Search PubMed⌕ Search

Biomedical subjects

W Wang

Publications and source records attributed to W Wang.

At least 613 records · Page 34Linked to original sources
PubMed1999

Molecular characterization and mapping of canine cGMP-phosphodiesterase delta subunit (PDE6D).

cGMP-phosphodiesterase (PDE) is composed of two catalytic (alpha and beta) and two identical inhibitory (gamma) subunits. The human gene (PDE6D) encoding a new subunit (delta) has been characterized and mapped to the long arm of chromosome 2 (HSA2q35-q36) where a new autosomal recessive retinitis pigmentosa (arRP) locus (RP26) has been localized. Characterization of the canine PDE6D shows the gene is about 4.2kb containing four exons interrupted by three introns; the size of the cDNA is 1059bp with an open reading frame (ORF) of 453bp. A single transcript of identical size (1.43kb) was detected in all tissues examined (liver, lung, spleen, kidney, heart, brain and retina), with the highest abundance in the retina. Canine PDE6D has been localized to canine radiation hybrid group 14-a, which extends conserved synteny between the dog, human chromosome 2q and mouse chromosome 1. The characterization of the canine PDE6D gene and its mapping provide important information for testing causal association of the gene with canine retinal degenerations, in particular rod-cone dysplasia 2 (rcd2) in collie dogs. This disease is characterized by abnormal retinal cGMP metabolism due to a deficiency in cGMP-PDE activity, yet the alpha, beta and gamma subunits of PDE have been excluded as candidate gene loci.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1999

Instability, stabilization, and formulation of liquid protein pharmaceuticals.

One of the most challenging tasks in the development of protein pharmaceuticals is to deal with physical and chemical instabilities of proteins. Protein instability is one of the major reasons why protein pharmaceuticals are administered traditionally through injection rather than taken orally like most small chemical drugs. Protein pharmaceuticals usually have to be stored under cold conditions or freeze-dried to achieve an acceptable shelf life. To understand and maximize the stability of protein pharmaceuticals or any other usable proteins such as catalytic enzymes, many studies have been conducted, especially in the past two decades. These studies have covered many areas such as protein folding and unfolding/denaturation, mechanisms of chemical and physical instabilities of proteins, and various means of stabilizing proteins in aqueous or solid state and under various processing conditions such as freeze-thawing and drying. This article reviews these investigations and achievements in recent years and discusses the basic behavior of proteins, their instabilities, and stabilization in aqueous state in relation to the development of liquid protein pharmaceuticals.

Animals↗
PubMed1999

Computational EST database analysis identifies a novel member of the neuropoietic cytokine family.

A novel member of the neuropoietic cytokine family has been cloned and the protein expressed and characterized. In an effort to identify novel secreted proteins, an algorithm incorporating neural network algorithms was applied to a large EST database. A full-length clone was identified that is 1710 bp in length and has a single open reading frame of 225 amino acids. This new cytokine is most homologous to cardiotrophin-1, having a similarity and an identity of 46 and 29%, respectively, and therefore we have named it cardiotrophin-like cytokine (CLC). Northern hybridization analysis identified a 1.4-kb messenger RNA that is highly expressed in spleen and peripheral leukocytes. Purified recombinant CLC induced the activation of NFkappaB and SRE reporter constructs in the TF-1, U937, and M1 cell lines. Furthermore, the signal transduction pathway for CLC was characterized in the neuroblastoma cell line SK-N-MC and found to involve tyrosine phosphorylation of gp130 and STAT-1.

Amino Acid Sequence↗
PubMed1999

Overexpression of urokinase-type plasminogen activator in pancreatic adenocarcinoma is regulated by constitutively activated RelA.

