Biomedical subjects

D Wen

Publications and source records attributed to D Wen.

At least 19 recordsLinked to original sources

PubMed2001

Chromatography of substituted benzoic acids with methanol-water-carbon dioxide mixtures.

The impact of the proportion of CO2 concentration in methanol-water-CO2 mobile phases on the separation of several substituted benzoic acids was explored by studying the variation of retention with mobile phase pH in these mixtures. As the amount of CO2 in methanol-aqueous buffer-CO2 mixtures increased, a more basic buffer was needed to control the dissociation of these acids. Differences in terms of retention, separation efficiency and peak asymmetry were shown for substituted benzoic acids with methanol-water-CO2 and methanol-aqueous buffer-CO2 mixtures. Variations of these chromatographic parameters with mobile phase pH were related to the dissociation of these acids and their interaction with methanol-aqueous buffer-CO2 mobile phases and the stationary phase. The addition of a buffer into methanol-aqueous solution-CO2 was an effective means to optimize separations of acidic analytes with high fluidity liquid mobile phases. The substituted benzoic acids had baseline separation in the least amount of time using the high fluidity liquid mobile phases.

Benzoates↗

PubMed2001

[Effect of low molecular weight heparin on unstable angina].

OBJECTIVE: To evaluate the effect of low molecular weight heparin (LMWH) and verus urokinase (UK) on unstable angina (UA). METHODS: Sixty seven patients with UA were divided in two groups by a single blind, controlled, randomized method: LMWH (anti-thrombi) group and UK (thromblytic therapy) group. Total ischemia load in 24 hours ambulatory ECG, mortality and cases of acute myocardial infarction (AMI) in 30 days were employed to compare the effect and safety between two groups. RESULTS: There was no significant difference in decreasing attacks of angina pectoris and total ischemia load between two groups. The UK group had two cases of AMI and five cases of complications of bleeding (two cases of epistaxis, two cases of bleeding gums, one case of hematochezia). CONCLUSION: Antithrombin therapy is prior to thromblitic therapy, treatment of LMWH is safer than that of low dose UK in UA.

Angina, Unstable↗

PubMed2001

[Clinical features of acute myocardial infarction in the young and middle-aged patients: analysis of 68 cases of coronary artery angiography].

OBJECTIVE: This study was to evaluate the clinical features of acute myocardial infarction (AMI) in young and middle-aged patients. METHODS: Eighty-seven patients were classified into two groups according to the age. Among them, 61 patients were 60 years old and above, 26 patients below 50 year old. Selective coronary artery angiography was performed on 68 cases and a scoring method which reflects the extent of narrowing of coronary artery was also used. RESULTS: The older group had more risk factors than the young group. The angina pectoris in the young group was less than the older group. The incidence of acute heart failure in the young group was higher than the older group. The mortality of both two groups had no significant differences. CONCLUSION: The high risk of AMI in the young and middle-aged patients should be paid more attention in the clinic.

Adult↗

PubMed2001

A novel chordin-like protein inhibitor for bone morphogenetic proteins expressed preferentially in mesenchymal cell lineages.

Chordin is a bone morphogenetic protein (BMP) inhibitor that has been identified as a factor dorsalizing the Xenopus embryo. A novel secreted protein, CHL (for chordin-like), with significant homology to chordin, was isolated from mouse bone marrow stromal cells. Injection of CHL RNA into Xenopus embryos induced a secondary axis. Recombinant CHL protein inhibited the BMP4-dependent differentiation of embryonic stem cells in vitro and interacted directly with BMPs, similar to chordin. However, CHL also weakly bound to TGFbetas. In situ hybridization revealed that the mouse CHL gene, located on the X chromosome, was expressed predominantly in mesenchyme-derived cell types: (1) the dermatome and limb bud mesenchyme and, later, the subdermal mesenchyme and the chondrocytes of the developing skeleton during embryogenesis and (2) a layer of fibroblasts/connective tissue cells in the gastrointestinal tract, the thick straight segments of kidney tubules, and the marrow stromal cells in adults. An exception was expression in the neural cells of the olfactory bulb and cerebellum. Interestingly, the spatiotemporal expression patterns of CHL were distinct from those of chordin in many areas examined. Thus, CHL may serve as an important BMP regulator for differentiating mesenchymal cells, especially during skeletogenesis, and for developing specific neurons.

Alternative Splicing↗

PubMed2001

Enhanced potency of human Sonic hedgehog by hydrophobic modification.

