Lectins for electron microscopic distinction of eosinophils from other blood cells

Colloidal gold-labeled soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), Dolichos biflorus agglutinin (DBA), and Griffonia simplicifolia lectin (GS-1) were used for electron microscopic observation of blood cells. Colloidal gold-labeled SBA, HPA, and DBA showed marked deposition on eosinophil granules at all stages of maturation. Gold particles were not deposited on basophils, neutrophils, monocytes, lymphocytes, or other blood cells. Only a few colloidal gold-labeled GS-1 were deposited on eosinophil granules. Eosinophil granules are rich in N-acetyl-D-galactosamine compounds, and the colloidal gold-labeled SBA, HPA, and DBA are useful for electron microscopic detection of eosinophil granules.     

Stimulatory effects of a short chain phosphatidate on superoxide anion production in guinea pig polymorphonuclear leukocytes

Treatment of guinea pig polymorphonuclear leukocytes (PMNL) with a phosphatidate containing short-chain fatty acids, 1,2-didecanoyl-3-sn-phosphatidate (PA10), induced substantial superoxide anion (O2-) production in a dose-dependent manner, whereas phosphatidates prepared from egg lecithin and 1,2-dioleoyl-3-sn-phosphatidate (PA18:1) had no such effect. Calcium was not involved in PA10-induced O2- production, since the production was also observed in the case of addition of EGTA prior to PA10 or pretreatment of PMNL with quin-2 and EGTA to eliminate contributions of both extracellular and intracellular calcium. We have reported in previous papers that the phosphorylation of 46K protein(s), which was commonly observed in parallel with an activation of NADPH oxidase in PMNL, was increased by treatment with 10 microM 1-oleoyl-2-acetylglycerol (OAG) with little change in the O2- production (Okamura et al. (1984) Arch. Biochem. Biophys. 228, 270-277; Ohtsuka et al. (1988) Arch. Biochem. Biophys. 260, 226-231). Treatment of PMNL with a combination of PA10, which slightly increased 46K protein phosphorylation, and such a low concentration of OAG induced a marked increase in the O2- production with the increase in 46K protein phosphorylation, which was probably due to OAG action. Thus, it is likely that this protein phosphorylation plays a significant role in the stimulation of the O2- production by phosphatidate in PMNL.     

Stabilization of ascorbate solution by chelating agents that block redox cycling of metal ions

Among the seven chelating agents tested, ethylenediamine di(o-hydroxyphenylacetic acid) and diethylenetriamine pentaacetic acid were found to almost completely inhibit ascorbate oxidation catalyzed by iron ions. The inhibition with the former chelator is due to the prevention of the reduction of Fe3+ by ascorbate, while the inhibition with the latter is caused by the strong inhibition of both this reductive reaction and the oxidation of Fe2+ by O2. These chelators almost completely inhibit ascorbate oxidation catalyzed by copper ions as well. These results indicate that the blocking of redox cycling of metal ions is important to prevent the oxidation of ascorbate.     

Fatty acid binding protein 3 as a potential mediator for diabetic nephropathy in eNOS deficient mouse

In human diabetic nephropathy, glomerular injury was found to comprise lipid droplets, suggesting that abnormal lipid metabolism might take place in the development of diabetic glomerular injury. However, its precise mechanism remains unclear. Fatty acid binding protein (FABP) is currently considered as a key molecule for lipid metabolism. Since diabetic eNOS knockout (KO) mouse is considered to be a good model for human diabetic nephropathy, we here investigated whether FABP could mediate glomerular injury in this model. We found that glomerular injuries were associated with inflammatory processes, such as macrophage infiltration and MCP-1 induction. Microarray assay with isolated glomeruli revealed that among 10 isoforms in FABP family, FABP3 mRNA was most highly expressed in diabetic eNOSKO mice compared to non-diabetic eNOSKO mice. FABP3 protein was found to be located in the mesangial cells. Overexpression of FABP3 resulted in a greater response to palmitate, a satulated FA, to induce MCP-1 in the rat mesangial cells. In turn, the heart, a major organ for FABP3 protein in normal condition, failed to alter its expression level under diabetic condition in either wild type or eNOSKO mice. In conclusion, FABP3 is induced in the mesangial cells and likely a mediator to induce MCP-1 in the diabetic nephropathy.     

Purification of hepatic N-hydroxyarylamine sulfotransferases and their regulation by growth hormone and thyroid hormone in rats.

From liver cytosols of male Sprague-Dawley rats, two N-hydroxyarylamine sulfotransferases (HASTs) which sulfate N-hydroxy-2-acetylaminofluorene and a phenolsulfotransferase (PST-I) have been purified about 290-, 690-, and 210-fold, respectively, by the use of DEAE-anion exchange, Blue-Sepharose CL-6B, DEAE-HPLC, and ATP-agarose affinity chromatography. All three enzymes showed almost the same molecular weight of 33 kDa on SDS-polyacrylamide gel electrophoresis. In Western blots using antibody raised against HAST I, the two HASTs, but not PST-I, were detected. The content of total HAST in male rats was estimated as 4-5 micrograms/mg cytosolic protein, about 4 times higher than that in female rats. Direct comparison of the DEAE-HPLC elution profiles of cytosol showed that HAST I and HAST II were male-dominant and male-specific, respectively, in their expression in the livers. Hypophysectomy decreased the level of HAST by 60% in male rats, but had no obvious effect in female rats. Intermittent injection of growth hormone to mimic the male secretory pattern raised the content in hypophysectomized rats of both sexes close to that in intact male rats, while the continuous infusion of growth hormone to mimic the female secretory pattern showed limited effects. In addition, the administration of triiodothyronine stimulated HAST in hypophysectomized rats of both sexes, and the extent of stimulation was nearly the same as observed in the male-type growth hormone treatment. PST-I, in contrast to HAST, showed no clear sex-related difference in hepatic content, and was not apparently affected by growth hormone or triiodothyronine.(ABSTRACT TRUNCATED AT 250 WORDS)