Dysphagia due to an aberrant left subclavian artery in a right-sided aortic arch.
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Aorta, Thoracic↗
Biomedical subjects
E McKenna
Publications and source records attributed to E McKenna.
Treatment of solid tumours in children with tumour-lysate-pulsed dendritic cells.
Dendritic cells are potent stimulators of antigen-specific immune responses, including antitumour responses. We explored the use of tumour-lysate-pulsed dendritic cells in children with relapsed solid tumours. Dendritic cell treatment in children was feasible and apparently not toxic. The treatment was able to produce significant tumour regression in a child with metastatic fibrosarcoma.
Adolescent↗
Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel.
We report here a characterization of two families of calcium-activated K(+) channel beta-subunits, beta2 and beta3, which are encoded by distinct genes that map to 3q26.2-27. A single beta2 family member and four alternatively spliced variants of beta3 were investigated. These subunits have predicted molecular masses of 27. 1-31.6 kDa, share approximately 30-44% amino acid identity with beta1, and exhibit distinct but overlapping expression patterns. Coexpression of the beta2 or beta3a-c subunits with a BK alpha-subunit altered the functional properties of the current expressed by the alpha-subunit alone. The beta2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the beta3a-c subunits resulted in only partial inactivation of the current, and the beta3b subunit conferred an apparent inward rectification. Furthermore, unlike the beta1 and beta2 subunits, none of the beta3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these beta-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.
Alternative Splicing↗
Physical interactions between phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPases are dissociated by elevated Ca2+, but not by phospholamban phosphorylation, vanadate, or thapsigargin, and are enhanced by ATP.
Previous co-immunoprecipitation studies (Asahi, M., Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 32855-32862) revealed that physical interactions between phospholamban (PLN) and the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA1a) were retained, even with PLN monoclonal antibody 1D11 bound to an epitope lying between PLN residues 7 and 17. Because the 1D11 antibody relieves inhibitory interaction between the two proteins, it was of interest to determine whether PLN phosphorylation or elevation of Ca(2+), which also relieves inhibitory interactions between PLN and SERCA, would disrupt physical interactions. Co-immunoprecipitation was measured in the presence of increasing concentrations of Ca(2+) or after phosphorylation of PLN by protein kinase A. Physical interactions were dissociated by elevated Ca(2+) but not by PLN phosphorylation. The addition of ATP enhanced interactions between PLN and SERCA. The further addition of vanadate and thapsigargin, both of which stabilize the E(2) conformation, did not diminish binding of PLN to SERCA. These data suggest that physical interactions between PLN and SERCA are stable when SERCA is in the Ca(2+)-free E(2) conformation but not when it is in the E(1) conformation and that phosphorylation of PLN does not dissociate physical interactions between PLN and SERCA.
Adenosine Triphosphate↗
Characterization and quantitation of phospholamban and its phosphorylation state using antibodies.
Quantitative immunoassays to discriminate and quantitate phospholamban and its phosphorylation states in heart homogenates were developed using known amounts of protein determined by amino acid analysis. Synthetic 1-52 phospholamban, the hydrophilic 1-25 peptide, and 1-25 phosphopeptides containing P-Ser(16), P-Thr(17), and dually phosphorylated (P-Ser(16), P-Thr(17)) were used to calibrate immunoblot systems. In addition, synthetic 1-52 peptide was phosphorylated using cAMP-dependent protein kinase (P-Ser(16)) or Ca(2+)-calmodulin protein kinase (P-Thr(17)) and then separated from unphosphorylated 1-52 by HPLC prior to quantitation. Further, canine cardiac sarcoplasmic reticulum was phosphorylated in vitro using [gamma-(32)P]-ATP with cAMP-dependent protein kinase and/or Ca(2+)-calmodulin-dependent protein kinase as well as sequential phosphorylation in both orders to assess the veracity of antibody recognition of phosphorylated forms. Western blots proved useful in characterizing the reactivity of the different antibodies to phospholamban and phosphorylated phospholamban, but were inefficient for accurate quantitation and problems with antibody recognition of dually phosphorylated phospholamban were found. mAb 1D11 recognized all forms of phospholamban, polyclonal antibodies 285 and PS-16 were highly selective for P-Ser(16) phospholamban but had diminished reactivity to diphosphorylated (P-Ser(16), P-Thr(17)) phospholamban, and polyclonal antibody PT-17, although selective for P-Thr(17) phospholamban, generated very weak signals on Western blots and reacted poorly with diphosphorylated phospholamban. Results in quantitative immunodot blot experiments were even more compelling. None of the phosphorylation specific antibodies reacted with the diphospho 1-25 phospholamban peptide. Transgenic mouse hearts expressing varying levels of PLB and ferret heart biopsy samples taken before and after isoproterenol perfusion were analyzed. In all samples containing phospholamban, a basal level of Ser(16) phosphorylation (about 4% of the total PLB population) and a lesser amount of Thr(17) phosphorylation was observed. Upon isoproterenol perfusion, Ser(16) phosphorylation increased only to 17% of the total phospholamban population with a similar change in Thr(17) phosphorylation. This suggests that phospholamban phosphorylation may serve as an electrostatic switch that dissociates inactive calcium pump complexes into catalytically active units. Thus, direct correlations between phospholamban phosphorylation state and contractile parameters may not be valid.
