Peroxidase activity in human red cell: a biological model for excited state molecules generation.

The presence of enzymically generated triplet acetone in red cells and energy transfer to eosin, rose bengal and 9,10-dibromoanthracene-2-sulfonate was indicate by: (1) product distribution; (2) K_ET τ_o, similar to the 2-methylpropanal/peroxidase/O₂ system; (3) correlation between hemolysis, oxygen uptake and photon emission; (4) membrane protection by energy acceptors, and (5) by comparison of the 2-methylpropanal/peroxidase/O₂ system with 2-methylpropanal/red cells/membranes/O₂ and 2-methylpropanal/acid extractable protein from red cells membrane/O₂ systems, which have a high peroxidase activity.This is the first report of a biological system producing a photohemolysis effect in the dark.     

Mdm20 Modulates Actin Remodeling through the mTORC2 Pathway via Its Effect on Rictor Expression

NatB is an N-terminal acetyltransferase consisting of a catalytic Nat5 subunit and an auxiliary Mdm20 subunit. In yeast, NatB acetylates N-terminal methionines of proteins during de novo protein synthesis and also regulates actin remodeling through N-terminal acetylation of tropomyosin (Trpm), which stabilizes the actin cytoskeleton by interacting with actin. However, in mammalian cells, the biological functions of the Mdm20 and Nat5 subunits are not well understood. In the present study, we show for the first time that Mdm20-knockdown (KD), but not Nat5-KD, in HEK293 and HeLa cells suppresses not only cell growth, but also cellular motility. Although stress fibers were formed in Mdm20-KD cells, and not in control or Nat5-KD cells, the localization of Trpm did not coincide with the formation of stress fibers in Mdm20-KD cells. Notably, knockdown of Mdm20 reduced the expression of Rictor, an mTORC2 complex component, through post-translational regulation. Additionally, PKCα^S657 phosphorylation, which regulates the organization of the actin cytoskeleton, was also reduced in Mdm20-KD cells. Our data also suggest that FoxO1 phosphorylation is regulated by the Mdm20-mTORC2-Akt pathway in response to serum starvation and insulin stimulation. Taken together, the present findings suggest that Mdm20 acts as a novel regulator of Rictor, thereby controlling mTORC2 activity, and leading to the activation of PKCα^S657 and FoxO1.     

Genetic analysis of a mutation affecting ribosomal protein S1 in Escherichia coli

Molecular Genetics and Genomics - Ribosomal protein S1 from a newly isolated Escherichia coli mutant has a molecular weight of about 54,000 which is smaller than the wild type S1 (M.W. 65,000). The...     

Purification and Properties of Prostaglandin H-E Isomerase from the Cytosol of Human Brain: Identification as Anionic Forms of Glutathione S-Transferase

Abstract: Prostaglandin H-E isomerase (EC 5.3.99.3) was purified from human brain cytosol. Purification was by ammonium sulfate fractionation, diethylaminoethyl-Sephar-ose chromatography, gel filtration on a BioGel P-100 column, GSH-agarose chromatography, and MonoQ chromatography. The activity was eluted in two peaks from the MonoQ column, which were designated peaks 1 and 2. The molecular weights of peaks 1 and 2, determined by gel filtration, were 42,000 and 44,000, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peak 1 showed two bands at the molecular weights of 24,500 and 25,000, and peak 2 showed a single band at the molecular weight of 25,000, results suggesting that both were dimeric proteins. The pI values of both enzymes were ∼5.4. The enzymes catalyzed selective conversion of prostaglandin H₂ to prostaglandin E₂. The K _m values for prostaglandin H₂ of peaks 1 and 2 were 147 and 308 μ M , respectively, and the V _max values were 380 and 720 nmol/min/mg of protein, respectively. GSH was required for the catalysis of both enzymes, and no other sulfhydryl compounds could support the reaction. A part of glutathione S -transferase (EC 2.5.1.18) was copurified with peaks 1 and 2 of prostaglandin H-E isomerase. Prostaglandin H-E isomerase activity of peak 2 enzyme was competitively inhibited by 1-chloro-2,4-dinitrobenzene, a substrate of glutathione S -transferase. These results suggested that prostaglandin H-E isomerases in human brain cytosol were identical with anionic forms of glutathione S -transferase.     

