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Biomedical subjects

D Sun

Publications and source records attributed to D Sun.

At least 145 records · Page 8Linked to original sources
PubMed1999

Preparation and Morphology Studies of Core-Shell Type Waterborne Polyacrylate-Polyurethane Microspheres.

Core-shell type waterborne polyacrylate-polyurethane microspheres in which polyurethane acts as the shell and acrylic polymer as the core were prepared. Hydroxyethyl acrylate and adipic dihydrazide were used as cross-linking agents introduced to the ends of the shell molecular chains, and two kinds of cross-linked structures between the core and the shell were formed. The photon correlation spectroscopy studies indicate that all the microspheres present continuous particle-size distributions and their mean particle sizes are smaller than 200 nm. The transmission electron photographs reveal that there are obvious core-shell structures for the noncross-linked type microsphere particles and different cross-linked networks between the core and shell for the two kinds of cross-linked type microsphere particles. These microspheres possess excellent storage stability, water resistance, and coating properties. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

Resistance to type 1 diabetes induction in 12-lipoxygenase knockout mice.

Leukocyte 12-lipoxygenase (12-LO) gene expression in pancreatic beta cells is upregulated by cytotoxic cytokines like IL-1beta. Recent studies have demonstrated that 12-LO inhibitors can prevent glutamate-induced neuronal cell death when intracellular glutathione stores are depleted. Therefore, 12-LO pathway inhibition may prevent beta-cell cytotoxicity. To evaluate the role of 12-LO gene expression in immune-mediated islet destruction, we used 12-LO knockout (12-LO KO) mice. Male homozygous 12-LO KO mice and control C57BL/6 mice received 5 consecutive daily injections of low-dose streptozotocin to induce immune-mediated diabetes. Fasting serum glucose and insulin levels were measured at 7-day intervals, and the mice were followed up for 28 days. 12-LO KO mice were highly resistant to diabetes development compared with control mice and had higher serum insulin levels on day 28. Isolated pancreatic islets were treated with IL-1beta, TNF-alpha, and IFN-gamma for 18 hours. Glucose-stimulated insulin secretion in cytokine-treated islets from C57/BL6 mice decreased 54% from that of untreated islets. In marked contrast, the same cytokine mix led to only a 26% decrease in islets from 12-LO KO mice. Furthermore, cytokine-induced 12-hydroxyeicosatetraenoic acid (12-HETE) production was absent in 12-LO KO islets but present in C57/BL6 islets. Isolated peritoneal macrophages were stimulated for 48 hours with IFN-gamma + LPS and compared for nitrate/nitrite generation. 12-LO KO macrophages generated 50% less nitrate/nitrite when compared with C57BL/6 macrophages. In summary, elimination of leukocyte 12-LO in mice ameliorates low dose streptozotocin-induced diabetes by increasing islet resistance to cytokines and decreasing macrophage production of nitric oxide.

Animals↗
PubMed1999

p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development.

The p63 gene, a homologue of the tumour-suppressor p53, is highly expressed in the basal or progenitor layers of many epithelial tissues. Here we report that mice homozygous for a disrupted p63 gene have major defects in their limb, craniofacial and epithelial development. p63 is expressed in the ectodermal surfaces of the limb buds, branchial arches and epidermal appendages, which are all sites of reciprocal signalling that direct morphogenetic patterning of the underlying mesoderm. The limb truncations are due to a failure to maintain the apical ectodermal ridge, a stratified epithelium, essential for limb development. The embryonic epidermis of p63-/- mice undergoes an unusual process of non-regenerative differentiation, culminating in a striking absence of all squamous epithelia and their derivatives, including mammary, lacrymal and salivary glands. Taken together, our results indicate that p63 is critical for maintaining the progenitor-cell populations that are necessary to sustain epithelial development and morphogenesis.

Animals↗
PubMed1999

Regulation of catalytic activity and processivity of human telomerase.

