Molecular and antigenic structure of nicotinic acetylcholine receptors.
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Animals↗
Biomedical subjects
R Anand
Publications and source records attributed to R Anand.
Water-soluble nicotinic acetylcholine receptor formed by alpha7 subunit extracellular domains.
Water-soluble models of ligand-gated ion channels would be advantageous for structural studies. We investigated the suitability of three versions of the N-terminal extracellular domain (ECD) of the alpha7 subunit of the nicotinic acetylcholine receptor (AChR) family for this purpose by examining their ligand-binding and assembly properties. Two versions included the first transmembrane domain and were solubilized with detergent after expression in Xenopus oocytes. The third was truncated before the first transmembrane domain and was soluble without detergent. For all three, their equilibrium binding affinities for alpha-bungarotoxin, nicotine, and acetylcholine, combined with their velocity sedimentation profiles, were consistent with the formation of native-like AChRs. These characteristics imply that the alpha7 ECD can form a water-soluble AChR that is a model of the ECD of the full-length alpha7 AChR.
Acetylcholine↗
Reactivation of inflammatory lesions in ocular histoplasmosis.
BACKGROUND: Active inflammation has not been traditionally associated with the ocular histoplasmosis syndrome. OBJECTIVE: To investigate the occurrence of presumed inflammatory chorioretinal lesions in patients with the ocular histoplasmosis syndrome. METHODS: Patients seen with acute symptoms and a clinical picture of ocular histoplasmosis were observed prospectively between August 13, 1993, and December 2, 1997. Symptoms, visual acuity, and fluorescein sodium angiography were used to document changes in inflammatory loci. RESULTS: Twelve patients were seen with active inflammatory lesions. Eleven had resolution of the loci with lessening of symptoms and improvement in acuity and angiographic findings. A typical subretinal neovascular membrane developed in 1 patient 8 months after the onset of symptoms. CONCLUSIONS: Inflammatory chorioretinal lesions can reactivate in the ocular histoplasmosis syndrome. In most of these patients, neovascularization did not develop and visual acuity was preserved.
Acute Disease↗
N-acetylcysteine prevents development of the hyperdynamic circulation in the portal hypertensive rat.
Partial portal vein ligation (PPVL) leads to the development of a hyperdynamic circulation. It is associated with elevated levels of tumor necrosis factor (TNF-alpha) and nitric oxide (NO) production, both of which can result in oxidant injury. In this study, we have investigated whether PPVL is associated with the development of oxidative stress, by measuring urinary F2-isoprostanes. In addition, we have examined whether N-acetylcysteine (NAC) can ameliorate oxidant injury and prevent the development of the hyperdynamic circulation. Urinary excretion of F2-isoprostanes increased sixfold following PPVL together with a significant increase in plasma nitrite and nitrate. Treatment with NAC inhibited the formation of F2-isoprostanes as well as the increase in plasma nitrite and nitrate. Hemodynamic studies in anesthetized rats showed that following PPVL, cardiac output and portal pressure increased, and systemic vascular resistance decreased, consistent with the development of a hyperdynamic circulation. These changes were prevented by chronic administration of NAC. We conclude that NAC prevents the development of the hyperdynamic circulation and that the formation of reactive oxygen species may be important in the pathogenesis of these hemodynamic changes.
Acetylcysteine↗
Cyclosporine to tacrolimus: effect on hypertension and lipid profiles in renal allografts.
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Blood Pressure↗
Conversion to tacrolimus to ameliorate cyclosporine toxicity in kidney recipients.
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Adult↗
Adenomyosis: diagnosis by hysteroscopic endomyometrial biopsy, correlation of incidence and severity with menorrhagia.
OBJECTIVE: To assess the possibility of diagnosing adenomyosis by hysteroscopic endomyometrial biopsy in patients with menorrhagia and to compare the incidence and depth of penetration in patients who did not have menorrhagia. STUDY DESIGN: The study group included 53 patients who had menorrhagia and the control group included patients who were to have hysterectomy for conditions like prolapse uterus. Hysteroscopic endomyometrial biopsy was done in the menorrhagia group. The incidence and depth of penetration of adenomyosis were compared in both the groups using Kruskal Wallis H test. For correlation of severity of menorrhagia with depth of adenomyosis: Student's t-test and ANOVA were used. RESULT: The incidence of adenomyosis was 60% (18 patients) by endomyometrial biopsy in the menorrhagia group as compared to 33.3% in the control group. The mean depth of invasion of myometrium was 4.103 mm in the menorrhagia group and 2.03 mm in the control group. CONCLUSION: There is a definite correlation between menorrhagia and adenomyosis both in incidence and depth of penetration.
Adult↗
Mutations in both gp120 and gp41 are responsible for the broad neutralization resistance of variant human immunodeficiency virus type 1 MN to antibodies directed at V3 and non-V3 epitopes.
