Region-dependent differences and alterations of protective thiol compound levels in cultured astrocytes and brain tissues.

We examined region-dependent differences and alterations in the levels of protective thiol compounds, glutathione (GSH) and metallothionein (MT)-I and -II, in cultured rat astrocytes under several culture conditions and in brain tissues of rats at postnatal and weaning periods. Regardless of culture conditions, both protein concentrations and mRNA expressions of MT-I and -II were much higher in the cerebral hemisphere than in cerebellar astrocytes, whereas no difference was observed in GSH concentration. In both astrocytes, the GSH concentrations did not change within 12 h but significantly increased 24 h after being maintained in a serum-free defined medium.

Acute cytotoxic effects of mercuric compounds in cultured astrocytes prepared from cerebral hemisphere and cerebellum of newborn rats.

We investigated acute cytotoxic effects and Hg accumulation after exposure to methylmercury (MeHg) or Hg(2+) in the presence or absence of serum in cultured astrocytes prepared from the cerebral hemisphere or cerebellum of newborn rats. Dose-related changes in viable cell numbers after exposure to mercuric compounds were not different between astrocytes from both regions under the specified conditions. Accumulation of each compound for 3 h was similar in both astrocytes but that for 24 h became different, especially that of Hg(2+).

Over expression of hypoxia-inducible factor-1alpha in renal and bladder cancer cells increases tumorigenic potency.

Purpose: Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcriptional factor that regulates genes involved in the response to hypoxia. We evaluated the effects of HIF-1alpha over expression on the tumorigenic potency of renal cell carcinoma VMRC cells and bladder cancer EJ cells in vitro and in vivo.

Materials And Methods: We introduced HIF-1alpha expression vectors into VMRC and EJ cells, and generated the HIF-1alpha over expressing cell lines VMRC-HIF1alpha and EJ-HIF1alpha, and the vector only transfected cell lines VMRC-neo and EJ-neo.

Methods of analysis for chemicals that disrupt cellular signaling pathways: risk assessment for potential endocrine disruptors.

Here we present a basic concept and several examples of methods of analysis for chemicals that disrupt cellular signaling pathways, in view of risk assessment for potential endocrine disrupting chemicals (EDCs). The key cellular signaling pathways include 1) ER/coactivator interaction, 2) AR translocation into the nucleus, 3) ER/NO/sGC/cGMP, 4) ER/Akt, 5) ER/Src, 6)ER/Src/Grb2, and 7) ER/Ca2+/CaM/CaMK pathways. These were visualized in relevant live cells using newly developed fluorescent and bioluminescent probes.

Influence of LIF and BMP-2 on differentiation and development of glial cells in primary cultures of embryonic rat cerebral hemisphere.

Cells prepared from the cerebral hemisphere of embryonic Day 18 rats were maintained for 2 days in serum-free modified Bottenstein-Sato (mBS) medium containing thyroid hormone (TH), with or without leukemia inhibitory factor (LIF) or bone morphogenetic protein (BMP)-2, and these influences on the differentiation and development of glial cells were investigated using the cells maintained in mBS medium containing TH as controls. The levels of glial fibrillary acidic protein (GFAP) expression and the number of GFAP-positive astrocytes increased markedly with the addition of LIF or BMP-2, and were enhanced further with the addition of both LIF and BMP-2. The number of O1-positive oligodendrocytes increased with the addition of LIF, whereas it decreased with the addition of BMP-2.

Transcriptional regulation of thioredoxin reductase 1 expression by cadmium in vascular endothelial cells: role of NF-E2-related factor-2.

Thioredoxin reductase (TrxR) is a selenoprotein that catalyzes the reduction of the active site disulfide of thioredoxin (Trx), which regulates the redox status of the cells. In the present study, we found that TrxR1, one of the three TrxR isozymes, was induced by cadmium as well as tumor necrosis factor alpha (TNFalpha) in bovine arterial endothelial cells (BAEC), and investigated the mechanism of cadmium-induced TrxR1 expression. We here showed that cadmium, differently from TNFalpha, enhanced the promoter activity of the 5'-flanking region of human TrxR1 gene (nucleotides -1692 to +49).

Beta-naphthoflavone disturbs astrocytic differentiation of C6 glioma cells by inhibiting autocrine interleukin-6.

Regulation of astrocyte differentiation is a key process in the development of the central nervous system (CNS), and disturbance of the differentiation can lead to brain system dysfunction. Here we show that beta-naphthoflavone (betaNF), an agonist of the aryl hydrocarbon receptor (AhR), disturbed the cAMP-induced astrocytic differentiation of C6 glioma by inhibiting autocrine interleukin-6 (IL-6). Treatment of cells with betaNF reduced the induction of an astrocyte marker glial fibrillary acidic protein (GFAP).

