Arterialization of the portal vein in orthotopic and auxiliary liver transplantation. A report of three cases.
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Adult↗
Biomedical subjects
H de Groot
Publications and source records attributed to H de Groot.
Inhibition of superoxide and nitric oxide release and protection from reoxygenation injury by Ebselen in rat Kupffer cells.
Luminol chemiluminescence in phorbolester-activated cultured rat liver Kupffer cells was strongly inhibited by the selenoorganic compound ebselen (IC50 = 2 mumol/L). Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]one) also diminished reduction of ferricytochrome c (IC50 = 10 mumol/L), indicating a suppression of superoxide anion formation. Likewise, in lipopolysaccharide-pretreated Kupffer cells, ebselen proved to be a potent inhibitor of the conversion of oxyhemoglobin to methemoglobin (IC50 = 3 mumol/L) as a measure of nitric oxide formation. The sulfur-containing analog (2-phenyl-1,2-benzisothiazol-3[2H]one) and the ebselen derivative, methylselenobenzanilide, were inactive. These results indicate that ebselen is a potent inhibitor of NADPH oxidase in Kupffer cells, as has been reported for other macrophages and granulocytes. In addition, they suggest a novel characteristic of ebselen, namely very effective inhibition of nitric oxide synthase of macrophages. In line with its inhibitory effects on the release of reactive oxygen species by macrophages, complemented by its antioxidant properties, ebselen was potent in the prevention of reoxygenation injury of Kupffer cells (IC50 approximately 5 mumol/L).
Amino Acid Oxidoreductases↗
Isolated cells in the study of the molecular mechanisms of reperfusion injury.
Isolated cells make it possible to study mechanisms of cell and tissue injury under well-defined conditions, including the interaction of different cells in coculture experiments. Isolated cells, either in suspension or in monolayer cultures, have also been used to study the mechanism of reperfusion injury--in this case better termed as reoxygenation injury in view of the experimental approach taken. In hepatocytes, Kupffer, and endothelial cells, reoxygenation injury resulted in necrosis primarily mediated by reactive oxygen species released by various sources such as mitochondria (hepatocytes) and NADPH oxidase (Kupffer cells). In contrast, contracture was a characteristic feature of reoxygenation injury occurring in cardiomyocytes without loss of cytosolic enzymes. Beside reactive oxygen species, Kupffer cells were activated to release prostanoids and a decrease in endothelial cell-mediated fibrinolysis occurred upon reoxygenation. Reoxygenation injury in endothelial cells was significantly increased when neutrophils were added at the time of reoxygenation, presumably due to additional generation of reactive oxygen species and the release of proteases. As exemplified for the liver, these experiments suggest a mechanism of reperfusion injury in which the various cell types of a given tissue differ significantly in their response to hypoxia-reoxygenation but in which they interact with each other in a complex pathobiochemical network via various mediators such as cytokines, and tissue damaging effector molecules such as reactive oxygen species. Future experiments with isolated cells will allow detailed analysis of the underlying molecular mechanisms.
Cells, Cultured↗
Role of reactive oxygen species in cell toxicity.
Several types of compound exert their cytotoxicity by generating reactive oxygen species, notably the superoxide anion radical. These include quinoid and nitroaromatic compounds serving as redox cyclers, i.e. producing superoxide at the expense of NADPH and oxygen catalyzed by cellular reductases. In specialized cell-types employed in defense such as granulocytes, eosinophils and macrophages, myeloperoxidase, NADPH oxidase and nitric oxide synthase have been identified as major sources of reactive oxygen species in cell toxicity. These include hypochlorite, singlet oxygen, superoxide, nitric oxide and hydrogen peroxide. The interaction of superoxide and nitric oxide generates further oxidants such as peroxynitrite. Lumino-amplified chemiluminescence generated by Kupffer cells is partially sensitive to inhibitors of NO synthase. Superoxide dismutase has been found to catalyze a novel reaction, the reversible conversion of nitric oxide to the nitroxyl anion, the latter being viewed as another form of EDRF. In the defense against oxidative damage, there are enzymatic and nonenzymatic antioxidants. Regarding compounds used pharmacologically, we have been interested in ebselen, a seleno-organic compound exhibiting GSH peroxidase activity, which protects against reactive oxygen species generated, for example, at reoxygenation following a period of hypoxia. Further, we have studied lipoate and dihydrolipoate as antioxidant redox system and as singlet oxygen quencher, e.g. protecting against damage of deoxyguanosines in plasmid DNA generated by singlet oxygen.
