Muscle Thickness and Echo Intensity of the Abdominal and Lower Extremity Muscles in Stroke Survivors.

BACKGROUND AND PURPOSE: This study compared the muscle thickness (MT) and echo intensity (EI) of the abdominal, thigh, and lower leg muscles between the paretic and nonparetic sides in chronic stroke survivors. METHODS: Thirty-two stroke survivors living in the community participated in this study. The MT and EI, which are indicators of muscle mass and intramuscular fat or connective tissue, were assessed in the rectus abdominis, external oblique, internal oblique, transversus abdominis, rectus femoris, vastus intermedius, vastus lateralis, vastus medialis, tibialis anterior, gastrocnemius, and soleus via transverse ultrasound imaging. In addition, a possible indicator of physical activity-the frequency of going out per week-was evaluated. RESULTS: All quadriceps muscles and the tibialis anterior were significantly thinner and the EI values of the vastus intermedius, vastus lateralis, vastus medialis, and soleus were significantly higher in the paretic limb than the nonparetic limb. The MT and EI values of abdominal muscles did not differ significantly between the two sides. The MT values of the paretic rectus femoris, vastus lateralis, and vastus medialis were significantly associated with the frequency of going out after adjusting confounding factors. The MT of the nonparetic vastus lateralis was significantly associated with latency from stroke onset after adjusting confounding factors. CONCLUSIONS: Our results indicate that quantitative and qualitative changes on the paretic side in stroke survivors were the most robust in the thigh muscles, whereas such changes might not occur in the abdominal muscles.

Effect of a postoperative outpatient pulmonary rehabilitation program on physical activity in patients who underwent pulmonary resection for lung cancer.

AIM: Physical activity (PA) has been associated with an improvement in survival for individuals with cancer. However, little is known about the effect of postoperative pulmonary rehabilitation on PA after lobectomy in patients with lung cancer. The present study investigated the effect of outpatient rehabilitation on PA in patients with cancer after lung resection. METHODS: A total of 19 patients with lung cancer were recruited for this study and completed a preoperative rehabilitation program. One group of nine patients completed a postoperative outpatient pulmonary rehabilitation program (rehabilitation) and another group of 10 patients did not (control), but were similarly followed up. Preoperative lung function, assessed by forced expiratory volume in 1 s (FEV1 ), body mass index (BMI) and St. George's Respiratory Questionnaire (SGRQ) score were not different between groups. PA was measured before and 2 months after surgery using a three-axis accelerometer for 5-6 days. PA level (PAL) was defined as total energy expenditure divided by basal metabolic rate. RESULTS: Preoperative PAL was not different between groups. However, postoperative versus preoperative PAL was significantly lower in the control versus the rehabilitation group (P < 0.01), and PAL decline was less for the rehabilitation versus the control group (P < 0.001). A subgroup analysis showed improvement in postoperative PAL in rehabilitation patients aged <75 years and older. CONCLUSIONS: Two months after lung resection surgery, patients had not recovered to the preoperative PAL. However, compared with the control group, there was an improvement in the postoperative PAL in patients, including older patients, who underwent outpatient pulmonary rehabilitation. Geriatr Gerontol Int 2016; 16: 550-555.

Monophasic Pulsed Microcurrent of 1-8†Hz Increases the Number of Human Dermal Fibroblasts.

OBJECTIVE: Pressure injuries seriously impact the quality of life of patients and increase public and private healthcare costs. Electrical stimulation therapy is recommended for wound contraction, and some clinical studies have shown that the application of a monophasic pulsed microcurrent can help to reduce the treatment period. However, the optimal stimulus conditions are unclear. The purpose of this study was to investigate the effect of different frequencies of monophasic pulsed microcurrent stimulation on the number and viability of human dermal fibroblasts. METHODS: Human dermal fibroblasts were electrically stimulated in vitro (intensity: 200 µA; frequency: 1, 2, 4, 8, 16, 32, and 64 Hz; duty factor: 50%) for 1 h three times every 24 h. Controls were unstimulated. Cell numbers and cell viability were assessed after each electrical stimulation session. RESULTS: In the 1-, 2-, 4-, and 8-Hz groups, cell numbers were significantly higher than those in the control group, whereas electrical stimulation at 64 Hz resulted in a decrease in cell numbers at 24 h after the third treatment (p < 0.05). Cell viability was high in both the control and low-frequency stimulation groups, with no significant differences between groups. CONCLUSION: Application of 1-8 Hz monophasic pulsed microcurrent stimulation increased the number of human dermal fibroblasts in vitro, and is proposed as the optimal condition for accelerating the healing of pressure injuries.

