Phospholipase Cε plays a crucial role in neutrophilic inflammation accompanying acute lung injury through augmentation of CXC chemokine production from alveolar epithelial cells.

BACKGROUND: We have shown that phospholipase Cε (PLCε), an effector of Ras and Rap1 small GTPases, plays pivotal roles in inflammation and inflammation-associated carcinogenesis by augmenting proinflammatory cytokine production from epithelial cells of various organs. The purpose of this study is to analyze its role in neutrophilic alveolar inflammation accompanying acute lung injury (ALI), focusing on that in alveolar epithelial cells (AECs), which are known to make a major contribution to the pathogenesis of ALI. METHODS: We examine the effect of the PLCε genotypes on the development of ALI induced by intratracheal administration of lipopolysaccharide (LPS) to PLCε wild-type (PLCε+/+) and knockout (PLCεΔX/ΔX) mice. Pathogenesis of ALI is analyzed by histological examination of lung inflammation and measurements of the levels of various cytokines, in particular neutrophil-attracting chemokines such as Cxcl5, by quantitative reverse transcription-polymerase chain reaction and immunostaining. Primary cultures of AECs, established from PLCε+/+ and PLCεΔX/ΔX mice, are used to analyze the roles of PLCε, protein kinase D (PKD) and nuclear factor-κB (NF-κB) in augmentation of LPS-induced Cxcl5 expression. RESULTS: Compared to PLCε+/+ mice, PLCεΔX/ΔX mice exhibit marked alleviation of lung inflammation as shown by great reduction in lung wet/dry weight ratios, accumulation of inflammatory cells in the alveolar space and thickening of alveolar walls as well as the number of neutrophils and the protein concentration in bronchoalveolar lavage fluid. Also, LPS-induced expression of the CXC family of chemokines, in particular Cxcl5, is substantially diminished in the total lung and AECs of PLCεΔX/ΔX mice. Moreover, LPS-induced Cxcl5 expression in primary cultured AECs is markedly suppressed on the PLCεΔX/ΔX background (p < 0.05 versus PLCε+/+ AECs), which is accompanied by the reduction in phosphorylation of inhibitor κB (IκB), PKD and nuclear translocation of NF-κB p65. Also, it is suppressed by the treatment with inhibitors of PKD and IκB kinase, suggesting the involvement of the PLCε-PKD-IκB-NF-κB pathway. CONCLUSIONS: PLCε-mediated augmentation of the production of the CXC family of chemokines, in particular Cxcl5, in AECs plays a crucial role in neutrophilic alveolar inflammation accompanying ALI, suggesting that PLCε may be a potential molecular target for the treatment of acute respiratory distress syndrome.

Molecular Basis for Allosteric Inhibition of GTP-Bound H-Ras Protein by a Small-Molecule Compound Carrying a Naphthalene Ring.

The ras oncogene products (H-Ras, K-Ras, and N-Ras) have been regarded as some of the most promising targets for anticancer drug discovery because their activating mutations are frequently found in human cancers. Nonetheless, molecular targeted therapy for them is currently unavailable. Here, we report the discovery of a small-molecule compound carrying a naphthalene ring, named KBFM123, which binds to the GTP-bound form of H-Ras. The solution structure of its complex with the guanosine 5'-(ß,γ-imide) triphosphate-bound form of H-RasT35S (H-RasT35S·GppNHp) indicates that the naphthalene ring of KBFM123 interacts directly with a hydrophobic pocket located between switch I and switch II and allosterically inhibits the effector interaction by inducing conformational changes in switch I and its flanking region in strand ß2, which are directly involved in recognition of the effector molecules, including c-Raf-1. In particular, Asp38 of H-Ras, a crucial residue for the interaction with c-Raf-1 via the formation of a salt bridge with Arg89 of the Ras-binding domain (RBD) of c-Raf-1, shows a drastic conformational change: its side chain orients toward the opposite direction. Consistent with these results, KBFM123 exhibits an activity to inhibit, albeit weakly, the association of H-RasG12V·GppNHp with the c-Raf-1 RBD. The binding of the naphthalene ring to the hydrophobic pocket of H-RasT35S·GppNHp is further supported by nuclear magnetic resonance analyses showing that two other naphthalene-containing compounds with distinct structures also exhibit similar binding properties with KBFM123. These results indicate that the naphthalene ring could become a promising scaffold for the development of Ras inhibitors.

