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Featured researches published by Ana Crane.


Journal of Biological Chemistry | 1999

The C Terminus of SUR1 Is Required for Trafficking of KATP Channels

Nidhi Sharma; Ana Crane; John P. Clement; Gabriela Gonzalez; Andrey P. Babenko; Joseph Bryan; Lydia Aguilar-Bryan

In beta cells from the pancreas, ATP-sensitive potassium channels, or KATP channels, are composed of two subunits, SUR1 and KIR6.2, assembled in a (SUR1/KIR6.2)4 stoichiometry. The correct stoichiometry of channels at the cell surface is tightly regulated by the presence of novel endoplasmic reticulum (ER) retention signals in SUR1 and KIR6.2; incompletely assembled KATPchannels fail to exit the ER/cis-Golgi compartments. In addition to these retrograde signals, we show that the C terminus of SUR1 has an anterograde signal, composed in part of a dileucine motif and downstream phenylalanine, which is required for KATPchannels to exit the ER/cis-Golgi compartments and transit to the cell surface. Deletion of as few as seven amino acids, including the phenylalanine, from SUR1 markedly reduces surface expression of KATP channels. Mutations leading to truncation of the C terminus of SUR1 are one cause of a severe, recessive form of persistent hyperinsulinemic hypoglycemia of infancy. We propose that the complete loss of beta cell KATP channel activity seen in this form of hyperinsulinism is a failure of KATPchannels to traffic to the plasma membrane.


Current Pharmaceutical Design | 2005

Insulin Secretagogues, Sulfonylurea Receptors and KATP Channels

Joseph Bryan; Ana Crane; Wanda H. Vila-Carriles; Andrey P. Babenko; Lydia Aguilar-Bryan

ATP-sensitive K+ channels, termed K(ATP) channels, provide a link between cellular metabolism and membrane electrical activity in a variety of tissues. Channel isoforms have been identified and are targets for compounds that both stimulate and inhibit their activity resulting in membrane hyperpolarization and depolarization, respectively. Examples include relaxation of vascular smooth muscle and stimulation of insulin secretion. This article reviews the cloning, molecular biology, and structure of K(ATP) channels, with particular focus on the SUR1/K(IR)6.2 neuroendocrine channels that are important for the regulation of insulin secretion. We integrate the extensive pharmacologic structure-activity-relationship data on these channels, which defines a bipartite drug binding pocket in the SUR (sulfonylurea receptor), with recent structure-function studies that identify domains of SUR and K(IR)6.2, the channel pore, which are critical for channel assembly, for gating, and for the ligand-receptor interactions that modulate channel activity. The atomic structure of a sulfonylurea in a protein pocket is used to develop insight into the recognition of these compounds. A homology model of K(ATP) channels, based on VC-MsbA, another member of the ABC protein family, is described and used to position amino acids important for the action of channel openers and blockers within the core of SUR. The model has a central chamber which could serve as a multifaceted binding pocket.


Cell Transplantation | 1995

Effective cryopreservation and long-term storage of primary human hepatocytes with recovery of viability, differentiation, and replicative potential.

R. Mark Adams; Mary Wang; Ana Crane; Bridgette Brown; Gretchen J. Darlington; Fred D. Ledley

Despite reports of successful cryopreservation of primary human hepatocytes, existing methods do not produce sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocellular transplantation. We now describe a protocol for efficient cryopreservation of primary human hepatocytes using University of Wisconsin (UW) solution, fetal bovine serum, and dimethyl sulfoxide (DMSO). This method provides > 90% viability of differentiated, primary human hepatocytes 8 mo after cryopreservation as measured by trypan blue exclusion, preserves hepatocyte morphology, liver-specific gene expression (alpha 1 antitrypsin), and replication. The effectiveness of UW solution as a cryopreservative agent suggests that metabolic as well as ultrastructural factors may be important in the effective cryopreservation of primary human hepatocytes. The present method represents an effective protocol for cryopreserving differentiated primary human hepatocytes for research. This method may allow characterization and banking of human hepatocytes for clinical applications, including hepatocellular transplantation and hepatic assist devices.


