[Therapeutic trials in general practice].
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Clinical Trials as Topic↗
Biomedical subjects
M Nguyen
Publications and source records attributed to M Nguyen.
Protein sorting between mitochondrial membranes specified by position of the stop-transfer domain.
Recently, we fused a matrix-targeting signal to a large fragment of vesicular stomatitis virus G protein, which contains near its COOH-terminus a well-characterized endoplasmic reticulum (ER) stop-transfer sequence; the hybrid G protein was sorted to the inner mitochondrial membrane (Nguyen, M., and G. C. Shore. 1987. J. Biol. Chem. 262:3929-3931). Here, we show that the 19 amino acid G stop-transfer domain functions in an identical fashion when inserted toward the COOH-terminus of an otherwise normal matrix precursor protein, pre-ornithine carbamyl transferase; after import, the mutant protein was found anchored in the inner membrane via the stop-transfer sequence, with its NH2 terminus facing the matrix and its short COOH-terminal tail located in the intermembrane space. However, when the G stop-transfer sequence was placed near the NH2 terminus, the protein was inserted into the outer membrane, in the reverse orientation (NH2 terminus facing out, with a large COOH-terminal fragment located in the intermembrane space). These observations for mitochondrial topogenesis can be explained by a simple extension of existing models for ER sorting.
Amino Acid Sequence↗
Evaluation of a functional index and an articular index in ankylosing spondylitis.
We describe an index of functional impairment and a system of scoring joint tenderness for use in the assessment of ankylosing spondylitis. The functional index consists of 20 questions and the articular index is based on the scoring of a total of 10 joint responses after movement or firm digital pressure. These indices are simple to establish and not time consuming. They have a high degree of intra- and interobserver reproducibility. The indices showed changes in short term clinical trials of antiinflammatory drugs; these changes were highly correlated with the patient's overall assessment of his own clinical condition.
Activities of Daily Living↗
Import of hybrid vesicular stomatitis G protein to the mitochondrial inner membrane.
cDNA fusions were employed to construct a 35-kDa hybrid protein bearing the amino-terminal signal sequence of pre-ornithine carbamoyltransferase (pOCT), a mitochondrial matrix enzyme, fused to the carboxyl-terminal half of vesicular stomatitis virus G protein (VSV G). Following transcription-translation in vitro, the hybrid protein was imported by purified mitochondria and processed at its amino terminus by the matrix processing enzyme. The protein, however, remained anchored in the mitochondrial inner membrane, apparently in a transmembrane configuration, via the hydrophobic VSV G stop-transfer domain; a small portion (approximately 2 kDa) of the G protein fragment was accessible to protease digestion only after selective permeabilization of the mitochondrial outer membrane with digitonin, a finding consistent with localization of the extreme carboxyl-terminal cytoplasmic tail of G in the intermembrane space. The results demonstrate that the membrane-anchor domain of VSV G can function in a post-translational manner and can operate in membranes other than those derived from the endoplasmic reticulum. However, it appears to be selectively recognized as a stop-transfer signal by the translocation machinery of the mitochondrial inner, rather than outer, membrane.
Animals↗
Variations in lipoproteins during aminoglutethimide therapy.
Plasma lipids, apolipoproteins, and gamma-glutamyl-transpeptidase (GGT) were measured in 28 patients receiving aminoglutethimide (500 mg) and hydrocortisone (30 or 40 mg) for advanced breast cancer. A rise in cholesterol (CHOL), LDL-CHOL, apoprotein B, apoprotein CIII, and GGT was observed after 45 days. When the patients were divided in two groups according to lipid basal plasma levels, those with high CHOL and triglyceride did not experience any modification of lipid parameters (only GGT were elevated). Conversely normolipidaemic patients experienced an increase in CHOL, triglycerides, LDL-CHOL, apoproteins B and CIII, and GGT.
Aminoglutethimide↗
A signal sequence domain essential for processing, but not import, of mitochondrial pre-ornithine carbamyl transferase.
Studies using deletion mutagenesis indicate that a processing recognition site lies proximal to the normal cleavage position between gln32 and ser33 of pre-ornithine carbamyl transferase (pOCT). pOCT cDNA was manipulated to delete codons specifying amino acids 22-30 of the signal sequence. The mutant precursor, designated pOCT delta 22-30, was imported to the matrix compartment by purified mitochondria, but remained largely unprocessed; the low level of processing that was observed did not involve the normal cleavage site. Several manipulations performed downstream of the normal pOCT processing site (deletion, substitution, and hybrid protein constructions) affected neither import nor correct processing. Our data suggest that domains specifying import and processing site recognition may be functionally segregated within the signal peptide; that processing is not requisite for import of pOCT; and that a proximal region, not involving the normal signal peptide cleavage site, is required for processing site recognition.
Amino Acid Sequence↗
Lung as a possible additional target organ for vitamin D during fetal life in the rat.
