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Biomedical subjects

Y Zhao

Publications and source records attributed to Y Zhao.

At least 559 records · Page 31Linked to original sources
PubMed1998

Suramin is an active site-directed, reversible, and tight-binding inhibitor of protein-tyrosine phosphatases.

The effect of suramin, a well known antitrypanosomal drug and a novel experimental agent for the treatment of several cancers, on protein-tyrosine phosphatases (PTPases) has been examined. Suramin is a reversible and competitive PTPase inhibitor with Kis values in the low microM range, whereas the Kis for the dual specificity phosphatase VHR is at least 10-fold higher. Although suramin can also inhibit the activity of the potato acid phosphatase at a slightly higher concentration, it is 2-3 orders of magnitude less effective against the protein Ser/Thr phosphatase 1alpha and the bovine intestinal alkaline phosphatase. Suramin binds to the active site of PTPases with a binding stoichiometry of 1:1. Furthermore, when suramin is bound to the active site of PTPases, its fluorescence is enhanced approximately by 10-fold. This property has allowed the determination of the binding affinity of suramin for PTPases and several catalytically impaired mutant PTPases by fluorescence titration techniques. Thus, the active site Cys to Ser mutants bind suramin with similar affinity as the wild type, while the active site Arg to Ala mutant exhibits a 20-fold reduced affinity toward suramin. Interestingly, the general acid deficient Asp to Ala mutant PTPases display an enhanced affinity toward suramin, which is in accord with their use as improved "substrate-trapping" agents. That suramin is a high affinity PTPase inhibitor is consistent with the observation that suramin treatment of cancer cell lines leads to an increase in tyrosine phosphorylation of several cellular proteins. Given the pleiotropic effects of suramin on many enzyme systems and growth factor-receptor interactions, the exact in vivo actions of suramin require further detailed structure-activity investigation of suramin and its structural analogs.

Binding Sites↗
PubMed1998

Spectroscopic, kinetic, and electrochemical characterization of heterologously expressed wild-type and mutant forms of copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3.

We report the development of a high-yield heterologous expression system for the copper-containing nitrite reductase from a denitrifying variant of Rhodobacter sphaeroides. Typical yields of wild-type protein are 20 mg L-1, which can be fully loaded with copper. Nitrite reductase contains an unusual blue-green Type 1 copper center with a redox/electron transfer function and a nearby Type 2 center where nitrite binds and is reduced to nitric oxide. The wild-type enzyme was characterized by: (1) its blue-green Type 1 optical spectrum; (2) its EPR spectrum showing rhombic character to its Type 1 center and nitrite perturbation to its Type 2 center; (3) its 247-mV Type 1 midpoint potential which is low relative to other Type 1 centers; and (4) its kinetics as measured by both steady-state and stopped-flow methods. The Type 2 copper reduction potential as monitored by EPR in the absence of nitrite was below 200 mV so that reduction of the Type 2 center by the Type 1 center in the absence of nitrite is not energetically favored. The mutation M182T in which the methionine ligand of Type 1 copper was changed to a threonine resulted in a blue rather than blue-green Type 1 center, a midpoint potential that increased by more than 100 mV above that of the wild-type Type 1 center, and a somewhat reduced nitrite reductase activity. The blue color and midpoint potential of M182T are reminiscent of plastocyanin, but the Type 1 cupric HOMO ground-state electronic g value and copper hyperfine properties of M182T (as well as cysteine and histidine ENDOR hyperfine properties; see next paper) were unchanged from those of the blue-green native Type 1 center. His287 is a residue in the Type 2 region whose imidazole ring was thought to hydrogen bond to the Type 2 axial ligand but not directly to Type 2 copper. The mutation H287E resulted in a 100-fold loss of enzyme activity and a Type 2 EPR spectrum (as well as ENDOR spectra; see next paper) which were no longer sensitive to the presence of nitrite.

Copper↗
PubMed1998

Benzo[a]pyrene diol epoxide-induced 3p21.3 aberrations and genetic predisposition to lung cancer.