Post-translational modifications of the developmental signaling protein Sonic hedgehog (Shh) by a long-chain fatty acid at the N-terminus and cholesterol at the C-terminus greatly activate the protein in a cell-based signaling assay. To investigate the structural determinants of this activation phenomenon, hydrophobic and hydrophilic moieties have been introduced by chemical and mutagenic methods to the soluble N-terminal signaling domain of Shh and tested in both in vitro and in vivo assays. A wide variety of hydrophobic modifications increased the potency of Shh when added at the N-terminus of the protein, ranging from long-chain fatty acids to hydrophobic amino acids, with EC(50) values from 99 nM for the unmodified protein to 0.6 nM for the myristoylated form. The N-myristoylated Shh was as active as the natural form having both N- and C-terminal modifications. The degree of activation appears to correlate with the hydrophobicity of the modification rather than any specific chemical feature of the adduct; moreover, substitution with hydrophilic moieties decreased activity. Hydrophobic modifications at the C-terminus of Shh resulted in only a 2-3-fold increase in activity, and no activation was found with hydrophobic modification at other surface positions. The N-terminal modifications did not appear to alter the binding affinity of the Shh protein for the transfected receptor protein, Patched, and had no apparent effect on structure as measured by circular dichroism, thermal denaturation, and size determination. Activation of Desert Hh through modification of its N-terminus was also observed, suggesting that this is a common feature of Hh proteins.

Acyl Coenzyme A↗

PubMed2001

Identification of anaplastic lymphoma kinase as a receptor for the growth factor pleiotrophin.

Pleiotrophin (PTN) is a secreted growth factor that induces neurite outgrowth and is mitogenic for fibroblasts, epithelial, and endothelial cells. During tumor growth PTN can serve as an angiogenic factor and drive tumor invasion and metastasis. To identify a receptor for PTN, we panned a phage display human cDNA library against immobilized PTN protein as a bait. From this we isolated a phage insert that was homologous to an amino acid sequence stretch in the extracellular domain (ECD) of the orphan receptor tyrosine kinase anaplastic lymphoma kinase (ALK). In parallel with PTN, ALK is highly expressed during perinatal development of the nervous system and down-modulated in the adult. Here we show in cell-free assays as well as in radioligand receptor binding studies in intact cells that PTN binds to the ALK ECD with an apparent Kd of 32 +/- 9 pm. This receptor binding is inhibited by an excess of PTN, by the ALK ECD, and by anti-PTN and anti-ECD antibodies. PTN added to ALK-expressing cells induces phosphorylation of both ALK and of the downstream effector molecules IRS-1, Shc, phospholipase C-gamma, and phosphatidylinositol 3-kinase. Furthermore, the growth stimulatory effect of PTN on different cell lines in culture coincides with the endogenous expression of ALK mRNA, and the effect of PTN is enhanced by ALK overexpression. From this we conclude that ALK is a receptor that transduces PTN-mediated signals and propose that the PTN-ALK axis can play a significant role during development and during disease processes.

Adrenal Gland Neoplasms↗

PubMed2001

[Soil biota and its role in soil ecology].

Soil organisms alter soil physical, chemical and biological properties in different ways. The composition and structure of biotic communities at one hierarchical level can influence the spatial heterogeneity of resource at other hierarchical levels, and the latter is supported by a number of biologically relevant spheres-functional domains in soil. In this paper, the role of soil biota in soil ecosystem processes was assessed, and the relationship between soil biodiversity and ecosystem function was presented. Soil ecosystem responses to global change were also discussed.

Ecosystem↗

PubMed2000

The Escherichia coli signal transducers PII (GlnB) and GlnK form heterotrimers in vivo: fine tuning the nitrogen signal cascade.

The PII protein is Escherichia coli's cognate transducer of the nitrogen signal to the NRII (NtrB)/NRI (NtrC) two-component system and to adenylyltransferase. Through these two routes, PII regulates both amount and activity of glutamine synthetase. GlnK is the recently discovered paralogue of PII, with a similar trimeric x-ray structure. Here we show that PII and GlnK form heterotrimers, in E. coli grown in nitrogen-poor medium. In vitro, fully uridylylated heterotrimers of the two proteins stimulated the deadenylylation activity of adenylyltransferase, albeit to a lower extent than homotrimeric PII-UMP. Fully uridylylated GlnK did not stimulate, or hardly stimulated, the deadenylylation activity. We propose that uridylylated PII/GlnK heterotrimers fine-regulate the activation of glutamine synthetase. The PII/GlnK couple is a first example of prokaryotic signal transducer that can form heterotrimers. Advantages of hetero-oligomer formation as molecular mechanism for fine-regulation of signal transduction are discussed.

