Ticlopidine-associated thrombotic thrombocytopenic purpura.
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Humans↗
Biomedical subjects
B Wu
Publications and source records attributed to B Wu.
TIMP-1 contact sites and perturbations of stromelysin 1 mapped by NMR and a paramagnetic surface probe.
Surfaces of the 173 residue catalytic domain of human matrix metalloproteinase 3 (MMP-3(DeltaC)) affected by binding of the N-terminal, 126 residue inhibitory domain of human TIMP-1 (N-TIMP-1) have been investigated using an amide-directed, NMR-based approach. The interface was mapped by a novel method that compares amide proton line broadening by paramagnetic Gd-EDTA in the presence and absence of the binding partner. The results are consistent with the X-ray model of the complex of MMP-3(DeltaC) with TIMP-1 (Gomis-Rüth et al. (1997) Nature 389, 77-81). Residues Tyr155, Asn162, Val163, Leu164, His166, Ala167, Ala169, and Phe210 of MMP-3(DeltaC) are protected from broadening by the Gd-EDTA probe by binding to N-TIMP-1. N-TIMP-1-induced exposure of backbone amides of Asp238, Asn240, Gly241, and Ser244 of helix C of MMP-3(DeltaC) to Gd-EDTA confirms that the displacement of the N-terminus of MMP-3(DeltaC) occurs not only in the crystal but also in solution. These results validate comparative paramagnetic surface probing as a means of mapping protein-protein interfaces. Novel N-TIMP-1-dependent changes in hydrogen bonding near the active site of MMP-3(DeltaC) are reported. N-TIMP-1 binding causes the amide of Tyr223 of MMP-3(DeltaC) bound by N-TIMP-1 to exchange with water rapidly, implying a lack of the hydrogen bond observed in the crystal structure. The backbone amide proton of Asn162 becomes protected from rapid exchange upon forming a complex with N-TIMP-1 and could form a hydrogen bond to N-TIMP-1. N-TIMP-1 binding dramatically increases the rate of amide hydrogen exchange of Asp177 of the fifth beta strand of MMP-3(DeltaC), disrupting its otherwise stable hydrogen bond.
Amides↗
Characterization of a highly conserved FAD-binding site in human monoamine oxidase B.
Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of biogenic and xenobiotic amines. The oxidative step is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the apoenzyme at Cys397. Our previous studies identified two noncovalent flavin-binding regions in MAO B (residues 6-34 and 39-46) (Kwan, S.-W., Lewis, D. A., Zhou, B. P., and Abell, C. W. (1995) Arch. Biochem. Biophys. 316, 385-391; Zhou, B. P., Lewis, D. A., Kwan, S.-W., Kirksey, T. J., and Abell, C. W. (1995) Biochemistry 34, 9526-9531). In these regions, Glu34 and Tyr44 were found to be required for the initial binding of FAD. By comparing sequences with enzymes in the oxidoreductase family, we now have found an additional FAD-binding site in MAO B (residues 222-227), which is highly conserved across species (human, bovine, and rat). This conserved sequence contains adjacent glycine and aspartate residues (Gly226 and Asp227). Based on the x-ray crystal structures of several oxidoreductases (Eggink, G., Engel, H., Vriend, G., Terpstra, P., and Witholt, B. (1990) J. Mol. Biol. 212, 135-142; Van Driessche, G., Kol, M., Chen, Z.-W., Mathews, F. S., Meyer, T. E., Bartsch, R. G., Cusanovich, M. A., and Van Beeumen, J. J. (1996) Protein Sci. 5, 1753-1764), the Gly residue at the end of a beta-strand facilitates a sharp turn and extends the beta-carbonyl group of Asp to interact with the 3'-hydroxyl group of the ribityl chain of FAD. To assess the hypothesis that Gly226 and Asp227 are involved in FAD binding in MAO B, site-specific mutants that encode substitutions at these positions were prepared and expressed in mammalian COS-7 cells. Our results indicate that Gly226 and the beta-carbonyl group of Asp227 are required for covalent flavinylation and catalytic activity of MAO B, but not for noncovalent binding of FAD. Our studies also reveal that mutagenesis at Glu34 and Tyr44 not only interferes with covalent flavinylation and catalytic activity of MAO B, but also with noncovalent binding of FAD. Based on these collective results, we propose that the coupling of FAD to the MAO B apoenzyme is a multistep process.
Amino Acid Sequence↗
Effects of NMDA receptor glycine recognition site antagonism on cerebral metabolic rate for glucose and cerebral blood flow in the conscious rat.
