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Polymer-infiltrated ceramic network (PICN) materials have gained considerable attention as tooth restorative materials owing to their mechanical compatibility with human teeth. However, the mechanical strength of contemporary PICN materials is lower than those of conventional resin composites and ceramics. This study aims to develop novel high-strength PICN for use as a dental restorative material. Zirconia-based PICN (EXP) was fabricated using 3 mol% yttria tetragonal polycrystalline zirconia powder and resin monomers via slip casting, followed by sintering and polymer infiltration. Comprehensive analyses of the microstructure, mechanical properties, and physicochemical properties of EXP were performed using scanning electron microscopy with energy-dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, inorganic content measurements, three-point bending test, Vickers hardness test, two-body wear test, shear bond strength (SBS) test, surface free energy analysis, and water sorption/solubility test. Commercially available computer-aided design/computer-aided manufacturing (CAD/CAM) materials, including resin composite (CERASMART), silicate-based PICN (ENAMIC), and zirconia ceramic (e.max ZirCAD), were used for comparison. The analyses highlight the dual network structure of EXP, which comprised a zirconia skeleton and an infiltrated resin phase. EXP exhibits a flexural strength of 346.0 ± 46.0 MPa, flexural modulus of 44.0 ± 3.7 GPa, and Vickers hardness of 440.1 ± 51.2 VHN. The mechanical properties of EXP are significantly higher than those of CERASMART and ENAMIC but lower than those of ZirCAD. Notably, the EXP hardness closely mimics that of the human enamel. The wear volume, SBS, and water sorption/solubility of EXP are comparable to those of CERASMART and ENAMIC. Therefore, EXP has potential applications as a tooth restorative material., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Objective: To evaluate whether serum matrix metallopeptidase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels predict response to adrenocorticotropic hormone (ACTH) therapy in patients with infantile spasms., Methods: We prospectively evaluated patients with infantile spasms who were referred to Saitama Children's Medical Center from January 2011 to December 2020. We measured Q-albumin and serum MMP-9 and TIMP-1 levels before ACTH therapy. Patients were divided into three groups based on the etiology of their infantile spasms: those with an unknown etiology and normal development (unknown-normal group); those with a structural and acquired etiology (structural-acquired group); and those with a structural and congenital, genetic, metabolic, or unknown etiology with developmental delay (combined-congenital group). Responders were defined as those having complete cessation of spasms for more than 3 months with the resolution of hypsarrhythmia on electroencephalography during ACTH therapy., Results: We collected serum from 36 patients with West syndrome and five patients with infantile spasms without hypsarrhythmia before ACTH therapy. Twenty-three of 41 patients (56.1%) were responders, including 8/8 (100%) in the unknown-normal group, 6/9 (66.7%) in the structural-acquired group, and 9/24 (37.5%) in the combined-congenital group. The serum MMP-9 level and MMP-9/TIMP-1 ratio were significantly higher in responders than in nonresponders (P = 0.001 for both)., Conclusion: A therapeutic response to ACTH was associated with a higher serum MMP-9 level and higher MMP-9/TIMP-1 ratio in patients with infantile spasms. Therefore, these biomarkers may predict responses to ACTH therapy in this patient population., (Copyright © 2021 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Purpose: We aimed to study the efficacy of adrenocorticotropic hormone (ACTH) treatment on infantile spasms with different aetiologies. In particular, we were interested in patients with structural-acquired aetiology., Methods: Patients with infantile spasms, who were treated with ACTH, were divided into three groups based on the aetiologies: unknown aetiology with normal development (unknown-normal), structural-acquired, and combined-congenital aetiologies that included genetic, metabolic, structural-congenital, or unknown aetiology with developmental delay., Results: Of the 107 patients included (58 males, 49 females), 25 patients had unknown-normal aetiology [median age at onset 5 months, standard deviation (SD) 3.12, range 2-16 months]; 20 patients had structural-acquired aetiology (median age at onset 6.5 months, SD 3.85 months, range 4-17 months); and 62 patients had combined-congenital aetiologies (median age at onset 5 months, SD 2.73 months, range 2-16 months). The efficacy of ACTH was 64.0 %, 65 %, and 30.6 % in the unknown-normal aetiology, structural-acquired aetiology, and combined-congenital aetiologies, respectively (p < 0.01). Multivariate analysis showed a statistically significant higher efficacy in the unknown-normal aetiology [Odds ratio (OR) 4.63, 95 % confidence interval (CI) 1.60-13.30] and structural-acquired aetiology (OR 3.41, 95 % CI 1.01-11.50) compared to that in the combined-congenital aetiologies., Conclusion: Infantile spasms with structural-acquired aetiology had greater response to ACTH treatment than those with combined-congenital aetiologies. The efficacy of standard therapy of infantile spasms should be considered based on aetiology., (Copyright © 2020 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

