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Article ; Online: Nanopodia--thin, fragile membrane projections with roles in cell movement and intercellular interactions.

Lin, Chi-Iou / Lau, Chun-Yee / Li, Dan / Jaminet, Shou-Ching

Journal of visualized experiments : JoVE

2014 , Issue 86

Abstract: Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 ( ... ...

Abstract	Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 μm diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per μm and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 μm from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 °C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.
MeSH term(s)	Antigens, Surface/analysis ; Cell Communication/physiology ; Cell Line, Tumor ; Cell Movement/physiology ; Cytological Techniques/methods ; Human Umbilical Vein Endothelial Cells ; Humans ; Neoplasm Proteins/analysis ; Octoxynol/chemistry ; Pseudopodia/physiology
Chemical Substances	Antigens, Surface ; Neoplasm Proteins ; TM4SF1 protein, human (147016-68-0) ; Octoxynol (9002-93-1)
Language	English
Publishing date	2014-04-03
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/51320
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Article ; Online: BCL6 mediates the effects of Gastrodin on promoting M2-like macrophage polarization and protecting against oxidative stress-induced apoptosis and cell death in macrophages.

Jia, Jing / Shi, Xiaojie / Jing, Xiaoqian / Li, Jianguo / Gao, Jie / Liu, Mengya / Lin, Chi-Iou / Guo, Xinzhi / Hua, Qian

Biochemical and biophysical research communications

2017 Volume 486, Issue 2, Page(s) 458–464

Abstract: Cerebral palsy (CP) is the most common childhood disability worldwide, yet biomarkers for predicting CP are lacking. By subjecting peripheral blood samples from 62 CP patients and 30 healthy controls to Affymetrix ... ...

Abstract	Cerebral palsy (CP) is the most common childhood disability worldwide, yet biomarkers for predicting CP are lacking. By subjecting peripheral blood samples from 62 CP patients and 30 healthy controls to Affymetrix GeneChip
MeSH term(s)	Animals ; Antioxidants/therapeutic use ; Apoptosis/drug effects ; Arginase/genetics ; Arginase/metabolism ; Benzyl Alcohols/therapeutic use ; Biomarkers/metabolism ; Case-Control Studies ; Caspase 3/genetics ; Caspase 3/metabolism ; Cell Differentiation/drug effects ; Cell Line ; Cerebral Palsy/diagnosis ; Cerebral Palsy/drug therapy ; Cerebral Palsy/genetics ; Cerebral Palsy/pathology ; Child, Preschool ; Female ; Gene Expression Regulation ; Glucosides/therapeutic use ; Humans ; Hydrogen Peroxide/antagonists & inhibitors ; Hydrogen Peroxide/pharmacology ; Infant ; Lectins, C-Type/genetics ; Lectins, C-Type/metabolism ; Macrophages/cytology ; Macrophages/drug effects ; Macrophages/metabolism ; Male ; Membrane Glycoproteins/genetics ; Membrane Glycoproteins/metabolism ; Mice ; Proto-Oncogene Proteins c-bcl-6/antagonists & inhibitors ; Proto-Oncogene Proteins c-bcl-6/genetics ; Proto-Oncogene Proteins c-bcl-6/metabolism ; Psychomotor Performance/drug effects ; RNA, Small Interfering/genetics ; RNA, Small Interfering/metabolism ; Receptors, Cell Surface/genetics ; Receptors, Cell Surface/metabolism ; Signal Transduction
Chemical Substances	Antioxidants ; Bcl6 protein, mouse ; Benzyl Alcohols ; Biomarkers ; Glucosides ; Lectins, C-Type ; MGL2 protein, mouse ; MRC1 protein, mouse ; Membrane Glycoproteins ; Proto-Oncogene Proteins c-bcl-6 ; RNA, Small Interfering ; Receptors, Cell Surface ; gastrodin (5YS9U2W3RQ) ; Hydrogen Peroxide (BBX060AN9V) ; Casp3 protein, mouse (EC 3.4.22.-) ; Caspase 3 (EC 3.4.22.-) ; Arginase (EC 3.5.3.1)
Language	English
Publishing date	2017-04-29
Publishing country	United States
Document type	Journal Article
ZDB-ID	205723-2
ISSN	1090-2104 ; 0006-291X ; 0006-291X
ISSN (online)	1090-2104 ; 0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2017.03.062
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Article: Nanopodia - thin, fragile membrane projections with roles in cell movement and intercellular interactions

