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Biomedical subjects

T M Roberts

Publications and source records attributed to T M Roberts.

At least 145 records · Page 8Linked to original sources
PubMed1988

Voltage-sensitive calcium channels in normal and transformed 3T3 fibroblasts.

Patch clamp recordings of whole-cell and single channel currents revealed the presence of two voltage-sensitive calcium channel types in the membrane of 3T3 fibroblasts. The two calcium channel types were identified by their unitary properties and pharmacological sensitivities. Both calcium channel types were present in all control 3T3 cells, but one type was selectively suppressed in 3T3 cells that had been transformed by activated c-H-ras, EJ-ras, v-fms, or polyoma middle T oncogenes. The presence of voltage-sensitive calcium channels in these nonexcitable cells and the control of their functional expression by transforming oncogenes raises questions about their role in the control of calcium-sensitive processes such as cell motility, cytoskeletal organization, and cell growth.

Animals↗
PubMed1988

Relationship between plasma membrane mobility and substrate attachment in the crawling movement of spermatozoa from Caenorhabditis elegans.

Caenorhabditis elegans sperm are nonflagellated cells that lack actin and myosin yet can form pseudopods to propel themselves over solid substrates. Surface-attached probes such as latex beads, lectins, and antimembrane protein monoclonal antibodies move rearward over the dorsal pseudopod surface of sessile cells. Using monoclonal antibodies against membrane proteins of C. elegans sperm to examine the role of localized membrane assembly and rearward flow in crawling movement, we determined that substrates prepared by coating glass with antimembrane protein antibodies, but not naked glass or other nonmembrane-binding proteins, promote sperm motility. Sperm locomotion is inhibited in a concentration-dependent fashion when cells are bathed with soluble antimembrane protein monoclonal antibodies but not with antimouse Ig antibodies or a monoclonal antibody against a sperm cytoplasmic protein. Our results suggest that C. elegans sperm crawl by gaining traction with substrate-attached ligands via their surface proteins and by using the motor that moves those proteins rearward on unattached cells to pull the entire cell forward. Continuous insertion of new proteins at the front of the cell and their subsequent adhesion to the substrate allows this process to continue.

Animals↗
PubMed1988

Effects of ozone on foliar leaching in Norway spruce (Picea abies L. Karst): confounding factors due to NOx production during ozone generation.

Previous experiments with conifers fumigated with O(3), produced by air-operated electric discharge ozonators, have provided evidence that O(3) increases the leaching of NO(3)(-), NH(4)(+), K(+), Ca(2+), Mg(2+) and some other cations from needles, when the trees are treated with acid mist. This evidence has provided the foundation of the ozone-acid mist hypothesis of spruce decline. We report experiments with Norway spruce saplings fumigated with purified and unpurified O(3). The results show that the accumulation of NO(3)(-) in the needles arises from the rapid deposition of N(2)O(5) and HNO(3) formed from N(2) in the ozonator. An increase in removal of NH(4)(+), Na(+), Ca(2+), Mg(2+), Zn(2+) and Mn(2+) from the needles during soaking in H(2)SO(4), pH3, was also observed, which was related to the increase in NO(3)(-) but was independent of O(3) concentration. It is concluded that results of previous experiments cited in support of the ozone-acid mist hypothesis arose from effects which were at least partly caused by N(2)O(5) produced as a contaminant, and were incorrectly attributed to ozone. Other effects, such as growth stimulations, visible symptons, enhanced frost sensitivity, and infestation by pests or pathogens, which have been attributed to O(3) generated by electric discharge in air, should be interpreted with caution. Future experiments with ozone must eliminate this problem by either using O(2)-driven ozonators, or by purifying the output from air-driven ozonators using cold and/or water traps.

Journal Article↗
PubMed1988

Signal transduction from membrane to cytoplasm: growth factors and membrane-bound oncogene products increase Raf-1 phosphorylation and associated protein kinase activity.

