A novel biosensor with high signal-to-noise ratio for real-time measurement of dopamine levels in vivo.

Fast-scan cyclic voltammetry (FSCV) is an established method for measuring dopamine (DA) levels in the brain in real time. However, it is difficult to discriminate DA from other monoamines such as serotonin (5-hydroxytryptamine, 5-HT) and norepinephrine (NE). We report a novel DA-specific biosensor consisting of a carbon-fiber electrode coated with an ion-exchange membrane, a layer containing monoamine oxidase B, and a cellulose membrane. We performed FSCV using the probe to monitor the amount of DA in vitro and in vivo. First, we measured currents in vitro in phosphate-buffered saline as we added one micromole each of DA, 5-HT, and NE. The results confirmed that the biosensor selectively detected DA. Next, we implanted the probe in the striatum of male rats to investigate whether it could selectively detect changes in the DA content in vivo. The probe detected both the tonic change induced by methamphetamine administration and the phasic change induced by electrical stimulation of the medial forebrain bundle. In contrast, the electrode in the 6-hydroxydopamine-lesioned striatum did not respond to systemic selective serotonin or serotonin/norepinephrine reuptake inhibitors, confirming its selectivity. Furthermore, the probe in the striatum could still detect changes in the DA level 1 week after electrode implantation. The results suggest that the novel biosensor can measure real-time changes in DA levels in vivo with a relatively high signal-to-noise ratio.

Na(+)-K(+)-2Cl(-) cotransporter-mediated fluid secretion increases under hypotonic osmolarity in the mouse submandibular salivary gland.

Water-handling epithelia are sensitive to the osmotic environment. In this study, the effects of a hypo-osmotic challenge on carbachol (CCh)-induced fluid secretion was investigated using an ex vivo submandibular gland perfusion technique and intracellular pH and Ca(2+) measurements. The osmolality of the perfusion solution was altered to examine the response of the gland to a hypotonic challenge. The flow rate was increased by 34% with a 30% hypotonic solution (225 mosmol/kgH2O), although the Ca(2+) response was unchanged. The lowering of the external Cl(-) by 50% abolished this increase in the 30% hypotonic solution. Furthermore, bumetanide, an inhibitor of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), completely inhibited the fluid secretion increase caused by the 30% hypotonic solution, and both the total amount of fluid and the flow rate were identical to those of the isotonic solution. This finding was confirmed by measuring the NKCC1 bumetanide-dependent NH4 (+) transport; Na(+)-K(+)-2Cl(-) transport was upregulated >40% by a 30% hypotonic challenge. Therefore, the increase in CCh-induced fluid secretion in response to hypotonic conditions can be attributed, to a large extent, to the specific activation of the NKCC1.

Endothelial nitric oxide synthase plays a main role in producing nitric oxide in the superacute phase of hepatic ischemia prior to the upregulation of inducible nitric oxide synthase.

BACKGROUND: The aim of this study was to determine the intrahepatic kinetics of different types of nitric oxide (NO) synthase, such as endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS), during repeated ischemia/reperfusion (I/R). METHODS: Three different protocols of hepatic I/R in rats were designed as follows: 60 min of ischemia and 30 min of reperfusion (I/R 60/30); 5 min of ischemia and 5 min of reperfusion (I/R 5/5); and repeating 15 min of ischemia and 5 min of reperfusion for four cycles (I/R 15/5 × 4). Intrahepatic NO levels were measured using a selective NO sensor. Changes in hepatic microcirculation during I/R 5/5 were investigated using intravital microscopy. Hepatic expression of eNOS, phospho-eNOS, and iNOS were evaluated during repeated I/R by Western blot, reverse transcription polymerase chain reaction, and immunohistochemistry. RESULTS: During I/R 60/30, intrahepatic NO levels gradually increased and then reached a plateau approximately 15 min after starting ischemia. During I/R 5/5, the sinusoids after 5 min reperfusion were dilated compared with the sinusoids before ischemia. The expression of phospho-eNOS during I/R 15/5 × 4 markedly increased during the first ischemia, and then the levels attenuated during the subsequent repeating I/R cycles; however, the expression of iNOS gradually increased, as observed by Western blot, reverse transcription polymerase chain reaction, and immunohistochemical analysis. An impact of NO production by phospho-eNOS activation during the superacute phase of I/R was also confirmed using pharmacologic NOS inhibitors. CONCLUSION: Our results firstly demonstrated an altered activation of the phospho-eNOS system and iNOS over the course of repeated hepatic I/R.

