Cute sayings, costly drugs.
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Costs and Cost Analysis↗
Biomedical subjects
G A Thompson
Publications and source records attributed to G A Thompson.
Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene.
Molecular gene transfer techniques have been used to engineer the fatty acid composition of Brassica rapa and Brassica napus (canola) oil. Stearoyl-acyl carrier protein (stearoyl-ACP) desaturase (EC 1.14.99.6) catalyzes the first desaturation step in seed oil biosynthesis, converting stearoyl-ACP to oleoyl-ACP. Seed-specific antisense gene constructs of B. rapa stearoyl-ACP desaturase were used to reduce the protein concentration and enzyme activity of stearoyl-ACP desaturase in developing rapeseed embryos during storage lipid biosynthesis. The resulting transgenic plants showed dramatically increased stearate levels in the seeds. A continuous distribution of stearate levels from 2% to 40% was observed in seeds of a transgenic B. napus plant, illustrating the potential to engineer specialized seed oil compositions.
Base Sequence↗
Biphasic changes in the level and composition of Dunaliella salina plasma membrane diacylglycerols following hypoosmotic shock.
Hypoosmotic shock has been shown to trigger an immediate and selective increase of plasma membrane diacylglycerols (DAG) in the green alga Dunaliella salina, coinciding with an approximately equivalent loss of phosphatidylinositol 4,5-bisphosphate from this membrane [Ha, K.S., & Thompson, G.A., Jr. (1991) Plant Physiol. 97, 921-927]. Following a slight decline in amount, DAG levels of the plasma membrane resumed their rise by 2 min after the shock and by 40 min had achieved a maximum concentration equivalent to 230% of DAG levels in unstressed cells. This second, more sustained increase of plasma membrane DAG was matched by a DAG increase in the microsome-enriched cytoplasmic membrane fraction, commencing at 2 min and peaking at 140% of control values. The changing pattern of DAG molecular species produced in the plasma membrane during the early phases of hypoosmotic stress was compatible with their derivation from phospholipase C hydrolysis of inositol phospholipids and phosphatidylcholine. From 8 min following hypoosmotic shock, as relatively larger scale DAG accumulations developed in the cytoplasmic membranes, the molecular species composition changed to reflect a marked increase in de novo synthesis of sn-1-oleoyl, sn-2-palmitoylglycerol, and dioleoylglycerol. The former molecular species appears to be synthesized in the chloroplast while the latter is produced in the endoplasmic reticulum. The radioisotope labeling data with Na2(14)CO3 confirmed that the biphasic formation of DAG triggered by hypoosmotic shock culminates in a large-scale de novo synthesis of DAG. This is the first clear evidence for de novo synthesis as a source of DAG following PIP2-mediated signaling.(ABSTRACT TRUNCATED AT 250 WORDS)
Cell Membrane↗
Pharmacokinetics of teicoplanin upon multiple dose intravenous administration to normal healthy male volunteers.
Teicoplanin pharmacokinetics were investigated upon multiple dose intravenous administration of 6 and 12 mg kg-1 in 10 normal, healthy, male volunteers, using a two-period, randomized, crossover design; six subjects completed both periods. On day 1, 6 or 12 mg kg-1 was administered every 12 h as a 30-min constant rate intravenous infusion (two doses). Starting on day 2, the same dose (6 or 12 mg kg-1) was administered every 24 h for an additional 13 days. Blood and urine samples were collected over 21 days. Serum and urine were analyzed using a microbiological assay. Following a minimum of 3 weeks after completion of the first period, subjects were crossed over to the other dose. Following multiple dose intravenous administration of 6 and 12 mg kg-1, median pharmacokinetic parameters included: steady-state volume of distribution of 1.4 and 1.2 l kg-1; total clearance of 12.2 and 14.0 ml h-1 kg-1; renal clearance of 11.1 and 10.3 ml h-1 kg-1; and terminal disposition half-life of 159 and 155 h, respectively. No statistically significant dose-related difference was observed. In addition, a cross-study comparison further supports dose proportionality of teicoplanin upon multiple dose intravenous administration of 3 to 12 mg kg-1.
Adult↗
Applicability of teicoplanin dosage adjustment guidelines for renally impaired patients over the range of 3 to 30 mg kg-1.
