Biomedical subjects

D Zhou

Publications and source records attributed to D Zhou.

At least 217 records · Page 12Linked to original sources

PubMed1997

[The reversion of multidrug resistance in tumour cell line MCF-7/Adr by ribozyme].

OBJECTIVE: To construct a specific hammerhead ribozyme possessing catalytic activity that cleaves the mdr1 mRNA for reversing the resistant phenotype in tumour cell line. METHODS: A DNA sequence encoding the ribozyme gene was incorporated into a eukaryotic expression vector (pH beta Apr-1 neu) and transfected into the human breast carcinoma cell line MCF-7/Adr, which is resistant to adriamycin and expresses the MDR phenotype. RESULTS: The ribozyme was stably expressed in the cell line, and decreased the level of mdr1 mRNA expression by 83.5%. The ribozyme inhibited the formation of P-glycoprotein and reduced the cell's resistance to adriamycin. CONCLUSION: The resistant cells were 1,000-fold more resistant than the parental cell line (MCF-7), whereas those cell clones that showed ribozyme expression were only 6-fold more resistant than the parental cell line.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Evaluation of immunoisolated insulin-secreting beta TC6-F7 cells as a bioartificial pancreas.

OBJECTIVE: To evaluate the growth and insulin secretion from microencapsulated beta TC6-F7 cells in vitro and to assess the in vivo function of microencapsulated cells transplanted in rats with steptozotocin (STZ)-induced diabetes. METHOD: Alginate-poly-L-lysine encapsulated beta TC6-F7 cells were exposed to glucose, isobutylmethylxanthine (IBMX) and glucagon-like peptide I (7-36 amide) in a static in vitro challenge. In vivo, 2.5-3.5 x 10(7) encapsulated cells were implanted into diabetic rats. Graft function was evaluated by monitoring blood glucose concentrations and by an intraperitoneal glucose tolerance test. RESULTS: The cell density (number of cells per capsule) of cultured microencapsulated beta TC6-F7 cells increased almost 35-fold over a 55 day observation period to reach a plateau of approximately 3500 cells/capsule. While insulin secretion per capsule remained unchanged over the first 21 days of culture, a 7-fold increase was observed during the last 14 days of the 55 day observation period. Intraperitoneal transplantation of 3.5 x 10(7) encapsulated cells into diabetic rats resulted, within 24 hours, in reversal of hyperglycemia for up to 60 days. Post-transplantation blood glucose concentrations varied between 2 and 4 mM. Glucose clearance rates evaluated by an intraperitoneal glucose tolerance test at 30 days post-transplantation resulted in a markedly flat glucose clearance curve with blood glucose never rising above 4 mM. The glucose challenge of microencapsulated cells recovered 30 days post-transplantation resulted in a 2-fold increase in insulin response at glucose concentrations greater than 5.5 mM as compared to glucose-free media. In addition, immunostaining of recovered grafted tissue for insulin, reveals a strong presence of the peptide within the cell population. CONCLUSIONS: These data demonstrate the potential use of an immunoisolated beta-cell line for the treatment of diabetes.

1-Methyl-3-isobutylxanthine↗

PubMed1996

Effect of all-trans-retinoic acid and phorbol 12-myristate 13-acetate on the activity of human hepatocellular carcinoma cell-surface beta-1,4-galactosyltransferase.

A human hepatocellular carcinoma cell line, SMMC-7721, was treated with all-trans-retinoic acid (RA) and phorbol 12-myristate 13-acetate (PMA) to induce its differentiation and proliferation respectively. A biantennary sugar chain fluorescently labelled with 2-aminopyridine (PA), GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc-PA, was used to detect the activity of beta-1,4-galactosyltransferase on the cell surface by HPLC. The results show that the activity of beta-1,4-galactosyltransferase on the cell surface increases when the cell is treated with RA, but decreases when it is treated with PMA, whereas the activities of the whole cell remain stable during the treatments.

