A sensitive method to screen for hydroxyl radical scavenging activity in natural food extracts using competitive inhibition ELISA for 8-hydroxy deoxyguanosine

Summary Representative endogenous antioxidants and natural food extracts were screened for hydroxyl radical scavenging activity by an ELISA. Whereas conventional assays for hydroxyl radical scavenging activity use either spin traps following the induction of Fenton reaction or measure thiobarbituric acid-reactive substances, this assay measures 8-hydroxy deoxyguanosine liberated from the hydroxylation of deoxyguanosine by Cu²⁺/ascorbate system.     

Development of a protein–ligand-binding site prediction method based on interaction energy and sequence conservation

We present a new method for predicting protein–ligand-binding sites based on protein three-dimensional structure and amino acid conservation. This method involves calculation of the van der Waals interaction energy between a protein and many probes placed on the protein surface and subsequent clustering of the probes with low interaction energies to identify the most energetically favorable locus. In addition, it uses amino acid conservation among homologous proteins. Ligand-binding sites were predicted by combining the interaction energy and the amino acid conservation score. The performance of our prediction method was evaluated using a non-redundant dataset of 348 ligand-bound and ligand-unbound protein structure pairs, constructed by filtering entries in a ligand-binding site structure database, LigASite. Ligand-bound structure prediction (bound prediction) indicated that 74.0 % of predicted ligand-binding sites overlapped with real ligand-binding sites by over 25 % of their volume. Ligand-unbound structure prediction (unbound prediction) indicated that 73.9 % of predicted ligand-binding residues overlapped with real ligand-binding residues. The amino acid conservation score improved the average prediction accuracy by 17.0 and 17.6 points for the bound and unbound predictions, respectively. These results demonstrate the effectiveness of the combined use of the interaction energy and amino acid conservation in the ligand-binding site prediction.     

A high-throughput sequence analysis of Japanese patients revealed 11 candidate genes associated with type 1 autoimmune pancreatitis susceptibility

The pathogenesis of autoimmune pancreatitis is unknown. In the present study we used high-throughput sequencing with next generation sequencing to identify the candidate genes associated with AIP. A total of 27 type 1 AIP patients and 30 healthy blood donors were recruited, and DNA samples were isolated from their mononuclear cells. A high-throughput sequencer with an original custom panel of 1031 genes was used to detect the genetic variants in each sample. Polymorphisms of CACNA1S (c.4642C>T), rs41554316, rs2231119, rs1042131, rs2838171, P2RX3 (c.195delG), rs75639061, SMAD7 (c.624delC) and TOP1 (c.2007delG), were identified as candidate genetic variants in patients with type 1 AIP. P2RX3 and TOP1 were significantly associated with AIP, even after adjusting bay means of Bonferroni's correction. In addition, we also identified eight candidate genetic variants that were associated with the relapse of type 1 AIP, namely: rs1143146, rs1050716, HLA-C (c.759_763delCCCCCinsTCCCG), rs1050451, rs4154112, rs1049069, CACNA1C (c.5996delC) and CXCR3 (c.630_631delGC). Finally polymorphisms of rs1050716 and rs111493987 were identified as candidate genetic variants associated with extra-pancreatic lesions in patients with type 1 AIP. These candidates might be used as markers of AIP susceptibility and could contribute to the pathogenesis of type 1 AIP.     

Phosphorylation of paramyosin

1. Myofibrils isolated from Mercenaria mercenaria were phosphorylated by endogenous kinase. Over a range of ionic strengths only paramyosin was phosphorylated. 2. Thiophosphorylation of paramyosin caused an inhibition of steady-state actin-activated ATPase activity of the myofibrils. 3. It is proposed that the endogenous kinase is the catalytic subunit of the cAMP-dependent protein kinase. 4. The sequence around the phosphorylation site was determined. 5. The phosphorylation site probably is close to the C-terminus of the paramyosin molecule.     

