Publications by authors named "Hiroshi Nakai"

31 Publications

Discovery of the Orally Effective Thyrotropin-Releasing Hormone Mimetic: 1-{-[(4,5)-(5-Methyl-2-oxooxazolidine-4-yl)carbonyl]-3-(thiazol-4-yl)-l-alanyl}-(2)-2-methylpyrrolidine Trihydrate (Rovatirelin Hydrate).

ACS Omega 2018 Oct 19;3(10):13647-13666. Epub 2018 Oct 19.

Pharmacovigilance Japan, Allergan Japan K.K., 4-20-3-35, Ebisu, Shibuya-ku, Tokyo 150-6035, Japan.

We have explored orally effective thyrotropin-releasing hormone (TRH) mimetics, showing oral bioavailability and brain penetration by structure-activity relationship (SAR) study on the basis of in vivo antagonistic activity on reserpine-induced hypothermia in mice. By primary screening of the synthesized TRH mimetics, we found a novel TRH mimetic: l-pyroglutamyl-[3-(thiazol-4-yl)-l-alanyl]-l-prolinamide with a high central nervous system effect compared with TRH as a lead compound. Further SAR optimization studies of this lead compound led to discovery of a novel orally effective TRH mimetic: 1-{-[(4,5)-(5-methyl-2-oxooxazolidine-4-yl)carbonyl]-3-(thiazol-4-yl)-l-alanyl}-(2)-2-methylpyrrolidine trihydrate (rovatirelin hydrate), which was selected as a candidate for clinical trials.
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http://dx.doi.org/10.1021/acsomega.8b01481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217654PMC
October 2018

How do user experiences with different transport modes affect the risk of traffic accidents? From the viewpoint of licence possession status.

Accid Anal Prev 2017 Feb 13;99(Pt A):242-248. Epub 2016 Dec 13.

Graduate School of Human Sciences, Osaka University, 1-2 Yamadaoka Suita Osaka, 565-0871, Japan.

Road accidents between different modes of transport-such as between automobiles and pedestrians, automobiles and bicycles, or automobiles and motorcycles-are frequent. In such cases, it is important to consider the other side's perspective. This involves the ability to correctly judge, for every given situation, how other people on the road perceive their surroundings and what they intend to do next. In this paper, we conduct two types of studies assuming that this kind of ability to consider perspectives is higher when the person is licenced to drive the mode of transport used by the other party. For Study 1, we analysed accidents involving senior citizens between the ages of 65 and 74 years, who collided with automobiles as pedestrians or cyclists (1656 and 3192 cases respectively), in terms of the accident category and type of road at the accident spot. The results indicate that possession or non-possession of a licence was irrelevant for accidents involving cyclists, but for accidents with pedestrians, senior citizens who did not possess a licence are likely to be involved in a greater number of accidents in places that require interaction with automobiles, such as while crossing at crosswalks or at intersections. For Study 2, we reviewed 875 ordinary first-class licence practical test examinees, categorised them according to their licence possession status (motorcycle licence, moped licence, or no licence), and made a category-wise comparison of the test instructor's assessment of their ability to make a left turn. The results showed that those who had a motorcycle or moped licence tended to make a left turn more safely. Thus, the results indicate that experience with different modes of transport is likely to reduce the risk of accidents. These findings may be used to popularise educational interventions encouraging users of various transport modes to consider the perspective of others (i.e. via perspective-taking).
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http://dx.doi.org/10.1016/j.aap.2016.12.010DOI Listing
February 2017

[Stress reactions among bus drivers: towards the development of an educational resource for better management of emotions].

Shinrigaku Kenkyu 2014 Oct;85(4):373-82

Interview and questionnaire surveys were conducted with bus drivers in Japan, with the goal of developing an educational program for better control of emotions among bus drivers. The interviews aimed at identifying stressors and ways in which stress negatively influenced bus services. The questionnaire survey. which was being developed as a self-diagnosis tool, further provided bus drivers with the opportunity to understand their own emotional tendencies. Factor analysis identified six factors underlying work-related stress: anger at unsafe behaviours of nearby road users, irritation caused by complaints from passengers, time pressures, anxiety about traffic accidents, impatience with slow passengers, and resentment of bad-mannered passengers. The influence of stress on the drivers comprised four factors: cognitive failure, sullen behaviour, abrupt acceleration/deceleration, and aggressive driving. Moreover, drivers with lower stress were relatively older and more experienced. Based on these results, educational materials were proposed with the aim of enhancing bus drivers' understanding of their emotional processes and coping skills.
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http://dx.doi.org/10.4992/jjpsy.85.13216DOI Listing
October 2014

Stringent response processes suppress DNA damage sensitivity caused by deficiency in full-length translation initiation factor 2 or PriA helicase.

Mol Microbiol 2014 Apr 28;92(1):28-46. Epub 2014 Feb 28.

Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Box 571455, 3900 Reservoir Rd. NW, Washington, DC, 20057-1455, USA.

