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Biomedical subjects

R Wang

Publications and source records attributed to R Wang.

At least 199 records · Page 11Linked to original sources
PubMed2000

Submicron-size particles of ultrahigh molecular weight polyethylene produced via nonsolvent and temperature-induced crystallization.

Submicrometer size particles of ultrahigh molecular weight polyethylene (UHMWPE) were produced by crystallization from dilute (0.1-1.0 wt % of UHMWPE) solvent/nonsolvent emulsions. The procedure consisted of mixing a hot solution of UHMWPE in decalin or decane with a nonsolvent (tetraglyme) at approximately 160 degrees C, followed by rapid cooling of the mixture to zero or subzero temperatures. The rapid cooling causes microphase separation between the two liquids, resulting in the formation of an emulsion, which consists of microdroplets of the supercooled UHMWPE solution dispersed in tetraglyme. The consequent crystallization of the polymer in the microdroplets produces a suspension of fine crystals of UHMWPE, which can easily be isolated. The particles were characterized using scanning electron microscopy, differential scanning calorimetry, and Raman spectroscopy. Their degree of crystallinity is between that of GUR 1050 original (powder) and processed (molded) polymer. By changing the polymer concentration, solvent to nonsolvent ratio, and temperature, the size (from 0.1-1.0 microm) and shape (spheroids or rods) of the particles can be controlled. These particles may be used for immunochemical investigations and the study of the influence of UHMWPE wear debris on cell response.

Biocompatible Materials↗
PubMed2000

IL-12 enhances IL-2 function by inducing CD25 expression through a p38 mitogen-activated protein kinase pathway.

We previously showed that an IL-2 mutant, Q126D, could induce T cells to proliferate to the same extent as wild-type IL-2 but was unable to sensitize T cells to activation-induced cell death (AICD). Here we show that the partial signaling of Q126D is attributable to its inability to up-regulate the IL-2 receptor alpha chain (CD25). IL-12, which can up-regulate CD25 expression, enhances the ability of Q126D to induce AICD sensitivity and proliferation. IL-12 synergism with Q126D is dependent on CD25 up-regulation because the synergism is absent in CD25-deficient T cells. Inhibition of IL-12-induced up-regulation of CD25 by a p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580, also ablates the synergism between IL-12 and Q126D. Although CD25 is important for IL-2-induced proliferation and AICD sensitivity, it is not absolutely required because a high concentration of IL-2 can overcome the requirement for CD25. Under physiological concentrations of IL-2, CD25 expression is critical for the function of IL-2. IL-12 can enhance the function of IL-2 by up-regulating CD25 in a p38 MAP kinase-dependent manner.

Animals↗
PubMed2000

Apoptosis of lung epithelial cells in response to TNF-alpha requires angiotensin II generation de novo.

Recent work from this laboratory demonstrated that apoptosis of pulmonary alveolar epithelial cells (AEC) in response to Fas requires angiotensin II (ANGII) generation de novo and binding to its receptor (Wang et al., 1999b, Am J Physiol Lung Cell Mol Physiol 277:L1245-L1250). These findings led us to hypothesize that a similar mechanism might be involved in the induction of AEC apoptosis by TNF-alpha. Apoptosis was detected by assessment of nuclear and chromatin morphology, increased activity of caspase 3, binding of annexin V, and by net cell loss inhibitable by the caspase inhibitor ZVAD-fmk. Purified human TNF-alpha induced dose-dependent apoptosis in primary type II pneumocytes isolated from rats or in the AEC-derived human lung carcinoma cell line A549. Apoptosis in response to TNF-alpha was inhibited in a dose-dependent manner by the nonselective ANGII receptor antagonist saralasin or by the nonthiol ACE inhibitor lisinopril; the inhibition of TNF-induced apoptosis was maximal at 50 microgram/ml saralasin (101% inhibition) and at 0.5 microgram/ml lisinopril (86% inhibition). In both cell culture models, purified TNF-alpha caused a significant increase in the mRNA for angiotensinogen (ANGEN), which was not expressed in unactivated cells. Transfection of primary cultures of rat AEC with antisense oligonucleotides against ANGEN mRNA inhibited the subsequent induction of TNF-stimulated apoptosis by 72% (P < 0.01). Exposure to TNF-alpha increased the concentration of ANGII in the serum-free extracellular medium by fivefold in A549 cell cultures and by 40-fold in primary AEC preparations; further, exposure to TNF-alpha for 40 h caused a net cell loss of 70%, which was completely abrogated by either the caspase inhibitor ZVAD-fmk, lisinopril, or saralasin. Apoptosis in response to TNF-alpha was also completely inhibited by neutralizing antibodies specific for ANGII (P < 0.01), but isotype-matched nonimmune immunoglobulins had no significant effect. These data indicate that the induction of AEC apoptosis by TNF-alpha requires a functional renin/angiotensin system (RAS) in the target cell. They also suggest that therapeutic control of AEC apoptosis in response to TNF-alpha is feasible through pharmacologic manipulation of the local RAS.

