Biomedical subjects

W Zou

Publications and source records attributed to W Zou.

At least 19 recordsLinked to original sources

PubMed2005

Candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: developmental chemistry and investigation of immunological responses following immunization of mice and rabbits.

Glycoconjugates were prepared by covalently linking the immunogenic protein carrier CRM(197) to O-deacylated lipopolysaccharide (LPS) derived from Neisseria meningitidis (strain H44/76), immunotype L3 galE LPS. This mutant strain elaborates a truncated LPS structure that displays immunological epitopes characteristic of 76% of Group B meningococcal (NmB) strains. CRM(197) was covalently linked either to the reducing glucosamine residue of the lipid A region of the O-deacylated LPS or to a 2-keto-3-deoxy-octulosonic acid (Kdo) residue in the inner core region of the O-deacylated LPS. In both rabbits and mice a much stronger IgG response to the immunising antigen was generated in those animals that received conjugates linked via the lipid A region. Sera from mice that were immunized with these conjugates were assayed for their reactivity with LPS, both mutant and wild-type, of several homologous and heterologous NmB strains. Sera obtained from mice immunized with conjugates in which the carrier protein was linked via the Kdo moiety were only able to react with O-deacylated, but not fully acylated (native), LPS from the homologous strain. However, sera obtained from mice that were immunized with conjugates, in which the carrier protein was coupled to the lipid A region, reacted predominately with inner core epitopes that contained phosphoethanolamine at the same 3-position of the distal heptose residue (HepII) of the inner core LPS as was present on the immunising antigen. Additionally it was observed that sera from rabbits immunised with lipid A linked conjugates, unlike the mice responses, were generally not as specific for LPS antigens that contained phosphoethanolamine at the same 3-position as was present on the immunising antigen, but showed a broader inner core recognition, whereas those rabbits that received the Kdo-linked conjugates gave only a very weak non-specific response to all immunotypes. Finally, the sera from two out of six mice that had received lipid A linked conjugates had bactericidal activity against L3 wild-type NmB strain 8047 and one of these was able to passively protect against meningococcal infection in an infant rat model. This study demonstrates evidence towards the proof-in-principle that by using Nm inner core LPS conjugates coupled via the lipid A region with an intact phosphoethanolamine at the O-3 position of the HepII of the inner core LPS, it is possible to elicit functional and protective antibodies against meningococcal infection.

Animals↗

PubMed2005

TNF-alpha expression is transcriptionally regulated by RANK ligand.

Tumor necrosis factor (TNF)-alpha is known for its osteoclastogenic and resorptive activities. Induction of osteoclastogenesis by receptor activator of NF-kappaB ligand (RANKL) is accompanied by increased TNF-alpha expression. In the present study we investigated the mechanism by which RANKL induces expression of TNF-alpha in osteoclast precursors. The macrophage-like cell-line, RAW 264.7 was used as a model for osteoclast precursors. To examine if RANKL-mediated increase in TNF-alpha expression involves increased stability of its transcript, RAW264.7 cells were treated with or without RANKL, and then a transcription inhibitor was added. At different time points, TNF-alpha and L32 mRNA levels were examined. TNF-alpha mRNA stability was not altered by RANKL. We next measured directly the transcription rate of TNF-alpha by a run-on assay and found that RANKL increases TNF-alpha transcription rate by 2.9-fold in RAW264.7 cells. We further characterized this transcriptional induction of TNF-alpha by RANKL. Gel shift assays using nuclear extracts derived from RANKL-treated RAW264.7 cells show increased specific NF-kappaB binding activity on the murine TNF-alpha promoter. Gliotoxin, known for its ability to inhibit NF-kappaB activation blocked RANKL-induced TNF-alpha expression. We finally used 1,260 bp of the murine TNF-alpha promoter fused to luciferase, as well as four mutants of this promoter carrying mutations in each of the four NF-kappaB sites to stably transfect RAW 264.7 cells. Reporter activity was increased in response to RANKL in wild type promoter transfected cells, whereas treatment of the mutants' transfected cells did not elicit reporter activity. In conclusion, RANKL induces TNF-alpha expression via a transcriptional mechanism, depending on the NF-kappaB sites in the TNF promoter.