The pharmacokinetics of teicoplanin were investigated in 13 subjects with various degrees of renal impairment using a randomized two-period crossover design; 11 subjects completed both periods. Doses of 3 and 30 mg kg-1 were administered as single dose, 60-min constant rate intravenous infusions. Blood samples were obtained over 28 days and urine was collected over 48 h. Serum and urine were analyzed using a microbiological assay. As previously observed in studies conducted in renally impaired subjects, teicoplanin total and renal clearance significantly decreased with decreasing creatinine clearance (p < 0.0001). However, for these parameters, no differences between doses were observed. Dosage adjustment guidelines for renally impaired patients are usually developed using the ratio of total clearance in renally impaired patients to the total clearance in patients with normal renal function. Since no dose-related differences existed in the relationship between teicoplanin total clearance and creatinine clearance, initial dosage adjustment guidelines for renally impaired patients developed at 3 or 30 mg kg-1 are applicable over the range of 3 to 30 mg kg-1.
Aged↗
Dosage regimen design: a pharmacokinetic approach.
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Drug Administration Schedule↗
Pharmacokinetic-pharmacodynamic modeling: time-dependent protein binding--an alternative interpretation of clockwise and counterclockwise hysteresis.
Development of effect compartment model theory has greatly enhanced our understanding of the relationship between pharmacokinetics and pharmacodynamics. When effect versus concentration in serum (usually total concentration) is plotted and counterclockwise hysteresis is observed, an initial disequilibrium between receptor(s) and serum is generally presumed and an effect compartment model is used; alternatively, clockwise hysteresis may infer tolerance, which may be characterized by an adaptation model. In this simulation study, the influence of time-dependent binding to serum protein on the relationship between effect and concentration in serum was investigated. In these simulations, time-dependent protein binding occurred as a result of an increase in protein concentration in serum or displacement by a metabolite. When concentration of free drug in serum was responsible for the pharmacological response, and response versus total drug concentration in serum was plotted, counterclockwise hysteresis, consistent with an effect compartment, occurred with a time-dependent decrease in binding to serum protein. Clockwise hysteresis, consistent with tolerance, occurred with a time-dependent increase in binding to serum protein. For both sets of simulations, no hysteresis was observed when response was plotted against concentration of free drug in serum. These results indicate that, when response is related to concentration of free drug, measurement of concentration of free drug may allow a clearer interpretation of the pharmacokinetic-pharmacodynamic relationship.
Blood Proteins↗
Sequence analysis of linked maize 22 kDa alpha-zein genes.
We have determined the nucleotide sequence of a 7343 bp zein genomic clone (gZ22.8H3) from the maize inbred W64A. Computer-aided analysis of the DNA sequence revealed two contiguous 22 kDa alpha-zein genes. The 5' gene (gZ22.8) encodes a complete polypeptide and contains putative regulatory sequences in both the 5' and 3' flanking regions that are typical of zein genes. In contrast, the 3' gene (psi gZ22.8) appears to be a pseudogene, because it contains numerous insertions and deletions that would prevent translation of the mRNA. Alignment of the 5' and 3' flanking sequences of both genes indicated that they resulted from a 3.3 kb DNA duplication event.
Amino Acid Sequence↗
Pumpkin phloem lectin genes are specifically expressed in companion cells.
Pumpkin phloem exudate contains two abundant phloem proteins: PP1 is a 96-kD protein that forms polymeric filaments in vivo, and PP2 is a 48-kD dimeric lectin. Polyclonal antibodies raised against pumpkin phloem exudate were used to isolate several cDNAs corresponding to PP1 and PP2. RNA gel blot analysis indicated that PP1 is encoded by an mRNA of approximately 2500 nucleotides, whereas PP2 subunits are encoded by an mRNA of 1000 nucleotides. Sequence analysis of PP2 cDNAs revealed a 654-bp open reading frame encoding a 218-amino acid polypeptide; this polypeptide had the carbohydrate binding characteristics of a PP2 subunit. The PP2 mRNA was localized within the phloem of pumpkin hypocotyl cross-sections based on in situ hybridization of a digoxigenin-labeled antisense probe. PP2 mRNA was found within the companion cells in both the bicollateral vascular bundles and the extrafascicular phloem network.