Lin, Chi-Iou / Lau, Chun-Yee / Li, Dan / Jaminet, Shou-Ching

Journal of visualized experiments. 2014 Apr. 03, , no. 86

2014

Abstract	Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 μm diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per μm and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 μm from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 °C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.
Keywords	cell communication ; cell movement ; fluorescent antibody technique ; neoplasm cells ; neoplasms ; octoxynol ; staining ; temperature
Language	English
Dates of publication	2014-0403
Size	p. e51320.
Publishing place	Journal of Visualized Experiments
Document type	Article
ZDB-ID	2259946-0
ISSN	1940-087X
ISSN	1940-087X
DOI	10.3791/51320
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Article ; Online: Strategic combination therapy overcomes tyrosine kinase coactivation in adrenocortical carcinoma.

Lin, Chi-Iou / Whang, Edward E / Moalem, Jacob / Ruan, Daniel T

Surgery

2012 Volume 152, Issue 6, Page(s) 1045–1050

Abstract: Background: Coactivation of tyrosine kinase limits the efficacy of tyrosine kinase inhibitors. We hypothesized that a strategic combination therapy could overcome tyrosine kinase coactivation and compensatory oncogenic signaling in patients with ... ...

Abstract	Background: Coactivation of tyrosine kinase limits the efficacy of tyrosine kinase inhibitors. We hypothesized that a strategic combination therapy could overcome tyrosine kinase coactivation and compensatory oncogenic signaling in patients with adrenocortical carcinoma (ACC). Methods: We profiled 88 tyrosine kinases before and after treatment with sunitinib in H295R and SW13 ACC cells. The effects of monotherapy and strategic combination regimens were determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (ie, MTS) assay. Results: The minimum inhibitory concentrations (IC(min)) of sunitinib quenched its primary targets: FLT-3, VEGFR-2, and RET. In contrast, ERK, HCK, Chk2, YES, CREB, MEK, MSK, p38, FGR, and AXL were hyperactivated. Monotherapy with sunitinib or PD98059 at their IC(min) reduced proliferation by 23% and 19%, respectively, in H295R cells and by 25% and 24%, respectively, in SW13 cells. Sunitinib and PD98059 in combination decreased proliferation by 68% and 64% in H295R and in SW13 cells, respectively (P < .05 versus monotherapy). The effects of combination treatment exceeded the sum of the effects observed with each individual agent alone. Conclusion: We describe the first preclinical model to develop strategic combination therapy to overcome tyrosine kinase coactivation in ACC. Because many tyrosine kinase inhibitors are readily available, this model can be immediately tested in clinical trials for patients with advanced ACC.
MeSH term(s)	Adrenal Cortex Neoplasms/drug therapy ; Adrenal Cortex Neoplasms/enzymology ; Adrenocortical Carcinoma/drug therapy ; Adrenocortical Carcinoma/enzymology ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; Calcium-Calmodulin-Dependent Protein Kinases/administration & dosage ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Dose-Response Relationship, Drug ; Enzyme Activation ; Flavonoids/administration & dosage ; Humans ; Indoles/administration & dosage ; Phosphorylation ; Protein-Tyrosine Kinases/antagonists & inhibitors ; Protein-Tyrosine Kinases/metabolism ; Pyrroles/administration & dosage ; Receptor Protein-Tyrosine Kinases/metabolism
Chemical Substances	Flavonoids ; Indoles ; Pyrroles ; Protein-Tyrosine Kinases (EC 2.7.10.1) ; Receptor Protein-Tyrosine Kinases (EC 2.7.10.1) ; Calcium-Calmodulin-Dependent Protein Kinases (EC 2.7.11.17) ; 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (SJE1IO5E3I) ; sunitinib (V99T50803M)
Language	English
Publishing date	2012-10-25
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	202467-6
ISSN	1532-7361 ; 0039-6060
ISSN (online)	1532-7361
ISSN	0039-6060
DOI	10.1016/j.surg.2012.08.035
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Article ; Online: Autophagic activation potentiates the antiproliferative effects of tyrosine kinase inhibitors in medullary thyroid cancer.

