Contaminating DNA in RNA amplification by polymerase chain reaction.
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Base Sequence↗
Biomedical subjects
M Morell
Publications and source records attributed to M Morell.
Renin-angiotensin system in thyroidectomized rats at different periods of development.
The relationship between the renal function and some components of the renin-angiotensin system has been studied in hypothyroid rats thyroidectomized surgically at different periods of their life. Changes in plasma renin concentration (PRC) depending on the period hypothyroidism were induced. Results showed that the renin release control could result from an equilibrium between the reduced beta-adrenergic activity and the marked natriuresis observed in hypothyroidism. A reduction in plasma angiotensinogen concentration (PAC), due to a decrease in its hepatic production, was observed in thyroidectomized animals. PAC reduction was independent of the hypothyroidism induction period. Alterations in plasma renin activity (PRA) were a consequence of PRC and PAC changes in thyroidectomized animals, as an increase in fractional sodium excretion (FENa) time course dependent, was found in these rats.
Aging↗
[L-thyroxine and L-triiodothyronine in the cerebral tissues of the adult rat. Effect of hypothyroidism].
The L-thyroxine and L-triiodothyronine concentrations in several brain areas (cerebral cortex, brain stem, hypothalamus, total brain and hypophysis) in normal and hypothyroid rats have been studied. Results show that L-thyroxine values at tissue level are inferior in the hypothyroid group, although non-significant with respect to the control group, whereas L-triiodothyronine presents values similar to the hypothyroid group and its control in all the brain regions studied with the exception of hypophysis. These results show that in hypothyroid situations exist a compensatory mechanism for maintaining the adequate L-triiodothyronine levels in several brain areas, although the serum levels are strongly decreased in hypothyroid animals.
Animals↗
Molecular Characterization of the Brittle-2 Gene Effect on Maize Endosperm ADPglucose Pyrophosphorylase Subunits.
Activity of the enzyme ADPglucose pyrophosphorylase is known to be reduced in maize (Zea mays L.) endosperm mutants at two independent loci, Shrunken-2 (Sh(2)) and Brittle-2 (Bt(2)). Spinach leaf ADPglucose pyrophosphorylase has previously been shown to comprise two subunits of 51 and 54 kilodaltons. Anti-bodies raised to each of the two subunits of spinach leaf ADPglucose pyrophosphorylase were found to cross-react to different bands on Western blots prepared from polyacrylamide gel electrophoresis separated wild-type maize endosperm proteins. The anti-spinach leaf 51 kilodalton subunit antibody cross-reacted with a 55 kilodalton maize endosperm protein and the anti-spinach leaf 54 kilodalton subunit antibody cross-reacted with a 60 kilodalton maize endosperm protein. These immunological reactions were observed in maize endosperm extracts and with a highly purified preparation of maize endosperm ADPglucose pyrophosphorylase. Mutant bt(2) endosperm lacked the 55 kilodalton subunit while mutant sh(2) endosperm lacked the 60 kilodalton subunit on Western blots. These results suggest that the maize endosperm ADPglucose pyrophosphorylase is made up of two immunologically dissimilar subunits and that the bt(2) and sh(2) mutations cause reduction in ADPglucose pyrophosphorylase activity through the lack of one of these two subunits. An ADPglucose pyrophosphorylase cDNA clone antigenically selected from a rice seed cDNA expression library was found to hybridize strongly with a cDNA corresponding to a maize endosperm transcript which is absent in a W64A bt(2) mutant. Thus, the bt(2) mutant causes the absence not only of the small subunit but of the corresponding transcript. Bt(2) is implicated as the structural gene for the small (54 kilodalton) subunit of maize endosperm ADPglucose pyrophosphorylase.
Journal Article↗
The Subunit Structure of Potato Tuber ADPglucose Pyrophosphorylase.
