Molecular genetic analysis of 30 families with Joubert syndrome.

Joubert syndrome (JS) is rare recessive disorders characterized by the combination of hypoplasia/aplasia of the cerebellar vermis, thickened and elongated superior cerebellar peduncles, and a deep interpeduncular fossa which is defined by neuroimaging and is termed the 'molar tooth sign'. JS is genetically highly heterogeneous, with at least 29 disease genes being involved. To further understand the genetic causes of JS, we performed whole-exome sequencing in 24 newly recruited JS families. Together with six previously reported families, we identified causative mutations in 25 out of 30 (24 + 6) families (83.3%). We identified eight mutated genes in 27 (21 + 6) Japanese families, TMEM67 (7/27, 25.9%) and CEP290 (6/27, 22.2%) were the most commonly mutated. Interestingly, 9 of 12 CEP290 disease alleles were c.6012-12T>A (75.0%), an allele that has not been reported in non-Japanese populations. Therefore c.6012-12T>A is a common allele in the Japanese population. Importantly, one Japanese and one Omani families carried compound biallelic mutations in two distinct genes (TMEM67/RPGRIP1L and TMEM138/BBS1, respectively). BBS1 is the causative gene in Bardet-Biedl syndrome. These concomitant mutations led to severe and/or complex clinical features in the patients, suggesting combined effects of different mutant genes.

Protective effects of estradiol against amyloid beta protein-induced inhibition of neuronal Cl(-)-ATPase activity.

Low concentrations of amyloid beta proteins (Abetas, 1-10 nM) were recently demonstrated to reduce Cl(-)-ATPase activity in parallel with an increase in the intracellular Cl(-) concentration ([Cl(-)]i) and decreases in plasma membrane phosphorylated phosphatidylinositol (PIP and PIP2) levels in cultured rat hippocampal neurons. In this study, 17 beta-estradiol (estradiol) at a therapeutic concentration (1.8 nM) for Alzheimer's disease was found to block these Abeta (Abeta25-35)-induced changes. This protective effect of estradiol on Cl(-)-ATPase activity was antagonized by a pure estrogen receptor antagonist, ICI182780 and inhibitors for cyclic GMP-dependent protein kinase (PKG) (KT5823), Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) (KN62) and phosphatidylinositol (PI) 4-kinase (wortmannin and quercetin). Estradiol recovered Abeta-induced decreases in plasma membrane phosphoinositide (PIP and PIP2) levels, this effect being inhibited by KT5823 and KN62. Glutamate toxicity was augmented in neurons with elevated [Cl(-)]i either by Abeta-treatment or carbachol+KCl+LiCl-treatment. The increased glutamate toxicity in the Abeta-treated neurons was attenuated by estradiol. Thus, a therapeutic concentration of estradiol protected Abeta-treated neurons against inhibition of Cl(-)-ATPase activity and an increase in [Cl(-)]i through its receptor, probably via PKG- and CaMKII(-)mediated recovery of PI4P formation. Elevated [Cl(-)]i may be related to enhancement of glutamate toxicity.

Molecular cloning and characterization of the Cl(-) pump-associated 55-kDa protein in rat brain.

The Cl(-)-ATPase/pump in the plasma membrane of the rat brain is a candidate for active outwardly directed Cl(-) translocating systems. We recently isolated a Cl(-) pump, 520- or 580-kDa protein complex, which consisted of 51-, 55-, 60-, and 62-kDa proteins. In this study, we cloned a cDNA encoding a 55-kDa glycoprotein, designated as ClP55, which contained an open reading frame of 1512 base pairs encoding a protein of 504 amino acids including a signal peptide of 28 amino acids. Northern and Western blot analyses demonstrated expression of ClP55 mainly in the cerebrum. Application of antisense phosphorothioate oligonucleotides to cultured neurons resulted in a marked increase in the intracellular Cl(-) concentration ([Cl(-)](i)). Immunohistochemical analysis indicated that ClP55 was localized to the plasma membranes of neurons such as hippocampal pyramidal neurons and cerebellar Purkinje cells. Taken together, these results suggest that ClP55 is one of the Cl(-) pump subunits responsible for Cl(-) pump activity.

Amyloid beta proteins inhibit Cl(-)-ATPase activity in cultured rat hippocampal neurons.

