Biomedical subjects

K Wang

Publications and source records attributed to K Wang.

At least 19 recordsLinked to original sources

PubMed1999

Monitoring gene expression profile changes in ovarian carcinomas using cDNA microarray.

The development of cancer is the result of a series of molecular changes occurring in the cell. These events lead to changes in the expression level of numerous genes that result in different phenotypic characteristics of tumors. In this report we describe the assembly and utilization of a 5766 member cDNA microarray to study the differences in gene expression between normal and neoplastic human ovarian tissues. Several genes that may have biological relevance in the process of ovarian carcinogenesis have been identified through this approach. Analyzing the results of microarray hybridizations may provides new leads for tumor diagnosis and intervention.

Cloning, Molecular↗

PubMed1999

Molecular cloning and characterization of prostase, an androgen-regulated serine protease with prostate-restricted expression.

The identification of genes with selective expression in specific organs or cell types provides an entry point for understanding biological processes that occur uniquely within a particular tissue. Using a subtraction approach designed to identify genes preferentially expressed in specific tissues, we have identified prostase, a human serine protease with prostate-restricted expression. The prostase cDNA encodes a putative 254-aa polypeptide with a conserved serine protease catalytic triad and an amino-terminal pre-propeptide sequence, indicating a potential secretory function. The genomic sequence comprises five exons and four introns and contains multiple copies of a chromosome 19q-specific minisatellite repeat. Northern analysis indicates that prostase mRNA is expressed in hormonally responsive normal and neoplastic prostate epithelial tissues, but not in prostate stromal constituents. Prostase shares 35% amino acid identity with prostate-specific antigen (PSA) and 78% identity with the porcine enamel matrix serine proteinase 1, an enzyme involved in enamel matrix degradation and with a putative role in the disruption of intercellular junctions. Radiation-hybrid-panel mapping localized prostase to chromosome 19q13, a region containing several other serine proteases, including protease M, pancreatic/renal kallikrein hK1, and the prostate-specific kallikreins hK2 and hK3 (PSA). The sequence homology between prostase and other well-characterized serine proteases suggests several potential functional roles for the prostase protein that include the degradation of extracellular matrix and the activation of PSA and other proteases.

Amino Acid Sequence↗

PubMed1999

Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death.

"BH3 domain only" members of the BCL-2 family including the pro-apoptotic molecule BID represent candidates to connect with proximal signal transduction. Tumor necrosis factor alpha (TNFalpha) treatment induced a caspase-mediated cleavage of cytosolic, inactive p22 BID at internal Asp sites to yield a major p15 and minor p13 and p11 fragments. p15 BID translocates to mitochondria as an integral membrane protein. p15 BID within cytosol targeted normal mitochondria and released cytochrome c. Immunodepletion of p15 BID prevents cytochrome c release. In vivo, anti-Fas Ab results in the appearance of p15 BID in the cytosol of hepatocytes which translocates to mitochondria where it releases cytochrome c. Addition of activated caspase-8 to normal cytosol generates p15 BID which is also required in this system for release of cytochrome c. In the presence of BCL-XL/BCL-2, TNFalpha still induced BID cleavage and p15 BID became an integral mitochondrial membrane protein. However, BCL-XL/BCL-2 prevented the release of cytochrome c, yet other aspects of mitochondrial dysfunction still transpired and cells died nonetheless. Thus, while BID appears to be required for the release of cytochrome c in the TNF death pathway, the release of cytochrome c may not be required for cell death.

Amino Acid Sequence↗

PubMed1998

Mapping the subsite preferences of protein tyrosine phosphatase PTP-1B using combinatorial chemistry approaches.

Protein tyrosine phosphatases (PTPases) are important regulators of signal transduction systems, but the specificity of their action is largely unexplored. We have approached this problem by attempting to map the subsite preferences of these enzymes using combinatorial chemistry approaches. Protein-tyrosine peptidomimetics containing nonhydrolyzable phosphotyrosine analogues bind to PTPases with high affinity and act as competitive inhibitors of phosphatase activity. Human PTP-1B, a PTPase implicated to play an important role in the regulation of growth factor signal transduction pathways, was used to screen a synthetic combinatorial library containing malonyltyrosine as a phosphotyrosine mimic. Using two cross-validating combinatorial chemistry screening approaches, one using an iterative method and the other employing library affinity selection-mass spectrometric detection, peptides with high affinity for PTP-1B were identified and subsite preferences were detailed by quantitatively comparing residues of different character. Consistent with previous observations, acidic residues were preferred in subsites X-3 and X-2. In contrast, aromatic substitutions were clearly preferred at the X-1 subsite. This information supports the concept that this class of enzymes may have high substrate specificity as dictated by the sequence proximal to the phosphorylation site. The results are discussed with regards to the use of combinatorial techniques in order to elucidate the interplay between enzyme subsites.

