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Biomedical subjects

H Paterson

Publications and source records attributed to H Paterson.

At least 19 recordsLinked to original sources
PubMed2003

Impact of an interdisciplinary low vision service on the quality of life of low vision patients.

AIM: To investigate the impact of an interdisciplinary low vision service on the vision related quality of life of service users. METHODS: 71 patients were interviewed 2 weeks before their appointment with the service and again 6 months later to assess any changes in their vision related quality of life. The majority of these patients had age related macular degeneration. RESULTS: After contact with the service the majority of patients indicated a reduction in concern about most quality of life issues. They were significantly less anxious about deterioration of their vision, safety within the home, and coping with everyday life. CONCLUSION: Improvements in many areas of their vision related quality of life indicate that this interdisciplinary low vision service has a positive impact on the lives of service users. However many patients were still unable to carry out their preferred everyday activities, and feelings of loneliness and isolation were unchanged. The identification of issues unrelieved by input from the service will be important in planning future service delivery.

Activities of Daily Living↗
PubMed2001

Appropriate subcellular localisation of prodrug-activating enzymes has important consequences for suicide gene therapy.

Escherichia coli B nitroreductase (NR) has been expressed stably in MDA-MB-361 human breast adenocarcinoma cells either as the wild-type protein (wtNR), which is distributed evenly between the cytoplasmic and nuclear compartments, or targeted to the mitochondrion (mtNR). Whereas bacterial NR is active as a dimer, a proportion of wtNR is monomeric. In contrast, mtNR is mostly dimeric, suggesting that it adopts a more stable, native conformation. Despite this, when tested in gene-directed enzyme prodrug therapy cell cytotoxicity studies, cells expressing wtNR or mtNR had similar sensitivity to the prodrug CB1954 and mounted similar bystander killing effects. Furthermore, when short prodrug exposures were given, wtNR was more efficient at killing cells than mtNR. These data demonstrate that the site of enzyme expression and prodrug activation is an important variable that requires consideration in suicide gene therapy approaches.

Adenocarcinoma↗
PubMed1999

Absence of Brca2 causes genome instability by chromosome breakage and loss associated with centrosome amplification.

Women heterozygous for mutations in the breast-cancer susceptibility genes BRCA1 and BRCA2 have a highly elevated risk of developing breast cancer [1]. BRCA1 and BRCA2 encode large proteins with no sequence similarity to one another. Although involvement in DNA repair and transcription has been suggested, it is still not understood how loss of function of these genes leads to breast cancer [2]. Embryonic fibroblasts (MEFs) derived from mice homozygous for a hypomorphic mutation (Brca2(Tr2014)) within the 3' region of exon 11 in Brca2 [3], or a similar mutation (Brca2(Tr)) [4], proliferate poorly in culture and overexpress the tumour suppressor p53 and the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). These MEFs have intact p53-dependent DNA damage G(1)-S [3] [4] and G(2)-M checkpoints [4], but are impaired in DNA double-strand break repair [3] and develop chromosome aberrations [4]. Here, we report that Brca2(Tr2014/Tr2014) MEFs frequently develop micronuclei. These abnormal DNA-containing bodies were formed through both loss of acentric chromosome fragments and by chromosome missegregation, which resulted in aneuploidy. Absence of Brca2 also led to centrosome amplification, which we found associated with the formation of micronuclei. These data suggest a potential mechanism whereby loss of BRCA2 may, within subclones, drive the loss of cell-cycle regulation genes, enabling proliferation and tumourigenesis.

Aneuploidy↗
PubMed1999

Interaction of axin and Dvl-2 proteins regulates Dvl-2-stimulated TCF-dependent transcription.

Axin promotes the phosphorylation of beta-catenin by GSK-3beta, leading to beta-catenin degradation. Wnt signals interfere with beta-catenin turnover, resulting in enhanced transcription of target genes through the increased formation of beta-catenin complexes containing TCF transcription factors. Little is known about how GSK-3beta-mediated beta-catenin turnover is regulated in response to Wnt signals. We have explored the relationship between Axin and Dvl-2, a member of the Dishevelled family of proteins that function upstream of GSK-3beta. Expression of Dvl-2 activated TCF-dependent transcription. This was blocked by co-expression of GSK-3beta or Axin. Expression of a 59 amino acid GSK-3beta-binding region from Axin strongly activated transcription in the absence of an upstream signal. Introduction of a point mutation into full-length Axin that prevented GSK-3beta binding also generated a transcriptional activator. When co-expressed, Axin and Dvl-2 co-localized within expressing cells. When Dvl-2 localization was altered using a C-terminal CAAX motif, Axin was also redistributed, suggesting a close association between the two proteins, a conclusion supported by co-immunoprecipitation data. Deletion analysis suggested that Dvl-association determinants within Axin were contained between residues 603 and 810. The association of Axin with Dvl-2 may be important in the transmission of Wnt signals from Dvl-2 to GSK-3beta.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Differentiation of separated mouse mammary luminal epithelial and myoepithelial cells cultured on EHS matrix analyzed by indirect immunofluorescence of cytoskeletal antigens.