Introduction: Hypomyelinating leukodystrophies (HLDs) are genetically heterogeneous syndromes, presenting abnormalities in myelin development in the central nervous system. Recently, a recurrent de novo mutation in TMEM106B was identified to be responsible for five cases of HLD. We report the first Japanese case of TMEM106B gene mutation., Case Study: A 3-year-old patient presented with nystagmus and muscle hypotonia in his neonatal period, followed by delayed psychomotor development. Brain magnetic resonance images showed delayed myelination. Wave III and subsequent components were not presented by his auditory brainstem response. These features were similar to those observed in Pelizaeus-Merzbacher disease (PMD)., Methods: Proteolipid protein 1 (PLP1) gene screening, Mendelian disease panel exome, and whole-exome sequencing (WES) were sequentially performed., Results: After excluding mutations in either PLP1 or other known HLD genes, WES identified a mutation c.754G > A, p.(Asp252Asn) in TMEM106B, which appeared to occur de novo, as shown by Sanger sequencing and SalI restriction enzyme digestion of PCR products., Discussion: This is the sixth case of HLD with a TMEM106B mutation. All six cases harbored the same variant. This specific TMEM106B mutation should be investigated when a patient shows PMD-like features without PLP1 mutation. Our PCR-SalI digestion assay may serve as a tool for rapid HLD diagnosis., (Copyright © 2020 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Objective: Absence status epilepticus (ASE) is a form of non-convulsive status epilepticus characterized by ongoing or intermittent epileptic activity accompanied by behavioral and cognitive changes. Herein, we assessed high-frequency oscillations in the ripple band in patients with ASE and typical absence seizures., Methods: We enrolled five patients with ASE, 26 patients with childhood absence epilepsy (CAE), and 15 patients with juvenile absence epilepsy (JAE). We performed time-frequency analysis of electroencephalogram data for ictal absence seizures at each electrode to assess the high frequency activity (HFA) rate, peak frequency, and peak power., Results: The average HFA rates were 60.7%, 20.8%, and 12.9% in ASE, CAE, and JAE patients, respectively. The average peak frequencies were 126.4 Hz, 120.9 Hz, and 126.1 Hz in ASE, CAE, and JAE patients, respectively. The average peak power values were 2,388.8 μV 2 , 120.9 μV 2 , and 126.1 μV 2 in ASE, CAE, and JAE patients, respectively, and all epilepsy groups exhibited frontal-dominant ripple distribution., Conclusion: ASE patients presented higher power and frontal dominant ripples of absence seizure, compared to CAE and JAE patients., Significance: Future studies should utilize scalp-recorded ripples as a biomarker of absence epilepsy. This may aid in the development of novel treatment strategies for ASE., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.)

Objective: Some pediatric patients with autoimmune encephalitis (AE) experience sequelae in spite of immunotherapy. In this study, we aimed to evaluate the association of serum matrix metallopeptidase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels with the neurological prognosis of AE., Methods: We retrospectively included 13 patients with AE who had been referred to Saitama Children's Medical Center from February 2011 to May 2019. We compared serum MMP-9 levels, TIMP-1 levels, and MMP-9/TIMP-1 ratio in the acute period (within 30 days from the onset of AE) and subacute period (30-day period following the acute period). We also compared these biomarker levels between patients with (group A) and without sequelae (group B). Sequelae were evaluated at discharge or the last visit., Results: Group A (median age, 7.8 years; range, 5.3-10.7 years) and group B (median age, 13.3 years; range, 11.1-15.4 years) had 6 patients each; 1 patient was excluded because the time of AE onset was unknown. In the acute period, there were no significant differences in MMP-9 levels, TIMP-1 levels, and MMP-9/TIMP-1 ratio between groups A and B. In the subacute period, serum MMP-9/TIMP-1 ratio was higher in group A than in group B (p < 0.01). There were no significant differences in MMP-9 and TIMP-1 levels between groups A and B., Conclusions: Patients with sequelae of AE showed a high MMP-9/TIMP-1 ratio in the subacute period. Our study demonstrates that elevation of serum MMP-9/TIMP-1 ratio in the subacute period may be a predictive factor of sequelae of AE., (Copyright © 2019 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Purpose: Perampanel (PER) is a selective, non-competitive antagonist of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor. In Japan, PER is approved for patients with epilepsy who are at least 12 years old for the adjunctive treatment of primary generalised tonic-clonic seizures and partial-onset seizures (with or without secondary generalization). We surveyed the efficacy, adverse effects, and serum concentrations of PER, focusing especially on patients younger than 12 years of age., Methods: We retrospectively surveyed the clinical information of patients treated with PER and assessed the efficacy at 6 months after treatment initiation. We compared efficacy, adverse effects, and serum concentration in patients younger or older than 12 years of age. Responders were defined as those who experienced a ≥50% seizure reduction., Results: Eighty-four patients were enrolled. The average age of the younger group was 7.1 ± 3.3 (standard deviation) years compared to 16.4 ± 3.7 years in the older group. The responder rate was 42.9% (36/84). The responder rate did not differ between the two age groups (<12 years, 20/44, 45.4%; >12 years, 16/40, 40.0%; p = 0.78). The younger age group had a significantly lower concentration-to-dose (CD) ratio than the older age group (<12 years, 1849.8 ± 2209.3; >12 years, 3076.3 ± 3352.2, p = 0.02). Treatment-emergent adverse events (TEAEs) were observed in 22.6% (19/84) of patients, with the most common being somnolence (8/84, 9.5%)., Conclusion: PER may be an alternative to treat seizures in paediatric drug-resistant epilepsy. Serum concentrations of PER might be lower in patients younger than 12 years than in older patients., (Copyright © 2019 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