Animals↗
The human inward rectifier K(+) channel subunit kir5.1 (KCNJ16) maps to chromosome 17q25 and is expressed in kidney and pancreas.
A novel human Kir5.1 (inward rectifier K+ channel subunit, gene name KCNJ16) was identified through database searches. This human KCNJ16 was mapped to chromosome 17q25. The full-length cDNA was identified and its genomic structure was determined. Tissue distribution studies showed that human KCNJ16 is significantly expressed in human kidney, pancreas and thyroid gland. In situ hybridization revealed expression in convoluted tubule cells of kidney and in the acinar and ductal cells of pancreas. These suggest that human Kir5.1 may be involved in the regulation of fluid and pH balance, thus making it a potential therapeutic target for hypertension, renal failure, or pancreatic disease.
Amino Acid Sequence↗
Relationship between phospholamban and nucleotide activation of cardiac sarcoplasmic reticulum Ca2+ adenosinetriphosphatase.
A strong connection with nucleotide activation of Ca2+ATPase and phospholamban inhibition has been found. Phospholamban decreases the number of activatable Ca2+ATPase without affecting substrate affinity or the ability of nucleotide to serve its dual modulatory roles, i.e., catalytic and regulatory. Low concentrations of certain nucleotide mimetics, quercetin, tannin, and ellagic acid, with structural similarity to adenine can unmask phospholamban's inhibitory effect while concurrently acting as competitive inhibitors of nucleotide binding. Micromolar concentrations of tannin (EC50 approximately 0.3 microM) and ellagic acid (EC50 approximately 3 microM) stimulated Ca2+ uptake and calcium-activated ATP hydrolysis at submicromolar Ca2+ in isolated cardiac sarcoplasmic reticulum (SR). Stimulation of Ca2+ATPase was followed by pronounced inhibiton at only slightly higher tannin concentrations (IC50 approximately 3 microM), whereas inhibitory effects by ellagic acid were observed at much greater concentrations (IC50 > 300 microM) than the EC50. A complex relationship between compound, SR protein, and MgATP concentration is a major determining factor in the observed effects. Stimulation was only observed under conditions of phospholamban regulation, while the inhibitory effects were observed in cardiac SR at micromolar Ca2+ and in skeletal muscle SR, which lacks phospholamban. Maximal stimulation of Ca2+ATPase was identical to that observed with the anti-phospholamban monoclonal antibody 1D11. Both compounds appear to relieve the Ca2+ATPase from phospholamban inhibition, thereby increasing the calcium sensitivity of the Ca2+ATPase like that observed with phosphorylation of phospholamban or treatment with monoclonal antibody 1D11. Tannin, even under stimulatory conditions, is a competitive inhibitor of MgATP with a linear Dixon plot. The subsequent inhibitory action of higher tannin concentrations results from competition of tannin with the nucleotide binding site of the Ca2+ATPase. In contrast, ellagic acid produced a curvilinear Dixon plot suggesting partial inhibition of nucleotide activation. The data suggest that nucleotide activation of Ca2+ATPase is functionally coupled to the phospholamban interaction site. These compounds through their interaction with the adenine binding domain of the nucleotide binding site prevent or dissociate phospholamban regulation. Clearly, this portion of Ca2+ATPase needs further study to elucidate its role in phospholamban inhibition.