When Escherichia coli grows in the presence of DNA-damaging agents such as methyl methanesulphonate (MMS), absence of the full-length form of Translation Initiation Factor 2 (IF2-1) or deficiency in helicase activity of replication restart protein PriA leads to a considerable loss of viability. MMS sensitivity of these mutants was contingent on the stringent response alarmone (p)ppGpp being at low levels. While zero levels (ppGpp°) greatly aggravated sensitivity, high levels promoted resistance. Moreover, M+ mutations, which suppress amino acid auxotrophy of ppGpp° strains and which have been found to map to RNA polymerase subunits, largely restored resistance to IF2-1- and PriA helicase-deficient mutants. The truncated forms IF2-2/3 played a key part in inducing especially severe negative effects in ppGpp° cells when restart function priB was knocked out, causing loss of viability and severe cell filamentation, indicative of SOS induction. Even a strain with the wild-type infB allele exhibited significant filamentation and MMS sensitivity in this background whereas mutations that prevent expression of IF2-2/3 essentially eliminated filamentation and largely restored MMS resistance. The results suggest different influences of IF2-1 and IF2-2/3 on the replication restart system depending on (p)ppGpp levels, each having the capacity to maximize survival under differing growth conditions.
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http://dx.doi.org/10.1111/mmi.12538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4008491PMC
April 2014

A new role for translation initiation factor 2 in maintaining genome integrity.

PLoS Genet 2012 19;8(4):e1002648. Epub 2012 Apr 19.

Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, DC, United States of America.

Escherichia coli translation initiation factor 2 (IF2) performs the unexpected function of promoting transition from recombination to replication during bacteriophage Mu transposition in vitro, leading to initiation by replication restart proteins. This function has suggested a role of IF2 in engaging cellular restart mechanisms and regulating the maintenance of genome integrity. To examine the potential effect of IF2 on restart mechanisms, we characterized its influence on cellular recovery following DNA damage by methyl methanesulfonate (MMS) and UV damage. Mutations that prevent expression of full-length IF2-1 or truncated IF2-2 and IF2-3 isoforms affected cellular growth or recovery following DNA damage differently, influencing different restart mechanisms. A deletion mutant (del1) expressing only IF2-2/3 was severely sensitive to growth in the presence of DNA-damaging agent MMS. Proficient as wild type in repairing DNA lesions and promoting replication restart upon removal of MMS, this mutant was nevertheless unable to sustain cell growth in the presence of MMS; however, growth in MMS could be partly restored by disruption of sulA, which encodes a cell division inhibitor induced during replication fork arrest. Moreover, such characteristics of del1 MMS sensitivity were shared by restart mutant priA300, which encodes a helicase-deficient restart protein. Epistasis analysis indicated that del1 in combination with priA300 had no further effects on cellular recovery from MMS and UV treatment; however, the del2/3 mutation, which allows expression of only IF2-1, synergistically increased UV sensitivity in combination with priA300. The results indicate that full-length IF2, in a function distinct from truncated forms, influences the engagement or activity of restart functions dependent on PriA helicase, allowing cellular growth when a DNA-damaging agent is present.
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http://dx.doi.org/10.1371/journal.pgen.1002648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334882PMC
September 2012

Psychosocial and cognitive consequences of major neonatal surgery.

J Pediatr Surg 2011 Dec;46(12):2250-3

Department of Pediatric Surgery, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan.

Purpose: To evaluate the long-term quality of life (QOL) of patients who had undergone major neonatal surgery, the psychosocial and cognitive consequences of neonatal surgical stress were assessed when the patients reached school age.

Materials And Methods: Seventy-two patients who had undergone major neonatal surgery were enrolled in this study. Their primary diseases were anorectal malformation (ARM) in 27 cases, esophageal atresia (EA) in 23, and congenital diaphragmatic hernia (CDH) in 22. Intelligence tests using Wechsler Intelligence Scale for Children III (WISC-III) or a developmental test and the Child Behavior Checklist were conducted through questionnaires and interviews with clinical psychologists.

Results: Mental retardation (MR) was apparent in 25% of EA, 20% of ARM, and 18% of CDH, significantly higher than the 2% to 3% commonly found in the general population. The clinical range (CR) of the Child Behavior Checklist was seen in 35% of EA, 59% of ARM, and 38% of CDH, which is also significantly higher than the 25% typically seen in the general population. No significant differences in MR and CR were seen among the primary diseases. The most important factors influencing MR and CR remain to be identified.

Conclusions: To ensure true quality of life after neonatal surgical stress, pediatric surgeons must consider not only physical assessments but also cognitive, emotional, and psychosocial assessments.
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http://dx.doi.org/10.1016/j.jpedsurg.2011.09.006DOI Listing
December 2011

Management of perianal abscess with hainosankyuto in neonates and young infants.

Pediatr Int 2011 Dec;53(6):892-6

Department of Pediatric Surgery, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan.

Background: Perianal abscess (PA) is a common condition acquired in infancy, yet its treatment method remains controversial. We reviewed the outcome of neonates and young infants with PA who were treated with the traditional Japanese medicine, hainosankyuto (TJ-122).

Methods: Fifteen male infants with PA under the age of 3 months were reviewed. The median age of infants at disease onset was 33 days (range, 18-88 days) and the median bodyweight was 4.1 kg (range, 2.5-6.4 kg). TJ-122 was administered at a dose of 0.20 g/kg/day (n= 13) or 0.25 g/kg/day (n= 2) orally in two or three divided doses before meals. Antibiotics were not used in any of the patients.

Results: Of the 15 patients, 14 were cured and had no recurrence, with a median TJ-122 administration of 28 days (range, 14-117 days). Eight patients were cured within 28 days (53%) and 12 were cured within 60 days (80%). One patient, who was later diagnosed with growth hormone deficiency, showed incomplete healing of PA with intermittent pus discharge and recurrence. The patient was cured by 1 year of age following repeated administration of TJ-122 and juzentaihoto (TJ-48).

Conclusion: Medical management with TJ-122 was effective in most neonates and young infants with PA. It appears prudent to manage these patients with hainosankyuto before resorting to surgical intervention.
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http://dx.doi.org/10.1111/j.1442-200X.2011.03395.xDOI Listing
December 2011

Meconium-related ileus in extremely low-birthweight neonates: etiological considerations from histology and radiology.