Ras inhibitors display an anti-metastatic effect by downregulation of lysyl oxidase through inhibition of the Ras-PI3K-Akt-HIF-1α pathway.

Metastasis stands as the major obstacle for the survival from cancers. Nonetheless most existing anti-cancer drugs inhibit only cell proliferation, and discovery of agents having both anti-proliferative and anti-metastatic properties would be more beneficial. We previously reported the discovery of small-molecule Ras inhibitors, represented by Kobe0065, that displayed anti-proliferative activity on xenografts of human colorectal cancer (CRC) cell line SW480 carrying the K-rasG12Vgene. Here we show that treatment of cancer cells carrying the activated ras genes with Kobe0065 or a siRNA targeting Ras downregulates the expression of lysyl oxidase (LOX), which has been implicated in metastasis. LOX expression is enhanced by co-expression of RasG12V through activation of phosphatidylinositol 3-kinase (PI3K)/Akt and concomitant accumulation of hypoxia-inducible factor (HIF)-1α. Furthermore, Kobe0065 effectively inhibits not only migration and invasion of cancer cells carrying the activated ras genes but also lung metastasis of human CRC cell line SW620 carrying the K-rasG12V gene. Collectively, these results indicate that Kobe0065 prevents metastasis through inhibition of the Ras-PI3K-Akt-HIF-1α-LOX signaling and suggest that Ras inhibitors in general might exhibit both anti-proliferative and anti-metastatic properties toward cancer cells carrying the activated ras genes.

Rapgef2, a guanine nucleotide exchange factor for Rap1 small GTPases, plays a crucial role in adherence junction (AJ) formation in radial glial cells through ERK-mediated upregulation of the AJ-constituent protein expression.

Rapgef2 and Rapgef6 define a subfamily of guanine nucleotide exchange factors for Rap1, characterized by possession of the Ras/Rap-associating domains and implicated in the etiology of schizophrenia. We previously found that dorsal telencephalon-specific Rapgef2 conditional knockout mice exhibits severe defects in formation of apical surface adherence junctions (AJs) and localization of radial glial cells (RGCs). In this study, we analyze the underlying molecular mechanism by using primary cultures of RGCs established from the developing cerebral cortex. The results show that Rapgef2-deficient RGCs exhibit a decreased ability of neurosphere formation, morphological changes represented by regression of radial glial (RG) fibers and reduced expression of AJ-constituent proteins such as N-cadherin, zonula occludens-1, E-cadherin and ß-catenin. Moreover, siRNA-mediated knockdown of Rapgef2 or Rap1A inhibits the AJ protein expression and RG fiber formation while overexpression of Rapgef2, Rapgef6, Rap1AG12V or Rap1BG12V in Rapgef2-deficient RGCs restores them. Furthermore, Rapgef2-deficient RGCs exhibit a reduction in phosphorylation of extracellular signal-regulated kinase (ERK) leading to downregulation of the expression of c-jun, which is implicated in the AJ protein expression. These results indicate a crucial role of the Rapgef2-Rap1A-ERK-c-jun pathway in regulation of the AJ formation in RGCs.

Structural basis for intramolecular interaction of post-translationally modified H-Ras•GTP prepared by protein ligation.

Ras undergoes post-translational modifications including farnesylation, proteolysis, and carboxymethylation at the C terminus, which are necessary for membrane recruitment and effector recognition. Full activation of c-Raf-1 requires cooperative interaction of the farnesylated C terminus and the activator region of Ras with its cysteine-rich domain (CRD). However, the molecular basis for this interaction remains unclear because of difficulties in preparing modified Ras in amounts sufficient for structural studies. Here, we use Sortase A-catalyzed protein ligation to prepare modified Ras in sufficient amounts for NMR and X-ray crystallographic analyses. The results show that the farnesylated C terminus establishes an intramolecular interaction with the catalytic domain and brings the farnesyl moiety to the proximity of the activator region, which may be responsible for their cooperative recognition of c-Raf-1-CRD.

Fatal pulmonary arterial hypertension in an infant girl with incontinentia pigmenti.

We report the case of an infant girl with incontinentia pigmenti (IP) complicated by fatal pulmonary arterial hypertension (PAH). She was diagnosed with IP, based on the presence of specific skin lesions, neonatal seizures, hypereosinophilia and a maternal family history of IP. At the age of 2 months, she was diagnosed with PAH on systolic heart murmur due to tricuspid valve regurgitation. Despite several treatments for PAH but not including epoprostenol, severe PAH persisted and she died of pulmonary hypertensive crisis at the age of 5 months. On postmortem histopathology the pulmonary artery had severe intimal thickening, with occlusion or stenosis of the vascular lumen of the small pulmonary arteries as well as partial plexiform lesions, all of which were compatible with PAH. Modulation of nuclear factor-κB signaling may be involved in the development of PAH in IP.

