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看護学生のコミュニケーション能力の縦断的変化について明らかにし、学習支援や授業改善の資料とするために、大学１年生から２年生にわたり縦断的に調査を行った。調査内容及び方法は、コミュニケーション技術評価尺度を、１年生で行うコミュニケーション授業の前後と基礎看護学実習Ⅰ終了後、加えて２年生時の基礎看護学実習Ⅱの終了後に測定した。毎回の授業後には理解度・関心度を調査し、授業終了後には授業評価を実施した。なお、１年生で行う基礎看護学実習Ⅰ終了後には、コミュニケーションに関する感想や学び、課題を自由記述で求めた。結果として、１年生ではコミュニケーションの基本技術が向上し、２年生では、対象との関係性に向かうため非言語コミュニケーション力が向上していた。学習支援上の課題は、グループ活動による学習では、学生の対人緊張を和らげる工夫が必要と考えられた。

Background Spinal muscular atrophy is a neurodegenerative disorder caused by the deficient expression of survival motor neuron protein in motor neurons. A major goal of disease-modifying therapy is to increase survival motor neuron expression. Changes in survival motor neuron protein expression can be monitored via peripheral blood cells in patients; therefore we tested the sensitivity and utility of imaging flow cytometry for this purpose. Methods After the immortalization of peripheral blood lymphocytes from a human healthy control subject and two patients with spinal muscular atrophy type 1 with two and three copies of SMN2 gene, respectively, we used imaging flow cytometry analysis to identify significant differences in survival motor neuron expression. A bright detail intensity analysis was used to investigate differences in the cellular localization of survival motor neuron protein. Results Survival motor neuron expression was significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Moreover, survival motor neuron expression correlated with the clinical severity of spinal muscular atrophy according to SMN2 copy number. The cellular accumulation of survival motor neuron protein was also significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Conclusions The benefits of imaging flow cytometry for peripheral blood analysis include its capacities for analyzing heterogeneous cell populations; visualizing cell morphology; and evaluating the accumulation, localization, and expression of a target protein. Imaging flow cytometry analysis should be implemented in future studies to optimize its application as a tool for spinal muscular atrophy clinical trials.

Neuromuscular disorders are clinically and genetically heterogeneous diseases with broadly overlapping clinical features. Progress in molecular genetics has led to the identification of numerous causative genes for neuromuscular disorders, but Sanger sequencing-based diagnosis remains labor-intensive and expensive because the genes are large, the genotypes and phenotypes of neuromuscular disorders overlap and multiple genes related to a single phenotype exist. Recently, the advent of next-generation sequencing (NGS) has enabled efficient, concurrent examination of several related genes. Thus, we used NGS for target resequencing of neuromuscular disease-related genes from 42 patients in whom undiagnosed early-onset neuromuscular disorders. Causative genes were identified in 19/42 (45.2%) patients (six, congenital muscular dystrophy; two, Becker muscular dystrophy (BMD); three, limb-girdle muscular dystrophy; one, concurrent BMD and Fukuyama congenital muscular dystrophy; three, nemaline myopathy; one, centronuclear myopathy; one, congenital fiber-type disproportion; one, myosin storage myopathy; and one, congenital myasthenic syndrome). We detected variants of uncertain significance in two patients. In 6/19 patients who received a definitive diagnosis, the diagnosis did not require muscle biopsy. Thus, for patients with suspected neuromuscular disorders not identified using conventional genetic testing alone, NGS-based target resequencing has the potential to serve as a powerful tool that allows definitive diagnosis.

To clarify the long-term natural history of SMA in Japanese patients by investigating the peak motor milestones of cases 7months through 57years of age, in efforts to contribute to evaluating outcomes of new therapeutic interventions.We sub-classified 112 SMA type I-III cases into type Ia, type Ib, type IIa, type IIb, type IIIa and type IIIb, according to peak motor milestone achieved, and analyzed the SMN1, SMN2 and NAIP genes in relation to clinical subtypes.In type I cases, there was a significant difference (p0.0001), depending on whether or not head control was obtained, in the time of ventilation support being required. In type II cases as well, the time at which the ability to maintain the sitting position independently was lost also differed significantly (p0.01) between those acquiring the ability to sit unaided within eight months after birth and those acquiring this ability after eight months of age. In type III cases, being able versus unable to climb stairs was associated with a significant difference (p=0.02) in the median time until loss of walking independently. Positive correlations were also seen between copy numbers and the clinical severity of SMA.Our long-term results show peak motor milestone evaluations distinguishing between subtypes to be useful not only as outcome measures for assessing treatment efficacy in clinical trials but also for predicting the clinical courses of Japanese SMA patients.

