Heme-dependent radical generation from antimalarial fungal metabolites, radicicol and heptelidic acid.
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Animals↗
Biomedical subjects
F Fang
Publications and source records attributed to F Fang.
Construction of human insulin gene expression recombinant and its effect on blood glucose of diabetic rats.
OBJECTIVE: To study the effect of human insulin gene modified fibroblasts on blood glucose in diabetic rats. METHODS: An expression vector containing human insulin gene was constructed by recombinant DNA technique and introduced into fibroblast Ltk- cells by lipofectin-mediated DNA transfection. Following G418 screening, the survived cells were selected and enriched. Finally a cell line, PRI-12 was generated for the highest insulin production. Then, these cells were injected into streptozocin (STZ)-induced diabetic rats. RESULTS: Insulin DNA transfected Ltk- cells were able to express insulin at a high level in a long-term culture. Furthermore, these Ltk- transfectants could decrease blood glucose significantly (P < 0.01) and obviously increase the body weight (P < 0.01) when they were injected into STZ-induced diabetic rats. CONCLUSIONS: These results suggest that the cell lines transfected insulin gene could secrete insulin and execute effect on diabetic rats. The study provides support for the view that somatic cell gene therapy offers a potential approach to delivery insulin into diabetes mellitus.
Animals↗
[Study on cis-acting elements of rat glutathione S-tranferase P1 gene].
OBJECTIVE: To investigate the cis-acting regulatory DNA elements in 3.0 kb 5' flanking upstream region of rat glutathione S-tranferase (rGSTP1) gene. METHODS: Using exonuclease III (Exo III) and S1 nuclease, a series of deletions were made in the 3.0 kb region, then fused into highly sensitive luciferase reporter vector. The deleted mutants were transfected into HeLa cells. Luciferase activities of the transfected cell lysates were assayed. RESULTS: Deleted from -2.9 kb to -2.3 kb, luciferase activity had dropped about 67% (P < 0.05). The element between -2.5 kb and -2.2 kb could increase gene expression activity regardless of orientation and position. Further deleted from -1.8 kb to -1.3 kb, luciferase activity increased about three times (P < 0.05). CONCLUSIONS: There are enhancer element between -2.5 kb and -2.2 kb and a negative regulatory sequence between -1.8 kb and -1.3 kb in the upstream region of rGSTP1 gene.
Animals↗
[Modulation of cGMP levels, soluble guanylate cyclase and phosphodiesterase activities in brain of morphine-dependent mice].
By inducing morphine dependence in mice, the changes of cGMP contents, phosphodiesterase (PDE) and soluble guanylate cyclase (sGC) activities and their phosphorylation regulated by protein kinase A (PKA) were observed. It was found that: (1) cGMP contents in cerebellum, striatum, hippocampus and cerebral cortex were significantly lower. (2) The sGC activities were apparently decreased in cerebellum and striatum. In the striatum and cerebral cortex the sGC activities and phosphorylation levels in vitro were significantly increased and were inhibited by PKA inhibitor. (3) The PDE activities showed no change in cerebellum and hippocampus, but in striatum and cerebral cortex PDE activities and phosphorylation levels in vitro were significantly increased and were inhibited by PKA inhibitor. (4) These changes described above were not observed in mice treated with naloxone 30 min prior to daily morphine injection. Our data indicate that the decrease of cGMP contents occurred generally in brain regions of morphine-dependent mice. The decrease of cGMP contents in cerebellum and hippocampus may be due to the decrease of sGC activities, but the decrease of cGMP contents in striatum and cerebral cortex may be mainly due to the increase of PDE activity. Both sGC and PDE activities were regulated by PKA.
Animals↗
[Effects of chronic morphine treatment on inositol phosphates contents and PKC activity in mouse brain].
By inducing morphine dependence in mice, changes in inositol phosphate contents, protein kinase C(PKC) activity in brain regions and effect of PKC inhibitor on the development of morphine dependence were investigated. It was found that: (1) IP(inositol phosphate) and IP3 (inositol trisphosphate) contents in striatum, IP in cerebral cortex and total inositol phosphates(IP + IP2 + IP3) in striatum and cerebral cortex were markedly higher than those of the control. But no similar changes in hippocampus and cerebellum were observed. (2) Cytosolic PKC activity was significantly increased in cerebellum and cerebral cortex but decreased in striatum. The membrane PKC activity was apparently enhanced in striatum but decreased in cerebellum and hippocampus. (3) PKC inhibitor was found to prevent the development of morphine dependence. (4) These changes described above were not observed in mice treated with naloxone 30 min prior to daily morphine injection. Our data indicate that the increase of inositol phosphate contents in striatum implied activation of phospholipase C, which might lead to PKC activation. This PKC activation may be involved in the development of morphine dependence.
