A short form of gross motor function measure for Fukuyama congenital muscular dystrophy.

OBJECTIVES: The objective of this study was to confirm the validity of a short form of gross motor function measure for Fukuyama congenital muscular dystrophy (GMFM for FCMD). METHODS: This study is a case series and was conducted at the Tokyo Women's Medical University. Fifteen patients with FCMD were assessed using both the GMFM for FCMD with 68 items, which was created as a motor function measure for patients with FCMD on the basis of Rasch analysis, and the original GMFM with 88 items. The correlation between the GMFM for FCMD and the Ueda classification was assessed. Time required for each assessment was also evaluated. RESULTS: We found significant correlation between the GMFM for FCMD and the Ueda classification (r = 0.935); furthermore, the mean assessment time tended to decrease when using the GMFM for FCMD. CONCLUSIONS: GMFM for FCMD may be an appropriate motor function scale for patients with FCMD and might help decrease the assessment time.

Sterepinic Acids A⁻C, New Carboxylic Acids Produced by a Marine Alga-Derived Fungus.

Sterepinic acids A⁻C (1⁻3), new carboxylic acids with two primary alcohols, have been isolated from a fungal strain of Stereum sp. OUPS-124D-1 attached to the marine alga Undaria pinnatifida. Dihydro-1,5-secovibralactone (4), a new vibralactone derivative, was isolated from the same fungal metabolites together with known vibralactone A (5), and 1,5-secovibralactone (6). The planar structures of these compounds have been elucidated by spectroscopic analyses using IR, HRFABMS, and NMR spectra. To determine the absolute configuration of the compounds, we used the phenylglycine methyl ester (PGME) method. These compounds exhibited less activity in the cytotoxicity assay against cancer cell lines.

Maximum rate of sweat ions reabsorption during exercise with regional differences, sex, and exercise training.

PURPOSE: It is recently reported that determining sweat rate (SR) threshold for increasing galvanic skin conductance (GSC) would represent a maximum rate of sweat ion reabsorption in sweat glands. We evaluate the maximum rate of sweat ion reabsorption over skin regions, sex, and long-term exercise training by using the threshold analysis in the present study. METHODS: Ten males (2 untrained, 4 sprinters, and 4 distance runners) and 12 females (5 untrained, 4 sprinters, and 3 distance runners) conducted graded cycling exercise for 45 min at low, middle, and high exercise intensities (heart rate 100-110, 120-130, and 140-150 beats/min, respectively) for 10, 15, and 20 min, respectively, at 30 °C and 50% relative humidity. Comparisons were made between males and females and among untrained individuals, distance runners, and sprinters on the back and forearm. RESULTS: SR threshold for increasing GSC on back was significantly higher than that of forearm (P < 0.05) without any sex differences (back 0.70 ± 0.08 and 0.61 ± 0.04, forearm 0.40 ± 0.05 and 0.45 ± 0.06 mg/cm2/min for males and females, respectively). Distance runners and sprinters showed higher SR threshold for increasing GSC than that of untrained subjects on back (P < 0.05) but not on forearm (back 0.45 ± 0.06, 0.83 ± 0.06, and 0.70 ± 0.04, forearm 0.33 ± 0.04, 0.49 ± 0.02, and 0.39 ± 0.07 mg/cm2/min for untrained subjects, distance runners, and sprinters, respectively). CONCLUSION: These results suggest that the maximum sweat ion reabsorption rate on the back is higher than that of forearm without sex differences. Furthermore, exercise training in distance runners and sprinters improves the maximum sweat ion reabsorption rate on the back.

Efficient Generation of Viral and Integration-Free Human Induced Pluripotent Stem Cell-Derived Oligodendrocytes.

Here we document three highly reproducible protocols: (1) a culture system for the derivation of human oligodendrocytes (OLs) from human induced pluripotent stem cells (hiPS) and their further maturation-our protocol generates viral- and integration-free OLs that efficiently commit and move forward in the OL lineage, recapitulating all the steps known to occur during in vivo development; (2) a method for the isolation, propagation and maintenance of neural stem cells (NSCs); and (3) a protocol for the production, isolation, and maintenance of OLs from perinatal rodent and human brain-derived NSCs. Our unique culture systems rely on a series of chemically defined media, specifically designed and carefully characterized for each developmental stage of OL as they advance from OL progenitors to mature, myelinating cells. We are confident that these protocols bring our field a step closer to efficient autologous cell replacement therapies and disease modeling. © 2016 by John Wiley & Sons, Inc.

