Biomedical subjects

D Chen

Publications and source records attributed to D Chen.

At least 181 records · Page 10Linked to original sources

PubMed2000

15-LOX-1: a novel molecular target of nonsteroidal anti-inflammatory drug-induced apoptosis in colorectal cancer cells.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) appear to act via induction of apoptosis-programmed cell death-as potential colorectal cancer chemopreventive agents. NSAIDs can alter the production of different metabolites of polyunsaturated fatty acids (linoleic and arachidonic acids) through effects on lipoxygenases (LOXs) and cyclooxygenases. 15-LOX-1 is the main enzyme for metabolizing colonic linoleic acid to 13-S-hydroxyoctadecadienoic acid (13-S-HODE), which induces apoptosis. In human colorectal cancers, the expression of this enzyme is reduced. NSAIDs can increase 15-LOX enzymatic activity in normal leukocytes, but their effects on 15-LOX in neoplastic cells have been unknown. We tested the hypothesis that NSAIDs induce apoptosis in colorectal cancer cells by increasing the protein expression and enzymatic activity of 15-LOX-1. METHODS: We assessed 15-LOX-1 protein expression and enzymatic activity, 13-S-HODE levels, and 15-LOX-1 inhibition in association with cellular growth inhibition and apoptosis induced by NSAIDs (primarily sulindac and NS-398) in two colorectal cancer cell lines (RKO and HT-29). All P values are two-sided. RESULTS: Sulindac and NS-398 progressively increased 15-LOX-1 protein expression in RKO cells (at 24, 48, and 72 hours) in association with subsequent growth inhibition and apoptosis. Increased 13-S-HODE levels and the formation of 15-hydroxyeicosatetraenoic acid on incubation of the cells with the substrate arachidonic acid confirmed the enzymatic activity of 15-LOX-1. Inhibition of 15-LOX-1 in RKO cells by treatment with caffeic acid blocked NS-398-induced 13-S-HODE production, cellular growth inhibition, and apoptosis (P =. 007, P<.0001, and P<.0001, respectively); growth inhibition and apoptosis were restored by adding exogenous 13-S-HODE (P<.0001 for each) but not its parent compound, linoleic acid (P = 1.0 for each). Similar results occurred with other NSAIDs and in HT-29 cells. CONCLUSIONS: These data identify 15-LOX-1 as a novel molecular target of NSAIDs for inducing apoptosis in colorectal carcinogenesis.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2000

Detection of DNA base-excision repair activity for oxidative lesions in adult rat brain mitochondria.

Endogenous oxidative damage to brain mitochondrial DNA and consequential disturbances of gene expression and mitochondrial dysfunction have long been implicated in aging and the pathogenesis of neurodegenerative diseases. It has yet to be determined, however, whether mitochondria in brain cells contain an active DNA repair system and, if so, how this system functions. Therefore, the capacity for the repair of defined types of oxidative DNA lesions has been investigated in adult rat brain mitochondria. Using in vitro DNA incorporation repair assay, we have detected base excision repair (BER) activity for the common oxidative DNA adduct 8-hydroxyl-2'-deoxyguanine (8-oxodG) in mitochondria protein extracts from cortical tissues and cultured primary cortical neurons and astrocytes. The levels of BER activity were both protein concentration-dependent and repair-incubation time-dependent. To resolve the BER pathway, the activity of essential BER enzymes was examined in mitochondria using oligonucleotide incision assay, DNA polymerase assay, and DNA ligase assay employing specific DNA substrates. Mitochondrial extracts were able to remove specifically 8-oxodG, uracil, and the apurinic/apyrimidinic abasic site from substrates. Moreover, a gamma-like DNA polymerase activity and a DNA ligase activity were detected in mitochondiral extracts, based on the formation of specific repair products. These results demonstrate that adult brain mitochondria possess an active BER system for repairing oxidative DNA lesions. This repair system appears to function by sequential actions of DNA repair enzymes that are homologous to, but not identical to, that in the nucleus. Thus, BER may represent an endogenous protective mechanism against oxidative damage to mitochondrial, as well as nuclear, genomes in brain cells.