We have previously demonstrated that purified virgin mouse mammary luminal epithelial and myoepithelial cells promiscuously express cell type-specific cytokeratins when they are cloned in vitro. Changes in cytokeratin expression may be indicators of the loss or change of the differentiated identity of a cell. To investigate the factors that may be responsible for the maintenance of differentiated cellular identity, specifically cell-cell and cell-matrix interactions, we cloned flow-sorted mouse mammary epithelial cells on the extracellular matrix (ECM) derived from the Engelbreth-Holm-Swarm murine sarcoma (EHS matrix). Changes in cell differentiation on EHS, compared with culture on glass, were analyzed by comparing patterns of cytokeratin expression. The results indicate that ECM is responsible for maintenance of the differentiated identity of basal/myoepithelial cells and prevents the inappropriate expression of luminal antigens seen on glass or plastic. Luminal cell identity in the form of retention of luminal markers and absence of basal/myoepithelial antigens, on the contrary, appears to depend on homotypic cell-cell contacts and interactions. The results also show that luminal cells (or a subpopulation of them) can generate a cell layer that expresses only basal cytokeratin markers (and no luminal cytokeratin markers) and may form a pluripotent compartment. (J Histochem Cytochem 47:1513-1524, 1999)

Actins↗
PubMed1999

Cardiac valvular papillary fibroelastoma: a report of 2 cases.

Papillary fibroelastomas are rare cardiac valve tumors with potential for life-threatening complications such as stroke or sudden death. We report 2 cases of papillary fibroelastoma that were found by echocardiography. The 1st tumor arose from the mitral valve in a patient who presented after multiple transient neurologic events. The 2nd tumor arose from the aortic valve and was found incidentally during coronary artery bypass grafting. Both patients underwent successful surgical removal of the tumor.

Adult↗
PubMed1998

Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase C.

Receptor tyrosine kinase-mediated activation of the Raf-1 protein kinase is coupled to the small guanosine triphosphate (GTP)-binding protein Ras. By contrast, protein kinase C (PKC)-mediated activation of Raf-1 is thought to be Ras independent. Nevertheless, stimulation of PKC in COS cells led to activation of Ras and formation of Ras-Raf-1 complexes containing active Raf-1. Raf-1 mutations that prevent its association with Ras blocked activation of Raf-1 by PKC. However, the activation of Raf-1 by PKC was not blocked by dominant negative Ras, indicating that PKC activates Ras by a mechanism distinct from that initiated by activation of receptor tyrosine kinases.

Animals↗
PubMed1997

Ras signalling is required for inactivation of the tumour suppressor pRb cell-cycle control protein.

Ras proteins act as molecular switches, responding to signals by entering the active GTP-bound, rather than the inactive GDP-bound, state. The inhibition of normal Ras proteins by microinjection of neutralizing antibody or expression of dominant-negative mutants has shown that Ras signalling is required for growth factors to stimulate DNA synthesis [1] [2], but the link between Ras and the cell-cycle machinery is not clear. Regulation of the phosphorylation state of the retinoblastoma protein (pRb), the product of the tumour suppressor gene Rb, is a key event in the progression of cells from G1 phase into S phase. In growth-arrested or early G1 cells, pRb is hypophosphorylated and binds to transcription factors of the E2F family [3]. These pRb-E2F complexes act to suppress gene transcription required for entry into DNA synthesis either by preventing E2F from stimulating transcription or by actively repressing transcription [4]. During G1, cyclin-dependent kinases (CDKs) become activated and phosphorylate pRb at multiple sites, leading to the dissolution of pRb-E2F complexes and gene transcription [5]. Here, we have tested the hypothesis that Ras signalling is required for the inactivation of pRb. A neutralizing antibody directed against p21Ras was microinjected into cells derived from mutant mouse embryos that lack Rb or CDK inhibitors (CDKIs). Cells without pRb or the p16 CDKI were more resistant to the inhibitory effects of the anti-Ras antibody. DNA synthesis in some tumour cell lines was completely resistant to the anti-Ras injection, indicating that p21Ras is required for pRb inactivation but also has other functions in cell-cycle progression.

