Diacylglycerol- and phosphatidic acid-kinase studies in plant cell suspension cultures.
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Cells, Cultured↗
Biomedical subjects
B Riedel
Publications and source records attributed to B Riedel.
Fully automated assay for cobalamin-dependent methylmalonyl CoA mutase.
We constructed a fully automated assay for the cobalamin-dependent enzyme methylmalonyl coenzyme A (CoA) mutase. The assay involves preincubation of the enzyme with adenosylcobalamin, incubation with substrate, termination of the reaction by adding trichloroacetic acid, filtration to remove precipitated protein, and finally analysis of the filtrate (containing methylmalonyl CoA and the product succinyl CoA) by HPLC. These steps were carried out by an inexpensive programmable autosampler equipped with thermostated sample racks and mobile disposable extraction column racks used here as a sample filtering device. A central element in the developmental work was to measure stability of reagents, enzyme, and product against the storage conditions during unattended analysis and the time table of the program. We evaluated the performance of the method by measuring methylmalonyl CoA mutase activity in rat liver, human fibroblasts, and human glioma cells. The within-run imprecisions (CV) were 2-10% for measuring enzyme activity in 20 replicate samples of a homogenate (test of the automated assay), and 7-12% for measuring enzyme activity in homogenates from 20 culture dishes (test of the total procedure). The method allows the unattended analysis of 56 samples per 24 h. This strategy for automation may be easily adapted for other enzyme assays.
Acyl Coenzyme A↗
Preoperative methionine loading enhances restoration of the cobalamin-dependent enzyme methionine synthase after nitrous oxide anesthesia.
BACKGROUND: Prolonged exposure to nitrous oxide causes adverse effects mimicking those of cobalamin deficiency. This is explained by irreversible oxidation of cobalamin bound to the enzyme methionine synthase. The inactivation of methionine synthase by nitrous oxide in cultured human fibroblasts is decreased at high concentrations of methionine in culture medium. METHODS: We investigated the possible protection against cobalamin inactivation by preoperative methionine loading in patients undergoing nitrous oxide anesthesia. Fourteen patients receiving anesthesia for 75-230 min were included. Half of these patients received a peroral methionine loading dose 2 h before anesthesia. RESULTS: After nitrous oxide exposure, a considerable inactivation of methionine synthase in mononuclear white blood cells was seen in all patients, reaching a nadir after 5-48 h. In the patients not subjected to a methionine load, recovery of enzyme activity was not complete within 7 days. In the patients receiving a methionine load, the kinetics of inactivation of methionine synthase were similar, but the rate and extent of enzyme recovery was higher than in patients not receiving methionine, and in four patients, the enzyme activity even exceeded the preoperative level. The inactivation of methionine synthase was associated with a transient increase in plasma homocysteine, and the homocysteine concentration was still increased (mean 28.7%) 7 days after anesthesia in the patients not receiving methionine. A marked peak in homocysteine concentration was observed immediately after anesthesia in the methionine-loaded patients, but the homocysteine level was still increased (mean of 30.5%) after 7 days. The activity of the other cobalamin-dependent enzyme, methylmalonyl coenzyme A mutase, in the mononuclear white blood cells, and the serum concentration of the cobalamin marker methylmalonic acid, were not altered after nitrous oxide anesthesia or methionine loading or both. CONCLUSIONS: Our data suggest that short time exposure to nitrous oxide selectively impairs the function of the cobalamin-dependent methionine synthase. Furthermore, preoperative administration of methionine should be considered as a means to counteract adverse effects of nitrous oxide.
5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
Increased fecal eosinophil cationic protein in inflammatory bowel disease.
Eosinophil cationic protein (ECP) is a cytotoxic substance released by eosinophilic granulocytes. Serum and body-fluid levels may be elevated in some allergic and inflammatory conditions characterized by heavy infiltration of eosinophils. ECP has not previously been measured in feces. Because pronounced infiltration with eosinophils is often seen in ulcerative colitis, we examined fecal excretion of ECP in 29 patients with active inflammatory bowel disease (IBD) and in 10 healthy persons. Mean fecal ECP levels were more than 14 times higher in the patients than in the healthy persons (p < 0.001). Fecal ECP was similarly elevated in ulcerative colitis and Crohn's disease. During storage, ECP in feces was relatively stable. Steroid treatment may suppress fecal ECP excretion. The possible value of fecal ECP as an IBD activity parameter warrants further studies. ECP may also be involved in the pathogenesis of IBD.
Adult↗
Interaction between heparin and acetylsalicylic acid on gastric mucosal and skin bleeding in humans.
