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.

Molecular diversity of Photobacterium damselae ssp. piscicida from cultured amberjacks (Seriola spp.) in Japan by pulsed-field gel electrophoresis and plasmid profiles.

AIMS: This study deals with a pulsed-field gel electrophoresis (PFGE) for discriminating between the genetic variants of Photobacterium damselae ssp. piscicida, and characterizing of Japanese field isolates by PFGE together with plasmid profiles and antimicrobial resistances. METHODS AND RESULTS: A total of 74 field isolates from cultured Japanese amberjacks were used for PFGE. SmaI and NotI enabled to clearly differentiate strains and we obtained 24 of combined PFGE profiles which were distinct from those of classical Japanese and USA reference strains, and classified them into three groups (Ia-Ic). By plasmid size, we could classify these field isolates into three plasmid types, pA-pC. The predominant PFGE-type Ia was closely associated with plasmid-type pA, and Ib showed a moderate association with pB. Ic was closely associated with pC, and multiresistant isolates were not observed in this type. Whole-genomic variations were also observed between isolates having identical detection areas, fish species and detection-date by PFGE. CONCLUSION: Molecular diversity of P. damselae ssp. piscicida could be detected by PFGE, and some relations among the PFGE-type, plasmid-type and antimicrobial resistances were observed in Japanese field isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicated that some genetic transition might have occurred in P. damselae ssp. piscicida around the Japanese seas, and PFGE can be a valuable tool for the epidemiological study of this highly homogeneous subspecies.

Characterization of Lactococcus garvieae isolated from radish and broccoli sprouts that exhibited a KG+ phenotype, lack of virulence and absence of a capsule.

AIMS: To identify Lactococcus garvieae isolates from radish and broccoli sprouts and compare them with virulent and less virulent mutant strains obtained from yellowtails with regard to KG phenotype, presence of a capsule and virulence towards yellowtails and mice. METHODS AND RESULTS: Comparative 16S rRNA gene sequence analysis of six isolates obtained from radish and broccoli sprouts indicated that they were L. garvieae (similarity >99%). They were compared with KG9502, Lg2 and ATCC49156 strains obtained from yellowtails. A less virulent mutant strain Lg2-S was obtained by Lg2 subculture. Biochemical characterization of the six strains resembled that of KG9502, Lg2, ATCC49156 and Lg2-S, except for saccharose and tagatose acidification and the presence of hippuricase. These six strains were nonpathogenic towards yellowtails and mice, nonsusceptible to bacteriophages and demonstrated heterogeneity on pulsed-field gel electrophoresis analysis. Using transmission electron microscopy, a capsule was observed in KG9502 and Lg2 but not in ATCC49156 and Lg2-S. CONCLUSIONS: We isolated L. garvieae strains that lacked pathogenicity towards yellowtails and mice from radish and broccoli sprouts; these were noncapsulated and exhibited KG(+) phenotype. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first documentation of L. garvieae isolated from terrestrial plants. These isolates exhibited genetic diversity; however, they were noncapsulated and nonpathogenic towards yellowtails and mice.

Morphological observations and the effects of artificial digestive fluids on the survival of Diploscapter coronata from a Japanese patient.

Unusual non-human parasitic nematodes and eggs were detected in the faeces of an 8-year-old Japanese female suffering from Henoch-Schönlein purpura. The worms were adult female rhabditiform nematodes measuring 325.6-441.2 micro m in length and 18.3-26.5 micro m in width. One pair of the labia oris was notched with many spiny projections, while the other pair was strongly curved outwards. The worms were identified using light and scanning electron microscopy as the free-living nematode Diploscapter coronata (Cobb) based on their characteristic morphology. The patient's faeces containing worms and eggs were cultured using a filter-paper culture technique and after 7 days of culture, male as well as female worms were recovered. Worm survival time and hatchability of the eggs were examined in vitro after treatment with an artificial gastric or intestinal fluid. Although adult worms survived for less than one minute, eggs hatched after treatment with artificial gastric fluid. This suggests that eggs accidentally ingested or produced by adult D. coronata could develop in the human gastro-intestinal tract. Some morphological features of male D. coronata are also described.

