Involvement of nitric oxide in glucose toxicity on differentiated PC12 cells: prevention of glucose toxicity by tetrahydrobiopterin,a cofactor for nitric oxide synthase

Effects of high concentrations of glucose on cell survival of differentiated PC12 cells were examined. Seven day-culture with D-glucose (9.0-27.0 mg/ml as 2-6-fold of the optimal level) induced cell death in a dose-related manner but 3-day culture with high concentrations of glucose had no effect on cell viability. L-glucose had no effect on viability of PC12 cells, suggesting that D-glucose toxicity was independent of its osmolarity effect. Seven-day culture with D-glucose (13.5 mg/ml as 3-fold of the optimal level) increased nitric oxide metabolites (NOx) in the culture medium. Glucose-induced increase in NOx was eliminated by 0.1 mM L-nitro-arginine methylester (L-NAME), a nitric oxide synthase (NOS) inhibitor. Intracellular Ca(2+) concentration was increased by D-glucose in a dose-related manner, suggesting that D-glucose activated NOS by increasing intracellular Ca(2+) concentration in PC12 cells. Glucose-induced cell death was blunted by 0.1 mM L-NAME, showing that nitric oxide (NO) was involved in the glucose toxicity to PC12 cells. Tetrahydrobiopterin (BH(4)), a cofactor for NOS, attenuated both glucose-induced cell death and NOx production at 1 microM but not at 10 microM. The effects of BH(4) on glucose-induced cell death and NOx production were not mimicked by reducing agents such as ascorbate and cysteine. These results taken together suggest that high concentrations of glucose induced cell death via NO production and that low concentration of BH(4) had a protective effect against glucose neurotoxicity in differentiated PC12 cells.     

Primary renal angiosarcoma: A case report and review of the literature

Primary renal angiosarcoma is very rare. To our knowledge, only 15 cases have been reported to date. A 77-year-old Japanese man with a unilateral kidney presented with massive hematuria followed by renal failure. A renal tumor was suspected and a left nephrectomy was performed. The histopathological diagnosis was angiosarcoma of the kidney. A hemorrhagic tumor measuring 10 × 5 cm and clotted blood was found in the modularly area. The atypical tumor cells had a sinusoidal and solid appearance, and showed Immunohistochemically positive reactions for some of the endothelial markers. The patient died about 21 months after the nephrectomy and the autopsy revealed massive metastases to the liver and retroperitoneum. One of the differential diagnoses of the case was anglomyolipoma, because the tumor cells were relatively bland in their histological appearance with entrapped fat cells in the pelvic area. Fifteen case reports with titles that included the term 'hemangiosarcoma/anglosarcoma', 'hemangioendothelloma/endothelloma' or 'vascular sarcoma' of the kidney were reviewed and compared to the present case.     

Quantitative ultrastructure of synapses on functionally identified primary afferent neurons in the cat trigeminal mesencephalic nucleus

Though a number of studies have reported the presence of synapses on neurons in the trigeminal mesencephalic nucleus (Vmes), there have been no quantitative studies of either the density of innervation, or the ultrastructure, of the synapses on single, physiologically identified neurons in this nucleus. In this study we recorded from single neurons in the Vmes, identified them as being either muscle spindle afferents (MS) or periodontal ligament mechanoreceptor afferents (PL), and then labeled the neurons by intra-axonal injection of horseradish peroxidase (HRP). The material was first processed to reveal the HRP activity, following which ultrathin sections through the labeled somata were cut and examined under the electron microscope. Complete serial reconstructions were made through the soma of one MS neuron and one PL neuron, and the contacts on the neurons reconstructed. Boutons were found on the soma, spines, appendages and the axon hillock and the initial segment of the axon. The numbers of boutons terminating on the two neurons were 198 (PL) and 424 (MS), giving a packing density of 4.4 and 10.7 boutons respectively (i.e., number of boutons/100 micron 2 of the postsynaptic membrane). Boutons could be separated into two types on the basis of their vesicles: those containing clear, round vesicles (i.e., S-type) and those containing a mixture of round, oval and flattened vesicles (P-type). Ninety-five (PL neuron) and 99% (MS neuron) of terminals on the two neurons were P-type. All the S-type boutons and 80% of the P-type boutons formed asymmetric synaptic contacts while 10% of the P-type boutons made symmetric contacts. Quantitative measurements of the P-type boutons on the labeled neurons, in which the data of MS and PL neurons were pooled, revealed that bouton volume was highly correlated with bouton surface area, active zone number, total active zone area, vesicle number, and mitochondrial volume. However, comparing the quantitative measurements of the P-type boutons with those of previously reported vibrissa afferent terminals and their associated axon terminals revealed that all the parameters were smaller for the P-type boutons (on Vmes neurons) than those of the vibrissa afferent terminals but similar to those of axon terminals presynaptic to the vibrissa afferents. Taken together, our results emphasize the wide scope for synaptic interactions in the Vmes and suggest that it may be more fruitful to view the Vmes as an integrating center.     

Microfilamentous type VI collagen in the hyalinized stroma of the hypertrophied ligamentum flavum

Summary Thickened ligamenta flava obtained from 14 patients with spinal canal stenosis were examined with special reference to type VI collagen. The characteristic histological finding in the thickened area was rupture of normal elastic fibre meshwork with resultant fibrosis which usually appeared hyaline. Using an immunohistological method, collagen types VI, I and III were found to be present in the hyaline matrix. Ultrastructural study revealed many microfilamentous structures of type VI collagen admixed in loosely packed, banded collagen fibres. With differential salt precipitation of pepsin-extracted collagen the existence of type VI collagen was confirmed by SDS-polyacrylamide gel electrophoresis analysis and Western blotting analysis using anti-type VI collagen antibody. Quantification of type VI collagen in pepsin-extracted crude collagen samples by an inhibition enzyme-linked immunosorbent assay showed an increasing amount of type VI collagen in the thickened ligamenta flava compared to the normal ligaments. Thus, increase of type VI collagen is the main contribution to the thickening of the ligamentum flavum. This may represent an adaptational and reparative process associated with disruption of elastic fibres.     

