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Biomedical subjects

M Wang

Publications and source records attributed to M Wang.

At least 757 records · Page 42Linked to original sources
PubMed1995

Cloning, nucleotide sequence and expression of the bifunctional dihydrofolate reductase-thymidylate synthase from Glycine max.

cDNAs encoding the bifunctional dihydrofolate reductase-thymidylate synthase from Glycine max were isolated and sequenced. The 1794 base full length cDNA contains a single open reading frame of 1593 bases. The predicted size of the encoded protein is 530 amino acids with a molecular weight of 59,707. The protein has two domains: a 226 residue DHFR domain in the N-terminus, which is over 30% identical to human DHFR or the DHFR domain of protozoal DHFR-TS, and a 304 residue thymidylate synthase (TS) domain, which is over 60% identical to eukaryotic TS enzymes. The whole protein sequence is greater than 75% identical to DHFR-TS sequences from two other plants, Daucus carota and Arabidopsis thaliana. The sequence of two tryptic peptides obtained from DHFR preparations matched the predicted amino acid sequence, one peptide lying in the DHFR domain and the other in the TS domain. These results indicate that DHFR and TS exist in a bifunctional polypeptide in Glycine max. The coding region of the cDNA was inserted downstream of the T7 promoter and translation initiation signals in the vector pET-3a. This construct (pDR-TS) was transformed into Escherichia coli BL21 (DE) [plysS] which produces T7 RNA polymerase upon induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). The expression of the bifunctional enzyme was confirmed by detection of both DHFR and TS activities. The purified enzyme has a subunit molecular mass of 60 kDa. This is the first report of expression of a plant DHFR-TS cDNA.

Amino Acid Sequence↗
PubMed1995

Crystallization and preliminary x-ray studies of NADPH-cytochrome P450 reductase.

NADPH-cytochrome P450 reductase (CPR; NADPH:ferrihemoprotein reductase, EC 1.6.2.4) catalyzes the transfer of electrons to all known microsomal cytochromes P450. CPR is unique in that it is one of only two mammalian enzymes known to contain both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the other being the various isoforms of nitric oxide synthase. Similarities in amino acid sequence and in functional domain arrangement with other key flavoproteins, including nitric oxide synthase, make CPR an excellent prototype for studies of interactions between two flavin cofactors. We have obtained diffraction-quality crystals of rat liver CPR, expressed in Escherichia coli and solubilized by limited proteolysis with trypsin. The crystals were grown in Hepes buffer (pH 7.0), containing polyethylene glycol 4500 and NaCl. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 103.3 A, b = 116.1 A, and c = 120.4 A. If we assume that there are two molecules of the 72-kDa CPR polypeptide per asymmetric unit, the calculated value of Vm is 2.54 A3/Da.

Amino Acid Sequence↗
PubMed1995

The role of hyaluronan in tumour neovascularization (review).

Tumour growth and metastasis are totally dependant upon neovascularization. The target cell for tumour neovascularization is the blood-vessel endothelial cell, and specific angiogenic molecules produced or induced by the tumour are believed to initiate the process. In this report, we review one of these angiogenic molecules, the glycosaminoglycan hyaluronan (HA), which appears to have differing roles in neovascularization depending on its molecular mass. High-molecular-mass HA is anti-angiogenic whereas oligosaccharides of HA, of specific size, actively stimulate endothelial-cell proliferation and migration, 2 of the key events associated with neovascularization, and induce angiogenesis in vivo. We provide details of the action of HA oligosaccharides on endothelial cells, from binding to cell-surface receptors, through activation of signal transduction pathways and gene expression to protein synthesis, cell proliferation and cell migration. We also suggest a model to account for HA of differing molecular mass being present, at different locations, within a single tumour and how this HA aids both general tumour growth and tumour metastasis.

Cell Division↗
PubMed1995

Transmission-ratio distortion of X chromosomes among male offspring of females with skewed X-inactivation.

We have begun a search for heritable variation in X-chromosome inactivation pattern in normal females to determine whether there is a genetic effect on the imprinting of X-chromosome inactivation in humans. We have performed a quantitative analysis of X-chromosome inactivation in lymphocytes from mothers in normal, three-generation families. Eight mothers and 12 grandmothers exhibited evidence of highly skewed patterns of X-chromosome inactivation. We observed that the male offspring of females with skewed X-inactivation patterns were three times more likely to inherit alleles at loci that were located on the inactive X chromosome (Xi) than the active X chromosome (Xa). The region of the X chromosome for which this phenomenon was observed extends from Xp11 to -Xq22. We have also examined X-chromosome inactivation patterns in 21 unaffected mothers of male bilateral sporadic retinoblastoma patients. Six of these mothers had skewed patterns of X-chromosome inactivation. In contrast to the tendency for male offspring of skewed mothers from nondisease families to inherit alleles from the inactive X chromosome, five of the six affected males inherited the androgen receptor alleles from the active X chromosome of their mother.