Animals↗

PubMed2000

alpha-fluoromethylhistidine depletes histamine from secreting but not from non-secreting rat stomach ECL cells.

Histamine in the oxyntic mucosa of the rat stomach occurs in mast cells (10%) and ECL cells (90%). Unlike the mast cells, the ECL cells operate under the control of gastrin. alpha-Fluoromethylhistidine, an irreversible inhibitor of the histamine-forming enzyme, histidine decarboxylase depletes ECL-cell but not mast-cell histamine. This report shows that the effectiveness by which histidine decarboxylase inhibition depletes ECL-cell histamine depends on the rate of histamine secretion. Rats received alpha-fluoromethylhistidine by continuous subcutaneous infusion for 24 h. Maximally effective doses (>/=3 mg/kg/h) inhibited histidine decarboxylase and reduced oxyntic mucosal histamine in fed rats by 80-90%. In fasted rats, the reduction was 50%. alpha-Fluoromethylhistidine greatly reduced the number of histamine-immunoreactive ECL cells (immunocytochemistry) and of secretory vesicles in the ECL cells (electron microscopy) in fed but not in fasted rats. The half-life of oxyntic mucosal histamine (determined upon histidine decarboxylase inhibition) was 2.6 h in fed rats and 19.4 h in fasted rats. The amount of histamine secreted in response to gastrin (monitored by gastric submucosal microdialysis) was greatly reduced by alpha-fluoromethylhistidine in fed rats but not in fasted rats. ECL cells were isolated from rat stomach by elutriation (80% purity). Their histamine content was determined after culture, with or without alpha-fluoromethylhistidine, in the presence of varying concentrations of gastrin. In a medium containing 10 nM gastrin, ECL cells responded to a maximally effective concentration of alpha-fluoromethylhistidine (0.1 nM) with 80% reduction in histamine content. In the absence of gastrin, ECL cells responded to alpha-fluoromethylhistidine with 45% reduction of histamine; the releasable histamine pool was unaffected. In conclusion, the combination of histidine decarboxylase inhibition and a high rate of histamine secretion will promptly exhaust the ECL-cell histamine pool, while histidine decarboxylase inhibition and a low secretion rate will affect the histamine pool much less.

Animals↗

PubMed2000

Expression of a chimeric farnesyl diphosphate synthase gene in Artemisia annua L. transgenic plants via Agrobacterium tumefaciens-mediated transformation.

An Agrobacterium tumefaciens-mediated transformation system was developed for Artemisia annua L. Using this system a cDNA encoding farnesyl diphosphate synthase (FDS placed under a CaMV 35S promoter) was transferred into A. annua via A. tumefaciens strain LB4404. Leaf or leaf discs were used as explants to be infected with A. tumefaciens and an optimal concentration of 20 mg/l kanamycin was applied to select kanamycin resistant shoots. Forty-five lines of resistance kanamycin shoots transformed with FDS were established. Analysis of PCR showed that at least 20 shoots transformed with the FDS gene were PCR positive. Southern blot analysis suggested the foreign FDS gene had been integrated into the A. annua genome, and Northern blot analysis revealed that the foreign FDS gene expressed at the transcriptional level in five shoot lines (F-1, F-4, F-61, F-62 and F-73 shoot lines). Analysis of artemisinin demonstrated that about 8 approximately 10 mg/g DW of artemisinin were then detected in transgenic plants regenerated from five shoot lines, this is about 2-3 times higher than that in the control.

Journal Article↗

PubMed2000

A novel lysine 2,3-aminomutase encoded by the yodO gene of bacillus subtilis: characterization and the observation of organic radical intermediates.