Angiotensin II↗
PubMed2000

Effects of extracellular matrix on phenotype modulation and MAPK transduction of rat aortic smooth muscle cells in vitro.

The transition of arterial smooth muscle cells (SMCs) from a contractile to a synthetic phenotype may play an essential role in the formation of atherosclerotic and restenotic lesions. This process includes a prominent structural reorganization and allows cells to acquire the ability to migrate, proliferate, and secrete extracellular matrix components. According to Western blotting analysis and immunohistochemical and morphological observations, laminin not only retains SMCs in a contractile state but also possibly stimulates cells to transform a synthetic to a contractile phenotype at an early stage, mediated by P38 MAPK signal transduction. However, fibronectin promotes SMCs to transform from a contractile to a synthetic phenotype, mediated by the ERK MAPK signal pathway. The localization of smooth muscle alpha -actin, myosin heavy chain isoform SM2, and vimentin in explant-isolated rat SMCs was affected by a substrate of fibronectin and laminin and also by ERK MAP kinase inhibitor (PD098059) and P38 MAPK inhibitor (SB203580). Furthermore, vimentin may play a much more important role in differentiation than desmin in phenotype modulation in rat aortic smooth muscle cells.

Actins↗
PubMed2000

Hypercholesterolemia accelerates the Alzheimer's amyloid pathology in a transgenic mouse model.

Recent data suggest that cholesterol metabolism is linked to susceptibility to Alzheimer's disease (AD). However, no direct evidence has been reported linking cholesterol metabolism and the pathogenesis of AD. To test the hypothesis that amyloid beta-peptide (Abeta) deposition can be modulated by diet-induced hypercholesterolemia, we used a transgenic-mouse model for AD amyloidosis and examined the effects of a high-fat/high-cholesterol diet on central nervous system (CNS) Abeta accumulation. Our data showed that diet-induced hypercholesterolemia resulted in significantly increased levels of formic acid-extractable Abeta peptides in the CNS. Furthermore, the levels of total Abeta were strongly correlated with the levels of both plasma and CNS total cholesterol. Biochemical analysis revealed that, compared with control, the hypercholesterolemic mice had significantly decreased levels of sAPPalpha and increased levels of C-terminal fragments (beta-CTFs), suggesting alterations in amyloid precursor protein processing in response to hypercholesterolemia. Neuropathological analysis indicated that the hypercholesterolemic diet significantly increased beta-amyloid load by increasing both deposit number and size. These data demonstrate that high dietary cholesterol increases Abeta accumulation and accelerates the AD-related pathology observed in this animal model. Thus, we propose that diet can be used to modulate the risk of developing AD.

Alzheimer Disease↗
PubMed2000

Decreased in vitro fertilization and cleavage rates after an equipment error during CO(2) calibration.

OBJECTIVE: To report the decreased IVF rate of oocytes and the reduced cleavage rate of pronuclear stage embryos after gas has backflowed from a gas analyzer into an incubator during the calibration of the CO(2) concentration. DESIGN: Case report. SETTING: Clinical research unit for reproductive medicine in a hospital. INTERVENTION(S): Backflow of gas from a gas analyzer into an incubator during calibration of the CO(2) concentration. MAIN OUTCOME MEASURE: The IVF rate of oocytes and the cleavage rate of pronuclear stage embryos. RESULT(S): Thirty-two oocytes were retrieved from four infertile female patients. Nine oocytes were fertilized and four fertilized oocytes were cleaved. In a related animal experiment, the cleavage rate was 57.1% (36/63) in the control group and 25.4% (16/63) in the study group (P =.00026). CONCLUSION(S): The backflow of gas from the gas analyzer adversely affected the fertilization of oocytes and the cleavage of pronuclear stage embryos.