Animals↗

PubMed2004

Sensitivity of 14-3-3 protein test varies in subtypes of sporadic Creutzfeldt-Jakob disease.

BACKGROUND: The increase of the 14-3-3 protein in CSF is used as a diagnostic test in Creutzfeldt-Jakob disease (CJD), but the sensitivity and specificity of the 14-3-3 test are disputed. One reason for the dispute may be the recently established heterogeneity of sporadic CJD. The relationship between CSF 14-3-3 protein and sporadic CJD subtypes, distinguished by electrophoretic mobility of proteinase K-resistant prion protein (PrP(Sc)) and genotype at codon 129 of the prion protein gene, has not been elucidated. METHODS: The authors examined the 14-3-3 protein test in 90 patients with sporadic CJD. PrP(Sc) type (type 1 or type 2) and the genotype at polymorphic codon 129 were determined in each patient. Mutations were excluded by prion gene sequencing. RESULTS: The authors' findings indicate that the sensitivity of the 14-3-3 test is higher in patients with molecular features of the classic sporadic CJD than in patients with the nonclassic CJD subtypes. The difference appears to be related to the PrP(Sc) type and not to the codon 129 genotype. Disease duration before 14-3-3 testing might also have an influence because it was shorter in classic sporadic CJD. CONCLUSION: The Creutzfeldt-Jakob disease clinical subtype should be considered when interpreting results of the 14-3-3 test.

14-3-3 Proteins↗

PubMed2003

Structure-function study of the amino-terminal stretch of the catalase subunit molecule in oligomerization, heme binding, and activity expression.

Analysis of the protein structure of bovine liver catalase suggested that the N-terminal region containing two alpha-helices may function as a linker binding to another subunit. The number of amino-acid residues in catalase from the n-alkane-assimilating yeast Candida tropicalis (CTC) is the lowest of any eukaryotic catalase molecule hitherto investigated, and only one helix, corresponding to the helix alpha2 in bovine liver catalase, is estimated to be present in the same region. In the present study, N-terminal-deleted mutants of CTC were characterized to evaluate the role of the alpha-helix structure in the N-terminal region. CTCDelta1-4 and CTCDelta1-24, whose N-terminal regions were shortened by four and 24 amino-acid residues, respectively, showed an 80% decrease in specific activity compared to wild-type CTC in spite of containing the same amount of heme as in the wild-type. Polyacrylamide gel electrophoresis under nondenaturing conditions revealed that the mutants contained large amounts of oligomeric forms with molecular masses less than 220 kDa (tetramer assembly). Although the smaller oligomers were found to be bound with heme, only the tetramer exhibited catalase activity in activity staining on nondenaturing gel. CTCDelta1-49, a mutant with deletion of the N-terminal 49 amino-acid residues which contain the conserved helix alpha2, showed no catalase activity and no heme binding. However, the CD spectrum profiles of CTCDelta1-49, CTCDelta1-4, and CTCDelta1-24 indicated that these mutant subunits could attain secondary conformations similar to that of wild-type CTC, regardless of their binding with heme. From these results, it was concluded that the N-terminal stretch of catalase is significant for complete assembly into active tetramer and that the conserved helix alpha2, although it has little effect on the formation of the subunit secondary structure, is indispensable not only in assembling tetramer but also in binding heme.

Amino Acid Sequence↗

PubMed2003

Epididymal phenotype in luteinizing hormone receptor knockout animals and its response to testosterone replacement therapy.

Previous studies reported that epididymis contains functional LH receptors. The LH receptor knockout mice, which have epididymal phenotypes, gave us an opportunity to test the hypothesis that testosterone replacement alone may not be sufficient to reverse phenotypes to wild-type epididymis. The morphological phenotype in knockout animals includes a decrease in luminal diameter of the proximal and distal caput and cauda epididymis, the absence of clear and halo cells in the epithelial lining, a decrease in the height of principal cells and the number of cells containing cilia, a decrease in cilia length, and a change from basal to central location of nuclei in the principal cells. The biochemical phenotype includes a decrease in periodic acid-Schiff reaction product, reflecting the glycogen and glycoprotein synthesis and secretion, a decrease in androgen receptor (AR) and estrogen receptor (ER)beta, and an increase in ERalpha levels. Twenty-one-day testosterone replacement therapy in 30-day-old knockout animals reversed some, but not all, morphological and biochemical phenotypes. Those that did not reverse include luminal diameters of proximal and distal caput and cauda epididymis, the percentage of ciliated principal cells in caput epididymis, and nuclear AR localization. In summary, while our results reaffirm that androgens are important for normal epididymal morphology and function, they indicate that LH could be required for certain facets of epididymal morphology and/or function.

