Yasuhiro Aoki

CYP2A7 Pseudogene Transcript Affects CYP2A6 Expression in Human Liver by Acting as a Decoy for miR-126*

Human cytochrome P450 (CYP)2A6 is responsible for the metabolic activation of tobacco-related nitrosamines, as well as the metabolism of nicotine and some pharmaceutical drugs. There are large interindividual differences in CYP2A6 activity and expression, largely attributed to genetic polymorphisms. However, the variability was observed within homozygotes of the wild-type CYP2A6 gene. In this study, we investigated the possibility that CYP2A6 might be regulated by microRNA. A luciferase assay revealed that a microRNA recognition element (MRE) of miR-126* found in the 3′-untranslated region (UTR) of CYP2A6 mRNA is functional. We established two HEK293 cell lines stably expressing CYP2A6, with one including and the other excluding the full-length 3′-UTR (HEK/2A6+UTR and HEK/2A6 cells, respectively). Overexpression of miR-126* markedly decreased CYP2A6 protein levels, enzyme activity, and mRNA level in HEK/2A6+UTR cells, whereas it marginally decreased those in HEK/2A6 cells, indicating that the 3′-UTR including the MRE is functional for the downregulation of CYP2A6 by miR-126*. The inhibition of miR-126* increased CYP2A6 protein levels in primary human hepatocytes, suggesting that miR-126* downregulates endogenous CYP2A6 expression. In 20 human liver samples, the expression ratios of CYP2A6 and a pseudogene transcript CYP2A7 mRNA were highly variable (CYP2A7/CYP2A6: 0.1 to 12). Interestingly, we found that CYP2A7 was another target of miR-126* and restored the miR-126*–dependent downregulation of CYP2A6 by acting as a decoy for miR-126*. In conclusion, this study demonstrates that human CYP2A6 is post-transcriptionally regulated by miR-126* and that CYP2A7 affects CYP2A6 expression by competing for miR-126* binding.

Analyses of sexual dimorphism of reconstructed pelvic computed tomography images of contemporary Japanese using curvature of the greater sciatic notch, pubic arch and greater pelvis

Three-dimensional pelvic images were reconstructed from multi-slice CT data of contemporary Japanese (males: 124; females: 104, 25-92 years old), and curvature analysis to examine sexual dimorphism was carried out in the great sciatic notch (GSN), the pubic arch and the greater pelvis in the images. Reconstructed pelvic CT images were visualized fairly well and anatomical landmarks were easily recognizable. When calculating the radii (curvature radii) of the best-fit circles for the spline curve lines set along the edges of the GSNs and of the pubic arches, sexes from these regions were correctly identified in 89.1% (males: 93.8%; females: 83.7%) and 94.7% (males: 97.3%; females: 91.8%) of cases, respectively, by setting an appropriate cut-off value. Furthermore, sexing was possible even in deeper regions of the GSN which are relatively resistant to postmortem damage. Curvature radii of the best-fit spheres of greater pelves showed no significant difference between sexes. However, curvature of the best-fit sphere for the left iliac fossa was significantly larger than that of the right one (p<10(-24)) in males, and the ratios were >1.0 in 88% of all male specimens analyzed. Meanwhile, no significant difference was observed among female samples. Although some left-sided dominancy has been reported in 2-dimensional measurements of the human pelvis, this 3-dimensional laterality in males was much more significant, and is a potential index of sex difference.

5-hydroxytryptamine- and dopamine-releasing effects of ring-substituted amphetamines on rat brain: A comparative study using in vivo microdialysis

Using in vivo microdialysis, a comparative study was conducted to examine the effects of amphetamine-related compounds (methamphetamine, MAP; 3,4-methylenedioxymethamphetamine, MDMA; p-methoxyamphetamine, PMA; p-methoxymethamphetamine, PMMA; 4-methylthioamphetamine, 4-MTA; 3,4,5-trimethoxyamphetamine, TMA; 2,5-dimethoxy-4-iodoamphetamine, DOI) on extracellular levels of serotonin (5-HT) and dopamine (DA). Dialysates were assayed using HPLC equipped with electrochemical detector following i.p. administration with each drug at a dose of 5 mg/kg. MAP was found to drastically and rapidly increase 5-HT and DA levels (870% and 1460%, respectively). PMA, PMMA, and 4-MTA slightly increased DA levels (150-290%) but remarkably increased 5-HT levels (540-900%). In contrast, TMA and DOI caused no detectable changes in levels of both monoamines. We observed that the potent DA-releasing action of MAP was remarkably decreased by introduction of methoxy or methylthio group at the para position (MAP vs. PMMA or 4-MTA), but introduction of two additional adjacent methoxy groups into PMA totally abolished its 5-HT-/DA-releasing action (PMA vs. TMA). In addition, para-mono-substituted compounds inhibited both monoamine oxidase (MAO) enzymes more strongly than other compounds; PMA and 4-MTA exhibited submicromolar IC50 values for MAO-A. On the other hand, TMA scarcely affected the activity of both MAO enzymes as well as extracellular levels of 5-HT and DA. In this comparative study, MDMA, PMA, and 4-MTA functioned similar to PMMA, a typical empathogen; these findings therefore could be helpful in clarifying the psychopharmacological properties of amphetamine-related, empathogenic designer drugs.

