Respiratory arrest after peak expiratory flow measurement.
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Adult↗
Biomedical subjects
A M Brown
Publications and source records attributed to A M Brown.
Identification and cloning of a developmentally regulated Cryptosporidium parvum gene by differential mRNA display PCR.
To identify Cryptosporidium parvum genes expressed during intracellular development, differential mRNA display was used to detect differences in gene expression between mock-infected and C. parvum-infected human epithelial cells. A reproducible band present only in C. parvum-infected cells, ddHC-23, was isolated and cloned. Southern blot analysis demonstrated that ddHC-23 represented a C. parvum gene. RT-PCR revealed that HC-23 mRNA levels decreased from 6 to 12h post-infection (pi), were maximally expressed at 24h pi, and returned to low levels at 48 and 72h pi. Northern blot analysis determined that the approx. 3.6kb transcript is expressed by sporozoites prior to invasion of epithelial cells. Screening of a C. parvum genomic library with ddHC-23 isolated a genomic subclone which contained a 2790bp ORF, uninterrupted by introns. Sequence analysis indicated that the encoded protein, which displayed no similarity to any sequences in the public databases, contained a high proportion of polar amino acids, with the most abundant being Asp (17.3%), Ser (15.8%) and Gly (8.1%). Numerous potential sites for posttranslational modification were present including: casein kinase II and protein kinase C phosphorylation sites, N-myristolation sites and N-glycosylation sites. These findings demonstrate the usefulness of differential mRNA display for identifying developmentally regulated C. parvum genes within the background of genes expressed by the host cell. 1998 Elsevier Science B.V.
Amino Acid Sequence↗
A recessive variant of the Romano-Ward long-QT syndrome?
BACKGROUND: The congenital long-QT syndrome (LQTS) is a genetically heterogeneous disease characterized by prolonged ventricular repolarization and life-threatening arrhythmias. Mutations of the KVLQT1 gene, a cardiac potassium channel, generate two allelic diseases: the Romano-Ward syndrome, inherited as a dominant trait, and the Jervell and Lange-Nielsen syndrome, inherited as an autosomal recessive trait. METHODS AND RESULTS: A consanguineous family with the clinical phenotype of LQTS was screened for mutations in the KVLQT1 gene. Complementary RNAs for injection into Xenopus oocytes were prepared, and currents were recorded with the double microelectrode technique. A homozygous missense mutation, leading to an alanine-to-threonine substitution at the beginning of the pore domain of the KVLQT1 channel, was found in the proband, a 9-year-old boy with normal hearing, a prolonged QT interval, and syncopal episodes during physical exercise. The parents of the proband were heterozygous for the mutation and had a normal QT interval. The functional evaluation of the mutant channel activity showed reduction in total current, a hyperpolarizing shift in activation, and a faster activation rate consistent with a mild mutation likely to require homozygosity to manifest the phenotype. CONCLUSIONS: These findings provide the first evidence for a recessive form of the Romano-Ward long-QT syndrome and indicate that homozygous mutations on KVLQT1 do not invariably produce the Jervell and Lange-Nielsen syndrome. The implications of this observation prompt a reconsideration of the penetrance of different mutations responsible for LQTS and suggest that mild mutations in LQTS genes may be present among the general population and may predispose to drug-induced ventricular arrhythmias.
Child↗
Changes in [Ca2+]0 during anoxia in CNS white matter.
Irreversible anoxic injury of axons in the rat optic nerve requires the presence of extracellular Ca2+. To test the hypothesis that Ca2+ enters an intracellular compartment during anoxia we monitored [Ca2+]0 in this CNS white matter tract using ion-sensitive microelectrodes. Periods of anoxia lasting 15 min resulted in a rapid, reversible increase in [Ca2+]0 accompanied by transient loss of nerve conduction. This increase in [Ca2+]0 was apparently the result of extracellular space shrinkage. Anoxic periods lasting 60 min resulted in an initial rise followed by a sustained fall in [Ca2+]0, indicative of net influx of Ca2+ into an intracellular compartment. Following reoxygenation after 60 min of anoxia, [Ca2+]0 slowly returned toward control levels but nerve conduction recovered incompletely, indicating irreversible loss of function. Removal of bath Ca2+ lowered [Ca2+]0 to about 100 microM, prevented the anoxia-induced fall in [Ca2+]0, and protected against irreversible loss of the compound action potential.