Animals↗

PubMed2002

Screening of a molecule endowing Saccharomyces cerevisiae with n-nonane-tolerance from a combinatorial random protein library.

A combinatorial random protein library was constructed from random DNA fragments generated by "DNA random priming", an improved method of "random-priming recombination" using random-sequence primers and template cDNA from the yeast Saccharomyces cerevisiae. In order to express this library on the yeast cell surface, a yeast multicopy cassette vector was constructed, in which the random-protein-encoding DNA fragments were fused to a gene encoding the C-terminal 320 amino acids of alpha-agglutinin. Fluorescent labeling of the immuno-reaction of RGS(His)(6) epitope confirmed the surface display of random proteins. The surface display of heterologous random proteins on yeast cells will have a wide application. As an example, an n-nonane-tolerant yeast strain that could grow very well in nonane-overlaid culture medium was screened out from transformants displaying this combinatorial library. n-Nonane tolerance was dependent on the transformed plasmid, and the related protein was confirmed to localize on the cell surface by papain treatment and immunofluorescent labeling. Analysis of this displayed protein was also carried out. This strain is the first one to have been endowed artificially with organic solvent tolerance. This is a good example of creating cells exhibiting new phenotypes using a combinatorial protein library.

Adaptation, Physiological↗

PubMed2001

Multifunctionalized alpha,beta-cyclopentenones from C-2 and C-4-ulopyranosyl compounds: a stereospecific rearrangement initiated by base.

Base treatment of O-benzyl protected C-2- or C-4-ulopyranosyl compounds (4 alpha, 4 beta, and 11) by either 10% Et(3)N or 1% K(2)CO(3) in MeOH initiated a beta elimination to afford alpha,beta-unsaturated C-ulopyranosyl compounds (5 alpha, 5 beta, and 12), which further rearranged in a stereocontrolled manner to multifuctionalized alpha,beta-cyclopentenones (6 and 14) in 70-80% yield. Both C-alpha- and C-beta-2-ulosides (5 alpha and 5 beta) produced the same cyclopentenone 6, indicating that a 1,2-enolate is formed prior to the cleavage of the C-5--O bond. Because 6 is racemic, it was probably formed by the intramolecular cycloaldolization of two equally populated enantiomeric intermediates. When treated with 90% Et(3)N in MeOH, 5 alpha yielded almost exclusively 15 (isomer of 6), which was formed by a migration of the double bond in 5 alpha during the previously described rearrangement. Thus either 6 or 15 was the major product, depending on the base used.

Cyclopentanes↗

PubMed2001

Cobalt chloride induces PC12 cells apoptosis through reactive oxygen species and accompanied by AP-1 activation.

Reactive oxygen species (ROS) are supposed to play an important role in hypoxia- and ischemia/reperfusion-mediated neuronal injury with the characteristics of apoptosis. There are many reports showing that cobalt chloride (CoCl(2)) could mimic the hypoxic responses in some aspects including production of ROS in cultured cells. The cytotoxicity of CoCl(2) and its molecular mechanisms have yet to be elucidated. We report that CoCl(2) triggered neuronal PC12 cells apoptosis in a dose- and time-dependent manner. Apoptosis was demonstrated by morphological changes and DNA fragmentation, and was dependent on macromolecular synthesis. Apoptosis was also confirmed by the decrease of the expression of Bcl-X(L). To our knowledge, this is the first documentation of the apoptotic induction of CoCl(2) on PC12 cells. Furthermore, ROS production in PC12 cells was increased during CoCl(2) treatment. Antioxidants, which could inhibit ROS production, significantly blocked CoCl(2)-induced apoptosis, suggesting that apoptosis is mediated by ROS production. We also observed a significant increase of the DNA-binding activity of AP-1 in response to CoCl(2) and this increase was blocked by antioxidants, showing that CoCl(2)-induced apoptosis is accompanied by ROS-activated AP-1. CoCl(2)-treated PC12 cells may serve as an in vitro model for studies of molecular mechanisms in ROS-linked neuronal disorders.