Characterization of neutral glycosphingolipids from fetal human brain: evidence for stage-specific expression of the globo, ganglio, and neolacto series in the central nervous system

Neutral glycosphingolipids were isolated from normal human fetal brains, at 22 to 23 weeks gestation. They were identified as monohexosylceramides, lactosylceramide, and glycolipids belonging to the globo (globotriaosylceramide) and ganglio (gangliotriaosylceramide) series. In addition, considerable amounts of neolactotetraosylceramide and III3-alpha-fucosyl-neolactotetraosylceramide were detected. Although neutral glycolipids of the globo, ganglio, and neolacto series have been demonstrated in the brains of cases with some sphingolipidoses, they are not present in appreciable amounts in differentiated normal brain. Therefore, the present and previous observations would imply that the metabolism of these glycolipid series actively occurs in the normal brain at an early stage of differentiation and continues thereafter in the brain in the case of some sphingolipidoses. The diseased brain is most probably accompanied by a disturbance of differentiation.     

Site-directed mutagenesis of hepatitis B surface antigen sequence at codon 160 from arginine to lysine for conversion of subtypic determinant from r to w

Site-directed mutagenesis from G to A was induced at nucleotide 479 in the S gene of hepatitis B virus DNA, cloned from an individual carrying the surface antigen of subtype ayr. HepG2 cells were transfected with the plasmid DNA containing the mutant. They produced surface antigen of subtype ayw, unlike HepG2 cells harboring the parent viral DNA that produced surface antigen of subtype ayr. These results indicate that a point mutation from G to A at nucleotide 479 in the S gene, changing codon 160 for arginine to that for lysine, can convert the subtypic determinant of hepatitis B surface antigen from r to its allelic determinant w.     

Characterization of neutral glycosphingolipids from porcine erythrocyte membranes

1. Six neutral GSL fractions were purified from porcine erythrocyte membranes. 2. They were identified to be LacCer (14% of total neutral GSLs), 2-hydroxy acid-rich and -poor Gb3Cer (3 and 7%, respectively) and Gb4Cer (71%) by means of NMR spectrometry. 3. Monohexosylceramides (5%) were composed of GlcCer and GalCer with near amount. 4. All these GSL classes contained a high concentration (more than 20% of total acids in each class) of 2-hydroxy fatty acids. 5. GalCer and GlcCer contained considerable amounts of C16- and C18-acids, and of C18-phytosphingosine, whereas C24-acids and C18-sphingosine were predominant in the other GSLs. 6. A minor GSL fraction (less than 1% of total neutral GSLs) which migrated more slowly than Gb5Cer on a thin layer plate and composed of several GSL components contained L-fucose.     

Purification and properties of GM2 synthase, UDP-N-acetylgalactosamine: GM3 beta-N-acetylgalactosaminyltransferase from rat liver

A UDP-N-acetylgalactosamine:ganglioside GM3 beta-N-acetylgalactosaminyltransferase which catalyzes the conversion of ganglioside GM3 to GM2 has been purified over 6300-fold from a Triton X-100 extract of rat liver particulate fractions by hydrophobic chromatography and affinity chromatography on GM3-acid-Sepharose. The purified enzyme has two identical subunits of 64,000 daltons. The enzyme has a pH optimum of pH 6.7-6.9 and requires divalent cations such as Mn2+ and Ni2+. In studies on substrate specificity GM3 containing N-acetylneuraminic acid (GM3(NeuAc] and GM3 containing N-glycolylneuraminic acid were both good acceptors for the purified enzyme. The plots of the activity of transferase as a function of GM3(NeuAc) showed sigmoidal relationships. The oligosaccharide of GM3, sialyllactose, was also a good acceptor, which indicates that the preferred acceptor substrate has the possible structure NeuAc alpha 2- or NeuGc alpha 2-3 Gal beta 1-4Glc-OR.