Adenosine Triphosphate↗
Intrapulmonary concentrations of pyrazinamide.
The objective of this study was to compare the steady-state plasma and intrapulmonary concentrations of orally administered pyrazinamide in normal volunteers and subjects with AIDS. Pyrazinamide was administered at 1 g once daily for 5 days to 40 adult volunteers (10 men with AIDS, 10 normal men, 10 women with AIDS, and 10 normal women). Subjects with AIDS and with more than four stools per day were excluded. Blood was obtained prior to administration of the first dose, 2 h after the last dose, and at the completion of bronchoscopy and bronchoalveolar lavage, which were performed 4 h after the last dose. Standardized bronchoscopy was performed without systemic sedation. The volume of epithelial lining fluid (ELF) recovered was calculated by the urea dilution method. The total number of alveolar cells (AC) was counted in a hemocytometer, and differential cell counting was performed after cytocentrifugation. Pyrazinamide was measured by high-performance liquid chromatography. The presence of AIDS or gender had no significant effect on the concentrations of pyrazinamide in plasma. The concentrations of pyrazinamide in ELF and AC were lower in the subjects with AIDS than in the subjects without AIDS, but the difference was not significant. The concentrations in plasma (mean +/- standard deviation) were 25.1 +/- 7.6 and 21.1 +/- 6.8 microg/ml at 2 and 4 h after the last dose, respectively, and were not significantly different from the concentration (17.4 +/- 16.9 microg/ml) in AC. The concentration of pyrazinamide in ELF was high (431 +/- 220 microg/ml) and was approximately 4 to 40 times the reported MIC for pyrazinamide-susceptible strains of Mycobacterium tuberculosis. The high concentration of pyrazinamide in ELF may contribute in part to the effectiveness of the drug in treating pulmonary tuberculosis.
Acquired Immunodeficiency Syndrome↗
Nucleotide mimetics reverse phospholamban regulation in cardiac sarcoplasmic reticulum.
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Animals↗
Dissociation of phospholamban regulation of cardiac sarcoplasmic reticulum Ca2+ATPase by quercetin.
Quercetin had a biphasic effect on Ca2+ uptake and calcium-stimulated ATP hydrolysis in isolated cardiac sarcoplasmic reticulum (SR). Stimulation of Ca2+ATPase was observed at low quercetin concentrations (<25 microM) followed by inhibition at higher concentrations. The effects were dependent upon the SR protein concentration, the MgATP concentration, and intact phospholamban regulation of cardiac Ca2+ATPase. Only the inhibitory effects at higher quercetin concentrations were observed in skeletal muscle SR which lacks phospholamban and in cardiac SR treated to remove phospholamban regulation. Stimulation was additive with monoclonal antibody 1D11 (directed against phospholamban) at submaximal antibody concentrations; however, the maximal antibody and quercetin stimulation were identical. Quercetin increased the calcium sensitivity of the Ca2+ATPase like that observed with phosphorylation of phospholamban or treatment with monoclonal antibody 1D11. In addition, low concentrations of quercetin increased the steady-state formation of phosphoenzyme from ATP or Pi, but higher quercetin decreased phosphoenzyme levels. Quercetin, even under stimulatory conditions, was a competitive inhibitor of ATP, but appears to relieve the Ca2+ATPase from phospholamban inhibition, thereby, producing an activation. The subsequent inhibitory action of higher quercetin concentrations results from competition of quercetin with the nucleotide binding site of the Ca2+ATPase. The data suggest that quercetin interacts with the nucleotide binding site to mask phospholamban's inhibition of the SR Ca2+ATPase and suggests that phospholamban may interact at or near the nucleotide binding site.
