Amyloid ␤-protein (A␤) assemblies are thought to play primary roles in Alzheimer disease (AD). They are considered to acquire surface tertiary structures, not present in physiologic monomers, that are responsible for exerting toxicity, probably through abnormal interactions with their target(s). Therefore, A␤ assemblies having distinct surface tertiary structures should cause neurotoxicity through distinct mechanisms. Aiming to clarify the molecular basis of neuronal loss, which is a central phenotype in neurodegenerative diseases such as AD, we report here the selective immunoisolation of neurotoxic 10 -15-nm spherical A␤ assemblies termed native amylospheroids (native ASPDs) from AD and dementia with Lewy bodies brains, using ASPD tertiary structure-dependent antibodies. In AD patients, the amount of native ASPDs was correlated with the pathologic severity of disease. Native ASPDs are anti-pan oligomer A11 antibody-negative, high mass (>100 kDa) assemblies that induce degeneration particularly of mature neurons, including those of human origin, in vitro. Importantly, their immunospecificity strongly suggests that native ASPDs have a distinct surface tertiary structure from other reported assemblies such as dimers, A␤-derived diffusible ligands, and A11-positive assemblies. Only ASPD tertiary structure-dependent antibodies could block ASPD-induced neurodegeneration. ASPDs bind presynaptic target(s) on mature neurons and have a mode of toxicity different from those of other assemblies, which have been reported to exert their toxicity through binding postsynaptic targets and probably perturbing glutamatergic synaptic transmission. Thus, our findings indicate that native ASPDs with a distinct toxic surface induce neuronal loss through a different mechanism from other A␤ assemblies.Neurodegenerative diseases, such as Alzheimer disease (AD), 2 Parkinson disease, prion diseases, and the polyglutamine diseases, arise from abnormal protein interactions in the central nervous system (1). In these diseases, complex multistep processes of protein conformational change and accretion produce various nonfibrillar assemblies, leading finally to fibrils (1-5). Recent studies have suggested that the early assemblies in this process might be the most toxic, possibly through the exposure of buried moieties and the formation of surface tertiary structures not present in physiologic monomers (6). These surface tertiary structures could mediate abnormal interactions with other cellular components (1).In AD, extensive studies have suggested that accumulation of amyloid ␤-protein (A␤), a physiologic derivative of amyloid precursor protein (APP), plays a primary pathogenic role (7-9). Various forms of assemblies ranging in mass from dimers up to multimers of ϳ1 MDa have been reported as neurotoxins (10 -13) as follows: protofibrils (14); dimers/trimers (natural low-n oligomers) (15); 3-24-mer A␤-(1-42) assemblies termed A␤-derived diffusible ligands (ADDLs) (16); 12-mers termed globulomers (17) or A␤*56 (18); 15-20-mer A␤ assemblies te...

ABSTRACT. Degenerative lesions were induced in the knee joint of Wistar rats by intraarticular injection of chondrocyte metabolism inhibitor mono-iodoacetate (MIA) at doses of 0, 0.3 or 3 mg/joint. Histopathological examination and the measurement of hind paw weight ratio as an index of joint pain by incapacitance tester were performed. Histological findings that are similar to those observe d in human osteoarthritis (OA), such as disorganization of chondrocytes, erosion and fibrillation of cartilage surface, and subchondral bon e exposure etc., were observed in a MIA-dose-dependent manner. Saflanin-O fast green staining revealed that marked diffuse reduction of proteoglycan in cartilage tissue of rats treated with MIA. The clinical scores of the joint pain were closely correlated to the grade of histological findings. We conclude that the present experimental model in combination with a novel dual channel weight averager would be very useful for the study of human OA, and could be applied for estimation of therapeutic effect of new anti-OA drugs. KEY WORDS: mono-iodoacetate, osteoarthritis, pain assessment, rat.J. Vet. Med. Sci. 65(11): 1195-1199, 2003 Osteoarthritis (OA) is a degenerative joint disease characterized by fibrillation and erosion in cartilage tissue, chondrocyte proliferation and osteophyte formation at the joint margins, and sclerosis of subchondral bone [13]. Reportedly, imbalance occurs between synthetic and degenerative process within chondrocytes that leads to the net loss of cartilage tissue and subsequent pathologic condition [2]. At late stage of human OA, articular damages eventually lead to clinical findings such as joint impairment and pain.Although human OA-like lesions may occur spontaneously in dogs and mice, they are not appropriate for the evaluation of new anti-OA therapeutic agents because of low incidence and variable onset [5,16]. There are number of animal OA models that has variety of etiologies such as surgical induction [11, 12, 15], collagenase-induced [9], extracellular matrix loss [18,19], or impact-induced trauma [10]. However, studies on a new therapeutic drug for human OA and associated pain have been hampered because of the lack of useful animal model that closely mimic the human OA. Some study groups have previously reported that chondrocyte metabolism inhibitor mono-iodoacetate (MIA) have been reported to induce the disruption of glycolysis and subsequent cell death, and the loss of chondrocytes results in histologic changes in the knee joint resembling to human OA [6,17]. The objective of the present study is to clarify the histopathologic changes in MIA-induced knee joint lesion in the rats and its correlation to the dose of MIA and clinical pain evaluated by dual channel weight averager, with a development trial of non-invasive rat OA model for new drug development. MATERIALS AND METHODS Animals:Seven weeks old female Wistar rats were purchased from Charles River Japan Inc. (Yokohama, Kanagawa) and kept in air-conditioned animal room at 22°C and given tap water...

