Functional evaluation of novel variants of B4GALNT1 in a patient with hereditary spastic paraplegia and the general population

Hereditary spastic paraplegia (HSP) is a heterogeneous group of neurological disorders that are characterized by progressive spasticity and weakness in the lower limbs. SPG26 is a complicated form of HSP, which includes not only weakness in the lower limbs, but also cognitive impairment, developmental delay, cerebellar ataxia, dysarthria, and peripheral neuropathy, and is caused by biallelic mutations in the B4GALNT1 (beta-1,4-N-acetylgalactosaminyltransferase 1) gene. The B4GALNT1 gene encodes ganglioside GM2/GD2 synthase (GM2S), which catalyzes the transfer of N-acetylgalactosamine to lactosylceramide, GM3, and GD3 to generate GA2, GM2, and GD2, respectively. The present study attempted to characterize a novel B4GALNT1 variant (NM_001478.5:c.937G>A p.Asp313Asn) detected in a patient with progressive multi-system neurodegeneration as well as deleterious variants found in the general population in Japan. Peripheral blood T cells from our patient lacked the ability for activation-induced ganglioside expression assessed by cell surface cholera toxin binding. Structural predictions suggested that the amino acid substitution, p.Asp313Asn, impaired binding to the donor substrate UDP-GalNAc. An in vitro enzyme assay demonstrated that the variant protein did not exhibit GM2S activity, leading to the diagnosis of HSP26. This is the first case diagnosed with SPG26 in Japan. We then extracted 10 novel missense variants of B4GALNT1 from the whole-genome reference panel jMorp (8.3KJPN) of the Tohoku medical megabank organization, which were predicted to be deleterious by Polyphen-2 and SIFT programs. We performed a functional evaluation of these variants and demonstrated that many showed perturbed subcellular localization. Five of these variants exhibited no or significantly decreased GM2S activity with less than 10% activity of the wild-type protein, indicating that they are carrier variants for HSP26. These results provide the basis for molecular analyses of B4GALNT1 variants present in the Japanese population and will help improve the molecular diagnosis of patients suspected of having HSP

Learning and teaching how to speak Japanese language

How students learn to speak Japanese in a Japanese language class is the main question for my research. There are many ways of/earning how to speak a foreign language. The students\u27 learning can be promoted by a teacher\u27s lecture, but also by many other elements (listening, reading, speaking, watching films, etc.) In addition, there is the possibility that the students learn different speaking styles by observing interactions of their teacher. From this observation, I learned that teaching how to speak the Japanese language is complicated. I observed that Ms. Smith was always thinking about when, where, what, and how she teaches and to whom. It is extremely difficult to take every factor into consideration. There arc many factors which relate to learning. A lack of time seemed to be the greatest barrier facing Ms. Smith. The students learn how to speak Japanese most effectively when they are presented a wide-range of activities that have been adapted to the individual classroom, school and class make-up. Through this project, as a teacher, I learned that I need to be aware of all the factors to promote students\u27 learning

Stimulation with the Aureobasidium pullulans-produced β-glucan effectively induces interferon stimulated genes in macrophage-like cell lines

A β-(1,3),(1,6)-D-glucan produced by A. pullulans (AP-PG) is known to be an immune stimulating agent. In this study, we demonstrate that the stimulation with AP-PG effectively induces the interferon (IFN) stimulated genes (ISGs) in macrophage-like cell lines. The ISGs, Mx1, ISG15, and viperin mRNAs were significantly increased in RAW264.7 cells after stimulation with AP-PG. The stimulation with AP-PG transiently induced IFN-β mRNA. However, the expression of viperin mRNA was also increased after stimulation with AP-PG even when new protein synthesis was completely blocked by treatment with cycloheximide. Further, in IFN-α receptor knockdown RAW264.7 cells, AP-PG stimulation more effectively induced viperin mRNA compared with that of IFN-α stimulation. The phosphorylation of Ser 727 in STAT1 involved in the enhancement of STAT1 activation was immediately increased after stimulation with AP-PG. In addition, viperin mRNA expression induced after stimulation with IFN-α was significantly increased by combined stimulation with AP-PG. These results suggest that stimulation with AP-PG effectively induces the ISGs through the induction of IFN and the enhancement of STAT1-mediated transcriptional activation

Antimicrobial Properties of a Copper/Silicone Composite Membrane Prepared Using a Two-Step Immersion Process in Iodine and Copper Sulfate Solutions

Silicone (polydimethylsiloxane) materials are widely used in various applications. Due to microbe adherence and biofilm formation at the surface of silicone materials, silicone materials must possess antibacterial properties. To achieve this, we prepared copper (Cu)–silicone composite membranes using a simple two-step process of immersion in iodine and copper sulfate solutions. Subsequent scanning electron microscopy revealed Cu nanoparticles (CuNPs) of 10 to 200 nanometers in diameter on the silicone membrane surface, which were identified as copper iodide using energy-dispersive X-ray spectroscopy. The mechanical strength of the material did not change significantly as a result of the two-step immersion treatment and the Cu/silicone membrane showed excellent antibacterial efficacy against Escherichia coli and Staphylococcus aureus, maintaining R > 2 even after a physical impact such as stomacher treatment. Additionally, the Cu ions eluted from the Cu/silicone membrane remained at very low concentrations, suggesting firm immobilization of CuNPs on the silicone membrane. This proposed antimicrobial treatment method does not require special equipment, can be performed at room temperature, and has the potential for use on silicone materials other than membranes

Oral administration of the β-glucan produced by Aureobasidium pullulans ameliorates development of atherosclerosis in apolipoprotein E deficient mice