Animals↗
PubMed2000

Identification, cloning, and characterization of a major cat flea salivary allergen (Cte f 1).

An 18 kDa protein isolated from saliva of the cat flea, Ctenocephalides felis, elicits a positive intradermal skin test (IDST) in 100 and 80% of experimental and clinical flea allergic dogs, respectively. Using solid-phase enzyme-linked immuno assay (ELISA), this protein detected IgE in 100 and 80% of experimental and clinical flea allergic dogs, respectively. A cDNA (pFSI) encoding a full-length Cte f 1 protein was isolated from a C. felis salivary gland cDNA library, using a combination of PCR and hybridization screening. This cDNA is 658 bp in length, and contains an open reading frame of 528 bp. The open reading frame encodes a protein of 176 amino acids, consisting of an 18 amino acid signal sequence and a 158 amino acid mature protein. The calculated molecular weight and pI of the mature protein are 18106 Da and 9.3, respectively. The protein, named Cte f 1, is the first novel major allergen described for canine flea allergy. Recombinant Cte f 1 (rCte f 1) was expressed in Escherichia coli, Pichia pastoris and baculovirus infected Trichoplusia ni cells. Approximately, 90% of the rCte f 1 expressed in E. coli accumulated in insoluble inclusion bodies, which could be refolded to a soluble mixture of disulfide isomers with partial IgE binding activity. Small quantities of an apparently correctly refolded form of rCte f 1, which had IgE binding activity equal to the native antigen, was isolated from the soluble fraction of E. coli cells. However, P. pastoris and baculovirus infected insect cells expressed and secreted a fully processed, correctly refolded and fully active form of rCte f 1. Mass spectrometry analysis of the active forms of rCte f 1confirmed that eight intact disulfide bonds were present, matching the number observed in the native allergen. The relative ability of rCte f 1 to bind IgE in the serum of flea allergic animals, produced in these three expression systems, matched that of the native allergen. Competition ELISA demonstrated that approximately 90% of the specific IgE binding to native Cte f 1 could be blocked by the different forms of rCte f 1.

Alkylation↗
PubMed2000

Antiepidermal growth factor receptor antibodies augment cytotoxicity of chemotherapeutic agents on squamous cell carcinoma cell lines.

Recent literature by Fan et al (1993) demonstrated that addition of cisplatin and monoclonal antibody to the epidermal growth factor receptor (EGFR MAb) of the human squamous cell carcinoma (SCC), A431, eradicated gross tumors in nude mice. To determine whether a combination of either cisplatin or 5-fluorouracil (5-FU) with EGFR MAb could affect an SCC other than the A431 tumor model, an assay using 2 human tongue SCC cell lines, BroTo and SCC-25, was performed. Cells were pretreated with 1.25 microgram/mL cisplatin or 10 microgram/mL 5-FU. After a 4-hour incubation period, cisplatin-treated cells were washed with phosphate-buffered saline solution. Various concentrations of EGFR MAb were then added, and after a 24-hour incubation period, an MTT cell growth assay was performed. SCC-25 cells exhibited a greater decrease in growth with the addition of 16 nmol/L EGFR MAb to cisplatin compared with the cytotoxicity of cisplatin alone (P < 0.001). However, this combination did not produce similar results with BroTo cells (P > 0.05). The combination of EGFR MAb and 5-FU produced a growth inhibition versus control (unexposed cells) in both cell lines (P < 0.05). These findings suggest the possible augmentation of the activity of chemotherapeutic agents, cisplatin and/or 5-FU, with the addition of EGFR MAb on SCC cell lines other than A431.

Antibodies, Monoclonal↗
PubMed2000

Orientation relationships of martensite variants determined by electron backscatter diffraction

Orientation relationships between the parent phase and 2H type martensite, and between different variants in a plate group of martensite in the CuAlNi shape memory alloy were determined by electron backscatter diffraction. The result reveals clearly that the basal planes of different correspondence variants of the 2H martensite originate from different 110P planes of the parent phase, and the [010]2H axis of each correspondence variant originates from one of the <001>P axes of the parent phase. Our result reveals also the typical relationships of four habit-variants A, B, C and D in a martensite plate group, and that the habit-variants A and C (and equivalenty B and D) are twin-related by an 1212H mirror plane, and the habit-variants A and D (and B and C) are twin-related by an 1012H mirror plane. Usually, variants A and C (and B and D) form spear morphology and variants A and D (and B and C) form fork morphology.

