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Biomedical subjects

J Chappell

Publications and source records attributed to J Chappell.

At least 19 recordsLinked to original sources
PubMed2003

Phase separation in polyfluorene-polymethylmethacrylate blends studied using UV near-field microscopy.

In this paper we present a near-field microscopy study of thin films of a phase-separated blend of the fluorescent conjugated-polymer poly(9,9-dioctylfluorene) [PFO] with the non-fluorescent polymer polymethylmethacrylate [PMMA]. A scanning near-field optical microscope (NSOM) was used to generate (blue) fluorescence from the PFO following UV excitation at 362 nm. A range of different concentrations of PFO in PMMA were studied ranging from 1 to 50% PFO in PMMA by mass. By studying both the shear force and fluorescence images we were able accurately to determine the distribution of PFO in the PMMA. We found that phase separation occurs over a number of different length-scales between 5 micro m and 250 nm. We show that at PFO concentrations of 1%, the PFO lies on top of the PMMA. At a PFO relative concentration of 50%, the PMMA phase extends through the whole thickness of the film to the underlying substrate. We use such samples to discuss the resolution of NSOM when imaging thick organic films. Furthermore, we confirm that the length-scales of phase separation can be modified via control over spin-casting protocols.

Equipment Design↗
PubMed2001

Cloning, heterologous expression, and functional characterization of 5-epi-aristolochene-1,3-dihydroxylase from tobacco (Nicotiana tabacum).

Capsidiol is a bicyclic, dihydroxylated sesquiterpene produced by several solanaceous species in response to a variety of environmental stimuli. It is the primary antimicrobial compound produced by Nicotiana tabacum in response to fungal elicitation, and it is formed via the isoprenoid pathway from 5-epi-aristolochene. Much of the biosynthetic pathway for the formation of this compound has been elucidated, except for the enzyme(s) responsible for the conversion of 5-epi-aristolochene to its dihydroxylated form, capsidiol. Biochemical evidence from previous studies with N. tabacum (Whitehead, I. M., Threlfall, D. R., and Ewing, D. F., 1989, Phytochemistry 28, 775-779) and Capsicum annuum Hoshino, T., Yamaura, T., Imaishi, H., Chida, M., Yoshizawa, Y., Higashi, K., Ohkawa, H., Mizutani, J., 1995, Phytochemistry 38, 609-613. suggested that the oxidation of 5-epi-aristolochene to capsidiol was mediated by at least one elicitor-inducible cytochrome P450 hydroxylase. In extending these observations, we developed an in vivo assay for 5-epi-aristolochene hydroxylase activity and used it to demonstrate a dose-dependent inhibition of activity by ancymidol and ketoconazole, two well characterized inhibitors of cytochrome P450 enzymes. Using degenerate oligonucleotide primers designed to the well conserved domains found within most P450 enzymes, including the heme binding domain, cDNA fragments representing four distinct P450 families (CYP71, CYP73, CYP82, and CYP92) were amplified from a cDNA library prepared against mRNA from elicitor-treated cells using PCR. The PCR fragments were subsequently used to isolate full-length cDNAs (CYP71D20 and D21, CYP73A27 and A28, CYP82E1 and CYP92A5), and these in turn were used to demonstrate that the corresponding mRNAs were all induced in elicitor-treated cells, albeit with different induction patterns. Representative, full-length cDNAs for each of the P450s were engineered into a yeast expression system, and the recombinant yeast assessed for functional expression of P450 protein by measuring the CO difference spectra of the yeast microsomes. Only microsomal preparations from yeast expressing the CYP71D20 and CYP92A5 cDNAs exhibited significant CO difference absorbance spectra at 450 nm and were thus tested for their ability to hydroxylate 5-epi-aristolochene and 1-deoxycapsidiol, a putative mono-hydroxylated intermediate in capsidiol biosynthesis. Interestingly, the CYP71D20-encoded enzyme activity was capable of converting both 5-epi-aristolochene and 1-deoxycapsidiol to capsidiol in vitro, consistent with the notion that this P450 enzyme catalyzes both hydroxylations of its hydrocarbon substrate.

Amino Acid Sequence↗
PubMed2001

Effect of insulin on cell cycle progression in MCF-7 breast cancer cells. Direct and potentiating influence.

