Biomedical subjects

D Wen

Publications and source records attributed to D Wen.

79 records · Page 5Linked to original sources

PubMed1987

Characterization of fetal human brain cultures. Development of a potential model for selectively purifying human glial cells in culture.

Seven fetal human brain and three fetal human leptomeningeal cultures were characterized according to cell morphology, ultrastructural features, antigen expression, and collagen biosynthesis capabilities. Primary cultures derived from mechanically and enzymatically dissociated samples of fetal human brain consisted of a heterogeneous cell population in which astrocytes, oligodendrocytes, neurons, mesenchymal (leptomeningeal) cells, and macrophages were identified by light and electron microscopy. With progressive subcultivation, a homogeneous, leptomeningeal cell-derived population predominated. Fetal human brain and leptomeningeal specimens embedded in paraffin were analyzed immunohistochemically for the distribution of glial fibrillary acidic protein (GFAP), vimentin, factor-VIII-related antigen, fibronectin, laminin, type IV collagen, and procollagen III. Only GFAP and vimentin identified astrocytes and radial glia in the developing human brain; fibronectin, laminin, and the collagen types were immunolocalized largely to the leptomeninges and to the cerebral vasculature. The percentage of cells positively identified by antiserum to GFAP was greatest in primary cultures of fetal human brain; by the fourth passage, none of the fetal brain cultures were GFAP positive. The progressive decrease in the percentage of GFAP-positive cells was accompanied by an increase in the percentage of cells identified by collagen immunomarkers. Furthermore, in double immunolabeling experiments, antibodies to GFAP recognized a population of cells that was not identified by antibodies to laminin, fibronectin, type IV collagen, or procollagen III. SDS-PAGE and DEAE-cellulose chromatography of [3H]-proline-labeled early-passage fetal human brain cultures revealed collagen profiles identical to those obtained from direct cultures of the leptomeninges. The characteristics of later-passage fetal human brain cultures were identical in all respects to those of the fetal human leptomeningeal cultures. The proliferation of leptomeningeal cells could be inhibited by exposing the cells to cis-hydroxyproline (200 micrograms/ml). Primary fetal human brain cultures similarly treated with the proline analogue were found to be highly enriched for glial cells; these cultures were more than 90% GFAP positive. We conclude that primary fetal human brain cultures consist of a heterogeneous population of cells, most of which under the present culture conditions can be identified as glial cells. Subcultivation of human fetal brain cultures results in the overgrowth of mesenchymal cells, which are presumably derived from the leptomeninges.(ABSTRACT TRUNCATED AT 400 WORDS)

Brain↗

PubMed1987

Prolonged fructose feeding and aldose reductase inhibition: effect on the polyol pathway in kidneys of normal rats.

The effects of diets with differing carbohydrate composition on the kidney polyol pathway were investigated. The diets employed were F = fructose rich, G = glucose rich, S = cornstarch rich, and were fed for 30 days to six groups of 12 normal male Sprague-Dawley rats with and without addition of the aldose reductase inhibitor tolrestat (T). Fructose feeding resulted in higher kidney sorbitol levels (F = 0.847 +/- 0.152, G = 0.354 +/- 0.087, S = 0.207 +/- 0.041 microM/g wet wt, P less than 0.05). This was not observed in the tolrestat-treated animals (F + T = 0.182 +/- 0.024, G + T = 0.149 +/- 0.021, S + T = 0.152 +/- 0.020 microM/g wet wt). Aldose reductase activity was reduced with tolrestat administration (F = 0.0208 +/- 0.0023, F + T = 0.0048 +/- 0.0005; G = 0.0210 +/- 0.0002, G + T = 0.0059 +/- 0.0008; S = 0.0227 +/- 0.0022, S + T = 0.0062 +/- 0.0007 microU). Myoinositol levels did not differ among groups (F = 1.973 +/- 0.182, G = 2.291 +/- 0.307, S = 2.066 +/- 0.155 microM/g wet wt), but tended to increase with aldose reductase inhibition (F + T = 2.253 +/- 0.186, G + T = 2.713 +/- 0.166, S + T = 2.618 +/- 0.221 microM/g wet wt). Plasma glucose was higher in the fructose-fed rats (F = 10.78 +/- 0.55, G = 9.09 +/- 0.058, S = 9.03 +/- 0.52, F + T = 9.75 +/- 0.61, G + T = 8.42 +/- 0.64, S + T = 8.81 +/- 0.49 mM/liter). It is concluded that prolonged fructose feeding results in the accumulation of sorbitol in the kidney, caused by increased flux of glucose through the polyol pathway. This can be prevented by aldose reductase inhibition.