3p deletion, a common chromosome defect in lung cancer, occurs more frequently in the lung tumor tissues of smoking patients than it does in those of nonsmoking patients. This pilot study evaluated whether 3p aberrations induced by benzo[a]pyrene diol epoxide (BPDE), the metabolic product of benzo[a]pyrene, a constituent of tobacco smoke, were more common in the peripheral blood lymphocytes of 40 lung cancer patients than they were in those of 54 matched controls. Our hypothesis was that 3p sensitivity to BPDE reflects the susceptibility of a specific locus to damage from carcinogens in tobacco smoke. BPDE-induced chromosome 3p21.3 aberrations were significantly more frequent in cases (34.1 per 1000) than they were in controls (22.1 per 1000; P < 0.0001). However, no such difference was observed for 6q27, a control locus. Using the median value in the controls (20 per 1000) as a cutoff point to classify BPDE-induced sensitivity at 3p21.3 and after adjustment by age, sex, ethnicity, and smoking status, 3p BPDE sensitivity was associated with an elevated risk of 14.1 (95% confidence interval: 3.5, 56.2) for lung cancer. There was also a dose-response relationship between the degree of BPDE sensitivity at 3p21.3 and increased risk for lung cancer. Therefore, 3p may be a molecular target for BPDE damage in lung cancer cases.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1998

Identification of histidine 105 in the beta1 subunit of soluble guanylate cyclase as the heme proximal ligand.

Soluble guanylate cyclase isolated from bovine and rat lung is a heterodimeric hemoprotein composed of alpha1 and beta1 subunits. The heme binding region has been localized to residues 1-385 of the beta1 subunit [beta1(1-385)], while the catalytic site(s) have been localized to the C-terminal region of sGC. There are four conserved histidine residues in the heme binding region of sGC. H220 and H346 are conserved among all known sGC subunits (alpha and beta), while H105 and H134 are conserved only in the beta subunits (beta1 and beta2). Site-directed mutagenesis was used to individually change each of the conserved histidines in sGC beta1(1-385) to alanine or glycine, and the resulting mutants were expressed in E. coli. All of the mutants except for H105A and H105G had heme bound as isolated. Imidazole (Im) was able to rescue heme binding to H105G when added to the growth medium and purification buffers. The heme in H105G isolated in the presence of imidazole [H105G(Im)] was ferric and a mixture of 5-coordinate, high-spin and 6-coordinate, low-spin complexes. After reduction, the ferrous heme in H105G(Im) was 5-coordinate, high-spin as indicated by resonance Raman spectroscopy. When imidazole in H105G(Im) was exchanged with N-methylimidazole (MeIm), the Fe-N(Im/MeIm) stretching frequency was shifted from 221 to 212 cm-1. A shift of this magnitude is expected when the ligand is directly coordinated to the heme iron. All of the data are consistent with the conclusion that H105 in the beta1 subunit is the heme proximal ligand.

Alanine↗
PubMed1998

Interleukin-6 production by human neutrophils after Fc-receptor cross-linking or exposure to granulocyte colony-stimulating factor.

Polymorphonuclear neutrophils (PMNs) are essential effector cells in host defense and tissue inflammatory responses. These responses may be initiated after cross-linking of cell surface Fc receptors that bind the constant portion of IgG (FcgammaR). We evaluated the effect of cross-linking FcgammaRI or FcgammaRII on interleukin-6 (IL-6) production by purified PMNs from normal donors or from patients being treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF). In PMNs from normal donors, IL-6 mRNA was detected by reverse transcriptase-polymerase chain reaction only after FcgammaRI or FcgammaRII cross-linking. We also found that IL-6 mRNA could be detected in PMNs after either in vitro or in vivo rhG-CSF treatment in the absence of FcgammaR cross-linking. IL-6 protein was found to be produced intracellularly and secreted by PMNs after cross-linking FcgammaRI or FcgammaRII or after rhG-CSF stimulation. Cross-linking FcgammaRI or FcgammaRII on PMNs from patients treated with rhG-CSF resulted in a synergistic increase in IL-6 secretion. Upregulation of IL-6 production by PMNs after rhG-CSF treatment may contribute to a clinical engraftment syndrome that occurs during periods of rapid increase in PMN numbers in patients receiving rhG-CSF.

