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Biomedical subjects

L Wu

Publications and source records attributed to L Wu.

At least 199 records · Page 11Linked to original sources
PubMed2000

CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen.

The dendritic cells (DC) of mouse spleen and thymus were examined for expression of CD4 and CD8. Provided care was taken to avoid selective extraction or selective depletion of DC subpopulations, three main types of DC were detected in mouse spleen: a major new population of CD4+8- DEC-205low CD11bhigh DC, together with the previously described CD4-8- DEC-205low CD11bhigh DC and CD4-8alphaalpha+ DEC-205high CD11blow DC. The CD4 on the surface of the CD4+ splenic DC subpopulation was produced by the DC themselves, and CD4 RNA transcripts were present. Likewise, the CD8alpha on the surface of the splenic CD8+ DC was shown to be a product of the DC themselves, in agreement with earlier evidence. All three spleen DC types would be considered as mature, based on expression of CD80, CD86, and CD40 as well as on T cell stimulating function. Mouse thymuses appeared to contain two DC types; both were DEC-205highCD11blow, but they differed in the level of CD8alphaalpha expression. However, as well as this authenticated marker expression, immunofluorescent staining was also found to reflect a series of artifacts, due to the autofluorescence of contaminating cells and due to pickup of CD4 and CD8alphabeta. By constructing mice chimeric for the hemopoietic lineages using mixtures of wild-type bone marrow with CD4null or CD8alphanull bone marrow, a marked pickup by thymic DC of Ags derived from thymocytes was demonstrated.

Animals↗
PubMed2000

Gene promoter hypermethylation in tumors and serum of head and neck cancer patients.

Promoter hypermethylation is an important pathway for repression of gene transcription in cancer cells. We analyzed aberrant DNA methylation at four genes in primary tumors from 95 head and neck cancer patients and then used the presence of this methylation as a marker for cancer cell detection in serum DNA. These four genes were tested by methylation-specific PCR and included: p16 (CDKN2A), O6-methylguanine-DNA-methyltransferase, glutathione S-transferase P1, and death-associated protein kinase (DAP-kinase). Fifty-five % (52 of 95) of the primary tumors displayed promoter hypermethylation in at least one of the genes studied: 27% (26/95) at p16, 33% (31 of 95) at O6-methylguanine-DNA-methyltransferase; and 18% (17 of 92) at DAP-kinase. No promoter hypermethylation was observed at the glutathione S-transferase P1 gene promoter. We detected a statistically significant correlation between the presence of DAP-kinase gene promoter hypermethylation and lymph node involvement (P = 0.014) and advanced disease stage (P = 0.016). In 50 patients with paired serum available for epigenetic analysis, the same methylation pattern was detected in the corresponding serum DNA of 21 (42%) cases. Among the patients with methylated serum DNA, 5 developed distant metastasis compared with the occurrence of metastasis in only 1 patient negative for serum promoter hypermethylation (P = 0.056). Promoter hypermethylation of key genes in critical pathways is common in head and neck cancer and represents a promising serum marker for monitoring affected patients.

Apoptosis Regulatory Proteins↗
PubMed2000

Structure-based discovery of small molecule inhibitors targeted to protein tyrosine phosphatase 1B.

Protein tyrosine phosphatases (PTPases) are involved in the control of tyrosine phosphorylation levels in the cell and are believed to be crucial for the regulation of a multitude of cellular functions. A detailed understanding of the role played by PTPases in various signaling pathways has not yet been achieved, and potent and selective PTPase inhibitors are essential in the quest to determine the functionality of individual PTPases. Using the DOCK methodology, we have carried out a structure-based, computer-assisted search of an available chemical database in order to identify low molecular weight, nonpeptidic PTP1B inhibitors. We have identified several organic molecules that not only possess inhibitory activity against PTP1B but which also display significant selectivity for PTP1B. This indicates that although structural features important for pTyr recognition are conserved among different PTPases, it is possible to generate selective inhibitors targeted primarily to the catalytic site. Kinetic analysis and molecular modeling experiments suggest that the PTP1B active site possesses significant plasticity such that substituted and extended aromatic systems can be accommodated. The newly identified molecules provide a molecular framework upon which therapeutically useful compounds can ultimately be based, and systematic optimization of these lead compounds is likely to further enhance their potency and selectivity.

