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J C Stone

Publications and source records attributed to J C Stone.

At least 19 recordsLinked to original sources
PubMed1997

Increased concentrations of phosphatidate, diacylglycerol and ceramide in ras- and tyrosine kinase (fps)-transformed fibroblasts.

Concentrations of the bioactive lipids, phosphatidate and diacylglycerol, increased with time in culture in ras- and tyrosine kinase (fps)-transformed fibroblasts but not in control fibroblasts. On Day 3, diacylglycerol and phosphatidate concentrations were about 3.3- and 5.5-fold higher respectively in the ras-transformed compared to control fibroblasts. These concentrations in fps-transformed fibroblasts were increased about twofold. The changes in phosphatidate and diacylglycerol resulted from enhanced phospholipid turnover rather than from synthesis de novo. The increased ratio of phosphatidate to diacylglycerol is explained by decreased activities of two distinct phosphatidate phosphohydrolases and increased diacylglycerol kinase in ras-transformed fibroblasts. Ceramide concentrations were about 2.5- and threefold higher in the fps- and ras-transformed cells respectively on Day 3 compared to the controls. Incubating control fibroblasts from Days 1 to 3 with phosphatidylcholine-specific phospholipase C increased diacylglycerol, phosphatidate and ceramide concentrations, and decreased Mg2+-independent-phosphatidate phosphohydrolase activity. 8-(4-chlorophenylthio)-cAMP had a cytostatic effect in ras-transformed cells, it decreased the concentrations of phosphatidate and diacylglycerol, but increased that of ceramide. The consequences of increased ceramide and phosphatidate concentrations in ras-transformed cells are discussed in relation to signal transduction, cell division and the transformed phenotype.

Animals↗
PubMed1997

Interaction of activated Ras with Raf-1 alone may be sufficient for transformation of rat2 cells.

v-H-ras effector mutants have been assessed for transforming activity and for the ability of the encoded proteins to interact with Raf-1-, B-Raf-, byr2-, ralGDS-, and CDC25-encoded proteins in the yeast two-hybrid system. Transformation was assessed in rat2 cells as well as in a mutant cell line, rv68BUR, that affords a more sensitive transformation assay. Selected mutant Ras proteins were also examined for their ability to interact with an amino-terminal fragment of Raf-1 in vitro. Finally, possible cooperation between different v-H-ras effector mutants and between effector mutants and overexpressed Raf-1 was assessed. Ras transforming activity was shown to correlate best with the ability of the encoded protein to interact with Raf-1. No evidence for cooperation between v-H-ras effector mutants was found. Signaling through the Raf1-MEK-mitogen-activated protein kinase cascade may be the only effector pathway contributing to RAS transformation in these cells.

Animals↗
PubMed1996

ras effector loop mutations that dissociate p120GAP and neurofibromin interactions.

ras proteins are positively regulated by nucleotide exchange factors and negatively regulated by GTPase-activating proteins (GAPs). Two GAPs have been found in mammalian cells, p120GAP and neurofibromin, the product of the type 1 neurofibromatosis (NF1) gene. A library of substitutions in the effector loop region of ras in an Escherichia coli plasmid expression system was screened for c-Ha-ras species with altered GAP interactions. Several substitutions preferentially disrupted the interaction of ras with p120GAP as compared with the interaction with the recombinant GAP-related domain of neurofibromin (NF1-GRD). The most extreme example, Tyr32His, encoded a ras species that was unaffected by p120GAP but was stimulated normally by NF1-GRD. Tyr32His was weakly transforming in Rat2 cells. Tyr32His ras was primarily GDP-bound in quiescent Rat2 cells, although it rapidly associated with GTP after treatment of cells with epidermal growth factor. These results show that the NF1 product has less stringent requirements than p120GAP for ras effector domain structure and that negative regulation of ras can be achieved in rat fibroblasts by the product of NF1.

