Increase in cathepsin K gene expression in Duchenne muscular dystrophy skeletal muscle.

Dystrophinopathy is caused by alterations in the dystrophin gene. The severe phenotype, Duchenne muscular dystrophy (DMD), is caused by a lack of dystrophin in skeletal muscles, resulting in necrosis and regenerating fibers, inflammatory cells, and muscle fibrosis. Progressive muscle weakness is a characteristic finding of this condition. Here, we encountered a rare case of a 10-year-old patient with asymptomatic dystrophinopathy with no dystrophin expression and investigated the reason for the absence of muscle weakness to obtain therapeutic insights for DMD. Using RNA-seq analysis, gene expression in skeletal muscles was compared among patients with asymptomatic dystrophinopathy, three patients with typical DMD, and two patients without dystrophinopathy who were leading normal daily lives. Cathepsin K (CTSK), myosin heavy chain 3 (MYH3), and nodal modulator 3-like genes exhibited a >8-fold change, whereas crystallin mu gene (CRYM) showed a <1/8-fold change in patients with typical DMD compared with their expression in the patient with asymptomatic dystrophinopathy. Additionally, CTSK and MYH3 expression exhibited a >16-fold change (P < 0.01), whereas CRYM expression showed a <1/16-fold change (P < 0.01) in patients with typical DMD compared with their expression in those without dystrophinopathy. CTSK plays an essential role in skeletal muscle loss, fibrosis, and inflammation in response to muscles injected with cardiotoxin, one of the most common reagents that induce muscle injury. Increased CTSK expression is associated with muscle injury or necrosis in patients with DMD. The lack of muscle weakness in the patient with asymptomatic dystrophinopathy might be attributed to the low CTSK expression in the muscles. To the best of our knowledge, this is the first report to demonstrate that CTSK expression was significantly higher in the skeletal muscles of patients with DMD with a typical phenotype than in those without dystrophinopathy.

[Hashimoto's encephalopathy presenting with vertigo and muscle weakness in a male pediatric patient].

Hashimoto's encephalopathy is an anti-thyroid antibody-positive autoimmune encephalopathy. We herein report the case of a 13-year-old male patient with subacute vertigo, muscle weakness in the extremities and gait disturbance who was diagnosed with Hashimoto's encephalopathy. He showed no severe impairment of consciousness and no seizures, and there were no abnormalities on the brain MRI. However, epileptic spike and wave complexes were observed on an electroencephalogram, and a decline in blood flow was diffusely observed on brain SPECT (single photon emission computed tomography). His thyroid function was normal, but he was positive for anti-thyroid antibodies, such as anti-TPO (thyroid peroxidase) antibodies. He was also positive for serum anti-NAE (NH2-terminal alpha-enolase) antibodies. Systemic corticosteroid therapy and high-dose intravenous immunoglobulin therapy were effective, greatly improving his quality of life.

Estimation of muscle strength from actigraph data in Duchenne muscular dystrophy.

BACKGROUND: The purpose of this study was to evaluate the utility of a wrist actigraph for estimating muscle strength in Duchenne muscular dystrophy patients. METHODS: Twenty-two patients aged 4-19 years wore a wrist actigraph to monitor activities of daily living, and underwent a test of knee extension strength and the 6 min walk test. These measures were made at baseline and at 1 year later. The actigraph data were quantified using the zero crossing mode (ZCM), which indicates the frequency of movement, and the proportional integration mode (PIM), which indicates activity level or vigor of motion. RESULTS: The ZCM and PIM scores of ambulatory patients were higher than those of non-ambulatory patients (P < 0.001). The correlation coefficient between ZCM score and 6 min walk distance, ZCM score and knee extension strength, PIM score and 6 minute walk distance, and PIM score and knee extension strength was -0.44, 0.25, 0.58, and 0.63, respectively. This indicates that the PIM score had a moderate-good association with 6 min walk distance and knee extension strength. CONCLUSION: Muscle strength can be estimated using the PIM score calculated from actigraph data. The PIM score is a good tool for the estimation of muscle strength.

A Pediatric Case of COLQ-Related Congenital Myasthenic Syndrome with Marked Fatigue.

