VSV-G). Higher-efficiency gene transfer into CD34+ cells was achieved with a VSV-G-pseudotyped HIV-1 vector than with a vector packaged in an amphotropic envelope. Concentration of virus without loss of viral infectivity permitted a higher multiplicity of infection, with a consequent higher efficiency of gene transfer, reaching 2.8 copies per cell. These vectors also showed remarkable stability during storage at 4 degrees C for a week. In addition, there was no significant loss of titer after freezing and thawing of the stock virus. The ability of VSV-G-pseudotyped retroviral vectors to achieve a severalfold increase in levels of transduction into CD34+ cells will allow high-efficiency gene transfer into hematopoietic progenitor cells for gene therapy purposes. Furthermore, since it has now become possible to infect CD34+ cells with pseudotyped HIV-1 with a high level of efficiency in vitro, many important questions regarding the effect of HIV-1 on lineage-specific differentiation of hematopoietic progenitors can now be addressed.

Animals↗
PubMed1995

Rapid generation of antibodies against the HTLV-II external envelope protein by growth of mouse plasmacytomas in SCID mice.

Recombinant human T-cell leukemia virus type II (HTLV-II) envelope external glycoprotein, gp46-II, was expressed using a vaccinia virus vector. A recombinant gp46-II fused to an epitope of the influenza virus hemagglutinin, YPYDVPDYA, was purified by immunoaffinity chromatography. The purified glycoprotein was used to immunize Balb/c mice, and antibodies against gp46-II were detected by Western blot analysis and syncytium inhibition assays. We transformed spleen cells from the immunized mice by retroviral infection with ABL-MYC (psi 2) and intraperitoneally transplanted the infected cells into syngeneic Balb/c and severe combined immunodeficient (SCID) mice. The plasmacytomas established ascitic tumors that produced antibodies directed against HTLV-II gp46-II. Ascites developed more rapidly in SCID mice than in normal syngeneic mice. This procedure provides a general means to generate antibodies rapidly.

Amino Acid Sequence↗
PubMed1995

Candidate gamma-secretases in the generation of the carboxyl terminus of the Alzheimer's disease beta A4 amyloid: possible involvement of cathepsin D.

beta A4 amyloid peptide, the main constituent of amyloid plaques and cerebrovascular amyloid deposits associated with Alzheimer's disease, derives from a large precursor protein (APP) by the action of beta- and gamma-secretases, the unidentified endoproteases which release the amino and carboxyl termini of beta A4, respectively. Several gamma-secretase cleavage sites exist which yield the more soluble (1-39/40) forms of beta A4 and the longer forms (1-42/43) which have a greater tendency to aggregate into amyloid plaques. gamma-Secretase activity may therefore be critical in amyloid formation. In this study, a synthetic peptide which encompasses the various gamma-secretase cleavage sites was used as a substrate to probe proteases of various classes and specificities. Elastase, collagenase, and cathepsin D cleaved at the amyloidogenic sites (after Ala42 or after Thr43) to release the carboxyl termini of the aggregating forms. In addition, collagenase and pepsin released the carboxyl terminus of the more soluble forms. Human brain fractions enriched in lysosomes contained a proteolytic activity that cleaved the substrate at the amyloidogenic site(s). This activity was more active at acidic pH and was inhibited by pepstatin, two characteristics of the lysosomal aspartyl proteinase cathepsin D. The same lysosomal fractions were found to contain APP carboxyl-terminal fragments which are potentially amyloidogenic. These were degraded, only in acidic conditions, by an endogenous protease activity inhibited by pepstatin. Thus, a cathepsin D-like activity from human brain is a candidate for APP gamma-secretase(s).

Alzheimer Disease↗
PubMed1995

Intracellular production of beta A4 amyloid of Alzheimer's disease: modulation by phosphoramidon and lack of coupling to the secretion of the amyloid precursor protein.

The amyloid precursor protein (APP) undergoes abnormal metabolism in Alzheimer's disease, resulting in the accumulation of beta A4 amyloid in the brain. Normal APP metabolism includes the release of a truncated form (sAPP) which has been cleaved at the alpha-secretase site within the beta A4 amyloidogenic domain. However, intact forms of beta A4 protein may also be generated by the beta- and gamma-secretases. Soluble forms of beta A4 have been detected in various cell lines and in cerebrospinal fluid. Previous studies of protein kinase C activation have suggested a reciprocal relationship between sAPP secretion and beta A4 production and release. We find that phorbol ester activation of protein kinase C in untransfected SH-SY5Y neuroblastoma cells increases the release of sAPP without affecting beta A4 secretion. We provide further evidence for intracellular beta A4 production. Treatment of SY5Y cells with the protease inhibitor phosphoramidon results in a 2-fold increase in beta A4 secretion and an increase in the amount of beta A4 recovered from cell lysates, yet it does not affect sAPP secretion. The protease inhibitors thiorphan and N-[(RS)-2-carboxy-3-phenylpropanoyl]-L-leucine had no effect on beta A4 or sAPP secretion. The lysosomotropic agents chloroquine and NH4Cl decreased beta A4 secretion, providing additional evidence for the involvement of intracellular acidic compartments in the production of beta A4. Our results therefore demonstrate a double dissociation between the secretion of sAPP and beta A4 in the SH-SY5Y cell line. The effect of phosphoramidon supports previous studies which show that metalloproteases are involved in the biogenesis of beta A4.