Gene Expression Regulation↗
PubMed1998

Altering the nucleophile specificity of a protein-tyrosine phosphatase-catalyzed reaction. Probing the function of the invariant glutamine residues.

Protein-tyrosine phosphatases (PTPases) catalysis involves a cysteinyl phosphate intermediate, in which the phosphoryl group cannot be transferred to nucleophiles other than water. The dual specificity phosphatases and the low molecular weight phosphatases utilize the same chemical mechanism for catalysis and contain the same (H/V)C(X)5R(S/T) signature motif present in PTPases. Interestingly, the latter two groups of phosphatases do catalyze phosphoryl transfers to alcohols in addition to water. Unique to the PTPase family are two invariant Gln residues which are located at the active site. Mutations at Gln-446 (and to a much smaller extent Gln-450) to Ala, Asn, or Met (but not Glu) residues disrupt a bifurcated hydrogen bond between the side chain of Gln-446 and the nucleophilic water and confer phosphotransferase activity to the Yersinia PTPase. Thus, the conserved Gln-446 residue is responsible for maintaining PTPases' strict hydrolytic activity and for preventing the PTPases from acting as kinases to phosphorylate undesirable substrates. This explains why phosphoryl transfer from the phosphoenzyme intermediate in PTPases can only occur to water and not to other nucleophilic acceptors. Detailed kinetic analyses also suggest roles for Gln-446 and Gln-450 in PTPase catalysis. Although Gln-446 is not essential for the phosphoenzyme formation step, it plays an important role during the hydrolysis of the intermediate by sequestering and positioning the nucleophilic water in the active site for an in-line attack on the phosphorus atom of the cysteinyl phosphate intermediate. Gln-450 interacts through a bound water molecule with the phosphoryl moiety and may play a role for the precise alignment of active site residues, which are important for substrate binding and transition state stabilization for both of the chemical steps.

Bacterial Proteins↗
PubMed1998

[Analysis of expression of FMR-1 gene in male patients with fragile X syndrome using RT-PCR].

OBJECTIVE: To search the relationship between fragile X male patients and expression of FMR-1 gene and to establish a rapid and simple method for screening male patients in general population. METHODS: Two typical fragile X families demonstrated by cytogenetics method were analyzed by using RT-PCR. RESULTS: No expression of FMR-1 gene in all male patients in the two families was noted, but all normal individuals showed expression of FMR-1 gene. There was an expression of HPRT gene as internal control in each of the individuals detected. CONCLUSION: There is a strong relationship between loss of expression of FMR-1 gene and clinical phenotype of the fragile X male patients. Further research of this method will introduce a more simple and rapid method for detecting male patients with fragile X syndrome. This method may be used for general population screening.

Fragile X Messenger Ribonucleoprotein 1↗
PubMed1998

Avoidance of self-reactivity results in skewed CTL responses to rare components of synthetic immunogens.

In studying the CTL recognition of peptide determinants derived from the nuclear Ag La (SS-B), we observed significant skewing of the response toward rare components present within the immunogen. Thus, priming of naive mouse lymphocytes in vitro with a synthetic H-2Kb-binding peptide comprising human La (hLa) residues 51-58 resulted in class I-restricted cytotoxic T cells that failed to recognize naturally presented hLa 51-58 peptide. Instead, the majority of T hybrids recognized a low abundance (< or = 1%) contaminant present at picomolar concentrations in the original synthesis and identified as a peptide adduct containing N,4-t-butyl asparagine at position 6 of the hLa 51-58 sequence. The preferred T cell recognition of the butyl adduct was not due to increased affinity of this peptide for the H-2Kb molecule or to the antagonism of CTL recognizing the unmodified determinant. Rather, the bias in the immune response appeared to be the result of partial self-tolerance to the homologous mouse La 51-58 determinant, which differs from its human counterpart by only a single amino acid at position 1 (T-->I). Accordingly, the CTL response appeared to be focused on "non-self" ligands present within the synthesis, even though they were present at very low concentrations. These observations have significant implications for the use of synthetic peptide vaccines, especially those designed to manipulate responses to self peptides such as tumor Ags in which self-tolerance may result in unexpected reactivity.

