Masaaki Tsuda

Plasmid DNAs directly injected into mouse brain with lipofectin can be incorporated and expressed by brain cells.

In this study, we demonstrated that lipofectin-treated DNAs which were injected into mouse brain could be incorporated and expressed by brain cells. When L7RH-beta gal plasmid DNA harboring E. coli beta-galactosidase gene fused with the nuclear location signal of SV40 T-antigen gene was injected into brains of 1-week-old mice, cells whose nuclei appeared to be densely stained with the chromogenic substrate X-gal were detected in several portions of the brain till 9 days after injection. Injection of pMLV-CAT plasmid DNA which contains the E. coli chloramphenicol acetyltransferase (CAT) gene also resulted in cells immunoreactive to the anti-CAT antibody.

Modulation of AP-1 activity by nitric oxide (NO) in vitro: NO-mediated modulation of AP-1

To understand the role of nitric oxide (NO) in controlling the specific DNA‐binding activities of transcriptional factors, we investigated the in vitro effect of the NO‐donor sodium nitroprusside (SNP) on the AP‐1 activity of cultured mouse cerebellar granule cells. A gel‐mobility assay showed that SNP inhibited AP‐1 activity in the presence, but not the absence of dithiothreitol (DTT). This DTT‐dependent inhibition of AP‐1 activity by SNP corresponded with the activation of the chemical reactivity of SNP with DTT, which can be monitored by the production of nitrite (NO− 2). In contrast, diamide, a typical sulfhydryl oxidizing agent, inhibited AP‐1 activity in the absence of DTT and its inhibitory effect was reversed competitively by DTT. Studies using structurally or functionally related analogues of SNP demonstrated that S‐nitrosylation of the AP‐1 moiety mediated by some NO‐carriers but not by free NO, which can be produced by the chemical reaction of SNP with DTT, was responsible for the inhibition of AP‐1 activity, suggesting NO‐mediated regulation of the AP‐1 transcriptional factor.

Rapid attenuation of AP-1 transcriptional factors associated with nitric oxide (NO)-mediated neuronal cell death

Stimulation of glutamate receptors causes several intracellular reactions including activation of activator protein-1 (AP-1) production and nitric oxide (NO) generation. Exposing mouse cerebellar granule cells to N-methyl-D-aspartate or kainate (KA) in culture induced an increase of AP-1 DNA binding activity that was blocked by further addition of sodium nitroprusside (SNP), a typical NO donor. Immunoblotting using anti-c-Fos antiserum revealed the specific attenuation of AP-1, although total protein synthesis was not affected. Since the level of c-fos mRNA expression stimulated by KA remained constant even after exposure to SNP, the AP-1 attenuation can be post-transcriptionally induced. SNP did not affect the Ca2+ influx into the cells stimulated by KA. The involvement of NO in the AP-1 attenuation was supported by the fact that potassium ferrocyanide (K4Fe(CN)6), an analogue of SNP but devoid of NO, failed to inhibit the AP-1 DNA binding activity stimulated by KA. SNP alone induced neuronal cell death, which was blocked by the simultaneous addition of antioxidants, superoxide dismutase and catalase, and an NO scavenger, suggesting a direct role of peroxynitrite in the cell death. In good agreement with these effects, the AP-1 attenuation by SNP was also blocked by antioxidants. These results indicated that post-transcriptional attenuation of AP-1 is involved in the early processes of NO-mediated neuronal cell death.