Animals↗
Cloning and expression of a novel K+ channel regulatory protein, KChAP.
Voltage-gated K+ (Kv) channels are important in the physiology of both excitable and nonexcitable cells. The diversity in Kv currents is reflected in multiple Kv channel genes whose products may assemble as multisubunit heteromeric complexes. Given the fundamental importance and diversity of Kv channels, surprisingly little is known regarding the cellular mechanisms regulating their synthesis, assembly, and metabolism. To begin to dissect these processes, we have used the yeast two-hybrid system to identify cytoplasmic regulatory molecules that interact with Kv channel proteins. Here we report the cloning of a novel gene encoding a Kv channel binding protein (KChAP, for K+ channel-associated protein), which modulates the expression of Kv2 channels in heterologous expression system assays. KChAP interacts with the N termini of Kvalpha2 subunits, as well as the N termini of Kvalpha1 and the C termini of Kvbeta subunits. Kv2.1 and KChAP were coimmunoprecipitated from in vitro translation reactions supporting a direct interaction between the two proteins. The amplitudes of Kv2. 1 and Kv2.2 currents are enhanced dramatically in Xenopus oocytes coexpressing KChAP, but channel kinetics and gating are unaffected. Although KChAP binds to Kv1.5, it has no effect on Kv1.5 currents. We suggest that KChAP may act as a novel type of chaperone protein to facilitate the cell surface expression of Kv2 channels.
Amino Acid Sequence↗
Ion permeation and conduction in a human recombinant 5-HT3 receptor subunit (h5-HT3A).
1. A human recombinant homo-oligomeric 5-HT3 receptor (h5-HT3A) expressed in a human embryonic kidney cell line (HEK 293) was characterized using the whole-cell recording configuration of the patch clamp technique. 2. 5-HT evoked transient inward currents (EC50 = 3.4 microM; Hill coefficient = 1.8) that were blocked by the 5-HT3 receptor antagonist ondansetron (IC50 = 103 pM) and by the non-selective agents metoclopramide (IC50 = 69 nM), cocaine (IC50 = 459 nM) and (+)-tubocurarine (IC50 = 2.8 microM). 3. 5-HT-induced currents rectified inwardly and reversed in sign (E5-HT) at a potential of -2.2 mV. N-Methyl-D-glucamine was finitely permeant. Permeability ratios PNa/PCs and PNMDG/PCs were 0.90 and 0.083, respectively. 4. Permeability towards divalent cations was assessed from measurements of E5-HT in media where Ca2+ and Mg2+ replaced Na+. PCa/PCs and PMg/PCs were calculated to be 1.00 and 0.61, respectively. 5. Single channel chord conductance (gamma) estimated from fluctuation analysis of macroscopic currents increased with membrane hyperpolarization from 243 fS at -40 mV to 742 fS at -100 mV. 6. Reducing [Ca2+]o from 2 to 0.1 mM caused an increase in the whole-cell current evoked by 5-HT. A concomitant reduction in [Mg2+]o produced further potentiation. Fluctuation analysis indicates that a voltage-independent augmentation of gamma contributes to this phenomenon. 7. The data indicate that homo-oligomeric receptors composed of h5-HT3A subunits form inwardly rectifying cation-selective ion channels of low conductance that are permeable to Ca2+ and Mg2+.
Calcium↗
(R)-3,N-dimethyl-N-[1-methyl-3-(4-methyl-piperidin-1-yl) propyl]benzenesulfonamide: the first selective 5-HT7 receptor antagonist.
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Adenylyl Cyclases↗
Molecular determinants of dofetilide block of HERG K+ channels.
