Role of the glycosyl-phosphatidylinositol anchor in membrane protein sorting in Madin-Darby canine kidney cells.
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Animals↗
Biomedical subjects
S Howell
Publications and source records attributed to S Howell.
Pharmacokinetic model-driven infusion of fentanyl in children.
BACKGROUND: This study determined the accuracy of previously defined adult fentanyl pharmacokinetics in children having surgery; from this population, the pharmacokinetics of fentanyl were characterized in children when administered via a computerized assisted continuous-infusion device. METHODS: Twenty children between the ages of 2.7 and 11 y scheduled to undergo elective noncardiac surgery were studied. After induction, anesthesia was maintained with 60% nitrous oxide in oxygen supplemented with fentanyl (n = 10) or fentanyl plus isoflurane (n = 10). Fentanyl was administered via computerized assisted continuous-infusion to target concentrations determined by clinical requirements. Plasma fentanyl concentrations were measured and used to evaluate the performance of the fentanyl pharmacokinetics and then to determine a new set of pharmacokinetic parameters and the variance in the context-sensitive half-times simulated for these patients. RESULTS: The original adult fentanyl pharmacokinetics resulted in a positive bias (10.4%), indicating that measured concentrations were mostly greater than predicted. A two-compartment model with age and weight as covariates provided the optimal pharmacokinetic parameters. These resulted in a residual performance error of -1.1% and a median absolute performance error of 17.4%. The context-sensitive times determined from this pediatric population were considerably shorter than the context-sensitive times previously published for adults. CONCLUSIONS: The pharmacokinetics of fentanyl administered by computerized assisted continuous-infusion differ between adults and children. The newly derived parameters are probably more suitable to determine infusion schemes of up to 4 h in children between the ages of 2 and 11 y.
Anesthetics, Intravenous↗
Creatine ingestion favorably affects performance and muscle metabolism during maximal exercise in humans.
Nine male subjects performed two bouts of 30-s maximal isokinetic cycling before and after ingestion of 20 g creatine (Cr) monohydrate/day for 5 days. Cr ingestion produced a 23.1 +/- 4.7 mmol/kg dry matter increase in the muscle total creatine (TCr) concentration. Total work production during bouts 1 and 2 increased by approximately 4%, and the cumulative increases in both peak and total work production over the two exercise bouts were positively correlated with the increase in muscle TCr. Cumulative loss of ATP was 30.7 +/- 12.2% less after Cr ingestion, despite the increase in work production. Resting phosphocreatine (PCr) increased in type I and II fibers. Changes in PCr before exercise bouts 1 and 2 in type II fibers were positively correlated with changes in PCr degradation during exercise in this fiber type and changes in total work production. The results suggest that improvements in performance were mediated via improved ATP resynthesis as a consequence of increased PCr availability in type II fibers.
Adenosine Triphosphate↗
Metabolic response of type I and II muscle fibers during repeated bouts of maximal exercise in humans.
Nine male subjects performed two bouts of 30-s maximal isokinetic cycling. Each bout of exercise was performed at 80 revolutions/min and was separated by 4 min of recovery. Mixed-muscle phosphocreatine (PCr) resynthesis during recovery (88.1 +/- 6.1%) was positively correlated with the restoration of total work production during bout 2 (r = 0.80, P < 0.05). During bout 1, ATP and PCr utilization were greater in type II compared with type I fibers (P < 0.01 and P < 0.05, respectively). The subsequent 4-min period of recovery was insufficient to allow total restoration of ATP and PCr in type II fibers, but restoration of ATP and PCr in type I fibers was almost complete. During the second bout of exercise, ATP and PCr utilization were reduced in type II fibers (P < 0.01), without a corresponding change in type I fibers, and performance was also significantly reduced. The reduction in work capacity observed during bout 2 may have been related to a slower resynthesis, and consequently a reduced availability, of ATP and PCr in type II fibers.
Adenosine Triphosphate↗
Carnitine metabolism in human muscle fiber types during submaximal dynamic exercise.