Congenital myasthenic syndrome (CMS) is a clinically and genetically heterogeneous inherited disorder that is treatable. Although the disease usually develops at birth or during infancy, some patients develop the disease in the second to third decades of life. Collagen-like tail subunit of asymmetric acetylcholinesterase (COLQ)-related CMS is CMS with mutations in the COLQ, which results in end-plate acetylcholinesterase deficiency. Diagnostic delay is common in patients with later-onset CMS due to slow progression and fluctuating symptoms. Understanding CMS with atypical and unusual presentations is important to treat this condition effectively. Here, we report a case of COLQ-related CMS. A 10-year-old girl presented with only marked fatigue, which was provoked by exercise but improved after 30-60 min of rest. While motor nerve conduction velocity was normal, a compound muscle action potential (CMAP) with four peaks was recorded. Repetitive stimulation of the accessory nerve exhibited a decrease in CMAP amplitude. Genetic tests revealed compound heterozygous mutations in COLQ (c.1196-1_1197delinsTG and c.1354C>T). Treatment with salbutamol improved fatigue but not the electrophysiological markers. Thus, significant fatigue is a hallmark of COLQ-related CMS; early diagnosis is essential for ensuring appropriate treatment.

Increase in the Number and Duration of Sleep Episodes During Class After Reopening of Schools Following Closure due to COVID-19.

Sleep is important for the well-being of school-aged children. Almost all schools in Hyogo prefecture in Japan were closed from April 7 to May 31, 2020, owing to the coronavirus disease 2019 pandemic. The pandemic restrictions resulted in the disruption of the sleep routines of children. The number of children who experienced sleepiness in class after school closure increased. The number of children who visited our hospital 1 year before and after the closure was 208 (11.73 ± 3.24 years of age) and 155 (11.45 ± 3.30 years), respectively. The number of chief complaints of sleep-related symptoms at the first visits showed no significant difference between the two time periods. The percentage of patients who slept during class increased (but not significantly) after the school closure. However, the mean number and duration of sleep episodes during class significantly increased from 0.31 ± 0.76 to 1.04 ± 1.14 episodes/day and from 15.8 ± 38.6 to 45.7 ± 46.9 min/day (each P < 0.001) before and after school closure, respectively. The total number of patients in our hospital with the primary central disorders of hypersomnolence, i.e., narcolepsy, idiopathic hypersomnia, and Kleine-Levin syndrome, and the number of patients with insufficient sleep syndrome after the school closure significantly increased compared with those before closure (P = 0.034 and 0.048, respectively). School closure was associated with an increased incidence of sleeping during class; therefore, maintaining a stable daily routine for children with sleep disorders could have an alleviating effect.

SARS-CoV-2 Omicron BA.2.75 Variant May Be Much More Infective than Preexisting Variants Based on In Silico Model.

Previously, we developed a mathematical model via molecular simulation analysis to predict the infectivity of six SARS-CoV-2 variants. In this report, we aimed to predict the relative risk of the recent new variants of SARS-CoV-2 based on our previous research. We subjected Omicron BA.4/5 and BA.2.75 variants of SARS-CoV-2 to the analysis to determine the evolutionary distance of the spike protein gene (S gene) of the variants from the Wuhan variant so as to appreciate the changes in the spike protein. We performed molecular docking simulation analyses of the spike proteins with human angiotensin-converting enzyme 2 (ACE2) to understand the docking affinities of these variants. We then compared the evolutionary distances and the docking affinities of these variants with those of the variants that we had analyzed in our previous research. As a result, BA.2.75 has both the highest docking affinity (ratio per Wuhan variant) and the longest evolutionary distance of the S gene from the Wuhan variant. These results suggest that BA.2.75 infection can spread farther than can infections of preexisting variants.

Prediction of infectivity of SARS-CoV2: Mathematical model with analysis of docking simulation for spike proteins and angiotensin-converting enzyme 2.