Journal Article↗
PubMed2000

Defocus convergent beam electron diffraction determination of Burgers vectors of dislocations in quasicrystals

Principles, method and some application examples of determining Burgers vectors of dislocations in crystals and quasicrystals by means of defocus convergent-beam electron diffraction (CBED) technique are described and reviewed and compared with contrast experiment techniques. By using defocus CBED technique, dislocation reactions during high-temperature plastic deformation of face-centered icosahedral quasicrystals have been studied. These studies lead to a preliminary understanding to the micromechanism of high-temperature plastic deformation of quasicrystals.

Journal Article↗
PubMed2000

Ribozyme-mediated resistance to rice dwarf virus and the transgene silencing in the progeny of transgenic rice plants.

A hammerhead ribozyme (Rz) with long hybridizing arms targeting the mRNA of rice dwarf virus (RDV) segment 5 and a mutated nonfunctional ribozyme (mRz) were constructed. As predicted, Rz transcribed in vitro cleaved the target mRNA of RDV segment 5 into two fragments of 138 and 238 nucleotides in length. The Rz and mRz genes were each placed under the control of the CaMV 35S promoter and used to transform Japonica rice variety 'Tongling No. 1' via Agrobacterium tumefaciens. A total of 32 independent lines containing Rz or mRz was obtained as demonstrated by Southern blot analysis. Challenge inoculation with RDV viruliferous leafhoppers (Nephotettix cincticeps) showed that T1 plants containing the Rz transgene displayed high resistance or delayed and attenuated viral symptoms. In contrast, transgenic lines expressing mRz showed severe symptoms similar to the control plants transformed with the vector alone. These results suggest that Rz confers RDV resistance in transgenic rice. Genomic DNA PCR analysis confirmed that all of the examined T6 progeny plants contained the Rz transgene. However, accumulation of the Rz transcripts was detectable by RT-PCR only in the plants that were resistant to RDV. This suggested that loss of RDV resistance in progeny plants containing the Rz transgene may result from silencing of the Rz transgene.

Base Sequence↗
PubMed2000

Molecular cloning, expression, and distribution of glomerular epithelial protein 1 in developing mouse kidney.

BACKGROUND: Glomerular epithelial protein 1 (GLEPP1) is a receptor-like membrane protein tyrosine phosphatase (RPTP) with a large ectodomain consisting of multiple fibronectin type III repeats, a single transmembrane segment, and a single cytoplasmic phosphatase active site sequence. In adult human and rabbit kidneys, GLEPP1 is found exclusively on apical membranes of podocytes and especially on surfaces of foot processes. Although neither ligand nor function for this protein is known, other RPTPs with similar topologies have been implicated in mediating adherence behavior of cells. METHODS: To evaluate potential roles of GLEPP1 further, we cloned the full-length mouse GLEPP1 cDNA and examined its expression patterns in developing kidney by Northern blot analysis, in situ hybridization, and immunofluorescence microscopy. RESULTS: Nucleotide sequencing showed that mouse GLEPP1 was approximately 80% identical to rabbit and human GLEPP1 and approximately 91% identical at the amino acid level. The membrane-spanning and phosphatase domains of mouse GLEPP1 shared> 99% homology with PTPphi, a murine macrophage cytoplasmic phosphatase. Northern analysis identified a single GLEPP1 transcript of approximately 5.5 kb in fetal kidney that became approximately threefold more abundant in adults. In situ hybridization of newborn mouse kidney revealed GLEPP1 mRNA in visceral epithelial cells (developing podocytes) of comma- and S-shaped nephric figures, and expression increased in capillary loop and maturing stage glomeruli. Beginning on embryonic day 14, GLEPP1 protein was first observed on cuboidal podocytes of capillary loop stage glomeruli, but nascent podocytes of earlier comma- and S-shaped nephric figures were negative. At later stages of glomerular maturation, where foot process elongation and interdigitation occurs, GLEPP1 immunolabeling intensified on podocytes and then persisted at high levels in fully developed glomeruli. CONCLUSION: Our findings are consistent with a role for GLEPP1 in mediating and maintaining podocyte differentiation specifically.

