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Find video protocols related to scientific articles indexed in Pubmed.
Identification and characterization of PKC?, a kinase associated with SCA14, as an amyloidogenic protein.
Hum. Mol. Genet.
PUBLISHED: 09-12-2014
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Amyloid assemblies are associated with a wide range of human disorders, including Alzheimer's and Parkinson's diseases. Here, we identify protein kinase C (PKC) ?, a serine/threonine kinase mutated in the neurodegenerative disease spinocerebellar ataxia type 14 (SCA14), as a novel amyloidogenic protein with no previously characterized amyloid-prone domains. We found that overexpression of PKC? in cultured cells, as well as in vitro incubation of PKC? without heat or chemical denaturants, causes amyloid-like fibril formation of this protein. We also observed that SCA14-associated mutations in PKC? accelerate the amyloid-like fibril formation both in cultured cells and in vitro. We show that the C1A and kinase domains of PKC? are involved in its soluble dimer and aggregate formation and that SCA14-associated mutations in the C1 domain cause its misfolding and aggregation. Furthermore, long-term time-lapse imaging indicates that aggregates of mutant PKC? are highly toxic to neuronal cells. Based on these findings, we propose that PKC? could form amyloid-like fibrils in physiological and/or pathophysiological conditions such as SCA14. More generally, our results provide novel insights into the mechanism of amyloid-like fibril formation by multi-domain proteins.
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Synthesis and biological activities of simplified analogs of the natural PKC ligands, bryostatin-1 and aplysiatoxin.
Chem Rec
PUBLISHED: 02-20-2014
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Protein kinase C (PKC) isozymes play central roles in signal transduction on the cell surface and could serve as promising therapeutic targets of intractable diseases like cancer, Alzheimer's disease, and acquired immunodeficiency syndrome (AIDS). Although natural PKC ligands like phorbol esters, ingenol esters, and teleocidins have the potential to become therapeutic leads, most of them are potent tumor promoters in mouse skin. By contrast, bryostatin-1 (bryo-1) isolated from marine bryozoan is a potent PKC activator with little tumor-promoting activity. Numerous investigations have suggested bryo-1 to be a promising therapeutic candidate for the above intractable diseases. However, there is a supply problem of bryo-1 both from natural sources and by organic synthesis. Recent approaches on the synthesis of bryo-1 have focused on its simplification, without decreasing the ability to activate PKC isozymes, to develop new medicinal leads. Another approach is to use the skeleton of natural PKC ligands to develop bryo-1 surrogates. We have recently identified 10-methyl-aplog-1 (26), a simplified analog of tumor-promoting aplysiatoxin (ATX), as a possible therapeutic lead for cancer. This review summarizes recent investigations on the simplification of natural PKC ligands, bryo-1 and ATX, to develop potential medicinal leads.
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A new lyngbyatoxin from the Hawaiian cyanobacterium Moorea producens.
Mar Drugs
PUBLISHED: 02-17-2014
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Lyngbyatoxin A from the marine cyanobacterium Moorea producens (formerly Lyngbya majuscula) is known as the causative agent of "swimmer's itch" with its highly inflammatory effect. A new toxic compound was isolated along with lyngbyatoxin A from an ethyl acetate extract of M. producens collected from Hawaii. Analyses of HR-ESI-MS and NMR spectroscopies revealed the isolated compound had the same planar structure with that of lyngbyatoxin A. The results of optical rotation and CD spectra indicated that the compound was a new lyngbyatoxin A derivative, 12-epi-lyngbyatoxin A (1). While 12-epi-lyngbyatoxin A showed comparable toxicities with lyngbyatoxin A in cytotoxicity and crustacean lethality tests, it showed more than 100 times lower affinity for protein kinase C? (PKC?) using the PKC?-C1B peptide when compared to lyngbyatoxin A.
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Identification of 6-octadecynoic acid from a methanol extract of Marrubium vulgare L. as a peroxisome proliferator-activated receptor ? agonist.
Biochem. Biophys. Res. Commun.
PUBLISHED: 08-30-2013
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6-Octadecynoic acid (6-ODA), a fatty acid with a triple bond, was identified in the methanol extract of Marrubium vulgare L. as an agonist of peroxisome proliferator-activated receptor ? (PPAR?). Fibrogenesis caused by hepatic stellate cells is inhibited by PPAR? whose ligands are clinically used for the treatment of diabetes. Plant extracts of Marrubium vulgare L., were screened for activity to inhibit fibrosis in the hepatic stellate cell line HSC-T6 using Oil Red-O staining, which detects lipids that typically accumulate in quiescent hepatic stellate cells. A methanol extract with activity to stimulate accumulation of lipids was obtained. This extract was found to have PPAR? agonist activity using a luciferase reporter assay. After purification using several chromatographic methods, 6-ODA, a fatty acid with a triple bond, was identified as a candidate of PPAR? agonist. Synthesized 6-ODA and its derivative 9-octadecynoic acid (9-ODA), which both have a triple bond but in different positions, activated PPAR? in a luciferase reporter assay and increased lipid accumulation in 3T3-L1 adipocytes in a PPAR?-dependent manner. There is little information about the biological activity of fatty acids with a triple bond, and to our knowledge, this is the first report that 6-ODA and 9-ODA function as PPAR? agonists.
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Intracellular accumulation of toxic turn amyloid-? is associated with endoplasmic reticulum stress in Alzheimers disease.
Curr Alzheimer Res
PUBLISHED: 08-22-2013
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Amyloid-? protein (A?) accumulates in the neurons of Alzheimers disease (AD) patients at an early stage of the disease. Recently, we found that A? with a toxic turn at positions 22 and 23 accumulates in neurons in AD brain. Here, we studied the accumulation of A?, toxic turn A? and high-molecular-weight A? oligomers in presenilin 1 (PS1) gene-transfected SH-SY5Y cells as well as in the brains of 3xTg-AD mice and AD patients. Immunostaining revealed that accumulation of toxic turn A? was promoted in G384A- and I143T-mutant PS1-transfected cells and further enhanced by co-transfection of cells with the A?-precursor protein (A?PP) gene. In contrast, accumulation of high-molecular-weight A? oligomers was promoted in mutant PS1 cells but attenuated by co-transfection of cells with the A?PP gene. Toxic turn A? was detected in the neurons of 3xTg-AD mice aged 2 months, when the mice were cognitively unimpaired. In contrast, high-molecular-weight A? oligomers were detected in the neurons of 7-month-old mice, when memory dysfunction is apparent. Furthermore, immunostaining and western blotting for Rab4, Rab6 and GRP78 revealed increased levels of these proteins in mutant PS1 cells and their accumulation in the neurons of 3xTg-AD mice. Remarkably, GRP78 immunoreactivity was increased at 2 months of age. Double-label immunostaining of AD brain revealed an apparent association between toxic turn A? and GRP78, an endoplasmic reticulum (ER) stress marker. Intraneuronal accumulation of toxic turn A? may be associated with ER stress in the brains of AD model mice and AD patients at an early stage.