The Rel/NF-kappaB transcription factors regulate the expression of many genes. The activity of RelA, a member of the Rel/NF-kappaB transcription factor family, is constitutively activated in the majority of pancreatic adenocarcinomas and cell lines. We report that the urokinase-type plasminogen activator (uPA), one of the critical proteases involved in tumor invasion and metastasis, is overexpressed in pancreatic tumor cells and its overexpression is induced by constitutive RelA activity. The uPA promoter contains an NF-kappaB binding site that directly mediates the induction of uPA expression by RelA. Expression of a dominant-negative IkappaBalpha mutant inhibits kappaB site-dependent transcriptional activation of a uPA promoter-CAT reporter gene. Treating the pancreatic tumor cell lines with the known NF-kappaB inhibitors, dexamethasone and n-tosylphenyalanine chloromethyl ketone (TPCK), abolishes constitutive RelA activity and uPA overexpression. These results show that uPA is one of the downstream target genes induced by constitutively activated RelA in human pancreatic tumor cells, and suggests that constitutive RelA activity may play a critical role in tumor invasion and metastasis. Inhibition of constitutive RelA in pancreatic tumor cells may reduce their invasive and metastatic potential.

Adenocarcinoma↗
PubMed1999

Apoptosis of medulloblastoma cells in vitro follows inhibition of farnesylation using manumycin A.

Medulloblastoma is a malignant cerebellar tumor usually manifesting in childhood. We have previously shown that blocking the mevalonate pathway with lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, inhibits medulloblastoma proliferation and induces apoptosis in vitro. The underlying mechanism may involve blocking post-translational modification of important mitogenic signal-transduction proteins. We show that p21 ras processing is blocked by lovastatin, suggesting that inhibition of isoprenylation may be important in lovastatin-induced apoptosis. To test this hypothesis, manumycin A, an antibiotic which inhibits farnesyl protein transferase and thus farnesylation, was administered to 4 medulloblastoma cell lines in vitro. We found that blocking protein farnesylation with manumycin A was followed by apoptosis in a time- and dose-dependent manner. However, cell death induced by manumycin A was uniformly more rapid and efficient, requiring only 12 to 24 hr of treatment, than lovastatin-induced apoptosis, which required 36 to 96 hr (depending on the cell line tested). In addition, unlike lovastatin, which caused cell-cycle arrest in G1 phase and HMG-CoA reductase gene up-regulation, manumycin A had no effect on the cell cycle and resulted in down-regulation of HMG-CoA reductase gene expression. In both lovastatin- and manumycin A-treated cells, cellular cysteine protease precursor (CPP32) was activated, confirming the occurrence of apoptosis.

Antineoplastic Agents↗
PubMed1999

Interlocked mismatch-aligned arrowhead DNA motifs.

BACKGROUND: Triplet repeat sequences are of considerable biological importance as the expansion of such tandem arrays can lead to the onset of a range of human diseases. Such sequences can self-pair via mismatch alignments to form higher order structures that have the potential to cause replication blocks, followed by strand slippage and sequence expansion. The all-purine d(GGA)n triplet repeat sequence is of particular interest because purines can align via G.G, A.A and G.A mismatch formation. RESULTS: We have solved the structure of the uniformly 13C,15N-labeled d(G1-G2-A3-G4-G5-A6-T7) sequence in 10 mM Na+ solution. This sequence adopts a novel twofold-symmetric duplex fold where interlocked V-shaped arrowhead motifs are aligned solely via interstrand G1.G4, G2.G5 and A3.A6 mismatch formation. The tip of the arrowhead motif is centered about the p-A3-p step, and symmetry-related local parallel-stranded duplex domains are formed by the G1-G2-A3 and G4-G5-A6 segments of partner strands. CONCLUSIONS: The purine-rich (GGA)n triplet repeat sequence is dispersed throughout the eukaryotic genome. Several features of the arrowhead duplex motif for the (GGA)2 triplet repeat provide a unique scaffold for molecular recognition. These include the large localized bend in the sugar-phosphate backbones, the segmental parallel-stranded alignment of strands and the exposure of the Watson-Crick edges of several mismatched bases.

Base Pair Mismatch↗
PubMed1999

Secondary structure formation is the earliest structural event in the refolding of an all beta-sheet protein.

