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Mikihiko Naito

Publications and source records attributed to Mikihiko Naito.

At least 19 recordsLinked to original sources
PubMed2006

Inhibition of transforming growth factor-beta production in brain pericytes contributes to cyclosporin A-induced dysfunction of the blood-brain barrier.

: 1. The present study was designed to clarify whether brain pericytes and pericyte-derived transforming growth factor-beta1 (TGF-beta1) participate in cyclosporin A (CsA)-induced dysfunction of the blood-brain barrier (BBB).2. The presence of brain pericytes markedly aggravated CsA-increased permeability of MBEC4 cells to sodium fluorescein and accumulation of rhodamine 123 in MBEC4 cells.3. Exposure to CsA significantly decreased the levels of TGF-beta1 mRNA in brain pericytes in pericyte co-cultures. Treatment with TGF-beta1 dose-dependently inhibited CsA-induced hyperpermeability and P-glycoprotein dysfunction of MBEC4 cells in pericyte co-cultures.4. These findings suggest that an inhibition of brain pericyte-derived TGF-beta1 contributes to the occurrence of CsA-induced dysfunction of the BBB.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Cell differentiation inducers derived from thalidomide.

5-Hydroxy- and 4-amino-2-(2,6-diisopropylphenyl)-1H-isoindole-1,3-dione (5HPP-33 and 4APP-33, respectively) have been shown to possess cell differentiation-inducing activity toward human leukemia cell line HL-60.

Antineoplastic Agents↗
PubMed2005

HtrA2 cleaves Apollon and induces cell death by IAP-binding motif in Apollon-deficient cells.

Apollon/BRUCE is a giant IAP protein that has BIR and UBC domains in its amino- and carboxy-terminals, respectively. Apollon binds and ubiquitylates SMAC/DIABLO and caspase9, and regulates apoptosis by facilitating proteasomal degradation of these proteins. Apollon overexpression inhibits apoptosis, while its downregulation sensitizes cells to apoptosis, suggesting that Apollon level is important for apoptosis regulation. Here we show that HtrA2/Omi catalytically cleaves Apollon with its serine protease activity. Conversely, Apollon ubiquitylates and facilitates proteasomal degradation of HtrA2 that binds to Apollon through IAP-binding motif. Thus, Apollon and HtrA2 mutually downregulate each other. Expression of catalytically active, but not inactive, HtrA2 induced apoptosis in Apollon-expressing cells. In Apollon-deficient cells, however, expression of catalytically inactive HtrA2 mutant with IAP-binding motif also induced apoptosis. These results indicate that HtrA2 induces apoptosis in two different mechanisms, one with serine protease domain and the other with IAP-binding motif, in Apollon-deficient cells.

Catalysis↗
PubMed2005

Brain pericytes contribute to the induction and up-regulation of blood-brain barrier functions through transforming growth factor-beta production.

The blood-brain barrier (BBB) is a highly organized multicellular complex consisting of an endothelium, brain pericytes and astrocytes. The present study was aimed at evaluating the role of brain pericytes in the induction and maintenance of BBB functions and involvement of transforming growth factor-beta (TGF-beta) in the functional properties of pericytes. We used an in vitro BBB model established by coculturing immortalized mouse brain capillary endothelial (MBEC4) cells with a primary culture of rat brain pericytes. The coculture with rat pericytes significantly decreased the permeability to sodium fluorescein and the accumulation of rhodamine 123 in MBEC4 cells, suggesting that brain pericytes induce and up-regulate the BBB functions. Rat brain pericytes expressed TGF-beta1 mRNA. The pericyte-induced enhancement of BBB functions was significantly inhibited when cells were treated with anti-TGF-beta1 antibody (10 microg/ml) or a TGF-beta type I receptor antagonist (SB431542) (10 microM) for 12 h. In MBEC4 monolayers, a 12 h exposure to TGF-beta1 (1 ng/ml) significantly facilitated the BBB functions, this facilitation being blocked by SB431542. These findings suggest that brain pericytes contribute to the up-regulation of BBB functions through continuous TGF-beta production.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Functional characterization of adenosine transport across the BBB in mice.

