Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An experimental cancer gene therapy model was employed to develop a non-invasive imaging procedure using radiolabelled 2'-fluoro-2'-deoxy-5-iodo-1-beta- d-arabinofuranosyluracil (FIAU) as an enzyme substrate for monitoring retroviral vector-mediated herpes simplex virus type 1 thymidine kinase gene ( HSV1-tk) transgene expression. Iodine-131 labelled FIAU was prepared by a no-carrier-added (n.c.a.) synthesis process and lyophilised to give "hot kits". The labelling yield was over 95%, with a radiochemical purity of more than 98%. The stability of [(131)I]FIAU in the form of lyophilised powder (the hot kit) was much better than that in the normal saline solution. The shelf life of the final [(131)I]FIAU hot kit product is as long as 4 weeks. Cellular uptake of [(131)I]FIAU after different periods of storage was investigated in vitro with HSV1-tk-retroviral vector transduced NG4TL4-STK and parental non-transduced NG4TL4 murine sarcoma cell lines over an 8-h incubation period. The NG4TL4-STK cells accumulated more radioactivity than NG4TL4 cells in all conditions, and accumulation increased with time up to 8 h. The kinetic profile of the cellular uptake of n.c.a. [(131)I]FIAU formulated from the lyophilised hot kit or from the stock solution was qualitatively similar. For animal model cancer gene therapy studies, FVB/N mice were inoculated subcutaneously with the HSV1-tk(+) and tk(-) sarcoma cells into the flank to produce tumours. Biodistribution studies showed that tumour/blood ratios were 2, 3.5, 8.2 and 386.8 at 1, 4, 8 and 24 h post injection, respectively, for the HSV1-tk(+) tumours, and 0.5, 0.5, 0.7 and 5.4, respectively, for the HSV1-tk(-) tumours. Radiotracer clearance from blood was completed in 24 h and was bi-exponential. A significant difference in radioactivity accumulation was revealed among the HSV1-tk(+) tumours, the tk(-) tumours and other tissues. At 24 h p.i., higher activity retention was observed in HSV1-tk(+) tumours (9.67%+/-3.89%ID/g) than in HSV1-tk(-) tumours (0.48%+/-0.19%ID/g). After seven consecutive daily treatments with the prodrug ganciclovir, planar gamma camera imaging showed HSV1-tk(+) tumour regression at day 4, and complete tumour regression at day 7. These results clearly demonstrate that the simplified n.c.a. synthesis process developed in this study is reliable and that the [(131)I]FIAU product is useful for in vivo monitoring of HSV1-tk gene transfer, expression and gene therapy.
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PMID:Non-invasive in vivo imaging with radiolabelled FIAU for monitoring cancer gene therapy using herpes simplex virus type 1 thymidine kinase and ganciclovir. 1451 92

Activation of kit-receptor tyrosine kinase occurs in all cases of gastrointestinal stromal tumors, regardless of the mutation status of kit. Imatinib mesylate (STI 571,Gleevec) is a selective inhibitor of certain protein tyrosine kinases. It has been shown in preclinical models and clinical studies to have activity against such tumors. The aim of the present study was to report the efficacy of imatinib mesylate in the treatment of advanced gastrointestinal stromal tumors. Two adults with histologically confirmed, unresectable, and metastatic gastrointestinal stromal tumors that expressed CD117 (a marker of kit-receptor tyrosine kinase) were identified at our institution during 2000-2002. As the diseases were advanced and not amenable to surgery, chemotherapy, or radiation therapy, imatinib mesylate was used, because this targeted inhibitor has been shown to be active against advanced gastrointestinal stromal tumors and has a mild toxicity profile. Imatinib mesylate induced a sustained response in both patients with advanced unresectable or metastatic gastrointestinal stromal tumors. Inhibition of the KIT signal-transduction pathway is a promising treatment for advanced gastrointestinal stromal tumors, which resist conventional chemotherapy.
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PMID:Advanced gastrointestinal stromal tumors successfully treated with imatinib mesylate: a report of two cases. 1471 79

