Golgi study on macular mutant mouse after copper therapy

Summary This study was undertaken to elucidate the clinical and neuropathological effects of copper administration on the macular mutant mouse. Its hemizygote, which is considered to be a model of Menkes kinky hair disease (MKHD), was injected intraperitoneally four times with 10, 20, 20 and 30 g of cupric chloride on days 4, 6, 8 and 10, respectively. The hemizygote's curly whiskers gradually straightened and the frequent tonic seizures and ataxia disappeared after the injections. The body weight also gradually increased. In the cerebral cortex, the dendritic arborization of the pyramidal neurons in both the normal littermate and the treated hemizygote developed with time and reached the maximum around day 60. In the treated hemizygote, however, the arborization of the dendrites was significantly poor in comparison with that in the normal littermate from day 20 to 90. In the cerebellum of the treated hemizygote, the abnormal Purkinje cells with the few somal sprouts, thick stem dendrite and/or poor arborization, which were seen in the non-treated hemizygote, were improved by day 30, while their focal dendritic swellings remained even on day 60. These results indicate that the copper therapy improves not only the clinical manifestations but also the neuropathological changes, especially in the cerebellum.Supported in part by Grant no. 86-05-02 from the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare, Japan     

Mutation (D472Y) in the type 3 repeat domain of cartilage oligomeric matrix protein affects its early vesicle trafficking in endoplasmic reticulum and induces apoptosis

Cartilage oligomeric matrix protein (COMP) is a large pentameric extracellular glycoprotein found in cartilage, tendon, and synovium, and plays structural roles in cartilage as the fifth member of the thrombospondin family. Familial mutations in type 3 repeats of COMP are known to cause pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). Although such mutations induce enlarged rough endoplasmic reticulum (rER) as a morphological change, the metabolic trafficking of mutated COMP remains unclear. In transfected COS7 cells, wild-type COMP was rapidly secreted into culture medium, while the great majority of COMP with the type 3 repeats mutation (D472Y) remained in the cells and a small portion of mutated COMP was secreted. This finding was followed up with a confocal study with an antibody specific to COMP, which demonstrated mutated COMP tightly associated with abnormally enlarged rER. Phosphorylated eIF2alpha, an ER stress protein, was expressed as a pathological reaction in virtually all COS7 cells expressing mutated but not wild-type COMP. Moreover, COS7 cells expressing mutated COMP exhibited significantly more apoptotic reaction than those expressing wild-type COMP. Pathological accumulation of COMP in rER and apoptosis in COS7 cells that were induced by the mutation (D472Y) in COMP imply that COMP mutations play a role in the pathogenesis of PSACH.     

Z-line structural diversity in frog single muscle fiber in the passive state

The structural changes of the Z-line between small square net (ss) and basket weave (bw) cross-sectional patterns were examined using intact single fibers and mechanically skinned fibers in the passive state to determine if the pattern is related to the sarcomere length (SL) and if the pattern undergoes a reversible transition in low- and high-osmotic medium.Frog single fibers were isolated from the anterior tibial muscle in Ringer's solution. Entirely or partially skinned single fibers were prepared in relaxing solution (also called low-osmotic medium).The high osmotic medium contained 10% polyvinylpyrrolidone (PVP) in relaxing solution.The sarcomere length (SL) of each fiber was measured directly by use of a laser beam or indirectly from electron micrographs with use of a correction factor. The ss and bw forms in cross sections were quantified by analysis of electron micrographs. The results show that the structural change of Z-line occurs around bw 2.3–2.4m ss (n = 25) and bw 3.1–3.2m ss (n = 13) in intact single fibers and skinned fibers, respectively. With the quick freeze-freeze substitution method, an intact single fiber with a SL of 2.35m showed almost 100% of ss form. The structural transition in cross section was also confirmed in four partially skinned fibers, where patterns went from mostly ss form (intact portion) to mostly bw form (skinned portion) at the SL between 2.40 to 3.20m.The reversibility of the change between ss and bw was proved by using low- and high-osmotic medium. The transition and reversion of cross-sectional patterns both occur in the passive state.     