Alzheimer Disease↗
PubMed1995

Proteolytic processing of Alzheimer's disease beta A4 amyloid precursor protein in human platelets.

The processing of amyloid precursor protein (APP) and production of beta A4 amyloid are events likely to influence the development and progression of Alzheimer's disease, since beta A4 is the major constituent of amyloid deposited in this disorder. Our previous studies showed that human platelets contain full-length APP (APPFL) and are a suitable substrate to study normal APP processing. In the present study, we show that a 22-kDa beta A4-containing carboxyl-terminal fragment (22-CTF) of APP is present in unstimulated platelets. Both APPFL and 22-CTF are proteolytically degraded when platelets are activated with thrombin, collagen, or calcium ionophore A23187. Complete cleavage of APPFL and 22-CTF require the presence of extracellular calcium. Following stimulation in the presence of calcium, a new CTF of 17 kDa is generated, and the NH2-terminal epitope of beta A4 amyloid is lost. Preincubation of platelets with the cell-permeable cysteine protease inhibitors calpeptin, (2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane ethyl ester (E64d), Na alpha-p-tosyl-L-lysine chloromethyl ketone, or calcium chelator EGTA before platelet stimulation inhibits the degradation of both APPFL and 22-CTF. Divalent metal ions including zinc, copper, and cobalt inhibit the degradation of APPFL and 22-CTF. This study suggests that a calcium-dependent neutral cysteine protease is involved in the proteolytic processing of an amyloidogenic species of APP in human platelets.

Amyloid beta-Protein Precursor↗
PubMed1995

Affinity probes for the GABA-gated chloride channel: selection of 5e-tert-butyl-2e-[4-(substituted-ethynyl)phenyl]-1,3-dithianes and optimization of linker moiety.

The noncompetitive blocker (NCB) site of the gamma-aminobutyric acid (GABA)-gated chloride channel is the target for many important insecticides and potent convulsants. This site is specifically blocked by 3H ethynylbicycloorthobenzoate (3H EBOB) and other trioxabicyclooctane radioligands and might be suitable for affinity probes with an appropriate heterocyclic substituent and linker moiety. Optimal potency at the NCB site is achieved with 5e-tert-butyl-2e-[4-(substituted-ethynyl)phenyl]-1,3-dithianes+ ++ compared with analogs in which the butyldithiane portion is replaced with butyldithiane-sulfoxide or -sulfone, n-propyltrioxabicyclooctane or dioxatricyclododecene. Three positions were examined for coupling the linker and dithiane: C-2 of the dithiane; a branched substituent within the alkynyl moiety; the terminus of a straight chain extension from the ethynyl group, which proved to be the best. Optimized linkers for addition to the ethynylphenyldithiane to achieve appropriate length and fit within the active site, i.e. receptor potency, are CH2OCH2C(O)SCH2CH2 (SH or NH2) and the corresponding thiolates and amides. Several compounds with these spacers block the chloride channel, measured as inhibition of 3H EBOB binding, at 4-50 nM.

Affinity Labels↗
PubMed1995

Affinity probes for the GABA-gated chloride channel: 5e-tert-butyl-2e-[4-(substituted-ethynyl)phenyl]-1,3-dithianes with photoactivatable, fluorescent, biotin, agarose and protein substituents.

Affinity probes for the noncompetitive blocker or picrotoxinin site of the gamma-aminobutyric acid (GABA)-gated chloride channel were designed for four types of applications: photoaffinity reagents to covalently label the binding site; fluorescent probes for receptor analysis; biotinylated compounds and agarose/sepharose conjugates for affinity chromatography; ligand-protein/ enzyme conjugates for immunoassay. These 5e-tert-butyl-2e-[4-(substituted-ethynyl) phenyl]-1,3-dithianes were optimized by structure-activity studies for potency as inhibitors of 3H ethynylbicycloorthobenzoate binding to bovine brain membranes, measured as the concentration for 50% inhibition (IC50). Preferred compounds are 5e-(CH3)3CCH(CH2S)2CH-2e-C6H6-4-C = CCH2OCH2C(O)R, wherein R confers the following properties and IC50 values: R = SCH2CH2SCH2C(O)C6H4-4-N3, photoaffinity, 9 nM; R = NHCH2CH2NHC(O)C6H2-2-OH,5-1,4-N3, photoaffinity, 105 nM: R = SCH2CH2S-4-benzofurazan-7-NO2, fluorescent, 13 nM: R = SCH2CH2SCH2-5-fluorescein, fluorescent, 27 nM; R = NHCH2CH2NH[C(O)(CH2)5NH]2-biotin, affinity chromatography, 190 nM. The most potent photoaffinity ligand (IC50 9 nM) was labeled at 7 Ci mmol-1 by reacting the appropriate thiol with 3H 4-azidophenacyl bromide (obtained by alumina-catalyzed tritium exchange of its enolizable hydrogens). The first steps have been taken in using the NCB site for affinity chromatography of the GABAA receptor in CHAPS-solubilized bovine brain membranes with the dithiane-biotin probe and an avidin-acrylic bead system or with an analogous dithiane-agarose/ sepharose column eluting with GABA or dithiane as above (R = OH). A protein conjugate of a related dithiane-monosulfone elicited production of specific antisera in rabbits. These findings illustrate the diversity and utility of new affinity probes prepared in the alkynylphenyldithiane series.

Affinity Labels↗