Search PubMed⌕ Search

Biomedical subjects

A M Brown

Publications and source records attributed to A M Brown.

At least 37 records · Page 2Linked to original sources
PubMed2001

A role for the Agouti-Related Protein promoter in obesity and type 2 diabetes.

The murine Agouti-Related Protein (mAGRP) is upregulated in obese and diabetic mice and can stimulate hyperphagia when overexpressed in transgenic models. Here we report upstream nucleotide sequences of the human hAGRP gene with putative recognition sites for transcription factors including a site for the STAT transactivators. A polymorphism (-38C-->T) was identified in the promoter region and the C/C genotype had significantly higher promoter activity and affinity for transcription factors as tested in periphery- and hypothalamus-derived cell lines. The polymorphic site could affect the expression levels of hAGRP and the high expressing C/C genotype was significantly associated with high BMI and type 2 diabetes in Africans.

Agouti-Related Protein↗
PubMed2001

Wnt signaling in breast cancer: have we come full circle?

Since the original identification of Wnt1 as a mammary oncogene in mouse mammary tumor virus infected mice, questions have been asked about its relevance to human breast cancer. Wnt1 is now known to be one of a large family of Wnt genes encoding structurally similar secreted signaling proteins, several of which are functionally redundant. The principal intracellular signaling pathway activated by these proteins has been elucidated in recent years. Components of this pathway include proto-oncogene products, such as beta-catenin, and tumor suppressor proteins such as APC. Although WNT1 itself has not been implicated in human breast neoplasms, it has been reported that other WNT genes are sometimes overexpressed in human breast cancer and there is growing evidence that downstream components of the Wnt signaling pathway are activated in a significant proportion of breast tumors.

Animals↗
PubMed2001

Selective dopaminergic vulnerability: 3,4-dihydroxyphenylacetaldehyde targets mitochondria.

Parkinson's disease (PD) is a major cause of age-related morbidity and mortality, present in nearly 1% of individuals at ages 70-79 and approximately 2.5% of individuals at age 85. L-DOPA (L-dihydroxyphenylalanine), which is metabolized to dopamine by dopa decarboxylase, is the primary therapy for PD, but may also contribute to disease progression. Association between mitochondrial dysfunction, monoamine oxidase (MAO) activity, and dopaminergic neurotoxicity has been repeatedly observed, but the mechanisms underlying selective dopaminergic neuron depletion in aging and neurodegenerative disorders remain unclear. We now report that 3,4-dihydroxyphenylacetaldehyde (DOPAL), the MAO metabolite of dopamine, is more cytotoxic in neuronally differentiated PC12 cells than dopamine and several of its metabolites. In isolated, energetically compromised mitochondria, physiological concentrations of DOPAL induced the permeability transition (PT), a trigger for cell death. Dopamine was > 1000-fold less potent. PT inhibitors protected both mitochondria and cells against DOPAL. Sensitivity to DOPAL was reduced > or = 30-fold in fully energized mitochondria, suggesting that mitochondrial respiration may increase resistance to PT induction by the endogenous DOPAL in the substantia nigra. These data provide a potential mechanism of action for L-DOPA-mediated neurotoxicity and suggest two potentially interactive mechanisms for the selective vulnerability of neurons exposed to dopamine.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2001

PEA3 is up-regulated in response to Wnt1 and activates the expression of cyclooxygenase-2.

The inducible prostaglandin synthase cyclooxygenase-2 (COX-2) is aberrantly expressed in intestinal tumors resulting from APC mutation, and is also transcriptionally up-regulated in mouse mammary epithelial cells in response to Wnt1 expression. beta-Catenin stabilization is a consequence of both APC mutation and Wnt signaling. We have previously observed coordinate regulation of the matrilysin promoter by beta-catenin and Ets family transcription factors of the PEA3 subfamily. Here we show that while beta-catenin only weakly activates the COX-2 promoter, PEA3 family transcription factors are potent activators of COX-2 transcription. Consistent with this, PEA3 is up-regulated in Wnt1-expressing mouse mammary epithelial cells, and PEA3 factors are highly expressed in tumors from Wnt1 transgenic mice, in which Cox-2 is also up-regulated. Promoter mapping experiments suggest that the NF-IL6 site in the COX-2 promoter is important for mediating PEA3 responsiveness. The NF-IL6 site is also important for COX-2 transcription in some colorectal cancer lines (Shao, J., Sheng, H., Inoue, H., Morrow, J. D., and DuBois, R. N. (2000) J. Biol. Chem. 275, 33951-33956), and PEA3 factors are highly expressed in colorectal cancer cell lines. Therefore, we speculate that PEA3 factors may contribute to the up-regulation of COX-2 expression resulting from both APC mutation and Wnt1 expression.