Background: Studies using continuous-access drug self-administration showed that cocaine seeking increases during abstinence (incubation of cocaine craving). Recently, studies using intermittent-access self-administration showed increased motivation to self-administer and seek cocaine. We examined whether intermittent cocaine self-administration would potentiate incubation of craving in male and female rats and examined the estrous cycle's role in this incubation., Methods: In experiment 1, male and female rats self-administered cocaine either continuously (8 hours/day) or intermittently (5 minutes ON, 25 minutes OFF × 16) for 12 days, followed by relapse tests after 2 or 29 days. In experiments 2 and 3, female rats self-administered cocaine intermittently for six, 12, or 18 sessions. In experiment 4, female rats self-administered cocaine continuously followed by relapse tests after 2 or 29 days. In experiments 3 and 4, the estrous cycle was measured using a vaginal smear test., Results: Incubation of cocaine craving was observed in both sexes after either intermittent or continuous drug self-administration. Independent of access condition and abstinence day, cocaine seeking was higher in female rats than in male rats. In both sexes, cocaine seeking on both abstinence days was higher after intermittent drug access than after continuous drug access. In female rats, incubation of craving after either intermittent or continuous drug access was significantly higher during estrus than during non-estrus; for intermittent drug access, this effect was independent of the training duration., Conclusions: In both sexes, intermittent cocaine access caused time-independent increases in drug seeking during abstinence. In female rats, the time-dependent increase in drug seeking (incubation) is critically dependent on the estrous cycle phase., (Published by Elsevier Inc.)

Objective: To evaluate the efficacy and safety of pyridoxal for treating West syndrome., Methods: We retrospectively investigated pyridoxal's efficacy and safety in 117 patients with West syndrome at Saitama Children's Medical Center from July 1993 to May 2016. Pyridoxal was administered at doses of 10-50 mg/kg/day. We evaluated seizure outcomes and electroencephalographic findings at 4 weeks after pyridoxal therapy. The responders were those with complete cessation of spasms for more than 4 weeks and those with resolution of hypsarrhythmia on EEG at 1-4 weeks after pyridoxal therapy., Results: Five of the 117 patients (4.3%) were responders. The median duration between pyridoxal therapy to spasm cessation was 6 (5-13) days. Among the responders, four had hypsarrhythmia resolution, no spasm relapse, and no other seizure types more than 2 years after pyridoxal therapy. One responder had partial seizures and spasm relapse. No serious adverse effects occurred. There were no significant differences in sex, etiologies, complication, other seizure types preceding the spasms, onset age of spasms, age of pyridoxal therapy, treatment lag, initial and maintenance doses of pyridoxal, and adverse effects between pyridoxal responders and non-responders., Conclusions: The efficacy rate of pyridoxal monotherapy as first-line treatment for West syndrome was low. However, pyridoxal therapy showed a rapid response within 1 week and was safe. We consider pyridoxal therapy as a kind of challenge therapy during the evaluation period concerning differential diagnosis and etiologies of West syndrome and immunological risks before adrenocorticotrophic hormone therapy or vigabatrin therapy., (Copyright © 2018 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Objective: To quantitatively evaluate regional cerebral blood flow (rCBF) and regional developmental changes during childhood using 123 I-N-isopropyl-iodoamphetamine single-photon emission computed tomography (SPECT) and autoradiography., Methods: We retrospectively analyzed quantitative values of rCBF in 75 children (29 girls) aged between 16 days and 178 months (median: 12 months), whose brain images, including magnetic resonance imaging and SPECT data, were normal under visual inspection at Saitama Children's Medical Center between 2005 and 2015. The subjects had normal psychomotor development, no focal neurological abnormalities, and neither respiratory nor cardiac disease at the time of examination. Regions of interest were placed automatically using a three-dimensional stereotactic template., Results: rCBF was lowest in neonates, who had greater rCBF in the lenticular nucleus, thalamus, and cerebellum than the cerebral cortices. rCBF increased rapidly during the first year of life, reaching approximately twice the adult levels at 8 years, and then fell to approximately adult levels in the late teenage years. Cerebral cortex rCBF sequentially increased in the posterior, central, parietal, temporal, and callosomarginal regions during infancy and childhood., Conclusions: rCBF changed dramatically throughout childhood and ranged from lower than adult values to approximately two times higher than adult values. It had different trajectories in each region during brain development. Understanding this dynamic developmental change is necessary for SPECT image evaluation in children., (Copyright © 2018 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Objective: To determine whether the characteristics of scalp-recorded high frequency oscillations, especially ripples, can predict the "atypical forms" of benign epilepsy of childhood with centrotemporal spikes (ABECTS), in BECTS., Methods: Seven patients with ABECTS and eighteen patients with BECTS underwent electroencephalography (EEG) in the secondary bilateral synchrony (SBS) and non-SBS periods for ABECTS patients. SBS period is that when more than 50% of the interictal epileptiform discharges (IEDs) are bilaterally synchronized. We determined the IED-ripple co-occurrence rate, performed time frequency analysis, and calculated the asymmetry index (AI)., Results: The IEDs-ripple co-occurrence rate increased in the SBS compared to the non-SBS period. Time frequency analysis showed higher high-frequency activity rate and peak power in the SBS than in the non-SBS period. The AI was lower in ABECTS than BECTS, both in the non-SBS and SBS periods., Conclusions: Ripples were enhanced in the SBS period of ABECTS, and bilaterally synchronized both in the non-SBS and SBS periods, whereas ripples in BECTS were localized unilaterally., Significance: Bilaterally synchronized ripples in the non-SBS period of ABECTS may distinguish ABECTS from BECTS in the non-SBS period of IEDs, and may be helpful for early detection of progressive neurophysiological regression leading to early intervention., (Copyright © 2018 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.)