Effect of expiratory resistive loading in expiratory muscle strength training on orbicularis oris muscle activity.

[Purpose] The purpose of this study was to elucidate the effect of expiratory resistive loading on orbicularis oris muscle activity. [Subjects] Subjects were 23 healthy individuals (11 males, mean age 25.5±4.3 years; 12 females, mean age 25.0±3.0 years). [Methods] Surface electromyography was performed to measure the activity of the orbicularis oris muscle during maximum lip closure and resistive loading at different expiratory pressures. Measurement was performed at 10%, 30%, 50%, and 100% of maximum expiratory pressure (MEP) for all subjects. The t-test was used to compare muscle activity between maximum lip closure and 100% MEP, and analysis of variance followed by multiple comparisons was used to compare the muscle activities observed at different expiratory pressures. [Results] No significant difference in muscle activity was observed between maximum lip closure and 100% MEP. Analysis of variance with multiple comparisons revealed significant differences among the different expiratory pressures. [Conclusion] Orbicularis oris muscle activity increased with increasing expiratory resistive loading.

Change in tongue morphology in response to expiratory resistance loading investigated by magnetic resonance imaging.

[Purpose] The purpose of this study was to investigate the effect of expiratory resistance load on the tongue area encompassing the suprahyoid and genioglossus muscles. [Subjects] The subjects were 30 healthy individuals (15 males, 15 females, mean age: 28.9 years). [Methods] Magnetic resonance imaging was used to investigate morphological changes in response to resistive expiratory pressure loading in the area encompassing the suprahyoid and genioglossus muscles. Images were taken when water pressure was sustained at 0%, 10%, 30%, and 50% of maximum resistive expiratory pressure. We then measured tongue area using image analysis software, and the morphological changes were analyzed using repeated measures analysis of variance followed by post hoc comparisons. [Results] A significant change in the tongue area was detected in both sexes upon loading. Multiple comparison analysis revealed further significant differences in tongue area as well as changes in tongue area in response to the different expiratory pressures. [Conclusion] The findings demonstrate that higher expiratory pressure facilitates greater reduction in tongue area.

Genetic and functional interaction between Ryh1 and Ypt3: two Rab GTPases that function in S. pombe secretory pathway.

We have previously isolated ypt3-i5 mutant and showed that Ypt3 GTPase functions in the fission yeast secretory pathway. Here, the same genetic screen led to the isolation of ryh1-i6, a mutant allele of the ryh1+ gene encoding a homolog of Rab6. The ryh1-i6 mutant showed phenotypes that support its role in retrograde traffic from endosome to the Golgi. Interestingly, ryh1+ gene deletion was synthetically lethal with ypt3-i5 mutation. Consistently, the over-expression of the GDP-conformational mutant, Ryh1T25 N, inhibited the growth of ypt3-i5 mutant but had no effect on that of wild-type cells. Furthermore, the over-expression of the Ryh1T25N mutant inhibited the acid phosphatase glycosylation and exacerbated the cell wall integrity of ypt3-i5 mutant, but had no effect on those of wild-type cells. GFP-Ryh1 and GFP-Ypt3 both localized at the Golgi/endosome, but showed distinct subcellular localizations. The localization of GFP-Ryh1 in ypt3-i5 mutant and that of GFP-Ypt3 in ryh1-i6 mutant were distinct from those in wild-type cells. In addition, Ryh1 as well as Ypt3 were shown to be involved in acid phosphatase secretion. These results suggest that Ryh1 is involved in the secretory pathway and may have a potential overlapping function with Ypt3 in addition to its role in recycling.

Phosphatidylinositol-4-phosphate 5-kinase regulates fission yeast cell integrity through a phospholipase C-mediated protein kinase C-independent pathway.

Fission yeast its3-1 mutant is an allele of the essential gene its3+ that encodes a phosphatidylinositol-4-phosphate 5-kinase (PIP5K) that produces phosphatidylinositol 4,5-bisphosphate. We found that the its3-1 mutant is sensitive to micafungin, a (1,3)-beta-D-glucan synthase inhibitor, suggesting a cell wall integrity defect. Consistently, its3-1 mutation caused synthetic lethality with a (1,3)-beta-D-glucan synthase mutant, bgs1-i2, and its3-1 mutant cells showed aberrant localization of green fluorescent protein-Bgs1. Similar aberrant localization of green fluorescent protein-tagged Rgf1, a putative phosphatidylinositol 4,5-bisphosphate-binding guanine nucleotide exchange factor for Rho protein, in its3-1 mutants was observed, suggesting a defective Rgf1/Rho pathway. To unravel the molecular mechanism(s), putative downstream components of PIP5K signaling were analyzed. Unexpectedly, overexpression of phospholipase C (Plc1), but not that of protein kinase C (PKC; Pck1 and Pck2), suppressed the phenotypes of the its3-1 mutant. These findings indicate that PKCs are not involved in the suppression, and further analysis revealed that PKCs are not downstream of Plc1 in fission yeast. Also, the enzymatic activity of Plc1 is essential for the suppression of the phenotypes and for the viability of the its3-1 mutant. These findings suggest that Its3 PIP5K regulates cell integrity through a Plc1-mediated PKC-independent pathway, in addition to the Rho/PKC pathway.

