Changes in brain function after manipulation of the cervical spine.
Explore the source record for details and available documents.
Brain Mapping↗
Biomedical subjects
M Roudebush
Publications and source records attributed to M Roudebush.
Neurofibromin colocalizes with mitochondria in cultured cells.
Mutations in neurofibromatosis type 1 target the gene coding for neurofibromin. While neurofibromin is able to accelerate the rate of GTP hydrolysis by cellular Ras proteins, its biological function is not well understood. To gain information regarding its function, the intracellular localization of neurofibromin was analyzed in cultured cell lines using polyclonal antisera raised against four neurofibromin-specific peptides, three from the carboxyl terminus and one from the amino terminus. In methanol-fixed cells distinct rod-like structures distributed throughout the cytoplasm were recognized by the antisera. Similar structures were seen with each antiserum, including affinity-purified antibodies, and in each of the cultured cell lines tested. Similar structures were seen in paraformaldehyde-fixed cells. Double staining experiments showed that these structures colocalize with mitochondria, but not with actin, beta-tubulin, or endoplasmic reticulum. When actin or tubulin structures within the cell were disrupted by separate antimitotic drugs, these stained structures retained their shape. Neurofibromin association with mitochondria was confirmed biochemically when highly purified mitochondrial fractions from bovine heart tissue were shown in Western analysis to contain neurofibromin. This association might be helpful in predicting identification of some of the cellular proteins with which neurofibromin interacts.
3T3 Cells↗
Reduced expression of neurofibromin in human meningiomas.
Meningiomas are common, mostly benign, tumours arising from leptomeningeal cells of the meninges, which frequently contain mutations in the neurofibromatosis type 2 (NF2) gene. In this study, we analysed a protein product of the neurofibromatosis type 1 (NF1) gene, neurofibromin, in human established leptomeningeal cells LTAg2B, in 17 sporadic meningiomas and in a meningioma from a patient affected by NF2. The expression level of neurofibromin was determined by immunoblotting and immunoprecipitation with anti-neurofibromin antibodies. The functional status of neurofibromin was analysed through its ability to stimulate the intrinsic GTPase activity of p21 ras. In the cytosolic extracts of four sporadic meningiomas and in the NF2-related meningioma, the expression level and the GTPase stimulatory activity of neurofibromin were drastically reduced compared with the level present in the human brain, human established leptomeningeal cells LTAg2B and the remaining 13 meningiomas. Our results suggest that neurofibromin is expressed in leptomeningeal cells LTAg2B and in most meningiomas, i.e. tumours derived from these cells. The reduced expression and GTPase stimulatory activity of neurofibromin was found in about 23% of meningiomas and in the single NF2-related meningioma analysed. These results suggest that decreased levels of neurofibromin in these tumours may contribute to their tumorigenesis.
Adult↗
Catalytic properties, tissue and intracellular distribution of neurofibromin.
The neurofibromatosis type 1 (NF1) gene encodes a protein, neurofibromin, that shows homology with members of the GTPase-activating protein (GAP) family. To study neurofibromin, rabbit polyclonal antisera were raised against two synthetic peptides. These antisera immunoprecipitated a specific protein of about 240 kDa in lysates of adult murine and rat tissues both in the soluble (S100) and to a lesser degree in the particulate (P100) fractions. The neurofibromin immunoprecipitated from the lysates of several murine organs stimulated the intrinsic GTPase activity of p21 c-Ha-ras protein. Based on immunoblotting, immunoprecipitation and GTPase assays, neurofibromin appears to be at least 10-fold more abundant in the brain than in the other murine organs. The GTPase-stimulatory activity of full-length neurofibromin, like the catalytic GAP-related domain, is inhibited by arachidonic acid and the detergent dodecyl maltoside, while phosphatidic acid, containing arachidonic and stearic acid, is non-inhibitory. Immunofluorescence analysis with anti-neurofibromin sera in NIH3T3 cells suggests that at least some of the cellular protein associates with cytoplasmic structures that are distinct from actin or tubulin filaments.
Amino Acid Sequence↗
Ras GTPase-activating protein physically associates with mitogenically active phospholipids.
The physical interaction between GTPase-activating protein (GAP) and lipids has been characterized by two separate analyses. First, bacterially synthesized GAP molecules were found to associate with detergent-mixed micelles containing arachidonic but not with those containing arachidic acid. This association was detected by a faster elution time during molecular exclusion chromatography. Second, GAP molecules within a crude cellular lysate were specifically retained by a column on which certain lipids had been immobilized. The lipids able to retain GAP on such columns were identical to those which were shown previously to be most active in blocking GAP activity. The association between lipids and GAP was dependent upon magnesium ions. Lipids unable to inhibit GAP activity were also unable to physically associate with GAP. The tight association of GAP with these lipids was predicted by and helps to rationalize their ability to inhibit GAP activity.
Animals↗
Dominant inhibitory Ras mutants demonstrate the requirement for Ras activity in the action of tyrosine kinase oncogenes.
Two inhibitory Ras mutant proteins [(Asn 17) Ras and RAST] were microinjected into NIH3T3 cells in order to compare their inhibitory activity with that of a neutralizing anti-ras antibody. Both mutants were able to block efficiently the mitogenic effects of serum added to quiescent NIH3T3 cells. Furthermore, each of the inhibitors blocked cell cycle progression at the same point as the injected anti-ras antibody, just prior to the initiation of a new round of DNA synthesis. Finally, as with the injected anti-ras antibody, each of the inhibitors was efficiently able to block proliferation and reverse the transformed morphology of cells transformed by tyrosine kinase oncogenes, while cells transformed by serine kinase oncogenes were unaffected. Therefore, results with all three reagents clearly indicate that cellular Ras activity is required in the late G1 phase of the cell cycle and is essential for the maintenance of the transformed phenotype induced by tyrosine but not serine kinase oncogenes. These studies demonstrate the utility of dominant inhibitory mutants as a means of interfering with the activity of cellular oncogenes.
3T3 Cells↗