Objective: To clarify changes in clinical practice for infantile spasms, including West syndrome, in Japan over the past two decades., Methods: We investigated common treatment strategies for infantile spasms among 157 pediatric neurologists from a designated training facility for pediatric neurology and/or a designated training facility for epilepsy in Japan. A questionnaire was used to investigate use of adrenocorticotropic hormone (ACTH) therapy including daily dose, treatment duration, and tapering off period, and preferred first to fifth-line treatment choices., Results: Among 119 responses (75.8%), 107 enabled analysis of ACTH therapy and 112 were used to determine preferred order of first to fifth-line treatments. Over 80% respondents reported an initial ACTH dose of ≤0.0125 mg/kg/day, with a treatment duration of 14 days and various tapering periods. Following an unfavorable response of seizures to ACTH, 80% respondents increased the dose and/or extended treatment duration. The same ACTH therapy regimen was performed for symptomatic and cryptogenic patients at 95 facilities (88.8%). Preferred orders of therapeutic agents were the same for both symptomatic and cryptogenic patients at 64 facilities (57.1%). Over half the respondents selected vitamin B6 or valproate as the first and second-line treatments instead of ACTH therapy, while ACTH therapy was the most frequently selected third-line treatment., Conclusions: Current ACTH therapy regimens have lower doses and shorter durations than previously reported. However, treatment strategies for infantile spasms have not changed much in two decades. ACTH therapy should be the first/second-line treatment rather than third-line or later, especially for cryptogenic infantile spasms., (Copyright © 2018 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Introduction: In some skeletal Class III adult patients with nasomaxillary hypoplasia, the LeFort I osteotomy provides insufficient correction. This case report describes a 20-year-old woman with a combination of nasomaxillary hypoplasia and a protrusive mandible with a congenitally missing mandibular second premolar., Methods: We performed a LeFort II osteotomy for maxillary advancement. Autotransplantation of a tooth was also performed; the donor tooth was used to replace the missing permanent tooth. To increase the chance of success, we applied light continuous force with an improved superelastic nickel-titanium alloy wire technique before extraction and after transplantation., Results: The patient's profile and malocclusion were corrected, and the autotransplanted tooth functioned well. The postero-occlusal relationships were improved, and ideal overbite and overjet relationships were achieved., Conclusions: The methods used in this case represent a remarkable treatment., (Copyright © 2015 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.)

This study examined the potential for osteogenesis via regenerative medicine using autologous tissues (umbilical cord (UC) and umbilical cord blood (UCB)) in nude mice. The study was designed to provide the three elements required for regenerative medicine (cell, scaffold, and growth factor) and autoserum for culture by means of autologous tissues. Mesenchymal stromal cells were obtained from UC (UC-MSCs). Fibrin, platelet-rich-plasma, and autoserum were obtained from UCB as scaffold, growth factor and serum for culture respectively. UC-MSCs were obtained from Wharton jelly and cultured with UCB-derived fibrin (UCB-fibrin) for 3-4 weeks to induce their differentiation into osteoblasts. They were implanted subcutaneously into the dorsum of male nude mice for 6 weeks prior to undergoing assessment. The assessments performed were haematoxylin and eosin, and alizarin red staining, immunohistochemical staining of human mitochondria, scanning electron microscopy, scanning electron microscopy with energy dispersive X-ray spectrometry and real-time reverse transcriptase-polymerase chain reaction to assess the expressions of osteoblast markers. Consequently, the differentiation of UC-MSCs into osteoblasts and the production of hydroxyapatite were verified. This study suggested the possible formation of bone tissue using biomedical materials obtained from UC and UCB., (Copyright © 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Objective: Osteogenesis in the bone defect at the site of an alveolar cleft is important to enable patients with cleft lip and palate to acquire dental articulation. The presence of umbilical cord-derived mesenchymal stem cells has been reported. In this study, we used autoserum derived from the umbilical cord blood (UCB) of neonates in an attempt to examine the osteoblastic differentiation potential of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) in nude mice., Materials and Methods: UCB, hydroxyapatite, and rhBMP were used as the supply source of autoserum, scaffold, and osteoinductive growth factor, respectively. MSCs, obtained from Wharton's jelly and cultured for 3-4weeks to induce their differentiation into osteoblasts, were implanted subcutaneously into the dorsum of male nude mice for 6weeks before the assessment by real-time reverse transcriptase chain reaction of osteoblast marker expression., Results: UCB-derived autoserum was a viable source for the culture and implantation of UC-MSCs. The osteoblastic differentiation potential of UC-MSCs was demonstrated in nude mice by performing immunohistochemical staining and by the presence of osteoblast marker expression., Conclusions: Our results confirm the osteogenic potential of UC-MSCs and provide basic evidence for the realization of regenerative medicine using autologous tissues., (Copyright © 2012 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Stochastic resonance (SR) is a phenomenon occurring in nonlinear systems by which the ability to process information, for instance the detection of weak signals is statistically enhanced by a non-zero level of noise. SR effects have been observed in a great variety of systems, comprising electronic circuits, optical devices, chemical reactions and neurons. In this paper we report the SR phenomena occurring in the execution of an extremely simple behavioral rule inspired from bacteria chemotaxis. The phenomena are quantitatively analyzed by using Markov chain models and Monte Carlo simulations., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: The pathogenesis of polycystic kidney disease (PKD) remains unclear despite the identification of the genes responsible for hereditary PKD. In this study, we investigated the alteration of gene expressions in an acquired PKD model induced by 2-amino-4,5-diphenylthiazole (DPT) using the differential display method., Methods: Kidney mRNA from a Sprague-Dawley rat fed with 1% DPT for 4 days and from a control rat was compared by the RT-PCR differential display method. Differentially expressed bands were re-amplified and subcloned. Using these subclones as probes, the changes in gene expressions were confirmed by Northern blot analysis. Subsequently, mouse kidney cDNA library was screened., Results: The isolated 1.5-kb cDNA contained an open reading frame encoding 296 amino acids, which shared 94.3% identity with rat SULT1C2 sulfotransferase, and was considered to be its mouse ortholog (GenBank Accession No. AY005469). Mouse SULT1C2 mRNA was abundant in the kidney and stomach among normal mouse tissues. The expression of SULT1C2 mRNA was decreased in the rat kidney after DPT feeding but not in the stomach. Mouse SULT1C2 was expressed successfully using pET plasmid vector and E. coli. The recombinant 34-kD protein was capable of catalyzing the sulfation of p-nitrophenol at a Km of 3.1 mmol/L, by utilizing 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor., Conclusions: Although the physiological substrate and function of SULT1C2 have yet to be elucidated, its down-regulation could be involved in the cystic changes of tubules by decreasing the sulfation of the tubular basement membrane components.