The human ether-a-go-go-related gene (HERG) encodes a K+ channel with biophysical properties nearly identical to the rapid component of the cardiac delayed rectifier K+ current (IKr). HERG/IKr channels are a prime target for the pharmacological management of arrhythmias and are selectively blocked by class III antiarrhythmic methanesulfonanilide drugs, such as dofetilide, E4031, and MK-499, at submicromolar concentrations. By contrast, the closely related bovine ether-a-go-go channel (BEAG) is 100-fold less sensitive to dofetilide. To identify the molecular determinants for dofetilide block, we first engineered chimeras between HERG and BEAG and then used site-directed mutagenesis to localize single amino acid residues responsible for block. Using constructs heterologously expressed in Xenopus oocytes, we found that transplantation of the S5-S6 linker from BEAG into HERG removed high-affinity block by dofetilide. A point mutation in the S5-S6 linker region, HERG S620T, abolished high-affinity block and interfered with C-type inactivation. Thus, our results indicate that important determinants of dofetilide binding are localized to the pore region of HERG. Since the loss of high-affinity drug binding was always correlated with a loss of C-type inactivation, it is possible that the changes observed in drug binding are due to indirect allosteric modifications in the structure of the channel protein and not to the direct interaction of dofetilide with the respective mutated site chains. However, the chimeric approach was not able to identify domains outside the S5-S6 linker region of the HERG channel as putative candidates involved in drug binding. Moreover, the reverse mutation BEAG T432S increased the affinity of BEAG K+ channels for dofetilide, whereas C-type inactivation could not be recovered. Thus, the serine in position HERG 620 may participate directly in dofetilide binding; however, an intact C-type inactivation process seems to be crucial for high-affinity drug binding.
Amino Acid Sequence↗
Visual field extent in children 3.5-30 months of age tested with a double-arc LED perimeter.
Visual field extent along the four diagonal meridia was measured cross-sectionally in 180 normal children (infants and toddlers), and 22 adults. Infants were tested monocularly at 3.5, 7, or 9 months, and toddlers were tested binocularly at 11, 17, or 30 months. Adult control data were obtained under monocular viewing. Three testing methods were investigated: static and hybrid static-kinetic perimetry, using LED arrays under computer control, and kinetic perimetry, using white styrofoam spheres manipulated by hand. Data analysis included corrections for false positives in the method of constant stimuli and for errors of anticipation in the ascending method of limits. Across all data sets from children, kinetic perimetry yielded larger, more adult-like fields, which approached adult levels around 17 months, whereas static and hybrid static-kinetic perimetry yielded smaller visual fields, approaching adult levels only at 30 months.
Adult↗
The use of SIMS, XPS and in situ AFM to probe the acid catalysed hydrolysis of poly(orthoesters).
Due to poly(orthoesters) being susceptible to acid catalysed hydrolysis, these polymers have attracted considerable interest for the controlled delivery of therapeutic agents within biodegradable matrices. The pH-sensitivity of the poly (orthoesters) has lead to several drug delivery systems being developed, whose rate of drug release is predominantly controlled by the rate of polymer hydrolysis. This study reports on the use of X-ray photoelectron spectroscopy (XPS), secondary ion mass spectrometry (SIMS), and atomic force microscopy (AFM) in a multitechnique approach to probe the effect of acid catalysed hydrolysis at the interface of poly(orthoesters). The molecular specificity of SIMS was successfully employed, suggesting that the preferred mechanism for hydrolysis was via the cleavage of an exocyclic alkoxy bond in the 3,9,-diethylidene-2,4,8,10-tetraoxaspiro [5,5] undecane(DETOSU) unit. The resulting change in the surface chemical structure of the partially hydrolysed poly(orthoester) is such that it was not detectable by XPS analysis. Images acquired from an in situ AFM study of the hydrolysis ofa poly(orthoester), showed changes in the surface morphology, seen as the formation of pits, and an overall thinning of the polymer film. The use of SIMS, XPS and AFM has enabled changes in surface chemistry to be compared with changes in surface morphology. These complementary data, on the behaviour of the polymer during degradation have important implications for the further design of novel biodegradable materials.
Biocompatible Materials↗
5-HT1B receptor-mediated contractions in human temporal artery: evidence from selective antagonists and 5-HT receptor mRNA expression.