Animals↗
Biochemical and biophysical comparison of native and chemically synthesized phospholamban and a monomeric phospholamban analog.
Phospholamban (PLB) was rapidly isolated from canine cardiac sarcoplasmic reticulum using immunoaffinity chromatography and prepared by solid phase peptide synthesis. The two proteins are indistinguishable when analyzed by SDS-polyacrylamide gel electrophoresis and exhibit pentameric oligomeric states. They are similarly detected on Western blots, are phosphorylation substrates, have identical amino acid compositions that directly reflect their predicted values, yield the same internal amino acid sequences upon CNBr digestion, and have molecular mass values agreeing with the expected value (approximately 6123 Da). Native and synthetic PLB reduced the calcium sensitivity of Ca2+ATPase, which is reversed by anti-PLB antibody. A Cys-to-Ser PLB analog, where the cysteines (36, 41, and 46) were substituted by serines, is monomeric on SDS-polyacrylamide gel electrophoresis, can be phosphorylated, and is recognized by polyclonal antisera. PLB migrates with a sedimentation coefficient of 4.8 S in sedimentation velocity ultracentrifugation experiments, whereas Cys-to-Ser PLB does not sediment, consistent with a monomeric state. Circular dichroism spectral analysis of PLB indicates about 70% alpha-helical structure, whereas Cys-to-Ser PLB manifests only about 30%. Because the physiochemical properties of native and synthetic PLB appear identical, the more readily available synthetic protein should be suitable for more extensive structural studies.
Adenosine Triphosphatases↗
Single-dose intrapulmonary pharmacokinetics of azithromycin, clarithromycin, ciprofloxacin, and cefuroxime in volunteer subjects.
The intrapulmonary pharmacokinetics of azithromycin, clarithromycin, ciprofloxacin, and cefuroxime were studied in 68 volunteers who received single, oral doses of azithromycin (0.5 g), clarithormycin (0.5 g), ciprofloxacin (0.5 g), or cefuroxime (0.5 g). In subgroups of four subjects each, the subjects underwent bronchoscopy and bronchoalveolar lavage at timed intervals following drug administration. Drug concentrations, including those of 14-hydroxyclarithromycin (14H), were determined in serum, bronchoalveolar lavage fluid, and alveolar cells (ACs) by high-pressure liquid chromatography. Concentrations in epithelial lining fluid (ELF) were calculated by the urea diffusion method. The maximum observed concentrations (mean +/- standard deviation) of azithromycin, clarithromycin, 14H, ciprofloxacin, and cefuroxime in serum were 0.13 +/- 0.07, 1.0 +/- 0.6, 0.60 +/- 0.41, 0.95 +/- 0.32, and 1.1 +/- 0.3 microgram/ml, respectively (all at 6 h). None of the antibiotics except clarithromycin (39.6 +/- 41.1 micrograms/ml) was detectable in ELF at the 6-h bronchoscopy. The movement into and persistence in cells was different for azithromycin and clarithromycin. In ACs azithromycin was not detectable at 6 h, reached its highest concentration at 120 h, and exhibited the greatest area under the curve (7,403 micrograms.hr ml-1). The peak concentration of clarithromycin (181 +/- 94.1 micrograms/ml) was greater and occurred earlier (6 h), but the area under the curve (2,006 micrograms.hr ml-1) was less than that observed for azithromycin. 14H was detectable in ACs at 6 h (40.3 +/- 5.2 micrograms/ml) and 12 h (32.8 +/- 57.2 micrograms/ml). The peak concentration of ciprofloxacin occurred at 6 h (4.3 +/- 5.2 micrograms/ml), and the area under the curve was 35.0 micrograms.hr ml-1. The data indicate that after the administration of a single dose, azithromycin, clarithromycin, and ciprofloxacin penetrated into ACs in therapeutic concentrations and that only clarithromycin was present in ELF. The correlation of these kinetic observations with clinical efficacy or toxicity was not investigated and is unclear, but the data provide a basis for further kinetic and clinical studies.
Administration, Oral↗
Intrapulmonary pharmacokinetics of clarithromycin and of erythromycin.