Aminopyridines↗

PubMed1996

Stimulating properties of 5-oxo-eicosanoids for human monocytes: synergism with monocyte chemotactic protein-1 and -3.

The newly described products of 5-hydroxyeicosanoid dehydrogenase, 5-oxo-6,8,11,14-eicosatetraenoic acid (ETE) and 5-oxo-15(OH)ETE, induced directional migration and actin polymerization of human monocytes in vitro. At peak concentrations, the two eicosanoids had a chemotactic activity of about 40% of that observed in the presence of an optimal concentration of FMLP and twice the activity elicited by the related eicosanoid 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE). 15-Oxo-ETE showed a very low but detectable chemotactic activity. All of these chemotactic responses were blocked by Bordetella pertussis toxin, but were resistant to LY255283, a leukotriene B4 (LTB4) receptor antagonist. 5-Oxo-ETEs and 5-HETE induced homologous desensitization of chemotactic response, but did not cross-desensitize to other chemotactic agonists (e.g., monocyte chemotactic protein (MCP)-1 and LTB4). 5-Oxo-ETEs increased in a synergistic fashion the monocyte migration to MCP-1 and MCP-3. In the same range of concentrations, 5-oxo-ETE increased MCP-1-induced release of arachidonic acid from labeled monocytes. No synergistic interaction was observed when FMLP was used as chemoattractant. Thus, this study identifies monocytes as cells responsive to 5-oxo-ETEs and shows that monocyte activation by 5-oxo-ETEs occurs through an LTB4 receptor-independent mechanism that associates with pertussis toxin-sensitive G proteins. The synergistic interaction between 5-oxo-ETEs and C-C chemokines, two families of mediators both synthesized by phagocytic cells, may be relevant in vivo for the regulation of monocyte accumulation at sites of allergic and inflammatory reactions.

Actins↗

PubMed1996

Self-Assembly in a Mixture of Two Poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) Copolymers in Water

The self-assembly behavior in water of a mixture of two poly (ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) copolymers, (EO)13(PO)30(EO)13 (L64) and (EO)37(PO)58(EO)37 (P105), was explored at 25°C. The phase boundaries were established using 2H-NMR and inspection under polarized light; the structure of the various lyotropic liquid crystalline (LLC) phases was determined with small-angle X-ray scattering, while viscosity and differential scanning calorimetry measurements were used to probe the isotropic water-rich solution region. Isotropic regions, similar to the neat polymers, are stable at high polymer content. The addition of water induces structure in the amphiphilic block copolymer system. An extended lamellar (D) LLC phase is formed at 20-25% water content; a hexagonal (E) and a cubic (I) LLC phases supersede D at higher water contents. In addition to the above, a narrow isotropic region (L') is observed on the L64-water binary axis, in equilibrium with the E and the D phases. The hexagonal and lamellar LLC phases extended all the way from the L64-rich to the P105-rich side of the ternary L64-P105-water phase diagram; the characteristic hexagonal and lamellar structural dimensions varied linearly with P105 content in the L64-P105 mixture at a constant water concentration. An isotropic (micellar) solution phase (L1) dominates the high-water content corner of the ternary phase diagram. Viscosity measurements in this region provided evidence for increased interactions between the micelles as the boundary to the LLC phases was approached.

Journal Article↗

PubMed1996

Gamma radiation effect on vascular smooth muscle cells in culture.