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Site-specific inhibitory mechanism for amyloid ?42 aggregation by catechol-type flavonoids targeting the Lys residues.
J. Biol. Chem.
PUBLISHED: 06-21-2013
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The aggregation of the 42-residue amyloid ?-protein (A?42) is involved in the pathogenesis of Alzheimer disease (AD). Numerous flavonoids exhibit inhibitory activity against A?42 aggregation, but their mechanism remains unclear in the molecular level. Here we propose the site-specific inhibitory mechanism of (+)-taxifolin, a catechol-type flavonoid, whose 3,4-dihydroxyl groups of the B-ring plays a critical role. Addition of sodium periodate, an oxidant, strengthened suppression of A?42 aggregation by (+)-taxifolin, whereas no inhibition was observed under anaerobic conditions, suggesting the inhibition to be associated with the oxidation to form o-quinone. Because formation of the A?42-taxifolin adduct was suggested by mass spectrometry, A?42 mutants substituted at Arg(5), Lys(16), and/or Lys(28) with norleucine (Nle) were prepared to identify the residues involved in the conjugate formation. (+)-Taxifolin did not suppress the aggregation of A?42 mutants at Lys(16) and/or Lys(28) except for the mutant at Arg(5). In addition, the aggregation of A?42 was inhibited by other catechol-type flavonoids, whereas that of K16Nle-A?42 was not. In contrast, some non-catechol-type flavonoids suppressed the aggregation of K16Nle-A?42 as well as A?42. Furthermore, interaction of (+)-taxifolin with the ?-sheet region in A?42 was not observed using solid-state NMR unlike curcumin of the non-catechol-type. These results demonstrate that catechol-type flavonoids could specifically suppress A?42 aggregation by targeting Lys residues. Although the anti-AD activity of flavonoids has been ascribed to their antioxidative activity, the mechanism that the o-quinone reacts with Lys residues of A?42 might be more intrinsic. The Lys residues could be targets for Alzheimer disease therapy.
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Inhibition of amyloid ? aggregation by acteoside, a phenylethanoid glycoside.
Biosci. Biotechnol. Biochem.
PUBLISHED: 06-07-2013
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We examined the effects of acteoside (1a), which was isolated from Orobanche minor, and its derivatives on the aggregation of a 42-mer amyloid ? protein (A?42) in our search for anti-amyloidogenic compounds for Alzheimers disease (AD) therapy. Acteoside (1a) strongly inhibited the aggregation of A?42 in a dose-dependent manner. The structure-activity relationship for acteoside (1a) and related compounds suggests the catechol moiety of phenylethanoid glycosides to be essential for this inhibitory activity.
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Non-toxic conformer of amyloid ? may suppress amyloid ?-induced toxicity in rat primary neurons: implications for a novel therapeutic strategy for Alzheimers disease.
Biochem. Biophys. Res. Commun.
PUBLISHED: 05-22-2013
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The 42-mer amyloid ?-protein (A?42) oligomers cause neurotoxicity and cognitive impairment in Alzheimers disease (AD). We previously identified the toxic conformer of A?42 with a turn at positions 22-23 ("toxic" turn) to form oligomers and to induce toxicity in rat primary neurons, along with the non-toxic conformer with a turn at positions 25-26. G25P-A?42 and E22V-A?42 are non-toxic mutants that disfavor the "toxic" turn. Here we hypothesize that these non-toxic mutants of A?42 could suppress A?42-induced neurotoxicity, and examined their effects on the neurotoxicity, aggregation, and levels of the toxic conformer, which was evaluated by dot blotting using a monoclonal antibody (11A1) against the toxic conformer. G25P-A?42 and E22V-A?42 suppressed the neurotoxicity and aggregation of A?42 as well as the formation of the toxic conformer. The neurotoxicity induced by A?42 was also significantly reduced by the treatment of 11A1, but not of A?-sequence specific antibodies (6E10 and 4G8). Since recent studies indicate that A? oligomers contain parallel ?-sheet, the present results suggest that the non-toxic mutants of A?42 without the "toxic" turn could prevent the propagation process of the toxic conformer of A?42 resulting in suppression of the formation of the toxic oligomers. This could be a promising strategy for AD therapeutics.
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Structure-activity relationship for (+)-taxifolin isolated from silymarin as an inhibitor of amyloid ? aggregation.
Biosci. Biotechnol. Biochem.
PUBLISHED: 05-07-2013
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Silymarin, the seed extract of Silybium marianum, has preventive effects against Alzheimers disease-like pathogenesis in vivo. We isolated (+)-taxifolin (4) from silymarin as an inhibitor of aggregation of the 42-residue amyloid ?-protein. Structure-activity relationship studies revealed the 3,4-dihydroxyl groups to be critical to the anti-aggregative ability, whereas the 7-hydroxyl group and the stereochemistry at positions 2 and 3 were not important.
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Effects of the methoxy group in the side chain of debromoaplysiatoxin on its tumor-promoting and anti-proliferative activities.
Bioorg. Med. Chem. Lett.
PUBLISHED: 05-01-2013
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Debromoaplysiatoxin (DAT) is a tumor promoter isolated from sea hare and exhibits anti-proliferative activity against several cancer cell lines. To clarify key residues that are responsible for its tumor-promoting activity, we focused on the chiral methoxy group in the side chain, whose role had not yet been discussed or examined before. Demethoxy-DAT (8) was derived from DAT and we evaluated its tumor-promoting activity, anti-proliferative activity, and ability to bind to protein kinase C (PKC) isozymes. Compound 8 showed somewhat weaker tumor-promoting activity than that of DAT both in vitro and in vivo, but showed higher anti-proliferative activity against several cancer cell lines. Although the affinity to novel PKC isozymes of 8 was comparable to that of DAT, the affinity to conventional PKC isozymes decreased slightly. These results suggest that the methoxy group of DAT is one of the key residues critical for tumor-promoting activity but not for anti-proliferative activity. Since the methoxy group has little influence on the molecular hydrophobicity, this is the first report showing that structural factors other than hydrophobicity in the side chain of DAT affected its biological activities.
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Non-specific protein modifications by a phytochemical induce heat shock response for self-defense.