We recently demonstrated that in MCF-7 breast cancer cells, insulin promoted the phosphorylation and activation of geranylgeranyltransferase I (GGTI-I), increased the amounts of geranylgeranylated Rho-A and potentiated the transactivating activity of lysophosphatidic acid (LPA) (Chappell, J., Golovchenko, I., Wall, K., Stjernholm, R., Leitner, J., Goalstone, M., and Draznin, B. (2000) J. Biol. Chem. 275, 31792-31797). In the present study, we explored the mechanism of this potentiating effect of insulin on LPA. Insulin (10 nm) potentiated the ability of LPA to stimulate cell cycle progression and DNA synthesis in MCF-7 cells. The potentiating effect of insulin appears to involve increases in the expression of cyclin E and decreases in the expression of the cyclin-dependent kinase inhibitor p27Kip1. All potentiating effects of insulin were inhibited in the presence of an inhibitor of GGTase I, GGTI-286 (3 microm) or by an expression of a dominant negative mutant of Rho-A. In contrast to its potentiating action, a direct mitogenic effect of insulin in MCF-7 cells involves activation of phosphatidylinositol 3-kinase and increased expression of cyclin D1. We conclude that the ability of insulin to increase the cellular amounts of geranylgeranylated Rho-A results in potentiation of the LPA effect on cyclin E expression and degradation of p27Kip1 and cell cycle progression in MCF-7 breast cancer cells.

Breast Neoplasms↗
PubMed2001

Sea level change through the last glacial cycle.

Sea level change during the Quaternary is primarily a consequence of the cyclic growth and decay of ice sheets, resulting in a complex spatial and temporal pattern. Observations of this variability provide constraints on the timing, rates, and magnitudes of the changes in ice mass during a glacial cycle, as well as more limited information on the distribution of ice between the major ice sheets at any time. Observations of glacially induced sea level changes also provide information on the response of the mantle to surface loading on time scales of 10(3) to 10(5) years. Regional analyses indicate that the earth-response function is depth dependent as well as spatially variable. Comprehensive models of sea level change enable the migration of coastlines to be predicted during glacial cycles, including the anthropologically important period from about 60,000 to 20,000 years ago.

Journal Article↗
PubMed2001

Variability in the El Niño-Southern Oscillation through a glacial-interglacial cycle.

The El Niño-Southern Oscillation (ENSO) is the most potent source of interannual climate variability. Uncertainty surrounding the impact of greenhouse warming on ENSO strength and frequency has stimulated efforts to develop a better understanding of the sensitivity of ENSO to climate change. Here we use annually banded corals from Papua New Guinea to show that ENSO has existed for the past 130,000 years, operating even during "glacial" times of substantially reduced regional and global temperature and changed solar forcing. However, we also find that during the 20th century ENSO has been strong compared with ENSO of previous cool (glacial) and warm (interglacial) times. The observed pattern of change in amplitude may be due to the combined effects of ENSO dampening during cool glacial conditions and ENSO forcing by precessional orbital variations.

Animals↗
PubMed2000

Potentiation of Rho-A-mediated lysophosphatidic acid activity by hyperinsulinemia.

We have shown previously that insulin promotes phosphorylation and activation of farnesyltransferase and geranylgeranyltransferase (GGTase) II. We have now examined the effect of insulin on geranylgeranyltransferase I in MCF-7 breast cancer cells. Insulin increased GGTase I activity 3-fold and augmented the amounts of geranylgeranylated Rho-A by 18%. Both effects of the insulin were blocked by an inhibitor of GGTase I, GGTI-286. The insulin-induced increases in the amounts of geranylgeranylated Rho-A resulted in potentiation of the Rho-A-mediated effects of lysophosphatidic acid (LPA) on a serum response element-luciferase construct. Preincubation of cells with insulin augmented the LPA-stimulated serum response element-luciferase activation to 12-fold, compared with just 6-fold for LPA alone (p < 0.05). The potentiating effect of insulin was dose-dependent, inhibited by GGTI-286 and not mimicked by insulin-like growth factor-1. We conclude that insulin activates GGTase I, increases the amounts of geranylgeranylated Rho-A protein, and potentiates the Rho-A-dependent nuclear effects of LPA in MCF-7 breast cancer cells.

Alkyl and Aryl Transferases↗
PubMed2000

Differential induction of sesquiterpene metabolism in tobacco cell suspension cultures by methyl jasmonate and fungal elicitor.

Jasmonates are well documented for their ability to modulate the expression of plant genes and to influence specific aspects of disease/pest resistance traits. We and others have been studying the synthesis of sesquiterpene phytoalexins in elicitor/pathogen-challenged plants and have sought to determine if methyl jasmonate (MeJA) could substitute for fungal elicitors in the induction of capsidiol accumulation by tobacco cell cultures. The current results demonstrate that MeJA does in fact induce phytoalexin accumulation, but with a much more delayed induction time course than elicitor. While elicitor treatment induced strong but transient changes in key enzymes of sesquiterpene biosynthesis, sesquiterpene cyclase, and aristolochene/deoxy-capsidiol hydroxylase, MeJA did not. Instead, MeJA caused a protracted induction of cyclase activity and only a low level of hydroxylase activity. MeJA induced the expression of at least two sesquiterpene cyclase genes, including one that had not been observed previously in elicitor-induced mRNA populations. Only a small portion of the total sesquiterpene cyclase mRNA induced by MeJA was associated with polysomal RNA, suggesting that the MeJA treatment imposed both transcriptional and posttranscriptional regulation in tobacco cells. These results are not consistent with MeJA playing a role in orchestrating defense responses in elicitor-treated tobacco cells, but do provide evidence that MeJA induces a subset of genes coding for the biosynthesis of sesquiterpene phytoalexins.