Amino Acid Sequence↗
PubMed2000

Evaluation of EGS4/PRESTA multiple-scattering algorithms for 90Sr/90Y intravascular brachytherapy dosimetry.

The purpose of this work is to evaluate the EGS4/PRESTA electron multiple-scattering (MS) algorithms for dose calculation in intravascular brachytherapy (IVBT) using a 90Sr/90Y source. The small source size and the small volume of interest in IVBT require very fine spatial resolution, which may break down the constraints of Molière's MS theory as implemented in EGS4. The theory is accurate only when the electron step sizes are large enough to allow the number of collisions omega0 to be much greater than e = 2.7183. When step sizes are too small to allow at least 2.7183 collisions, as may be necessitated by the fine geometry, the algorithm may switch off MS, producing dosimetric artefacts. This study showed that switching off MS could produce a dose deviation of up to 6% when the half-thickness (d/2) of the dose scoring region is comparable with the Moliere minimum step size (t(min) = 2.7183). The effect of switching off MS is negligible if d/2 >> t(min) For the case of omega0 > e, if the electron step sizes are chosen to allow five to 40 collisions, with increasing step size, the doses surrounding the source increase and the error decreases. On the other hand, when larger step sizes are chosen, the dose calculation voxel size must also be increased in order for the calculations to converge. A good compromise between accuracy and applicability for IVBT simulation can be made, if the thickness of the scoring region is 0.1 mm and the electron step sizes are in the range allowing 10 to 30 collisions.

Algorithms↗
PubMed2000

Transforming growth factor-beta mRNA and protein in hypertrophic scar tissues and fibroblasts: antagonism by IFN-alpha and IFN-gamma in vitro and in vivo.

Hypertrophic scarring (HSc) following burn injury is a common, disfiguring, and functionally limiting form of dermal fibrosis, compromising recovery. Previously, elevated levels of transforming growth factor-beta1 (TGF-beta1), a fibrogenic cytokine, were found in wounds and serum of severely injured patients, antagonized in part by treatment with systemic interferon-alpha2b (IFN-alpha2b) both in vitro and in vivo. It is hypothesized that in wound healing after injury, platelets are an initial source of TGF-beta, but wound fibroblasts may be capable, after activation, of autoamplification of the initial response to injury by increasing TGF-beta mRNA and protein that may subsequently be responsive to IFN therapy with IFN-alpha or IFN-gamma or both. Using three pairs of site-matched HSc and normal fibroblasts from the same individuals, nonconfluent and near confluent fibroblasts were treated with TGF-beta, and cell proliferation and collagen production were assayed using cell counting and 18O2 isotopic uptake into hydroxyproline before analysis by gas chromatography-mass spectrometry (GC-MS). HSc and normal fibroblasts were assayed for the production of TGF-beta protein secretion using ELISA for TGF-beta1, TGF-beta2, and TGF-beta3 after acidification of medium samples from 96-h cultures. HSc and normal fibroblasts were treated with IFN-alpha2b or IFN-gamma or both for 96 h. Quantitative RT-PCR and Northern analysis were performed using newly synthesized internal standards for human TGF-beta1. TGF-beta stimulates both HSc and normal fibroblast proliferation. Collagen synthesis is greater in HSc than in normal fibroblasts and is maximally stimulated at 75 pM TGF-beta. TGF-beta stimulated collagen metabolism is antagonized by IFN-alpha or IFN-gamma or both in an additive fashion. HSc and normal fibroblasts not only possess the mRNA for TGF-beta1 but also secrete mature TGF-beta protein. Treatment of HSc and normal fibroblasts with IFN-alpha2b or IFN-gamma antagonizes TGF-beta protein production, and additive effects occur. RT-PCR demonstrates that after IFN treatment, downregulation of TGF-beta1 mRNA accounts in part for the reduction in protein secretion in HSc fibroblasts. Elevations of systemic TGF-beta may be due to wound fibroblasts. TGF-beta synthesis and antagonism of fibroblast TGF-beta protein secretion occurs with either IFN-alpha or IFN-gamma, in part by downregulation of TGF-beta1 mRNA levels.

