scholarly journals Predominant role of catalase in the disposal of hydrogen peroxide within human erythrocytes

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1595-1599 ◽  
Author(s):  
GF Gaetani ◽  
AM Ferraris ◽  
M Rolfo ◽  
R Mangerini ◽  
S Arena ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Glutathione Peroxidase ◽  
Human Erythrocytes ◽  
Predominant Role ◽  
Intact Cells ◽  
Free System ◽  
Major Function ◽  
Relative Contribution ◽  
A Cell

Purified enzymes were mixed to form a cell-free system that simulated the conditions for removal of hydrogen peroxide within human erythrocytes. Human glutathione peroxidase disposed of hydrogen peroxide (H2O2) at a rate that was only 17% of the rate at which human catalase simultaneously removed hydrogen peroxide. The relative rates observed were in agreement with the relative rates predicted from the kinetic constants of the two enzymes. These results confirm two earlier studies on intact erythrocytes, which refuted the notion that glutathione peroxidase is the primary enzyme for removal of hydrogen peroxide within erythrocytes. The present findings differ from the results with intact cells, however, in showing that glutathione peroxidase accounts for even less than 50% of the removal of hydrogen peroxide. A means is proposed for calculating the relative contribution of glutathione peroxidase and catalase in other cells and species. The present results raise the possibility that the major function of glutathione peroxidase may be the disposal of organic peroxides rather than the removal of hydrogen peroxide.

scholarly journals Hyaluronate synthetase inhibition by normal and transformed human fibroblasts during growth reduction

1987 ◽  
Vol 104 (4) ◽  
pp. 1105-1115 ◽  
Author(s):  
K Matuoka ◽  
M Namba ◽  
Y Mitsui
Keyword(s):  
Cell Proliferation ◽  
Cell Density ◽  
Human Fibroblasts ◽  
Transformed Cells ◽  
Synthetase Activity ◽  
Intact Cells ◽  
Free System ◽  
Glycosaminoglycan Synthesis ◽  
Cell Free System ◽  
A Cell

To establish the relation of glycosaminoglycan synthesis to cell proliferation, we investigated the synthesis of individual glycosaminoglycan species by intact cells and in a cell-free system, using normal and transformed human fibroblasts under differing culture conditions. Reducing serum concentration brought about a marked decline in the synthesis of hyaluronate (HA) as well as cell proliferation on both normal and transformed cells. Both HA synthesis and proliferation decreased with increasing cell densities markedly (in inverse proportion to cell density) in normal cells but gradually in transformed cells. This noticeable congruity of the changes in HA synthesis and proliferation indicates that the change in HA synthesis is related primarily to cell proliferation rather than to cell density or cellular transformation. Examination of HA synthesis in a cell-free system demonstrated that the activity of HA synthetase also fluctuated in conjunction with cell proliferation. Furthermore, growth-reduced cells (except crowded transformed cells) inhibited cell-free HA synthesis and this inhibition was induced coincidentally with a decrease in both HA synthetase activity and proliferation. These findings suggest that the change in HA synthesis is significant in the regulation of cell proliferation.

Photoreaktivierung von UV-inaktivierter Polyuridylsäure

1964 ◽  
Vol 19 (5) ◽  
pp. 406-408 ◽  
Author(s):  
Adolf Wacker ◽  
Makoto Ishimoto ◽  
Prakash Chandra ◽  
Reinhold Selzer
Keyword(s):  
Hydrogen Peroxide ◽  
Visible Light ◽  
Uv Irradiation ◽  
Trichloroacetic Acid ◽  
Uranyl Acetate ◽  
Free System ◽  
E Coli ◽  
Polyuridylic Acid ◽  
A Cell ◽  
The One