Distribution of Aconitum alkaloids in autopsy cases of aconite poisoning

Aconite is a well-known toxic-plant containing Aconitum alkaloids such as aconitines, benzoylaconines, and aconins. We describe here the distribution of Aconitum alkaloids detected by liquid chromatography-tandem mass spectrometry (LC/MS/MS) in three autopsy cases of suicide by aconite poisoning. Case 1: a male in his fifties had eaten aconite leaves. The concentrations of jesaconitine in cardiac blood, urine, and kidney were 12.1 ng/ml, 993.0 ng/ml, and 114.2 ng/g, respectively. Case 2: a female in her fifties had eaten aconite root. The aconite root in the stomach included a high level of mesaconitine. The concentrations of mesaconitine in cardiac blood, liver, and kidney were 69.1 ng/ml, 960.9 ng/g, and 776.9 ng/g, respectively. Case 3: a male in his sixties had drunk liquor in which aconite root had been soaked. The concentrations of mesaconitine and aconitine in cardiac blood were 259.5 and 228.5 ng/ml, respectively. The Aconitum alkaloid levels were very high in the liver. The absorption of ethanol and Aconitum alkaloids might have been increased because of his having undergone total gastrectomy. In all three cases, the Aconitum alkaloid levels were high in the liver and kidney and low in the heart and cerebrum. The level in the cerebrum was lower than that in blood. Data on the distribution of the Aconitum alkaloids in the body in cases of aconite poisoning is useful to elucidate various actions of aconite alkaloids.

Three dimensional surface analyses of pubic symphyseal faces of contemporary Japanese reconstructed with 3D digitized scanner

Three dimensional pubic bone images were analyzed to quantify some age-dependent morphological changes of the symphyseal faces of contemporary Japanese residents. The images were synthesized from 145 bone specimens with 3D measuring device. Phases of Suchey-Brooks system were determined on the 3D pubic symphyseal images without discrepancy from those carried out on the real bones because of the high fidelity. Subsequently, mean curvatures of the pubic symphyseal faces to examine concavo-convex condition of the surfaces were analyzed on the 3D images. Average values of absolute mean curvatures of phase 1 and 2 groups were higher than those of phase 3-6 ones, whereas the values were approximately constant over phase 3 presumably reflecting the inactivation of pubic faces over phase 3. Ratio of the concave areas increased gradually with progressing phase or age classes, although convex areas were predominant in every phase.

Distinct breakpoints in two cases with deletion in the Yp11.2 region in Japanese population

The amelogenin gene on the Y chromosome (AMELY) is a homolog of the X chromosome amelogenin gene (AMELX), and the marker is employed for sexing in forensic casework. Deletion of the sequences in the Yp11.2 region containing the AMELY locus has been found in males from various ethnic populations. Two cases of AMELY null males found in the Japanese population had different Y haplogroups and deletion mapping. Proximal and distal breakpoints of a sample of haplogroup D2* were located in TSPYA and TSPYB arrays, respectively, suggesting that the deletion mechanism was non-allelic homologous recombination (NAHR). On the other hand, a sample of haplogroup O3a3c* had the distal breakpoint in the TSPYB array and the proximal breakpoint at position 7.94 Mb, not in the TSPYA array. The likely deletion mechanism is non-homologous end-joining. High-resolution STS mapping in the TSPYB array showed the distal breakpoints differed according to the haplogroups. The deletion length was estimated as 3.1–3.7 Mb and 1.6–1.7 Mb for the sample of haplogroup D2* and O3a3c*, respectively. These deletion events should have occurred independently.

Simultaneous time course analysis of multiple markers based on DNA microarray in incised wound in skeletal muscle for wound aging.

Assessment of incised wound age in skeletal muscles is important because fatal injuries are often complicated with muscle involvement. Transcriptome of injured skeletal muscle along with histopathological and immunohistochemistry staining, were analyzed to explore the biological effect of incised injuries using a mouse incised injury model. An incisional wound was made at the biceps femoris muscle of anesthetized mice, and the muscles were sampled at 6, 12, 24, 36 and 48h post-injury. DNA microarray analysis using RNA extracted from the muscle samples of 12h post-injury identified 3,655 upregulated and 3,583 downregulated genes. Referring to the results of the gene ontology and gene expression pathway analysis, time course expression of five cytokines, namely chemokine (C-C motif) ligand 4 (CCL4), chemokine (C-X-C motif) ligand 5 (CXCL5), interleukin-1 beta (IL-1β), interleukin- 6 (IL-6) and interleukin-7 (IL-7), were analyzed by quantative reverse transcription PCR (qRT-PCR). CXCL5 was the most upregulated gene throughout the post-injury period with higher expression from 6 through 36h post injury. Upregulation of CCL4 and IL-1β was also persisted until 36h post injury. IL-6 mRNA was highly and rapidly expressed at 6h post-injury followed by significant decrease at 12h. Unlike other four cytokines, IL-7 showed slow and steady increasing over time until 48h post-injury. Immunohistochemical staining of post-injury samples showed gradual mild increase of staining intensity proportional to increasing time points especially around the wound edges. The present study highlights the unique dynamics of each cytokine and reflects their roles in the process of muscle wound healing, and suggests the potential of them as a tool for forensic wound age estimation.