We have examined the phosphorylation and the serine/threonine-specific kinase activity of the protooncogene product Raf-1 (formerly c-raf) in response to oncogenic transformation or growth-factor treatment of mouse 3T3 cells. Expression of the membrane-bound oncogene products encoded by v-fms, v-src, v-sis, polyoma virus middle-sized tumor antigen, and Ha-ras increased the apparent molecular weight and phosphorylation of the Raf-1 protein, while expression of the nuclear oncogene and protooncogene products encoded by v-fos and c-myc did not. Changes in electrophoretic mobility and phosphorylation occurred rapidly in response to treatment of cells with platelet-derived growth factor, acidic fibroblast growth factor, epidermal growth factor, and the protein kinase C activator phorbol 12-myristate 13-acetate, but not insulin. The phosphorylation of the Raf-1 protein occurred primarily on serine and threonine residues. However, a subpopulation of Raf-1 molecules was phosphorylated on tyrosine residues in cells transformed by v-src or stimulated with platelet-derived growth factor. Transformation by v-src, or treatment with platelet-derived growth factor or phorbol 12-myristate 13-acetate, activated the Raf-1-associated serine/kinase activity as measured in immune-complex kinase assays. These findings suggest that proliferative signals generated at the membrane result in the phosphorylation of the Raf-1 protein and the activation of its serine/threonine kinase activity. Raf-1 activation may thus serve to transduce signals from the membrane to the cytoplasm and perhaps on to the nucleus.

Animals↗
PubMed1988

Cellular proteins that associate with the middle and small T antigens of polyomavirus.

We have used two-dimensional gel electrophoresis to analyze in more detail the cellular proteins which associate with the middle and small tumor antigens (MT and ST, respectively) of polyomavirus. Proteins with molecular masses of 27, 29, 36, 51, 61, 63, and 85 kilodaltons (kDa) that specifically coimmunoprecipitated with MT were identified on these gels. The 36-, 51-, 61-, 63-, and 85-kDa proteins are probably the same as the proteins of similar sizes previously reported by a number of groups, whereas the 27- and 29-kDa proteins represent proteins that are heretofore undescribed. The 27- and 29-kDa proteins were abundant cellular proteins, whereas the others were minor cellular constituents. The association of each of these proteins with MT was sensitive to one or more mutations in MT that rendered it transformation defective. The association of the 85-kDa protein was the most sensitive indicator of the transformation competence of MT mutants. In addition, the 85-kDa protein was the only associated protein whose association with MT changed consistently in parallel with MT-associated phosphatidylinositol kinase activity. Furthermore, the fraction of the 85-kDa protein which was found associated with the MT complex contained 15 to 20% of its phosphate content on tyrosine. The 36- and 63-kDa proteins complexed with both polyomavirus MT and ST and comigrated on two-dimensional gels with two simian virus 40 ST-associated proteins originally described by Rundell and coworkers (K. Rundell, E. O. Major, and M. Lampert, J. Virol. 37:1090-1093, 1981). None of the other MT-associated proteins associated significantly with ST.

Antigens, Polyomavirus Transforming↗
PubMed1988

Recombinant retroviruses that transduce middle T antigen cDNAs derived from polyomavirus mutants: separation of focus formation and soft-agar growth in transformation assays and correlations with kinase activities in vitro.

To study correlations between cellular transformation and the biochemical properties of polyomavirus middle T antigen, middle T cDNAs have been derived from the polyomavirus mutants dl1015, dl23, and NG59b and have been introduced into rodent fibroblast cell lines by using a retrovirus vector. It was found that all three mutants are completely defective in inducing growth in soft agar but possess a range of activities in assays of focus formation on cell monolayers. Furthermore, when assays of middle T antigen-associated kinase activities were performed in vitro, a correlation between the level of associated phosphatidylinositol kinase activity and the ability of mutant middle T antigens to induce focus formation was observed. However, the association of this activity with middle T antigen does not appear to be sufficient to bring about full transformation, since the middle T antigen derived from dl1015 is completely defective for soft-agar growth but is associated with a level of phosphatidylinositol kinase activity which is comparable to that of the wild type. Therefore, some other unidentified middle T antigen function may also be required for full transformation.

