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H Paterson

Publications and source records attributed to H Paterson.

At least 37 records · Page 2Linked to original sources
PubMed1992

Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC12 cells. Comparison of the effects of nerve growth factor and epidermal growth factor.

Stimulation of PC12 cells with nerve growth factor (NGF) increased mitogen-activated protein kinase kinase (MAPKK) activity > 20-fold after 5 min to a level that was largely sustained for at least 90 min. MAPKK activity was stimulated to a similar level by epidermal growth factor (EGF), but peaked at 2 min, declining thereafter and returning to basal levels after 60-90 min. Activation of MAPKK by either growth factor occurred prior to the activation of MAP kinase, consistent with MAPKK being the physiological activator of MAP kinase. The results demonstrate that the transient activation of MAPKK by EGF and its sustained activation by NGF underlies the transient and sustained activation of MAP kinase induced by EGF and NGF respectively. NGF or EGF induced the same two forms of MAPKK that were resolved on a Mono Q column. The Peak-1 MAPKK was activated initially and partially converted into the more acidic peak-2 MAPKK after prolonged growth-factor stimulation. The Peak-2 MAPKK was 20-fold more sensitive to inactivation by the catalytic subunit of protein phosphatase 2A. Stimulation with NGF caused a striking translocation of MAP kinase from the cytosol to the nucleus after 30 min, but not nuclear translocation of MAP kinase occurred after stimulation with EGF. The results suggest that sustained activation of the MAP kinase cascade may be required for MAP kinase to enter the nucleus, where it may initiate the gene transcription events required for neuronal differentiation of PC12 cells.

Animals↗
PubMed1991

Characterization of recombinant human Kirsten-ras (4B) p21 produced at high levels in Escherichia coli and insect baculovirus expression systems.

Kirsten-ras is the oncogene most frequently activated in human tumors. Studies of its biological function have been limited by the nonavailability of significant amounts of the major protein product, Kirsten-ras (4B) p21. When expressed in Escherichia coli K12, the recombinant protein was rapidly cleaved upon cell lysis in the lysine-rich C terminus region, probably by the ompT protease. However, soluble full-length protein was obtained when the Kirsten-ras gene was expressed in an E. coli strain lacking the ompT gene, and also in a baculovirus/insect cell expression system. Additionally, the baculovirus/insect cell system produced about half of the Kirsten-ras protein in a membrane-associated form, which was post-translationally modified by polyisoprenylation and carboxyl-methylation. A C-terminally truncated form (residues 1-166) was also expressed at high levels in E. coli for x-ray crystallographic studies. The kinetics of GDP release and of GTP hydrolysis of the purified proteins are similar to those of the corresponding Harvey-ras proteins, though there are small differences in the relative affinities for GDP and GTP. Biological activity of full-length Kirsten Val-12 p21 was demonstrated by microinjection into Swiss 3T3 cells, resulting in morphological transformation, with a lower potency than that of Harvey Val-12 protein.

Animals↗
PubMed1991

A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins.

Mutational analysis of p21ras has shown that plasma membrane targeting requires the combination of a CAAX motif with a polybasic domain of six lysine residues or a nearby palmitoylation site. However, it is not known from these studies whether these signals alone target p21ras to the plasma membrane. We now show that these C-terminal sequences are sufficient to target a heterologous cytosolic protein to the plasma membrane. Interestingly, the key feature of the p21K-ras(B) polybasic domain appears to be a positive charge, since a polyarginine domain can function as a plasma membrane targeting motif in conjunction with the CAAX box and p21K-ras(B) with the polylysine domain replaced by arginines is biologically active. Since some ras-related proteins are modified by geranylgeranyl rather than farnesyl we have investigated whether modification of p21ras with geranylgeranyl affects its subcellular localization. Geranylgeranyl can substitute for farnesyl in combining with a polybasic domain to target p21K-ras(B) to the plasma membrane, but such geranylgeranylated proteins are more tightly bound to the membrane. This increased avidity of binding is presumably due to the extra length of the geranylgeranyl alkyl chain.

