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Y Wu

Publications and source records attributed to Y Wu.

At least 847 records · Page 47Linked to original sources
PubMed1996

Quantitation of putative activator-target affinities predicts transcriptional activating potentials.

We quantitate the 'activating potentials' of deletion and point mutation variants of a 42 amino acid yeast transcriptional activating region excised from the yeast activator GAL4 and, using surface plasmon resonance, we measure the relative affinities of these molecules for a variety of proteins, including plausible target proteins as well as GAL80, a specific inhibitor of GAL4. We find a remarkable correlation between the relative activating potentials of the derivatives and their relative affinities for yeast TBP and for yeast TFIIB; other tested proteins interacted significantly more weakly, if at all. These results provide an especially strong argument that TBP and TFIIB are activating region targets. We also show, using one set of yeast activating region mutants, that activator-target interactions are strongly correlated with the length of the activating region, that the effect of point mutants is highly dependent on the length of the activating region mutated and that, unlike interactions with TBP and TFIIB, interaction with the specific inhibitor GAL80 is destroyed by deletion of certain critical residues in the C-terminal half of the 42 amino acid activating region.

Amino Acid Sequence↗
PubMed1996

Pertussis toxin-sensitive activation of phospholipase C by the C5a and fMet-Leu-Phe receptors.

Signal transduction pathways that mediate C5a and fMet-Leu-Phe (fMLP)-induced pertussis toxin (PTx)-sensitive activation of phospholipase C (PLC) have been investigated using a cotransfection assay system in COS-7 cells. The abilities of the receptors for C5a and fMLP to activate PLC beta2 and PLC beta3 through the Gbetagamma subunits of endogenous Gi proteins in COS-7 cells were tested because both PLC beta2 and PLC beta3 were shown to be activated by the betagamma subunits of G proteins in in vitro reconstitution assays. Neither of the receptors can activate endogenous PLC beta3 or recombinant PLC beta3 in transfected COS-7 cells. However, both receptors can clearly activate PLC beta2 in a PTx-sensitive manner, suggesting that the receptors may interact with endogenous PTx-sensitive G proteins and activate PLC beta2 probably through the Gbetagamma subunits. These findings were further corroborated by the results that PLC beta3 could only be slightly activated by Gbeta1gamma1 or Gbeta1gamma5 in the cotransfection assay, whereas the Gbetagamma subunits strongly activated PLC beta2 under the same conditions. PLC beta3 can be activated by Galphaq, Galpha11, and Galpha16 in the cotransfection assay. In addition, the Ggamma2 and Ggamma3 mutants with substitution of the C-terminal Cys residue by a Ser residue, which can inhibit wild type Gbetagamma-mediated activation of PLC beta2, were able to inhibit C5a or fMLP-mediated activation of PLC beta2. These Ggamma mutants, however, showed little effect on m1-muscarinic receptor-mediated PLC activation, which is mediated by the Gq class of G proteins. These results all confirm that the Gbetagamma subunits are involved in PLC beta2 activation by the two chemoattractant receptors and suggest that in COS-7 cells activation of PLC beta3 by Gbetagamma may not be the primary pathway for the receptors.

Amino Acid Sequence↗
PubMed1996

Dietary antioxidant vitamins and death from coronary heart disease in postmenopausal women.

BACKGROUND: The role of dietary antioxidant vitamins in preventing coronary heart disease has aroused considerable interest because of the knowledge that oxidative modification of low-density lipoprotein may promote atherosclerosis. METHODS: We studied 34,486 postmenopausal women with no cardiovascular disease who in early 1986 completed a questionnaire that assessed, among other factors, their intake of vitamins A, E, and C from food sources and supplements. During approximately seven years of follow-up (ending December 31, 1992), 242 of the women died of coronary heart disease. RESULTS: In analyses adjusted for age and dietary energy intake, vitamin E consumption appeared to be inversely associated with the risk of death from coronary heart disease. This association was particularly striking in the subgroup of 21,809 women who did not consume vitamin supplements (relative risks from lowest to highest quintile of vitamin E intake, 1.0, 0.68, 0.71, 0.42, and 0.42; P for trend 0.008). After adjustment for possible confounding variables, this inverse association remained (relative risks from lowest to highest quintile, 1.0, 0.70, 0.76, 0.32, and 0.38; P for trend, 0.004). There was little evidence that the intake of vitamin E from supplements was associated with a decreased risk of death from coronary heart disease, but the effects of high-dose supplementation and the duration of supplement use could not be definitely addressed. Intake of vitamins A and C did not appear to be associated with the risk of death form coronary heart disease. CONCLUSIONS: These results suggest that in postmenopausal women the intake of vitamin E from food is inversely associated with the risk of death from coronary heart disease and that such women can lower their risk without using vitamin supplements. By contrast, the intake of vitamins A and C was not associated with lower risks of dying from coronary disease.

