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.

The reeler gene-associated antigen on Cajal-Retzius neurons is a crucial molecule for laminar organization of cortical neurons.

In the neurological mutant mouse reeler, the histological organization of the neocortex develops abnormally and essentially results in an inversion of the relative positions of the cortical layers. The reeler mutation, therefore, provides an insight into the molecular mechanisms underlying the formation of the cortical layers. We have generated a monoclonal antibody (CR-50) that probes a distinct allelic antigen present in wild-type but not in reeler mutant mice. CR-50 reacted specifically with Cajal-Retzius neurons, one of the first cortical neurons to differentiate in the neocortex, but whose functional role is not known. When dissociated cerebral cortical cells were incubated with CR-50 in reaggregation culture, the genotype-dependent histogenetic assembly of wild-type cortical cells resembled that of reeler mutants. These findings revealed that the selective expression of a distinct molecule on Cajal-Retzius neurons is critical for the normal lamination of cortical neurons in the mammalian neocortex.

Swelling-induced O2- generation in guinea-pig neutrophils.

Without the addition of any exogenous stimuli, neutrophils generated O2- and then ceased in a reversible manner that correlated with cellular swelling and contraction. The nature of the possible mechanism responsible for this O2- generation was studied and compared with that observed in the triggering of stimulant-dependent O2- generation (respiratory burst). The swelling-induced O2- generation was inhibited by diphenyliodonium, and was independent of the functional distortion of mitochondrial and/or microsomal electron transport and xanthine oxidase. This suggested that such generation was involved in respiratory-burst oxidase activation; however, this generation was not accompanied by any new phosphorylation of the 47-kDa protein or of tyrosine proteins. Dihydrocytochalasin B potentiated the O2- generation. The cellular swelling produced a priming effect on the triggering of respiratory burst with different stimuli. Cellular contraction, conversely, suppressed the respiratory burst. The structural specificity of the swelling-induced plasma membrane modulation for the O2- generation was suggested by the finding that modulation of plasma membrane structures by various non-ionic detergents per se inhibited O2- generation. Lipophilic and positively-charged agents inhibited the generation and this inhibition was abrogated by negatively-charged, but not by non-ionic agents. Negatively-charged agents potentiated the O2- generation. These results suggest that both the interaction of the plasma membrane with the cytoskeleton and an increase in net negative charges at the plasma membrane play important role in evoking O2- generation; this is discussed and compared with the signal transduction reported previously for respiratory burst.

Identification of two highly homologous presynaptic proteins distinctly localized at the dendritic and somatic synapses.

Through screening of a murine brain cDNA library, we have isolated two brain specific cDNAs encoding highly homologous proteins, named 921-L and 921-S, comprised of 134 amino acids with 80% identity. Immunohistological study with the mAbs raised against the bacterially expressed 921 proteins showed that 921-L protein is distributed at the dendritic region and 921-S at the neuronal somatic surface. Immuno-electron microscopic study revealed that both 921 proteins are localized at the presynaptic terminal, indicating that the 921 proteins are differentially expressed at the dendritic and somatic presynapses.

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.

Panning and immune electron-microscopic study of mucinous ovarian-cancer antigen-specific cells derived from malignantly transformed bloom-syndrome B-lymphoblastoid cells.

We separated characteristic mucinous ovarian cancer (OVC) antigen cells from malignantly transformed Bloom syndrome (BS) cell line (BS-SHI-4M) with the panning procedure using OVC patients sera. We undertook an immune electron-microscopic and scanning electron-microscopic study to acquire information regarding the antigenic determinant of the membrane using pre-embedding method, as well as immunofluorescence (IF) study. The distribution of Protein A colloidal gold (PAG) grains on the cell membrane of mucinous OVC antigen cells paralleled that of fluorescein-conjugated anti-human IgG observed in the IF study. The three patterns of PAG labeling of uniform labeling, uniform partial labeling, and partial labeling of one side of the cell paralleled the three patterns of IF labeling observed under IF. These findings strongly suggest the immunological reaction of BS-SHI-4M OVC-MU antigen cells with the antibody of mucinous OVC patient serum. Western blot analysis demonstrated that the antigen which characterizes mucinous OVC has a band at 84000 MW.

Midterm results of the rotation-advancement flap method for correction of partial anomalous pulmonary venous drainage into the superior vena cava.

