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Biomedical subjects

A M Brown

Publications and source records attributed to A M Brown.

At least 73 records · Page 4Linked to original sources
PubMed2000

Novel characteristics of a misprocessed mutant HERG channel linked to hereditary long QT syndrome.

Hereditary long QT syndrome (hLQTS) is a heterogeneous genetic disease characterized by prolonged QT interval in the electrocardiogram, recurrent syncope, and sudden cardiac death. Mutations in the cardiac potassium channel HERG (KCNH2) are the second most common form of hLQTS and reduce the delayed rectifier K(+) currents, thereby prolonging repolarization. We studied a novel COOH-terminal missense mutation, HERG R752W, which segregated with the disease in a family of 101 genotyped individuals. When the mutant cRNA was expressed in Xenopus oocytes it produced enhanced rather than reduced currents. Simulations using the Luo-Rudy model predicted minimal shortening rather than prolongation of the cardiac action potential. Consequently, a normal or shortened QT interval would be expected in contrast to the long QT observed clinically. This anomaly was resolved by our observation that the mutant protein was not delivered to the plasma membrane of mammalian cells but was retained intracellularly. We found that this trafficking defect was corrected at lower incubation temperatures and that functional channels were now delivered to the plasma membrane. However, trafficking could not be restored by chemical chaperones or E-4031, a specific blocker of HERG channels. Therefore, HERG R752W represents a new class of trafficking mutants in hLQTS. The occurrence of different classes of misprocessed channels suggests that a unified therapeutic approach for altering HERG trafficking will not be possible and that different treatment modalities will have to be matched to the different classes of trafficking mutants.

Action Potentials↗
PubMed2000

Mechanisms of I(Ks) suppression in LQT1 mutants.

Mutations in the cardiac potassium ion channel gene KCNQ1 (voltage-gated K(+) channel subtype KvLQT1) cause LQT1, the most common type of hereditary long Q-T syndrome. KvLQT1 mutations prolong Q-T by reducing the repolarizing cardiac current [slow delayed rectifier K(+) current (I(Ks) )], but, for reasons that are not well understood, the clinical phenotypes may vary considerably even for carriers of the same mutation, perhaps explaining the mode of inheritance. At present, only currents expressed by LQT1 mutants have been studied, and it is unknown whether abnormal subunits are transported to the cell surface. Here, we have examined for the first time trafficking of KvLQT1 mutations and correlated the results with the I(Ks) currents that were expressed. Two missense mutations, S225L and A300T, produced abnormal currents, and two others, Y281C and Y315C, produced no currents. However, all four KvLQT1 mutations were detected at the cell surface. S225L, Y281C, and Y315C produced dominant negative effects on wild-type I(Ks) current, whereas the mutant with the mildest dysfunction, A300T, did not. We examined trafficking of a severe insertion deletion mutant Delta544 and detected this protein at the cell surface as well. We compared the cellular and clinical phenotypes and found a poor correlation for the severely dysfunctional mutations.

Animals↗
PubMed2000

Wnt-1 regulation of connexin43 in cardiac myocytes.

Gap junction channels composed of connexin43 (Cx43) are essential for normal heart formation and function. We studied the potential role of the Wnt family of secreted polypeptides as regulators of Cx43 expression and gap junction channel function in dissociated myocytes and intact hearts. Neonatal rat cardiomyocytes responded to Li(+), which mimics Wnt signaling, by accumulating the effector protein beta-catenin and by inducing Cx43 mRNA and protein markedly. Induction of Cx43 expression was also observed in cardiomyocytes cocultured with Rat-2 fibroblasts or N2A neuroblastoma cells programmed to secrete bioactive Wnt-1. By transfecting a Cx43 promoter-reporter gene construct into cardiomyocytes, we demonstrated that the inductive effect of Wnt signaling was transcriptionally mediated. Enhanced expression of Cx43 increased cardiomyocyte cell coupling, as determined by Lucifer Yellow dye transfer and by calcium wave propagation. Conversely, in a transgenic cardiomyopathic mouse model that exhibits ventricular arrhythmias and gap junctional remodeling, beta-catenin and Cx43 expression were downregulated concordantly. In response to Wnt signaling, the accumulating Cx43 colocalized with beta-catenin in the junctional membrane; moreover, forced expression of Cx43 in cardiomyocytes reduced the transactivation potential of beta-catenin. These findings demonstrate that Wnt signaling is an important modulator of Cx43-dependent intercellular coupling in the heart, and they support the hypothesis that dysregulated signaling contributes to altered impulse propagation and arrhythmia in the myopathic heart.