The effect of prolonged exhaustive exercise on free carnitine and acetylcarnitine concentrations in mixed-fiber skeletal muscle and in type I and II muscle fibers was investigated in humans. Needle biopsy samples were obtained from the vastus lateralis of six subjects immediately after exhaustive one-legged cycling at approximately 75% of maximal O2 uptake from both the exercised and nonexercised (control) legs. In the resting (control) leg, there was no difference in the free carnitine concentration between type I and II fibers (20.36 +/- 1.25 and 20.51 +/- 1.16 mmol/kg dry muscle, respectively) despite the greater potential for fat oxidation in type I fibers. However, the acetylcarnitine concentration was slightly greater in type I fibers (P < 0.01). During exercise, acetylcarnitine accumulation occurred in both muscle fiber types, but accumulation was greatest in type I fibers (P < 0.005). Correspondingly, the concentration of free carnitine was significantly lower in type I fibers at the end of exercise (P < 0.001). The sum of free carnitine and acetylcarnitine concentrations in type I and II fibers at rest was similar and was unchanged by exercise. In conclusion, the findings of the present study support the suggestion that carnitine buffers excess acetyl group formation during exercise and that this occurs in both type I and II fibers. However, the greater accumulation of acetylcarnitine in type I fibers during prolonged exercise probably reflects the greater mitochondrial content of this fiber type.
Acetylcarnitine↗
Compartmental analysis of Ca2+ kinetics in long-lasting diaphragm fatigue: loss of t-tubular membrane Ca2+.
An analytic method based on simulation and modeling of long-term 45Ca2+ efflux data was used to estimate Ca2+ contents (nmol Ca2+/g tissue wet wt) and exchange fluxes (nmol Ca2+.min-1.g-1) for extracellular and intracellular compartments in in vitro hamster diaphragm. Three physiological states were studied: control (n = 5), acute fatigue (after repeated tetany; n = 5), and long-lasting fatigue (1-h recovery; n = 5). Experimental muscles were loaded with 45Ca2+ for 1 h, and efflux data were collected for 8 h by use of a flow-through tissue chamber. Induction of acute diaphragm fatigue led to a uniform 200% elevation of the 8-h efflux curve (expressed as dpm.min-1.mg-1) relative to control. Conversely, in long-lasting fatigue the early component of the efflux curve was depressed compared with control, whereas the balance of the curve was restored to baseline. Analysis of control efflux data revealed that the early curve (0-2 h) contained data on two rapidly exchanging extracellular Ca2+ compartments, whereas the late curve (2-8 h) reflected information on two slowly exchanging intracellular compartments. Modeling of acute fatigue efflux data estimated a 239% increase in one extracellular Ca2+ compartment (putative t-tubular membrane) and a 546% increase in one intracellular Ca2+ compartment (putative terminal cisternae). These increase accounted for the model prediction of a twofold rise in total diaphragm Ca2+. The kinetic data were quantitatively consistent with the hypothesis that diaphragm Ca2+ overload in acute fatigue required sarcolemmal Ca2+ permeability to double and Ca2+ diffusion into the t-tubular and terminal cisternal compartments to escalate nearly threefold. Fitting of long-lasting fatigue efflux data was associated with the sole prediction that t-tubular membrane Ca2+ was reduced to less than one-half of the control value.
Animals↗
Type VI membrane proteins?
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Antigens, Helminth↗
Vasopressin modulation of peritoneal, lymphatic, and plasma drug exposure following intraperitoneal administration.
i.p. administration of cytotoxic drugs for the treatment of regionally confined cancers results in a greater total drug exposure [area under the concentration x time curve (AUC)] for the peritoneal fluid and regional lymphatics than for plasma. We sought to augment the relative advantage of i.p. administration further through modulation of peritoneal clearance by reduction in splanchnic blood flow. Pigs were treated with 5-fluorouracil, etoposide (VP-16), and carboplatin (CBDCA) alone by the i.p. route or with the same drugs in combination with i.v. lypressin, a synthetic vasopressin analogue, which reduces splanchnic blood flow. Drug concentrations in peritoneal fluid, plasma, and thoracic duct lymph were monitored over the ensuing 6 h. The pharmacokinetics of 5-fluorouracil were not altered by vasopressin; however, vasopressin increased the peritoneal fluid:plasma AUC ratio for CBDCA from 30.6 +/- 5.6 to 70. 6 +/- 7.4 (P < 0.01) and increased the lymph:plasma AUC ratio from 1.1 +/- 0.4 to 2.6 +/- 0.22 (P < 0.05). In the case of VP-16, vasopressin increased the peritoneal fluid:plasma AUC ratio from 129 +/- 35 to 350 +/- 76 (P < 0.05) and the lymph:plasma AUC ratio from 2.1 +/- 0.6 to 10.6 +/- 3.5 (P < 0.05). Concurrent i.v. administration of vasopressin can increase the pharmacokinetic advantage of the i.p. route of administration of CBDCA and VP-16 markedly in the pig model. These data suggest that the strategy of concurrent i.p. administration of CBDCA or VP-16 plus an agent that reduces splanchnic blood flow may increase the dose intensity in the abdominal cavity and intraabdominal lymphatic tissue substantially without increasing systemic toxicity.