Animals↗

PubMed2001

Construction of multivalent sialyl Le(x) from the type Ia group B Streptococcus capsular polysaccharide.

The type Ia group B Streptococcus (GBSIa) capsular polysaccharide was specifically degraded by partial Smith oxidation of 2,3-diol of the Glc in the backbone to fragments representing asialo core repeating units. Sialylation of these oligomers furnished GBSIa multiple repeating units. One, two and three repeating units of GBSIa were obtained pure, and the higher oligomers were obtained as mixtures. After enzymatic fucosylation oligosaccharides carrying bivalent, trivalent and other multivalent sialyl Le(x) epitopes presented as appendages on an oligolactoside scaffold were obtained.

Antigens, Bacterial↗

PubMed2001

Critical sites for the interaction between IL-2Rgamma and JAK3 and the following signaling.

JAK3 is the only known protein tyrosine kinase associating with IL-2Rgamma. This interaction is supposed to be very important to IL-2 signaling. In order to identify the critical residues for these two molecular interactions and the following signal events, various mutants of gammac and JAK3 were constructed on the basis of computer analysis. The direct interaction was determined via the yeast two-hybrid system, while the signaling was analyzed with reporter genes under the control of the c-fos, c-myc, or tnf-beta promoters, respectively. Results showed that there are two key sites on gammac involved in this interaction and the following signal transduction: the critical one is E327 via electrostatic interaction, the other is L293 via hydrophobic interaction. As to JAK3, the data indicated that Y100 is important for the interaction with gammac. These results also document that the requirement for interaction between gammac and JAK3 is different to activate different signaling pathways mediated by gammac, such as c-fos, c-myc, and JAK-STAT.

Animals↗

PubMed2001

Reciprocal regulation of plasmacytoid dendritic cells and monocytes during viral infection.

Reciprocal regulation of opposing functions characterizes biological systems. We now show that adenovirus-infected plasmacytoid dendritic cells (PDC) inhibit monocyte to myeloid dendritic cell (MDC) differentiation and function, and that adenovirus-infected monocytes inhibit PDC type I interferon secretion. Adenovirus-infected PDC secreted IFN-alpha, beta and omega in an 86:2:1 ratio. PDC type I interferons inhibited MDC differentiation and function (reduced IL-12 secretion, IFN-gamma induction, MLR and CD40 expression, and increased CD1a(+)CD14(+) cells). Type I interferon receptor blocking antibody reversed all PDC effects, and recombinant IFN-alpha, beta or omega replicated all effects, except reduced CD40. Adenovirus-infected monocytes suppressed PDC type I interferon secretion, which was reversed with anti-IL-10 neutralizing antibodies. Exogenous IL-10 suppressed PDC type I interferon secretion without reducing PDC viability. Therefore, monocyte IL-10 regulates PDC type I interferon secretion, and PDC type I interferons inhibit MDC differentiation and function. Such reciprocal regulation of potentially opposing influences may help modulate anti-pathogen immunity.

Adenovirus Infections, Human↗

PubMed2001

The conformational epitope of type III group B Streptococcus capsular polysaccharide.

In order to further characterize the conformational epitope of GBSPIII, we synthesized various oligosaccharides with the GBSPIII-related structures by a tailor-assembly synthetic scheme and a more traditional block-wise chemo-enzymatic approach. The oligosaccharides were used to probe the conformational epitope of GBSPIII using number of complementary techniques. The protective epitope of GPSPIII was further defined as length-dependent and conformational. The results of the studies confirmed that two repeating units (2RU) is the minimum binding unit and the epitope optimization mainly takes place between chain length 2RU to 7RU. Epitope optimization and multivalency were observed between 7RU and 20RU. The data support our hypothesis that the conformational epitope is an extended helical segment of the GBSPIII. GBSPIII exists mainly in the random coil form, which structurally mimics short oligosaccharide self-antigens, but it can infrequently and spontaneously form extended helices. Although not prevalent in GBSPIII the immune system preferentially selects these helical epitopes because they are unique to the polysaccharide.