PURPOSE: Prior work in our laboratory demonstrated that external gamma irradiation administered within 48 h following balloon catheter carotid artery injury in rats produced a marked inhibition of intimal hyperplasia and restenosis. The current study used smooth muscle cells (SMC) in vitro to examine the radiation dose response and to investigate the cellular mechanism by which radiation inhibits SMC proliferation. METHODS AND MATERIALS: Quiescent rat aortic SMC in plasma were refed with whole blood serum to stimulate synchronous proliferation and immediately irradiated with single fraction doses of 1.25-20 Gy. RESULTS: Comparison between a micronucleus assay and a clonogenic assay indicated a dose-dependent inhibition of SMC growth, with an ED50 at 2-3 Gy. The micronucleus assay also demonstrated a dose-dependent increase in apparent chromosomal damage at 72 h after irradiation. Inhibition of SMC growth by radiation did not correlate with changes in intracellular or released mitogenic activity. Furthermore, there was no evidence of apoptosis in irradiated SMC up to 96 h after treatment. CONCLUSION: Radiation likely inhibits SMC proliferation after arterial injury by a dose-dependent mechanism of lethal and/or sublethal cellular injury leading to clonogenic cell death.

Animals↗

PubMed1996

Normalization of diabetes in spontaneously diabetic cynomologus monkeys by xenografts of microencapsulated porcine islets without immunosuppression.

Porcine pancreatic islets were microencapsulated in alginate-polylysine-alginate capsules and transplanted intraperitoneally into nine spontaneously diabetic monkeys. After one, two, or three transplants of 3-7 x 10(4) islets per recipient, seven of the monkeys became insulin independent for periods ranging from 120 to 804 d with fasting blood glucose levels in the normoglycemic range. Glucose clearance rates in the transplant recipients were significantly higher than before the graft administration and the insulin secretion during glucose tolerance tests was significantly higher compared with pretransplant tests. Porcine C-peptide was detected in all transplant recipients throughout their period of normoglycemia while none was found before the graft administration. Hemoglobin A1C levels dropped significantly within 2 mo after transplantation. While ketones were detected in the urine of all recipients before the graft administration, all experimental animals became ketone free 2 wk after transplantation. Capsules recovered from two recipients 3 mo after the restoration of normoglycemia were found physically intact with enclosed islets clearly visible. The capsules were free of cellular overgrowth. Examination of internal organs of two of the animals involved in our transplantation studies for the duration of 2 yr revealed no untoward effect of the extended presence of the microcapsules.

Animals↗

PubMed1996

Beta-amyloid (25-35) peptide and IFN-gamma synergistically induce the production of the chemotactic cytokine MCP-1/JE in monocytes and microglial cells.

The molecular mechanisms involved in the development of senile plaques characteristic of aging and Alzheimer's disease are poorly understood. In this study, we examined whether human monocytes and murine microglial cells stimulated with the active fragment of amyloid beta-protein (Abeta(25-35)) express the monocyte chemotactic protein-1 (MCP-1)/JE. We show that upon incubation with Abeta(25-35), human monocytes accumulate MCP-1 mRNA and produce significant amounts of MCP-1. The effect of Abeta(25-35) on MCP-1 secretion was neither mimicked by a scrambled analogue nor affected by polymyxin B sulfate, even though the latter almost completely abolished the effect of LPS on MCP-1 expression. Murine microglial cells stimulated with Abeta(25-35) also expressed high levels of JE mRNA (the murine counterpart of MCP-1) and released bioactive chemotactic factors. In addition, we report that IFN-gamma significantly synergizes with Abeta(25-35) either in human monocytes or in murine microglial cells, and that Abeta(25-35) plus/minus IFN-gamma-mediated early induction of MCP-1 mRNA does not require new protein synthesis. Finally, we provide evidence that the Abeta(25-35)- and Abeta plus IFN-gamma-induced production of MCP-1 is, in large part, mediated in an autocrine fashion by endogenous TNF-alpha. Taken together, our findings uncover another novel biologic action of Abeta(25-35) and might help in better understanding the mechanisms underlying mononuclear phagocyte recruitment and activation into amyloid deposits.

Amyloid beta-Peptides↗

PubMed1996

Binding characteristics of seven inhibitors of human aromatase: a site-directed mutagenesis study.