Loss of Apm1, the micro1 subunit of the clathrin-associated adaptor-protein-1 complex, causes distinct phenotypes and synthetic lethality with calcineurin deletion in fission yeast.

Calcineurin is a highly conserved regulator of Ca(2+) signaling in eukaryotes. In fission yeast, calcineurin is not essential for viability but is required for cytokinesis and Cl(-) homeostasis. In a genetic screen for mutations that are synthetically lethal with calcineurin deletion, we isolated a mutant, cis1-1/apm1-1, an allele of the apm1(+) gene that encodes a homolog of the mammalian micro1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex. The cis1-1/apm1-1 mutant as well as the apm1-deleted (Deltaapm1) cells showed distinct phenotypes: temperature sensitivity; tacrolimus (FK506) sensitivity; and pleiotropic defects in cytokinesis, cell integrity, and vacuole fusion. Electron micrographs revealed that Deltaapm1 cells showed large vesicular structures associated with Golgi stacks and accumulated post-Golgi secretory vesicles. Deltaapm1 cells also showed the massive accumulation of the exocytic v-SNARE Syb1 in the Golgi/endosomes and a reduced secretion of acid phosphatase. These phenotypes observed in apm1 mutations were accentuated upon temperature up-shift and FK506 treatment. Notably, Apm1-GFP localized to the Golgi/endosomes, the spindle pole bodies, and the medial region. These findings suggest a role for Apm1 associated with the Golgi/endosome function, thereby affecting various cellular processes, including secretion, cytokinesis, vacuole fusion, and cell integrity and also suggest that calcineurin is involved in these events.

Lub1 participates in ubiquitin homeostasis and stress response via maintenance of cellular ubiquitin contents in fission yeast.

Ubiquitin-dependent proteolysis plays a pivotal role in stress responses. To investigate the mechanisms of these cellular processes, we have been studying Schizosaccharomyces pombe mutants that have altered sensitivities to various stress conditions. Here, we showed that Lub1, a homologue of Ufd3p/Zzz4p/Doa1p in budding yeast, is involved in the regulation of ubiquitin contents. Disruption of the lub1+ gene resulted in monoubiquitin as well as multiubiquitin depletion without change in mRNA level and in hypersensitivity to various stress conditions. Consistently, overexpression of genes encoding ubiquitin suppressed the defects associated with lub1 mutation, indicating that the phenotypes of the lub1 mutants under stress conditions were due to cellular ubiquitin shortage at the posttranscriptional level. In addition, the lub1-deleted cells showed aberrant functions in ubiquitin/proteasome-dependent proteolysis, with accelerated degradation of ubiquitin. Also Cdc48, a stress-induced chaperon-like essential ATPase, was found to interact with Lub1, and this association might contribute to the stabilization of Lub1. Our results indicated that Lub1 is responsible for ubiquitin homeostasis at the protein level through a negative regulation of ubiquitin degradation.

Pmr1, a P-type ATPase, and Pdt1, an Nramp homologue, cooperatively regulate cell morphogenesis in fission yeast: the importance of Mn2+ homeostasis.

Schizosaccharomyces pombe pmr1+ gene is homologous to Saccharomyces cerevisiae PMR1 gene, which encodes the P-type Ca2+/Mn2+-ATPase. Addition of Mn2+, as well as Ca2+, to the medium induced pmr1+ gene expression in a calcineurin-dependent manner. The pmr1 knockout (Deltapmr1) cells exhibited hypersensitivity to EGTA. A screen for high gene dosage-suppressors of the EGTA-hypersensitive phenotype of Deltapmr1 led to the identification of pdt1+ gene, which encodes an Nramp-related metal transporter. The Deltapmr1 cells showed round cell morphology. Although Deltapdt1 cells appeared normal in the regular medium, it showed round cell morphology similar to that of the Deltapmr1 cells when Mn2+ was removed from the medium. The removal of Mn2+ also exacerbated the round morphology of the Deltapmr1 cells. The Deltapmr1Deltapdt1 double mutants grew very slowly and showed extremely aberrant cell morphology with round, enlarged and depolarized shape. The addition of Mn2+, but not Ca2+, to the medium completely suppressed the morphological defects, while both Mn2+ and Ca2+ markedly improved the slow growth of the double mutants. These results suggest that Pmr1 and Pdt1 cooperatively regulate cell morphogenesis through the control of Mn2+ homeostasis, and that calcineurin functions as a Mn2+ sensor as well as a Mn2+ homeostasis regulator.