Molecular characterisation of glutamate dehydrogenase gene defects in Japanese patients with congenital hyperinsulinism/hyperammonaemia

Congenital hyperinsulinism and hyperammonaemia (CHH) is caused by dysregulation of glutamate dehydrogenase (GDH). We characterised the GDH gene in two Japanese patients with CHH. Patient 1 showed late-onset and mild hypoglycaemic episodes and mild hyperammonaemia, compared with patient 2. In GDH activity of lymphoblasts, patient 1 showed twofold higher basal GDH activity than control subjects and mild insensitivity for GTP inhibition. Patient 2 showed severe insensitivity for GTP inhibition, and similar allosteric stimulation by ADP in the controls. Genetic studies identified heterozygous and de novo L413V and G446D mutations in patients 1 and 2, respectively. COS cell expression study confirmed that both mutations were disease-causing gene. The insensitivity for GTP inhibition in L413V and G446D was emphasised in COS cell expression system as a result of the dosage effect of mutant GDH gene. L413V showed less impairment of GDH than G446D based on biochemical and genetic results, which was consistent with the clinical phenotype. Based on the structure of bovine GDH, G446D was located in GTP binding site of pivot helix and its surroundings, while L413V was located in alpha-helix of antenna-like structure. These different locations of mutations gave different effects on GDH enzyme. The antenna-like structure plays an important role in GDH activity.     

Enkephalin-like immunoreactive neurons and fibers in the ventromedial hypothalamic nucleus

Enkephalin-like immunoreactive (ENK-IR) neurons and fibers in the rat ventromedial hypothalamic nucleus (VMH) were examined by light and electron microscopy using the peroxidase-antiperoxidase immunocytochemistry. There were groups of ENK-IR neurons present in the ventrolateral part of the VMH, and such neurons were scattered elsewhere. These neurons had perikarya 10-25 microns in diameter with moderately developed cell organelles and enfolded nuclei that were often distributed eccentrically placed in the cell. The perikarya and dendrites contained diffuse, large-cored vesicles (LCV) (60-230 nm with a predominance in the 60-80 nm). ENK-IR neurons received synaptic inputs on the soma and dendrites from unlabeled axonal boutons containing many small, clear vesicles and occasional LCV. The ultrastructural features of the ENK-IR cells in the VMH seemed to correspond to the "common cells" described by Millhouse. Dense ENK-IR fibers were distributed in this nucleus throughout the rostrocaudal and ventrodorsal areas. Axonal boutons containing numerous small, clear vesicles, and dispersed LCV generally made synaptic contacts with the cell bodies and dendrites of unlabeled neurons. The findings suggest that opioid peptides directly influence VMH neurons through synaptic contacts.     

Study on the developing potentiality of mouse morula produced in vitro or in vivo after vitrification

Great horovement haS been made in the cryopreservation Of inalnlnalian elnbmp since wnttingham['] reported his Pioneering wold about successful cryOPreservationof mouse embryo. It results in the availability of cryOPreserVation of embryos of all stages[']. HOwever, cryopreserved embryos can not avoid the damages by ice crystal,which is a major cause of cen death in the freezing andthawing ProceduresL',']. SO it is necesseq to seareh for anew and more efficient method for embryO cryOPrese…     

Suppression of neointimal thickening by a newly developed HMG-CoA reductase inhibitor,BAYw6228, and its inhibitory effect on vascular smooth muscle cell growth

