Novel biallelic mutations in the PNPT1 gene encoding a mitochondrial-RNA-import protein PNPase cause delayed myelination.

Recent studies suggest that impaired transcription or mitochondrial translation of small RNAs can cause abnormal myelination. A polynucleotide phosphorylase (PNPase) encoded by PNPT1 facilitates the import of small RNAs into mitochondria. PNPT1 mutations have been reported in patients with neurodevelopmental diseases with mitochondrial dysfunction. We report here 2 siblings with PNPT1 mutations who presented delayed myelination as well as mitochondrial dysfunction. We identified compound heterozygous mutations (c.227G>A; p.Gly76Asp and c.574C>T; p.Arg192*) in PNPT1 by quartet whole-exome sequencing. Analyses of skin fibroblasts from the patient showed that PNPase expression was markedly decreased and that import of the small RNA RNaseP into mitochondria was impaired. Exogenous expression of wild-type PNPT1, but not mutants, rescued ATP production in patient skin fibroblasts, suggesting the pathogenicity of the identified mutations. Our cases expand the phenotypic spectrum of PNPT1 mutations that can cause delayed myelination.

The gamete fusion process is defective in eggs of Cd9-deficient mice.

The cell-surface molecule Cd9, a member of the transmembrane-4 superfamily, interacts with the integrin family and other membrane proteins. and is postulated to participate in cell migration and adhesion. Expression of Cd9 enhances membrane fusion between muscle cells and promotes viral infection in some cells. Fertilization also involves membrane fusion, between gametes. In mammals, the sperm binds to microvilli on the egg surface, and sperm-egg membrane fusion first occurs around the equatorial region of the sperm head12. The fused membrane is then disrupted, and the sperm nucleus as well as the cytoplasm is incorporated into the egg. Cd9 is expressed on the plasma membrane of the mouse egg, and an anti-Cd9 monoclonal antibody inhibits sperm-egg surface interactions. We generated Cd9 mice and found that homozygous mutant females were infertile. Sperm-egg binding was normal, but sperm-egg fusion was almost entirely inhibited in eggs from Cd9 females. Intracellular Ca2 oscillations, which signal fertilization, were absent in almost all mutant eggs; in rare cases, a response occurred after a long time period. In normal animals, Cd9 molecules were expressed on the egg microvilli and became densely concentrated at the sperm attachment site. Thus, our results show that Cd9 is important in the gamete fusion process at fertilization.

Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning.

1 alpha,25-Dihydroxyvitamin D3[1 alpha,25(OH)2D3], an active form of vitamin D, has roles in many biological phenomena such as calcium homeostasis and bone formation, which are thought to be mediated by the 1 alpha,25(OH)2D3 receptor (VDR), a member of the nuclear hormone receptor superfamily. However, the molecular basis for the actions of 1 alpha,25(OH)2D3 in bone formation, its role during development and VDR genetic polymorphisms for predicting bone mineral density are uncertain. To investigate the functional role of VDR, we generated mice deficient in VDR by gene targeting. We report here that in VDR null mutant mice, no defects in development and growth were observed before weaning, irrespective of reduced expression of vitamin D target genes. After weaning, however, mutants failed to thrive, with appearance of alopoecia, hypocalcaemia and infertility, and bone formation was severely impaired as a typical feature of vitamin D-dependent rickets type II (refs 8, 9). Unlike humans with this disease, most of the null mutant mice died within 15 weeks after birth, and uterine hypoplasia with impaired folliculogenesis was found in female reproductive organs. These defects, such as alopoecia and uterine hypoplasia, were not observed in vitamin D-deficient animals. The findings establish a critical role for VDR in growth, bone formation and female reproduction in the post-weaning stage.

Metabolic Syndrome and Cognitive Function: Cross-Sectional Study on Community-Dwelling Non-Demented Older Adults in Japan.