We describe a Gorlin syndrome (GS) case with two different second hit mutations of PTCH1, one in a keratocystic odontogenic tumor (KCOT) and the other in an ovarian leiomyoma. GS is a rare genetic condition manifesting as multiple basal cell nevi associated with other features such as medulloblastomas, skeletal abnormalities, and ovarian fibromas. A 21-year-old Japanese woman with a history of two KCOTs was diagnosed with GS according to clinical criteria. A PTCH1 mutation, c.1427del T, was detected in peripheral blood. A novel PTCH1 mutation, c.264_265insAATA, had been found in the maxillary KCOT as a second hit mutation. More recently, the ovarian tumor was detected during a gynecological examination. Laparoscopic adnexectomy was performed, and the pathological diagnosis of the ovarian tumor was leiomyoma. Interestingly, another novel mutation, loss of heterozygosity spanning from 9q22.32 to 9q31.2, including PTCH1 and 89 other genes, was detected in this ovarian tumor, providing evidence of a second hit mutation. This is the first report describing a GS-associated ovarian tumor carrying a second hit in the PTCH1 region. We anticipate that accumulation of more cases will clarify the importance of second hit mutations in ovarian tumor formation in GS.

Cardiac remodeling and angiotensin II-forming enzyme activity of the left ventricle on chronic pressure overload were studied in male Syrian hamsters, whose chymase activity is similar to that of dogs. Pressure overload was achieved by banding at the ascending aorta (aortic stenosis). Echocardiography, histological analysis, and analysis of cardiac angiotensin-converting enzyme and chymase-like activities were performed. At 10 weeks after banding, concentric hypertrophy of the left ventricle was evident. At 20 weeks after banding, the ventricular weight-to-body ratio, cardiac fibrosis, and cardiac chymase-like activity were significantly increased, while cardiac angiotensin-converting enzyme activity was significantly decreased. This suggests that cardiac chymase, compared with cardiac angiotensin-converting enzyme, was activated against the chronic pressure overload and was responsible for the cardiac remodeling through the formation of angiotensin II. Considering the utility of the rodents, the interspecies similarity of the Ang II-forming pathway, and the effect of chymase in the hamsters, the present model is considered useful for studies evaluating the effect of Ang II and chymase in the canine heart with chronic pressure overload.

ATF3 (Activating transcription factor 3), a member of the CREB/ATF family, can be induced by stress and growth factors in mammalian cells, and is thought to play an important role in the cardiovascular system. However, little is currently known about how the induction of ATF3 is regulated, except that the JNK pathway is involved. Here, we investigated the differential roles of the MAPK pathways involved in TNFalpha (tumour necrosis factor alpha)-induced ATF3 expression in vascular endothelial cells. In human umbilical vein endothelial cells, the expression of constitutively active MKK7 (MAPK kinase 7) increased the number of ATF3-positive cells, and dominant negative MKK7 suppressed the TNFalpha-induced expression of ATF3, indicating a requirement for the JNK pathway. In contrast, the expression of constitutively active or dominant negative MEK1/2 (MAPK/ERK kinase 1/2) suppressed or enhanced TNFalpha-mediated induction of ATF3, respectively. In support of this, the MEK1/2 specific inhibitor U0126 enhanced the expression of ATF3 induced by TNFalpha. Furthermore, the ERK pathway inhibits the TNFalpha-mediated induction of ATF3 mRNA, but not its stability, suggesting the involvement of ERK activity in the transcriptional regulation of the ATF3 gene. Our results suggest that TNFalpha-induced ATF3 gene expression is bidirectionally regulated by the JNK and ERK pathways in vascular endothelial cells.