Cl(-)-ATPase in the CNS is a candidate for an outwardly directed neuronal Cl(-) transporter requiring phosphatidylinositol-4-phosphate (PI4P) for its optimal activity. To test its pathophysiological changes in a phosphatidylinositol (PI) metabolism disorder, the effects of neurotoxic factors in Alzheimer's disease (AD), amyloid beta proteins (Abetas), on the Cl(-)-ATPase activity were examined using primary cultured rat hippocampal neurons. Amyloid beta proteins (1-40, 1-42 and 25-35) concentration-dependently (1-100 nM) and time-dependently (from 1 h to 6 day) decreased Cl(-)-ATPase activity and elevated intracellular Cl(-) concentrations ([Cl(-)]i), Abeta25-35 being the most potent. Addition of inositol or 8-Br-cyclic GMP completely reversed these Abeta-induced changes. The recoveries in enzyme activity were attenuated by an inhibitor of PI 4-kinase, 10 microM wortmannin or 20 microM quercetin, but not by a PI 3-kinase inhibitor, 50 nM wortmannin or 10 microM LY294002. The PI, PIP and PIP2 levels of the plasma membrane-rich fraction were lower in the Abeta-treated cells as compared with each control. In the Abeta-exposed culture, but not in control, stimulation by 10 microM glutamate for 10 min significantly increased fragmentation of DNA and decreased cell viability. Addition of inositol or 8-Br-cyclic GMP prevented the effect of Abeta-treatment on the neurotoxicity of glutamate. Thus, Abetas reduce neuronal Cl(-)-ATPase activity, resulting in an increase in [Cl(-)]i probably by lowering PI4P levels, and this may reflect a pre-apoptotic condition in early pathophysiological profiles of AD.

Hemolysis after mitral valve replacement with mechanical valve prostheses.

OBJECTIVE: We evaluated effects of type, size, and orientation of mechanical mitral valve prostheses on hemolysis. METHODS: Subjects were 84 patients who had undergone mitral valve replacement. Lactate dehydrogenase was mainly used as a marker of hemolysis and was measured before surgery, 1 month after surgery, and in the late postoperative period. RESULTS: Valves used included 16 Medtronic-Hall, 32 St. Jude Medical, and 36 CarboMedics valves. Medtronic-Hall valves caused less hemolysis than St. Jude Medical or CarboMedics valves in the late postoperative period. This resulted because hemolysis due to Medtronic-Hall valves was more severe 1 month after surgery than in the late postoperative period and because hemolysis due to St. Jude Medical or CarboMedics valves was more severe in the late postoperative period than 1 month after surgery. One reason for this finding is that cardiac output was greater in the late postoperative period than 1 month after surgery, making regurgitation through the pivots of bileaflet valves more severe. The orifice area and the orientation of prostheses did not affect hemolysis. CONCLUSION: St. Jude Medical or CarboMedics valves caused more severe hemolysis than Medtronic-Hall valves in the late postoperative period.

Developmental changes in Cl(-)-ATPase activity in rat brains.

Developmental changes in brain Cl(-)-ATPase activity were examined using fetal, neonatal and adult rats. The Cl(-)-ATPase activity rapidly increased over 20 postnatal days to a level four-fold higher than that in an 18-day-old fetus. On Western blot analysis using an anti-Cl(-)-ATPase/pump 51 kDa subunit (ClP51) antibody, the amount of ClP51 protein increased in parallel with Cl(-)-ATPase activity. Immunohistochemistry using the same antibody showed Cl(-)-ATPase-like immunoreactivity on the cell membranes of neurons such as cerebral and hippocampal pyramidal cells and cerebellar Purkinje cells, where the immunoreactivity increased with developmental changes in the size and shape of the neurons. These findings suggest that neuronal Cl(-)-ATPase activity markedly increases during early postnatal development with an increase in the amount of Cl(-)-ATPase protein, which may support the formation of inwardly directed neuronal Cl(-) gradients.

Cl(-)-ATPase in rat brain and kidney.