Bacterial Proteins↗

PubMed2000

Characterization of pH in liquid mixtures of methanol/H2O/CO2.

The presence of H2O and CO2 in enhanced-fluidity liquids changes the pH in these mixtures due to the formation of carbonic acid. The acid-base equilibria in enhanced-fluidity liquids will also be affected by the reduction in the dielectric constant with the addition of CO2. The pH of enhanced-fluidity liquid mixtures at room temperature was determined from the UV/visible absorption spectra of several pH indicators. pH values of methanol/H2O/CO2 mixtures with CO2 proportions as high as 19.2 mol% are reported. The effect of adding buffer to methanol/H2O/CO2 mixtures on pH was also studied. It was also shown that pressure variation did not significantly influence the pH of enhanced-fluidity liquids.

Buffers↗

PubMed2000

The effect of intraislet somatostatin immunoneutralization on insulin secretion in the isolated perfused rat pancreas.

BACKGROUND: There is evidence of a local inhibitory effect of somatostatin on insulin secretion in the isolated human pancreas, but this has not been shown in a rat model. The possible phasic effect of somatostatin on insulin secretion has not been demonstrated. AIMS: This study was undertaken to determine if somatostatin has a local regulatory effect on phasic insulin secretion within a rat pancreas model. METHODS: The basal and glucose stimulated secretion of insulin was compared with and without immunoneutralization of somatostatin using a somatostatin antibody in an isolated perfused rat pancreas model. High concentration, high affinity monoclonal somatostatin antibody was perfused through isolated rat pancreata. Radioimmunoassay for insulin was performed on the portal effluent. RESULTS: Immunoneutralization of somatostatin during basal insulin secretion produced a rise in insulin secretion of 551 +/- 163% that approached significance. Immunoneutralization during glucose stimulated insulin secretion produced a significant rise in insulin secretion compared to the control group of 2,678 +/- 187% vs. 535 +/- 39% (p < 0.05). The phase I vs. the phase II response in the glucose stimulated pancreas was similar in the presence of control antibody, 867 +/- 351% vs. 900 +/- 398% (p = NS). With somatostatin immunoneutralization, the glucose stimulated pancreas had a significantly higher phase II response than phase I; 3,832 +/- 688% vs. 2,516 +/- 431% (p < 0.05). CONCLUSION: These data indicate that intraislet somatostatin is an inhibitor of insulin secretion in the isolated perfused rat pancreas. This effect occurs primarily in phase II of insulin secretion.

Animals↗

PubMed1999

Zinc-dependent structural stability of human Sonic hedgehog.

The role of the zinc site in the N-terminal fragment of human Sonic hedgehog (ShhN) was explored by comparing the biophysical and functional properties of wild-type ShhN with those of mutants in which the zinc-coordinating residues H140, D147, and H182, or E176 which interacts with the metal ion via a bridging water molecule, were mutated to alanine. The wild-type and E176A mutant proteins retained 1 mol of zinc/mol of protein after extensive dialysis, whereas the H140A and D147A mutants retained only 0.03 and 0.05 mol of zinc/mol of protein, respectively. Assay of the wild-type and mutant proteins in two activity assays indicated that the wild-type and E176A mutant proteins had similar activity, whereas the H140A and D147A mutants were significantly less active. These assays also indicated that the H140A and D147A mutants were susceptible to proteolysis. CD, fluorescence, and (1)H NMR spectra of the H140A, D147A, and E176A mutants measured at 20 or 25 degrees C were very similar to those observed for wild-type ShhN. However, CD measurements at 37 degrees C showed evidence of some structural differences in the H140A and D147A mutants. Guanidine hydrochloride (GuHCl) denaturation studies revealed that the loss of zinc from the H140A and D147A mutants destabilized the folded proteins by approximately 3.5 kcal/mol, comparable to the effect of removing zinc from wild-type ShhN by treatment with EDTA. Thermal melting curves of wild-type ShhN gave a single unfolding transition with a midpoint T(m) of approximately 59 degrees C, whereas both the H140A and D147A mutants displayed two distinct transitions with T(m) values of 37-38 and 52-54 degrees C, similar to that observed for EDTA-treated wild-type ShhN. Addition of zinc to the H140A and D147A mutants resulted in a partial restoration of stability against thermal and GuHCl denaturation. The ability of these mutants to bind zinc was confirmed using a fluorescence-based binding assay that indicated that they bound zinc with K(d) values of approximately 1.6 and approximately 15 nM, respectively, as compared to a value of </=100 pM for wild-type ShhN. The properties of the E176A mutant were indistinguishable from those of wild-type ShhN in all biophysical and functional assays, indicating that this residue does not contribute significantly to stabilization of the zinc-binding site and that ShhN does not require hydrolase activity for in vitro biological function.