AIM: This is a cross-sectional study of relation between metabolic syndrome and cognitive function in community-dwelling non-demented older adults in Japan. We examine the effect of metabolic syndrome and its components on global cognitive function. We also aim to clarify differences of specific cognitive domains between the subjects with and without metabolic syndrome. METHODS: We studied 2150 subjects aged between 60 and 90 years whose scores on mini mental state examination (MMSE) were over 23 points. We analyzed difference in MMSE scores between the subjects with and without metabolic syndrome. Logistic regression analysis was performed with MMSE score as the dependent variable and metabolic syndrome components as the independent variable adjusted with age. We also examined differences in attention, logical memory, and verbal and category fluency between the subjects with and without metabolic syndrome. RESULTS: MMSE scores were not significantly different between subjects with and without metabolic syndrome. In logistic regression analysis, the score of MMSE was significantly negatively associated with triglycerides in males and significantly negatively associated with abdominal circumference in females. Subjects with metabolic syndrome showed significantly lower performance of attention tasks compared to subjects without metabolic syndrome. CONCLUSIONS: Our results suggest that in community-dwelling non-demented Japanese older adults, attention but not global cognitive function may be impaired by metabolic syndrome. Inverted association between some components of metabolic syndrome and global cognitive function indicate necessity of further studies on the relation between undernutrition and cognitive function.

Binding of hepatitis C virus to CD81.

Chronic hepatitis C virus (HCV) infection occurs in about 3 percent of the world's population and is a major cause of liver disease. HCV infection is also associated with cryoglobulinemia, a B lymphocyte proliferative disorder. Virus tropism is controversial, and the mechanisms of cell entry remain unknown. The HCV envelope protein E2 binds human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Binding of E2 was mapped to the major extracellular loop of CD81. Recombinant molecules containing this loop bound HCV and antibodies that neutralize HCV infection in vivo inhibited virus binding to CD81 in vitro.

Long-term protection in hamsters against human parainfluenza virus type 3 following mucosal or combinations of mucosal and systemic immunizations with chimeric alphavirus-based replicon particles.

No licensed vaccines are available to protect against parainfluenza virus type 3 (PIV3), a significant health risk for infants. In search of a safe vaccine, we used an alphavirus-based chimeric vector, consisting of Sindbis virus (SIN) structural proteins and Venezuelan equine encephalitis virus (VEE) replicon RNA, expressing the PIV3 hemagglutinin-neuraminidase (HN) glycoprotein (VEE/SIN-HN). We compared different routes of intramuscular (i.m.), intranasal (i.n.), or combined i.n. and i.m. immunizations with VEE/SIN-HN in hamsters. Six months after the final immunization, all hamsters were protected against live PIV3 i.n. challenge in nasal turbinates and lungs. This protection appeared to correlate with antibodies in serum, nasal turbinates and lungs. This is the first report demonstrating mucosal protection against PIV3 for an extended time following immunizations with an RNA replicon delivery system.

Intron retention generates a novel isoform of the murine vitamin D receptor that acts in a dominant negative way on the vitamin D signaling pathway.

We identified and characterized a novel rat vitamin D receptor isoform (rVDR1), which retains intron 8 of the canonical VDR (rVDR0) during alternative splicing. In this isoform protein directed by the stop codon in this newly identified exon, a part of the ligand binding domain (86 amino acids) is truncated at the C-terminal end but contains 19 extra amino acids. The rVDR1 transcript was expressed at a level 1/15 to 1/20 of that of rVDR0 in the kidney and intestine in adult rats but not in embryos. The recombinant rVDR1 protein showed no ligand binding activity. Homo- and heterodimers of the recombinant rVDR0 and rVDR1 proteins bound to a consensus vitamin D response element (VDRE) but not to consensus response elements for thyroid hormone and retinoic acid. However, unlike rVDR0, rVDR1 did not form a heterodimeric complex with RXR on the VDRE. A transient expression assay showed that this isoform acted as a dominant negative receptor against rVDR0 transactivation. Interestingly, the dominant negative activities of rVDR1 differed among VDREs. Thus, the present study indicates that this new VDR isoform negatively modulates the vitamin D signaling pathway, through a particular set of target genes.

Aberrant growth plate development in VDR/RXR gamma double null mutant mice.