Amino Acid Substitution↗
PubMed1998

PCR display identifies tamoxifen induction of the novel angiogenic factor adrenomedullin by a non estrogenic mechanism in the human endometrium.

Tamoxifen is currently the most widely used drug for the treatment of breast cancer, but there now exists considerable evidence that tamoxifen can also induce endometrial hyperplasia in pre menopausal women. We have used PCR differential display on primary human endometrial isolates in an attempt to identify genes induced by tamoxifen but not estrogen. Eight such differentially expressed bands were cloned and sequenced, one of which was found to be the peptide adrenomedullin. We have shown that adrenomedullin is a novel growth factor for endothelial cells and is angiogenic in vivo in the chick chorioallantoic membrane assay. Immunohistochemical analysis of endometrial sections have shown that while macrophages in the endometrium express adrenomedullin at a low level, endometrial macrophages of women receiving tamoxifen strongly express adrenomedullin (P=0.008). We postulate that endometrial induction of the angiogenic factor adrenomedullin by tamoxifen is part of the mechanism by which tamoxifen results in endometrial hyperplasia.

Adrenomedullin↗
PubMed1998

Starvation and diabetes increase the amount of pyruvate dehydrogenase kinase isoenzyme 4 in rat heart.

This study investigated whether conditions known to alter the activity and phosphorylation state of the pyruvate dehydrogenase complex have specific effects on the levels of isoenzymes of pyruvate dehydrogenase kinase (PDK) in rat heart. Immunoblot analysis revealed a remarkable increase in the amount of PDK4 in the hearts of rats that had been starved or rendered diabetic with streptozotocin. Re-feeding of starved rats and insulin treatment of diabetic rats very effectively reversed the increase in PDK4 protein and restored PDK enzyme activity to levels of chow-fed control rats. Starvation and diabetes also markedly increased the abundance of PDK4 mRNA, and re-feeding and insulin treatment reduced levels of the message to that of controls. In contrast with the findings for PDK4, little or no changes in the amounts of PDK1 and PDK2 protein and the abundance of their messages occurred in response to starvation and diabetes. The observed shift in the relative abundance of PDK isoenzymes probably explains previous studies of the effects of starvation and diabetes on heart PDK activity. The results indicate that control of the amount of PDK4 is important in long-term regulation of the activity of the pyruvate dehydrogenase complex in rat heart.

Animals↗
PubMed1998

Hydrogen peroxide-induced cytoskeletal rearrangement in cultured pulmonary endothelial cells.

Although the signaling pathways leading to hydrogen peroxide (H2O2)-induced endothelial monolayer permeability remain ambiguous, cytoskeletal proteins are known to be essential for maintaining endothelial integrity and regulating solute flux through the monolayer. We have recently demonstrated that thrombin-induced actin reorganization in bovine pulmonary artery endothelial cells (BPAEC) requires activation of both myosin light chain kinase (MLCK) and protein kinase C (PKC). Therefore, the present study was designed to investigate the effects of H2O2 on actin reorganization in BPAEC. H2O2 initiated sustained recruitment of actin to the cytoskeleton and transient myosin recruitment in a time- and concentration-dependent manner. The H2O2-induced actin recruitment was significantly inhibited by the calmodulin antagonists, W7 and TFP, but not by the MLCK inhibitor, KT5926, nor the PKC inhibitors, H7 and calphostin C. H2O2 also caused actin filament rearrangement in BPAEC with disruption of the dense peripheral bands and formation of stress fibers. These alterations occurred prior to actin translocation to the cytoskeleton and are prevented by inhibition of either MLCK or PKC. High concentrations of H2O2 transiently attenuated PKC activity but slightly increased the phosphorylation of the prominent PKC substrate and actin-binding protein, myristoylated alanine-rich C kinase substrate (MARCKS), by 5 min. However, MARCKS phosphorylation was reduced to below basal levels by 30 min. On the other hand, H2O2 induced a time- and dose-dependent phosphorylation of myosin light chains which was eliminated by both MLCK and PKC inhibitors. These data suggest that MLCK contributes to H2O2-induced myosin light chain phosphorylation and actin rearrangement and that PKC may play a permissive role. Neither of these enzymes appears to be involved in the H2O2-induced recruitment of actin to the cytoskeleton.