PLoS ONE
PUBLISHED: 02-05-2013
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Accumulated evidence shows that some phytochemicals provide beneficial effects for human health. Recently, a number of mechanistic studies have revealed that direct interactions between phytochemicals and functional proteins play significant roles in exhibiting their bioactivities. However, their binding selectivities to biological molecules are considered to be lower due to their small and simple structures. In this study, we found that zerumbone, a bioactive sesquiterpene, binds to numerous proteins with little selectivity. Similar to heat-denatured proteins, zerumbone-modified proteins were recognized by heat shock protein 90, a constitutive molecular chaperone, leading to heat shock factor 1-dependent heat shock protein induction in hepa1c1c7 mouse hepatoma cells. Furthermore, oral administration of this phytochemical up-regulated heat shock protein expressions in the livers of Sprague-Dawley rats. Interestingly, pretreatment with zerumbone conferred a thermoresistant phenotype to hepa1c1c7 cells as well as to the nematode Caenorhabditis elegans. It is also important to note that several phytochemicals with higher hydrophobicity or electrophilicity, including phenethyl isothiocyanate and curcumin, markedly induced heat shock proteins, whereas most of the tested nutrients did not. These results suggest that non-specific protein modifications by xenobiotic phytochemicals cause mild proteostress, thereby inducing heat shock response and leading to potentiation of protein quality control systems. We considered these bioactivities to be xenohormesis, an adaptation mechanism against xenobiotic chemical stresses. Heat shock response by phytochemicals may be a fundamental mechanism underlying their various bioactivities.
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Structure-activity studies on the side chain of a simplified analog of aplysiatoxin (aplog-1) with anti-proliferative activity.
Bioorg. Med. Chem.
PUBLISHED: 01-30-2013
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We have recently developed a simplified analog of aplysiatoxin (aplog-1) as an activator of protein kinase C (PKC) with anti-proliferative activity like bryostain 1. To identify sites in aplog-1 that could be readily modified to optimize therapeutic performance and to develop a molecular probe for examining the analogs mode of action, substituent effects on the phenol ring were systematically examined. Whereas hydrophilic acetamido derivatives were less active than aplog-1 in inhibiting cancer cell growth and binding to PKC?, introduction of hydrophobic bromine and iodine atoms enhanced both biological activities. The anti-proliferative activity was found to correlate closely with molecular hydrophobicity, and maximal activity was observed at a logP value of 4.0-4.5. On the other hand, an induction test with Epstein-Barr virus early antigen demonstrated that these derivatives have less tumor-promoting activity in vitro than aplog-1 regardless of the hydrophobicity of their substituents. These results would facilitate rapid preparation of molecular probes to examine the mechanism of the unique biological activities of aplog-1.
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Modeling Alzheimers disease with iPSCs reveals stress phenotypes associated with intracellular A? and differential drug responsiveness.
Cell Stem Cell
PUBLISHED: 01-18-2013
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Oligomeric forms of amyloid-? peptide (A?) are thought to play a pivotal role in the pathogenesis of Alzheimers disease (AD), but the mechanism involved is still unclear. Here, we generated induced pluripotent stem cells (iPSCs) from familial and sporadic AD patients and differentiated them into neural cells. A? oligomers accumulated in iPSC-derived neurons and astrocytes in cells from patients with a familial amyloid precursor protein (APP)-E693? mutation and sporadic AD, leading to endoplasmic reticulum (ER) and oxidative stress. The accumulated A? oligomers were not proteolytically resistant, and docosahexaenoic acid (DHA) treatment alleviated the stress responses in the AD neural cells. Differential manifestation of ER stress and DHA responsiveness may help explain variable clinical results obtained with the use of DHA treatment and suggests that DHA may in fact be effective for a subset of patients. It also illustrates how patient-specific iPSCs can be useful for analyzing AD pathogenesis and evaluating drugs.
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Geranyl modification on the tryptophan residue of ComXRO-E-2 pheromone by a cell-free system.
FEBS Lett.
PUBLISHED: 11-28-2011
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ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in the gram-positive bacterium Bacillus. Since posttranslational prenylation on the tryptophan residue has not been reported except in ComX pheromone, the universality of this modification has not yet been elucidated. In this paper, we established a cell-free system, whereby the tryptophan residue in peptides is modified with a geranyl group by modifying enzyme ComQ. In addition, we investigated enzymatic reaction conditions using an in vitro enzyme reaction system. This is the first report of in vitro geranylation on the tryptophan residue. This system is potentially a useful tool for elucidating the universality of prenylation on the tryptophan residue.
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SOD1 (copper/zinc superoxide dismutase) deficiency drives amyloid ? protein oligomerization and memory loss in mouse model of Alzheimer disease.
J. Biol. Chem.
PUBLISHED: 11-09-2011
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Oxidative stress is closely linked to the pathogenesis of neurodegeneration. Soluble amyloid ? (A?) oligomers cause cognitive impairment and synaptic dysfunction in Alzheimer disease (AD). However, the relationship between oligomers, oxidative stress, and their localization during disease progression is uncertain. Our previous study demonstrated that mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn-SOD, SOD1) have features of drusen formation, a hallmark of age-related macular degeneration (Imamura, Y., Noda, S., Hashizume, K., Shinoda, K., Yamaguchi, M., Uchiyama, S., Shimizu, T., Mizushima, Y., Shirasawa, T., and Tsubota, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 11282-11287). Amyloid assembly has been implicated as a common mechanism of plaque and drusen formation. Here, we show that Sod1 deficiency in an amyloid precursor protein-overexpressing mouse model (AD mouse, Tg2576) accelerated A? oligomerization and memory impairment as compared with control AD mouse and that these phenomena were basically mediated by oxidative damage. The increased plaque and neuronal inflammation were accompanied by the generation of N(?)-carboxymethyl lysine in advanced glycation end products, a rapid marker of oxidative damage, induced by Sod1 gene-dependent reduction. The Sod1 deletion also caused Tau phosphorylation and the lower levels of synaptophysin. Furthermore, the levels of SOD1 were significantly decreased in human AD patients rather than non-AD age-matched individuals, but mitochondrial SOD (Mn-SOD, SOD2) and extracellular SOD (CuZn-SOD, SOD3) were not. These findings suggest that cytoplasmic superoxide radical plays a critical role in the pathogenesis of AD. Activation of Sod1 may be a therapeutic strategy for the inhibition of AD progression.
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1,2-Di-O-?-linolenoyl-3-O-?-galactosyl-sn-glycerol as a superoxide generation inhibitor from Perilla frutescens var. crispa.
Biosci. Biotechnol. Biochem.
PUBLISHED: 11-07-2011
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Using a superoxide (O(2)(-)) generation assay system with differentiated HL-60 cells, 1,2-di-O-?-linolenoyl-3-O-?-galactosyl-sn-glycerol (DLGG) was identified as an O(2)(-) generation inhibitor from Perilla frutescens var. crispa (a local variety, kida-chirimen shiso). DLGG suppressed the O(2)(-) level in a dose-dependent manner with an IC(50) value of 21 µM, comparable to those of rosmarinic acid (RoA, IC(50) = 29 µM) and caffeic acid (CA, IC(50) = 30 µM). While RoA and CA also dose-dependently inhibited O(2)(-) generation in a xanthine-xanthine oxidase system, DLGG had no effect in the same system. Thus DLGG appeared to decrease the O(2)(-) level in the HL-60 assay system by mechanisms different from those of RoA and CA, which appeared to act as O(2)(-) scavengers.
