Biomedical subjects
Publications and source records attributed to Y Wu.
Five new diprenylated flavonols, broussonol A (1), broussonol B (2), broussonol C (3), broussonol D (4), and broussonol E (5), along with two known compounds, were isolated from an ethanolic extract of the leaves of Broussonetia kazinoki. Their structures were elucidated by chemical and spectral methods. Cytotoxic activities were evaluated against several different cell lines.
During rice seed development, prolamine RNAs are localized to the surface of the prolamine storage protein bodies (PBs), organelles bounded by the endoplasmic reticulum (ER). The exact mechanism by which prolamine RNAs are enriched on this ER subdomain is not known but recent evidence indicates the directed transport and targeting of prolamine RNAs to the prolamine PBs. As such a process involves RNA signal determinants and cytoskeleton-interacting proteins that recognize these signals, we obtained an enriched cytoskeleton-PB fraction and identified a prominent RNA-binding activity, Rp120, by RNA-binding UV-cross-linking assay. Recombinant cDNA clones of Rp120 revealed that the primary sequence shared considerable structural homology to the human transcriptional coactivator p100 and possessed a modular organization, four nucleic acid-binding SN domains, a tudor domain and a coil-coil domain. Consistent with the presence of SN domains, Rp120 binds a variety of RNAs including prolamine RNA. Interaction with the latter RNA, however, was specific as binding activity was evident only to the prolamine 3' UTR and not to the 5' UTR or coding sequences. Rp120 is also able to interact with other proteins as its sedimentation behavior in sucrose density gradient suggests an association with the cytoskeleton. The presence of a tudor domain, suggested to have a role in RNA processing or transport, together with the SN and coiled-coil domains are consistent with the view that Rp120 may be involved in RNA sorting in rice endosperm.
A novel cDNA fragment was identified from a human fetal brain cDNA library by using the coding sequence of human BRI3 gene (Accession No. NM015379) as bait in a yeast two-hybrid screening. Then by 5'-RACE (rapid amplification of cDNA end) and electronic hybridization, we obtained a 1.9 kb contig which consists of a novel gene. It was designated as BRI3BP by the HUGO Nomenclature Committee. It contains an open reading frame encoding 251 amino acids. The calculated molecular weight of the deduced protein is 27.8 kU. The predicted isoelectric point is 9.48. Northern hybridization showed its mRNA was highly expressed in brain, kidney, and liver. By RH mapping, the BRI3BP gene was mapped to human chromosome 12q24.2-qter
Different forms of vascular endothelial growth factor (VEGF) and their cellular receptors (VEGFR) are associated with angiogenesis, as demonstrated by the lethality of VEGF-A, VEGFR-1 or VEGFR-2 knockout mice. Here we have used an in vitro angiogenesis model, consisting of human microvascular endothelial cells (hMVEC) cultured on three-dimensional (3D) fibrin matrices to investigate the roles of VEGFR-1 and VEGFR-2 in the process of VEGF-A and VEGF-C-induced tube formation. Soluble VEGFR-1 completely inhibited the tube formation induced by the combination of VEGF-A and TNF alpha (VEGF-A/TNF alpha). This inhibition was not observed when tube formation was induced by VEGF-C/TNF alpha or bFGF/TNF alpha. Blocking monoclonal antibodies specific for VEGFR-2, but not antibodies specifically blocking VEGFR-1, were able to inhibit the VEGF-A/TNF alpha-induced as well as the VEGF-C/TNF alpha-induced tube formation in vitro. P1GF-2, which interacts only with VEGFR-1, neither induced tube formation in combination with TNF alpha, nor inhibited or stimulated by itself the VEGF-A/TNF alpha-induced tube formation in vitro. These data indicate that VEGF-A or VEGF-C activation of the VEGFR-2, and not of VEGFR-1, is involved in the formation of capillary-like tubular structures of hMVEC in 3D fibrin matrices used as a model of repair-associated or pathological angiogenesis in vitro.
Multiple exostosis, biparietal foramina, minor craniofacial abnormalities, and mental retardation are characteristic of the syndrome associated with a proximal deletion of 11p (MIM # 601224), which has been shown to be a true contiguous gene deletion syndrome. The presence of multiple exostosis is associated with deletion of the EXT2 gene. Similarly, the presence of biparietal foramina has been shown to be associated with the deletion of ALX4 located proximally to EXT2. Specific genes related to mental retardation and craniofacial abnormalities, however, have yet to be identified. We report on a family with a microdeletion of 11(pll.2p11.2) with multiple exostosis and biparietal foramina without mental retardation or craniofacial abnormalities. Our results suggest that genes related to mental retardation and craniofacial development must be located outside of the D11S1785-D11S1385 region.