The presence of specific cytosol binding sites for 1,25-(OH)2D3 was evaluated in rat fetal tissues during the last quarter of gestation (days 17-21). The content of 1,25-(OH)2D3-binding sites was low in intestine, brain, liver, spleen, pancreas, sternum and thymus during the period of gestation studied. It was highest in skeleton (ribs and vertebral bodies), kidney and lung from day 19 onwards. In the cytosol of these latter tissues, a high affinity (Kd 0.7-3.6 X 10(-10) M, low capacity [3H]1,25-(OH)2D3 binding was demonstrated and a distinct 2.9- to 3.5-S [3H]1,25-(OH)2D3-binding component was observed. These findings suggest that fetal lung, skeleton and kidney are possibly major target tissues for 1,25-(OH)2D3.
Animals↗
[Anti-LAV/HTLV III antibodies in wives of seropositive hemophilic patients].
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Acquired Immunodeficiency Syndrome↗
Import and processing of hybrid proteins by mammalian mitochondria in vitro.
Normal and hybrid proteins were synthesized following transcription of pSP64 recombinant plasmids with SP6 polymerase and translation of resultant mRNAs in a rabbit reticulocyte lysate. The precursor to the rat liver mitochondrial matrix enzyme, ornithine carbamyltransferase (Mr = approximately 40,000), was efficiently imported by rat heart mitochondria in vitro and processed to mature protein. Import of the precursor to a second matrix enzyme, carbamyl-phosphate synthetase I (Mr = approximately 165,000), could not be demonstrated. However, a 33-kDa hybrid protein, bearing the precarbamyl-phosphate synthetase I signal sequence (38 amino acids) and 55 amino acids from the amino terminus of the mature enzyme, followed by the carboxyl-terminal 209 amino acids of ornithine carbamyltransferase, was imported and processed under identical conditions. The topogenic function of the preornithine carbamyltransferase signal sequence (32 amino acids) was confirmed by constructing a hybrid protein bearing the signal sequence and five amino acids of mature enzyme, followed by 250 amino acids of the cytosolic enzyme of Escherichia coli, asparagine synthetase; the hybrid was transported to the matrix compartment of heart mitochondria where processing, albeit incorrect, took place. A hybrid asparagine synthetase bearing the pre-ornithine carbamyltransferase signal sequence plus 28 amino acids of mature ornithine carbamyltransferase, however, was imported and processed with apparent fidelity.
Amino Acid Sequence↗
Expression in Escherichia coli of functional precursor to the rat liver mitochondrial enzyme, ornithine carbamyl transferase. Precursor import and processing in vitro.
cDNA encoding the full-length cytosolic precursor of rat liver ornithine carbamyl transferase, a mitochondrial matrix enzyme, was inserted into pKK223-3 and expressed under the control of the tac promoter. Following transformation of E. coli strain JM105 and induction by isopropylthiogalactoside, a polypeptide was synthesized which reacted with antibody against ornithine carbamyl transferase and co-migrated in SDS-polyacrylamide gels with authentic enzyme precursor (pOCT, Mr = 40,000 kDa); it constituted approximately 0.1% of total E. coli protein. pOCT was synthesized in vitro by coupled transcription-translation of the recombinant plasmid in an E. coli S30 extract; upon subsequent addition of purified rat liver or heart mitochondria, the precursor was imported and processed to mature form. Synthesis of pOCT in a bacterial system, therefore, results in production of a functional precursor polypeptide which does not require additional cytosolic factors from eukaryotic cells to support its uptake and processing by mitochondria in vitro.
Animals↗
Morphine-augmented cholescintigraphy in the diagnosis of acute cholecystitis.
Cholescintigraphy is a sensitive procedure for diagnosing or excluding acute cholecystitis. However, when rapid diagnosis is critical, the requirement for delayed images (4 hr or more after injection) to minimize the false-positive rate diminishes its utility. We prospectively evaluated 40 cholescintigraphic examinations that did not visualize the gallbladder 1 hr after injection of 99mTc diisopropyliminodiacetic acid. These examinations were then augmented by administration of IV morphine, followed by an additional 30 min of imaging. After the morphine, 18 of these examinations demonstrated visualization of the gallbladder; none subsequently required surgical exploration. Of the remaining 22, who demonstrated persistent nonvisualization of the gallbladder post-morphine, 11 were explored surgically and found to be abnormal. The 11 others were treated medically. Low-dose morphine administered when the gallbladder fails to visualize after 1 hr is a useful adjunct to conventional cholescintigraphy because it reduces the time required to obtain a diagnostic result and decreases the number of false-positive results.
Acute Disease↗
Adenylate cyclase activity is absent in inactive and motile sperm in the nematode parasite, Ascaris suum.
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Adenylyl Cyclases↗
Expression of nuclear genes encoding the urea cycle enzymes, carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase, in rat liver and intestinal mucosa.
