Serum amyloid A (SAA)-induced remodeling of CSF-HDL

Inflammation is a risk factor for Alzheimer's disease. Serum amyloid A (SAA) is an acute phase protein that dissociates apolipoprotein AI (apoAI) from plasma HDL. In cerebrospinal fluid (CSF), the SAA concentration is much higher in subjects with Alzheimer's disease than in controls. CSF-HDL is rich in apoE, which plays an important role as a ligand for lipoprotein receptors in the central nervous system (CNS). To clarify whether SAA dissociates apoE from CSF-HDL, we added recombinant SAA to CSF and determined the apoE distribution in the CSF using native two-dimensional gel electrophoresis. We found that SAA dissociated apoE from CSF-HDL in a dose-dependent manner. This effect was more evident in apoE4 carriers than in apoE3 or apoE2 carriers. After a 24-h incubation at 37 degrees C, SAA continuously dissociated apoE from CSF-HDL. Amyloid beta (Abeta) fragments (1-42) were bound to large CSF-HDL but not to apoE dissociated by SAA. In conclusion, SAA dissociates apoE from CSF-HDL. We postulate that inflammation in the CNS may impair Abeta clearance due to the loss of apoE from CSF-HDL.     

Interaction study between synthetic glycoconjugate ligands and endocytic receptors using flow cytometry

Flow cytometric analysis of synthetic galactosyl polymers, asialofetuin and LDL derivatives labeled with FITC (Fluorescein Isothiocyanate) was carried out to determine the phenotypes of endocytic receptors, such as asialoglycoprotein (ASPG) and the LDL receptor, on various types of cells. When FITC-labeled galactosyl polystyrene (GalCPS), being a synthetic ligand of ASPG, was applied to rat hepatocytes and human cancer cells (Hep G2 and Chang Liver), surface fluorescence intensities varied according to receptor expression on the cells. The fluorescence intensity originates from the calcium-dependent binding of the FITC-labeled GalCPS. Although unaltered by pre-treatment with glucosyl polystyrene (GluCPS), fetuin and LDL, the fluorescence intensity was suppressed by pre-treatment with (non-labeled) GalCPS and asialofetuin. Flow cytometry allowed us to demonstrate that the calcium-dependent binding of FITC-labeled LDL (prepared from rabbits) upon the addition of 17alpha-ethinyl estradiol enhances LDL receptor expression, and the expression is suppressed upon the addition of a monoclonal antibody to the LDL receptor. The binding efficiency based on the combination of FITC-labeled ligands suggests a possible application for the classification of cell types and conditions corresponding to endocytic receptor expression without the need for immuno-active antibodies or radiolabeled substances. Furthermore, the synthetic glycoconjugate (GalCPS) is shown to be a sensitive and useful marker for classification based on cell phenotype using flow cytometry.     

Cytotoxic activity toward KB cells of 2-substituted naphtho[2,3-b]furan-4,9-diones and their related compounds

We investigated the cytotoxic activity of 2-substituted naphtho[2,3-b]furan-4,9-diones. We have previously synthesized 33 types of 2-substituted and related compounds, and the cytotoxic activity of these compounds was then examined by a KB cell culture assay. 2-(3-Furanoyl)benzoic acids and 1,4-naphthoquinones had no activity. 2-Acetyl-4,9-dimethoxynaphtho[2,3-b]furan 4 showed low activity. However, parent naphtho[2,3-b]furan-4,9-dione 2 and most 2-substituted derivatives exhibited cytotoxic activity. The parent structure was therefore for cytotoxicity. 2-Formylnaphtho[2,3-b]furan-4,9-dione 11 had particularly potent activity (ED50=0.09 microg/ml).     

Two pathways of apoptosis are simultaneously induced in the embryonal brains of neural cell-specific HIF-1α-deficient mice

The aim of this study was to clarify the mechanism of apoptosis seen in the cortex of neural cell-specific hypoxia inducible factor-1α (HIF-1α)-deficient embryos. A previous study showed that the neural cells in the cortical area of the mutant embryos underwent apoptosis coincident with vascular regression. Through histological, immunohistochemical, and electron microscopic technique, two kinds of apoptotic cells were detected in the mutant embryonal cortex. Apoptotic cells of one type were clustered in small round structures, 10–20 μm in diameter, whereas the others, present in large numbers, were distributed in a group at the cortical plate located more to the outer side than the round structures. The histochemical and electron microscopic findings indicate that the former represented the appearance of macrophages, in which cellular fragments including vascular cells underwent oxidative stress-related, TNF receptor-mediated, caspase-2-induced apoptosis, while the latter showed c-Myc-related, caspase-3-activated apoptosis of the neural cells. These results suggest that two pathways of apoptosis are induced in neuronal and vascular cells of the cortex in the neural cell-specific HIF-1α-deficient mouse.     