The ends of eukaryotic chromosomes are specialized sequences, called telomeres comprising tandem repeats of simple DNA sequences. Those sequences are essential for preventing aberrant recombination and protecting genomic DNA against exonucleolytic DNA degradation. Telomeres are maintained at a stable length by telomerase, an RNA-dependent DNA polymerase. Recently, human telomerase has been recognized as a unique diagnostic marker for human tumors and is potentially a highly selective target for antitumor drugs. In this study, we have examined the major factors affecting the catalytic activity and processivity of human telomerase. Specifically, both the catalytic activity and processivity of human telomerase were modulated by temperature, substrate (dNTP and primer) concentration, and the concentration of K+. The catalytic activity of telomerase increased as temperature (up to 37 degrees C), concentrations of dGTP, primer, and K+ were increased. However, the processivity of human telomerase decreased as temperature, primer concentration, and K+ were increased. Our results support the current model for human telomerase reaction and strengthen the hypothesis that a G-quadruplex structure of telomere DNA plays an important role in the regulation of the telomerase reaction.

Catalysis↗
PubMed1999

The intermediate filament protein peripherin is the specific interaction partner of mouse BPAG1-n (dystonin) in neurons.

The dystonia musculorum (dt) mouse suffers from severe degeneration of primary sensory neurons. The mutated gene product is named dystonin and is identical to the neuronal isoform of bullous pemphigoid antigen 1 (BPAG1-n). BPAG1-n contains an actin-binding domain at its NH2 terminus and a putative intermediate filament-binding domain at its COOH terminus. Because the degenerating sensory neurons of dt mice display abnormal accumulations of intermediate filaments in the axons, BPAG1-n has been postulated to organize the neuronal cytoskeleton by interacting with both the neurofilament triplet proteins (NFTPs) and microfilaments. In this paper we show by a variety of methods that the COOH-terminal tail domain of mouse BPAG1 interacts specifically with peripherin, but in contrast to a previous study (Yang, Y., J. Dowling, Q.C. Yu, P. Kouklis, D.W. Cleveland, and E. Fuchs. 1996. Cell. 86:655-665), mouse BPAG1 fails to associate with full-length NFTPs. The tail domains interfered with the association of the NFTPs with BPAG1. In dt mice, peripherin is present in axonal swellings of degenerating sensory neurons in the dorsal root ganglia and is downregulated even in other neural regions, which have no obvious signs of pathology. Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.

Animals↗
PubMed1999

Effects of cationic porphyrins as G-quadruplex interactive agents in human tumor cells.

A series of cationic porphyrins has been identified as G-quadruplex interactive agents (QIAs) that stabilize telomeric G-quadruplex DNA and thereby inhibit human telomerase; 50% inhibition of telomerase activity was achieved in HeLa cell-free extract at porphyrin concentrations in the range < or = 50 microM. Cytotoxicity of the porphyrins in vitro was assessed in normal human cells (fibroblast and breast) and human tumor cells representing models selected for high telomerase activity and short telomeres (breast carcinoma, prostate, and lymphoma). In general, the cytotoxicity (EC50, effective concentration for 50% inhibition of cell proliferation) against normal and tumor cells was > 50 microM. The porphyrins were readily absorbed into tumor cell nuclei in culture. Inhibition of telomerase activity in MCF7 cells by subcytotoxic concentrations of TMPyP4 showed time and concentration dependence at 1-100 microM TMPyP4 over 15 days in culture (10 population doubling times). The inhibition of telomerase activity was paralleled by a cell growth arrest in G2-M. These results suggest that relevant biological effects of porphyrins can be achieved at concentrations that do not have general cytotoxic effects on cells. Moreover, the data support the concept that a rational, structure-based approach is possible to design novel telomere-interactive agents with application to a selective and specific anticancer therapy.

Antineoplastic Agents↗
PubMed1999

Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene.