Glycine is a requisite cofactor for glutamatergic activation of the N-methyl-D-aspartate (NMDA) receptor. Antagonism of glutamate at the NMDA receptor has been shown to cause substantial changes in regional cerebral metabolic rate for glucose utilization (CMRglu) and blood flow (CBF). This study examined CMRglu and CBF changes caused by antagonism of glycine at the NMDA receptor recognition site. Rats were anesthetized with halothane and vascular access was obtained. The animals were then awakened. One hour later, either vehicle (control) or ACEA 1021 (5 mg/kg followed by 3.5 mg x kg(-1) x h(-1) or 10 mg/kg followed by 7 mg x kg(-1) x h(-1)) was infused intravenously. CMRglu and CBF were then determined. Autoradiographic analysis of 25 regions revealed effects of ACEA 1021 on CMRglu in the frontal, sensory, parietal and auditory cortices and the anteroventral and subthalamic nuclei. These changes deviated less than 15% from control. Effects on CBF were also small. The CMRglu and CBF effects of ACEA 1021 are substantially less than those previously observed for either competitive or non-competitive glutamate NMDA antagonists. We conclude that inhibition of the NMDA glycine recognition site has little or no effect on CMRglu or CBF at the doses examined. This is consistent with the absence of psychotomimetic effects observed for this class of drugs.
Animals↗
Activation regimens to prepare bovine oocytes for intracytoplasmic sperm injection.
Activation of bovine oocytes to produce a single haploid pronucleus in preparation for intracytoplasmic sperm injection (ICSI) has been investigated with various combinations of ionomycin and 6-dimethylaminopurine (DMAP). Effects were evaluated by immunocytochemical staining, chromosomal analysis and assessment of development in vitro. Oocytes matured in vitro were exposed to: ionomycin alone (single or repeated treatments, Groups 1 and 2 respectively), ionomycin followed by DMAP (immediately or after a 3-h delay, Groups 3 and 4), or no treatment (control, Group 5). They were then co-cultured in M199 with bovine oviductal epithelial cells. Activation rates were not significantly different among groups but significantly fewer oocytes in Group 3 extruded a second polar body than in Groups 1, 2, and 4. Most parthenotes (60% to 80%) in Groups 1, 2, and 4 were haploid, whereas 82% in Group 3 were mixoploid or polyploid. Most of the parthenotes (88%) in Group 4 formed a single pronucleus besides extruding the second polar body and were therefore more suitable for ICSI than those of Groups 1 and 2 in which condensed chromosomes predominated. The respective rates of oocyte cleavage in Groups 1 to 4 were 24%, 36%, 70%, and 75%; corresponding blastocyst rates were 1%, 5%, 17%, and 8%. There were significantly fewer cells in the parthenotes of Groups 1, 2, and 4 than of Group 3, or of embryos produced by in vitro fertilization. Thus, delaying the addition of DMAP after ionomycin decreases chromosomal abnormalities and produces a high proportion of activated oocytes suitable for ICSI.
Adenine↗
The effect of B cell deficiency on the immune response to acetylcholine receptor and the development of experimental autoimmune myasthenia gravis.
To study the involvement of B cells in the immune response to acetylcholine receptor (AChR), B-cell-deficient (mu mutant) and control wild-type C57BL/6 mice were immunized with AChR and assessed for clinical and immunopathological manifestations of experimental autoimmune myasthenia gravis (EAMG). The mu mutant mice failed to generate anti-AChR antibodies and were completely resistant to the induction of EAMG. However, mu mutant mice developed clinical EAMG when antibodies to the AChR main immunogenic region were passively transferred. Further, the in vivo expansion of lymph node cells after AChR immunization was greatly impaired in mu mutant mice. The mu mutant mice gave an effective in vitro T cell immune response to the immunodominant pathogenic AChR alpha chain peptide 146-162 (alpha 146-162) and to the whole AChR protein when tested on day 90 after immunization with AChR, whereas the response to both AChR and its alpha 146-162 peptide was reduced when tested on day 7 after immunization. The in vitro production of IFN-gamma and IL-2 by AChR-specific and alpha 146-162 peptide-specific lymphocytes was lower in mu mutant mice. The AChR immune mu mutant T cells proliferated and produced IFN-gamma when AChR or alpha 146-162 peptide was presented by wild-type irradiated AChR-primed antigen-presenting cells (APCs). This indicates that B cells are important in the processing and presentation of AChR dominant peptide in vitro during the initial immune response to AChR. However, APCs of non-B-cell lineage are sufficient to process AChR and prime the T cells to AChR dominant T cell epitope peptides.