Computer Simulation↗

PubMed1998

Single electron reduction of cytochrome c oxidase compound F: resolution of partial steps by transient spectroscopy.

The final step of the catalytic cycle of cytochrome oxidase, the reduction of oxyferryl heme a3 in compound F, was investigated using a binuclear polypyridine ruthenium complex (Ru2C) as a photoactive reducing agent. The net charge of +4 on Ru2C allows it to bind electrostatically near CuA in subunit II of cytochrome oxidase. Photoexcitation of Ru2C with a laser flash results in formation of a metal-to-ligand charge-transfer excited state, Ru2C, which rapidly transfers an electron to CuA of cytochrome oxidase from either beef heart or Rhodobacter sphaeroides. This is followed by reversible electron transfer from CuA to heme a with forward and reverse rate constants of k1 = 9.3 x 10(4) s-1 and k-1 = 1.7 x 10(4) s-1 for R. sphaeroides cytochrome oxidase in the resting state. Compound F was prepared by treating the resting enzyme with excess hydrogen peroxide. The value of the rate constant k1 is the same in compound F where heme a3 is in the oxyferryl form as in the resting enzyme where heme a3 is ferric. Reduction of heme a in compound F is followed by electron transfer from heme a to oxyferryl heme a3 with a rate constant of 700 s-1, as indicated by transients at 605 and 580 nm. No delay between heme a reoxidation and oxyferryl heme a3 reduction is observed, showing that no electron-transfer intermediates, such as reduced CuB, accumulate in this process. The rate constant for electron transfer from heme a to oxyferryl heme a3 was measured in beef cytochrome oxidase from pH 7.0 to pH 9.5, and found to decrease upon titration of a group with a pKa of 9.0. The rate constant is slower in D2O than in H2O by a factor of 4.3, indicating that the electron-transfer reaction is rate-limited by a proton-transfer step. The pH dependence and deuterium isotope effect for reduction of isolated compound F are comparable to that observed during reaction of the reduced, CO-inhibited CcO with oxygen by the flow-flash technique. This result indicates that electron transfer from heme a to oxyferryl heme a3 is not controlled by conformational effects imposed by the initial redox state of the enzyme. The rate constant for electron transfer from heme a to oxyferryl heme a3 is the same in the R. sphaeroides K362M CcO mutant as in wild-type CcO, indicating that the K-channel is not involved in proton uptake during reduction of compound F.

2,2'-Dipyridyl↗

PubMed1998

BID, a proapoptotic BCL-2 family member, is localized to mouse chromosome 6 and human chromosome 22q11.

BID is a proapoptotic member of the BCL-2 family of cell death regulators. BID shares sequence homology with other members of the family within a single alpha-helical domain, BH3. BH3 is required for BID to interact with BCL-2 and BAX, as well as for its function as a death agonist. We have isolated and characterized mouse Bid and human BID genomic clones. The sequence for BID is encoded within five exons. We used interspecific backcross analysis to localize Bid to the distal region of mouse chromosome 6 near the Atp6e locus. Fluorescence in situ hybridization analysis localized human BID to a syntenic human region, chromosome 22q11, close to the BCR-1 gene.

Animals↗

PubMed1998

Prostaglandin E2 and dexamethasone inhibit IL-12 receptor expression and IL-12 responsiveness.

