Oxidative damage to RNA and expression patterns of MTH1 in the hippocampi of senescence-accelerated SAMP8 mice and Alzheimer's disease patients.

Mammalian MTH1 protein, a MutT-related protein, catalyzes the hydrolysis of 8-oxo-7,8-dihydroguanosine triphosphate (8-oxoGTP) to monophosphate, thereby preventing incorporation of 8-oxo-7,8-dihydroguanine (8-oxoguanine) into RNA. In this study, we applied immunohistochemistry to follow the expression of MTH1 and the amount of 8-oxoguanine in RNA during aging. There were increased amounts of 8-oxoguanine in RNA in the CAl and CA3 subregions of hippocampi of 8- and 12-month-old SAMP8 mice, which exhibited early aging syndromes and declining learning and memory abilities compared to those of age-matched control SAMR1 mice. The expression levels of MTH1 in the hippocampi of 8- and 12-month-old SAMP8 mice were significantly lower than those of control mice. Therefore, in this mouse model, age-related accumulation of 8-oxoguanine in RNA is correlated with decreased expression of MTH1. Increased amounts of 8-oxoguanine in the RNA, and decreased expression of MTH1 were also observed in the hippocampi of patients suffering from Alzheimer's disease. These results suggest that MTH1 deficiency might be a causative factor for aging and age-related disorders.

Increased Oxidative Damage of RNA in Early-Stage Nephropathy in db/db Mice.

To evaluate RNA oxidation in the early stage of diabetic nephropathy, we applied an accurate method based on isotope dilution high-performance liquid chromatography-triple quadruple mass spectrometry to analyze the oxidatively generated guanine nucleosides in renal tissue and urine from db/db mice of different ages. We further investigated the relationship between these oxidative stress markers, microalbumin excretion, and histological changes. We found that the levels of 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were increased in the urine and renal tissue of db/db mice and db/db mice with early symptoms of diabetic nephropathy suffered from more extensive oxidative damage than lean littermate control db/m mice. Importantly, in contrast to the findings in db/m mice, the 8-oxoGuo levels in the urine and renal tissue of db/db mice were higher than those of 8-oxodGuo at four weeks. These results indicate that RNA oxidation is more apparent than DNA oxidation in the early stage of diabetic nephropathy. RNA oxidation may provide new insight into the pathogenesis of diabetic nephropathy, and urinary 8-oxoGuo may represent a novel, noninvasive, and easily detected biomarker of diabetic kidney diseases if further study could clarify its source and confirm these results in a large population study.

Development of a method for concentrating and purifying SARS coronavirus RNA by a magnetic bead capture system.

Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel coronavirus, which has been designated the SARS coronavirus (SARS-CoV). To date, molecular assays for the detection of SARS-CoV has focused mainly on reverse transcriptase-PCR (RT-PCR) analysis of specimens. However, RT-PCR assays currently available have low sensitivity during the early stage of the disease in which the viral load in specimens is very low. A method for concentrating and purifying SARS-CoV RNA by a magnetic bead capture system was developed and followed by an RT-PCR assay in this study with the goal of improving the sensitivity of the RT-PCR method. This approach takes advantage of the cooperative interaction between adjacently hybridized oligonucleotides. A capture probe was covalently coupled to magnetic beads and a second probe, which anneals adjacent to the capture probe site, was prehybridized in solution to the target. It was shown that, when applied to SARS RNA samples, the sensitivity of nucleic acid capture RTPCR was about 10-fold greater than routine RT-PCR. This nucleic acid capture system was effective in improving the sensitivity of the RT-PCR, due to enriching and purifying SARS-CoV RNA. The method will be helpful for the early detection of the SARS-associated coronavirus.

High-Resolution Analyses of Human Leukocyte Antigens Allele and Haplotype Frequencies Based on 169,995 Volunteers from the China Bone Marrow Donor Registry Program.

Allogeneic hematopoietic stem cell transplantation is a widely used and effective therapy for hematopoietic malignant diseases and numerous other disorders. High-resolution human leukocyte antigen (HLA) haplotype frequency distributions not only facilitate individual donor searches but also determine the probability with which a particular patient can find HLA-matched donors in a registry. The frequencies of the HLA-A, -B, -C, -DRB1, and -DQB1 alleles and haplotypes were estimated among 169,995 Chinese volunteers using the sequencing-based typing (SBT) method. Totals of 191 HLA-A, 244 HLA-B, 146 HLA-C, 143 HLA-DRB1 and 47 HLA-DQB1 alleles were observed, which accounted for 6.98%, 7.06%, 6.46%, 9.11% and 7.91%, respectively, of the alleles in each locus in the world (IMGT 3.16 Release, Apr. 2014). Among the 100 most common haplotypes from the 169,995 individuals, nine distinct haplotypes displayed significant regionally specific distributions. Among these, three were predominant in the South China region (i.e., the 20th, 31st, and 81sthaplotypes), another three were predominant in the Southwest China region (i.e., the 68th, 79th, and 95th haplotypes), one was predominant in the South and Southwest China regions (the 18th haplotype), one was relatively common in the Northeast and North China regions (the 94th haplotype), and one was common in the Northeast, North and Northwest China (the 40th haplotype). In conclusion, this is the first to analyze high-resolution HLA diversities across the entire country of China, based on a detailed and complete data set that covered 31 provinces, autonomous regions, and municipalities. Specifically, we also evaluated the HLA matching probabilities within and between geographic regions and analyzed the regional differences in the HLA diversities in China. We believe that the data presented in this study might be useful for unrelated HLA-matched donor searches, donor registry planning, population genetic studies, and anthropogenesis studies.

Age-related alterations in the expression of MTH2 in the hippocampus of the SAMP8 mouse with learning and memory deterioration.

MutT-related proteins degrade 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), a mutagenic substrate for DNA synthesis in the nucleotide pool, thereby preventing DNA replication errors. MTH2 (Mut T homolog 2), which belongs to this family of proteins, possesses 8-oxo-7,8-dihydro-2'-deoxyguanosine triphosphatase (8-oxo-dGTPase) activity and appears to function in the protection of the genetic material from the untoward effects of endogenous oxygen radicals. To examine the roles of MTH2 in the aging process, we used the senescence-accelerated prone mouse 8 (SAMP8), which exhibits early aging syndromes and declining abilities of learning and memory. Immunohistochemical and western blot analysis revealed that the level of MTH2 protein in the hippocampus of the SAMP8 mouse progressively decreases beginning from four months after birth, whereas no such change was observed in the control senescence-accelerated resistant mouse 1 (SAMR1). Under these conditions, 8-oxoguanine accumulates in the nuclear DNA in the CA1 and CA3 subregions of the hippocampus of SAMP8 in an age-dependent manner. In SAMR1 mice, accumulation of 8-oxoguanine in the DNA was not observed. These results suggest that the MTH2 deficiency might be one of the causative factors for accelerated aging.