The escape of human immunodeficiency virus type 1 from effects of neutralizing antibodies was studied by using neutralization-resistant (NR) variants generated by growing the neutralization-sensitive (NS) wild-type MN virus in the presence of human serum with neutralizing antibodies, more than 99% of which were directed at the V3 region of gp120. The variants obtained had broad neutralization resistance to human sera, without limitation with respect to the V3 specificity of the sera. The molecular basis for the resistance was evaluated with molecularly cloned viruses, as well as with pseudoviruses expressing envelope glycoproteins of the NS and NR phenotypes. Nucleotide sequence analyses comparing NS and NR clones revealed a number of polymorphisms, including six in the V1/V2 region, two in C4/V5 of gp120, three in the leucine zipper (LZ) domain of gp41, and two in the second external putative alpha-helix region of gp41. A series of chimeras from NS and NR env genes was constructed, and each was presented on pseudoviruses to locate the domain(s) which conferred the phenotypic changes. The neutralization phenotypes of the chimeric clones were found to be dependent on mutations in both the C4/V5 region of gp120 and the LZ region of gp41. Additionally, interaction between mutations in gp120 and gp41 was demonstrated in that a chimeric env gene consisting of a gp120 coding sequence from an NS clone and a gp41 sequence from an NR clone yielded a pseudovirus with minimal infectivity. The possible significance of predicted amino acid changes in these domains is discussed. The results indicate that polyvalent antibodies predominantly directed against V3 can induce NR through selection for mutations that alter interactions of other domains in the envelope complex.
Amino Acid Sequence↗
Expression and stable germline transmission of neomycin-resistance gene in transgenic mice.
Transgenic mice were produced to study the expression of amino-3' glycosyl phosphotransferase gene (neomycin resistance gene) in the embryonic fibroblast cells. A 1.9 Kb linear fragment of neomycin resistance gene under the control of pPGK promoter was microinjected into the pronucleus of mouse embryos. Out of 64 potential founders born, 5 were identified to be transgenic by the polymerase chain reaction (PCR) and southern hybridization. Multiple mice from first and second generation from two transgenic founders (N-10 and N-32) were analysed to determine the germline transmission. It was found to be 24.6 and 71.4% in first and second generation respectively. Results were also further confirmed by RT-PCR, sequencing and in vitro bioassays.
Animals↗
Experimental autoimmune uveitis as animal model for human posterior uveitis.
Uveitis is an intraocular inflammatory disease that mostly affects children and young adults. It is one of the major causes of blindness in young individuals in India and the world. It is responsible for about 10 per cent of total visual impairment. Unfortunately, etiological diagnosis is not evident in a majority of these patients. It is generally felt that autoimmune mechanism may be involved in so called 'idiopathic' cases which has led to search for the putative autoantigens in experimental animal models. It has been demonstrated that experimental autoimmune uveitis (EAU) can be elicited against several retinal proteins in rats, mice and sub-human primates. These include the S-antigen, a major protein on retinal photoreceptor cell, interphotoreceptor retinoid binding protein (IRBP) and several others. There are many similarities between clinical entities and the EAU, but the EAU differs from the clinical conditions in being self-limited, and requiring complete Freund's adjuvant for induction of the disease. The disease can be induced only in susceptible strains. Nevertheless, use of the EAU model has allowed for identification of disease causing epitopes of antigens and evaluation of disease modifying strategies which could be applied in clinical situations. There has been significant progress in this field, but still a lot more is required to be learnt to translate it into clinical practice.
Adult↗
Determinants of channel gating located in the N-terminal extracellular domain of nicotinic alpha7 receptor.
We identified regions within the N-terminal extracellular domain of alpha7 nicotinic acetylcholine receptors that affect channel gating. By single-channel analysis of alpha7 nicotinic acetylcholine receptors currents, we show that the difference in efficacy between the two agonists acetylcholine and 1,1-dimethyl-4-phenylpiperazinium (DMPP) is due to a slower channel activation rate by DMPP. The partial efficacy of DMPP was not caused by channel block or faster desensitization of alpha7 AChRs by DMPP. In addition, the efficacy and, by inference, the activation rate were found to be voltage dependent. Using chimeras of the two closely related subunits alpha7 and alpha8, we map residues that affect channel activation rate and agonist affinity to two different regions of the extracellular domain. Residues that affect channel activation rate are within the sequence 1-179, whereas residues that affect agonist affinity are within the sequence 180-208.
Animals↗
Natural history of asymptomatic valvar pulmonary stenosis diagnosed in infancy.