VDR forms heterodimers with one of three RXRs, RXR alpha, RXR beta, and RXR gamma, and it is thought that RXR ligands can also modulate the trans-activation function of VDR/RXR heterodimers. In the present study we generated VDR/RXR gamma double null mutant mice to examine the convergent actions of vitamin D and vitamin A signaling and to explore the possibility of a functionally redundant VDR. Although RXR gamma(-/-) mice exhibited no overt abnormalities, VDR(-/-)/RXR gamma(-/-) mice appeared similar to VDR(-/-) mice, showing features typical of vitamin D-dependent rickets type II, including growth retardation, impaired bone formation, hypocalcemia, and alopecia. However, compared to VDR(-/-) mice, growth plate development in VDR(-/-)/RXR gamma(-/-) mutant mice was more severely impaired. Normalizing mineral ion homeostasis through dietary supplementation with high calcium and phosphorous effectively prevented rachitic abnormalities, except for disarranged growth plates in VDR(-/-)/RXR gamma(-/-) mutant mice, and alopecia in both VDR(-/-) and VDR(-/-)/RXR gamma(-/-) mutant mice. Histological analysis of VDR(-/-)/RXR gamma(-/-) growth plates revealed that development of the hypertrophic chondrocytes was selectively impaired. Thus, our findings indicated that the combined actions of VDR- and RXR gamma-mediated signals are essential for the normal development of growth plate chondrocytes, and raised the possibility that a functionally redundant VDR is present on chondrocytes as a heterodimer with RXR gamma.

The small extracellular loop of CD81 is necessary for optimal surface expression of the large loop, a putative HCV receptor.

Human tetraspanin CD81 is a putative receptor for hepatitis C virus (HCV), because it has been shown to bind 'bona fide' HCV particles. CD81, as all tetraspanins, spans the membrane four times forming two extracellular loops: a small (SEL) and a large one (LEL). We have shown previously that a recombinant form of LEL is sufficient for binding HCV through the major envelope glycoprotein E2. The role of SEL in the CD81-HCV interaction was questioned. We found that transfectants expressing LEL alone bind the recombinant HCV-E2 protein at much lower levels than cells expressing the wild type CD81. And therefore whether SEL contributes to the CD81-HCV interaction or whether it influences the expression of LEL was examined. We have found that in the absence of SEL, LEL is expressed at significantly reduced levels on the cell surface because it is retained intracellularly, while HCV-E2 still binds LEL. Our data suggest that SEL of CD81 does not mediate interaction with HCV, but contributes to optimal cell surface expression of LEL by mediating translocation of the whole CD81 molecule to the cell surface.

Hepatocellular carcinoma with tumor thrombus extending into the right atrium: report of a successful resection with the use of cardiopulmonary bypass.

Hepatocellular carcinoma with a tumor thrombus extending into the right atrium has been considered beyond the reach of resection. These patients usually die within a short period because of pulmonary embolism, heart failure, or cancer progression. The only treatment is hepatic resection with removal of the tumor thrombus. A 38-year-old woman underwent left lobectomy with removal of the tumor thrombus with the use of cardiopulmonary bypass. The patient had an uneventful course and is doing well 15 months after surgery, without signs of recurrence. We have proved that hepatic resection with removal of a tumor thrombus extending into the right atrium can be carried out successfully. The next problem is whether the lives of these patients can be prolonged by this operation.

Long-lasting salivation induced by a novel muscarinic receptor agonist SNI-2011 in rats and dogs.

The sialogogic effect of SNI-2011, a novel muscarinic receptor agonist, (+/-)-cis-2-methylspilo [1,3-oxathiolane-5,3'-quinuclidine] hydrochloride, hemihydrate, was compared with that of pilocarpine hydrochloride in a dose range in which the two muscarinic agonists exhibited approximately similar efficacy in eliciting salivation. Pilocarpine (0.66-2.0 mg/kg, i.d.) induced a marked but short-lasting salivation in rats, whereas the salivation induced by SNI-2011 (20-60 mg/kg, i.d.) lasted 1.4- to 1.8-fold longer. In dogs, the sialogogic effect of SNI-2011(1-3 mg/kg, i.v.) also lasted about 2-fold longer than that of pilocarpine (0.1-0.3 mg/kg, i.v.). The plasma SNI-2011 level that caused salivation at a rate of 0.4 ml/min was about 100 ng/ml and higher rates of salivation (over 0.4 ml/min) induced by 1 mg/kg SNI-2011 lasted for about 90 min in dogs. The plasma pilocarpine level that caused salivation at a rate of 0.4 ml/min was about 25 ng/ml and the higher rate of salivation (over 0.4 ml/min) induced by 0.1 mg/kg pilocarpine lasted only for 20 min in dogs. Effective plasma levels of SNI-2011 persisted longer than those of pilocarpine. These results indicate that SNI-2011 may be useful in the treatment of xerostomia because of its long-lasting sialogogic action.