Regulation of the factors governing IL-12R expression and IL-12 responsiveness has been shown to be important in the generation and stability of Th1- and Th2-type responses. In this regard, cytokines have been shown to have a prominent role in regulating IL-12R expression. In this study, the role that PGE2 and dexamethasone (DXM) have in regulating IL-12R expression was evaluated. Addition of PGE2 or DXM to human PBMCs stimulated with immobilized anti-CD3 plus IL-12 inhibited the production of IFN-gamma in a dose-responsive manner. Moreover, PBMCs stimulated with immobilized anti-CD3 in the presence of PGE2 or DXM for 3 days, washed extensively, and restimulated in the presence of IL-12 still did not produce IFN-gamma. This lack of IL-12 responsiveness from cells cultured in either PGE2 or DXM was correlated with diminished surface expression of IL-12Rbeta1, IL-12Rbeta2 mRNA expression, and IL-12 binding. Finally, the PGE2- and DXM-mediated inhibition of IL-12R expression was not affected significantly by addition of neutralizing Abs against either IL-4, IL-10, or TGF-beta. By contrast, addition of dibutyryl cAMP, 8-bromoadenosine 3:5 cAMP (8-Br-cAMP), or cholera toxin substantially reduced IL-12R expression, suggesting that PGE2 may be mediating its effects through enhancement of cAMP.

Cells, Cultured↗

PubMed1998

Hematopoietic malignancies demonstrate loss-of-function mutations of BAX.

The BCL-2 gene family regulates the susceptibility to apoptotic cell death in many cell types during embryonic development and normal tissue homeostasis. Deregulated expression of anti-apoptotic BCL-2 can be a primary aberration that promotes malignancy and also confers resistance to chemotherapeutic agents. Recently, studies of Bax-deficient mice have indicated that the pro-apoptotic BAX molecule can function as a tumor suppressor. Consequently, we examined human hematopoietic malignancies and found that approximately 21% of lines possessed mutations in BAX, perhaps most commonly in the acute lymphoblastic leukemia subset. Approximately half were nucleotide insertions or deletions within a deoxyguanosine (G8) tract, resulting in a proximal frame shift and loss of immunodetectable BAX protein. Other BAX mutants bore single amino acid substitutions within BH1 or BH3 domains, demonstrated altered patterns of protein dimerization, and had lost death-promoting activity. Thus, mutations in the pro-apoptotic molecule BAX that confer resistance to apoptosis are also found in malignancies.

Animals↗

PubMed1998

A conserved sequence block in murine and human T cell receptor (TCR) Jalpha region is a composite element that enhances TCR alpha enhancer activity and binds multiple nuclear factors.

A conserved sequence block (CSB) located in a noncoding region of the mouse and human TCR alpha/delta loci, showing six differences over 125 nucleotide positions (95% similar), was subjected to detailed analyses in this study. Transient transfection results showed that the CSB-containing element in conjunction with the TCR alpha enhancer up-regulated the alpha enhancer activity, whereas no enhancer activity was detected when CSB alone was assayed. In vitro occupancy analyses of CSB by nuclear factors reveal the existence of an unexpectedly intricate network of CSB-protein and protein-protein interactions. Lymphoid-specific as well as T-lineage-specific nuclear factors are involved to differentially form CSB-bound complexes in extracts of various tissues and cell lines. Liver was shown to contain factor(s) sequestering thymic CSB-binding factors. Furthermore, the putative binding sites for transcription factors known to be important for lymphoid-lineage development are present in CSB and are targeted by nuclear factors. On the basis of these results, we propose that the CSB element may play a role in shaping the chromatin structure by which the accessibility of TCR alpha/delta loci to the recombinase complex and/or to the transcriptional apparatus can be controlled.

Animals↗

PubMed1998

2-amino-4H-3,1-benzoxazin-4-ones as inhibitors of C1r serine protease.

A series of 2-amino-4H-3,1-benzoxazin-4-ones have been synthesized and evaluated as inhibitors of the complement enzyme C1r. C1r is a serine protease at the beginning of the complement cascade, and complement activation by beta-amyloid may represent a major contributing pathway to the neuropathology of Alzheimer's disease. Compounds such as 7-chloro-2-[(2-iodophenyl)-amino]benz[d][1,3]oxazin-4-one (32) and 7-methyl-2-[(2-iodophenyl)amino]benz[d][1,3]oxazin-4-one (37) show improved potency compared to the reference compound FUT-175. Many of these active compounds also possess increased selectivity for C1r compared to trypsin and enhanced hydrolytic stability relative to 2-(2-iodophenyl)-4H-3,1-benzoxazin-4-one (1).

Complement C1r↗

PubMed1998

The reaction of lanthanide ions with n-doxyl stearic acids and its utilization for the ESR study on the permeability of lipid-bilayer of erythrocyte membrane to gadolinium ions.

