Showing papers in "Biomedica biochimica acta in 1983"

Mechanism of autocatalytic oxidation of oxyhemoglobin by nitrite.

Abstract: Oxidation of oxyhemoglobin by nitrite is characterized by the presence of a lag phase followed by the autocatalysis. In phosphate buffer, an asymmetric ESR signal is detected at g = 2.005 (hereafter referred to as the g = 2 radical) during the oxidation which is similar to that of the methemoglobin free radical generated from methemoglobin and H2O2. Catalase and KCN prolong the oxidation, indicating the involvement of H2O2 and methemoglobin in the reaction. Superoxide dismutase, on the other hand, does not modify the oxidation. The present results suggest a chain reaction mechanism for the oxidation in which the g = 2 radical catalyzes the formation of NO.2 from NO-2 by a peroxidase action and NO.2 oxidizes oxyhemoglobin. However, in N,M-bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (bistris) buffer, superoxide dismutase markedly elongates the lag phase and accelerates the autocatalysis: bistris scavenges the g = 2 radical and a radical derived from bistris reduces O2 to O-2.

Senescent red cell-bound IgG is attached to band 3 protein.

Abstract: Senescent red cell-bound IgG autoantibody-antigen complexes were precipitated from extracts of surface-125I-iodinated red cells with an antibody to human IgG. Immunoprecipitates contained labeled band 3 protein and some higher molecular weight complexes.

Transport of amino acids for glutathione biosynthesis in human and dog red cells.

Abstract: Cysteine and glycine for glutathione biosynthesis in human red cells enter via specific amino acid transport systems, principally system ASC and gly respectively. In contrast, human red cells are impermeable to glutamate, but glutamine enters via a Na-dependent, saturable pathway, which apparently obeys Michaelis-Menten kinetics with appKm and Vmax values of 90-160 microM and 140-240 mumol/1.cells/h respectively. Dog red cells however, do show a Na-dependent glutamate uptake which apparently obeys Michaelis-Menten kinetics with appKm and Vmax values of 4-6 microM and 70-110 mumol/1.cells/h respectively.

Bilayer couple hypothesis of red cell shape transformations and osmotic hemolysis.

Abstract: It is shown how according to the bilayer couple hypothesis the cell volume and cell shape at hemolysis depend on the difference between areas of the two leaflets of the membrane bilayer. The result is used to interpret the time course of the hemolytic process.

Structural and kinetic differences between the M2 type pyruvate kinases from lung and various tumors.

Abstract: The kinetic and structural properties of purified, homogeneous pyruvate kinase type M2 from chicken lung and tumors, including that from Rous sarcoma virus-transformed chicken fibroblasts, have been compared. The "tumor enzyme" is characterized by a low affinity for phosphoenolpyruvate, pronounced serine activation, and strong alanine inhibition as compared to the "lung type". In contrast to the rat lung enzyme, which is not affected by serine, the chicken lung enzyme is slightly activated by serine. The serine metabolites phosphoserine and glycine do not activate the "tumor type M2 pyruvate kinase", but L-alpha-glycerophosphorylcholine and phosphatidylserine do slightly activate at physiological concentrations. Studies with substances structurally related to serine reveal that the hydroxyl group of serine is a prerequisite for the activation and that the amino and carboxyl groups determine the affinity of the "tumor type M2 pyruvate kinase" for serine. Two different fragmentation methods (CNBr-cleavage and V-8 proteolysis) and two different methods for separation of the resulting peptide fragments (polyacrylamide gel isoelectric focussing and SDS-polyacrylamide electrophoresis) indicated a high degree of homology between the type M2 pyruvate kinases from lung and tumors as well the type M1 from muscle. Each type of pyruvate kinase, however, contains one or two unique protein fragments which are characteristic for its type. We have termed those fragments L (lung), M (muscle), and T (tumor).

Does cell density correlate with red cell age

Abstract: We have examined age-related changes in red cells by employing a serial hypertransfusion protocol to generate populations of in vivo-aged mouse red cells. Studies of these old cells have revealed alterations in membrane components; namely, a conversion of band 4.1b to 4.1a and an increase in the amount of membrane-associated, Triton-insoluble globin. Furthermore, we have found that the erythrocyte does change in density, but only during the earliest stages of its life-span. As the cell continues to age, there is no longer a correlation between age and density.