Sinus Histiocytosis With Massive Lymphadenopathy A Histogenic Analysis of Histiocytes Found in the Fourth Japanese Case

The present paper deals with immunohistochemical and ultrastructural study of the lymph nodes of sinus histiocytosis with massive lymphadenopathy (Rosai and Dorfman, SHML) of a 12-year-old Japanese boy. This is the fourth case in Japan. Osseous manifestation was also found in the bilateral ulnae. With hallmarks of S-100 protein and interdigitating cytoplasmic extensions, the phagocytizing histiocytes proliferating in the sinuses were considered to be derived mostly from interdigitating cells in the paracortex or T cell dependent area, which have heretofore been regarded as nonphagocytizing. Furthermore, it is most interesting that lymphoid cells bearing thymic cortical cell-antigen (OKT 6) were increasingly recognized in the patient's peripheral blood. These results suggested that SHML is a specialized reactive histiocytosis analogous to histiocytosis X and histiocytic medullary reticulosis.     

Pathogenesis of Rinderpest Virus Infection in Rabbits II. Effect of Rinderpest Virus on the Immune Functions of Rabbits

Rinderpest virus infection was shown to induce marked suppression of both humoral antibody response and cell-mediated immunity in rabbits. The virus exhibited a suppressive effect on primary antibody response as indicated by a decrease in numbers of plaque-forming cells (immunoglobulin [Ig]M) and hemagglutinating antibody titers of both IgM and IgG types to sheep red blood cells, whereas there was no detectable effect of the virus on the production of memory cells. Virus-induced suppression of cell-mediated immunity was demonstrated by a decreased rate of proliferative response of peripheral lymphocytes to phytohemagglutinin stimulus and by a depression of delayed-type skin reactions to purified protein derivative. Such suppressive effects were indicated to persist for 14 days or longer. Alteration in phagocytic activity of the reticuloendothelial system was not observed. The relevance of the virus-induced histological lesions in the lymphoid tissues to the virus-induced immunosuppression was discussed.     

Identification and characterization of temperature-sensitive mild mutations in three Japanese patients with nonsevere forms of very-long-chain acyl-CoA dehydrogenase deficiency

Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is clinically classified into severe, intermediate, and myopathic forms. We identified mutations in three unrelated Japanese patients with VLCAD deficiency: two with the myopathic form and one with the intermediate form, all compound heterozygotes of K264E/M437V, A416T/1798delA, and P89S/IVS16-3delAA, respectively. We characterized four missense mutations, K264E, M437V, A416T, and P89S, by transisent expression analysis, using SV40-transformed fibroblasts derived from a VLCAD-null patient, as recipient cells. In transient expression of the wild-type VLCAD cDNA, VLCAD activity at 30 degrees C was higher than at 37 degrees C. Moreover, this temperature-sensitive character is more evident in all the mutant proteins tested than in wild type. Based on characterization of the five missense mutations identified in four Japanese patients, including data on one patient with the myopathic form previously reported, patients with the nonsevere forms (intermediate or myopathic forms) have missense mutations with residual activities in at least one allele. Expression analysis at 30 degrees C may be more useful for evaluating these missense mutations, compared with that at 37 degrees C.     

No association of GSK3beta gene (GSK3B) with Japanese schizophrenia.

Several lines of evidence indicate that glycogen synthase kinase-3beta (GSK3beta) is one of the candidates for schizophrenia-susceptibility factor. However, it has not been reported the association analysis between GSK3beta gene (GSK3B) and Japanese schizophrenia based on linkage disequilibrium (LD). We provide an association analysis using relatively large samples (381 schizophrenia, and 352 controls) after determination of "tag single nucleotide polymorphisms (SNPs)." In this LD mapping, we selected and genotyped for eight polymorphisms (seven SNPs and one diallelic (CAA)(n) repeat), which covered the entire region of GSK3B, and determined two "tag SNPs." In the following association analysis using these two "tag SNPs," we could not find association with Japanese schizophrenia. Furthermore, we also include subgroup analysis considering age-at-onset and subtypes, neither could we find associations. Because our samples provided quite high power, these results indicate that GSK3B may not play a major role in Japanese schizophrenia.     

Ridge filter design for proton therapy at Hyogo Ion Beam Medical Center

At the Hyogo Ion Beam Medical Center (HIBMC) we have developed a new design method for the bar ridge filter used in proton therapy, taking into consideration the scattering and nuclear interaction effects within the filter itself, which are introduced in the design. In our beam delivery system, the bar ridge filter is employed as the range modulator. It is combined with the wobbler system, and produces a three-dimensionally uniform spread-out Bragg peak (SOBP). The design program predicts the three-dimensional dose distribution. Ridge filters of 3-12 cm SOBP in 1 cm increments were designed in the maximum radiation field for 150 MeV and 190 MeV proton beams so that a uniform physical dose area is obtained in the SOBP region three-dimensionally. Measurements were performed with the constructed ridge filters to verify the uniformity and these were compared with the predictions of the design program. The predictions and measurements were found to be in agreement except for the 12 cm SOBP. The uniformities were better than +/- 3.0% for all SOBPs produced. The ridge filters are now clinically in use.