Levodopa-Carbidopa Intestinal Gel Injection for Patient with Severe Parkinson's Disease Followed by Total Hip Arthroplasty: A Case Report and Literature Review.

BACKGROUND: Patients with Parkinson's disease have a high dislocation rate after total hip arthroplasty (THA). This study describes a case with severe Parkinson's disease who developed rapidly destructive coxarthrosis (RDC) and underwent THA using a dual mobility cup after a levodopa-carbidopa intestinal gel (LCIG) infusion. CASE PRESENTATION: The patient is a 59-year-old female with a ten-year history of Parkinson's disease, which was first treated with oral levodopa. The patient developed RDC of the right hip joint. However, THA was difficult owing to Parkinson's disease and its treatment side effects, such as wearing-off, dyskinesia, and freezing of the gait, Thus, LCIG was initiated, and improvement in wearing-off and dyskinesia was observed. Two months after the LCIG therapy, the disease was controlled well. THA was subsequently performed using a dual mobility cup to prevent postoperative dislocation. Postoperatively, LCIG therapy was continuously administered to carefully manage the disease, which was controlled well with no increase in wearing-off and dyskinesia after surgery. At 1 year after surgery, the walking speed, stride length, and the Harris hip score improved compared to preoperatively. The UPDRS III motor score improved to eight without signs of wearing-off or dyskinesia. The Hoehn-Yahr scale was II in the "on" period and remained unchanged 1 year after surgery. The patient could walk without a cane and had satisfactory functional outcomes. CONCLUSION: This case proved that LCIG treatment performed preoperatively, followed by THA using a dual mobility cup, and strict management of Parkinson's disease could result in a satisfactory clinical course without recurrence of wearing-off and dyskinesia. Similar procedures may benefit other patients with Parkinson's disease who have previously been deemed unsuitable for THA.

Novel deletion at Xq24 including the UBE2A gene in a patient with X-linked mental retardation.

By using an in-house bacterial artificial chromosome-based X-tilling array, we detected a 0.4 Mb novel deletion at Xq24 that included UBE2A in a 4-year-old and 10-month-old boy with mental retardation and various other characteristics inherited from his mother; for example, marked developmental delay, synophrys, ocular hypertelorism, esotropia, low nasal bridge, marked generalized hirsutism and seizure. Although additional nine transcripts around UBE2A were also defective, a phenotypic similarity with a recently reported X-linked familial case involving a novel X-linked mental retardation syndrome and a nonsense mutation of UBE2A indicates a functional defect of UBE2A to be responsible for most of the abnormalities in these cases. Because some characteristics, such as congenital heart disease and proximal placement of the thumb, were not described in the family reported previously, suggesting genes other than UBE2A within the deleted region to be responsible for those abnormalities.

A novel homozygous mutation of CLCN2 in a patient with characteristic brain MRI images - A first case of CLCN2-related leukoencephalopathy in Japan.

Chloride channel 2 (ClC-2) is one of nine ClC family proteins and is encoded by CLCN2. We report the first patient with a CLCN2 mutation in Japan. A 22-month-old female had generalized tonic-clonic convulsions at the age of 3 months. Brain MRI showed high signals in the bilateral cerebellar white matter including the dentate nucleus, dorsal midbrain, and posterior limbs of the internal capsules in diffusion-weighted images, and apparent diffusion coefficient values were low in the same areas. Antiepileptic drugs were effective, and she had neither intellectual disabilities nor motor disturbance. A homozygous frameshift mutation (c.61dup, p.Leu21Profs∗27) of CLCN2 was identified in the patient. Homozygous mutations of CLCN2 are known to be associated with CLCN2-related leukoencephalopathy (CC2L). The clinical findings of this patient were different from other patients with CC2L. Therefore, mutations in CLCN2 may cause various phenotypes. Further accumulation of cases with CLCN2-mutations is required to explore the clinical spectrum of CC2L.

The first report of Japanese patients with asparagine synthetase deficiency.

