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Biomedical subjects

J A Watson

Publications and source records attributed to J A Watson.

At least 19 recordsLinked to original sources
PubMed2004

Macrophages restrain contraction of an in vitro wound healing model.

Significant numbers of macrophages are present during all stages of dermal wound repair, but the functional significance of these macrophages, especially during the later contraction and remodelling stages of repair, remains unclear. We investigated the effect of macrophages on wound contraction using a novel in vitro model based upon the contracting dermal equivalent (DE). Macrophages were found to reversibly restrain DE contraction, a rapid and sustained effect that was enhanced by lipolysaccharide (LPS) treatment of macrophages and partially inhibited by hydrocortisone. Prolonged inhibition of contraction was strongly correlated with an inhibition of fibroblast proliferation. The rapid contraction-inhibiting effect of the macrophages was mediated through activation of protein kinase C (PKC). These results suggest that inflammatory macrophages restrain the later stages of wound repair, namely matrix contraction and remodeling. The novel in vitro model established here provides a useful system for examining fibroblast-macrophage interactions in the healing wound.

Adolescent↗
PubMed2000

Cyclopamine inhibition of Sonic hedgehog signal transduction is not mediated through effects on cholesterol transport.

Cyclopamine is a teratogenic steroidal alkaloid that causes cyclopia by blocking Sonic hedgehog (Shh) signal transduction. We have tested whether this activity of cyclopamine is related to disruption of cellular cholesterol transport and putative secondary effects on the Shh receptor, Patched (Ptc). First, we report that the potent antagonism of Shh signaling by cyclopamine is not a general property of steroidal alkaloids with similar structure. The structural features of steroidal alkaloids previously associated with the induction of holoprosencephaly in whole animals are also associated with inhibition of Shh signaling in vitro. Second, by comparing the effects of cyclopamine on Shh signaling with those of compounds known to block cholesterol transport, we show that the action of cyclopamine cannot be explained by inhibition of intracellular cholesterol transport. However, compounds that block cholesterol transport by affecting the vesicular trafficking of the Niemann-Pick C1 protein (NPC1), which is structurally similar to Ptc, are weak Shh antagonists. Rather than supporting a direct link between cholesterol homeostasis and Shh signaling, our findings suggest that the functions of both NPC1 and Ptc involve a common vesicular transport pathway. Consistent with this model, we find that Ptc and NPC1 colocalize extensively in a vesicular compartment in cotransfected cells.

Androstenes↗
PubMed1999

Phage display identifies thioredoxin and superoxide dismutase as novel protein kinase C-interacting proteins: thioredoxin inhibits protein kinase C-mediated phosphorylation of histone.

Using phage display we identify the redox proteins thioredoxin and superoxide dismutase (SOD) as novel protein kinase C (PKC)-interacting proteins. Overlay assays demonstrated that PKC bound to immobilized thioredoxin, providing supporting evidence for the phage display results. Kinase assays demonstrated that SOD and thioredoxin were not direct substrates for PKC but that both proteins blocked autophosphorylation of PKC. Moreover, thioredoxin inhibited PKC-mediated phosphorylation of histone (IC(50) of approx. 20 ng/ml).

Cloning, Molecular↗
PubMed1999

Down-regulation of mammalian 3-hydroxy-3-methylglutaryl coenzyme A reductase activity with highly purified liposomal cholesterol.