The refolding kinetics of cobrotoxin (CBTX), a small-molecular-weight ( approximately 7 kDa) all beta-sheet protein, has been monitored using a variety of biophysical techniques. The secondary structure formation and hydrophobic collapse occur as distinct events during the refolding of the protein. Complete secondary structure formation occurs prior to the clustering of the hydrophobic residues. The late stage(s) of the refolding pathway of CBTX is characterized by change(s) in the local environment and optical asymmetry of the indole ring of the sole tryptophan residue. The results obtained in the present study, to our knowledge, represent the first unambiguous experimental support for the framework model of protein folding.

Cobra Neurotoxin Proteins↗
PubMed1999

Characterization of the isoforms of PIXY321, a granulocyte-macrophage-colony stimulating factor-interleukin-3 fusion protein, separated by preparative isoelectric focusing on immobilized pH gradients.

We present here the purification and the characterization of the isoforms of PIXY321, a genetically engineered fusion of granulocyte-macrophage-colony stimulating factor and interleukin-3 expressed in yeast. The isoforms of PIXY321 were isolated using preparative isoelectric focusing (IEF) on immobilized pH gradients. Analysis of the collected fractions on analytical IEF gels showed that PIXY321 was resolved into four discrete isoforms of isoelectric point (pI) 5.0, 5.1, 5.2 and 5.3 with excellent yields. Subsequent analysis of purified isoforms of PIXY321 by peptide mapping and mass spectrometry linked the microheterogeneity of the original molecule to three parameters, the presence of deamidated residues, charged glycans and the pattern of O-linked glycosylation along the peptide sequence. This last parameter emphasizes the role of conformational aspects as key factors influencing the apparent isoelectric point of protein isoforms.

Amino Acid Sequence↗
PubMed1999

Extracellular matrix inhibits apoptosis and enhances endothelial cell differentiation by a NfkappaB-dependent mechanism.

Hormonal and environmental factors that control the growth, differentiation, and regression of the vasculature are of fundamental importance in tumorigenesis and in the choice of therapeutic strategies. To test the hypothesis that estradiol (E2) and basement membrane proteins would affect the survival of vascular endothelial cells (EC), immortalized human umbilical vein endothelial cells (ECV304) were examined for their response to the chemotherapeutic drugs taxol and etoposide. ECV cell apoptosis was inhibited by E2 (taxol only) or attachment to extracellular matrix (ECM) (taxol or etoposide). E2 increased ECV growth, while ECM binding resulted in growth arrest and differentiation. Apoptosis was associated with decreased levels of Bcl-2 and p21 proteins. E2 prevented down-regulation of p21 and Bcl-2 induced by taxol but did not prevent the down-regulation of p21 induced by etoposide, consistent with the failure of E2 to inhibit etoposide-induced cell death. However, ECM prevented p21 and Bcl-2 down-regulation induced by taxol or etoposide. Persistent activation of NFkappaB occurred after attachment of ECV cells to ECM, suggesting a role in survival or differentiation. IkappaBalpha levels were not affected by taxol but were reduced by etoposide treatment, while IkappaBbeta levels did not change with drug treatment. E2 did not alter the levels of IkappaBalpha or IkappaBbeta. Interestingly, levels of IkappaBalpha and IkappaBbeta declined in etoposide-treated ECV cells on ECM concomitant with the elevation of NFkappaB, suggesting that in these cells degradation of IkappaB may be responsible for NFkappaB activation. In agreement with these data, anti-sense NFkappaB treatment of ECV cells inhibited differentiation on ECM, but did not affect cell survival. In conclusion, culture of ECV cells on ECM or treatment with E2 inhibited apoptosis. NFkappaB activation by ECM was necessary for cellular differentiation, rather than inhibition, of apoptosis.

