Search PubMed⌕ Search

Biomedical subjects

M A White

Publications and source records attributed to M A White.

At least 55 records · Page 3Linked to original sources
PubMed1998

Differential effects of protein kinase A on Ras effector pathways.

Ras mutants with the ability to interact with different effectors have played a critical role in the identification of Ras-dependent signaling pathways. We used two mutants, RasS35 and RasG37, which differ in their ability to bind Raf-1, to examine Ras-dependent signaling in thyroid epithelial cells. Wistar rat thyroid cells are dependent upon thyrotropin (TSH) for growth. Although TSH-stimulated mitogenesis requires Ras, TSH activates protein kinase A (PKA) and downregulates signaling through Raf and the mitogen-activated protein kinase (MAPK) cascade. Cells expressing RasS35, a mutant which binds Raf, or RasG37, a mutant which binds RalGDS, exhibited TSH-independent proliferation. RasS35 stimulated morphological transformation and anchorage-independent growth. RasG37 stimulated proliferation but not transformation as measured by these indices. TSH exerted markedly different effects on the Ras mutants and transiently repressed MAPK phosphorylation in RasS35-expressing cells. In contrast, TSH stimulated MAPK phosphorylation and growth in cells expressing RasG37. The Ras mutants, in turn, exerted differential effects on TSH signaling. RasS35 abolished TSH-stimulated changes in cell morphology and thyroglobulin expression, while RasG37 had no effect on these activities. Together, the data indicate that cross talk between Ras and PKA discriminates between distinct Ras effector pathways.

Animals↗
PubMed1997

Partial purification and characterization of the DNA polymerase from the cyanelles of Cyanophora paradoxa.

A DNA polymerase was partially purified and characterized from the photosynthetic organelles (cyanelles) of the protist, Cyanophora paradoxa. While cyanelles have several cyanobacterial features, such as a lysozyme-sensitive cell wall, unstacked thylakoids and light harvesting phycobilisomes, their genome size and structure resemble those of chloroplasts, suggesting that cyanelles occupy a unique intermediate position between chloroplasts and their phylogenetic ancestors, the cyanobacteria. When comparing the biochemical characteristics of the cyanelle DNA polymerase to those of its counterparts from higher plant chloroplasts and from a cyanobacterium, it is clear that the cyanelle enzyme resembles chloroplast DNA polymerases which are eukaryotic gamma-type enzymes.

Animals↗
PubMed1997

An investigation of occupational metal exposure in thermal spraying processes.

A cross-sectional study of 34 workers engaged in thermal spraying at six worksites was undertaken in order to determine levels of exposure to and uptake of metals during different metal spraying activities. Levels of exposure to cobalt, chromium and nickel were highest in plasma sprayers and, on occasions exceeded UK Occupational Exposure Limits. Exposure to metals during detonation gun and electric arc spraying was better controlled and levels remained below the relevant Occupational Exposure Limits throughout the study period. Urinary levels of cobalt and nickel mirrored the airborne concentrations and the highest urine concentrations were again found in plasma sprayers. Urinary chromium levels were highest in electric arc sprayers, which may also reflect an increased body burden in this group due to a longer history of exposure. The findings clearly indicate that exposure to and uptake of metals may exceed UK Occupational Limits or Standards when spraying is performed manually or semi-automatically and where control relies on local exhaust ventilation (LEV) and personal respiratory protective equipment (RPE).

Adult↗
PubMed1997

Physical association with ras enhances activation of membrane-bound raf (RafCAAX).

The transforming activity of artificially membrane-targeted Raf1 suggests that Ras-mediated recruitment of Raf1 to the plasma membrane is an important step in Raf1 activation. Cellular Ras is concentrated in the caveolae, a microdomain of the plasma membrane that is highly enriched in caveolin, glycosylphosphatidylinositol-anchored proteins, and signal transduction molecules. Growth factor stimulation recruits Raf1 to this membrane domain. Whether Ras simply promotes Raf1 association with caveolae membranes or also modulates subsequent activation events is presently unclear. We have identified a ras variant, ras12V,37G, that does not interact with Raf1 but does interact with a mutant raf1, raf1(257L). To examine the role of Ras in the activation of membrane-bound Raf1, raf1CAAX, and raf1(257L)CAAX, membrane-targeted variants of Raf1 and raf1(257L), respectively, were expressed in fibroblasts with or without coexpression of ras12V, 37G. Cell fractionation localized both raf1CAAX and raf1(257L)CAAX to caveolae membranes independent of ras12V,37G expression; however, coexpression of ras12V,37G enhanced the activation of raf(257L)CAAX, but not raf1CAAX, as monitored by induction of cellular transformation, increased Raf kinase activity, and induction of activated MAP kinase. These results suggest that the Ras/Raf1 interaction plays a role in Raf1 activation that is distinct from membrane recruitment.