Animals↗
Expression and characterization of maize ZBP14, a member of a new family of zinc-binding proteins.
A maize gene (Mz2-12), with a deduced amino acid sequence similar to that of a protein kinase C (PKC) inhibitor from bovine brain, has been expressed in Escherichia coli and the protein (ZBP14) purified to homogeneity. The bovine protein was originally identified by Walsh's group and named PKC inhibitor-1 (PKCI-1). The recombinant maize protein (ZBP14) shares characteristics of bovine PKCI-1: it has similar secondary structure, is dimeric, and has a similar affinity for zinc. However, the maize ZBP14 had very little activity as an inhibitor of mammalian brain PKC, thus precluding zinc sequestration as the mechanism of inhibition. The biological role for the maize protein in plant kinase regulation is therefore unclear. In the presence of both maize ZBP14 and 14-3-3 protein (which inhibits PKC in the absence of diacylglycerol), the effects on PKC appeared to be synergistic.
Animals↗
14-3-3 alpha and delta are the phosphorylated forms of raf-activating 14-3-3 beta and zeta. In vivo stoichiometric phosphorylation in brain at a Ser-Pro-Glu-Lys MOTIF.
The 14-3-3 protein family has received considerable attention recently in the literature, because of the finding that beta and zeta isoforms interact with and activate Raf. We had previously shown that these 14-3-3 isoforms also exist as phosphorylated forms in mammalian and avian brain. The presence of this modification enhances the activity of 14-3-3 as an inhibitor of protein kinase C nearly 2-fold. In this report we show by a combination of electrospray mass spectrometry and protein microsequencing that alpha and delta are in vivo post-translationally modified forms of beta and zeta, respectively, and the site of phosphorylation, serine 185, is in a consensus sequence motif for proline-directed kinases.
14-3-3 Proteins↗
Direct targeting of neutral endopeptidase (EC 3.4.24.11) to the apical cell surface of transfected LLC-PK1 cells and unpolarized secretion of its soluble form.
LLC-PK1 cells were transfected with a cDNA encoding rabbit neutral endopeptidase (NEP; EC 3.4.24.11), an abundant enzyme of the kidney proximal brush border. Clones of cells expressing high levels of the protein were isolated. Selective biotinylation and radioimmunolabelling were used to determine that 85-95% of NEP was localized in the apical domain of filter-grown LLC-PK1 cells. Pulse-chase and selective biotinylation studies revealed that the majority (85%) of newly made NEP was directly targeted to the apical membrane. However, a soluble form of NEP was found to be secreted in approximately equal amounts from both sides of the monolayer when expressed in LLC-PK1 cells. Transfected pro-opiomelanocortin, a pituitary hormone precursor, was secreted almost exclusively into the basolateral medium, suggesting that the bulk flow is to the basolateral membrane. This behaviour contrasts with that observed in MDCK cells, where both the transmembrane and secreted forms of NEP are directly targeted to the apical membrane and where the secretion of pro-opiomelanocortin is unpolarized.
Animals↗
Neutral endopeptidase can hydrolyze beta-amyloid(1-40) but shows no effect on beta-amyloid precursor protein metabolism.
High performance liquid chromatographic analyses of incubations of beta-amyloid(1-40) with neutral endopeptidase revealed at least nine product peaks, indicating that neutral endopeptidase can cleave beta-amyloid at multiple sites. Mass spectroscopic analysis of hydrolyzed beta-amyloid identified at least five cleavage sites, between residues Glu3-Phe4, Gly9-Trp10, Phe19-Phe20, Ala30-Ile31, and Gly33-Leu34. In contrast, amyloid precursor protein metabolism in Neuro2A cells was unaffected by the expression of recombinant neutral endopeptidase in the same cells or by the addition of a secreted form of neutral endopeptidase to spent Neuro2A cell media.
Amino Acid Sequence↗
14-3-3 proteins: biological function and domain structure.
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14-3-3 Proteins↗
Measured context-sensitive half-times of remifentanil and alfentanil.