This study investigated salivary anticandidal activity and salivary composition in stimulated whole saliva of 18 advanced HIV-infected patients and compared these values to healthy controls. Stimulated whole saliva from HIV-infected patients showed decreased anticandidal activity. The flow rate was reduced by 40% as compared with controls. The saliva flow rate for HIV-infected patients who had recoverable yeast in their saliva was reduced as compared to HIV-infected patients without recoverable yeast. For HIV-infected patients, the saliva concentrations of lactoferrin, secretory IgA and Cl- were increased while the secretion rate of lysozyme, total protein and K+ were reduced. There was no difference in any parameter as a function of taking the antifungal drug fluconazole. There was no association between salivary anticandidal activity and any salivary component. This study shows reduced anticandidal activity and salivary flow rate in HIV-infected patients. These alterations may contribute to their increased incidence of oral candidal infections.
To maintain quality control in cells, mechanisms distinguish among improperly folded peptides, mature and functional proteins, and proteins to be targeted for degradation. The molecular chaperones, including heat-shock protein Hsp90, have the ability to recognize misfolded proteins and assist in their conversion to a functional conformation. Disruption of Hsp90 heterocomplexes by the Hsp90 inhibitor geldanamycin leads to substrate degradation through the ubiquitin-proteasome pathway, implicating this system in protein triage decisions. We previously identified CHIP (carboxyl terminus of Hsc70-interacting protein) to be an interaction partner of Hsc70 (ref. 4). CHIP also interacts directly with a tetratricopeptide repeat acceptor site of Hsp90, incorporating into Hsp90 heterocomplexes and eliciting release of the regulatory cofactor p23. Here we show that CHIP abolishes the steroid-binding activity and transactivation potential of the glucocorticoid receptor, a well-characterized Hsp90 substrate, even though it has little effect on its synthesis. Instead, CHIP induces ubiquitylation of the glucocorticoid receptor and degradation through the proteasome. By remodelling Hsp90 heterocomplexes to favour substrate degradation, CHIP modulates protein triage decisions that regulate the balance between protein folding and degradation for chaperone substrates.
Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf-/-). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf-/- mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf-/- mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders.
We investigated a possible role of the mismatch-repair gene MLH3 in hereditary nonpolyposis colorectal cancer by scanning for mutations in 39 HNPCC families and in 288 patients suspected of having HNPCC. We identified ten different germline MLH3 variants, one frameshift and nine missense mutations, in 12 patients suspected of HNPCC. Three of the 12 also carried a mutation in MSH6.
The role of bone marrow (BM)-derived precursor cells in tumor angiogenesis is not known. We demonstrate here that tumor angiogenesis is associated with recruitment of hematopoietic and circulating endothelial precursor cells (CEPs). We used the angiogenic defective, tumor resistant Id-mutant mice to show that transplantation of wild-type BM or vascular endothelial growth factor (VEGF)-mobilized stem cells restore tumor angiogenesis and growth. We detected donor-derived CEPs throughout the neovessels of tumors and Matrigel-plugs in an Id1+/-Id3-/- host, which were associated with VEGF-receptor-1-positive (VEGFR1+) myeloid cells. The angiogenic defect in Id-mutant mice was due to impaired VEGF-driven mobilization of VEGFR2+ CEPs and impaired proliferation and incorporation of VEGFR1+ cells. Although targeting of either VEGFR1 or VEGFR2 alone partially blocks the growth of tumors, inhibition of both VEGFR1 and VEGFR2 was necessary to completely ablate tumor growth. These data demonstrate that recruitment of VEGF-responsive BM-derived precursors is necessary and sufficient for tumor angiogenesis and suggest new clinical strategies to block tumor growth.
The Bcl-2 family of proteins has been characterized by either anti-apoptotic or pro-apoptotic activity. Insight into how Bcl-2 family members function has been gained by determining their intracellular localization. We have generated a monoclonal anti-A1-a antibody and used a COS-7 overexpression system to study the localization of the murine anti-apoptotic Bcl-2 family member, A1-a. A1-a overexpressed in COS-7 cells localized to the nucleus as determined by subcellular fractionation and immunofluorescent microscopy. A1-a in the COS-7 nucleus bound tightly to the nuclear matrix as evidenced by resistance to treatment with DNAse I and RNAse A and sequential extraction with 1.0% Triton X-100, 0.15 M NaCl, 0.25 M HCl, 0.5 M Tris pH 7.4 and 6 M urea. HPLC analysis of A1-a, subsequent to SDS extraction, produced fractions that gave multiple bands when analyzed by Western blot analysis suggesting a propensity to form multimers. COS-7 cells transfected with A1-a were protected from apoptotic induction by staurosporine treatment.