Cicatrix, Hypertrophic↗
PubMed2000

Accuracy of free thyroxine measurements across natural ranges of thyroxine binding to serum proteins.

Systemic inaccuracies, proportional to the concentrations of serum proteins and the thyroxine (T4) they carry, have been reported in direct free T4 immunoassays. However, analytical recoveries of free T4 have not been carefully examined in most current methods, and they have not previously been examined across the pathophysiological range of serum T4 binding. In the present study we investigated ranges of serum T4 binding using free and total T4 measurements from 1359 individuals. Carefully characterized, gravimetrically calibrated, serum-based free T4 test solutions were then prepared with a constant normal free T4 concentration (12 ng/L) and varied serum T4 binding (approximately 300:1 to 24,000:1, ng protein bound T4: ng free T4). These standardized test solutions were analyzed using five T4 analog based free T4 methods. Analytical recoveries were calculated as ratios of actual free T4 measurements to the target value, and expressed as a percent of the target. Analytical recoveries were directly proportional to the extent of serum T4 binding and ranged 2% to 155%, 25% to 131%, 53% to 106%, 37% to 93%, and 37% to 73%, lowest to highest, in different methods. These systemic inaccuracies will confound interpretations of free T4 test results in clinical conditions with altered T4 binding. Future investigations into free T4 status must examine the analytical recovery of the free T4 method(s) used, as they relate to the extent of serum T4 binding in the clinical condition(s) studied.

Adolescent↗
PubMed2000

Cell-free assays for gamma-secretase activity.

The amyloid b-protein (Ab) deposited in Alzheimer's disease (AD) is a normally secreted proteolytic product of the amyloid b-protein precursor (APP). Generation of Ab from the APP requires two sequential proteolytic events: an initial b-secretase cleavage at the amino terminus of the Ab sequence followed by g-secretase cleavage at the carboxyl terminus of Ab. We describe the development of a robust in vitro assay for g-secretase cleavage by showing de novo Ab production in vitro and establish that this assay monitors authentic gamma-secretase activity by documenting the production of a cognate g-CTF, confirming the size of the Ab produced by mass spectrometry, and inhibiting cleavage in this system with multiple inhibitors that alter g-secretase activity in living cells. Using this assay, we demonstrate that the g-secretase activity 1) is tightly associated with the membrane, 2) can be solubilized, 3) has a pH optimum of 6.8 but is active from pH 6.0 to pH >8.4, and 4) ascertain that activities of the g-40 and g-42 are indeed pharmacologically distinct. These studies should facilitate the purification of the protease or proteases that are responsible for this unusual activity, which is a major therapeutic target for the treatment of AD.

Amyloid Precursor Protein Secretases↗
PubMed2000

Cell loss in isolated human islets occurs by apoptosis.

Purified islet allografts have largely failed to maintain long-term glucose homeostasis in human recipients, and the reasons for this are unclear. It is noteworthy, however, that islet isolation destroys or removes cellular and noncellular elements of the pancreas that could play an important role in supporting islet survival. The purpose of this study was to determine whether human islet isolation leads to the induction of programmed cell death. Human islets were enzymatically isolated from cadaveric donor pancreata using Liberase or Collagenase P, purified over a discontinuous BSA gradient, then cultured in RPMI 1640 at 37 degrees C in 5% CO2 for < or = 7 days. Islets were examined daily by routine histology and immunocytochemistry for islet hormones, DNA fragmentation [cell death; enzyme-linked immunosorbent assay (ELISA) and TUNEL assay] and for transglutaminase (TG) activity, two indicators of apoptosis. TG activity and DNA fragmentation increased by 1,000% and 1,890%, respectively (p < 0.05) This corresponded to the appearance of pyknotic nuclei on light microscopy, the presence of apoptotic bodies on electron microscopy, and the demonstration of TUNEL-positive cells. These were present primarily in a distribution that corresponded to the insulin-immunoreactive cells. At 5 days, 31.4 +/- 2.2% of islet cells were TUNEL positive. In summary, apoptosis of islet cells appears soon after islet isolation, and involves primarily the beta cell. This is the first report of apoptosis of islet cells after human islet isolation. The loss of beta-cell mass could be implicated in the failure of islet transplantation and merits further investigation.

Adolescent↗