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Biomedical subjects

C M Fuller

Publications and source records attributed to C M Fuller.

At least 19 recordsLinked to original sources
PubMed2004

A molecular epidemiological investigation of isolates of the variant avian paramyxovirus type 1 virus (PPMV-1) responsible for the 1978 to present panzootic in pigeons.

A sequence of 375 nucleotides, which included the region encoding the cleavage activation site and signal peptide of the fusion protein gene, was determined for 178 isolates of the pigeon variant strain of Newcastle disease virus (PPMV-1). These were compared with the sequences of 47 similar isolates published by GenBank, which included 30 isolates from pigeons and 17 representatives from each sublineage of avian paramyxovirus type 1. The resulting alignment was analysed phylogenetically using maximum likelihood and the results are presented as unrooted phylogenetic trees. By phylogenetic analysis all the PPMV-1 isolates except one were placed in lineage 4b (VIb). Within this lineage there was considerable genetic heterogeneity, which appears to be predominantly influenced by the date of isolation and, to a lesser extent, geographical origins of the isolates. There were two large distinguishable groups, 4bi and 4bii. The earliest isolate available, PIQPI78442, isolated in 1978 in Iraq, was situated at the node from which the two groups diverge.

Animals↗
PubMed2004

Acceptance and completion of hepatitis B vaccination among drug users in New York City.

Hepatitis B (HBV) vaccination rates remain low among drug users. We examined correlates of vaccine acceptance and completion in two ongoing prospective studies of young injecting and non-injecting drug users in New York City. Street recruited drug users were enrolled at one of two neighbourhood locations (Harlem and the Bronx) between 2000 and 2004 and completed risk behaviour questionnaires and HBV testing. Free HBV vaccination was offered. Among 1117 participants, 26.1% (275) had a previous HBV infection, 57.9% (610) were susceptible to HBV, and 16.0% (169) had serological evidence of previous vaccination. Of the 610 participants susceptible to HBV, 466 (76.4%) returned for their results and were offered vaccination; 53.9% (251) received at least one dose of the vaccine (acceptors). Correlates of vaccine acceptance included older age, public assistance as main income source, and being recruited in the Bronx. Daily crack users were significantly less likely to initiate the vaccine series. Among 240 vaccine acceptors, 98 (40.8%) completed all three doses. Daily injectors, Hispanics, and those recruited in Harlem were less likely to complete the vaccination series. HBV vaccination acceptance among drug users seems likely in programmes that are convenient and offer remuneration; however, extended efforts are needed to improve series completion.

Adult↗
PubMed2001

pH alterations "reset" Ca2+ sensitivity of brain Na+ channel 2, a degenerin/epithelial Na+ ion channel, in planar lipid bilayers.

Members of the degenerin/epithelial Na(+) channel superfamily of ion channels subserve many functions, ranging from whole body sodium handling to mechanoelectrical transduction. We studied brain Na(+) channel 2 (BNaC-2) in planar lipid bilayers to examine its single channel properties and regulation by Ca(2+). Upon incorporation of vesicles made from membranes of oocytes expressing either wild-type (WT) BNaC-2 or BNaC-2 with a gain-of-function (GF) point mutation (G433F), functional channels with different properties were obtained. WT BNaC-2 resided in a closed state with short openings, whereas GF BNaC-2 was constitutively activated; a decrease in the pH in the trans compartment of the bilayer activated WT BNaC-2 and decreased its permeability for Na(+) over K(+). Moreover, these maneuvers made the WT channel more resistant to amiloride. In contrast, GF BNaC-2 did not respond to a decrease in pH, and its amiloride sensitivity and selectivity for Na(+) over K(+) were unaffected by this pH change. Buffering the bathing solutions with EGTA to reduce the free [Ca(2+)] to <10 nm increased WT single channel open probability 10-fold, but not that of GF BNaC-2. Ca(2+) blocked both WT and GF BNaC-2 in a dose- and voltage-dependent fashion; single channel conductances were unchanged. A drop in pH reduced the ability of Ca(2+) to inhibit these channels. These results show that BNaC-2 is an amiloride-sensitive sodium channel and suggest that pH activation of these channels could be, in part, a consequence of H(+) "interference" with channel regulation by Ca(2+).

Acid Sensing Ion Channels↗
PubMed2001

Ca(2+)-activated Cl(-) channels: a newly emerging anion transport family.