The intrapulmonary pharmacokinetics of orally administered clarithromycin (500 mg every 12 h for five doses) or erythromycin (250 mg every 6 h for nine doses) were studied in 32 healthy adult volunteers. Four of the subjects, two in the clarithromycin group and two in the erythromycin group, were smokers. Bronchoscopy, bronchoalveolar lavage, and venipuncture were performed at 4, 8, 12, 24, and 48 h after administration of the last dose of clarithromycin and at 4, 8, and 12 h after administration of the last dose of erythromycin. Clarithromycin was measured by high-performance liquid chromatography, and erythromycin was measured by a microbiological assay. No systemic sedation was used. There were no major adverse events. The concentrations of antibiotics in epithelial lining fluid (ELF) were calculated by the urea dilution method. The volumes (mean +/- standard deviation) of ELF were 1.9 +/- 2.0 ml and 1.5 +/- 0.7 ml in the clarithromycin and erythromycin groups, respectively (P > 0.05). There was no effect of smoking on the amount of bronchoalveolar lavage fluid recovered, the volume of ELF, or the number of erythrocytes present in the lavage fluid (P > 0.05 for all comparisons). The total number of alveolar cells, however, was almost threefold greater in the smokers versus that in the nonsmokers (P < 0.05). Clarithromycin was concentrated in ELF (range, 72.1 +/- 73.0 micrograms/ml at 8 h to 11.9 +/- 3.6 micrograms/ml at 24 h) and alveolar cells (range, 505.8 +/- 293.1 micrograms/ml at 4 h to 17.0 +/- 34.0 micrograms/ml at 48 h). 14-(R)-Hydroxyclarithromycin was also present in these compartments, but at lower concentrations than the parent compound. The concentrations of erythromycin in ELF and alveolar cells were low at 4, 8, and 12 h following the last dose of drug (range, 0 to 0.8 +/- microgram/ml in ELF and 0 to 0.8 +/- 1.3 microgram/ml in alveolar cells). The clinical significance of any antibiotic concentrations in these compartments in unclear. The data suggest, and we conclude, that clarithromycin may be a useful drug in the treatment of pulmonary infections, particularly those caused by intracellular organisms.
Adult↗
Expression of lactose permease in contiguous fragments as a probe for membrane-spanning domains.
The lactose permease of Escherichia coli is a membrane transport protein containing 12 transmembrane hydrophobic domains connected by hydrophilic loops. Coexpression of lacY gene fragments encoding contiguous polypeptides corresponding to the first and second halves of the permease [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325-4329] or the first two transmembrane domains and the remainder of the molecule [Wrubel, W., Stochaj, U., Sonnewald, U., Theres, C., & Ehring, R. (1990) J. Bacteriol. 172, 5374-5381] leads to active lactose transport. It is shown here that contiguous permease fragments with discontinuities in loop 1 (periplasmic), loop 6 (cytoplasmic), or loop 7 (periplasmic) exhibit transport activity; however, fragments with discontinuities in transmembrane domains III or VII fail to do so. The results are consistent with the interpretation that contiguous permease fragments with discontinuities in hydrophilic loops form functional duplexes, while fragments with discontinuities in transmembrane alpha-helical domains do not. On the basis of this notion, a series of contiguous, nonoverlapping permease fragments with discontinuities at various positions in loop 6, putative helix VII, and loop 7 were coexpressed to approximate the boundaries of putative transmembrane domain VII. Contiguous fragments with a discontinuity between Leu222 and Trp223 or between Gly254 and Glu255 are functional, but fragments with a discontinuity between Cys234 and Thr235, between Gln241 and Gln242, or between Phe247 and Thr248 are inactive. Therefore, it is likely that Leu222 and Gly254 are located in hydrophilic loops 6 and 7, respectively, while Cys234, Gln241, and Phe247 are probably located within transmembrane domain VII.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Insertional mutagenesis of hydrophilic domains in the lactose permease of Escherichia coli.