The yodO gene product of Bacillus subtilis has been cloned and overexpressed in Escherichia coli and purified. The nucleotide sequence encodes a protein of 471 amino acids with a calculated molecular mass of 54071 Da. The translated amino acid sequence is more than 60% identical to that of the lysine 2,3-aminomutase from Clostridium subterminale SB4. Analytical HPLC gel-permeation chromatography leads to an estimate of an over all molecular mass of 224000+/-21000 Da, which corresponds to a tetrameric protein. The purified protein contains iron, sulphide and pyridoxal 5'-phosphate (PLP) and displays an optical absorption band extending to 700 nm, suggesting the presence of an iron-sulphide cluster. After reductive incubation with L-cysteine anaerobically, the protein catalyses the transformation of L-lysine into beta-lysine in the presence of S-adenosylmethionine (AdoMet) and sodium dithionite. The K(m) value for L-lysine is estimated to be 8.0+/-2.2 mM. The iron-sulphur centre is stable in air,allowing aerobic purification. EPR spectroscopy at 10 K of the purified enzyme revealed an EPR signal similar to that of the [4Fe-4S](3+) cluster observed in the clostridial lysine 2, 3-aminomutase. Incubation with cysteine under anaerobic conditions converts the iron-sulphur centre into the EPR-silent [4Fe-4S](2+). Unlike the clostridial enzyme, the fully reduced [4Fe-4S](+) could not be characterized by further reduction with dithionite in the presence of AdoMet, although both dithionite and AdoMet were required to activate the enzyme. Upon addition of L-lysine, dithionite and AdoMet to the reduced enzyme and freezing the solution to 77 K, the EPR spectrum revealed the presence of an organic free-radical signal (g=2.0023), which displayed multiple hyperfine transitions very similar to the spectrum of the beta-lysine-related radical in the mechanism of the clostridial lysine 2,3-aminomutase. Experiments with isotopically substituted L-lysine and lysine analogues verified the association of spin density with the carbon skeleton of lysine. The data indicate that the protein encoded by the yodO gene of B. subtilis is a novel lysine 2,3-aminomutase. The E. coli homologue of clostridial lysine 2,3-aminomutase was also expressed in E. coli and purified. This protein contained ironand sulphide but not PLP, it did not display lysine 2,3-aminomutase activity, and addition of PLP did not induce 2,3-aminomutase activity.

Amino Acid Sequence↗

PubMed2000

MIR16, a putative membrane glycerophosphodiester phosphodiesterase, interacts with RGS16.

We have identified the protein MIR16 (for Membrane Interacting protein of RGS16) from a yeast two-hybrid screen by using RGS16 as bait. MIR16 shares strong homology with bacterial glycerophosphodiester phosphodiesterases. It interacts with RGS16 and, more weakly, with several other selected RGS proteins. Analysis of deletion mutants showed that the N-terminal region of the RGS domain in RGS16 is required for its interaction with MIR16. MIR16 is an integral membrane glycoprotein, because it remained associated with membrane fractions after alkaline treatment and because, in some cells, it is sensitive to digestion with endoglycosidase H. By immunofluorescence and immunoelectron microscopy, MIR16 was localized on the plasma membrane in liver and kidney and on intracellular membranes in rat pituitary and cultured pituitary cells. MIR16 represents the only integral membrane protein identified thus far to interact with an RGS domain and, to our knowledge, is the only mammalian glycerophosphodiester phosphodiesterase that has been cloned. The putative enzymatic activity of MIR16 and its interaction with RGS16 suggest that it may play important roles in lipid metabolism and in G protein signaling.

Amino Acid Sequence↗

PubMed2000

Effects of soaking temperature and soaking time during preparation of water extract of tea on anticlastogenicity against environmental tobacco smoke in the sister-chromatid exchange assay.

Water extract of tea (WET) was prepared by soaking green tea at different temperatures for various periods of time and was used to test whether the soaking temperature and soaking time during the preparation of WET influence the content of polyphenols and the anticlastogenicity of WET against environmental tobacco smoke (ETS). Five major polyphenols in WET were measured. Extractable-respirable particulate (ERP) was obtained from ETS-contaminated indoor air (ERP-ETS). The sister-chromatid exchange assay (SCE) was utilized to evaluate the clastogenic effects of ERP-ETS and the anticlastogenic effects of WET. The results indicate that ERP-ETS is clastogenic and WET has significant anticlastogenic effects on ERP-ETS. The content of polyphenols and the anticlastogenic potential of WET depended on the soaking temperature and soaking time during WET preparation. At the soaking temperature of 80 degrees C, an increased soaking time was correlated with a higher percentage of polyphenols and a concomitantly enhanced anticlastogenic efficacy. By contrast, at the soaking temperature of 100 degrees C, a longer soaking time was associated with a higher percentage of polyphenols concomitant with a lower anticlastogenic efficacy. The data suggest that, besides polyphenols, and additional material(s), which may be partially inactivated at 100 degrees C, is contributing to the anticlastogenicity of WET.