Differences between Lactococcus garvieae isolated from the genus Seriola in Japan and those isolated from other animals (trout, terrestrial animals from Europe) with regard to pathogenicity, phage susceptibility and genetic characterization.

AIMS: To clarify the epidemiological relationship between Lactococcus garvieae isolates from the Seriola in Japan and isolates from other animals. METHODS AND RESULTS: A total of 32 isolates obtained from aquatic (the genus Seriola and trout) and terrestrial animals (cow, pig, cat, dog and horse) was used to evaluate its pathogenicity to yellowtail and mouse, phenotype (KG+ and KG-), its susceptibility to three bacteriophages and the pattern of pulsed field gel electrophoresis (PFGE). Lactococcus garvieae isolated from Seriola showed strong pathogenicity to yellowtail, while isolates from trout showed weak pathogenicity and those obtained from terrestrial animals showed no distinct pathogenicity. Only, the isolates from the genus Seriola in Japan showed susceptibility to the bacteriophages. The results of PFGE pattern indicate that the isolates obtained from the Seriola predict homogeneity, while there is no similarity among the isolates obtained from different animals. CONCLUSION: This experiment indicates that L. garvieae isolated from Seriola in Japan appears to be very different from the isolates obtained from other animals, and the isolates prevalent among the genus Seriola in Japan might be homogeneous. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that a particular genetic group that has specially adapted and acquired virulence toward yellowtail were prevalent among the genus Seriola in Japan.

Drug resistance and random amplified polymorphic DNA analysis of Photobacterium damselae ssp. piscicida isolates from cultured Seriola (yellowtail, amberjack and kingfish) in Japan.

AIMS: To investigate the antimicrobial susceptibility and genetic characteristics of Photobacterium damselae ssp. piscicida isolates obtained from cultured Seriola in Japan. METHODS AND RESULTS: Minimal inhibitory concentrations of 14 antimicrobials for 74 isolates from Seriola in Japan in 2002 were determined. Isolates showed high frequencies of resistance to sulfamonomethoxine (SMMX) (97.3%), oxytetracycline (OTC) (77.0%), flumequine (FMQ) (77.0%), chloramphenicol (CP) (75.7%), kanamycin (KM) (63.5%) and oxolinic acid (OA) (62.0%), but low to ampicillin (ABPC) (2.8%). All isolates were susceptible to bicozamycin (BCM), fosfomycin (FOM) and florfenicol (FF). Of these isolates, 45 (60.8%) showed same resistance pattern (SMMX-OTC-FMQ-OA-CP-KM). In random amplified polymorphic DNA (RAPD) analysis, no difference was observed among our 74 field isolates and ATCC51736 isolated from Seriola in 1974 in Japan, but different from ATCC 17911 isolated from white perch in USA. CONCLUSIONS: FF, BCM, FOM and ABPC were useful antimicrobials for treating pseudotuberculosis. However, the frequency of multidrug resistance was high. RAPD analysis showed homogeneity of isolates from Seriola in Japan. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that some antimicrobials were still useful for treating pseudotuberculosis and that P. damselae ssp. piscicida strains of same origin might have spread among Seriola in Japan since 1974.

Congenital pulmonary-systemic collateral vein without obstructed left atrial egress is associated with conotruncal anomalies.

We present a case of congenital pulmonary systemic collateral vein associated with truncus arteriosus. Pulmonary systemic collateral vein with nonobstructed left atrial egress is different from those with obstructed left atrial egress in that it is functionally redundant. Including this case, 8 patients among 33 reported cases with pulmonary systemic collateral veins have had nonobstructed left atrial egress. Association with conotruncal anomalies in 4 of these 8 patients, as well as the reported finding that neural crest cells are distributed not only in the conotruncus but also in the cardinal vein, indicates that neural crest cells play roles in remodeling of the systemic venous system.