Evolution of teleostean hatching enzyme genes and their paralogous genes

We isolated genes for hatching enzymes and their paralogs having two cysteine residues at their N-terminal regions in addition to four cysteines conserved in all the astacin family proteases. Genes for such six-cysteine-containing astacin proteases (C6AST) were searched out in the medaka genome database. Five genes for MC6AST1 to 5 were found in addition to embryo-specific hatching enzyme genes. RT-PCR and whole-mount in situ hybridization evidenced that MC6AST1 was expressed in embryos and epidermis of almost all adult tissues examined, while MC6AST2 and 3 were in mesenterium, intestine, and testis. MC6AST4 and 5 were specifically expressed in jaw. In addition, we cloned C6AST cDNA homologs from zebrafish, ayu, and fugu. The MC6AST1 to 5 genes were classified into three groups in the phylogenetic positions, and the expression patterns and hatching enzymes were clearly discriminated from other C6ASTs. Analysis of the exon–intron structures clarified that genes for hatching enzymes MHCE and MAHCE were intron-less, while other MC6AST genes were basically the same as the gene for another hatching enzyme MLCE. In the basal Teleost, the C6AST genes having the ancestral exon–intron structure (nine exon/eight intron structure) first appeared by duplication and chromosomal translocation. Thereafter, maintaining such ancestral exon–intron structure, the LCE gene was newly diversified in Euteleostei, and the MC6AST1 to 5 gene orthologs were duplicated and diversified independently in respective fish lineages. The HCE gene lost all introns in Euteleostei, whereas in the lineage to zebrafish, it was translocated from chromosome to chromosome and lost some of its introns.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.The nucleotide sequence data reported in the present paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with accession numbers from AB256940 to AB256952.     

Design and evaluation of new antipsoriatic antedrug candidates having 16-en-22-oxa-vitamin D3 structures

Design, synthesis, and in vitro and in vivo evaluation of a series of antipsoriatic antedrugs having 16-en-22-oxa-vitamin D3 are described. Among the seven compounds examined, two are promising: ester 5c and amide 5f, both of which exhibit greater potent antiproliferation activity with lessened calcemic activity than the presently prescribed maxacalcitol (2).     

Identification of new secreted proteins and secretion of heterologous amylase by <Emphasis Type="Italic">C. glutamicum</Emphasis>

In this study, secreted Corynebacterium glutamicum proteins were investigated by two-dimensional gel electrophoresis. Around 100 spots observed in the pH range 4.5–5.5 had molecular masses that varied from 10 to 50 kDa. Upon N-terminal amino acid sequence analysis by Edman degradation, two of them were hits to two hypothetical proteins encoded by cgR_1176 and cgR_2070 on C. glutamicum R genome, respectively. Active-form α-amylase derived from Geobacillus stearothermophilus was successfully secreted by using the predicted cgR_1176 and cgR_2070 signal sequences, indicating that these hypothetical proteins were secreted proteins. Analysis using a disruption mutant of the twin-arginine translocation (Tat) export pathway machinery of C. glutamicum suggested that one is Tat pathway dependent secretion while the other is independent of the pathway. Our results demonstrate that C. glutamicum can secrete exoproteins by using its own signal sequences, indicating its potential as a host for protein productions.     