A study on the effect of UV-irradiated polyuridylic acid on the incorporation of phenylalanine into the polypeptide precipitable through trichloroacetic acid, in a cell-free system from E. coli was made. Attempts were made to reactivate the UV-inactivated polyuridylic acid through hydrogen peroxide, uranyl acetate and visible light. We could show that polyuridylic acid irradiated at a dose of 1.2 ×105 ergs/mm2 could be completely reactivated, while the one irradiated at a higher dose of 2.4 ×105 ergs/mm2 could not be completely reactivated under the conditions of our experiment. We have studied the effects of hydrogen peroxide and uranyl acetate on UV-irradiated polyuridylic acid chemically as well. Our results altogether show that the photoreactivating effect of uranyl acetate and hydrogen peroxide is due to their ability to split the uracil dimers formed during UV-irradiation.

Initiation of protein synthesis in a cell-free system prepared from rat hepatocytes

1989 ◽  
Vol 256 (1) ◽  
pp. C28-C34 ◽  
Author(s):  
S. R. Kimball ◽  
W. V. Everson ◽  
K. E. Flaim ◽  
L. S. Jefferson
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Hepatocytes ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Free System ◽  
Cell Free System ◽  
A Cell

A cell-free system, which maintained a linear rate of protein synthesis for up to 20 min of incubation, was prepared from isolated rat hepatocytes. The rate of protein synthesis in the cell-free system was approximately 20% of the rate obtained in isolated hepatocytes or perfused liver. More than 70% of total protein synthesis in the cell-free system was due to reinitiation, as indicated by addition of inhibitors of initiation, i.e., edeine or polyvinyl sulfate. The rate of protein synthesis and formation of 43S initiation complexes in the cell-free system were reduced to 60 and 30% of the control values, respectively, after incubation of hepatocytes in medium deprived of an essential amino acid. Therefore, the cell-free system maintained the defect in initiation induced in the intact cells by amino acid deprivation. The defect in initiation was corrected by addition of either rat liver eukaryotic initiation factor 2 or the guanine nucleotide exchange factor (GEF) to the cell-free system. A role for GEF in the defect in initiation was further implicated by experiments that showed that the activity of the factor was decreased in extracts from livers perfused with medium deficient in amino acids. The cell-free system should provide a valuable tool for investigation of mechanisms involved in the regulation of initiation of protein synthesis.

scholarly journals Synthesis of 8-epi-prostaglandin F2α by human endothelial cells: role of prostaglandin H2 synthase

1999 ◽  
Vol 344 (3) ◽  
pp. 747-754 ◽  
Author(s):  
Michael T. WATKINS ◽  
George M. PATTON ◽  
Hiram M. SOLER ◽  
Hassan ALBADAWI ◽  
Donald E. HUMPHRIES ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Endothelial Cells ◽  
Umbilical Artery ◽  
Prostaglandin F2α ◽  
Human Endothelial Cells ◽  
Microcarrier Beads ◽  
A Cell ◽  
Superoxide Scavenger ◽  
Negative Cell Line

The experiments described in this paper were designed to determine the mechanism underlying the increase in 8-isoprostaglandin F2α (8-epi-PGF2α) production by cultured human endothelial cells during reoxygenation following hypoxia. Human umbilical artery endothelial cells were grown on microcarrier beads and exposed to sequential periods of normoxia, hypoxia, and reoxygenation. The amount of 8-epi-PGF2α in the medium was determined by ELISA. The production of 8-epi-PGF2α decreased by greater than 90% during hypoxia. Upon reoxygenation 8-epi-PGF2α production increased linearly for 90 min reaching nearly 3 times normoxic levels. When added to the medium during reoxygenation, neither superoxide dismutase nor Tiron, a cell-permeable superoxide scavenger, inhibited 8-epi-PGF2α production. However, 8-epi-PGF2α production was inhibited by catalase. The production of 8-epi-PGF2α was also inhibited by indomethacin and aspirin. Exogenous hydrogen peroxide stimulated 8-epi-PGF2α production by normoxic cells, and aspirin inhibited the hydrogen peroxide-mediated increase in 8-epi-PGF2α production. These results indicate that the reactive oxygen species responsible for 8-epi-PGF2α synthesis during reoxygenation is hydrogen peroxide and that in endothelial cells 8-epi-PGF2α synthesis is mediated by prostaglandin H2 synthase (PGHS). To verify the role of PGHS in 8-epi-PGF2α synthesis, human PGHS-1 was expressed in COS-7 cells, a PGHS negative cell line that does not synthesize 8-epi-PGF2α. In the presence of exogenous arachidonic acid the COS-7 cells expressing human PGHS-1 produced substantial amounts of PGE2 and 8-epi-PGF2α. These data indicate that human PGHS-1 can support the synthesis of 8-epi-PGF2α and that 8-epi-PGF2α synthesis by cultured human endothelial cells during reoxygenation is dependent on the activity of PGHS-1.