Animals↗
PubMed2001

Mutations in the Kv beta 2 binding site for NADPH and their effects on Kv1.4.

Kv beta 2 enhances the rate of inactivation and level of expression of Kv1.4 currents. The crystal structure of Kv beta 2 binds NADP(+), and it has been suggested that Kv beta 2 is an oxidoreductase enzyme (). To investigate how this function might relate to channel modulation, we made point mutations in Kv beta 2 in either the NADPH docking or putative catalytic sites. Using the yeast two-hybrid system, we found that these mutations did not disrupt the interaction of Kv beta 2 with Kv alpha 1 channels. To characterize the Kv beta 2 mutants functionally, we coinjected wild-type or mutant Kv beta 2 cRNAs and Kv1.4 cRNA in Xenopus laevis oocytes. Kv beta 2 increased both the amplitude and rate of inactivation of Kv1.4 currents. The cellular content of Kv1.4 protein was unchanged on Western blot, but the amount in the plasmalemma was increased. Mutations in either the orientation or putative catalytic sites for NADPH abolished the expression-enhancing effect on Kv1.4 current. Western blots showed that both types of mutation reduced Kv1.4 protein. Like the wild-type Kv beta 2, both types of mutation increased the rate of inactivation of Kv1.4, confirming the physical association of mutant Kv beta 2 subunits with Kv1.4. Thus, mutations that should interfere with NADPH function uncouple the expression-enhancing effect of Kv beta 2 on Kv1.4 currents from its effect on the rate of inactivation. These results suggest that the binding of NADPH and the putative oxidoreductase activity of Kv beta 2 may play a role in the processing of Kv1.4.

Animals↗
PubMed2001

Pharmacological assessment of the role of the glycine transporter GlyT-1 in mediating high-affinity glycine uptake by rat cerebral cortex and cerebellum synaptosomes.

Two distinct types of glycine transporter, GlyT-1 and GlyT-2, have been characterised. GlyT-1 and GlyT-2 are known to be differentially expressed amongst CNS areas, but direct functional evidence for their relative contributions to high-affinity glycine uptake by brain tissues is lacking. In the present study, we have used the selective GlyT-1 inhibitor N[3-(4"-fluorophenyl)-3-(4"-phenylphenoxy)propyl]sarcosine (NFPS) to investigate the role of GlyT-1 in mediating glycine uptake. HEK293 cells expressing human GlyT-1c or GlyT-2 showed high levels of Na(+)-dependent glycine uptake, with K(m) values of 117+/-13 and 200+/-22 microM, respectively. NFPS potently inhibited uptake in GlyT-1c cells (IC(50) value 0.22+/-0.03 microM), being around 500-fold more potent than glycine or sarcosine, but had no effect on uptake in GlyT-2 cells (IC(50) >10 microM). Efflux of pre-loaded [3H]-glycine from GlyT-1c cells was increased by glycine or sarcosine, whereas NFPS had no effect on its own but blocked the effects of glycine or sarcosine. These results confirm that NFPS is a potent, selective and non-transportable GlyT-1 inhibitor. Rat cortex and cerebellum synaptosomes also showed a high-affinity Na(+)-dependent component of glycine uptake, with affinities similar to those observed for uptake in GlyT-1c or GlyT-2 cells. In cortex synaptosomes, NFPS and sarcosine produced the same maximal inhibition of uptake as glycine itself. However, in cerebellum synaptosomes, the maximal inhibition produced by NFPS and sarcosine was only half that produced by glycine. In both tissues NFPS was around 1000-fold more potent than glycine or sarcosine. Overall, our findings indicate that high-affinity glycine uptake in cerebral cortex occurs predominantly via GlyT-1. However, in cerebellum, only a part of the high-affinity uptake is mediated by GlyT-1, with the remaining NFPS-insensitive component most likely mediated by GlyT-2.