Objectives: Variants of a coronavirus (SARS-CoV-2) have been spreading in a global pandemic. Improved understanding of the infectivity of future new variants is important so that effective countermeasures against them can be quickly undertaken. In our research reported here, we aimed to predict the infectivity of SARS-CoV-2 by using a mathematical model with molecular simulation analysis, and we used phylogenetic analysis to determine the evolutionary distance of the spike protein gene (S gene) of SARS-CoV-2. Methods: We subjected the six variants and the wild type of spike protein and human angiotensin-converting enzyme 2 (ACE2) to molecular docking simulation analyses to understand the binding affinity of spike protein and ACE2. We then utilized regression analysis of the correlation coefficient of the mathematical model and the infectivity of SARS-CoV-2 to predict infectivity. Results: The evolutionary distance of the S gene correlated with the infectivity of SARS-CoV-2 variants. The calculated biding affinity for the mathematical model obtained with results of molecular docking simulation also correlated with the infectivity of SARS-CoV-2 variants. These results suggest that the data from the docking simulation for the receptor binding domain of variant spike proteins and human ACE2 were valuable for prediction of SARS-CoV-2 infectivity. Conclusion: We developed a mathematical model for prediction of SARS-CoV-2 variant infectivity by using binding affinity obtained via molecular docking and the evolutionary distance of the S gene.

Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential.

Muscle-derived stem cells (MDSCs) can differentiate into multiple lineages, including haematopoietic lineages. However, it is unknown whether MDSCs preserve their myogenic potential after differentiation into other lineages. To address this issue, we isolated from dystrophic muscle a population of MDSCs that express stem-cell markers and can differentiate into various lineages. After systemic delivery of three MDSC clones into lethally irradiated mice, we found that differentiation of the donor cells into various lineages of the haematopoietic system resulted in repopulation of the recipients' bone marrow. Donor-derived bone-marrow cells, isolated from these recipients by fluorescence-activated cell sorting (FACS), also repopulated the bone marrow of secondary, lethally irradiated, recipients and differentiated into myogenic cells both in vitro and in vivo in normal mdx mice. These findings demonstrate that MDSC clones retain their myogenic potential after haematopoietic differentiation.

Effective drug delivery system for duchenne muscular dystrophy using hybrid liposomes including gentamicin along with reduced toxicity.

It is known that gentamicin (GM) could be a possible treatment for Duchenne Muscular Dystrophy (DMD). However, GM therapy has been hindered by several problems such as severe side effects of GM. In order to resolve these problems, we developed the drug delivery system (DDS) of GM using hybrid liposomes (HL) composed of L-α-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(23) lauryl ether (C12(EO)23). The hydrodynamic diameters of HL including GM (GM-HL) were 60-90 nm with a narrow range of the size distribution and the sizes were kept almost constant for over 4 weeks, suggesting that GM-HL could avoid the reticuloendothelial system in vivo. Furthermore, GM-HL accumulated more to the skeletal muscle cells of X chromosome-linked muscular distrophy (mdx) mice as compared to those of normal mice. Significantly, we succeeded in increasing dystrophin positive fibers in skeletal muscle cells of mdx mice using GM-HL along with the reduction of ototoxicity. It is suggested that GM should be carried more efficiently into the muscular cells of mdx mice by HL. These results indicate that HL could be an effective carrier in the DDS of GM therapy for DMD.

Exon-skipping events in candidates for clinical trials of morpholino.

BACKGROUND: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out-of-frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in-frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino-mediated exon skipping, which changes out-of-frame deletions to in-frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon-skipping event in cells of DMD patients before the clinical trial. METHODS: Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon-skipping event was analyzed on reverse transcription-polymerase chain reaction. RESULTS: Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon 51-skipping event in all deletion patterns of cells tested. Morpholino I25, which is located at the exon donor, induced two different exon-skipping patterns, which are total or partial exon 51-skipping events. According to the sequence analysis, the unexpected unskipped regions were the 95 bp section and the 188 bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out-of-frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon 51-skipping event. CONCLUSION: An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.

Core body temperature changes in school-age children with circadian rhythm sleep-wake disorder.

OBJECTIVE/BACKGROUND: Core body temperature (CBT) is considered a valuable marker for circadian rhythm. This study aimed to investigate the changes in CBT that are associated with the symptoms of circadian rhythm sleep-wake disorder (CRSWD) post-treatment in children. PATIENTS/METHODS: Twenty-eight school-age children [10 boys and 18 girls; mean age (±standard deviation), 13.68 ± 0.93 years] who were admitted to our hospital with CRSWD underwent treatment for 6-8 weeks according to the following protocol: lights-out for sleep at 21:00; phototherapy for waking at 6:00 or 7:00; light exercise everyday (eg, a 20- to 30-min walk). CBT was continuously measured for 24 h on the first day of admission and on the first day after treatment. RESULTS: The mean time of sleep onset/offset (±standard deviation; in hours:minutes) 1 week before admission and 1 week after treatment were 23:53 ± 2:26/9:58 ± 2:15 and 21:17 ± 0:19/6:46 ± 0:32, respectively. The mean times of sleep onset and offset measured post-treatment were significantly earlier than those measured pre-treatment (p < 0.001). The mean CBT and mean minimum CBT during sleep were significantly lower on the first day post-treatment than on the first day of admission (p = 0.011 and p < 0.001, respectively). CONCLUSIONS: Symptom improvements in patients with CRSWD were associated with a decrease in CBT during sleep, suggesting that CBT may be a biomarker for improvements in CRSWD. These results help elucidate the cause of this sleep disorder.