The reaction of lanthanide ions with n-doxyl stearic acid (nDS) spin labels (n = 5,7,12,16) was investigated by the electron spin resonance technique in aqueous solution. Among the lanthanides, the Gd3+, Tb3+, Tm3+ and Ce3+ ions strongly quenched the ESR signal of spin labels, but the effects of La3+, Eu3+ and Lu3+ are very weak. The quenching effects are featured by: (1) the dependence on the concentration of lanthanide ions; (2) no obvious changes of the ESR line shape in the presence of lanthanide ions; (3) the quenching constant decreases in the order: Gd3+ > Tb3+ > Tm3+ > Ce3+; (4) the quenching effects of lanthanide ions are found to strikingly correlate with their magnetic properties. These findings indicate that the interaction of lanthanide ions with nitroxide oxygen leading to the reduction of ESR signal amplitude is dominated by their magnetic characteristics rather than the coordination effect. By labeling erythrocyte membrane with nDS, n = 5,7,12,16 at different depths, we studied the diffusion of Gd3+ into the lipid-bilayer of erythrocyte membrane by monitoring the reduction processes of the ESR signals of nitroxide spin labels located at different depths of membrane lipid-bilayer after addition of Gd3+. These results revealed that the Gd3+ ions can penetrate into the lipid-bilayer, though the entry rate is slow. It was shown that the Gd3+ ions bind to the membrane and enhance the permeability of extracellular ascorbate into erythrocyte membrane. The transport mechanism of Gd3+ ions through the lipid-bilayer might be involved in the Gd3+ cation-induced pore formation in the surface of membrane.

Ascorbic Acid↗

PubMed1998

Hevin, an antiadhesive extracellular matrix protein, is down-regulated in metastatic prostate adenocarcinoma.

Hevin, a gene closely related to the extracellular matrix protein SPARC, is an acidic cysteine-rich glycoprotein shown to be important for the adhesion and trafficking of cells through the endothelium. Through the use of differential display and differential EST analysis, we identified Hevin as a gene whose transcription is down-regulated in transformed prostate epithelial cell lines and metastatic prostate adenocarcinoma. These results were confirmed by comparing expression levels between normal and neoplastic human prostate tissues using Northern analysis. In situ hybridization with an 35S-labeled antisense riboprobe demonstrated the loss of Hevin expression in metastatic prostate carcinoma. The expression pattern of Hevin in transformed and metastatic epithelium may provide further insights into the complex cell adhesion events involved in the metastatic progression of prostate carcinoma.

Adenocarcinoma↗

PubMed1998

Fluorescence study of the multiple binding equilibria of the galactose repressor.

A fluorescence assay has been developed to study the multiple linked equilibria which function in regulation of the Escherichia coli galactose operon. Fluorescein 5-isothiocyanate was attached to Amino-Modifier C6dT at different positions in an oligonucleotide containing the sequence for the OE site of the galactose operon. These fluorescently labeled oligonucleotides were used to study OEDNA-GalR-d-galactose interactions. The data were analyzed and fit to various models including the classical competitive binding model as well as models involving the formation of a ternary DNA-repressor-inducer complex. Examination of the reduced chi-square of the various fits and comparison of fitted parameters with those obtained in independent experiments were used to distinguish different models. Since the ternary complex is likely to exist under physiological conditions, our results suggest that obligatory dissociation of GalR from DNA may not be required for induction of the gal operon. Rather, induction may involve the formation of a ternary complex of OEDNA with GalR and D-galactose with a different conformation than the GalR-OEDNA binary repressor complex.

Amino Acid Sequence↗

PubMed1998

Effects of thiol protease inhibitors on myoblast fusion and myofibril assembly in vitro.

To investigate the roles of thiol proteases such as cathepsins and calpains in muscle differentiation, we have treated primary cultures of pectoralis muscle with a variety of protease inhibitors and examined the effects these agents have on myoblast fusion and myofibrillogenesis. We have found that a membrane-permeable inhibitor, E64D, has dramatic effects on both events of muscle differentiation. Cells treated with this inhibitor display gross morphological changes, severe delays in myofibril assembly, and reduced ability to fuse in culture. These morphological changes are correlated with a build up of beta1-integrin throughout the cytoplasm. These effects could also be produced using NH4Cl, a lysosomotrophic agent. In addition, we show that two nonpermeable inhibitors (leupeptin and E64) slightly decrease myoblast fusion, but have no effects on the ability of the cells to form mature myofibrils. These results are discussed in terms of their relevance to the inheritable disease of muscular dystrophy and I-cell disease (mucolipodosis II).