Amino Acid Transport Systems, Neutral↗
PubMed2001

A step-by-step guide to non-linear regression analysis of experimental data using a Microsoft Excel spreadsheet.

The objective of this present study was to introduce a simple, easily understood method for carrying out non-linear regression analysis based on user input functions. While it is relatively straightforward to fit data with simple functions such as linear or logarithmic functions, fitting data with more complicated non-linear functions is more difficult. Commercial specialist programmes are available that will carry out this analysis, but these programmes are expensive and are not intuitive to learn. An alternative method described here is to use the SOLVER function of the ubiquitous spreadsheet programme Microsoft Excel, which employs an iterative least squares fitting routine to produce the optimal goodness of fit between data and function. The intent of this paper is to lead the reader through an easily understood step-by-step guide to implementing this method, which can be applied to any function in the form y=f(x), and is well suited to fast, reliable analysis of data in all fields of biology.

Computational Biology↗
PubMed2001

Electrocardiographic prediction of abnormal genotype in congenital long QT syndrome: experience in 101 related family members.

INTRODUCTION: Previous studies showed that diagnosing congenital long QT syndrome (LQTS) is difficult due to variable penetrance and genetic heterogeneity, especially when subjects from multiple families with diverse mutations are combined. We hypothesized that a combination of clinical and ECG techniques could identify gene carriers within a single family with congenital LQTS. METHODS AND RESULTS: One hundred one genotyped members of a family with LQTS, including 26 carriers of a HERG mutation, underwent history and ECG analysis. Forty-eight family members also underwent exercise testing with QT and T wave alternans (TWA) analysis and 24-hour Holter monitoring with QT and heart rate variability analysis. A logistic regression model, which included age, gender, QTc, and QTc by age, provided the best prediction of gene carrier status, although there was substantial overlap (78%) of QTc among subjects with and without the mutation. QTc was not helpful as a discriminator in children < or = 13 years. TWA (observed infrequently) did not add significantly to the model's ability to predict abnormal genotype. CONCLUSION: Even in this homogeneous LQTS population, the phenotype was so variable that clinical and detailed ECG analyses did not permit an accurate diagnosis of gene carrier status, especially in children. Sustained microvolt TWA was a specific (100%) but insensitive (18%) marker for LQTS. Its ability to predict risk of arrhythmia in this population remains to be determined. Genetic testing serves an essential role in screening for carriers of LQTS.

Adolescent↗
PubMed2001

Ionic mechanisms of aglycemic axon injury in mammalian central white matter.

The authors investigated ionic mechanisms underlying aglycemic axon injury in adult rat optic nerve, a central white matter tract. Axon function was assessed using evoked compound action potentials (CAPs). Glucose withdrawal led to delayed CAP failure, an alkaline extracellular pH shift, and an increase in extracellular [K(+)]. Sixty minutes of glucose withdrawal led to irreversible axon injury. Aglycemic axon injury required extracellular calcium; the extent of injury progressively declined as bath [Ca(2+)] was decreased. To evaluate Ca(2+) movements during aglycemia, the authors recorded extracellular [Ca(2+)] ([Ca(2+)](o)) using Ca(2+)-sensitive microelectrodes. Under control conditions, [Ca(2+)](o) fell with a similar time course to CAP failure, indicating extracellular Ca(2+) moved to an intracellular position during aglycemia. The authors quantified the magnitude of [Ca(2+)]o decrease as the area below baseline [Ca(2+)]o during aglycemia and used this as a qualitative measure of Ca(2+) influx. The authors studied the mechanisms of Ca(2+) influx. Blockade of Na(+) influx reduced Ca(2+) influx and improved CAP recovery, suggesting Na(+)-Ca(2+) exchanger involvement. Consistent with this hypothesis, bepridil reduced axon injury. In addition, diltiazem or nifedipine decreased Ca(2+) influx and increased CAP recovery. The authors conclude aglycemic central white matter injury is caused by Ca(2+) influx into intracellular compartments through reverse Na(+)-Ca(2+) exchange and L-type Ca(2+) channels.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2001

Molecular determinants of inactivation and dofetilide block in ether a-go-go (EAG) channels and EAG-related K(+) channels.