BACKGROUND AND HYPOTHESIS: Valvar pulmonary stenosis is a common congenital heart defect. Progression of stenosis over time, even when mild initially, has been shown by serial cardiac catheterization studies in children and adults. We studied the natural history of asymptomatic valvar pulmonary stenosis diagnosed in infancy with two-dimensional echocardiography and Doppler method. METHODS: Between November 1986 and March 1993, 51 infants in the Northeast Tennessee and Southwest Virginia region were clinically diagnosed to have isolated valvar pulmonary stenosis. In 40 patients, the diagnosis was confirmed by two-dimensional echocardiogram/Doppler and color-flow mapping study at the time of presentation, and only their course is reported. Of 40 infants, six asymptomatic infants (15%) showed rapid progression of pulmonary stenosis over a relatively short period of time. Within the first 6 months of life, three of the six infants showed worsening of the stenosis needing intervention (one had surgical valvectomy and the others had percutaneous balloon valvuloplasty). The three other infants showed a more gradual increase of pulmonary stenosis over the first 2 years of life. RESULTS: Pulmonary stenosis even when mild can worsen in infancy, and it is not possible to predict which patients will follow this course. In our group of asymptomatic infants with initial mild pulmonary stenosis, 15% developed significant stenosis that needed intervention. CONCLUSION: We recommend frequent follow-up of asymptomatic infants with mild pulmonary stenosis during the first 2 years of life to detect rapid progression that may need intervention.
Disease Progression↗
Mapping of the gene encoding the B56 beta subunit of protein phosphatase 2A (PPP2R5B) to a 0.5-Mb region of chromosome 11q13 and its exclusion as a candidate gene for multiple endocrine neoplasia type 1 (MEN1).
The multiple endocrine neoplasia type 1 (MEN1) locus has been previously localised to 11q13 by combined tumour deletion mapping and recombination studies, and a 0.5-Mb region, flanked by PYGM and D11S449, has been defined. In the course of constructing a conting, we have identified the location of the gene encoding the B56 beta subunit of protein phosphatase 2A (PP2A), which is involved in cell signal transduction pathways and thus represents a candidate gene for MEN1. We have searched for mutations in the PP2A-B56 beta coding region, together with the 5' and 3' untranslated regions in six MEN1 patients. DNA sequence abnormalities were not identified and thus the PP2A-B56 beta gene is excluded as the candidate gene for MEN1. However, our precise localisation of PP2A-B56 beta to this region of 11q13 may help in elucidating the basis for other disease genes mapping to this generich region.
Chromosome Mapping↗
8-Isoprostaglandin F2 alpha, a product of lipid peroxidation, increases portal pressure in normal and cirrhotic rats.
BACKGROUND & AIMS: The F2-isoprostanes are a recently described class of prostaglandins formed by free radical-mediated lipid peroxidation. 8-Isoprostaglandin F2 alpha (8-iso-PGF2 alpha), an F2-isoprostane, has previously been shown to be a potent renal vasoconstrictor acting via a thromboxane-like receptor. The aim of this study was to investigate whether 8-iso-PGF2 alpha increases portal pressure. METHODS: Livers from normal and bile duct-ligated cirrhotic rats were perfused, and portal pressure response to infused agonist was monitored continuously. RESULTS: Infusion of 8-iso-PGF2 alpha increased portal pressure in both groups, with a significantly greater response in cirrhotic rats. At a dose of 2.5 nmol/min, the mean portal pressure increased from a baseline of 8.2 +/- 0.6 to 9.8 +/- 1.3 mm Hg, whereas in cirrhotic animals, the increase was from 12.0 +/- 0.9 to 18.6 +/- 1.8 mm Hg. This response was completely blocked by SQ29548, a thromboxane receptor antagonist. A similar response pattern was observed with the thromboxane receptor agonist U46619. CONCLUSIONS: 8-iso-PGF2 alpha can increase portal pressure in cirrhotic rats. If extrapolated to patients with cirrhosis, lipid peroxidation secondary to alcoholic liver injury, sepsis, or other liver pathology may cause an acute increase in portal pressure such as that observed in acute liver injury.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Influence of the cannulated vitrectomy system on the occurrence of iatrogenic sclerotomy retinal tears.
PURPOSE: To determine whether the use of the cannulated vitrectomy system decreases the incidence of sclerotomy-related retinal tears relative to traditional vitrectomy techniques. METHODS: Forty-one eyes of 77 patients in this study were randomly selected to undergo treatment with the cannulated port system. RESULTS: We demonstrated a statistically significant decreased incidence of sclerotomy tears in the cannulated group relative to the noncannulated group (1% vs. 7.7%, P < 0.05). The benefit of the cannulated port system appears to be greatest in cases in which an inexperienced surgeon is learning the techniques of vitreoretinal surgery, in eyes with a preoperative diagnosis of tractional diabetic detachment, and in surgery requiring membrane delamination (simple and extensive). CONCLUSION: The three-port vitrectomy system decreases the incidence of sclerotomy-related retinal tears.