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Solid-state NMR analysis of interaction sites of curcumin and 42-residue amyloid ?-protein fibrils.
Bioorg. Med. Chem.
PUBLISHED: 07-12-2011
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Aggregation of 42-residue amyloid ?-protein (A?42) plays a pivotal role in the etiology of Alzheimers disease (AD). Curcumin, the yellow pigment in the rhizome of turmeric, attracts considerable attention as a food component potentially preventing the pathogenesis of AD. This is because curcumin not only inhibits the aggregation of A?42 but also binds to its aggregates (fibrils), resulting in disaggregation. However, the mechanism of interaction between curcumin and the A?42 fibrils remains unclear. In this study, we analyzed the binding mode of curcumin to the A?42 fibrils by solid-state NMR using dipolar-assisted rotational resonance (DARR). To improve the quality of 2D spectra, 2D DARR data were processed with the covariance NMR method, which enabled us to detect weak cross peaks between carbons of curcumin and those of the A?42 fibrils. The observed (13)C-(13)C cross peaks indicated that curcumin interacts with the 12th and 17-21st residues included in the ?-sheet structure in the A?42 fibrils. Interestingly, aromatic carbons adjacent to the methoxy and/or hydroxy groups of curcumin showed clear cross peaks with the A?42 fibrils. This suggested that these functional groups of curcumin play an important role in its interaction with the A?42 fibrils.
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Synthesis and biological evaluation of the 12,12-dimethyl derivative of Aplog-1, an anti-proliferative analog of tumor-promoting aplysiatoxin.
Biosci. Biotechnol. Biochem.
PUBLISHED: 06-13-2011
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Aplog-1 is a unique analog of tumor-promoting aplysiatoxin that inhibits tumor-promotion by phorbol diesters and proliferation of tumor cells. While the structural features relevant to the biological activities of Aplog-1 remain to be identified, recent studies by us have suggested that local hydrophobicity around the spiroketal moiety of Aplog-1 is a crucial determinant of its anti-proliferative activity. This hypothesis led us to design 12,12-dimethyl-Aplog-1 (3), in which a hydrophobic geminal dimethyl group is installed proximal to the spiroketal moiety to improve biological potency. As expected, 3 was more effective than Aplog-1 in inhibiting cancer cell growth and binding to protein kinase C?, a putative receptor responsible for the biological response of Aplog-1. Moreover, an induction test on Epstein-Barr virus early antigen demonstrated 3 to be a better anti-tumor promoter than Aplog-1. These results indicate that 3 is a superior derivative of Aplog-1, and thus a more promising lead for anti-cancer drugs.
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Insulin receptor mutation results in insulin resistance and hyperinsulinemia but does not exacerbate Alzheimers-like phenotypes in mice.
Biochem. Biophys. Res. Commun.
PUBLISHED: 04-20-2011
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Obesity is a risk factor for Alzheimers disease (AD), which is characterized by amyloid ? depositions and cognitive dysfunction. Although insulin resistance is one of the phenotypes of obesity, its deleterious effects on AD progression remain to be fully elucidated. We previously reported that the suppression of insulin signaling in a mouse with a heterozygous mutation (P1195L) in the gene for the insulin receptor showed insulin resistance and hyperinsulinemia but did not develop diabetes mellitus [15]. Here, we generated a novel AD mouse model carrying the same insulin receptor mutation and showed that the combination of insulin resistance and hyperinsulinemia did not accelerate plaque formation or memory abnormalities in these mice. Interestingly, the insulin receptor mutation reduced oxidative damage in the brains of the AD mice. These findings suggest that insulin resistance is not always involved in the pathogenesis of AD.
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Generation of Unnatural Natural Product library and identification of a small molecule inhibitor of XIAP.
Bioorg. Med. Chem.
PUBLISHED: 04-08-2011
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Natural products have been utilized for drug discovery. To increase the source diversity, we generated a new chemical library consisting of chemically modified microbial metabolites termed Unnatural Natural Products by chemical conversion of microbial metabolites in crude broth extracts followed by purification of reaction products with the LC-photo diode array-MS system. Using this library, we discovered an XIAP inhibitor, C38OX6, which restored XIAP-suppressed enzymatic activity of caspase-3 in vitro. Furthermore, C38OX6 sensitized cancer cells to anticancer drugs, whereas the unconverted natural product did not. These findings suggest that our library could be a useful source for drug seeds.
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Formation of the 42-mer Amyloid ? Radical and the Therapeutic Role of Superoxide Dismutase in Alzheimers Disease.
J Amino Acids
PUBLISHED: 01-16-2011
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Oxidative stress is closely involved in age-related diseases and ageing itself. There is evidence of the leading contribution of oxidative damage to neurodegenerative disease, in contrast to other diseases where oxidative stress plays a secondary role. The 42-mer amyloid ? (A?42) peptide is thought to be a culprit in the pathogenesis of Alzheimers disease (AD). A?42 aggregates form the oligomeric assembly and show neurotoxicity, causing synaptic dysfunction. A?42 also induces tissue oxidation (DNA/RNA, proteins, and lipids) through trace metals (Cu, Zn, and Fe), which can be protected by antioxidant enzymes, vitamin C, and vitamin E. Superoxide dismutase catalyzes the conversion of toxic superoxide radical to less reactive hydrogen peroxide, contributing to protection from AD. Here we review the involvement of oxidative stress in AD progression induced from an imbalance between the radical formation of A?42 itself together with unique turn structure at positions Glu22 and Asp23 and several defense systems.
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Challenges to the development of bryostatin-type anticancer drugs based on the activation mechanism of protein kinase C?.
Med Res Rev
PUBLISHED: 11-09-2010
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Protein kinase C (PKC) isozymes are widely recognized as targets for anticancer therapy, and recent investigations demonstrated that PKC activators are potential therapeutic candidates for Alzheimers disease and acquired immune deficiency syndrome. However, concerns exist about their therapeutic uses because most PKC activators are potent tumor promoters. Bryostatin 1 (bryo-1) is a unique PKC activator with little tumor-promoting activities. Bryo-1 is currently undergoing clinical trials for the treatment of cancer. However, its limited availability from natural sources and difficulty in the synthesis hamper further studies on its mode of action and structural optimization. Although excellent practical methods for synthesizing several bryo-1-related compounds have been developed, the identification of synthetically more accessible compounds with bryo-1-like activity also provides a promising way to circumvent the problem of supply. The authors focused on the bryo-1s unique mechanism of activating PKC? that plays a tumor suppressor role, and found that a simple and less lipophilic analogue (aplog-1) of the tumor-promoting aplysiatoxin showed PKC?-activating behavior similar to bryo-1. Aplog-1 was easily synthesized in only 22 steps using standard reactions. Moreover, its tumor-promoting activity in vitro was very weak, and its cell growth-inhibitory activities were comparable to those of bryo-1. These data suggest that aplog-1 could become another therapeutic lead for cancer.