Y-700 (1-[3-Cyano-4-(2,2-dimethylpropoxy)phenyl]-1H-pyrazole-4-carboxylic acid) is a newly synthesized inhibitor of xanthine oxidoreductase (XOR). Steady-state kinetics with the bovine milk enzyme indicated a mixed type inhibition with K i and K i Ј values of 0.6 and 3.2 nM, respectively. Titration experiments showed that Y-700 bound tightly both to the active sulfo-form and to the inactive desulfo-form of the enzyme with K d values of 0.9 and 2.8 nM, respectively. X-ray crystallographic analysis of the enzyme-inhibitor complex revealed that Y-700 closely interacts with the channel leading to the molybdenum-pterin active site but does not directly coordinate to the molybdenum ion. In oxonate-treated rats, orally administered Y-700 (1-10 mg/kg) dose dependently lowered plasma urate levels. At a dose of 10 mg/kg, the hypouricemic action of Y-700 was more potent and of longer duration than that of 4-hydroxypyrazolo(3,4-d)pyrimidine, whereas its action was approximately equivalent to that of 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid, a nonpurine inhibitor of XOR. In normal rats, orally administered Y-700 (0.3-3 mg/kg) dose dependently reduced the urinary excretion of urate and allantoin, accompanied by an increase in the excretion of hypoxanthine and xanthine. Y-700 (1 mg/kg) was absorbed rapidly by the oral route with high bioavailability (84.1%). Y-700 was hardly excreted via the kidneys but was mainly cleared via the liver. These results suggest that Y-700 will be a promising candidate for the treatment of hyperuricemia and other diseases in which XOR may be involved.XOR catalyzes the hydroxylation of hypoxanthine and xanthine, the last two steps in urate biosynthesis. The enzyme is found in a wide range of organisms from bacteria to man. It exists as a homodimer, each subunit of which contains one molybdo-pterin cofactor, two iron-sulfur clusters, and one FAD molecule (Bray, 1975;Hille, 1996). Mammalian XOR is synthesized as the dehydrogenase form, XDH (EC 1.1.1.204), and exists mostly as such in the cell, but can be readily converted to the oxidase form, XO (EC 1.1.3.22), by oxidation of sulfhydryl residues or by proteolysis (Nishino, 1994;Hille and Nishino, 1995). XDH displays a preference for NAD ϩ reduction at the FAD site, whereas XO fails to react with NAD ϩ and exclusively uses molecular oxygen as its substrate, leading to the formation of superoxide anion and hydrogen peroxide (Hille and Nishino, 1995).XOR is a target of drugs against gout and hyperuricemia ( Rundles et al., 1969), and the conversion of XDH to XO is of major interest because it has been implicated in diseases characterized by superoxide anion-induced tissue damage, such as postischemic-reperfusion injury (McCord, 1985). Allopurinol (Fig. 1), a purine analog, is a well-known inhibitor

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