Apoptosis↗
PubMed1999

Wheat prolamine crosslinking through dityrosine formation catalyzed by peroxidases: improvement in the modification of a poorly accessible substrate by "indirect" catalysis

"Enzyme-assisted" oxidative polymerization of wheat gliadins was performed in an attempt to obtain new protein-based networks. Two plant peroxidases (soybean and horseradish) were used to induce the dimerization of tyrosine residues. The results show that tyrosines are poorly modified by these enzymes in an aqueous medium (dityrosine corresponded to 2% of the total amount of tyrosine). Two approaches were tested to overcome problems relating to accessibility to the target tyrosines: First, the efficiency of protein crosslinking via tyrosine-tyrosine aromatic ring condensation was enhanced in water when the proteins were oxidized by a fungus peroxidase (manganese-dependent peroxidase from Phanerochaete chrysosporium), which acts according to an indirect catalysis mechanism (up to 12% of the total amount of tyrosine is recovered under a dimeric form). Second, when the gliadins were dispersed in a water/dioxane (3/1) mixed solvent system, the tyrosines were more accessible on the protein surface, and similar yields were obtained with both types of peroxidase. The two types of catalysis (contact and indirect) are considered from the standpoint of the accessibility of the target residues. Enzymatic oxidations were also performed on synthetic peptides mimicking the repeatitive domains of gliadins. The results show that exposure of tyrosine to the solvent may not be sufficient to induce dityrosine formation. The mechanical properties of some films obtained from peroxidase-treated gliadins were investigated to correlate protein crosslinking with a potential application. One effect of the enzymatic treatment was to increase the tensile strength of the films. Copyright 1999 John Wiley & Sons, Inc.

Journal Article↗
PubMed1999

Kinetic evidence for the formation of a Michaelis-Menten-like complex between horseradish peroxidase compound II and di-(N-acetyl-L-tyrosine).

The formation of a reversible adsorption complex between a dimer of N-acetyl-L-tyrosine [di-(N-acetyl-L-tyrosine), (NAT)2] and horseradish peroxidase (HRP) compound II (CII) was demonstrated using a kinetic approach. A specific KIIm value (0.58 mM) was deduced for this step from stopped-flow measurements. The dimerization of the dipeptide Gly-Tyr was analysed at the steady state and compared with (NAT)2 dimerization [(NAT)2-->(NAT)4]. A saturation of the enzyme was observed for both substrates within their range of solubility. In each case the rate of dimerization reflected the rate-limiting step of compound II reduction to the native HRP (E) (kappcat/Kappm approximately kII-->E). The kappcat values for (Gly-Tyr)2 and (NAT)4 formation were 254 s-1 and 3.6 s-1 respectively. The KappM value of Gly-Tyr was 24 mM. It was observed that the value (0.7 mM) for (NAT)2 was close both to its specific KIIm value for the second step of reduction (CII-->E) and to its thermodynamic dissociation constant (Kd=0.7 mM) with the resting form of the enzyme. As (NAT)2 was a tighter ligand but a poorer substrate than Gly-Tyr, a steady-state kinetic study was performed in the presence of both substrates. A kinetic model which includes an enzyme-substrate adsorption prior to each of the two steps of reduction was derived. This one agreed reasonably well with the experimental data.

Adsorption↗
PubMed1999

Lungkine, a novel CXC chemokine, specifically expressed by lung bronchoepithelial cells.

We describe a novel mouse CXC chemokine that is selectively expressed in lung epithelial cells and up-regulated in various lung inflammation models. Although this chemokine clusters with other ELR-CXC chemokines, none of them can confidently be assigned to be its human homologue based on sequence identity. In addition, the highly restricted mRNA tissue distribution of this chemokine differentiates it from all previously described chemokines: Lungkine could not be detected in any of the 70 cDNA libraries analyzed corresponding to specific murine cell populations and tissues. High levels of Lungkine mRNA were specifically detected in the lung and at lower levels in fetal lung tissue by Northern blot and in situ hybridization, suggesting a potential role for this chemokine during lung development. Moreover, Lungkine protein is secreted into the airway spaces and induces the in vitro and in vivo migration of neutrophils, suggesting that it is involved in lung-specific neutrophil trafficking. Using fluorescent in situ hybridization, we show that Lungkine maps to mouse chromosome 5.

Amino Acid Sequence↗
PubMed1999

Bidirectional transmembrane modulation of integrin alphaIIbbeta3 conformations.