ADPglucose pyrophosphorylase has been extensively purified from potato (Solanum tuberosum L.) tuber tissue to study its structure. By employing a modified published procedure (JR Sowokinos, J Preiss [1982] Plant Physiol 69: 1459-1466) together with Mono Q chromatography, a near homogeneous enzyme preparation was obtained with substantial improvement in enzyme yield and specific activity. In single dimensional sodium dodecyl sulfate polyacrylamide gels, the enzyme migrated as a single polypeptide band with a mobility of about 50,000 daltons. Analysis by two-dimensional polyacrylamide gel electrophoresis, however, revealed the presence of two types of subunits which could be distinguished by their slight differences in net charge and molecular weight. The smaller potato tuber subunit was recognized by antiserum prepared against the smaller spinach leaf 51 kilodalton ADPglucose pyrophosphorylase subunit. In contrast, the anti-54 kilodalton raised against the spinach leaf subunit did not significantly react to the tuber enzyme subunits. The results are consistent with the hypothesis that the potato tuber ADPglucose pyrophosphorylase is not composed of a simple homotetramer as previously suggested, but is a product of two separate and distinct subunits as observed for the spinach leaf and maize enzymes.
Journal Article↗
Serum activity of the key gluconeogenic enzymes in carbon-tetrachloride-induced experimental hepatotoxicity.
Serum activity has been measured in three of the key enzymes in the gluconeogenic pathway in rats subjected to experimental hepatotoxicity after intraperitoneal administration of carbon tetrachloride. The levels of phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-biphosphatase (FBPase) showed a similar behavior to the transaminase (AST and ALT), increasing markedly with respect to the controls at 12 h after administration of the poison, reaching their maximum peak of activity at between 24 and 36 h, and returning to normal values at 96 h. The activity of glucose-6-phosphatase was not significantly modified throughout the treatment. These results seem to demonstrate that the determination of the serum activity of PEPCK and FBPase could be a sensitive and specific marker of hepatic cytolysis.
Alanine Transaminase↗
Immunological characterization of L-triiodothyronine and L-thyroxine labelled with rhodamine B isothiocyanate.
In the present work the label of L-T3 and L-T4 with RBITC have been developed by means of a relatively easy procedure. A purification by TLC, previously optimized, offers the possibility of obtaining a good recovery of conjugates T3 and T4-RBITC. The immunoreactivity of conjugates was calculated by a formulas especially designed for this purpose. This formulas could be applied to any molecule labelled with fluorescent compounds.
Chromatography, Thin Layer↗
The encoded primary sequence of a rice seed ADP-glucose pyrophosphorylase subunit and its homology to the bacterial enzyme.
Rice seed ADP-glucose pyrophosphorylase cDNA clones were isolated by screening a lambda expression library prepared from rice endosperm poly(A+) RNA with a heterologous antibody raised against the spinach leaf enzyme and subsequently by nucleic acid hybridization. One cDNA plasmid, possessing about 1650 nucleotides, was shown by both DNA and RNA sequence analysis to contain the complete ADP-glucose pyrophosphorylase coding sequence of 483 amino acids. The primary sequence displayed a putative leader peptide presumably required for transport of this nuclear encoded protein into the amyloplasts, a differentiated starch containing plastid. The leader peptide, however, showed little sequence homology with transit peptides displayed by other known nuclear encoded proteins localized in the chloroplasts. A comparison of the primary sequence of the putative mature subunit to the Escherichia coli pyrophosphorylase showed two regions displaying significant homology. These two conserved regions contain residues shown previously to be essential for the allosteric regulation and catalytic activity of the E. coli enzyme. Differences in the primary sequences of the plant and bacterial enzyme may reflect the distinct nature of the allosteric effectors that control these enzymes.
Amino Acid Sequence↗
Influence of thyroidectomy on serum and pituitary FSH in control and orchidectomized rats.
The influence of hypothyroidism on both pituitary and serum FSH has been studied in male rats thyroidectomized in early stages of development. To know whether FSH alterations are produced by changes of the gonadal feed-back system or by a direct effect of thyroid hormones upon pituitary synthesis, estradiol, testosterone and testicular weight were measured. The result showed a decrease of biological and immunological activity of pituitary FSH in thyroidectomized rats. Serum FSH levels were also low. The deficiency of FSH production cannot be attributed to an alteration of the gonadal feed-back systems, since (1) in thyroidectomized rats, estradiol and testosterone were lower than in the controls, (2) signs of tubular atrophy were found in the testes and hence it could be expected that inhibin is low, and (3) in the absence of gonadal factors (induced by orchidectomy) thyroidectomized rats also showed a decrease of pituitary FSH. The present data suggest that the decrease of FSH can be attributed to a direct effect of thyroid hormones on pituitary FSH production.