Amino Acid Sequence↗
Pharmacokinetics of teicoplanin upon multiple-dose intravenous administration of 3, 12, and 30 milligrams per kilogram of body weight to healthy male volunteers.
Teicoplanin pharmacokinetics were evaluated after multiple-dose intravenous administration to healthy male volunteers by using a randomized, double-blind, parallel design. Doses of 3, 12, or 30 mg of teicoplanin per kg of body weight were administered every 24 h for 14 days as 60-min constant-rate intravenous infusions. Blood and urine samples were collected over 21 days and analyzed by a microbiological assay. Twenty-three subjects were included in the pharmacokinetic analysis. The median pharmacokinetic parameters upon multiple-dose intravenous administration of 3, 12, and 30 mg/kg included steady-state volumes of distribution of 0.94, 0.77, and 0.68 liter/kg; total clearances of 11.9, 12.0, and 13.2 ml/h/kg; and terminal disposition half-lives of 143, 166, and 96 h, respectively. Renal clearance accounted for approximately 95% of total clearance. No dose-related differences existed for teicoplanin total or renal clearance. The steady-state volume of distribution decreased significantly with increasing doses. As a result of the decrease in the volume of distribution, the terminal disposition half-life at 30 mg/kg was significantly decreased. However, the decreases in the volume of distribution and terminal disposition half-life are of limited clinical importance, since steady-state trough concentrations in serum increase in proportion to dose. Combined results of all multiple-dose studies with similar durations of sample collection indicate no dose-related differences for any pharmacokinetic parameters from 3 to 12 mg/kg. As observed in the present study, no dose-related differences exist for teicoplanin total and renal clearances from 3 to 30 mg/kg. However, at 30 mg/kg, a significant decrease in the steady-state volume of distribution is observed. As a consequence of the reduction in the volume of distribution at 30 mg/kg with no change in clearance, the terminal disposition half-life is decreased.
Adult↗
Structural characterization of a novel glycosyl-phosphatidylinositol from the protozoan Tetrahymena mimbres.
A glycolipid metabolically labelled with [14C]GlcN was isolated from the free-living protozoan Tetrahymena mimbres. The glycolipid was sensitive to a bacterial phosphatidylinositol-specific phospholipase C, and the headgroup was shown to contain a phosphorylated Man alpha 1-2Man alpha 1-4Man alpha 1-4GlcN glycan. The Tetrahymena glycolipid is structurally unique among the glycosylphosphatidylinositols that have so far been characterized, including those from several protozoan parasites of humans.
Animals↗
Phosphatidylinositol glycan formation and utilization by the ciliate Tetrahymena mimbres.
Ryals et al. (Ryals, P.E., Pak, Y., and Thompson, G. A., Jr., (1991) J. Biol. Chem. 266, 15048-15053) have described and partially characterized phosphatidylinositol glycans (PI glycans) present in Tetrahymena mimbres. We now describe the time course of PI glycan labeling from exogenous [3H]myristate, [14C]glucosamine, and [3H]ethanolamine. Over the first 2-12 h following pulse radioisotope addition a sizeable proportion of the radioactivity associated with the protein pellet remaining after cell delipidation existed as PI glycans. These compounds were distributed throughout the cell, with the largest proportion at 12 h being associated with a fraction containing mitochondria, lysosomes, and peroxisomes. However, by 24 h radioactivity had nearly disappeared from the PI glycans and had become associated with proteins by a process that was almost totally inhibited by cycloheximide or tunicamycin. PI glycans appeared to be incorporated mainly into a protein migrating on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a relatively broad band with an apparent molecular mass of 24-29 kDa. The exact mobility of the protein band within this molecular weight range was dependent upon the growth temperature of the cells. The apparent molecular masses of the principal PI-anchored proteins formed by other closely related Tetrahymena species varied widely, ranging from 22 to 76 kDa. The PI-anchored proteins may belong to a group of surface proteins known as immobilization antigens. Treatment of 24-h-labeled T. mimbres cells with phosphatidylinositol-specific phospholipase C in vivo released labeled proteins from the cells. Some labeled proteins were present even in the medium of control, non-phospholipase-treated cells. Tetrahymena PI glycans appear to accumulate in a metabolic pool from which they are gradually removed for attachment to externally oriented PI-anchored proteins. Tetrahymena is a versatile system well suited for studying the regulation of PI-anchored protein biochemistry.