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Silymarin attenuated the amyloid ? plaque burden and improved behavioral abnormalities in an Alzheimers disease mouse model.
Biosci. Biotechnol. Biochem.
PUBLISHED: 11-07-2010
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Alzheimers disease (AD) is characterized by progressive cognitive impairment and the formation of senile plaques. Silymarin, an extract of milk thistle, has long been used as a medicinal herb for liver diseases. Here we report marked suppression of amyloid ?-protein (A?) fibril formation and neurotoxicity in PC12 cells after silymarin treatment in vitro. In vivo studies had indicated a significant reduction in brain A? deposition and improvement in behavioral abnormalities in amyloid precursor protein (APP) transgenic mice that had been preventively treated with a powdered diet containing 0.1% silymarin for 6 months. The silymarin-treated APP mice also showed less anxiety than the vehicle-treated APP mice. These behavioral changes were associated with a decline in A? oligomer production induced by silymarin intake. These results suggest that silymarin is a promising agent for the prevention of AD.
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Monoclonal antibody against the turn of the 42-residue amyloid ?-protein at positions 22 and 23.
ACS Chem Neurosci
PUBLISHED: 08-04-2010
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Aggregation of the 42-mer amyloid ?-protein (A?42) plays a critical role in the pathogenesis of Alzheimers disease (AD). We have proposed a toxic conformer with a turn at positions 22 and 23, as well as a nontoxic conformer with a turn at positions 25 and 26, in A?42 aggregates from systematic proline scanning and solid-state NMR studies. Although recent clinical trials of immunization targeting A?42 aggregates have proved useful, some adverse effects were reported. One of the reasons was hypothesized to be excessive immunoreactions derived from the unintended removal of nontoxic A?42, which plays an important role in the physiological function. To develop a monoclonal antibody for toxic A?42, E22P-A?10-35, a minimum moiety for neurotoxicity containing the turn at positions 22 and 23, was used for the generation of antibodies, following the selection of clones using A?42 mutants of E22P (turn-inducing) and E22V (turn-preventing). The obtained clone (11A1) showed a high binding affinity (K(D) = 10.3 nM) for A?42 using surface plasmon resonance. 11A1 also inhibited the neurotoxicity of A?42 in PC12 cells. Immunohistochemical studies showed that not only extracellular but intracellular amyloid was stained in human AD brains. In Western blotting analyses using human brains, low-molecular weight-oligomers rather than the monomer of A? were readily recognized by 11A1. These results imply that 11A1 could detect toxic A?42 oligomers with the turn at positions 22 and 23 and that 11A1 could be applicable for the therapeutic targeting of toxic A?42 in AD.
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Downregulation of programmed cell death 4 by inflammatory conditions contributes to the generation of the tumor promoting microenvironment.
Mol. Carcinog.
PUBLISHED: 07-08-2010
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Ample evidence has shown key roles of inflammation in tumor promotion and carcinogenesis, and tumor-associated macrophages are known to promote tumor growth and dissemination. Programmed cell death 4 (Pdcd4) is a novel tumor suppressor, and although various studies have revealed that the functions and expression mechanisms of Pdcd4 in tumor promotion, those in regard to inflammation remain unclear. In the present study, we examined whether inflammatory stimuli regulate Pdcd4 expression. 12-O-tetradecanoylphorbol 13-acetate (TPA) suppressed expression of pdcd4 mRNA in human monocytic cell lines (U937, THP-1). Similarly, the bacterial endotoxin lipopolysaccharide (LPS) downregulated pdcd4 level in mouse RAW264.7 and peritoneal macrophages. Furthermore, conditioned medium from LPS-stimulated RAW264.7 macrophages suppressed pdcd4 mRNA in RAW264.7 macrophages, and findings obtained with recombinant tumor necrosis factor-alpha (TNF-alpha) and TNF-alpha-specific siRNA suggested that TNF-alpha partly mediates LPS-triggered Pdcd4 downregulation via an autocrine mechanism. Specific inhibitors of phosphoinositide-3-kinase (PI3K) and c-jun N-terminus kinase (JNK) restored LPS-abolished pdcd4 mRNA. Consistently, in MCF7 mammary carcinoma cells, conditioned medium from TPA-differentiated/activated U937 cells suppressed pdcd4 mRNA. Additionally, knockdown of pdcd4 in RAW264.7 macrophages using siRNA significantly enhanced LPS-induced TNF-alpha protein production, and interferon-gamma, CC chemokine ligand (Ccl) 1, Ccl20, and interleukin-10 mRNA expression. These results suggest that Pdcd4 suppresses the induction of these inflammatory mediators. Taken together, loss of Pdcd4 in macrophages may be a critical step in establishing the inflammatory environment while that in tumor cells contributes to tumor progression.
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Role of the phenolic hydroxyl group in the biological activities of simplified analogue of aplysiatoxin with antiproliferative activity.
Bioorg. Med. Chem. Lett.
PUBLISHED: 07-03-2010
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The 18-deoxy derivative (3) of a simplified analogue (1) of aplysiatoxin with antiproliferative activity was synthesized to examine the role of the phenolic hydroxyl group at position 18 in the biological activities of 1. Compound 3 as well as 1 showed significant affinity for protein kinase C? (PKC?), and the antiproliferative activity of 3 was slightly higher than that of 1. However, the anti-tumor-promoting activity of 3 was less than that of 1 in vitro, suggesting that the phenolic hydroxyl group of 1 is necessary for the anti-tumor-promoting activity but not for the binding of PKC? and antiproliferative activity. Moreover, PKC isozyme selectivity of 3 was similar to that of 1, suggesting non-PKC receptors for these compounds to play some roles in the anti-tumor-promoting activity of 1.
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The turn formation at positions 22 and 23 in the 42-mer amyloid beta peptide: the emerging role in the pathogenesis of Alzheimers disease.
Geriatr Gerontol Int
PUBLISHED: 07-02-2010
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One hallmark of Alzheimers disease (AD) is the accumulation of amyloid beta (Abeta) peptides in the brain; Abeta mainly consists of 42-mer and 40-mer peptides (Abeta42 and Abeta40). Abeta42 plays a more critical role in the pathogenesis of AD because Abeta42 aggregates much faster and is more toxic than Abeta40. Therefore, there is an urgent need to elucidate the mechanism of aggregation and neurotoxicity of Abeta42 to develop therapeutic agents. Here, we introduce the pathological role of Abeta42 in AD and review our recent findings of the structural analysis of Abeta42 using systematic proline replacement, electron spin resonance and solid-state nuclear magnetic resonance, and the new mechanism of neurotoxicity of Abeta42 through the formation of radicals.
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Suppression of CD74 expression and Helicobacter pylori adhesion by auraptene targeting serum starvation-activated ERK1/2 in NCI-N87 gastric carcinoma cells.