We examine susceptibility to subsequent spontaneous recurrences of methamphetamine psychosis (i.e. flashbacks) in 11 flashbackers with a single episode and in nine flashbackers with subsequent episodes. All had undergone frightening stressful experiences during previous MAP use. Mild psychosocial stressors then triggered flashbacks. During flashbacks, the nine flashbackers with subsequent episodes had more markedly increased norepinephrine levels, with slightly increased 3-methoxytyramine levels. The duration of imprisonment in this subgroup approached significantly long levels than in the 11 flashbackers with a single episode. Robust noradrenergic hyperactivity with slightly increased dopamine release may therefore predict subsequent flashbacks. Longer exposure to distressing situations may also contribute to robust noradrenergic hyperactivity.

We examined increased sensitivity to stress in relation to spontaneous recurrences of methamphetamine (MAP) psychosis (i.e., flashbacks). Plasma monoamine metabolite levels were assayed in: 26 flashbackers, of whom 11 were on neuroleptics before and during the study, and the other 15 received neuroleptics in the course of the study; 18 non-flashbackers with a history of MAP psychosis; eight subjects with persistent MAP psychosis; and 23 MAP user and 11 non-user controls. The 26 flashbackers had experienced stressful events and/or MAP-induced fear-related psychotic symptoms during previous MAP use. Mild psychosocial stressors then triggered flashbacks. During flashbacks plasma norepinephrine levels increased markedly; among the flashbackers, those with a history of stressful events, whether or not they had experienced fear-related symptoms, showed a further increase in 3-methoxytyramine levels. Stressful experiences, together with MAP use, may therefore induce sensitization to stress associated with noradrenergic hyperactivity, involving increased dopamine release, and so triggering flashbacks.

The present study examined the effects of activating GABA(A) receptors in the anterior and posterior regions of the ventral tegmental area (VTA) on operant reinforcement behavior, using the technique of intracranial self-administration. Rats were given the opportunity to self-administer vehicle alone (artificial CSF) and vehicle containing 25, 50, and 100 microM muscimol, a GABA(A) agonist, into the anterior or posterior VTA during four sessions (3 h/session) in standard two-lever operant chambers. Rats received five times greater infusions of 50 and 100 microM muscimol than vehicle into the posterior VTA; both doses significantly increased responding above vehicle levels on the active and inactive (control) levers equally. When the response requirement for muscimol infusions was increased from a fixed-ratio 1 (FR1) to FR3 in a single-lever chamber, the total session responses increased approximately twofold. Muscimol was not self-infused when cannula placements were in the anterior VTA. The self-infusion of muscimol into the posterior VTA was attenuated by coadministration of picrotoxin. Overall, the results suggest that the activation of GABA(A) receptors in the posterior VTA produces goal-directed behavior.

In skeletal muscles, increased utilization of lipids and carbohydrates accompanied with increased energy expenditure has been observed during and after exercise. UCP3, mitochondrial uncoupling protein, is expressed in skeletal muscles. We investigated UCP3 mRNA levels in exercise training mice which increased energy expenditure and in sciatic nerve-denervated mice which decreased energy expenditure. Mice exercised by 2 wk swimming had 14- to 18-fold increases of UCP3 mRNA in skeletal muscles 3 h after the last swimming, but no increases of UCP1 mRNA in BAT and of UCP2 mRNA in WAT. However, 22 h after exercise, UCP3 mRNA increases observed in skeletal muscles 3 h after exercise returned to sedentary levels. Similar transient increases of UCP3 mRNA were observed in 1 wk treadmill running training or a single exercise bout. In denerved gastrocnemius, GLUT4 and UCP3 mRNA decreased by 58 and 45%, respectively. These data indicate that UCP3 may have a role for fine adjustments of energy expenditure and that up-regulation of UCP3 mRNA may be a defense mechanism against extra energy supply to consume extra energy in skeletal muscles., (Copyright 1998 Academic Press.)

Fourteen kb murine GLUT4 minigene (= -7395 GLUT4) contains DNA sequence that confers tissue specific, exercise-induced up-regulation of the GLUT4 gene in skeletal muscle and high-fat diet induced-down-regulation in white adipose tissue. To identify the DNA sequences required for regulated expression, we generated GLUT4 minigene transgenic mice harboring 3237, 2000, 1000, and 442 bp of 5'-flanking region, all exons and introns, and 1 kb of 3'-flanking sequence of the mouse GLUT4 gene. The -3237-, -2000-, and -1000-GLUT4 constructs were expressed in a tissue-specific manner identical to the endogenous GLUT4. Exercise-induced up-regulation and high-fat diet-induced down-regulation of these constructs also paralleled those of the endogenous GLUT4 gene. In contrast, the -442 GLUT4 construct was expressed substantially in skeletal muscle (gastrocnemius and quadriceps) and heart, but was only expressed very weakly in white adipose tissue and was not expressed in brown adipose tissue. Furthermore, this -442 GLUT4 construct failed to respond to exercise or a high-fat diet in either muscle or adipose tissue. These results indicate that brown and white adipocyte-specific enhancer(s) and exercise- and high-fat diet-responsive elements are located between bases -1000 and -442 of the murine GLUT4 5'-flanking region., (Copyright 1997 Academic Press.)