Current status of the development of Ras inhibitors.

Despite the importance of ras as driver genes in many cancers, clinically effective anti-cancer drugs targeting their products, Ras, have been unavailable so far, which was in part ascribable to the apparently 'undruggable' nature of their tertiary structures. Nonetheless, recent studies in academia and industry have identified novel surface pockets accepting small-molecule ligands in both their active GTP-bound and inactive GDP-bound forms (Ras•GTP and Ras•GDP, respectively), which has led to a surge of investigations into the discovery of Ras-specific inhibitors particularly by utilizing their structural information for structure-based drug design (SBDD). We have been developing Ras inhibitors by SBDD targeting a novel conformation of Ras•GTP called state 1, possessing 'druggable' surface pockets, which emerges from the conformational dynamics. In this article, we will survey Ras functions from the structural biological point of view and summarize the current status of the development of Ras inhibitors including our own.

Electrocardiographic and chronobiological features of paroxysmal AV block recorded by ambulatory electrocardiography.

The goal of this study was to investigate the electrocardiographic and chronobio-logical features of paroxysmal atrioventricular (AV) block (PAVB) using data from ambulatory electrocardiography (AECG). The study population consisted of five men and six women aged from 47 to 82 years of age. Main presenting symptoms were pre-syncope in five patients (45.5%) and syncope in three patients (27.3%). Organic cardiovascular diseases were seen in eight patients (72.7%), and AV conduction disturbances were seen in six patients (54.5%), such as right bundle branch block, first to second degree AV block on standard 12-lead electrocardiography. Incidence of PAVB events were 1-329 (37.9 ± 98.0) episodes/patient/day, and the maximum pause during Holter recordings was 3.3-12.4 (6.39 ± 3.09) seconds. This maximum pause caused by intrinsic AV block was longer than that of vagally mediated AV block (8.4 ± 3.2 sec vs 4.7 ± 1.0 sec, p<0.05). In chronobiological analysis, episodes of PAVB exhibited a circadian rhythm characterized by a peak between 2:00 am and 4:00 am and a trough between 0:00 pm and 2:00 pm. AECG is a useful tool to detect the maximum pause occurring during sleep and provides critical data necessary to prevent the sudden cardiac death caused by PAVB.

A new treatment for retroperitoneal fibrosis: initial experiences of using Seprafilm® to wrap the ureter.

OBJECTIVE: To confirm the efficacy of using Seprafilm® (Genzyme Corp., Cambridge, MA, USA) for wrapping the ureter to treat the ureteric stenosis caused by retroperitoneal fibrosis (RPF). PATIENTS AND METHODS: Between August 2010 and September 2012, 11 ureters in eight patients with RPF (seven males and one female, mean age 65 years) were treated. The mean (range) length of the narrow segment of the ureter was 30 (10-90) mm. During surgery, after having been released from adhesive tissue, the stenotic segment of the ureter was wrapped with Seprafilm to isolate it from the surrounding tissue. A radiographic follow-up was performed every 6 months using computed tomography, i.v. pyelography and/or (99m) Tc-mercapto-acetylglycyl-glycyl-glycine ((99m) Tc-MAG3) renal scintigraphy. RESULTS: For the unilateral operations, the mean estimated blood loss was 39 mL, and the mean operating time was 154 min. All ureters were isolated from the fibrotic tissue and wrapped with Seprafilm successfully without major complications. During the mean follow-up period of 17 months, no ureteric restenoses were observed in the affected sides, but new stenosis occurred in the contralateral side of the ureter in one patient. CONCLUSIONS: Although the follow-up period is still limited, we believe that the use of Seprafilm has the potential to become an effective option in the treatment of ureteric stenosis caused by RPF, when the omentum cannot be used. To establish the relative advantages of using Seprafilm over performing a standard omental wrap, further experimentation will be required to compare the two techniques.

Evaluation of seed chemical quality traits and sensory properties of natto soybean.