Animals↗
PubMed2000

Shh and Wnt signaling pathways converge to control Gli gene activation in avian somites.

The regulation of the Gli genes during somite formation has been investigated in quail embryos. The Gli genes are a family encoding three related zinc finger transcription factors, Gli1, Gli2 and Gli3, which are effectors of Shh signaling in responding cells. A quail Gli3 cDNA has been cloned and its expression compared with Gli1 and Gli2. These studies show that Gli1, Gli2 and Gli3 are co-activated at the time of somite formation, thus providing a mechanism for regulating the initiation of Shh signaling in somites. Embryo surgery and paraxial mesoderm explant experiments show that each of the Gli genes is regulated by distinct signaling mechanisms. Gli1 is activated in response to Shh produced by the notochord, which also controls the dorsalization of Gli2 and Gli3 following their activation by Wnt signaling from the surface ectoderm and neural tube. This surface ectoderm/neural tube Wnt signaling has both negative and positive functions in Gli2 and Gli3 regulation: these signals repress Gli3 in segmental plate mesoderm prior to somite formation and then promote somite formation and the somite-specific activation of Gli2 and Gli3. These studies, therefore, establish a role for Wnt signaling in the control of Shh signal transduction through the regulation of Gli2 and Gli3, and provide a mechanistic basis for the known synergistic actions of surface ectoderm/neural tube and notochord signaling in somite cell specification.

Amino Acid Sequence↗
PubMed2000

Measurement of cytochrome P450 gene induction in human hepatocytes using quantitative real-time reverse transcriptase-polymerase chain reaction.

Drug-induced changes in expression of cytochrome (CYP) P450 genes are a key cause of drug-drug interactions. Consequently, preclinical prediction of these changes by novel compounds is an integral component of drug development. To date, in vitro models of CYP induction have used mRNA measurement, immunodetection, and substrate metabolism as reporters. Here, we describe the application of quantitative real-time reverse transcriptase polymerase chain reaction to study CYP1A1 and CYP3A4 gene induction in 5-day-old cultures of human hepatocytes by known CYP inducers. After 5 days in culture, CYP1A1 expression was significantly elevated (5.1- to 26-fold; P <.01) in all four livers studied. In direct contrast, CYP3A4 mRNA levels consistently decreased during culture (80- to 300-fold; P <.001). In three independent experiments, a 48-h exposure to 3-methylcholanthrene, omeprazole, and lansoprazole significantly induced CYP1A1 expression in comparison to untreated cultures (P <.05). Rifampicin and solvent were without effect on CYP1A1 expression. Under identical experimental conditions, rifampicin and lansoprazole significantly elevated CYP3A4 mRNA expression (P <.05), whereas 3-methylcholanthrene, omeprazole, and dimethyl sulfoxide were without significant effect. These data demonstrate the applicability of quantitative reverse transcriptase polymerase chain reaction to the determination of gene dynamics in human hepatocytes. This offers a highly specific alternative to quantification of drug effects on CYP expression using immunodetection and substrate metabolism.

Base Sequence↗
PubMed2000

Interactions of the 5-hydroxytryptamine 3 antagonist class of antiemetic drugs with human cardiac ion channels.

Administration of the 5-hydroxytryptamine 3 receptor class of antiemetic agents has been associated with prolongation in the QRS, JT, and QT intervals of the ECG. To explore the mechanisms underlying these findings, we examined the effects of granisetron, ondansetron, dolasetron, and the active metabolite of dolasetron MDL 74,156 on the cloned human cardiac Na(+) channel hH1 and the human cardiac K(+) channel HERG and the slow delayed rectifier K(+) channel KvLQT1/minK. Using patch-clamp electrophysiology we found that all of the drugs blocked Na(+) channels in a frequency-dependent manner. At a frequency of 3 Hz, the IC(50) values for block of Na(+) current measured 2.6, 88.5, 38.0, and 8.5 microM for granisetron, ondansetron, dolasetron, and MDL 74,156, respectively. Block was relieved by strong hyperpolarizing potentials, suggesting a possible interaction with an inactivated channel state. Recovery from inactivation was impaired at -80 mV compared with -100 mV, and the fractional recovery was impaired by drug in a concentration-dependent manner. IC(50) values for block of the HERG cardiac K(+) channel measured 3.73, 0.81, 5.95, and 12.1 microM for granisetron, ondansetron, dolasetron, and MDL 74,156, respectively. Ondansetron (3 microM) also slowed decay of HERG tail currents. In contrast, none of these drugs (10 microM) produced greater than 30% block of the slow delayed rectifier K(+) channel KvLQT1/minK. We concluded that the antiemetic agents tested in this study block human cardiac Na(+) channels probably by interacting with the inactivated state. This may lead to clinically relevant Na(+) channel blockade, especially when high heart rates or depolarized/ischemic tissue is present. The submicromolar affinity of ondansetron for the HERG K(+) channel likely underlies the prolongation of cardiac repolarization reported for this drug.