Animals↗
Production of reactive oxygen by mitochondria from normoxic and hypoxic rat heart tissue.
Reactive oxygen species (ROS), which may be involved in ischemic or reperfusion heart injury, can be produced by mitochondria. Previous work indicated that coupled mitochondria from ischemic heart tissue incubated in calcium-free medium produced less ROS than normal. The effects of calcium, which may be elevated in hypoxic or ischemic tissue, were not examined. The relative production of ROS by mitochondria from normoxic or hypoxic rat heart tissue was estimated by measuring the oxidation of dichlorofluorescin to the fluorescent compound, dichlorofluorescein. ROS were detectable during succinate-stimulated State 4 respiration. In the absence of calcium, mitochondria from hypoxic (60 min) heart tissue produced less ROS than mitochondria from normoxic heart tissue. In the presence of 0.1, 1 or 10 microM calcium, ROS produced by hypoxic mitochondria were increased to normoxic levels. While function was depressed in mitochondria from hypoxic tissue, the presence of 0.1 and 1 microM calcium had no further effect. Respiration was uncoupled in the presence of 10 microM calcium in mitochondria from both normoxic and hypoxic heart tissue. ROS production was increased in mitochondria from hypoxic tissue with both increasing concentrations of calcium and increasing duration of exposure. ROS production in mitochondria from normoxic heart tissue was only stimulated after 200 or more seconds of exposure to 1 or 10 microM calcium. Production of ROS in mitochondria from hypoxic tissue in the presence of 1 microM calcium was inhibited by rotenone (80%), ruthenium red (69%), and a combination of these agents (96%). In contrast, ruthenium red had no effect on ROS production by mitochondria from normoxic heart tissue.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Magic angle spinning NMR studies on the metarhodopsin II intermediate of bovine rhodopsin: evidence for an unprotonated Schiff base.
Magic angle spinning (MAS)13C-NMR spectra of the metarhodopsin II intermediate have been obtained using bovine rhodopsin regenerated with retinal 13C-labeled at the C-13 and C-15 positions to investigate the protonation state of the retinal Schiff base linkage. The 13C-labeled rhodopsin was reconstituted into 1,2-dipalmitoleoylphosphatidylcholine bilayers to increase the amount of meta II trapped at low temperature. Both the 13C-15 (159.2 ppm) and 13C-13 (144.0 ppm) isotropic chemical shifts are characteristic of an unprotonated Schiff base, while the 13C-15 shift is significantly different from that of retinal (191 ppm) or a tetrahedral carbinolamine group (70-90 ppm) previously proposed as an intermediate in the hydrolysis of the Schiff base at the meta II stage. This rules out the possibility that meta II non-covalently binds retinal or is a carbinolamine intermediate and provides convincing evidence that Schiff base deprotonation occurs in the meta I-meta II transition, an event that is likely to be important in triggering the activation of transducin.
Animals↗
Release of reactive oxygen by hepatocytes on reoxygenation: three phases and role of mitochondria.
Reoxygenation of isolated hepatocytes in primary culture resulted in a three-phase response in the release of reactive oxygen species (ROS) as determined by peroxidase-dependent luminol chemiluminescence. Release of ROS within the first and second phase correlated well with the extent of reoxygenation injury, both being most significant after approximately 4 h of hypoxic incubation. During the third phase, some of the ROS were released by already nonviable cells. Both antimycin A and rotenone significantly increased release of ROS, indicating severe alterations of the mitochondrial respiratory chain caused by hypoxia and suggesting that the altered mitochondrial respiratory chain represents an important source for the release of ROS on reoxygenation. Generation of ROS rose sharply when the O2 content was increased from 0 to 2%, whereas a further increase in the O2 content, of up to 95%, resulted in only small but steady increases in the formation of ROS. The latter suggests that, in addition to enzymatic sources such as the mitochondrial respiratory chain, nonenzymatic reactions may also contribute to the formation of ROS on reoxygenation.
Animals↗
Morphological changes of cultured rat hepatocytes exposed to methylglyoxal. Calcium-independence of injury.
Methylglyoxal-induced morphological changes were studied in cultured rat hepatocytes. Hepatocytes incubated either in the presence or absence of 2.5 mM Ca2+ were injured by 10 mM methylglyoxal to a similar extent, while 1 mM of the alpha-oxoaldehyde caused a moderate damage only in the absence of Ca2+. In the absence of Ca2+, however, hepatocytes were already injured, even without the presence of methylglyoxal hepatotoxicity proceeds independently of the Ca2+ influx.