Journal of Biological Chemistry | 2004

Assembly, Maturation, and Turnover of KATP Channel Subunits

Ana Crane; Lydia Aguilar-Bryan

ATP-sensitive K+, or KATP, channels are comprised of KIR6.x and sulfonylurea receptor (SUR) subunits that assemble as octamers, (KIR/SUR)4. The assembly pathway is unknown. Pulse-labeling studies show that when KIR6.2 is expressed individually, its turnover is biphasic; ∼60% is lost with t½ ∼36 min. The remainder converts to a long-lived species (t½ ∼26 h) with an estimated half-time of 1.2 h. Expressed alone, SUR1 has a long half-life, ∼25.5 h. When KIR6.2 and SUR1 are co-expressed, they associate rapidly and the fast degradation of KIR6.2 is eliminated. Based on changes in the glycosylation state of SUR1, the half-time for the maturation of KATP channels, including completion of assembly, transit to the Golgi, and glycosylation, is ∼2.2 h. Estimation of the turnover rates of mature, fully glycosylated SUR1 associated with KIR6.2 and of KIR6.2 associated with Myc-tagged SUR1 gave similar values for the half-life of KATP channels, a mean value of ∼7.3 h. KATP channel subunits in INS-1 β-cells displayed qualitatively similar kinetics. The results imply the octameric channels are stable. Two mutations, KIR6.2 W91R and SUR1 ΔF1388, identified in patients with the severe form of familial hyperinsulinism, profoundly alter the rate of KIR6.2 and SUR1 turnover, respectively. Both mutant subunits associate with their respective partners but dissociate freely and degrade rapidly. The data support models of channel formation in which KIR6.2-SUR1 heteromers assemble functional channels and are inconsistent with models where SUR1 can only assemble with KIR6.2 tetramers.


Journal of Clinical Investigation | 1992

Cloning and expression of a mutant methylmalonyl coenzyme A mutase with altered cobalamin affinity that causes mut- methylmalonic aciduria.

Ana Crane; Ruud Jansen; E. Andrews; Fred D. Ledley

Distinct genotypic and phenotypic forms of methylmalonyl CoA mutase (MCM) apoenzyme deficiency can be delineated by biochemical analysis of mutant fibroblasts. One form, designated mut-, expresses a phenotype in which residual enzyme activity is evident in cultured cells exposed to high concentrations of hydroxycobalamin. We describe cloning of an MCM cDNA from cells exhibiting a mut- phenotype and characterization of the mutant gene product overexpressed in primary muto human fibroblasts and Saccharomyces cerevisiae. Three novel base changes were observed. Recombinant clones containing one of these base changes (G717V) express four characteristics of the mut- phenotype: failure to constitute [14C]propionate incorporation activity in fibroblasts assayed under basal cell culture conditions, constitution of [14C]propionate incorporation activity in fibroblasts stimulated with 0.1-1.0 micrograms/ml hydroxycobalamin, interallelic complementation with alleles bearing an R93H mutation, and an apparent Km (adenosylcobalamin) 1,000-fold higher than normal. These results demonstrate that the G717V mutation produces the mut- phenotype and localizes determinants for adenosylcobalamin binding near the carboxyl terminus of MCM.


Journal of Clinical Investigation | 1991

Genetic characterization of a MUT locus mutation discriminating heterogeneity in mut0 and mut- methylmalonic aciduria by interallelic complementation.

M L Raff; Ana Crane; Ruud Jansen; Fred D. Ledley; David S. Rosenblatt

Genetic complementation of fibroblasts from patients with methylmalonic aciduria (MMA) defines a unique class of allelic mutations arising from mutations at the locus encoding the methylmalonyl coenzyme A (CoA) mutase apoenzyme. Various phenotypes of MMA have been delineated including complete absence of enzyme activity (mut0) and abnormal enzyme activity with an elevated Km for adenosylcobalamin (mut-). We describe genetic studies on a cell line (WG1130) from a patient with mut0 MMA which exhibited an unusual complementation phenotype, complementing with three of nine mut0 cell lines and four of five mut- cell lines. This suggests that interallelic complementation occurs between mutant alleles in WG1130 and subsets of alleles associated with both mut0 and mut- phenotypes. The methylmalonyl CoA mutase cDNA was cloned from WG1130 and found to contain a G354----A (Arg93----His) mutation. Gene transfer of this mutant clone into primary fibroblasts from patients with MMA confirms that this mutation expresses a mut0 phenotype when transferred into a mut0 cell line with low levels of mRNA but can contribute to apoenzyme function when transferred into mut cell lines which show correction with WG1130 by somatic cell complementation. These results point to further heterogeneity within both mut0 and mut- and may enable identification of mutations affecting discrete components of apoenzyme function.