Pediatr Int 2011 Dec;53(6):887-91

Department of Pediatric Surgery, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan.

Background: A nationwide survey on neonatal surgery conducted by the Japanese Society of Pediatric Surgeons has demonstrated that the mortality of neonatal intestinal perforation has risen over the past 15 years. The incidence of intestinal perforation in extremely low-birthweight (ELBW) neonates has been increasing as more ELBW neonates survive and as the live-birth rate of ELBW has increased. In contrast to necrotizing enterocolitis (NEC) and focal intestinal perforation (FIP), the pathogenesis of meconium-related ileus, defined as functional bowel obstruction characterized by delayed meconium excretion and microcolon, remains unclarified.

Methods: The histology of 13 ELBW neonates with intestinal perforation secondary to meconium-related ileus was reviewed, and the radiology of 33 cases of meconium-related ileus diagnosed on contrast enema was reviewed. Specimens obtained from 16 ELBW neonates without gastrointestinal disease served as age-matched controls for histological assessment.

Results: The size of the ganglion cell nucleus in meconium-related ileus and in control subjects was 47.3 ± 22.0 µm(2) and 37.8 ± 11.6 µm(2), respectively, which was not significantly different. In all cases of meconium-related ileus, contrast enema demonstrated a microcolon or small-sized colon, with a gradual caliber change in the ileum and filling defects due to meconium in the ileum or colon, showing not-identical locations of caliber changes and filling defects.

Conclusion: Morphological immaturity of ganglia was not suggested to be the pathogenesis of meconium-related ileus. Impaction of inspissated meconium is not the cause of obstruction, but the result of excessive water absorption in the hypoperistaltic bowel before birth, although the underlying mechanism responsible for the fetal hypoperistalsis remains unclear.
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http://dx.doi.org/10.1111/j.1442-200X.2011.03381.xDOI Listing
December 2011

Therapeutic strategy for persistent cloaca: the efficacy of antegrade continent enema as a salvage surgery.

Pediatr Surg Int 2011 May;27(5):505-8

Department of Pediatric Surgery, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Osaka, 594-1101, Japan.

Purpose: The aim of this study is to evaluate our therapeutic strategy for persistent cloaca from the viewpoint of long-term functional outcome.

Materials And Methods: This study covers 17 cases of persistent cloaca treated at our institution and followed for more than 3 years. As a definitive repair for anorectal and urogenital systems, simultaneous surgery with posterior sagittal approach or anorecto-urethrovagino-plasty (PSARUVP) was performed. The length of the common channel and the shape of the vagina determined the vaginoplasty methods. Fecal function was assessed with the scoring system of the Japan Study Group of Anorectal Anomalies.

Results: Anorectoplasty was performed with the posterior sagittal approach in 15 cases and with the perineal approach in two. Vaginoplasty was performed with total urogenital mobilization in nine cases, rectal interposition in four, vaginal flap in two and with other methods. Fecal function was classified as good in three cases, moderate in ten, and poor in four. In the poor cases, Malone's antegrade continence enema (MACE) was performed, which improved fecal function significantly.

Conclusion: PSARUVP might be the optimal surgery for persistent cloaca at present; however, satisfactory fecal function could not be achieved in those cases with a longer common channel. MACE effectively compensated for the poor outcome and was especially successful at eliminating incontinence.
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http://dx.doi.org/10.1007/s00383-010-2819-7DOI Listing
May 2011

Should fundoplication be added at the time of gastrostomy placement in patients who are neurologically impaired?

J Pediatr Surg 2010 Dec;45(12):2373-6

Department of Pediatric Surgery, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho Izumi, Osaka 594-1101, Japan.

Background/purpose: Patients who have advanced neurologic impairment (NI) and require gastrostomy placement (GP) frequently have symptomatic gastroesophageal reflux. We investigated the outcomes of GP without fundoplication in patients who had NI.

Methods: This was a retrospective review of 54 patients with NI (median, 7 years; range, 1-18 years) undergoing GP alone. The operative criteria included medically controllable or no reflux symptoms. The patients were divided into 2 groups based on the percentage of total esophageal time with a pH less than 4.0 (reflux index, or RI): group I (GI, n = 33), RI less than 5.0% (median age, 6 years; range, 2-15 years); group II (GII, n = 21), RI 5.0% or greater (median age, 10 years; range, 1-18 years). Data are expressed as medians and ranges.

Results: Nutritional management was successfully conducted after GP with or without the administration of lansoprazole, famotidine, or rikkunshito in all but 2 patients. One GI patient with alpha-thalassemia required fundoplication, and one GII patient with Cockayne syndrome required gastrojejunal tube feeding. The RI increased significantly in GI patients (2.1% [0%-4.8%] vs 4.5% [0.2%-11.4%], P = .004), whereas it decreased significantly in GII patients (11.2% [5.9%-41.6%] vs 9.8% [1.05-26.6%], P = .04).

Conclusion: Gastroesophageal reflux and related symptoms rarely deteriorate to require additional treatment after GP in patients with NI. Gastrostomy placement is a less invasive and effective procedure for improving the quality of life in those patients.
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http://dx.doi.org/10.1016/j.jpedsurg.2010.08.034DOI Listing
December 2010

Physiological and clinical characteristics of gastroesophageal reflux after congenital diaphragmatic hernia repair.

J Pediatr Surg 2010 Dec;45(12):2346-50

Department of Pediatric Surgery, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, 594-1101, Japan.