Familial Overexpression of β Antithrombin Caused by an Asn135Thr Substitution

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4242-4247
Author(s):  
T.A. Bayston ◽  
A. Tripodi ◽  
P.M. Mannucci ◽  
E. Thompson ◽  
H. Ireland ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Consensus Sequence ◽  
Wild Type ◽  
Antithrombin Deficiency ◽  
Free System ◽  
Heparin Interaction ◽  
A Cell ◽  
Variant Protein ◽  
Very High

We have investigated the basis of antithrombin deficiency in an asymptomatic individual (and family) with borderline levels (≈70% antigen and activity) of antithrombin. Direct sequencing of amplified DNA showed a mutation in codon 135, AAC to ACC, predicting a heterozygous Asn135Thr substitution. This substitution alters the predicted consensus sequence for glycosylation, Asn-X-Ser, adjacent to the heparin interaction site of antithrombin. The antithrombin isolated from plasma of the proband by heparin-Sepharose chromatography contained amounts of β antithrombin (the very high affinity fraction) greatly increased (≈20% to 30% of total) above the trace levels found in normals. Expression of the residue 135 variant in both a cell-free system and COS-7 cells confirmed altered glycosylation arising as a consequence of the mutation. Wild-type and variant protein were translated and exported from COS-7 cells with apparently equal efficiency, in contrast to the reduced level of variant observed in plasma of the affected individual. This case represents a novel cause of antithrombin deficiency, removal of glycosylation concensus sequence, and highlights the potentially important role of β antithrombin in regulating coagulation.

scholarly journals Stimulatory and inhibitory actions of lysophosphatidylcholine, depending on its fatty acid residue, on the phospholipase C/Ca2+ system in HL-60 leukaemia cells

1998 ◽  
Vol 336 (2) ◽  
pp. 491-500 ◽  
Author(s):  
Fumikazu OKAJIMA ◽  
Koichi SATO ◽  
Hideaki TOMURA ◽  
Atsushi KUWABARA ◽  
Hiromi NOCHI ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Phospholipase C ◽  
Inositol Phosphate ◽  
Inhibitory Effect ◽  
Fatty Acid Residue ◽  
Stimulatory Action ◽  
Intact Cells ◽  
Free System ◽  
A Cell ◽  
G Protein Coupled

