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D W Foster

Publications and source records attributed to D W Foster.

At least 37 records · Page 2Linked to original sources
PubMed1991

Mitochondrial import and processing of rat liver carnitine palmitoyltransferase II defines the amino terminus of the mature protein. Possibility of differential modification of the rat and human isoforms.

[35S]Methionine-labeled porcine heart citrate synthase (used here as a positive control) and rat liver carnitine palmitoyltransferase II (CPT II) were generated by in vitro transcription and translation of their cDNA constructs in appropriate Bluescript plasmids. Each product was imported into rat liver mitochondria in an energy-dependent manner to yield an immunoprecipitable protein of smaller size that comigrated with the corresponding purified enzyme. The size shift occurring with citrate synthase was consistent with the removal of the postulated 27-amino acid leader peptide. To determine the amino terminus of mature CPT II, [35S]methionine- or [3H]leucine-labeled material (after import and processing) was subjected to Edman degradation, followed by counting of the radioactivity released on each cycle. The results established that the precursor targeting peptide was cleaved between leucine 25 and serine 26 in the previously deduced amino acid sequence. Taken in conjunction with the recent report of Finocchiaro et al. (Finocchiaro, G., Taroni, F., Rocchi, M., Martin, A. L., Colombo, I., Tarelli, G. T., and DiDonato, S. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 661-665), the present results establish three key points concerning the rat and human forms of CPT II. First, it appears that in both species the initial translation product contains 658 amino acids and, upon mitochondrial import, is reduced in length by 25 residues through cleavage at an identical site. Second, the difference in electrophoretic mobility between the two mature proteins (documented earlier) presumably reflects either anomalous behavior of one of them on polyacrylamide gels or differential covalent modification. Finally, the recent suggestion by Brady et al. (Brady, P. S., Liu, J. S., Park, E. A., Hanson, R. W., and Brady, L. J. (1991) FASEB J. 5, A817) that our CPT II cDNA construct is incomplete in the 5'-coding region is refuted.

Amino Acid Sequence↗
PubMed1991

New insights into the mitochondrial carnitine palmitoyltransferase enzyme system.

Dissection of the mitochondrial carnitine palmitoyltransferase (CPT) enzyme system in terms of its structure/function relationships has proved to be a formidable task. Although no one formulation has gained universal agreement we believe that the weight of evidence supports a model with the following features: a) in any given tissue CPT I and CPT II are distinct proteins; b) CPT I, unlike CPT II, is detergent labile; c) within a species CPT II is expressed body wide, whereas CPT I exists as tissue specific isoforms; d) malonyl-CoA and other CPT I inhibitors probably interact at the catalytic center of the enzyme, not with a regulatory subunit. The amino acid sequences of rat and human CPT II (deduced from cDNA clones) show them to be similar proteins (greater than 80% identity) but encoded by mRNAs of significantly different sizes. Efforts to clone and sequence the cDNA for rat liver CPT I are presently underway.

Amino Acid Sequence↗
PubMed1990

[A new technique of pulmonary arterial angioscopy: capnoangioscopy. An experimental study].

From animal experiments (n = 10), the authors describe a new technique of pulmonary arterial angioscopy. A balloon-fitted catheter is introduced into the trunk of one of the pulmonary arteries. A small caliber angioscope with an end capable of being propped up is then pushed within the lumen of the catheter. Intraluminal visualization of the pulmonary artery and its branches cannot begin until after the balloon is inflated to arrest blood flow and the visualization chamber is cleared by sterile carbon dioxide. The quality of the image obtained is similar to that of bronchial endoscopy, making is possible to examine at length the walls of the proximal and distal pulmonary arteries up to a caliber of 2.5 mm, and to detect blood clots. Pulmonary angioscopy is tolerated to the same extent as right heart catheterization. Potential applications of this technique are diagnosis of chronic cor pulmonale and treatment of pulmonary embolism.

Animals↗
PubMed1990

Inter-tissue and inter-species characteristics of the mitochondrial carnitine palmitoyltransferase enzyme system.

