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Biomedical subjects

James R Howe

Publications and source records attributed to James R Howe.

At least 19 recordsLinked to original sources
PubMed2006

RIZ1 is epigenetically inactivated by promoter hypermethylation in thyroid carcinoma.

BACKGROUND: Allelotype studies have suggested that chromosome 1p is frequently lost in thyroid cancers, thus suggesting that there is an important tumor suppressor at this location. RIZ1 (PRDM2), located on 1p36, is a recently described tumor suppressor gene and is a member of the protein methyltransferase superfamily. RIZ1 expression is lost in a variety of tumors, primarily by means of epigenetic mechanisms that involve promoter hypermethylation. METHODS: RIZ1 expression was examined in a panel of thyroid tumor cell lines and primary thyroid tissues (14 normal, 19 benign, and 31 cancerous) by using real-time polymerase chain reaction (PCR). Methylation status of the RIZ1 promoter was studied using bisulfite sequencing and methylation-specific PCR. RESULTS: The authors demonstrated that RIZ1 expression is lost in thyroid tumor cell lines and is also significantly reduced in thyroid carcinomas, when compared with normal thyroid tissues (P < .0001) and benign tumors (P = .0003). The current study results also showed that loss of RIZ1 is mediated by aberrant cytosine methylation of the RIZ1 promoter. One hundred percent of carcinomas were methylated, compared with 33% of normal thyroid tissues (P = .001). RIZ1 mRNA expression was significantly higher (P = .02) in unmethylated (1.22 +/- 1.2, mean +/- standard deviation [SD]), compared with methylated tissues (0.37 +/- 0.42, mean +/- SD). Last, treatment with a DNA methyltransferase inhibitor led to reactivation of RIZ1 expression in cell lines that had negligible RIZ1 expression at baseline. CONCLUSIONS: The current study suggested an important role for RIZ1 expression in thyroid tumorigenesis and identified a potential novel therapeutic target for tumors unresponsive to other therapies.

Antimetabolites, Antineoplastic↗
PubMed2006

Desensitization at the interface.

Normal brain function requires the faithful transmission and integration of information on a timescale of milliseconds, and this rapid signaling is mediated by cell membrane receptors that are ligand-gated ion channels. Fast excitatory transmission occurs when synaptically released glutamate opens channels in neighboring neurons, but these channels desensitize rapidly during sustained high-frequency firing. Recent structural data have begun to provide important insights into the molecular mechanisms that underlie channel activation and desensitization.

Animals↗
PubMed2006

The relationship between agonist potency and AMPA receptor kinetics.

AMPA-type glutamate receptors are tetrameric ion channels that mediate fast excitatory synaptic transmission in the mammalian brain. When agonists occupy the binding domain of individual receptor subunits, this domain closes, triggering rearrangements that couple agonist binding to channel opening. Here we compare the kinetic behavior of GluR2 channels activated by four different ligands, glutamate, AMPA, quisqualate, and 2-Me-Tet-AMPA, full agonists that vary in potency by up to two orders of magnitude. After reduction of desensitization with cyclothiazide, deactivation decays were strongly agonist dependent. The time constants of decay increased with potency, and slow components in the multiexponential decays became more prominent. The desensitization decays of agonist-activated currents also contained multiple exponential components, but they were similar for the four agonists. The time course of recovery from desensitization produced by each agonist was described by two sigmoid components, and the speed of recovery varied substantially. Recovery was fastest for glutamate and slowest for 2-Me-Tet-AMPA, and the amplitude of the slow component of recovery increased with agonist potency. The multiple kinetic components appear to arise from closed-state transitions that precede channel gating. Stargazin increases the slow kinetic components, and they likely contribute to the biexponential decay of excitatory postsynaptic currents.

Cell Line↗
PubMed2005

Stargazin modulates AMPA receptor gating and trafficking by distinct domains.

AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors mediate fast excitatory synaptic transmission in the brain. These ion channels rapidly deactivate and desensitize, which determine the time course of synaptic transmission. Here, we find that the AMPA receptor interacting protein, stargazin, not only mediates AMPA receptor trafficking but also shapes synaptic responses by slowing channel deactivation and desensitization. The cytoplasmic tail of stargazin determines receptor trafficking, whereas the ectodomain controls channel properties. Stargazin alters AMPA receptor kinetics by increasing the rate of channel opening. Disrupting the interaction of stargazin ectodomain with hippocampal AMPA receptors alters the amplitude and shape of synaptic responses, establishing a crucial function for stargazin in controlling the efficacy of synaptic transmission in the brain.

Animals↗
PubMed2005

AMPA receptor binding cleft mutations that alter affinity, efficacy, and recovery from desensitization.