Efficient Generation of Viral and Integration-Free Human Induced Pluripotent Stem Cell-Derived Oligodendrocytes.

Here we document three highly reproducible protocols: (1) a culture system for the derivation of human oligodendrocytes (OLs) from human induced pluripotent stem cells (hiPS) and their further maturation-our protocol generates viral- and integration-free OLs that efficiently commit and move forward in the OL lineage, recapitulating all the steps known to occur during in vivo development; (2) a method for the isolation, propagation and maintenance of neural stem cells (NSCs); and (3) a protocol for the production, isolation, and maintenance of OLs from perinatal rodent and human brain-derived NSCs. Our unique culture systems rely on a series of chemically defined media, specifically designed and carefully characterized for each developmental stage of OL as they advance from OL progenitors to mature, myelinating cells. We are confident that these protocols bring our field a step closer to efficient autologous cell replacement therapies and disease modeling. © 2016 by John Wiley & Sons, Inc.

Pituitary adenylyl cyclase-activating peptide counteracts hedgehog-dependent motor neuron production in mouse embryonic stem cell cultures.

Pituitary adenylyl cyclase-activating peptide (PACAP; ADCYAP1) is a neuropeptide that regulates a wide array of functions within the brain and periphery. We and others have previously demonstrated that PACAP and its high-affinity receptor PAC1 are expressed in the embryonic mouse neural tube, suggesting that PACAP plays a role in early brain development. Moreover, we previously showed that PACAP antagonizes the mitotic action of Sonic hedgehog (Shh) in postnatal cerebellar granule precursors. In the present study, we demonstrate that PACAP completely blocked Shh-dependent motor neuron generation from embryonic stem cell cultures and reduced mRNA levels of the Shh target gene Gli-1 and several ventral spinal cord patterning genes. In vivo examination of motor neuron and other patterning markers in embryonic day 12.5 spinal cords of wild-type and PACAP-deficient mice by immunofluorescence, on the other hand, revealed no obvious alterations in expressions of Islet1/2, MNR2, Lim1/2, Nkx2.2, or Shh, although the Pax6-positive area was slightly expanded in PACAP-deficient spinal cord. Caspase-3 staining revealed low, and similar, numbers of cells undergoing apoptosis in embryonic wild-type vs. PACAP-deficient spinal cords, whereas a slight but significant increase in number of mitotic cells was observed in PACAP-deficient mice. Thus, although PACAP has a strong capacity to counteract Shh signaling and motor neuron production in vitro, corresponding patterning defects associated with PACAP loss may be obscured by compensatory mechanisms.

A case of reversible posterior leukoencephalopathy syndrome in a patient on peritoneal dialysis.

Reversible posterior leukoencephalopathy syndrome (RPLS) is a recently identified clinical and radiologic entity. The characteristic radiologic findings are bilateral gray and white matter edema in the posterior regions of the cerebral hemispheres. The typical clinical syndrome includes headache, confusion, visual symptoms, and seizures. RPLS most often occurs in the setting of hypertensive crisis, preeclampsia, or with cytotoxic immunosuppressive therapy, but many other clinical settings are described, such as cryoglobulinemia, hemolytic uremic syndrome, systemic lupus erythematosus, and use of erythropoietin. A 24-year-old man, diagnosed as having anaphylactoid purpura nephritis at 12 years of age and who started peritoneal dialysis (PD) at 23 years of age, was admitted to our hospital with a seizure and consciousness disturbance. His blood pressure (BP) and body fluid volume had not been controlled well because of poor compliance with medication and PD. T2-weighted magnetic resonance imaging (MRI) revealed high signal intensity changes restricted to the cortex and subcortical white matter of the cerebellum. On the other hand, diffusion-weighted imaging showed an isointense signal. From these findings, he was diagnosed as having RPLS. With appropriate control of BP and volume control by PD and hemodialysis, his symptoms improved, and a follow-up cranial MRI 1 month later was almost normal. To the best of our knowledge, this is the first report of RPLS in an adult PD patient.

Genetic materials at the gene engineering division, RIKEN BioResource Center.