Biosci. Biotechnol. Biochem.
PUBLISHED: 05-07-2010
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Helicobacter pylori (H. pylori) is a major human pathogen and plays a central role in chronic gastritis and gastric cancer. Since the adhesion of H. pylori to the human gastric epithelium is the initial and critical step of its infection, anti-H. pylori adhesion agents may be effective for the prevention and therapy of H. pylori-associated diseases. CD74 has recently been identified as a new receptor for H. pylori urease, and we have previously reported that several citrus components strongly suppressed CD74 expression in NCI-N87 gastric carcinoma cells. We found in this present study that auraptene (citrus coumarin) disrupted serum starvation-induced extracellular signaling-regulated kinase (ERK) 1/2 activation and attenuated H. pylori adhesion and IL-8 production in a co-culture system. In addition, the knockdown of CD74 expression led to a significant decrease of H. pylori adhesion, but unexpectedly increased IL-8 production. However, PD98059 (a MEK1/2 inhibitor) dramatically down-regulated this cytokine, suggesting MEK/ERK-dependent IL-8 production. Our results suggest that auraptene suppressed H. pylori adhesion and resulting chemokine production by disrupting ERK1/2 activation.
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Inhibition by genistein of the lipopolysaccharide-induced down-regulation of programmed cell death 4 in RAW 264.7 mouse macrophages.
Biosci. Biotechnol. Biochem.
PUBLISHED: 05-07-2010
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Programmed cell death 4 (Pdcd4), a novel tumor suppressor, has recently emerged as an anti-inflammatory protein. We assessed in the present study the effects of 18 different food factors with anti-inflammatory activity on the lipopolysaccharide-induced down-regulation of pdcd4 mRNA expression in mouse RAW 264.7 macrophages. Genistein, a soy isoflavone, significantly inhibited pdcd4 down-regulation.
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Stomatal density is controlled by a mesophyll-derived signaling molecule.
Plant Cell Physiol.
PUBLISHED: 12-09-2009
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Stomata are composed of a pair of guard cells and a pore between them, and their density and positions are regulated by developmental and environmental signals. In a screen in which we overexpressed many genes coding for putative secretory proteins one by one in Arabidopsis, we identified a gene named STOMAGEN, which increases stomatal density when overexpressed. The STOMAGEN gene encodes a small peptide with a putative secretory signal sequence at its N-terminus and is expressed preferentially in mesophyll cells. This peptide belongs to the EPIDERMAL PATTERNING FACTOR (EPF) family of the cysteine-rich peptides superfamily. The mature form was a 45-amino-acid peptide (stomagen) with three intramolecular disulfide bonds. Stomagen treatment at very low concentrations, as low as 10 nM, increased the stomatal density of wild-type Arabidopsis plants. We propose that stomagen is a mesophyll-to-epidermis signaling molecule that positively regulates stomatal density. We also suggest that stomagen increases stomatal density by competing with negative regulators EPF1 and EPF2 for the receptor-like protein TOO MANY MOUTHS.
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In vitro covalent binding proteins of zerumbone, a chemopreventive food factor.
Biosci. Biotechnol. Biochem.
PUBLISHED: 08-07-2009
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Zerumbone (ZER), a sesquiterpene in Zingiber zerumbet Smith, has been implicated as a promising chemopreventive agent. Here we found that ZER suppressed expression of pro-inflammatory genes (COX-2 and iNOS) and induced detoxification genes (GSTP1 and NQO1) in RAW264.7 macrophages. Using the ZER-bound Sepharose gel, it appeared ZER was covalently bound to proteins, Keap1 and HuR, that play key roles in these molecular mechanisms.
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A simple analogue of tumor-promoting aplysiatoxin is an antineoplastic agent rather than a tumor promoter: development of a synthetically accessible protein kinase C activator with bryostatin-like activity.
J. Am. Chem. Soc.
PUBLISHED: 05-20-2009
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Protein kinase C (PKC) is widely recognized as a therapeutic target in intractable diseases such as cancer, Alzheimers disease (AD), and acquired immune deficiency syndrome (AIDS). While inhibition of PKC is a general therapeutic strategy for the treatment of cancer, PKC activators are potential therapeutic agents for AD and AIDS. However, concerns have been raised about their therapeutic use since PKC activators such as phorbol esters exhibit potent tumor-promoting activities. Naturally occurring bryostatin 1 (bryo-1), prostratin, and 12-deoxyphorbol 13-phenylacetate (DPP) are fascinating PKC activators without tumor-promoting activities. Bryo-1 is currently in clinical trials for the treatment of cancer and is also effective against AD. Prostratin and DPP are attractive candidates for the adjunctive treatment of human immunodeficiency virus (HIV) infection. However, their limited availability from natural sources and synthetic complexity have hampered further development as therapeutic agents. We report here easy access (22 steps) to a simple analogue (1) of the tumor-promoting aplysiatoxin (ATX) as a novel PKC activator with anticancer and anti-tumor-promoting activities. Anticancer activities of 1 against several human cancer cell lines were comparable to those of bryo-1. Moreover, 1 as well as bryo-1 significantly inhibited the Epstein-Barr virus early antigen (EBV-EA) induction by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), whereas ATX strongly induced EBV-EA. This inhibitory effect is characteristic of antitumor promoters. Compound 1 as well as bryo-1 displayed significant binding and activation of PKCdelta and induced its translocation to the nuclear membrane in CHO-K1 cells. This study provides a synthetically accessible PKC activator with bryo-1-like activities, which could be another therapeutic lead for cancer, AD, and AIDS.
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Identification of physiological and toxic conformations in Abeta42 aggregates.
Chembiochem
PUBLISHED: 03-06-2009
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Aggregation of the 42-residue amyloid beta-protein (Abeta42) plays a crucial role in the pathogenesis of Alzheimers disease (AD). Despite numerous structural studies on Abeta aggregates, the relationship between tertiary structure and toxicity remains unclear. Our proline scanning and solid-state NMR studies suggested that aggregates both of wild-type Abeta42 and of E22K-Abeta42 (one of the mutants related to cerebral amyloid angiopathy) contain two conformers: a major one with a turn at positions 25 and 26, and a minor one with a turn at positions 22 and 23. To identify the toxic conformer, the derivative Abeta42-lactam(22K-23E), in which the side chains at positions 22 and 23 were covalently linked, was synthesized as a minor conformer surrogate, along with Abeta42-lactam(25K-26E) as a major conformer surrogate. The Abeta42-lactam(22K-23E) showed stronger aggregation, neurotoxicity, radical generation, and oligomerization than wild-type Abeta42, whereas in Abeta42-lactam(25K-26E) were weak. The transition from the physiological conformation with a turn at positions 25 and 26 to the toxic conformation with a turn at positions 22 and 23 might be a key event in the pathogenesis of AD.