Animals↗

PubMed2001

Expression of P53 during lens epithelial cell apoptosis induced by ultraviolet.

The apoptosis of lens epithelial cells (LECs) induced by ultraviolet and the expression of P53 were investigated. Wistar rats received 100 mW/m2 ultraviolet irradiation (UVR) (lambda = 280 nm-315 nm) for 15 min. One, 6, 24 h after irradiation the lens capsules were dissected. The percentages of apoptotic cells were evaluated by the TdT-dUTP terminal nick-end labeling (TUNEL) technique and the expression of P53 was detected by using immunohistochemical assay. The results showed that the percentages of TUNEL-positive nuclei at 24 h after irradiation was significantly higher than in the control group and those 1 h, 6 h after irradiation. The percentages of P53-positive cells at 6 h, 24 h after irradiation were significantly higher than in the control group and those 1 h after irradiation. It was concluded that UVR could induce the apoptosis of lens epithelial cell. The expression of P53 might be responsible for the apoptosis of lens epithelial cells.

Animals↗

PubMed2001

Epoxyeicosatrienoic acids (EET(11,12)) may partially restore endothelium-derived hyperpolarizing factor-mediated function in coronary microarteries.

BACKGROUND: Endothelial cells derive nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). The cytochrome P-450-monooxygenase metabolites of arachidonic acid (epoxyeicosatrienoic acids [EETs]) have been suggested to be EDHF. This study was designed to examine the effect of EET(11,12) with regard to the possibility of restoring EDHF function when added into hyperkalemic cardioplegic solution. METHODS: Porcine coronary microartery rings were studied in a myograph. In groups 1 and 2, paired arteries were incubated in either hyperkalemic solution (K+ 20 mmol/L) or Krebs' solution (control). In group 3, the paired arteries were incubated in hyperkalemia plus EET(11,12) (1 x 10(-6.5) mol/L) or hyperkalemia alone (control) at 37 degrees C for 1 hour, followed by Krebs' washout and then precontracted with 1 x 10(-8.5) mol/L U46619. The EDHF-mediated relaxation to EET(11,12) (group 1) or bradykinin (groups 2 and 3) was studied in the presence of N(G)-nitro-L-arginine, indomethacin, and oxyhemoglobin. RESULTS: After exposure to hyperkalemia, the EDHF-mediated maximal relaxation by bradykinin (72.5% +/- 7.8% versus 41.6% +/- 10.6%; p < 0.05), but not by EET(11,12) (18.4% +/- 3.3% versus 25.1% +/- 4.9%; p > 0.05) was significantly reduced. Incubation with EET(11,12) partially restored EDHF function (33.3% +/- 9.5% versus 62.0% +/- 8.5%; p < 0.05). CONCLUSIONS: In coronary microarteries, hyperkalemia impairs EDHF-mediated relaxation, and EET(11,12) may partially mimic the EDHF function. Addition of EET(11,12) into cardioplegic solution may partially restore EDHF-mediated function reduced by exposure to hyperkalemia.

8,11,14-Eicosatrienoic Acid↗

PubMed2001

Stromal-derived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells.

Dendritic-cell (DC) trafficking and function in tumors is poorly characterized, with studies confined to myeloid DCs (DC1s). Tumors inhibit DC1 migration and function, likely hindering specific immunity. The role of plasmacytoid DCs (DC2s) in tumor immunity is unknown. We show here that malignant human ovarian epithelial tumor cells express very high levels of stromal-derived factor-1, which induces DC2 precursor (preDC2) chemotaxis and adhesion/transmigration, upregulates preDC2 very late antigen (VLA)-5, and protects preDC2s from tumor macrophage interleukin-10-induced apoptosis, all through CXC chemokine receptor-4. The VLA-5 ligand vascular-cell adhesion molecule-1 mediated preDC2 adhesion/transmigration. Tumor preDC2s induced significant T-cell interleukin-10 unrelated to preDC2 differentiation or activation state, and this contributed to poor T-cell activation. Myeloid precursor DCs (preDC1s) were not detected. Tumors may weaken immunity by attracting preDC2s and protecting them from the harsh microenvironment, and by altering preDC1 distribution.