Genetic materials are one of the most important and fundamental research resources for studying biological phenomena. Scientific need for genetic materials has been increasing and will never cease. Ever since it was established as RIKEN DNA Bank in 1987, the Gene Engineering Division of RIKEN BioResource Center (BRC) has been engaged in the collection, maintenance, storage, propagation, quality control, and distribution of genetic resources developed mainly by the Japanese research community. When RIKEN BRC was inaugurated in 2001, RIKEN DNA Bank was incorporated as one of its six Divisions, the Gene Engineering Division. The Gene Engineering Division was selected as a core facility for the genetic resources of mammalian and microbe origin by the National BioResource Project (NBRP) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan in 2002. With support from the scientific community, the Division now holds over 3 million clones of genetic materials for distribution. The genetic resources include cloned DNAs, gene libraries (e.g., cDNA and genomic DNA cloned into phage, cosmid, BAC, phosmid, and YAC), vectors, hosts, recombinant viruses, and ordered library sets derived from animal cells, including human and mouse cells, microorganisms, and viruses. Recently genetic materials produced by a few MEXT national research projects were transferred to the Gene Engineering Division for further dissemination. The Gene Engineering Division performs rigorous quality control of reproducibility, restriction enzyme mapping and nucleotide sequences of clones to ensure the reproducibility of in vivo and in vitro experiments. Users can easily access our genetic materials through the internet and obtain the DNA resources for a minimal fee. Not only the materials, but also information of features and technology related to the materials are provided via the web site of RIKEN BRC. Training courses are also given to transfer the technology for handling viral vectors. RIKEN BRC supports scientists around the world in the use of valuable genetic materials.

Stable embryonic stem cell lines in rabbits: potential small animal models for human research.

Although embryonic stem (ES) cell lines derived from mice and primates are used extensively, the development of such lines from other mammals is extremely difficult because of their rapid decline in proliferation potential and pluripotency after several passages. This study describes the establishment of rabbit ES cell lines with indefinite proliferation potential. It was found that the feeder cell density determines the fate of rabbit ES cells, and that maximum proliferation potential was obtained when they were cultured on a feeder cell density of one-sixth of the density at confluency. Higher and lower densities of feeder cells induced ES cell differentiation or division arrest. Under optimized conditions, rabbit ES cells were passaged 50 times, after which they still possessed high telomerase activity. This culture system enabled efficient gene transduction and clonal expansion from single cells. During culture, rabbit ES cells exhibited flattened monolayer cell colonies, as reported for monkey and human ES cells, and expressed pluripotency markers. Embryoid bodies and teratomas formed readily in vitro and in vivo respectively. These ES cell lines can be safely cryopreserved for later use. Thus, rabbit ES cells can be added to the list of stable mammalian ES cells, enabling the rabbit to be used as a small animal model for the study of human cell transplantation therapy.

[The range of activities of home enteral and infusion therapy for home medical care at Hyogo College of Medicine].

Our nutrition department has consolidated to become a clinical nutrition department by integrating NST and home enteral and intravenous nutrition supporting nurses into the nutrition department, which deals with a total control of nutrition therapy including diet, infusion and care at hospital and home. The advantages are that nutrition therapy is feasible in addition to nutritional control by the involvement (participation) of physicians, and that it becomes available for a national registered dietitian to counsel on a nutritional control on more clinical bases. Further, a framework has been constructed to perform all the nutritional control and nutrition therapy with proper coordination on hospitalized patients as well as home-care patients.

Hepatic gene expression profile of lipid metabolism in rats: Impact of caloric restriction and growth hormone/insulin-like growth factor-1 suppression.

We investigated the role of the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis on caloric restriction (CR) using male wild-type and transgenic homozygous dwarf rats bearing an antisense GH transgene and their F1 heterozygous progeny fed either ad libitum or subjected to 30% CR. CR predominantly altered expression of hepatic genes involved in the stress response, xenobiotic metabolism, and lipid metabolism. Most gene expressions involved in stress response and xenobiotic metabolism were regulated in a GH/IGF-1-dependent manner, and those involved in lipid metabolism were regulated in a GH/IGF-1-independent manner. Moreover, CR enhanced the gene expression involved in fatty acid synthesis after feeding and those encoding mitochondrial beta-oxidation enzymes during food shortage, probably via transcriptional regulation by peroxisome proliferator-activated receptor alpha. These results, taken together with serum biochemical measures and hepatic triglyceride content, suggest that CR promotes lipid utilization through hepatic transcriptional alteration and prevents hepatic steatosis in a GH/IGF-1-independent manner.

