Aya Miyoshi

Multiplex amplified product-length polymorphism analysis for rapid detection of human mitochondrial DNA variations

A number of mutations in coding and noncoding regions of mitochondrial DNA (mtDNA) have previously been studied. In the present study, we simultaneously typed six mutation sites in the coding region by use of amplified product‐length polymorphism (APLP) analysis. The mtDNA variations of 2471 individuals from 20 populations of Japanese, Korean, Chinese, and German were examined and classified into 18 haplotypes. Two of these haplotypes, B1 (estimated ancestral haplotype) and C1, were distributed among all populations tested. However, the haplotypes A1, A2, B2, B3, and C2 were mostly restricted to the Mongoloid populations, whereas haplotypes B5 and C5 appeared almost exclusively in the German population. Phylogenetic analysis by the neighbor‐joining method revealed that the Japanese populations were more closely related to each other than to the other East Asian populations surveyed. The multiplex APLP method is suitable for large‐scale screening studies of mtDNA variability because it is both rapid and economical.

Ligand-activated PPARβ efficiently represses the induction of LXR-dependent promoter activity through competition with RXR

Angiopoietin-like protein 3 (angptl3), a member of the vascular endothelial growth factor family, was shown to play an important role in regulating lipid metabolism. To elucidate the mechanism by which PPARbeta represses angptl3 promoter activity, reporter constructs were prepared and transfection analysis carried out. PPARbeta repressed angptl3-Luc promoter activity and activation of PPARbeta by L-165041, a PPARbeta-specific ligand, increased the extent of repression. The repression by L-165041 was lost in angptl3-Luc plasmids having a deleted or mutated LXRalpha binding site (DR4). PPARbetaL405R, deficient in RXRalpha binding, had no effect on angptl3-Luc promoter activity. PPARbeta did not repress the activity of GAL4-LXRalpha which activates of GAL4DBD TK-Luc independent of RXR. Addition of RXRalpha completely abolished the repression of angptl3-Luc activity by PPARbeta. Mammalian two-hybrid analysis revealed that PPARbeta ligand binding enhanced the dissociation of the LXRalpha-RXRalpha heterodimer. Gel shift assays also indicated that PPARbeta ligand binding increased dissociation of LXRalpha/RXRalpha binding to a DR4 oligonucleotide probe; addition of RXRalpha restored the binding lost by addition of PPARbeta. Collectively, these results suggest that the binding of PPARbeta-specific ligand enhances the affinity between RXRalpha and activated PPARbeta and thus may regulate angptl3 gene expression through a DR4 element by competing with LXRalpha for RXRalpha.

Distribution of the F374 Allele of the SLC45A2 (MATP) Gene and Founder‐Haplotype Analysis

The membrane‐associated transporter protein (MATP) plays an important role in melanin synthesis. The L374F mutation in the SLC45A2 gene encoding MATP has been suggested to be associated with skin colour in major human populations. In this study more detailed distribution of the F374 allele was investigated in 1649 unrelated subjects from 13 Eurasian populations and one African population. The highest allele frequency was observed in Germans (0.965); French and Italians showed somewhat lower frequencies; and Turks had an intermediate value (0.615). Indians and Bangladeshis from South Asia were characterized by low frequencies (0.147 and 0.059, respectively). We also found the F374 allele in some East and Southeast Asian populations, and explained this by admixture. Haplotype analysis revealed that the haplotype diversity was much lower in Germans than in Japanese, and suggest that the L374F mutation occurred only once in the ancestry of Caucasians. The large differences in distribution of the F374 allele and its haplotypes suggest that this allele may be an important factor in hypopigmentation in Caucasian populations.

Headspace solid-phase microextraction and gas chromatographic-mass spectrometric screening for volatile hydrocarbons in blood.

Optimization for headspace solid-phase microextraction (SPME) was studied with a view to performing gas chromatographic-mass spectrometric (GC-MS) screening of volatile hydrocarbons (VHCs) in blood. Twenty hydrocarbons comprising aliphatic hydrocarbons ranging from n-hexane to n-tridecane, and aromatic hydrocarbons ranging from benzene to trimethylbenzenes were used in this study. This method can be used for examining a burned body to ascertain whether the victim had been alive or not when the burning incident took place. n-Hexane, n-heptane and benzene, the main indicators of gasoline components, were found as detectable peaks through the use of cryogenic oven trapping upon SPME injection into a GC-MS instrument. The optimal screening procedure was performed as follows. The analytes in the headspace of 0.2 g of blood mixed with 0.8 ml of water plus 0.2 microg of toluene-d8 at -5 degrees C were adsorbed to a 100-microm polydimethylsiloxane (PDMS) fiber for 30 min, and measured using the full-mass-scanning GC-MS method. The lower detection limits of all the compounds were 0.01 microg per 1 g of blood. Linearities (r2) within the range 0.01 to 4 microg per 1 g of blood were only obtained for the aromatic hydrocarbons at between 0.9638 (pseudocumene) and 0.9994 (toluene), but not for aliphatic hydrocarbons at between 0.9392 (n-tridecane) and 0.9935 (n-hexane). The coefficients of variation at 0.2 microg/g were less than 8.6% (n-undecane). In conclusion, this method is feasible for the screening of volatile hydrocarbons from blood in forensic medicine.