Hemorrhagic infarction after vasospasm due to ruptured cerebral aneurysm.

Hemorrhagic infarction after vasospasm is a rare condition in patients with aneurysmal subarachnoid hemorrhage (SAH). Induced hypertensive therapy is used for patients with vasospasm, but this treatment has a risk of inducing hemorrhagic infarction. A total of 221 patients whose first computed tomographic (CT) scans were examined within 2 weeks after SAH were investigated for this study. There was symptomatic vasospasm in 99 (45%), cerebral infarction in 37 (17%), and hemorrhagic infarction in 13 (6%). Hemorrhagic infarction usually occurred 20 to 30 days after aneurysmal rupture; this period corresponds with the remission stage of the vasospasm. On CT scans, the hemorrhagic infarction was revealed as a leaky hemorrhage in a low density area in 11 cases, and a massive hemorrhage with mass effect was seen in 2 cases. These findings suggest that hemorrhagic infarction after vasospasm may sometimes be fatal. Cerebral blood flow autoregulation in patients with vasospasm was normal or of a hypertensive type during the remission stage of vasospasm, when hemorrhagic infarction usually appeared. This finding shows that induced hypertension therapy is ineffective during this stage; it should be stopped by this stage because it is ineffective and also may aggravate hemorrhagic infarction.

A possible involvement of central atrial natriuretic peptide in cerebral cortical microcirculation.

The possible involvement of atrial natriuretic peptide (ANP) in cerebral cortical microcirculation was investigated in rats by means of laser-Doppler flowmetry and immunohistochemistry. In the laser-Doppler study, local cerebral blood flow (LCBF) changes after the administration of 10(-6) to 10(-8) mol/LANP solution or vehicle (saline solution) as an intracortical injection for 5 minutes were continuously monitored throughout the 30 minutes of the study and were expressed as percentages of preinjection values represented as 0%. The administration of 10(-6) to 10(-8) mol/LANP caused a significant decrease in LCBF; the onset of LCBF responses occurred within a few minutes after the start of the injection and the decrease in LCBF reached the maximum level within 7 to 10 minutes after the completion of the administration, after which LCBF gradually recovered. In the immunohistochemical study, no specific ANP immunoreactivity was found associated with the intraparenchymal blood vessels; however, ANP-immunoreactive neurons were observed primarily in the hypothalamus and septum, in which high concentrations of ANP-containing neurons have been identified. The data from the laser-Doppler study suggest that central ANP may produce a vasoconstriction of the intraparenchymal blood vessels, regardless of whether through direct action on these vessels or through the mediation by some system in the central nervous system. Because there is no evidence for ANP-containing nerves around these vessels, the role of central ANP in the cerebral circulation must await identification of the source of perivascular ANP.

Pineoblastoma with an unusually long survival. Case report.

The authors report a case of pineoblastoma with a 9-year follow-up period after stereotaxic biopsy, a shunting procedure, and radiotherapy. Light and electron microscopic studies of biopsy and autopsy specimens revealed no cell differentiation of the pineoblastoma. The possible factors predisposing to long survival are discussed in comparison with the course in patients with medulloblastoma.

Bilateral morning glory syndrome associated with sphenoid encephalocele. Case report.

