scholarly journals Subcellular localization of NAD(P)H oxidase(s) in human neutrophilic polymorphonuclear leucocytes

1978 ◽  
Vol 176 (1) ◽  
pp. 175-178 ◽  
Author(s):  
D B Iverson ◽  
P Wang-Iverson ◽  
J K Spitznagel ◽  
L R DeChatelet
Keyword(s):  
Cell Membrane ◽  
Nadph Oxidase ◽  
Subcellular Localization ◽  
Density Gradient ◽  
Membrane Fraction ◽  
Sucrose Density Gradient ◽  
Human Neutrophils ◽  
Polymorphonuclear Leucocytes

NADH and NADPH oxidase activities in a homogenate of human neutrophils co-sediment in a linear sucrose density gradient under either velocity or isopycnic conditions of centrifugation. The position of these activities in the gradient does not correspond to any known subcellular granule or to the cell-membrane fraction. These data suggest that the oxidase activities may reside in a unique granule that has previously not been recognized.

GANGLIOSIDES IN DEVELOPING RAT BRAIN: ISOLATION AND COMPOSITION OF SUBCELLULAR MEMBRANES ENRICHED IN GANGLIOSIDES

1967 ◽  
Vol 45 (5) ◽  
pp. 671-688 ◽  
Author(s):  
M. W. Spence ◽  
L. S. Wolfe
Keyword(s):  
Rat Brain ◽  
Density Gradient ◽  
Membrane Fraction ◽  
Dry Weight ◽  
Sucrose Density Gradient ◽  
Particulate Material ◽  
Neonatal Rat ◽  
Mitochondrial Fractions ◽  
Developing Rat ◽  
Neonatal Rat Brain

Neurones in the central nervous system of vertebrates contain considerable amounts of a complex group of acidic glycosphingolipids, the gangliosides. Determination of gangliosides, cholesterol, phospholipid phosphorus, neutral glycolipid hexose, and protein in developing rat brain showed that the deposition of gangliosides is predominantly a premyelin event. A light membrane fraction enriched in gangliosides relative to protein was obtained from neonatal rat-brain crude mitochondrial fractions by sucrose density-gradient centrifugations. Adult rat-brain fractions of similar density were not enriched in gangliosides because of the presence of myelin. If the initial homogenization and the separation of the crude mitochondrial fraction were carried out in 0.32 M sucrose at pH 9.2, the ganglioside enrichment of the light membrane fraction from both adult and neonatal rat brain was doubled. By further separation of the light membrane fraction on a second density gradient, particulate material was obtained from neonatal rat brain which consisted almost entirely of vesicular membrane elements. Based on dry weight, it contained gangliosides 7–9%, phospholipids 36–40%, cholesterol 5–7%, neutral glycolipids 1–3%, protein 28–29%, and cations (Na+, K+, Ca2+, Mg2+) 8%. This membrane fraction is likely derived from the neuronal plasma membrane or the endoplasmic reticulum.

scholarly journals Subcellular localization of ferritin and iron taken up by rat hepatocytes

1989 ◽  
Vol 262 (2) ◽  
pp. 685-688 ◽  
Author(s):  
J C Sibille ◽  
M Ciriolo ◽  
H Kondo ◽  
R R Crichton ◽  
P Aisen
Keyword(s):  
Acid Phosphatase ◽  
Cytochrome C ◽  
Subcellular Localization ◽  
Cytochrome C Oxidase ◽  
Density Gradient ◽  
Rat Hepatocytes ◽  
Sucrose Density Gradient ◽  
Gel Chromatography ◽  
Density Gradient Ultracentrifugation

The subcellular localization of ferritin and its iron taken up by rat hepatocytes was investigated by sucrose-density-gradient ultracentrifugation of cell homogenates. After incubation of hepatocytes with 125I-labelled [59Fe]ferritin, cells incorporate most of the labels into structures equilibrating at densities where acid phosphatase and cytochrome c oxidase are found, suggesting association of ferritin and its iron with lysosomes or mitochondria. Specific solubilization of lysosomes by digitonin treatment indicates that, after 8 h incubation, most of the 125I is recovered in lysosomes, whereas 59Fe is found in mitochondria as well as in lysosomes. As evidenced by gel chromatography of supernatant fractions, 59Fe accumulates with time in cytosolic ferritin. To account for these results a model is proposed in which ferritin, after being endocytosed by hepatocytes, is degraded in lysosomes, and its iron is released and re-incorporated into cytosolic ferritin and, to a lesser extent, into mitochondria.