Serum concentrations of insulin-like growth factor-1 as a biomarker of improved circadian rhythm sleep-wake disorder in school-aged children.

STUDY OBJECTIVES: We aimed to investigate whether improvements in the symptoms of circadian rhythm sleep-wake disorder after treatment were associated with an increase in serum insulin-like growth factor-1 (IGF-1) concentration. METHODS: Eighty-seven school-aged children (32 males, 55 females), aged 14.31 ± 1.50 years (mean ± standard deviation), who were admitted to our hospital with circadian rhythm sleep-wake disorder received treatment for 6-8 weeks consisting of the following protocol: (1) lights-out for sleep occurred at 21:00, (2) phototherapy for waking started at 06:00 or 07:00, and (3) light exercise was required every day (eg, a 20- to 30-minute walk). Blood samples were collected at 08:00 am to measure the serum concentrations of IGF-1, pre- and posttreatment. RESULTS: The mean times of day of sleep onset and offset at the pre- and posttreatment timepoints were 23:32 ± 4.21 and 10:27 ± 2.98, and 21:26 ± 0.55 and 06:50 ± 0.70, respectively. The mean times of day of sleep onset and offset measured at the posttreatment timepoint were significantly earlier compared with the pretreatment baselines (P < .01). The mean serum levels of IGF-1 significantly increased from 315.59 ± 68.26 ng/mL at pretreatment to 335.09 ± 69.78 ng/mL at posttreatment (P < .01). CONCLUSIONS: Improvements in the symptoms of patients with circadian rhythm sleep-wake disorders were associated with increased serum concentrations of IGF-1, suggesting that serum IGF-1 may be a biomarker of improvements in school-aged children with circadian rhythm sleep-wake disorder.

Clinical outcomes after long-term treatment with alglucosidase alfa in infants and children with advanced Pompe disease.

PURPOSE: A clinical trial was conducted to evaluate the safety and efficacy of alglucosidase alfa in infants and children with advanced Pompe disease. METHODS: Open-label, multicenter study of IV alglucosidase alfa treatment in 21 infants 3-43 months old (median 13 months) with minimal acid alpha-glucosidase activity and abnormal left ventricular mass index by echocardiography. Patients received IV alglucosidase alfa every 2 weeks for up to 168 weeks (median 120 weeks). Survival results were compared with an untreated reference cohort. RESULTS: At study end, 71% (15/21) of patients were alive and 44% (7/16) of invasive-ventilator free patients remained so. Compared with the untreated reference cohort, alglucosidase alfa reduced the risk of death by 79% (P < 0.001) and the risk of invasive ventilation by 58% (P = 0.02). Left ventricular mass index improved or remained normal in all patients evaluated beyond 12 weeks; 62% (13/21) achieved new motor milestones. Five patients were walking independently at the end of the study and 86% (18/21) gained functional independence skills. Overall, 52% (11/21) of patients experienced infusion-associated reactions; 95% (19/20) developed IgG antibodies to recombinant human lysosomal acid alpha-glucosidase; no patients withdrew from the study because of safety concerns. CONCLUSIONS: In this population of infants with advanced disease, biweekly infusions with alglucosidase alfa prolonged survival and invasive ventilation-free survival. Treatment also improved indices of cardiomyopathy, motor skills, and functional independence.

Novel mutation in splicing donor of dystrophin gene first exon in a patient with dilated cardiomyopathy but no clinical signs of skeletal myopathy.