Neuronal nitric oxide synthase (NNOS) catalyzes one-electron reduction of 2,4,6-trinitrotoluene, resulting in decreased nitric oxide production and increased nNOS gene expression: implication for oxidative stress.

To determine the mechanism of 2,4,6-trinitrotoluene (TNT)-induced oxidative stress involving neuronal nitric oxide synthase (nNOS), we examined alterations in enzyme activity and gene expression of nNOS by TNT, with an enzyme preparation and rat cerebellum primary neuronal cells. TNT inhibited nitric oxide formation (IC(50) = 12.4 microM) as evaluated by citrulline formation in a 20,000 g cerebellar supernatant preparation.

Bisphenol A causes hyperactivity in the rat concomitantly with impairment of tyrosine hydroxylase immunoreactivity.

We examined the effects of bisphenol A, an endocrine disruptor, on rat behavioral and cellular responses. Single intracisternal administration of bisphenol A (0.2-20 microg) into 5-day-old male Wistar rats caused significant hyperactivity at 4-5 weeks of age.

Subcellular localization and regulation of hypoxia-inducible factor-2alpha in vascular endothelial cells.

The hypoxia-inducible factors 1alpha (HIF-1alpha) and 2alpha (HIF-2alpha) have extensive structural homology and have been identified as transcription factors that mediate hypoxia-inducible gene expression through hypoxia-responsive element (HRE). They play critical roles not only in normal development, but also in tumor progression. Endothelial cells (EC) express both HIF-1alpha and -2alpha.

cAMP-induced astrocytic differentiation of C6 glioma cells is mediated by autocrine interleukin-6.

Elevation in the level of intracellular cAMP is known to induce the astrocytic differentiation of C6 glioma cells by unknown mechanisms. In this report, we show that cAMP-induced autocrine interleukin 6 (IL-6) promoted astrocytic differentiation of C6 cells. Treatment of cells with N(6),2'-O-dibutyryl cAMP (Bt(2)AMP) and theophylline caused the delayed phosphorylation of signal transducer and activator of transcription 3 (STAT3), as well as the expression of an astrocyte marker, glial fibrillary acidic protein (GFAP).

Overexpression of thioredoxin reductase 1 regulates NF-kappa B activation.

Thioredoxin reductase (TrxR) is a flavoprotein that contains a C-terminal penultimate selenocysteine (Sec) and has an ability to reduce thioredoxin (Trx), which regulates the activity of NF-kappa B. To date, three TrxR isozymes, TrxR1, TrxR2, and TrxR3, have been identified. In the present study, we found that among these isozymes only TrxR1 was induced by tumor necrosis factor-alpha (TNF alpha) in vascular endothelial cells.

Selective disappearance of an axonal protein, 440-kDa ankyrinB, associated with neuronal degeneration induced by methylmercury.

The 440-kDa isoform of brain ankyrin, 440-kDa ankyrinB, is a neuron-specific protein and is confined to axons. Cerebellum is one of the areas characteristically altered by methylmercury intoxication both in the adult and during development. When rat cerebellar neurons matured for 7 days in vitro were exposed to methylmercury at 0.

Influence of cell density and thyroid hormone on glial cell development in primary cultures of embryonic rat cerebral hemisphere.

The influence of cell density and thyroid hormone (TH) on the development of astrocytes and oligodendrocytes was investigated in primary cultures prepared from rat cerebral hemisphere on embryonic day (E)18. At the beginning of the culture, most of the cells were microtubule-associated protein 2 (MAP2)-positive neurons, whereas O1-positive oligodendrocytes and glial fibrillary acidic protein (GFAP)-positive astrocytes were rarely observed. After the cells were maintained in serum-free defined medium, astrocytes developed at high cell density but rarely at a low one.

Attenuation of both apoptotic and necrotic actions of cadmium by Bcl-2.

We examined the effects of cadmium on the bcl-2 family of proteins--bcl-2, bax, bad, and bcl-xS/L--in cadmium-induced cytotoxicity. Addition of 10 microM cadmium to cultured porcine kidney LLC-PK(1) cells caused apoptosis. Western blot analyses revealed that cadmium markedly increased endogenous bcl-2 protein (to 3-4 times the level in wild-type cells) earlier than metallothionein induction, but that the metal did not enhance the induction of bax, bad, or bcl-xS proteins.