Animals↗
PubMed1996

Stimulation of ras GTPase activity by an anti-ras monoclonal antibody.

Wild-type ras has GTPase activity, and this activity is accelerated substantially by GTPase-activating proteins (GAPs). Oncogenic ras species have an abnormally low intrinsic GTPase activity, and this activity is insensitive to GAPs. We confirmed that the anti-ras monoclonal antibody Y13-238 inhibited GAP activity in vitro, but we also noted that this antibody had GAP activity of its own. We studied the GAP activity of Y13-238 in circumstances in which ras GTPase activity was influenced by the GTPase-inhibitory antibody Y13-259 or by substitutions in ras. The GTPase-inhibitory antibody Y13-259 blocked the GAP associated with Y13-238. A ras species with a substitution in the effector loop that blocked conventional GAP activity was sensitive to stimulation by Y13-238. Both Y13-238 and Y13-259 stimulated the autophosphorylation of Ala59Thr ras. We interpreted these data in terms of a model in which the extrinsic factors influence the ras GTPase reaction by affecting the balance between "committed" and "uncommitted" states. We suggest that there is a mechanism distinct from that exploited by conventional GAPs for stimulating ras GTPase activity.

Amino Acid Sequence↗
PubMed1995

RAS signalling is abnormal in a c-raf1 MEK1 double mutant.

A mutant rat cell clone that suppresses the transformation defects of RAS effector loop substitutions is heterozygous for mutations in c-raf1 and MEK1. The mutant cells can be transformed by many otherwise defective RAS effector mutants, including RAS genes with the effector regions of distantly related GTPases, even though the encoded RAS proteins do not interact with either the mutant or wild-type RAF in Saccharomyces cerevisiae. While the significance of the c-raf1 mutation is unclear, the MEK1 mutation increases MEK1 activity and leads to activation of mitogen-activated protein kinase. The mutant MEK1 is coupled to the epidermal growth factor pathway but exhibits decreased physical interaction with RAF. When overexpressed, the MEK1 mutation is transforming and causes hyperphosphorylation of RAF. Signalling from RAS to MEK1 may be mediated by something other than RAF alone, but signalling through MEK1 is probably sufficient for RAS transformation.

Amino Acid Sequence↗
PubMed1994

Insertion mutations of the RIIA Na+ channel reveal novel features of voltage gating and protein kinase A modulation.

A linker insertion mutagenesis strategy was developed to probe functional subdomains of the RIIA Na+ channel alpha-subunit. We describe mutations within the first two repeat domains that provide new functional information for three segments of the channel structure. 1. The insertion of two alanine residues within the short peptide segment joining helices S4 and S5 in domain II had two effects: a depolarizing shift of steady-state activation and reduced single-channel currents. These results suggest that the peptide segment following the S4 voltage sensor is involved in the activation process and is facing the ion pore. 2. An insertion immediately N-terminal to the proposed transmembrane helix S1 in domain II shifted the steady-state activation in the depolarizing direction, suggesting a functional role in channel gating. 3. Insertions in the large, cytoplasmic loop between domains I and II affect two channel functions: inactivation and protein kinase A modulation. These results demonstrate that the linker insertion approach can provide novel insights into the structure-function relationships of large, multi-domain ion channel proteins.

Amino Acid Sequence↗
PubMed1993

Decreased activities of phosphatidate phosphohydrolase and phospholipase D in ras and tyrosine kinase (fps) transformed fibroblasts.