Acetates↗
PubMed2000

Molecular characterization of squalene synthase from the green microalga Botryococcus braunii, race B.

The green microalga Botryococcus braunii produces large amounts of liquid hydrocarbons and is classified into three races, depending on the type of the hydrocarbon produced. The B race produces two types of triterpenoid hydrocarbons, squalene and botryococcene, both of which are putative condensation products of farnesyl diphosphate. In an attempt to better understand the regulation involved in the production of squalene and botryococcene, we have isolated and characterized a squalene synthase (SS) gene from the B race of B. braunii. A 366-bp cDNA fragment was initially obtained from the B race utilizing a reverse transcription/polymerase chain reaction and degenerate primers based on conserved amino acid sequences found in all SS enzymes. Using this putative SS fragment as a probe, a 2632-bp cDNA clone was isolated from a cDNA library. This cDNA contained an open reading frame coding for a protein with 461 amino acids and a predicted molecular mass of 52.5 kDa. Comparison of the Botryococcus SS (BSS) with SS from different organisms showed 52% identity with Nicotiana tabacum, 51% with Arabidopsis thaliana, 48% with Zea mays, 40% with rat, 39% with yeast, and 26% with Zymomonas mobilis. Expression of full-length and carboxy-terminus truncated BSS cDNA in Escherichia coli resulted in significant levels of bacterial SS enzyme activity but no botryococcene synthase activity. RNA blot hybridization analysis of algal cultures during a culture cycle indicated that BSS gene expression is preferential during rapid growth. Given that the DNA blot analysis indicated only a single copy of the SS gene in the algal genome, these results suggest either that there exists coordinate expression of separate synthase genes for squalene and botryococcene biosynthesis or that there might be unique physiological conditions controlling the SS vs botryococcene synthase activity of a single peptide species.

Algal Proteins↗
PubMed2000

Gastric volvulus associated with wandering spleen in a child.

Wandering spleen is an uncommon entity in childhood and has been described only rarely in association with gastric volvulus. Wandering spleen and gastric volvulus were diagnosed in a 5-year-old boy who presented with acute abdominal pain and distension. Intraoperatively, normal ligamentous connections between the stomach, spleen, and posterior abdominal wall were absent. Developmental anomalies that result in wandering spleen may lead to hypermobility of the stomach and a predisposition to gastric volvulus. In such patients, prophylactic gastropexy should be considered.

Child, Preschool↗
PubMed1999

Farnesol is utilized for isoprenoid biosynthesis in plant cells via farnesyl pyrophosphate formed by successive monophosphorylation reactions.

The ability of Nicotiana tabacum cell cultures to utilize farnesol (F-OH) for sterol and sesquiterpene biosynthesis was investigated. [(3)H]F-OH was readily incorporated into sterols by rapidly growing cell cultures. However, the incorporation rate into sterols was reduced by greater than 70% in elicitor-treated cell cultures whereas a substantial proportion of the radioactivity was redirected into capsidiol, an extracellular sesquiterpene phytoalexin. The incorporation of [(3)H]F-OH into sterols was inhibited by squalestatin 1, suggesting that [(3)H]F-OH was incorporated via farnesyl pyrophosphate (F-P-P). Consistent with this possibility, N. tabacum proteins were metabolically labeled with [(3)H]F-OH or [(3)H]geranylgeraniol ([(3)H]GG-OH). Kinase activities converting F-OH to farnesyl monophosphate (F-P) and, subsequently, F-P-P were demonstrated directly by in vitro enzymatic studies. [(3)H]F-P and [(3)H]F-P-P were synthesized when exogenous [(3)H]F-OH was incubated with microsomal fractions and CTP. The kinetics of formation suggested a precursor-product relationship between [(3)H]F-P and [(3)H]F-P-P. In agreement with this kinetic pattern of labeling, [(32)P]F-P and [(32)P]F-P-P were synthesized when microsomal fractions were incubated with F-OH and F-P, respectively, with [gamma-(32)P]CTP serving as the phosphoryl donor. Under similar conditions, the microsomal fractions catalyzed the enzymatic conversion of [(3)H]GG-OH to [(3)H]geranylgeranyl monophosphate and [(3)H]geranylgeranyl pyrophosphate ([(3)H]GG-P-P) in CTP-dependent reactions. A novel biosynthetic mechanism involving two successive monophosphorylation reactions was supported by the observation that [(3)H]CTP was formed when microsomes were incubated with [(3)H]CDP and either F-P-P or GG-P-P, but not F-P. These results document the presence of at least two CTP-mediated kinases that provide a mechanism for the utilization of F-OH and GG-OH for the biosynthesis of isoprenoid lipids and protein isoprenylation.

Catalysis↗