3T3 Cells↗
PubMed1991

Immunoelectron microscopic localization of the p21 protein in HT1080 human fibrosarcoma cell lines with altered N-ras gene expression.

The cellular localization of the p21 protein was studied by immunoelectron microscopy in human fibrosarcoma cells with different N-ras gene expression. The tumorigenic HT1080 cells--expressing equally the normal and mutant N-ras genes--contained the p21 in the plasma membrane, in pinocytotic areas as well as in the intracellular vesicles. The non-tumorigenic revertants--expressing more normal N-ras gene than the mutant one--contained p21 in the plasma membrane, especially at the cell junctions. These results suggest, that the mutant p21 may locate to the membrane areas connected with pino- or endocytotic functions.

Fibrosarcoma↗
PubMed1991

Immunoelectron microscopic localization of the p21 protein in HT1080 human fibrosarcoma cell lines with altered N-ras gene expression.

The cellular localization of the p21 protein was studied by immunoelectron microscopy in human fibrosarcoma cells with different N-ras gene expression. The tumorigenic HT1080 cells-expressing equally the normal and mutant N-ras genes-contained the p21 in the plasma membrane, in pinocytotic areas as well as in the intracellular vesicles. The non-tumorigenic revertants-expressing more normal N-ras gene than the mutant one-contained p21 in the plasma membrane, especially at the cell junctions. These results suggest, that the mutant p21 may locate to the membrane areas connected with pino- or endocytotic functions.

Cell Division↗
PubMed1990

A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane.

The C-terminal CAAX motif of ras proteins undergoes a triplet of posttranslational modifications that are required for membrane association. The CAAX motif lies immediately C-terminal to the hypervariable domain, a region of 20 amino acids that distinguishes the ras proteins from each other. The hypervariable domains of p21H-ras, p21N-ras, and p21K-ras(A) contain sites for palmitoylation, which we now show must combine with the CAAX motif to target specific plasma membrane localization. Within the hypervariable domain of p21K-ras(B), which is not palmitoylated, we have identified a novel plasma membrane targeting signal consisting of a polybasic domain that also acts in combination with the CAAX motif. One function of the hypervariable domains of p21ras is therefore to provide different signals for plasma membrane localization.

Amino Acid Sequence↗
PubMed1990

Localization and production of proteoglycans by HT1080 cell lines with altered N-ras expression.

The alterations in the production of proteoglycans in relation to the expression of the malignant phenotype - controlled by the level of expression of activated N-ras gene - was studied in HT1080 human fibrosarcoma cells and in its revertant variants rev.1c and rev.10a. A decreased production of radiolabelled PGs - especially HSPGs - was observed in HT1080 cells, compared to the revertant lines. By immunofluorescence, the HSPG epitopes were localized mainly into the putative endoplasmic reticulum and/or Golgi zone in HT1080 cells, and to the diffuse cytoplasmic and membrane localization in the revertant lines. It is suggested, that the altered expression of PGs represents an important aspect of the transformed phenotype of HT1080 fibrosarcoma cells.

Cell Division↗
PubMed1990

A novel sequence-specific DNA binding protein which interacts with three regularly spaced direct repeats of the CCCTC-motif in the 5'-flanking sequence of the chicken c-myc gene.

The chicken c-myc 5'-flanking sequence has previously been shown to bind multiple proteins present in undifferentiated and differentiated red blood cells. In this report the protein binding to one specific region within a hypersensitive site approximately 200 base pairs upstream of the start of transcription has been analysed in detail. Using a combination of a modified agarose gel retardation assay with O-phenanthroline-copper footprinting in situ, missing contact point and methylation interference techniques, two proteins were found to bind to overlapping sequences within 180-230 bp upstream of the start of transcription. One protein resembles the transcription factor Sp1, the other is a protein which binds to three regularly spaced repeats of the core sequence CCCTC. This CCCTC-binding factor was termed CTCF. It requires additional sequences outside the three recognition motifs for tight binding. CTCF was purified to near homogeneity by sequence-specific DNA chromatography. The approximate molecular weight of the CTCF was estimated to be 130,000. Removal of 110 bp sequence binding both CTCF and Sp1-like proteins leads to a 4 to 8-fold increase in transcription of stably transfected c-myc fusion constructs in chicken embryonic fibroblasts, suggesting that the CTCF is likely to be one of multiple nuclear factors involved in the transcriptional regulation of the chicken c-myc gene.