The activity of N-ethylmaleimide-insensitive phosphatidate phosphohydrolase (PAP-2) was characterized in control, ras-transformed, and tyrosine kinase-(fps) transformed rat fibroblasts. PAP-2 was assayed in two different ways: 1) within its natural membrane using liposomes of phosphatidate and 2) in the presence of sufficient Triton X-100 to solubilize PAP-2, and to form mixed micelles with the phosphatidate. Harvesting the fibroblasts in medium containing orthovanadate and Zn2+ gave up to 3-fold higher PAP-2 activities when measured in the absence, but not in the presence, of Triton X-100. PAP-2-specific activities from both assays increased in the control fibroblasts as the cells reached confluence. Both specific activities were lower in the oncogenically transformed fibroblasts than in controls at all cell densities tested. The specific activities of PAP-2 did not increase with time in culture in transformed cells which continued to divide. The relative increase in activity of phospholipase D after stimulation with serum or phorbol myristate acetate was lower in the transformed fibroblasts compared to control cells. This indicates a coordinated decrease in the phospholipase D/phosphatidate phosphohydrolase pathway at the level of both enzymes in ras and fps transformed fibroblasts. The ratio of the production of diacylglycerol relative to phosphatidate, after stimulation with serum, or phorbol ester, was lower in both transformed fibroblasts relative to the controls. This is compatible with the decreased specific activity of PAP-2 and indicates functional significance for the differences in PAP-2 activity in regulating the balance between the two mitogenic lipids, phosphatidate and diacylglycerol. Control of PAP-2 activity could be an important factor in regulating appropriate signals for cell division.

Animals↗
PubMed1993

Effector domain mutations dissociate p21ras effector function and GTPase-activating protein interaction.

The GTPase activity of p21ras is stimulated by GTPase-activating proteins (GAPs) such as p120GAP and the product of the neurofibromatosis 1 gene, which may negatively regulate p21 function. GAPs are also proposed effectors of ras. We have sought activating substitutions in c-H-ras in the region encoding the effector domain, on the rationale that such mutations would dissociate effector function from negative regulation by GAP. One such activating mutation, Pro-34-->Arg, encodes protein that is substantially bound to GTP in vivo. In vitro, this protein is not stimulated by GAPs, and its binding to p120GAP is grossly impaired. The results support the idea that the p21 structural requirements for effector function and GAP interaction are quite different and suggest that some molecule(s) other than p120GAP serves as the ras effector. In contrast to the results obtained with p120GAP, the Pro-34-->Arg p21 species is effectively coupled to the raf-1 product, as judged from electrophoretic mobility shifts of the Raf-1 phosphoprotein.

Amino Acid Sequence↗
PubMed1993

Evaluation of the National Cancer Institute's clinical trials booklet.

Evaluating the impact of written materials is a means to enhance the effectiveness of patient education, yet few controlled studies of publications have been completed. In 1984, as a result of a needs assessment, the National Cancer Institute (NCI) developed and pretested the booklet "What Are Clinical Trials All About?" The booklet was designed to help cancer patients make informed decisions about participation in clinical trials, which are critical for improving cancer treatment. The booklet, which is currently available, has been used internationally as a model for communicating information on clinical trials. Since 1985, the booklet has been used by the Cancer Information Service (CIS) as an educational tool for answering questions from cancer patients about treatment and clinical trials. The CIS, which has traditionally assisted NCI in the development and testing of educational materials, was involved in the pretesting and particularly the posttesting of this booklet. The CIS regional offices at Fox Chase Cancer Center and Sylvester Comprehensive Cancer Center together with National Institutes of Health Clinical Center and North Memorial Medical Center conducted a posttest evaluation of the booklet's effectiveness for cancer patients. Two hospitals tested the booklet on patients who were eligible for a specific clinical trial, and two hospitals tested the booklet on patients who were theoretically eligible for a clinical trial (with a cancer site and stage for which a trial existed). Patients were randomly assigned: 203 experimental subjects received the booklet, and 194 control subjects were not given the booklet until after a 2-week posttest examining attitudes, knowledge, and beliefs about clinical trials.(ABSTRACT TRUNCATED AT 250 WORDS)

Attitude↗
PubMed1992

Isolation and phenotypic analysis of conditional-lethal, linker-insertion mutations in the gene encoding the largest subunit of RNA polymerase II in Saccharomyces cerevisiae.