Alkaline Phosphatase↗

PubMed1999

Neuregulins and ErbB receptors in cultured neonatal astrocytes.

Neuregulins (NRGs) are a family of growth factors involved in signaling between neurons and glial cells of the peripheral and central nervous system. NRGs are synthesized and secreted by a number of cell types including Schwann cells, neurons, and oligodendrocytes. NRG transduction signals are mediated by the erbB family of receptor tyrosine kinases. These NRGs may be important for paracrine or autocrine signaling during development, injury, and the normal functioning of the central nervous system. In this study, we characterize the NRGs and erbB receptors expressed by cultured neonatal rat astrocytes. Using immunoblotting protocols with pan-specific antibodies, we identified eleven NRG molecular weight isoforms from approximately 16 kDa to 105 kDa in cultured neonatal rat astrocytes. Immunocyotchemistry with isoform-specific antibodies revealed the expression of both major isoform families (NRGalpha, NRGbeta). Additionally, astrocyte-conditioned media contained two molecular weight isoforms of NRGs. We detected mRNA expression of NRGalpha and NRGbeta in astrocytes by amplifying mRNA transcripts with reverse transcription polymerase chain reaction. Furthermore, we confirm that cultured astrocytes express all four erbB receptors as detected by immunocytochemical and immunoblotting techniques. These data indicate that astrocytes contain and secrete NRGs.

Animals↗

PubMed1999

Fractionation of polyclonal antibodies to fragments of a neuroreceptor using three increasingly chaotropic solvents.

We have developed specific antibodies against fragments of anaplastic lymphoma kinase (ALK) in order to develop tools for characterizing the expression and biological function of this orphan receptor. The first fragment consisted of residues 280 to 480 of the murine extracellular domain, was expressed in Escherichia coli (E. coli), purified in the presence of urea from the pellet of mechanically lysed cells and injected into rabbits as an unfolded protein in urea. The second fragment consisted of residues 1519 to 1619 of the murine sequence, corresponding to the C-terminal side of the kinase domain. It was expressed in E. coli as a soluble glutathione-S-transferase fusion protein, purified from the supernatant of broken cells and injected into rabbits as a folded protein. Both antisera were purified using antigen affinity chromatography, with the polyclonal antibodies eluted stepwise using three different buffers, 0.1 M glycine, pH 2.9, followed by 7 M urea, pH 4, followed by 6 M guanidine-HCl (GdnHCl), pH 4. Antisera prepared against either antigen contained antibodies that eluted in each of the three pools, indicating that solvents more chaotropic than acid were required to elute antibody populations that were tightly bound to the antigen column. All three antibody pools were reactive towards their respective antigens upon Western blot analysis. Purified polyclonal antibodies (pAbs) to both fragments also recognized the full-length protein expressed in Chinese hamster ovary cells. In every case, the pAbs eluting in GdnHCl were the most sensitive for detecting full-length ALK.

Anaplastic Lymphoma Kinase↗

PubMed1999

Induced fit activation mechanism of the exceptionally specific serine protease, complement factor D.

We have investigated the mechanism by which the complement protease, Factor D, achieves its high specificity for the cleavage of Factor B in complex with C3(H2O). Kinetic experiments showed that Factor B and C3(H2O) associate with a KD of >/=2.5 microM and that Factor D acts on this complex with a second-order rate constant of kcat/KM >/= 2 x 10(6) M-1 s-1, close to the rate of a diffusion-controlled reaction for proteins of this size. In contrast, Factor D, which is a member of the trypsin family of serine proteases, was 10(3)-10(4)-fold less active than trypsin toward both thioester and p-nitroanilide substrates containing an arginine at P1. Furthermore, peptides spanning the Factor B cleavage site were not detectably cleaved by Factor D (kcat/KM </= 0.5 M-1 s-1). These results imply that contacts between Factor D and the C3(H2O)B complex, outside the vicinity of the cleavage site in Factor B, generate >/=9 kcal/mol of binding energy to stabilize the transition state for reaction. In support of this, we demonstrate that chemical modification of Factor D at a single lysine residue that is distant from the active site abolishes the activity of the enzyme toward Factor B while not affecting activity toward small synthetic substrates. We propose that Factor D may exemplify a special case of the induced fit mechanism in which the requirement for conformational activation of the enzyme results in a substantial increase in substrate specificity.