1. In the human temporal artery both 5-HT1-like and 5-HT2A receptors mediate the contractile effects of 5-hydroxytryptamine (5-HT) and we have suggested that the 5-HT1-like receptors resemble more closely recombinant 5-HT1B than 5-HT1D receptors. To investigate further which subtype is involved, we investigated the blockade of 5-HT-induced contractions by the 5-HT1B-selective antagonist SB-224289 (2,3,6,7-tetrahydro-1'-methyl-5-[2-methyl-4'[(5-methyl-1,2,4-oxadiazole- 3-yl) biphenyl-4-yl] carbonyl] furo[2,3-f]indole-3-spiro-4'-piperidine oxalate) and the 5-HT1D-selective antagonist BRL-15572 (1-phenyl-3[4-3-chlorophenyl piperazin-1-yl] phenylpropan-2-ol). We also used RT-PCR to search for the mRNA of 5-HT1B, 5-HT1D and other 5-HT receptors. 2. The contractile effects of 5-HT in temporal artery rings were partially antagonized by SB-224289 (20, 200 nM) (apparent KB = 1 nM) and ketanserin (1 microM) but not by BRL-15572 (500 nM). 3. Sumatriptan evoked contractions (EC50, 170 nM) that were resistant to blockade by BRL-15572 (500 nM) but antagonized by SB-224289 (20, 200 nM). 4. The potency of 5-HT (EC50) was estimated to be 94 nM for the ketanserin-sensitive receptor and 34 nM for the SB-224289-sensitive receptor. The fraction of maximal 5-HT response mediated through SB-224289-sensitive receptors was 0.20-0.67, the remainder being mediated through ketanserin-sensitive receptors. 5. We detected arterial receptor mRNA for the following receptors (incidence): 5-HT1B (8/8), 5-HT1D (2/8), 5-HT1F (0/4), 5-HT2A (0/8) 5-HT2B (0/8), 5-HT2C (0/8), 5-HT4 (4/8) and 5-HT7 (4/8). 6. We conclude that the ketanserin-resistant fraction of the 5-HT effects and the effects of sumatriptan are mediated by 5-HT1B receptors. The lack of antagonism by BRL-15572 rules out 5-HT1D receptors as mediators of the contractile effects of 5-HT and sumatriptan.
Adolescent↗
Functional characterisation of the human cloned 5-HT7 receptor (long form); antagonist profile of SB-258719.
1. The functional profile of the long form of the human cloned 5-HT7 receptor (designated h5-HT7(a)) was investigated using a number of 5-HT receptor agonists and antagonists and compared with its binding profile. Receptor function was measured using adenylyl cyclase activity in washed membranes from HEK293 cells stably expressing the recombinant h5-HT7(a) receptor. 2. The receptor binding profile, determined by competition with [3H]-5-CT, was consistent with that previously reported for the h5-HT7(a) receptor. The selective 5-HT7 receptor antagonist SB-258719 ((R)-3,N-Dimethyl-N-[1-methyl-3-(4-methylpiperidin-1-yl)propyl]ben zene sulfonamide) displayed high affinity (pKi 7.5) for the receptor. 3. In the adenylyl cyclase functional assay, 5-CT and 8-OH-DPAT were both full agonists compared to 5-HT and the rank order of potency for agonists (5-CT > 5-HT > 8-OH-DPAT) was the same in functional and binding studies. 4. Risperidone, methiothepin, mesulergine, clozapine, olanzapine, ketanserin and SB-258719 antagonised surmountably 5-CT-stimulated adenylyl cyclase activity. Schild analysis of the antagonism by SB-258719 gave a pA2 of 7.2+/-0.2 and slope not significantly different from 1, consistent with competitive antagonism. 5. The same antagonists also inhibited basal adenylyl cyclase activity with a rank order of potency in agreement with those for antagonist potency and binding affinity. Both SB-258719 and mesulergine displayed apparent partial inverse agonist profiles compared to the other antagonists tested. These inhibitory effects of antagonists appear to be 5-HT7 receptor-mediated and to reflect inverse agonism. 6. It is concluded that in this expression system, the h5-HT7(a) receptor shows the expected binding and functional profile and displays constitutive activity, revealing inverse agonist activity for a range of antagonists.
Adenylyl Cyclases↗
Functional effects of the muscarinic receptor agonist, xanomeline, at 5-HT1 and 5-HT2 receptors.