A new family of chloride transport proteins has recently emerged. These proteins have extensive homology to a protein previously isolated from bovine tracheal epithelium that acts as a Ca(2+)-sensitive Cl(-) channel (CaCC) when heterologously expressed or when reconstituted into planar lipid bilayers. Several new members of this family have been identified in human, murine, and bovine epithelia, in addition to some other tissues, and are associated with Ca(2+)-sensitive conductive chloride transport when heterologously expressed in Xenopus oocytes or HEK 293 cells. The expressed current is also sensitive to inhibitors such as DIDS and niflumic acid. In addition, at least one family member acts as an endothelial cell adhesion molecule. This emerging family may underlie the Ca(2+)-mediated Cl(-) conductance responsible for rescue of the cystic fibrosis (CF) knockout mouse from significant airway disease.

Animals↗
PubMed2001

Differential gene expression profiling in human brain tumors.

Gene expression profiling of three human temporal lobe brain tissue samples (normal) and four primary glioblastoma multiforme (GBM) tumors using oligonucleotide microarrays was done. Moreover, confirmation of altered expression was performed by whole cell patch clamp, immunohistochemical staining, and RT-PCR. Our results identified several ion and solute transport-related genes, such as N-methyl-d-aspartate (NMDA) receptors, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-2 receptors, GABA(A) receptor subunits alpha3, beta1, beta2, and beta3, the glutamate transporter, the glutamate/aspartate transporter II, the potassium channel K(V)2.1, hK(V)beta3, and the sodium/proton exchanger 1 (NHE-1), that are all downregulated in the tumors compared with the normal tissues. In contrast, aquaporin-1, possibly aquaporins-3 and -5, and GLUT-3 message appeared upregulated in the tumors. Our results also confirmed previous work showing that osteopontin, nicotinamide N-methyltransferase, murine double minute 2 (MDM2), and epithelin (granulin) are upregulated in GBMs. We also demonstrate for the first time that the cytokine and p53 binding protein, macrophage migration inhibitory factor (MIF), appears upregulated in GBMs. These results indicate that the modulation of ion and solute transport genes and heretofore unsuspected cytokines (i.e., MIF) may have profound implications for brain tumor cell biology and thus may identify potential useful therapeutic targets in GBMs.

Aquaporin 1↗
PubMed2001

Factors associated with adolescent initiation of injection drug use.

OBJECTIVE: The purpose of this study was to evaluate the extent to which demographic, sexual, and non-injection drug use practices predict adolescent initiation of injection drug use. METHODS: Street recruited injection drug users 15-30 years of age in Baltimore, Maryland, who initiated injection within five years of study enrollment, completed a questionnaire that included a year-by-year history regarding the five years prior to initiation of injection. Factors associated with initiation during adolescence (< or = 21 years of age) versus young adulthood (>21 ) were determined using logistic regression. RESULTS: Of 226 participants, most were female (61%) and African American (64%). Median age of participants was 25; median age at initiation of injection was 23. Factors significantly associated with adolescent initiation in multivariate analysis included race other than African American, and practices prior to initiating injection including condom use, lack of cocaine use, exclusive crack smoking just prior to initiation, and smoking marijuana. Adolescent initiates also had shorter durations of illicit drug use prior to initiating injection. CONCLUSION: Short-term non-injection drug use, particularly exclusive crack smoking, was associated with adolescent initiation of injection drug use. Early prevention efforts targeting this high-risk group of younger drug users are warranted in order to delay or prevent onset of injection drug use.

Adolescent↗
PubMed2001

Regions in the carboxy terminus of alpha-bENaC involved in gating and functional effects of actin.

Gating differences occur between the alpha-subunits of the bovine and rat clones of an amiloride-sensitive epithelial Na+ channel (ENaC). Deletion of the carboxy terminus of bovine alpha-ENaC (alpha-bENaC) at R567 converted the gating properties to that of rat alpha-ENaC (alpha-rENaC). The equivalent truncation in alpha-rENaC was without effect on the gating of the rat homologue. The addition of actin to ENaC channels composed of either alpha-rENaC or alpha-bENaC alone produced a twofold reduction in conductance and an increase in open probability. Neither alpha-rENaC (R613X) nor alpha-bENaC (R567X) was responsive to actin. Using a chimera consisting of alpha-rENaC1-615 and alpha-bENaC570-650, we examined several different carboxy-terminal truncation mutants plus and minus actin. When incorporated into planar bilayers, the gating pattern of this construct was identical to wild-type (wt) alpha-bENaC. Premature stop mutations proximal to E685X produced channels with gating patterns like alpha-rENaC. Actin had no effect on the E631X truncation, whereas more distal truncations all interacted with actin, as did wt alpha-bENaC. Key findings were confirmed using channels expressed in Xenopus oocytes and studied by cell-attached patch-clamp recording. Our results suggest that the site of actin regulation at the carboxy terminus of the chimera is located between residues 631 and 644.