The process that triggers flashbacks due to previous methamphetamine (MAP) psychosis was studied in 28 flashbackers, along with 84 non-flashbackers with a history of MAP psychosis. We measured plasma monoamine metabolite levels in 12 of the flashbackers and eight of the non-flashbackers, along with 28 normal controls. Most flashbackers had undergone frightening experiences during previous MAP use. The dominant triggering factor was a mild fear of other people. Plasma norepinephrine (NE) levels in the 12 flashbackers were increased during flashbacks. Thus, MAP use associated with threatening experiences may have increased sensitivity to stressors, leading to the occurrence of flashbacks, including exaggerated noradrenergic activity.

The Duffy gene has been shown not to be split by introns, even in its 5' untranslated region, and to be expressed not only in erythroid but in postcapillary venule endothelium of almost every organ in the body. To further investigate the transcriptional start position in erythroid and postcapillary venule endothelium, we performed 5'-rapid amplification of cDNA ends (5'-RACE). While every positive clone of 5'-RACE encoded the identical sequence of previously identified cDNA downstream from nucleotide 203, the upstream sequences were different. The upstream sequences corresponded to the sequence from nucleotide -279 to -308/-357 in erythroblasts and from -279 to -355/-383 in lung and were regarded as comprising a novel exon. This novel exon encoded seven residues initiated with a methionine, linked to nucleotide 203 in-frame and in agreement with the GT-AG splicing rule. The major erythroid transcriptional start position was identified in human erythroleukemia cells by primer extension and in bone marrow by ribonuclease protection analysis at 34 bases upstream from the first ATG codon. Distinctively, in lung and kidney, the transcription was started at 82 bases upstream from the ATG. Both Northern blotting and reverse transcription-polymerase chain reaction followed by Southern analysis indicated a predominance of the novel spliced form of mRNA of about 50- to 200-fold comparing with the unspliced form, in every studied organ and erythroid lineage cells. The spliced form of cDNA has been transfected into a human erythroleukemic cell line, K562, and the expressed protein reacted with Duffy-specific murine monoclonal antibody Fy6. These studies indicate that the product from the spliced form of mRNA is the major product of the Duffy gene in the erythroid lineage and postcapillary venule endothelium.

The Duffy blood group antigen has been characterized by its roles on red blood cells: as a receptor for the malarial parasites and as a promiscuous receptor for chemokine superfamily. Recently, the Duffy blood group associated glycoprotein D (gpFy) cDNA has been cloned (Chaudhuri et al: Proc Natl Acad Sci USA 90:10793, 1993). In this report we describe the organization of genomic DNA coding for the gpFy and elucidate the molecular nature of Fya/b polymorphisms. By a Southern blotting analysis probed with gpFy cDNA, gpFy gene was shown to be composed of three DNA fragments; 1.1-kb Sac I, 1.9-kb EcoRI, and their intervening 47-bp fragments. We cloned the 1.1-kb Sac I and 1.9-kb EcoRI fragments by inverted polymerase chain reaction (IPCR) procedure. The promoter region of the gpFy gene was cloned by IPCR of 1.1-kb Sac I fragment and the 3' flanking sequence was cloned by IPCR of 1.9 kb EcoRI fragment. The both IPCR products contained on both side the known gpFy cDNA sequence without introns, as expected. Although no TATA or CCAAT boxes are present in the promoter sequence, several transcription factor binding site motifs are contained, including AP-1, HNF-5, TCF-1, ApoE B2, W-element, H-APF-1, and Sp-1. The 3' flanking region has two additional polyadenylation signals, other than that used in the cDNA, and also has an indirect and a direct repeat sequence clustered with the 5' flanking region. These facts indicate a possibility that the gpFy gene has been evolved by multiple retrotransposition events. By comparing the coding area of the gpFy gene in 28 Duffy-positive individuals, we elucidated that one base change that results in an amino acid substitution [GA-T(Asp44)-->GGT(Gly)] is in accordance with the Fya/Fyb polymorphism. This fact proves that the gpFy cDNA and its gene described in this report encode the Duffy blood group system.

Melatonin (0.1-1.0 mg/kg) reduced isolation-induced distress vocalizations (DVs) in young domestic chickens in a dose-dependent manner. This effect was unaffected by the administration of d-amphetamine (1.0 mg/kg) suggesting that melatonin's effects were not merely due to fatigue. The melatonin reduction in DVs was not naloxone reversible, indicating an action independent of the endogenous opioid system. However, chronic pretreatment with naltrexone facilitated the melatonin effect, suggesting a complex relationship between melatonin and the endogenous opioids in regulating distress vocalizations. Chickens exhibited a marked reduction in DVs when isolation chambers were darkened, suggesting endogenous, as well as exogenous, melatonin mediation of isolation distress; however; pinealectomy only partially reversed the darkness effect. Pinealectomized animals, like control animals, exhibited a reduction in DVs following melatonin treatment; however, the melatonin effect was shorter lasting. The implications that these results may have for socialization and emotional distress are discussed.

