Search

Amorphous nanoparticles of supramolecular coordination polymer networks are spontaneously self-assembled from nucleotides and lanthanide ions in water. They show intrinsic functions such as energy transfer from nucleobase to lanthanide ions and excellent performance as contrast enhancing agents for magnetic resonance imaging (MRI). Furthermore, adaptive inclusion properties are observed in the self-assembly process: functional materials such as fluorescent dyes, metal nanoparticles, and proteins are facilely encapsulated. Dyes in these nanoparticles fluoresce in high quantum yields with a single exponential decay, indicating that guest molecules are monomerically wrapped in the network. Gold nanoparticles and ferritin were also wrapped by the supramolecular shells. In addition, these nucleotide/lanthanide nanoparticles also serve as scaffolds for immobilizing enzymes. The adaptive nature of present supramolecular nanoparticles provides a versatile platform that can be utilized in a variety of applications ranging from material to biomedical sciences. As examples, biocompatibility and liver-directing characteristics in in vivo tissue localization experiments are demonstrated.

Gold nanorods can be used as photothermal converters, permitting near-infrared (NIR) light to be transmitted deep into tissues without causing damage. We prepared hybrid nanorods with a core-shell structure, i.e., a single gold nanorod encapsulated in a poly (N-isopropylacrylamide) nanogel. Hybrid nanorods demonstrated remote, reversible, pulsatile phase transition and in vivo action after irradiation using a NIR laser.

Emerging evidence has suggested that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) protects neurons from glutamate-induced neurotoxicity. In this study, we examined the effect of AMPA on the cell surface expression of the N-methyl-D-aspartate (NMDA) receptor, a central player in glutamate-induced neurotoxicity, using rat cortical neurons. AMPA (10 microM, 24 h) attenuated the expression of cell surface NR1, an NMDA receptor subunit, and also inhibited glutamate-induced increases in intracellular Ca2+. SB216763, an inhibitor of glycogen synthase kinase 3beta (GSK3beta), had effects similar to those of AMPA. We have earlier shown that AMPA treatment attenuated GSK3beta activity. Our data suggest that AMPA reduces the cell surface expression of NMDA receptors through the regulation of GSK3beta and, consequently, reduces the amount of intracellular Ca2+.

Syndiotactic poly(N-n-propylacrylamide)s (PNNPAM) with various racemo (r) diad contents were synthesized, and their phase transition behaviors were studied by high-sensitivity differential scanning calorimetry and FT-IR spectroscopy. The cooperativity of the phase transition increased with the increase in the r diad content. Compared with the atactic PNNPAM, the number of cooperative units, which shows the monomeric units length undergoing cooperative phase transition, increased 2.5- to 4-fold in the syndiotactic polymers. From the results of FT-IR spectroscopy and quantum chemical calculations, it was determined that the high cooperativity of the syndiotactic polymers resulted from local formation of an ordered structure in a dehydrated state.

Hsp16.5, a small heat-shock protein (sHSP) from hyperthermophilic archaeon, forms a homogeneous complex comprised of 24 subunits with a molecular mass of 400 kDa. This complex self-organizes under physiological conditions, and the structure of the complex is a nanoscale spherical capsule with small pores. Furthermore, this natural nanocapsule exhibits very high thermal stability. In this paper, we functionalized the nanocapsule to control the structure in response to external stimuli such as a protease signal and temperature. For this purpose, several mutations (Mut1-10) to create a cleavage site for a specific protease, Factor Xa, were introduced on the outer surface of the nanocapsule using a genetic engineering strategy. The resulting mutants were expressed to high levels in Escherichia coli. One of these mutants, Mut6, which has the most accessible cleavage site located at the triangular pore on the surface of the capsule, formed a spherical assembly similar to that observed for the wild-type protein. Mut6 showed the highest sensitivity to Factor Xa, and the structure of the protease digested Mut6 disassembled irreversibly after heating. In contrast, the nanocapsule comprising the wild-type Hsp16.5 was not influenced by the dual stimuli. These results suggest that Mut6 acts as a stimulus-responsive nanocapsule. Such a characteristic of the protein-based nanocapsule has attractive potential as a versatile intelligent system.

We previously reported a novel disease-site-specific gene targeting system that can release plasmid DNA (pDNA) from polymeric carriers responding to abnormally activated signal proteins in disease cells. In this study, the molecular mechanism of the gene targeting system responding to Caspase-3 activity was studied in detail. The polymeric carrier used was composed of a neutral main chain polymer and a grafted oligocationic peptide which contains the substrate sequence of Caspase-3. The polyplex formed from the polymeric carrier and pDNA was stable in physiological saline solution and protected from access of RNA polymerase and the transcriptional factors. These results indicate that the polyplex adopts a core-shell-like structure with a polyion complex core surrounded by neutral main chain polymers. In spite of the inert character of the polyplex to transcription, the polyplex afforded the access of Caspase-3 to the substrate peptide because the electrostatic interaction between each peptide and DNA is essentially weak. After the Caspase-3 reaction, the polyplex was weakened and then became available as a template for transcription.

Gold nanorods, which have a strong surface plasmon band at the near-infrared region, absorb light energy which is then converted to heat. Since near-infrared light can penetrate deeply into tissue, gold nanorods are expected to be useful as photosensitizers for photothermal therapy. In this study, the length of the poly(ethylene glycol) (PEG) chain was optimized in order to stabilize the gold nanorods in the blood circulation after intravenous injection. PEG(5000)- and PEG(10000)-modified gold nanorods showed higher stability in the blood circulation compared with PEG(2000)- and PEG(20000)-modified gold nanorods. As a demonstration of photothermal tissue damage, PEG(5000)-modified gold nanorods were injected into the muscle in the hind limbs of a mouse, and then irradiated with near-infrared pulsed laser light. Significant tissue damage was observed only in the presence of gold nanorods and laser irradiation. We next injected the gold nanorods directly into subcutaneous tumors in mice, and then irradiated the tumor with near-infrared pulsed laser light. Significant suppression of tumor growth was observed. In the case of the intravenous injection of gold nanorods, the suppression of tumor growth was weaker than for the case of direct injection, indicating that the targeted delivery of gold nanorods to the tumor tissue is an important key to improve the therapeutic effect.