We examined the mechanism of action of lysophosphatidylcholine (LPC), which is suggested to be involved in the pathogenesis of atherosclerosis and inflammatory disorders, in HL-60 leukaemia cells. Extracellular 1-palmitoyl LPC increased the intracellular Ca2+ concentration in association with production of inositol phosphate. These actions of LPC were markedly inhibited by treatment of the cells with pertussis toxin and U73122, a phospholipase C inhibitor. The lipid-induced stimulation of the phospholipase C/Ca2+ system was also attenuated in the dibutyryl cAMP-induced differentiated (neutrophil-like) cells, in which phospholipase C activation induced by NaF or formyl-Met-Leu-Phe was enhanced. In contrast with the stimulatory action of 1-palmitoyl LPC, 1-stearoyl LPC was inhibitory for the phospholipase C/Ca2+ system stimulated by NaF as well as by 1-palmitoyl LPC or other Ca2+-mobilizing agonists. In a cell-free system, only an inhibitory effect on phospholipase C activity was observed even by 1-palmitoyl LPC; 1-stearoyl LPC was more inhibitive than 1-palmitoyl LPC. Taken together, these results suggest that atherogenic and inflammatory LPC exerts both stimulatory and inhibitory actions on the phospholipase C/Ca2+ system depending on the species of fatty acid residue of the lipid; the stimulatory effect is possibly mediated through G-protein-coupled receptors; the inhibitory effect might be caused by dysfunction of the components involved in the enzyme system owing to the amphiphilic nature of the lipid. 1-Palmitoyl LPC prefers the former receptor stimulation at least in intact cells, but 1-stearoyl LPC preferentially exerts the latter inhibitory action.

scholarly journals Aster migration determines the length scale of nuclear separation in the Drosophila syncytial embryo

2012 ◽  
Vol 197 (7) ◽  
pp. 887-895 ◽  
Author(s):  
Ivo A. Telley ◽  
Imre Gáspár ◽  
Anne Ephrussi ◽  
Thomas Surrey
Keyword(s):  
Early Embryo ◽  
Length Scale ◽  
Free System ◽  
Nuclear Separation ◽  
A Cell ◽  
Distinct Distance ◽  
Spindle Elongation ◽  
Insect Embryos ◽  
Microtubule Aster

In the early embryo of many species, comparatively small spindles are positioned near the cell center for subsequent cytokinesis. In most insects, however, rapid nuclear divisions occur in the absence of cytokinesis, and nuclei distribute rapidly throughout the large syncytial embryo. Even distribution and anchoring of nuclei at the embryo cortex are crucial for cellularization of the blastoderm embryo. The principles underlying nuclear dispersal in a syncytium are unclear. We established a cell-free system from individual Drosophila melanogaster embryos that supports successive nuclear division cycles with native characteristics. This allowed us to investigate nuclear separation in predefined volumes. Encapsulating nuclei in microchambers revealed that the early cytoplasm is programmed to separate nuclei a distinct distance. Laser microsurgery revealed an important role of microtubule aster migration through cytoplasmic space, which depended on F-actin and cooperated with anaphase spindle elongation. These activities define a characteristic separation length scale that appears to be a conserved property of developing insect embryos.

Role of calreticulin in the sensitivity of myocardiac H9c2 cells to oxidative stress caused by hydrogen peroxide

2006 ◽  
Vol 290 (1) ◽  
pp. C208-C221 ◽  
Author(s):  
Yoshito Ihara ◽  
Yoshishige Urata ◽  
Shinji Goto ◽  
Takahito Kondo
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Molecular Chaperone ◽  
Vital Role ◽  
H9c2 Cells ◽  
Cardiac Physiology ◽  
Caspase 12 ◽  
A Cell ◽  
Induced Apoptosis

Calreticulin (CRT), a Ca2+-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac apoptosis in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In the present study, the effect of overexpression of CRT on susceptibility to apoptosis under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. Under oxidative stress due to H2O2, the CRT-overexpressing cells were highly susceptible to apoptosis compared with controls. In the overexpressing cells, the levels of cytoplasmic free Ca2+ ([Ca2+]i) were significantly increased by H2O2, whereas in controls, only a slight increase was observed. The H2O2-induced apoptosis was enhanced by the increase in [Ca2+]i caused by thapsigargin in control cells but was suppressed by BAPTA-AM, a cell-permeable Ca2+ chelator in the CRT-overexpressing cells, indicating the importance of the level of [Ca2+]i in the sensitivity to H2O2-induced apoptosis. Suppression of CRT by the introduction of the antisense cDNA of CRT enhanced cytoprotection against oxidative stress compared with controls. Furthermore, we found that the levels of activity of calpain and caspase-12 were elevated through the regulation of [Ca2+]i in the CRT-overexpressing cells treated with H2O2 compared with controls. Thus we conclude that the level of CRT regulates the sensitivity to apoptosis under oxidative stress due to H2O2 through a change in Ca2+ homeostasis and the regulation of the Ca2+-calpain-caspase-12 pathway in myocardiac cells.