Aged↗
PubMed1996

Role of the N terminus in RNase A homologues: differences in catalytic activity, ribonuclease inhibitor interaction and cytotoxicity.

A number of biochemical properties differ dramatically among homologues within the pancreatic ribonuclease superfamily. Human pancreatic ribonuclease (hRNase) has high enzyme activity, extreme sensitivity to ribonuclease inhibitor (RI) and is non-toxic, whereas a homologous RNase from frog eggs, called onconase, has much lower enzyme activity, is not sensitive to RI and is cytotoxic to cancer cell lines and animals. To explore the structural basis of these differences among members in the RNAse family we synthesized genes for onconase, hRNase, a mutant onconase (K9Q) and onconase-hRNase N-terminal hybrids and expressed the proteins in Escherichia coli with final yields of 10 to 50 mg per liter of culture after purification. A recombinant version of onconase with an N-terminal methionine instead of the native pyroglutamyl residue had decreased cytotoxicity and enzyme activity. Cleavage of the recombinant onconase Met-1 residue, and cyclization of the Gln1 residue to reform the pyroglutamyl N terminus, reconstituted cytotoxicity and enzyme activity. Thus a unique role of the pyroglutamyl residue in the active site of amphibian RNases is indicated. Replacement of one to nine residues of onconase with the homologous residues of hRNase increased the enzymatic activity against most of the substrates tested with a simultaneous shift in the enzyme specificity from high preference for poly(U) to slight preference for poly(C). Cytotoxicity of the chimera decreased, dissociating cytotoxicity from enzymatic activity. The molecular basis for the low binding affinity of onconase for RI has been examined experimentally with the recombinant RNases and by fitting onconase and RNase A structures to the coordinates from the recently published RNase A-RI complex.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of the mouse obesity gene tubby: a member of a novel gene family.

The mutated gene responsible for the tubby obesity phenotype has been identified by positional cloning. A single base change within a splice donor site results in the incorrect retention of a single intron in the mature tub mRNA transcript. The consequence of this mutation is the substitution of the carboxy-terminal 44 amino acids with 24 intron-encoded amino acids. The normal transcript appears to be abundantly expressed in the hypothalamus, a region of the brain involved in body weight regulation. Variation in the relative abundance of alternative splice products is observed between inbred mouse strains and appears to correlate with an intron length polymorphism. This allele of tub is a candidate for a previously reported diet-induced obesity quantitative trait locus on mouse chromosome 7.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Identification of a phospholipase C beta2 region that interacts with Gbeta-gamma.

To delineate the phospholipase C (PLC; EC 3.1.4.3) beta2 sequences involved in interactions with the beta-gamma subunits of G proteins, we prepared a number of mammalian expression plasmids encoding a series of PLC beta2 segments that span the region from the beginning of the X box to the end of the Y box. We found the sequence extending from residue Glu-435 to residue Val-641 inhibited Gbeta-gamma-mediated activation of PLC beta2 in transfected COS-7 cells. This PLC beta2 sequence also inhibited ligand-induced activation of PLC in COS-7 cells cotransfected with cDNAs encoding the complement component C5a receptor and PLC beta2 but not in cells transfected with the alpha1B-adrenergic receptor, suggesting that the PLC beta2 residues (Glu-435 to Val-641) inhibit the Gbeta-gamma-mediated but not the Galpha-mediated effect. The inhibitory effect on Gbeta-gamma-mediated activation of PLC beta2 may be the result of the interaction between Gbeta-gamma and the PLC beta2 fragment. This idea was confirmed by the observation that a fusion protein comprising these residues (Glu-435 to Val-641) of PLC beta2 and glutathione S-transferase (GST) bound to Gbeta-gamma in an in vitro binding assay. The Gbeta-gamma-binding region was further narrowed down to 62 amino acids (residues Leu-580 to Val-641) by testing fusion proteins comprising various PLC beta2 sequences and GST in the in vitro binding assay.

Amino Acid Sequence↗
PubMed1996

Evidence for different chemosensory signal transduction pathways in olfactory and vomeronasal neurons.