Cl(-)-stimulated ATPase/ATP-dependent Cl(-) pump (Cl(-)-ATPase/pump) has been found as a candidate for an active outwardly directed Cl(-) transporter in brain neurons. (1) A 520-kDa protein complex with Cl(-)-ATPase/pump activity was isolated from rat brain. It consisted of four protein subunits (51, 55, 60, and 62 kDa proteins), the 51-kDa protein being a covalent phosphorylenzyme subunit. (2) An antiserum against the 51-kDa protein inhibited Cl(-)-ATPase/pump activity. Western blot analysis showed an immunoreactive 51-kDa protein in the brain, spinal cord, and kidney. By enzyme histochemistry and immunohistochemistry, Cl(-)-ATPase-like activity or immunoreactivity was observed on the plasma membranes of brain neurons, and on the baso-lateral membranes of type A intercalated cells of renal collecting ducts. (3) Reconstituted Cl(-)-ATPase/pump activity was highest in liposomes with phosphatidylinositol-4-monophosphate. LiCl, an inhibitor of inositolphosphatase, reduced Cl(-)-ATPase activity and increased intracellular Cl(-) concentrations in cultured rat hippocampal neurons with increased phosphatidylinositol turnover. (4) In the brains of patients with Alzheimer's disease (AD), where phosphatidylinositol 4-kinase activity is reduced, Cl(-)-ATPase activity was also reduced. Thus, Cl(-)-ATPase is likely an outwardly directed ATP-dependent Cl(-) transporter that consists of four subunits and is regulated by phosphatidylinositol-4-monophosphate. Changes in Cl(-)-ATPase activity may be related to the pathophysiology of human neurodegenerative diseases. J. Exp. Zool. 289:224-231, 2001.

Lithium decreases Cl--ATPase activity and increases intracellular Cl- concentration in cultured rat hippocampal neurons.

Under the conditions of stimulated phosphatidylinositol turnover (0. 1 mM carbachol plus 20 mM KCl), LiCl (0.1-10 mM) reduced the activity of Cl--ATPase in cultured rat hippocampal neurons without affecting Na+/K+- or anion-insensitive Mg2+-ATPase. This inhibition of Cl--ATPase was attenuated by the addition of 0.5 mM inositol to culture media. The intracellular Cl- concentrations of the LiCl-treated neurons increased in an inositol-sensitive manner.

[Coronary artery fistula with left atrial myxoma: report of a case].

A 64-year-old male was referred for surgical treatment of left atrial myxoma. Preoperative coronary angiography revealed coronary artery fistula from the left anterior descending artery and the circumflex artery draining into the main pulmonary artery. Operative treatment was performed including resection of the myxoma, patch closure of the atrial septal defect, and closure of the fistula with pledgeted mattress sutures from within the main pulmonary artery on cardiopulmonary bypass. His postoperative course was uneventful, and disappearance of the left atrial myxoma and the coronary artery fistula was ascertained by echocardiography and coronary angiography.

Persistent hemolysis after coil occlusion of a patent ductus arteriosus in a patient with aortic regurgitation.

We report a rare case of hemolysis after coil occlusion of a patent ductus arteriosus (PDA), which was treated by surgical removal of the coil and closure of PDA. A 65-year-old woman was admitted to our hospital with congestive heart failure due to severe aortic regurgitation associated with PDA. Before undergoing open heart surgery she underwent closure of the PDA using a Jackson coil as an adjunct of treatment to improve her hemodynamic state. However, a small residual shunt resulted in severe hemolysis. Two weeks after the intervention she underwent aortic valve replacement and PDA closure after removal of the coil through the main pulmonary artery under moderate hypothermia and temporary circulatory arrest. Hemolysis is always secondary to a residual leak and several methods have been reported to manage this complication. Our report suggests that early surgical retrieval of the coil before the organized thrombus is formed, can be safely performed even in an elderly patient whose ductus is usually fragile.

CI-ATPase and Na+/K(+)-ATPase activities in Alzheimer's disease brains.

The enzyme activities and the protein levels of Cl(-)-ATPase and Na+/K(+)-ATPase were examined in Alzheimer's disease (AD) brains. Cl(-)-ATPase and Na+/K(+)-ATPase activities in AD brains (n = 13) were significantly lower than those in age-matched control brains (n = 12). In contrast, there was no significant difference in anion-insensitive Mg2(+)-ATPase activity between the two groups. Western blot analysis revealed that the protein levels of Cl(-)-ATPase, Na+/K(+)-ATPase and neuron specific Na+/K(+)-ATPase alpha3 isoform were also significantly reduced in AD brains, while the amount of protein disulfide isomerase, one of the house keeping membrane proteins, was not different between the two groups. The data first demonstrated that Cl(-)-ATPase and Na+/K(+)-ATPase are selectively impaired in AD brains, which may reduce the gradients of Na(+), K(+) and Cl(-) across the cell membranes to cause excitotoxic cellular response and the resulting neuronal death.

