New Safe Method for Preparation of Sarin-Exposed Human Erythrocytes Acetylcholinesterase Using Non-Toxic and Stable Sarin Analogue Isopropyl p-Nitrophenyl Methylphosphonate and its Application to Evaluation of Nerve Agent Antidotes

Introduction A non-toxic and stable sarin analogue, isopropyl p-nitrophenyl methylphosphonate (INMP), was synthesized for safe preparation of sarin-exposed acetylcholinesterase (AChE). Results and Discussion This agent was stable for years, able to be handled in an ordinary laboratory without special care, and its 50% inhibitory concentration (IC₅₀) on 0.04 U/ml human erythrocytes AChE was 15 nM. This reagent was thought to be especially useful since it enables experiments that require sarin-inhibited AChE, such as the development of antidotes for sarin, in a usual laboratory. To demonstrate the usefulness of this method, 40 known and novel pyridinealdoxime methiodide (PAM)-type oxime antidotes were synthesized, and their reactivation activities to INMP-exposed AChE and structure–activities correlation were studied. Conclusion Among the antidotes tested in this experiment except for 2-PAM, the compound found to have the highest reactivation activity, was the novel hydrophobic 2-PAM-type compound, 2-[(hydroxyimino)methyl]-1-[4-(tert-butyl)benzyl] pyridinium bromide.     

Disturbance of response to acute thermal pain in naturally occurring cholecystokinin-a receptor gene knockout Otsuka Long-Evans Tokushima Fatty (OLETF) rats

Otsuka Long-Evans Tokushima Fatty (OLETF) rats lack cholecystokinin-A receptor (CCK-AR) because of a genetic abnormality. We observed that body temperature homeostasis in response to changes in ambient temperature was deteriorated in OLETF rats, while the functions of the signal outputs from the hypothalamus to effectors were not impaired. Deteriorated homeostasis was also seen in CCK-AR deficient (-/-) mice. In the present study, we examined whether the sensory pathway involved in transmitting signals about temperature from the skin to the brain was impaired in OLETF rats. To elucidate the involvement of CCK-AR function, we conducted the same experiment in CCK-AR(-/-) mice. Responses to thermal pain were assessed using the Hargreaves' plantar test apparatus. Shortening of withdrawal latency was observed in OLETF rats compared to control rats, indicating thermal hyperalgesia. Behavioral responses following paw withdrawal were disturbed in OLETF rats. The 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid contents in the hippocampus and frontal cortex of OLETF rats were significantly higher than in those of the controls. CCK-AR(-/-) mice did not show any differences from wild-type mice. In conclusion, OLETF rats showed thermal hyperalgesia and disturbed responses to thermal pain, and an alteration of 5-HT function might have a role in this disturbance.     

Histopathological examination and analysis of mortality in DBA/2 mouse vasculitis induced with CAWS, a water-soluble extracellular polysaccharide fraction obtained from Candida albicans

CAWS, a water-soluble extracellular polysaccharide fraction obtained from the culture supernatant of Candida albicans, is one of the fungal pathogen-associated molecular patterns (PAMPs). It has been reported to show potent activity inducing arteritis and coronaritis in mice. Especially, CAWS-induced arteritis has a 100% incidence and severe mortality in the DBA/2 mouse strain. This artificial vasculitis was reported to provide a good murine model of Kawasaki disease and other inflammatory vascular disease. However, severe mortality was observed only in DBA/2 mice, which is a CAWS-sensitive strain. In this study, to clarify the mechanisms of CAWS-induced arteritis and mortality, we investigated microscopic histopathological changes in cardiovascular tissues in DBA/2 mice. Severe inflammatory infiltration was observed from the external elastic lamina in the aorta and proximal coronary arteries within 1 week after CAWS administration. Severe stenosis of the aorta and coronary arteries was observed more than 3 weeks after CAWS administration. Fibrinoid necrosis was observed in these vessel walls. All CAWS-treated mice died between the fifth and twelfth week after administration. Severe inflammatory change with aortic valve transformation suggested that CAWS-treated mice died of valvular endocarditis or cardiac dysfunction. Based on the simple induction method and complete incidence, these data suggest that CAWS-induced arteritis is a good model of not only Kawasaki disease but also other cardiovascular diseases such as valvular endocarditis.     