The major subunit of the cardiac delayed rectifier current I(Kr) is encoded by the human ether a-go-go related gene (HERG). HERG/I(Kr) channels are blocked selectively by class III antiarrhythmic methanesulfonanilide drugs such as dofetilide. The binding site for methanesulfonanilides is believed to be similar for nonantiarrhythmic drugs such as antihistamines, antibiotics, and antipsychotics. To gain further insight into the binding site, we examined the minimal structural changes necessary to transform low-affinity binding of dofetilide by the related bovine ether a-go-go channel bEAG to high-affinity binding of HERG. Previously, it was shown that high-affinity binding in HERG required intact C-type inactivation; the bovine ether a-go-go K(+) channel (bEAG), unlike HERG, is noninactivating. Therefore, we introduced C-type inactivation into noninactivating bEAG using site-directed mutagenesis. Two point mutations in the pore region, T432S and A443S, were sufficient to produce C-type inactivation. Low concentrations of dofetilide produced block of bEAG T432S/A443S; unlike HERG, block was almost irreversible. Substitution of an additional amino acid in transmembrane domain S6 made the block reversible. Dofetilide blocked the triply mutated bEAG T432S/A443S/A453S with an IC(50) value of 1.1 microM. The blocking potency was 30-fold greater than bEAG WT and about one third that of HERG WT. We conclude that high affinity methanesulfonanilide binding to HERG channels is strongly dependent on C-type inactivation.

Amino Acid Substitution↗
PubMed2001

Two nonredundant SecA homologues function in mycobacteria.

The proper extracytoplasmic localization of proteins is an important aspect of mycobacterial physiology and the pathogenesis of Mycobacterium tuberculosis. The protein export systems of mycobacteria have remained unexplored. The Sec-dependent protein export pathway has been well characterized in Escherichia coli and is responsible for transport across the cytoplasmic membrane of proteins containing signal sequences at their amino termini. SecA is a central component of this pathway, and it is highly conserved throughout bacteria. Here we report on an unusual property of mycobacterial protein export--the presence of two homologues of SecA (SecA1 and SecA2). Using an allelic-exchange strategy in Mycobacterium smegmatis, we demonstrate that secA1 is an essential gene. In contrast, secA2 can be deleted and is the first example of a nonessential secA homologue. The essential nature of secA1, which is consistent with the conserved Sec pathway, leads us to believe that secA1 represents the equivalent of E. coli secA. The results of a phenotypic analysis of a Delta secA2 mutant of M. smegmatis are presented here and also indicate a role for SecA2 in protein export. Based on our study, it appears that SecA2 can assist SecA1 in the export of some proteins via the Sec pathway. However, SecA2 is not the functional equivalent of SecA1. This finding, in combination with the fact that SecA2 is highly conserved throughout mycobacteria, suggests a second role for SecA2. The possibility exists that another role for SecA2 is to export a specific subset of proteins.

Adenosine Triphosphatases↗
PubMed2001

KChAP/Kvbeta1.2 interactions and their effects on cardiac Kv channel expression.

KChAP and voltage-dependent K+ (Kv) beta-subunits are two different types of cytoplasmic proteins that interact with Kv channels. KChAP acts as a chaperone for Kv2.1 and Kv4.3 channels. It also binds to Kv1.x channels but, with the exception of Kv1.3, does not increase Kv1.x currents. Kvbeta-subunits are assembled with Kv1.x channels; they exhibit "chaperone-like" behavior and change gating properties. In addition, KChAP and Kvbeta-subunits interact with each other. Here we examine the consequences of this interaction on Kv currents in Xenopus oocytes injected with different combinations of cRNAs, including Kvbeta1.2, KChAP, and either Kv1.4, Kv1.5, Kv2.1, or Kv4.3. We found that KChAP attenuated the depression of Kv1.5 currents produced by Kvbeta1.2, and Kvbeta1.2 eliminated the increase of Kv2.1 and Kv4.3 currents produced by KChAP. Both KChAP and Kvbeta1.2 are expressed in cardiomyocytes, where Kv1.5 and Kv2.1 produce sustained outward currents and Kv4.3 and Kv1.4 generate transient outward currents. Because they interact, either KChAP or Kvbeta1.2 may alter both sustained and transient cardiac Kv currents. The interaction of these two different classes of modulatory proteins may constitute a novel mechanism for regulating cardiac K+ currents.