Background/purpose: Gastroesophageal reflux (GER) is an important sequela of congenital diaphragmatic hernia (CDH) repair. This study investigated the physiological and clinical characteristics of GER in CDH survivors.

Methods: A total of 52 CDH survivors were investigated retrospectively. Esophageal acid exposure was evaluated with 24-h esophageal pH monitoring in all patients, and esophageal anatomical and motor functional abnormalities were examined with videomanometry in 16 patients.

Results: Fundoplication was necessary in 1 patient. Medical treatment with acid suppression or rikkunshito, a traditional Japanese medicine, was successful in nine patients, and the reflux symptoms were ameliorated at the age of 3 years. The percentage of total time the esophageal pH was below 4.0 (reflux index: RI) ranged from 0.1 to 44.3%. No patient with an RI < 10% had reflux symptoms requiring treatment. The basal lower esophageal sphincter (LES) tone ranged from 15 to 35 mmHg (median 25 mmHg). Esophageal peristalsis was preserved in all of the patients examined, except one who had failed peristalsis and poor clearance in the dilated esophagus.

Conclusion: The motor function of the esophageal body and LES is usually preserved in CDH survivors despite the wide range of esophageal acid exposure in early infancy. Those with symptomatic GER outgrow it, unless associated with advanced respiratory distress or neurological impairment.
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http://dx.doi.org/10.1016/j.jpedsurg.2010.08.029DOI Listing
December 2010

Activation of a dormant ClpX recognition motif of bacteriophage Mu repressor by inducing high local flexibility.

J Biol Chem 2008 Apr 28;283(14):9060-70. Epub 2008 Jan 28.

Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, D. C. 20057, USA.

The C-terminal domain (CTD) of bacteriophage Mu immunity repressor (Rep) regulates DNA binding by the N-terminal domain and degradation by ClpXP protease. Five residues at the Rep C terminus (CTD5) can serve as a ClpX recognition motif, but it is dormant unless activated, a state that can be induced by the presence of dominant-negative mutant repressors (Vir). Conversion of Rep to ClpXP-sensitive form was associated with not only increased exposure of CTD5 to solvent but also increased CTD motion or flexibility as measured by fluorescence anisotropy. CTD mutations (V183S, K193S, and V196S) promoting ClpXP resistance without destroying the recognition motif prevented increased CTD motion induced by Vir. Suppression of ClpXP protease resistance conferred by the V196S mutation also correlated with restoration of CTD motion. The temperature-sensitive R47Q mutation present in cis within the DNA-binding domain restored ClpXP protease sensitivity to the V196S mutant, and anisotropy analysis indicated that R47Q allows the V196S CTD to gain increased flexibility when Vir was present. The results indicate that the CTD functions to turn the recognition motif on and off, most likely by modulating flexibility of the domain that harbors the ClpX recognition motif, suggesting a general mechanism by which proteins can regulate their own degradation.
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http://dx.doi.org/10.1074/jbc.M705508200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2431027PMC
April 2008

Trans-targeting of protease substrates by conformationally activating a regulable ClpX-recognition motif.

Mol Microbiol 2008 Feb 7;67(4):920-33. Epub 2008 Jan 7.

Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Rm. 331 Basic Science Bldg., 3900 Reservoir Rd. NW, Washington, DC 20057-1455, USA.

Conversion of bacteriophage Mu repressor to ClpXP-sensitive form correlates with induced local flexibility at the ClpX recognition motif located at the C-terminal end. Changing the C-terminal valine to an alanine (RepV196A) caused the degradation tag to be constitutively active like that of mutant repressors called Vir, which have a dominant ClpXP-sensitive conformation. However, unlike Vir, RepV196A was unable to convert wild-type repressor (Rep) to the ClpXP-sensitive form. In mixtures with Rep, only RepV196A was rapidly degraded by ClpXP. Unlike Rep, RepV196A was ClpXP sensitive without induced C-terminal flexibility. And unlike adaptor proteins that tether and deliver substrates to ClpX for trans-targeting, Vir promoted rapid degradation of Rep by ClpX deleted for the tethering site that binds adaptor proteins. Therefore, Rep's ClpX recognition motif has regulable properties, allowing an alternative trans-targeting mechanism in which an inactive degradation tag is turned on by induced conformational changes.
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http://dx.doi.org/10.1111/j.1365-2958.2007.06099.xDOI Listing
February 2008

Translation factor IF2 at the interface of transposition and replication by the PriA-PriC pathway.

Mol Microbiol 2007 Dec 19;66(6):1566-78. Epub 2007 Nov 19.

Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Rm. 331 Basic Science Bldg., 3900 Reservoir Road NW, Washington, DC 20057-1455, USA.

Bacteriophage Mu DNA synthesis is initiated during transposition by replication restart proteins PriA, DnaT and either PriB or PriC. The PriA-PriC pathway requires PriA's helicase activity and other host factors that promote the orderly transition from transpososome to replisome on the Mu DNA template. The host factor MRFalpha-PR, which removes obstacles to PriA binding and promotes the PriA-PriC pathway, was identified to be the translation initiation factor IF2. Purified isoform IF2-2, which is truncated at the N-terminal end, had full MRFalpha-PR activity whereas full-length IF2-1 was inactive. IF2-2 was bound to the Mu DNA template specifically at the step for prereplisome assembly. Prior steps in the orderly transition from transpososome were essential to promote efficient IF2-2 binding. Moreover, PriA helicase activity was subsequently needed to displace IF2-2, remodelling the template to permit replisome assembly. IF2's role in the transition mechanism as well as its function as G protein and translation factor suggest its potential to regulate DNA synthesis by this pathway.
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http://dx.doi.org/10.1111/j.1365-2958.2007.06022.xDOI Listing
December 2007

Medical treatment of infantile hypertrophic pyloric stenosis: should we always slice the "olive"?