1-Phosphatidylinositol 4-Kinase↗
PubMed1988

Tyrosine phosphorylations in vivo associated with v-fms transformation.

The role of tyrosine-specific phosphorylation in v-fms-mediated transformation was examined by immunoblotting techniques together with a high-affinity antibody that is specific for phosphotyrosine. This antiphosphotyrosine antibody detected phosphorylated tyrosine residues on the gp140v-fms molecule, but not gP180v-fms or gp120v-fms, in v-fms-transformed cells. This antibody also identified a number of cellular proteins that were either newly phosphorylated on tyrosine residues or showed enhanced phosphorylation on tyrosine residues as a result of v-fms transformation. However, the substrates of the v-fms-induced tyrosine kinase activity were not the characterized pp60v-src substrates. The phosphorylation of some of these cellular proteins and of the gp140fms molecule was found to correlate with the ability of v-fms/c-fms hybrids to transform cells. In addition, immunoblotting with the phosphotyrosine antibody allowed a comparison to be made of the substrates phosphorylated on tyrosine residues in various transformed cell lines. This study indicates that the pattern of tyrosine phosphorylation in v-fms-transformed cells is strikingly similar to that in v-sis-transformed cells.

Animals↗
PubMed1988

Separation of simian virus 40 large-T-antigen-transforming and origin-binding functions from the ability to block differentiation.

Wild-type simian virus 40 large T antigen is very effective at blocking adipocyte differentiation in 3T3-F442A cells as assayed by triglyceride accumulation, induction of glycerophosphate dehydrogenase activity, and expression of mRNAs for glycerophosphate dehydrogenase, the adipocyte serine protease adipsin, and the putative lipid-binding protein adipocyte P2. Point mutants defective for either origin-specific DNA binding or transformation blocked differentiation as completely as wild type.

Adipose Tissue↗
PubMed1988

The carboxy terminus of pp60c-src is a regulatory domain and is involved in complex formation with the middle-T antigen of polyomavirus.

A large number of mutations were introduced into the carboxy-terminal domain of pp60c-src. The level of phosphorylation on Tyr-416 and Tyr-527, the transforming activity (as measured by focus formation on NIH 3T3 cells), kinase activity, and the ability of the mutant pp60c-src to associate with the middle-T antigen of polyomavirus were examined. The results indicate that Tyr-527 is a major carboxy-terminal element responsible for regulating pp60c-src in vivo. A good but not perfect correlation exists between lack of phosphorylation at Tyr-527 and increased phosphorylation at Tyr-416, between elevated phosphorylation on Tyr-416 and activated kinase activity, and between activated kinase activity and transforming activity. Phosphorylation of Tyr-527 was insensitive to the mutation of adjacent residues, indicating that the primary sequence only has a minor role in recognition by kinases or phosphatases which regulate it in vivo. Three mutants which have in common a modified Glu-524 residue were phosphorylated on Tyr-416 and Tyr-527 and were weakly transforming. This suggests that other mechanisms besides complete dephosphorylation of Tyr-527 can lead to increased phosphorylation of Tyr-416 and activation of the transforming activity of pp60c-src. Furthermore, the residues between Asp-518 and Pro-525 were required to form a stable complex with middle-T antigen. The proximity of these sequences to Tyr-527 suggests a model in which middle-T activates pp60c-src by binding directly to this region of the molecular and thereby preventing phosphorylation of Tyr-527. Alternatively, middle-T binding may mediate a conformational change in this region, which in turn induces an alteration in the level of phosphorylation at Tyr-527 and Tyr-416.

Amino Acid Sequence↗
PubMed1988

A Moloney MLV-rat somatotropin fusion gene produces biologically active somatotropin in a transgenic pig.