Animals↗
PubMed2001

Axonal L-type Ca2+ channels and anoxic injury in rat CNS white matter.

We studied the magnitude and route(s) of Ca2+ flux from extra- to intracellular compartments during anoxia in adult rat optic nerve (RON), a central white matter tract, using Ca2+ sensitive microelectrodes to monitor extracellular [Ca2+] ([Ca2+]o). One hour of anoxia caused a rapid loss of the stimulus-evoked compound action potential (CAP), which partially recovered following re-oxygenation, indicating that irreversible injury had occurred. After an initial increase caused by extracellular space shrinkage, anoxia produced a sustained decrease of 0.42 mM (29%) in [Ca2+]o. We quantified the [Ca2+]o decrease as the area below baseline [Ca2+]o during anoxia and used this as a qualitative index of suspected Ca2+ influx. The degree of RON injury was predicted by the amount of Ca2+ leaving the extracellular space. Bepridil, 0 Na+ artificial cerebrospinal fluid or tetrodotoxin reduced suspected Ca2+ influx during anoxia implicating reversal of the Na+/Ca2+ exchanger as a route of Ca2+ influx. Diltiazem reduced suspected Ca2+ influx during anoxia, suggesting that Ca2+ influx via L-type Ca2+ channels is a route of toxic Ca2+ influx into axons during anoxia. Immunocytochemical staining was used to demonstrate and localize high-threshold Ca2+ channels. Only alpha1(C) and alpha1(D) subunits were detected, indicating that only L-type Ca2+ channels were present. Double labeling with anti-neurofilament antibodies or anti-glial fibrillary acidic protein antibodies localized L-type Ca2+ channels to axons and astrocytes.

Animals↗
PubMed2001

Insulin inhibits the maturation phase of VLDL assembly via a phosphoinositide 3-kinase-mediated event.

LY 294002 (80 micromol/L), an inhibitor of phosphoinositide 3-kinase, was used to investigate the involvement of this enzyme in the insulin-mediated regulation of very low density lipoprotein (VLDL) apolipoprotein B (apoB) output from cultured rat hepatocytes. Newly synthesized apoB was pulse-labeled with [(35)S]methionine and was then allowed to assemble, via an intermediate precursor stage, into mature VLDL during subsequent chase periods. Brefeldin A (BFA, 0.2 microgram/mL) was used to discriminate between the role of insulin in the regulation of the early, compared with the later, events of VLDL assembly, including apoB degradation. Insulin (78 nmol/L), when present during the pulse-labeling and subsequent chase periods, inhibited the secretion of apoB-100 and apoB-48 as VLDL by 53% and 56%, respectively. Degradation of both was concomitantly increased. Secretion of high density lipoprotein apoB, derived from VLDL precursors, was relatively unaffected under these conditions, as was the net synthesis of apoB-100 and apoB-48. The presence of BFA during the pulse-labeling period and subsequent chase period prevented the maturation of VLDL in the insulin-treated and the non-insulin-treated cells. BFA was then removed, allowing the maturation of VLDL to proceed. Removal of insulin at this stage reversed the overall inhibitory effect of insulin. Furthermore, when insulin remained present during this period, the simultaneous presence of LY 294002 also reversed the inhibitory effect of insulin on VLDL apoB output and abolished the increase in apoB degradation. The results suggest that insulin signaling via phosphoinositide 3-kinase inhibited the maturation phase of VLDL assembly by preventing bulk lipid transfer to a VLDL precursor, thus enhancing the degradation of apoB. There was no inhibition of the conversion of newly synthesized apoB into the VLDL precursor form.