We investigated the effect of serofendic acid, a neuroprotective substance derived from fetal calf serum, on the morphological changes in cultured cortical astrocytes. Cultured astrocytes developed a stellate morphology with several processes following exposure to dibutylyl cAMP (dbcAMP), a membrane-permeable cAMP analog; 8-Br-cGMP, a membrane-permeable cGMP analog; or phorbol-12-myristate-13-acetate (PMA), a protein kinase C activator. Serofendic acid significantly accelerated the stellation induced by dbcAMP- and 8-Br-cGMP. In contrast, the PMA-induced stellation was not affected by serofendic acid. Next, we attempted to elucidate the mechanism underlying the dbcAMP-induced stellation and explore the site of action of serofendic acid. Both the stellation induced by dbcAMP and the promotional effect of serofendic acid were partially inhibited by KT5720, a specific protein kinase A (PKA) inhibitor. Furthermore, serofendic acid failed to facilitate the stellation induced by Y-27632, an inhibitor of Rho-associated kinase (ROCK). These results indicate that serofendic acid promotes dbcAMP- and 8-Br-cGMP-induced stellation and the promotional effect on dbcAMP-induced stellation is mediated at least partly by the regulation of PKA activity and not by controlling ROCK activity.

Recently, we have proposed a novel strategy for a cell-specific gene therapy system based on responses to intracellular signals. In this system, an intracellular signal that is specifically and abnormally activated in the diseased cells is used for the activation of transgene expression. In this study, we used protein kinase C (PKC)alpha as a trigger to activate transgene expression. We prepared a PKCalpha-responsive polymer conjugate [PPC(S)] and a negative control conjugate [PPC(A)], in which the phosphorylation site serine (Ser) was replaced with alanine (Ala). The phosphorylation for polymer/DNA complexes was determined with a radiolabel assay using [gamma-(32)P]ATP. PPC(S)/DNA complexes were phosphorylated by the addition of PKCalpha, but no phosphorylation of the PPC(A)/DNA complex was observed. Moreover, after microinjection of polymer/GFP-encoding DNA complexes into HepG2 cells at cation/anion (C/A) ratios of 0.5 to 2.0, significant expression of GFP was observed in all cases using PPC(S)/DNA complexes, but no GFP expression was observed in the negative control PPC(A)/DNA complex-microinjected cells at C/A ratios of 1.0 and 2.0. On the other hand, GFP expression from PPC(S)/DNA complexes was completely suppressed in cells pretreated with PKCalpha inhibitor (Ro31-7549). These results suggest that our gene regulation system can be used for tumor cell-specific expression of a transgene in response to PKCalpha activity.

Abnormal proteolytic processing of amyloid precursor protein (APP) is a pathologic feature of Alzheimer's disease. Recent studies have demonstrated that serine/threonine phosphorylation specifically at amino-acid residue Thr668 (APP695 numbering) regulates APP processing. In this study, we investigated the possibility that tyrosine phosphorylation of APP regulates APP processing. A tyrosine kinase inhibitor decreased expression of the C83 fragment which is a cleaved product of APP by alpha-secretase. By overexpressing APP mutant proteins, Tyr687 was found to be the major tyrosine kinase phosphorylation site. Expression of the C83 fragment was decreased in APPY687A-expressing cells relative to APP wild-type (APPWT)-expressing cells, which likely reflects the different cellular localization patterns of these two proteins. Expression of APP intracellular domain (AICD) which is a cleaved product of the C83 fragment by gamma-secretase was decreased in C83Y687A-expressing cells. These results suggest that phosphorylation of APP at Tyr687 regulates APP processing by alpha- and gamma-secretases, determining the expression level of AICD.

Previously, we showed that in rat cortical neurons, chronic donepezil treatment (10 microM, 4 days) up-regulates nicotinic receptors (nAChR) and makes neurons more sensitive to the neuroprotective effect of donepezil. Here we examined the mechanism of donepezil-induced neuroprotection in neurons chronically treated with donepezil. The mechanism of neuroprotection was examined under different conditions of exposure to glutamate, acute and moderate, that induce cell death associated with necrotic and apoptotic cell death, respectively. Concomitant treatment with antagonists of nAChRs but not muscarinic receptors inhibited donepezil pretreatment-induced neuroprotection against acute glutamate treatment-induced death. Donepezil pretreatment prevented acute glutamate- and ionomycin-induced neurotoxicity, but not S-nitrosocysteine-induced neurotoxicity, suggesting that donepezil protects neurons via nAChR at levels before nitric oxide synthase activation against acute glutamate neurotoxicity. Concomitant treatment with antagonists of nAChR or phosphatidylinositol 3-kinase (PI3K) signaling inhibitors significantly inhibited neuroprotection against moderate glutamate neurotoxicity and decreased the phosphorylation level of Akt. Neuroprotection was also inhibited by treatment with inhibitor of mitogen-activated protein kinase (MAPK) kinase. These results suggest that donepezil protects neurons against moderate glutamate neurotoxicity via nAChR-PI3K-Akt and MAPK signaling pathways. This study provides novel insight into the mechanism of donepezil-induced neuroprotection that involves nAChR up-regulation., ((c) 2008 Wiley-Liss, Inc.)

To investigate whether dopamine agonists induce neurite outgrowth, we examined the effects of dopamine D(2) receptor agonists such as bromocriptine, talipexole, and pramipexole in PC12 cells, a well-studied model of neurite outgrowth. Bromocriptine significantly induced neurite outgrowth in a concentration-dependent manner. Neither talipexole nor pramipexole induced neurite outgrowth. Domperidone and sulpiride, dopamine D(2) receptor antagonists, did not have any effect on the bromocriptine-induced neurite outgrowth. These results suggest that the stimulation of dopamine D(2) receptors would not affect neurite outgrowth in nerve regeneration. Next, we investigated how bromocriptine-induced neurite outgrowth. Bromocriptine is not only a dopamine D(2) receptor agonist but also an ergot alkaloid. We examined the involvement of the structure of ergot alkaloids in the effect of bromocriptine. Ergot alkaloids have been divided into two groups: amino acid ergot alkaloids, including bromocriptine, and amine ergot alkaloids. Amino acid ergot alkaloids, such as ergocornine and ergotamine, induced neurite outgrowth, but amine ergot alkaloids, including ergometrine and methylergometrine, did not. These results indicate that the structure of amino acid ergot alkaloids is important for the effect of bromocriptine. Moreover, the effect of bromocriptine was inhibited by a src inhibitor and a mitogen-activated protein kinase kinase (MEK) inhibitor. Taken together, bromocriptine-induced neurite outgrowth via src and the MEK/extracellular signal-regulated kinase 1/2 signaling pathway in PC12 cells.