The promoter of the rat pgp2/mdr1b gene has a GC-rich region (pgp2GC) that is highly conserved in mdr genes and contains an consensus Sp1 site. Sp1's role in transactivation of the pgp2/mdr1b promoter was tested in Drosophila Schneider cells. The pgp2/mdr1b promoter was strongly activated by co-transfected wild type Sp1 but not mutant Sp1 and mutation of the Sp1 site abrogated Sp1-dependent transactivation. In gel shift assays, the same mutations abolished Sp1-DNA complex formation. Moreover, basal activity of the pgp2/mdr1b Sp1 mutant promoter was dramatically lower. Enforced ectopic overexpression of Sp1 in H35 rat hepatoma cells revealed that cell lines overexpressing Sp1 had increased endogenous pgp2/mdr1b mRNA, demonstrating that Sp1 activates the endogenous pgp2/mdr1b gene. Pgp2GC oligonucleotide also bound Egr-1 in gel shift assays and Egr-1 competitively displaced bound Sp1. In transient transfections of H35 cells (and human LS180 and HepG2 cells) Egr-1 potently and specifically suppressed pgp2/mdr1b promoter activity and mutations in the Egr-1 site decreased Egr-1 binding and correlated with pgp2/mdr1b up-regulation. Ectopic overexpression of Egr-1 in H35 cells decreased Pgp expression and selectively increased vinblastine sensitivity. In conclusion, Sp1 positively regulates while Egr-1 negatively regulates the rat pgp2/mdr1b gene. Moreover, competitive interactions between Sp1 and Egr-1 in all likelihood determine the constitutive expression of the pgp2/mdr1b gene in H35 cells.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

Crystallization and characterization of a fragment of pseudouridine synthase RluC from Escherichia coli.

RluC from E. coli is the enzyme responsible for catalyzing the isomerization of uridines 955, 2504 and 2580 in 23S rRNA to pseudouridine. Histidine-tagged RluC was cloned, overexpressed and purified by nickel-affinity chromatography. A proteolytically derived fragment of the enzyme consisting of residues 89-319 has been shown to retain catalytic activity. Crystals of this fragment, grown by precipitation with sodium acetate at pH 8.0, belong to space group P321, with unit-cell dimensions a = b = 97.1, c = 86.3 A and have two molecules in the crystallographic asymmetric unit. The flash-frozen crystals diffract X-rays to at least 2.3 A resolution and appear suitable for crystal structure determination.

Crystallization↗
PubMed1999

Regulatory T cells in experimental allergic encephalomyelitis. III. Comparison of disease resistance in Lewis and Fischer 344 rats.

Rats of the Fischer 344 (F344) strain are resistant to experimental allergic encephalomyelitis (EAE) induced by active immunization with guinea pig myelin basic protein (MBP) in complete Freund's adjuvant whereas Lewis (LEW) rats are susceptible even though both strains share the same I-A-like class II alleles of the MHC RT1.B locus. To determine factors that might contribute to this difference in disease susceptibility, we have compared in these two strains (1) the frequency of MBP-reactive T cells in the lymph nodes and spleens of MBP-immunized animals, (2) the dominant MBP epitopes recognized by responding T cells, (3) the ability of MBP-reactive T cells to enter the central nervous system (CNS), and (4) the frequency of CD8+ regulatory T cells (RTC) whose activity is functionally antagonistic to MBP-reactive T cells. The results indicate that MBP-reactive T cell numbers are similar in MBP-immunized F344 and LEW rats, they both recognize p68-88 as the dominant encephalitogenic epitope of MBP, and MBP-reactive T cells isolated from immunized rats and adoptively transferred to naive animals are similarly effective in penetrating the blood-brain barrier and entering the CNS, leading to pathogenesis in EAE. However, the frequency of RTC that functionally inhibit MBP-reactive T cells is greater in F344 rats.

Animals↗
PubMed1999

Allelic loss in human papillomavirus-positive and -negative vulvar squamous cell carcinomas.