Properties of the carnitine palmitoyltransferase (EC 2.3.1.21) (CPT) enzyme system were compared in isolated mitochondria from a range of tissues in rodents, monkey, and man. Common features were as follows: (a) while membrane-bound, CPT I, but not CPT II, was inhibited reversibly by malonyl-coenzyme A (CoA) and irreversibly by CoA esters of certain oxirane carboxylic acids; (b) the detergent, Tween-20, readily solubilized CPT II in active form while leaving CPT I membrane associated and catalytically functional; (c) octyl glucoside and Triton X-100 released active CPT II but caused essentially complete loss of CPT I activity. Use of [3H]tetradecylglycidyl-CoA, a covalent ligand for CPT I, yielded estimates of the enzyme's monomeric molecular size: approximately 86 kDa in non-hepatic tissues and approximately 90-94 kDa in liver, depending upon species. A polyclonal antibody to purified rat liver CPT II recognized a single protein in each tissue; its apparent molecular mass was approximately 70 kDa in all rat tissues and approximately 68 kDa in all mouse tissues as well as monkey and human liver. On Northern blot analysis a rat liver CPT II cDNA probe detected a single approximately 2.5-kilobase mRNA in all rat and mouse tissues examined. The following points are emphasized. First, CPT I and II are different proteins. Second, within a species CPT II, but not CPT I, is probably conserved across tissue lines. Third, slight variations in size of both enzymes were found in different species, although, at least in the case of CPT II, significant amino acid identity exists among the various isoforms. Fourth, CPT I, unlike CPT II, requires membrane integrity for catalytic function. Finally, the strategic use of detergents provides a simple means of discriminating between the two enzyme activities.

Acyltransferases↗
PubMed1990

Cloning, sequencing, and expression of a cDNA encoding rat liver mitochondrial carnitine palmitoyltransferase II.

We report the isolation and characterization of a full-length cDNA encoding rat liver carnitine palmitoyltransferase II (CPT II). Beginning with the purified protein CNBr fragments were generated and sequenced. Corresponding oligonucleotides were used to screen a rat liver cDNA library constructed in the plasmid cloning vector, pcDV. The clone ultimately obtained consisted of a 62 nucleotide 5'-untranslated region, a single open reading frame of 1,974 bases predicting a protein of 658 amino acids (Mr = 74,119), and a 3'-untranslated segment of 260 nucleotides followed by the poly (A) tail. The identity of the cDNA was confirmed by the findings that (a) the open reading frame encoded all three peptides found in the original protein; (b) a fourth peptide synthesized from a portion of the deduced amino acid sequence and used to immunize a rabbit resulted in the generation of an antibody that recognized pure CPT II on a Western blot; (c) in vitro transcription and translation of the cDNA (ligated into pBlue-script KS (+] generated a protein that was specifically immunoprecipitated by anti-CPT II antibody and having a Mr slightly greater than that of mature CPT II; (d) transfection of COS cells with the cDNA subcloned into the expression vector, pCMV4, resulted in a 6-fold induction of mitochondrial CPT II catalytic activity. It seems likely that the de novo synthesized enzyme gains entry into the mitochondrion via a targeting peptide that is subsequently cleaved. The mature protein probably associates (relatively loosely) with the inner membrane through a limited number of membrane spanning domains. The predicted amino acid sequence of CPT II shows strong identity with those of two other acyltransferases, namely, rat liver peroxisomal carnitine octanoyltransferase and porcine choline acetyltransferase.

Acyltransferases↗
PubMed1989

Exercise intolerance, lactic acidosis, and abnormal cardiopulmonary regulation in exercise associated with adult skeletal muscle cytochrome c oxidase deficiency.

A 27-yr-old woman with lifelong severe exercise intolerance manifested by muscle fatigue, lactic acidosis, and prominent symptoms of dyspnea and tachycardia induced by trivial exercise was found to have a skeletal muscle respiratory chain defect characterized by low levels of reducible cytochromes a + a3 and b in muscle mitochondria and marked deficiency of cytochrome c oxidase (complex IV) as assessed biochemically and immunologically. Investigation of the pathophysiology of the exercise response in the patient revealed low maximal oxygen uptake (1/3 that of normal sedentary women) in cycle exercise and impaired muscle oxygen extraction as indicated by profoundly low maximal systemic arteriovenous oxygen difference (5.8 ml/dl; controls = 15.4 +/- 1.4, mean +/- SD). The increases in cardiac output and ventilation during exercise, normally closely coupled to muscle metabolic rate, were markedly exaggerated (more than two- to threefold normal) relative to oxygen uptake and carbon dioxide production accounting for prominent tachycardia and dyspnea at low workloads. Symptoms in our patient are similar to those reported in other human skeletal muscle respiratory chain defects involving complexes I and III, and the exaggerated circulatory response resembles that seen during experimental inhibition of the mitochondrial respiratory chain. These results suggest that impaired oxidative phosphorylation in working muscle disrupts the normal regulation of cardiac output and ventilation relative to muscle metabolic rate in exercise.

Acidosis, Lactic↗
PubMed1988

Regulation of lipoprotein lipase in different rat tissues.