We succeeded in cancer cell specific gene expression by using a polyplex responsive to protein kinase Calpha, which is activated in various types of cancer cells.

Dibutyryl cyclic AMP (dbcAMP) and retinoic acid (RA) have been demonstrated to be the inducers of morphological differentiation in SH-SY5Y cells, a human catecholaminergic neuroblastoma cell line. However, it remains unclear whether morphologically differentiated SH-SY5Y cells by these compounds acquire catecholaminergic properties. We focused on the alteration of tyrosine hydroxylase (TH) expression and intracellular content of noradrenaline (NA) as the indicators of functional differentiation. Three days treatment with dbcAMP (1mM) and RA (10microM) induced morphological changes and an increase of TH-positive cells using immunocytochemical analysis in SH-SY5Y cells. The percentage of TH-expressing cells in dbcAMP (1mM) treatment was larger than that in RA (10microM) treatment. In addition, dbcAMP increased intracellular NA content, whereas RA did not. The dbcAMP-induced increase in TH-expressing cells is partially inhibited by KT5720, a protein kinase A (PKA) inhibitor. We also investigated the effect of butyrate on SH-SY5Y cells, because dbcAMP is enzymatically degraded by intracellular esterase, thereby resulting in the formation of butyrate. Butyrate induced the increase of NA content at lower concentrations than dbcAMP, although the increase in TH-expressing cells by butyrate was smaller than that by dbcAMP. The dbcAMP (1mM)- and butyrate (0.3mM)-induced increase in NA content was completely suppressed by alpha-methyl-p-tyrosine (1mM), an inhibitor of TH. These results suggest that dbcAMP induces differentiation into the noradrenergic phenotype through both PKA activation and butyrate.

Neurogenesis occurs in two neurogenic regions of the adult mammalian brain: the subgranular zone and the subventricular zone. We have recently demonstrated that the number of bromodeoxyuridine-positive and doublecortin-positive cells is decreased in the subgranular zone of amyloid precursor protein with a Swedish mutation and presenilin-1 with a deletion of exon 9 transgenic mice, an animal model of Alzheimer's disease. In this study, we characterized neurogenesis in the subventricular zone of amyloid precursor protein with a Swedish mutation and presenilin-1 with a deletion of exon 9 transgenic mice at 9 months of age and compared it with neurogenesis in the subgranular zone. In the subventricular zone, the number of proliferating cell nuclear antigen-positive and bromodeoxyuridine-positive cells were normal. In the subgranular zone, the number of proliferating cell nuclear antigen-positive cells was normal; however, the number of bromodeoxyuridine-positive cells was significantly decreased. These results suggest that neurogenesis, probably reflecting the survival of neural progenitor cells, differs between the subgranular zone and the subventricular zone.

The mechanisms of colorimetric assays based on aggregation of gold nanoparticles (GNPs) have been separated into two categories, crosslinking, and noncrosslinking aggregation. The noncrosslinking aggregation has recently been emerging as a simple and rapid mechanism and has been applied to enzymatic activity assays and DNA detection. We report here the detailed study of an enzymatic activity assay for protein kinases based on noncrosslinking aggregation. The principle of the assay is to detect kinase activity by utilizing the difference of coagulating ability of a cationic substrate peptide and its phosphorylated form toward GNPs with anionic surface charge. The critical coagulation concentrations (CCCs) of the peptides were about 10(3) times lower than those of the metal cations with the same cationic charges. The multivalent coordination bonds of the functional groups of the peptides with the GNP surface will strongly support the adsorption of the peptide on the GNP surface. The effect of the GNP size (10, 20, 40, 60 nm) on the dynamic range of OD before and after aggregation was studied. The dynamic range became a maximum for 20 nm GNP among those studied. The difference of CCC between the phosphorylated and nonphosphorylated peptides was governed by (1) the ratio between the peptide concentration and the surface area concentration of GNP and (2) the net charge of the peptides. When the assay system was applied to the activity assessment of protein kinase A, the dynamic range of OD was largest for 20 nm GNPs. However, when the peptide concentration was lowered, the largest 60 nm GNP was advantageous because of its smaller specific surface area.

Activated astrocytes show various patterns of Ca(2+) mobilization under pathological conditions. In the present study we revealed a novel function of astrocytic Ca(2+) dynamics through investigation of thrombin-induced unique Ca(2+) entry. Using 1321N1 human astrocytoma cells, which have been shown to be a good model for detecting morphological dynamics, we observed rapid retraction of bipolar protrusions that were reversibly evoked by 0.03-3 U/mL thrombin. Morphological changes were predominantly dependent on a specific thrombin receptor subtype, proteinase-activated receptor 1 (PAR-1). In parallel, Fura-2 imaging of intracellular Ca(2+) concentration ([Ca(2+)](i)) showed that thrombin induced heterogeneous Ca(2+) responses with asynchronous repetitive peaks. These oscillations were found to be a result of repetitive Ca(2+) release from intracellular stores, followed by refilling of Ca(2+) from the extracellular region without a direct [Ca(2+)](i) increase. Pharmacological manipulation with BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate indicated that Ca(2+) mobilization was involved in thrombin-induced morphological changes. We further addressed the role of Ca(2+) entry using small interfering RNA (siRNA) for transient receptor potential canonical 3 (TRPC3). As a result, both thrombin-induced morphological changes and oscillatory Ca(2+) responses were significantly attenuated in siRNA-transfected cells. Inhibition of TRPC3 with pyrazole-3 also provided support for the contribution of Ca(2+) influx. Moreover, TRPC3-mediated Ca(2+) dynamics regulated thrombin-induced phosphorylation of myosin light chain 2. These results suggest a novel function of astrocytic Ca(2+) dynamics, including Ca(2+) entry, in the pathophysiological effects of PAR-1-mediated astrocytic activation. TRPC3 forms a functional Ca(2+) channel and might modulate astrocytic activation in response to brain hemorrhaging., ((c) 2008 Wiley-Liss, Inc.)

