Augmentation of retinoic acid-induced granulocytic differentiation in HL-60 leukemia cells by serine/threonine protein phosphatase inhibitors.

To evaluate the involvement of protein phosphatases (PP) in differentiation of human myelogenous leukemia HL-60 cells, we made use of potent inhibitors of PP1 and PP2A, calyculin-A (CAL-A) and okadaic acid (OKA). CAL-A and OKA could augment all-trans retinoic acid (ATRA)-induced granulocytic differentiation, whereas the differentiation toward macrophage lineage by 12-o-tetradecanoylphorbol acetate (TPA) was unchanged in the presence of CAL-A. CAL-A augmented the phosphorylation of 18K, 23K and 30K proteins induced by ATRA. The PP1 and PP2A were identified and were present mainly in the cytosol of HL-60 cells. These results suggest that either PP1 or PP2A or both may be involved in regulating granulocytic differentiation of HL-60 cells.     

Neurocalcin: a novel calcium-binding protein from bovine brain.

A novel calcium-binding protein (molecular weight 23,000-24,000, pI 5.3-5.5), which we term neurocalcin, was identified in bovine brain. Using calcium-dependent drug affinity chromatography ((S)-P-(2-aminoethyloxy)-N-[2-(4-benzyloxycarbonylpiperazinyl++ +)-1-(P- methoxybenzyl)ethyl]-N-methylbenzene-sulfonamide dihydrochloride, W-77, -coupled Sepharose 6B), we purified neurocalcin from bovine brain. The partial amino acid sequence of neurocalcin revealed it to be an as yet unidentified protein with three putative calcium binding sites (EF-hands). Further purification and sequence analysis demonstrated the presence of four isoprotein forms designated alpha, beta, gamma 1, and gamma 2. When the 165 sequenced residues of neurocalcin beta are compared with sequences of other proteins, neurocalcin beta has a 38.2% sequence homology with visinin and 45.5% with recoverin (Yamagata, K., Goto, K., Kuo, C.-H., Kondo, H., and Miki, N. (1990) Neuron 2, 469-476; Dizhoor, A. M., Ray, S., Kumar, S., Niemi, G., Spencer, M., Brolley, D., Walsh, K. A., Philipov, P. P., Hurley, J. B., and Stryer, L. (1991) Science 251, 915-918). Both visinin and recoverin are expressed specifically in retinal photoreceptors and are not found in brain. Unlike visinin and recoverin, neurocalcin is purified not only from retina but also from bovine brain. Our results suggest that neurocalcin is a recoverin-like protein expressed in bovine brain.     

Genetic polymorphism of alpha-2-HS-glycoprotein: four new alleles and allele frequencies in Japanese

alpha 2-HS-glycoprotein (AHSG) phenotyping was done in 655 Japanese from the Goto Islands, western Japan, using isoelectric focusing followed by immunoblotting. Four new AHSG alleles were encountered, AHSG*G1-G4, whose genetic transmissions were established in family studies. The allele frequencies were: AHSG*1 = 0.7221; AHSG*2 = 0.2748, and AHSG*G1-G4 = 0.0008, respectively.     

Neutralization of microcystin shock in mice by tumor necrosis factor alpha antiserum.

Microcystis aeruginosa is a common cyanobacterium in water blooms that appear widely in nutrient-rich, fresh, and brackish waters, and its toxic blooms cause the death of domestic animals. The administration of a crude toxic cell extract of M. aeruginosa K-139 to mice can produce tumor necrosis factor (TNF) and prompt severe physiological disturbances, especially liver damage, which can lead to death. The in vitro production of TNF-alpha by peritoneal macrophages was observed after stimulation with the cell extract or the purified toxin from K-139 cells. The expression of a TNF-alpha mRNA was also detected in spleen cells and peritoneal macrophages after stimulation with the cell extract. However, a previous injection of rabbit anti-murine TNF-alpha serum could prevent the liver damage to some extent and protect the mice from death. These findings indicate the involvement of TNF in microcystin shock.     

