Comparative chromosome mapping of sex-linked genes and identification of sex chromosomal rearrangements in the Japanese wrinkled frog (Rana rugosa, Ranidae) with ZW and XY sex chromosome systems

There are regional variations of sex chromosome morphologies in the Japanese wrinkled frog, Rana rugosa (2n = 26): heterogametic ZZ/ZW-type and XX/XY-type sex chromosomes, and two different types of homomorphic sex chromosomes. To search for homology between the ZW and XY sex chromosomes and the chromosome rearrangements that have occurred during sex chromosomal differentiation in R. rugosa, we performed chromosome mapping of sexual differentiation genes for R. rugosa by FISH. Three genes, AR, SF-1/Ad4BP and Sox3, were localized to both the ZW and XY chromosomes, and their locations were all different between the Z and W and between the X and Y. AR and SF-1/Ad4BP were located on the short arms of the W and X and the long arms of Z and Y, and Sox3 was mapped to the different locations on the long arms between the Z and W and between the X and Y, probably as a result of multiple rearrangements that occurred during the process of sex chromosome differentiation. However, the chromosomal locations of three genes were almost consistent between the Z and Y and between the W and X, indicating that the Z and Y chromosomes and the W and X chromosomes were respectively derived from the same origins. Dmrt1, which is located on avian sex chromosomes, was localized to autosomes in R. rugosa with both the ZW and XY sex chromosomes, suggesting that Dmrt1 might not be related to sex determination in this species.     

Unexpected mitochondrial matrix localization of Parkinson's disease‐related DJ‐1 mutants but not wild‐type DJ‐1

DJ‐1 has been identified as a gene responsible for recessive familial Parkinson's disease (familial Parkinsonism), which is caused by a mutation in the PARK7 locus. Consistent with the inferred correlation between Parkinson's disease and mitochondrial impairment, mitochondrial localization of DJ‐1 and its implied role in mitochondrial quality control have been reported. However, the mechanism by which DJ‐1 affects mitochondrial function remains poorly defined, and the mitochondrial localization of DJ‐1 is still controversial. Here, we show the mitochondrial matrix localization of various pathogenic and artificial DJ‐1 mutants by multiple independent experimental approaches including cellular fractionation, proteinase K protection assays, and specific immunocytochemistry. Localization of various DJ‐1 mutants to the matrix is dependent on the membrane potential and translocase activity in both the outer and the inner membranes. Nevertheless, DJ‐1 possesses neither an amino‐terminal alpha‐helix nor a predictable matrix‐targeting signal, and a post‐translocation processing‐derived molecular weight change is not observed. In fact, wild‐type DJ‐1 does not show any evidence of mitochondrial localization at all. Such a mode of matrix localization of DJ‐1 is difficult to explain by conventional mechanisms and implies a unique matrix import mechanism for DJ‐1 mutants.     

Characterization of a homologue of mammalian serine racemase from Caenorhabditis elegans: the enzyme is not critical for the metabolism of serine in vivo

Free d ‐serine (d ‐Ser) plays a crucial role in regulating brain function in mammals. In various organisms, including mammals, d ‐Ser is biosynthesized by Ser racemase, a synthetic enzyme that produces d ‐Ser from l ‐Ser. Ser racemase also exhibits dehydratase activity toward several hydroxyamino acids. Thus, this enzyme is unique in that it possesses the capability to both synthesize and degrade d ‐Ser; however, the physiological significance of its degradative activity remains unclear. In contrast to the physiological roles of d ‐Ser in mammals, little is known about the role of this amino acid in lower organisms, including the nematode Caenorhabditis elegans. It is known that a mammalian Ser racemase homologue (T01H8.2) from C. elegans exhibits racemase activity. Here, the enzymatic properties of recombinant T01H8.2 were characterized and compared with those of recombinant human Ser racemase. Furthermore, the levels of several d ‐ and l ‐amino acids were measured in wild‐type C. elegans and in a mutant in which the T01H8.2 gene is partially deleted and thereby inactivated. The results indicate that T01H8.2 also shows dehydratase activity toward several hydroxyamino acids, although the enzyme is not critical for Ser metabolism in vivo. The possible physiological roles of T01H8.2 are discussed.     

Emergence of a Multidrug-Resistant Shiga Toxin-Producing Enterotoxigenic Escherichia coli Lineage in Diseased Swine in Japan

Enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing E. coli (STEC) are important causes of diarrhea and edema disease in swine. The majority of swine-pathogenic E. coli strains belong to a limited range of O serogroups, including O8, O138, O139, O141, O147, O149, and O157, which are the most frequently reported strains worldwide. However, the circumstances of ETEC and STEC infections in Japan remain unknown; there have been few reports on the prevalence or characterization of swine-pathogenic E. coli. In the present study, we determined the O serogroups of 967 E. coli isolates collected between 1991 and 2014 from diseased swine in Japan, and we found that O139, O149, O116, and OSB9 (O serogroup of Shigella boydii type 9) were the predominant serogroups. We further analyzed these four O serogroups using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, and virulence factor profiling. Most of the O139 and O149 strains formed serogroup-specific PFGE clusters (clusters I and II, respectively), whereas the O116 and OSB9 strains were grouped together in the same cluster (cluster III). All of the cluster III strains belonged to a single sequence type (ST88) and carried genes encoding both enterotoxin and Shiga toxin. This PFGE cluster III/ST88 lineage exhibited a high level of multidrug resistance (to a median of 10 antimicrobials). Notably, these bacteria were resistant to fluoroquinolones. Thus, this lineage should be considered a significant risk to animal production due to the toxigenicity and antimicrobial resistance of these bacteria.     