Glutamate binds to AMPA receptors within a deep cleft between two globular protein domains (domains 1 and 2). Once glutamate binds, the cleft closes, and agonist-bound structures of the isolated ligand binding core suggest that closure of the binding cleft is sufficiently complete that it essentially prevents ligand dissociation. There is also considerable evidence supporting the view that cleft closure is the initial conformational change that triggers receptor activation and desensitization, and it has been clearly demonstrated that there is a correlation between the degree of cleft closure and agonist efficacy. It is unknown, however, whether the stability of binding cleft closure also influences receptor-channel properties. The crystallographic structures indicate that closed-cleft conformations are stabilized by the formation of hydrogen bonds that involve amino acid side chains of residues in domains 1 and 2. We show here that mutations that disrupt one such cross-cleft hydrogen bond (in the AMPA receptor subunit GluR2) decrease both agonist affinity and efficacy. The same mutations also hasten recovery from desensitization. We conclude that the stability of binding cleft closure has a significant impact on AMPA receptor function and is a major determinant of the apparent affinity of agonists. The results suggest that the stability of cleft closure has been tuned so that glutamate dissociates as rapidly as possible yet remains a full agonist.

Binding Sites↗
PubMed2004

Genetic conditions associated with intestinal juvenile polyps.

Juvenile polyps are hamartomatous polyps found primarily in infants and children, and in association with juvenile polyposis (JP; OMIM #174900), Cowden syndrome (CS; OMIM #158350), and Bannayan-Riley-Ruvalcaba syndrome (BRRS; OMIM# 153480). Although solitary juvenile polyps are benign lesions, when present in JP patients they may lead to gastrointestinal cancers. Germline mutations in MADH4 and BMPR1A predispose to JP, and both genes are involved in TGF-beta superfamily signaling pathways. In CS and BRRS, juvenile polyps are a less consistent feature, and CS patients are at risk for breast and thyroid cancers. Mutations of the tumor suppressor gene PTEN have been found in the germline of both CS and BRRS patients. Despite different underlying genetic mechanisms, these and other syndromes share the same phenotypic feature of juvenile polyps.

Animals↗
PubMed2004

Reaction mechanism determines NMDA receptor response to repetitive stimulation.

At central excitatory synapses, N-methyl-D-aspartate (NMDA) receptors, which have a high affinity for glutamate, produce a slowly rising synaptic current in response to a single transmitter pulse and an additional current after a second, closely timed stimulus. Here we show, by examining the kinetics of transmitter binding and channel gating in single-channel currents from recombinant NR1/NR2A receptors, that the synaptic response to trains of impulses is determined by the molecular reaction mechanism of the receptor. The rate constants estimated for the activation reaction predict that, after binding neurotransmitter, receptors hesitate for approximately 4 ms in a closed high-affinity conformation before they either proceed towards opening or release neurotransmitter, with about equal probabilities. Because only about half of the initially fully occupied receptors become active, repetitive stimulation elicits currents with distinct waveforms depending on pulse frequency. This high-affinity/low-efficiency activation mechanism might serve as a link between stimulation frequency and the directionality of the ensuing synaptic plasticity.

Animals↗
PubMed2004

Effects of the lurcher mutation on GluR1 desensitization and activation kinetics.

Previous studies of the lurcher mutation in GluR1 channels concluded that its main effect is to create constitutively active channels (Kohda et al., 2000; Taverna et al., 2000). GluR1Lc channels also exhibit slowed kinetics and a shift in their apparent affinity for glutamate (Kohda et al., 2000; Taverna et al., 2000). Here, we have undertaken a kinetic analysis of GluR1Lc channels to quantify the effects of lurcher and to determine the relative contribution of these effects to the lurcher phenotype. Analysis of GluR1Lc leak current demonstrated that the 2,3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulfonamide (NBQX)-sensitive portion of the leak current corresponded to a current generated by glutamate concentrations similar to the levels of contaminating glutamate measured in our normal external solutions. This result, and the small size of the leak current relative to the currents evoked by saturating glutamate, indicates that GluR1Lc channels exhibit little or no constitutive activity. Our results indicate that the primary effect of the lurcher mutation is to increase the affinity of GluR1 for glutamate and reduce the desensitization of GluR1 at nanomolar concentrations. We also found that the mutation makes both the rate and extent of GluR1Lc channel desensitization depend strongly on subunit occupancy. We conclude that the poor survival of GluR1Lc-transfected cells, and presumably cerebellar neurons in lurcher mice, results because channels carrying the lurcher mutation open and do not desensitize at ambient levels of glutamate.

Animals↗
PubMed2004

Challenging colonic polyposis pedigrees: differential diagnosis, surveillance, and management concerns.