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Zerumbone, an electrophilic sesquiterpene, induces cellular proteo-stress leading to activation of ubiquitin-proteasome system and autophagy.
Biochem. Biophys. Res. Commun.
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Zerumbone, a sesquiterpene present in Zingiber zerumbet Smith, has been implicated as a promising chemopreventive agent. Interestingly, a number of studies have revealed that its potent bioactivities are dependent on the electrophilic moiety of its ?,?-unsaturated carbonyl group, while our recent findings showed its chemical potential for binding to cellular proteins through a Michael reaction. In the present study, modifications of proteins by zerumbone led to their insolubilization in vitro. In living cell models, zerumbone induced ubiquitination and aggregation of cellular proteins, which demonstrated its substantial proteo-toxicity. On the other hand, it was also revealed that zerumbone possesses potential for activating intracellular proteolysis mechanisms of the ubiquitin-proteasome system and autophagy. Furthermore, it up-regulated expressions of pro-autophagic genes including p62, which is known as a cargo receptor of aggrephagy, the selective autophagic process for protein aggregates. Pretreatment of Hepa1c1c7 cells with zerumbone conferred a phenotype resistant to cytotoxicity and protein modifications by 4-hydroxy-2-nonenal, an endogenous lipid peroxidation product, in a p62-dependent manner. Together, these results suggest that protein modifications by zerumbone cause mild proteo-stress, thereby activating intracellular proteolysis machineries to maintain protein homeostasis. We consider these effects on proteolysis mechanisms to be hormesis, which provides beneficial functions through mild biological stresses.
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Solid-state NMR analysis of the ?-strand orientation of the protofibrils of amyloid ?-protein.
Biochem. Biophys. Res. Commun.
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Alzheimers disease (AD) is caused by abnormal deposition (fibrillation) of a 42-residue amyloid ?-protein (A?42) in the brain. During the process of fibrillation, the A?42 takes the form of protofibrils with strong neurotoxicity, and is thus believed to play a crucial role in the pathogenesis of AD. To elucidate the supramolecular structure of the A?42 protofibrils, the intermolecular proximity of the Ala-21 residues in the A?42 protofibrils was analyzed by (13)C-(13)C rotational resonance experiments in the solid state. Unlike the A?42 fibrils, an intermolecular (13)C-(13)C correlation was not found in the A?42 protofibrils. This result suggests that the ?-strands of the A?42 protofibrils are not in an in-register parallel orientation. A?42 monomers would assemble to form protofibrils with the ?-strand conformation, then transform into fibrils by forming intermolecular parallel ?-sheets.
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Toxicity in rat primary neurons through the cellular oxidative stress induced by the turn formation at positions 22 and 23 of A?42.
ACS Chem Neurosci
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The 42-mer amyloid ?-protein (A?42) aggregates to form soluble oligomers that cause memory loss and synaptotoxicity in Alzheimers disease (AD). Oxidative stress is closely related to the pathogenesis of AD. We previously identified the toxic conformer of A?42 with a turn at positions 22 and 23 ("toxic turn") by solid-state NMR and demonstrated that a monoclonal antibody (11A1) against the toxic turn in A?42 mainly detected the oligomer in the brains of AD patients. Our recent study suggested that oxidative stress is a key factor of the oligomerization and cognitive impairment induced by A? overproduction in vivo. However, the involvement of the toxic conformer in A?42-induced oxidative damage remains unclear. To investigate this mechanism, we examined the levels of intracellular reactive oxygen species (ROS) and neurotoxicity in rat primary neurons using E22P-A?42, a mutant that induces a turn at positions 22 and 23, and E22V-A?42, a turn-preventing mutant. E22P-A?42, but not E22V-A?42, induced greater ROS production than Wt-A?42 in addition to potent neurotoxicity. Interestingly, the formation of the toxic conformer in both E22P-A?42 and Wt-A?42 probed by the 11A1 antibody preceded A?42-induced neurotoxicity. Trolox (a radical scavenger) and Congo red (an aggregation inhibitor) significantly prevented the neurotoxicity and intracellular ROS induced by E22P-A?42 and Wt-A?42, respectively. These results suggest that A?42-mediated toxicity is caused by the turn that favors toxic oligomers, which increase generation of ROS.
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Glyceraldehyde-3-phosphate dehydrogenase regulates cyclooxygenase-2 expression by targeting mRNA stability.
Arch. Biochem. Biophys.
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Cyclooxygenase (COX)-2 is an inducible inflammatory protein whose expression is partially regulated at the post-transcriptional level. We investigated whether glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds to the AU-rich element (ARE) of COX-2 mRNA for its degradation. Knockdown of GAPDH in hepa1c1c7 cells significantly enhanced COX-2 expressions. Recombinant GAPDH bound to the COX-2 ARE within the first 60 nucleotides of the 3-UTR via the NAD(+) binding domain. Interestingly, a C151S GAPDH mutant retained binding activity. Confocal microscopy observation revealed that LPS exposure reduced the localization of GAPDH in nuclei. Our results indicate that GAPDH negatively regulates COX-2 by binding to its ARE.
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Protective effects of caffeoylquinic acids on the aggregation and neurotoxicity of the 42-residue amyloid ?-protein.
Bioorg. Med. Chem.
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Alzheimers disease (AD), a neurodegenerative disorder, is characterized by aggregation of 42-mer amyloid ?-protein (A?42). A?42 aggregates through ?-sheet formation and induces cytotoxicity against neuronal cells. A?42 oligomer, an intermediate of the aggregates, causes memory loss and synaptotoxicity in AD. Inhibition of A?42 aggregation by small molecules is thus a promising strategy for the treatment of AD. Caffeoylquinic acid (CQA), a phenylpropanoid found widely in natural sources including foods, shows various biological activities such as anti-oxidative ability. Previously, our group reported that 3,5-di-O-caffeoylquinic acid (3,5-di-CQA) rescued the cognitive impairment in senescence-accelerated-prone mice 8. However, structure-activity relationship of CQA derivatives on the aggregation and neurotoxicity of A?42 remains elusive. To evaluate the anti-amyloidogenic property of CQA-related compounds for AD therapy, we examined the effect of CQA and its derivatives on the aggregation and neurotoxicity of A?42. In particular, 4,5-di-O-caffeoylquinic acid (4,5-di-CQA) and 3,4,5-tri-O-caffeoylquinic acid (3,4,5-tri-CQA) strongly inhibited the aggregation of A?42 in a dose-dependent manner. Structure-activity relationship studies suggested that the caffeoyl group in CQA is essential for the inhibitory activity. These CQAs also suppressed the transformation into ?-sheet and cytotoxicity against human neuroblastoma cells of A?42. Furthermore, 3,4,5-tri-CQA blocked the formation of A?42 oligomer. These results indicate that 3,4,5-tri-CQA could be a potential agent for the prevention of AD.