Lin, Chi-Iou / Whang, Edward E / Lorch, Jochen H / Ruan, Daniel T

Surgery

2012 Volume 152, Issue 6, Page(s) 1142–1149

Abstract: Background: We hypothesized that autophagy inhibition would enhance the anticancer efficacy of ret protooncogene-targeted therapy in medullary thyroid cancer.: Methods: Medullary thyroid cancer-1.1 and TT cells were treated with sunitinib or ... ...

Abstract	Background: We hypothesized that autophagy inhibition would enhance the anticancer efficacy of ret protooncogene-targeted therapy in medullary thyroid cancer. Methods: Medullary thyroid cancer-1.1 and TT cells were treated with sunitinib or sorafenib in the presence or absence of everolimus, trehalose, or small interfering RNA directed against autophagy protein 5. Results: Sunitinib and sorafenib each robustly induced light chain 3-II expression, indicating autophagy activation. Autophagy protein 5 silencing diminished the antiproliferative effects of sunitinib and sorafenib by 44% (P < .05) and 41% (P < .05), respectively, in medullary thyroid cancer-1.1 cells and by 43% (P < .01) and 39% (P < .05), respectively, in TT cells. In contrast, everolimus increased the antiproliferative effects of sunitinib and sorafenib by 24% (P < .01) and 27% (P < .01), respectively, in medullary thyroid cancer-1.1 cells and by 20% (P < .05) and 23% (P < .05), respectively, in TT cells. Trehalose increased the antiproliferative effects of sunitinib and sorafenib by 26% (P < .01) and 27% (P < .01), respectively, in medullary thyroid cancer-1.1 cells and by 28% (P < .05) and 29% (P < .05), respectively, in TT cells. Autophagy protein 5 silencing abrogated both everolimus- and trehalose-induced increases in tyrosine kinase inhibitor efficacy. Conclusion: Loss (gain) of autophagy diminishes (improves) the efficacy of sunitinib and sorafenib. Our findings suggest that autophagic activation should be combined with targeted ret protooncogene therapy for patients with advanced medullary thyroid cancer.
MeSH term(s)	Antineoplastic Agents/therapeutic use ; Autophagy/drug effects ; Benzenesulfonates/therapeutic use ; Benzimidazoles/pharmacology ; Carcinoma, Neuroendocrine ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Dose-Response Relationship, Drug ; Drug Synergism ; Everolimus ; Humans ; Immunosuppressive Agents/pharmacology ; Indoles/therapeutic use ; Niacinamide/analogs & derivatives ; Phenylurea Compounds ; Protein Kinase Inhibitors/therapeutic use ; Protein-Tyrosine Kinases/antagonists & inhibitors ; Proto-Oncogene Proteins c-ret/metabolism ; Pyridines/therapeutic use ; Pyrroles/therapeutic use ; Sirolimus/analogs & derivatives ; Sirolimus/pharmacology ; Thyroid Neoplasms/metabolism ; Thyroid Neoplasms/pathology ; Thyroid Neoplasms/physiopathology
Chemical Substances	AZD 6244 ; Antineoplastic Agents ; Benzenesulfonates ; Benzimidazoles ; Immunosuppressive Agents ; Indoles ; Phenylurea Compounds ; Protein Kinase Inhibitors ; Pyridines ; Pyrroles ; Niacinamide (25X51I8RD4) ; Everolimus (9HW64Q8G6G) ; sorafenib (9ZOQ3TZI87) ; Protein-Tyrosine Kinases (EC 2.7.10.1) ; Proto-Oncogene Proteins c-ret (EC 2.7.10.1) ; sunitinib (V99T50803M) ; Sirolimus (W36ZG6FT64)
Language	English
Publishing date	2012-12
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	202467-6
ISSN	1532-7361 ; 0039-6060
ISSN (online)	1532-7361
ISSN	0039-6060
DOI	10.1016/j.surg.2012.08.016
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Article: BCL6 mediates the effects of Gastrodin on promoting M2-like macrophage polarization and protecting against oxidative stress-induced apoptosis and cell death in macrophages

Jia, Jing / Chi-Iou Lin / Jianguo Li / Jie Gao / Mengya Liu / Qian Hua / Xiaojie Shi / Xiaoqian Jing / Xinzhi Guo