Hereditary polyposis syndromes show extensive phenotypic and genotypic heterogeneity within and among families, a situation that may hinder diagnosis. In these settings, germline mutation testing may be the sine qua non for diagnosis if such a mutation is identified in a patient or family. We provide examples of phenotypically differing polyposis pedigrees depicting various challenges in hereditary polyposis syndrome diagnosis. Our purpose is to augment physician understanding of phenotypic variation and thus help identify high-risk presymptomatic family members who could benefit from highly targeted surveillance and management strategies. We describe nine familial polyposis pedigrees displaying anecdotal clinical problems that can confound the differential diagnosis. Emphasis was given to a multidisciplinary approach focusing on pathological confirmation with respect to number, histology, and location of polyps in the gastrointestinal tract; a detailed family history of cancer at all anatomic sites; noncancer phenotypic features of hereditary polyposis syndromes; and appropriate molecular genetic testing in concert with genetic counseling. Improved physician understanding of the clinical natural history features, genetic transmission patterns, and appropriate gene testing will help in diagnosis and, ultimately, surveillance and management for the various hereditary polyposis syndromes.

Adenomatous Polyposis Coli↗
PubMed2003

How AMPA receptor desensitization depends on receptor occupancy.

AMPA-type glutamate receptors mediate fast excitatory transmission at many central synapses, and rapid desensitization of these receptors can shape the decay of synaptic currents and limit the fidelity of high-frequency synaptic transmission. Here we use a combination of fast glutamate application protocols and kinetic simulations to determine how AMPA receptor desensitization depends on the number of subunits occupied by glutamate. We show that occupancy of a single subunit is sufficient to desensitize AMPA-type channels and that receptors with one to four glutamates bound enter desensitization at similar rates. We find that recovery from desensitization follows a similar sigmoid time course for channels with two to four glutamates bound but is faster and exponential for singly occupied channels. The results suggest that desensitization, at intermediate and high glutamate concentrations, is accompanied by two conformational changes that slow glutamate dissociation. We propose a kinetic scheme that accurately predicts several types of experimental results and differs significantly from previous models in the assignment of affinities for binding to closed and desensitized states. We conclude that desensitization involves a rearrangement that stabilizes the binding domains of one subunit in each dimer in a partially closed conformation. This stabilization likely results from an interaction at the dimer-dimer interface between the binding domains of adjacent subunits.

Binding Sites↗
PubMed2003

Role of the DPC4 tumor suppressor gene in adenocarcinoma of the ampulla of Vater: analysis of 140 cases.

The K-ras oncogene is activated in approximately 90% of pancreatic adenocarcinomas, and the DPC4 (MADH4/SMAD4) tumor suppressor gene is inactivated in approximately 55% of pancreatic adenocarcinomas. The contributions of these genetic alterations to the development of adenocarcinoma of the ampulla of Vater have not been fully established. One hundred forty surgically resected ampullary adenocarcinomas (76 with associated adenomas with high-grade dysplasia) were immunohistochemically labeled for the DPC4 gene product, and in 85 cases the results were correlated with the status of the K-ras oncogene from previously reported data. The results were correlated with clinical outcome and with other pathologic predictors of prognosis. Complete loss of Dpc4 labeling was identified in 34% (95% confidence interval [CI]: 26%, 43%) of the invasive carcinomas and in none (upper 95% CI: 6%) of the associated adenomas. Focal loss of Dpc4 was seen in three (4%; 95% CI: 1%, 14%) of the areas of high-grade dysplasia. Complete loss of Dpc4 expression was seen in 28/77 intestinal-type tumors, in 17/46 pancreaticobiliary-type tumors, and in 0/10 colloid carcinomas. Activating point mutations in the K-ras gene were identified in 40% of the invasive cancers. There was no correlation between K-ras gene mutations and Dpc4 expression and no correlation between these variables and survival. The overall 5-year survival rate was 38%. Lymph node metastases were associated with shorter survival (P =.03). Loss of Dpc4 expression occurs in approximately one third of invasive ampullary cancers but is not seen in adenomas; thus, loss of Dpc4 expression occurs late in ampullary carcinogenesis. Although ampullary and pancreatic adenocarcinomas share histologic and molecular features, ampullary carcinomas are less likely to show loss of Dpc4 expression or K-ras gene mutations.

Adenocarcinoma↗
PubMed2002

A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction.

BACKGROUND: The jellyfish green fluorescent protein (GFP) can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins. RESULTS: We tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, alphas. All of the in-frame GFP insertions produced a fluorescent protein, consistent with the idea that GFP will fold and form a fluorophore when inserted into virtually any domain of another protein. Some of the proteins retained their signaling function, and the random nature of the transposition process revealed permissive sites for insertion that would not have been predicted on the basis of structural or functional models of how that protein works. CONCLUSION: This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs.