Both the olfactory and vomeronasal epithelia mediate chemosensory reception. Here we report that several molecules that are highly expressed in the olfactory epithelium and therefore are likely to be important mediators of olfactory signal transduction (Golfalpha, adenylyl cyclase III and the olfactory cyclic nucleotide gated ion channel) are not present in the vomeronasal epithelium. Therefore it appears that distinct molecules mediate chemosensory signal transduction in the olfactory and vomeronasal epithelia. The genes for Golfalpha, adenylyl cyclase III, the olfactory cyclic nucleotide gated ion channel, Ggamma8 and olfactory marker protein which are all expressed in the olfactory epithelium have consensus Olf-1 binding sites. The transcription factor Olf-1 was found to be highly expressed in the olfactory epithelium and was detected at a similar level in the vomeronasal epithelium. The expression pattern of Olf-1 did not correlate with that of molecules involved in olfactory signaling but was more similar to the expression pattern of Ggamma8 and olfactory marker protein which are found both in olfactory and vomeronasal neurons. Therefore, expression of Olf-1 in the olfactory epithelium and the presence of Olf-1 binding sites in a number of different genes found to be expressed in the olfactory epithelium are not sufficient to explain the observed gene expression patterns.

Adenylyl Cyclases↗
PubMed1996

Selective G protein coupling by C-C chemokine receptors.

The C-C chemokines are major mediators of chemotaxis of monocytes and some T cells in inflammatory reactions. The pathways by which the C-C chemokine receptors activate phospholipase C (PLC) were investigated in cotransfected COS-7 cells. The C-C chemokine receptor-1 (CKR-1), the MCP-1 receptor-A (MCP-1Ra), and MCP-1Rb can reconstitute ligand-induced accumulation of inositol phosphates with PLC beta2 in a pertussis toxin-sensitive manner, presumably through G beta gamma released from the Gi proteins. However, these three receptors demonstrated different specificity in coupling to the alpha subunits of the Gq class. While none of the receptors can couple to Galphaq/11, MCP-1Rb can couple to both Galpha14 and Galpha16, but its splicing variant, MCP-1Rb, cannot. Since MCP-1Ra and -b differ only in their C-terminal intracellular domains, the C-terminal ends of MCP-1Rs determine G protein coupling specificity. CKR-1 can couple to Galpha14 but not to Galpha16, suggesting some of the C-C chemokine receptors, unlike the C-X-C chemokine receptors, discriminate against Galpha16, a hematopoietic-specific Galpha subunit. The intriguing specificity in coupling of the Gq class of G proteins implies that the chemokines may be involved in some distinct functions in vivo. The commonality of the chemokine receptors in coupling to the Gi-Gbetagamma-PLC beta2 pathway provides a potential target for developing broad spectrum anti-inflammatory drugs.

Animals↗
PubMed1996

delta Opioid receptor in neuronal cells undergoes acute and homologous desensitization.

Using delta opioid receptor as a model system, acute desensitization of neuronal opioid receptor was studied in detail in neuroblastoma x glioma NG108-15 cells and primarily-cultured mouse cortical cells. The opioid desensitization could occur in as short as 3 minutes of agonist treatment and the half-life of the desensitized state was about 90 minutes. This acute opioid desensitization was homologous in nature in both neuronal cells. The acute desensitization was almost abolished by treatment of the neuronal cells with staurosporine, a nonspecific protein kinase inhibitor. Treatment with the protein kinase C-selective inhibitor, calphostin C, however, caused partial block. In conclusion, neuronal opioid receptor undergoes acute, agonist-dependent, and homologous desensitization, during which protein kinases appear to play an important role.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine.

Plasmodium falciparum malaria parasites were transformed with plasmids containing P. falciparum or Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (dhfr-ts) coding sequences that confer resistance to pyrimethamine. Under pyrimethamine pressure, transformed parasites were obtained that maintained the transfected plasmids as unrearranged episomes for several weeks. These parasite populations were replaced after 2 to 3 months by parasites that had incorporated the transfected DNA into nuclear chromosomes. Depending upon the particular construct used for transformation, homologous integration was detected in the P. falciparum dhfr-ts locus (chromosome 4) or in hrp3 and hrp2 sequences that were used in the plasmid constructs as gene control regions (chromosomes 13 and 8, respectively). Transformation by homologous integration sets the stage for targeted gene alterations and knock-outs that will advance understanding of P. falciparum.

Animals↗
PubMed1996

Rhinovirus stimulation of interleukin-6 in vivo and in vitro. Evidence for nuclear factor kappa B-dependent transcriptional activation.