Biochemical and biophysical research communications. 2017 Apr. 29, v. 486

2017

Abstract	Cerebral palsy (CP) is the most common childhood disability worldwide, yet biomarkers for predicting CP are lacking. By subjecting peripheral blood samples from 62 CP patients and 30 healthy controls to Affymetrix GeneChipÂ® PrimeViewâ¢ HumanGene Expression Microarray analysis, we identified the novel biomarker B-cell lymphoma 6 (BCL6) as the most upregulated gene in the CP samples. Gastrodin is a traditional Chinese medicine and bioactive compound that promotes adductor angle release, as well as gross and fine motor performance by increasing Gross Motor Function Measure-66 and Fine Motor Function Measure-45 scores. Gastrodin upregulates the mRNA expression of Mgl2 and Mrc1, M2 macrophage markers, and arginase activity, an M2 polarization indicator, in murine RAW264.7 macrophages. Moreover, these effects were blocked by BCL6 siRNA, which also abrogated the protective effects of Gastrodin against hydrogen peroxide-induced apoptosis and death in RAW264.7Â cells. Our work identified BCL6 as a novel biomarker for early prediction of CP. Moreover, we demonstrated that Gastrodin not only stimulated polarization toward M2-like macrophages, which promote tissue repair, but also rescued macrophages from oxidative stress, apoptosis and death by inducing BCL6 expression. BCL6-targeted therapeutic strategies have promise for improving motor performance in CP patients.
Keywords	apoptosis ; arginase ; B-cell lymphoma ; bioactive compounds ; biomarkers ; blood sampling ; cerebral palsy ; childhood ; gene expression ; gene expression regulation ; genes ; hydrogen peroxide ; macrophages ; messenger RNA ; mice ; microarray technology ; Oriental traditional medicine ; oxidative stress ; patients ; prediction ; protective effect ; small interfering RNA ; tissue repair
Language	English
Dates of publication	2017-0429
Size	p. 458-464.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	205723-2
ISSN	0006-291X ; 0006-291X
ISSN (online)	0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2017.03.062
Database	NAL-Catalogue (AGRICOLA)

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Article: Intracellular distribution of TM4SF1 and internalization of TM4SF1-antibody complex in vascular endothelial cells

Sciuto, Tracey E / Merley, Anne / Lin, Chi-Iou / Richardson, Douglas / Liu, Yu / Li, Dan / Dvorak, Ann M / Dvorak, Harold F / Jaminet, Shou-Ching S

Biochemical and biophysical research communications. 2015 Sept. 25, v. 465

2015

Abstract: Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane-associated glycoprotein that is highly and selectively expressed on the plasma membranes of tumor cells, cultured endothelial cells, and, in vivo, on tumor-associated endothelium. ... ...

Abstract	Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane-associated glycoprotein that is highly and selectively expressed on the plasma membranes of tumor cells, cultured endothelial cells, and, in vivo, on tumor-associated endothelium. Immunofluorescence microscopy also demonstrated TM4SF1 in cytoplasm and, tentatively, within nuclei. With monoclonal antibody 8G4, and the finer resolution afforded by immuno-nanogold transmission electron microscopy, we now demonstrate TM4SF1 in uncoated cytoplasmic vesicles, nuclear pores and nucleoplasm. Because of its prominent surface location on tumor cells and tumor-associated endothelium, TM4SF1 has potential as a dual therapeutic target using an antibody drug conjugate (ADC) approach. For ADC to be successful, antibodies reacting with cell surface antigens must be internalized for delivery of associated toxins to intracellular targets. We now report that 8G4 is efficiently taken up into cultured endothelial cells by uncoated vesicles in a dynamin-dependent, clathrin-independent manner. It is then transported along microtubules through the cytoplasm and passes through nuclear pores into the nucleus. These findings validate TM4SF1 as an attractive candidate for cancer therapy with antibody-bound toxins that have the capacity to react with either cytoplasmic or nuclear targets in tumor cells or tumor-associated vascular endothelium.
Keywords	cytoplasmic vesicles ; drugs ; endothelial cells ; endothelium ; fluorescence microscopy ; glycoproteins ; microtubules ; monoclonal antibodies ; neoplasm cells ; neoplasms ; plasma membrane ; surface antigens ; therapeutics ; toxins ; transmission electron microscopy
Language	English
Dates of publication	2015-0925
Size	p. 338-343.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	205723-2
ISSN	0006-291X ; 0006-291X
ISSN (online)	0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2015.07.142
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Article: Sphingosine 1-phosphate regulates inflammation-related genes in human endothelial cells through S1P1 and S1P3.