Animals↗
PubMed2001

Increased cholesterol efflux in apolipoprotein AI (ApoAI)-producing macrophages as a mechanism for reduced atherosclerosis in ApoAI((-/-)) mice.

The concentration of apolipoprotein (apo) AI in the artery wall is thought to enhance cellular cholesterol efflux and protect against atherosclerosis. It has been shown that although macrophages do not make apoAI, they respond to it by increased cholesterol efflux. We hypothesized that macrophage production of apoAI would increase cholesterol efflux and reduce atherogenesis. In this study, we produced mice expressing human apoAI under the control of the macrophage-specific scavenger receptor-A promoter (mphi-AI). Human apoAI was detectable in the serum HDL fraction of mphi-AI transgenic mice at concentrations too low to affect serum cholesterol or HDL levels. Immunoblotting showed the presence of human apoAI in transgenic macrophage culture supernatants, mostly as lipoprotein-free protein, with a small component associated with HDL-like particles. Atherosclerosis studies using apoAI((-/-)) mice transplanted with mphi-AI bone marrow showed that in the absence of macrophage-derived apoE, local expression of apoAI reduced diet-induced lesions in the proximal aorta. Additionally, mphi-AI macrophages showed a 40% increase in cholesterol efflux compared with control macrophages. These data support the hypothesis that apoAI production by macrophages in the artery wall is protective against atherosclerosis. This protection is likely mediated by increased cholesterol efflux and decreased foam cell formation in vivo.

Animals↗
PubMed2001

Correlation of the clinical severity of Alzheimer's disease with an aberration in mitochondrial DNA (mtDNA).

Controversy exists about which of the well-established neurobiological abnormalities in Alzheimer's disease (AD) relate directly to the clinical disabilities. Because of an interest in the mitochondrial lesion in AD, we tested the correlation between clinical disability (measured by the Clinical Dementia Rating [CDR] scale) and an anomaly in mitochondrial DNA (mtDNA) in AD brain. Simultaneous polymerase chain reaction (PCR) amplification of the CO1 gene in mtDNA and CO1 pseudogenes in nuclear DNA (nDNA) were performed in samples from AD and non-AD brain, and the ratios of mtDNA/nDNA amplicons calculated. This approach utilizes PCR amplification of endogenous nDNA as a normalization standard for the amplification of mtDNA. We examined total DNA from the brains of Caucasian residents of a Jewish nursing home (86 AD and 26 non-AD "controls"). These patients had been closely followed clinically until death and then autopsied. In this sample, the degree of cognitive impairment in the AD patients correlated with the reduction in the amplification of the mtDNA gene (p = 0.23; p = 0.034), but not with the density of neuritic plaques (p = 0.109). These results agree with the suggestion that the well-documented impairment in brain-energy metabolism in AD may be a direct cause of the clinical disability.

Aged↗
PubMed2001

Cyclooxygenase-2: a target for the prevention and treatment of breast cancer.

Cyclooxygenase-2 (COX-2), an inducible prostaglandin synthase, is normally expressed in parts of the kidney and brain. Aberrant COX-2 expression was first reported in colorectal carcinomas and adenomas, and has now been detected in various human cancers, including those of the breast. Strikingly, COX-2 overexpression in murine mammary gland is sufficient to cause tumour formation. To date, the role of COX-2 in tumorigenesis has been most intensively studied in the colon. Thus, the relationship between COX-2 and neoplasia can best be illustrated with reference to intestinal tumorigenesis. Here we consider the potential utility of selective COX-2 inhibitors for the prevention and treatment of breast cancer. Data for cancers of the colon and breast are compared where possible. In addition, the mechanisms by which COX-2 is upregulated in cancers and contributes to tumorigenesis are discussed. Importantly, several recent studies of mammary tumorigenesis in animal models have found selective COX-2 inhibitors to be effective in the prevention and treatment of breast cancer. Clinical trials will be needed to determine whether COX-2 inhibition represents a useful approach to preventing or treating human breast cancer.

Animals↗