Morning glory syndrome is a congenital anomaly of the optic disc in which the disc is enlarged and excavated, with white glial tissue in the center. A case is presented of morning glory syndrome associated with sphenoid encephalocele, median cleft lip, and agenesis of the corpus callosum. A 22-day-old boy was referred to the Wakayama Medical College Hospital for management of dyspnea due to a soft-tissue mass in the oral cavity. Magnetic resonance imaging revealed a mass extending through a bone defect in the sphenoid region and into the oral cavity. Surgical repair was attempted through a bifrontal craniotomy. A bone defect was identified in the sphenoid plate, through which the arachnoid membrane was connected to the oral cavity. Both optic nerves were elongated and adhered to the encephalocele. The wall of the meningocele was compressed digitally through the oral cavity and sutured to the dura mater of the bone defect. The operative findings suggest that a basal encephalocele protruding from a bone defect in the sphenoid plate may disturb the normal development of the optic nerve.

Meningeal melanocytoma: magnetic resonance imaging characteristics and pathological features. Case report.

A case of meningeal melanocytoma at the foramen magnum is reported in a 62-year-old man. Magnetic resonance (MR) imaging revealed characteristic signal patterns: homogeneous high intensity on the T1-weighted image and low intensity on the T2-weighted image. Light microscopy showed a histological appearance similar to that of melanotic meningioma. The ultrastructural features of the neoplastic cells were compatible with those of melanocytes, but they contained no features of arachnoidal cells. Immunohistochemical bromodeoxyuridine study revealed low proliferative activity among the neoplastic cells. The MR appearance and pathological features in this rare case of meningeal melanocytoma are demonstrated and discussed.

Enhancement of the response to levodopa therapy after intrastriatal transplantation of autologous sympathetic neurons in patients with Parkinson disease.

OBJECT: There is growing evidence to indicate that tissue transplantation can potentially be a restorative neurosurgical treatment for patients with Parkinson disease (PD). In this study the authors investigated the clinical effect of unilateral intrastriatal grafting of autologous sympathetic neurons in patients with PD. METHODS: Four patients with PD who had been observed for 1 year after graft placement of autologous sympathetic neurons were selected for an analysis of the effect of that procedure. Sympathetic ganglion tissue was endoscopically excised from the thoracic sympathetic trunk and grafted into the unilateral caudate head and putamen of the PD patients. No changes were made in the patients' preoperative regimens of antiparkinsonian medications, and clinical evaluations were made principally according to those established by the Core Assessment Program for Intracerebral Transplantation Committee. Whereas the sympathetic neuron grafts failed to affect clinical scores reflecting the patients' motor performance, which was evaluated during either the "on" or "off' phases, the grafts significantly increased the duration of the levodopa-induced on period with consequent reduction in the percentage of time spent in the off phase. This beneficial effect may be explained by the results of the present in vitro experiment, which show that human sympathetic neurons have the ability to convert exogenous levodopa to dopamine and to store this synthesized dopamine. CONCLUSIONS: Sympathetic neuron autografts were found to improve performance status in patients with PD by reducing the time spent in the off phase. This clearly indicates that sympathetic ganglion tissue, the use of which involves few ethical issues, can be an efficacious donor source in cell transplantation therapy for PD. Further studies are needed to determine whether the grafts may provide long-lasting clinical benefits.

Molecular analysis of V(H)I+ B lymphocytes in hepatitis C patients.

BACKGROUND AND AIMS: Hepatitis C virus infection is often associated with lymphoproliferative disorders such as essential mixed cryoglobulinemia and B-cell non-Hodgkin lymphoma, which show preferential expression of VHI family products. By analyzing immunoglobulin heavy chain usage, we addressed the question of whether or not clonal B-cell expansion occurrs in patients free of essential mixed cryoglobulinemia or non-Hodgkin lymphoma. PATIENTS AND METHODS: Four hepatitis C virus-positive patients, all undergoing liver transplantation, were studied. Peripheral blood, intra-hepatic, and lymph node lymphocytes were used as a source of B cells. A patient with hepatocellular carcinoma and fresh blood from four healthy donors were used as negative controls. VHI family sequences were cloned and analyzed by reverse transcription-polymerase chain reaction. RESULTS: Immunoglobulin heavy chain sequences from clonally expanded B lymphocytes were identified in three out of four hepatitis C virus-infected patients. The clonally expanded B lymphocyte populations showed a broad spectra of immunoglobulin heavy chain gene usage. CONCLUSIONS: HCV infection can induce B-cell expansion with larger clonal variation. The restricted V gene usage in hepatitis C virus-associated non-Hodgkin lymphoma suggests that there may be selection mechanisms to develop non-Hodgkin lymphoma from non-malignant, clonally expanded B-cell populations in hepatitis C virus-infected patients.