Stimulation of Cultured Cerebellar Granule Cells via Glutamate Receptors Induces TRE‐ and CRE‐Binding Activities Mediated by Common DNA‐Binding Complexes

Abstract: By use of nuclear mini‐extracts prepared from cultured cerebellar granule cells in a gel‐mobility assay, exogenous N‐methyl‐D‐aspartate (NMDA) or kainate was shown to increase both 12‐O‐tetradecanoylphorbol 13‐acetate‐responsive element (TRE)‐ and cyclic AMP‐responsive element (CRE)‐binding activity. These increases were specifically prevented by the NMDA receptor antagonist D,L‐2‐amino‐5‐phosphonovalerate and the non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, respectively. The increase of TRE‐binding activity was dependent on de novo protein synthesis, and its inductions by both NMDA and kainate required extracellular Ca2+. TRE‐binding activity was competitively inhibited by the CRE, and vice versa, showing higher DNA‐binding affinity to the CRE than to the TRE. A proteolytic clipping bandshift assay demonstrated that the increase in CRE‐binding activity could be mediated by the TRE‐binding activity. Thus, the TRE‐binding activity cross‐binding to the CRE could be activated by NMDA or kainate stimulation. The involvement of c‐Fos or Fos‐related proteins in the TRE‐ and CRE‐binding complexes was shown by a supershift gel‐mobility assay using anti‐c‐Fos antiserum.

Inactivation of aconitase during the apoptosis of mouse cerebellar granule neurons induced by a deprivation of membrane depolarization

During the excitotoxic neuronal cell death which accompanies an overflow of extracellular Ca2+ into neurons, aconitase, an oxidative stress‐sensitive enzyme of the tricarboxylic acid (TCA)‐cycle in mitochondria, is inactivated due to the generation of oxidative stress (Patel et al. [1996] Neuron 16:345–355). In this study, we investigated whether aconitase could be inactivated during the apoptosis of mouse cerebellar granule cells (CGCs), which was caused by a deprivation of membrane depolarization followed by a stoppage of Ca2+ influx into CGCs. Upon lowering the potassium (K+) concentration in medium from 25 to 5 mM (low K+), aconitase was inactivated in accordance with the decrease in methylthiazoletetrazolium (MTT)‐reducing activity although its mRNA expression did not change. The blockade of Ca2+ influx into CGCs mediated by nicardipine at 25 mM KCl also caused the inactivation of aconitase, accompanying induction of the apoptosis of CGCs. Suppression of the apoptosis of CGCs mediated by the Ca2+ influx or neurotrophic factors such as brain‐derived neurotrophic factor (BDNF) and adenylate cyclase activating polypeptide‐38 (PACAP‐38) attenuated the aconitase inactivation as well as the lactate dehydrogenase (LDH)‐release and the decrease in MTT reduction. On the other hand, the levels of intracellular glutathione and manganese superoxide dismutase‐2 mRNA decreased under the low K+ condition, supporting a cause for oxidative stress at low K+ due to a loss of anti‐oxidant activity. Thus, the inactivation of aconitase is also caused by a deprivation of Ca2+ influx into neurons, suggesting that aconitase is a key mitochondrial enzyme influencing the viability of neurons in response to oxidative stress.

Properties of a Na^+-coupled serine-threonine transport system in Escherichia coli

Based on the following experimental results we conclude that the serine-threonine transport system in Escherichia coli is a Na+-coupled cotransport system. (1) Addition of serine to cell suspensions induced H+ efflux in the presence of Na+. (2) Addition of serine to cell suspensions induced Na+ uptake by cells. (3) Imposition of an artificial electrochemical potential of Na+ in starved cells induced serine uptake. Some of these phenomena were observed when threonine was added instead of serine or inhibited when cells were preincubated with threonine. The Na+/serine (threonine) cotransport system was considerably repressed when cells were grown on a mixture of amino acids. Serine transport in cells grown in the absence of amino acids mixture was stimulated by Na+. The half maximum concentration of Na+ was 21 microM. Sodium ion increased the Vmax of serine transport without affecting the Km.