3T3 Cells↗
PubMed1997

RalGDS functions in Ras- and cAMP-mediated growth stimulation.

Thyroid-stimulating hormone stimulates proliferation through both the cAMP-dependent protein kinase and Ras but not through Raf-1 and mitogen-activated and extracellular signal-related kinase kinase. We now report that thyroid-stimulating hormone represses mitogen-activated protein kinase activity and that microinjection of an effector domain mutant Ha-Ras protein, Ras(12V,37G), defective in Raf-1 binding and mitogen-activated protein kinase activation, stimulates DNA synthesis in quiescent and thyroid-stimulating hormone-treated thyrocytes. A yeast two-hybrid screen identified RalGDS as a Ras(12V,37G) binding protein and therefore a potential effector of Ras in these cells. Associations between Ras and RalGDS were observed in extracts prepared from thyroid cells. Microinjection of a mutant RalA(28N) protein thought to sequester RalGDS family members reduced DNA synthesis stimulated by Ras as well as cAMP-mediated DNA synthesis in two cell lines which respond to cAMP with mitogenesis. These results support the idea that RalGDS may be an effector of Ras in cAMP-mediated growth stimulation.

Cyclic AMP↗
PubMed1997

Dynamics of families during the third trimester of pregnancy in southwest Finland.

Transition to a changed family structure during pregnancy brings into focus the family's dynamics. To describe these dynamics during the third trimester of a healthy pregnancy, the Family Dynamics Measure was administered to 136 mothers and 131 fathers in southwest Finland. Mothers and fathers reported differences in 4 of the 6 dimensions of family dynamics. Clearer communication was perceived by mothers of higher social status. Second-time parents perceived greater isolation and role conflict than first-time parents. Findings contribute cues for specific family guidance by nurses during the transition to parenthood regarding partner differences, parity and social status.

Adolescent↗
PubMed1997

Competition between adjacent meiotic recombination hotspots in the yeast Saccharomyces cerevisiae.

In a wild-type strain of Saccharomyces cerevisiae, a hotspot for meiotic recombination is located upstream of the HIS4 gene. An insertion of a 49-bp telomeric sequence into the coding region of HIS4 strongly stimulates meiotic recombination and the local formation of meiosis-specific double-strand DNA breaks (DSBs). When strains are constructed in which both hotspots are heterozygous, hotspot activity is substantially less when the hotspots are on the same chromosome than when they are on opposite chromosomes.

Alcohol Oxidoreductases↗
PubMed1997

Signaling through mitogen-activated protein kinase and Rac/Rho does not duplicate the effects of activated Ras on skeletal myogenesis.

The ability of basic helix-loop-helix muscle regulatory factors (MRFs), such as MyoD, to convert nonmuscle cells to a myogenic lineage is regulated by numerous growth factor and oncoprotein signaling pathways. Previous studies have shown that H-Ras 12V inhibits differentiation to a skeletal muscle lineage by disrupting MRF function via a mechanism that is independent of the dimerization, DNA binding, and inherent transcriptional activation properties of the proteins. To investigate the intracellular signaling pathway(s) that mediates the inhibition of MRF-induced myogenesis by oncogenic Ras, we tested two transformation-defective H-Ras 12V effector domain variants for their ability to alter terminal differentiation. H-Ras 12V,35S retains the ability to activate the Raf/MEK/mitogen-activated protein (MAP) kinase cascade, whereas H-Ras 12V,40C is unable to interact directly with Raf-1 yet still influences other signaling intermediates, including Rac and Rho. Expression of each H-Ras 12V variant in C3H10T1/2 cells abrogates MyoD-induced activation of the complete myogenic program, suggesting that MAP kinase-dependent and -independent Ras signaling pathways individually block myogenesis in this model system. However, additional studies with constitutively activated Rac1 and RhoA proteins revealed no negative effects on MyoD-induced myogenesis. Similarly, treatment of Ras-inhibited myoblasts with the MEK1 inhibitor PD98059 revealed that elevated MAP kinase activity is not a significant contributor to the H-Ras 12V effect. These data suggest that an additional Ras pathway, distinct from the well-characterized MAP kinase and Rac/Rho pathways known to be important for the transforming function of activated Ras, is primarily responsible for the inhibition of myogenesis by H-Ras 12V.