Effective administration route for the deleted form of hepatocyte growth factor To exert its pharmacological effects.

The pharmacokinetics and the pharmacological effects of the deleted form of hepatocyte growth factor (dHGF) after intravenous (iv), subcutaneous (sc), or intramuscular (im) administration (0.25 and 2. 5 mg/kg) were studied in rats. After single iv administration (2.5 mg/kg), dHGF in serum rapidly decreased (alpha- and beta-phase half-life: 3.2 and 26.5 min, respectively). Two to four hours after single sc or im administration (2.5 mg/kg), the serum level of dHGF reached a maximum and then gradually declined (half-life: 2.7 h). The serum levels were not changed by repetitive iv administration, but were dramatically decreased by repetitive sc or im administration. Liver weight and serum levels of total protein, albumin, and HDL-cholesterol were significantly increased by iv administration of dHGF (twice daily for 4 days at 0.25 mg/kg). Sc or im administration of dHGF did not increase these parameters at the same dose, but did significantly at 2.5 mg/kg. These observations suggest that iv administration is the most effective in exerting the pharmacological effects of dHGF among three administration routes. dHGF after iv administration was distributed mainly and rapidly into liver (53.6% of the injected dHGF within 5 min) and was sustained at a higher level in the liver than in plasma. In infusion (0.5 mg/kg/3 h), dHGF level in plasma and liver reached a steady-state 15 and 60 min after starting the infusion, respectively. The steady-state level of dHGF was 7- to 9-fold higher in liver than in plasma, and the higher level in liver was sustained beyond the steady-state.

Radiculopathy after laminoplasty of the cervical spine.

STUDY DESIGN: The risk factors of patients with and without radiculopathy after laminoplasty of the cervical spine were compared retrospectively. OBJECTIVES: To study the association between risk variables and postlaminoplastic radiculopathy to clarify the pathogenesis of radiculopathy and to devise preventive measures. SUMMARY OF BACKGROUND DATA: Radiculopathy after cervical laminoplasty on the expanded side has been attributed mainly to traumatic surgical techniques, whereas radiculopathy on the hinged side has been attributed to traction, tethering, or kinking of the nerve root that has resulted from posterior shift of the spinal cord from the preoperative position. There is still much divergence of opinion concerning the risk factors for the outbreak as well as the prevention. METHODS: Of 365 patients who had undergone laminoplasty, 20 patients (5.5%) developed postoperative radiculopathy. Using data from postoperative computed tomography scans and other sources, these patients were compared with 211 patients with no radiculopathy, who had undergone laminoplasty during the same period, to identify risk factors related to patient characteristics and surgical techniques. RESULTS: Of various risk factors studied, the narrowest level of the spinal canal, preoperative symptomatic severity, flatness of the spinal cord assessed by computed tomography myelography at C4-C5, cervical curvature, anterior protrusion of the superior articular process as assessed by computed tomography scan, laterality of the osteophytes, and ossification of the posterior longitudinal ligament could not significantly discriminate between patients with and without postoperative radiculopathy. The angle of lamina as measured by using computed tomography scans obtained after expansion in the patients with radiculopathy was greater than 68 degrees on the opened and hinged sides and was significantly greater than the angle in patients without radiculopathy (P < 0.05). The incidence of radiculopathy on both the opened and hinged sides was significantly higher in patients in whom the bony gutter had been cut on the lateral side of the medial aspect of the zygapophyseal joint. CONCLUSION: Any one of patients' characteristics could not be correlated with postoperative cervical radiculopathy in this study. To prevent postoperative radiculopathy, it may be important during surgery to place the bony gutter on the medial side of the zygapophyseal joint and to keep the slope of the opened lamina within 60 degrees.