Role of FGF10 on tumorigenesis by MS‐K

Murine MS‐K and NFSA cell lines formed tumor after inoculation into mouse and both cell lines expressed high level of vascular endothelial growth factor‐A (vegf‐A) and produced same level of VEGF‐A. However, poor blood vessel formation, and necrosis was significantly observed in NFSA‐tumor, contrary to well‐developed blood vessel formation in MS‐K tumor. The microarray analysis showed high expression of fibroblast growth factor‐10 (fgf‐10) in MS‐K than NFSA. In this report, the role of fgf‐10 on tumor growth was studied. MS‐K enhanced more proliferation of endothelial cells by direct co‐culture than NFSA, and rFGF10 supported the proliferation of HUVEC in combination with VEGF‐A. fgf‐10‐knocked down MS‐K, MS‐K (fgf‐10‐KD), proliferated slower in vitro and the tumorigenicity of them was also slower than control. The blood vessel formation in these MS‐K (fgf‐10‐KD) clones was reduced compared with the MS‐K (normal). qPCR analysis showed the suppression of vegf‐A, vegf‐C and fgfr‐1‐expression in the MS‐K (fgf‐10‐KD) clones. Taken together, these results indicated that FGF10, which was produced from tumor cells, was essential for the proliferation of tumor cell itself and also supports proliferation of endothelial cells. Thus, FGF10 plays an important role for tumor growth by both paracrine and autocrine manner.     

Enhancement of reactive oxygen species and induction of apoptosis in streptozotocin-induced diabetic rats under hyperbaric oxygen exposure

An important source of reactive oxygen species (ROS) production is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which on activation induces superoxide production via oxidation in the mitochondria, inflammation and stress; such ROS are implicated in the pathogenesis of diabetic complications, including neuropathy. Hyperbaric oxygen (HBO) treatments are applied various diseases including diabetic patients with unhealing foot ulcers, however, and also increases the formation of ROS. In a previous study, we showed that a clinically recommended HBO treatment significantly enhanced oxidative stress of pancreatic tissue in the diabetic rats. However, no study has been undertaken with regard to the effects of HBO on the activity and gene expression of the NADPH oxidase complex and on apoptosis in the pancreas of diabetic animals. The purpose of this study was to investigate the effect of HBO exposure on gene expression of the NADPH complex, and pancreatic expression of genes related to apoptosis via the mitochondria, using the NADPH oxidase inhibitor apocynin. The mRNA expression of genes related to NADPH oxidase complex and apoptosis increased significantly (P < 0.05) in the pancreas of diabetic rats under HBO exposure. Similarly, activities of NADPH oxidase and caspase-3 changed in parallel with mRNA levels. These results suggest that oxidative stress caused by HBO exposure in diabetic animals induces further ROS production and apoptosis, potentially through the up-regulation of NADPH oxidase complex. Thus, this study can contribute to development of a better understanding of the molecular mechanisms of apoptosis via the mitochondria in diabetes, under HBO exposure.     

Applicability of automated tractography during acute care stroke rehabilitation

[Purpose] To assess the clinical applicability of a novel automated tractography tool named XTRACT during acute stroke rehabilitation. [Participants and Methods] Three patients with left hemisphere stroke were sampled. Diffusion tensor images were acquired on the second week, and automated tractography was then applied. Tractography images and fractional anisotropy (FA) values in the corticospinal tract (CST) and arcuate fasciculus (AF) were assessed in relation to hemiparesis and aphasia. [Results] Patient 1 was nearly asymptomatic; FA in the left CST was 0.610 and that in the AF was 0.509. Patient 2 had severe hemiparesis and mild motor aphasia. Tractography images of the CST and AF were blurred; FA in the left CST was 0.295 and that in the AF was 0.304. Patient 3 showed no hemiparesis or aphasia at initial assessment. Tractography image of the CST was intact but that of the AF was less clear; FA in the left CST was 0.586 and that in the AF was 0.338. Considering the less clear images of the AF and lower FA value in Patients 2 and 3, further examinations for aphasia were performed, which revealed agraphia. [Conclusion] Visualization and quantification of neural fibers using automated tractography promoted planning acute care rehabilitative treatment in patients with stroke.     

Evaluation of 4-hand reduction for obturator hernia with the guidance of sonography as a new treatment strategy: A retrospective study

To evaluate the effectiveness of 4-hand reduction for obturator hernia with the guidance of sonography (FROGS) as a new treatment strategy for obturator hernia. Since November 2019, FROGS was performed for all patients with obturator hernia at our emergency department. We retrospectively compared the clinical data of 20 patients who underwent FROGS (after FROGS group) to those of 23 patients who did not (before FROGS group). All patients except one were female. The male-to-female ratio, age, duration of symptoms, lesion site, and predisposing factors did not significantly differ between groups. However, the diameter of the prolapsed bowel and the body mass index of the after FROGS group were significantly larger and lower, respectively. Manual reduction was successfully performed for all 20 patients in the after FROGS group, and bowel resection was avoided for all 20 cases. However, 14 patients in the before FROGS group underwent manual reduction; of these, only one was successfully treated using a method other than FROGS, and 8 patients underwent bowel resection. There were no significant differences between groups in terms of postprocedural complications or mortality within 30 days of hernia presentation. The FROGS technique was safe and reproducible and could be used as the first choice of treatment for patients with obturator hernia.