[Reoperation for relief of valvular and supravalvular stenosis late after aortic valve replacement with Manouguian's anulus enlargement: report of a case].

Thirteen year-old boy who received aortic valve replacement with Manouguian's anulus enlargement for congenital aortic stenosis 7 years ago developed valvular and supravalvular stenosis. Pressure gradient between left ventricle and aorta was 55 mmHg. The etiology of supravalvular stenosis was extensive patch plasty of ascending aorta and severe degeneration of expanded polytetrafluoroethylene patch. Moreover, white hard mass existed just outside the patch and was one of the components of the stenosis. This mass was acellular calcification without foreign body reaction. Degenerated prosthetic patch was thoroughly resected, and redo Manouguian's procedure with autogeneous pericardium was performed. We tried to use two sizes larger 21 mm valve, but, after all, 19 mm valve was sewn into place. His postoperative course was uneventful.

[Reoperation for mitral regurgitation 13 years after aortic valve replacement and manouguian's anulus enlargement: report of a case].

The patient was 22-year-old female. She had undergone aortic valve replacement and Manouguian's anulus enlargement with low porosity woven Dacron patch for congenital aortic stenosis 13 years ago, and developed mitral regurgitation 9 years after that operation. Two regurgitant flow were observed. One was originated from the orifice due to mitral prolapse. The other was from a tear in the anterior leaflet. It was around the tip of the prosthetic patch, approximately 7 mm in size, and was repaired easily. But the mitral valve itself was found to be malformed and prolapsed, requiring mitral valve replacement. Her postoperative course was uneventful.

The stiffness of lymph nodes containing lung carcinoma metastases: a new diagnostic parameter measured by a tactile sensor.

BACKGROUND: It is believed that the stiffness or hardness of a lymph node containing a metastasis differs from that of lymph node without a metastasis because of the difference in tissue density, which is derived from the lymph node's histopathologic features. Prior to this study, however, there had been no attempts to quantify the hardness or stiffness of lymph nodes. The authors developed a new tactile sensor and system for measuring the stiffness (g/cm) of lymph nodes accurately, and they studied its utility as a tool for diagnosing lymph node metastases. METHODS: Clinical specimens were obtained from 14 patients who underwent lobectomy or pneumonectomy with hilar and mediastinal lymph node dissection for nonsmall cell lung carcinoma at the University of Tokyo between January and July 1996. With the tactile sensor developed by the authors, 212 resected lymph nodes were measured for their stiffness. RESULTS: Among these 212 resected lymph nodes, 57 were diagnosed as containing metastases (38 from adenocarcinomas and 19 from squamous cell carcinomas). The mean stiffness of the lymph nodes that contained metastases was 3.35 +/- 1.57 g/cm, and that of lymph nodes without metastases was 1.23 +/- 0.50 g/cm (P < 0.001). Receiver operating characteristic analysis revealed that the area under the curve was 0.93, indicating excellent accuracy of the method. When the cutoff was 1.5 g/cm, the sensitivity was 91.2% and the specificity was 78.1% for detection of lymph node metastases. CONCLUSIONS: Measurement of the stiffness of resected lymph nodes was confirmed as an accurate approach to diagnosing lymph node metastases without knowledge of other factors, such as lymph node size or color.

[Improvement of cardiac function by aortic valve replacement for chronic aortic regurgitation with carbomedics heart valve].

Cardiac function after aortic valve replacement for chronic aortic regurgitation with CarboMedics heart valve was evaluated repeatedly by echocardiography and some examinations in 27 cases. There was no late death and all patients belonged to NYHA I functional class postoperatively. Systolic and diastolic dimensions of left ventricle, left ventricular mass index, cardiothoracic ratio and SV1+RV5 on electrocardiography improved significantly within half a year and those improved results remained thereafter. Fractional shortening of left ventricle also improved gradually and the difference reached statistical significance one year later. This investigation revealed reduced systolic function or eccentric myocardial hypertrophy were often reversible. Average values of systolic diameter and fractional shortening late after operation were within normal range even in cases whose preoperative systolic function were severely depressed. Left ventricular mass index decreased markedly although postoperative values were still out of normal range in most cases. These results suggest that aortic valve replacement can be indicated even in cases of aortic regurgitation with severely impaired preoperative cardiac function.