Mitogen-activated protein kinases (MAPKs) are activated in response to various extracellular stimuli, and their activities are regulated by upstream activating kinases and protein phosphatases such as MAPK phosphatases (MKPs). We report the identification and characterization of a novel MKP termed SKRP1 (SAPK pathway-regulating phosphatase 1). It contains an extended active site sequence motif conserved in all MKPs but lacks a Cdc25 homology domain. Immunoblotting analysis revealed that SKRP1 is constitutively expressed, and its transcripts of 4.0 and 1.0 kb were detected in almost tissues examined. SKRP1 was highly specific for c-Jun N-terminal kinase (JNK) in vitro and effectively suppressed the JNK activation in response to tumor necrosis factor alpha or thapsigargin. Endogenous SKRP1 was present predominantly in the cytoplasm and co-localized with JNK. However, SKRP1 does not bind directly to its target JNK, but co-precipitation of SKRP1 with the MAPK kinase MKK7, a JNK activator, was found in vitro and in vivo. Furthermore, we found that SKRP1 did not interfere with the co-precipitation of MKK7 with JNK. Together, our findings indicate that SKRP1 interacts with its physiological substrate JNK through MKK7, thereby leading to the precise regulation of JNK activity in vivo.

Spinal muscular atrophy (SMA) is caused by mutations within the survival motor neuron 1 (SMN1) gene. These mutations result in the reduction of survival motor neuron (SMN) protein expression and SMN complex in spinal motor neurons and other tissues. SMN protein has been used as a therapeutic biomarker in recent SMA clinical studies using enzyme-linked immunosorbent assay (ELISA). Here, we investigated whether imaging flow cytometry can be a viable source of quantitative information on the SMN protein. Using a FlowSight imaging flow cytometer (Merck-Millipore, Germany), we demonstrated that imaging flow cytometry could successfully identify different expression patterns and subcellular localization of SMN protein in healthy human fibroblasts and SMA patient-derived fibroblasts. In addition, we could also evaluate the therapeutic effects of SMN protein expression by valproic acid treatment of SMA patient-derived cells in vitro. Therefore, we suggest that imaging flow cytometry technology has the potential for identifying SMN protein expression level and pattern as an evaluation tool of clinical studies.

We have developed a method for visualizing phosphorylation of proteins in living cells using a novel fluorescent indicator composed of two green fluorescent protein (GFP) variants joined by the kinase-inducible domain (KID) of the transcription factor cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB). Phosphorylation of KID by the cAMP-dependent protein kinase A (PKA) decreased the fluorescence resonance energy transfer (FRET) among the flanking GFPs. By transfecting COS-7 cells with an expression vector encoding this indicator protein (termed ART for cAMP-responsive tracer), we were able to visualize activation dynamics of PKA in living cells.

First-trimester chorionic-villi-derived cells (FTCVs) are the earliest fetal material that can be obtained for prenatal diagnosis of fetal disorders such as Duchenne muscular dystrophy (DMD). DMD is a devastating X-linked disorder characterized by the absence of dystrophin at the sarcolemma of muscle fibers. Currently, a limited number of treatment options are available for DMD, although cell therapy is a promising treatment strategy for muscle degeneration in DMD patients. A novel candidate source of cells for this approach is FTCVs taken between the 9th and 11th weeks of gestation. FTCVs might have a higher undifferentiated potential than any other tissue-derived cells because they are the earliest fetal material. We examined the expression of mesenchymal stem cell and pluripotent stem cell markers in FTCVs, in addition to their myogenic potential. FTCVs expressed mesenchymal stem cell markers and Nanog and Sox2 transcription factors as pluripotent stem cell markers. These cells efficiently differentiated into myotubes after myogenic induction, at which point Nanog and Sox2 were down-regulated, whereas MyoD, myogenin, desmin and dystrophin were up-regulated. To our knowledge, this is the first demonstration that FTCVs can be efficiently directed to differentiate in vitro into skeletal muscle cells that express dystrophin as the last stage marker of myogenic differentiation. The myogenic potential of FTCVs reveals their promise for use in cell therapy for DMD, for which no effective treatment presently exists. Electronic supplementary material The online version of this article (doi:10.1007/s00441-012-1340-9) contains supplementary material, which is available to authorized users.