BACKGROUND: Asparagine synthetase (ASNS) deficiency was recently discovered as a metabolic disorder of non-essential amino acids, and presents as severe progressive microcephaly, intellectual disorder, dyskinetic quadriplegia, and intractable seizures. METHODS: Two Japanese children with progressive microcephaly born to unrelated patients were analyzed by whole exome sequencing and novel ASNS mutations were identified. The effects of the ASNS mutations were analyzed by structural evaluation and in silico predictions. RESULTS: We describe the first known Japanese patients with ASNS deficiency. Their clinical manifestations were very similar to reported cases of ASNS deficiency. Progressive microcephaly was noted during the prenatal period in patient 1 but only after birth in patient 2. Both patients had novel ASNS mutations: patient 1 had p.L145S transmitted from his mother and p.L247W which was absent from his mother, while patient 2 carried p.V489D and p.W541Cfs*5, which were transmitted from his mother and father, respectively. Three of the four mutations were predicted to affect protein folding, and in silico analyses suggested that they would be pathogenic. CONCLUSION: We report the first two Japanese patients with ASNS deficiency. Disease severity appears to vary among patients, as is the case for other non-essential amino acid metabolic disorders.

MR imaging and 1H-MR spectroscopy of a case of van der Knaap disease.

Van der Knaap disease, characterized by megalencephalic leukoencephalopathy and subcortical cysts, is a rare and recently defined condition. We discuss here the MR image (MRI) and MR spectroscopy (MRS) features in a 30-year-old man with S93L homozygous mutation in the MLC1 gene. MRI demonstrated high intensity diffuse white matter with T2-weighted image and subcortical cysts in the parietal and temporal lobes and MRS showed mildly reduced N-acetylaspartate (NAA) in areas of severe T2 elongation with a long TE sequence. A peak of lactate/lipid was indicated at a chemical shift of 1.3 ppm with a short TE sequence. The peak for myo-inositol was normal in areas of severe and mild T2 elongation with short TE MRS. These findings suggest that demyelination progresses slowly in van der Knaap disease and that MRS with long and short TE is useful for the evaluation of neural metabolization associated with van der Knaap disease.

Agenesis of the gallbladder with hypoplastic cystic duct diagnosed at laparoscopy.

An 86-year-old man was admitted to our department with complaints of intermittent upper abdominal pain. Ultrasonography of the abdomen showed dilated extrahepatic bile ducts containing stones; however, the gallbladder was not clearly identified. Magnetic resonance cholangiopancreatography showed dilated extrahepatic ducts and choledocholithiasis without gallbladder visualization. The stone extraction was performed with endoscopic sphincterotomy. Three-dimensional images using spiral-computed tomography after intravenous-infusion cholangiography clearly demonstrated an obstruction of the cystic duct. The patient was scheduled for laparoscopic cholecystectomy. At laparoscopy, the gallbladder fossa was not identified on the undersurface of the liver. Despite a thorough examination of the intrahepatic (left-sided within the lesser omentum), retroperitoneal, retrohepatic (within the falciform ligament), retroduodenal, and retropancreatic areas using laparoscopic ultrasonography, the gallbladder was not found. After careful dissection of the hepatoduodenal ligament, the dilated extrahepatic bile duct and a 1-cm length of hypoplastic cystic duct were found. Gallbladder agenesis is usually accompanied by the lack of the cystic duct. The present case is the third report of gallbladder agenesis with a patent or hypoplastic cystic duct.

Serial MR imaging and 1H-MR spectroscopy of unidentified bright objects in a case of neurofibromatosis type 1.

Serial study using MR imaging and 1H-MR spectroscopy (1H-MRS) of unidentified bright objects (UBO) in a 9-year-old boy with neurofibromatosis type 1 (NF1) is described. UBO was indicated in the left globus pallidum at 3 years old, then appeared in the right globus pallidum at 5 years old along with left hemiconvulsion. These UBO gradually disappeared over several years. On 1H-MRS, the ratios of N-acetylaspartate (NAA)/creatine and that of choline/creatine were each mildly reduced in the UBO. These ratios improved in parallel with disappearance of the UBO. In addition, elevation of the lactate/lipid peak was shown in UBO during the early stage, however, it disappeared in the latter. The longitudinal follow-up using MR image and 1H-MRS was useful for metabolic evaluation of UBO in patients with NF1.

Postobstructive pulmonary edema that developed immediately after the removal of an endobronchial foreign body.

The patient was a 5-year-old boy who was transported to our hospital for a paroxysmal cough, disturbance of consciousness, tonic-clonic convulsions and labored breathing. The patient's respiratory failure persisted after the convulsions remitted, and the presence of an endobronchial foreign body was suspected based on the findings of chest CT performed the following day. A peanut was subsequently removed from the right main bronchus using a bronchoscope with tracheal intubation and bag valve mask ventilation. Immediately after removal, the patient rapidly developed exacerbated hypoxemia, and a reduction in right lung lucency was noted on chest radiography. He was therefore diagnosed with type II postobstructive pulmonary edema, and his condition improved within a short period of time.