Chinese hamster ovary-215 cells (CHO-215) cannot synthesize C27 and C28 sterols because of a defect in the reaction that decarboxylates 4-carboxysterols [Plemenitas, A., Havel, C.M. & Watson, J.A. (1990) J. Biol. Chem. 265, 17012-17017]. Thus, CHO-215 cell growth is dependent on an exogenous metabolically functional source of cholesterol. We used CHO-215 cells to (a) determine whether highly purified (> 99.5%) cholesterol, in egg lecithin liposomes, could down-regulate derepressed 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and if so (b) determine whether the loss in reductase catalytic activity correlated kinetically with the synthesis and accumulation of detectable oxycholesterol derivatives. Liposomal cholesterol (26-39 microM) supported maximum CHO-215 growth and initiated suppression of HMG-CoA reductase activity at concentrations greater than 50 microM. Maximum suppression (50-60%) of reductase activity was achieved with 181.3 microM liposomal cholesterol in 6 h. Also, regulatory concentrations of highly purified liposomal [3H]cholesterol were not converted (biologically or chemically) to detectable levels of oxy[3H]cholesterol derivatives during 3-6 h incubations. Lastly, a broad-spectrum cytochrome P450 inhibitor (miconazole) had no effect on liposomal cholesterol-mediated suppression of HMG-CoA reductase activity. These observations established that (a) highly purified cholesterol, incorporated into egg lecithin liposomes, can signal the down-regulation of derepressed mammalian cell HMG-CoA reductase activity and (b) if oxycholesterol synthesis was required for liposomal cholesterol-mediated down-regulation, the products had to be more potent than 24-, 25-, or 26-/27-hydroxycholesterol.

Animals↗
PubMed1997

Induction of the cholesterol metabolic pathway regulates the farnesylation of RAS in embryonic chick heart cells: a new role for ras in regulating the expression of muscarinic receptors and G proteins.

We propose a novel mechanism for the regulation of the processing of Ras and demonstrate a new function for Ras in regulating the expression of cardiac autonomic receptors and their associated G proteins. We have demonstrated previously that induction of endogenous cholesterol synthesis in cultured cardiac myocytes resulted in a coordinated increase in expression of muscarinic receptors, the G protein alpha-subunit, G-alphai2, and the inward rectifying K+ channel, GIRK1. These changes in gene expression were associated with a marked increase in the response of heart cells to parasympathetic stimulation. In this study, we demonstrate that the induction of the cholesterol metabolic pathway regulates Ras processing and that Ras regulates expression of G-alphai2. We show that in primary cultured myocytes most of the RAS is localized to the cytoplasm in an unfarnesylated form. Induction of the cholesterol metabolic pathway results in increased farnesylation and membrane association of RAS. Studies of Ras mutants expressed in cultured heart cells demonstrate that activation of Ras by induction of the cholesterol metabolic pathway results in increased expression of G-alphai2 mRNA. Hence farnesylation of Ras is a regulatable process that plays a novel role in the control of second messenger pathways.

Animals↗
PubMed1997

Strengths and needs of working-class African-American and Anglo-American grandparents.

This study examined gender and racial differences in the grandparenting strengths and needs of working class grandparents. A total of 192 African-American and Anglo-American grandmothers and grandfathers from the Washington, D.C. metropolitan area were administered the Grandparent Strengths and Needs Inventory. Grandmothers perceived themselves to be significantly more involved in teaching their grandchildren and significantly more successful in the grandparent role than grandfathers. African-American grandparents perceived themselves to be significantly more involved in teaching their grandchildren than Anglo-American grandparents, but were also significantly more likely than their Anglo-American counterparts to express frustration and need for information about the grandparenting role. A significantly greater percentage of African-American grandparents expressed interest in taking a grandparent education course than Anglo-American grandparents. Implications of the findings for grandparent education are discussed.

Adolescent↗
PubMed1995

Serotonin elevates intracellular Ca2+ in rat choroid plexus epithelial cells by acting on 5-HT2C receptors.

The effects of serotonin (5-HT) on intracellular calcium activity ([Ca2+]i) in epithelial cells from rat choroid plexuses were examined. Experiments were performed on isolated cells which had been maintained in primary culture. ([Ca2+]i) was measured using micro-spectrofluorimetric techniques and the fluorescent indicator Fura-2. 5-HT was found to increase [Ca2+]i in a dose dependent manner. The [Ca2+]i response was biphasic, with an initial peak of [Ca2+]i (due to release from intracellular stores), followed by an elevated plateau phase (the result of calcium influx). The effect of 1 microM 5-HT was inhibited by mesulergine and mianserin (50 nM), which are antagonists of the 5-HT2C receptor. Spiperone and ketanserin (200 nM), less specific 5-HT2 receptor blockers, caused only a slight reduction in the response to 1 microM 5-HT. The [Ca2+]i response decreased upon repeated challenges with 1 microM 5-HT, probably as a result of receptor desensitisation. Taken together, the data suggest that 5-HT acts at 5-HT2C receptors to increase [Ca2+]i in choroid plexus epithelial cells, both by liberating Ca2+ from intracellular stores and by activating a Ca2+ influx pathway.