Animals↗
Formation and amplification of a novel tombusvirus defective RNA which lacks the 5' nontranslated region of the viral genome.
Defective interfering (DI) RNAs of tomato bushy stunt virus (TBSV) are small, subgenomic, helper-dependent replicons that are believed to be generated primarily by aberrant events during replication of the plus-sense RNA genome. Prototypical TBSV DI RNAs contain four noncontiguous segments (regions I through IV) derived from the 5' nontranslated region (NTR) (I), an internal section (II), and the 3'-terminal portion (III and IV) of the viral genome. We have studied the formation of these molecules by using engineered precursor DI RNA transcripts and report here the consistent accumulation of a novel defective RNA species, designated RNA B. Northern blot, primer extension, and sequence analyses indicated that, unlike prototypical DI RNAs, RNA B lacks region I. In vitro transcripts corresponding to the region II-III-IV structure of RNA B were amplified when coinoculated with helper, indicating that the 5' NTR of the genome does not harbor cis-acting replication elements essential for viral RNA replication. Region I is, however, important for DI RNA fitness, since molecules lacking it accumulated to significantly lower levels ( approximately 10-fold reduction). Analysis of the minus-strand sequence of region I led to the identification of an RNA undecamer sequence, arranged in tandem, at its very 3' terminus. Additional variants of the undecamer motif were also identified at internal positions in region I and in the negative strands of regions II, III, and IV. Features of the undecamer motif, the consensus of which is (-)3'-CCCAAAGAGAG, are consistent with a role as a cis-acting replication element. It is proposed that the ability of RNA B to be amplified is due, in part, to compensatory effects of a strategically positioned undecamer motif in region II. Possible replicase-mediated mechanisms for the generation of this novel viral RNA are also presented.
5' Untranslated Regions↗
Uncoupled expression of p33 and p92 permits amplification of tomato bushy stunt virus RNAs.
Tomato bushy stunt virus (TBSV) is a plus-sense RNA virus which encodes a 33-kDa protein in its 5'-most open reading frame (ORF). Readthrough of the amber stop codon of the p33 ORF results in the production of a 92-kDa fusion protein. Both of these products are expressed directly from the viral genome and are suspected to be involved in viral RNA replication. We have investigated further the roles of these proteins in the amplification of viral RNAs by using a complementation system in which p33 and p92 are expressed from different viral RNAs. Our results indicate that (i) both of these proteins are necessary for viral RNA amplification; (ii) translation of these proteins can be uncoupled while maintaining amplification of viral RNAs; (iii) if compatibility requirements exist between p33 and p92, they are not exceptionally strict; and (iv) the C-terminal approximately 6% of p33 is necessary for its functional activity. Interestingly, no complementation was observed when a p33-encoding replicon containing a deletion of a 3'-located segment, region 3.5, was tested. However, when 5'-capped transcripts of the same replicon were analyzed, complementation allowing for RNA amplification was observed. This ability to compensate functionally for the absence of region 3.5 by the addition of a 5' cap suggests that this RNA segment may act as a translational enhancer for the expression of virally encoded products.
Gene Amplification↗
Integration of surface modification and 3D fabrication techniques to prepare patterned poly(L-lactide) substrates allowing regionally selective cell adhesion.
Regeneration of organizationally complex tissue requires regulation of spatial distributions of particular cell types in three dimensions. In this paper we demonstrate an integration of polymer processing and selective polymer surface modification using methods suitable for construction of three-dimensional polymer scaffolds which may aid such cell organization. Specifically, the surfaces of degradable polyesters were modified with poly(ethylene-oxide) (PEO)-poly(propylene-oxide) (PPO) copolymers using a process compatible with a solid free-form fabrication technique, the 3DP printing process. We demonstrate inhibition of cell (hepatocyte and fibroblast) adhesion to regions of two-dimensional poly(lactide) (PLA) substrates modified with PEO-PPO-PEO copolymers. We further show that PEO-PPO-PEO-modified surfaces which are not adhesive for hepatocytes or fibroblasts can be made selectively adhesive for hepatocytes by covalent linkage of a carbohydrate ligand specific for the hepatocyte asialoglycoprotein receptor to the PEO chain ends. Our approach may be generally useful for creating regionally selective, microarchitectured scaffolds fabricated from biodegradable polymers, for spatial organization of diverse cell types.
3T3 Cells↗
Effect of interleukin-6 on the growth of human lung cancer cell line.
