Porcine α-1,3-galactosyltransferase: full length cDNA cloning, genomic organization, and analysis of splicing variants

Full length cDNA and genomic DNA of porcine -1,3-galactosyltransferase were isolated, and their structures were analysed. The coding region was encoded by six exons as in the mouse, and the length of each exon was conserved between the two species. The porcine exons were designated Exon 4–9, since in the mouse coding exons started from Exon 4. Introns tended to be longer in the porcine gene; the distance from Exon 4 to the 3-end of Exon 9 was 24 kb, while this region was 18 kb in the mouse gene. The cDNA structure was extended from the previous data to the 3-end and to the 5 side of the cDNA. In addition to a cDNA clone with all coding exons, clones lacking parts of these exons were isolated and their structures were determined. One variant lacked Exon 5; the second, Exons 5 and 6; and the third, Exons 5, 6 and 7. The last variant was not found in the mouse, and cDNA transfection of this variant yielded scarcely any enzymatic activity using asialo 1-acid glycoprotein as a substrate, and decreased activity using N-acetyllactosamine as a substrate.     

Melting Curve Analysis after T Allele Enrichment (MelcaTle) as a Highly Sensitive and Reliable Method for Detecting the JAK2V617F Mutation

Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.     

In Vivo Regulation of Erythropoiesis by Chemically Inducible Dimerization of the Erythropoietin Receptor Intracellular Domain

Erythropoietin (Epo) and its receptor (EpoR) are required for the regulation of erythropoiesis. Epo binds to the EpoR homodimer on the surface of erythroid progenitors and erythroblasts, and positions the intracellular domains of the homodimer to be in close proximity with each other. This conformational change is sufficient for the initiation of Epo-EpoR signal transduction. Here, we established a system of chemically regulated erythropoiesis in transgenic mice expressing a modified EpoR intracellular domain (amino acids 247–406) in which dimerization is induced using a specific compound (chemical inducer of dimerization, CID). Erythropoiesis is reversibly induced by oral administration of the CID to the transgenic mice. Because transgene expression is limited to hematopoietic cells by the Gata1 gene regulatory region, the effect of the CID is limited to erythropoiesis without adverse effects. Additionally, we show that the 160 amino acid sequence is the minimal essential domain of EpoR for intracellular signaling of chemically inducible erythropoiesis in vivo. We propose that the CID-dependent dimerization system combined with the EpoR intracellular domain and the Gata1 gene regulatory region generates a novel peroral strategy for the treatment of anemia.     

Osteological Variation among Extreme Morphological Forms in the Mexican Salamander Genus Chiropterotriton (Amphibia: Plethodontidae): Morphological Evolution And Homoplasy

Osteological variation is recorded among and within four of the most distinctive species of the Mexican salamander genus Chiropterotriton. Analysis of the data is consistent with the monophyletic status of the genus and documents previously unrecorded intraspecific and interspecific variation. Most of the recorded variation involves qualitative and quantitative proportional differences, but four fixed differences constitute autapomorphic states that affirm and diagnose some species (C. dimidiatus, C. magnipes). Osteological variation in 15 characters is analyzed with respect to predictions generated from four hypotheses: 1) phylogeny, 2) adaptation to specific habitats (the four species include cave-dwelling, terrestrial, and arboreal forms), 3) size-free shape, and 4) size. High levels of intraspecific variation suggest that the characters studied are not subject to rigid functional constraints in salamanders, regardless of size. The pattern predicted by the hypothesis based on size differences seen among these four Chiropterotriton species matches most closely the observed pattern of relative skull robustness. Since size change and heterochrony are often associated in plethodontid evolution, it is likely that changes in developmental timing play a role in the morphological transitions among these morphologically diverse taxa. Webbed feet, miniaturization, body shape, and an unusual tarsal arrangement are morphologies exhibited in species of Chiropterotrition that are shown to be homoplastic with other clades of tropical plethodontids. Although extensive homoplasy in salamanders might be seen as a roadblock to unraveling phylogenetic hypotheses, the homologous developmental systems that appear to underlie such homoplasy may reveal common and consistent evolutionary processes at work.     