Possible presence of the difference peptide in alkali light chain 1 of fish fast skeletal myosin

1. Presence of N-terminal peptide ("difference peptide") in alkali light chain 1 (A1) of fish fast skeletal myosin was examined by comparing two kinds of light chain-based myosin subfragment 1 (S1) isozymes from the yellowtail Seriola quinqueradiata. 2. On tryptic digestion, A1 was cleaved to a smaller fragment (mol. wt decrement by 2000) along with the cleavage of S1 heavy chain, while A2 was resistant to trypsin. Two-dimensional gel electrophoresis showed that A1 released a basic peptide by tryptic digestion. 3. Both S1 isozymes showed clear kinetic differences in actin-activated Mg-ATPase activity, suggesting a higher affinity of A1 for actin. Affinity of A2 for heavy chain was also estimated to be about 2-fold higher than that of A1, as judged by the model experiments in which rabbit S1 isozymes were hybridized with heterologous alkali light chains.     

Covalent binding of a mercaptan S-sulfate to hepatic cytosolic proteins and its inhibition by glutathione

4-Nitrobenzyl mercaptan (NBM) S-sulfate, a new type of the sulfate conjugate enzymatically formed from NBM in the presence of 3'-phosphoadenosine 5'-phosphosulfate in rat liver cytosol, bound covalently to rat liver cytosolic proteins at pH 7.4. The protein binding of NBM S-sulfate was strongly retarded by GSH. GSH not only played a role as a scavenger for NBM S-sulfate with formation of NBM and GSSG via S-(4-nitrobenzyl)thioglutathione, but also cleaved the covalent bonds, possibly disulfides formed from NBM S-sulfate and sulfhydryl groups of the cytosolic proteins. Thus, evidence was provided that NBM S-sulfate be a new type of the reactive metabolite.     

Epoxidation of androsta-5,16-dien-3 beta-ol by hepatic microsomal lipid peroxidation

Male rat liver microsomes oxidized androsta-5,16-dien-3 beta-ol (delta 16-ANDO) to delta 16-ANDO-5,6 alpha-, -5,6 beta-, -16,17 alpha-, and -16,17 beta-epoxides and delta 16-ANDO-5 alpha,6 beta-, -16 alpha,17 beta-, and -16 beta,17 alpha-glycols in the presence of an NADPH-generating system and the microsomal lipid peroxidation accelerator, Fe2+-ADP. The hepatic microsomes hydrolyzed all the delta 16-ANDO epoxides to the glycols. delta 16-ANDO-5 alpha,6 beta-glycol was the sole metabolite from both 5,6 alpha- and 5,6 beta-epoxides. Microsomal epoxide hydrolase also hydrolyzed delta 16-ANDO-16,17 alpha-epoxide specifically to the 16 beta,17 alpha-glycol and the isomeric 16,17 beta-epoxide to the 16 alpha,17 beta- and 16 beta,17 alpha-glycols approximately in the equal ratio. The delta 5-epoxidation of delta 16-ANDO by microsomes occurred only under the conditions that lipid peroxidation took place. Direct evidence was obtained for the participation of microsomal lipid hydroperoxides in the epoxidation of delta 16-ANDO by using photochemically prepared hydroperoxides of phospholipids separated from the hepatic microsomes. The hydroperoxides generated active oxygens, tentatively assigned as alk(ylper)oxy radicals, by the action of ferrous ion and epoxidized delta 16-ANDO to afford the 5,6- and 16,17-epoxides. The Fe2+-ADP-mediated epoxidation of delta 16-ANDO by the phospholipid hydroperoxides occurred preferentially at delta 5 to delta 16 and afforded the 5,6 beta-epoxide in a higher ratio than the 5,6 alpha-epoxide, similar to the Fe2+-ADP-mediated microsomal epoxidation, while the alpha-epoxide was preferentially formed to the beta-epoxide for delta 16 in the epoxidation by both systems.