Yoichi Kiuchi

β-amyloid induces caspase-dependent early neurotoxic change in PC12 cells: correlation with H2O2 neurotoxicity

We examined neurotoxic effects of Abeta(25-35), an active fragment of beta-amyloid (Abeta), and compared the effect with H2O2 neurotoxicity in PC12 cells. Abeta(25-35) induced the loss of mitochondria function as detected using a tetrazolium salt (WST-1) reduction assay and decreased the number of cells adhering to collagen type 1-coated plates. Abeta(25-35) did not induce cell death, as detected by Hoechst 33342/propidium iodide staining. The caspase tetrapeptide inhibitor z-IETD-fluoromethylketone (FMK) and z-LEHD-FMK inhibited the attenuation of WST-1 reduction induced by Abeta(25-35) and H2O2, while the caspase-3 inhibitor z-DEVD-FMK afforded protection only against H2O2 neurotoxicity. Caspase-3 protease activity was increased by treatment of H2O2 but not Abeta(25-35). Thus, Abeta(25-35) induces early neurotoxic events by activating caspases other than caspase-3. H2O2 -induced oxidative stress may not be implicated in Abeta-induced neurotoxic pathways.

The anti-emetic activity of GK-128 in Suncus murinus.

In Suncus murinus, various emetic responses and the anti-emetic activity of a new 5-hydroxytryptamine3 (5-HT3) receptor antagonist, GK-128 (2-[(2-methylimidazol-1-yl) methyl benzo[f]thiochromen-1-one monohydrochloride hemihydrate), were investigated. Cancer chemotherapeutic agents, cisplatin and cyclophosphamide, dose-dependently induced emesis of long-lasting duration. The 5-HT3 receptor agonist, 2-methyl-5-HT, and copper sulfate also induced emesis of short duration. However, another 5-HT3 receptor agonist, m-chlorophenylbiguanide, was not consistently emetic. GK-128 inhibited the emetic responses induced by chemotherapeutic agents and 2-methyl-5-HT with similar potency. The anti-emetic action of GK-128 was more potent than that of ondansetron, Y-25130, granisetron and metoclopramide. The order of potency of these drugs, except granisetron, was consistent with that of their 5-HT3 receptor binding affinity in rat cortex. GK-128 failed to inhibit copper sulfate-induced emesis. These data suggest that GK-128 has a potent inhibitory effect on emesis via the 5-HT3 receptor, and that the 5-HT3 receptor involved in emesis in Suncus murinus may be different from the classically defined 5-HT3 receptor in other animals such as rats, dogs and ferrets.

Type IV collagen prevents amyloid β-protein fibril formation

The potential of targeting through molecular therapeutics the underlying amyloid beta-protein (A beta) fibrillogenesis causing the initiation and progression of Alzheimers disease (AD) offers an opportunity to improve the disease. Type IV collagen (collagen IV) is localized in senile plaques in patients with AD. By using thioflavin T fluorescence spectroscopy and electron microscopy, we found that collagen IV inhibited A beta1-40 (A beta40) fibril formation. The critical concentration of collagen IV for this inhibition was 5 microg/mL. Circular dichroism data indicate that collagen IV prevents formation of a beta-structured aggregate of A beta40. These studies demonstrated that collagen IV is apparently a potent inhibitor of A beta fibril formation.

Disassembly of amyloid β-protein fibril by basement membrane components

Amyloid beta-protein (A3) fibril in senile plaque may be related to the pathogenesis of Alzheimers disease (AD). Basement membrane (BM) components are associated with the plaques in AD brain. It suggests that the BM components may play an important role in the deposition of the plaque. We investigated the potential of BM components, such as type IV collagen (collagen IV) and entactin, to induce disassembly of preformed Abeta1-42 (Abeta42) fibrils in direct comparison to laminin. Thioflavin T assays revealed that these BM components disrupted preformed Abeta42 fibrils in a dose-dependent manner. The high concentration of BM components, 100 microg/mL laminin, 50 microg/mL collagen IV and 50 microg/mL entactin, had most effect on disassembly of preformed Abeta42 fibrils (Molar ratio; Abeta42:laminin = 90:1, Abeta42:collagen IV = 34:1, Abeta42:entactin = 20:1). Circular dichroism spectroscopy data indicated that the high concentration of BM components induced structural transition in Abeta42 from beta-sheet to random structures. These results suggest that collagen IV and entactin, as well as laminin, are effective inducers of disassembly of Abeta42 fibrils. The ability of these BM components to induce random structures may be linked to the disassembly of preformed Abeta42 fibrils.

Entactin-induced inhibition of human amyloid β-protein fibril formation in vitro

Abstract Amyloid β-protein (Aβ) fibril in senile plaques may possibly be related to the pathogenesis of Alzheimers disease (AD). Basement membrane (BM) components are localized to the plaques. Entactin binds the plaque associated BM components. We investigated the potential of entactin to prevent Aβ fibril formation. Thioflavin T fluorometric assay and electron microscopy revealed that entactin significantly inhibited Aβ1–40 (Aβ40) fibril formation at an Aβ40:entactin molar ratio of 50:1. The inhibitory effect of entactin was displayed in a dose-dependent manner. Circular dichroism spectroscopy data indicated that entactin induced a random coil structure in Aβ40. We propose that the ability of entactin to induce random structure is linked to the inhibition of Aβ fibril formation. Entactin may be related to the pathogenesis of AD by regulating Aβ40 fibril formation.

Role of nitric oxide and prostaglandin in the protective effect of pibutidine hydrochloride, a novel histamine H2-receptor antagonist, on the gastric mucosal lesions in rats.

1. The role of endogenous nitric oxide (NO) and prostaglandins (PGs) in the gastric mucosal protective action of pibutidine hydrochloride (IT-066), a novel histamine H2-receptor antagonist, was investigated in a 0.15 N hydrochloride (HCl) + 60% ethanol (EtOH)-induced gastric lesion model. 2. The 0.15 N HCl + 60% EtOH-induced lesion formation was reduced significantly by IT-066 (3 mg/kg, PO), NOR3 (spontaneous NO releaser; 0.03-0.1 mg/kg, SC) or PGE2 (0.01 mg/kg, PO) but was not reduced by famotidine (1-10 mg/kg, PO). 3. Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME) (3 mg/kg, IV), an inhibitor of NO synthase, inhibited the protective action of IT-066 (3 mg/kg, PO), and the inhibitory effect of L-NAME was reversed by L-arginine (300 mg/kg, IV). The protective effect of PGE2 (0.01 mg/kg, PO) was not affected by the pretreatment with L-NAME (3 mg/kg, IV). 4. Infusion of carboxy-PTIO (1 mg/kg/min), a direct NO scavenger, inhibited the protective effect of IT-066 (3 mg/kg, SC) or NOR3 (0.1 mg/kg, SC). Pretreatment with indomethacin (5 mg/kg, SC) markedly reduced the protective effect of IT-066 (3 mg/kg, PO) or NOR3 (0.1 mg/kg, SC). 5. These results suggest that endogenous NO and PGs may be implicated in the gastric mucosal protection induced by IT-066 and that the endogenous PGs may contribute to the protective effect of NO.