A fatal case of hemoperitoneum after ultrasound-guided liver biopsy in a patient with intravascular large B-cell lymphoma

An autopsy case of fatal complication of percutaneous liver biopsy targeting a rare type of large B-cell lymphoma is presented. A 79 year-old man was referred to the hematology unit of a hospital because of anemia with thrombocytopenia and hepatosplenomegaly. Results of inguinal lymph node biopsy were inconclusive. To investigate a mass lesion in the liver visualized by ultrasonography, image-guided liver biopsy was performed following platelet infusion. The patient became unresponsive 6h post procedure because of hypotension due to intraperitoneal hemorrhage of undetermined origin. Autopsy revealed hemoperitoneum but failed to identify any macroscopic intra- or extrahepatic vascular injuries. Histopathological investigation disclosed infiltration of atypical lymphocytes into the systemic organs including the lymph nodes, spleen, liver, and lungs. Prominent hemophagocytosis was also noted. The lymphoma cells were exclusively accumulated within vascular and sinusoidal structures, and diagnosed with immunohistochemistry as Asian variant of intravascular large B-cell lymphoma. Massive blood extravasation was presumed to originate directly from the markedly dilated liver sinusoids filled with erythrocytes, macrophages and tumor cells, under the condition of impaired hemostasis. Although the biopsy was thought to have been correctly performed, this case would be instructive for evaluation of the indications and risks associated with liver biopsy.

Time-Dependent Changes in Local and Serum Levels of Inflammatory Cytokines as Markers for Incised Wound Aging of Skeletal Muscles

Wound age estimation is an important research field in forensic pathology. The expression levels of cytokines in the incised skeletal muscle were analyzed using a mouse model to explore the applicability for wound aging. A 5-mm long incisional wound was made at the biceps femoris muscle, and the muscle and serum were sampled at 6, 12, 24 and 48 hours after injury. Using a multiplex bead-based immunoassay, we measured the tissue levels of nine cytokines (IL-1β, IL-6, IL-7, CCL2, CCL3, CCL4, CXCL1, CXCL2, and CXCL10), which are all involved in the pathways of inflammatory response and tissue injury. Immunoassay of post-injury muscle samples revealed significant increases in the levels of six cytokines, except for CCL3, CCL4 and IL-7, at 6 hours after injury. The elevated tissue levels of these six cytokines were maintained during 48 hours after injury, although the levels of IL-6 and CXCL1 were significantly decreased at 12 hours. In case of CCL3, its tissue levels were increased only at 12 hours. By contrast, CCL4 and IL-7 levels were increased only at 48 hours. Moreover, serum levels of most cytokines, except for CXCL1, remained unchanged during 24 hours after injury, followed by significant increases at 48 hours. Serum CXCL1 levels were increased at 6 hours and then decreased to the basal levels. Thus, the significant increase in the muscle levels of CXCL1 and IL-7 was observed at 6 and 48 hours after injury, respectively. Measuring muscle CXCL1 and IL-7 levels is helpful for estimating incised wound aging.

A simple method for calculating the likelihood ratio in a kinship test using X-chromosomal markers incorporating linkage, linkage disequilibrium, and mutation

X-chromosomal short tandem repeats (X-STRs) are useful for personal identification and kinship tests. However, it has not yet been fully established how to incorporate linkage and linkage disequilibrium (LD) into the calculation of the likelihood ratio (LR). In this paper, we describe a simple calculation method of LR for X-STRs that incorporated linkage, LD, and mutations. Initially, Japanese population data of 27 X-STRs (DXS6807, DXS9902, DXS6795, DXS6810, DXS10076, DXS10077, DXS10078, DXS10162, DXS10163, DXS10164, DXS7132, DXS981, DXS6800, DXS6803, DXS6809, DXS6789, DXS6799, DXS7424, DXS101, DXS7133, GATA172D05, DXS10103, HPRTB, GATA31E08, DXS8377, DXS10147, and DXS7423) were collected from 748 unrelated individuals to estimate the influence of LD. Significant LD was observed on six pairs of loci. Subsequently, using the population data, a simulation study was performed to evaluate the validity of the present calculation method for LR in cases of father-daughter, full-sisters, paternal half-sisters, maternal half-sisters, and unrelated pairs of females (FD, FS, PHS, MHS, and UR, respectively). As a result, the distribution of LR among FD was completely separated from that among UR. In the sibship test, 98.6% of FS and 98.0% of PHS surpassed the maximum value of UR in combined LR. Even in the FS versus MHS setting, 60.5% of FS had a higher LR than any MHS. We conclude that the present model is powerful in discriminating the relationship and is able to obtain a reasonable LR with fewer computations.