Animals↗
Phosphatidylinositol-linked glycans and phosphatidylinositol-anchored proteins of Tetrahymena mimbres.
The insoluble residue from Tetrahymena mimbres cells that had been preincubated in vivo for 2 h with [3H]myristic acid and then exhaustively delipidated with organic solvents retained radioactivity, principally in material which migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 10-14 kDa. This material was extractable from the delipidated cell residue with organic solvents known to solubilize phosphatidylinositol glycans (PI glycans). The same material could also be labeled with [3H]inositol, [14C]glucosamine, and [3H] ethanolamine. When the delipidated residue of cells labeled for 2 h with [3H]myristate was treated with phosphatidylinositol-specific phospholipase C or nitrous acid, much of the associated radioactivity was released. A similar release was obtained using the putative PI glycan fraction extracted from the cell residue. After further purification by thin layer chromatography, this latter material was hydrolyzed with HCl and shown to contain fatty acids, alkylglyceryl ethers, phosphate, inositol, glucosamine, mannose, and ethanolamine. The findings indicate that T. mimbres contains PI glycans resembling in structure those recently characterized in trypanosomes and mammalian cells. As the time of incubation with the radiotracers enumerated above was increased to 6-24 h, increasing amounts of radioactivity appeared in the 22-27-kDa region of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. This higher molecular weight material is shown in the companion paper (Pak, Y., Ryals, P.E., and Thompson, G.A., Jr. (1991) J. Biol. Chem. 266, 15054-15059) to be released by in vivo phosphatidylinositol-specific phospholipase C treatment. Thus T. mimbres contains a pool of free PI glycans and at least one phosphatidylinositol-anchored protein.
Animals↗
Biosynthesis of the unique trans-delta 3-hexadecenoic acid component of chloroplast phosphatidylglycerol: evidence concerning its site and mechanism of formation.
As in most higher plants, chloroplast membranes of the green alga Dunaliella salina contain phosphatidylglycerol (PG) that is rich in trans-delta 3-hexadecenoic acid (16:1t), a fatty acid found nowhere else in the cell. After labeling D. salina with exogenous [3H]myristic acid [( 3H]14:0), the cis-unsaturated fatty acids of monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol as well as PG had higher specific radioactivities in chloroplast envelopes than in thylakoids. In contrast, 16:1t was very slow to become radioactive, and its specific radioactivity was several times higher in isolated thylakoids than in envelopes after brief (3-20 min) labeling with [3H]14:0. Analysis of individual PG molecular species revealed that the fatty acid paired with 16:1t was also labeled slowly. Thus linoleate (18:2) released from a 16:1t-containing PG had a 350-fold (at 3 min) to 20-fold (at 60 min) lower specific radioactivity than did 18:2 from a palmitate (16:0)-containing PG. The findings suggest that the substrates for trans-desaturation are 16:0-containing PG molecular species which are readily labeled from [3H]14:0 in the envelope but are diluted by the large pool of thylakoid PG before penetrating to the desaturation site. By examining the labeling patterns of individual PG molecular species classes, it was concluded that D. salina 16:1t is formed from 16:0 linked to 18:2/16:0 PG and 18:3/16:0 PG by a trans-desaturase located within the inner recesses of the thylakoid compartment.
Chlorophyta↗
Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity.
Stearoyl-acyl carrier protein (ACP) desaturase (EC 1.14.99.6) catalyzes the principal conversion of saturated fatty acids to unsaturated fatty acids in the synthesis of vegetable oils. Stearoyl-ACP desaturase was purified from developing embryos of safflower seed, and extensive amino acid sequence was determined. The amino acid sequence was used in conjunction with polymerase chain reactions to clone a full-length cDNA. The primary structure of the protein, as deduced from the nucleotide sequence of the cDNA, includes a 33-amino-acid transit peptide not found in the purified enzyme. Expression in Escherichia coli of a gene encoding the mature form of stearoyl-ACP desaturase did not result in an altered fatty acid composition. However, active enzyme was detected when assayed in vitro with added spinach ferredoxin. The lack of significant activity in vitro without added ferredoxin and the lack of observed change in fatty acid composition indicate that ferredoxin is a required cofactor for the enzyme and that E. coli ferredoxin functions poorly, if at all, as an electron donor for the plant enzyme.