Vulvar squamous cell carcinoma (VSCC) is a biologically and morphologically diverse disease, consisting of human papillomavirus (HPV)-positive and -negative tumors that differ in their morphological phenotypes and associated vulvar mucosal disorders. This study analyzed the frequencies of allelic loss (loss of heterozygosity (LOH)) in HPV-positive and -negative VSCCs to identify potential targets for the study of preinvasive diseases, to determine whether HPV status influenced patterns of LOH, and to determine whether these patterns differed from HPV-positive tumors of another genital site, cervical squamous cell carcinomas (CSCC). DNA extracted from microdissected archival sections of two index tumors, one each HPV negative and positive, was analyzed for LOH at 65 chromosomal loci. Loci scoring positive with either sample were included in an analysis of 14 additional cases that were also typed for HPV. Frequencies of LOH at loci were computed in a panel of HPV-positive and -negative VSCCs. Twenty-nine loci demonstrated LOH on the initial screen and were used to screen the remaining 14 tumors. High frequencies of LOH were identified, some of which were similar to a prior karyotypic study (3p, 5q, 8p, 10q, 15q, 18q, and 22q) and others of which had not previously been described in VSCC (1q, 2q, 8q, 10p, 11p, 11q, 17p, and 21q). With the exception of 5q and 10p, there were no significant associations between frequency of LOH and HPV status in VSCC. LOH at 3p and 11q were frequent in both VSCC and CSCC; however, allelic losses at several sites, including 5q, 8q, 17p, 21q, and 22q, were much more common in VSCC. VSCCs exhibit a broad range of allelic losses irrespective of HPV status, with high frequencies of LOH on certain chromosomal arms. This suggests that despite their differences in pathogenesis, both HPV-positive and -negative VSCCs share similarities in type and range of genetic losses during their evolution. Whether the different frequencies of LOH observed between VSCC and CSCC are real or reflect differences in stage and/or tumor size remains to be determined by further comparisons. The role of these altered genetic loci in the genesis of preinvasive vulvar mucosal lesions merits additional study.

Carcinoma, Squamous Cell↗
PubMed1999

Atypical immature metaplastic-like proliferations of the cervix: diagnostic reproducibility and viral (HPV) correlates.

Although some immature squamous lesions (papillary immature metaplasias) of the cervix have been described and associated with human papillomaviruses (HPV), nonpapillary atypical immature squamous proliferations (AISPs) are a poorly defined entity and range from atypical reactive metaplasias to squamous intraepithelial lesions resembling immature metaplasia. This study examined the diagnostic reproducibility of AISPs and their relationship to HPV nucleic acids. Forty-four diagnostically problematic AISPs were studied. Based on nuclear density (crowding), chromasia, variation (anisokaryosis) in nuclear size, and surface cytoplasmic maturation, cases were independently scored by 2 observers as (1) probably reactive (Rx), (2) not otherwise specified (NOS), and (3) squamous intraepithelial lesion (SIL). Extracted archival DNA was scored for HPV by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Interobserver reproducibility (kappa statistic) and HPV correlates (chi square) were computed. Approximately one third of cases were classified in each category by the observers. Interobserver reproducibility was excellent (0.80), poor (0.23), and fair to good (0.41) for a diagnosis of Rx, NOS, and SIL, respectively. Differences in HPV DNA positivity between Rx and SIL were significant for both observers (5.8% to 6.7% v 38.4% to 50.0%, respectively); however, differences between NOS and SIL (30.7% to 42.8% v 38.4% to 50.0%) were not, even when cases were limited to those in which both observers agreed (28.6% v 37.5%). By light microscopy, AISPs exceeding the threshold for presumed reactive changes (NOS or SIL) are a morphologically heterogeneous group that defy precise classification. Furthermore, their histopathologic appearance, even when there is diagnostic agreement, does not consistently correlate with their HPV status. The laboratory management of AISPs should take into account the uncertainty of this diagnosis.