Antiemetics↗
PubMed1999

Endogenous mutations in human uncoupling protein 3 alter its functional properties.

Human uncoupling protein (UCP3) is a mitochondrial transmembrane carrier that uncouples oxidative phosphorylation and is a candidate gene for obesity. Expression of native human UCP3 mutations in yeast showed complete loss (R70W), significant reduction (R143X), or no effect (V102I and IVS6+1G > A) on the uncoupling activity of UCP3. It is concluded that certain mutations in UCP3 alter its functional impact on membrane potential (deltaphi), possibly conferring susceptibility to develop metabolic diseases.

Carrier Proteins↗
PubMed1999

Apoptogenic ganglioside GD3 directly induces the mitochondrial permeability transition.

Early events in apoptotic cascades initiated by ceramides or by activation of the surface receptor CD95 (Fas/APO-1) include the formation of ganglioside GD3. GD3 appears to be both necessary and sufficient to propagate this lipid-mediated apoptotic pathway. Later events common to many apoptotic pathways include induction of the mitochondrial permeability transition (PT) and cytochrome c release, which in turn triggers downstream caspases and cell death. The links between GD3 formation and downstream stages of apoptosis are unknown. We report that ganglioside GD3 directly induces the PT in isolated rat liver mitochondria at 30-100 microM in the presence of exogenous substrate (succinate) and at approximately 3 microM in the absence of exogenous substrate. In contrast, other gangliosides tested (e.g. GM1) have only weak stimulatory effects in the presence of succinate and protect against PT induction in the absence of respiratory substrates. GD3-mediated induction of PT was antagonized by known PT inhibitors, namely cyclosporin A, ADP, trifluoperazine, and Mg(2+). GD3 induced PT even in the presence of submicromolar Ca(2+); GD3 is therefore the first biological PT inducer identified that does not require elevated Ca(2+). Exposure to GD3 also led to mitochondrial cytochrome c release. In contrast, C(2)-ceramide, which can initiate the lipid-mediated apoptotic cascade in susceptible cells, failed to either induce PT or release cytochrome c. These observations suggest that GD3 propagates apoptosis by inducing the PT and cytochrome c release. This model provides a mechanistic link between the earlier and later stages of CD95-induced/ceramide-mediated apoptosis.

Adenosine Diphosphate↗
PubMed1999

Decreased sodium and increased transient outward potassium currents in iron-loaded cardiac myocytes. Implications for the arrhythmogenesis of human siderotic heart disease.

BACKGROUND: Patients with chronic iron overload may develop a cardiomyopathy manifested by ventricular arrhythmias and heart failure. We hypothesized that iron-loaded cardiomyocytes may have abnormal excitability. METHODS AND RESULTS: We examined a new model of human iron overload, the Mongolian gerbil given repeated injections of iron dextran. In ventricular myocytes, we measured iron concentration and distribution, action potential, sodium and potassium currents, and sodium channel protein. We showed for the first time that (1) the iron content of gerbil ventricular cardiomyocytes was increased to amounts similar to those of patients with iron-induced cardiomyopathy; (2) the overshoot and duration of the cardiac action potential decreased; (3) sodium current was reduced, steady-state inactivation was enhanced, and single-channel currents were unchanged; and (4) transient outward potassium current was increased, but inwardly rectifying potassium current was unchanged. Neonatal rat cardiomyocytes incubated with iron for 1 to 3 days showed similar changes, and levels of cardiac sodium channel proteins were unchanged. CONCLUSIONS: Abnormal excitability and heterogeneous cardiac iron deposition may cause the arrhythmogenesis of human siderotic heart disease.