Aromatase, a cytochrome P450, catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids. In this study, eight human aromatase mutants (I133Y, I133W, F235L, I395F, I474Y, I474W, I474M, and I474N) were prepared to evaluate the active site and a proposed hydrophobic pocket of the enzyme that exists in an aromatase model based on the X-ray structure of cytochrome P450cam. In addition, the binding characteristics of three steroidal inhibitors [4-hydroxyandrostenedione, 7alpha-(4'-amino)phenylthio-1,4-androstandiene-3,17-dione, and bridge (2,19-methyleneoxy)androstene-3,17-dione (MDL 101,003)] and four nonsteroidal inhibitors [aminoglutethimide, CGS 20267, ICI D1033, and vorozole (R83842)] were investigated through inhibitory profile studies on the eight new and three previously generated mutants (P308F, D309A, and T310S). The latter analyses have provided a molecular basis regarding how seven aromatase inhibitors with different structures bind to the active site of aromatase.

Amino Acid Sequence↗

PubMed1996

Identification of a promoter that controls aromatase expression in human breast cancer and adipose stromal cells.

Aromatase, a cytochrome P450, catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogens. In this study, the regulatory properties of a 696-base pair region, that contains the promoter II and is situated immediately upstream of exon II of the human aromatase gene, were investigated. Chloramphenicol acetyltransferase (CAT) functional studies with DNA segments derived from this genomic region and primer-extension analysis revealed the presence of a second promoter which is functional in adipose stromal cells and in breast cancer cells. Detailed DNase-1 footprinting analysis, DNA mobility shift assays, and CAT functional studies of this genomic region were performed and led to the identification of a segment (B1) that could act as a promoter (probably promoter I.3) in adipose stromal and breast cancer cells. The study revealed further that the B1 region could be divided into two domains which were designated RE1 and RE2. RE1 was found to have the promoter activity, and RE2 was found to regulate the promoter activity of RE1, but in different manners in MCF-7 cells (as an example of breast cancer cells) and in adipose stromal cells. RE2 was found to function as a positive regulatory element in MCF-7 cells and as a negative regulatory element in adipose stromal cells, respectively. DNA mobility shift and UV-cross-linking experiments with BrUrd-substituted B1 fragment and nuclear extracts isolated from two types of cells were performed. The experiments identified DNA-bound proteins with molecular masses around 50 kDa. These findings serve as the basis for further examination of the regulatory mechanism of aromatase expression in human breast cancer and adipose stromal cells.

Adenocarcinoma↗

PubMed1996

Plasmids with erythromycin resistance and catechol 2,3-dioxygenase- or beta-galactosidase-encoding gene cassettes for use in Neisseria spp.

Four new plasmids containing the ermC' (encoding a methyltransferase which confers resistance to erythromycin), xylE-ermC' (xylE, encoding catechol 2,3-dioxygenase) and lacZ-ermC' cassettes have been constructed. The 10-bp gonococcal uptake sequence has been placed downstream from ermC' to facilitate the delivery of these cassettes into pathogenic Neisseria spp. Several restriction sites have been placed to flank the cassettes to allow their excision and directional cloning. These plasmids will provide valuable tools for constructing insertional mutants and transcriptional fusions in Neisseria spp. or other bacteria.

Anti-Bacterial Agents↗

PubMed1996

Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1.

Treatment of human monocytes with vascular endothelial growth factor (VEGF) isolated from tumor cell supernatants was reported to induce monocyte activation and migration. In this study we show that recombinant human VEGF165, and VEGF121 had a maximal effect on human monocyte migration at 65 to 250 pmol/L. Chemotactic activity of VEGF165 was inhibited by a specific antiserum against VEGF, by heat treatment of VEGF165, and by protein kinase inhibitors. In addition, we could show that VEGF-stimulated monocyte migration is mediated by a pertussis toxin-sensitive GTP-binding protein. Placenta growth factor (PlGF152), a heparin-binding growth factor related to VEGF, was also chemotactic for monocytes at concentrations between 2.5 and 25 pmol/L. In accordance with these findings, human monocytes showed specific and saturable binding for 125I-VEGF165 (half-maximal binding at 1 to 1.5 nmol/L). Using Northern blot analysis, we further could show that human monocytes express only the gene for the VEGF receptor type, flt-1, but not for the second known VEGF receptor, KDR. Resting monocytes expressed low levels of flt-1 gene only. Brief exposure (2 to 4 hours) of human monocytes to lipopolysaccharide, a prototypic monocyte activator, led to a significant upregulation of the flt-1 mRNA level. The results presented here suggest that monocyte chemotaxis in response to VEGF and most likely to PlGF152 is mediated by flt-1 and thus show a possible function for the VEGF-receptor flt-1.