Beta-site APP cleaving enzyme-1 (BACE-1), is a rate-limiting enzyme for beta amyloid production. Beta amyloid induces the production of radical oxygen species and neuronal injury. Oxidative stress plays a key role in various neurological diseases such as ischemia and Alzheimer's disease. Recent studies suggest that oxidative stress induces BACE-1 protein upregulation in neuronal cells. Here, we demonstrate that naturally occurring compounds (-)-epigallocatechin-3-gallate and curcumin suppress beta amyloid-induced BACE-1 upregulation. Exposure of beta amyloid 1-42 to neuronal culture increased BACE-1 protein levels. (-)-Epigallocatechin-3-gallate or curcumin significantly attenuated beta amyloid-induced radical oxygen species production and beta-sheet structure formation. These two compounds have novel pharmacological effects that may be beneficial for Alzheimer's disease treatment.

alpha7-nicotinic acetylcholine receptors are one of the most abundant subtypes of nicotinic receptors in the brain and have been shown to be involved in the neuroprotective effect of donepezil. Recently, we showed that in primary culture of rat cortical neurons, chronic donepezil treatment (10 muM, 4 days) (1) induces the up-regulation of alpha7-nicotinic receptors, (2) enhances the nicotine-induced increase in [Ca(2+)](i) and (3) enhances the sensitivity to the neuroprotective effect of donepezil. Here we demonstrate the involvement of alpha7-nicotinic receptors in these three effects. Concomitant treatment with nicotinic receptor antagonist inhibited the up-regulation of alpha7-nicotinic receptor, enhancement of the increase in [Ca(2+)](i) induced by nicotine, and enhancement of sensitivity to the neuroprotective effect of donepezil. Next, using inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways, we demonstrate the involvement of these pathways in the up-regulation of alpha7-nicotinic receptors and in making the neurons more sensitive to the neuroprotective effects of donepezil. Concomitant chronic donepezil treatment with inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways inhibited nicotinic receptor up-regulation and enhancement of the response to nicotine, and enhanced the sensitivity to donepezil. This study increases understanding of the less-studied mechanism of chronic donepezil treatment-induced nicotinic receptor up-regulation and increased sensitivity to donepezil.

Electroporation is a useful means for non-viral gene delivery. Here, we investigated the use of electroporation to deliver polymer-DNA complexes into living cells using a protein kinase C (PKC)alpha-responsive polymer. The polymer was complexed with a luciferase-encoding DNA and electroporated into B16 melanoma cells. Gene expression from polymer-DNA complexes was 3- to 5-fold higher than from naked DNA. Moreover, after introduction of the polymer-DNA complex into tissues, luciferase levels were >2-fold higher in B16 melanoma tumors than in normal skin tissue. These results suggest that the combination of our polymer and electroporation is useful for the effective delivery of DNA into tumors.

Gold nanorods showing surface plasmon (SP) bands in the near-IR region are used as bioimaging probes that respond to near-IR light in mice. The SP bands of intravenously injected polyethylene glycol-modified gold nanorods are directly monitored from the mouse abdomen by using a spectrophotometer equipped with an integrating sphere. The absorbance at 900 nm from the gold nanorods immediately increases after injection and reaches a plateau. The injection of phosphatidylcholine-modified gold nanorods also increases the absorbance at 900 nm, but the absorbance decreases single exponentially with a 1.3-min half-life. In vivo spectral changes of gold nanorods depend on the surface characteristics, and can be observed in real time using simple spectroscopic measurements.

Gene therapy is a growing topic in the medical arena. Since the safety system of gene therapy has not been sufficiently established, its clinical use is limited. Recently, we developed a cell-specific gene regulation system based on a new concept, D-RECS, or Drug and Gene Delivery System Responding to Cellular Signals. We hoped here to apply this D-RECS concept to gene therapy for virus infections. In the present study, we report the design, synthesis and characterization of the functional polymers, which are able to discriminate normal and human immunodeficiency virus type 1 (HIV-1) infected cells. In the D-RECS concept, certain intracellular signals, which are extraordinary activated in the target disease cells specifically, are used as a trigger to activate a transgene expression. Thus, we paid attention to HIV protease as a target signal in this case, because HIV protease is essential for the proliferation of HIV. This protease is therefore an indicator of HIV infection. Two types of polymers were designed and synthesized using methacryloyl peptide and acrylamide with radical copolymerization as a functional gene regulator. The grafted peptide possesses a cationic protein transduction domain (PTD) sequence of HIV-Tat protein, GRKKRRQRRRPPQ for cell permeation, which are connected with polyacrylamide backbone via a consensus substrate sequence for HIV protease, SQNY/PIVQ. At first, the polymers were evaluated to see whether they possess DNA binding ability and HIV protease responsibility using gel retardation assay. The results suggested that a polymer could form a stable complex with DNA and release the DNA specifically responding to HIV protease activity. Furthermore, it was shown that this controlled release of DNA by the HIV protease signal-responsive intelligent polymer actually regulated the gene expression in the cell-free system. This system would be a useful tool for gene therapy in HIV infection, and this methodology will be applicable if the cationic peptide is replaced by another virus-specific protease, which is critical for the replication of a corresponding virus.

The excitatory neurotransmitter glutamate can accumulate in the brain and is thought to be involved in the etiology of many neurodegenerative disorders, including ischemia and Alzheimer disease. Therefore, it is important to search for compounds that reduce glutamate neurotoxicity. This glutamate-mediated excitotoxicity is caused by intracellular Ca2+ overload via the N-methyl-D-aspartate receptor NMDAR), reactive oxygen species (ROS) generation, and caspase-3 activation. Here we show that the natural flavonoid myricetin inhibited glutamate-induced excitotoxicity and protected neurons by multiple, distinct pathways. First, myricetin affected modulation of the NMDAR by phosphorylation, causing a subsequent reduction in glutamate-induced intracellular Ca2+ overload. Second, myricetin inhibited the ROS production caused by glutamate. Finally, glutamate-induced activation of caspase-3 was reduced by myricetin treatment. Moreover, myricetin directly interacted with the active site of caspase-3 via three hydrogen bonds and inhibited its activity. We conclude that myricetin inhibited glutamate-induced neuronal toxicity by multiple biochemical pathways. These results show that myricetin is a potent antineurodegenerative compound and may contribute to the discovery of a drug with which to combat neurodegeneration., ((c) 2008 Wiley-Liss, Inc.)