Fission yeast epsin, Ent1p is required for endocytosis and involved in actin organization.

In this study, we have characterized an essential gene ent1+ encoding fission yeast epsin, which is similar to mammalian and budding yeast endocytic protein epsins. The S. pombe Ent1p contains ENTH (epsin amino-terminal homology) domain at its amino terminus, two copies of a ubiquitin-interacting motif (UIM) immediately carboxyl-terminal to the ENTH domain, three NPF motifs in the carboxyl-terminal half, and the clathrin-binding motif at the carboxyl terminal. When repressed the expression of ent1+ gene, the conditional ent1 gene knockout cells showed a marked defect in internalization of fluorescent dyes, suggesting that Ent1p is essential for endocytosis. Changes in conserved amino acid residues within ENTH domain in ent1 mutant cells revealed temperature-sensitive defect in actin organization and cell morphology. The Ent1p bound PI(4,5)P2 and PI(3,5)P2 immobilized onto nitrocellulose in vitro and also weakly bound PI(3,4)P2, PI(3,4,5)P3, PI4P and PI5P. Surprisingly, the localization of Ent1p-GFP was not affected even in the its3-1 cells, in which the level of PI(4,5)P2 was severely reduced, suggesting that PI(4,5)P2 may not be essential for proper localization of Ent1p at endocytic sites. Our findings indicate that S. pombe Ent1p is an essential component in endocytic process, and involved in actin organization and cell morphogenesis.

Feedback regulation of MAPK signalling by an RNA-binding protein.

Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes that convert extracellular signals into various outputs such as cell growth, differentiation and cell death. MAPK phosphatases selectively inactivate MAPKs by dephosphorylating critical phosphothreonine and phosphotyrosine residues. The transcriptional induction of MAPK phosphatase expression by various stimuli, including MAPK activation, has been well documented as a negative-feedback mechanism of MAPK signalling. Here we show that Rnc1, a novel K-homology-type RNA-binding protein in fission yeast, binds and stabilizes Pmp1 messenger RNA, the MAPK phosphatase for Pmk1 (refs 10, 11). Rnc1 therefore acts as a negative regulator of Pmk1 signalling. Notably, Pmk1 phosphorylates Rnc1, causing enhancement of the RNA-binding activity of Rnc1. Thus, Rnc1 is a component of a new negative-feedback loop that regulates the Pmk1 pathway through its binding to Pmp1 mRNA. Our findings--the post-transcriptional mRNA stabilization of a MAPK phosphatase mediated by an RNA-binding protein--provide an additional regulatory mechanism for fine-tuning of MAPK signalling pathways.

Zinc finger protein Prz1 regulates Ca2+ but not Cl- homeostasis in fission yeast. Identification of distinct branches of calcineurin signaling pathway in fission yeast.

Calcineurin is an important mediator that connects the Ca(2+)-dependent signaling to various cellular responses in a wide variety of cell types and organisms. In budding yeast, activated calcineurin exerts its function mainly by regulating the Crz1p/Tcn1 transcription factor. Here, we cloned the fission yeast prz1(+) gene, which encodes a zinc finger transcription factor highly homologous to Crz1/Tcn1. Similar to the results in budding yeast, calcineurin dephosphorylated Prz1 and resulted in the trans-location of Prz1 from the cytoplasm to the nucleus. Prz1 expression was stimulated by high extracellular Ca(2+) in a calcineurin-dependent fashion. However, unlike in budding yeast, the prz1-null cells did not show any phenotype similar to those previously reported in calcineurin deletion such as aberrant cell morphology, mating defect, or hypersensitivity to Cl(-). Instead, the prz1-null cells showed hypersensitivity to Ca(2+), consistent with a dramatic decrease in transcription of Pmc1 Ca(2+) pump. Interestingly, overexpression of Prz1 did not suppress the Cl(-) hypersensitivity of calcineurin deletion, and overexpression of Pmp1 MAPK phosphatase suppressed the Cl(-) hypersensitivity of calcineurin deletion but not the Ca(2+) hypersensitivity of prz1 deletion. In addition, mutations in the its2(+)/cps1(+), its8(+), and its10(+)/cdc7(+) genes that showed synthetic lethal genetic interaction with calcineurin deletion did not exhibit synthetic lethality with the prz1 deletion. Our results suggest that calcineurin activates at least two distinct signaling branches, i.e. the Prz1-dependent transcriptional regulation and an unknown mechanism, which functions antagonistically with the Pmk1 MAPK pathway.