Activation of blood platelets by physiological stimuli (e.g. thrombin, ADP) at sites of vascular injury induces inside-out signaling, resulting in a conformational change of the prototype integrin alphaIIbbeta3 from an inactive to an active state competent to bind soluble fibrinogen. Furthermore, ligand occupancy of alphaIIbbeta3 initiates outside-in signaling and additional conformational changes of the receptor, leading to the exposure of extracellular neoepitopes termed ligand-induced binding sites (LIBS), which are recognized by anti-LIBS monoclonal antibodies. To date, the mechanism of bidirectional transmembrane signaling of alphaIIbbeta3 has not been established. In this study, using our newly developed anti-LIBScyt1 monoclonal antibody, we showed that extracellular ligand binding to alphaIIbbeta3 on blood platelets induces a transmembrane conformational change in alphaIIbbeta3, thereby exposing the LIBScyt1 epitope in the alphaIIb cytoplasmic sequence between Lys994 and Asp1003. In addition, a point mutation at this site (P998A/P999A) renders alphaIIbbeta3 constitutively active to bind extracellular ligands, resulting in fibrinogen-dependent cell-cell aggregation. Taken collectively, these results demonstrated that the extracellular ligand-binding site and a cytoplasmic LIBS epitope in integrin alphaIIbbeta3 are conformationally and functionally coupled. Such bidirectional modulation of alphaIIbbeta3 conformation across the cell membrane may play a key role in inside-out and outside-in signaling via this integrin.

Amino Acid Sequence↗
PubMed1999

Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha.

A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

Amino Acid Sequence↗
PubMed1999

Cooperative binding of Smad proteins to two adjacent DNA elements in the plasminogen activator inhibitor-1 promoter mediates transforming growth factor beta-induced smad-dependent transcriptional activation.

Transforming growth factor beta (TGFbeta) activates transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene through a major TGFbeta-responsive region (-740 and -647) in the PAI-1 promoter. This process requires the Smad family of signaling molecules. Upon phosphorylation by the TGFbeta receptors, Smad2 and Smad3 homoligomerize and heteroligomerize with Smad4, translocate to the nucleus and activate transcription of TGFbeta responsive genes. Smad3 and Smad4 have been shown to bind to various sites in the PAI-1 promoter. To determine the number of Smad-binding sites within the 94-base pair major TGFbeta-responsive region and the mechanism of Smad-mediated transactivation, we systematically mapped the Smad-binding sites and show that Smad4 and Smad3 bind cooperatively to two adjacent DNA elements in this region. Both elements were required for TGFbeta-induced, Smad3- and Smad4-dependent activation of PAI-1 transcription. Contrary to previous reports, transactivation of the PAI-1 promoter was mediated by the amino- but not carboxyl-terminal domains of the Smads. Furthermore, oligomerization of Smad3 markedly enhanced its binding to the two binding sites. Finally, a Smad4 mutation identified in a human pancreatic carcinoma that inactivates Smad4 signaling abolished Smad4 DNA binding activity, hence preventing transactivation of TGFbeta-responsive genes. These results underscore the importance of the Smad4 DNA binding activity in controlling cell growth and carcinogenesis.

Base Sequence↗
PubMed1999

Phosphorylation of TRAF2 inhibits binding to the CD40 cytoplasmic domain.

TRAF2 is a signal transducing adaptor molecule which binds to the CD40 cytoplasmic domain. We have found that it is phosphorylated, predominantly on serine residues, when transiently overexpressed in 293 cells. The phosphorylation appears to be related to the signaling events that are activated by TRAF2 under these circumstances, since two nonfunctional mutants were found to be phosphorylated significantly less than the wild-type protein. Furthermore, the phosphorylation status of TRAF2 had significant effects on the ability of the protein to bind to CD40, as evidenced by our observations that the CD40 cytoplasmic domain interacted preferentially with underphosphorylated TRAF2 and that phosphatase treatment significantly enhanced the binding of TRAF2 to CD40. We conclude from these studies that the phosphorylation of TRAF2 is likely to play an important role in regulating signaling by virtue of its ability to influence the CD40-TRAF2 interaction.

Amino Acid Substitution↗