Apoptosis↗

PubMed2001

Expression of the chemokine RANTES in pulmonary Wegener's granulomatosis.

Wegener's granulomatosis (WG) is an inflammatory, destructive, angiotropic lesion. The inflammatory process involves accumulation of macrophages, lymphocytes, and polymorphonuclear neutrophils. We studied 6 lung biopsy specimens from patients with WG to characterize the cellular infiltrate and to analyze the mechanism of immune cell recruitment. We show that lymphocytes accumulating in WG lesions are mostly memory CD4(+)CD45RO(+) T lymphocytes and, although less numerous, CD8(+)CD45RO(+) T lymphocytes. Few if any B lymphocytes or natural killer cells are present within lesions. The chemokine RANTES (regulated upon activation in normal T cells, expressed and secreted) has been reported to recruit memory T lymphocytes and macrophages selectively. We used reverse-transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry to study its production in WG. RANTES was expressed at a higher level in WG lungs than in normal controls, especially around microabscesses. As visualized immunohistochemically in serial sections with anti-RANTES monoclonal antibody, RANTES production was produced mainly by macrophages. Expression of the gene coding for interferon-gamma (IFN-gamma), a potent RANTES inducer, was also studied. Its expression was also much stronger in WG than in controls. Our observations are consistent with a cascade of events leading to the recruitment of immune cells in WG, sequentially involving production of IFN-gamma by T lymphocytes and RANTES production by macrophages, leading to the homing of memory T-helper lymphocytes and macrophages. HUM PATHOL 32:320-326.

Adult↗

PubMed2001

Antibody-antigen interactions measured by surface plasmon resonance: global fitting of numerical integration algorithms.

The interactions between adenylate kinase (AK) and a monoclonal antibody against AK (McAb3D3) were examined by means of optical biosensor technology, and the sensograms were fitted to four models using numerical integration algorithms. The interaction of a solution of McAb3D3 with immobilized AK follows a double exponential function and the data fitted well to an inhomogeneous ligand model. The interaction of a solution AK with immobilized McAb3D3 follows a single exponential function and the data fitted well to a pseudo-first order reaction model. The true association constants of AK binding to McAb3D3 in solution were obtained from competition BIAcore measurements. The difference in results obtained with solid-phase BIAcore and competition BIAcore may be due to rebinding of the dissociated analyte to the immobilized surface. The results obtained with BIAcore are compared to those obtained by ELISA methods. We suggest that the best method for analysis of BIAcore data is direct, global fitting of sensorgrams to numerical integration algorithms corresponding to the different possible models for binding.

Adenylate Kinase↗

PubMed2001

Genetically controlled self-aggregation of cell-surface-engineered yeast responding to glucose concentration.

We constructed an arming (cell-surface-engineered) yeast displaying two types of agglutinin (modified a-agglutinin and alpha-agglutinin) on the cell surface, with agglutination being independent of both mating type and pheromones. The modified a-agglutinin was artificially prepared by the fusion of the genes encoding Aga1p and Aga2p. The modified a-agglutinin could induce agglutination of cells displaying Agalpha1p (alpha-agglutinin). The upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL), active at a low glucose concentration, was used as the promoter to express the modified a-agglutinin- and alpha-agglutinin-encoding genes. The arming yeast displaying both agglutinins agglutinated and sedimented in response to decreased glucose concentration. When the glucose concentration was high, the arming yeast grew normally. In the late log phase, when the glucose concentration became very low, agglutination occurred suddenly and drastically and yeast cells sedimented completely. Sedimentation was confirmed by weighing the aggregated cells after filtration of the broth. Strains in which aggregation can be genetically controlled can be used in industrial processes in which the separation of yeast cells from the supernatant is necessary.

Agglutination↗