OBJECTIVE: To investigate the effect of interleukin-6 (IL-6) on the growth of human lung cancer in vivo as well as in vitro. METHODS: To examine the mRNA level of IL-6 receptor (IL-6R) in high-metastatic human lung giant cell carcinoma cell line PG by means of reverse transcription polymerase chain reaction (RT-PCR). To assess the existence of IL-6 receptor complex (including IL-6R and gp130) with the treatment of PG cells by use of recombinant human IL-6 (rhIL-6), recombinant human oncostatin M (rhOSM), and recombinant human leukemia inhibitory factor (rhLIF), respectively. To detect the expression of IL-6 by Northern blotting hybridization and bioactive assay. To identify the effect of IL-6 secreted by PG cells by use of IL-6 and IL-6R antisense oligodeoxynucleotides (ODNs), and specific neutralizing antibody to IL-6. To document the influence of IL-6 on PG cells growth in vivo through the strategy of the transfection of expression vector inserted antisense IL-6 cDNA. RESULTS: RT-PCR analysis revealed that PG cells expressed IL-6R mRNA. Any one of the recombinant cytokine IL-6, OSM and LIF stimulated the growth of PG cells in vitro in a concentration-dependent manner. These results demonstrated IL-6 receptor complex exist in PG cells. At the same time, PG cells expressed IL-6 mRNA and secreted bioactive IL-6. Both IL-6 antisense ODNs and IL-6R ODNs inhibited PG cells proliferation. Treatment of PG cells with IL-6 antibodies reduced the growth of PG cells in vitro. PG cells transfected with IL-6 antisense expression vector showed a decreased growth in nude mice. CONCLUSION: IL-6 functions as an autocrine growth stimulator for PG cells in vivo as well as in vitro.
Animals↗
[The role of diet consultation on improvement of the diet nutrition in pregnant women].
OBJECTIVE: To study the improvement of the intakes of various nutrients including protein, fat, carbohydrate, fibers, calcium, iron, zinc et al, in pregnant women after appropriate diet consultation by doctors. METHODS: 100 cases of pregnant women coming to our diet consultation clinic were randomly selected, in which 50 cases (control group) came for the first time and another 50 cases (study group) were for the third visit at least. The diet nutrition of the study group after the second consultation were compared with that before consultation and with concurrent control group. RESULTS: After diet consultation, except for retinol, the intake of various nutrients, including calorie intake, protein, fibers, magnesium, calcium, phosphorus, iron, zinc, vitamin E, riboflavin, thiamine, nicotinic acid, in study group were significantly improved compared with that of the study group before diet consultation and of the control group (P < 0.01). CONCLUSION: Diet consultation plays excellent role in promoting scientific and appropriate nutrients intake of pregnant women.
Adult↗
[Clinical applications of computer system for diet consultation of pregnant women].
OBJECTIVE: To investigate nutritional status of women in different gestational ages and seasons and the role of diet consultation by a computer system on the improvement of intakes of nutrients of pregnant women. METHODS: 712 women in different gestational ages and 747 cases in different seasons received diet consultation by the computer system. A comparison was done for nutritional status between pregnant women with consultation and the women without consultation. RESULTS: Major pregnant women in various gestational ages and seasons was deficient in nutrient intake. However, the nutritional status was significantly improved in women following diet consultation in comparison with the women not received diet consultation. CONCLUSION: Computer system really plays an excellent role in promoting diet consultation and improvement of diet condition of pregnant women.
Diet Surveys↗
[Liver metastasis models of human colo-rectal carcinoma established in nude mice by orthotopic transplantation and their biologic characteristics].
OBJECTIVES: To establish a liver metastasis models of human colorectal carcinomas in nude mice. METHODS: Orthotopic transplantation of histologically intact colo-rectal cancer tissues from patients into colo-rectal mucosa of nude mice. Tumorgenicity, invasion, metastasis and morphological characteristics of the transplanted tumors were studied by light microscopy, electron microscopy and immunuhistochemistry. RESULTS: Liver metastasis models of human colon carcinoma (HCA-HMN-1) and human rectal carcinoma (HRA-HMN-2) were established after screening from 34 colorectal carcinomas. They had been passaged in vivo for 18 and 21 gererations respectively. There were lymphatic, hematogenous, and implanting metastases. CEA secretion was maintained after transplantation. The primary and liver metastatic tumors were similar to the original human carcinoma in histopathological and ultrastructural features, DNA content and chromosomal karyotype. CONCLUSION: The liver metastasis models provide useful tools for the study of mechanism of metastasis and its treatment of human colo-rectal cancer.