We investigated transport characteristics of adenosine across the blood-brain barrier (BBB) in mice. Uptake clearance across the BBB was measured by using an in situ mouse brain perfusion technique and cultured mouse brain capillary endothelial cell line (MBEC4 cells). Nucleoside transporter was cloned by RT-PCR and expressed on Xenopus laevis oocyte. Both in situ and in vitro studies revealed that the adenosine uptake is concentration-dependent, Na(+)-independent and S-(p-nitrobenzyl)-6-thioinosine (NBMPR)-sensitive. The K(t) values of in situ and in vitro studies were 31.7 +/- 13.8 microM and 11.9 +/- 2.84 microM, respectively. A good correlation was found for the inhibitory effects of nucleoside analogs to adenosine uptake between in situ and in vitro studies. RT-PCR revealed the expression of RNA of mouse equilibrative nucleoside transporter (mENT1) in mouse brain capillary and MBEC4 cells. In mENT1 expressed on X. laevis oocyte, K(t) value of adenosine transport was 6.9 +/- 2.7 microM (and comparable to those in situ and in vitro studies). In conclusion, we characterized the adenosine transport across the BBB in mice by using in situ brain perfusion technique and MBEC4 cells and found that these transports share common characteristics with mENT1-mediated transport. Transport of adenosine across the BBB in mice may be attributable to mENT1.

Adenosine↗
PubMed2004

Nitric oxide mediates cyclosporine-induced impairment of the blood-brain barrier in cocultures of mouse brain endothelial cells and rat astrocytes.

The present study was designed to clarify the involvement of nitric oxide (NO) signaling in the adverse effect of cyclosporine on the blood-brain barrier. Cyclosporine increased the permeability of sodium-fluorescein and the cellular accumulation of rhodamine 123, a substrate of P-glycoprotein, in mouse brain endothelial (MBEC4) cells. This effect was markedly enhanced two- to threefold when MBEC4 cells were cocultured with rat astrocytes or C6 glioma cells. Direct and continuous electrochemical measurement of NO demonstrated that cyclosporine dose-dependently increased histamine- and phenylephrine-evoked NO production in MBEC4 cells and astrocytes, respectively. A NO synthase inhibitor (NG-monomethyl-L-arginine) blocked slightly and markedly cyclosporine-induced impairment of the endothelial barrier in the monolayer and coculture system, respectively. These findings suggest that cyclosporine impairs the brain endothelial barrier function by accelerating NO production in the brain endothelial and astroglial cells. This event may be interpreted as triggering the occurrence of cyclosporine neurotoxicity.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2004

Transport of somatostatin and substance P by human P-glycoprotein.

P-glycoprotein is an efflux pump for a broad spectrum of hydrophobic agents. We found that bioactive peptides including somatostatin and substance P inhibit ATP-dependent vincristine binding to P-glycoprotein-overexpressing K562/ADM membrane vesicles. Some of these bioactive peptides including somatostatin stimulate basal ATPase activity of P-glycoprotein; in contrast, other peptides including substance P inhibit it. The K562/ADM membrane vesicles showed an ATP-dependent, osmotically sensitive uptake of somatostatin and substance P, which was inhibited by valspodar, an inhibitor of P-glycoprotein. These findings suggested that certain bioactive peptides such as somatostatin and substance P directly interact with human P-glycoprotein as endogenous substrates for P-glycoprotein-mediated transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Apollon ubiquitinates SMAC and caspase-9, and has an essential cytoprotection function.

Apollon (also known as BRUCE or BIRC6) is a large protein containing baculoviral-IAP-repeat (BIR) and ubiquitin-conjugating enzyme (UBC) domains at the amino- and carboxy termini, respectively. Apollon inhibits apoptosis, but its molecular and physiological function remains unclear. Here we report that Apollon binds to, ubiquitinates and facilitates proteasomal degradation of SMAC and caspase-9, which both contain IAP-binding motifs. Targeted disruption of Apollon in mice caused embryonic and neonatal lethality. Notably, SMAC induced apoptosis in Apollon-deficient cells, but not in Apollon-expressing cells. Furthermore, the IAP-binding motif of SMAC was required to induce apoptosis in Apollon-deficient cells. These results suggest that Apollon has an essential function in preventing SMAC-induced apoptosis.

Animals↗
PubMed2004

Hypoxic up-regulation of triosephosphate isomerase expression in mouse brain capillary endothelial cells.

A protein with a molecular mass of 27kDa was induced by hypoxia in a mouse brain capillary endothelial cell line and identified as triosephosphate isomerase (TPI) by amino-terminal sequencing. Hypoxia caused an elevation of the TPI protein level, concomitant with an increase of the TPI mRNA level. However, hypoxia resulted in an insufficient elevation of TPI activity level, compared to an increase of TPI protein level. When cells expressing the recombinant TPI protein with histidine tag were exposed to hypoxia and the TPI protein was affinity-purified, the catalytic activity (specific activity) of the TPI protein purified from hypoxic cells was substantially lower than that obtained from normoxic cells. In addition, three TPI isoforms with an electrophoretic multiplicity were found; two of the three isoforms were substantially increased in response to the hypoxia, but the level of the most acidic isoform was barely changed. The induction of TPI gene expression by hypoxia was suppressed by (1) a chelator of intracellular Ca(2+), (2) a blocker of non-selective cation channels, (3) a blocker of Na(+)/Ca(2+) exchangers, (4) an inhibitor of Ca(2+)/calmodulin-dependent protein kinases, and (5) an inhibitor of c-jun/AP-1 activation.