Since 1986, a rotation-advancement flap method has been used in 11 patients with partial anomalous pulmonary venous drainage into the superior vena cava. This method consists of atrial partitioning, enlargement of the superior vena cava, and protection of the sinus node. The midterm postoperative sinus node function and hemodynamic changes were examined in this study. Postoperative angiograms showed normal pulmonary venous pathway and no stenosis in the superior vena cava. Cardiac rhythm was normal and no clinical symptoms appeared.

Reactivation of rat cytomegalovirus in lung allografts: an experimental and immunohistochemical study in rats.

Reactivation of latent rat cytomegalovirus (RCMV) from a lung allograft or from a recipient was studied in RCMV-mismatched combinations (donor [D]-/recipient [R]+, D+/R-, and D+/R+) with latently infected lung grafts and chronically infected rats in an inbred rat model. Nineteen transplants in a major histocompatibility complex different strain combination (Brown-Norway/Lewis) were immunosuppressed daily (cyclosporine, azathioprine, and prednisolone) from day 3 after orthotopic left lung transplantation and killed on days 3, 6, and 21. Control groups consisted of nine chronically RCMV-infected rats with immunosuppression without transplantation and six allografts with immunosuppression without RCMV infection. Reactivation of latent RCMV was tested by immunohistochemical staining with monoclonal antibodies against RCMV-induced antigens and by plaque assays of the virus in the salivary glands. The following results were obtained: (1) All allotransplants developed acute ongoing rejection on days 3 and 6, and the rejection was resolved on day 21 by immunosuppression. (2) Reactivation was observed in allotransplanted groups, but not in the control rats. (3) In the D+/R+ and D-/R+ groups on days 3 and 6, the number of RCMV-related antigen-positive cells increased in the recipient spleen and lymph nodes and in the bronchus-associated lymphoid tissue of the donor lung in the D-/R+ group, but not in the chronically RCMV-infected controls. (4) In the D+/R- group on day 6, RCMV-induced antigen-positive cells were observed in the spleen and lymph nodes of the recipient and also around the vessels in the recipient lung. (5) In the D-/R+ group, vascular endothelial cells or mildly infiltrated mononuclear cell subpopulations around the vessels in the lung allograft showed weakly positive staining against RCMV-related antigens on day 6. (6) After the initial acute rejection on days 3 and 6 was treated by immunosuppressive drugs, reactivated acute RCMV infection became chronic or latent again on day 21. We conclude that RCMV infection could be transferred with latently infected lung allografts by reactivation of latent RCMV. In rats, as in man, alloimmune responses seemed to have a definite influence on the reactivation of latent RCMV after lung transplantation.

Tuberculous pseudoaneurysm of the thoracic aorta.

Tuberculous pseudoaneurysm has been reported to be a fatal, but rare complication of tuberculosis. We report a case of a 68-year-old man who underwent successful surgical treatment for a tuberculous pseudoaneurysm of the thoracic aorta with bronchial communication, and review previous reports of patients who also underwent operation for similar lesions, focusing especially on the pathway of infection to the aorta.

Application of a new tactile sensor to thoracoscopic surgery: experimental and clinical study.

BACKGROUND: We developed a new tactile sensor that could quantify the hardness of objects as changes in the resonance frequency of the sensor (delta f). We have applied it to thoracoscopic operations for the localization of small invisible nodules in the lung. METHODS: When the sensor probe was moved over the lung surface, a delta f curve was depicted on the computer screen. When the sensor tip reached a point directly above a hard object, a sudden upward jump of the delta f curve was evoked. After experimental studies using pigs, the sensor was applied in 8 patients. More recently we produced a needle sensor to distinguish small nodules from bronchi that may evoke similar upward jumps of the delta f curve. Eight nodules and four bronchi in resected human lungs were probed directly using this sensor. RESULTS: In all of the patients, the hardness of various thoracic structures could be quantified. A total of 10 nodules were found using the sensor and resected thoracoscopically. The needle sensor distinguished nodules from bronchi, as the mean delta f of the bronchial walls (-64 +/- 45.9 Hz) was significantly higher than that of nodules (-526 +/- 168 Hz, p < 0.001). CONCLUSIONS: Thoracoscopic detection of small and invisible pulmonary nodules using our new tactile sensor is feasible.

Successful management of massive pulmonary tumor embolism from renal cell carcinoma.