RNA dot-blot, quantitative electron microscope immunocytochemistry, and electrophoretic immunoblotting techniques were employed to investigate the expression of carbamoyl-phosphate synthetase I (CPS) and ornithine carbamoyl transferase (OCT) genes in rat liver and intestinal mucosa. Comparing only those cell types in the two tissues which express these enzymes, we show that the concentration of CPS and OCT in hepatocyte mitochondria is 2.3-times and 1.2-times greater, respectively, than in intestinal epithelial cell mitochondria. As a percentage of total tissue protein, however, liver homogenates contain 10-20 times more CPS and 5-10 times more OCT than is found in intestinal mucosa. These relatively large differences in enzyme protein levels between the two tissues are not reflected by differences in their mRNA levels. As a percentage of total translational activity in vitro (based on incorporation of [35S]methionine), total liver mRNA directed synthesis of about twice as much precursor CPS (pCPS) and precursor OCT (pOCT) than did equivalent amounts of mRNA from intestinal mucosa. The ratio of pCPS and pOCT mRNA levels between the two tissues (2:1, liver:intestinal mucosa) was confirmed by dot-blot and Northern hybridizations employing specific cDNA probes. The sizes of the respective mRNAs were the same for the two tissues: about 6000 residues for pCPS mRNA and about 1700 residues for pOCT mRNA.
Animals↗
Expression of carbamoyl-phosphate synthetase I mRNA in Reuber hepatoma H-35 cells. Regulation by glucocorticoid and insulin.
Reuber hepatoma H-35 cells actively synthesize the urea cycle enzyme, carbamoyl-phosphate synthetase I. Treatment of H-35 cells with dexamethasone (0.14 microM), however, enhanced synthesis of the enzyme (as measured by incorporation of [35S]methionine) by 4-5-fold. Insulin (0.18 microM) completely inhibited dexamethasone-dependent stimulation of enzyme synthesis. In vitro translation and cDNA hybridization assays were employed to measure effects of dexamethasone plus or minus insulin on levels of mRNA encoding the biosynthetic precursor of carbamoyl-phosphate synthetase I (pCPS) in Reuber H-35 cells. Both measurements yielded similar results: dexamethasone increased pCPS mRNA levels by 4-5-fold and insulin suppressed this response, but only by 50%. Specific cDNA hybridization assays also demonstrated that Reuber H-35 cells, even after hormone treatments, contain only very low levels of albumin mRNA, and no detectable ornithine carbamoyl-transferase mRNA.
Albumins↗
[Aminoglutethimide-induced dyslipemia. Clinical study].
Plasma levels of cholesterol, HDL cholesterol (HDL chol), LDL cholesterol (LDL chol), triglycerides, Al apoprotein and B apoprotein were studied in 73 patients receiving 500 mg aminoglutethimide (AG)/day and 40 mg hydrocortisone/day for advanced breast cancer. These dosages were done before treatment and then repeated during AG therapy. When all patients were considered together, a significant increase of cholesterol, HDL chol, LDL chol and Al apoprotein was noted. If one considers two groups of patients: group A (where cholesterol and triglycerides plasma levels were normal before treatment) and group B (where cholesterol and/or triglycerides plasma levels were high before treatment), it appears that variations differ in both groups. In group A patients we found an increase in cholesterol, chol LDL, B apoprotein and to a lesser extent Al apoprotein plasma levels. In group B patients there was an increase in cholesterol, HDL chol and Al apoprotein plasma levels.
Aminoglutethimide↗
[Aminoglutethimide-induced dyslipidemia. Experimental study].
Aminoglutethimide (AG) is a drug that inhibits steroid synthesis; it is used in advanced breast cancer. After the observation of abnormalities in lipid metabolism in patients, we realized an experimental study to try to look for a pathogenetic hypothesis. Three groups of rats received respectively AG, Hydrocortisone (HC) or both (AG + HC) and were compared to controls. Animals treated with AG (with or without HC) had a greater liver content of triglycerides, cholesterol and phospholipids than controls. Cholesterol plasma level was higher in animals treated with AG + HC. The bile flow was higher in rats receiving AG and AG + HC whereas biliary salt concentrations were lower. These variations could be the consequence of both an enzymatic induction and an inhibition of some cytochrome P450 dependent hydroxylases.
Aminoglutethimide↗
Regulation and expression of carbamyl phosphate synthetase I mRNA in developing rat liver and Morris hepatoma 5123D.
A cDNA clone complementary to mRNA encoding the precursor (Mr = 165,000) to the rat liver mitochondrial matrix enzyme carbamyl phosphate synthetase I (Mr = 160,000) was employed to compare relative amounts of the messenger in adult and fetal liver and in Morris hepatoma 5123D and 3924A cells. Northern blot analysis gave a size estimate for the messenger of 6,500-6,700 nucleotides. Carbamyl phosphate synthetase mRNA levels in 15-day-old fetal liver were less than 10% of adult levels; 5123D cells expressed the messenger at levels about 2-fold higher than normal adult liver, but the messenger was undetectable in 3924A cells. Albumin mRNA was also expressed in the former but not in the latter. Maintaining rats for 5 days on a diet containing 60% casein augmented the relative amount of carbamyl phosphate synthetase mRNA by about 2-fold, while a protein-free diet resulted in reduced levels of the mRNA (about 50% compared to animals on a normal diet). Finally, the pattern of hybridization of carbamyl phosphate synthetase cDNA to HindIII-digested genomic DNA showed no differences between normal liver and its corresponding hepatoma; however, a HindIII site polymorphism was observed between Buffalo and ACI rats.
Animals↗