Linker-insertion mutagenesis was used to isolate mutations in the Saccharomyces cerevisiae gene encoding the largest subunit of RNA polymerase II (RPO21, also called RPB1). The mutant rpo21 alleles carried on a plamid were introduced into a haploid yeast strain that conditionally expresses RPO21 from the inducible promoter pGAL10. Growth of this strain on medium containing glucose is sustained only if the plasmid-borne rpo21 allele encodes a functional protein. Of nineteen linker-insertion alleles tested, five (rpo21-4 to -8) were found that impose a temperature-sensitive (ts) lethal phenotype on yeast cells. Four of these five ts alleles encode mutant proteins in which the site of insertion lies near one of the regions of the largest subunit that have been conserved during evolution. Two of the ts mutants (rpo21-4 and rpo21-7) display pleiotropic phenotypes, including an auxotrophy for inositol and a decreased proliferation rate at the permissive temperature. The functional relationship between RPO21 and RPO26, the gene encoding the 17.9 kDa subunit shared by RNA polymerases I, II, and III was investigated by determining the ability of increased dosage of RPO26 to suppress the ts phenotype imposed by rpo21-4 to -8. Suppression of the ts defect was specific for the rpo21-4 allele and was accompanied by co-suppression of the inositol auxotrophy. These results suggest that mutations in the largest subunit of RNA polymerase II can have profound effects on the expression of specific subsets of genes, such as those involved in the metabolism of inositol. In the rpo21-4 mutant, these pleiotropic phenotypes can be attributed to a defective interaction between the largest subunit and the RPO26 subunit of RNA polymerase II.

Alleles↗
PubMed1992

Organization and chromosomal locations of Rap1a/Krev sequences in the mouse.

The human RAP1A gene encodes a protein that apparently can antagonize the function of oncogenic ras genes in gene transfer experiments, but its normal function is unknown. To understand the function of this gene, we have undertaken a study of the mouse homolog, Rap1a. The complete coding sequence of a mouse Rap1a cDNA has been determined, and genomic clones representing three distinct Rap1a species were recovered. We find that Rap1a is located on distal mouse Chromosome (Chr) 3 near Nras, Ampd-1, Tshb, Ngfb, and Atp1a1. Two related sequences (Rap1a-rs1 and Rap1a-rs2) were also characterized. Rap1a-rs1, which was not localized, has a sequence very similar to the Rap1a cDNA, suggesting that it has been recently acquired by the mouse genome. Rap1a-rs2 is more distantly related to the gene sequence and is located on Chr 2 near Actc-1.

Amino Acid Sequence↗
PubMed1992

The murine leukemia inhibition factor gene (Lif) is located on proximal chromosome 11, not chromosome 13.

Lif, the murine gene encoding leukemia inhibition factor (LIF), has been previously localized to proximal Chromosome (Chr) 11. Hilda, the murine gene encoding "human interleukin in DA cells" (HILDA) has been localized to Chr 13. Since these two growth factors are identical, the proposal for two different structural loci is intriguing. To address this issue, blot hybridization methods have been used to establish the position of the structural gene sequence unambiguously. DNAs from somatic cell hybrids, recombinant inbred mice, and backcross mice have been probed with a sequence that encodes LIF/HILDA. The results support the assignment of this sequence to proximal Chr 11. These studies also establish a synteny group, including Lif and Tcn-2, the structural gene for transcobalamin 2, that is conserved between man and mouse.

Animals↗
PubMed1992

The impact of tailored self-help smoking cessation guides on young mothers.