Effect of levetiracetam in acute encephalitis with refractory, repetitive partial seizures during acute and chronic phase.

AIM: To clarify the effect of levetiracetam (LEV) for acute and chronic seizure control in acute encephalitis with refractory, repetitive partial seizures (AERRPS). METHODS: We retrospectively reviewed the clinical course of six AERRPS cases treated with LEV, and explored the acute phase termination by withdrawal from barbiturate-induced coma under artificial ventilation, and the reduction in seizure frequency during the chronic phase. LEV was administrated orally or via nasogastric tubes as an add-on agent during acute (n=3; age 8-10 years) and chronic (n=3; age 19-30 years) AERRPS. RESULTS: In the acute phase, administration of LEV (50-60 mg/kg/d) in combination with phenobarbital (n=3; peak 57.9-76.1 µg/ml) and potassium bromide (n=2; 30-36 mg/kg/d)) resulted in successful reduction of intravenous barbiturate dosage and withdrawal from artificial ventilation. In the chronic phase, seizure frequency reduced by >75% for 5-18 months with LEV 750-1500 mg/d. CONCLUSION: LEV may affect seizure control in AERRPS, particularly during the chronic phase, through its unique action of inhibition of excitatory neurotransmitter release. The regimen of oral barbiturate, potassium bromide and LEV would be worth for trial during the acute phase of AERRPS.

A case of a teenage boy with eosinophilic gastroenteritis with esophageal involvement developing a hemorrhagic duodenal ulcer.

A boy in his early teens visited our hospital with chief complaints of hematemesis and tarry stools. Upper gastrointestinal endoscopy identified a hemorrhagic duodenal ulcer, for which hemostasis was performed using a clip. Proton pump inhibitor (PPI) administration diminished the ulcer but relapse occurred after PPI discontinuation. The esophagus showed concentric rings and longitudinal linear furrows considered to be characteristic of eosinophilic esophagitis. Biopsies of the duodenal ulcer and the esophagus revealed marked infiltration of eosinophils, leading to a diagnosis of eosinophilic gastroenteritis with esophageal involvement. Steroid treatment was initiated, and the duodenal ulcer and esophagitis resolved. Endoscopic findings characteristic of eosinophilic esophagitis were key to the diagnosis of eosinophilic gastroenteritis.

Infantile Cases of Sitosterolaemia with Novel Mutations in the ABCG5 Gene: Extreme Hypercholesterolaemia is Exacerbated by Breastfeeding.

Few data exists regarding the clinical impact of breastfeeding in infantile sitosterolaemic cases. We report four Japanese infantile cases of sitosterolaemia, an extremely rare inherited disease characterised by increased serum levels of plant sitosterol, presenting with severe hypercholesterolaemia and systemic xanthomas exacerbated by breastfeeding. In these four cases, genetic analyses were performed for low-density lipoprotein (LDL) receptor, proprotein convertase subtilisin/kexin type 9 (PCSK9), LDL receptor adaptor protein 1 and ATP-binding cassette (ABC) subfamily G member 5 and 8 (ABCG5 and ABCG8) genes. We assessed their clinical manifestations, including responsiveness to a variety of treatments, especially to weaning from breastfeeding and use of ezetimibe. Two pairs of mutations in the ABCG5 gene in each case, including two novel mutations (c.130C>T or p.Ser44Ala and c.1813_1817delCTTTT or p.Pro558GlufsX14) and two known mutations (c.1306G>A or p.Arg389His and c.1336C>T or p.Arg446X), were identified. Significant reductions in cholesterol levels were obtained by means of weaning from breastfeeding alone. Substantial reductions in sitosterol levels, without any apparent side effects, were observed with ezetimibe. In conclusion, we have identified infantile Japanese sitosterolaemic subjects with extreme hypercholesterolaemia exacerbated by breastfeeding. Their unique response to weaning from breastfeeding, as well as to use of ezetimibe, could provide insights into the metabolic basis of sterols in humans.

Laparoscopic treatment using an argon beam coagulator for nonparasitic liver cysts.