Binding, Competitive↗

PubMed1996

Normalization of diabetes by xenotransplantation of cryopreserved microencapsulated pancreatic islets. Application of a new strategy in islet banking.

To develop a requisite islet bank for the clinical implementation of an injectable bioartificial endocrine pancreas, microencapsulated islets were cryopreserved and assessed both in vitro by static glucose challenge and in a transplantation study. The insulin response of cryopreserved encapsulated rat islets was comparable with fresh islets. Transplantation of 800-900 banked rat islets resulted in the normalization of the metabolic blood glucose perturbation, body weight, and general health characteristics in 8 out of 8 diabetic mice for the study duration of 90 days. Whereas free islets are easily fragmented and lost during the freezing process, the capsule protects the fragile islets from freezing damage, increasing the retrieval rate from 79.5 +/- 9.8% to 97.2 +/- 1.3.

Animals↗

PubMed1996

IL-1 beta primes IL-8-activated human neutrophils for elastase release, phospholipase D activity, and calcium flux.

Interleukin-8 (IL-8), the prototype of the alpha (e.i., C-X-C branch) chemokine family, induced elastase release in a concentration-dependent manner (50-1000 ng/mL) in cytochalasin B-treated human polymorphonuclear leukocytes (PMNs). This response was potentiated about twofold if PMNs were preexposed to interleukin-1 beta (IL-1 beta) at concentrations that were by themselves inactive. The effect of IL-1 beta was clearly observed after 5 min and was maximal after a 30-min preincubation of the cells. The effect was present over the whole active concentration range of IL-8 and was completely blocked by the presence of IL-1 receptor antagonist. Priming of elastase release by IL-1 beta was not associated with a change in receptor number or affinity for IL-8. On the contrary, it was correlated with priming of phospholipase D activity and calcium flux activated by IL-8. Preincubation of the cells with ethanol and/or La3+ inhibited IL-8-induced degranulations, suggesting that activation of phospholipase D and increase of [Ca2+]i were important for this response. In contrast, ethanol and La3+ did not decrease the priming effect of IL-1 beta. IL-8 and IL-1 beta have been shown to be released by the same cell types and may be concomitantly present at sites of inflammation, giving rise to an amplification of the inflammatory response.

Calcium↗

PubMed1996

Migratory Response of Human NK Cells to Monocyte-Chemotactic Proteins

NK cells are present mostly in blood and spleen but under certain pathological and physiological conditions rapidly accumulate at extrahematic sites. The present study investigates the responsiveness of NK cells to C-C chemokines and the mechanisms of emigration from the bloodstream. MCP-1 induced migration across polycarbonate filters of IL-2-activated NK cells, whereas it was a weak attractant for unstimulated cells. The related chemokines MCP-2 and MCP-3 were also active. IL-2-activated NK cells showed specific binding sites for labeled MCP-1, and cell migration was inhibited by both cholera and Bordetella pertussis toxins. In agreement with functional assays the expression of mRNA specific for MCP-1 receptors was detectable only in IL-2-activated NK cells. The ability of NK cells to respond to MCP-1 and related chemokines may be one important determinant of NK cell emigration and recruitment in tissues.

Journal Article↗

PubMed1996

The mRNA for an ETR1 homologue in tomato is constitutively expressed in vegetative and reproductive tissues.