We previously reported that a sulfur-containing neuroprotective substance named serofendic acid purified and isolated from fetal calf serum prevented glutamate neurotoxicity in rat cortical cultured neurons. In the present study, we investigated the effect of serofendic acid on ischemic injury induced by a transient occlusion of the middle cerebral artery in rats. Serofendic acid was intracerebroventricularly administered 30 min after the onset of the occlusion. Serofendic acid (30 nmol) significantly reduced total infarct volume, similar to edaravone (30 nmol), a free radical scavenger. Treatment with serofendic acid (1-30 nmol) reduced the infarct volume in a dose-dependent manner. Moreover, serofendic acid (30 nmol) improved neurological deficit scores. These results suggest that intracerebroventricular administration of serofendic acid prevents the neurodegeneration induced by a transient focal cerebral ischemia and reperfusion.

We have recently demonstrated that microglia as multipotential stem cells give rise to microtubule-associated protein 2 (MAP2)-positive and glial fibrillary acidic protein (GFAP)-positive cells and that microglia-derived MAP2-positive cells possess properties of functional neurons. In this study, we investigated the molecular pathways involved in the generation of microglia-derived MAP2-positive and GFAP-positive cells. Western blot analyses demonstrated that expression levels of Id2 protein, an inhibitory basic helix-loop-helix transcription factor of the inhibitor of differentiation and DNA binding family, and Smad proteins were upregulated under differentiation conditions. Immunocytochemical analyses demonstrated that the generation of MAP2-positive and GFAP-positive cells from microglia was promoted by bone morphogenetic proteins (BMPs) and was inhibited by noggin which is a BMP antagonist, Smad4 siRNA and Id2 siRNA. These results indicate that activation of BMP signaling through Smad and Id2 proteins is one of the molecular pathways involved in the generation of microglia-derived MAP2-positive and GFAP-positive cells.

Generation and accumulation of the amyloid beta peptide (Abeta) following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 (Beta-site APP Cleaving Enzyme-1, beta-secretase) and gamma-secretase is a main causal factor of Alzheimer's disease (AD). Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Abeta, is an attractive therapeutic approach for the treatment of AD. In this study, we discovered that natural flavonoids act as non-peptidic BACE-1 inhibitors and potently inhibit BACE-1 activity and reduce the level of secreted Abeta in primary cortical neurons. In addition, we demonstrated the calculated docking poses of flavonoids to BACE-1 and revealed the interactions of flavonoids with the BACE-1 catalytic center. We firstly revealed novel pharmacophore features of flavonoids by using cell-free, cell-based and in silico docking studies. These results contribute to the development of new BACE-1 inhibitors for the treatment of AD.

The purpose of this study was to find protein kinase C (PKC) isozyme-specific peptides. A peptide library containing 1772 sequences was designed using Scansite and screened by MALDI-TOF MS and kinase activity assays for PKC isozyme-specificity. A peptide (Alphatomega; H-FKKQGSFAKKK-NH(2)) with high specificity for PKC alpha relative to other isozymes was identified. The peptide was phosphorylated to a greater extent by tissue lysates from B16 melanoma, HepG2, and human breast cancer, which had higher levels of activated PKC alpha, when compared to normal skin, liver, and human breast tissue lysates, respectively. Moreover, addition of Ro-31-7549, an inhibitor with great specificity for PKC alpha, to the phosphorylation reaction caused a dose-dependent reduction in phosphorylation, but no inhibition was identified with the addition of rottlerin and H-89. These results show that this peptide has great potential as a PKC alpha-specific substrate.

Microglia are believed to play an important role in the regulation of phagocytosis, neuronal survival, neuronal cell death, and inflammation. Recent studies have demonstrated that microglia are multipotential stem cells that give rise to neurons, astrocytes, and oligodendrocytes. However, the functional properties of neurons derived from microglia are poorly understood. In this study, we investigated the possibility that microglia differentiate into functional neurons. Immunocytochemical study demonstrated that microtubule-associated protein 2 (MAP2)-positive cells were derived from microglia under differentiation conditions. Intracellular Ca(2+) imaging study demonstrated that KCl caused no significant changes in [Ca(2+)](i) in microglia, whereas it caused a remarkable increase in [Ca(2+)](i) in microglia-derived cells. Furthermore, electrophysiological study demonstrated that the spike waveform, firing rate, and tetrodotoxin sensitivity of extracellular action potentials evoked by 4-aminopyridine from microglia-derived MAP2-positive cells were nearly identical to those from cultured cortical neurons. These results suggest that microglia-derived MAP2-positive cells possess properties of functional neurons.

We developed a peptide microarray based on surface plasmon resonance (SPR) imaging for monitoring protein kinase activities in cell lysates. The substrate peptides of kinases were tethered to the microarray surface modified with a self-assembled monolayer of an alkanethiol with triethylene glycol terminus to create a low nonspecific binding surface. The phosphorylation of the substrate peptides immobilized on the surface was detected with the following phosphate specific binders by amplifying SPR signals: anti-phosphotyrosine antibody for tyrosine kinases and Phos-tag biotin (a phosphate-specific ligand with biotin tag) for serine/threonine kinases. Using the microarray, 9 kinds of protein kinases were evaluated as a pattern of phosphorylation of 26 kinds of substrate peptides. The pattern was unique for each protein kinase. The microarray could be used to evaluate the inhibitory activities of kinase inhibitors. The microarray was applied successfully for kinase activity monitoring of cell lysates. The chemical stimuli responsive activity changes of protein kinases in cell lysates could also be monitored by the peptide microarray. Thus, the peptide microarray based on SPR imaging would be applicable to cell-based drug discovery, diagnosis using tissue lysates, and biochemical studies to reveal signal transduction pathways.

