Kanamycin ototoxicity in glutamate transporter knockout mice

Glutamate-aspartate transporter (GLAST), a powerful glutamate uptake system, removes released glutamate from the synaptic cleft and facilitates the re-use of glutamate as a neurotransmitter recycling system. Aminoglycoside-induced hearing loss is mediated via a glutamate excitotoxic process. We investigated the effect of aminoglycoside ototoxicity in GLAST knockout mice using the recorded auditory brainstem response (ABR) and number of hair cells in the cochlea. Kanamycin (100 mg/mL) was injected directly into the posterior semicircular canal of mice. Before the kanamycin treatment, there was no difference in the ABR threshold average between the wild-type and knockout mice. Kanamycin injection aggravated the ABR threshold in the GLAST knockout mice compared with the wild-type mice, and the IHC degeneration was more severe in the GLAST knockout mice. These findings suggest that GLAST plays an important role in preventing the degeneration of inner hair cells in aminoglycoside ototoxicity.     

Repair of infarcted myocardium mediated by transplanted bone marrow-derived CD34+ stem cells in a nonhuman primate model

Rodent and human clinical studies have shown that transplantation of bone marrow stem cells to the ischemic myocardium results in improved cardiac function. In this study, cynomolgus monkey acute myocardial infarction was generated by ligating the left anterior descending artery, and autologous CD34(+) cells were transplanted to the peri-ischemic zone. To track the in vivo fate of transplanted cells, CD34(+) cells were genetically marked with green fluorescent protein (GFP) using a lentivirus vector before transplantation (marking efficiency, 41% on average). The group receiving cells (n = 4) demonstrated improved regional blood flow and cardiac function compared with the saline-treated group (n =4) at 2 weeks after transplant. However, very few transplanted cell-derived, GFP-positive cells were found incorporated into the vascular structure, and GFP-positive cardiomyocytes were not detected in the repaired tissue. On the other hand, cultured CD34(+) cells were found to secrete vascular endothelial growth factor (VEGF), and the in vivo regional VEGF levels showed a significant increase after the transplantation. These results suggest that the improvement is not the result of generation of transplanted cell-derived endothelial cells or cardiomyocytes; and raise the possibility that angiogenic cytokines secreted from transplanted cells potentiate angiogenic activity of endogenous cells.     

Clinical application of rapid assay of interleukin-6 in influenza-associated encephalopathy

The characteristics of influenza-associated encephalopathy is the high mortality and nimble progress with coma which appears in general cases within 48 hours. Most of patients show no abnormalities in the standard blood checks on admission or in early stage. In this study we investigated if a rapid assay of interleukin (IL)-6 is useful in influenza-associated encephalopathy in early stages. The levels of IL-6 in patients with influenza-associated encephalopathy did not show any significant difference compared with those in patients with febrile convulsion and rotavirus-associated convulsion. However the levels of IL-6 in severe cases were significantly higher than those of mild cases with influenza-associated encephalopathy. Consequently the rapid assay of serum IL-6 is useful to evaluate and decide the therapies.     

Gaps and fragmentation of the superficial cortex in the abdominal and pelvic lymph nodes of elderly Japanese

Gaps and fragmentation of the superficial lymph node cortex are considered to provide intranodal shunt flow between the afferent and efferent vessels. Using serial sections of 205 nodes obtained from 27 donated cadavers more than 70 years of age, we examined the histological architecture of the abdominal and pelvic nodes in elderly Japanese. Secondary follicles were rare in the specimens. Cortex gaps were, to a greater or lesser degree, found in all nodes. We classified these nodes into three types according to how often the gap occurred. Type 1 nodes, with a relatively complete shield for the afferent lymph, were most frequently found in gastric nodes, whereas type 3 nodes, with numerous gaps, were often observed in the colic, para-aortic and pelvic nodes. The type 3 nodes showed a specific architecture characterized by a fragmented superficial cortex, three-dimensionally assembled cords and a common sinus between them. Primary follicles were located in the assembled cord structures as well as at the superficial cortex. Irrespective of the type, B and T lymphocyte areas were intermingled in the cortex-like areas. The present results reveal region-specific histological heterogeneity in aged human visceral nodes. Due to increased surface areas, the type 3 architecture seemed to accelerate systemic immunity rather than act as a local barrier in the para-aortic and pelvic nodes, which are located centrally along the lymphatic drainage routes. However, thick trabeculae often seemed to develop in the type 3 sinus to decrease nodal function with aging.     

Characterization of a G14 equine rotavirus (strain CH3) isolated in Japan

Summary Antigenic and genomic properties of equine rotavirus strain CH3 isolated in Japan were studied by cross-neutralization tests and nucleotide sequence determination of the VP4 and VP7 genes. It was shown that the strain CH3 belongs to G14 and shares VP4 genotype with strain H2.The nucleotide sequence data reported in this paper appear in the DDBJ, EMBL and GeneBank nucleotide sequence detabases under the accession numbers D25228 (VP4 of strain CH3) and D25229 (VP7 of strain CH3).     