Xanomeline [3(3-hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-me thylpyridine] has been reported to act as a functionally selective muscarinic partial agonist with potential use in the treatment of Alzheimer's disease. This study examined the functional activity of xanomeline at 5-HT1 and 5-HT2 receptors in native tissue and/or human cloned receptors. Xanomeline had affinity for muscarinic receptors in rat cortical membranes where the ratio of the displacement affinity of [3H]-Quinuclidinyl benzilate vs that of [3H]-Oxotremorine-M was 16, indicative of partial agonist activity. Radioligand binding studies on human cloned receptors confirmed that xanomeline had substantial affinity for M1, M2, M3, M4, M5 receptors and also for 5-HT1 and 5-HT2 receptor subtypes. Carbachol and xanomeline stimulated basal [35S]-GTPgammaS binding in rat cortical membranes with micromolar affinity. The response to carbachol was attenuated by himbacine and pirenzepine with pA2 of 8.2, 6.9 respectively consistent with the response being mediated, predominantly, via M2 and M4 receptors. Xanomeline-induced stimulation of [35S]-GTPgammaS binding was inhibited by himbacine with an apparent pKb of 6.3, was not attenuated by pirenzepine up to 3 microM and was inhibited by the selective 5-HT1A antagonist WAY100635 with an apparent pKb of 9.4. These data suggest the agonist effect of xanomeline in this tissue is, in part, via 5-HT1A receptors. Similar studies on human cloned receptors confirmed that xanomeline is an agonist at human cloned 5-HT1A and 5-HT1B receptors. In studies using the fluorescent cytoplasmic Ca2+ indicator FLUO-3AM, xanomeline induced an increase in cytoplasmic Ca2+ concentration in SH-SY5Y cells expressing recombinant human 5-HT2C receptors. Atropine antagonized this response, consistent with mediation via endogenously-expressed muscarinic receptors. In the presence of atropine, xanomeline antagonized 5-HT-induced cytoplasmic changes in Ca2+ concentration in cells expressing h5-HT2A, h5-HT2B and h5-HT2c receptors with potencies similar to its affinity at these receptors. These studies indicate that xanomeline is a potent agonist at 5-HT1A and 5-HT1B receptors and an antagonist at 5-HT2 receptor subtypes.
Animals↗
Progesterone stimulates the activity of the promoters of peripheral myelin protein-22 and protein zero genes in Schwann cells.
To understand better the mechanisms by which progesterone (PROG) promotes myelination in the PNS, cultured rat Schwann cells were transiently transfected with reporter constructs in which luciferase expression was controlled by the promoter region of either the peripheral myelin protein-22 (PMP22) or the protein zero (P0) genes. PROG stimulated the P0 promoter and promoter 1, but not promoter 2, of PMP22. The effect of PROG was specific, as estradiol and testosterone only weakly activated promoters. Dose-response curves for stimulation of both promoter constructs by PROG were biphasic. RU486, a PROG antagonist, did not abolish the effect of PROG, but stimulated promoter activities by itself. In the human carcinoma cell line T47D expressing high levels of PROG receptor, PROG did not stimulate the P0 and PMP22 promoters, whereas the promoter region of the mouse mammary tumor virus was fully activated. Thus, the activation by PROG of promoter activity of two peripheral myelin protein genes is Schwann-cell specific.
Animals↗
Induction of exocytosis from permeabilized mast cells by the guanosine triphosphatases Rac and Cdc42.
We applied recombinant forms of the Rho-related small guanosine triphosphatases (GTPases) Rac2 and Cdc42/G25K to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells permeabilized with streptolysin-O leak soluble (cytosol) proteins over a period of 5 min and become refractory to stimulation by Ca2+ and guanosine triphosphate (GTP)gammaS over about 20-30 min. This loss of sensitivity is likely to be due to loss of key regulatory proteins that are normally tethered at intracellular locations. Exogenous proteins that retard this loss of sensitivity to stimulation may be similar, if not identical, to those secretory regulators that are lost. Recombinant Rac and Cdc42/G25K, preactivated by binding GTPgammaS, retard the loss of sensitivity (run-down) and, more importantly, enable secretion to be stimulated by Ca2+ alone. Investigation of the concentration dependence of each of these two GTPases applied individually to the permeabilized cells, and of Cdc42/G25K applied in the presence of an optimal concentration of Rac2, has provided evidence for a shared effector pathway and also a second effector pathway activated by Cdc42/G25K alone. Dominant negative mutant (N17) forms of Rac2 and Cdc42/G25K inhibit secretion induced by Ca2+ and GTPgammaS. Our data suggest that Rac2 and Cdc42 should be considered as candidates for GE, GTPases that mediate exocytosis in cells of hematopoeitic origin.