Long-term depression (LTD) at parallel fibre-Purkinje cell synapse of the cerebellum is thought to be a cellular substrate for motor learning. LTD requires activation of metabotropic glutamate receptor subtype 1 (mGluR1) and its downstream signalling pathways, which invariably involves phospholipase Cbetas (PLCbetas). PLCbetas consist of four isoforms (PLCbeta1-4) among which PLCbeta4 is the major isoform in most Purkinje cells in the rostral cerebellum (lobule 1 to the rostral half of lobule 6). We studied mutant mice deficient in PLCbeta4, and found that LTD was deficient in the rostral but not in the caudal cerebellum of the mutant. Basic properties of parallel fibre-Purkinje cell synapses and voltage-gated Ca2+ channel currents appeared normal. The mGluR1-mediated Ca2+ release induced by repetitive parallel fibre stimulation was absent in the rostral cerebellum of the mutant, suggesting that their LTD lesion was due to the defect in the mGluR1-mediated signalling in Purkinje cells. Importantly, the eyeblink conditioning, a simple form of discrete motor learning, was severely impaired in PLCbeta4 mutant mice. Wild-type mice developed the conditioned eyeblink response, when pairs of the conditioned stimulus (tone) and the unconditioned stimulus (periorbital shock) were repeatedly applied. In contrast, PLCbeta4 mutant mice could not learn the association between the conditioned and unconditioned stimuli, although their behavioural responses to the tone or to the periorbital shock appeared normal. These results strongly suggest that PLCbeta4 is essential for LTD in the rostral cerebellum, which may be required for the acuisition of the conditioned eyeblink response.
Xp95, a protein recently identified in Xenopus laevis, is potentially involved in progesterone-induced Xenopus oocyte maturation. In this study, we cloned a human homologue of Xp95, designated Hp95, and examined the effect of its overexpression on the growth properties of human malignant HeLa cells which have lost the contact inhibition of cell proliferation. We observed that although HeLa cells did not undergo G1 phase arrest at any stage after confluence, they were able to downregulate their G1 phase CDK activities in response to confluence. When Hp95 was overexpressed in HeLa cells by transfection with a constitutive or an inducible expression vector containing a full-length Hp95 transgene, HeLa cells became able to undergo G1 phase arrest and form a monolayer culture after confluence. However, the G1 phase CDK activities in these Hp95 overexpressing cells were not inhibited further as compared to control cells after confluence. These results indicate that the defects in HeLa cells that cause the loss of contact inhibition of cell proliferation are in components downstream of the G1 phase CDKs and that overexpression of Hp95 counteracts some of these defects.
beta-Arrestins regulate opioid receptor-mediated signal transduction and play an important role in opiate-induced analgesia and tolerance/dependence. This study was carried out to measure the direct interaction between beta-arrestins and opioid receptor. Immunoprecipitation experiments demonstrated that beta-arrestin 1 physically interacts with delta opioid receptor (DOR) co-expressed in human embryonic kidney 293 cells in an agonist-enhanced manner and truncation of the carboxyl terminus of DOR partially impairs the interaction. In vitro data from glutathione-S-transferase pull-down assay showed that the carboxyl terminus (CT) and the third intracellular loop (I3L) of DOR are both capable of and either domain is sufficient for binding to beta-arrestin 1 and 2. Surface plasmon resonance determination further revealed that binding of CT and I3L of DOR to beta-arrestin is additive, suggesting these two domains bind at distinctly different sites on beta-arrestin without considerable spatial hindrance. This study demonstrated for the first time the direct binding of beta-arrestins to the two distinct domains, the carboxyl terminus and the third intracellular loop, of DOR.
AIMS: The effect of phage concentration on the activity of adenylate kinase (AK) released from the cells lysed during infection was investigated in order to optimize a bioluminescent phage-mediated method for bacterial enumeration. METHODS AND RESULTS: The number of bacteria lysed by phages specific to Salmonella enteritidis and E. coli was determined using a bioluminescent method for the detection of AK released. In order to optimize the assay, the effect of phage concentration and time of infection on the amount of AK released was investigated. The release of AK was greatest at a multiplicity of infection (moi) of 10-100. CONCLUSION: The amount of AK released from Salmonella enteritidis and E. coli G2-2 cells by specific phages, SJ2 and AT20, respectively, depended on the type of bacteria, the stage of growth, the nature of phage, moi and time. SIGNIFICANCE AND IMPACT OF THE STUDY: An assay is described which allows detection of E. coli and Salmonella Enteritidis within 2 h at levels of 103 cfu ml-1.