scholarly journals PREPARATION OF PLASMA MEMBRANE FROM ISOLATED NEURONS

1972 ◽  
Vol 53 (3) ◽  
pp. 654-661 ◽  
Author(s):  
Fritz A. Henn ◽  
Hans-Arne Hansson ◽  
Anders Hamberger
Keyword(s):  
Plasma Membrane ◽  
Density Gradient ◽  
Electron Micrographs ◽  
Membrane Fraction ◽  
Neuronal Cell ◽  
Sucrose Density Gradient ◽  
Isolated Neurons ◽  
Fold Enrichment ◽  
Neuronal Cell Bodies ◽  
Neuronal Plasma Membrane

A bulk fraction enriched with respect to neuronal cell bodies was used as starting material for the isolation of neuronal plasma membrane The cells were gently homogenized in isotonic sucrose and a crude membrane containing fraction sedimented at 3000 g. Subsequently, the membrane fraction was purified on a discontinuous sucrose density gradient between 35% and 25 5% sucrose (w/w). Enzymatic analyses showed a 4–5-fold enrichment in plasma membrane markers, and a 10–15% contamination of mitochondrial and microsomal material. Electron micrographs of the membrane fraction confirmed the enzymatic data Fragmented membranes were found, mainly in vesicular form No ribosomes, but a few mitochondria and some multilamellar membranes were seen

scholarly journals Activation of NADPH oxidase of human neutrophils involves the phosphorylation and the translocation of cytosolic p67phox

1993 ◽  
Vol 296 (2) ◽  
pp. 367-371 ◽  
Author(s):  
S Dusi ◽  
F Rossi
Keyword(s):  
Nadph Oxidase ◽  
Membrane Fraction ◽  
Direct Evidence ◽  
Disease Patient ◽  
Human Neutrophils ◽  
Maximal Rate ◽  
Granulomatous Disease ◽  
Chronic Granulomatous Disease Patient ◽  
Phosphorylated Form ◽  
Activated Neutrophils

Activation of human neutrophil NADPH oxidase requires the interaction of cytosolic and membrane-associated components. Evidence has been accumulated that in phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils, the translocation to the plasma membrane of the cytosolic components p47phox and p67phox and the phosphorylation of p47phox are essential steps in activation of NADPH oxidase. No direct evidence has been presented to date as to whether p67phox is also phosphorylated. To address this problem we have immunoprecipitated p67phox from neutrophil cytosol and membrane fractions. The results indicate that, very soon after activation with PMA (20 s), p67phox was present in a phosphorylated form in the cytosol and in the membranes. At later times (1-3 min) the extent of p67phox phosphorylation continuously increased both in the cytosol and in the membrane fraction, while oxygen consumption reached the maximal rate within 40 s, and then remained linear. p67phox was also phosphorylated in formyl-methionyl-leucyl-phenylalanine-activated neutrophils. That the phosphorylated p67 protein we identified in immunoprecipitation experiments was p67phox was confirmed by the observation that no phosphorylated band of 67 kDa was immunoprecipitated from the cytosol and membranes of PMA-stimulated neutrophils from a p67phox-deficient chronic granulomatous disease patient. In this case, p47phox was normally phosphorylated. These data demonstrate that: (1) the phosphorylation of p67phox is correlated with activation of NADPH oxidase, and (2) continuous phosphorylation of p67phox is required in order to maintain the linearity of the respiratory burst.

scholarly journals Subcellular localization of oestrogen-induced uterine peroxidase

1976 ◽  
Vol 160 (2) ◽  
pp. 237-241 ◽  
Author(s):  
C R Lyttle ◽  
P H Jellinck
Keyword(s):  
Acid Phosphatase ◽  
Subcellular Localization ◽  
Succinate Dehydrogenase ◽  
Density Gradient ◽  
Peroxidase Activity ◽  
Sucrose Density Gradient ◽  
Urate Oxidase ◽  
Mitochondrial Marker ◽  
Membrane Bound