Actins↗
PubMed1998

Casemix-based funding of Northern Territory public hospitals: adjusting for severity and socio-economic variations.

The Northern Territory intends to make use of Australian National Diagnosis Related Groups (DRGs) and their cost relativities as the basis for the allocation of budgets among public hospitals. The study reported here attempted to assess the extent to which there are variations in severity of illness and socio-economic status which are not adequately explained by DRG alone and, if so, to develop a DRG payment adjustment index by use of routinely available data items. The investigation was undertaken by use of a database containing all discharges between July 1992 and June 1995. Hospital length of stay was used as a proxy for cost. Multivariate analysis was undertaken and it was found that several variables were associated with cost variations within DRGs. Stepwise multiple linear regression was used to develop a model in which 14 variables were able to explain 45% of the variations. Index values were subsequently computed from the regression model for each of eight categories of admitted patient episodes which are the intersections of three binary variables: Aborigine or non-Aborigine, rural or urban usual place of residence of the patient and hospital type (teaching or other). It is intended that these index values will be used to compute differential funding rates for each hospital in the Territory.

Aged↗
PubMed1998

Capillary electrophoresis and capillary electrophoresis-electrospray-mass spectroscopy for the analysis of heterocyclic amines.

Fourteen heterocyclic amines (HAs) were analyzed by capillary electrophoresis (CE) on a polyvinyl alcohol (PVA)-coated capillary column. The optimized electrolyte is composed of 20 mM ammonium acetate, pH 3.0, and 20% methanol. Similar conditions were applied in electrospray-mass spectrometry (CE-ES-MS). The CE-ES-MS optimization procedure includes the position of the capillary in the stainless steel interface, the flow of the sheath liquid and its composition.

Acetates↗
PubMed1998

Immunological and molecular analysis of three monoclonal lupus anticoagulant antibodies from a patient with systemic lupus erythematosus.

Antiphospholipid antibodies (APA), including lupus anticoagulants (LAC; as detected by in vitro blood clotting tests) and anti-cardiolipin antibodies (ACA; as assayed by solid-phase immunoassay), are strongly associated with recurrent thrombosis, thrombocytopenia, and recurrent fetal loss in some patients with systemic lupus erythematosus (SLE). The combined presence of APA and these clinical manifestations is termed antiphospholipid syndrome (APS). LAC and ACA comprise heterogeneous and somewhat overlapping autoantibody subsets. To date, it is unclear what degree of heterogeneity is present in an individual patient and between patients. To begin to address these issues, we generated three monoclonal LAC antibodies from a patient with SLE and APS. These antibodies were studied for their binding specificities and variable (V) region nucleotide sequences. All three LAC were unreactive with DNA, cardiolipin or other phospholipids. Sequence analysis of these antibodies revealed extensive overlap in their Ig V genes with anti-DNA antibodies and other autoantibodies characteristic of lupus. These data provide the first V gene sequence information on a group of SLE-derived LAC without ACA activity, representative of a similar subset of LAC found in patients with APS.

Adult↗
PubMed1998

Purification, amino acid sequence, and cDNA sequence of a novel calcium-precipitating proteolipid involved in calcification of corynebacterium matruchotii.