Natto is a popular soyfood in Japan, and the U.S. is the largest supplier of natto soybeans. However, information on natto seed chemical and sensory properties is very limited. The objectives of this study were to evaluate differences of seed chemical and sensory properties among natto types and determine heritability and correlation. A total of 15 small-seeded natto genotypes (three superior, nine moderate and three inferior) were evaluated for protein, oil, calcium, manganese, boron and sugar content and processed into a natto product to evaluate appearance, stickiness, flavor, texture and shelf-life. The superior natto group had a higher sugar content but lower protein plus oil, calcium, manganese and boron content than other two groups. Most seed quality traits exhibited high heritability. The natto sensory preference was positively correlated with sucrose and oil content, but negatively correlated with seed hardness, protein, protein plus oil, calcium, manganese, and boron contents. Selecting soybean lines with low protein, protein plus oil, calcium, manganese, and boron content while with high sucrose will be an effective approach for soybean breeding for natto production.

[Pre-evening meal administration of tacrolimus improved refractory ocular symptoms in two young children with latent general myasthenia gravis].

We report two patients with latent general myasthenia gravis (MG) with refractory ocular symptoms who were successfully treated with pre-evening meal administration of tacrolimus. Patient 1 was a 4-year-old girl with persistent ocular symptoms despite high-dose steroid therapy and thymectomy. Oral tacrolimus was initiated at the age of 3 years, which was resulted in complete resolution of symptoms. After one year, hemilateral ptosis recurred. The plasma consentration of tacrolimus was very low, probably due to sudden weight gain. Increasing the dose and a change from post- to pre-evening meal administration of tacrolimus enabled maintenance of its concentration and complete control of ocular symptoms. Patient 2 was a 2-year-old boy whose symptoms were refractory to steroid therapy after his first relapse. Since post-meal administration of tacrolimus provided partial benefit, the closing schedule was changed to pre-evening meal administration, with good results. Neither patient had adverse effects of tacrolimus. It is difficult to maintain an effective tacrolimus concentration in children due to marked growth and rapid metabolic rates. Pre-evening meal administration of tacrolimus is an easy, safe and useful method of treatment in MG young children.

[Assessing weight and other factors associated with the urinary incontinence of elderly women: a cross-sectional study].

OBJECTIVES: Urination diseases--particularly urinary incontinence (UI)--strongly affect individuals' quality of life (QOL). It is important to maintain overall QOL in societies with an increasing number of long-living elderly people, such as in Japan. Thus, this study aims to clarify the risk factors concerning UI in elderly women. METHODS: For this study, we obtained the approval of the Ethical Committee of Sapporo Medical University. In October 2010, we randomly selected 1,600 women, aged between 65 and 74 years, from the registry of Sapporo city residents and mailed out a self-administered questionnaire. Our questionnaire consisted of five subsections (24 items in total), including fundamental attributes, health condition, past history, present illness, and the International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF). The self-reported prevalence of UI was defined as frequent UI being at least once a week. A logistic regression analysis was used to assess the risk factors for UI. RESULTS: Among those who received the questionnaire, 802 women (response rate: 50.1%) returned a completed questionnaire with written informed consent. Their mean age (+/- standard deviation) was 69.8 +/- 2.6 years, and the prevalence of UI was found to be 29.7%. The mean scores (+/- standard deviation) of ICIQ-SF were 1.7 +/- 2.9 for all participating subjects, and 5.55 +/- 2.50 and 0.09 +/- 0.53 for the groups with or without UI, respectively. The results of the multiple logistic regression analysis were as follows: the odds ratio (95% confidence interval) was 1.94 (1.32, 2.85) for a past maximum weight heavier than or equal to 60 kg; 1.98 (1.18, 3.32) for a smoking index more than or equal to 300; 2.54 (1.47, 4.39) for a poor self-perceived health status; 1.62 (1.09, 2.40) for having a past history of bladder diseases; 1.72 (1.11, 2.69) for having a past history of hemorrhoidal disease; and 2.05 (1.36, 3.10) for a history of UI in one's mother. CONCLUSION: The self-reported prevalence rate of UI was 29.7% in women aged 65-74 years. In the future, we plan to conduct a follow-up survey to further clarify the risk factors of UI that have been implicated in this cross-sectional study.

In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras-effector interaction.

Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of Ras⋅GTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-Ras⋅GTP carrying an H-Ras-type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-Ras⋅GTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H-ras(G12V)-transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras(G12V) gene by oral administration. The NMR structure of a complex of the compound with H-Ras⋅GTP(T35S), exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple Ras⋅GTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target Ras⋅GTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.