Developmental changes of glycogen phosphorylase b isozymes in rat tissues.

Abstract: The isozyme distribution of glycogen phosphorylase b was studied in various fetal, neonatal, and adult rat tissues by means of discontinuous polyacrylamide gel electrophoresis in the presence of glycogen in the separating gel. The brain-type isozyme BB being the predominant form in fetal rat tissues is replaced during maturation either partially (brain and kidney) or completely (skeletal muscle and liver) by the isozymes MM or LL, respectively. In the various organs, the developmental formation of the adult phosphorylase isozyme pattern does not proceed simultaneously. The definitive isozyme pattern is expressed in heart muscle at the 19th day of gestation. The isozyme transitions in liver and skeletal muscle are finished about two days after birth, while at this time in the brain just the muscle-type subunit M appears and forms the hybrid MB. The isozyme distribution of the maturated rat brain (BB, MB, MM) is not expressed before the 9th day of postnatal life.

Cell shape and total adenylate concentration as important factors for posttransfusion survival of erythrocytes.

Abstract: Red cells were stored for 42 days in a CPD/SAGM plastic bag system and then rejuvenated with bicarbonate, pyruvate and slow addition of adenosine. The reversibility and the time dependence of metabolic and morphologic changes were compared with autologous, posttransfusion survival. Total adenylates correlated better (r = 0.88) than ATP with viability in vivo ATP was better maintained when the blood was stored under anaerobic than aerobic conditions. A high correlation between morphology and viability was found in rejuvenated cells (r = 0.95). It is suggested that the time needed for reversal of changes is essential for viability.

Vasopressin and oxytocin in cerebrospinal fluid and plasma of conscious rabbits--response to dehydration and haemorrhage.

Abstract: Vasopressin and oxytocin were determined simultaneously by a radioimmunoassay in cerebrospinal fluid and plasma of conscious, unrestrained rabbits. An elevation of neuropeptide levels in plasma, but not in cerebrospinal fluid, in response to dehydration or haemorrhage suggests an independent regulation of vasopressin and oxytocin concentration in both body fluids.

Status of glutathione in the rat liver. Enhanced formation of oxygen radicals at low oxygen tension.

Abstract: Rat livers were initially perfused and then stored at various temperatures up to 4 h. The intra- and extrahepatic status of glutathione, the accumulation of hydrogen peroxide in the preservation medium, the action of a OH-scavenger and of a xanthine oxidase-inhibitor were investigated as candidates for the assessment of oxidative alterations due to ischemia. Furthermore respiratory functions of mitochondria were measured. The increased efflux of GSSG from the liver tissue, the increase of the GSSGtot/GSHtot-ratio and the favourable effects of formate as OH-scavenger and of allopurinol as xanthine oxidase-inhibitor confirm the hypothesis about the oxidative damage under conditions of oxygen deficiency. The nucleotide degradation, especially the steps catalyzed by xanthine oxidase and uricase, is the main metabolic pathway for the generation of oxygen radicals under ischemic conditions.

A rapid procedure for the preparation of highly purified pyruvate decarboxylase from brewer's yeast.

Abstract: A rapid purification procedure for pyruvate decarboxylase (E.C. 4.1.1.1.) from fresh cells of brewer's yeast (Saccharomyces carlsb.) is reported. The preparation of a crude enzyme (30-45 U/mg) by the use of fractionation steps with protamine sulfate, acetone, and ammonium sulfate takes about 6-7 h. A stable pyruvate decarboxylase (70-85 U/mg) was obtained from such preparations after purification on CM Sephadex C 50 after another 2-3 h. Stability and structural properties are compared for enzymes prepared from fresh and dried yeast.

Cathepsin D from human brain: purification and multiple forms.