Animals↗
Opiate receptors in the periaqueductal gray mediate analgesic effect of nitrous oxide in rats.
The site of action and the pathways which are activated by nitrous oxide (N2O) to produce an analgesic effect are not well defined. Experiments were designed to determine whether N2O produces analgesia by activating opiate receptors or alpha2-adrenoceptors in periaqueductal gray. The analgesic effect of N2O was determined using the tail flick response to noxious radiant heat in lightly anesthetized rats. Different antagonists were bilaterally microinjected into ventrolateral periaqueductal gray to determine whether the analgesic effect produced by N2O was reversed. The increase in the tail flick latencies produced by N2O was reversed by bilateral microinjection into the ventrolateral part of periaqueductal gray with the opiate receptor antagonist naloxone 2.5 microg/0.5 microl, but not with the alpha2-adrenoceptors antagonist yohimbine 1.5 microg/0.5 microl. These results indicate that the N2O analgesic effect is mediated by activation of opiate receptors, but not alpha2-adrenoceptors, in the periaqueductal gray. Combined with the previous experiments that the N2O analgesic effect is reversed by intrathecal injection of an alpha2-adrenoceptor antagonist but not by an opiate receptor antagonist, it seems likely that N2O causes activation of the opiate receptors in the periaqueductal gray, which in turn activate the noradrenergic descending pathways to the spinal cord to produce the analgesic effect.
Adrenergic alpha-Antagonists↗
Detection of Duffy antigen in the plasma membranes and caveolae of vascular endothelial and epithelial cells of nonerythroid organs.
The nonerythroid expression of the Duffy blood group protein (gp-Fy) was confined to certain cell types. Immunocytochemistry studies of the kidney showed gp-Fy in the endothelium of glomeruli, peritubular capillaries, vasa recta, and the principal cells (epithelial) of collecting ducts. Gp-Fy was also produced in the endothelial cells of large venules and epithelial cells (type-I) of pulmonary alveoli. In the thyroid, only the endothelial cells of capillaries produced gp-Fy. In the spleen, the endothelial cells of capillaries, high endothelial venule, and sinusoids produced abundant gp-Fy. Ultrastructural studies showed that apical and basolateral plasma membrane domains, including caveolae, had gp-Fy. Immunoblot analysis showed substantially less gp-Fy in nonerythroid cells than in erythrocytes. Moreover, the analyzed nonerythroid organs of Duffy-negative individuals did not produce more gp-Fy to compensate for the lack of this protein in their erythrocytes. The nucleotide sequence and the size of kidney mRNA from a Duffy-positive individual were the same as that of bone marrow. It is assumed, therefore, that nonerythroid Duffy protein is the product of the same gene as that of bone marrow. This notion is reinforced by the fact that nonerythroid and erythroid gp-Fy have the same antigenic domains.
Cell Membrane↗
Immune bioactivity in shellfish toward serum-free cultured human cell lines.
The biologically functional effect of eight kinds of hot-water extracts of shellfish on cultured human cell lines was examined in a serum-free medium model. Meretrix lusoria and Sinonovacula constricta extracts enhanced IgM secretion of both hybridoma HB4C5 and SI102 cells when cultured with the respective extracts. The purified principle exhibited remarked activity in the adsorbed fraction in hydroxyapatite and Concanavalin A columns. The extracts of Corbicula fluminea, Crassostreas gigas, Meretrix lusoria, Anadara granosa, and Sinonovacula constricta enhanced in nitroblue tetrazolium (NBT)-reducing ability of macrophage U-M cells. Meretrix lusoria, Anadara granosa, and Sinonovacula constricta were specifically cytotoxic to both cultures of MCF-7 breast cancer cells and HuH-6KK hepatoblastoma. These findings imply that the extracts of shellfish that were examined exhibited a differential effect on immune cells and tumor cells.
Animals↗
Two new components of the aspochalasins produced by Aspergillus sp.
Aspergillus sp. FO-4282 was found to produce two new components of the aspochalasins. Their structures were determined by spectroscopic analyses.
Animals↗
[Relation between pulmonary surfactants and asthma].
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Animals↗
[Experimental studies on somatic gene therapy for diabetes. I. Structuring of recombinant from human insulin gene and ammalian expression vector PRC/CMV].
To develop a model somatic gene therapy system for diabetes, we constructed a human insulin expression vector in non B cells. As the first step, an insulin cDNA fragment of 260 bp, generated by a complet digestion of PBCA with EcoR I and BamH I, was inserted into the EcoR I/BamH I site of plasmid PBS.SK by ligation of cohevise-ended DNA to construct transition plasmid PBS.INS. Then the plasmid PBS.INS was completly digested by Hind III and Xbal I. The small DNA fragment containing insulin cDNA gene was subcloned to the expression plasmid PRC/CMV to form recombinant PRC/CMV.INS.