One cause of X-linked dilated cardiomyopathies is mutation of the dystrophin gene. We report the case of a young boy who suffered from dilated cardiomyopathy caused only by dystrophin-deficient cardiac muscle, but who did not present with any clinical signs of skeletal myopathy. Sequence analysis of the patient's dystrophin gene revealed the presence of a novel single point mutation at the first exon-intron boundary, inactivating the 5' splice site consensus sequence of the first intron. The lack of muscle weakness observed clinically can be explained by expression of the brain and Purkinje dystrophin isoforms in skeletal muscle.

A Skeletal Muscle Model of Infantile-onset Pompe Disease with Patient-specific iPS Cells.

Pompe disease is caused by an inborn defect of lysosomal acid α-glucosidase (GAA) and is characterized by lysosomal glycogen accumulation primarily in the skeletal muscle and heart. Patients with the severe type of the disease, infantile-onset Pompe disease (IOPD), show generalized muscle weakness and heart failure in early infancy. They cannot survive over two years. Enzyme replacement therapy with recombinant human GAA (rhGAA) improves the survival rate, but its effect on skeletal muscle is insufficient compared to other organs. Moreover, the patho-mechanism of skeletal muscle damage in IOPD is still unclear. Here we generated induced pluripotent stem cells (iPSCs) from patients with IOPD and differentiated them into myocytes. Differentiated myocytes showed lysosomal glycogen accumulation, which was dose-dependently rescued by rhGAA. We further demonstrated that mammalian/mechanistic target of rapamycin complex 1 (mTORC1) activity was impaired in IOPD iPSC-derived myocytes. Comprehensive metabolomic and transcriptomic analyses suggested the disturbance of mTORC1-related signaling, including deteriorated energy status and suppressed mitochondrial oxidative function. In summary, we successfully established an in vitro skeletal muscle model of IOPD using patient-specific iPSCs. Disturbed mTORC1 signaling may contribute to the pathogenesis of skeletal muscle damage in IOPD, and may be a potential therapeutic target for Pompe disease.

A novel approach to identify Duchenne muscular dystrophy patients for aminoglycoside antibiotics therapy.

Aminoglycoside antibiotics have been found to suppress nonsense mutations located in the defective dystophin gene in mdx mice, suggesting a possible treatment for Duchenne muscular dystrophy (DMD). However, it is very difficult to find patients that are applicable for this therapy, because: (1) only 5-13% of DMD patients have nonsense mutations in the dystrophin gene, (2) it is challenging to find nonsense mutations in the gene because dystrophin cDNA is very long (14 kb), and (3) the efficiency of aminoglycoside-induced read-through is dependent on the kind of nonsense mutation. In order to develop a system for identifying candidates that qualify for aminoglycoside therapy, fibroblasts from nine DMD patients with nonsense mutation of dystrophin gene were isolated, induced to differentiate to myogenic lineage by AdMyoD, and exposed with gentamicin. The dystrophin expression in gentamicin-exposed myotubes was monitored by in vitro dystrophin staining and western blotting analysis. The results showed that gentamicin was able to induce dystrophin expression in the differentiated myotubes by the read-through of the nonsense mutation TGA in the gene; a read-through of the nonsense mutations TAA and TAG did not occur and consequently did not lead to dystrophin expression. Therefore, it is speculated that the aminoglycoside treatment is far more effective for DMD patients that have nonsense mutation TGA than for patients that have nonsense mutation TAA and TAG. In this study, we introduce an easy system to identify patients for this therapy and report for the first time, that dystrophin expression was detected in myotubes of DMD patients using gentamicin.

Disease modeling and lentiviral gene transfer in patient-specific induced pluripotent stem cells from late-onset Pompe disease patient.

Pompe disease is an autosomal recessive inherited metabolic disease caused by deficiency of acid α-glucosidase (GAA). Glycogen accumulation is seen in the affected organ such as skeletal muscle, heart, and liver. Hypertrophic cardiomyopathy is frequently seen in the infantile onset Pompe disease. On the other hand, cardiovascular complication of the late-onset Pompe disease is considered as less frequent and severe than that of infantile onset. There are few investigations which show cardiovascular complication of late onset Pompe disease due to the shortage of appropriate disease model. We have generated late-onset Pompe disease-specific induced pluripotent stem cell (iPSC) and differentiated them into cardiomyocytes. Differentiated cardiomyocyte shows glycogen accumulation and lysosomal enlargement. Lentiviral GAA rescue improves GAA enzyme activity and glycogen accumulation in iPSC. The efficacy of gene therapy is maintained following the cardiomyocyte differentiation. Lentiviral GAA transfer ameliorates the disease-specific change in cardiomyocyote. It is suggested that Pompe disease iPSC-derived cardiomyocyte is replicating disease-specific changes in the context of disease modeling, drug screening, and cell therapy.