In rats maintained on a fat-free diet, hepatic production of very low density lipoprotein (VLDL) fell by only 18% after 20 hours of fasting (as judged by rates of triglyceride accumulation in plasma following administration of Triton WR1339). Infusion of heparin into fed rats caused a rapid accumulation in plasma of two distinct lipase activities in approximately equal amounts: heparin releasable lipoprotein lipase (HR-LPL) and heparin releasable triglyceride lipase (HR-TGL). Fasting reduced the former by 67%, but the latter by only 20 to 25%. To gain insight into the effects of nutritional status on the interorgan distribution of these enzymes, experiments were designed for the perfusion of liver and heart by standard procedures, and of epididymis, gastrocnemius muscle (red fiber), and psoas muscle (white fiber) by micro techniques. After addition of heparin, perfusates were analyzed for each species of lipase. The objective was to selectively monitor the capillary-bound, physiologically functional lipase without significant contamination by intracellular enzyme. HR-TGL was found only in liver, where it represented the major lipase activity; it was only minimally affected by fasting. In epididymis HR-LPL fell by 85% in fasted animals and, remarkably, rebounded almost to ad libitum fed values after only two hours of refeeding. In both heart and gastrocnemius muscle, directional changes in HR-LPL were exactly reciprocal to those seen in epididymis. By contrast, HR-LPL of psoas muscle was unresponsive to fasting. Qualitatively, the data were in keeping with documented nutritionally-induced shifts in tissue disposal of VLDL-triglycerides.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Characterization of the mitochondrial carnitine palmitoyltransferase enzyme system. I. Use of inhibitors.

The effects of various inhibitors of carnitine palmitoyltransferase I were examined in mitochondria from rat liver and skeletal muscle. Three types of inhibitors were used: malonyl-CoA (reversible), tetradecylglycidyl-CoA and three of its analogues (irreversible), and 2-bromopalmitoyl-CoA (essentially irreversible when added with carnitine). Competitive binding studies between labeled and unlabeled ligands together with electrophoretic analysis of sodium dodecyl sulfate-solubilized membranes revealed that in mitochondria from both tissues all of the inhibitors interacted with a single protein. While the binding capacity for inhibitors was similar in liver and muscle (6-8 pmol/mg of mitochondrial protein) the proteins involved were of different monomeric size (Mr 94,000 and 86,000, respectively). Treatment of mitochondria with the detergent, octyl glucoside, yielded a soluble form of carnitine palmitoyltransferase and residual membranes that were devoid of enzyme activity. The solubilized enzyme displayed the same activity regardless of whether carnitine palmitoyltransferase I of the original mitochondria had first been exposed to an irreversible inhibitor or destroyed by chymotrypsin. It eluted as a single activity peak through four purification steps. The final product from both liver and muscle migrated as single band on sodium dodecyl sulfate-polyacrylamide electrophoresis with Mr of approximately 80,000. The data are consistent with the following model. The inhibitor binding protein is carnitine palmitoyltransferase I itself (as opposed to a regulatory subunit). The hepatic monomer is larger than the muscle enzyme. Each inhibitor interacts via its thioester group at the palmitoyl-CoA binding site of the enzyme but also at a second locus that is probably different for each agent and dictated by the chemical substituent on carbon 2. Disruption of the mitochondrial inner membrane by octyl glucoside causes inactivation of carnitine palmitoyltransferase I while releasing carnitine palmitoyltransferase II in active form. The latter is readily purified, is a smaller protein than carnitine palmitoyltransferase I, and has the same molecular weight in liver and muscle. It is insensitive to inhibitors where on or off the mitochondrial membrane.

Acyl Coenzyme A↗
PubMed1987

Characterization of the mitochondrial carnitine palmitoyltransferase enzyme system. II. Use of detergents and antibodies.

Exposure of rat liver mitochondrial membranes to octyl glucoside, Triton X-100, or Tween 20 solubilized an active and tetradecylglycidyl-CoA (TG-CoA)-insensitive carnitine palmitoyltransferase (presumed to be carnitine palmitoyltransferase II). The residual membranes after octyl glucoside or Triton X-100 treatment were devoid of all transferase activity. By contrast, Tween 20-extracted membranes were still rich in transferase; this was completely blocked by TG-CoA and thus was presumed to be carnitine palmitoyltransferase I. The residual carnitine palmitoyltransferase activity disappeared from the membranes upon subsequent addition of octyl glucoside or Triton X-100 and could not be recovered in the supernatant fraction. Antibody raised against purified rat liver transferase II (Mr 80,000) recognized only this protein in immunoblots from untreated liver mitochondrial membranes containing both transferases I and II. Tween 20-extracted membranes, which contained only transferase I, did not react with the antibody. Purified transferase II from skeletal muscle (also of Mr 80,000) was readily recognized by the antiserum, suggesting antigenic similarity with the liver enzyme. These and other studies on the effects of detergents on the mitochondrial [3H]TG-CoA binding protein provide further support for the model of carnitine palmitoyltransferase proposed in the preceding paper. They suggest that: 1) carnitine palmitoyltransferases I and II in rat liver are immunologically distinct proteins; 2) transferase I is more firmly anchored into its membrane environment than transferase II; 3) association of carnitine palmitoyltransferase I with a membrane component(s) is necessary for catalytic activity. While carnitine palmitoyltransferase I is a different protein in liver and muscle, it seems likely that both tissues share the same transferase II.