Acetylation↗

PubMed1999

[Studies on chemical constituents of Ilex Merr].

OBJECTIVE: To further probe into the chemical composition of Ilex hainanensis. METHOD: Compound isolation was affected on the basis of the EtOAc soluble fraction of the ethanolic extract from the leaves of the plant. RESULT: Four compounds were obtained and by physico-chemical and spectroscopic methods identified as ursolic acid, ilexgenin A, ilexsaponin A1 and beta-sitosterol-3-O-beta-D-glucoside. CONCLUSION: All the four compounds were isolated from the plant for the first time.

Ilex↗

PubMed1999

[Expression of P53 protein in premalignant lesion and carcinoma of larynx].

To investigate the expression of P53 protein in the premalignant lesion and carcinoma of laryngeal epithelium, the authors used DO-7, an antibody to wild and mutant type P53 protein. Eleven cases of simple hyperplasia of epithelium (SHE), 32 cases of atypical hyperplasia of epithelium (AHE) and 42 cases of laryngeal squamous cell carcinoma (LSCC) were examined by ABC technique. The results showed that the overexpression of P53 protein were 0%, 31.25% and 52.38% in SHE, AHE and LSCC, respectively. The positive rate of P53 protein were 0%, 33.33% and 75.00% in mild, moderate and severe AHE, respectively. In well, moderately and poorly differentiated carcinomas of larynx, the positive rates were 62.50%, 76.92% and 15.39%, respectively. There were highly significant differences (P < 0.01). No correlation was found between the P53 protein overexpression and the clinical stages, neck lymph node metastasis, local recurrence and, survival of LSCC. The results suggest that the overexpression of P53 protein may play a role in the pathogenesis and development of LSCC.

Carcinoma, Squamous Cell↗

PubMed1998

Release of the neuregulin functional polypeptide requires its cytoplasmic tail.

Based on both in vivo and in vitro studies, we have shown previously that the intracellular domain of a membrane-bound isoform of the growth factor, neuregulin, regulates proteolytic release of its extracellular domain ErbB receptor-activating ligand. To investigate the mechanism(s) involved in this regulation, a series of intracellular domain mutants were constructed and tested for susceptibility to proteolytic processing after transient transfection in COS-7 cells. These studies revealed that regulation of extracellular domain cleavage by the intracellular domain is sequence-specific and involves three distinct 30-60-residue segments. The presence of any two of these three segments is both necessary and sufficient for proteolytic processing, and resistance to proteolysis is not due to an alteration in cellular localization or transport. Evidence was also obtained that regulation of extracellular domain processing involves initial intracellular domain dimerization. Thus, with expression of a construct encoding only the intracellular domain, dimerization could be demonstrated in cross-linking experiments. Furthermore, resistance to proteolytic processing of a construct lacking a large portion of the intracellular domain was rescued with a chimera, in which the intracellular domain was replaced with a spontaneously dimerizing Fc fragment. Taken together these studies indicate that intracellular domain interactions are critically involved in the spacial and temporal control of growth and development by membrane-bound neuregulin isoforms.

Animals↗

PubMed1998

Transmembrane neuregulins interact with LIM kinase 1, a cytoplasmic protein kinase implicated in development of visuospatial cognition.

The neuregulins are receptor tyrosine kinase ligands that play a critical role in the development of the heart, nervous system, and breast. Unlike many extracellular signaling molecules, such as the neurotrophins, most neuregulins are synthesized as transmembrane proteins. To determine the functions of the highly conserved neuregulin cytoplasmic tail, a yeast two-hybrid screen was performed to identify proteins that interact with the 157-amino acid sequence common to the cytoplasmic tails of all transmembrane neuregulin isoforms. This screen revealed that the neuregulin cytoplasmic tail interacts with the LIM domain region of the nonreceptor protein kinase LIM kinase 1 (LIMK1). Interaction between the neuregulin cytoplasmic tail and full-length LIMK1 was demonstrated by in vitro binding and co-immunoprecipitation assays. Transmembrane neuregulins with each of the three known neuregulin cytoplasmic tail isoforms interacted with LIMK1. In contrast, the cytoplasmic tail of TGF-alpha did not interact with LIMK1. In vivo, neuregulin and LIMK1 are co-localized at the neuromuscular synapse, suggesting that LIMK1, like neuregulin, may play a role in synapse formation and maintenance. To our knowledge, LIMK1 is the first identified protein shown to interact with the cytoplasmic tail of a receptor tyrosine kinase ligand.

Animals↗