DNA, Complementary↗
[Determination of gamma-aminobutyric acid and glutamic acid in human cerebrospinal fluid by high performance liquid chromatography].
Gamma-aminobutyric acid (Gaba) and glutamic acid (Glu) are believed to be the major neurotransmitter. Levels in cerebrospinal fluid (CSF) may reflect their metabolism in various neurotic and psychiatric diseases. Measurements of Gaba and Glu in body fluids will help to elucidate their metabolic role and diagnostic value. In the present work, the concentrations of glutamic acid and gamma-aminobutyric acid in human cerebrospinal fluid were determined by reversed-phase high performance liquid chromatography using pre-column derivatized with dansyl chloride and UV detection at 254 nm. The mobile phase was A:CH3OH and B: THF/CH3OH/0.05 mol/L NaAc (pH 6.2) (5/75/420, V/V) with gradient elution. The flow rate was 1 mL/min. CSF samples were deproteinizated with methanol. After centrifugation at 15000 r/min for 10 min, the supernatant was introduced into a screw-capped vial and evaporated to near dryness at 80 degrees C. Derivatization was carried out by the addition of 250 microL of bicarbonate solution (pH 9.8) and 250 microL of dansyl chloride solution (4 g/L) followed by heating at 40 degrees C water bath for 30 min. Extraction was performed with 300 microL of ethyl acetate and the organic layer separated was dried at room temperature under nitrogen. The dry residue was dissolved and injected into the HPLC system. The linear range of the method was 5-1000 micromol/L (r = 0.9988) for Glu and 1-600 micromol/L (r = 0.9984) for Gaba. The lower limit of detection were 0.002 micromol/L and 0.001 micromol/L respectively. The recoveries were 96.9%-98.8% and 97.5%-98.8% and the coefficients of variation of peak height measurements were 2.2%-3.4% and 3.8%-5.6% respectivery. The method enables a simple, rapid and reproducible quantification of Glu and Gaba neurotransmitter.
Chromatography, High Pressure Liquid↗
Spinal cholinergic and monoamine receptors mediate the antinociceptive effect of morphine microinjected in the periaqueductal gray on the rat tail, but not the feet.
The antinociceptive effects of morphine (5 micrograms) microinjected into the ventrolateral periaqueductal gray were determined using both the tail flick and the foot withdrawal responses to noxious radiant heating in lightly anesthetized rats. Intrathecal injection of appropriate antagonists was used to determine whether the antinociceptive effects of morphine were mediated by alpha 2-noradrenergic, serotonergic, opioid, or cholinergic muscarinic receptors. The increase in the foot withdrawal response latency produced by microinjection of morphine in the ventrolateral periaqueductal gray was reversed by intrathecal injection of the cholinergic muscarinic receptor antagonist atropine, but was not affected by the alpha 2-adrenoceptor antagonist yohimbine, the serotonergic receptor antagonist methysergide, or the opioid receptor antagonist naloxone. In contrast, the increase in the tail flick response latency produced by morphine was reduced by either yohimbine, methysergide or atropine. These results indicate that microinjection of morphine in the ventrolateral periaqueductal gray inhibits nociceptive responses to noxious heating of the tail by activating descending neuronal systems that are different from those that inhibits the nociceptive responses to noxious heating of the feet. More specifically, serotonergic, muscarinic cholinergic and alpha 2-noradrenergic receptors appear to mediate the antinociception produced by morphine using the tail flick test. In contrast, muscarinic cholinergic, but not monoamine receptors appear to mediate the antinociceptive effects of morphine using the foot withdrawal response.
Animals↗
Influence of temperature on the retention behaviour of proteins in cation-exchange chromatography.
The chromatographic behaviour of several amino acid derivatives, peptides and proteins has been investigated with the so called "tentacle-type" LiChrospher-100 SO3- adsorbent and an analogous poly(2-sulphoethylaspartamide) cation-exchange adsorbent, PolySulphoethyl A. In particular, the dependences of the retention properties of a range of biosolutes on temperature and the chromatographic residence time were evaluated with the objective of gaining further insight into the influence of ligand type and flexibility and the role of solute conformation on the chromatographic behaviour of proteins with these two strong cation-exchange chromatographic adsorbents. The results indicate that significant differences in the chromatographic retention behavior between proteins and low-molecular-mass solutes occur as a function of temperature and the type of co- and counter ions present in the mobile phase with both adsorbents. Moreover, the dependences of the Zc and log Kc values on temperature for most of the proteins examined exhibited significant changes in magnitude between 4 and 75 degrees C, whilst no equivalent changes were evident for low-molecular-mass solutes. With the "tentacle-type" LiChrospher-1000 SO3- adsorbent at higher temperatures, e.g., at 75 degrees C, most of the proteins could still be eluted although several exhibited very large increases in their retention parameters. With the PolySulphoethyl A adsorbent, on the other hand, none of the proteins examined were eluted at 75 degrees C. The results moreover indicate that hydrophobic interactions play an increasingly important role in protein retention with both types of ion-exchange adsorbents at higher temperatures, but are more dominant with the PolySulphoethyl A ligand. In general, the Zc values for the proteins with the "tentacle-type" LiChrospher-1000 SO3- adsorbent were greater than those obtained with the PolySulphoethyl A adsorbent, suggesting that the "tentacular" ligands present on this strong cation-exchange adsorbent interact with protein molecules through larger contact areas. Collectively, these investigations provide further support for the concept that the adsorption behaviour of proteins with the "tentacle-type" LiChrospher-1000 SO3- adsorbent and similar types of "tentacular" ligand systems involves a multilayer dissolution mechanism with the protein interacting with a more diffuse or extended Donnan double layer in the ion-exchange environment, resulting in multi-site binding processes.