Cell Division↗
PubMed1999

Regulation of guinea pig hepatic acyl-coa:cholesterol acyltransferase activity by dietary fat saturation and cholesterol.

We measured the interactive effects of dietary cholesterol and fat on the regulation of hepatic acyl-CoA:cholesterol acyltransferase (ACAT) activity and its relationship to hepatic microsomal lipid composition in guinea pigs fed 15 g/100 g (w/w) fat diets (corn oil, olive oil, or lard) with 0.01, 0.08, 0.17, or 0.33 g/100 g (w/w) added cholesterol. Guinea pigs exhibited a dose dependent increase in hepatic microsomal ACAT activity, with increasing levels of cholesterol intake (P < 0.001) in all dietary fat groups. Animals fed monounsaturated olive oil had the highest hepatic ACAT activity with the exception of the 0.33 g/100 g cholesterol diet (P < 0.001). There were no differences in ACAT activity with intake of polyunsaturated corn oil or saturated lard. Dietary cholesterol resulted in increased microsomal free cholesterol (FC) concentrations in a dose dependent manner but had no effects on microsomal phosphatidylcholine (PC) concentrations. Guinea pigs fed olive oil generally had the highest microsomal FC/PC molar ratios, and hepatic ACAT activities correlated significantly with this parameter. After modification of the lipid compositions of the microsomes from guinea pigs fed the 12 test diets with FC/PC liposome treatment, microsomal ACAT activities remained significantly related to the microsomal FC/PC molar ratios, and dietary fat type did not affect this correlation. Our findings do not support the hypothesis that the stimulation of hepatic ACAT activity with cholesterol intake is enhanced by polyunsaturated fat intake. The data demonstrate that although dietary fat type and cholesterol amount have differential effects on hepatic ACAT activity, substrate availability, expressed as microsomal FC/PC molar ratio, is a major regulator of hepatic microsomal ACAT activity.

Journal Article↗
PubMed1999

Kinetic compensation and the role of cations in pectinesterase catalysis.

The catalytic rate constant of thermostable pectinesterase (TS-PE) from Marsh grapefruit pulp was determined at pH 7 at temperatures between 25 and 60 degrees C. TS-PE activity was measured at NaCl concentrations of 0.05, 0.10, 0.15, and 0.20 M and at CaCl(2) concentrations of 0.005, 0.010, 0.015, and 0.020 M. For sodium- and calcium-added conditions, the kinetic functions of this reaction agreed with kinetic compensation relations. The isokinetic temperatures for sodium- and calcium-added conditions were 327.8 and 312.4 K, respectively. Different isokinetic temperatures and compensation parameters suggest that sodium and calcium uniquely affect TS-PE catalysis. This is the first demonstration of kinetic compensation in an enzyme-catalyzed reaction.

Calcium Chloride↗
PubMed1999

Grapefruit gland oil composition is affected by wax application, storage temperature, and storage time.

The effect of wax application, storage temperature (4 or 21 degrees C), and storage time (14 or 28 days after wax application) on grapefruit gland oil composition was examined by capillary gas chromatography. Wax application decreases nonanal and nootkatone levels. beta-Pinene, alpha-phellandrene, 3-carene, ocimene, octanol, trans-linalool oxide, and cis-p-mentha-2,8-dien-1-ol levels increase, but limonene levels decrease, with temperature. Levels of alpha-pinene, limonene, linalool, citronellal, alpha-terpineol, neral, dodecanal, and alpha-humulene decrease with time. Levels of alpha-phellandrene, 3-carene, ocimene, and trans-linalool oxide increase with time. No compound level was affected by the interactive action of temperature and wax application, suggesting that these two factors cause grapefruit oil gland collapse (postharvest pitting) through means other than changing gland oil composition. Compounds that are toxic to the Caribbean fruit fly (alpha-pinene, limonene, alpha-terpineol, and some aldehydes) decrease with time, thus suggesting grapefruit becomes increasingly susceptible to the fly during storage.