BACKGROUND: With changing demographics of the United States population and the continuous need to recruit new donors, it is important to monitor the demographic profile of first-time donors and to evaluate changes in the donor pool to improve recruitment targeting. STUDY DESIGN AND METHODS: First-time whole blood (n = 901,862) donors at five United States blood centers between 1991 and 1996 were analyzed. RESULTS: The total number of first-time donors appears to be decreasing. Over the 6-year period, there was an overall increase in the proportion of Hispanic and other minority first-time donors and a concurrent decrease in the proportion of white donors at Retrovirus Epidemiology Donor Study centers. Other variables, including age, sex, and education, did not show a consistent trend. CONCLUSION: The demographic profile of first-time donors is changing. These data highlight the importance for blood centers to continuously monitor the donor population. A better understanding of the donor population may help blood centers adjust their donor outreach, recruitment, and retention programs. New recruitment efforts appear needed to counter general apathy toward donating blood, and minority groups appear to be receptive to becoming blood donors.
BACKGROUND: The COBAS AmpliScreen HIV-1 test, version 1.5 (v1.5) (Roche Molecular Systems), is designed for screening pools composed of samples from 24 individual units of blood or plasma. A specimen-processing procedure (Multiprep) simultaneously concentrates and extracts HIV-1, HCV, and HBV particles from plasma and incorporates an HIV-1 internal control (IC) RNA. Processed samples are amplified by RT-PCR using HIV-1-specific primers and detected by hybridization of the amplified products to HIV-1- and IC-specific oligonucleotide probes. STUDY DESIGN AND METHODS: Plasma samples containing known quantities of HIV-1 were used to evaluate analytical sensitivity and precision and to validate a pool testing algorithm. Analytical specificity was evaluated by adding various viruses and bacteria to HIV-1-negative plasma. Seroconversion panels were tested to estimate the window-period reduction achieved by RNA testing. RESULTS: The analytical sensitivity of the test (concentration that yields > or = 95% positive results in a set of replicate tests) was 25 copies of HIV-1 RNA per mL of pooled plasma. Representative strains from all HIV-1 group M subtypes were reproducibly detected (> 95% positive results) at concentrations of 20 to 200 viral particles per mL. The test did not cross-react with a set of 31 viral and 5 bacterial isolates, and it yielded negative results on a panel of 500 blood samples from HIV-1-seronegative donors. Plasma samples containing abnormally high levels of Hb, albumin, triglycerides, or bilirubin did not interfere with the test. HIV-1 RNA was detected 2 to 14 days before HIV-1 antibody and 0 to 28 days before p24 antigen. The test specifically detected pools containing a single positive unit with 2400 HIV-1 RNA copies per mL and correctly identified the positive unit. CONCLUSION: The COBAS AmpliScreen HIV-1 test, v1.5, has sufficient sensitivity to detect a single infected unit containing 600 copies of HIV-1 per mL in a pool with 23 uninfected units and should reduce the window period between infection and seroconversion by at least 2 to 14 days.
BACKGROUND & AIMS: Germline mutations in one of four mismatch repair genes have been found in the majority of families with hereditary nonpolyposis colorectal cancer (HNPCC), but only in a small part of families with atypical HNPCC. The recently cloned EXO1 gene might be involved in the pathogenesis of HNPCC because the EXO1 protein strongly interacts with the MSH2 protein. To determine its role in HNPCC, EXO1 was scanned for germline mutations. METHODS: All 14 exons of EXO1 were scanned for mutations in index patients from 33 families with HNPCC fulfilling the Amsterdam criteria and in 225 index patients suspected of HNPCC. RESULTS: Germline variants of EXO1 were detected in 14 patients, including one splice-site mutation in a family with HNPCC and 13 missense mutations in patients with atypical HNPCC. These variants did not occur in more than 200 control individuals. From 13 of these 14 patients, tumors were available for analysis of microsatellite instability and loss of heterozygosity. Six of the tumors showed microsatellite instability. Heterozygosity analysis showed one case without EXO1 allelic loss and 12 tumors with loss of the mutant allele and retention of the normal one. CONCLUSIONS: The results indicate a possible association of germline EXO1 variants with HNPCC and atypical HNPCC.