The lactose permease of Escherichia coli is a membrane transport protein postulated to contain a hydrophilic N terminus (hydrophilic domain 1), 12 hydrophobic transmembrane alpha-helices that traverse the membrane in zigzag fashion connected by hydrophilic domains, and a hydrophilic C terminus (hydrophilic domain 13). To test whether the hydrophilic domains are important for function, each domain was independently disrupted by insertion of two or six contiguous histidine residues, and the mutants were characterized with respect to initial rate of lactose transport and steady-state level of accumulation. Remarkably, histidine insertions into 10 out of 13 hydrophilic domains result in molecules that catalyze lactose accumulation effectively, although the initial rate of transport is compromised in certain cases. In contrast, insertions into hydrophilic domain 3, 9, or 10 cause a marked decrease in transport activity. As judged by immunoblots and [35S]methionine pulse-chase experiments, diminished activity is not due to decreased expression of the mutated permeases, defective insertion into the membrane, or increased rates of proteolysis after insertion. The results (i) suggest that most of the hydrophilic domains in the permease do not play an essential role in the transport mechanism and (ii) focus on the region of the permease containing putative helices IX and X as being particularly important for activity.
Amino Acid Sequence↗
Evidence that the final turn of the last transmembrane helix in the lactose permease is required for folding.
Although truncation of the hydrophilic C-terminal tail of the lactose (lac) permease of Escherichia coli (residues 401-417) has no significant effect on membrane insertion, stability, or transport activity, sequential substitution of stop codons for amino acid codons 398-401 leads to a progressive increase in transport activity and in the lifetime of the permease in the membrane (McKenna, E., Hardy, D., Pastore, J. C., and Kaback, H. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2969-2973). Thus, either the last turn of putative helix XII or the region immediately distal to helix XII is important for proper folding, and hence, activity and resistance to proteolysis. In an effort to determine whether this 3-4-amino acid sequence comprises the final turn of the last transmembrane helix of the permease or the beginning of the hydrophilic C-terminal tail, we deleted residues 401-417 and replaced amino acid residues 397-400 with either 4 Leu residues ("helix making") or Gly-Pro-Gly-Pro ("helix breaking"). Permease with 4 Leu residues at positions 397-400 is fully functional with respect to transport and completely stable, as judged by [35S]methionine labeling experiments. In marked contrast, permease with Gly-Pro-Gly-Pro at the same positions exhibits minimal activity and is unstable. The results imply that the amino acid sequence ... Val397Phe398Thr399 Leu400 ... in lac permease may comprise the last turn of transmembrane helix XII, rather than the beginning of the C-terminal tail.
Amino Acid Sequence↗
Sequential truncation of the lactose permease over a three-amino acid sequence near the carboxyl terminus leads to progressive loss of activity and stability.
Previous experiments are consistent with the notion that residues 396-401 (... SVFTLS ...) at the carboxyl terminus of the last putative transmembrane helix of the lactose (lac) permease of Escherichia coli are important for protection against proteolytic degradation and suggest that this region of the permease may be necessary for proper folding. Stop codons (TAA) have now been substituted sequentially for amino acid codons 396-401 in the lacY gene, and the termination mutants were expressed from the plasmid pT7-5. With respect to transport, permease truncated at residue 396 or 397 is completely defective, while molecules truncated at residues 398, 399, 400, and 401, respectively, exhibit 15-25%, 30-40%, 40-45%, and 70-100% of wild-type activity. As judged by pulse-chase experiments with [35S]methionine, wild-type permease or permease truncated at residue 401 is stable, while permease molecules truncated at position 400, 399, 398, 397, or 396 are degraded at increasingly rapid rates. The findings indicate that either the last turn of putative helix XII or the region immediately distal to helix XII is important for proper folding and protection against proteolytic degradation.
Amino Acid Sequence↗
Toward an understanding of the dihydropyridine-sensitive calcium channel.
The dihydropyridine-sensitive calcium channel continues to fascinate scientific investigators. Each new discovery leads to more complexity. Further work elucidating the molecular structures and functions of the various isoforms of the channel will lead us to a better understanding of its nature. We are well on our way towards understanding the molecular structure and mechanisms involved in calcium permeability, and the coming decade promises to reveal numerous breakthroughs in our understanding of this channel.
Amino Acid Sequence↗