Inhibition of self-renewal and induction of neural differentiation by PACAP in neural progenitor cells.

Several lines of evidence have suggested roles for pituitary adenylate cyclase-activating polypeptide (PACAP) in the developing nervous system. Previously, we showed that mRNA for PACAP, vasoactive intestinal peptide (VIP), and their three receptor subtypes, is differentially expressed in embryonic stem (ES) cells, ES cell-derived, neural stem cell-enriched cultures, and differentiated neurons, by using the five steps of the in vitro neuronal culture model of ES cell differentiation. Here, we examined the effects of PACAP on self-renewal and cell lineage determination of neural progenitor/stem cells. PACAP inhibited the basic fibroblast growth factor-induced proliferation (self-renewal), as assessed by neurosphere formation. PACAP increased microtubule-associated protein 2-positive neurons without affecting the number of cells positive for the neural stem cell marker nestin, astrocyte marker glial fibrillary acidic protein, and oligodendrocyte marker CNPase. These results suggest that PACAP inhibits self-renewal but, instead, induces early neuronal differentiation of neural progenitor cells.

A database of recombinant viruses and recombinant viral vectors available from the RIKEN DNA bank.

BACKGROUND: Viral vectors are required as gene-delivery systems for gene therapy and basic research. Recombinant adenoviruses (rAds) expressing genes of interest are being developed as research tools and many studies in vitro and in vivo have already been performed with such rAds. METHODS: Shuttle vectors for rAds were constructed with full-length cDNAs and rAds were generated in HEK293 cells by the COS-TPC method. The rAds and shuttle vectors were developed by the Japanese research community and deposited in the RIKEN DNA Bank (RDB; http://www.brc.riken.jp/lab/dna/en/) for distribution to the scientific community. The Recombinant Virus Database (RVD; http://www.brc.riken.jp/lab/dna/rvd/) was established at the RIKEN BioResource Center (BRC) in Japan as the source of information about and distribution of the various resources. RESULTS: The RIKEN BRC is releasing more than 300 recombinant viruses (RVs) and 500 shuttle vectors, as well as all related information, which is included in a newly established database, the RVD. The RVD consists of (i) information about the RVs, the inserted cDNAs and the shuttle vectors; (ii) data about sequence-tagged sites (STSs) that are markers of viral DNAs; and (iii) experimental protocols for the use of RVs. CONCLUSIONS: The new database and available resources should be very useful to scientists who are studying human gene therapy and performing related basic research. It is a web-interfaced flat-file database that can be accessed through the internet. Moreover, all of the resources deposited in the RDB, which is a public facility in Japan, are available to researchers around the world.

Purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration.

Adenoviruses are excellent vectors for gene transfer and are used extensively for high-level expression of the products of transgenes in living cells. The development of simple and rapid methods for the purification of stable infectious recombinant adenoviruses (rAds) remains a challenge. We report here a method for the purification of infectious adenovirus type 5 (Ad5) that involves ultracentrifugation on a cesium chloride gradient at 604,000g for 15 min at 4 degrees C and tangential flow filtration. The entire procedure requires less than two hours and infectious Ad5 can be recovered at levels higher than 64% of the number of plaque-forming units (pfu) in the initial crude preparation of viruses. We have obtained titers of infectious purified Ad5 of 1.35x10(10) pfu/ml and a ratio of particle titer to infectious titer of seven. The method described here allows the rapid purification of rAds for studies of gene function in vivo and in vitro, as well as the rapid purification of Ad5.

Differential expression of mRNAs for PACAP and its receptors during neural differentiation of embryonic stem cells.

The expressions of mRNAs for pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and their receptors (PAC1, VPAC1 and VPAC2) were examined in the five steps of the in vitro neuronal culture model of embryonic stem (ES) cell differentiation. mRNAs for PACAP, VIP, PAC1 receptor, and VPAC2 receptor were moderately expressed in neural stem cell-enriched cultures, while VPAC1 receptor mRNA was most prominently expressed in embryoid bodies (EBs). The expression of PAC1 receptor mRNA was further upregulated after terminal differentiation into neurons. In contrast, the expressions of PAC1 receptor and PACAP mRNAs were markedly decreased after glial differentiation. These results suggest that this in vitro neuronal culture system will be a useful model for future studies on the functional role of the PACAPergic system during different stages of neuronal development.