Effectiveness of Trivalent Inactivated Influenza Vaccine in Children Estimated by a Test-Negative Case-Control Design Study Based on Influenza Rapid Diagnostic Test Results

We assessed vaccine effectiveness (VE) against medically attended, laboratory-confirmed influenza in children 6 months to 15 years of age in 22 hospitals in Japan during the 2013–14 season. Our study was conducted according to a test-negative case-control design based on influenza rapid diagnostic test (IRDT) results. Outpatients who came to our clinics with a fever of 38°C or over and had undergone an IRDT were enrolled in this study. Patients with positive IRDT results were recorded as cases, and patients with negative results were recorded as controls. Between November 2013 and March 2014, a total of 4727 pediatric patients (6 months to 15 years of age) were enrolled: 876 were positive for influenza A, 66 for A(H1N1)pdm09 and in the other 810 the subtype was unknown; 1405 were positive for influenza B; and 2445 were negative for influenza. Overall VE was 46% (95% confidence interval [CI], 39–52). Adjusted VE against influenza A, influenza A(H1N1)pdm09, and influenza B was 63% (95% CI, 56–69), 77% (95% CI, 59–87), and 26% (95% CI, 14–36), respectively. Influenza vaccine was not effective against either influenza A or influenza B in infants 6 to 11 months of age. Two doses of influenza vaccine provided better protection against influenza A infection than a single dose did. VE against hospitalization influenza A infection was 76%. Influenza vaccine was effective against influenza A, especially against influenza A(H1N1)pdm09, but was much less effective against influenza B.     

Association of Psychosocial Conditions,Oral Health,and Dietary Variety with Intellectual Activity in Older Community-Dwelling Japanese Adults

Background

This study examined the factors related to intellectual activity in community-dwelling elderly persons.

Methods

Self-administered questionnaires mailed to all people aged ≥65 years in a dormitory suburb in Japan (n = 15,210). The response rate was 72.2%. Analytical subjects (n = 8,910) were those who lived independently and completely answered questions about independent and dependent variables and covariates. Independent variables included psychosocial conditions (i.e., social activities, hobbies, and a sense that life is worth living (ikigai)), oral health (i.e., dental health behaviors and oral function evaluated by chewing difficulties, swallowing difficulties, and oral dryness), and dietary variety measured using the dietary variety score (DVS). A dependent variable was intellectual activity measured using the Tokyo Metropolitan Institute of Gerontology Index of Competence. Covariates included age, gender, family structure, pensions, body mass index, alcohol, smoking, medical history, self-rated health, medications, cognitive function, depression, and falling. Logistic regression was used to estimate the odds ratio (OR) for poor intellectual activity.

Results

Poor intellectual activity was reported by 28.9% of the study population. After adjustment for covariates and independent variables, poor intellectual activity was significantly associated with nonparticipation in social activities (OR = 1.90, 95%CI = 1.61–2.24), having neither hobbies nor ikigai (3.13, 2.55–3.84), having neither regular dental visits nor daily brushing (1.70, 1.35–2.14), the poorest oral function (1.61, 1.31–1.98), and the lowest DVS quartile (1.96, 1.70–2.26).

Conclusion

These results indicate that psychosocial conditions, oral health, and dietary variety are independently associated with intellectual activity in elderly persons. The factors identified in this study may be used in community health programs for maintaining the intellectual activity ability of the elderly.     

Rapid discrimination of <Emphasis Type="Italic">Porphyra tenera</Emphasis> Kjellman var. <Emphasis Type="Italic">tamatsuensis</Emphasis> Miura by PCR-RFLP

To allow to discriminate rapidly the strains of Porphyra tenera var. tamatsuensis, cultivars of which grow more vigorously than strains of P. tenera var. tenera, strains of both varieties were examined by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis using mitochondrial DNA related to the ATP synthase F0 subunit 6 (ATP6) gene. The lengths of all sequences in this region of three strains of each variety were 670 bp and had just a single nucleotide substitution. Digestion with the restriction enzyme of TaaI yielded three visible bands which appeared in the three strains of P. tenera var. tamatsuensis, whereas two bands appeared in the three strains of P. tenera var. tenera. We therefore conclude that PCR-RFLP analysis is a valuable tool for discrimination of P. tenera var. tamatsuensis among the stock of P. tenera strains used for mariculture.