Base Sequence↗
PubMed1995

Pulsed field separation of large supercoiled and open-circular DNAs and its application to bacterial artificial chromosome cloning.

We have studied the separation of large (80-300 kbp) supercoiled (SC) DNA in conventional agarose gel electrophoresis, field inversion gel electrophoresis (FIGE) and pulsed field gel electrophoresis (PFGE). DNA migration was measured under a variety of electrophoretic conditions including different switch times, temperatures, agarose concentrations, and voltage gradients. The migration of SC DNA was found to be inversely proportional to its molecular weight in the three electrophoresis systems tested. In conventional agarose electrophoresis, voltage gradient was found to be the determining parameter in the separation of SC DNA. Unlike large linear DNAs, the migration of SC DNA was found to be independent of switch time in PFGE and FIGE. Broad DNA bands were observed in prolonged FIGE runs. In addition, we have also studied the migration of open-circular (OC) DNA (80 and 100 kbp) in pulsed field gel electrophoresis. Eighty kbp OC DNA can migrate into agarose gels under certain pulsed field conditions whereas 100 kbp OC DNA was trapped at the wells. Based on electrophoretic conditions described in this report, we can determine the size of bacterial artificial chromosome (BAC) clones without restriction enzyme digestion and have enriched the percentage of larger size clones in BAC cloning.

Animals↗
PubMed1995

Prenatal and presymptomatic diagnosis of the Marfan syndrome using fluorescence PCR and an automated sequencer.

The Marfan syndrome (MFS) is a heritable connective tissue disorder characterized by skeletal, ocular, and cardiovascular abnormalities. Defects in fibrillin, an elastin-associated microfibrillar protein, are now known to cause MFS. Since the discovery of fibrillin as the gene responsible for MFS, requests for prenatal and presymptomatic diagnosis have become common-place. Here we report the use of the polymerase chain reaction (PCR), using fluorescence labelled primers and an automated sequencer, to establish linkage data for 'molecular diagnosis'. The mistaken clinical diagnosis of MFS based on the appearance of a common cardiovascular manifestation, mitral valve prolapse, and a positive family history is also discussed.

Autoanalysis↗
PubMed1995

The effect of intracellular pH on the regulation of the Rab 16A and the alpha-amylase 1/6-4 promoter by abscisic acid and gibberellia.

Intracellular pH (pHi) of barley aleurone cells is known to be affected by hormones and plant growth conditions. The possible mechanisms by which these pHi shifts influence the actions of abscisic acid (ABA) or gibberellin (GA) is being investigated. Here we report an attempt to study the effect of pHi on hormone-induced gene expression. We used weak acids and weak bases to artificially mimic the pHi changes brought about by ABA and GA and found that chloramphenicol acetyltransferase (CAT) expression controlled by the Rab promoter was affected while the alpha-amylase promoter seemed insensitive. CAT fused to the 35S promoter was used as a control which is not inducible by ABA or GA3. The expression of this construct was not significantly affected by artificial pHi changes.

Abscisic Acid↗
PubMed1995

Products of cells from gliomas: IX. Evidence that two fundamentally different mechanisms change extracellular matrix expression by gliomas.

Four human astrocytic gliomas of high grade of malignancy were each evaluated in tissue and in vitro for percentages of cells expressing glial fibrillary acidic protein (GFAP), collagen type IV, laminin and fibronectin assessed by immunofluorescence with counterstaining of nuclear DNA. Percentages of cells with reticulin and cells binding fluorescein-labeled Ulex europaeus agglutinin were also assessed. In tissue, each extracellular matrix (ECM) component was associated with cells in the walls of abnormal proliferations of glioma vessels, and all four tumors had the same staining pattern. Two strikingly different patterns of conversion of gene product expression emerged during in vitro cultivation. (1). In the most common pattern, percentages of all six markers consistently shifted toward the exact phenotype of mesenchymal cells in abnormal vascular proliferations: increased reticulin, collagen type IV, laminin and fibronectin; markedly decreased glial marker GFAP and absent endothelial marker Ulex europaeus agglutinin. The simplest explanation of this constellation of changes coordinated toward expression of vascular ECM markers is that primary glioma cell cultures are overgrown by mesenchymal cells from the abnormal vascular proliferations of the original glioma. These cell cultures were tested for in situ hybridization (ISH) signals of chromosomes 7 and 10. Cells from one glioma had diploid signals. Cells from the other glioma had aneuploid signals indicating they were neoplastic; however, their signals reflected different numerical chromosomal aberrations than those common to neoplastic glia. (2). The second pattern was different. Cells with ISH chromosomal signals of neoplastic glia retained GFAP, and gained collagen type IV. Their laminin and fibronectin diminished, but persisted among a lower percentage of cells. Cloning and double immunofluorescence confirmed the presence of individual cells with glial and mesenchymal markers. A cell expressing GFAP in addition to either fibronectin, reticulin or collagen type IV is not a known constituent of glioblastoma tissue. This provides evidence of a second mechanism of conversion of gene expression in gliomas.