Animals↗
PubMed1989

Suppression of transformation and immortality in human/Chinese hamster fibroblast hybrids--a model for suppressor gene isolation.

Somatic cell hybrids were produced by fusion of normal human (foreskin) fibroblasts and a transformed Chinese hamster fibroblast line V79-8. Overall, approximately 30% of hybrid clones showed stable reversion to normal morphology and growth control in vitro as shown by serum and anchorage dependence. In one-third of these clones, senescence was observed after a number of generations similar to that required for the human fibroblast parent cells to senesce. The remainder appear to be immortal. Normal human chromosomes can therefore restore growth control with or without finite life-span to this transformed cell. V79 cells were found to be transfectable at an efficiency compatible with detection of single-copy gene transfer from genomic DNA. Furthermore, these cells were exceptionally sensitive to negative ("suicide") selection. Taken together, our data suggest that the V79 line represents an ideal system for isolation of human tumour suppressor genes.

Animals↗
PubMed1989

Flat revertants of EJ human bladder carcinoma cells show two different mechanisms of reversion.

To investigate the way in which ras proteins cause transformation, we have isolated revertants from human tumour cell lines which contain transforming ras genes. Two types of revertant have been isolated from the human fibrosarcoma cell line, HT1080. One class has normal and mutant alleles in a ratio of 2:1, compared to 1:1 in the parental cells, showing that reversion can be a dosage phenomenon. The other class has lost the transforming allele. All the HT1080 revertants isolated can be re-transformed by transforming ras proteins. To test whether reversion is due to a change in the relative amounts of normal and mutant proteins, or to a reduction in the absolute amount of the transforming protein, mixtures of the purified proteins were microinjected into 208F (Rat-1) cells, chosen because they are less sensitive to transformation by p21ras. Normal H-ras p21 was unable to suppress the transforming effects of the mutant ras protein when co-injected at up to ninefold excess. Revertants of EJ human bladder carcinoma cells were of two types: one was sensitive to re-transformation by oncogenically activated ras proteins, the other was not. The EJ revertants that are resistant to re-transformation fall into two classes, since hybrids of one revertant with the parental EJ cells are non-transformed, whereas hybrids of another revertant with the parental cells are transformed.

Bacterial Proteins↗
PubMed1989

Signal transduction by p21ras.

Experiments using the physical loading of purified recombinant p21 ras proteins into quiescent normal cells to analyze how the proteins stimulate DNA synthesis and morphological transformation are reviewed. The results indicate that oncogenic p21ras proteins rapidly activate a protein-kinase-C-dependent pathway and a protein-kinase-C-independent pathway. The activation of protein kinase C is absolutely required for p21ras to stimulate DNA synthesis but is not required for morphological transformation.

Animals↗
PubMed1989

N-ras dependent revertant phenotype in human HT1080 fibrosarcoma cells is associated with loss of proliferation within normal tissues and expression of an adult membrane antigenic phenotype.

To investigate how the activated N-ras oncogene contributes to the tumorigenic potential of malignant human fibrosarcoma HT1080 cells we analysed the behavior of the parental cell line and of two flat revertants (1c and 10a) in an organ culture assay for invasion. In this assay the two revertants retain the ability of HT1080 cells to migrate within the chick cardiac muscle but lose the capacity to proliferate and to replace the normal tissue. Moreover the reversion of tumorigenic potential is associated with an evolution from an oncofoetal membrane antigenic pattern towards expression of a normal adult phenotype. Both the 4F2 antigen, which is implicated in the control of HT1080 cell proliferation, and heterodimers of the two chains (alpha and beta) of the IL2 receptor (IL2-R) are expressed in embryonic and HT1080 cells, but not in normal adult fibroblasts or in the revertant cell lines. For the first time in a non-lymphoid environment, we have detected a complex between the two IL2-R chains, together with a new species of mRNA (2.8 kB) from the IL2-R alpha gene. The behavior of these membrane markers strengthens the hypothesis that HT1080 cells may represent a block in the differentiation pathway of fibroblastic cells.