The Rh blood group system was recognized as one of the most complex polymorphisms in human. Whether or not the Rh antigens are present on human leukocytes is still an unresolved question. The expression of the Rh gene at the mRNA was analyzed in purified populations of human leukocytes by using reverse transcription and polymerase chain reaction (RT-PCR) followed by Southern blot analysis. The PCR products of the 5'-terminal region showed a single band as expected in the CD19+TCR-1 and CD14+ cells but doubtfully in the CD13+CD71- and CD190-TCR-1+ cells. On the other hand, in all these cells, the PCR products of the 3' region exhibited multiple additional bands migrating ahead of the band as expected, which showed distinctly different sets of bands in each cell. The additional bands appeared to consist of RhPI- and RhPII-cDNA splicing isoforms. These results indicated that the expression of the Rh gene is not restricted to human erythroid lineage. Additionally, it was suggested that different transcription initiation sites might be utilized preferentially for Rh gene expression.

The alternatively spliced from mRNA of platelet glycoprotein IIb (GPIIb) with a deletion of exon 28 (GPIIb-28) has been isolated from the HEL cell cDNA library. The defective expression on the surface of DNA cotransfected COS-1 cells with GPIIb-28 and GPIIIa cDNAs was described in an earlier report. We studied siblings with Glanzmann's thrombasthenia who expressed only the GPIIb-28 mRNA in their platelets. Flow cytometry showed that the patients' platelets failed to bind GPIIb/IIIa complex-specific and GPIIb-specific monoclonal antibody. Western blotting showed that the patients' platelets had defective GPIIb and have trace amounts of GPIIIa. Sequence analysis was performed after polymerase chain amplification of the patients' GPIIb and GPIIIa mRNAs. The patients' GPIIb cDNA had a deletion of the exon 28 nucleotides. The polymerase chain reaction (PCR) from exon 27 to 29 showed that the GPIIb-28 mRNA was 3% +/- 1.6% of the normally, spliced form in control platelets, and 61% in the megakaryoblastic cell line UT-7. The patients' platelets showed only the GPIIb-28. Family study and quantitative PCR studies showed that these patients were compound heterozygotes of two GPIIb gene defects. The father's allele is described in this report and involves skipping exon 28 secondary to a base substitution at codon Gln948, CAG-->TAG. The mother's allele appears to involve decreasing GPIIb mRNA levels in platelets. Our results indicate that the GPIIb-28 is not expressed on platelet membranes as a stable GPIIb/IIIa heterodimer or left as a monomer in platelets. Our studies confirm the previous data observed in COS-1 cells expressing recombinant GPIIb-28.

Two isoforms of RhPI cDNA encoding an Rh polypeptide, RhPI-alpha and RhPI-beta, were isolated from mRNAs of immature erythroblasts cultured in a selective two-phase liquid culture system for human erythroid progenitors in peripheral blood. The RhPI-alpha and RhPI-beta cDNA clones encoded the hydrophobic membrane polypeptides of 242 and 244 amino acids which were missing in amino acid residues 227 to 383 and 212 to 384 of RhPI polypeptide, respectively. These peptide deletions produce unexpected structural properties resulting in the generation of a new open reading frame with a new stop codon caused by frameshift translations in RhPI-alpha cDNA and in the reverse orientation of topology in RhPI-beta cDNA. Both isoforms are abundantly present in immature erythroblasts, but not in mature erythroblasts. These results indicate that the two Rh-related mRNAs identified are expressed at the immature erythroblast stage on the development of erythroid series.

The genotoxic and mutagenic activation of aflatoxin B1 (AFB1) by hepatic, renal, and pulmonary microsomes and purified cytochrome P450s was investigated in Salmonella typhimurium TA1535/pSK1002 cells in which an umu response shows DNA damage. The activity of the hepatic microsomes was greatest. Pulmonary microsomes had moderate activity and renal microsomes had low activity. P450 2C11, 2B1, 3A2, 4A2, 4B1, K-2, and K-4 were assayed in a reconstituted system with dilauroylphosphatidylcholine (DLPC). P450 2C11 (a major hepatic cytochrome P450 in male rats) had high activity. P450 2B1 (a major form as well as P450 4B1 in pulmonary microsomes) and K-2 (a minor form in renal microsomes) had moderate activity. P450 4A2 (a major form in renal microsomes), P450 K-4 (a renal form), and P450 4B1 had low activity. P450 3A2 did not have high activity in these conditions but it had high activity toward AFB1 in a modified reconstituted system with a lipid mixture and sodium cholate instead of DLPC only. The activities of other forms were not enhanced by the modification of reconstituted system. Anti-P450 2C11 or 3A2 antibodies inhibited the bioactivation of AFB1 by hepatic microsomes to 50%. These results suggest that the greater ability of hepatic microsomes as compared with pulmonary and renal microsomes to metabolize AFB1 to mutagenic products is a function of the relative proportions of the highly active cytochrome P450s, P450 2C11 and 3A2, in the liver.

A method was devised for detecting both the molecular mass and the isoelectric point (pI) of the lectin in the seed extract of Momordica charantia on a nitrocellulose membrane. It was associated with the electrophoretic blotting technique that produced replicas of proteins separated on micro two-dimensional polyacrylamide gels. The red blood cell adherence procedure on the blotted membrane exhibited only one red spot with molecular mass 107.10(3) and pI 5.3, which indicated the lectin activity. Additionally, the lectin appeared to be a glycoprotein with mannose and/or glucose, because it was stained by concanavalin A-peroxidase staining.