Animals↗
PubMed2004

Early processing of Bid and caspase-6, -8, -10, -14 in the canine brain during cardiac arrest and resuscitation.

A clinically relevant model of transient global brain ischemia involving cardiac arrest followed by resuscitation in dogs was utilized to study the expression and proteolytic processing of apoptosis-regulatory proteins. In the hippocampus, an increase in pro-apoptotic Bcl-2 family proteins Bcl-XS and Bak was detected, concomitant with proteolysis of Bcl-XL and Bcl-2, following ischemia-reperfusion injury. Also, biphasic cleavage of Bid was found in this region of the brain, with early generation of tBid-p11 within 10 min of cardiac arrest, followed by generation of tBid-p15 within 30-min reperfusion, consistent with activation of this pro-apoptotic protein. In addition, cardiac arrest and resuscitation induced early, reperfusion-dependent proteolytic processing of pro-caspase-6, -8, -10, and -14, which preceded caspase-3 activation. Immunohistochemical analysis using antibodies, which preferentially recognize processed caspase-3, -6, -8, and -10, provided evidence of time-dependent activation of these proteases in both neurons and glia in ischemia-sensitive regions of the brain. In conclusion, extremely rapid, cell-selective processing of apoptosis-regulatory proteins occurs in a clinically relevant model of ischemic brain injury caused by cardiac arrest and resuscitation. The early cleavage of Bid and rapid depletion of 32-kDa pro-caspase-14 from the canine hippocampus after induction of ischemia suggests the involvement of calpains in the processing of these proteins. Demonstration of in vitro cleavage of recombinant mouse caspase-14 by calpain I in the present study lends support to this hypothesis, further implicating cross-talk between different protease families in the pathophysiology of ischemic neural cell death.

Animals↗
PubMed2004

Uptake and efflux of quinacrine, a candidate for the treatment of prion diseases, at the blood-brain barrier.

1. A clinical trial of quinacrine in patients with Creutzfeldt-Jakob disease is now in progress. The permeability of drugs through the blood-brain barrier (BBB) is a determinant of their therapeutic efficacy for prion diseases. The mechanism of quinacrine transport across the BBB was investigated using mouse brain endothelial cells (MBEC4). 2. The permeability of quinacrine through MBEC4 cells was lower than that of sodium fluorescein, a BBB-impermeable marker. The basolateral-to-apical transport of quinacrine was greater than its apical-to-basolateral transport. In the presence of P-glycoprotein (P-gp) inhibitor, cyclosporine or verapamil, the apical-to-basolateral transport of quinacrine increased. The uptake of quinacrine by MBEC4 cells was enhanced in the presence of cyclosporine or verapamil. 3. Quinacrine uptake was highly concentrative, this event being carried out by a saturable and carrier-mediated system with an apparent Km of 52.1 microM. Quinacrine uptake was insensitive to Na+-depletion and changes in the membrane potential and sensitive to changes in pH. This uptake was decreased by tetraethylammonium and cimetidine, a substrate and an inhibitor of organic cation transporters, respectively. 4. These findings suggest that quinacrine transport at the BBB is mediated by the efflux system (P-gp) and the influx system (organic cation transporter-like machinery).

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Transforming growth factor-beta1 upregulates the tight junction and P-glycoprotein of brain microvascular endothelial cells.

1. The present study was aimed at elucidating effects of transforming growth factor-beta (TGF-beta) on blood-brain barrier (BBB) functions with mouse brain capillary endothelial (MBEC4) cells. 2. The permeability coefficients of sodium fluorescein and Evans blue albumin for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4 cells were dose-dependently decreased after a 12-h exposure to TGF-beta1 (0.01-10 ng/mL). 3. The present study demonstrates that TGF-beta lowers the endothelial permeability and enhances the functional activity of P-gp, suggesting that cellular constituents producing TGF-beta in the brain may keep the BBB functioning.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Caspase-independent necrotic cell death induced by a radiosensitizer, 8-nitrocaffeine.