Human Genetics | 1992

Phenotype of disease in three patients with identical mutations in methylmalonyl CoA mutase

Ana Crane; Laura S. Martin; David Valle; Fred D. Ledley

SummaryWe have previously identified a mutation in the gene for methylmalonyl CoA mutase in a patient with the mut- phenotype of methylmalonic aciduria. This mutation (G717V) interferes with the binding of the deoxyadenosylcobalamin cofactor to the apoenzyme producing a mutant holoenzyme that is defective, but not completely inactive, in vitro. This report describes the clinical phenotype associated with this mutation in the original patient and two additional patients who are homozygous for this allele. All three patients presented in the first years of life with multiple episodes of life-threatening organic acidosis and hyperammonemia. None had evidence of disease in the perinatal period, and all three have low-normal intelligence. These three children exhibit a distinctive phenotype of disease that is intermediate between the fulminant and benign forms of methylmalonic aciduria. These data suggest that this phenotype is the specific consequence of the G717V mutation, and that the degree of residual enzyme activity associated with the G717V mutation is close to the threshold required in vivo for maintaining metabolic homeostasis.


Journal of Clinical Investigation | 1991

Differential diagnosis of mut and cbl methylmalonic aciduria by DNA-mediated gene transfer in primary fibroblasts.

Michael F. Wilkemeyer; Ana Crane; Fred D. Ledley

Methylmalonic aciduria can be caused by mutations in the gene encoding the methylmalonyl coenzyme A mutase apoenzyme (mut) or genes required for the provision of cofactor B12 (cbl). The mut and cbl forms are classically differentiated by somatic cell complementation. We describe a novel method for differential diagnosis of mut and cbl methylmalonic aciduria using DNA-mediated gene transfer of a methylmalonyl CoA mutase cDNA clone. Gene transfer of a functional methylmalonyl CoA mutase cDNA clone into mut fibroblasts reconstitutes holoenzyme activity measured by metabolism of [14C]-propionate in culture. Identical gene transfers into cbl fibroblasts have no effect. This method is used for the differential diagnosis of mut and cbl genotypes in cells from patients with a clinical diagnosis of methylmalonic aciduria and is shown to be a facile, sensitive, and specific method for genetic diagnosis. This work establishes the principle of using DNA-mediated gene transfer to identify the genotype of diseases which can result from mutations at several different genetic loci. This type of differential genotypic diagnosis will be particularly important for establishing the applicability of somatic gene therapy in individual patients.


Biochemical and Biophysical Research Communications | 1991

Is there methylmalonyl CoA mutase in Aspergillus nidulans

Fred D. Ledley; Ana Crane; Kathryn T. Klish; Gregory S. May

In most animal species and many prokaryotes, methylmalonyl CoA mutase catalyzes isomerization between methylmalonyl CoA and succinyl CoA using adenosylcobalamin as a cofactor. We describe the absence of this enzyme in Aspergillus nidulans based on the absence of enzyme activity in vitro and the failure to metabolize methylmalonate or grow in media containing this organic acid as the sole carbon source. These data contrast previous assumptions that propionate may be metabolized through propionyl CoA and methylmalonyl CoA to the TCA cycle in this organism. This is consistent with the separate evolution of these pathways in animals and lower eukaryotes due to the distinct endosymbiotic origin of their mitochondria.


Human Gene Therapy | 1994

Overexpression of Human Methylmalonyl CoA Mutase in Mice after In Vivo Gene Transfer with Asialoglycoprotein/Polylysine/DNA Complexes

Jozsef Stankovics; Ana Crane; Elizabeth Andrews; Cathy H. Wu; George Y. Wu; Fred D. Ledley

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Fred D. Ledley

Baylor College of Medicine

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Joseph Bryan

Baylor College of Medicine

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Nidhi Sharma

Baylor College of Medicine

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Gabriela Gonzalez

Baylor College of Medicine

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Ruud Jansen

Baylor College of Medicine

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Andrey P. Babenko

Baylor College of Medicine

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Arupa Ganguly

University of Pennsylvania

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Bridgette Brown

Baylor College of Medicine

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Cathy H. Wu

University of Delaware

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