To further understand the biology of rhinovirus (RV), we determined whether IL-6 was produced during RV infections and characterized the mechanism by which RV stimulates lung cell IL-6 production. In contrast to normals and minimally symptomatic volunteers, IL-6 was detected in the nasal washings from patients who developed colds after RV challenge. RV14 and RV1A, major and minor receptor group RVs, respectively, were potent stimulators of IL-6 protein production in vitro. These effects were associated with significant increases in IL-6 mRNA accumulation and gene transcription. RV was also a potent stimulator of IL-6 promoter-driven luciferase activity. This stimulation was modestly decreased by mutation of the nuclear factor (NF)-IL-6 site and abrogated by mutation of the NF-kappa B site in this promoter. An NF-kappa B-DNA binding activity, mediated by p65, p50, and p52 NF-kappa B moieties, was rapidly induced in RV-infected cells. Activator protein 1-DNA binding was not similarly altered. These studies demonstrate that IL-6 is produced during symptomatic RV infections, that RVs are potent stimulators of IL-6 elaboration, and that RV stimulation IL-6 production is mediated by an NF-kappa B-dependent transcriptional stimulation pathway. IL-6 may play an important role in the pathogenesis of RV infection, and NF-kappa B activation is likely to be an important event in RV-induced pathologies.

Adult↗
PubMed1996

Determination of LTE4 in human urine by liquid chromatography coupled with ionspray tandem mass spectrometry.

A sensitive and specific high-performance liquid chromatographic/ionspray tandem mass spectrometric (LC/ionspray MS/MS) method was developed and validated to quantitate leukotriene E4 (LTE4) in human urine. This method involves solid-phase extraction with Empore membrane disks to isolate LTE4 and its internal standard, LTE4-d3, from the urine stabilized with antioxidant and metal ion chelating agent. The reconstituted extracts were analyzed by LC/ionspray MS/MS in the selected reaction monitoring (SRM) mode. The assay has a lower level of quantitation (LOQ) of 50 pg ml-1 for LTE4 based on 5 ml aliquots of urine. The calibration graphs were linear from 50 pg ml-1 to 10 ng ml-1 for LTE4 extracted from urine. The inter- and intra-assay precision (RSD) and accuracy (DEV) did not exceed 11% and 8% at any level of the calibration standards and quality control (QC) samples, respectively. The recovery of LTE4 using the Empore disk solid-phase extraction technology was independent of LTE4 concentration in human urine. The overall extraction recovery for LTE4 was 72% (RSD = 2.14%).

Antioxidants↗
PubMed1996

Activation of the insulin-like growth factor-I receptor inhibits tumor necrosis factor-induced cell death.

The effect of insulin-like growth factor (IGF) on tumor necrosis factor (TNF)-induced cell killing was determined for mouse BALB/c3T3 fibroblasts in vitro. Cells maintained in 0.5% fetal bovine serum (FBS) were killed by TNF within 6 h in a concentration-dependent manner, an effect that was prevented by IGF-I. TNF-induced cytotoxicity of 3T3 cells that overexpress the human IGF-I receptor (p6 cells) was prevented by IGF-I alone in the absence of serum. TNF-induced cell death was associated with the morphologic features of apoptosis and the release of low-molecular-weight DNA, both of which were prevented by IGF-I. Neither epidermal growth factor (EGF) nor platelet-derived growth factor (PDGF) protected p6 cells from TNF-induced apoptosis. The specific protective action of the IGF-I receptor was demonstrated further by the marked sensitivity to TNF of embryo fibroblasts derived from mice with targeted disruption of the IGF-I receptor (R-cells) but not of fibroblasts derived from wild-type littermates or R-cells transfected with the cDNA for the human IGF-I receptor. Cycloheximide or actinomycin D markedly reduced the protection offered by IGF-I. IGF-I protection of BALB/c3T3 cells persisted for up to 5 days in the presence of PDGF and EGF, whereas IGF-I lost its effectiveness after 2 days in the absence of growth factors. IGF-I did not prevent TNF-induced release of arachidonic acid. The results demonstrate a specific role for the IGF-I receptor in the protection against TNF cytotoxicity. This action of the IGF-I receptor is mediated by protective cytosolic proteins that exhibit a high rate of turnover and whose levels are regulated principally by factors within serum other than IGF-I.

3T3 Cells↗
PubMed1996

Comprehensive and accurate mutation scanning of the CFTR gene by two-dimensional DNA electrophoresis.