Cell Line↗
PubMed2002

RNA editing of neurotransmitter receptors in the mammalian brain.

RNA editing refers to various posttranscriptional mechanisms that alter the nucleotide sequence of RNA. In the mammalian brain, RNA editing results in significant changes in the functional properties of receptors for the important neurotransmitters glutamate and serotonin. These changes result from site-specific deamination of single adenosines in the pre-messenger RNA encoding these receptors. Here, we review what is known about the mechanisms underlying this editing, the consequences of RNA editing for glutamate and serotonin receptor function, and recent studies on transgenic mice and human post-mortem tissue that have begun to elucidate the role of RNA editing in the intact mammalian brain.

Animals↗
PubMed2002

Common deletion of SMAD4 in juvenile polyposis is a mutational hotspot.

Juvenile polyposis (JP) is an autosomal dominant syndrome in which affected patients develop upper- and/or lower-gastrointestinal (GI) polyps. A subset of families with JP have germline mutations in the SMAD4 (MADH4) gene and are at increased risk of GI cancers. To date, six families with JP have been described as having the same SMAD4 deletion (1244-1247delAGAC). The objective of the present study is to determine whether this deletion is a common ancestral mutation or a mutational hotspot. DNA from members of four families with JP, from Iowa, Mississippi, Texas, and Finland, that had this 4-bp deletion was used to genotype 15 simple tandem repeat polymorphism (STRP) markers flanking the SMAD4 gene, including 2 new STRPs within 6.3 and 70.9 kb of the deletion. Haplotypes cosegregating with JP in each family were constructed, and the distances of the closest markers were determined from the draft sequence of the human genome. No common haplotype was observed in these four families with JP. A 14-bp region containing the deletion had four direct repeats and one inverted repeat. Because no common ancestor was suggested by haplotype analysis and the sequence flanking the deletion contains repeats frequently associated with microdeletions, this common SMAD4 deletion in JP most likely represents a mutational hotspot.

Adenomatous Polyposis Coli↗
PubMed2002

The expression of dominant-negative subunits selectively suppresses neuronal AMPA and kainate receptors.

Glutamate-gated ion channels are widely expressed in neurons where they serve a host of cellular functions. An appealing, but yet unexplored, way to delineate the functions of particular glutamate receptor subtypes is to direct the expression of dominant-negative and gain-of-function mutant subunits. We tested the ability of two dominant-negative subunits, an alpha-amino-3-hydroxy-5-methyl-isoxazolproprionic acid receptor subunit and a kainate receptor subunit, to silence recombinant and neuronal glutamate receptors. Co-expression studies in non-neuronal cells indicated that the inclusion of a single mutant subunit was sufficient to silence the receptor. When expressed in cerebellar granule cells, the dominant-negative subunits silenced native channels in a subtype-specific fashion. Immunocytochemical staining of control and transfected neurons, as well as studies with a gain-of-function glutamate receptor-1 mutant, indicated that the mutant subunits were expressed at levels roughly equal to the total abundance of related native subunits, and both dominant-negatives suppressed native channel expression 60-65% when tested 24 h post-transfection. If co-assembly of the mutant subunits with related native subunits is combinatorial, this level of suppression gives receptor half-lives of approximately 20 h.

Animals↗
PubMed2002

The speeding of EPSC kinetics during maturation of a central synapse.

Several factors contribute to the shape of excitatory postsynaptic currents (EPSCs) in CNS neurons, among them the kinetics of presynaptic release, transmitter clearance, and the properties and distribution of postsynaptic receptors. The decays of AMPA receptor-mediated EPSCs at rat cerebellar mossy fibre-granule cell (MF-gc) synapses follow a bi-exponential time-course. The fast component dominates the decay, accounting for 84-94% of the peak amplitude. Here we show that both components of decay, and also the risetimes, became faster during postnatal maturation. At adult, but not immature, synapses, the risetimes and decays of evoked multiquantal EPSCs were similar to those of monoquantal miniature (m)EPSCs. The faster risetimes at mature synapses reflected increased synchrony of multivesicular release, whereas the faster decays appeared to reflect changes in the properties of postsynaptic receptors. Inhibition of glutamate uptake was without effect on evoked EPSCs at both ages. Furthermore, after slowing receptor desensitization with cyclothiazide, the EPSCs at mature synapses decayed as slowly as EPSCs at immature synapses, suggesting that faster glutamate clearance does not account for the developmental speeding of EPSC decay. Our results support previous conclusions that glutamate clearance and receptor deactivation are important determinants of the fast decay component at immature synapses. Desensitization becomes increasingly important during development and plays a major role in shaping EPSC decay at mature synapses.

Aging↗