Adenocarcinoma↗
[A study on the correlation between the activity of 72,000 type IV collagenase and the metastatic potential of cancer cells].
OBJECTIVE: To evaluate the enzyme activity of 72,000 type IV collagenase and its relationship with the metastatic potential of cancer cells. METHODS: The levels of secreted 72,000 type IV collagenase in the conditioned media of five human cancer cell lines with different metastatic potential and a normal lung fibroblast strain treated with cancer cell culture media were examined by gelatin zymography and densitometric analysis. RESULTS: The levels of 72,000 type IV collagenase secreted by cancer cells with high metastatic potential (PG, WM451 and WM983a) were higher than those secreted by cancer cells with low metastatic potential (PAa and WM35). In the conditioned media of fibroblasts which were treated with the culture media of PG and WM451, enhanced levels of activation were observed. CONCLUSION: The secretion of 72,000 type IV collagenase is closely correlated to the metastatic potential of cancer cells. The cancer cells with high metastatic potential may possibly through certain soluble mediators stimulate normal fibroblasts to activate 72,000 type IV procollagenase.
Adenocarcinoma↗
Effect of splenectomy on CCl4-induced liver fibrosis in rats.
OBJECTIVE: To assess the effects of splenectomy on CCl4-induced liver fibrosis in rats. METHODS: Wistar rats were injected with CCl4 subcutaneously for 3 to 15 weeks to develop liver fibrosis. Two hundred and thirty-six rats were divided into 9 groups: (1) model group without splenectomy; (2) splenectomized group in which operation was performed before induction of fibrosis; (3) sham-operated group in which caparotomy was done before induction of fibrosis; (4) (5) (6) groups in which splenectomy was performed at the 6th, 9th and 12th week after induction of fibrosis, respectively; and (7) (8) (9) sham operation was also performed at the 6th, 9th and 12th week after induction of fibrosis, respectively. The spleen and liver biopsy, hepatic hydroxyproline, liver homogenate levels of procollagen type III (PC III), hyaluronic acid (HA), laminin (LN) and volume density of collagen were examined at different times. RESULTS: The results showed that the degree of hepatic damage and liver fibrosis in splenectomized group was significantly milder than that in model group and sham-operated groups. Splenectomy might delay the progress of liver fibrosis for about 3 weeks. The role of preventing rat liver fibrosis was also observed when splenectomy was performed after CCl4 induction of fibrosis for 6 and 9 weeks. CONCLUSIONS: These results suggest that splenectomy may be of a preventive role against CCl4-induced rat liver fibrosis to a certain degree.
Animals↗
[Matrix metalloproteinases and neoplasm invasiveness and metastasis].
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Animals↗
[A study on the tumor suppressing effect of a specific point mutant p53 minigene in the expression regulated model with a tetracycline-transactivative response promoter].
OBJECTIVE: To establish a tetracycline-regulated expression model and to determine and verify whether a specific point mutant type p53 minigene, containing an Arg-->Leu substitution at amino acid 172, possesses a suppressing effect on human lung cancer. METHODS: The tumor suppressing effects of inducing apoptosis and inhibition of the formation of G418 resistant colonies of the specific point mutated p53 minigene in a structural expression vector on a human cancer cell line PG with preexisting dominant negative p53 were preliminarily verified. Then the specific p53 minigene was sub-cloned into a tetracycline-transactivative controlled expression vector pBPSTR1 by gene recombination methods. Through LipofectaMINE, the vector was transfected into PG cells under the presence of tetracycline (1.0 mg/ml), and the transfectants were screened in the selecting medium containing 1.5 micrograms/ml puromycin, the p53 minigene expression and tumor suppressing effects were studied dynamically in presence/absence (1.0/0 mg/ml) of tetracycline. RESULTS: The specific mutant p53 minigenes had a stronger tumor suppressing effect than wild type p53 minigene on colony formation and transient expression could induce PG cell apoptosis (P < 0.05). The tetracycline transactivative p53 minigene-regulated transgene model was successfully established. When tetracycline was absent, a large amount of apoptosis cells in transgenic passage colonies could be detected. Therefore the tumor suppressing effects were further verified. CONCLUSION: The specific point mutant p53 minigene may be a good candidate for cancer gene therapy. The tetracycline transactivative response promoter was found to be a good regulator of down stream gene expression, this may be useful in future gene therapy.
Animals↗
[Telomere, telomerase and cancers].
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Animals↗