Animals↗
PubMed1996

A role for the Ral guanine nucleotide dissociation stimulator in mediating Ras-induced transformation.

Oncogenic Ras transforms cells through the activation of multiple downstream pathways mediated by separate effector molecules, one of which is Raf. Here we report the identification of a second ras-binding protein that can induce cellular transformation in parallel with activation of the Raf/mitogen-activated protein kinase cascade. The Ral guanine nucleotide dissociation stimulator (RalGDS) was isolated from a screen for Ras-binding proteins that specifically interact with a Ras effector-loop mutant, ras(12V,37G), that uncouples Ras from activation of Raf1. RalGDS, like ras(12V, 37G), cooperates synergistically with mutationally activated Raf to induce foci of growth and morphologically transformed NIH 3T3 cells. RalGDS does not significantly enhance MAP kinase activation by activated Raf, suggesting that the cooperativity in focus formation is due to a distinct pathway acting downstream of Ras and parallel to Raf.

3T3 Cells↗
PubMed1996

Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of RAS.

The RAS guanine nucleotide binding proteins activate multiple signaling events that regulate cell growth and differentiation. In quiescent fibroblasts, ectopic expression of activated H-RAS (H-RASV12, where V12 indicates valine-12) induces membrane ruffling, mitogen-activated protein (MAP) kinase activation, and stimulation of DNA synthesis. A mutant of activated H-RAS, H-RASV12C40 (where C40 indicates cysteine-40), was identified that was defective for MAP kinase activation and stimulation of DNA synthesis, but retained the ability to induce membrane ruffling. Another mutant of activated H-RAS, H-RASV12S35 (where S35 indicates serine-35), which activates MAP kinase, was defective for stimulation of membrane ruffling and induction of DNA synthesis. Expression of both mutants resulted in a stimulation of DNA synthesis that was comparable to that induced by H-RASV12. These results indicate that membrane ruffling and activation of MAP kinase represent distinct RAS effector pathways and that input from both pathways is required for the mitogenic activity of RAS.

Animals↗
PubMed1996

Ras interaction with two distinct binding domains in Raf-1 may be required for Ras transformation.

Although Raf-1 is a critical Ras effector target, how Ras mediates Raf-1 activation remains unresolved. Raf-1 residues 55-131 define a Ras-binding domain essential for Raf-1 activation. Therefore, our identification of a second Ras-binding site in the Raf-1 cysteine-rich domain (residues 139-184) was unexpected and suggested a more complex role for Ras in Raf-1 activation. Both Ras recognition domains preferentially associate with Ras-GTP. Therefore, mutations that impair Ras activity by perturbing regions that distinguish Ras-GDP from Ras-GTP (switch I and II) may disrupt interactions with either Raf-1-binding domain. We observed that mutations of Ras that impaired Ras transformation by perturbing its switch I (T35A and E37G) or switch II (G60A and Y64W) domain preferentially diminished binding to Raf-1-(55-131) or the Raf-1 cysteine-rich domain, respectively. Thus, these Ras-binding domains recognize distinct Ras-GTP determinants, and both may be essential for Ras transforming activity. Finally, since Ha-Ras T35A and E37G mutations prevent Ras interaction with full-length Raf-1, we suggest that Raf-Cys is a cryptic binding site that is unmasked upon Ras interaction with Raf-1-(55-131).

3T3 Cells↗
PubMed1996

The determination of silver in whole blood and its application to biological monitoring of occupationally exposed groups.