Preconditioning of sublethal ischemia exhibits neuroprotection against subsequent ischemia-induced neuronal death. It has been indicated that glutamate, an excitatory amino acid, is involved in the pathogenesis of ischemia-induced neuronal death or neurodegeneration. To elucidate whether prestimulation of glutamate receptor could counter ischemia-induced neuronal death or neurodegeneration, we examined the effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), an ionotropic subtype of glutamate receptor, on excess glutamate-induced excitotoxicity using primary cortical neuronal cultures. We found that AMPA exerted a neuroprotective effect in a time- and concentration-dependent manner. A blocker of phosphatidylinositol-3 kinase (PI3K), LY294002 (10 microM), significantly attenuated AMPA-induced protection. In addition, Ser473 of Akt/PKB, a downstream target of PI3K, was phosphorylated by AMPA administration (10 microM). Glycogen synthase kinase 3beta (GSK3beta), which has been reported to be inactivated by Akt, was phosphorylated at Ser9 by AMPA. Ser9-phosphorylated GSK3beta or inactivated form would be a key molecule for neuroprotection, insofar as lithium chloride (100 microM) and SB216763 (10 microM), inhibitors of GSK3beta, also induced phosphorylation of GSK3beta at Ser9 and exerted neuroprotection, respectively. Glutamate (100 microM) increased cleaved caspase-3, an apoptosis-related cysteine protease, and caspase-3 inhibitor (Ac-DEVD-CHO; 1 microM) blocked glutamate-induced excitotoxicity in our culture. AMPA (10 microM, 24 hr) and SB216763 (10 microM) prominently decreased glutamate-induced caspase-3 cleavage. These findings suggest that AMPA activates PI3K-Akt and subsequently inhibits GSK3beta and that inactivated GSK3beta attenuates glutamate-induced caspase-3 cleavage and neurotoxicity.

Enzymatic activity monitoring of caspases, which are a class of cysteine protease, was performed by using peptide arrays based on surface plasmon resonance (SPR) imaging. The strategy of the detection is straightforward, using streptavidin to amplify the SPR signals of the surface-immobilized substrate peptides labeled with biotin at the C termini. Thus, the cleavage of the substrate peptides by caspases was detected as a signal decrease. Using this method, we succeeded in monitoring the activities of purified caspases and caspases in cell lysates. The SPR imaging-based peptide array would be applicable to cell-based drug screening and biochemical studies to reveal signal transduction processes.

Cholinergic hypothesis and amyloid cascade hypothesis are mainly proposed for Alzheimer's disease; however, the relationship between these hypotheses is poorly understood. To address the question of whether amyloid beta-peptide pathology affects cholinergic neurotransmission, we examined the effect of a cholinesterase inhibitor, physostigmine, on field excitatory postsynaptic potentials (EPSPs) evoked by single-pulse stimulation in the CA1 region of the hippocampus of various APPswe/PS1dE9 transgenic mice with different degrees of amyloid beta-peptide pathology. Reduced field EPSPs by physostigmine in transgenic mice at 3 months of age, when the mice had negligible amyloid beta-peptide levels and no amyloid beta-peptide deposits, were indistinguishable from those in age-matched wild-type mice. In contrast, reduced field EPSPs by physostigmine in transgenic mice at 5 months of age, when the mice had low amyloid beta-peptide levels and subtle amyloid beta-peptide deposits, were significantly lower than those in age-matched wild-type mice. Next, we characterized acetylcholine receptors, which play important roles in cholinergic neurotransmission, because physostigmine resulted in increased acetylcholine levels in the synaptic cleft. Different reductions of field EPSPs by physostigmine between transgenic and wild-type mice at 5 months of age were not affected by a nicotinic receptor antagonist, mecamylamine; however, reduced field EPSPs by physostigmine in both transgenic and wild-type mice were restored to basal levels by a muscarinic receptor antagonist, atropine. These results indicate that cholinergic modulation of glutamatergic transmission is already impaired at the onset of the formation of amyloid beta-peptide deposits, and muscarinic receptor dysfunction is one of the causes of this impairment.

The vascular endothelium plays an important role in regulating vascular homeostasis. Damage to the endothelium can lead to cardiovascular diseases such as arteriosclerosis. Therefore, early-stage detection and evaluation of vascular endothelium dysfunction would be very important for effective diagnosis and therapy. We synthesized a polymeric drug carrier bearing an Evans blue analogue as a probing unit for endothelium injury. The polymeric carrier spontaneously formed stable nanoparticles with micelle-like structure in aqueous media and could encapsulate hydrophobic doxorubicin (DOX). The encapsulated DOX showed a sustainable release profile over a period of 10-60 h depending on the loaded DOX concentration. The polymeric carrier specifically adsorbed against the endothelium-injured site in extracted porcine aorta. These properties of the polymeric drug carrier will be suitable for specific drug delivery to endothelium dysfunctional region.

RNA interference (RNAi) is a valuable tool for the validation of gene identification and functional genomics. Previously, it was reported that 6th generation dendritic poly(L-lysine) (KG6) transfected DNA into several cultivated cell lines with high efficiency and without any cytotoxic effects. In this study, the potential of KG6 to be an efficient siRNA carrier is investigated. KG6 showed effective knockdown of GAPDH with low cytotoxicity in combination with the weak-base amphiphilic peptide, Endo-Porter. In addition, the knockdown of PEPCK, which is the rate-limiting enzyme for gluconeogenesis, led to a reduction in glucose production in rat hepatoma H4IIEC3 cells. Knockdown of organic cation transporter 1 (OCT1), which is thought to be the gene that influences metformin action, was shown to successfully diminish the ability of metformin to inhibit gluconeogenesis in H4IIEC3 cells. In conclusion, using a combination of KG6 and Endo-Porter, a model system in which genes that influence metformin action can be identified was successfully constructed.