The haemorrhagic effect of unfractionated heparin and of the low molecular weight heparin, enoxaparin, on gastric mucosal bleeding induced by acetylsalicylic acid (ASA) and on skin bleeding induced by the Simplate technique was investigated in healthy human volunteers. Endoscopic estimation of gastric bleeding by visual analogue scores was more sensitive than biochemical quantitation of blood in the gastric washing by a modified HaemoQuant method. Contrary to what was expected, the ASA-induced gastric mucosal bleeding was not increased by heparin pretreatment, whereas heparin in combination with ASA, but not ASA alone, significantly increased the skin bleeding time. In the interaction with ASA, enoxaparin and unfractionated heparin appeared to act similarly.
Adult↗
Haemorrhagic effect of enoxaparin, a low molecular weight heparin. Comparison with unfractionated heparin in humans.
The haemorrhagic effects of unfractionated heparin (UFH) and the low molecular weight heparin (LMWH) enoxaparin were investigated and compared in the gastric mucosa (haemorrhage induced by biopsy) and skin (haemorrhage induced by Simplate) of 12 healthy volunteers. Administration of UFH and LMWH (given in a dose of 75 anti-Xa U/kg intravenously) increased median gastric bleeding time (3.5 min) and geometric mean blood loss (11.5 microliters) to 19 min (p = 0.00003) and 54.1 microliters (p = 0.0021) after UFH and to 13 min (p = 0.008) and 29.0 microliters (p = 0.275) after LMWH. Median skin bleeding time (4.25 min) increased to 6.0 min after UFH (p = 0.003) and to 6.75 min after LMWH (p = 0.0008). Mean heparin activity in plasma was 20% higher after LMWH than after UFH. The calculated gastric bleeding time to heparin activity ratio was significantly lower for LMWH than for UFH (p < 0.05).
Adult↗
Epidermal growth factor up-regulates intestinal Na+/H+ exchange activity.
The present studies were designed to examine the regulation of Na+/H+ exchange activity by epidermal growth factor (EGF) in an in vitro system. Na+/H+ exchange activity was determined in brush-border membranes isolated from rat jejunal enterocytes incubated with epidermal growth factor and a number of second messengers. EGF at physiological concentrations stimulated Na+/H+ exchange activity without affecting vesicle size. The stimulation of Na+/H+ activity was the result of increasing Vmax of Na+/H+ (6.0 +/- 0.4 compared with 3.3 +/- 0.27 nmol/mg protein/5 sec, P < 0.01). Km values of the Na+/H+ exchanger in brush-border membrane from cells stimulated with EGF and controls were similar (16.0 +/- 3.0 vs 13.0 +/- 3.0, respectively). Na+/H+ activity was inhibited by phorbol esters, calmodulin, and cyclic AMP. The effects of EGF, calmodulin, cyclic AMP, and phorbol esters were dependent on ATP, because depleting the cells from ATP masked the effects on Na+/H+ exchange activity. The results suggest that EGF stimulates Na+/H+ exchange activity in the enterocytes. This stimulation is most likely not via activation of the phosphatidylinositol pathway.
Adenosine Triphosphate↗
[The subacute and subchronic toxicity of chlorophacinone in Japanese quail (Coturnix c. japonica)].
Accordingly to the subacute toxicity of chlorphacinone to Japanese quail (LC 50: 60 mg/kg feed), the uptake of Delicia-chlorphacinone-baits for several days can be lethal to birds as a matter of principle. A subchronic study resulted in a "no-observed-effect-level" of 1 mg chlorphacinon/kg feed. Reproduction is not affected until dose levels which resulted in lethality or are accompanied by physiological changes (coagulability of blood, microcytic anaemia). Increased sensibility of descendants to chlorphacinone cannot be derived from the findings.
Animals↗
[Occurrence of Campylobacter in feces of birds kept in human living areas].
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Animals↗
Differences in the distribution of methyl mercury in erythrocytes, plasma, and brain of Japanese quails and rats after a single oral dose.
Distribution of a single oral dose of methyl mercury (10 mg Hg/kg body weight) was followed from 90 min up to 120 h in plasma, erythrocytes, and brain of Japanese quails and rats. Significantly higher Hg concentrations were observed in plasma and brain of quails and red blood cells of rats. Blood/brain ratio decreased in quails from 6 to 2 at 24 h and 120 h respectively, whereas it increased in rats. Erythrocyte/plasma ratio in quails was about three times lower and averaged 54. The differences in Hg distribution were accompanied by a more than 3-fold higher acute toxicity in quails under adequate experimental conditions.
Animals↗
Chemical effects on the activity of eight enzymes: a review and a discussion relevant to environmental monitoring.
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Enzyme Inhibitors↗
Effect of water pollutants and other chemicals upon the activity of lipase in vitro.