Animals↗
PubMed1994

Mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase function in alpha-toxin-perforated cells.

The regulation of mevalonic acid synthesis requires both nonsterol isopentenoid and sterol regulatory signal molecules. A primary target of this multivalent control process is the enzyme which catalyzes mevalonate synthesis: 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34). In this report Staphylococcus aureus alpha-toxin perforated Chinese hamster ovary cells were used to facilitate the identification of isopentenoidogenic reactions and metabolites required for mevalonate-mediated loss of HMG-CoA reductase activity. alpha-Toxin-perforated cells retained the capacity to decrease, upon demand, HMG-CoA reductase activity and protein in response to mevalonate or isopentenoid pyrophosphate esters. Also, it was deduced with highly specific metabolic inhibitors, that conversion of farnesyl 1-diphosphate to squalene was required for mevalonate-mediated suppression of reductase activity. Since squalene (2 microM) did not downregulate reductase activity, pre-squalene pyrophosphate or a derivative, or polyprenyl-1-pyrophosphate-generated inorganic pyrophosphate, or a combination of these metabolites are proposed as candidate regulatory nonsterol isopentenoid signal molecules.

Animals↗
PubMed1993

The mitotic feedback control gene MAD2 encodes the alpha-subunit of a prenyltransferase.

The mad2-1 mutation inactivates the cell-cycle feedback control that prevents budding yeast cells from leaving mitosis until spindle assembly is complete. The gene product of MAD2 shows significant sequence similarity to the alpha-subunit of prenyltransferases. Here we isolate a new temperature-sensitive mad2 mutant, mad2-2ts, and find that Mad2p is required for the membrane association of Ypt1p and Sec4p, two prenylated small GTP-binding proteins involved in protein trafficking. Extracts from mad2-2ts mutant cells fail to geranylgeranylate a number of substrates at the non-permissive temperature. mad2-2ts is synthetically lethal with bet2-1, a mutation in the gene that encodes for the beta-subunit of the Ypt1p and Sec4p geranylgeranyl transferase. Therefore MAD2 and BET2 gene products may physically interact to form a geranylgeranyl transferase complex. In addition, the difference between the phenotypes of mad2-1 and mad2-2ts suggests that MAD2 has distinct roles in protein transport and the mitotic feedback control.

Calcium-Binding Proteins↗
PubMed1993

Isopentenoid synthesis in eukaryotic cells. An initiating role for post-translational control of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Using insect (Drosophila) and rodent mammalian cell cultures we demonstrate that acute mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A was initiated in the absence of protein synthesis. In addition, insect and mammalian cells depleted (1 h) of putative regulatory post-isopentenyl-1-pyrophosphate metabolites by incubation with an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A synthase (L-659,699) accumulated 3-hydroxy-3-methylglutaryl coenzyme A reductase enzyme units. This accumulation resulted primarily from a decrease in the loss of 3-hydroxy-3-methylglutaryl coenzyme A reductase enzyme units rather than from an increase in enzyme synthesis. These observations suggest that the initial phase of mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity was governed primarily by post-translational processes. An unknown rapidly turning over post-isopentenyl-1-pyrophosphate metabolite(s) is proposed as the agonist for these post-translational processes.

Cell Line↗
PubMed1993

Preschool embedded figures test performance of young children: age and gender differences.

An analysis of the literature was the basis for a set of predictions regarding the Preschool Embedded Figures Test performance of a small, cross-sectional sample of 37 3- to 5-year-old children. The test scores were modestly reliable. Predicted age-related differences in scores for boys and girls were observed, including an interaction of age with gender; however, other predictions regarding those scores were not supported. Based on a small sample, it was tentatively concluded that the evidence for continued use of the Preschool Embedded Figures Test as a measure of field independence for young children was weak.