Myricetin (3,3',4',5,5',7-hexahydroxyflavone) is classified as a flavonoid with strong antioxidant effects. Oxidative stress plays a key role in various neurological diseases such as ischemia and Alzheimer's disease (AD). To elucidate whether myricetin could counter the progress of AD, we examined the effects of myricetin on neurotoxicity induced by beta-amyloid (A beta), a component of senile plaques in the AD brain. We found that cultured rat primary cortical neurons treated for 48 hr with A beta1-42 (1 microM) induced significant neuronal injury. Conformationally altered A beta1-42 caused apoptotic changes, such as nuclear fragmentation, as shown by DAPI staining. Pre- and simultaneous administration of myricetin and A beta1-42 reduced A beta neurotoxicity in a concentration-dependent manner. By using circular dichroism spectroscopy and a thioflavin T binding assay, we show that myricetin (10 microM, 48 hr) prevented structural changes in A beta1-42 from a random coil to a beta-sheet-rich structure. A beta1-42-induced apoptotic changes and caspase-3 activation were reduced by myricetin treatment. Furthermore, we determined that administration of myricetin significantly decreased A beta1-40 and A beta1-42 levels in culture media. These effects were based on two mechanisms: the activation and up-regulation of alpha-secretase (ADAM10) protein levels as indicated by fluorescence resonance energy transfer (FRET) assay and immunoblot analysis and the direct binding and inhibition of beta-secretase (BACE-1) indicated by cell-free FRET assays. Evidently, myricetin has multiple functions to counter the progress of AD by the reduction of A beta production and the detoxification of A beta through a structural change., ((c) 2007 Wiley-Liss, Inc.)

To identify the correlation between the phosphorylation ratios by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) analysis and enzyme kinetics (Km, Vmax, and Vmax/Km) is important to understand whether MALDI-TOF MS can be applied for monitoring the properties of peptides that are substrates of protein kinases. The correlation between phosphorylation ratios and enzyme kinetics was examined using peptides for protein kinase C (PKC) and for 60 kDa phosphoprotein, encoded by the cellular sarcoma gene (c-Src). Phosphorylation ratios, analyzed by MALDI-ToF MS, showed higher correlation coefficient (r = or > +0.7) for Vmax/Km compared with that (r = or < -/+0.6) for Km or Vmax. For ion modes, a higher correlation coefficient between phosphorylation ratios and Vmax/Km was identified in the positive mode (r = or > +0.7) compared to that in the negative mode (r = or < +0.5). These results suggest that MALDI-ToF MS is a useful tool to evaluate Vmax/Km of peptides for protein kinases.

The purpose of this study was to investigate whether plasma can influence the phosphorylation of protein kinase C (PKC). Lysate samples were prepared from normal skin or melanoma tissue and were reacted with a PKC peptide substrate in the presence or absence of plasma. In normal skin tissue lysates, the phosphorylation rates were much lower than those in melanoma tissue lysates. However, the level of phosphorylated peptide was increased in both normal skin and melanoma tissue lysates if plasma was present. Phosphorylation rates in the samples taken from the centre of B16 melanoma tissue were lower than those in samples taken from the edge. Moreover, addition of activator and/or cofactors (diacylglycerol, phosphatidylserine and/or Ca2+) of PKC, or plasma to the lysates contaminated by plasma had no effect on phosphorylation rates for the peptide substrate. These results indicate that plasma can play a role of activator and cofactor for substrate phosphorylation., (2007 John Wiley & Sons, Ltd.)

A recent hypothesis suggests that there is impaired hippocampal neurogenesis in Alzheimer's disease. Here we examined the proliferation, the first stage in neurogenesis, of hippocampal progenitor cells in amyloid precursor protein with Swedish mutation and presenilin-1 with deletion of exon 9 (APPswe/PS1dE9) transgenic mice. Compared with age-matched wild-type mice, transgenic mice at 5 months of age with low amyloid beta-peptide (Abeta) levels and subtle Abeta deposits showed normal proliferation of hippocampal progenitor cells; however, transgenic mice at 9 months of age with high Abeta levels and numerous Abeta deposits showed decreased proliferation of these cells. The number of proliferating cells in male transgenic mice was indistinguishable from that in female transgenic mice. These results indicate that neurogenesis is decreased with degrees of Abeta pathology, and that there is no gender difference in their proliferation in APPswe/PS1dE9 transgenic mice.

We present efficient syntheses of serofendic acids A and B (SA-A and SA-B), novel neuroprotective substances isolated from fetal calf serum. Biological and pharmacological evaluation showed that SA-A and SA-B have potent protective action against glutamate-induced neurotoxicity, but do not interact directly with glutamate receptors. A pharmacokinetic study showed that they have good oral bioavailability in rats. The results indicate that SA-A and SA-B are potential lead compounds for candidate drugs to treat various neurological disorders.

The ionization of phosphorylated peptides is usually suppressed by non-phosphorylated peptides when alpha-cyano-4-hydroxycinnamic acid (CHCA) is used as a matrix for matrix-assisted laser desorption/ionization-time-of-Flight (MALDI-TOF) mass spectrometry analysis. In the present study, we examined the effect of diammonium citrate addition to the CHCA matrix on the detection of phosphorylated peptides. Substrates for protein kinase C (PKC) and c-Src were synthesized and phosphorylated by reaction with cell and tissue lysate samples. The addition of diammonium citrate to the CHCA matrix increased the sensitivity for distinguishing phosphorylated peptides from background noise. However, the effect depended on substrate concentration.

Donepezil is a potent and selective acetylcholinesterase inhibitor developed for the treatment of Alzheimer's disease. To elucidate whether donepezil causes neuronal differentiation, we examined its effect on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Donepezil (10 microM) significantly potentiated the neurite outgrowth evoked by low (1 ng/ml) and high (50 ng/ml) concentrations of NGF. The effect of donepezil (1 - 10 microM) was concentration-dependent. The enhancement of neurite outgrowth caused by donepezil was not blocked by the acetylcholine receptor (AChR) antagonists mecamylamine and scopolamine. Furthermore, the AChR agonists nicotine and carbachol did not affect the neurite outgrowth induced by NGF. Donepezil (10 microM) also significantly potentiated neurite outgrowth evoked by dibutyryl cyclic AMP. Moreover, donepezil potentiated the NGF-induced phosphorylation of extracellular signal-regulated kinase (ERK). These results suggest that donepezil potentiated neuronal differentiation by enhancing the activation of ERK.