[Clinical study of organic acidemias and fatty acid oxidation disorders detected in adults].

Adult-onset inborn errors of metabolism (IEM) are very rare and their details remain unknown. Diagnosis, age at onset, clinical findings, and outcome of patients with IEM over 20 years old whose diagnosis were made at Shimane University between 2001 and 2010 were investigated. Out of 386 IEM cases identified, 24 cases (6.4%) were diagnosed during adulthood, among which 15 patients were adult onset. There were 11 cases with alkaptonuria, 6 patients with organic acidemia without alkaptonuria, 4 cases with urea cycle disorders and 3 subjects with fatty acid oxidation disorders. Outcome of adult-onset IEM are better than those of child-onset; however, some patients suffered from progressive neurological disorders because of the time lag before the diagnosis are determined after the onset. Blood acylcarnitines and urine organic acids should be analyzed for adult patients with unknown regression or mental retardation for early diagnosis.

Randomized controlled trial of pelvic floor muscle training with or without biofeedback for urinary incontinence.

INTRODUCTION AND HYPOTHESIS: To compare the effects of pelvic floor muscle training (PFMT), with or without biofeedback (BF), for stress urinary incontinence (SUI), focusing on condition-specific quality of life (QOL) outcomes. METHODS: Women with SUI were randomized to PFMT with BF (BF group, n = 23) or without BF (PFMT group, n = 23) for 12 weeks. As primary outcome measures, subjective symptoms and QOL were assessed by the King's Health Questionnaire (KHQ) and International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF). A voiding diary, 1-h pad test, and measurement of PFM strength were secondary outcome measures. Changes in the primary and secondary outcomes were assessed before and after 12 weeks' exercise training. RESULTS: Of the 9 domains of the KHQ, the scores of 5 significantly decreased in the PFMT group, and the scores of 7 significantly decreased in the BF group. All ICIQ-SF items and the total score significantly decreased in both groups after therapy. The number of incontinence episodes significantly decreased in the PFMT group, and tended to decrease in the BF group, but this was not significant (P = 0.054). The leakage volume in the 1-h pad test tended to decrease in both groups, but was not significant. Maximum vaginal squeeze pressure significantly increased in both groups. There were no significant inter-group differences in the changes in any of the parameters assessed. CONCLUSIONS: The results indicate that PFMT is effective for treating SUI. There is no apparent add-on effect of BF training in short-term follow-up.

Discovery of small-molecule Ras inhibitors that display antitumor activity by interfering with Ras·GTP-effector interaction.

Ras proteins, particularly their active GTP-bound forms (Ras·GTP), were thought "undruggable" owing to the absence of apparent drug-accepting pockets in their crystal structures. Only recently, such pockets have been found in the crystal structures representing a novel Ras·GTP conformation. We have conducted an in silico docking screen targeting a pocket in the crystal structure of M-Ras(P40D)·GTP and obtained Kobe0065, which, along with its analogue Kobe2602, inhibits binding of H-Ras·GTP to c-Raf-1. They inhibit the growth of H-rasG12V-transformed NIH3T3 cells, which are accompanied by downregulation of not only MEK/ERK but also Akt, RalA, and Sos, indicating the blockade of interaction with multiple effectors. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma carrying K-rasG12V. The nuclear magnetic resonance structure of a complex of the compound with H-Ras(T35S)·GTP confirms its insertion into the surface pocket. Thus, these compounds may serve as a novel scaffold for the development of Ras inhibitors with higher potency and specificity.

Alexander disease with mild dorsal brainstem atrophy and infantile spasms.

We present the case of a Japanese male infant with Alexander disease who developed infantile spasms at 8 months of age. The patient had a cluster of partial seizures at 4 months of age. He presented with mild general hypotonia and developmental delay. Macrocephaly was not observed. Brain magnetic resonance imaging (MRI) findings fulfilled all MRI-based criteria for the diagnosis of Alexander disease and revealed mild atrophy of the dorsal pons and medulla oblongata with abnormal intensities. DNA analysis disclosed a novel heterozygous missense mutation (c.1154 C>T, p.S385F) in the glial fibrillary acidic protein gene. At 8 months of age, tonic spasms occurred, and electroencephalography (EEG) revealed hypsarrhythmia. Lamotrigine effectively controlled the infantile spasms and improved the abnormal EEG findings. Although most patients with infantile Alexander disease have epilepsy, infantile spasms are rare. This comorbid condition may be associated with the distribution of the brain lesions and the age at onset of Alexander disease.