Absorption↗
Heterodimers of placenta growth factor/vascular endothelial growth factor. Endothelial activity, tumor cell expression, and high affinity binding to Flk-1/KDR.
Here we show that the Escherichia coli expressed monomers of placenta growth factor (PLGF)129 and vascular endothelial growth factor (VEGF)165 can be re-folded in vitro to form PLGF/VEGF heterodimers. The purified recombinant PLGF/VEGF heterodimers and VEGF homodimers have potent mitogenic and chemotactic effects on endothelial cells. However, PLGF/VEGF heterodimers display 20-50-fold less mitogenic activity than VEGF165 homodimers. In contrast, PLGF129 homodimers have little or no effect in these in vitro assays. We also demonstrate the presence of natural PLGF/VEGF heterodimers in the conditioned media of various human tumor cell lines. While PLGF/VEGF heterodimers bind with high affinity to a soluble Flk-1/KDR receptor, PLGF129 homodimers fail to bind to this receptor. Cross-linking of 125I-ligands to human umbilical vein endothelial cells reveals that PLGF/VEGF heterodimers and VEGF165 homodimers, but not PLGF129 homodimers, form complexes with membrane receptors. VEGF165 homodimers and PLGF/VEGF heterodimers stimulate tyrosine phosphorylation of a 220-kDa protein, the expected size for the KDR receptor in human umbilical vein endothelial cells, whereas PLGF129 homodimers are unable to induce tyrosine phosphorylation of this protein. These data indicate that PLGF may modulate VEGF-induced angiogenesis by the formation of PLGF/VEGF heterodimers in cells producing both factors.
Angiogenesis Inducing Agents↗
Dependence of cyclin E-CDK2 kinase activity on cell anchorage.
Most nonmalignant cells are anchorage-dependent; they require substrate attachment for growth and, in some instances, survival. This requirement is lost on oncogenic transformation. The cyclin E-CDK2 complex, which is required for the G1-S transition of the cell cycle, was activated in late G1 phase in attached human fibroblasts, but not in fibroblasts maintained in suspension. In transformed fibroblasts the complex was active regardless of attachment. The lack of cyclin E-CDK2 activity in suspended cells appeared to result from increased expression of CDK2 inhibitors and a concomitant decrease in phosphorylation of CDK2 on threonine-160. Suppression of cyclin E-CDK2 activity may thus underlie the anchorage dependence of cell growth.
CDC2-CDC28 Kinases↗
[Construction of recombinant glutathione S-transferase pi cDNA and retrovirus vector].
In order to study effect of expression of glutathione S-transferase pi gene on transformation induced by chemical Carcinogens in cellular experiments and explore possibility of gene prevention of tumor, as the first step, we have constructed recombinant glutathione S-transferase pi cDNA and retrovirus vector pXT1.
Animals↗
[Proto-oncogene point mutation of c-Ha-ras in bronchogenic carcinoma].
11 bronchogenic carcinoma and 8 corresponding paracarcinoma specimens were detected for activating point mutation in codon 12 of c-Ha-ras gene by Southern blotting hybridization technique. The c-Ha-ras gene was found to be activated by point mutation in codon 12 in 8 of bronchogenic carcinoma and 1 of paracarcinoma tissues. The mutative and unmutative hybrid band was both discovered in 2 of paracarcinoma tissues. Analysis of clinical and pathological features, we conclude that: (1) The point mutation of c-Ha-ras gene codon 12 is found in 77.8% bronchogenic carcinoma among Chinese; (2) At least in part cases, mutation of c-Ha-ras gene codon 12 is not the "initiation" event, but may be a early-stage event in the pathogenesis; (3) No apparent association between the c-Ha-ras gene codon 12 mutation and pathological types, disease stages and history of smoking is present.
Adenocarcinoma↗