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Lack of the consensus sequence necessary for tryptophan prenylation in the ComX pheromone precursor.
Biosci. Biotechnol. Biochem.
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ComX, an oligopeptide pheromone that stimulates the natural genetic competence controlled by quorum sensing in Bacillus subtilis and related bacilli, contains a prenyl-modified tryptophan residue. Since ComX is the only protein known to contain prenylated tryptophan, the universality of this unique posttranslational modification has yet to be determined. Recently, we developed a cell-free assay system in which the tryptophan residue in the ComX(RO-E-2) pheromone precursor derived from B. subtilis strain RO-E-2 can be geranylated by the ComQ(RO-E-2) enzyme. We report here our attempt to identify the consensus sequence surrounding the geranylated tryptophan residue by using the cell-free system with various ComX(RO-E-2) pheromone precursor analogs. We found that [47-58]ComX(RO-E-2), corresponding to the C-terminal 12-residue peptide of the pheromone precursor, contained a short sequence essential for geranylation. We also found that the length of the sequence between the tryptophan residue and the C-terminus was important for geranylation, and that some [47-58]ComX(RO-E-2) pheromone precursor amino acids were involved in the geranylation reaction. However, we could not identify a consensus sequence surrounding the geranylated tryptophan. Our evidence suggests that, like Rab which lacks a consensus sequence yet is geranylgeranyl-modified on a cysteine residue, the ComX pheromone and its precursor also lack a consensus sequence.
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Stimulation of the amyloidogenic pathway by cytoplasmic superoxide radicals in an Alzheimers disease mouse model.
Biosci. Biotechnol. Biochem.
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Oxidative stress is involved in the pathogenesis of neurodegeneration. Amyloid ? (A?) oligomer as an intermediate of aggregates causes memory loss in Alzheimers disease (AD). We have suggested that oxidative stress plays an important role in A? oligomerization and cognitive impairment using a human amyloid precursor protein (hAPP) transgenic AD mice lacking cytoplasmic superoxide dismutase (hAPP/Sod1-/-). Recently, clinical trials revealed inhibitors of A? production from hAPP as promising therapeutics, but the relationship between oxidative stress and A? metabolism remains unclear. Here we found that Sod1 deficiency enhanced ?-cleavage of hAPP, suggesting that it increased A? production in hAPP/Sod1-/- mice. In contrast, A? degradation did not decrease in hAPP/Sod1-/- as compared with hAPP/Sod1+/+ mice. Furthermore, we successfully detected in situ superoxide radicals associated with increased protein carbonylation in hAPP/Sod1-/-. These results suggest that cytoplasmic oxidative stress is involved in A? production as well as aggregation during AD progression.
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Isolation, identification, and biological evaluation of Nrf2-ARE activator from the leaves of green perilla (Perilla frutescens var. crispa f. viridis).
Free Radic. Biol. Med.
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The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a cellular defense system against oxidative stress. Activation of this pathway increases expression of antioxidant enzymes. Epidemiological studies have demonstrated that the consumption of fruits and vegetables is associated with reduced risk of contracting a variety of human diseases. The aim of this study is to find Nrf2-ARE activators in dietary fruits and vegetables. We first attempted to compare the potency of ARE activation in six fruit and six vegetables extracts. Green perilla (Perilla frutescens var. crispa f. viridis) extract exhibited high ARE activity. We isolated the active fraction from green perilla extract through bioactivity-guided fractionation. Based on nuclear magnetic resonance and mass spectrometric analysis, the active ingredient responsible for the ARE activity was identified as 2,3-dihydroxy-4,6-dimethoxychalcone (DDC). DDC induced the expression of antioxidant enzymes, such as ?-glutamylcysteine synthetase (?-GCS), NAD(P)H: quinone oxidoreductase-1 (NQO1), and heme oxygenase-1. DDC inhibited the formation of intracellular reactive oxygen species and the cytotoxicity induced by 6-hydroxydopamine. Inhibition of the p38 mitogen-activated protein kinase pathway abolished ARE activation, the induction of ?-GCS and NQO1, and the cytoprotective effect brought about by DDC. Thus, this study demonstrated that DDC contained in green perilla enhanced cellular resistance to oxidative damage through activation of the Nrf2-ARE pathway.
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Identification and biological activities of bryostatins from Japanese bryozoan.
Biosci. Biotechnol. Biochem.
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Six bryostatins were isolated from Japanese bryozoan by evaluating their binding to the C1B domain of protein kinase C? (PKC?). Structure-activity studies of bryostatins 4, 10, and 14 suggested that the ester group at C20 was not necessary for binding to and activating PKC?. These bryostatins showed significant anti-tumor-promoting activity in induction tests with the Epstein-Barr virus early antigen.
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Structure-activity studies on the spiroketal moiety of a simplified analogue of debromoaplysiatoxin with antiproliferative activity.
J. Med. Chem.
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Aplog-1, a simplified analogue of tumor-promoting debromoaplysiatoxin, is antiproliferative but not tumor-promoting. Our recent study has suggested that local hydrophobicity around the spiroketal moiety is a crucial determinant for antiproliferative activity. To further clarify the structural features relevant to the activity, we synthesized two methyl derivatives of aplog-1, where a methyl group was installed at position 4 or 10 of the spiroketal moiety. 10-Methyl-aplog-1 (5) bound to the C1B domains of novel PKCs (?, ?, and ?) with subnanomolar K(i) values, approximately 10-20 times stronger than aplog-1, and markedly inhibited the growth of many human cancer cell lines, while 4-methyl-aplog-1 (4) had levels of activity similar to those of aplog-1. Interestingly, 5 showed little tumor-promoting activity unlike the tumor promoter debromoaplysiatoxin. These results suggest that 5 is a potent PKC ligand without tumor-promoting activity and could be a therapeutic lead for the treatment of cancer, like bryostatins.
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Auraptene attenuates gastritis via reduction of Helicobacter pylori colonization and pro-inflammatory mediator production in C57BL/6 mice.
J Med Food
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Helicobacter pylori is a major human pathogen that plays central roles in chronic gastritis and gastric cancer. Recently, we reported that auraptene suppressed H. pylori adhesion via expression of CD74, which has been identified as a new receptor for H. pylori urease. In this study, we attempted to clarify the effects of oral feeding of auraptene on H. pylori infection and resultant inflammatory responses in C57BL/6 mice and found that it remarkably attenuated H. pylori colonization and gastritis. Biochemical analyses revealed that auraptene inhibited H. pylori-induced expression and/or production of CD74, macrophage migration inhibitory factor, interleukin-1?, and tumor necrosis factor-? in gastric mucosa, together with serum macrophage inhibitory protein-2. It is notable that treatment with this coumarin during the pretreatment period was more effective than that during posttreatment. Our results suggest that auraptene is a promising phytochemical for reducing the risk of H. pylori-induced gastritis and carcinogenesis.
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