Abstract: Cathepsin D was purified about 1000-fold from human brain cortex by a procedure involving ammonium sulfate fractionation (30-70%), Sephadex G-75 chromatography, affinity chromatography on pepstatin-Sepharose and isoelectric focusing. The enzyme was assayed fluorometrically at pH 3.2, the substrates used were globin or haemoglobin modified with pyridoxal-5'-phosphate. 6 multiple forms of cathepsin D were resolved in the isoelectric focusing step with pI values 4.4, 4.8, 5.3, 6.2, 6.5 and 6.8. Km of pyridoxal-globin and pyridoxal-haemoglobin for all 6 multiple forms is 1.8-2.0 X 10(-5) M and 1.3 to 4 X 10(-6) M, respectively, and Ki of pepstatin is 2-4 X 10(-9) M. Gel filtration of the multiple forms on Sephadex G-100 column showed that each has a molecular weight of about 50 000. Human brain cathepsin D has a pH optimum of 3.2 with a smaller second optimum at pH 4.0 (pyridoxal-haemoglobin being used as substrate). All the multiple forms have the same pH-dependence curve. On SDS-polyacrylamide gel electrophoresis the purified enzyme produced 3 bands approximately corresponding to Mr 50 000, 35 000 and 15 000. Study of the breakdown of substance P and its C-terminal heptapeptide by cathepsin D shows that cleavage occurs at the Phe-Phe linkages of both substrates tested.

Haemoglobin oxidation and free radical production in the red cell.

Abstract: The production and fate of superoxide and other free radicals, and the role of superoxide dismutase in the red cell is considered. Superoxide reacts slowly and at a comparable rate with oxyhaemoglobin and methaemoglobin, but superoxide dismutase markedly accelerates oxidation of oxyhaemoglobin by oxidizing drugs or xenobiotics such as quinones or paraquat. The mechanism involves inhibition by the enzyme (via its reaction with superoxide) of methaemoglobin reduction by the drug radicals. The possibility that such inhibition of organic radical reactions by superoxide dismutase is one of its protective functions is discussed.

(E)-5-(2-Bromovinyl)-2'-deoxyuridine: a good substrate for mammalian pyrimidine nucleoside phosphorylases.

Abstract: Specific antiherpetic thymidine analogues are split by mammalian pyrimidine nucleoside phosphorylases with the following order of activity: VUdR greater than BVUdR greater than thymidine greater than EtUdR.

Bilirubin-induced cytomorphological changes in guinea-pig leucocytes.

Abstract: The in vitro influence of bilirubin on guinea-pig peritoneal exudate cells was investigated at concentrations comparable to the conditions of neonatal hyperbilirubinaemia. In in vitro cultures of granulocytes and macrophages bilirubin manifested itself macroscopically by yellow colouring and microscopically by bilirubin aggregation near the nucleus. The intracellular bilirubin content was determined to be 40 nmol per 3 . 10(7) cells. The deposit of bilirubin was irreversible even after subsequent incubation with albumin. Subsequent incubation in heat-inactivated serum reverses binding of bilirubin to cells. Moreover, adding serum to the bilirubin sample prevents the influx of bilirubin. Simultaneously with the deposition of bilirubin there were conspicuous cytomorphological changes in the granulocytes and macrophages observed. The changes consist in a large number of rigid plasma extensions on the whole cell surface. The morphological changes were also reversible by subsequent incubation with serum and their formation was prevented by addition of serum. Since albumin as a well known bilirubin binding protein could not prevent the deposition of bilirubin or the morphological changes, it is suggested that serum factors can regulate the transport of bilirubin across cell membranes.

Substance P-like immunoreactivity in plasma and adrenal medulla of rats with spontaneous hypertension and WKY rats under acute stress.

Abstract: The substance P-like immunoreactivity (SPLIR) of plasma and adrenal medulla from spontaneously hypertensive (SHR) and normotensive WKY rats was measured by a sensitive and highly specific radioimmunoassay for substance P (SP). With increasing age the level of substance P in the adrenal medulla decreased, whereas the SP content of the plasma remained constant in both groups. The SPLIR in plasma of SHR was significantly lower than in normotensive WKY rats. After acute stress the SPLIR, especially of the adrenal medulla of WKY rats, increases, and decreases in SHR. The reason for this different behaviour is discussed and it is assumed that hyperreactions in the sympathetic system may be responsible.

Dopaminergic innervation of the hippocampus: evidence for midbrain raphe neurons as the site of origin.