Animals↗
Immunocytochemical Localization of ADPglucose Pyrophosphorylase in Developing Potato Tuber Cells.
The subcellular localization of ADPglucose pyrophosphorylase, a key regulatory enzyme in starch biosynthesis, was determined in developing potato tuber cells by immunocytochemical localization techniques at the light microscopy level. Specific labeling of ADPglucose pyrophosphorylase by either immunofluorescence or immunogold followed by silver enhancement was detected only in the amyloplasts and indicates that this enzyme is located exclusively in the amyloplasts in developing potato tuber cells. Labeling occurred on the starch grains and, in some instances, specific labeling patterns were evident which may be related to sites active in starch deposition.
Journal Article↗
Effect of low-iodine diet and propylthiouracil on glycolytic enzymes in brain areas of adult rats.
Glycolytic activity of five brain areas in the rat was studied under two hypothyroid states: (1) induced by low-iodine diet from weaning, and (2) induced by propylthiouracil. The areas studied were the anterior cortex, amygdala, hypothalamus, septum and hippocampus. A low-iodine diet induced a decrease of pyruvate kinase activity in three region and of phosphofructokinase in the hippocampus, while hexokinase increased in both the amygdala and septum. Propylthiouracil treatment produced an increase in hexokinase activity in the hypothalamus and septum, and a decrease in the anterior cortex, while phosphofructokinase decreased significantly in the hippocampus. No significant changes of lactate dehydrogenase activity were observed. The correlation between the results and type of hypothyroidism is discussed.
Animals↗
Mental stress and the induction of silent myocardial ischemia in patients with coronary artery disease.
To assess the causal relation between acute mental stress and myocardial ischemia, we evaluated cardiac function in selected patients during a series of mental tasks (arithmetic, the Stroop color--word task, simulated public speaking, and reading) and compared the responses with those induced by exercise. Thirty-nine patients with coronary artery disease and 12 controls were studied by radionuclide ventriculography. Of the patients with coronary artery disease, 23 (59 percent) had wall-motion abnormalities during periods of mental stress and 14 (36 percent) had a fall in ejection fraction of more than 5 percentage points. Ischemia induced by mental stress was symptomatically "silent" in 19 of the 23 patients with wall-motion abnormalities (83 percent) and occurred at lower heart rates than exercise-induced ischemia (P less than 0.05). In contrast, we observed comparable elevations in arterial pressure during ischemia induced by mental stress and ischemia induced by exercise. A personally relevant, emotionally arousing speaking task induced more frequent and greater regional wall-motion abnormalities than did less specific cognitive tasks causing mental stress (P less than 0.05). The magnitude of cardiac dysfunction induced by the speaking task was similar to that induced by exercise. Personally relevant mental stress may be an important precipitant of myocardial ischemia--often silent--in patients with coronary artery disease. Further examination of the pathophysiologic mechanisms responsible for myocardial ischemia induced by mental stress could have important implications for the treatment of transient myocardial ischemia.
Adult↗
Affinity labeling of the allosteric activator site(s) of spinach leaf ADP-glucose pyrophosphorylase.
Pyridoxal-P has been shown to be an activator of the spinach leaf ADP-glucose pyrophosphorylase. It has a higher apparent affinity than the physiological activator 3-phosphoglycerate but only activates the enzyme activity 6-fold whereas 3-phosphoglycerate gives a 25-fold activation. Reductive phosphopyridoxylation of the spinach leaf enzyme results in enzyme having less dependence on the presence of activator for activity. Labeled pyridoxal-P is incorporated into both the 54- and 51-kilodalton subunits of the spinach leaf enzyme. The incorporation is inhibited by the presence of either 3-phosphoglycerate or the allosteric inhibitor, inorganic phosphate, thus suggesting that pyridoxal phosphate is covalently bound to the allosteric activator site. The pyridoxal phosphate is bound to an epsilon-amino group of a lysine residue. The phosphopyridoxylated enzyme is more resistant to phosphate inhibition than the unmodified form. The modified 51-kDa subunit has been digested with trypsin, and the peptide containing the labeled pyridoxal phosphate has been purified via high performance liquid chromatography and sequenced. Comparison of this sequence with the deduced amino acid sequence of a rice endosperm cDNA clone indicates that the putative allosteric site of the 51-kDa subunit is close to the carboxyl-terminal. This is in contrast to what had been demonstrated for the position of the activator site of the Escherichia coli ADP-glucose pyrophosphorylase which was shown to be close to the amino-terminal of the subunit.