Age Factors↗
PubMed1992

Identification of a prenylation site in delta virus large antigen.

During replication, hepatitis delta virus (HDV) switches from production of small to large delta antigen. Both antigen isoforms have an HDV genome binding domain and are packaged into hepatitis B virus (HBV)-derived envelopes but differ at their carboxy termini. The large antigen was shown to contain a terminal CXXX box and undergo prenylation. The large, but not the small, antigen formed secreted particles when expressed singly with HBV surface antigen. Mutation of Cys211 in the CXXX box of the large antigen abolished both prenylation and particle formation, suggesting that this site is important for virion morphogenesis.

3T3 Cells↗
PubMed1992

Isopentenoid synthesis in isolated embryonic Drosophila cells: absolute, basal mevalonate synthesis rate determination.

Embryonic Drosophila cells (Kc cells) and [5-3H]mevalonate (less than or equal to 10 microM) were used to determine the absolute basal in vivo rate of total mevalonic acid synthesis/utilization. An absolute in vivo mevalonic acid synthesis rate of 0.69 nmol/h/mg total cell protein was measured. Absolute mevalonate utilization was obtained by correcting for the extent of endogenous dilution of exogenous [3H]mevalonate at isotopic equilibrium. Cellular [3H]farnesol specific radioactivity was used as representative of a rapidly turning over isopentenoid pool. Although our previous Kc cell study (Havel, C. M., Rector, E. R. II, Watson, J. A., 1986, J. Biol. Chem. 261, 10,150-10,156) demonstrated that greater than or equal to 40% of the metabolized [3H]mevalonate appeared as 3H-labeled media water, this report established that t,t-3,7,11-[3H]trimethyl-2,6,10-dodecatriene-1,12 dioic acid was also secreted. Media accumulation of the C15-alpha,omega-prenyl dioic acid and 3H2O was related directly to [3H]mevalonic acid availability. This is the first mevalonate carbon balance study reported for a eukaryotic organism. It was concluded that (i) Kc cells synthesized more mevalonate than needed for normal growth and essential isopentenoids and (ii) excess mevalonate carbon accumulated intra- and extracellularly as isopentenoid compounds distal to C5 products. Finally, this study emphasized the need to measure total mevalonate utilization and not mevalonate conversion to a single isopentenoid end product in carbon balance investigations.

Animals↗
PubMed1992

Isopentenoid synthesis in isolated embryonic Drosophila cells: absolute mevalonic acid utilization and 3-hydroxy-3-methylglutaryl-coenzyme A reductase modulation.

The relationship between absolute isopentenoidogenesis (total and specific) and 3-hydroxy-3-methylglutaryl-coenzyme A suppression in response to increased mevalonate availability is unknown. We determined absolute isopentenoidogenesis values for the nonsterologenic Drosophila Kc cell incubated (2 h) with increasing [3H]mevalonate concentrations. At least 80% of the maximum suppression of 3-hydroxy-3-methyl glutaryl-co-enzyme A activity was achieved when total isopentenoidogenesis was increased only 2-fold. However, a 12-fold increase in total isopentenoidogenesis was achieved at higher exogenous [3H]mevalonate concentrations. Thus, modulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity was coupled to physiological changes in mevalonate/nonsterol isopentenoid availability. In contrast, isopentenoid accumulation, oxidation, and secretion were enhanced with pharmacological increases in mevalonate availability. Furthermore, an apparent constancy of total isopentenoidogenesis values plus increased metabolism of exogenous mevalonate and a significant (35-45%) suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity, in response to exogenous substrate concentrations (less than 150 microM), supported a partial or complete compensatory dimunition in endogenous substrate synthesis. Since these responses occurred within the 2-h study, earlier time periods must be assessed to (i) define the initial nonsterol-mediated regulatory response and (ii) to trap the nonsterol isopentenoid regulatory signal molecule(s).

Animals↗