Abstract: The purpose of the study was to verify the site of origin of a postulated dopaminergic (DA) innervation of the hippocampus (HPC) in rats. The retrograde labeling of hippocampal afferents by Granular Blue (GB) was combined with the fluorescence histochemical identification of biogenic amines. Among the dopaminergic cell groups A8--A14 there was no one neuron labeled with GB. Some of the retrograde labeled neurons of the midbrain raphe nuclei (mainly B8-region) were identified as catecholaminergic, probably dopaminergic. It is concluded that mesencephalic dopaminergic fibers innervate the HPC, but that they arise from raphe nuclei rather than from the ventral tegmental area. In order to confirm these data neurophysiologically, hippocampal responses to electrical stimulation of midbrain raphe nuclei were recorded in freely moving Wistar rats. The intrahippocampal injection of the DA antagonist haloperidol through a chronic microcannula caused two types of changes in average evoked potentials: A significant decrease in the amplitude of an early, negative component (peak latency 8 ms). An increase in the amplitude of a second, positive component, which occurred only after a preceding inhibition of serotonine synthesis. The haloperidol sensitivity of the early component of hippocampal responses is interpreted as support of the morphological data about an ascending dopaminergic input from the median raphe nucleus to the hippocampus.

Differences in toxicity and antigenicity between mistletoe lectin I and viscotoxin A 3.

Abstract: In contrast to mistletoe lectin I (ML I), viscotoxin A 3 does not inhibit protein synthesis in cell-free systems. From the immunological studies it is concluded that ML I and viscotoxin do not share identical structural domains.

Dopamine release from striatum slices of rats at different age: influence of hypoxia.

Abstract: During the maturation and aging of rats the dopamine release from striatum slices shows different dynamics in dependence on the stimuli used. Electrically evoked release is very constant. Amphetamine induced release is fully developed at the age of day 14 and declines after 9 months. The K+ stimulated release develops to a maximum at 10 weeks and decreases thereafter. The stimulus and age dependent inhibition of dopamine release after an 18 h hypoxic exposure may be the result of peroxidative reactions of membranous phospholipid components induced by free radicals. Adult rats exposed to a 200 h hypoxia reveal no change in dopamine release after the last exposure probably in consequence of adaptive processes of physiological protection, among other things, by scavenging of free radicals. Adult rats exposed to long term hypoxia postnatally during the growth spurt period of neuronal structures show an increased dopamine release and no posthypoxic release inhibition. Our data indicate that different mechanisms are operative during hypoxia in animals of different age.

Lung strips from guinea pigs as test system for lipoxygenase inhibitors. Inhibition of arachidonic acid-induced contractions by 3-t-butyl-4-hydroxyanisole and nordihydroguaiaretic acid.

Abstract: Lung strips and tracheal spirals from guinea pigs and rats were compared as test systems for lipoxygenase inhibitors. The contraction of the smooth muscle was induced by addition of arachidonic acid. Lung strips of guinea pigs proved to be the most suitable in vitro model. Under basal conditions half-maximal contraction was produced by 13 microM arachidonic acid (AA) which was strongly inhibited by 50 microM nordihydroguaiaretic acid (NDGA) or 100 microM 3-t-butyl-4-hydroxyanisole (BHA). NDGA and BHA caused a shift of response towards higher concentrations of AA. BHA also produced pronounced dilatations of both basal tonus and carbachol-induced contractions of the preparations from the respiratory tract. It is concluded that the actions of NDGA and BHA on the airway preparations are due to inhibition of the lipoxygenase reaction and, therefore, of leukotriene synthesis. The assumption of the lipoxygenase inhibition is supported by their action on purified lipoxygenase from rabbit reticulocytes which was inhibited by 50% by 0.5 microM NDGA and 160 microM BHA. In contrast, the antioxidant 2,6-di-t-butyl-4-hydroxytoluene (BHT) did not inhibit at all at 1 mM. To the knowledge of the authors this is the first evidence so far reported for the bronchodilating action of BHA.

Phosphoglycerate kinase abnormalities: functional, structural and genomic aspects.

Abstract: Phosphoglycerate kinase (PGK) deficiency is associated with hemolytic anemia and mental disorders in man. Complete amino acid sequence of normal human PGK was determined and its three-dimensional structure could be deduced from that of horse and yeast enzymes. Specific amino acid substitutions of several PGK variants associated with clinical problems were elucidated, and their functional abnormalities were correlated to their structural abnormalities. Full-length cDNA clones for normal human PGK were isolated and it is possible to examine PGK variants at the genomic level.

