Astrocytes release glutamate via cystine/glutamate antiporter upregulated in response to increased oxidative stress related to sporadic amyotrophic lateral sclerosis.

A vast body of evidence implicates increased oxidative stress and extracellular glutamate accumulation in the pathomechanism of sporadic amyotrophic lateral sclerosis (ALS). Cystine/glutamate antiporter (xCT) carries extracellular cystine uptake and intracellular glutamate release (cystine/glutamate exchange) in the presence of oxidative stress. The aim of the present study was to determine the involvement of xCT in ALS. Immunohistochemical observations in the spinal cord sections demonstrated that xCT was mainly expressed in astrocytes, with staining more intense in 12 sporadic ALS patients as compared to 12 age-matched control individuals. Western blot and densitometric analyses of the spinal cord samples revealed that the relative value of xCT/ß-actin optical density ratio was significantly higher in the ALS group as compared to the control group. Next, we conducted cell culture experiments using a human astrocytoma-derived cell line (1321N1) and a mouse motor neuron/neuroblastoma hybrid cell line (NSC34). In 1321N1 cells, the normalized xCT expression levels in cell lysates were significantly increased by H2 O2 treatment. Glutamate concentrations in 1321 N1 cell culture-conditioned media were significantly elevated by H2 O2 treatment, and the H2 O2 -driven elevations were completely canceled by the xCT inhibitor erastin pretreatment. In motor neuron-differentiated NSC34 cells (NSC34d cells), both the normalized xCT expression levels in the cell lysates and glutamate concentrations in the cell-conditioned media were constant with or without H2 O2 treatment. The present results provide in vivo and in vitro evidence that astrocytes upregulate xCT expression to release glutamate in response to increased oxidative stress associated with ALS, contributing to extracellular glutamate accumulation.

Soluble iron accumulation induces microglial glutamate release in the spinal cord of sporadic amyotrophic lateral sclerosis.

Previous studies on sporadic amyotrophic lateral sclerosis (SALS) demonstrated iron accumulation in the spinal cord and increased glutamate concentration in the cerebrospinal fluid. To clarify the relationship between the two phenomena, we first performed quantitative and morphological analyses of substances related to iron and glutamate metabolism using spinal cords obtained at autopsy from 12 SALS patients and 12 age-matched control subjects. Soluble iron content determined by the Ferrozine method as well as ferritin (Ft) and glutaminase C (GLS-C) expression levels on Western blots were significantly higher in the SALS group than in the control group, while ferroportin (FPN) levels on Western blots were significantly reduced in the SALS group as compared to the control group. There was no significant difference in aconitase 1 (ACO1) and tumor necrosis factor-alpha (TNFα)-converting enzyme (TACE) levels on Western blots between the two groups. Immunohistochemically, Ft, ACO1, TACE, TNFα, and GLS-C were proven to be selectively expressed in microglia. Immunoreactivities for FPN and hepcidin were localized in neuronal and glial cells. Based on these observations, it is predicted that soluble iron may stimulate microglial glutamate release. To address this issue, cell culture experiments were carried out on a microglial cell line (BV-2). Treatment of BV-2 cells with ferric ammonium citrate (FAC) brought about significant increases in intracellular soluble iron and Ft expression levels and conditioned medium glutamate and TNFα concentrations. Glutamate concentration was also significantly increased in conditioned media of TNFα-treated BV-2 cells. While the FAC-driven increases in glutamate and TNFα release were completely canceled by pretreatment with ACO1 and TACE inhibitors, respectively, the TNFα-driven increase in glutamate release was completely canceled by GLS-C inhibitor pretreatment. Moreover, treatment of BV-2 cells with hepcidin resulted in a significant reduction in FPN expression levels on Western blots of the intracellular total protein extracts. The present results provide in vivo and in vitro evidence that microglial glutamate release in SALS spinal cords is enhanced by intracellular soluble iron accumulation-induced activation of ACO1 and TACE and by increased extracellular TNFα-stimulated GLS-C upregulation, and suggest a positive feedback mechanism to maintain increased intracellular soluble iron levels, involving TNFα, hepcidin, and FPN.

Casting of MOD inlay using rings with holes on both sides: 12∼18 wt%Au-20∼26Pd-14.48∼26.48Cu-40Ag-1.5Zn-0.02Ir alloys.

Using a casting ring with openings on both sides and a water-absorbent polymer, heterogeneity is maintained in a single casting and a precise MOD inlay can be produced. We produced 9 different kinds of gold-silver-palladium (Au-Ag-Pd) alloys by changing the ratio of palladium, gold, and copper and investing them, and changing parameters such as the angulation of the casting ring openings and the water:powder ratios to produce MOD inlay castings. We measured the expansion and shrinkage percentage of the castings in both the buccolingual and mesiodistal directions. From this experiment, we learned that precise MOD inlay castings can be produced using rings with 240° openings when invested in a thick mix having a standard water:powder ratio or using rings with 200° openings when invested in a thick mix having a water:powder ratio for a 12 wt%Au-20∼26Pd-20.48∼26.48Cu-40Ag-1.5Zn-0.02Ir alloyes.

The grinding efficiency by diamond points developed for yttria partially stabilized zirconia.

Recently, the use of all-ceramic restorations has increased and been applied for many types of clinical treatment such as bridges which can be made with high strength ceramic material: yttria partially stabilized zirconia (Y-PSZ) employing the CAD/CAM system. Consequently, points that can grind high strength ceramic material effectively are necessary for modifying the shape of restorations or removing them. So in our research, we fabricated new diamond points used to grind Y-PSZ. These points were made by electrodeposition of diamond grains. The diameter of the diamond grains are larger (200 µm) than that of traditional points (100 µm). High strength ceramic material was ground to assess the grinding weight and grinding depth various types of points. As a result, Y-PSZ could be ground efficiently with the diamond point which has a larger diameter and employs lager grains (200 µm).