Aldehyde Reductase↗

PubMed1986

Expression of genes encoding vesicular stomatitis and Sindbis virus glycoproteins in yeast leads to formation of disulfide-linked oligomers.

Saccharomyces cerevisiae strains transformed with plasmids containing cDNAs coding for the glycoproteins of vesicular stomatitis or Sindbis viruses can be induced to produce large amounts of glycosylated virus glycoproteins. Studies reported here show that these proteins from high molecular weight disulfide-linked oligomers in the yeast endoplasmic reticulum. Oligomers were also found for two genetically altered forms of VSV G; one of these was lacking the membrane anchor domain and the other had the cysteine in the cytoplasmic tail replaced with serine. These oligomers can be separated from the bulk of yeast proteins by brief high-speed centrifugation of yeast extracts prepared by boiling cells with 1% sodium dodecyl sulfate. Treatment with thiol-reducing agents converts the oligomers to soluble monomeric forms, and this procedure leads to a substantial purification of glycoproteins from bulk yeast protein.

Disulfides↗

PubMed1984

Release of fatty acids from virus glycoproteins by hydroxylamine.

The fatty acids bound to the glycoproteins of Sindbis and vesicular stomatitis viruses can be released by treating the protein with 1 M hydroxylamine at pH 8.0, but the rates of release vary greatly among the three proteins. The most labile fatty acyl bonds were in the Sindbis virus PE2/E2 proteins and the most stable were in the E1 protein. Some of the fatty acids in Sindbis virus glycoproteins were reduced to the alcohol after treatment with sodium borohydride, indicating that protein-bound fatty acids could be in thiolester linkage. Sindbis virus PE2/E2 has several cysteine residues near the carboxy terminus, a region of the protein postulated to be localized on the inside (cytoplasmic face) of the bilayer, and protease digestion of microsomal membranes containing E2 protein removed a small portion of this cytoplasmic tail as well as significant amounts of the fatty acid. For the vesicular stomatitis virus G protein, the sensitivity of fatty acid hydrolysis appeared to depend on the conformation of the protein and a significant fraction of G protein was converted to a disulfide-linked dimer by hydroxylamine. These data implicate cysteinyl groups on these proteins as sites involved in fatty acid acylation.

Animals↗

PubMed1984

Mapping midbrain sites for circling using current-frequency trade off data.

Electrical stimulation applied near the medial longitudinal fasciculus (MLF) produces ipsiversive circling. In this experiment we examined the spatial and temporal properties of this substrate in rats by determining the current required to produce a constant amount of circling at frequencies from 15 to 500 Hz. In general, when frequency was high, the required current was low and vice versa, but there were deviations from a reciprocal relationship. Frequencies above 200 Hz failed to reduce the current needed to reach criterion. Electrodes placed distant from the MLF required more current, and deviated from reciprocity in a systematic way. A geometric model of the overlap between substrate and stimulation field is proposed that allows estimation of spatial properties of the two regions: (1) the location of the center of the substrate for circling (0.7 mm lateral, 7.0 mm below dura in the coronal plane 1.0 mm posterior to ear bars); (2) the radius of the substrate (0.77 mm); and (3) the radius of excitation at each current. Current-frequency trade off data can provide an in vivo estimate of the behavioral effectiveness of a given electrode, and hence its placement relative to a behavioral substrate.

Animals↗

PubMed1984

Fatty acid-acylated proteins in secretory mutants of Saccharomyces cerevisiae.