Lin, Chi-Iou / Chen, Chiung-Nien / Lin, Po-Wei / Lee, Hsinyu

Biochemical and biophysical research communications

2007 Volume 355, Issue 4, Page(s) 895–901

Abstract: Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid (LPL) ligand that binds endothelial differentiation gene (Edg) family G-protein-coupled receptors and has been implicated as an important regulator in endothelial cells during inflammation ... ...

Abstract	Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid (LPL) ligand that binds endothelial differentiation gene (Edg) family G-protein-coupled receptors and has been implicated as an important regulator in endothelial cells during inflammation processes. In this study, we attempt to determine which S1P receptors mediating the inflammatory response in human endothelial cells. Our results indicated that introduction of siRNA against S1P(1) significantly suppressed S1P-induced ICAM-1 mRNA, total protein, and cell surface expressions in human umbilical vein endothelial cells (HUVECs). Moreover, U937 cells adhesion to S1P-treated HUVECs was profoundly reduced by knock-down of S1P(1) in HUVECs. By knock-down of S1P(1) or S1P(3) in HUVECs, S1P-enhanced IL-8, MCP-1 mRNA expression, and THP-1 cell chemotaxis toward S1P-treated HUVEC-conditioned media was profoundly reduced. These results suggested that S1P-induced inflammatory response genes expression is mediated through S1P(1) and S1P(3). Our findings suggest the possible utilization of S1P(1) or S1P(3) as drug targets to treat severe inflammation.
MeSH term(s)	Cell Adhesion/drug effects ; Cells, Cultured ; Chemotaxis ; Endothelial Cells/cytology ; Endothelial Cells/drug effects ; Endothelial Cells/metabolism ; Gene Expression Regulation/genetics ; Humans ; Inflammation Mediators/metabolism ; Intercellular Adhesion Molecule-1/genetics ; Intercellular Adhesion Molecule-1/metabolism ; Lysophospholipids/metabolism ; Lysophospholipids/pharmacology ; RNA, Messenger/genetics ; RNA, Small Interfering/genetics ; Receptors, Lysosphingolipid/genetics ; Receptors, Lysosphingolipid/metabolism ; Sphingosine/analogs & derivatives ; Sphingosine/metabolism
Chemical Substances	Inflammation Mediators ; Lysophospholipids ; RNA, Messenger ; RNA, Small Interfering ; Receptors, Lysosphingolipid ; Intercellular Adhesion Molecule-1 (126547-89-5) ; sphingosine 1-phosphate (26993-30-6) ; Sphingosine (NGZ37HRE42) ; lysophosphatidic acid (PG6M3969SG)
Language	English
Publishing date	2007-04-20
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	205723-2
ISSN	0006-291X ; 0006-291X
ISSN (online)	0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2007.02.043
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Article ; Online: Intracellular distribution of TM4SF1 and internalization of TM4SF1-antibody complex in vascular endothelial cells.

Sciuto, Tracey E / Merley, Anne / Lin, Chi-Iou / Richardson, Douglas / Liu, Yu / Li, Dan / Dvorak, Ann M / Dvorak, Harold F / Jaminet, Shou-Ching S

Biochemical and biophysical research communications

2015 Volume 465, Issue 3, Page(s) 338–343

Abstract	Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane-associated glycoprotein that is highly and selectively expressed on the plasma membranes of tumor cells, cultured endothelial cells, and, in vivo, on tumor-associated endothelium. Immunofluorescence microscopy also demonstrated TM4SF1 in cytoplasm and, tentatively, within nuclei. With monoclonal antibody 8G4, and the finer resolution afforded by immuno-nanogold transmission electron microscopy, we now demonstrate TM4SF1 in uncoated cytoplasmic vesicles, nuclear pores and nucleoplasm. Because of its prominent surface location on tumor cells and tumor-associated endothelium, TM4SF1 has potential as a dual therapeutic target using an antibody drug conjugate (ADC) approach. For ADC to be successful, antibodies reacting with cell surface antigens must be internalized for delivery of associated toxins to intracellular targets. We now report that 8G4 is efficiently taken up into cultured endothelial cells by uncoated vesicles in a dynamin-dependent, clathrin-independent manner. It is then transported along microtubules through the cytoplasm and passes through nuclear pores into the nucleus. These findings validate TM4SF1 as an attractive candidate for cancer therapy with antibody-bound toxins that have the capacity to react with either cytoplasmic or nuclear targets in tumor cells or tumor-associated vascular endothelium.
MeSH term(s)	Antibodies, Monoclonal/immunology ; Antigens, Surface/immunology ; Cells, Cultured ; Clathrin/immunology ; Dynamins/immunology ; Endothelial Cells/immunology ; Endothelial Cells/ultrastructure ; Humans ; Neoplasm Proteins/immunology ; Subcellular Fractions/immunology
Chemical Substances	Antibodies, Monoclonal ; Antigens, Surface ; Clathrin ; Neoplasm Proteins ; TM4SF1 protein, human (147016-68-0) ; Dynamins (EC 3.6.5.5)
Language	English
Publishing date	2015-09-25
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	205723-2
ISSN	1090-2104 ; 0006-291X ; 0006-291X
ISSN (online)	1090-2104 ; 0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2015.07.142
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Global tyrosine kinome profiling of human thyroid tumors identifies Src as a promising target for invasive cancers.