Antigens, Surface↗
PubMed1987

Activated N-ras controls the transformed phenotype of HT1080 human fibrosarcoma cells.

To investigate whether the activated N-ras oncogene of HT1080 human fibrosarcoma cells contributes to the expression of the transformed phenotype, we have isolated flat revertants. In two independent revertant lines, an increase in chromosomal ploidy occurred without a concomitant increase in the number of copies of the N-ras transforming allele. Immunoprecipitation confirms that the level of the mutant N-ras p21 gene product in the revertants is correspondingly lower than in HT1080. Analysis of sporadic tumors derived from the revertant cells reveals an increased dosage of the transforming allele. The revertants also retransform after transfection of cloned activated ras oncogenes. These results imply direct participation of an N-ras oncogene in maintaining the transformed phenotype of a human tumor cell line.

Alleles↗
PubMed1987

Partial transformation of mouse fibroblastic and epithelial cell lines with the v-myc oncogene.

To investigate the role of the myc gene in mammalian cell transformation, plasmid constructs containing the v-myc oncogene and a co-selectable G418 resistance marker were introduced into both mouse fibroblasts (NIH-3T3) and bladder epithelial cells (BBN3 and BBN7). After transfection or microinjection of DNA, no transformed foci could be detected on confluent monolayers but, when the cells were cultured under conditions in which individual cells were allowed to grow and form colonies, morphological transformation was observed. Unlike ras-transformed NIH-3T3 cells, v-myc-transformed cells were unable to grow in serum-free medium and therefore still required exogenous growth factors. v-myc-transformed NIH-3T3 cells were poor at forming foci when co-cultivated with untransformed cells; however, the efficiencies could be increased by addition of EGF to the medium. Both v-myc-transformed fibroblasts and epithelial cells acquired the ability to grow in soft agar, though at efficiencies lower than the corresponding ras transformants. Subcutaneous inoculation of v-myc-transformed NIH-3T3 cells into nude mice resulted in no tumours within 6 weeks. After protracted periods (2-3 months) a few tumours were detected, but at a frequency barely above that for spontaneous tumour formation. Epithelial cells transformed by v-myc were either non-tumorigenic or gave a very low incidence of tumours. We conclude that the v-myc oncogene induces morphological changes and anchorage independence in immortal mouse fibroblasts and epithelial cell lines but further events are required for the cells to become tumorigenic.

Animals↗
PubMed1987

Biochemical and biological properties of the human N-ras p21 protein.

We characterized the normal (Gly-12) and two mutant (Asp-12 and Val-12) forms of human N-ras proteins produced by Escherichia coli. No significant differences were found between normal and mutant p21 proteins in their affinities for GTP or GDP. Examination of GTPase activities revealed significant differences between the mutant p21s: the Val-12 mutant retained 12% of wild-type GTPase activity, whereas the Asp-12 mutant retained 43%. Both mutant proteins, however, were equally potent in causing morphological transformation and increased cell motility after their microinjection into quiescent NIH 3T3 cells. This lack of correlation between transforming potency and GTPase activity or guanine nucleotide binding suggests that position 12 mutations affect other aspects of p21 function.

Animals↗
PubMedDate not supplied

Management of enuresis in children.

A postal survey was conducted to gather information on the enuresis service of Oxfordshire Health Authority Community Unit. This article discusses the size of the problem, treatment and knowledge and expertise. The findings are summarized in the form of a list of recommendations.

Child↗