J Pediatr Surg 2005 Dec;40(12):1848-51

Department of Pediatric Surgery, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Osaka 594-1101, Japan.

Background/purpose: Laparoscopic pyloromyotomy has recently gained wide acceptance as the optimum treatment of infantile hypertrophic pyloric stenosis (IHPS). However, medical treatment may be superior to laparoscopic surgery in invasiveness. The efficacy of our regimen of intravenous atropine therapy for IHPS was assessed in comparison with surgical treatment.

Methods: Medical treatment was initially chosen for 52 (61%) of 85 infants with IHPS at our institute between 1996 and 2004. Atropine was given intravenously at 0.01 mg/kg 6 times a day before feeding. When vomiting ceased and the infants were able to ingest 150 mL/kg per day of formula after stepwise increases in the feeding volume, they were given 0.02 mg/kg atropine 6 times a day orally, and the dose was decreased stepwise.

Results: Of the 52 patients, 45 (87%) ceased projectile vomiting with treatment using intravenous (median, 7 days) and subsequent oral (median, 44 days) atropine administration. The median hospital stay was 13 days (6-36), and no significant complications were encountered during atropine therapy. The remaining 7 patients required surgery. Of 40 who underwent surgery, 4 had wound infections and 1 with hemophilia had postoperative hemorrhagic shock. The patients who underwent successful atropine therapy had body weights comparable with those who underwent surgery at the age of 1 year.

Conclusions: The high success rate of intravenous atropine therapy for IHPS suggests that this therapy is an effective alternative to pyloromyotomy if the length of the hospital stay and the necessity of continuing oral atropine medication are accepted.
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http://dx.doi.org/10.1016/j.jpedsurg.2005.08.025DOI Listing
December 2005

Host factors that promote transpososome disassembly and the PriA-PriC pathway for restart primosome assembly.

Mol Microbiol 2005 Jun;56(6):1601-16

Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Room 331 Basic Science Bldg., 3900 Reservoir Road NW, Washington, DC 20057-1455, USA.

Initiation of bacteriophage Mu DNA replication by transposition requires the disassembly of the transpososome that catalyses strand exchange and the assembly of a replisome promoted by PriA, PriB, PriC and DnaT proteins, which function in the host to restart stalled replication forks. Once the molecular chaperone ClpX weakens the very tight binding of the transpososome to the Mu ends, host disassembly factors (MRFalpha-DF) promote the dissociation of the transpososome from the DNA template and the assembly of a new nucleoprotein complex. Prereplisome factors (MRFalpha-PR) further alter the complex, allowing PriA binding and loading of major replicative helicase DnaB onto the template promoted by the restart proteins. MRFalpha-PR is essential for DnaB loading by restart proteins even on the deproteinized Mu fork whereas MRFalpha-DF is not required on the deproteinized template. When the transition from transpososome to replisome was reconstituted using MRFalpha-DF and MRFalpha-PR, initiation of Mu DNA replication was strictly dependent upon added PriC and PriA helicase. In contrast, initiation on the deproteinized template was predominantly dependent upon PriB and did not require PriA's helicase activity. The results indicate that transition mechanisms beginning with the transpososome disassembly can determine the pathway of replisome assembly by restart proteins.
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http://dx.doi.org/10.1111/j.1365-2958.2005.04639.xDOI Listing
June 2005

Properties of the PriA helicase domain and its role in binding PriA to specific DNA structures.

J Biol Chem 2004 Sep 13;279(37):38503-12. Epub 2004 Jul 13.

Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D. C. 20057, USA.

Primosome assembly protein PriA functions in the assembly of the replisome at forked DNA structures. Whereas its N-terminal DNA binding domain (DBD) binds independently to DNA, the affinity of DBD protein for forked structures is relatively weak. Although the PriA helicase domain (HD) is required for high affinity fork binding, HD protein had very low affinity for DNA. It had only low levels of ATPase activity, and it hydrolyzed ATP when DNA was absent whereas PriA did not. HD catalyzed unwinding of a minimal substrate composed of a duplex with a 3' single-stranded tail. Single-strand binding protein (SSB) bound to the tail of this substrate inhibited this reaction by full-length PriA but enhanced the reaction by HD. SSB stabilized binding of PriA but not of DBD or HD to duplexes with a 5' or 3' single-stranded tail. On forked substrates SSB enhanced helicase action on the lagging-strand arm by PriA but not by HD. The results indicate that synergy of the DBD and HD allows stable binding at the interface between duplex and single-stranded DNA bound by SSB. This mode of binding may be analogous to fork binding, which orients the helicase to act on the lagging-strand side of the fork.
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http://dx.doi.org/10.1074/jbc.M404769200DOI Listing
September 2004

A context-dependent ClpX recognition determinant located at the C terminus of phage Mu repressor.

J Biol Chem 2003 Dec 14;278(52):52333-9. Epub 2003 Oct 14.

Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D.C. 20057, USA.