Cho, Nancy L / Lin, Chi-Iou / Du, Jinyan / Whang, Edward E / Ito, Hiromichi / Moore, Francis D / Ruan, Daniel T

Biochemical and biophysical research communications

2012 Volume 421, Issue 3, Page(s) 508–513

Abstract: Background: Novel therapies are needed for the treatment of invasive thyroid cancers. Aberrant activation of tyrosine kinases plays an important role in thyroid oncogenesis. Because current targeted therapies are biased toward a small subset of tyrosine ...

Abstract	Background: Novel therapies are needed for the treatment of invasive thyroid cancers. Aberrant activation of tyrosine kinases plays an important role in thyroid oncogenesis. Because current targeted therapies are biased toward a small subset of tyrosine kinases, we conducted a study to reveal novel therapeutic targets for thyroid cancer using a bead-based, high-throughput system. Methods: Thyroid tumors and matched normal tissues were harvested from twenty-six patients in the operating room. Protein lysates were analyzed using the Luminex immunosandwich, a bead-based kinase phosphorylation assay. Data was analyzed using GenePattern 3.0 software and clustered according to histology, demographic factors, and tumor status regarding capsular invasion, size, lymphovascular invasion, and extrathyroidal extension. Survival and invasion assays were performed to determine the effect of Src inhibition in papillary thyroid cancer (PTC) cells. Results: Tyrosine kinome profiling demonstrated upregulation of nine tyrosine kinases in tumors relative to matched normal thyroid tissue: EGFR, PTK6, BTK, HCK, ABL1, TNK1, GRB2, ERK, and SRC. Supervised clustering of well-differentiated tumors by histology, gender, age, or size did not reveal significant differences in tyrosine kinase activity. However, supervised clustering by the presence of invasive disease showed increased Src activity in invasive tumors relative to non-invasive tumors (60% v. 0%, p<0.05). In vitro, we found that Src inhibition in PTC cells decreased cell invasion and proliferation. Conclusion: Global kinome analysis enables the discovery of novel targets for thyroid cancer therapy. Further investigation of Src targeted therapy for advanced thyroid cancer is warranted.
MeSH term(s)	Adenocarcinoma, Papillary/drug therapy ; Adenocarcinoma, Papillary/enzymology ; Adenocarcinoma, Papillary/pathology ; Cell Proliferation ; Dasatinib ; High-Throughput Screening Assays ; Humans ; Metabolome ; Molecular Targeted Therapy ; Mutation ; Neoplasm Invasiveness ; Phosphorylation ; Protein Kinase Inhibitors/pharmacology ; Pyrimidines/pharmacology ; Thiazoles/pharmacology ; Thyroid Neoplasms/drug therapy ; Thyroid Neoplasms/enzymology ; Thyroid Neoplasms/pathology ; Tumor Cells, Cultured ; Tyrosine/metabolism ; Up-Regulation ; src-Family Kinases/antagonists & inhibitors ; src-Family Kinases/genetics ; src-Family Kinases/metabolism
Chemical Substances	Protein Kinase Inhibitors ; Pyrimidines ; Thiazoles ; Tyrosine (42HK56048U) ; src-Family Kinases (EC 2.7.10.2) ; Dasatinib (RBZ1571X5H)
Language	English
Publishing date	2012-05-11
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	205723-2
ISSN	1090-2104 ; 0006-291X ; 0006-291X
ISSN (online)	1090-2104 ; 0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2012.04.034
Database	MEDical Literature Analysis and Retrieval System OnLINE

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