Involvement of endogenous PACAP expression in the activity-dependent survival of mouse cerebellar granule cells

Membrane depolarization causes Ca2+ influx through L-type voltage-dependent calcium channels (L-VDCC), which promotes the activity-dependent survival of mouse cerebellar granule cells (CGCs). Although exogenously added pituitary adenylate cyclase activating polypeptide (PACAP) is effective in promoting the survival of CGCs, it is unknown whether PACAP is synthesized in CGCs and involved in the activity-dependent survival of CGCs. In this study, we found that the PACAP gene was activated in depolarized CGCs cultured at 25 mM KCl (high K+), independently of de novo protein synthesis. In addition, the PACAP immunoreactivity increased through the activation of L-VDCC in depolarized CGCs, indicating that PACAP is concomitantly produced with PACAP mRNA in an activity-dependent manner. Exogenously added PACAP attenuated the apoptosis of CGCs through a specific interaction with PACAP receptors. Furthermore, a PACAP receptor antagonist, PACAP(6-38), reduced the survival of CGCs at high K+. These findings indicate that endogenous PACAP production induced by Ca2+ signals exerts a survival effect on CGCs via PACAP receptors, which, at least in part, accounts for the activity-dependent survival of CGCs.

Effects of single cyclosporin A pretreatment on pentylenetetrazol-induced convulsion and on TRE-binding activity in the rat brain

Using electrophoretic mobility-shift assay (EMSA), we examined changes in DNA-binding activities of transcriptional factor-activated protein-1 (AP-1), which is a Fos-Jun protein complex, onto its responsive element TRE in the hippocampus and amygdaloid nucleus of rats stimulated with pentylenetetrazol (PTZ) injection, and also investigated the effects of a single administration of the immunosuppressant cyclosporin A (CsA). In EMSA with nuclear extracts from the rat brain, the TRE-binding activity of AP-1 in the hippocampus and amygdaloid nucleus markedly increased 2 h after the PTZ injection (75 mg/kg, i.p.). These PTZ-induced increases of the TRE-binding protein in these regions were completely suppressed, by pretreatment with CsA (5 mg/kg, s.c.) 1 h before the PTZ injection. In addition, the administration of CsA significantly ameliorated PTZ-induced convulsion. This therapeutic effect of single CsA pretreatment may be based, in part, on the effects on the TRE-binding activity of AP-1 in the brain. Since single pretreatment of CsA in the present study had no effect on the PTZ-induced induction of c-fos mRNA, c-jun mRNA, Fos protein nor Jun protein, the inhibitory effects of single CsA administration on PTZ-induced TRE-binding activity in the brain may be related to the effects of CsA on AP-1 itself. These results suggest that an immune response via activation of transcriptional factor in the brain tissue is involved in the convulsion.

Neurite outgrowth from mouse neuroblastoma and cerebellar cells induced by the protein kinase inhibitor H-7

The protein kinase inhibitor H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) or staurosporine was found to induce neurite outgrowth from mouse neuroblastoma N18TG2 cells and cultured cerebellar cells by light microscopy. Examination by electron microscopy revealed some morphological differences between the neurites of N18TG2 cells induced by H-7 and dB-cAMP, in which H-7 formed longer and thinner neurites and more abundant localized varicose structures along neurites than dB-cAMP. These results indicate that protein kinase inhibitor itself can act as a potent stimulus for inducing neurite outgrowth in a different manner from dB-cAMP.

Target of serine inhibition in Escherichia coli.

L-serine has long been known to inhibit growth of Escherichia coli cells cultured in minimal medium supplemented with glucose, lactate, or another carbohydrate as the sole source of carbon. However, the target of serine inhibition was not known. The growth inhibition was released by adding isoleucine, 2-ketobutyric acid, threonine or homoserine, but not by aspartate. Thus the inhibition site must be between aspartate and homoserine in the isoleucine biosynthetic pathway. We found that homoserine dehydrogenase I was strongly inhibited by serine. We isolated serine-resistant mutants, and found that in these mutants homoserine dehydrogenase I was resistant to serine. Thus, we conclude that the target of serine inhibition in Escherichia coli is homoserine dehydrogenase I.