Hyperinsulinemic hypoglycemia of infancy in Sotos syndrome.

Sotos syndrome (OMIM #117550) is a congenital syndrome characterized by overgrowth with advanced bone age, macrocephaly, and learning difficulties. Endocrine complications of this syndrome have not yet been fully described in previous reports. We here investigated the clinical manifestations of Sotos syndrome in Japanese patients who presented with hyperinsulinemic hypoglycemia of infancy. We recruited patients diagnosed as having Sotos syndrome who presented with the complication of hyperinsulinemia during the neonatal period using a survey of the abstracts of Pediatric Meetings in domestic areas of Japan from 2007 to 2011. As a result, five patients (four females and one male) were recruited to evaluate the clinical presentation of Sotos syndrome by reference to the clinical record of each patient. A 5q35 deletion including the NSD1 gene was detected in all patients. Major anomalies in the central nervous, cardiovascular, and genito-urinary systems were frequently found. Hypoglycemia occurred between 0.5 and 3 hr after birth and high levels of insulin were initially found within 3 days of birth. The patients were treated with intravenous glucose infusion at a maximum rate of 4.6-11.0 mg/kg/min for 12-49 days. Three of the five patients required nasal tube feeding. One patient received medical treatment with diazoxide. This study shows that patients with Sotos syndrome may present with transient hyperinsulinemic hypoglycemia in the neonatal period.

High-density CT of muscle and liver may allow early diagnosis of childhood-onset Pompe disease.

Pompe disease is classified into infantile-, childhood- and adult-onset forms based on onset age and the degree of organ involvement. Differing from the infantile-onset form which is characterized by marked organ involvement, the childhood-onset form usually presents with muscle weakness and elevation of serum creatine kinase (CK), mimicking those of progressive muscular dystrophy. We report our successful early diagnosis and initiation of enzyme replacement therapy (ERT) in a young girl with childhood-onset Pompe disease before the development of skeletal muscle symptoms. She was referred to our hospital at the age of 2 years 4 months because of hyperCKemia detected incidentally. She was active and lacked developmental delay and muscle weakness; however, hepatomegaly was noted. The combination of high-density changes in the liver and skeletal muscle on computed tomography (CT) images was suggestive of glycogen storage disorder, especially childhood-onset Pompe disease. Low alpha-glucosidase (GAA) activity on dried blood spots facilitated the diagnostic process, and genetic analysis of GAA allowed a definitive diagnosis, without performing muscle biopsy. We promptly started ERT at the age of 2 years 6 months. After 1 year, she still had not developed any skeletal muscle symptoms, and serum CK level was almost normal. Since the efficacy of ERT is thought to depend on the extent of muscle damage at its commencement, we expect that ERT may have prevented the manifestation of skeletal muscle involvement in this patient.

Solution structure of the state 1 conformer of GTP-bound H-Ras protein and distinct dynamic properties between the state 1 and state 2 conformers.

Ras small GTPases undergo dynamic equilibrium of two interconverting conformations, state 1 and state 2, in the GTP-bound forms, where state 2 is recognized by effectors, whereas physiological functions of state 1 have been unknown. Limited information, such as static crystal structures and (31)P NMR spectra, was available for the study of the conformational dynamics. Here we determine the solution structure and dynamics of state 1 by multidimensional heteronuclear NMR analysis of an H-RasT35S mutant in complex with guanosine 5'-(ß, γ-imido)triphosphate (GppNHp). The state 1 structure shows that the switch I loop fluctuates extensively compared with that in state 2 or H-Ras-GDP. Also, backbone (1)H,(15)N signals for state 2 are identified, and their dynamics are studied by utilizing a complex with c-Raf-1. Furthermore, the signals for almost all the residues of H-Ras·GppNHp are identified by measurement at low temperature, and the signals for multiple residues are found split into two peaks corresponding to the signals for state 1 and state 2. Intriguingly, these residues are located not only in the switch regions and their neighbors but also in the rigidly structured regions, suggesting that global structural rearrangements occur during the state interconversion. The backbone dynamics of each state show that the switch loops in state 1 are dynamically mobile on the picosecond to nanosecond time scale, and these mobilities are significantly reduced in state 2. These results suggest that multiconformations existing in state 1 are mostly deselected upon the transition toward state 2 induced by the effector binding.