Action Potentials↗
PubMed1999

A DLST genotype associated with reduced risk for Alzheimer's disease.

Recent studies suggest that variants of the DLST gene alter the risk of AD. DLST encodes the core subunit of the mitochondrial alpha-ketoglutarate dehydrogenase complex, which is deficient in AD. The authors report that in 247 US white subjects, homozygosity for DLST A19,117, T19,183 was associated with a reduced risk of AD (odds ratio [OR] = 0.35, p = 0.018). The reduced risk was marked in subjects who did not carry the apolipoprotein (APOE)-4 allele (OR = 0.16, p = 0.014). Further study of DLST in AD appears warranted.

Acyltransferases↗
PubMed1999

In vitro characterisation of the muscarinic receptor partial agonist, sabcomeline, in rat cortical and heart membranes.

We have investigated the pharmacology of the functionally selective muscarinic M1 receptor partial agonist, sabcomeline [SB-202026 (R-(Z)-(+)-alpha-(methoxyamino)-1-azabicyclo[2.2.2] octane-3-acetonitrile)], in rat cortex and heart using radioligand binding and functional studies. The Quinuclidinyl benzilate/Oxotremorine-M acetate ratio from radioligand binding studies suggested that sabcomeline and xanomeline [3(3-hexyloxy-1,25-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-met hylpyridine] are muscarinic receptor partial agonists in cortical and heart membranes. In [35S]GTPgammaS binding studies in rat cortex, carbachol stimulated binding via muscarinic M2/M4 receptors which could be blocked by sabcomeline with a pA2 of 7.2. In rat heart membranes, carbachol also stimulated [35S]GTPgammaS binding studies through muscarinic M2 receptors. Sabcomeline caused a small stimulation of basal [35S]GTPgammaS binding in both rat and heart tissues. Sabcomeline did not stimulate phosphoinositide hydrolysis in rat cortical slices, but did block the muscarinic M1 receptor-mediated response caused by carbachol with apparent pKb of 6.9. Xanomeline and milameline also had no effect on phosphoinositide hydrolysis up to 100 microM. In adenylyl cyclase studies in rat atria, sabcomeline inhibited forskolin-stimulated adenylyl cyclase activity to a similar extent to that of carbachol, xanomeline and milameline. The present study, using the techniques of radioligand binding, supports previous publications which have claimed that sabcomeline is a muscarinic receptor partial agonist. As expected, this study shows that the functional actions of this compound at muscarinic receptor subtypes and in different tissues will depend on receptor reserve.

Adenylyl Cyclases↗
PubMed1999

Transcriptional activation of cyclooxygenase-2 in Wnt-1-transformed mouse mammary epithelial cells.

Wnt-1 acts as a mammary oncogene when ectopically expressed in the mouse mammary gland. APC is a tumor suppressor gene, mutations in which cause intestinal tumorigenesis in humans and rodents. Both Wnt-1 expression and APC mutation activate a common signaling pathway involving transcriptional activation mediated by beta-catenin/Tcf complexes, but few targets relevant to carcinogenesis have yet been identified. Expression of the inducible prostaglandin synthase cyclooxygenase-2 appears critical for intestinal tumorigenesis resulting from APC mutation, suggesting that cyclooxygenase-2 might be a transcriptional target for beta-catenin/Tcf complexes. Here, we have investigated the effect of Wnt-1 on cyclooxygenase-2 expression. Wnt-1 expression in the mouse mammary epithelial cell lines RAC311 and C57MG induces stabilization of cytosolic beta-catenin and morphological transformation. Expression of Wnt-1 in these cells caused transcriptional up-regulation of the cyclooxygenase-2 gene, resulting in increased levels of cyclooxygenase-2 mRNA and protein. Prostaglandin E2 production was increased as a consequence of the elevated cyclooxygenase-2 activity and could be decreased by treatment with a selective cyclooxygenase-2 inhibitor. Cyclooxygenase-2 thus appears to be a common downstream target for APC mutation and Wnt-1 expression. In view of the critical role of cyclooxygenase-2 in intestinal tumorigenesis, cyclooxygenase-2 up-regulation in response to Wnt signaling may contribute to Wnt-induced mammary carcinogenesis.