Astrocytoma↗
PubMed1995

Visualization of dipole solutions to the neuromagnetic inverse problem.

We have developed a computer program that computes and displays current-dipole solutions to the neuromagnetic inverse problem. The three-dimensional positions, orientations, and corresponding magnetic field values of the detection coils can be visualized with respect to a realistic representation of the subject's head shape. The topographies of the experimental data, theoretical fit, and residual field are represented without the use of data smoothing or interpolation, allowing more direct comparisons between them. A simple method for displaying dipole source location was implemented.

Brain Mapping↗
PubMed1995

Inhibition of secretion from isolated rat alveolar epithelial type II cells by the cell permeant calpain inhibitor II (N-acetyl-leucyl-leucyl-methioninal).

Although several signal transduction pathways, including activation of specific protein kinases have been proposed and studied for the secretory processes of lung surfactant from alveolar epithelial type II cells, the role of proteolytic processing by calpains (calcium-activated neutral proteases) in secretion has not been investigated. Therefore, we examined the effect of cell permeable calpain inhibitor I (N-acetyl-leucyl-leucyl-norleucinal) and II (N-acetyl-leucyl-leucyl-methioninal) on secretion to test the hypothesis that calpains participate in the secretory processes of alveolar epithelial type II cells. Calpain inhibitor I preferentially inhibits micro (mu)-calpain while inhibitor II inhibits milli (m)-calpain. Isolated type II cells were prelabelled with [3H]-choline for 18-24 h. To measure secretion, [3H]-labelled disaturated phosphatidylcholine (DSPC) released in the medium was monitored. Basal secretion of DSPC was maximally (87%) depressed by the presence of 10 microM inhibitor II. Secretagogue-stimulated secretion was also modulated by inhibitor II treatment. Stimulation with calcium ionophore A23187 enhanced secretion 3-fold. However, cells pre-exposed to inhibitor II displayed a 90% reduction of calcium-stimulated secretion. Terbutaline (10 microM) and ATP (1 mM) each increased secretion 2- and 4-fold, respectively. However, the inhibitor-treated cells, exposed to the same stimuli, attained only 53 or 62% of these increases. Calpain inhibitor I, on the other hand, inhibited neither basal nor stimulated secretion. The results suggest that m-calpain, the major isozyme of lung calpain requiring mM calcium for activity in vitro, is involved in the secretory pathways of alveolar epithelial type II cells.

Amino Acid Sequence↗
PubMed1995

DNA adducts induced by lipids and lipid peroxidation products: possible relationships to I-compounds.

A methanol-extractable lipid fraction of oats has been found previously to induce three specific I-compounds (age-dependent covalent DNA modifications) in female rat liver DNA, as detected by the 32P-postlabeling assay. The current report used an in vitro system to explore the possible mechanisms involved in the formation of these DNA derivatives. Ground oats or commercial oatmeal were extracted with methanol, and the extracts were incubated with rat lung DNA in vitro. DNA was recovered and analyzed by the nuclease P1-enhanced version of the 32P-postlabeling assay. A number of adducts were induced by the in vitro reaction but none of them was identical by chromatographic analysis to oats-specific I-compounds detected in vivo. Addition of rat liver microsomes and cofactors (NADPH or cumene hydroperoxide) to the in vitro reaction also failed to induce any of the oats-specific I-compounds. Pretreatment of oat lipids with soybean lipoxidase and oxygen enhanced formation of most adducts formed in vitro in a dose- and time-dependent manner. Several of these adducts were related to peroxide derivatives of linoleic acid. Chromatographic evidence suggests that one of the major adducts is derived from 4-hydroxynonenal, a reactive intermediate lipid peroxidation product. This adduct was detectable in liver and kidney DNA of untreated rats and its level increased with age. These results were in line with previous in vivo results, suggesting that the oats-specific I-compounds are presumably formed via an indirect mechanism rather than by direct binding of oats components to DNA.