The bacteriophage Mu immunity repressor is a conformationally sensitive sensor that can be interconverted between forms resistant to and sensitive to degradation by ClpXP protease. Protease-sensitive repressor molecules with an altered C-terminal sequence promote rapid degradation of the wild-type repressor by inducing its C-terminal end to become exposed. Here we determined that the last 5 C-terminal residues (CTD5) of the wild-type repressor contain the motif required for recognition by the ClpX molecular chaperone, a motif that is strongly dependent upon the context in which it is presented. Although attachment of the 11-residue ssrA degradation tag to the C terminus of green fluorescent protein (GFP) promoted its rapid degradation by ClpXP, attachment of 5-27 C-terminal residues of the repressor failed to promote degradation. Disordered peptides derived from 41 and 35 C-terminal residues of CcdA (CcdA41) and thioredoxin (TrxA35), respectively, activated CTD5 when placed as linkers between GFP and repressor C-terminal sequences. However, when the entire thioredoxin sequence was included as a linker to promote an ordered configuration of the TrxA35 peptide, the resulting substrate was not degraded. In addition, a hybrid tag, in which CTD5 replaced the 3-residue recognition motif of the ssrA tag, was inactive when attached directly to GFP but active when attached through the CcdA41 peptide. Thus, CTD5 is sufficient to act as a recognition motif but has requirements for its presentation not shared by the ssrA tag. We suggest that activation of CTD5 may require presentation on a disordered or flexible domain that confers ligand flexibility.
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http://dx.doi.org/10.1074/jbc.M308724200DOI Listing
December 2003

Toriello-Carey syndrome associated with respiratory failure and non-mechanical ileus.

Am J Med Genet A 2003 Aug;120A(4):537-41

Department of Pathology, Osaka University Medical School/Graduate School of Frontier Bioscience, Suita, Osaka, Japan.

Toriello-Carey syndrome comprises agenesis of the corpus callosum, telecanthus, small palpebral fissures, Pierre Robin sequence, abnormal ears, and cardiac defects. We report a boy who has some additional findings, including a severe respiratory failure and intestinal dysmotility. The boy died of these two disorders at age 13 months. Histological examination revealed pulmonary immaturity and a defect of smooth muscle cells in the longitudinal muscle coat of the intestinal musculature, both of which might explain some aspects of the pathophysiology of the patient.
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http://dx.doi.org/10.1002/ajmg.a.20094DOI Listing
August 2003

FTY720 reduces T-cell recruitment into murine intestinal allograft and prevents activation of graft-infiltrating cells.

Transplantation 2003 May;75(9):1469-74

Department of Pediatric Surgery, Osaka University Graduate School of Medicine, Japan.

Background: Effective immunosuppression is a critical determinant of graft survival in small-bowel transplantation (SBTx). The present study was designed to determine the potency of FTY720, a newly synthesized immunosuppressant, in rat SBTx and examine the phenotype of graft-infiltrating cells to evaluate its effect on intestinal allografts.

Materials And Methods: A segment of intestine of Dark Agouti rats was transplanted heterotopically into Lewis rats. The recipients were treated with or without oral FTY720 at a dose of 1 mg/kg per day. Six days after surgery, peripheral blood lymphocytes and lymphocytes from the mesenteric lymph nodes, Peyer's patches, intraepithelial site, and lamina propria of the intestinal allograft were isolated. After the number of lymphocytes in each site was counted, the lymphocyte subpopulations in the intestinal allograft were evaluated by means of a FACScan flow cytometer using several monoclonal antibodies.

Results: FTY720 treatment significantly prolonged recipient survival and strongly inhibited rejection histologically in comparison with control rats. FTY720 immunosuppression resulted in a marked reduction of lymphocyte number in the graft epithelium and lamina propria and the proportion of CD8+ and CD25+ cells. FTY720 also significantly decreased T-cell receptors and increased B cells in the graft Peyer's patches.

Conclusion: FTY720 promoted long-term SBTx recipient survival and maintained the architecture of intestinal allografts. FTY720 immunosuppression may be associated with a reduction of T-cell recruitment subsequent to the redistribution of lymphocyte subpopulations to control the proliferation and activation of graft-infiltrating cells in intestinal allografts.
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http://dx.doi.org/10.1097/01.TP.0000058816.13525.92DOI Listing
May 2003

Three novel mutations responsible for Cockayne syndrome group A.

Genes Genet Syst 2003 Feb;78(1):93-102

Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.

Cockayne syndrome (CS) is a rare autosomal recessive disease, which shows diverse clinical symptoms such as photosensitivity, severe mental retardation and developmental defects. CS cells are hypersensitive to killing by UV-irradiation and defective in transcription-coupled repair. Two genetic complementation groups in CS (CS-A and CS-B) have been identified. We analyzed mutations of the CSA gene in 5 CS-A patients and identified 3 types of mutations. Four unrelated CS-A patients (CS2OS, CS2AW, Nps2 and CS2SE) had a deletion including exon 4, suggesting that there is a founder effect on the CSA mutation in Japanese CS-A patients. Patient CS2SE was a compound heterozygote for this deletion and an amino acid substitution at the 106th glutamine to proline (Q106P) in the WD-40 repeat motif of the CSA protein, which resulted in a defective nucleotide excision repair. Patient Mps1 had a large deletion in the upstream region including exon 1 of the CSA gene. Our results indicate that a rapid and reliable diagnosis of CSA mutations could be achieved in CS-A patients by PCR or PCR-RFLP and that the Q106P mutation could alter the propeller structure of the CSA protein which is important for the formation of the CSA protein complex.
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http://dx.doi.org/10.1266/ggs.78.93DOI Listing
February 2003

Modulation of phage Mu repressor DNA binding and degradation by distinct determinants in its C-terminal domain.

Mol Microbiol 2003 Jan;47(1):171-82

Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Rm. 331, Basic Science Bldg., 3900 Reservoir Road NW, Washington, DC 20057, USA.