Stress-activated protein kinase (SAPK) pathway-regulating phosphatase 1 (SKRP1) has been identified as a member of the mitogen-activated protein kinase (MAPK) phosphatase (MKP) family that interacts physically with the MAPK kinase (MAPKK) MKK7, a c-Jun N-terminal kinase (JNK) activator, and inactivates the MAPK JNK pathway. Although these findings indicated that SKRP1 contributes to the precise regulation of JNK signaling, it remains to be elucidated how SKRP1 is integrated into this pathway. We report that SKRP1 also plays a scaffold role for the JNK signaling, judged by the following observations. SKRP1 selectively formed the stable complexes with MKK7 but not with MKK4 and biphasically regulated the MKK7 activity and MKK7-induced gene transcription in vivo. Co-precipitation analysis between SKRP1 and MKK7-activating MAPKK kinases (MAPKKKs) revealed that SKRP1 also interacted with the MAPKKK, apoptosis signal-regulating kinase 1 (ASK1), but not with MAP kinase kinase kinase 1 (MEKK1). Consistent with these findings, SKRP1 expression increased the ASK1-MKK7 complexes in a dose-dependent manner and specifically enhanced the activation of MKK7 by ASK1. Thus, our findings are, to our knowledge, the first evidence to show that an MKP also functions as a scaffold protein for the particular MAPK signaling.

Mitogen-activated protein kinases (MAPKs) are activated in response to various extracellular stimuli, and their activities are regulated by upstream activating kinases and protein phosphatases such as MAPK phosphatases (MKPs). We report the identification and characterization of a novel MKP termed SKRP1 (SAPK pathway-regulating phosphatase 1). It contains an extended active site sequence motif conserved in all MKPs but lacks a Cdc25 homology domain. Immunoblotting analysis revealed that SKRP1 is constitutively expressed, and its transcripts of 4.0 and 1.0 kb were detected in almost tissues examined. SKRP1 was highly specific for c-Jun N-terminal kinase (JNK) in vitro and effectively suppressed the JNK activation in response to tumor necrosis factor alpha or thapsigargin. Endogenous SKRP1 was present predominantly in the cytoplasm and co-localized with JNK. However, SKRP1 does not bind directly to its target JNK, but co-precipitation of SKRP1 with the MAPK kinase MKK7, a JNK activator, was found in vitro and in vivo. Furthermore, we found that SKRP1 did not interfere with the co-precipitation of MKK7 with JNK. Together, our findings indicate that SKRP1 interacts with its physiological substrate JNK through MKK7, thereby leading to the precise regulation of JNK activity in vivo.

Mitosis utilizes a number of kinesin-related proteins (KRPs). Here we report the identification of a novel KRP termed KRMP1, which has a deduced 1780-amino acid sequence composed of ternary domains. The amino-terminal head domain is most similar to the kinesin motor domain of the MKLP-1 subfamily and has an intrinsic ATPase activity that is diminished by substituting the consensus Lys-168 with Arg. The central stalk domain is predicted to form a long alpha-helical coiled-coil, and can interact with each other in vivo. An in vivo labeling experiment revealed that KRMP1 is phosphorylated, and we also found that the region within the tail domain containing Thr-1604 as the cdc2 kinase phosphorylation site differs from the bimC box conserved in the bimC subfamily of KRPs. Immunofluorescence analysis showed that endogenous KRMP1 was localized predominantly to the cytoplasm during interphase and dispersed throughout the cell during mitosis. Consistent with this finding, overexpressed KRMP1 was detected in a complicated nuclear or cytoplasmic pattern reflecting multiple nuclear localization/export signals. Furthermore, KRMP1 interacted with the mitotic peptidyl-prolyl isomerase Pin1 in vivo, and an in vitro interaction was detected between the tail domain of KRMP1 and the WW domain of Pin1. Overexpression of KRMP1 caused COS-7 cells to arrest at G(2)-M, and co-expression of Pin1 reversed this effect, indicating their physiological interaction. Together, our results suggest that KRMP1 is a mitotic target regulated by Pin1 and vice versa.