We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods. Study cases (27 carcinomas and 3 ductal breast carcinoma in situ (DCIS) cases) showed varying Her-2 expression as determined by IHC (HercepTest). In carcinomas, there was a good correlation between HER-2 DNA amplification and strong HER-2 protein expression detected by FISH and IHC, respectively. A single DCIS case was amplified in FISH, but not in IHC. Both HER-2 gene amplification and expression could be quantified in microdissected paraffin-embedded tumors using real-time PCR, DNA and RNA being successfully detected in 146 of 150 (97%) and 141 of 150 (94%) samples, respectively. PCR analysis for HER-2 DNA amplification using the LightCycler HER2/neu DNA Quantification kit (Roche Molecular Biochemicals, Mannheim, Germany) correlated fairly well with IHC and FISH. All IHC HER-2 3+ tumors were amplified according to the kit, as was the FISH-amplified DCIS case. DNA-PCR identified five additional tumors as being amplified. Interestingly, all these scored 2+ with the HercepTest, but were negative using FISH. We believe that real-time quantitative PCR analysis of HER-2 DNA amplification following microdissection represents a useful supplementary or perhaps even an alternative technique for establishing HER-2 status in paraffin-embedded tumors.
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PMID:Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma: an alternative method for HER-2/neu analysis. 1473 26

Sea urchin embryotoxicity tests are widely used for evaluating the biological effects of contaminants in marine environments. The currently used traditional and standardized protocols are quite slow and laborious. The present work shows a modified bioassay (new embryotoxicity test; NET) in an attempt to speed up laboratory work using a limited number of fertilized eggs. Several experiments have been conducted both with a traditional bioassay and with the NET, using the same test conditions, in order to evaluate the reliability of the proposed simplified bioassay. Adult Paracentrotus lividus (Lamark) were collected from the Tyrrenian Sea (Bay of Naples) and embryos, reared in filtered seawater, were exposed to increasing potassium dichromate and copper sulfate concentrations. Then the EC(50) was calculated. The analysis of the results evidenced good repeatability. The confidence limits in all tests overlapped; moreover, data correlation analysis between the results of both tests showed a high significant accordance (chromium, R2 = 0.93, P < 0.01; copper, R2 = 0.86, P < 0/05). In conclusion, the NET seems to be a good alternative to the traditional tests; it could be a first step toward a new routine ecotoxicological kit for seawater.
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PMID:Sea urchin embryotoxicity test: proposal for a simplified bioassay. 1475 57

The proto-oncogene, C-KIT (KIT), encodes a tyrosine kinase receptor, and mutations in this gene are causative for several mammalian diseases, including cancer and a form of pigmentation-associated hereditary deafness. Our laboratories are interested in a form of hereditary deafness that is associated with abnormalities in pigmentation and is common in the Dalmatian. Thus, KIT is being analyzed as a candidate gene for deafness in this breed. In addition to our interest in deafness, we are involved in mapping gene loci in the canine genome. Reported here is the identification of two isoforms of canine C-kit and radiation hybrid mapping of KIT to CFA13.
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PMID:Identification of isoforms and RH mapping of canine KIT. 1497 Jul 13

The expression of Sprouty4 (Spry4), an intracellular FGF receptor antagonist, shows a temporally and spatially restricted pattern in embryonic lung and is induced by ERK signaling. To clarify the molecular mechanisms regulating Spry4 transcription, the genomic structure of the human Sprouty4 (hSpry4) gene was first determined by using the GenomeWalker kit. The hSpry4 gene spans > 14 kb and is organized in three exons and two introns. Multiple transcription start sites were subsequently mapped by 5'-rapid amplification of cDNA ends. Analysis of up to 4 kb of sequence in the 5'-flanking region of the gene showed the presence of multiple potential transcription factor binding sites but no TATA or CAAT boxes. Transient transfection using luciferase reporter gene constructs with progressive deletions of the hSpry4 5'-flanking region revealed that the core promoter activity is located within the proximal 0.4-kb region, whereas the minimal ERK-inducible promoter activity is between -69 and -31. Homology analysis further showed that the core promoter region of the hSpry4 gene exhibits significant similarity to the 5'-flanking region of the mouse gene.
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PMID:Genomic structure and promoter characterization of the human Sprouty4 gene, a novel regulator of lung morphogenesis. 1497 31

The aim of this study is to investigate whether Ginkgo biloba extract can augment endothelial progenitor cells numbers, and promote the cells' proliferative, migratory, adhesive, and in vitro vasculogenesis capacity. Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with Ginkgo biloba extract (to make a series of final concentrations: 10 mg/L, 25 mg/L, and 50 mg/L) or vehicle control for the respective time points (6 hours, 12 hours, 24 hours, and 48 h). Endothelial progenitor cells were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. They were further documented by demonstrating the expression of KDR, VEGFR-2, and AC133 with flow cytometry. Endothelial progenitor cells proliferation, migration, and in vitro vasculogenesis activity were assayed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, modified Boyden chamber assay, and in vitro vasculogenesis kit, respectively. Endothelial progenitor cells adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. Incubation of isolated human mononuclear cells with Ginkgo biloba extract dose- and time-dependently increased the number of endothelial progenitor cells, maximum at 25 mg/L, 24 hours (approximately 1-fold increase, P < 0.01). In addition, Ginkgo biloba extract also dose- and time-dependently promoted endothelial progenitor cells proliferative, migratory, adhesive, and in vitro vasculogenesis capacity. The results of the present study defined a novel functional effect of Ginkgo biloba extract: the augmentation of endothelial progenitor cells with enhanced functional activity.
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PMID:Effects of Ginkgo biloba extract on number and activity of endothelial progenitor cells from peripheral blood. 1507 17