Catalytic Domain↗
PubMed2000

Effects on general acid catalysis from mutations of the invariant tryptophan and arginine residues in the protein tyrosine phosphatase from Yersinia.

General acid catalysis in protein tyrosine phosphatases (PTPases) is accomplished by a conserved Asp residue, which is brought into position for catalysis by movement of a flexible loop that occurs upon binding of substrate. With the PTPase from Yersinia, we have examined the effect on general acid catalysis caused by mutations to two conserved residues that are integral to this conformation change. Residue Trp354 is at a hinge of the loop, and Arg409 forms hydrogen bonding and ionic interactions with the phosphoryl group of substrates. Trp354 was mutated to Phe and to Ala, and residue Arg409 was mutated to Lys and to Ala. The four mutant enzymes were studied using steady state kinetics and heavy-atom isotope effects with the substrate p-nitrophenyl phosphate. The data indicate that mutation of the hinge residue Trp354 to Ala completely disables general acid catalysis. In the Phe mutant, general acid catalysis is partially effective, but the proton is only partially transferred in the transition state, in contrast to the native enzyme where proton transfer to the leaving group is virtually complete. Mutation of Arg409 to Lys has a minimal effect on the K(m), while this parameter is increased 30-fold in the Ala mutant. The k(cat) values for R409K and for R409A are about 4 orders of magnitude lower than that for the native enzyme. General acid catalysis is rendered inoperative by the Lys mutation, but partial proton transfer during catalysis still occurs in the Ala mutant. Structural explanations for the differential effects of these mutations on movement of the flexible loop that enables general acid catalysis are presented.

Acids↗
PubMed2000

Intratumoral administration of adenoviral interleukin 7 gene-modified dendritic cells augments specific antitumor immunity and achieves tumor eradication.

In two murine lung cancer models adenoviral interleukin 7-transduced dendritic cells (DC-AdIL-7) were administered intratumorally, resulting in complete tumor regression. Intratumoral DC-AdIL-7 therapy was as effective as DCs pulsed with specific tumor peptide antigens. Comparison with other intratumoral therapies including recombinant IL-7, AdIL-7 vector alone, unmodified DCs, IL-7-transduced fibroblasts, or DCs pulsed with tumor lysates revealed DC-AdIL-7 therapy to be superior in achieving antitumor responses and augmenting immunogenicity. Mice with complete tumor eradication as a result of either DC-AdIL-7 or AdIL-7 therapy were rechallenged with parental tumor cells 30 days or more after complete tumor eradication. All the DC-AdIL-7-treated mice completely rejected a secondary rechallenge, whereas the AdIL-7-treated mice had sustained antitumor effects in only 20-25% of the mice. DC-AdIL-7 therapy was more effective than AdIL-7 in achieving systemic antitumor responses and enhancing immunogenicity. After complete tumor eradication, those mice treated with DC-AdIL-7 evidenced significantly greater release of splenocyte GM-CSF and IFN-gamma than did controls or AdIL-7-treated mice. After intratumoral injection, gene-modified DCs trafficked from the tumor to lymph node sites and spleen. DCs were detected in nodal tissues for up to 7 days after intratumoral injection. We report that intratumoral DC-AdIL-7 leads to significant systemic immune responses and potent antitumor effects in murine lung cancer models.

Adenoviridae↗
PubMed2000

Monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor CCR5: implications for the tertiary structure of the receptor and for an N-terminal binding site for HIV-1 gp120.