BACKGROUND: Laparoscopic deroofing has been shown to produce good patient satisfaction and to have results similar to those of open surgical techniques. We evaluated the feasibility and efficacy of laparoscopic deroofing using an argon beam coagulator (ABC) in the patients with nonparasitic liver cysts. METHODS: Laparoscopic deroofing for the treatment of liver cysts was attempted on 14 patients. After the deroofing, the secreting epithelium within the residual cystic cavity wall was destroyed using the ABC. RESULTS: Laparoscopic deroofing was successful in all patients. No deaths or surgical morbidity occurred, and no postoperative complications were recorded. The median postoperative hospital stay was 7 days. The median follow-up was 56 months for all patients, and all patients have remained completely asymptomatic for 6 months after the surgery, with no recurrence of the cysts. CONCLUSIONS: Our results indicate that laparoscopic deroofing using the ABC method in patients with nonparasitic liver cysts was effective in preventing cyst recurrence.

MR imaging and 1H-MR spectroscopy in a case of cerebral infarction with transient cerebral arteriopathy.

A case of cerebral infarction with transient cerebral arteriopathy in a 12-year-old female is described. The child showed with an acute onset of left hemiplegia, central facial palsy and aphasia. She was diagnosed with right internal carotid artery obstruction with no predisposing factors for cerebral infarction. A serial MR image and MR spectroscopy of the brain was performed. MR angiography revealed reversible vascular changes in the right middle cerebral artery after several months. The ratio of choline/creatine was elevated and that of N-acetylaspartate (NAA)/creatine was depressed in the cerebral lesion, however, they were normalized during the remission phase with long echo time-MR spectroscopy. The elevation of the lactate peak was clarified with short echo time-MR spectroscopy. The longitudinal follow up using MR angiography, and MR spectroscopy with long and short echo time was useful for the evaluation of reversible vascular change with cerebral infarction in children.

MR imaging and 1H-MR spectroscopy in a case of juvenile Alexander disease.

The serial MR image and MR spectroscopy in the brain were examined in a young male diagnosed as having juvenile Alexander disease. He had megalencephaly, psychomotor retardation, seizures, and increasing elevation of increasing alpha-B crystallin and heat shock protein 27 in the cerebrospinal fluid. Serial MR images demonstrated increased demyelination of the bilateral frontal region to left occipital region over several years. The myo-inositol/creatine ratio was significantly increased in both the demyelinated white matter and normal area in the MR spectroscopy. These results suggested that demyelination very slowly progressed from the frontal to occipital region and that glial degeneration may occur even in the unaffected white matter of patients with juvenile Alexander disease.

Molecular genetic study in Japanese patients with Alexander disease: a novel mutation, R79L.

Since the first report by Brenner et al. of mutations in the glial fibrillary acidic protein (GFAP) gene in patients with Alexander disease, several molecular genetic studies have been performed in different ethnic groups. We previously reported a Japanese patient with a mutation, R239C, which is identical to one commonly found in American patients. Here we have analyzed four additional Japanese patients by screening for known mutations or, if no known mutation was found, by sequencing of all exons of the GFAP gene. We detected three missense mutations; one was a novel mutation, R79L, and two were previously reported mutations, R239C and R79C. All of our patients were heterozygous for their mutations. Together with the novel mutation, R79L, four different nucleotide changes altering the R79 residue have been reported, implying that any alternation of this arginine residue can give the GFAP protein a dominant negative effect, leading to accumulation of GFAP as Rosenthal fibers. We conclude that molecular genetic analysis of the GFAP gene is feasible for antemortem diagnosis of Alexander disease in Japanese patients.

Postoperative atypical hemolytic uremic syndrome associated with complement c3 mutation.

Atypical hemolytic uremic syndrome (aHUS) can be distinguished from typical or Shiga-like toxin-induced HUS. The clinical outcome is unfavorable; up to 50% of affected patients progress to end-stage renal failure and 25% die during the acute phase. Multiple conditions have been associated with aHUS, including infections, drugs, autoimmune conditions, transplantation, pregnancy, and metabolic conditions. aHUS in the nontransplant postsurgical period, however, is rare. An 8-month-old boy underwent surgical repair of tetralogy of Fallot. Neurological disturbances, acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia developed 25 days later, and aHUS was diagnosed. Further evaluation revealed that his complement factor H (CFH) level was normal and that anti-FH antibodies were not detected in his plasma. Sequencing of his CFH, complement factor I, membrane cofactor protein, complement factor B, and thrombomodulin genes was normal. His ADAMTS-13 (a disintegrin-like and metalloprotease with thrombospondin-1 repeats 13) activity was also normal. However, he had a potentially causative mutation (R425C) in complement component C3. Restriction fragment length polymorphism analysis revealed that his father and aunt also had this mutation; however, they had no symptoms of aHUS. We herein report a case of aHUS that developed after cardiovascular surgery and was caused by a complement C3 mutation.