Mulberry leaf has been reported to possess medicinal properties, including hypoglycemic, hypotensive and diuretic effects. Little is known, however, about its medicinal properties for central nervous system disorders, including Alzheimer's disease. Accumulating evidence suggests that amyloid beta-peptide (1-42) plays an important role in the etiology of Alzheimer's disease. Here we show that mulberry leaf extract inhibits the amyloid beta-peptide (1-42) fibril formation by both the thioflavin T fluorescence assay and atomic force microscopy. Furthermore, mulberry leaf extract protected hippocampal neurons against amyloid beta-peptide (1-42)-induced cell death in a concentration-dependent manner. These results suggest that mulberry leaf extract provides a viable treatment for Alzheimer's disease through the inhibition of amyloid beta-peptide (1-42) fibril formation and attenuation of amyloid beta-peptide (1-42)-induced neurotoxicity.

Little is known on mechanisms involved in transport of complex of DNA and gene carrier molecules from the cytosol to the nucleus. We aimed to identify cytosolic proteins interacting with the polyethylenimine (PEI)/DNA complex, using 2-D gel electrophoresis and peptide mass fingerprinting. Fifteen proteins including actin, beta-tubulin, and other metabolic proteins were identified. They demonstrated various molecular weights and isoelectric points, and were categorized into 3 groups: early binding, late binding, and transient binding proteins. Protein binding caused DNA release from the PEI/DNA complex with a cation/anion (C/A) ratio of 2, where complex formation was weak. Knowledge on interactions between cytosolic proteins and DNA/carrier complexes will help understand intracellular gene delivery mechanisms.

In this study, we prepared gold nanorod (NR)-embedded N-isopropylacrylamide (NIPAM) hydrogels and studied their volume phase transition behavior induced by near-infrared (near-IR) laser irradiation utilizing the photothermal conversion characteristics of the NRs. When poly(ethylene glycol)-modified NRs were used for the preparation of composite gels, the NRs showed marked dispersion stability in the gel. Near-IR laser irradiation of the gel (cylindrical shape, diameter = 140 microm) under the following conditions, NR concentrations in the gel > or =100 microM and laser irradiation power > or =490 mW, resulted in shrinkage of the gel in the following manner: (1) waist formation around the irradiation spot and (2) growth of the waist along the axial directions of the gel. The gel shrinking induced by near-IR irradiation occurred much more rapidly than that afforded by a temperature jump, because the former was not accompanied by the skin layer formation, which disturbs the rapid shrinking of the gels. When a composite gel containing the model drug (rhodamine-labeled dextran) was irradiated with a near-IR laser, the rapid release of the drug was observed. Taking advantage of the high spatial resolution of the irradiation point, we further achieved the irradiation-point-specific release of the drug from one such gel.

DNA microarray enables the analysis of DNA or mRNA expression levels, but it has not been possible to completely understand life using obtained information. Consequently, protein or peptide arrays have attracted much interest. Since the development of a practical protein microarray is still far away in light of handling difficulties, the peptide microarray is a promising tool for analyzing protein functions. We have developed a peptide microarray to detect protein kinase activity in cell lysate. All substrate peptides for kinases were immobilized chemoselectively on amino-coated glass slides. After phosphorylation of the immobilized peptides, phosphorylation was detected by fluorescence imaging. We detected the protein kinase activities, including that in cell lysate, in response to drug stimulation. Therefore, this peptide microarray would be useful for a high-throughput kinase assay of intracellular signals and would be applicable to drug screening.

A nonamer peptide containing a diene group in the center of the sequence was synthesized. When the peptide forms an antiparallel beta-sheet, the diene groups align ca. 5 A apart on the beta-sheet. The diene groups successfully photopolymerized without distorting the beta-sheet structure. The obtained beta-sheet showed high stability against acid denaturation and addition of 1,1,1,3,3,3-hexafluoroisopropanol.

Neural stem/progenitor cells (NSPCs) proliferate and differentiate depending on their intrinsic properties and local environment. It has been recognized that astrocytes promote neurogenic differentiation of NSPCs, suggesting the importance of cell-cell interactions between glial cells and NSPCs. Recent studies have demonstrated that microglia, one type of glial cells, play an important role in neurogenesis. However, little is known about how activated microglia control the proliferation and differentiation of NSPCs. In this study, we investigated the possibility that microglia-derived soluble factors regulate the behaviour of NSPCs. To this end, NSPCs and microglial cultures were obtained from rat embryonic day 16 subventricular zone (SVZ) and rat postnatal 1 day cortex, respectively, and the conditioned medium from microglia was prepared. Microglial-conditioned medium had no significant effect on the proliferation of NSPCs. In contrast, it increased the percentage of cells positive for a marker of astrocytes, glial fibrillary acidic protein (GFAP) during differentiation. The induction of astrocytic differentiation by microglial-conditioned medium was reduced by the inhibition of the Janus kinase/signal transducer and activation of transcription (JAK/STAT) and mitogen-activated protein kinase (MAPK) pathways. Furthermore, microglia-derived interleukin (IL)-6 and leukaemia inhibitory factor (LIF) were identified as essential molecules for this astrocytic differentiation using neutralizing antibodies and recombinant cytokines. Our results suggest that microglia as well as astrocytes contribute to the integrity of the local environment of NSPCs, and at least IL-6 and LIF released by activated microglia promote astrocytic differentiation of NSPCs via the activation of the JAK/STAT and MAPK pathways.

Rho-associated kinase (Rho-kinase/ROCK/ROK) is a serine/threonine kinase and plays an important role in various cellular functions. The cAMP-dependent protein kinase (protein kinase A/PKA) and protein kinase C (PKC) are also serine/threonine kinases, and directly and/or indirectly take part in the signal transduction pathways of Rho-kinase. They have similar phosphorylation site motifs, RXXS/T and RXS/T. The purpose of this study was to identify whether sites phosphorylated by Rho-kinase could be targets for PKA and PKC and to find peptide substrates that are specific to Rho-kinase, i.e., with no phosphorylation by PKA and PKC. A total of 18 substrates for Rho-kinase were tested for phosphorylation by PKA and PKC. Twelve of these sites were easily phosphorylated. These results mean that Rho-kinase substrates can be good substrates for PKA and/or PKC. On the other hand, six Rho-kinase substrates showing no or very low phosphorylation efficiency (<20%) for PKA and PKC were identified. Kinetic parameters (K(m) and k(cat)) showed that two of these peptides could be useful as substrates specific to Rho-kinase phosphorylation.