Corynebacterium matruchotii is a microbial inhabitant of the oral cavity associated with dental calculus formation. It produces membrane-associated proteolipid capable of inducing hydroxyapatite formation in vitro. This proteolipid was purified from chloroform:methanol extracts by chromatography on Sephadex LH-20 and migrated on SDS-polyacrylamide gel electrophoresis at 6-9 kDa. Removal of covalently attached acyl moieties by methanolic KOH decreased its molecular mass to approximately 5.5 kDa. The amino acid sequence of the apoproteolipid indicated a peptide of 50 amino acids, a calculated molecular weight of 5354 Da, and an isoelectric point of 4.28. Sequence analysis revealed an 8 amino acid sequence with homology to human phosphoprotein phosphatase 2A as well as several potential acylation sites and one phosphorylation site. The purified proteolipid induced calcium precipitation in vitro. Deacylation of the proteolipid by hydroxylamine treatment resulted in >50% loss of calcium-precipitating activity, suggesting that covalently attached lipids are required. Degenerate oligonucleotide primers, based on the amino acid sequence, were used to amplify the gene for the 5.5 kDa proteolipid from total chromosomal DNA of C. matruchotii by PCR. A 166 bp cDNA was isolated and sequenced, confirming the amino acid sequence of the proteolipid. Thus, we have sequenced a unique bacterial proteolipid that is involved in the formation of dental calculus by precipitating Ca2+ and possibly in transport of inorganic phosphate, necessary for hydroxyapatite formation.

Amino Acid Sequence↗
PubMed1998

Application of "one-bead one-compound" combinatorial library methods in signal transduction research.

Using a "split-synthesis" solid phase synthetic approach, bead libraries can be generated such that each bead displays only one chemical entity. This "one-bead one-compound" combinatorial library can then be assayed for specific biological properties using either a solid-phase on-bead binding or functional assay, or a releasable solution phase assay. Positive compound-beads can then be isolated for structure determination. Various assay systems to screen such a "one-bead one-compound" library are described. We have used this combinatorial library method to discover peptides that bind to the cell surface immunoglobulins of murine lymphoma cells. Such peptides, when presented in an oligomeric form to a lymphoma cell are able to induce signal transduction. Additionally, we have also applied the "one-bead one-compound" combinatory library approach to elucidate peptide substrate motifs for protein tyrosine kinases. Multiple distinct peptide motifs were identified for p60(c-src) protein tyrosine kinase. Using the identified peptide substrates as templates, potent and highly specific pseudosubstrate-based peptide inhibitors were developed.

Animals↗
PubMed1998

Antigenic characterization of HIV-1 gp41 binding proteins.

The envelope protein HIV-1 gp41 has been shown to exert various effects on human T-cells, B-cells and monocytes like inhibition of cell proliferation, modulation of MHC expression and cytokine production. In contrast to gpl20, where several receptor molecules have been identified, the receptor for gp41 is still unknown. Using a sepharose column, coupled with recombinant soluble gp41, (rsgp41; Env amino acids 539-684), five gp41-binding proteins of 37, 45, 50, 62 and 100 kDa had been isolated from lysates of the B-cell line Raji. Two mouse antiserums were generated against the proteins P45 and P62 and were tested against the binding specificity of both antiserums. In Western blot analysis the antiserums recognized two protein bands of 45 and 62 kDa in complete Raji cell lysates, as well as the purified proteins P45 and P62, respectively, but did not show any cross-reaction, indicating that the two proteins do not share any immunological epitopes. Besides, the polyclonal antiserums did not recognize the other gp41-binding proteins P37, P50 and P100. Using the P62 antiserum proteins of the same size as in Raji cell lysates were stained in the lysates of the monocytic cell line U937 and the T-cell line H9, demonstrating distribution of P62 in different blood cells. P45 seems not to be identical to HLA-C which had been shown to bind to gp41. These results indicate, that P45 and P62 are two separate gp41-binding proteins without homology to each other or to the other gp41-binding proteins.

Animals↗