The chemokine receptor CCR5 contains seven transmembrane-spanning domains. It binds chemokines and acts as co-receptor for macrophage (m)-tropic (or R5) strains of HIV-1. Monoclonal antibodies (mAb) to CCR5, 3A9 and 5C7, were used for biopanning a nonapeptide cysteine (C)-constrained phage-displayed random peptide library to ascertain contact residues and define tertiary structures of possible epitopes on CCR5. Reactivity of antibodies with phagotopes was established by enzyme-linked immunosorbent assay (ELISA). mAb 3A9 identified a phagotope C-HASIYDFGS-C (3A9 / 1), and 5C7 most frequently identified C-PHWLRDLRV-C (5C7 / 1). Corresponding peptides were synthesized. Phagotopes and synthetic peptides reacted in ELISA with corresponding antibodies and synthetic peptides inhibited antibody binding to the phagotopes. Reactivity by immunofluorescence of 3A9 with CCR5 was strongly inhibited by the corresponding peptide. Both mAb 3A9 and 5C7 reacted similarly with phagotopes and the corresponding peptide selected by the alternative mAb. The sequences of peptide inserts of phagotopes could be aligned as mimotopes of the sequence of CCR5. For phage 3A9 / 1, the motif SIYD aligned to residues at the N terminus and FG to residues on the first extracellular loop; for 5C7 / 1, residues at the N terminus, first extracellular loop, and possibly the third extracellular loop could be aligned and so would contribute to the mimotope. The synthetic peptides corresponding to the isolated phagotopes showed a CD4-dependent reactivity with gp120 of a primary, m-tropic HIV-1 isolate. Thus reactivity of antibodies raised to CCR5 against phage-displayed peptides defined mimotopes that reflect binding sites for these antibodies and reveal a part of the gp120 binding sites on CCR5.

Amino Acid Sequence↗
PubMed2000

Human intestinal lamina propria and intraepithelial lymphocytes express receptors specific for chemokines induced by inflammation.

To determine which chemokine receptors might be involved in T lymphocyte localization to the intestinal mucosa, we examined receptor expression on human intestinal lamina propria lymphocytes (LPL), intraepithelial lymphocytes (IEL) and CD45RO+beta7hi gut homing peripheral blood lymphocytes (PBL). Virtually all LPL and IEL expressed CXCR3 and CCR5, receptors that have been associated with Th1(Tc1)/Th0 lymphocytes, while CCR3 and CCR4, receptors associated with Th2 (Tc2)lymphocytes, CCR7, CXCR1 and CXCR2 were not expressed. CXCR3 and CCR5 receptors were functional, as LPL and IEL migrated to their respective ligands I-TAC and RANTES. In addition, most alphaEbeta7- LPL and IEL expressed high levels of CCR2. While the majority of CD45RO(-)beta7hi PBL also expressed CXCR3 and CCR5, a proportion of these cells were CXCR3- and/or CCR5- and some expressed CCR4 and/or CCR7, indicating that lymphocytes recruited to the intestinal mucosa represent a subset of these cells. In summary, our results show that LPL and IEL within the normal intestine express a specific and similar array of chemokine receptors whose ligands are constitutively expressed in the intestinal mucosa and whose expression is up-regulated during intestinal inflammation. These results support the view that CXCR3, CCR5 and CCR2 may play an important role in lymphocyte localization within the intestinal mucosa.

Animals↗
PubMed2000

RNA and protein expression of the murine autoimmune regulator gene (Aire) in normal, RelB-deficient and in NOD mouse.

Mutations in the putative transcription factor autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiosis-ectodermal dystrophy (APECED; OMIM#240300), a monogenic recessively inherited disease characterized by destructive autoimmune diseases of the endocrine organs, chronic candidiosis of mucous membranes and ectodermal dystrophies. In this study the expression of murine homolog for AIRE protein, Aire, was detected in a fraction of thymic medullary epithelial cells. Subcellularly, in thymus the protein appears as concentrated into nuclear dot-like structures, whereas in transfected cells the protein is also bound along a cytosolic fibrillar network. By RT-PCR Aire mRNA was detected in thymus, lymph node, spleen and testis although the second round PCR amplified Aire specific band from most mouse tissues analyzed. Furthermore, the Aire mRNA was detected in dendritic cell (DC) populations isolated from thymus and spleen, representing both myeloid- and lymphoid-related lineages of DC. We also demonstrate that the Aire protein is absent in the thymus of RelB-deficient mouse and in NOD thymus most of the Aire positive cells showed an abnormal morphology. These results suggest that the Aire protein is associated with the normal development and/or action of a subset of thymic medullary stromal cells involved in tolerance induction.

Alternative Splicing↗
PubMed2000

Proper developmental control of human globin genes reproduced by transgenic mice containing a 160-kb BAC carrying the human beta-globin locus.