Biological activities of Bacteroides forsythus lipoproteins and their possible pathological roles in periodontal disease

Bacteroides forsythus is a gram-negative, anaerobic, fusiform bacterium and is considered to be an etiological agent in periodontal disease. A lipoprotein fraction prepared from B. forsythus cells by Triton X-114 phase separation (BfLP) activated human gingival fibroblasts and a human monocytic cell line, THP-1, to induce interleukin-6 production and tumor necrosis factor alpha production. BfLP was found to be capable of inducing nuclear factor-kappaB translocation in human gingival fibroblasts and THP-1 cells. By using Chinese hamster ovary K1 cells transfected with Toll-like receptor genes together with a nuclear factor-kappaB-dependent CD25 reporter plasmid, it was found that signaling by BfLP was mediated by Toll-like receptor 2 but not by CD14 or Toll-like receptor 4. BfLP induced apoptotic cell death in human gingival fibroblasts, KB cells (an oral epithelial cell line), HL-60 cells (a human myeloid leukemia cell line), and THP-1 cells but not in MOLT4 cells (a T-cell leukemia cell line). Caspase-8, an initiator caspase in apoptosis, was found to be activated in these cells in response to BfLP stimulation. Thus, this study suggested that BfLP plays some etiological roles in oral infections, especially periodontal disease, by induction of cell activation or apoptosis.     

The ultrasound examination of the deep vein thrombosis

Ultrasonography is very useful for detection of deep vein thrombosis. The purpose of this paper is to show a method for detecting them efficiently by high resolution transducer and color Doppler system. We examined patients in the supine and prone positions. To detect the venous flow easily and differentiate thrombi from simple venous dilatation, some maneuvers are useful; one is pushing the vein area using the transducer on examination, the second is breathing overload, and the last is so-called milking. We can find throombi in the external iliac or femoral veins of patients who have symptoms of lower leg swelling, however, we need to better detect venous thrombi in the lower leg in patients with a history of pulmonary embolism. Because deep venous thrombi are increasing, the role of ultrasound will expand in the future.     

Production and release of infectious hepatitis C virus from human liver cell cultures in the three-dimensional radial-flow bioreactor

Lack of efficient culture systems for hepatitis C virus (HCV) has been a major obstacle in HCV research. Human liver cells grown in a three-dimensional radial-flow bioreactor were successfully infected following inoculation with plasma from an HCV carrier. Subsequent detection of increased HCV RNA suggested viral replication. Furthermore, transfection of HCV RNA transcribed from full-length cDNA also resulted in the production and release of HCV virions into supernatant. Infectivity was shown by successful secondary passage to a new culture. Introduction of mutations in RNA helicase and polymerase regions of HCV cDNA abolished virus replication, indicating that reverse genetics of this system is possible. The ability to replicate and detect the extracellular release of HCV might provide clues with regard to the persistent nature of HCV infection. It will also accelerate research into the pathogenicity of HCV, as well as the development of prophylactic agents and new therapy.     

Co-expression in CHO cells of two muscle proteins involved in excitation-contraction coupling

Summary Ryanodine receptors and dihydropyridine receptors are located opposite each other at the junctions between sarcoplasmic reticulum and either the surface membrane or the transverse tubules in skeletal muscle. Ryanodine receptors are the calcium release channels of the sarcoplasmic reticulum and their cytoplasmic domains form the feet, connecting sarcoplasmic reticulum to transverse tubules. Dihydropyridine receptors are L-type calcium channels that act as the voltage sensors of excitation-contraction coupling: they sense surface membrane and tranverse tubule depolarization and induce opening of the sarcoplasmic reticulum release channels. In skeletal muscle, ryanodine receptors are arranged in extensive arrays and dihydropyridine receptors are grouped into tetrads, which in turn are associated with the four subunits of ryanodine receptors. The disposition allows for a direct interaction between the two sets of molecules.CHO cells were stably transformed with plasmids for skeletal muscle ryanodine receptors and either the skeletal dihydropyridine receptor, or a skeletal-cardiac dihydropyridine receptor chimera (CSk3) which can functionally substitute for the skeletal dihydropyridine receptor, in addition to plasmids for the ₂, and subunits. RNA blot hybridization gave positive results for all components. Immunoblots, ryanodine binding, electron microscopy and exposure to caffeine show that the expressed ryanodine receptors forms functional tetrameric channels, which are correctly inserted into the endoplasmic reticulum membrane, and form extensive arrays with the same spacings as in skeletal muscle. Since formation of arrays does not require coexpression of dihydropyridine receptors, we conclude that self-aggregation is an independent property of ryanodine receptors. All dihydropyridine receptor-expressing clones show high affinity binding for dihydropyridine and immunolabelling with antibodies against dihydropyridine receptor. The presence of calcium currents with fast kinetics and immunolabelling for dihydropyridine receptors in the surface membrane of CSk3 clones indicate that CSk3-dihydropyridine receptors are appropriately targeted to the cell's plasmalemma. The expressed skeletal-type dihydropyridine receptors, however, remain mostly located within perinuclear membranes. In cells coexpressing functional dihydropyridine receptors and ryanodine receptors, no junctions between feet-bearing endoplasmic reticulum elements and surface membrane are formed, and dihydropyridine receptors do not assemble into tetrads. A separation between dihydropyridine receptors and ryanodine receptors is not unique to CHO cells, but is found also in cardiac muscle, in muscles of invertebrates and, under certain conditions, in skeletal muscle. We suggest that failure to form junctions in co-transfected CHO cell may be due to lack of an essential protein necessary either for the initial docking of the endoplasmic reticulum to the surface membrane or for maintaining the interaction between dihydropyridine receptors and ryanodine receptors. We also conclude that formation of tetrads requires a close interaction between dihydropyridine receptors and ryanodine receptors.