Catheterization↗
Treatment of cytomegalovirus retinitis with the intraocular ganciclovir implant.
The treatment of cytomegalovirus retinitis (CMV-R) has improved considerably in the past two years. Local ocular therapy has proved to be effective and provides significant advantages over systemic therapy with regards to better control, preventing progression, avoiding bone marrow and renal toxicity. Direct intraocular injections of anti-CMV drugs and the sustained-release ganciclovir device are the mainstay of local therapy. The ganciclovir intraocular device has undergone rigorous testing and has recently been approved for clinical use. This review describes the surgical procedure, recommendations for use, and exchange and management of associated retinal detachment. In addition, the results of the initial studies and the multicenter, randomized clinical trial are discussed. Current data available from the Study for the Ocular Complications of AIDS (SOCA) and AIDS Clinical Trials Group (ACTG) studies are summarized in the context of CMV-R management.
Antiviral Agents↗
Chronic nicotine treatment up-regulates alpha3 and alpha7 acetylcholine receptor subtypes expressed by the human neuroblastoma cell line SH-SY5Y.
Chronic exposure to nicotine has been reported to increase the number of nicotinic acetylcholine receptors (AChRs) in brain. The mechanism of up-regulation for the alpha4beta2 AChR subtype, which accounts for the majority of high affinity nicotine binding in mammalian brain, has previously been shown to involve a decrease in the rate of alpha4beta2 AChR turnover. Here, we report an investigation of the extent and mechanism of nicotine-induced up-regulation of alpha3 AChRs and alpha7 AChR subtypes expressed in the human neuroblastoma cell line SH-SY5Y. Up-regulation of human alpha3 AChRs and alpha7 AChRs, unlike alpha4beta2 AChRs, requires much higher nicotine concentrations than are encountered in smokers; the extent of increase of surface AChRs is much less; and the mechanisms of up-regulation are different than with alpha4beta2 AChRs. The mechanisms of up-regulation may be different for alpha3 AChRs or alpha7 AChRs. Chronic treatment with nicotine or carbamylcholine, but not d-tubocurarine, mecamylamine, or dihydro-beta-erythroidine, induced a 500-600% increase in the number of alpha3 AChRs but only a 30% increase in alpha7 AChRs. Chronic nicotine treatment did not increase affinity for nicotine or increase the amount of RNA for alpha3 or alpha7 subunits. The effect of nicotine on up-regulation of alpha7 AChRs was partially blocked by either d-tubocurarine or mecamylamine. The effect of nicotine treatment on the number of alpha3 AChRs was only slightly blocked by the antagonists d-tubocurarine, mecamylamine, or dihydro-beta-erythroidine at concentrations that efficiently block alpha3 AChR function. Most of the nicotine-induced increase in alpha3 AChRs was found to be intracellular. The alpha3 AChRs, which accumulate intracellularly, were shown to have been previously exposed on the cell surface by their susceptibility to antigenic modulation. The data suggest that chronic exposure to nicotine may induce a conformation of cell surface alpha3 AChRs that at least in this cell line are consequently internalized but not immediately destroyed.
Endocytosis↗
"Orphan" alpha6 nicotinic AChR subunit can form a functional heteromeric acetylcholine receptor.
Previously, a rat brain cDNA was reported that was designated alpha6 because of its homology with nicotinic acetylcholine receptor (AChR) alpha subunits, being especially similar to alpha3, but no acetylcholine-gated cation channels were detected when it was expressed in Xenopus laevis oocytes alone or in combination with other known rat AChR subunits. We cloned chicken alpha6 and human beta4 AChR subunits and tested for acetylcholine-gated cation channels with alpha6 by expression in X. laevis oocytes alone or in pairwise combination with chicken alpha3, beta2, or beta4 or with human alpha3, beta2, or beta4 AChR subunits. Chicken alpha6 formed detectable functional AChRs only when expressed together with the human beta4 subunit. The alpha6beta4 AChR-mediated currents show strong inward rectification and dependence on extracellular Ca2+. It exhibited a distinct pharmacological profile with an EC50 value of 28 microM for acetylcholine, 24 nM for (+)-epibatidine, 6.6 microM cytisine, and 15 microM 1,1-dimethyl-4-phenylpiperazinium. Both cytisine and 1,1-dimethyl-4-phenylpiperazinium behaved as partial (approximately 30%) agonists. Remarkably, nicotine (EC50 = 22 microM) was an even weaker partial agonist (approximately 18%) and had a relatively long-lasting inhibitory effect. Coexpression of the previously cloned rat alpha6 subunit with the human the beta4 subunit also resulted in functional alpha6beta4 AChRs with properties resembling those of the chicken/human alpha6beta4 AChRs. Therefore, alpha6 can function as part of AChRs with unusual pharmacological properties.
Acetylcholine↗