Yeast secretory (sec) mutants that are blocked in the transport of secretory proteins and accumulate membrane organelles were used to study the biosynthesis of fatty acid-acylated proteins. Four proteins were labeled with [3H]palmitate in sec mutants accumulating endoplasmic reticulum membranes. Three of these (molecular weights approximately equal to 20,000, 50,000, and 120,000) were N-linked glycoproteins, based on their ability to be labeled with [3H]mannose and their sensitivity to endoglycosidase H. The fourth protein (molecular weight approximately equal to 30,000) also was labeled with [3H]mannose but was insensitive to endoglycosidase H; it appeared to contain O-linked sugars. In sec mutants accumulating Golgi membranes or post-Golgi vesicles, a 35-kilodalton protein was labeled with [3H]palmitate. Analysis of Staphylococcus aureus protease V8 digests and pulse-chase experiments indicated that the 30-kilodalton protein was a precursor of 35 kilodaltons. None of these proteins was labeled with [3H]palmitate in a sec mutant that blocked the penetration of nascent polypeptides into endoplasmic reticulum; thus, acylation occurred in endoplasmic reticulum. All four proteins could be recovered from fractions enriched for yeast membranes. Fatty acids were not released from proteins by boiling in sodium dodecyl sulfate or extraction with organic solvents but were recovered as methyl esters after proteins were treated with KOH-methanol, a reaction characteristic of an acyl ester linkage.

Acylation↗

PubMedDate not supplied

Tissue expression of neu differentiation factor/heregulin and its receptor complex in prostate cancer and its biologic effects on prostate cancer cells in vitro.

BACKGROUND: Prostate cancer is the most common cancer in American men and the second leading cause of cancer death. All clinical observations correlate poorly differentiated high-grade prostate cancer with disease-specific mortality. The HER2 cell membrane tyrosine kinase, a member of the epidermal growth factor receptor family that is the transcription product of the erbB2neu oncogene, and HER3, a receptor protein of the same family, are overexpressed in prostate cancer and prostatic intraepithelial neoplasia. The ligand for these receptors and another related family member, HER4, has recently been identified by independent investigator groups and called neu differentiation factor (NDF) or heregulin. In vitro treatment of HER2- and HER3- or HER2- and HER4-expressing breast cancer cells stimulates tyrosine phosphorylation of HER2 and produces changes in the rate of proliferation, degree of cellular differentiation, and synthesis of physiologic secretion products. There are no published reports on the expression of NDF and HER4 in prostate cancer or the in vitro effects of NDF in prostate cancer cells. METHODS: Expression of NDF, HER2, HER3, and HER4 was studied in 24 frozen prostatectomy specimens by immunohistochemistry. The biologic effect of human recombinant NDF was studied in vitro, using the LNCaP, PC3, and DU145 human prostate cancer cell lines. HER and NDF protein expression was studied by immunohistochemistry. NDF mRNA was analyzed using reverse transcriptase polymerase chain reaction from whole RNA. The biologic effects of NDF on prostate cancer cells in vitro included cell proliferation, thymidine synthesis, induction of prostate-specific antigen mRNA, anchorage-dependent and anchorage-independent cell growth, and ploidy analysis. Data analysis was performed using Student's t test. RESULTS: Observations in clinical prostatectomy specimens: Immunohistochemistry studies in clinical prostatectomy specimens demonstrate absence of significant NDF expression in prostate cancer, whereas it is expressed in 100% of the stroma, 100% of basal epithelial cells, and 58% of luminal cells in normal and benign hyperplastic prostatic tissue. The HER4 receptor protein is strongly expressed by normal prostate luminal cells, but not prostate cancer. Benign prostate tissue exhibits strong expression of HER2, HER3, and HER4 by basal cells, but only luminal cells significantly express HER4. Only 23% of prostate cancer specimens express HER4, while 95% express HER3 and 82% HER2. Prostatic intraepithelial neoplasia stained similarly to cancer for all proteins studied. Observations in prostate cancer cell lines: In vitro treatment with NDF significantly reduces aneuploidy and proliferation and growth of androgen-sensitive prostate cancer cells. Incubation with NDF also induces prostate-specific antigen mRNA in prostate cancer cells. In spite of displaying NDF mRNA, prostate cancer cells do not produce detectable NDF protein, but express HER2 and HER3 proteins. DISCUSSION: These data suggest that NDF may be a paracrine differentiation factor involved in normal adult prostate physiology and that functional loss of the NDF/HER ligand/ receptor loop may be an early event associated with prostate tumorigenesis.

Antineoplastic Agents↗