A sensitive and rapid technique for directly measuring silver in blood, using electrothermal atomization atomic absorption spectrophotometry (ETAAS) is described. The method can be used to analyse precisely up to 40 blood samples a day in duplicate. Well-mixed, whole blood samples, collected in EDTA, were diluted 1 + 4 with a diluent containing 40 g l.-1 ammonium dihydrogen orthophosphate and 0.5 ml l.-1 Triton X-100. Aliquots of diluted blood were then analysed by ETAAS using wall atomization with a pyrolytically coated tube. The coefficient of variation for within-run precision was 4.55% at 10 micrograms 1.-1 and 5% at 25 micrograms 1.-1 Between-run variation, it was 4.1% at 25 micrograms l.-1 The analytical recovery for the method was 98% +/- 3% at both 8 and 30 micrograms 1. -1 The detection limit of the method was 0.1 microgram 1. -1, which was sufficiently sensitive to distinguish exposed from non-exposed individuals. Blood silver levels in unexposed subjects were found to be between < 0.1 and 0.2 micrograms 1. -1. Blood silver levels were determined in 98 occupationally exposed workers involved in bullion production, cutlery manufacture, chemical manufacture, jewelery production and silver reclamation. Blood silver levels ranged from 0.1 to 23 micrograms 1.-1, with some of the highest levels found in silver reclaimers.

Environmental Monitoring↗
PubMed1996

Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation.

Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.

3T3 Cells↗
PubMed1995

Multiple Ras functions can contribute to mammalian cell transformation.

We have developed a generalized approach, using two hybrid interactions, to isolate Ha-Ras effector loop mutations that separate the ability of Ha-Ras to interact with different downstream effectors. These mutations attenuate or eliminate Ha-ras(G12V) transformation of mammalian cells, but retain complementary activity, as demonstrated by synergistic induction of foci of growth-transformed cells, and by the ability to activate different downstream components. The transformation defect of Ha-ras(G12V, E37G) is rescued by a mutant, raf1, that restores interaction. These results indicate that multiple cellular components, including Raf1, are activated by Ha-Ras and contribute to Ha-Ras-induced mammalian cell transformation.

Animals↗
PubMed1995

Toxicologic evaluations of an immunotoxin, H65-RTA.

A series of acute and multiple dose toxicology studies were performed to support the clinical dose and to evaluate the systemic toxicity of an immunotoxin, H65-RTA. H65-RTA consists of a murine anti-CD5 monoclonal antibody and ricin A chain (RTA). The LD50 of H65-RTA was estimated to be between 60 and 62.5 mg/kg in the rat. H65-RTA was administered to the rat and the monkey as a bolus injection at doses of 0.1, 0.5, and 2 mg/kg and over 1-hr infusion at 0.2 and 2 mg/kg, respectively. Two to three weeks of postdosing recovery was included in the study design. Following repeated doses of H65-RTA, the following findings were demonstrated: peripheral edema, decreased body weight, decreased body temperature (monkey only) in addition to a general inflammatory reaction evidenced by changes in hematology, clinical chemistry, and urinalysis parameters. Histopathologically, chronic inflammation in the nonarticular soft tissue was found in the rat at doses of 0.1 mg/kg and higher and monkeys developed much more severe toxicity when compared to the rat at the same doses. Inflammation, hemorrhage, and/or edema were evident in a variety of tissues. Myeloid hyperplasia was also evident. Additional findings resulting from the drug-related stress involved adrenals, spleen, thymus, and lymph nodes. All toxicity was reversible. The antibody response was evident in rats at doses of 0.5 mg/kg and higher and in all monkeys at doses of 0.2 and 2 mg/kg. In conclusion, since H65 antibody does not cross-react with the T cells from either the rat or the cynomolgus monkey, the toxicity observed in the studies described above was not related to T lymphocytes and was probably due to a series of acute to subacute inflammatory reactions caused largely by the RTA moiety of H65-RTA.

Animals↗
PubMed1995

The lithium test dose prediction method in aggressive children.

Cooper and associates (1973) developed a method of ascertaining the lithium dosage required to attain a therapeutic serum level of 0.6 to 1.2 mEq/L. However, reports about the safety and accuracy of their method in children are limited (Geller & Fetner 1989). This study relates our experience with using this method in children. Subjects were 16 conduct-disordered children (13 males, 3 females), ages 8.97 to 17.14 years (mean, 12.73 +/- 2.12), who were treated with lithium to decrease aggressive behavior. Following a lithium 600-mg loading dose, a 24-hour serum lithium level was drawn from which a "predicted" lithium dosage was established. These dosages ranged from 600 to 1,800 mg/day (mean, 1,312.5 +/- 450) and the corresponding serum lithium levels at steady-state ranged from 0.58 to 1.13 mEq/L (mean, 0.87 +/- 0.15). No severe side effects were encountered. This suggests that the method is safe and useful for predicting lithium dosages in children.

Adolescent↗