We investigated the protein pattern of a surgically resected tumour in a case where it was difficult to distinguish between duodenal and pancreatic cancer. The investigation was performed using two-dimensional polyacrylamide gel electrophoresis with silver staining. Samples of the duodenal tumour, normal duodenal mucosa and normal pancreatic tissue from the same patient were compared. Each gel had ca. 250-300 protein spots, and the tumour sample pattern more closely resembled that of normal duodenal mucosa than that of pancreatic tissue, suggesting that the tumour had arisen from duodenal mucosa. There were three proteins identified only in the tumour sample gel, and these may have been tumour-specific proteins.

Patterns of proteins of five surgically resected esophageal carcinomas were studied by two-dimensional polyacrylamide gel electrophoresis with silver staining. The samples of normal esophageal mucosa and esophageal carcinoma from the same patient were compared. Each gel had ca. 300 protein spots and had a similar pattern of proteins. Four spots were observed in all of the esophageal carcinomas that were not present in any of the normal mucosae. The molecular weights and isoelectric points were 46,000 and 5.3, 46,000 and 5.2, 36,000 and 4.7 and 33,000 and 5.1, respectively. One spot was observed in all of the normal mucosae but not in any of the esophageal carcinomas. Its molecular weight and isoelectric point were 27,000 and 5.3, respectively.

In order to examine the sequential expression of major blood group antigens on human erythroblasts, a selective two phase liquid culture system for erythroid progenitors was established. After mononuclear cells obtained from peripheral blood were cultured in the presence of phytohemagglutinin stimulated-leukocyte conditioned medium (PHA-LCM) for 7 days (the first phase), nonphagocytic cells were recultured under hypoxic culture conditions containing 30% fetal calf serum, 1% bovine serum albumin, 300 micrograms/mL transferrin and 2 U/mL recombinant erythropoietin (the second phase). Mature (orthochromatic) erythroblasts were observed on day 4 of the second phase, and reached 57.1 +/- 3.1% of total cells on day 8, followed by the appearance of denucleated red cells, equivalent to mature red cells in peripheral blood. Hemoglobin contents reached the level of 16.8 +/- 0.7 micrograms/10(6) cells on day 8. Flow cytometric analyses revealed that, on day 3 of the second phase, cells became blood type M-positive, corresponding to the maturation of erythroid cells. Regarding the expression of ABH blood group antigens, a small number of blood type H-positive cells were initially detected on day 0 of the second phase, while blood type A-positive cells, which essentially were not observed on day 0, increased gradually corresponding to the extent of erythroid maturation. In the present system, Lewis and P1 blood group antigens were expressed at day 5 of the second phase, although autologous plasma was required to determine the expression of Lewis blood group antigens. This culture system is beneficial for studies on normal and abnormal human red cell membranes, because the erythroid progenitors in human peripheral blood were used, and a reasonable number of erythroid cells (0.5 to 1.5 x 10(7] was obtained with good maturation.

Proteins of the mouse saliva are resolved into about 20 discrete bands by polyacrylamide gel electrophoresis. Sexual dimorphism and monomorphism were found in a subset (Msp-1) of these salivary proteins from different inbred strains. This sexual dimorphism involves a fast moving band (F-type) and a slow moving one (S-type). Mature males of seven strains (A/J, AKR, CBA/J, C3H/HeN, A/Sn, B10.A, and B10.BR) exhibit the S phenotype while mature females of these strains were typed as F. Sexually immature males and females of these strains were uniformly typed as F, but at puberty (5-6 weeks of age) the phenotype of the males switched to type S, while the phenotype of the females remained the same. This switch to type S at puberty did not take place in males of four strains (BALB/cAnn, B10.D2, C57BL/6, and C57BL/10); therefore, we conclude that these strains were sexually monomorphic with regard to Msp-1. The phenotype of mature males of C3H/HeN reverted to type F following castration, whereas castrated males and mature females switched to type S in response to testosterone administration. The testosterone treatment had no effect on the type S phenotype of males and females of the sexually monomorphic BALB/cAnn strain. The male-specific type S phenotype of Msp-1 was seen only in mice with H-2 haplotype a or k; thus an association with H-2 haplotype was suggested. All F1 males of reciprocal crosses involving the sexually dimorphic and monomorphic strains (e.g., C3H/HeN X BALB/cAnn) demonstrated the type S phenotype at puberty.

The supernatants of dispase-treated platelets from normal persons and patients with myeloproliferative disorders were analysed by capillary isotachophoresis. The examined myeloproliferative disorders were three cases of chronic myelogeneous leukemia, one case of chronic neutrophilic leukemia, one case of essential thrombocythemia and one case of polycythemia vera. An additional peak was revealed in the samples from the patients with chronic myelogeneous leukemia. Platelets from the other three patients showed no additional bands. Isotachophoresis will be useful not only to analyse the superficial peptide on the cell membrane but also to define the peptide associated with malignant transformation.

Antigen analysis of the red cell membrane in a patient with angio-immunoblastic lymphadenopathy with dysproteinemia (AILD) with red cell autoantibody revealed that four blood group antigens had been acquired. These four antigens consisted of S of MNSs blood group, Lua of Lutheran blood group, and K and Kpa of Kell-Cellano blood group. These antigens disappeared and the Coombs' test became negative after complete remission induced by combination chemotherapy. Free amino acid analysis after Dispase treatment of red cell membrane suggested that the antigenic modifications were associated with abnormal composition of amino acids.