Extragastrointestinal stromal tumor (EGIST) is a unique tumor that occurs outside the gastrointestinal tract. EGIST shows a c-kit expression and histologic appearance similar to those of gastrointestinal stromal tumor (GIST). Most GISTs have gain-of-functional mutation of the c-kit gene, and some have mutation of the platelet-derived growth factor receptor-alpha (PDGFRA) gene. However, the frequency of mutation of those genes in EGISTs remains unclear. We examined the clinicopathologic features, prognostic factors, and c-kit and PDGFRA mutation in 39 cases of EGIST. Tumors with high mitotic counts (>or=5/50 high power fields) or a high Ki-67 labeling index (>or=10%) were significantly correlated with worse prognoses. The c-kit mutation was found in the juxtamembrane domain (exon 11) and the extracellular domain (exon 9) in 12 of 29 cases (41.4%) and 2 of 29 cases (6.9%), respectively. The PDGFRA gene mutation was found at the juxtamembrane domain (exon 12) and the tyrosine kinase domain (exon 18) in one case each. The pattern of kit and PDGFRA mutation in EGIST was essentially similar to that in GIST. Our results suggest that the c-kit and PDGFRA mutations play an important role in the tumorigenesis of EGIST. High mitotic counts and a high Ki-67 labeling index may be useful for predicting the aggressive biologic behavior in EGIST. Furthermore, STI-571, targeting c-kit and PDGFR tyrosine kinase, seems to be a possible therapeutic strategy for EGISTs, especially advanced cases.
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PMID:c-kit and PDGFRA mutations in extragastrointestinal stromal tumor (gastrointestinal stromal tumor of the soft tissue). 1508 67

Hypercholesterolaemia contributes to atherosclerosis and coronary artery diseases by inducing endothelial cell injury and dysfunction. Recent studies have provided increasing evidence that EPCs (endothelial progenitor cells) participate in ongoing endothelial repair and postnatal neovascularization. However, the changes in EPCs in patients with hypercholesterolaemia have not been elucidated to date. Therefore we investigated the number and functional activity of EPCs in patients with hypercholesterolemia. Total MNCs (mononuclear cells) were isolated from 20 patients with hypercholesterolaemia and 20 matched control subjects. EPCs were characterized as adherent cells double-positive for DiI-LDL (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide percholate-labelled low-density lipoprotein) uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope, and were characterized further by demonstrating the expression of KDR (kinase insert domain-containing receptor), CD34 and AC133 by flow cytometry. Proliferation, migration and in vitro vasculogenesis activity of EPCs were assayed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, modified Boyden chamber assay and an in vitro vasculogenesis kit respectively. EPC adhesion assay was performed by replating cells on fibronectin-coated dishes and then counting the adherent cells. As a result, the number of EPCs was significantly reduced in patients with hypercholes-terolaemia compared with that in control subjects (41.8 +/- 8.7 compared with 64.5 +/- 16.6 EPCs/x 200 field respectively; P < 0.05). The number of EPCs was inversely correlated with total cholesterol (r = -0.659, P < 0.001) and LDL-cholesterol (r = -0.611, P < 0.001) levels. In addition, the functional activities of isolated EPCs, such as proliferative, migratory, adhesive and in vitro vasculogenesis capacity, were also impaired. In conclusion, the results of the present study may state a novel pathophysiological mechanism of hypercholesterolaemia: the reduction of EPCs with decreased functional activity.
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PMID:Number and activity of endothelial progenitor cells from peripheral blood in patients with hypercholesterolaemia. 2095 66

Urticaria pigmentosa (UP), resulting from the accumulation of excessive numbers of mast cells in the skin, is the most common form of cutaneous mastocytosis. Observations highlight the diversity of this disease. Clonal expansion of early hematopoietic progenitor cells carrying activating mutations in KIT seems to be the basis of adult-onset UP. New pathogenetic findings are leading to the development of new diagnostic surrogate markers of disease and therapeutic approaches targeting neoplastic mast cells. Promising strategies may arise from an increased understanding about the cause of mastocytosis and the signaling pathways initiated by kit activation.
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PMID:Urticaria pigmentosa. 1512 Jan 52


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