Antimutagenic Agents↗

PubMed2000

Calculated equation of state of al, cu, ta, mo, and W to 1000 GPa

Calculated Hugoniots and 293-K isotherms at pressures up to 1 TPa (10 Mbar) for the five reference metals Al, Cu, Ta, Mo, and W are reported using the classical mean-field approach where both the cold and the thermal parts of the Helmholtz free energy are derived entirely from the 0-K total energies and electronic density of states calculated with the full-potential linearized augmented plane wave method within the generalized gradient approximation to exchange correlational functional. Our approach permits efficient computation and invokes no empirical parameters. Both the experimental Hugoniots and 293-K isotherms are reproduced excellently.

Journal Article↗

PubMed2000

Roles of topoisomerases in maintaining steady-state DNA supercoiling in Escherichia coli.

DNA supercoiling is essential for bacterial cell survival. We demonstrated that DNA topoisomerase IV, acting in concert with topoisomerase I and gyrase, makes an important contribution to the steady-state level of supercoiling in Escherichia coli. Following inhibition of gyrase, topoisomerase IV alone relaxed plasmid DNA to a final supercoiling density (sigma) of -0.015 at an initial rate of 0.8 links min(-1). Topoisomerase I relaxed DNA at a faster rate, 5 links min(-1), but only to a sigma of -0.05. Inhibition of topoisomerase IV in wild-type cells increased supercoiling to approximately the same level as in a mutant lacking topoisomerase I activity (to sigma = -0.08). The role of topoisomerase IV was revealed by two functional assays. Removal of both topoisomerase I and topoisomerase IV caused the DNA to become hyper-negatively supercoiled (sigma = -0.09), greatly stimulating transcription from the supercoiling sensitive leu-500 promoter and increasing the number of supercoils trapped by lambda integrase site-specific recombination.

DNA Topoisomerase IV↗

PubMed2000

Activation of antitumor cytotoxic T lymphocytes by fusions of human dendritic cells and breast carcinoma cells.

We have reported that fusions of murine dendritic cells (DCs) and murine carcinoma cells reverse unresponsiveness to tumor-associated antigens and induce the rejection of established metastases. In the present study, fusions were generated with primary human breast carcinoma cells and autologous DCs. Fusion cells coexpressed tumor-associated antigens and DC-derived costimulatory molecules. The fusion cells also retained the functional potency of DCs and stimulated autologous T cell proliferation. Significantly, the results show that autologous T cells are primed by the fusion cells to induce MHC class I-dependent lysis of autologous breast tumor cells. These findings demonstrate that fusions of human breast cancer cells and DCs activate T cell responses against autologous tumors.

Breast Neoplasms↗

PubMed2000

Molecular mechanisms of platelet exocytosis: role of SNAP-23 and syntaxin 2 in dense core granule release.