Citrus↗
PubMed1999

MRP4: A previously unidentified factor in resistance to nucleoside-based antiviral drugs.

Dideoxynucleosides, which are potent inhibitors of HIV reverse transcriptase and other viral DNA polymerases, are a common component of highly active anti-retroviral therapy (HAART) (ref. 1). Six reverse transcriptase inhibitors have been approved for human use: azidothymidine; 2'3'-dideoxycytidine; 2'3'-dideoxyinosine; 2', 3'-didehydro-3'deoxythymidine; 2',3'-dideoxy-3'-thiacytidine; and 4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-++ +metha nol. Although drug-resistant HIV strains resulting from genetic mutation have emerged in patients treated with HAART (ref. 1), some patients show signs of drug resistance in the absence of drug-resistant viruses. In our study of alternative or additional mechanisms of resistance operating during antiviral therapy, overexpression and amplification of the MRP4 gene correlated with ATP-dependent efflux of PMEA (9-(2-phosphonylmethoxyethyl)adenine) and azidothymidine monophosphate from cells and, thus, with resistance to these drugs. Overexpression of MRP4 mRNA and MRP4 protein severely impaired the antiviral efficacy of PMEA, azidothymidine and other nucleoside analogs. Increased resistance to PMEA and amplification of the MRP4 gene correlated with enhanced drug efflux; transfer of chromosome 13 containing the amplified MRP4 gene conferred resistance to PMEA. MRP4 is the first transporter, to our knowledge, directly linked to the efflux of nucleoside monophosphate analogs from mammalian cells.

Adenine↗
PubMed1999

A human breast cancer model for the study of telomerase inhibitors based on a new biotinylated-primer extension assay.

Telomerase is an RNA-dependent polymerase that synthesizes telomeric DNA (TTAGGG)n repeats. The overall goal of our work was to establish human cancer models that can be used to design clinical trials with telomerase inhibitors. The objectives of this study were (1) to set up a human breast cancer system that allows evaluation of the effects of telomerase inhibitors in cultured cells using a non-amplified telomerase assay and (2) to test this system using two drugs (cisplatin and TMPyP4) that affect the telomerase expression in breast cancer cells in culture. We first compared the telomerase activity in a variety of human breast cancer cell lines to that of other tumour types using a new biotinylated-primer extension assay. Our method, based on a non-amplified primer extension assay shows the direct incorporation of 32P-labelled nucleotides induced by telomerase on human telomeric primers. The 32P-dGTP labelled telomerase-extended 5'-biotinylated (TTAGGG)3 primer can subsequently be separated using streptavidin-coated magnetic beads. As compared to other non-amplified method, we showed that this procedure improved the characterization and the quantification of the banding pattern resulting from telomerase extension by reducing the radioactive background. Using this method, we observed that telomerase activity varies markedly in a panel of 39 human cancer cell lines. For example, MCF7 breast cancer cells in culture showed intermediate telomerase activity corresponding to 33.8+/-3.4% of that of the HeLa cells (reference cell line). Similarly, the telomere length varied with each cell line (average: 6.24+/-6.16). No correlation between the level of telomerase and telomere length was observed, suggesting that a high processivity is not required to maintain telomeres and that, in some cell lines, another mechanism of telomere elongation can maintain telomere length. From this study, we selected MCF7 and MX1 models that showed reproducible telomerase activity and a relatively limited telomere length for the testing of potential telomere-telomerase interacting agents. Using cisplatin and a new porphyrin-derived compound TMPyP4, we showed that our model was able to detect a down-regulation of the telomerase activity in MCF7 cells in culture and in a human MX1 tumour xenografts. Based on these results, a breast cancer model for evaluating telomerase and telomere interactive agents is proposed.

Animals↗