Genetic Evidence Linking Age-Dependent Attenuation of the 26S Proteasome with the Aging Process

The intracellular accumulation of unfolded or misfolded proteins is believed to contribute to aging and age-related neurodegenerative diseases. However, the links between age-dependent proteotoxicity and cellular protein degradation systems remain poorly understood. Here, we show that 26S proteasome activity and abundance attenuate with age, which is associated with the impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen, we characterized Rpn11, which encodes a subunit of the 19S RP, as a suppressor of expanded polyglutamine-induced progressive neurodegeneration. Rpn11 overexpression suppressed the age-related reduction of the 26S proteasome activity, resulting in the extension of flies'' life spans with suppression of the age-dependent accumulation of ubiquitinated proteins. On the other hand, the loss of function of Rpn11 caused an early onset of reduced 26S proteasome activity and a premature age-dependent accumulation of ubiquitinated proteins. It also caused a shorter life span and an enhanced neurodegenerative phenotype. Our results suggest that maintaining the 26S proteasome with age could extend the life span and suppress the age-related progression of neurodegenerative diseases.Ubiquitin-conjugated, misfolded protein aggregates are observed in the brain during normal aging and in late-onset human neurodegenerative diseases, such as Alzheimer''s, Parkinson''s, and polyglutamine diseases (e.g., Huntington''s disease or spinocerebellar ataxias) (9). Many of the mutations that cause dominantly inherited neurodegenerative diseases dramatically increase the amount of protein aggregates in vitro and in vivo, supporting the widely accepted hypothesis that proteotoxicity caused by the aggregates underlies the pathogenesis of many neurodegenerative diseases (32). Proteotoxicity can have many effects, including disruption of microtubule-dependent axonal transport (10), perturbation of membrane permeability (23), and impaired function of the ubiquitin-proteasome system (UPS) (1, 17). Aggregation-mediated toxicity has also been suggested in normal aging, because recent reports show that the impairment of autophagy in the central nervous system causes accumulation of ubiquitinated proteins and leads to neurodegenerative diseases (12, 21). These observations suggest that the continuous clearance of misfolded proteins through cellular degradation systems, including the UPS and autophagy, is important for preventing aggregation-mediated proteotoxicity both in age-related neurodegenerative diseases and in normal aging.Clinical symptoms of neurodegenerative diseases generally do not appear or progress until advanced ages, not only in sporadic forms but also in inherited forms of neurodegenerative diseases (26). These observations suggest that aggregation-mediated toxicity appears in a late-onset manner both in normal aging and in neurodegenerative diseases. Furthermore, a link between the aging process and aggregation-mediated proteotoxicity has been suggested by evidence that Huntington''s disease-associated proteotoxicity was ameliorated when the aging process slowed, that is, the life span extension via decreased insulin/insulin growth factor-1-like signaling in Caenorhabditis elegans (13, 31).A possible mechanism for the late onset of aggregation-mediated toxicity is age-related impairment of the UPS, because loss-of-function mutations in genes encoding UPS components can enhance the cytotoxicity of protein aggregation in dominantly inherited neurodegenerative diseases (4, 5, 18). In addition, an age-related decline of proteasome activity has been observed in the tissues of humans and other mammals (8) and in aged flies (36). Considering the role of the proteasome in neuroprotection and the age dependence of most neurodegenerative diseases, the age-related decline of proteasome activity could well be a key factor both in normal aging and in the late onset and/or progression of neurodegenerative diseases. However, the mechanism underlying the age-related decline of proteasome activity remains to be elucidated, and there is no direct genetic evidence showing that the age-related decline of proteasome activity causes age-related aggregation-mediated toxicity in normal aging and in age-related neurodegenerative diseases.Here, we studied the age-related decline of proteasome activity by using Drosophila melanogaster and found age-related attenuation of the 26S proteasome activity and abundance that was associated with impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen, we identified Rpn11, which encodes one of the lid subunits in the 19S RP, as a suppressor of the age-dependent progression of a polyglutamine-induced neurodegenerative phenotype. The overexpression of Rpn11 prevented the age-related reduction of the 26S proteasome activity, which suppressed the age-dependent accumulation of ubiquitinated proteins and extended the life span. On the other hand, the loss of function of Rpn11 enhanced the age-related reduction of 26S proteasome activity, leading to a shorter life span, a premature age-dependent accumulation of ubiquitinated proteins, and an early onset of a neurodegenerative phenotype. Our results demonstrate for the first time that the age-related reduction of the 26S proteasome activity is a key factor in the induction of certain age-related biological changes and in the increased risk for the onset or progression of neurodegenerative diseases. Our findings imply that improving the amount and/or activity of the 26S proteasome by overexpressing a lid subunit, such as Rpn11, could provide an extension to the mean life span and prevent the age-dependent onset or progression of neurodegeneration.