To characterize the molecular mechanisms of platelet secretion, we focused on the calcium-induced exocytosis of dense core granules. Platelets contain several known t-SNAREs (soluble N-ethylmaleimide sensitive factor [NSF] attachment protein receptors) such as syntaxins 2, 4, and 7 and SNAP-23 (synaptosomal associated protein 23). By using an in vitro exocytosis assay, we have been able to assign roles for some of these t-SNAREs in dense core granule release. This calcium-induced secretion relies on the SNARE proteins because it is stimulated by the addition of recombinant alpha-SNAP and inhibited by a dominant negative alpha-SNAP-L294A mutant or by anti-alpha-SNAP and anti-NSF antibodies. SNAP-23 antibodies and an inhibitory C-terminal SNAP-23 peptide both blocked dense core granule release, demonstrating a role for SNAP-23. Unlike other cell types, platelets contain a significant pool of soluble SNAP-23, which does not partition into Triton X-114. Of the anti-syntaxin antibodies tested, only anti-syntaxin 2 antibody inhibited dense core granule release. Immunoprecipitation studies showed that the 2 t-SNAREs syntaxin 2 and SNAP-23 do form a complex in vivo. These data clearly show that SNAPs, NSF, and specific t-SNAREs are used for dense core granule release; these data provide a greater understanding of regulated exocytosis in platelets.

Antigens, Surface↗

PubMed2000

Molecular mechanisms of platelet exocytosis: requirements for alpha-granule release.

Platelets function by secreting components necessary for primary clot formation. This report describes an in vitro assay that measures alpha-granule secretion. Using permeabilized platelets, it is possible to recreate Ca(2+)-stimulated release of platelet factor 4 (PF4) that is ATP- and temperature-dependent. Though other divalent cations can replace Ca(2+) (i.e., Sr(2+), Mn(2+), Zn(2+)), there is no effect of Ba(2+). Analysis by electron microscopy indicates that the in vitro assay also mimics the cytoskeletal rearrangements and granule centralization that occurs upon platelet activation in vivo. Antibody inhibition studies show that PF4 release requires the general membrane fusion protein N-ethylmaleimide-sensitive factor (NSF) and well as the target membrane SNAP receptors (t-SNAREs), syntaxin 2, 4, and SNAP-23. As shown by electron microscopy, the anti-t-SNARE antibodies block granule to target membrane fusion. This finding is unique in that it is the first report of a role for two syntaxins in the same exocytosis event.

Adenosine Triphosphate↗

PubMed2000

Requirement for a kinase-specific chaperone pathway in the production of a Cdk9/cyclin T1 heterodimer responsible for P-TEFb-mediated tat stimulation of HIV-1 transcription.

Tat activation of HIV-1 transcription is mediated by human transcription elongation factor P-TEFb, which interacts with Tat and phosphorylates the C-terminal domain of RNA polymerase II. The catalytic subunit of the P-TEFb complex, Cdk9, has been shown to interact with cyclin T and several other proteins of unknown identity. Consequently, the exact subunit composition of active P-TEFb has not been determined. Here we report the affinity purification and identification of the Cdk9-associated proteins. In addition to forming a heterodimer with cyclin T1, Cdk9 interacted with the molecular chaperone Hsp70 or a kinase-specific chaperone complex, Hsp90/Cdc37, to form two separate chaperone-Cdk9 complexes. Although the Cdk9/cyclin T1 dimer was exceptionally stable and produced slowly in the cell, free and unprotected Cdk9 appeared to be degraded rapidly. Several lines of evidence indicate the heterodimer of Cdk9/cyclin T1 to be the mature, active form of P-TEFb responsible for phosphorylation of the C-terminal domain of RNA polymerase II interaction with the Tat activation domain, and mediation of Tat activation of HIV-1 transcription. Pharmacological inactivation of Hsp90/Cdc37 function by geldanamycin revealed an essential role for the chaperone-Cdk9 complexes in generation of Cdk9/cyclin T1. Our data suggest a previously unrecognized chaperone-dependent pathway involving the sequential actions of Hsp70 and Hsp90/Cdc37 in the stabilization/folding of Cdk9 as well as the assembly of an active Cdk9/cyclin T1 complex responsible for P-TEFb-mediated Tat transactivation.

Benzoquinones↗

PubMed2000

The 2(3)Delta(g) State of (7)Li(2).

Using pulsed perturbation-facilitated optical-optical double resonance (PFOODR) spectroscopy, the 2(3)Delta(g) state of (7)Li(2) (electronic configuration (varsigma(g)2s) (4ddelta(g)), effective principal quantum number n* = 4.101) has been observed and assigned. Molecular constants and a RKR potential energy curve were obtained. The major molecular constants are Copyright 2000 Academic Press.