Lipase preparations were treated in vitro with 100 chemicals of various classes, many of which are environmental pollutants, to determine their effect upon enzyme activity. The greatest inhibition was caused by mercuric ion and certain heavy metal cations; almost as inhibiting were pentachlorophenol, dicofol, 2,4,5-trichlorophenol, and DDT. Less inhibition was found with other inorganic salts, organo-metals, other organic chemicals, and some pesticides. Apart from dichromate ion, the anions studied had a low to negligible effect on lipase. A mixture of 10 inorganic salts had an approximately cumulative inhibitory effect on enzyme activity. None of the chemicals studied caused a measurable activation of the enzyme.
Lipase↗
[Progesterone and estradiol levels in blood plasma of calves following superovulation treatment].
Studies into quantitative behaviours of oestradiol and progesterone in the blood plasma of six heads of prepuberal cattle, following superovulation treatment, using pregnant-mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) have shown the oestradiol concentrations to double, as early as two days after PMSG, and to go up to ten times their original values, after another four days had elapsed. Progesterone values increased, as early as along with induced oestrus, and, consequently, suggested scatter of the ovulation process. The maximum progesterone levels reached something in the vicinity of 100 ng/ml, following superovulation, and, consequently, were far in excess of anything recordable from spontaneously ovulating cyclic cattle. Hormone analyses were continued over six weeks from induced oestrus and superovulation, with evidence being produced to the effect that no permanent cyclic activity had been introduced. All calves rather returned to their prepuberal condition.
Animals↗
Effect of selected water toxicants and other chemicals upon adenosine triphosphatase activity in vitro.
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Adenosine Triphosphate↗
Novel antiviral activity found in the media of Sindbis virus-persistently infected mosquito (Aedes albopictus) cell cultures.
Aedes albopictus (mosquito) cells persistently infected with Sindbis virus for a period of 6 months release into the medium a low-molecular-weight material capable of specifically reducing the yields of Sindbis virus during the "acute phase" of infection in mosquito cells. The antiviral activity was produced in detectable levels at 3 days after infection, and its concentration in the extracellular medium increased thereafter. The antiviral activity was inactivated by treatment with the enzyme protease K and heat. It was not activated by treatment with antibody prepared against extracts of Sindbis virus-infected BHK-21 cells. The antiviral activity differs from interferon produced by vertebrate cells in that it is virus specific as well as cell specific.
Aedes↗
Morphogenesis of vesicular stomatitis virus: electron microscope observations with freeze-fracture techniques.
The morphogenesis of vesicular stomatitis virus was examined using freeze-fracture techniques, and the results obtained were compared with those from previously published experiments carried out with influenza viruses and togaviruses. The process of conversion of the host cell plasma membrane into the vesicular stomatitis virus envelope was accompanied by a loss of the intramembranal particles abundant in cell membranes. Frequently a dense accumulation of intramembranal particles could be seen at the base of the developing virion, suggesting that these structures might play some role in the generation of viral envelope. In addition to the viral structures that were seen to develop in the classical fashion, with their long axis perpendicular to the cell surface, structures were also found that suggested the initiation of a process similar to budding, with the long axis of the viral capsid parallel to the plasma membrane. In this situation, as in the "perpendicular" process, intramembranal particles were excluded from the viral structure, and an accumulation of these particles could be seen adjacent to the developing viral membrane.
Cell Line↗
Role of extracellular virus on the maintenance of the persistent infection induced in Aedes albopictus (mosquito) cells by Sindbis virus.
Sindbis virus infection of cultured mosquito cells was found to have no effect on the growth of these cells; instead, a persistent infection of the culture followed an initial acute phase of rapid virus synthesis. Nearly all of the cells in the acute stage of infection were found to actively release virus in an infectious-center assay and to contain significant amounts of virus antigen as determined by immunofluorescence. Cells in the persistent phase of infection released few virions into the media, and only a small percentage of the cultured cells could be demonstrated to contain detectable amounts of virus antigen by immunofluorescence assay. In spite of the fact that nearly 100% of the cells in the persistent phase of infection were found to be virus negative by the two assays described above, the culture as a whole totally excluded the expression of superinfecting virus, as did cells in the acute phase, suggesting that most of the persistently infected cells did, indeed, contain virus information. Prevention of reinfection of the cells in the persistent phase by eliminating extracellular virus resulted in a curing of the culture such that it responded to infection by added virus much as would an uninfected culture.
Aedes↗
Denudation of the urinary bladder mucosa in the cat by formaldehyde.
Chemical Stripping of the urinary bladder mucosa was studied in 38 cats using 5 to 25% formaldehyde solutions. The contact time varied from 1 to 20 min. With a 20% solution and contact time of 1 min, total denudation was possible without necrosis of subepithelial layers. In such cases, complete reepithelialisation and normal bladder dynamics were seen within 3-4 weeks after formaldehyde instillation. Signs of formaldehyde intoxication due to vesical resorption were not observed.
Animals↗