Dominant mutations in the Arabidopsis ETR1 gene block the ethylene signal transduction pathway. The ETR1 gene has been cloned and sequenced. Using the ETR1 cDNA as a probe, we identified a cDNA homologue (eTAE1) from tomato. eTAE1 contains an open reading frame encoding a polypeptide of 754 amino acid residues. The nucleic acid sequence for the coding sequence in eTAE1 is 74% identical to that for ETR1, and the deduced amino acid sequence is 81% identical and 90% similar. Genomic Southern blot analysis indicates that three or more ETR1 homologues exist in tomato. RNA blots show that eTAE1 mRNA is constitutively expressed in all the tissues examined, and its accumulation in leaf abscission zones was unaffected by ethylene, silver ions (an inhibitor of ethylene action) or auxin.

Abscisic Acid↗

PubMed1996

Water molecule binding and lifetimes on the DNA duplex d(CGCGAATTCGCG)2.

Measurements of the water proton spin-lattice relaxation rate for aqueous solutions of the palindromic dodecamer, d(CGCGAATTCGCG)2, are reported as a function of the magnetic field strength. The magnitude of the relaxation rates at low magnetic field strengths and the shape of the relaxation dispersion curve permit assessment of the number of water molecules which may be considered bound to the DNA for a time equal to or longer than the rotational correlation time of the duplex. The data are examined using limiting models that arbitrarily use the measured rotational correlation time of the polynucleotide complex as a reference point for the water molecule lifetime. If it is assumed that water molecules are bound at DNA sites for times as long as or longer than the rotational correlation time of the duplex, then the magnitude of the relaxation rates at low field require that there may be only two or three such water sites. However, if the lifetime constraint is relaxed, and we assume that the number of water molecules bound to the DNA is more nearly the number identified in the X-ray structures, then the average water molecule lifetime is on the order of 1 ns. Measurements of 1H NOESY spectra demonstrate that some water molecules must have lifetimes sufficiently long that negative Overhauser effects are observed. Taken together, these results suggest a distribution of water molecule lifetimes in which most of the DNA-bound water molecule lifetimes are shorter than the rotational correlation time of the duplex, but where some have lifetimes of at least 1 ns under these concentrated conditions.

Binding Sites↗

PubMed1996

Rat strain differences in the vulnerability of serotonergic nerve endings to neurotoxic damage by p-chloroamphetamine.

Substituted amphetamines are known to selectively destroy serotonin (5-HT) nerve endings in distant projection fields of the dorsal raphe nuclei and the systemic administration of these drugs is widely used in investigations of the role of the central 5-HT system and of the mechanisms involved in their toxicity. Until now Sprague-Dawley rats were almost exclusively used for this purpose and the findings were thought to apply to other strains as well. We compared the long-term effects of the administration of different doses of para-chloroamphetamine (PCA) on three specific markers of the density of 5-HT presynapses, [3H]-paroxetine binding to 5-HT-transporters, tryptophan hydroxylase apoenzyme contents, and 5-HT levels in the frontal cortex of Sprague-Dawley and Wistar rats. PCA-treatment caused a dose dependent decline of all three parameters which was much more pronounced in Sprague-Dawley compared to Wistar rats. An i.p. dose of 4 mg PCA/kg body weight, which caused a severe, about 90% reduction of all three parameters of 5-HT innervation in Sprague-Dawley rats was almost ineffective in Wistar rats. The dose of 8 mg/kg which was required to eliminate about 80% of cortical 5-HT presynapses in Wistar rats was already lethal to Sprague-Dawley rats. The reasons of this different susceptibility of the 5-HT system in the two rat strains are unknown. Their elucidation will contribute to a better understanding of inherited differences in individual vulnerability to the neurotoxic effects of substituted amphetamines. The combined measurements of transporter density, of tryptophan hydroxylase apoenzyme contents, and of 5-HT levels is a powerful tool for the assessment of experimentally induced changes in the density of 5-HT innervation in distant projection fields of the raphe nuclei.

Animals↗