Aging↗
PubMed1995

Problems and suggested solutions in creating an archive of clinical trials data to permit later meta-analysis: an example of methotrexate trials in rheumatoid arthritis.

Because data archives contain patient-based rather than study-based data, they can address meta-analytic questions on uncommon outcomes and on predefined patient subsets, questions that are difficult to address using the traditional meta-analytic approach based on grouped data. We report the tasks involved in establishing the first data archive of rheumatoid arthritis trials. In general, problems stem from the heterogeneity of trials in the archive and we suggest some solutions. In the initial phases, difficulties include recruitment and incomplete participation of trial investigators, whereas later on, other issues arise, such as quality control, coping with different dataset designs, and incomplete documentation. Other issues include heterogeneous measures, missing variables, and comparing data across different visit intervals and trial lengths. Suggested solutions include requesting trial data in predefined archive-wide structures and asking for all possible documentation for each dataset. Data cleaning is necessary, as is rescaling of variables or developing unit-free outcomes, and estimating data for missing variables. Archive design should allow for referencing a patient's data among various datasets. Although one goal is to reduce the quantity of data in the archive while retaining information content, data from early stages of archive building must be accessible for developing new analysis datasets. Documentation of archive building and software choices are discussed. Our experience suggests data archiving for meta-analysis is time consuming and expensive, yet it provides a useful method for analyzing data from multiple trials.

Archives↗
PubMed1995

Relationship between intact 1-84 parathyroid hormone and bone histomorphometric parameters in dialysis patients without aluminum toxicity.

With the markedly reduced usage of aluminum salts in renal failure, parathyroid hormone (PTH) has become the major determinant of currently seen bone disease. Clinicians now must consider what PTH level should be sought. Too low a level may lead to the aplastic bone lesion (low turnover bone), and too high a level may cause osteitis fibrosa. Furthermore, conventional normal PTH levels may not be a suitable target because of the well-known resistance to PTH in uremic patients. In this report, we derive the PTH levels that best distinguish patients with low and high bone formation states from those with normal bone formation in a group of 175 dialysis patients without aluminum toxicity. Using bone histological parameters, we propose that ideally PTH levels should be maintained between 10 pmol/L (100 pg/mL) and 20 to 30 pmol/L (200 to 300 pg/mL) in chronic dialysis patients, levels two to four times the upper limit of values found in normal subjects.

Adult↗
PubMed1995

Effects of nonsteroidal antiinflammatory drugs on oxidative pathways in A/J mice.

The tobacco-specific N-nitrosamine, NNK, is a potent carcinogen in laboratory animals. The authors have shown previously that NNK-induced lung tumorigenesis in A/J mice can be reduced significantly by certain nonsteroidal antiinflammatory drugs (NSAIDs), such as sulindac, ibuprofen, or piroxicam treatments. In this study, the authors investigated whether NSAIDs could reduce NNK-induced oxidative, DNA damage and/or inhibit endogenous lipid peroxidation, or prostaglandin E2 (PGE2) synthesis in A/J mice. In the first experiment, A/J mice were gavaged with NNK (112 mumol/kg b.w.) three times a week while being maintained on a diet to which either ibuprofen (263 mg/kg diet), naproxen (230 mg/kg), sulindac (123 mg/kg), piroxicam (25 mg/kg), indomethacin (5 mg/kg), or no NSAIDs had been added. Levels of 8-OH-dG in the DNA of lung and liver were measured by high-performance liquid chromatography with electron capture detector. Treatment with NSAIDs had no significant effects on the endogenous or NNK-induced formation of 8-OH-dG in the lung of the mice. In a second experiment, after treatment of A/J mice with NSAIDs for 2 weeks, lipid peroxidation was assayed by determining thiobarbituric acid-reactive substances (TBA-RS) in lung tissues, and prostaglandin E2 levels were measured in plasma by an enzyme immunoassay. Treatments with some NSAIDs lowered the levels of lipid peroxidation and plasma levels of PGE2 below basal levels. Taken together, these results suggest that the inhibition of NNK-induced lung tumorigenesis by NSAIDs is more likely related to an inhibition of prostaglandin synthesis than to a direct inhibition of lipid peroxidation or oxidative DNA damage induced by NNK.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1995

Inhibition of head and neck squamous cell carcinoma cell lines by transforming growth factor-beta.