Superoxide radicals in the metabolism of the red cell.

Abstract: The formation of O2- radicals as primary products of O2 reduction by human erythrocytes is demonstrated. O2- radicals react directly with GSH and OxyHb as 1e-donors forming GSSG and MetHb. After exhaustion of the glutathione system MetHb increases greatly and secondary radicals formation ensues with breakdown of Hb. The toxicity of O2- radicals is caused mainly by secondary radicals.

Electron microscopic investigations on the location of rat liver ribosomal proteins S3a, S5, S6, S7 and S9 by means of antibody labeling.

Abstract: The location of ribosomal proteins S3a, S5, S6, S7 and S9 on the surface of 40S subunits of rat liver ribosomes has been determined by means of antibody labeling and electron microscopy. Multiple antigenic determinants could be detected for proteins S3a, S6 and S7, whereas protein S9 shows only one and protein S5 two closely neighboured antigenic determinants. Antigenic determinants of proteins S3a, S6 and S7 have been mapped in the head and the body region of the 40S subunits. Protein S5 could be localized in the head and protein S9 in the body of the 40S subunits. These findings are discussed with regard to the location of functional sites on the 40S ribosomal subunit.

Membrane processes during 'in vivo' aging of human erythrocytes.

Abstract: Membrane modifications occurring during in vitro and in vivo aging in human erythrocytes have been investigated. The structural damages are the consequence of proteolytic and oxidative processes and involve both glycoprotein and protein component of the membrane. The target of these processes seems to be the same during in vitro and in vivo aging.

Transferrin endocytosis and iron uptake during erythroid cell development.

Abstract: Electron microscope autoradiography was used to quantitate the uptake of transferrin and transferrin-bound iron by rat erythroid cells at the various stages of development The capacity of the cells to take up iron closely paralleled their ability to bind transferrin, suggesting that the level of transferrin receptors is the major factor which regulates the rate at which immature erythroid cells can accumulate iron Maximal transferrin and iron uptake occurred at the basophilic normoblast stage and then decreased progressively during maturation to the reticulocyte During development, immature rat erythroid cells acquire about twice as much iron from transferrin as is present in the haemoglobin of mature erythrocytes

Influence of polyethylene glycol on the structure of the erythrocyte membrane: an ESR study.

Abstract: Using a fatty acid spin label (I (10, 3)) it was found that the membrane fluidity of intact human erythrocytes is not influenced by polyethylene glycol (PEG) up to a concentration of 10 wt%. On the contrary a drastic effect of PEG on the spectrum of maleimide spin labeled membrane proteins of erythrocyte ghosts were detected. It is suggested that aggregation and/or conformational changes of membrane proteins were induced by PEG.

Adrenergic and antiadrenergic activity of cycloheximide in cultured rat heart cells.

Abstract: Cycloheximide at concentrations between 10(-5) to 10(-3) M increased the rate of beating of rocker-cultured neonatal rat myocardial cells through activation of alpha 1-adrenoceptors. At concentrations between 3 X 10(-10) and 10(-6) M, at which [14C]-leucine incorporation into the cultured cells was not or not greatly affected, the antibiotic inhibited surmountably the positive chronotropic action of the rather nonselective beta-adrenergic agonist isoprenaline. It also antagonized the cyclic AMP response to isoprenaline. A powerful positive chronotropic action of the beta 2-selective agonist clenbuterol was not opposed by cycloheximide. Neither did cycloheximide (10(-8) and 10(-6) M) influence the acceleration of beating by phenylephrine, dibutyryl cyclic AMP, and elevation of the extracellular calcium concentration. Displacement of the nonselective beta-adrenoceptor ligand [125I]iodopindolol from its specific binding sites on the cultured heart cells by cycloheximide was diphasic, with 15 to 35 per cent of the displacement taking place below 10(-8) M cycloheximide (IC50 = = 2 X 10(-9) M) and the remaining 65 to 85 per cent above 10(-5) M. The action of low concentrations of cycloheximide on beta-adrenoceptors remains to be delineated.