Distribution of two Asian-related coding SNPs in the MC1R and OCA2 genes.

Very little is known about the genes and mechanisms affecting skin lightening in Asian populations. In this study, two coding SNPs, c.G1129A (R163Q) at the MC1R (melanocortin 1 receptor) gene and c.A1962G (H615R) at the OCA2 (oculocutaneous albinism type II) gene, were investigated in a total of 1,809 individuals in 16 populations from various areas. The Q163 and R615 alleles prevailed almost exclusively in East and Southeast Asian populations. Wright’s FST was 0.445 for R163Q and 0.385 for H615R among the 16 populations. The frequency of the Q163 allele was higher in Northeast Asians than in Southeast Asians. The frequency of the R615 allele was highest in South China and unlikely to be associated with levels of ultraviolet radiation. This allele may be a good marker to study the genetic affinity among East Asians because of its restricted distribution and marked difference in allele frequency.

OCA2*481Thr, a hypofunctional allele in pigmentation, is characteristic of northeastern Asian populations

AbstractAsians as well as Europeans have light skin, for which no genes to date are known to be responsible. A mutation, Ala481Thr (c.G1559A), in the oculocutaneous albinism type II (OCA2) gene has approximately 70% function of the wild type allele in melanogenesis. In this study, the distribution of the mutation was investigated in a total of 2,615 individuals in 20 populations from various areas. OCA2*481Thr prevailed almost exclusively in a northeastern part of Asia. The allele frequency was highest in Buryat (0.24) in Mongolia and showed a north-south downward geographical gradient. These findings suggest that OCA2*481Thr arose in a region of low ultraviolet radiation and thereafter spread to neighboring populations.

Successful DNA Typing of Urine Stains Using a DNA Purification Kit Following Dialfiltration

To evaluate the utility of DNA polymorphism typing of urine stains in forensic investigations, the amplifiable amount of DNA was estimated in 20 urine specimens obtained from 10 male and 10 female volunteers using a DNA purification kit following dialfiltration. DNA obtained from both urine and urine stains was amplified with the AmpflSTR Profiler PCR Amplification Kit, and was analyzed by capillary electrophoresis using the Genetic Analyzer. The amount of male and female urine necessary for obtaining a complete DNA profile was 0.2 mL and 0.08 mL, respectively. When 0.2 mL of male urine were used to create urine stains, complete DNA profiles could be obtained from just some of the stains. However, when only 0.1 mL of female urine was used, complete profiles could be successfully obtained from all of the stains. DNA on bleached cotton remained amplifiable for 3-6 weeks. This method using a DNA purification kit following dialfiltration can be recommended for the genotyping of urine stains.

Two simple methods for enantiomeric analyses of urinary amphetamines by GC/MS using deuterium-labeled l-amphetamines as internal standards

Two simple methods for enantiomeric analyses of amphetamines in urine by gas chromatography-mass spectrometry (GC-MS) using l-amphetamine-d3 and l-methamphetamine-d6 as internal standards are presented. One method (method A) employs extractive derivatization on a diatomaceous column with (S)-(-)-N-(trifluoroacetyl)prolyl chloride (TPC) followed by separation with a conventional capillary column. The second method (method B) uses headspace solid-phase microextraction (HD-SPME) after derivatization with heptafluoro-n-butyryl chloride (HFB), followed by separation with an enantiomeric capillary GC column. By the two methods, all enantiomers were well separated in each chromatogram, and good linearity was obtained in practical concentration ranges (0.1–1.6μg/ml for method A and 0.05–1μg/ml for method B) for every compound by selected-ion monitoring. The precision studies indicated satisfactory coefficients of variation (<5%) for every enantiomer at 0.1μg/ml by both methods. Both methods were also evaluated by applying them to an actual poisoning case. Both methods are recommended for use in forensic analysis, because of their simplicity, high precision, and sufficient sensitivity.

Rapid analysis of acetaminophen in serum by gas chromatography-mass spectrometry with extractive derivatization using a diatomaceous earth tube

A new analytical method for acetaminophen (ACAP) in serum was developed by modifying an existing method used for amphetamines, which used extractive derivatization followed by gas chromatographymass spectrometry. After a serum sample was adjusted to pH 12.8, it was applied onto a diatomaceous earth tube; the analyte was simultaneously extracted and heptafluorobutyrylated during elution with a solvent containing a derivatizing reagent. Three internal standard (IS) candidates were tested: N-acetyl-d3-paminophenol, N-acetyl-m-aminophenol, and N-acetyl-4-amino-m-cresol. All ISs gave good linear relationships (r2 > 0.999) for ACAP in the concentration range from 1 to 200μg/ml. The detection limit for ACAP using each IS was estimated to be 0.05–0.1μg/ml. Intraday precision was satisfactory with a coefficient of variation of less than 8.3%. The use of this method with any of the three ISs is recommended in forensic and clinical toxicology because of its rapidity and good reproducibility.