Rapid degradation of the bacteriophage Mu immunity repressor can be induced in trans by mutant, protease-hypersensitive repressors (Vir) with an altered C-terminal domain (CTD). Genetic and biochemical analysis established that distinct yet overlapping determinants in the wild-type repressor CTD modulate Vir-induced degradation by Escherichia coli ClpXP protease and DNA binding by the N-terminal DNA-binding domain (DBD). Although deletions of the repressor C-terminus resulted in both resistance to ClpXP protease and suppression of a temperature-sensitive DBD mutation (cts62), some cysteine-replacement mutations in the CTD elicited only one of the two phenotypes. Some CTD mutations prevented degradation induced by Vir and resulted in the loss of intrinsic ClpXP protease sensitivity, characteristic of wild-type repressor, and at least two mutant repressors protected Vir from proteolysis. One protease-resistant mutant became susceptible to Vir-induced degradation when it also contained the cts62 mutation, which weakens DNA binding but apparently facilitates conversion to a protease-sensitive conformation. Conversely, this CTD mutation was able to suppress temperature sensitivity of DNA binding by the cts62 repressor. The results suggest that determinants in the CTD not only provide a cryptic ClpX recognition motif but also direct CTD movement that exposes the motif and modulates DNA binding.
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http://dx.doi.org/10.1046/j.1365-2958.2003.03286.xDOI Listing
January 2003

Novel antibacterial macrolides: synthesis of 15-membered diolides.

J Org Chem 2002 Dec;67(26):9146-52

Discovery Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553-0002, Japan.

Novel 15-membered macrolides possessing the dilactone skeleton, diolides 13a and 13b, have been synthesized in our research program aimed at finding new antibacterial macrolides. Key strategic elements of the approach include the ring-expanding reaction of 13-membered dilactones, prepared from erythromycin A (Ery-A), to 15-membered dilactones via intramolecular translactonization. The absolute configuration at the regenerated C-8 position of the new diolides was determined by chemical transformation, leading to the corresponding lactam analogues, whose stereochemistry is known in the literature. For further confirmation, X-ray analysis was performed. The X-ray structure determination of 13a revealed a backbone conformation similar to that of Ery-A. Novel 15-membered diolide 13a and the 11,12-diol 18 exhibited antibacterial activities comparable to that of Ery-A.
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http://dx.doi.org/10.1021/jo020458eDOI Listing
December 2002

Trans-targeting of the phage Mu repressor is promoted by conformational changes that expose its ClpX recognition determinant.

J Biol Chem 2003 Jan 6;278(3):1612-7. Epub 2002 Nov 6.

Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20057, USA.

Dominant negative forms of the phage Mu repressor, including the mutant Vir repressors, are not only rapidly degraded by the ClpXP protease but also promote degradation of the unmodified, wild-type repressor. This trans-targeting of the wild-type repressor depends upon a determinant within its C-terminal domain, which is needed for recognition by ClpX. An environmentally sensitive fluorescent probe (2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS)) attached to the C terminus of the full-length repressor indicated that Vir induces the movement of this domain into a more exposed configuration. Vir also promoted attachment of MIANS to the C terminus of the repressor at an accelerated rate, and it greatly increased the rate of phosphorylation of a cAMP-dependent protein kinase motif attached to the repressor C terminus. While an excess of Vir was needed to promote repressor phosphorylation at maximal rates, the presence of ClpX could increase phosphorylation rates at lower Vir levels. trans-Targeting of the Mu repressor is therefore promoted by exposing its ClpX recognition determinant, and the action of ClpX can assist Vir in exposing these determinants.
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http://dx.doi.org/10.1074/jbc.M209352200DOI Listing
January 2003

A novel class of endothelin-A receptor antagonists, (R)-2-(benzo[1,3]dioxol-5-yl)-6-isopropyloxy-2H-chromene-3-carboxylic acids (S-1255). Conformational analysis of basic structure, crucial for ET(A) antagonism, in solution and solid states.

Bioorg Med Chem 2002 Dec;10(12):3965-72

Shionogi Research Laboratories, Shionogi & Co., Ltd., 12-4, Sagisu 5-chome, Fukushima-ku, Osaka, Japan.

Conformational studies of potent and selective endothelin-A (ET(A)) receptor antagonists, 4-substituted (R)-2-(benzo[1,3]-dioxol-5-yl)-6-isopropoxy-2H-chromene-3-carboxylic acids, are reported. X-ray crystallography and NMR studies of the 4-anisyl derivative 2 (S-1255), the stable atropisomers 3 and the 4-n-butyl derivative 4 reveal that the A-, B- and C-rings in these compounds adopt a L-like conformation in both solution and solid states. Molecular mechanics calculation shows that this L-like conformation is an inevitable conformation as determined by intramolecular steric repulsions. These 2H-chromene derivatives bound to an ET(A) receptor with IC(50) values of less than 1 nM, whereas the dihydro compounds 7 and 9 not having the L-like conformation showed weaker affinities. These results suggest that the L-like conformation is specifically recognized by the active site of the ET(A) receptor. The roles of the L-like conformation in the receptor binding are discussed.
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http://dx.doi.org/10.1016/s0968-0896(02)00300-0DOI Listing
December 2002

Follow-up studies of anorectal malformations after posterior sagittal anorectoplasty.

J Pediatr Surg 2002 Nov;37(11):1529-33

Department of Pediatric Surgery, Osaka University Graduate School of Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, Suita, Osaka, Japan.

Background/purpose: There are few follow-up studies comparing posterior sagittal anorectoplasty (PSARP) with conventional procedures for patients with anorectal malformations (ARM). The authors have examined retrospectively postoperative anorectal function of patients with ARM treated with PSARP compared with those treated with conventional methods.