Animals↗
PubMed1996

A member of the MAP kinase phosphatase gene family in mouse containing a complex trinucleotide repeat in the coding region.

We have identified a novel mouse gene encoding a protein that shows high homology to the dual-specificity tyrosine/threonine phosphatase family of proteins. The gene encodes a 5 kb transcript which is expressed predominantly in brain and lung and contains a translated complex trinucleotide repeat within the coding region. Using interspecific mouse backcross analysis, the gene has been localised to distal mouse chromosome 7. In human, homologous sequences are located in the syntenic region on distal chromosome 11p as well as to chromosome 10q11.2 and 10q22. The presence of a CG-rich trinucleotide repeat in the coding region provides a target for mutation which might result in loss of function or altered properties of this phosphatase.

Amino Acid Sequence↗
PubMed1994

EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor.

BACKGROUND: Mitogen-activated protein (MAP) kinase is the central component of a signal transduction pathway that is activated by growth factors interacting with receptors that have protein tyrosine kinase activity. The stimulation of PC12 phaeochromocytoma cells with nerve growth factor leads to the sustained activation and nuclear translocation of the p42 and p44 isoforms of MAP kinase and induces the differentiation of these chromaffin cells to a sympathetic-neuron-like phenotype. In contrast, stimulation with epidermal growth factor induces a transient activation of p42 and p44 MAP kinases without pronounced nuclear translocation and does not trigger cell differentiation. We have examined whether the differential activation of MAP kinases forms the basis of the differential response of the cells to the two factors. RESULTS: By overexpressing either wild-type or mutant receptors for epidermal growth factor in PC12 cells, we found that p42 and p44 MAP kinase activity remains elevated for longer in cells that overexpress receptors than in untransfected cells. Epidermal growth factor promotes both a striking nuclear translocation of p42 MAP kinase and the differentiation of the overexpressing cells. CONCLUSIONS: Our results strongly suggest that the distinct effects of nerve growth factor and epidermal growth factor on PC12 cell differentiation can be explained by differences in the extent and duration of activation of p42 and p44 MAP kinases in response to the two factors, without invoking a signal transduction pathway specific to nerve growth factor.

Animals↗
PubMed1994

Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells.

The MAP kinase pathway is activated by a wide variety of external signals leading to cell proliferation or differentiation. However, it is not clear whether activation of this pathway is required for cellular responses or whether it is only one branch point in signal transduction. To investigate these questions, we generated constitutively activated and interfering mutants of MAP kinase kinase 1. The activated mutants stimulated PC12 cell neuronal differentiation and transformed NIH 3T3 cells. The interfering mutants inhibited growth factor-induced PC12 differentiation, growth factor stimulation of proliferation, and reverted v-src- and ras-transformed cells. These results therefore show that, depending on cellular context, activation of MAP kinase kinase is necessary and sufficient for cell differentiation or proliferation.

3T3 Cells↗
PubMed1994

The effector domain of Rab6, plus a highly hydrophobic C terminus, is required for Golgi apparatus localization.

C-terminal lipid modifications are essential for the interaction of Ras-related proteins with membranes. While all Ras proteins are farnesylated and some palmitoylated, the majority of other Ras-related proteins are geranylgeranylated. One such protein, Rab6, is associated with the Golgi apparatus and has a C-terminal CXC motif that is geranylgeranylated on both cysteines. We show here that farnesylation alone cannot substitute for geranylgeranylation in targeting Rab6 to the Golgi apparatus and that whereas Ras proteins that are farnesylated and palmitoylated are targeted to the plasma membrane, mutant Rab proteins that are both farnesylated and palmitoylated associate with the Golgi apparatus. Using chimeric Ras-Rab proteins, we find that there are sequences in the N-terminal 71 amino acids of Rab6 which are required for Golgi complex localization and show that these sequences comprise or include the effector domain. The C-terminal hypervariable domain is not essential for the Golgi complex targeting of Rab6 but is required to prevent prenylated and palmitoylated Rab6 from localizing to the plasma membrane. Functional analysis of these mutant Rab6 proteins in Saccharomyces cerevisiae shows that wild-type Rab6 and C-terminal mutant Rab6 proteins which localize to the Golgi apparatus in mammalian cells can complement the temperature-sensitive phenotype of ypt6 null mutants. Interestingly, therefore, the C-terminal hypervariable domain of Rab6 is not required for this protein to function in S. cerevisiae.