BACKGROUND: The context-sensitive half-time, rather than the terminal elimination half-life, has been proposed as a more clinically relevant measure of decreasing drug concentration after a constant infusion of a given duration. The context-sensitive half-time is derived from computer modelling using known pharmacokinetic parameters. The modelled context-sensitive half-time for a 3-h infusion of alfentanil is 50-55 min and is 3 min for remifentanil. The terminal elimination half-life is 111 min for alfentanil and 12-30 min for remifentanil. It has not been tested whether the modelled context-sensitive half-time reflects the true time for a 50% decrease in drug concentration or drug effect. METHODS: Thirty volunteers received a 3-h infusion of remifentanil or alfentanil at equieffective concentrations. Depression of minute ventilation to 7.5% ETCO2 was used as a measure of drug effect. Minute ventilation response was measured, and blood samples for drug concentration were taken during and after drug infusion. The recovery of minute ventilation (drug effect) and decrease in blood drug concentration was plotted, and the time for a 50% change was determined. RESULTS: The measured pharmacokinetic context-sensitive half-time for remifentanil after a 3-h infusion was 3.2 +/- 0.9 min, and its pharmacodynamic offset was 5.4 +/- 1.8 min. Alfentanil's measured pharmacokinetic context-sensitive half-time was 47.3 +/- 12 min, and its pharmacodynamic offset was 54.0 +/- 48 min. The terminal elimination half-life modelled from the volunteers was 11.8 +/- 5.1 min for remifentanil and 76.5 +/- 12.6 min for alfentanil. CONCLUSIONS: The measured context-sensitive half-times were in close agreement with the context-sensitive half-times previously modelled for these drugs. The results of this study confirm the value of the context-sensitive half-time in describing drug offset compared to the terminal elimination half-life.
Adult↗
Clinical drug level predictions and practice.
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Analgesics↗
Congestive heart failure: differential adaptation of the diaphragm and latissimus dorsi.
Diaphragm and latissimus dorsi muscle functions, histochemistries, and morphometries were studied in anesthetized male Yucatan minipigs with congestive heart failure (CHF) induced by supraventricular tachycardia (n = 5). Sham-operated animals served as a control group (n = 5). In CHF animals, transdiaphragmatic pressure measured during supramaximal phrenic stimulation was reduced by 40% at low frequencies (< or = 20 Hz) and by 60% at higher frequencies. Twitch amplitude and half-relaxation time were also decreased. The cross-sectional areas of type I, IIa, and IIb fibers were reduced in the diaphragm. The proportion of type I fibers increased, whereas type IIa fibers decreased. Succinate dehydrogenase activity was elevated in type IIa and IIb fibers, but diaphragmatic fatigability was not altered. CHF reduced latissimus dorsi isometric force by 40% for stimulation frequencies > or = 30 Hz. The cross-sectional area of latissimus dorsi type IIb fibers was decreased, but twitch characteristics, fiber type composition, succinate dehydrogenase activity, and fatigability were unchanged. Experimental CHF appears to cause greater intrinsic adaptive changes in the diaphragm compared with those in the latissimus dorsi in the minipig. For both muscles, reduced contractile function was associated with atrophy. Impaired performance of the diaphragm may also be attributed to an increase in the relative contribution of type I fibers to the total tension-generating capacity of the muscle and to the pathophysiological mechanisms underlying the shortened relaxation time of the twitch response.
Adaptation, Physiological↗
Purification of 14-3-3 protein and analysis of isoforms in chicken brain.
14-3-3 proteins are apparently ubiquitous eukaryotic proteins that comprise a large number of isoforms. We have used specific antibodies raised against each mammalian isoform to probe for 14-3-3 isoforms in adult hen brains. The results suggest that there is a remarkable degree of similarity in primary structure (at least in the regions containing the epitopes). Reverse-phase HPLC of the purified avian 14-3-3 proteins indicates a high overall degree of similarity in sequence and levels of expression of each isoform that are remarkably similar to their mammalian counterparts.
14-3-3 Proteins↗
A cleavable N-terminal signal peptide is not a prerequisite for the biosynthesis of glycosylphosphatidylinositol-anchored proteins.
During the biosynthesis of glycosylphosphatidylinositol (GPI)-anchored proteins, an N-terminal signal peptide is used to direct biosynthesis to the endoplasmic reticulum. It was previously unknown whether or not this signal must be removed during the biosynthesis of GPI-anchored proteins. Using neutral endopeptidase (EC 3.4.24.11), a well characterized type II membrane protein that is attached to the membrane via an uncleaved N-terminal signal peptide, we extended its C terminus with 33 of the 37 amino acids of the GPI anchor signal sequence of decay-accelerating factor. When expressed in COS-1 and Chinese hamster fibroblast (CHW) cells, the protein was shown to possess both transmembrane and GPI anchors, indicating that a cleavable N-terminal signal peptide is not a prerequisite for the biosynthesis of GPI-anchored proteins.
Animals↗