Bacterial Proteins↗
Functional consequences of ROMK mutants linked to antenatal Bartter's syndrome and implications for treatment.
The antenatal variant of Bartter's syndrome is an autosomal recessive kidney disease characterized by polyhydramnios, premature delivery, hypokalemic alkalosis and hypercalciuria. It is genetically heterogeneous, having been linked recently to mutations in an ATP-sensitive, renal outer medullary K+channel, ROMK, and earlier to mutations in the Na-K-2Cl co-transporter, NKCC2. We characterized four of the mutations reported in three heterozygous ROMK variants of antenatal Bartter's and found that each expressed a distinct phenotype in Sf9 cells. One mutation expressed normal function and appears to be an allelic polymorphism. The other three mutations produced channels with significantly reduced K+fluxes. However, the mechanisms in each case were different and reflected abnormalities in phosphorylation, proteolytic processing or protein trafficking. The different mechanisms may be important in the design of appropriate therapy for patients with this disease.
Animals↗
Expression of cadherins and catenins in oral epithelial dysplasia and squamous cell carcinoma.
The immunocytochemical expression of cadherins and catenins was examined during the process of oral carcinogenesis by comparing their expression in normal and dysplastic epithelium with primary and metastatic carcinomas. While control epithelium showed normal distribution for P and E cadherin and the catenins, in severe dysplasia P-cadherin was upregulated. In other cases and in carcinoma-in-situ adjacent to infiltrating carcinomas, membranous expression of the cadherins and catenins was reduced or lost. The changes in expression of E-cadherin and the catenins suggest that disruption of the E-cadherin/catenin complex is a late event associated with invasion. In primary carcinomas reduced membranous and cytoplasmic staining were observed for both cadherins and catenins. Abnormal localisation of E-cadherin occurred in the more superficial parts of the better differentiated carcinomas, suggesting abnormality to the E-cadherin complex(es). In contrast, membranous expression of cadherins and catenins was reduced or lost in the deep invasive margin of primary carcinomas and in most poorly differentiated carcinomas. For E-cadherin at least, this reduction appears associated with differentiation, invasion and possibly prognosis. Possible mechanisms involved for changes in expression of the cadherins and associated catenins and areas for further study are discussed.
Cadherins↗
Modulation of potassium channel gating by coexpression of Kv2.1 with regulatory Kv5.1 or Kv6.1 alpha-subunits.
We have determined the effects of coexpression of Kv2.1 with electrically silent Kv5.1 or Kv6.1 alpha-subunits in Xenopus oocytes on channel gating. Kv2.1/5.1 selectively accelerated the rate ofinactivation at intermediate potentials (-30 to 0 mV), without affecting the rate at strong depolarization (0 to +40 mV), and markedly accelerated the rate of cumulative inactivation evoked by high-frequency trains of short pulses. Kv5.1 coexpression alsoslowed deactivation of Kv2.1. In contrast, Kv6.1 was much less effective in speeding inactivation at intermediate potentials, had a slowing effect on inactivation at strong depolarizations, and had no effect on cumulative inactivation. Kv6.1, however, had profound effects on activation, including a negative shift of the steady-state activation curve and marked slowing of deactivation tail currents. Support for the notion that the Kv5.1's effects stem from coassembly of alpha-subunits into heteromeric channels was obtained from biochemical evidence of protein-protein interaction and single-channel measurements that showed heterogeneity in unitary conductance. Our results show that Kv5.1 and Kv6.1 function as regulatory alpha-subunits that when coassembled with Kv2.1 can modulate gating in a physiologically relevant manner.
Action Potentials↗