Animals↗

PubMed1998

Relation between ventricular fibrillation voltage and probability of defibrillation shocks. Analysis using Hilbert transforms.

We used Hilbert transforms to re-evaluate the previously reported correlation between defibrillation shock outcome and absolute ventricular fibrillation voltage (AVFV). Previously in the literature, single values of AVFV acquired just prior to shock delivery were used to show a correlation between AVFV and shock outcome. In subsequent studies, a modified analysis procedure was used to show a correlation between shock outcome and moving average of AVFV. The use of single values of AVFV makes the AVFV sensitive to the local phase of the electrocardiographic (ECG) waveform, whereas moving averages are weighted by previous values of the ECG. The envelope is independent of the phase of the ECG and is not weighted by previous values of ECG as is the moving average. We explored, therefore, whether the AVFV computed from the envelopes of the ECG showed a stronger correlation with shock outcome than single values and moving averages of the AVFV. We estimated envelopes using the Hilbert transform. Orthogonal ECGs (sagittal, X; transverse, Y; and longitudinal, Z) were recorded from 11 dogs during 10 seconds of electrically induced ventricular fibrillation followed by a defibrillation shock with 50% probability of success. We used transvenous (right ventricular apex to subcutaneous patch) leads to deliver defibrillation shocks. Results from 236 successful and 249 unsuccessful trials showed, in contrast with the previously reported correlation, that moving average of AVFV was not higher for successful trials. In the Z direction, unsuccessful trials had higher voltage than successful trials (P < .05). Comparison of envelope voltages between successful and unsuccessful trials did not show any consistent and statistically significant differences. Although there were some methodological differences between ours and the previously reported studies, they are unlikely to have caused the discrepant observations. Our results suggest, therefore, that the absolute voltage of ECG during ventricular fibrillation is not robustly correlated with shock outcome.

Animals↗

PubMed1998

Optimal harvesting policy for single population with periodic coefficients.

In this paper, we examine the exploitation of single population modeled by time-dependent Logistic equation with periodic coefficients. First, it is shown that the time-dependent periodic Logistic equation has a unique positive periodic solution, which is globally asymptotically stable for positive solutions, and we obtain its explicit representation. Further, we choose the maximum annual-sustainable yield as the management objective, and investigate the optimal harvesting policies for constant harvest and periodic harvest. The optimal harvest effort that maximizes the annual-sustainable yield, the corresponding optimal population level, the corresponding harvesting time-spectrum, and the maximum annual-sustainable yield are determined, and their explicit expressions are obtained in terms of the intrinsic growth rate and the carrying capacity of the considered population. Our interesting and brief results generalize the classical results of Clark for a population described by the autonomous logistic equation in renewable resources management.

Conservation of Natural Resources↗

PubMed1998

Relationship between cellular uptake rate and chemical behavior of diammine/diaminocyclohexane platinum (II) complexes with oxygen-ligating anionic groups.

The uptake kinetics of the platinum (II) complexes of the formula Pt(NH3)2X, Pt(dach)X by human erythrocyte in the plasma isotonic buffer was studied. The results showed that across-membrane transport of all the platinum complexes studied follows a first-order kinetic process. The uptake rate constants decrease with the change of oxygen-ligating anionic group in the sequence: sulfato > selenato > anion of squaric acid > oxalato > anion of demethylcantharic acid > malonato and increase with increasing lipophilicity of carrier group. The relationship between uptake rate and reactivity of these complexes was established. The stereochemistry of dach isomers was shown without effect on the reactivity and the sequence.

Actins↗

PubMed1998

The bidirectional effect of vanadyl ion on the oxygen affinity of human hemoglobin.

The bidirectional effects of vanadyl on the oxygen affinity of hemoglobin depend on the mole ratio of vanadyl to Hb(R). In low R, the vanadyl ion increases Hb's oxygen affinity due to DPG hydrolysis. The lowered oxygen affinity in higher R is mainly due to the reactive-oxygen-species, probably superoxide, induced oxidation of Fe(II)-Hb to Fe(III)-Hb. The conformation change due to vanadyl binding contribute also to the lowered oxygen binding, but is monotonously decreasing with increasing of R values.

Circular Dichroism↗