Calcineurin is implicated in the regulation of the septation initiation network in fission yeast.

BACKGROUND: In fission yeast, calcineurin has been implicated in cytokinesis because calcineurin-deleted cells form multiple septa and cell separation is impeded. However, this mechanism remains unclear. RESULTS: We screened for mutations that confer synthetic lethality with calcineurin deletion and isolated a mutant, its 10-1/cdc7-i10, a novel allele of the cdc7+ gene involved in the septation initiation network (SIN). The mutation created a termination codon, resulting in the truncation of Cdc7 by 162 amino acids, which is not localized in the spindle pole body. Following treatment with the immune suppressive drug FK506, cdc7-i10 and the original cdc7-24 mutant cells showed highly elongated multinuclear morphology with few visible septa, closely resembling the phenotype at the restrictive temperature. Other SIN mutants, cdc11, spg1, sid2 and mob1 showed similar phenotypes following FK506 treatment. Consistent with this, expression of the constitutively active calcineurin suppressed the growth defects and septum initiation deficiency of these SIN mutants at the restrictive temperature. Moreover, electron microscopy revealed that calcineurin-deleted cells had very thick multiple septa which were partially and ectopically formed. CONCLUSION: These results suggest that calcineurin is involved in the regulation of the SIN pathway, and is required for the proper formation and maturation of the septum in fission yeast.

Role of the Rab GTP-binding protein Ypt3 in the fission yeast exocytic pathway and its connection to calcineurin function.

A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1(+) leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.

Genetic interaction between calcineurin and type 2 myosin and their involvement in the regulation of cytokinesis and chloride ion homeostasis in fission yeast.

Calcineurin plays a critical role in Ca(2+) signaling in various cell types. In fission yeast, calcineurin is required for cytokinesis and chloride ion homeostasis. However, most of its physiological functions remain obscure. A genetic screen was performed to identify genes that share an essential function with calcineurin. We screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and to a high concentration of chloride ion and isolated a mutant, cis2-1/myp2-c2, which contains a novel allele of the myp2(+)/myo3(+) gene that encodes a type 2 myosin heavy chain. The myp2-c2 mutant showed morphological defects similar to those associated with a calcineurin deletion mutant, such as multiseptated and branched cells. Consistently, myp2-null cells were hypersensitive to chloride ion and showed the multiseptated phenotype in the presence of immunosuppressants or at high chloride concentrations. Overexpression of constitutively active calcineurin suppressed the chloride ion-sensitive growth defect and cytokinesis abnormality of the myp2-c2 mutant and myp2-null cells. Interestingly, the essential myosin light chain mutant cdc4-8 failed to grow and could not form a normal contractile ring in the presence of immunosuppressants. Furthermore, calcineurin-null cells exhibited aberrant contractile rings, suggesting impaired contraction of the rings. These results indicate that calcineurin is involved in the regulation of cytokinesis and that chloride ion homeostasis is mediated by type 2 myosin.

Calcineurin phosphatase in signal transduction: lessons from fission yeast.

Calcineurin (protein phosphatase 2B), the only serine/threonine phosphatase under the control of Ca2+/calmodulin, is an important mediator in signal transmission, connecting the Ca2+-dependent signalling to a wide variety of cellular responses. Furthermore, calcineurin is specifically inhibited by the immunosuppressant drugs cyclosporin A and tacrolimus (FK506), and these drugs have been a powerful tool for identifying many of the roles of calcineurin. Calcineurin is enriched in the neural tissues, and also distributes broadly in other tissues. The structure of the protein is highly conserved from yeast to man. The combined use of powerful genetics and of specific calcineurin inhibitors in fission yeast Schizosaccharomyces pombe (S. pombe) identified new components of the calcineurin pathway, and defined new roles of calcineurin in the regulation of the many cellular processes. Recent data has revealed functional interactions in which calcineurin phosphatase is involved, such as the cross-talk between the Pmk1 MAP kinase signalling, or the PI signalling. Calcineurin also participates in membrane traffic and cytokinesis of fission yeast through its functional connection with members of the small GTPase Rab/Ypt family, and Type II myosin, respectively. These findings highlight the potential of fission yeast genetic studies to elucidate conserved elements of signal transduction cascades.