It has been suggested that tailoring self-help materials for specific target populations will increase their effectiveness. This study tested the value of a self-help guide tailored specifically for women with young children. These women were recruited through a media campaign that encouraged smokers to call the Cancer Information Service (CIS) for assistance in stopping smoking. Women smokers with young children (under the age of 6) who called the CIS were given telephone counseling on quitting and were mailed one of three stop smoking guides. One third of callers received Quitting Times, a guide written specifically for women with young children; one third received the American Lung Association guide, Freedom from Smoking for You and Your Family; and one third received Clearing the Air, a guide developed by the National Cancer Institute. Six months after calling the CIS, these women were contacted by telephone to assess changes in smoking behavior. Overall, 12.5% of the women reported not smoking for at least 1 week at the time of the 6-month follow-up interview. There were no significant differences between subjects in the three groups in use of the self-help guides, methods used to attempt quitting, and quitting behavior. Findings from this study do not support the hypothesis that using a tailored stop smoking guide increases the targeted audience's cessation rate or affects quitting-related behavior. However, it should be noted that the smokers who called were predominantly in the contemplation or action stages.

Adult↗
PubMed1991

Genetic definition of ras effector elements.

The products of ras genes may function as GTP-binding signal transducers, but the nature of their targets is largely unknown. To define genetically the cellular effector(s) of ras in rat fibroblast transformation, somatic variants that suppress the nontransforming phenotype of v-H-ras effector domain mutations were sought. Variant cell lines perturbed in the ras effector pathway were recovered, and the properties of one suggest that the primary target of ras action may be altered. In this cell variant, no single residue in the ras protein effector domain must be wild type to bring about transformation. In parental rat cells, conservative substitutions are tolerated in six of nine residues. Functional interaction with the target may not require a high degree of structural specificity in the ras protein effector domain.

Alleles↗
PubMed1991

A ras effector domain mutant which is temperature sensitive for cellular transformation: interactions with GTPase-activating protein and NF-1.

A series of v-rasH effector domain mutants were analyzed for their ability to transform rat 2 cells at either low or high temperatures. Three mutants were found to be significantly temperature sensitive: Ile-36 changed to Leu, Ser-39 changed to Cys (S39C), and Arg-41 changed to Leu. Of these, the codon 39 mutant (S39C) showed the greatest degree of temperature sensitivity. When the same mutation was analyzed in the proto-oncogene form of ras(c-rasH), this gene was also found to be temperature sensitive for transformation. Biochemical analysis of the proteins encoded by v-rasH(S39C) and c-rasH(S39C) demonstrated that the encoded p21ras proteins were stable and bound guanine nucleotides in vivo at permissive and nonpermissive temperatures. On the basis of these findings, it is likely that the temperature-sensitive phenotype results from an inability of the mutant (S39C) p21ras to interact properly with the ras target effector molecule(s) at the nonpermissive temperature. We therefore analyzed the interaction between the c-rasH(S39C) protein and the potential target molecules GTPase-activating protein (GAP) and the GAP-related domain of NF-1, on the basis of stimulation of the mutant p21ras GTPase activity by these molecules in vitro. Assays conducted across a range of temperatures revealed no temperature sensitivity for stimulation of the mutant protein, compared with that of authentic c-rasH protein. We conclude that for this mutant, there is a dissociation between the stimulation of p21ras GTPase activity by GAP and the GAP-related domain NF-1 and their potential target function. Our results are also consistent with the existence of a distinct, as-yet-unidentified effector for mammalian ras proteins.

Animals↗
PubMed1991

Fox Chase Cancer Center Referral Resource Directory.

The Cancer Information Service at the Fox Chase Cancer Center receives over 8,000 calls per year requesting referrals to local health care services. We developed a multi-user microcomputer-based system for entering, storing, updating and retrieving cancer referral resource information. Counselors use a menu-based system to retrieve referral resources by agency name, subject, state, county, city, telephone area code, zip code, or fee code. The system also includes menus for expediting database management: data entry, updating, browsing, and report generation. A data entry screen allows records to be entered or edited. The system can print mailing labels and update sheets for each agency. This system enhances our ability to disseminate high-quality information to the public.

Cancer Care Facilities↗