Four independent bacterial artificial chromosome (BAC) clones containing the human Beta-globin gene locus were obtained from a human genomic BAC library. A 160-kb clone (186D7) carrying the entire human Beta-globin locus including the Beta-globin gene family, locus control region (LCR), and 3' regulatory elements was used to transform mice. Four transgenic lines were generated by microinjecting the purified BAC DNA into the fertilized eggs. RNase protection analysis showed that the expression of human Beta-globin genes is tissue- and developmental stage-specific and the expression level is similar among the three independent transgenic lines which carry the entire human Beta-globin locus; however, no Beta-globin gene expression was detected in the transgenic mice lacking the LCR region. The results suggest that the transgenic mouse model system that we have produced and that uses BAC to study the complex human Beta-globin gene cluster is stable and reproducible. Our results also indicate that some newly characterized HSs upstream from the LCR appear not to play an important role in globin gene expression and switching, while the traditional LCR can ensure correct human Beta-globin gene expression in transgenic mice. The BAC-mediated transgenic system can be used for further studies to determine which kinds of cis-acting elements are included in regulating the developmental timing and the level of human Beta-globin gene expression.

Animals↗
PubMed2000

Changes of soil and plant tissue selenium status in an upland grassland contaminated by selenium-rich agricultural drainage sediment after ten years transformed from a wetland habitat.

A field survey was conducted in 1989, 1994, and 1999 in order to monitor the soil and vegetation Se concentrations at the Kesterson upland grassland contaminated by Se-rich drainage sediment. The rate of Se dissipation estimated by the change of soil Se concentration, via volatilization, found to be about 1.1% per year. Soil water-extractable Se increased in 1994, but greatly reduced in 1999. The increase of soil Se concentration in the top 15 cm of soil at the fresh-soil fill sites indicates that the plants were able to effectively take up the soluble soil Se from the lower soil profile and to deposit it on the top of the field. This process may reduce the rate of leaching of soil Se. Overall, the concentration of soil water-soluble Se was relatively low and it is unlikely that problems of transport of Se from the Kesterson soil to the adjacent uncontaminated environment by leaching can occur. Plant tissue Se concentration was found to coincide with the soil water-extractable Se concentration. The average plant tissue Se concentration and soil water-extractable Se detected in 1999 was about 10 microg Se g(-1) and 110 microg Se kg(-1) dry weight, respectively, and the estimated bioaccumulation value of this upland grassland is less than 10% of the previous wetland habitat. Therefore, the existing Kesterson grassland should not be at high risk to the environment.

Agriculture↗
PubMed2000

Fusion protein vectors to increase protein production and evaluate the immunogenicity of genetic vaccines.

Genetic immunization is a method for vaccination and laboratory antibody production where antigen-expressing plasmids are introduced into animals to elicit immune responses. Although genetic immunization works well for many antigens, problems can arise with protein sequences that (i) are toxic to host cells, (ii) are difficult to translate by mammalian cells, or (iii) evade immune presentation. We demonstrate here the ability to increase protein production and antigen secretion by the simple method of fusing poorly expressed sequences to well-expressed heterologous proteins. Proof-of-principle is demonstrated here using the poorly translated HIV-1 envelope whose protein production is rescued by fusing this antigen to the carboxy-termini of two well-expressed proteins: the cytoplasmic green fluorescent protein and the secreted human protein a1-antitrypsin. This approach represents a simple and substantially less expensive method to increase protein and antigen production than codon-optimization strategies. It may therefore be more useful than whole gene codon replacement to enable inexpensive laboratory antibody production of poorly expressed antigens and for large-scale genomic protein or antigen screening efforts. Finally, we demonstrate a second benefit of this antigen fusion strategy in which the test antigen is "sandwiched" between two positive control antigens. By this approach, we demonstrate the intrinsic lack of immunogenicity of HIV-1 envelope under conditions when robust antibody responses are generated against its fusion protein partners, but not against this evasive antigen. These fusion protein vectors therefore represent a simple approach to not only increase antigen production, but also assess antigen production and immunogenicity in vivo.

Cell Line↗
PubMed2000

Identification of a hybrid-specific expressed gene encoding novel RNA-binding protein in wheat seedling leaves using differential display of mRNA.