Actins↗
PubMed2001

Point mutations in the post-M2 region of human alpha-ENaC regulate cation selectivity.

We tested the hypothesis that an arginine-rich region immediately following the second transmembrane domain may constitute part of the inner mouth of the epithelial Na+ channel (ENaC) pore and, hence, influence conduction and/or selectivity properties of the channel by expressing double point mutants in Xenopus oocytes. Double point mutations of arginines in this post-M2 region of the human alpha-ENaC (alpha-hENaC) led to a decrease and increase in the macroscopic conductance of alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, respectively, but had no effect on the single-channel conductance of either double point mutant. However, the apparent equilibrium dissociation constant for Na+ was decreased for both alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, and the maximum amiloride-sensitive Na+ current was decreased for alphaR586E,R587Ebetagamma-hENaC and increased for alphaR589E,R591Ebetagamma-hENaC. The relative permeabilities of Li+ and K+ vs. Na+ were increased 11.25- to 27.57-fold for alphaR586E,R587Ebetagamma-hENaC compared with wild type. The relative ion permeability of these double mutants and wild-type ENaC was inversely related to the crystal diameter of the permeant ions. Thus the region of positive charge is important for the ion permeation properties of the channel and may form part of the pore itself.

Animals↗
PubMed2001

Chlorzoxazone or 1-EBIO increases Na(+) absorption across cystic fibrosis airway epithelial cells.

Previous studies demonstrated that chlorzoxazone or 1-ethyl-2-benzimidazolinone (1-EBIO) enhances transepithelial Cl(-) secretion by increasing basolateral K(+) conductance (G(K)) (Singh AK, Devor DC, Gerlach AC, Gondor M, Pilewski JM, and Bridges RJ. J Pharmacol Exp Ther 292: 778-787, 2000). Hence these compounds may be useful to treat cystic fibrosis (CF) airway disease. The goal of the present study was to determine whether chlorzoxazone or 1-EBIO altered ion transport across Delta F508-CF transmembrane conductance regulator homozygous CFT1 airway cells. CFT1 monolayers exhibited a basal short-circuit current that was abolished by apical amiloride (inhibition constant 320 nM) as expected for Na(+) absorption. The addition of chlorzoxazone (400 microM) or 1-EBIO (2 mM) increased the amiloride-sensitive I(sc) approximately 2.5-fold. This overlapping specificity may preclude use of these compounds as CF therapeutics. Assaying for changes in the basolateral G(K) with a K(+) gradient plus the pore-forming antibiotic amphotericin B revealed that chlorzoxazone or 1-EBIO evoked an approximately 10-fold increase in clotrimazole-sensitive G(K). In contrast, chlorzoxazone did not alter epithelial Na(+) channel-mediated currents across basolateral-permeabilized monolayers or in Xenopus oocytes. These data further suggest that alterations in basolateral G(K) alone can modulate epithelial Na(+) transport.

Amiloride↗
PubMed2000

Expression and regulation of normal and polymorphic epithelial sodium channel by human lymphocytes.

Gene expression, protein expression, and function of amiloride-sensitive sodium channels were examined in human lymphocytes from normal individuals and individuals with Liddle's disease. Using reverse transcriptase polymerase chain reactions, expression of all three cloned epithelial sodium channel (ENaC) subunits was detected in lymphocytes. Polyclonal antibodies to bovine alpha-ENaC bound to the plasma membrane of normal and Liddle's lymphocytes. A quantitative analysis of fluorescence-tagged ENaC antibodies indicated a 2.5-fold greater surface binding of the antibodies to Liddle's lymphocytes compared with normal lymphocytes. The relative binding intensity increased significantly (25%; p < 0.001) for both normal and Liddle's cells after treatment with 40 microM 8-CPT-cAMP. Amiloride-sensitive whole cell currents were recorded under basal and cAMP-treated conditions for both cell types. Liddle's cells had a 4.5-fold larger inward sodium conductance compared with normal cells. A specific 25% increase in the inward sodium current was observed in normal cells in response to cAMP treatment. Outside-out patches from both cell types under both treatment conditions revealed no obvious differences in the single channel conductance. The P(open) was 4.2 +/- 3.9% for patches from non-Liddle's cells, and 27.7 +/- 5.4% in patches from Liddle's lymphocytes. Biochemical purification of a protein complex, using the same antibodies used for the immunohistochemistry, yielded a functional sodium channel complex that was inhibited by amiloride when reconstituted into lipid vesicles and incorporated into planar lipid bilayers. These four independent methodologies yielded findings consistent with the hypotheses that human lymphocytes express functional, regulatable ENaC and that the mutation responsible for Liddle's disease induces excessive channel expression.