Molecular mechanisms of apoptosis have been extensively studied, but little is known about non-apoptotic cell death. To study the mechanism of non-apoptotic cell death, we searched for non-apoptotic cell death inducers for U937 cells, which are highly sensitive to apoptosis induction by various stimuli. We found that 8-nitrocaffeine and its analog, which are candidate radiosensitizers for cancer therapy, induced exclusively caspase-independent necrotic cell death in cell lines such as U937, HL-60, K562 and Jurkat. The 8-nitrocaffeine-induced necrotic cell death was mediated by reactive oxygen species (ROS) because (i) ROS were produced in the 8-nitrocaffeine-treated cells, (ii) ROS scavengers inhibited the caspase-independent necrotic cell death induced by 8-nitrocaffeine, and (iii) the necrotic cell death was completely suppressed in hypoxic cells. Cells selected for resistance to nitrocaffeine showed cross resistance to CH-11, an anti-Fas antibody, suggesting that the necrotic process plays an important role in Fas-mediated cell death in this cell line. Since cancer cells are often derived from a selected population of cells resistant to apoptosis, inducers of necrotic cell death could be beneficial to kill cancer cells that have acquired resistance to apoptosis-induction therapy.

Antibodies, Monoclonal↗
PubMed2004

Cellular FLIP inhibits beta-catenin ubiquitylation and enhances Wnt signaling.

Cellular FLIP (cFLIP) is a close homologue of caspase 8 without caspase activity that inhibits Fas signaling. The cFLIP protein is often expressed in human tumors and is believed to suppress antitumor immune responses involving the Fas system. Here, we report that a long form of cFLIP (cFLIP-L) inhibits beta-catenin ubiquitylation and increases endogenous cytosolic beta-catenin, which results in translocation of beta-catenin into nuclei and induction of beta-catenin-dependent gene expression in cFLIP-L-expressing cells. When cells stably expressing cFLIP-L were stimulated with Wnt3a, enhanced Wnt signaling was observed compared with the control cells. Conversely, depletion of endogenous cFLIP results in reduced Wnt signaling. Furthermore, cFLIP-L increases secondary-body axis formation when coinjected with suboptimal doses of beta-catenin into early Xenopus embryos. Down-regulation of FADD by RNA-mediated interference abolishes the beta-catenin-dependent gene expression induced by cFLIP-L. These results indicate that cFLIP-L, in cooperation with FADD, enhances canonical Wnt signaling by inhibiting proteasomal degradation of beta-catenin, thus suggesting an additional mechanism involved with tumorgenesis, in addition to inhibiting Fas signaling.

Animals↗
PubMed2004

Tolbutamide uptake via pH- and membrane-potential-dependent transport mechanism in mouse brain capillary endothelial cell line.

UNLABELLED: The purpose of this study was to investigate the transport mechanism of tolbutamide across the blood-brain barrier (BBB) using MBEC4 cells as an in vitro BBB model. METHODS: The BBB transport of tolbutamide was studied by using a mouse brain capillary endothelial cell line, MBEC4, cultured on dishes with their luminal membrane facing the culture medium. RESULTS: The uptake of [14C]tolbutamide by MBEC4 cells was dependent on temperature and energy. The uptake coefficient of [14C]tolbutamide increased markedly with decreasing pH of the external medium from neutral to acidic. Valinomycin and replacement of chloride with sulfate or gluconate significantly increased the initial uptake of [14C]tolbutamide, while replacement with nitrate significantly decreased it. The uptake was significantly reduced by a proton ionophore, FCCP, and an anion-exchange inhibitor, DIDS. The initial uptake of [14C]tolbutamide was saturable with Kt of 0.61+/-0.03 mM (pH 7.4) and 1.76+/-0.19 mM (pH 6.5). At pH 6.5, the initial uptake of [14C]tolbutamide was significantly reduced by several sulfa drugs, salicylic acid, valproic acid and probenecid, and was competitively inhibited by sulfaphenazole (Ki=3.47+/-0.50 mM) and valproic acid (Ki=2.29+/-0.43 mM). CONCLUSION: These observations indicate the existence of a pH- and membrane-potential-dependent anion exchange and/or proton-cotransport system(s) for concentrative uptake of tolbutamide and sulfa drugs in MBEC4 cells.

Animals↗
PubMed2004

[Screening for apoptosis inducers].

We carried out a screening for drugs that can induce apoptosis in human monocytic leukemia U937 cells. In the screening, we found that 8-nitrocaffeine induces cell death distinct from typical apoptosis. Morphological and biochemical analysis revealed that reactive oxygen species mediates the 8-nitrocaffeine-induced necrotic cell death.

Apoptosis↗