Expression of a Moloney murine leukemia virus (MLV) rat somatotropin fusion gene was examined in a transgenic pig. The fusion gene was integrated in a single site within the genome in a tandem array with approximately eight copies per cell. The integrated in a single site within the genome in a tandem array with approximately eight copies per cell. The integrated MLV-rat somatotropin fusion gene produced high levels of circulating rat somatotropin and resulted in an elevation in the circulating levels of insulin-like growth factor I. Although there was no increase in the rate of growth of the transgenic animal during the rapid growth phase, several phenotypic changes were evident. Skeletal growth was markedly increased and fat deposition was reduced throughout the animal. Blood glucose levels were elevated without ketosis. Northern blot analyses of rat somatotropin RNA revealed that expression of the fusion gene was highest in the spleen, lung, intestine, lymph nodes, and bone marrow. These results show that the MLV promoter can be used to express high levels of biologically active rat somatotropin in transgenic swine.

Animals↗
PubMed1987

The in vitro behavior of hemopoietic cells transformed by polyoma middle T antigen parallels that of primary human myeloid leukemic cells.

A retrovirus encoding polyoma middle T antigen has been used to infect a murine hemopoietic cell line (FDC-P1) dependent on either granulocyte-macrophage colony-stimulating factor (GM-CSF) or multipotential colony-stimulating factor (Multi-CSF). A number of cell lines have been established on the basis of their initial ability to proliferate in the absence of added colony-stimulating factor (CSF). The transformed lines display one of three patterns of growth in vitro: those able to grow fully autonomously; those whose proliferation depends on cell density; and those displaying dependence on added CSF regardless cell density. This latter class of cells are reminiscent of the majority of primary myeloid leukemic cells. Unlike parental FDC-P1 cells, all three classes of transformed cells are leukemogenic in syngeneic mice; moreover, they produce variable amounts of GM-CSF which we believe underlies their neoplastic behavior.

Animals↗
PubMed1987

Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity.

The phosphorylation of proteins on tyrosine in vivo and in vitro was examined in 3T3 cells stimulated by platelet-derived growth factor (PDGF) and transformed by polyoma middle T antigen (MTAg) by using an antibody directed against phosphotyrosine (P-tyr). Two common events were observed upon PDGF stimulation or MTAg transformation of cells: the appearance in the immunoprecipitates of an 85 kd phosphoprotein, and increased phosphatidylinositol (PI) kinase activity. In PDGF-stimulated cells, the 85 kd phosphoprotein and PI kinase activity appeared rapidly, within 1 min of growth factor addition. The PI kinase activity and 85 kd phosphorylation were also increased in anti-P-tyr immunoprecipitates from cells transformed by v-fms and v-sis, but not by SV40 T antigen. The presence of the tyrosine-phosphorylated 85 kd protein correlated with PI kinase activity during several purification steps. These results suggest that the 85 kd phosphoprotein, a putative PI kinase, is a substrate for both the PDGF receptor and MTAg/pp60c-src tyrosine kinase activities.

1-Phosphatidylinositol 4-Kinase↗
PubMed1987

Heterogeneous mutations in the beta subunit common to the LFA-1, Mac-1, and p150,95 glycoproteins cause leukocyte adhesion deficiency.

Leukocyte adhesion deficiency (LAD) is a heritable disease involving deficient expression of three related leukocyte adhesion glycoproteins: LFA-1, Mac-1, and p150,95. These proteins are alpha beta heterodimers containing identical 95,000 dalton beta subunits. Here we demonstrate that the primary defect in LAD is in the beta subunit gene. We identified five distinct beta subunit phenotypes in LAD patients: undetectable beta subunit mRNA and protein precursor; low levels of beta subunit mRNA and precursor; an aberrantly large beta subunit precursor, probably due to an extra glycosylation site; an aberrantly small precursor; and a grossly normal precursor. Mutant beta subunit precursors from LAD patients failed to associate with the LFA-1 alpha subunit. In family studies, inheritance of the aberrant precursors correlates with the known inheritance of the LAD defect.