Neuroprotective action of endogenous PACAP in cultured rat cortical neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts neurotrophic effects both in vitro and in vivo. Here we demonstrate the upregulation of PACAP mRNA expression in cultured rat cortical neurons after excitotoxic glutamate exposure, and the exacerbating effect of the PACAP receptor antagonist, PACAP(6-38), on neuronal viability. PACAP mRNA levels were increased up to 3.5-fold 8 h after glutamate exposure. PACAP(6-38) decreased the viability of cortical neurons, irrespective of whether the cells were exposed to glutamate or not. PACAP(6-38) also inhibited glutamate-induced expression of PACAP mRNA, suggesting that PACAP acts via an autocrine or paracrine mechanism to enhance PACAP expression itself. Glutamate exposure is known to increase brain-derived neurotrophic factor (BDNF) mRNA expression. This increased expression was markedly suppressed by PACAP(6-38). Our previous study has shown that PACAP stimulates the PACAP gene transcription in PC12 cells. Taken together, these data may suggest that endogenous PACAP regulates the expression of PACAP itself and BDNF. Although it may also be possible that PACAP(6-38)-induced death of PACAP and BDNF mRNA-expressing cells, per se, results in reduced levels of these mRNAs, the present results support the idea that endogenous PACAP has a neuroprotective action.

A simple method for the simultaneous detection of E1A and E1B in adenovirus stocks.

Recombinant adenoviral vectors have been developed for use as therapeutic agents and for the introduction of exogenous genes into living cells. However, the occurrence of replication-competent adenoviruses (RCA) in adenovirus stocks produced in 293 cells remains a major problem in terms of the safe use of such vectors. To overcome the problems associated with the occurrence of RCA, we have established a simple method for the simultaneous detection of amplified E1A and E1B from RCA that might contaminate adenoviral stocks. The products amplified by polymerase chain reaction (PCR) were fractionated by regular electrophoresis on agarose gels and visualized by staining with ethidium bromide. This method is rapid and inexpensive for detection of RCA in the preparation of adenoviruses.

Possible involvement of a cyclic AMP-dependent mechanism in PACAP-induced proliferation and ERK activation in astrocytes.

In cultured astrocytes, PACAP activates extracellular signal-regulated kinase (ERK) and induces cell proliferation at picomolar concentrations. Here, we examined the role of cyclic AMP signaling underlying the effects of PACAP. PACAP38 induced accumulation of cyclic AMP in astrocytes at concentrations as low as 10(-12)M. PACAP38 (10(-12)-10(-9)M)-stimulated cell proliferation was completely abolished by the cyclic AMP antagonist Rp-cAMP, whereas the protein kinase A (PKA) inhibitor H89 had no effect. This PACAP38-mediated effect was also abolished by the ERK kinase inhibitor PD98059, suggesting the involvement of ERK in PACAP-induced proliferation. PACAP38 (10(-12)M)-stimulated phosphorylation of ERK lasted for at least 60 min. This effect was completely abolished by Rp-cAMP but not by H89. Dibutyryl cyclic AMP maximally stimulated the incorporation of thymidine and activation of ERK at 10(-10)M. These results suggest that PACAP-mediated stimulation of ERK activity and proliferation of astrocytes may involve a cyclic AMP-dependent, but PKA-independent, pathway.

Defects in reproductive functions in PACAP-deficient female mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved neuropeptide and widely expressed in both brain and peripheral tissues, including several reproductive organs (e.g., testis and ovary). PACAP stimulates syntheses of several sexual hormones and steroids, suggesting it has possible roles in reproductive function. In this study, the role of PACAP in female reproductive functions such as fertility, mating behavior and maternal behaviors were investigated by using mice lacking PACAP (PACAP(-/-)). PACAP(-/-) females showed reduced fertility (the number of parturitions relative to the number of pairings). Mating experiments using vasectomized males revealed that mating frequency and its intervals in some PACAP(-/-) females were quite different (zero to eight times/4 weeks), whereas the frequency was relatively constant (two to three times/4 weeks) in wild-type females. In PACAP(-/-) females, maternal crouching behavior tended to decrease compared to wild-type females, although the influence of litter size on maternal behavior needs to be considered. These data suggest a role for endogenous PACAP in female reproductive processes.