Animals↗
Contribution of nitric oxide synthase to luminol-dependent chemiluminescence generated by phorbol-ester-activated Kupffer cells.
Phorbol 12-myristate 13-acetate-induced luminol chemiluminescence in rat Kupffer cells was doubled by the addition of L-arginine and significantly (up to 70%) inhibited by NG-nitro-L-arginine and NG-monomethyl-L-arginine, competitive inhibitors of L-arginine-dependent nitric oxide (NO) formation. The release of superoxide anion (O2-) by NADPH oxidase was neither affected by L-arginine nor by the inhibitors. Only very slight luminol chemiluminescence was detectable in lipopolysaccharide-pretreated Kupffer cells, a condition in which significant amounts of NO were formed but no O2-. In a cell-free system, significant luminol chemiluminescence only occurred when both authentic NO and the O2-/H2O2- generating system xanthine/xanthine oxidase were present. The results indicate that luminol chemiluminescence in phorbol-ester-activated Kupffer cells largely depends on L-arginine metabolism by NO synthase, requiring the concurrent formation of NO and O2-/H2O2.
Acridines↗
13C magic-angle spinning NMR studies of bathorhodopsin, the primary photoproduct of rhodopsin.
Magic-angle spinning NMR spectra have been obtained of the bathorhodopsin photointermediate trapped at low temperature (less than 130 K) by using isorhodopsin samples regenerated with retinal specifically 13C-labeled at positions 8, 10, 11, 12, 13, 14, and 15. Comparison of the chemical shifts of the bathorhodopsin resonances with those of an all-trans-retinal protonated Schiff base (PSB) chloride salt show the largest difference (6.2 ppm) at position 13 of the protein-bound retinal. Small differences in chemical shift between bathorhodopsin and the all-trans PSB model compound are also observed at positions 10, 11, and 12. The effects are almost equal in magnitude to those previously observed in rhodopsin and isorhodopsin. Consequently, the energy stored in the primary photoproduct bathorhodopsin does not give rise to any substantial change in the average electron density at the labeled positions. The data indicate that the electronic and structural properties of the protein environment are similar to those in rhodopsin and isorhodopsin. In particular, a previously proposed perturbation near position 13 of the retinal appears not to change its position significantly with respect to the chromophore upon isomerization. The data effectively exclude charge separation between the chromophore and a protein residue as the main mechanism for energy storage in the primary photoproduct and argue that the light energy is stored in the form of distortions of the bathorhodopsin chromophore.
Chlorides↗
Generation of developmental patterns in the neuroepithelium of the developing mammalian eye: the pigment epithelium of the eye.
Culture experiments with eye anlages of mouse embryos were performed to study developmental traits of the neuroepithelial cells of the prospective pigment epithelium in the eye anlage of pigmented mice. Between the neural plate stage on embryonic day 8 (ED 8, developmental stage 12) and the neural tube stage on embryonic day 9 1/2 (stage 15), the cultured neuroepithelium of the eye generated neurons and glia, identified by morphological and immunocytochemical evidence, but no pigmented cells. In contrast, eye anlages did produce pigment epithelium when cultured in their natural position in a head tissue fragment. A minority of developing neurons displayed tyrosine hydroxylase immunoreactivity, whereas GABAergic, serotoninergic and substance P-ergic neurons, which are common in the mature neuroretina, were not observed. When neuroepithelial cells from embryonic eyes older than stage 15 (ED 9 1/2) were cultured, they differentiated into pigment cells but not into nerve cells or glia. This developmental sequence indicates that the pigment cells derive from the neural lineage. Pigment cell fate dominates over the neural fate beginning at about stage 15 (ED 9 1/2-10). That is at least 2 days before the pigment cell phenotype becomes apparent in vivo (ED 11 1/2-12).
Animals↗
Modulation of PTH-stimulated osteoclastic resorption by bisphosphonates in fetal mouse bone explants.
We examined the effects of the bisphosphonates Cl2MDP, APD, and Me2APD on osteoclastic resorption in the absence and presence of PTH using fetal mouse osteoclast-free bone explants cocultured with fetal liver as a source of osteoclast precursors. Results revealed qualitative and quantitative differences among the bisphosphonates tested. With Cl2MDP and APD fractional inhibition of resorption (measured as 45Ca release) in the presence of PTH was proportional to that obtained in its absence. In contrast, Me2APD, which is the most potent inhibitor of the three, was found at low concentrations (less than or equal to 5 x 10(-7) M) to enhance the PTH-stimulated osteoclastic resorption. APD as well, at concentrations that could not inhibit resorption, had a similar effect, but Cl2MDP did not. These studies describe a new phenomenon, that low doses of nitrogen-containing bisphosphonates can act synergistically with PTH and enhance osteoclastic resorption. These findings may have clinical implications in the management of patients with increased osteoclastic resorption due to parathyroid overactivity.