The Aureobasidium pullulans-produced β-glucan (AP-PG) is an immune stimulator, and believed to exhibit beneficial effects on health through its immune stimulating activity. Here, the effect of oral administration of AP-PG on high-fat diet (HFD)-induced atherosclerosis was evaluated using apolipoprotein E deficient mice, a widely used mouse model for atherosclerosis. The results demonstrated that HFD-induced development of atherosclerosis was significantly reduced in the AP-PG-treated mice when compared with that of the control mice. In serological analysis, blood levels of oxidized low-density lipoprotein cholesterol, a well-known risk factor for the development of atherosclerosis, were significantly reduced in the AP-PG-treated group of mice. Further, immunohistochemical analysis using MOMA-2 antibody showed that oral administration of AP-PG is effective in ameliorating vascular accumulation of macrophages. These data suggest the possibility that oral administration of AP-PG is effective in ameliorating HFD-induced development of atherosclerosis

Oral administration of the Aureobasidium pullulans-derived β-glucan effectively prevents the development of high fat diet-induced fatty liver in mice

Aureobasidium pullulans-derived β-glucan (AP-PG) consisting of a β-(1,3)-linked glucose main chain and β-(1,6)-linked glucose branches is taken as a supplement to improve health. This study demonstrates that oral administration of AP-PG is effective to prevent the development of high-fat diet (HFD)-induced fatty liver in mice. Here, C57BL/6N mice were fed with a normal diet or HFD, and AP-PG diluted in drinking water was administered orally. After 16 weeks, the serological analysis showed that HFD-induced high blood cholesterol and triglyceride levels were reduced by the oral administration of AP-PG. Further, HFD induced-fatty liver was significantly reduced by the oral administration of AP-PG. The triglyceride accumulation in the liver was also significantly reduced in mice administered AP-PG. Liver injury as indicated by an increase in serum alanine aminotransferase (ALT) in the HFD-fed mice was significantly reduced in the mice administered AP-PG orally, and the gene expression of cholesterol 7 alpha-hydroxylase (CYP7A1) which is known to be involved in cholesterol degradation in the liver was significantly increased in the AP-PG administered mice. These results suggest the possibility that the oral administration of AP-PG is effective to prevent the development of non-alcoholic fatty liver disease (NAFLD)

β-Glucan Derived from Aureobasidium pullulans Is Effective for the Prevention of Influenza in Mice

β-(1→3)-D-glucans with β-(1→6)-glycosidic linked branches produced by mushrooms, yeast and fungi are known to be an immune activation agent, and are used in anti-cancer drugs or health-promoting foods. In this report, we demonstrate that oral administration of Aureobasidium pullulans-cultured fluid (AP-CF) enriched with the β-(1→3),(1→6)-D-glucan exhibits efficacy to protect mice infected with a lethal titer of the A/Puerto Rico/8/34 (PR8; H1N1) strain of influenza virus. The survival rate of the mice significantly increased by AP-CF administration after sublethal infection of PR8 virus. The virus titer in the mouse lung homogenates was significantly decreased by AP-CF administration. No significant difference in the mRNA expression of inflammatory cytokines, and in the population of lymphocytes was observed in the lungs of mice administered with AP-CF. Interestingly, expression level for the mRNA of virus sensors, RIG-I (retinoic acid-inducible gene-I) and MDA5 (melanoma differentiation-associated protein 5) strongly increased at 5 hours after the stimulation of A. pullulans-produced purified β-(1→3),(1→6)-D-glucan (AP-BG) in murine macrophage-derived RAW264.7 cells. Furthermore, the replication of PR8 virus was significantly repressed by pre-treatment of AP-BG. These findings suggest the increased expression of virus sensors is effective for the prevention of influenza by the inhibition of viral replication with the administration of AP-CF

A small scale study on the effects of oral administration of the β-glucan produced by Aureobasidium pullulans on milk quality and cytokine expressions of Holstein cows, and on bacterial flora in the intestines of Japanese black calves

Background: The β–(1→3),(1→6)-D-glucan extracellularly produced by Aureobasidium pullulans exhibits immunomodulatory activity, and is used for health supplements. To examine the effects of oral administration of the β–(1→3),(1→6)-D-glucan to domestic animals, a small scale study was conducted using Holstein cows and newborn Japanese Black calves. Findings: Holstein cows of which somatic cell count was less than 3 x 105/ml were orally administered with or without the β-(1→3),(1→6)-D-glucan-enriched A. pullulans cultured fluid (AP-CF) for 3 months, and the properties of milk and serum cytokine expression were monitored. Somatic cell counts were not significantly changed by oral administration of AP-CF, whereas the concentration of solid non fat in the milk tended to increase in the AP-CF administered cows. The results of cytokine expression analysis in the serum using ELISA indicate that the expressions of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in all cows which were orally administered with AP-CF became slightly lower than that of control cows after the two-month treatment. On the other hand, IL-8 expression tended to indicate a moderately higher level in all treated cows after the three-month administration of AP-CF in comparison with that of the control cows. Peripartum Japanese Black beef cows and their newborn calves were orally administered with AP-CF, and bacterial flora in the intestines of the calves were analyzed by T-RFLP (terminal restriction fragment length polymorphism). The results suggest that bacterial flora are tendentiously changed by oral administration of AP-CF. Conclusions: Our data indicated the possibility that oral administration of the β–(1→3),(1→6)-D- glucan produced by A. pullulans affects cytokine expressions in the serum of Holstein cows, and influences bacterial flora in the intestines of Japanese Black calves. The findings may be helpful for further study on the efficacies of oral administration of β-(1→3),(1→6)-D-glucans on domestic animals