Transforming growth factor-beta is known to be a potent autocrine growth inhibitor produced by a wide variety of cells, including cells of the immune system. Other investigators have noted that the growth of nontransformed keratinocytes is inhibited by transforming growth factor-beta, whereas various carcinoma cell lines are resistant to these effects. Head and neck squamous cell carcinoma cells are known to have surface receptors for this cytokine. We thus assessed the effect of transforming growth factor-beta on the growth of head and neck squamous cell carcinoma cell lines. Four head and neck squamous cell carcinoma cell lines were incubated with varying concentrations of transforming growth factor-beta, and cytotoxicity was evaluated with a methylene blue colorimetric assay. After culturing in transforming growth factor-beta for 4 days, inhibition of growth was detected in CAL-27 (maximal inhibition at 5.0 ng/ml), UMSCC-1, and UMSCC-19 (maximal inhibition at 50 ng/ml) cell lines. One other cell line, UMSCC-8 was found resistant to the inhibitory effects of transforming growth factor-beta. Kinetics analysis experiments revealed minimal inhibition before day 2 of incubation, at which time inhibition increased linearly to day 4. Assessment of double-stranded DNA fragmentation suggested that DNA fragmentation occurs before significant cytotoxicity. Electron microscopic analysis and gel electrophoresis of extracted DNA revealed morphologic features consistent with apoptotic cell death. Our findings indicate that transforming growth factor-beta significantly inhibits the growth of head and neck squamous cell carcinoma cell lines by inducing apoptotic cell death.

Carcinoma, Squamous Cell↗
PubMed1995

Reactions of alpha-acetoxy-N-nitrosopyrrolidine and alpha-acetoxy-N-nitrosopiperidine with deoxyguanosine: formation of N2-tetrahydrofuranyl and N2-tetrahydropyranyl adducts.

The goal of this study was to compare the reactions of alpha-acetoxy-N-nitrosopyrrolidine (alpha-acetoxyNPYR) and alpha-acetoxy-N-nitrosopiperidine (alpha-acetoxyNPIP) with deoxyguanosine (dG). alpha-AcetoxyNPYR and alpha-acetoxyNPIP are stable precursors to the alpha-hydroxynitrosamines which are formed metabolically from NPYR and NPIP. These alpha-hydroxynitrosamines are believed to be the proximate carcinogens of NPYR and NPIP. NPYR and NPIP, although structurally similar, have remarkably different carcinogenic properties, and a comparison of the reactions of their metabolically activated forms with dG and ultimately DNA could provide insights on their mechanisms of carcinogenicity. Reactions of alpha-acetoxyNPYR and alpha-acetoxyNPIP with dG were carried out at 37 degrees C and pH 7.0. The products were analyzed by HPLC and characterized by their spectral properties and by comparison to standards. In each reaction, one of the major products was a new type of dG adduct: N2-(tetrahydrofuran-2- yl)dG (THF-dG) from alpha-acetoxyNPYR and N2-(3,4,5,6-tetrahydro-2H-pyran-2-yl)dG (THP-dG) from alpha-acetoxyNPIP. THF-dG was synthesized independently by reaction of either 2-chlorotetrahydrofuran or 2,3-dihydrofuran with dG. Similarly, THP-dG was prepared by reaction of 2-chloro-3,4,5,6-tetrahydro-2H-pyran with dG. The structures of THF-dG and THP-dG were established by their UV and 1H-NMR spectra. THF-dG was less stable than THP-dG, but could be readily converted to a stable derivative, N2-(4-hydroxybutyl)dG, by reaction with NaBH4. THF-dG and THP-dG were converted to dG and 2-hydroxytetrahydrofuran or 2-hydroxy-3,4,5,6-tetrahydro-2H-pyran, respectively, upon neutral thermal or acid hydrolysis. This reaction was found to be reversible, with the adducts being produced in substantial amounts by reaction of 2-hydroxytetrahydrofuran or 2-hydroxy-3,4,5,6-tetrahydro-2H-pyran with dG. The latter reaction accounts for part of the THF-dG and THP-dG produced from the alpha-acetoxynitrosamines; stable oxonium ion-derived electrophiles may also be involved in the formation of THF-dG and THP-dG. Comparisons of the yields of various adducts in the reaction of alpha-acetoxyNPYR and alpha-acetoxyNPIP with dG showed some major differences. Whereas yields of THF-dG and THP-dG were similar, adducts formed from open chain diazonium ion or related intermediates were formed more extensively from alpha-acetoxyNPYR than from alpha-acetoxyNPIP. Adducts formed from enal products of the two nitrosamines were also different. Adduct formation as characterized in this study may account for some of the contrasting carcinogenic properties of NPYR and NPIP.

Carcinogens↗