Amino Acid Sequence↗
PubMed1994

N-terminally myristoylated Ras proteins require palmitoylation or a polybasic domain for plasma membrane localization.

Plasma membrane targeting of Ras requires CAAX motif modifications together with a second signal from an adjacent polybasic domain or nearby cysteine palmitoylation sites. N-terminal myristoylation is known to restore membrane binding to H-ras C186S (C-186 is changed to S), a mutant protein in which all CAAX processing is abolished. We show here that myristoylated H-ras C186S is a substrate for palmitoyltransferase, despite the absence of C-terminal farnesylation, and that palmitoylation is absolutely required for plasma membrane targeting of myristoylated H-ras. Similarly, the polybasic domain is required for specific plasma membrane targeting of myristoylated K-ras. In contrast, the combination of myristoylation plus farnesylation results in the mislocalization of Ras to numerous intracellular membranes. Ras that is only myristoylated does not bind with a high affinity to any membrane. The specific targeting of Ras to the plasma membrane is therefore critically dependent on signals that are contained in the hypervariable domain but can be supported by N-terminal myristoylation or C-terminal prenylation. Interestingly, oncogenic Ras G12V that is localized correctly to the plasma membrane leads to mitogen-activated protein kinase activation irrespective of the combination of targeting signals used for localization, whereas Ras G12V that is mislocalized to the cytosol or to other membranes activates mitogen-activated protein kinase only if the Ras protein is farnesylated.

Amino Acid Sequence↗
PubMed1993

Motility of intracellular particles in rat fibroblasts is greatly enhanced by phorbol ester and by over-expression of normal p21N-ras.

Particle motility in cultured rat fibroblasts was studied using video-enhanced differential interference contrast microscopy. The average velocity of large bright particles (apparent diameter about 0.5-0.7 micron) was measured in control cells and in cells treated with agents which affected targets related to signal transduction pathways. A Rat-2-derived fibroblast line transfected with a construct containing multiple copies of the N-ras proto-oncogene under the control of dexamethasone-sensitive promoter was used as a main experimental model. Dexamethasone treatment was shown to induce high levels of N-ras expression in these cells. This treatment greatly increased the average particle velocity. At the same time dexamethasone did not influence the particle motility in the non-transfected parent cells and in the cells transfected with a construct which did not contain N-ras. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), also induced an approximate eightfold increase in the particle rate after several hours of incubation, while sphingosine, an inhibitor of PKC, prevented this activation. Sphingosine alone reduced the particle motility after a 20 min incubation. The particle movements were inhibited also by colcemid. These data show that the activation of N-ras and PKC produced dramatic activation of microtubule-dependent particle motility. A possible role of this activation in signal-induced alterations of cell morphology is discussed.

Animals↗
PubMed1993

Specific association of activated MAP kinase kinase kinase (Raf) with the plasma membranes of ras-transformed retinal cells.

Plasma membrane-enriched fractions were prepared from human embryonic retinal cells transformed with either adenovirus E1A and oncogenic ras DNA, or E1A and E1B DNA. Ras comprised 5-10% of the membrane protein from the E1A/ras transformed cells, whereas the membranes from E1A/E1B transformed cells did not overexpress Ras. The membranes from E1A/ras cells contained MAP kinase kinase kinase (MAPKKK) activity, even after washing in 0.5 M NaCl, whereas the membranes from E1A/E1B cells did not. Neither membrane fraction contained MAP kinase kinase or MAP kinase activity after washing with 0.5M NaCl. Immunoblotting experiments revealed about 10-fold more c-Raf in the membranes from E1A/ras cells than from E1A/E1B cells, and 50-60% of the MAPKKK activity in Triton X100-solubilised membranes from E1A/ras cells was immunoprecipitated with anti-Raf antibodies. A striking enrichment of c-Raf in the plasma membranes of E1A/ras cells was also demonstrated by immunocytochemistry, where it was co-localized with Ras. The MAPKKK activity in E1A/ras membranes was unaffected by incubation with protein phosphatases or by inclusion of protein phosphatase inhibitors during isolation, nor was it activated by GTP-Ras or inhibited by GDP-Ras. The results support the view that Ras and c-Raf interact with one another, but that neither c-Raf phosphorylation nor its interaction with GTP-Ras are alone sufficient for activation. The identification of MAPKKK activity in the membranes of ras-transformed cells may prove useful in elucidating the mechanism by which Raf is activated by Ras.

Amino Acid Sequence↗