Oxidative modification of glycated low density lipoprotein in the presence of iron

This is the first observation for contributing to the glycation of low density lipoprotein (LDL) to oxidative modification of its own lipids and protein. Human plasma LDL was glycated by incubation with glucose (G-LDL). Glucose incorporated into apoprotein B was approximately 10 mol/mol of apoprotein (2.8% modification of lysine residues) and 84% of G-LDL was adsorbed on phenylboronate affinity column. G-LDL incubated with Fe3+ for 4 h caused a significantly higher level of lipid peroxidation than U-LDL (untreated with glucose), and a higher molecular weight protein was observed in apoprotein B on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), increasing with incubation period. Corresponding to change on SDS-PAGE, G-LDL exposed to Fe3+ moved faster than G-LDL per se or U-LDL to anode on agarose gel electrophoresis. The higher the Fe3+ concentration, the more lipid peroxidation was caused. Alpha-tocopherol or probucol suppressed the lipid peroxidation of G-LDL exposed to Fe3+.     

Isolation of two forms of decay-accelerating factor (DAF) from human urine.

Decay-accelerating factor (DAF) was purified from human pooled urine by conventional techniques. The urine DAF was separated into two peaks, pool I and pool II, by gel chromatography. DAF-U1 was isolated from pool I by hydrophobic chromatography, and DAF-U2 from pool II by anti-DAF IgG column. The specific activities of DAF-U1 and DAF-U2 to decay membrane-phase C5 convertase were about 3% and 70% of membrane form DAF, respectively. However, both urine DAFs revealed a similar activity to each other and slightly higher activity than that of membrane form DAF in decay-accelerating fluid-phase C3 convertase of the alternative pathway.     

Establishment and characterization of new cell lines from human renal cell carcinoma

We have characterized seven human renal cell carcinoma cell lines established from primary sites of five patients between 1987 and 1989. Two lines, OUR-20P and OUR-20S, were derived from the OUR-20 cells by cloning with a dilution method 3 months after the primary culture. These three cell lines were tumorigenic in athymic nude mice when inoculated subcutaneously. Examined by a dye uptake method, OUR-20 was highly sensitive to interferon-alpha (IFN-alpha); OUR-20P, OUR-20S and OUR-30 showed slight sensitivities, while the other three cell lines were insensitive. All seven cell lines have been maintained for more than 2 years and over 50 passages in vitro. Cytogenetic analyses performed 1.5 to 3 years after the starts of primary cultures indicated that all seven cell lines, which exhibited different morphologies in phase-contrast micrographs, were aneuploid with modal chromosome numbers 41 to 89.     

Further fractionation of the glycoprotein families of porcine zona pellucida by anion-exchange HPLC and some characterization of the separated fractions

The glycoproteins of porcine zonae pellucidae have been fractionated into three families (PZP1-3) by gel filtration HPLC [Nakano et al. (1987) Biochem. Int. 14, 417-423]. However, they still comprise heterogeneous molecular species differing in electric charge. We found that sulfate, but not phosphate, is contained in PZP1-3 by a simple and rapid method for microanalysis of the anionic groups. These families were efficiently separated into many fractions by anion-exchange HPLC. When elution was performed by stepwise increase in NaCl concentration in 8 M urea/20 mM Tris-HCl, pH 8.0, a single distinctive peak emerged for each step. The analyses of amino acids, monosaccharides, and anions of the eight separated fractions of the major family, PZP3, showed that larger amounts of sulfated lactosamine linked to the constituent proteins are present in the fractions that are eluted later: the chain length and/or the chain number of these polylactosamines and the sulfate content increased with stepwise increase in NaCl concentration. Composition analyses also revealed that twice as much N-glycolylneuraminic acid is present as N-acetylneuraminic acid in all fractions. The contents of these sialic acids in the fractions slightly increased in the order of elution. These results together with those of the analyses of endo-beta-galactosidase digests showed that the charge heterogeneity of the porcine zona proteins is due mainly to differences in the amount of sulfated lactosamine, which is predominantly distributed in the non-reducing regions of the sugar chains.