Methods: Anorectal function in 23 patients with high and intermediate type anorectal malformations (PSARP group), who underwent PSARP more than 4 years previously, were assessed by Kelly's clinical scoring system and objective studies. These results were compared with those in 14 cases (5 high and 9 intermediate type cases; control group), who underwent other conventional surgical procedures.

Results: Using Kelly's clinical scoring system, scores of the PSARP group compared with the control group were good in 48% versus 21%, fair in 48% versus 58%, and poor in 4% versus 21%, respectively. Barium enema studies suggested better anorectal sphincteric function in patients with high anorectal malformation in the PSARP group. Magnetic resonance imaging (MRI) studies showed more correct placement of the rectum through the striated muscle complex in the PSARP group at the I-line level. Manometric studies showed no difference in maximum resting pressure, anal canal length, and the incidence of anorectal reflex between the two groups.

Conclusions: The favorable results of MRI and barium enema studies can be explained by direct visualization of the striated muscle complex with the aid of electrical stimulation as well as no harmful effects of amputation of the sphincter muscle in PSARP. However, manometric studies suggest anorectal function in patients with high and intermediate anorectal malformations is limited even after PSARP. Long-term postoperative follow-up with adequate bowel management is required for all patients with high or intermediate anorectal malformation.
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http://dx.doi.org/10.1053/jpsu.2002.36178DOI Listing
November 2002

Derepression of bacteriophage mu transposition functions by truncated forms of the immunity repressor.

J Mol Biol 2002 Sep;322(2):311-24

Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20007, USA.

To trigger bacteriophage Mu transposition and replication in response to physiological signals, its immunity repressor must be synchronously inactivated. Two repressor mutants (Vir), which have an altered C-terminal domain and are highly susceptible to degradation by ClpXP protease, confer a dominant negative phenotype by promoting degradation of the wild-type repressor. To search for other modified repressors that can induce Mu derepression in vivo and to determine what part of the inducing repressor molecules are needed to target the unmodified repressor population, repressor peptides with nested deletions starting at the C-terminal end were constructed. Such peptides with a C-terminal ssrA degradation tag promoted a sharp reduction in cellular levels of full-length unmodified repressor, a process largely dependent upon the clpP protease function. Only the repressor DNA-binding domain, located at the N-terminal end, was required in tagged peptides to target unmodified repressor. In addition, some repressor peptides containing the DNA-binding domain promoted derepression without the clpP function, being able to promote repressor inactivation without promoting its degradation. None of the modified repressors could promote derepression if immunity was established by a mutant repressor lacking 18 residues at its C-terminal end. The results indicate that inducing repressor peptides influence the function of the C-terminal domain of the intact repressor, a domain that regulates its degradation and DNA binding. They suggest the possibility that tagged repressor molecules, produced by stalled ribosomes, can be inducers of transposition under starvation conditions.
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http://dx.doi.org/10.1016/s0022-2836(02)00755-6DOI Listing
September 2002

Effects of isolated small bowel transplantation on liver dysfunction caused by intestinal failure and long-term total parenteral nutrition.

Pediatr Transplant 2002 Jun;6(3):235-9

Department of Pediatric Surgery, Osaka University Medical School, Suita City, Osaka, Japan.

It has not been fully determined whether isolated small bowel transplantation (ISBTx) can reverse liver dysfunction caused by intestinal failure requiring long-term total parenteral nutrition (TPN). A boy with congenital microvillus inclusion disease presented with vomiting and severe diarrhea since the first day of life and had been managed by TPN since then. He suffered from catheter-related sepsis several times. At 14 yr of age he developed progressive hepatosplenomegaly with thrombocytopenia and coagulopathy. He underwent ISBTx with an ileal graft from his blood-identical grandmother at the age of 16 yr. Oral feeding was started on the 14th day after ISBTx and gradually increased. TPN was completely withdrawn after 5 months. Liver was palpated 5 cm below the costal margin before ISBTx, while it became non-palpable 5 months after ISBTx. Serum liver enzyme levels and prothrombin time normalized in the 5 months following ISBTx. Liver biopsy showed marked steatosis, slight cholestasis, and mild bridging fibrosis before ISBTx. Although histological examination of liver biopsy revealed complete disappearance of steatosis 7 and 11 months after ISBTx, liver fibrosis remained unchanged. This clinical experience has shown that although steatosis and cholestasis are reversible after successful ISBTx and withdrawal of TPN, liver fibrosis may remain unchanged.
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http://dx.doi.org/10.1034/j.1399-3046.2002.01074.xDOI Listing
June 2002

Stachyflin and acetylstachyflin, novel anti-influenza A virus substances, produced by Stachybotrys sp. RF-7260. I. Isolation, structure elucidation and biological activities.

J Antibiot (Tokyo) 2002 Feb;55(2):155-64

Shionogi Research Laboratories, Shionogi & Co, Ltd, Osaka, Japan.

Two novel compounds, stachyflin and acetylstachyflin, have been isolated by solid-state fermentation of Stachybotrys sp. RF-7260. The structures of both metabolites, determined by detailed NMR analyses and X-ray crystallographic analysis, are novel with a pentacyclic moiety including cis-fused decalin. The absolute stereochemistry of stachyflins was determined by circular dichroism analysis. Stachyflin showed antiviral activity against influenza A virus (H1N1) in vitro with an IC50 value of 0.003 microM. Acetylstachyflin was about 77-fold less active than stachyflin.
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http://dx.doi.org/10.7164/antibiotics.55.155DOI Listing
February 2002