Metastable decomposition at the peptide C-terminus: Possible use in protein identification.

RATIONALE: The b n-1 ion of a peptide, as well as a [b n-1 + 18] ion, can be observed not only as normal product ions, but also as prominent metastable ions in a reflectron-embedded matrix-assisted laser desorption ionization time-of-flight spectrometer. The m/z values for the peaks are slightly shifted compared with the ordinary product ions and appear as relatively broad peaks, which permits them to be discriminated from other ions. METHODS: A standard protein mixture and gel-derived proteins digested with LysN protease, which cleaves peptide linkages in proteins at the N-terminal side of Lys residues, were examined. The collected data were used for protein identification using in-house software, iD-plus (http://coco.protein.osaka-u.ac.jp/id-plus/), which was developed for searching for proteins in the peptide database, based on enzyme specificity (N-terminal Lys in this study), peptide masses and C-terminal amino acids. RESULTS: The b n-1 as well as [b n-1 + 18] ions were observed as broad ion peaks for all of the peptides (86 peptides) examined in this study. In silico calculations using the database of LysN digested peptides (11 969 470), created from 553 941 protein sequences (SwissProt: 2017_03), indicate that the use of no less than four peptides permits a protein to be identified without the need of any probability-based scoring. CONCLUSIONS: The preference for b n-1 ion formation is probably due to the higher propensity of the C-terminal peptide bond to be cleaved than other internal bonds. The fact that such C-terminal fragmentation takes place for most of the peptides examined suggests that the use of an N-terminal specific enzyme would allow the C-terminal amino acids to be more reliably read out than other internal sequences, information that could be efficiently used for protein identification.

Effects of Molecular Hydrogen Assessed by an Animal Model and a Randomized Clinical Study on Mild Cognitive Impairment.

BACKGROUND: Oxidative stress is one of the causative factors in the pathogenesis of neurodegenerative diseases including mild cognitive impairment (MCI) and dementia. We previously reported that molecular hydrogen (H2) acts as a therapeutic and preventive antioxidant. OBJECTIVE: We assess the effects of drinking H2-water (water infused with H2) on oxidative stress model mice and subjects with MCI. METHODS: Transgenic mice expressing a dominant-negative form of aldehyde dehydrogenase 2 were used as a dementia model. The mice with enhanced oxidative stress were allowed to drink H2-water. For a randomized double-blind placebo-controlled clinical study, 73 subjects with MCI drank ~300 mL of H2-water (H2-group) or placebo water (control group) per day, and the Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog) scores were determined after 1 year. RESULTS: In mice, drinking H2-water decreased oxidative stress markers and suppressed the decline of memory impairment and neurodegeneration. Moreover, the mean lifespan in the H2-water group was longer than that of the control group. In MCI subjects, although there was no significant difference between the H2- and control groups in ADAS-cog score after 1 year, carriers of the apolipoprotein E4 (APOE4) genotype in the H2-group were improved significantly on total ADAS-cog score and word recall task score (one of the sub-scores in the ADAS-cog score). CONCLUSION: H2-water may have a potential for suppressing dementia in an oxidative stress model and in the APOE4 carriers with MCI.

Construction of new primer sets for corresponding to genetic evolution of human adenoviruses in major capsid genes through frequent recombination.

A number of novel recombinant human adenoviruses (HAdVs) have recently been identified through sequencing of the complete genomes. The recombinant HAdV sequences share similarity with other types in the major capsid genes, namely the hexon, penton base, and fiber genes, implying recombination events, which may result in escape from the immune response and the acquisition of different organotropisms. Therefore, a surveillance system of HAdVs that considers the effect of frequent recombination on genetic evolution in these genes must be constructed. In this study, we designed new primer sets that can amplify the partial penton base and fiber genes from species HAdV-A to HAdV-F and proteotype HAdVs on the basis of sequence analyses, including previously reported primers that amplify loop 1 of the hexon. Phylogenetic analysis through sequencing with these primers correctly classified clinical HAdV isolates in loop 1 of the hexon gene, the Arg-Gly-Asp (RGD) loop of the penton base gene, and the knob of the fiber gene, which contain neutralizing, hemagglutination, and receptor binding epitopes associated with immunogenicity and tissue tropisms of HAdVs. This study contributes to the accumulation of correct information regarding genetic diversity and evolution in the worldwide HAdV surveillance.