Animals↗
[Primary hepatocyte cultures as a model of experimental study of liver preservation].
Primary hepatocyte cultures have been used to evaluate data concerning hypoxic liver cell injury. To show the suitability of this method in liver preservation studies hepatocyte cultures were incubated under different conditions: warm normoxia (37 degrees C, pO2 greater than 70 mm Hg), warm hypoxia (37 degrees C, pO2 less than 0.1 mm Hg), cold normoxia (4 degrees C, pO2 greater than 70 mm Hg) and cold hypoxia (4 degrees C, pO2 less than 0.1 mm Hg). Incubations were performed in Euro Collins solution (EC), University of Wisconsin solution of Belzer (UW) and histidine ketoglutarat tryptophan solution of Bretschneider (HTK) as well as in Krebs Henseleit buffer (KH) for control incubations. During 12 h of incubation hepatocyte cultures under warm normoxia lost viability continuously in EC, UW and HTK while in KH they remained stable. Under warm normoxia all cultures lost 50% of their viability during 12 h of incubation while in cold normoxia loss of viability was mild but significant. Under cold anoxia which is the standard condition of liver preservation the cultured hepatocytes remained unchanged for 12 h in KH, UW and HTK, while in EC most of the cells were dead after 6 h. It is concluded that incubations of primary hepatocyte cultures under different pO2 and temperatures are well suited to contribute to liver preservation studies on a preclinical level and thus may help to save animal experiments.
Animals↗
Affinity purification of a major and a minor allergen from dog extract: serologic activity of affinity-purified Can f I and of Can f I-depleted extract.
Most dog-allergic patients react to a major 25 kd component on sodium dodecyl sulfate blots, Can f I (Ag 13). We initially raised monoclonal antibodies (Cf-3 and Cf-2) reactive with IgE-binding components distinct from Can f I. After a slight modification, we immunized other strains of mice and produced monoclonal antibodies coded Cf-1a and Cf-1b reactive with Can f 1. We affinity purified the allergens, Can f I and "dog allergen 2" with Cf-1a and Cf-2 ascites, respectively, and house dust-rich dog dander. Comparison of purified Can f I with dog saliva in RAST demonstrated that Can f I is a potent allergen for most dog-allergic patients (average response, 70%). After depletion of dog saliva of Can f I, a slightly lower contribution for Can f I was found, but the overall results supported the conclusion that Can f I is a major allergen in dog saliva. Comparison of purified dog allergen 2 with dog dander in RAST demonstrated that dog allergen 2 is less important for dog-allergic patients (average response, 23%). We radiolabeled the purified allergens and developed assays to measure Can f I and dog allergen 2 in allergen extracts and dust samples. Dog saliva was a strong allergen source, dog urine and feces contained very little of the allergens, and both allergens were found to a variable degree in the nine dog breeds tested.
Allergens↗
Oxygen partial pressure and free intracellular adenosine of isolated cardiomyocytes.
Adenosine formation by the heart is known to critically depend on the ratio of oxygen supply to oxygen demand, but the sensitivity of cardiomyocytes to defined changes in PO2 is not known. Isolated metabolically stable rat cardiomyocytes were incubated up to 45 min at constant PO2 values ranging from 0.1 to 100 mmHg using a feedback-controlled incubation system (oxystat system). Changes of the free intracellular adenosine concentration were measured after trapping of adenosine by cytosolic S-adenosylhomocysteine (SAH) hydrolase in the presence of 200 microM L-homocysteine thiolactone. Rate of SAH formation was constant at a PO2 between 3 and 100 mmHg and gradually increased at PO2 less than 3 mmHg. Cellular ATP decreased only at PO2 less than 1 mmHg, and this was accompanied by a decline of oxygen consumption. Treatment of cells with 5.5 mM deoxyglucose and 4 micrograms/ml oligomycin increased SAH formation 60-fold and was associated with elevated intra- and to a lesser extent extracellular adenosine levels. Inhibition of nucleoside transport with 20 microM S-(p-nitrobenzyl)-6-thioinosine steepened the transmembrane adenosine gradient. Our findings suggest that the cardiomyocyte responds to metabolic poisoning and oxygen deprivation with an enhanced formation of adenosine. This adenosine is mainly formed intracellularly and reaches the extracellular space by diffusion. Threshold for adenosine formation is as low as 3 mmHg.