Journal Article↗

PubMed2000

The relationship between apoptosis and the expression of proliferating cell nuclear antigen and the clinical stages in gastric carcinoma.

The relationship between the apoptosis and the expression of proliferating cell nuclear antigen (PCNA) and the clinical stages in gastric cancers was studied. By using terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique and PCNA immunohistochemical staining, the apoptosis and the expression of PCNA in tissue of gastric carcinoma were assayed in situ, the index of apoptosis (AI), index of PCNA (PI) and the rate of AI/PI were calculated. AI and PI in gastric cancer tissues were (6.5 +/- 3.7)% and (49.8 +/- 15.9)% respectively, and the rate of AI/PI was 0.13 +/- 0.05, which were obviously different from those of normal gastric mucosa in paragastric cancer (P < 0.01). With the advanced TNM stages of gastric carcinoma, the AI was decreased, PI was increased and the rate of AI/PI decreased in gastric carcinoma. There was significant difference in them between the gastric cancer tissues and normal gastric mucosa in pericarcinoma in TNM stage II to IV (P < 0.05). It was suggested that the decreased apoptotic cells and the increased proliferating cells were obviously related to the tumor genesis and tumor progression in gastric carcinoma. The AI, PI and the rate of AI/PI would become the prognostic factors in advanced gastric carcinoma.

Adenocarcinoma↗

PubMed2000

Study on angiogenesis factor of human osteosarcoma.

Angiogenesis factor of human osteosarcoma was partially purified and its biological features were studied. The active peptide with 8000 to 10,000 u molecular weight in the conditioned medium obtained from the cultivation of human osteosarcoma cells were partially purified by ultrafiltration, chromatography and dialysis. The angiogenic effects of the fractions were assessed by proliferation assay of human umbilical vein and pig aorta thoracic endothelial cells. The results showed that the chromatography fractions of 4 to 6 could significantly promote the proliferation of the endothelial cells. It was suggested that the human osteosarcoma cells could synthesize and secrete angiogenesis factor with a molecular weight of 8000 to 10,000 u.

Angiogenesis Inducing Agents↗

PubMed2000

Effect of vagotomy on acute pancreatitis in rats.

One hundred and eighty-two SD rats were randomly divided into the normal control group, fast operating group and food-intake operating group. The experimental model of acute pancreatitis (AP) in rats was established by injecting 5% sodium taurocholate into the pancreatic duct of rat according to Aho's method. The sandostatin was used for positive contrast. The concentrations of serum amylase, calcium, C reaction protein (CRP) and interleukin-6 (IL-6) were assayed respectively at different time points. The pathological sections were observed. Each operating group contained 10 rats. The mortality of the operating groups was observed during the 24 h. The serum amylase level in the AP rats was reduced after receiving vagotomy (VG, P < 0.05). Although the serum calcium level in most groups was decreased, the reduction in the group with VG plus sandostatin was not obvious (P > 0.05). The increase of CRP and IL-6 was not obvious after VG (P > 0.05). The change of mortality was not significant (P > 0.05). The pathological sections showed that the AP pathological change was mild after VG. The disease condition of food-intake operating group was more serious than that of fast operating group. It was suggested that VG had some influence on the prognosis of AP in rats.

Acute Disease↗

PubMed2000

Toxicity of magnetic albumin microspheres bearing adriamycin.

Magnetic albumin microspheres bearing adriamycin (ADM-MAM) is a novel chemotherapeutic compound with site-specific drug delivery characteristics. The acute and subacute toxic tests of the compound, local irritating test and anaphylactic test were performed on mice and guinea pigs. The results showed there was no macroscopically and microscopically direct cytotoxic injuries of the compound to the animal organs or to the cells. The LD50 value of the compound was higher than that of the single used adriamycin, indicating that the compound was less toxic than the single adriamycin and quite safe in its therapeutic dosage. Furthermore, there was also no side effects or toxic reactions to be observed on clinical patients with advanced carcinoma or gastric cancer.

Albumins↗