Renal cell carcinoma occasionally invades the inferior vena cava and rarely extends to the right atrium. However, despite the frequency of venous extension, it is unusual to recognize patients with massive pulmonary tumor embolus clinically. We describe a 60-year-old man who underwent pulmonary tumor embolectomy using cardiopulmonary bypass combined with profound hypothermia and intermittent low-flow perfusion. The patient is currently alive and well without implantation metastasis 6 months after the operation.

Retrograde gastric varices in a patient with total cavopulmonary shunt.

Gastric varices formed in a patient who had undergone a total cavopulmonary shunt operation 7 years previously. The varices were found to be due to development of collaterals from high-pressure systemic vein to low-pressure portal vein. Bleeding gastric varix can be a late complication of total cavopulmonary shunt.

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.

Impaired responsiveness of paraventricular neurosecretory neurons to osmotic stimulation in rats after local anesthesia of the subfornical organ.

Extracellular recordings were obtained from 32 phasically active neurosecretory cells in the hypothalamic paraventricular nucleus (PVN) of urethane-anesthetized male rats. None of the PVN cells changed their activity to intracarotid infusions of isotonic saline (0.15 M NaCl solution, 0.05 ml). Of these PVN neurons, 26 displayed an increase in neuronal activity following intracarotid infusions of hypertonic saline (0.2 M NaCl solution, 0.05 ml), while the remainder were unresponsive. Microinjection of the local anesthetic lidocaine into the subfornical organ (SFO) reversibly diminished the excitatory response to the infusions of hypertonic saline in 10 out of 15 PVN neurons tested, whereas the injection of lidocaine into the vicinity of the SFO (n = 4) or the third ventricle (n = 4) did not cause a marked change. These results show an involvement of the SFO in the mechanism of osmotic activation of putative vasopressin (AVP)-secreting neurons in the PVN.

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.

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.

Purification of infectious bronchitis coronavirus by Sephacryl S-1000 gel chromatography.

A procedure was developed to purify infectious bronchitis virus (IBV) by gel chromatography (GC) with a Sephacryl S-1000 column. Virus samples concentrated by centrifugation were applied to a Sephacryl S-1000 column and eluted by 0.02 M phosphate buffer (pH 7.2) containing 0.15 M NaCl. Virus particles were recovered mainly in the first peak. Purity of the samples was evaluated by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy. Using electron microscopy, it was found that there were more spike-rich particles in the virus samples purified by GC than in those purified by sucrose density gradient centrifugation (SDGC). In addition, the hemagglutination unit [log10 (infectivity titer/hemagglutination titer)] of GC-purified virus samples was approximately 10 times lower than that of SDGC-purified virus samples. These results indicate that Sephacryl S-1000 gel chromatography is useful for purification of IBV.

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.

Detection of infectious bronchitis virus antigen from experimentally infected chickens by indirect immunofluorescent assay with monoclonal antibody.

Infectious bronchitis virus (IBV) was detected by indirect immunofluorescent assay with a monoclonal antibody (MAb-IFA). The monoclonal antibody was specific for the nucleocapsid protein of IBV strain M41. The MAb-IFA clearly detected IBV with high specificity in infected chicken kidney cells. The assay furthermore detected IBV in tracheal smears and sliced tracheas from experimentally infected chickens. The positive reaction was found to be longer than that in the virus recovery test. These results indicate that MAb-IFA is a useful method for the detection of IBV from chickens suspected to have infectious bronchitis.

Clinical experience of Hall-Kaster valve. Operative results and hemodynamic studies.

The Hall-Kaster pivotal disc prosthetic valve (HK valve) was introduced in 1977. Since October 1979, we have used the HK valve in 137 patients for valve replacement. The over-all operative mortality was 5.1%. During 36 months follow-up, there were 3 late deaths due to congestive heart failure, but no valve related death, no mechanical failure and no thromboembolic episode have been encountered. Intraoperative hemodynamic studies revealed its valvular function to be excellent. The mean pressure gradients across the prosthesis were 11.0 +/- 1.6 and 4.2 +/- 2.0 mmHg in aortic and mitral position, respectively. The mean calculated valve areas were 1.7 +/- 0.6 and 3.7 +/- 1.2 cm2 in aortic and mitral position, respectively. Thus, the Hall-Kaster prosthetic valve was found to be an efficient and favorable prosthetic heart valve for aortic and mitral valve replacement.