Adenine Nucleotides↗
O2-. release by activated Kupffer cells upon hypoxia-reoxygenation.
Primary cultures of rat liver Kupffer cells generated large amounts of superoxide anion radical (O2-.) when subjected to reoxygenation after a hypoxic period of at least 2 h. O2-. formation reached its maximum rate of approximately 25 nmol/10(6) cells within 1 h after reoxygenation. Two to four hours after reoxygenation, the number of injured cells began to increase and after 10 h approximately 60% of the cells were dead. During the period of O2-. release no significant difference in cell viability was observed between reoxygenated and hypoxically incubated cells, indicating a distinct time lag between O2-. release and onset of cell damage. Addition of diphenyliodonium, a specific inhibitor of the neutrophilic NADPH oxidase, to the Kupffer cells just before reoxygenation diminished both O2-. formation and cell injury up to 70%. Reoxygenation injury was completely prevented when superoxide dismutase and catalase were added immediately before reoxygenation. The results indicate that Kupffer cells subjected to hypoxia-reoxygenation generate a burst of reactive oxygen species and that this kind of "activation," probably by activating the NADPH oxidase, contributes to the self-destruction of the cells.
Animals↗
Two distinct effects of recombinant human tumor necrosis factor-alpha on osteoclast development and subsequent resorption of mineralized matrix.
The multifunctional cytokine tumor necrosis factor-alpha (TNF alpha) stimulates osteoclastic resorption. It is not known which steps in osteoclast formation are affected by TNF alpha. We have investigated the effects of recombinant human TNF alpha (rhTNF alpha) on osteoclast development and osteoclastic resorption in two different in vitro resorption systems which are each characterized by a different stage of development of the osteoclast. The effects were further compared to those of bovine PTH-(1-84). rhTNF alpha at concentrations between 0.01-50 ng/ml (3 x 10(-13) to 1.5 x 10(-9) M) did not alter the activity of mature osteoclasts, measured as 45Ca release in fetal mouse radii. In the osteoclast precursor-dependent system (fetal mouse metacarpals) rhTNF alpha had a biphasic effect. It stimulated resorption dose-dependently from 0.01 ng/ml onward, with a maximal response at 0.5 ng/ml. At concentrations above 10 ng/ml rhTNF alpha, resorption was inhibited. In experiments in which irradiation was used to block replication, it was found that TNF alpha stimulates the proliferation of osteoclast progenitors at both low and high concentrations. As a result, at relatively low concentrations, more osteoclasts were formed in the calcified matrix, coinciding with an increased release of 45Ca. However, at relatively high concentrations, the increase in osteoclast progenitors did not lead to increased resorption, since the putative osteoclast progenitors were arrested in the periosteum. In comparison, bovine PTH-(1-84) stimulated resorption independent of proliferation by enhancing the differentiation of postmitotic osteoclast precursors and activating mature osteoclasts. In conclusion, the effects of TNF alpha on osteoclastic resorption are dependent on the stage of osteoclast development and the concentrations applied.
Animals↗
Reoxygenation injury in rat hepatocytes: mediation by O2/H2O2 liberated by sources other than xanthine oxidase.
The mechanism of reoxygenation injury was studied in primary cultures of isolated hepatocytes from rat liver. Reoxygenation injury, which affected up to 80% of the hepatocytes, was only inducible within a certain time window of the anaerobic incubation. Reintroduction of oxygen before this vulnerable period ensured the survival of the hepatocytes. After the vulnerable period upon reintroduction of oxygen the hepatocytes continued to die in the same way as the anaerobic control. Allopurinol had no effect on reoxygenation injury. From the inhibitors of the mitochondrial respiratory chain, both cyanide and antimycin A increased injury while rotenone was without significant effect on injury. Reoxygenation injury was significantly diminished by superoxide dismutase, but not by catalase. When added together, superoxide dismutase and catalase completely prevented reoxygenation injury. The results demonstrate that reoxygenation injury in hepatocytes is mediated by the combined action of both O2- and H2O2. These reduced oxygen species are not liberated by xanthine oxidase but possibly originate from the mitochondrial respiratory chain.
Allopurinol↗