1. The aim of this study was to determine whether BAYw6228 (BAYw), a newly developed 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, could suppress an atherogenic process such as intimal thickening by a mechanism other than lowering the level of serum cholesterol.
2. First, we evaluated the in vitro effect of BAYw on the proliferation of vascular smooth muscle cells (SMC) from various species: Sprague-Dawley (SD) rats, New Zealand (NZ) white rabbits, intimal cells from Watanabe hereditary hyperlipidemic (WHHL) rabbit and SMC from the new-born human aorta. The increasing rate of total protein content of these cells was inhibited by the addition of BAYw in a dose-dependent fashion. In the presence of 2% foetal calf serum (FCS), the value of IC₅₀ was 1.0 μM in SD rats. 2.1 μM in NZ white rabbits, and 0.3 μM in WHHL rabbits. With human SMC, the value was 0.02 μM in the presence of 10% FCS and 0.2 μM with a mixture of growth factors.
3. Based on these above in vitro findings, we next examined the in vivo effect of the agent to determine whether it could suppress rabbit intimal thickening induced by balloon catheterization. A balloon catheter was inserted from a peripheral branch of the left external carotid artery to the aorta to denude the endothelium of the left common carotid artery in Japanese white rabbits. After 12 days they were divided into control and BAYw groups. The former were subcutaneously injected with saline and the latter with BAYw 1 mg kg⁻¹ day⁻¹. Two days after the beginning of treatment, a second balloon injury was performed to the previously injured left common carotid artery in both groups. After another two weeks, the left common carotid artery was removed and variously stained. Although the total serum cholesterol in the BAYw group was significantly lower than in the control (P<0.05), the difference was not enough to affect intimal thickening. In addition, the BAYw group had a smaller intima/media ratio than the control group, decreasing to 45% of control (P<0.05). By anti-α smooth muscle actin antibody staining, these intimal thickening areas were entirely occupied by SMCs, and their amount was attenuated by BAYw. By anti-rabbit macrophage antibody (RAM 11) staining, the number of positive cells in the intimal thickening was markedly decreased in the BAYw group compared to control (P<0.01).
4. These results indicate that BAYw has an inhibitory effect on intimal thickening by attenuating intimal SMC proliferation and infiltration of macrophages, suggesting that BAYw could be effective in the prevention of the progression of atherosclerotic plaque-like restenosis after angioplasty.

     

The relation between superoxide dismutase in cancer tissue and clinico pathological features in breast cancer

The localization of Cu/Zn- and Mn-superoxide dismutase (SOD) in breast cancer tissue (12 papillotubular carcinomas, 21 solid-tubular carcinomas, 16 scirrhous carcinomas, 1 medullary carcinoma, 1 secreting carcinoma, 1 lobular carcinoma, 1 Paget's disease) was investigated via an immunohistochemical technique using antihuman Cu/Zn- and Mn-SOD antibodies in 10%formalin fixed-paraffin embedded thin sections. Both SODs stained strongly in the normal breast gland, but not clearly in many cancer tissues. Furthermore, Cu/Zn-SOD stained more strongly in well differentiated tubular carcinomas than in poorly differentiated tubular carcinomas. It tended to stain less in tumors which recurred or had a poor outcome, and in tumors with a diploid pattern on DNA flow cytometry. Mn-SOD staining was similar to that of Cu/Zn-SOD, but no significant differences among subgroups was found, since the incidence of positively staining tumors was too small in all groups. The intensity of SOD staining seems to change in relation to cell proliferation and differentiation in breast carcinoma, and may be a prognostic indicator, since SOD decreased in poorly differentiated carcinoma and in tumors which developed distant metastasis. Thus, the localization of SOD in breast cancer tissue can provide useful information for cancer treatment.     

Prevalence of second generation antibody to hepatitis C virus among voluntary blood donors in Osaka,Japan

To clarify the demographic characteristics of the prevalence of hepatitis C virus (HCV) infection in Osaka, Japan, where hepatocellular carcinoma is common, we investigated the screening data of antibody to HCV (anti-HCV, DAINABOTHCVPHA, second generation assay) in 197,600 voluntary blood donors residing in Osaka. The study found that age-standardized prevalence of anti-HCV was significantly higher than that of HBsAg (2.25cf 0.86 percent among males,P<0.001; 2.17cf 0.55 percent among females,P<0.001. It was much higher in the blood donors aged 55–64 years than in those aged 16–54 years (8.49cf 1.32 percent among males,P<10^–5; 7.26cf 1.42 percent among females,P<10^–5). The prevalence of anti-HCV among males was significantly higher than that of females in the younger (25–34 years) generations (1.02 to 1.49 percentcf 0.71 to 1.13 percent,P<0.05). A similar tendency was observed in the prevalence of high-titer (2¹²) anti-HCV. The number of coinfection (both HBsAg and anti-HCV seropositive) was very small, and it was not statistically different from the expected number.