The distribution of oestrogen-induced peroxidase in the resuspended 8000g pellet of rat uterine homogenates was examined by centrifugation in a sucrose density gradient. Within 10h of treatment with oestradiol, peroxidase activity was found in a region devoid of catalase or urate oxidase (peroxisomal markers) which did not overlap the fractions containing succinate dehydrogenase (mitochondrial marker) or acid phosphatase (lysosomal marker). The induced uterine enzyme was localized in reticular membrane-bound vesicles with isopycnic density of 1.28g/ml from which it could be released by treatment with detergent.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

Characterization and Partial Purification of Heterodisperse Polysomal RNAs in Normal and Neoplastic Cells

1972 ◽  
Vol 58 (2) ◽  
pp. 71-94
Author(s):  
Ada Sacchi ◽  
Gianni Chinali ◽  
Susetta Pons ◽  
Michela Galdieri ◽  
Piero Cammarano
Keyword(s):  
Size Distribution ◽  
Density Gradient ◽  
Rat Liver ◽  
Sucrose Density Gradient ◽  
Partial Purification ◽  
Messenger Rnas ◽  
Alkaline Ph ◽  
Sedimentation Profile ◽  
Molecular Weights ◽  
Phenol Extraction

The size distribution of cytoplasmic messenger RNAs (m-RNA) has been studied in rat liver and in monodifferentiated cells (mouse reticulocytes and myelomas). It has been found that the RNA which exhibits a « rapid turnover » and a polydisperse profile of radioactivity is refractory to phenol extraction. This property has been exploited to selectively isolate m–RNA from the phenol residue by means of an extraction at an alkaline pH. The sucrose density gradient profiles of m–RNA isolated from monodifferentiated cells show monodisperse peaks having the sedimentation coefficients expected on the basis of the molecular weights of monocistronic messages for α and β chains of hemoglobin (reticulocytes) and L and H chains of immunoglobulin (myelomas). The sedimentation profile of cytoplasmic m–RNA associated with rat liver polysomes shows a much broader distribution, with sedimentation coefficients ranging from 8 S to 28 S.

scholarly journals Sex-dependent dysregulation of human neutrophil responses by bisphenol A

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Wioletta Ratajczak-Wrona ◽  
Marzena Garley ◽  
Malgorzata Rusak ◽  
Karolina Nowak ◽  
Jan Czerniecki ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Bisphenol A ◽  
Total Concentration ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Human Neutrophils ◽  
Boyden Chamber ◽  
No Production ◽  
Pathogen Elimination ◽  
Latex Beads

Abstract Background In the present study, we aimed to investigate selected functions of human neutrophils exposed to bisphenol A (BPA) under in vitro conditions. As BPA is classified among xenoestrogens, we compared its action and effects with those of 17β-estradiol (E2). Methods Chemotaxis of neutrophils was examined using the Boyden chamber. Their phagocytosis and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase activity were assessed via Park’s method with latex beads and Park’s test with nitroblue tetrazolium. To assess the total concentration of nitric oxide (NO), the Griess reaction was utilized. Flow cytometry was used to assess the expression of cluster of differentiation (CD) antigens. The formation of neutrophil extracellular traps (NETs) was analyzed using a microscope (IN Cell Analyzer 2200 system). Expression of the investigated proteins was determined using Western blot. Results The analysis of results obtained for both sexes demonstrated that after exposure to BPA, the chemotactic capacity of neutrophils was reduced. In the presence of BPA, the phagocytic activity was found to be elevated in the cells obtained from women and reduced in the cells from men. Following exposure to BPA, the percentage of neutrophils with CD14 and CD284 (TLR4) expression, as well as the percentage of cells forming NETs, was increased in the cells from both sexes. The stimulatory role of BPA and E2 in the activation of NADPH oxidase was observed only in female cells. On the other hand, no influence of E2 on the expression of CD14 and CD284, chemotaxis, phagocytosis, and the amount of NET-positive neutrophils was found for both sexes. The study further showed that BPA intensified NO production and iNOS expression in the cells of both sexes. In addition, intensified expression of all tested PI3K-Akt pathway proteins was observed in male neutrophils. Conclusions The study demonstrated the influence of BPA on neutrophil functions associated with locomotion and pathogen elimination, which in turn may disturb the immune response of these cells in both women and men. Analysis of the obtained data showed that the effect of this xenoestrogen on the human neutrophils was more pronounced than E2.

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