A hybrid-specific expressed cDNA fragment, designated as AG5, has been identified in wheat seedling leaves using differential mRNA display. AG5 contains an open reading frame (ORF) encoding 183 amino acid residues. Comparison with amino acid sequences in GenBank revealed that the AG5 protein is homologous to a group of Gly-rich proteins with consensus sequence-type RNA-binding domains (CS-RBD). Structural analysis showed that AG5 protein contains five motifs, including a consensus sequence-type RNA-binding domain near its N-terminus, arginine/aspartic acid repeats and a Gly-rich region in its center, a Cys-X2-Cys-X4-His-X4-Cys (CCHC) zinc finger motif in the Gly-rich region, and TrySer2ArgAsp2Arg repeats towards its C-terminus. Of all previously described RNA-binding proteins, only RZ-1 from tobacco has a similar structure to the AG5 protein, but RZ-1 lacks a TrySer2ArgAsp2Arg repeat motif, indicating that the two proteins may belong to a family of closely related proteins in plants. The possible role of AG5 and its relation to wheat heterosis are discussed.

Amino Acid Sequence↗
PubMed2000

Effects of water stress on selenium accumulation in tall fescue (Festuca arundinacea Schreb) from a selenium-contaminated soil.

A greenhouse experiment was conducted evaluating the effects of water stress on uptake and accumulation of selenium (Se) as well as on uptake of calcium (Ca), potassium (K), and magnesium (Mg) in tall fescue (Festuca arundinacea Schreb). Also, this study has documented changes in soil Se inventory of Se-contaminated field soil. The findings of this experiment suggest that low soil moisture and plant water stress had a positive effect on plant tissue Se concentration. However, the negative effect of water stress on plant growth superimposed the increase of plant tissue Se concentration. Therefore, Se accumulation and soil Se removal by tall fescue became largely dependent on plant dry weight production. Strategies utilizing for remediation of Se-contaminated soils should maximizing the growth rates of tall fescue by maintaining soil moisture levels above water-stress conditions (greater than -1.0 MPa). The results of this study also suggest that the relative sensitivity of net uptake of Se and the three macronutrients to reduced soil moisture is: Ca uptake < Se uptake < Mg uptake < K uptake.

Calcium↗
PubMed2000

Phlegmonous colitis: a specific and severe complication of chronic hepatic disease.

Phlegmonous colitis (PC) is an acute infectious entity caused by bacteria. In this study, we reviewed 8,822 autopsy cases and found 13 cases of PC (0.15%). PC affected 2.43% of patients with hepatic cirrhosis or subacute liver atrophy, both of which were considered to be due to hepatitis viral infection. Before autopsy, none of the cases studied was suspected to involve PC, irrespective of the immediate cause of patient death. Thirteen autopsy cases showed some or all of the following pathohistologic characteristics: (1) involvement of the cecum (9 cases, 76.9%), (2) phlegmonous inflammatory changes and edema in the submucosa (100%), (3) bacterial infection (100%), (4) no microscopically detectable mucosal injuries (12 cases, 92.3%), and (5) acute serositis (peritonitis) (2 cases, 15.4%). These results suggest that PC is an unrecognized, but fatal complication of patients with some hepatic diseases and that PC has pathohistologic characteristics in common with previously reported spontaneous bacterial peritonitis in animal models. PC probably arises due to spontaneous infection in patients with hepatic cirrhosis.

Adult↗
PubMed2000

Direct detection of infectious HIV-1 in blood using a centrifugation-indicator cell assay.

Plasma HIV RNA is a useful surrogate marker for predicting HIV-1 disease progression in infected individuals but provides no information regarding the infectious viral titer. Traditional assays of infectious HIV-1 are, however, time consuming, insensitive, use non-standardized reagents and are subject to selection bias introduced by prolonged cultivation. In this pilot study infectious HIV-1 was detected directly in patient plasma using the indicator line HeLa-CD4-CCR5-LTR/beta-gal in a centrifugation-culture method. Replication competent HIV-1 was identified within 2 days of tissue culture inoculation in six (26%) of 23 plasma specimens. The capability of a new cell line, MT4-CCR5-tat, to amplify plasma HIV-1 was also tested. HIV was cultivated from ten (71%) of 14 specimens using MT4-CCR5-tat cells before titering the virus with the indicator cell assay. Using these stable cell lines in refined versions of this assay it may be feasible to develop rapid, simple methods for titering infectious plasma HIV-1 and for testing the susceptibility of the virus to antiretroviral drugs.

Blood↗