Animals↗
PubMed1999

Tumorigenesis in Mlh1 and Mlh1/Apc1638N mutant mice.

An3 1 KAL I MutL homologue 1 (MLH1) is a member of the family of proteins required for DNA mismatch repair. Germ-line mutations in MLH1 lead to the cancer susceptibility syndrome hereditary nonpolyposis colorectal cancer (HNPCC). We generated mice carrying a null mutation in the Mlh1 gene. We showed that mice heterozygous and homozygous for the Mlh1 gene are predisposed to developing tumors of the gastrointestinal (GI) tract, lymphomas, and a number of other tumor types. We also examined the role of adenomatous polyposis coli gene (Apc) gene mutations in the GI tumors of Mlh1 mutant mice by different methods and showed that the GI tumors in Mlh1 mice express little or no adenomatous polyposis coli protein. When an Apc gene mutation was bred into the Mlh1 mutant mice, the GI tumor incidence increased 40-100-fold. The wild-type Apc allele in these tumors was found to contain mutations. Together, these results show that we have developed two mouse models for human HNPCC and that the mechanisms of tumor development in the GI tract of these mice involve loss of Apc gene function in a manner very similar to that seen in the GI tumors of HNPCC.

Adaptor Proteins, Signal Transducing↗
PubMed1999

A methodology for simulating biological systems using Microsoft Excel.

The objective of this present study was to develop a simple, easily understood methodology for solving biologically based models using a Microsoft Excel spreadsheet. The method involves the use of in-cell formulas in which Rows and Columns of new data are generated from data typed into the spreadsheet, but does not require any programming skills or use of the macro language. The approach involves entering the key parameter values into the spreadsheet and conducting the simulation by solving a set of equations based on these parameter values. The examples used in this paper are firstly, a simple voltage clamp simulation in which initial parameter values are used to calculate a system in steady state. The second example is a current clamp simulation where steady state is not reached and the solution of the equations for each time increment is used as the input for the next time increment in the simulation. The calculations are based on the Hodgkin Huxley mathematical equations that describe the voltage dependence of ion channel behavior. The problems and flexibility of the method are briefly discussed. The methodology developed in this present study should help novice modelers to create simple simulations without the need to learn a programming language or purchase expensive software.

Computer Graphics↗
PubMed1999

Wnt signalling: pathway or network?

Members of the Wnt family of secreted glycoproteins participate in many signalling events during development. Recent findings suggest that Wnt signals can sometimes play a permissive role during cell-fate assignment. Wnt proteins have been shown to interact with a number of extracellular and cell-surface proteins, whereas many intracellular components of the Wnt-signalling pathway are also involved in other cellular functions. The consequences of Wnt signalling can be affected by members of the MAP kinase family. These observations suggest that the future understanding of Wnt signalling may require models that are based on a signalling network rather than a single linear pathway.

Animals↗
PubMed1999

Pharmacological characterization of extracellular acidification rate responses in human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells.

This study characterized pharmacologically the functional responses to agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) receptors separately expressed in cloned cells using the cytosensor microphysiometer. Dopaminergic receptor agonists caused increases in extracellular acidification rate in adherent Chinese hamster ovary (CHO) clones expressing hD2, hD3 or hD4 receptors. Acidification rate responses to agonists in other cell lines expressing these receptors were smaller than those in adherent CHO cells. The time courses and maximum increases in acidification rate of the agonist responses in adherent CHO cells were different between the three dopamine receptor clones. Responses were blocked by pretreatment of cells with pertussis toxin or amiloride analogues. Most agonists had full intrinsic activity at each of the dopamine receptor subtypes, as compared to quinpirole, however both enantiomers of UH-232 and (-)3-PPP were partial agonists in this assay system. The functional potency of full agonists at each of the three receptors expressed in CHO cells was either higher than, or similar to, the apparent inhibition constants (Ki) determined in [125I]-iodosulpride competition binding studies. Functional selectivities of the agonists were less than radioligand binding selectivities. The rank orders of agonist potencies and selectivities were similar, but not identical, to the rank orders of radioligand binding affinities and selectivities. The dopamine receptor antagonists, iodosulpride and clozapine, had no effect on basal acidification rates but inhibited acidification responses in CHO cells to quinpirole in an apparently competitive manner. Antagonist potencies closely matched their radioligand binding affinities in these cells.

Amiloride↗