The large number of possible disease-causing mutations in the 27 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has severely limited direct diagnosis of cystic fibrosis (CF) patients and carriers by mutation detection. Here we show that in principle testing for mutations in the CFTR gene can be both substantially facilitated and made virtually complete, by two-dimensional DNA electrophoretic separation of polymerase chain reaction (PCR) amplified exons on the basis of size and basepair sequence in denaturing gradient gels. Under a single optimized set of conditions we were able to obtain a pattern of spots representing all 27 exons of the CFTR gene and to readily detect 17 out of 17 identified sequence variations in 9 different exons in DNA from 11 CF patients and carriers. Our results demonstrate the potential of 2-dimensional DNA electrophoresis for comprehensive mutation analysis of the CFTR gene. The approach serves as a model for comprehensive diagnosis of the many other large disease genes for which a variety of mutations have also which been reported.

Cystic Fibrosis↗
PubMed1996

Epitope analysis of the major reactive region of the 100-kd protein of PM-Scl autoantigen.

OBJECTIVE: To localize the epitope(s) bound by anti-PM-Scl antibodies in the N-terminal half of the 100-kd protein, the major antigen of the PM-Scl complex. METHODS: Investigations were performed by immunoblotting 20 anti-PM-Scl positive sera against bacterially expressed, polymerase chain reaction-derived deletion mutants of the S1 fragment (amino acids 11-437), enzyme-linked immunosorbent assay (ELISA) screening against synthesized serial octapeptides, and ELISA screening, with anti-PM-Scl positive sera, against a synthesized 21-amino acid peptide covering the active region. RESULTS: Anti-PM-Scl positive sera retained full immunoblot activity with fragment 207-436 and most activity with fragment 11-241, but had markedly decreased activity against fragments 236-436 and 11-212, indicating a major epitope in the aa 207-241 region. Fusion proteins with smaller fragments localized this activity between aa 226 and aa 246. Of 42 anti-S1-positive, anti-PM-Scl positive sera tested by ELISA against a synthetic peptide of this region, 36 were definitely positive, 4 borderline, and 2 negative. Similar activity was seen with a peptide from which proline 228 was deleted. Three additional epitope areas were found in S1, but each reacted with only a few sera. Anti-PMScl positive sera did not react with any octapeptide spanning the major epitope area (aa 207-246). CONCLUSION: The main immunoblot epitope of the PM-Scl 100-kd protein is within a central area of 21 aa (aa 226-246), but is longer than the usual linear epitope. This peptide may be useful in patient testing. Three minor epitopes in S1 may also be recognized by some sera.

Epitopes↗
PubMed1996

Single molecule detection of RNA reporter probes by amplification with Q beta replicase.

The addition of target-specific probe sequences within MDV RNA, an otherwise efficient template for Q beta replicase, generally resulted in RNA molecules with inferior replication properties, including reduced replication rates and poor sensitivities. We have discovered that the replication characteristics of MDV RNA molecules with internally placed probe sequences are dramatically affected by short RNA sequences (spacer elements) at the 5' and 3' ends of the probe sequence. For a given probe sequence, the sequences of the flanking spacer elements effected replication sensitivity by six orders of magnitude and replication rate by three fold. By taking advantage of spacer elements, internal MDV probes were developed that permitted the reproducible, real time, fluorescence detection of a single RNA molecule in less than 25 min through amplification with Q beta replicase. RNA structural analysis of such probes suggested that the spacer elements functioned by allowing the RNA to fold in a way which substantially maintained the tertiary structure of the MDV domain. MDV reporter probes with suitable replication properties were obtained from libraries of RNA molecules in which the probe sequence was flanked by many different spacer elements (generated by random nucleotide synthesis). We demonstrated that this is a general method for developing RNA reporter molecules which are rapidly and reproducibly amplified by Q beta replicase, even from a single molecule.

Base Sequence↗
PubMed1996

Isolation and characterization of full-length cDNA clones of the giant taro (Alocasia macrorrhiza) trypsin/chymotrypsin inhibitor.

A full-length cDNA encoding the 206 amino acid open reading frame of a trypsin/chymotrypsin inhibitor abundant in the corms of giant taro (Alocasia macrorrhiza) was isolated. An internal fragment was cloned using degenerate primers corresponding to a region of the mature protein sequence and the 'rapid amplification of cDNA ends' (RACE) method used to generate a composite cDNA sequence. The length of the cDNA was close to the predicted size of the corresponding transcript deduced from northern blot analysis of corm mRNA. The inhibitor was expressed strongly in the mature corm, at low levels in leaf blades and petioles but not in roots. Southern blot analysis of the giant taro DNA indicated that this inhibitor is encoded by a small multigene family and this was further supported by the isolation of two different sequence classes from corm cDNA using primers to the ends of the composite sequence.

Amino Acid Sequence↗