Amiloride↗
PubMed2000

The cytosolic termini of the beta- and gamma-ENaC subunits are involved in the functional interactions between cystic fibrosis transmembrane conductance regulator and epithelial sodium channel.

Epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) are co-localized in the apical membrane of many epithelia. These channels are essential for electrolyte and water secretion and/or reabsorption. In cystic fibrosis airway epithelia, a hyperactivated epithelial Na(+) conductance operates in parallel with defective Cl(-) secretion. Several groups have shown that CFTR down-regulates ENaC activity, but the mechanisms and the regulation of CFTR by ENaC are unknown. To test the hypothesis that ENaC and CFTR regulate each other, and to identify the region(s) of ENaC involved in the interaction between CFTR and ENaC, rENaC and its mutants were co-expressed with CFTR in Xenopus oocytes. Whole cell macroscopic sodium currents revealed that wild type (wt) alphabetagamma-rENaC-induced Na(+) current was inhibited by co-expression of CFTR, and further inhibited when CFTR was activated with a cAMP-raising mixture (CKT). Conversely, alphabetagamma-rENaC stimulated CFTR-mediated Cl(-) currents up to approximately 6-fold. Deletion mutations in the intracellular tails of the three rENaC subunits suggested that the carboxyl terminus of the beta subunit was required both for the down-regulation of ENaC by activated CFTR and the up-regulation of CFTR by ENaC. However, both the carboxyl terminus of the beta subunit and the amino terminus of the gamma subunit were essential for the down-regulation of rENaC by unstimulated CFTR. Interestingly, down-regulation of rENaC by activated CFTR was Cl(-)-dependent, while stimulation of CFTR by rENaC was not dependent on either cytoplasmic Na(+) or a depolarized membrane potential. In summary, there appear to be at least two different sites in ENaC involved in the intermolecular interaction between CFTR and ENaC.

Amiloride↗
PubMed2000

Electrophysiological characteristics of the Ca2+-activated Cl- channel family of anion transport proteins.

1. A protein isolated from the bovine tracheal epithelium behaves as a Ca2+-activated Cl- channel (CaCC) when incorporated into planar lipid bilayers. 2. An antibody raised against this protein was used to screen a cDNA expression library and resulted in the isolation of a cDNA clone that exhibited nearly identical electrophysiological characteristics to the isolated endogenous protein when expressed. 3. Recent cloning of several related proteins has revealed that the cloned bovine CaCC is one of a large and growing family. All new family members so far examined are associated with the appearance of a novel Ca2+-mediated Cl- conductance when heterologously expressed. 4. This new group of proteins may underlie the Ca2+-mediated Cl- conductance upregulated in the cystic fibrosis (CF) knockout mouse and thought to be responsible for the escape from the significant airway pathology associated with CF.

Animals↗
PubMed2000

Cost effectiveness analysis of screening of high school athletes for risk of sudden cardiac death.

Sudden cardiac death of a high school athlete is an alarming tragedy. Three preparticipation screening methods have been recommended to reduce its occurrence: specific cardiovascular history and physical examination, 12-lead ECG, and two-dimensional (2D) echocardiography. This study analyzes the cost effectiveness of each of these methods. The cost to perform each test and to evaluate abnormal screening findings were approximated. The years of life gained through detection of athletes with potential causes of sudden cardiac death were estimated. Overall, the approximate costs per year of life saved for the preparticipation cardiovascular screening examinations are: specific cardiovascular history and physical examination, $84,000; 12-lead ECG, $44,000; and 2D echocardiography, $200,000. The 12-lead ECG is the most cost effective preparticipation cardiovascular modality of the three currently recommended methods. Similar cost effectiveness for history and physical examination or 2D echocardiography would require respectively a 2-fold increase in sensitivity or 4.5-fold decrease in cost.

Adolescent↗
PubMed2000

Molecular characterisation of pancreatic zymogen granule ion channel and regulator proteins involved in exocytosis.