Four-channeled aortic dissection and rupture.

A four-channeled aortic dissection is quite rare, which is a highly life-threatening situation predisposing to aortic rupture. We report a successful management of a four-channeled aortic dissection and an aortic rupture in a 59-year-old woman with Marfan's syndrome 11 years after an initial Bentall procedure for DeBakey type I dissection. The total arch and the descending thoracic aorta were replaced under deep hypothermia and circulatory arrest.

Fibrous tissue overgrowth and prosthetic valve endocarditis: report of a case.

Mechanical valve stenosis without restricted occluder motion and paravaluvular leakage developed in a patient who had undergone patch closure of partial atrioventricular septal defect and replacement of the left atrioventricular valve 13 years previously. Dense calcification of the supravalvular region was shown in a cineradiogram, whereas transthoracic and transesophageal echocardiography failed to reveal any obstructive mechanism. Elevated transprosthetic pressure gradient with unrestricted occluder motion suggested prosthetic valve stenosis resulting from fibrous tissue overgrowth, although this was not visualized by the modern diagnostic imaging tools. Reoperation confirmed calcified fibrous tissue overgrowth obstructing the mechanical valve inflow. Examination of resected tissue revealed prosthetic valve endocarditis due to alpha-streptococcus. Paravalvular leakage accompanying fibrous tissue overgrowth may indicate the presence of prosthetic valve infection even if the clinical manifestations are scarce.

Thoracoscopic implantation of a pacemaker lead: experimental study.

We have performed five thoracoscopic pacemaker lead implantations into the porcine myocardium. A small opening, approximately 2 cm in diameter, was made thoracoscopically in the pericardium overlying the apex of the left ventricle, avoiding phrenic nerve injury. Three screw-in type or two stab-in type standard sutureless unipolar epicardial electrodes were introduced through a trocar, and then screwed or stabbed into the myocardium near the apex of the left ventricle, avoiding the coronary vessels. Hemorrhage and arrhythmia were negligible during the procedures. Electrical tests of the pacing threshold and lead impedance after implantation of the leads revealed acceptable values in each lead. We conclude that thoracoscopic implantation of pacemaker leads is feasible, and may be used as a minimally invasive option when conventional transvenous techniques are unavailable or contraindicated.

Relationship between serum carotenoid levels and cancer death rates in the residents, living in a rural area of Hokkaido, Japan.

The relationship between serum carotenoid levels and cancer death in rural Japanese residents, aged more than 39 years, was examined epidemiologically. The follow-up subjects participated in the comprehensive health examinations, every August from 1986 to 1989, were 929 males and 1424 females, living in a rural area of Hokkaido, Japan. During 2-8 years to December 1994, the ninety-eight deaths were observed among the cohort; that is, deaths from all causes were 67 males and 31 females, and cancer deaths were 34 males and 10 females. Serum samples at fasting were collected at the entry of the cohort and serum levels of beta-carotene, alpha-carotene and lycopene were measured by the HPLC method. The relation between the mortality rates and serum carotenoid levels was estimated statistically using the Cox's proportional hazard model. The results were as follows; the hazard ratio with the overall on high serum beta-carotene levels to those of lower was 0.46 (95% C.I.: 0.27-0.78) for all causes and 0.33 (95% C.I.: 0.14-0.75) for cancer deaths. This result suggested that high levels of serum beta-carotene may play some roles on preventing cancer death.

New tactile sensor techniques for localization of pulmonary nodules.

We developed a new method for thoracoscopically localizing pulmonary nodules using a tactile sensor. This sensor was originally designed to probe the hardness of an object, which is represented by changes in resonance frequency of the sensor (delta f: Hz). By moving over the lung surface, the sensor probe, introduced through a 10 mm trocar, was able to search for nodules within the pulmonary parenchyma thoracoscopically. When the sensor probe passed just above a nodule, which was harder than the adjacent tissue, a sudden sharp increase in the curve was observed. We have successfully utilized this method in 16 cases since August 1994. A representative case is that of a 68-year-old male who was admitted with an indeterminate nodule, demonstrated preoperatively in the left lung by chest computed tomography, which was localized with this technique and resected under thoracoscopic surgery.