Increased levels of plasma p3-alcα35, a major fragment of Alcadeinα by γ-secretase cleavage, in Alzheimer's disease.

p3-Alcα is a metabolic fragment of Alcadeinα (Alcα). Similar to the generation of the p3 fragment from amyloid-ß protein precursor (AßPP) processing, Alcα is cleaved by α- and γ-secretases, leading to the secretion of p3-Alcα peptides into cerebrospinal fluid (CSF). p3-Alcα is also detected in the plasma, similar to amyloid-ß (Aß), which is a metabolic fragment of AßPP cleaved by amyloidogenic ß- and γ-secretases. Because p3-Alcα is a non-aggregatable and stable peptide, unlike aggregatable Aß and metabolically labile p3 of AßPP, the changes of p3-Alcα in quality and/or quantity in CSF and plasma are expected to be a marker for assessing alteration of substrate cleavage by γ-secretase, such as Aß generation from AßPP. The present study describes a sandwich enzyme-linked immunosorbent assay for quantifying levels of p3-Alcα35, the major form of the p3-Alcα species, and examines levels of p3-Alcα35 in the plasma of three independent Japanese cohorts. In two of the three cohorts, the p3-Alcα35 levels were significantly increased with a concomitant decrease in the Mini-Mental State Examination score, or in clinically diagnosed Alzheimer's disease (AD) patients, when compared with age-matched non-demented subjects. The values were significantly lower in AD subjects who were administered donepezil, when compared to AD subjects without donepezil treatment. The increase in plasma p3-Alcα35 levels may indicate an endophenotype in subjects in whom AD is due to a progressing cognitive impairment in subjects with a γ-secretase malfunction, or a disorder of the clearance of peptides.

Genome sequence of a novel virus of the species human adenovirus d associated with acute gastroenteritis.

A novel virus of the species human adenovirus D, HAdV-67 (P-New/H9/F25), was first isolated from diarrheal feces of six children in Dhaka City, Bangladesh. The genome of this novel virus may be composed of multiple recombinations among HAdV-9, HAdV-25, HAdV-26, HAdV-33, HAdV-46, and an unknown human adenovirus D which was an origin of HAdV-67.

Genome sequence of an unusual human G10P[8] rotavirus detected in Vietnam.

A rare human G10P[8] rotavirus with a reassortment between bovine and human viruses was detected from a patient with acute gastroenteritis in Vietnam. Genetic analysis using complete coding sequences of all segments showed a genomic constellation of this virus of G10-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Its VP7 region was genetically related to that of a bovine rotavirus derived from Australia (strain VICG10.01), whereas all other genes were identical to those of a human rotavirus derived from Australia (strain Victoria/CK00047). These results indicate a possibility that the reassortment of the rotavirus was caused by immune escape in Australia and the rotavirus was carried to Vietnam. Additionally, this finding will help further understanding the evolution of rotaviruses circulating in Vietnam.

A molecular epidemiologic study of human adenovirus type 8 isolates causing epidemic keratoconjunctivitis in Kawasaki City, Japan in 2011.

A local outbreak of epidemic keratoconjunctivitis (EKC) caused by human adenovirus type 8 (HAdV-D8) occurred in Kawasaki city, Japan in July-August 2011. Since the cases were sporadic in nature, the source of the infection could not be identified. The results of PCR analysis and the appearance of cytopathic effects in the samples indicated that 22 patients were positive for HAdV. The mean age of the patients (10 men and 12 women) was 64.3 ± 17.3 years (median, 68 years; range, 11-86 years). The sequences of hexon, which included hypervariable loop 1; the penton, which included RGD loops; and the fiber, which included the knob-coding regions, were identical in all the HAdV-positive cases. Phylogenetic analysis of the major capsid protein-encoding regions of HAdV confirmed that the isolates were HAdV-D8. Although the incidence of HAdV-D8 outbreaks has decreased in Japan since 1997, the results of our study imply that HAdV-D8 is still a causative agent for EKC outbreaks in Japan.

Novel human adenovirus strain, Bangladesh.

We report a novel human adenovirus D (HAdV-65) isolated from feces of 4 children in Bangladesh who had acute gastroenteritis. Corresponding genes of HAdV-65 were related to a hexon gene of HAdV-10, penton base genes of HAdV-37 and HAdV-58, and a fiber gene of HAdV-9. This novel virus may be a serious threat to public health.