Scintillation proximity assay to measure binding of soluble fibronectin to antibody-captured alpha 5 beta 1 integrin.

A scintillation proximity assay (SPA) has been developed to measure binding of alpha 5 beta 1 integrin, a heterodimeric cell-surface adhesion receptor, to fibronectin. This assay utilizes an anti-beta 1 integrin monoclonal antibody to simultaneously capture alpha 5 beta 1 from a cellular lysate and couple the integrin to anti-mouse IgG antibody-coated SPA beads for detection of 125I-fibronectin binding. The assay does not require prior purification of alpha 5 beta 1 nor physical separation of bound and free 125I-fibronectin. Chinese hamster ovary cells that stably overexpress human alpha 5 integrin (CHO#7 cells) were used as a source of alpha 5 beta 1 fibronectin receptor. Using the anti-hamster beta 1 monoclonal antibody 7E2 to capture alpha 5 beta 1 from a CHO#7 cell lysate, this SPA assay allowed measurement of specific 125I-fibronectin binding as defined by displacement by the Arg-Gly-Asp containing peptide GRGDSP or the anti-human alpha 5 antibody P1D6. IC50 values for displacement of 125I-fibronectin binding by GRGDSP and the novel cyclic peptides cRGDGF, cRGEGF, and cRRETAWA were 2.6, 0.045, 3.2, and 37 microM, respectively. Specific 125I-fibronectin binding to alpha 5 beta 1 from C8161 human melanoma cells was also measured using anti-human beta 1 antibodies. This method should be generally useful to measure cell-free ligand binding to receptors that are difficult to purify.

Endothelin (ETA) receptor number and calcium signalling are up-regulated by protein kinase C-beta 1 overexpression.

To evaluate the role of protein kinase C (PKC) in regulation of cellular responsiveness to mitogens, we used rat 6 (R6) fibroblasts that stably overexpress the beta 1 isoenzyme of protein kinase C (PKC-beta 1). The potent vasoconstrictor and mitogen endothelin-1 (ET-1; 100 nM) was substantially more effective in stimulating InsP3 accumulation in PKC-beta 1-overexpressing fibroblasts (PKC3 cells) than in control fibroblasts lacking the PKC-beta 1 cDNA insert. PKC3 cells were found to express a 7-fold greater number of endothelin receptors than did control cells, whereas both cell lines showed equivalent Kd values. These receptors were of the ETA subtype, as defined by a 1000-fold greater affinity for ET-1 than for ET-3. Changes in intracellular free Ca2+ levels ([Ca2+]i) in response to ET-1 measured with the fluorescent Ca2+ indicator fura-2 showed that ET-1 was more potent and efficacious in stimulating [Ca2+]i in PKC3 cells than in control fibroblasts. The ET-1-induced Ca2+ rise was completely blocked by the selective ETA antagonist BQ123, but only slightly diminished by extracellular application of 2 mM EGTA. In contrast with the effects of PKC-beta 1 overexpression on responsiveness to ET-1, alpha-thrombin, which was previously found to have a weaker effect on InsP3 accumulation in PKC-beta 1-overexpressing cells, was also a less effective stimulator of [Ca2+]i in PKC3 cells than in control cells. These results demonstrate that, although the Ca2+ response to alpha-thrombin is diminished by PKC-beta 1 overexpression, ETA receptor number and cellular responsiveness to ET-1 are increased in PKC-beta 1-overexpressing cells.

Differential regulation of phosphoinositide and phosphatidylcholine hydrolysis by protein kinase C-beta 1 overexpression. Effects on stimulation by alpha-thrombin, guanosine 5'-O-(thiotriphosphate), and calcium.

Rat 6 fibroblasts that stably overexpress cDNA for the beta 1 isozyme of protein kinase C (PKC3 cells) were used to determine the effect of protein kinase C (PKC) overexpression on hormonal stimulation of phospholipid hydrolysis. In control Rat 6 cells, inositol trisphosphate levels (InsP3) were increased 9-fold in 15 s in response to 10 nM alpha-thrombin, compared with only a 2-fold increase in PKC3 cells. PKC overexpression also inhibited thrombin-stimulated production of 1,2-diacylglycerol, the other product of phosphatidylinositol 4,5-bisphosphate hydrolysis, by 73% at 15 s. In permeabilized cells, PKC overexpression greatly reduced guanosine thiotriphosphate-stimulated InsP3 accumulation, but did not affect InsP3 stimulation by increased free calcium concentration. These data suggest that desensitization of thrombin-stimulated phosphoinositide-phospholipase C is enhanced by PKC-beta 1 overexpression and may involve modulation of G-protein/phospholipase C coupling. In contrast, thrombin was 4.5-fold more effective in stimulation of phosphatidylcholine-phospholipase D activity in PKC3 cells than in control cells, as determined by phosphatidylethanol formation. In permeabilized cells, guanosine thiotriphosphate also stimulated phospholipase D activity more effectively in PKC3 cells than in control cells, suggesting that upregulation of phospholipase D activity by PKC overexpression occurs distal to the thrombin receptor. These results suggest that PKC may act as a switch to up-regulate phosphatidylcholine-phospholipase D and down-regulate phosphoinositide-phospholipase C stimulations.

NCTR computer systems designed for toxicologic experimentation. V. Post-experiment information system.

The Post-Experiment Information System (PEIS) is an automated data collection and reporting system composed of three specialized subsystems: Pathology, Chemistry and Microbiology. These subsystems function either independently or collectively to construct and maintain a comprehensive data base of all experimental values derived from, or associated with, an animal carcass. All data are retrievable by the unique Carcass Identification (CID) number assigned at death, which is the correlative of the Unique Identification Number (UIN) assigned to the animal at birth and used throughout its lifespan. Elements processed under the PEIS include gross and microscopic pathological observations, organ weights, hematologic data, chemical data, and microbiological analyses. The ability of the system to integrate the post-experiment data with the information collected on an animal from birth (BIS) and during the experiment (EIS) provides a complete animal history to the Principal Investigator or other requestor.

NCTR computer systems designed for toxicologic experimentation. IV. Experiment information system.

The Experiment Information System (EIS) is a computerized data collection, maintenance, and reporting system for specified information values collected during the lifespan of animals assigned to toxicologic investigations at NCTR. The system records and/or controls experimental variables, which might ultimately affect the results, through the operation and integration of the Diet Preparation Subsystem (DPS), the Environmental Monitoring Subsystem (EMS), the Microbiology Subsystem (MBS), and the Chemistry Data Subsystem (CDS). The fifth component of the EIS, the Experimental Data Collection Subsystem (EDCS), is responsible for handling all data generated by, or attributed to, the animals from assignment until death or removal. Through integration of these five subsystems, the history of an animal while on study is recorded and stored for later recall. In addition, "routine" and "special" reports are made available through the system software which enables stringent control of the experiment by the Principal Investigator, Animal Husbandry, and NCTR Management.