Affinity Labels↗
Postnatal development of the renin-angiotensin system in thyroidectomized rats.
Postnatal changes in plasma renin activity (PRA), plasma renin concentration (PRC), plasma renin substrate (PRS) and plasma angiotensin II concentration (AII) were studied in young thyroidectomized rats from the 4th to the 10th week of life. Although there were no differences in the PRA pattern between thyroidectomized and euthyroid animals, an increase in PRC from the 6th week of life, together with a reduction of the glomerular filtration rate (GFR) and an increase in the fractional excretion of sodium (FENa) from the 8th week of life, was observed in young hypothyroid animals. Moreover, in thyroidectomized animals, PRS and AII declined until the 10th week of age, while in euthyroid animals an increase of PRS and AII was observed between the 8th and 10th weeks of life. These results show that the changes in sodium renal handling following thyroidectomy could have an influence on the RAS components.
Aging↗
Regulation of starch synthesis: enzymological and genetic studies.
Significant advances have been made with respect to elucidating the structure, the allosteric site, interactions of effectors, covalent modifications, and the amino acid sequence of the ADPG synthetase. It is hoped that in the near future, sufficient information will be obtained to enable facile manipulation of the plant tissue ADPG synthetase gene and its product.
Allosteric Site↗
Angiotensin converting enzyme in hyper- and hypothyroid rats.
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Animals↗
Effect of various components of the renin-angiotensin-aldosterone system on angiotensinogen production in a monolayer culture of adult rat hepatocytes.
A monolayer culture of adult rat hepatocytes was used to study the effect of various components of the renin-angiotensin-aldosterone system (angiotensin II, aldosterone and angiotensinogen), and intracellular sodium depletion (by ouabaine addition) on the angiotensinogen production rate. Normal hepatocytes synthesized linearly angiotensinogen for 6 h at a mean rate of 110 pg angiotensin I generated/mg intracellular protein each h. The addition of hydrocortisone (0.1 microM) to the culture medium significantly enhanced the angiotensinogen production after 2 h of incubation (P less than 0.05), being about 2-fold higher than normal control values at the 6th h of incubation. In contrast to this increase, the addition of angiotensin II (70 nM) to the medium produced a higher stimulatory effect on angiotensinogen synthesis, being the differences with the control significants after 1 h of incubation (P less than 0.01). At the 6th h of incubation, angiotensin II enhanced the angiotensinogen production over 2 fold in relation to the control group. No change in the angiotensinogen production rate was observed in monolayer culture of hepatocytes in the presence of aldosterone (1 microM), angiotensinogen (0.1 microM) or ouabaine (10 microM). These results provide further evidence that angiotensinogen synthesis is subject to a positive feed-back mechanism by angiotensin II, indicating that this mechanism takes place at physiological levels of plasma AII.
Aldosterone↗
Activities of glycolytic enzymes in some brain areas of thyroidectomized rats and their response to replacement therapy.
Glycolytic metabolism has been assessed by studying a set of key enzymes, in anterior cortex, amygdala, hypothalamus septum and hippocampus, in thyroidectomized rats. The reversibility of the changes induced by the thyroidectomy has been assessed by replacement therapy. In thyroidectomized rats the hexokinase activity was significantly decreased in anterior cortex and hypothalamus. The increase in phosphofructokinase and pyruvate kinase activity was probably due to an increase in cellular energy requirements. Hexokinase activity was best restored by treatment with L-thyroxine (T4) or T4+ propylthiouracil (PTU). The low response of pyruvate kinase activity in all treated animals could suggest that this metabolic step is the least reversible.
Animals↗