Antibodies↗
PubMed1987

lac repressor can regulate expression from a hybrid SV40 early promoter containing a lac operator in animal cells.

The E. coli lac operator and repressor were adapted for function in mammalian cells. Plasmids containing an SV40 early region (pSVlacO) or a chloramphenicol acetyl transferase gene (pSVlacOCAT) linked to a hybrid SV40 early promoter bearing a lac operator were tested for function. Identical plasmids lacking an operator (pX-8 and pX-8CAT) were controls. In vitro, early transcription from pSVlacO, but not from pX-8, was inhibited by lac repressor, and repression was overcome by IPTG. Repression of large T synthesis or CAT activity occurred in vivo only when the respective operator-containing plasmid was cotransfected with a plasmid encoding lac repressor, or when the recipient cells stably synthesized lac repressor. IPTG substantially relieved repression in both cases. CAT enzyme repression was paralleled by a decrease in CAT mRNA abundance. Thus regulatory elements of the lac operon function physiologically in mammalian cells.

Acetyltransferases↗
PubMed1987

Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src.

To investigate the importance of tyrosine phosphorylation in the regulation of pp60c-src, we have substituted phenylalanine for tyrosine at positions 416, 519, and 527. Cells expressing the 527 or the 519/527 mutant but not the 416 or the 519 mutant were morphologically transformed, grew in soft agar, and formed foci. In addition, the 527 and 519/527 mutants had elevated kinase activities in vitro. Modifying Tyr 416 to phenylalanine in the 527 or the 519/527 mutants only partially inhibited their kinase activities yet abolished their ability to induce focus formation and promote growth in soft agar. These results suggest that two events must occur to activate the full transforming potential of pp60c-src: hypophosphorylation at Tyr 527 and hyperphosphorylation at Tyr 416.

Animals↗
PubMed1987

Cloning of the beta subunit of the leukocyte adhesion proteins: homology to an extracellular matrix receptor defines a novel supergene family.

We have isolated cDNA clones encoding the beta subunit of the human LFA-1, Mac-1, and p150,95 family of leukocyte adhesion proteins. The deduced 769-amino-acid sequence defines a cysteine-rich polypeptide with the characteristic features of an integral membrane protein. Peptide sequence data, Northern blot analysis, and Southern blot analysis suggest that a single gene encodes the beta subunit of all three leukocyte adhesion proteins. There is 45% homology between the beta subunit sequence and band III of integrin, a chick fibronectin and laminin receptor. This homology defines a new supergene family of cellular adhesion proteins.

Amino Acid Sequence↗
PubMed1987

Caenorhabditis elegans spermatozoa assemble membrane proteins onto the surface at the tips of pseudopodial projections.

The crawling movement of nematode sperm, like that of many other crawling metazoan cells, is accompanied by movement of membrane components from the leading edge of the cell rearward. We used colloidal gold conjugates of monoclonal antibodies (CGP-ABY) to membrane proteins on Caenorhabditis elegans sperm to examine this surface movement by electron microscopy. Antibody binding sites on fixed sperm are distributed uniformly over the cell surface. However, blocking these sites on live sperm with unlabelled antibody or removing them with protease and then pulse-labelling the cell with CGP-ABY revealed that new antigen is assembled onto the surface at the tips of the stubby projections that stud the pseudopod surface. These proteins then move rearward rapidly so that the pseudopod surface pool of antigen is replaced within 2 min. The same pattern of surface movement was observed when live cells were labelled with CGP-ABY and then washed with buffer before fixation. Bound CGP-ABY was cleared first from the tips of the projections and subsequently from the entire pseudopod surface. These gold particles accumulated at the base of the pseudopod without moving onto the cell body or being internalized. We did, however, detect a pool of antigen in the pseudopod cytoplasm that may be available for assembly onto the pseudopod surface. We propose that the localized assembly of new membrane and its subsequent rearward movement may play an important role in sperm locomotion.

Animals↗