Reactive oxygen species in phagocytic leukocytes

Phagocytic leukocytes consume oxygen and generate reactive oxygen species in response to appropriate stimuli. The phagocyte NADPH oxidase, a multiprotein complex, existing in the dissociated state in resting cells becomes assembled into the functional oxidase complex upon stimulation and then generates superoxide anions. Biochemical aspects of the NADPH oxidase are briefly discussed in this review; however, the major focus relates to the contributions of various modes of microscopy to our understanding of the NADPH oxidase and the cell biology of phagocytic leukocytes

Light and electron microscopical demonstration of the ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity (K-NPPase) using a Ce-Mg-double capture technique

The cerium-based method of Kobayashi et al. for the histochemical demonstration of K-NPPase activity was improved. Besides Ce3+ additionally Mg2+ ions as orthophosphate capture were employed (double capture technique). For light microscopical purposes the Mg-phosphate was converted into Ce-phosphate by treatment of the sections with Ce-citrate yielding higher quantity of reaction product. Unspecific background staining was eliminated by EGTA. In the electron microscope this technique brought about fine granular reaction products without diffusion artefacts

Extralysosomal localisation of acid phosphatase in the rat kidney

There is strong evidence that acid phosphatase (AcPase) plays an important role in the catabolism of the glomerular basement membrane (GEM) and the removal of macromolecular debris resulting from ultrafiltration. Recent enzyme histochemical investigations provide new evidence of the antithrombotic and anti-inflammatory function of ADPase and on the distribution of AcPase in mouse kidney tubule cells. By means of 3 mM cerium as the trapping agent and 1 mM p-nitrophenyl phosphate as the substrate, extralysosomal AcPase could be demonstrated at the ultrastructural level. Following a mild per fusion fixation (2% formaldehyde + 0.07% glutaraldehyde), an effective postfixation and short enzyme incubations (20 min) with microwave irradiation, highly specific enzyme histochemical reaction product and reasonable structural preservation were obtained. Extralysosomal, membrane-bound AcPase was observed along the endoplasmic reticulum, the trans-Golgi cisternae, the nuclear envelope, basal infoldings of the proximal and distal tubular cells and on glomerular profiles, e.g. cell membranes of podocytes, endothelium and basement membrane. Large amounts of extralysosomal AcPase were observed in the basement membrane of glomeruli, in contrast to no AcPase activity in the tubular and mesangial basement membrane. The observed difference in AcPase activity in the tubular epithelial basement membrane and the GEM supports the idea that AcPase in GEM specifically serves in the clearance of macromolecular debris to facilitate ultrafiltration. In the GEM a laminar distribution is observed, suggesting that both epithelial and endothelial cells are involved in the production of AcPase.</p

Cerium as amplifying agent--an improved cerium-perhydroxide-DAB-nickel (Ce/Ce-H2O2-DAB-Ni) method for the visualization of cerium phosphate in resin sections

A new visualization (Ce/Ce-H2O2-DAB-Ni) procedure for cerium (Ce III) phosphate in semithin and ultrathin plastic sections (Epon 812, Lowicryl K4M, glycol methacrylate) of rat kidney tissues that had been incubated before embedding for the demonstration of phosphatases (alkaline and acid phosphatase, 5(1)-nucleotidase, Mg-dependent ATPase) is described. For this purpose the hydrophobic Epon resin was removed in NaOH-ethanol solution, whereas the hydrophilic Lowicryl and methacrylate sections did not required any etching. The primary reaction product Ce III-phosphate was amplified in a Ce III-citrate solution, subsequently oxidized with H2O2 and then visualized in a H2O2 containing DAB-nickel medium (Ce IV-perhydroxy induced DAB polymerization principle). The method yielded a very clear localization of enzyme activity. The final reaction product (DAB-nickel polymers) in in 0.5 - 2.0-mu-m semithin sections is blue-black; the background staining is completely prevented. An increase of the staining contrast was obtained by posttreatment with O(s)O4 (osmium black formation). Furthermore, the enzyme reaction product could be demonstrated in 40 nm thick ultrathin sections by silver intensification, which utilized the high argyrophilia of the polymerized DAB-nickel complexes. This procedure replaces the earlier published technique