scholarly journals Direct Evidence for Catalase as the Predominant H2O2 -Removing Enzyme in Human Erythrocytes

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4973-4978 ◽  
Author(s):  
Sebastian Mueller ◽  
Hans-Dieter Riedel ◽  
Wolfgang Stremmel
Keyword(s):  
Hydrogen Peroxide ◽  
Glutathione Peroxidase ◽  
Enzyme Inhibition ◽  
Direct Evidence ◽  
Human Erythrocytes ◽  
H2o2 Concentration ◽  
H2o2 Decomposition ◽  
Erythrocyte Catalase ◽  
Normal Human ◽  
Kinetics Of

Abstract Decomposition of hydrogen peroxide (H2O2 ) at physiological levels was studied in human erythrocytes by means of a recently developed sensitive H2O2 assay. The exponential decay of H2O2 in the presence of purified erythrocyte catalase was followed down to 10−9 mol/L H2O2 at pH 7.4. H2O2 decomposition by purified erythrocyte glutathione peroxidase (GPO) could be directly observed down to 10−7 mol/L H2O2 . No enzyme inhibition was observed at these low H2O2 concentrations. Catalase and GPO activities can be determined separately in a titrated mixture of purified enzymes, which simulates the conditions of H2O2 removal by the erythrocyte. Experiments with fresh human hemolysate allowed us to determine H2O2 decomposition by catalase and GPO using these enzymes in their original quantitative ratio. The different kinetics of these enzymes are shown: H2O2 decomposition by catalase depends linearly on H2O2 concentration, whereas that by GPO becomes saturated at concentrations above 10−6 mol/L H2O2 . Even at very low H2O2 concentrations GPO reaches only approximately 8% of the rate at which catalase simultaneously degrades H2O2 . These data indicate an almost exclusive role for catalase in the removal of H2O2 in normal human erythrocytes.

Familial Overexpression of β Antithrombin Caused by an Asn135Thr Substitution

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4242-4247 ◽  
Author(s):  
T.A. Bayston ◽  
A. Tripodi ◽  
P.M. Mannucci ◽  
E. Thompson ◽  
H. Ireland ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Consensus Sequence ◽  
Wild Type ◽  
Antithrombin Deficiency ◽  
Free System ◽  
Heparin Interaction ◽  
A Cell ◽  
Variant Protein ◽  
Very High

Abstract We have investigated the basis of antithrombin deficiency in an asymptomatic individual (and family) with borderline levels (≈70% antigen and activity) of antithrombin. Direct sequencing of amplified DNA showed a mutation in codon 135, AAC to ACC, predicting a heterozygous Asn135Thr substitution. This substitution alters the predicted consensus sequence for glycosylation, Asn-X-Ser, adjacent to the heparin interaction site of antithrombin. The antithrombin isolated from plasma of the proband by heparin-Sepharose chromatography contained amounts of β antithrombin (the very high affinity fraction) greatly increased (≈20% to 30% of total) above the trace levels found in normals. Expression of the residue 135 variant in both a cell-free system and COS-7 cells confirmed altered glycosylation arising as a consequence of the mutation. Wild-type and variant protein were translated and exported from COS-7 cells with apparently equal efficiency, in contrast to the reduced level of variant observed in plasma of the affected individual. This case represents a novel cause of antithrombin deficiency, removal of glycosylation concensus sequence, and highlights the potentially important role of β antithrombin in regulating coagulation.

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