Type 3 (adult) GM1 gangliosidosis: case report

GM1 gangliosidosis is usually a pediatric disease caused by hereditary acid beta-galactosidase deficiency. There have been a few cases in adults. We saw a 51-year-old Japanese man with type 3 GM1 gangliosidosis who was manifesting dementia, dysarthria, gait disturbance, and limb rigidity. Radiologic studies showed platyspondylia, acetabular hypoplasia, and flattened femoral heads. Biochemical analysis revealed generalized acid beta-galactosidase deficiency.     

Audiologic evaluation of patients with localized intracranial lesions.

A battery of audiological tests for retrocochlear disorders was performed of 79 cases with localized intracranial lesions confirmed surgically or neurologically. The Type III trace of Békésy audiometry was found to originate from pathologies in the brainstem and the Type IV from abnormalities in the first neuron of the VIIIth nerve or in the brainstem. It was suggested that an unusually large amplitude peak in the Békésy tracing indicates a lesion in the temporal lobe. Large values of differential limen for short increment (DLSI) were found in patients with brainstem lesions. A good speech discrimination with a poor filtered speech discrimination apparently indicated supratentorial lesions. The binaural separation test using dichotically presented digit sounds was found to be helpful for differential diagnosis between the supratentorial and subtentorial lesions.     

OECD validation of the Hershberger assay in Japan: Phase 3. Blind study using coded chemicals

The Organization for Economic Co-operation and Development (OECD) has initiated the development of new guidelines for the screening and testing of potential endocrine disrupters. The Hershberger assay is one of the assays selected for validation based on the need for in vivo screening to detect androgen agonists or antagonists by measuring the response of five sex accessory organs and tissues of castrated juvenile male rats: the ventral prostate, the seminal vesicles with coagulating glands, the levator ani and bulbocavernosus muscle complex (LABC), Cowper's glands, and the glans penis. The Phase 1 feasibility demonstration stage of the Hershberger validation program has been successfully completed with a single androgen agonist and a single antagonist as reference substances. The Phase 2 validation study was performed, employing a range of additional androgen agonists and antagonists. Recently, the Phase 3 validation study was conducted and performed in several International laboratories. Three Japanese laboratories have contributed to the blind study using coded materials of Phase 3 validation. Four coded test substances in the agonistic version and seven substances in the antagonistic version were orally administered by gavage for 10 consecutive days, respectively. In the antagonist version of the assay, 0.2mg/kg/day of testosterone propionate (TP) was coadministered by subcutaneous injection. All five accessory sex reproductive organs and tissues consistently responded with statistically significant changes in weight within a narrow window in both versions. Therefore, the Japanese studies support the Hershberger assay as a reliable and reproducible screening assay for the detection of androgen agonistic and antagonistic effects.     

Transactivation of the ICAM-1 gene by CD30 in Hodgkin's lymphoma

The ICAM-1/LFA-1 complex mediates cell-cell interaction. ICAM-1 is overexpressed in Hodgkin/Reed-Sternberg (H/RS) cells, and serum levels of its soluble form are higher in Hodgkin's lymphoma (HL) patients than in controls. There are no data, however, regarding the regulation of expression of ICAM-1 in H/RS cells. CD30 was identified in H/RS cells of HL and has attracted much interest as a molecular marker of HL. To analyze ICAM-1 expression in H/RS cells, we examined the expression of ICAM-1, LFA-1, CD30 and CD30L in HL-derived cell lines. All cell lines expressed ICAM-1 and CD30, but not LFA-1 or CD30L. CD30 induced ICAM-1 expression. Analysis of the ICAM-1 promoter showed the importance of NF-kappaB binding site for CD30-induced ICAM-1 gene expression. Coexpression of IkappaB, IKK, NIK and TRAF dominant-negative constructs with CD30 inhibited CD30-induced activation of ICAM-1 promoter, suggesting that CD30 induces ICAM-1 via NF-kappaB signalling. The ICAM-1 promoter was activated by the C-terminal region of CD30, which activated NF-kappaB signalling. A decoy CD30 lacking the cytoplasmic region inhibited ICAM-1 promoter activity in HL cell lines. Thus, in H/RS cells, ligand-independent activation of CD30 signalling activates NF-kappaB and this leads to constitutive ICAM-1 expression, suggesting a link between 2 well known phenotypic characteristics of HL, CD30 and ICAM-1 overexpression.