Dystrophin delivery in dystrophin-deficient DMDmdx skeletal muscle by isogenic muscle-derived stem cell transplantation.

Duchenne's muscular dystrophy (DMD) is a lethal muscle disease caused by a lack of dystrophin expression at the sarcolemma of muscle fibers. We investigated retroviral vector delivery of dystrophin in dystrophin-deficient DMD(mdx) (hereafter referred to as mdx) mice via an ex vivo approach using mdx muscle-derived stem cells (MDSCs). We generated a retrovirus carrying a functional human mini-dystrophin (RetroDys3999) and used it to stably transduce mdx MDSCs obtained by the preplate technique (MD3999). These MD3999 cells expressed dystrophin and continued to express stem cell markers, including CD34 and Sca-1. MD3999 cells injected into mdx mouse skeletal muscle were able to deliver dystrophin. Though a relatively low number of dystrophin-positive myofibers was generated within the gastrocnemius muscle, these fibers persisted for up to 24 weeks postinjection. The injection of cells from additional MDSC/Dys3999 clones into mdx skeletal muscle resulted in varying numbers of dystrophin-positive myofibers, suggesting a differential regenerating capacity among the clones. At 2 and 4 weeks postinjection, the infiltration of CD4- and CD8-positive lymphocytes and a variety of cytokines was detected within the injected site. These data suggest that the transplantation of retrovirally transduced mdx MDSCs can enable persistent dystrophin restoration in mdx skeletal muscle; however, the differential regenerating capacity observed among the MDSC/Dys3999 clones and the postinjection immune response are potential challenges facing this technology.

Improvement of Germ Line Transmission by Targeting ß-galactosidase to Nuclei in Transgenic Mice: (lacZ, nuclear location signal/sexual dimorphism/transgenic mouse/toxicity).

The Escherichia coli lacZ gene has frequently been used as a reporter in cell lineage analysis, in determining the elements regulating spatial and temporal gene expression, and in enhancer/gene trap detection of developmentally regulated genes. However, it is uncertain whether lacZ expression affects eukaryotic cell growth and development. By using a gene trap, we previously isolated the promoter, Ayu1, which is active in ES cells and in several tissues including the gonads. We used this promoter and the nuclear location signal of the SV40 large T gene to locate ß-galactosidase either in the cytoplasm or the nucleus. Transgenic lines containing ß-galactosidase in the cytoplasm of a wide variety of cell types did not transmit the transgene to their offspring. In contrast, transgenic mice, containing ß-galactosidase in the nucleus, did transmit the transgene successfully. Interestingly, lacZ expression in the brain was more restricted when ß-galactosidase activity was detected in the cytoplasm. These data suggested that cytoplasmic ß-galactosidase affects certain developmental processes or gametogenesis resulting in transmission distortion of the transgene, and that this effect can be reduced by targeting ß-galactosidase to the nucleus. We also found that Ayu1-driven lacZ expression in the duodenum of adult transgenic mice was sexually dimorphic, being positive in females and negative in males.

Parathyroid hormone and parathyroid hormone type-1 receptor accelerate myocyte differentiation.

The ZHTc6-MyoD embryonic stem cell line expresses the myogenic transcriptional factor MyoD under the control of a tetracycline-inducible promoter. Following induction, most of the ZHTc6-MyoD cells differentiate to myotubes. However, a small fraction does not differentiate, instead forming colonies that retain the potential for myocyte differentiation. In our current study, we found that parathyroid hormone type 1 receptor (PTH1R) expression in colony-forming cells at 13 days after differentiation was higher than that in the undifferentiated ZHTc6-MyoD cells. We also found that PTH1R expression was required for myocyte differentiation, and that parathyroid hormone accelerated the differentiation. Our analysis of human and mouse skeletal muscle tissues showed that most cells expressing PTH1R also expressed Pax7 and CD34, which are biomarkers of satellite cells. Furthermore, we found that parathyroid hormone treatment significantly improved muscle weakness in dystrophin-deficient mdx mice. This is the first report indicating that PTH1R and PTH accelerate myocyte differentiation.