In pancreatic acinar cells Ca(2+)-dependent secretagogues promote the fusion of zymogen granules (ZG) with the apical plasma membrane (PM) and exocytosis of digestive enzymes. In addition to exocytotic fusion complexes between SNARE proteins in the ZG membrane (ZGM) and the apical PM, enzyme secretion elicited by Ca(2+)-dependent secretagogues requires cytosolic Cl and K+ and is inhibited by blockers of Cl- and K+-channels. We have identified a Cl-conductance activated by ATP, and a K+-conductance (with properties similar to ATP-sensitive K+-channels), regulated by the granule matrix protein Zg-16p in the ZGM. Both conductances are inversely regulated by a 65-kD mdr1 gene product. We have also identified a novel Ca(2+)-activated anion conductance in ZGM, the Ca(2+)-sensitivity of which increases 50-fold when Cl is replaced by 1. This conductance is blocked by micromolar H2-DIDS or DTT, reminiscent of a family of epithelial Ca(2+)-activated Cl -channels (CaCC). Expression of a CaCC in exocrine pancreas has been confirmed by RT-PCR analysis, and by immunoblotting and immunogold labeling of ZG membranes. These data suggest that ion channels in the ZGM are essential elements in pancreatic exocytosis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Peptide inhibition of constitutively activated epithelial Na(+) channels expressed in Xenopus oocytes.

The hypothesis that 30-amino acid peptides corresponding to the C-terminal portion of the beta- and/or gamma-rat epithelial sodium channel (rENaC) subunits block constitutively activated ENaC was tested by examining the effects of these peptides on wild-type (wt) rENaC (alphabetagamma-rENaC), truncated Liddle's mutants (alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC), and point mutants (alphabeta(Y)gamma-, alphabetagamma(Y)-rENaC) expressed in Xenopus oocytes. The chord conductances of alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC were 2- or 3-fold greater than for wt alphabetagamma-rENaC. Introduction of peptides into oocytes expressing alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC produced a concentration-dependent inhibition of the amiloride-sensitive Na(+) conductances, with apparent dissociation constants (K(d)) ranging from 1700 to 160 microM, depending upon whether individual peptides or their combination was used. Injection of peptides alone or in combination into oocytes expressing wt alphabetagamma-rENaC or single-point mutants did not affect the amiloride-sensitive whole-cell currents. The single channel conductances of all the mutant ENaCs were the same as that of wild type (alphabetagamma-). The single channel activities (N.P(o)) of the mutants were approximately 2.2-2.6-fold greater than wt alphabetagamma-rENaC (1.08 +/- 0.24, n = 7) and were reduced to 1.09 +/- 0.17 by 100 microM peptide mixture (n = 9). The peptides were without effect on the single channel properties of either wt or single-point mutants of rENaC. Our data demonstrate that the C-terminal peptides blocked the Liddle's truncation mutant (alphabeta(T)gamma(T)) expressed in Xenopus oocytes but not the single-point mutants (alphabeta(Y)gamma or alphabetagamma(Y)). Moreover, the blocking effect of both peptides in combination on alphabeta(T)gamma(T)-rENaC was synergistic.

Amino Acid Sequence↗
PubMed1999

Regulation of epithelial Na(+) channels by actin in planar lipid bilayers and in the Xenopus oocyte expression system.

The hypothesis that actin interactions account for the signature biophysical properties of cloned epithelial Na(+) channels (ENaC) (conductance, ion selectivity, and long mean open and closed times) was tested using planar lipid bilayer reconstitution and patch clamp techniques. We found the following. 1) In bilayers, actin produced a more than 2-fold decrease in single channel conductance, a 5-fold increase in Na(+) versus K(+) permselectivity, and a substantial increase in mean open and closed times of wild-type alphabetagamma-rENaC but had no effect on a mutant form of rENaC in which the majority of the C terminus of the alpha subunit was deleted (alpha(R613X)betagamma-rENaC). 2) When alpha(R613X)betagamma-rENaC was heterologously expressed in oocytes and single channels examined by patch clamp, 12.5-pS channels of relatively low cation permeability were recorded. These characteristics were identical to those recorded in bilayers for either alpha(R613X)betagamma-rENaC or wild-type alphabetagamma-rENaC in the absence of actin. Moreover, we show that rENaC subunits tightly associate, forming either homo- or heteromeric complexes when prepared by in vitro translation or when expressed in oocytes. Finally, we show that alpha-rENaC is properly assembled but retained in the endoplasmic reticulum compartment. We conclude that actin subserves an important regulatory function for ENaC and that planar bilayers are an appropriate system in which to study the biophysical and regulatory properties of these cloned channels.

Actins↗