Amino Acid Sequence↗
Pharmacokinetics and bioavailability of a new formulation of teicoplanin following intravenous and intramuscular administration to humans.
Pharmacokinetics, bioavailability, and local tolerance (at the site of intramuscular administration) of a new formulation of teicoplanin (400 mg/3 mL) were investigated in 24 normal, healthy, male volunteers. A single dose of 6 mg/kg was administered intravenously and intramuscularly using a randomized crossover design. Volunteers and investigator were blinded as to the route of administration; placebo was administered by the other route. Blood and urine samples were collected for 21 days and were analyzed for microbiological activity. The median (range) pharmacokinetic parameters of teicoplanin following single-dose iv administration were as follows: steady-state volume of distribution of 1.6 (1.2-2.8) L/kg; total clearance of 10.2 (8.6-15.1) mL/h/kg; renal clearance of 10.0 (7.9-13.8) mL/h/kg; and terminal disposition half-life of 168 (111-278) h. Following single-dose im administration, significantly more subjects complained of pain following administration of teicoplanin (58%) compared with placebo (4%). Teicoplanin was completely absorbed with a median (range) peak serum concentration of 12.3 (6.6-37.5) micrograms/mL occurring at a median (range) time of 4.1 (0.7-6.1) h. Since the 90% confidence interval for the ratio of areas under the serum concentration-time curve falls within the range of 80 to 120%, the extent of systemic absorption of teicoplanin following im administration is equivalent to that following iv administration.
Adult↗
Normal and lysine-containing zeins are unstable in transgenic tobacco seeds.
Chimeric genes composed of the beta-phaseolin promoter, an alpha-zein coding sequence and its modified versions containing lysine codons, and a beta-zein polyadenylation signal were inserted into the genome of tobacco by Agrobacterium-mediated transformation. alpha-Zein mRNA levels in the transgenic tobacco seeds 20 days after self-pollination varied between 1.0% and 2.5% of the total mRNA population. At 25 days after pollination the 19 kDa alpha-zein was immunologically detected with a polyclonal antiserum in protein extracts from the seeds of transgenic plants. The transgenic plant with the highest level of zein gene expression had an alpha-zein content that was approximately 0.003% of the total seed protein. The amount of alpha-zein in other transgenic plants varied between 1 x 10(-4)% and 1 x 10(-5)% of the total seed protein. The differences in the amounts of mRNA and protein did not correlate with the lysine substitutions introduced into the alpha-zein protein. Polysomes translating alpha-zein mRNA isolated from tobacco seeds contained fever ribosomes than those from maize endosperm, but this did not appear to be the cause of the inefficient protein synthesis. In vivo labelling and immunoprecipitation indicated that newly synthesized alpha-zein was degraded in tobacco seeds with a half-life of less than 1 hour.
Lysine↗
Teicoplanin pharmacokinetics in intravenous drug abusers being treated for bacterial endocarditis.
The pharmacokinetics of teicoplanin were determined after multiple 30-min intravenous infusions of 10 to 15 mg/kg every 12 to 24 h in 11 intravenous drug abuse (IVDA) patients being treated for bacterial endocarditis. Multiple serum samples were obtained over 7 to 14 days. Twenty-four-hour urine collections were obtained on days 1 and 5. Serum concentration-time data were analyzed by using multiple-dose pharmacokinetic analysis (NONLIN84). Results were compared with pharmacokinetic parameters derived from previous studies in normal healthy volunteers following multiple intravenous infusions of teicoplanin (3 to 6 mg/kg/day). Total and renal clearances of teicoplanin in IVDA patients were found to be significantly greater and more highly variable than those observed previously in normal healthy volunteers. As a result, predicted steady-state trough concentrations in serum may vary up to fivefold. The mechanism responsible for this variation appears to be related to the glomerular filtration rate. In IVDA patients, individualized teicoplanin dosage may be required in the treatment of bacterial endocarditis.
Adult↗