Acyltransferases↗
PubMed1986

The glucose-phosphorylating capacity of liver as measured by three independent assays. Implications for the mechanism of hepatic glycogen synthesis.

In the fasted to fed transition liver glycogen derives mainly from gluconeogenic precursors. Why glucose is not used efficiently as a direct precursor of glycogen has become a controversial issue, in part because of disagreement over the question of how well liver can phosphorylate glucose under conditions prevailing postprandially. To try to resolve the matter the relative merits of two recently described assays, one spectrophotometric (A), the other isotopic (B), for monitoring rates of glucose phosphorylation in the high speed supernatant fraction of liver have been rigorously evaluated. A third method, also isotopic (C), was developed for use with unfractionated as well as fractionated liver homogenates. Using fasted rats and mice from different nutritional backgrounds the glucose-phosphorylating capacity of liver extracts was measured and compared with rates of hepatic glycogen synthesis observed during refeeding. Two of the assays (A and C) provided reliable data at all concentrations of glucose tested (5-100 mM), while method B exhibited shortcomings at lower substrate concentrations. The results clearly establish that in both rats and mice the ability of the liver to phosphorylate glucose at physiological concentrations is sufficient to support only 25-30% of postprandial glycogen synthesis. A limited capacity for glucose phosphorylation probably accounts for the fact that two-thirds of glycogen synthesized with refeeding after a fast is formed by the indirect (gluconeogenic) pathway.

Animals↗
PubMed1986

Active hepatic glycogen synthesis from gluconeogenic precursors despite high tissue levels of fructose 2,6-bisphosphate.

When fasted rats ate regular lab chow there was a lag time of about 2 h before the concentration of fructose 2,6-bisphosphate (Fru-2,6-P2) in liver began to rise from its low basal level. By contrast, in animals refed on a sucrose-based diet hepatic [Fru-2,6-P2] increased 20-fold (to a value of approximately 12 nmol/g wet weight) during the first hour. These responses correlated with differences in the ability of the two diets to increase the circulating [insulin]/[glucagon] ratio and thus to elevate the ratio of 6-phosphofructo-2-kinase to fructose-2, 6-bisphosphatase. Liver glycogen was deposited briskly in both groups of rats. To assess its mechanism of synthesis (directly from glucose versus indirectly via the gluconeogenic pathway), animals eating the chow or sucrose diets received intravenous infusions of [14C]bicarbonate, [1-14C] fructose, and 3H2O. After isolation, the glycogen was subjected to positional isotopic analysis of its glucose residues. The results established that regardless of the diet the bulk of liver glycogen was gluconeogenic in origin. The fact that with sucrose feeding carbon flow through hepatic fructose-1,6-bisphosphatase remained active despite high levels of Fru-2,6-P2 (a potent inhibitor of this enzyme in vitro) presents a metabolic paradox. Conceivably, the suppressive effect of Fru-2, 6-P2 on hepatic fructose-1,6-bisphosphatase is overridden in vivo by some unknown factor or factors generated in response to sucrose feeding. Alternatively, metabolic zonation in liver might result in the coexistence of hepatocytes rich in Fru-2,6-P2 (high glycolytic, low gluconeogenic, low glycogenic capacitites) with cells depleted of Fru-2,6-P2 (low glycolytic, high gluconeogenic, high glycogenic capacities).

Animals↗
PubMed1986

[To visualize the posterolateral segment of the external meniscus. Value of the position of internal flexion rotation].

Interpretation of images of the posterolateral segment of external meniscus, during arthrography with contrasts of knee, is known to be difficult and this explains the possibility of false positives and the frequency of false negatives observed during exploration on this meniscus. A flexion-internal rotation projection of knee was used to improve the image of the posterior horn of the external meniscus, and analysis of results of about 100 examinations showed significant benefits. Use of the method is illustrated in a normal knee, and one with a rupture of posterior horn of external meniscus masked in extension and detected only in the flexion-internal rotation projection.

Humans↗