Left innominate vein-pulmonary artery shunt with Glenn anastomosis in a Fontan candidate with central pulmonary artery stenosis.

Heart failure developed 9 years after Fontan takedown with systemic-pulmonary artery shunt in a 12-year-old girl with pulmonary atresia, intact ventricular septum, and obstruction in the proximal pulmonary artery. Surgical scar after multiple operations complicated direct repair of the pulmonary artery, and thus she was not eligible for definitive palliation. Left innominate vein-to-left pulmonary artery shunt using an expanded polytetrafluoroethylene conduit in association with Glenn anastomosis functionally established an unobstructive superior cavopulmonary connection without direct repair of the central pulmonary artery, later facilitating one and a half ventricle repair. Use of an extraanatomical shunt may functionally relieve central pulmonary artery obstruction in candidates for Fontan-like circulation.

IGF-I gene transfer by electroporation promotes regeneration in a muscle injury model.

The goal of this study was to determine whether insulin-like growth factor-I (IGF-I) gene delivery by electroporation promotes repair after muscle injury. An injury-repair model was created using mice in which a hamstring muscle was cut and sutured. A total of 50 microg of IGF-I DNA or green fluorescent protein (GFP) DNA (both in pCAGGS) was injected into the lesion and introduced into muscle cells by electrostimulation using an electric pulse generator. The number of regenerating muscle fibers in the IGF-I DNA group was significantly more than that in the GFP DNA group at 2 weeks after injection. The diameter of regenerating muscle fibers from the IGF-I DNA group was larger than that of the GFP DNA group at 4 weeks after injection. There was no significant difference in the serum IGF-I concentration between the IGF-I DNA group and the GFP DNA group at 1, 2, and 4 weeks after injection. However, muscle IGF-I concentration in the IGF-I DNA injection group was significantly greater than that in the GFP DNA injection group at 2 weeks after injection. These results demonstrated that the effects of enhanced IGF-I production were local and limited to the injected area. The ratio (injected/uninjected; intact) of the amplitude of compound muscle action potentials (CMAP) in the IGF-I DNA injection group was greater than that in the GFP DNA injection group at 4 weeks after injection and of the control group. In conclusion, IGF-I gene transfer by electroporation proved to be a simple, safe, inexpensive, and effective method to promote the regeneration of injured muscles in our injury model.

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.

Changes in the cell structure of Flavobacterium psychrophilum with length of culture.

Flavobacterium psychrophilum, the pathogen of bacterial cold-water disease, causes serious problems in ayu Plecoglossus altivelis culture. This study investigated the effect of the culture period of F. psychrophilum and on the structure of its cells. From the SDS-PAGE of total proteins of cellular components, much difference was found between the 36 hr culture and the 48 and 72 hr cultures. A SEM observation of the cells showed many fragments, especially on the cell surface of the 36 hr culture. These fragments consisted of an outer membrane, seen by TEM observation, and may contain substances causing the virulence. Specific proteins observed by the SDS-PAGE and fragments in the 36 hr culture may be related to the virulence of F. psychrophilum.

Reduced hippocampal LTP in mice lacking a presynaptic protein: complexin II.

The SNAP receptor (SNARE) complex is a core complex specialized for synaptic vesicle exocytosis, and the binding of SNAPs to the complex is an essential step for neurotransmitter release. Complexin I and II have been identified as SNARE-complex-associated proteins. Importantly, complexins compete with alpha-SNAP for binding to the complex, suggesting that complexins may modulate neurotransmitter release process. To examine this possibility and to understand the physiological function of complexins, we generated complexin II knockout mice. The complexin-II-deficient mice (-/-) were viable and fertile, and appeared normal. Electrophysiological recordings in the mutant hippocampus showed that ordinary synaptic transmission and paired-pulse facilitation, a form of short-term synaptic plasticity, were normal. However, long-term potentiation (LTP) in both CA1 and CA3 regions was impaired, suggesting that complexin II may not be essential for synaptic vesicle exocytosis, but it does have a role in the establishment of hippocampal LTP.

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.