In vitro characterization of missense mutations associated with quantitative protein Sdeficiency

名古屋大学NAGOYA University博士(医療技術学)Objective: To elucidate the molecular consequences of hereditary protein S (PS) deficiency, we investigated the in vitro synthesis of the PS missense mutants in COS-1 cells and their activated protein C (APC) cofactor activities. Patients: Four patients with quantitative PS deficiency suffering from venous thrombosis were examined. Results: We identified three distinct novel missense mutations, R275C, P375Q and D455Y, and two previously reported missense mutations, C80Y and R314H. The P375Q and D455Y mutations were found in one patient and observed to be in linkage on the same allele. The R314H mutant showed the lowest level of expression (32.7%), and the C80Y, P375Q + D455Y, and R275C mutants exhibited a moderate impairment of expression, that is, 43.8%, 49.5%, and 72.3% of the wild type, respectively. Furthermore, pulse-chase experiments demonstrated that all mutants showed impaired secretion and longer half-lives in the cells than the wild type PS. In the APC cofactor assays, the C80Y mutant showed no cofactor activity, and the R275C mutant showed reduced activity, 62.3% of the wild type PS, whereas the R314H and P375Q + D455Y mutants exhibited normal cofactor activity. Conclusion: These data indicate that the C80Y and R275C mutations affect the secretion and function of the PS molecule, and that the R314H and P375Q + D455Y mutations are responsible for only secretion defects, causing the phenotype of quantitative PS deficiency observed in the patients.名古屋大学博士学位論文 学位の種類:博士（医療技術学）（課程）学位授与年月日:平成19年3月23日In vitro characterization of missense mutations associated with quantitative protein Sdeficiency Schattauer, v.4, iss.9, pp.2003-2009を、博士論文として提出したもの。doctoral thesi

Activity of secondary bacterial metabolites in the control of citrus canker.

This study investigated the protective effects of secondary bacterial metabolites, produced by Pseudomonas sp. (bacterium strain LN), on citrus canker disease caused by Xanthomonas axonopodis pv. citri (Xac 306). The LN bacteria strain was cultured in liquid medium and the supernatant free-cells was treated with methanol (AMF) and ethyl acetate (AEF), respectively, and then the extract was concentrated, filtrated, lyophilized and fractionated by vacuum liquid chromatography (VLC). After VLC, eight fractions were obtained. All fractions? activity against Xac 306 by agar well diffusion assay and minimum inhibitory concentration but in different concentrations were tested. Cytotoxicity effects were observed in all fractions in 50 ?g?mL?1 concentration. The comet assay demonstrated that the fractions EAF, VLC2 and VLC3 presented no genotoxic effects at tested concentrations. In plants only VLC3 showed significant results (p < 0.05), reducing the incidence of citrus canker lesions

Activation of the SPHK/S1P signalling pathway is coupled to muscarinic receptor-dependent regulation of peripheral airways

BACKGROUND: In peripheral airways, acetylcholine induces contraction via activation of muscarinic M2-and M3-receptor subtypes (M(2)R and M(3)R). Cholinergic hypersensitivity is associated with chronic obstructive pulmonary disease and asthma, and therefore the identification of muscarinic signaling pathways are of great therapeutic interest. A pathway that has been shown to be activated via MR and to increase [Ca(2+)](i )includes the activation of sphingosine kinases (SPHK) and the generation of the bioactive sphingolipid sphingosine 1-phosphate (S1P). Whether the SPHK/S1P signaling pathway is integrated in the muscarinic control of peripheral airways is not known. METHODS: To address this issue, we studied precision cut lung slices derived from FVB and M(2)R-KO and M(3)R-KO mice. RESULTS: In peripheral airways of FVB, wild-type, and MR-deficient mice, SPHK1 was mainly localized to smooth muscle. Muscarine induced a constriction in all investigated mouse strains which was reduced by inhibition of SPHK using D, L-threo-dihydrosphingosine (DHS) and N, N-dimethyl-sphingosine (DMS) but not by N-acetylsphingosine (N-AcS), a structurally related agent that does not affect SPHK function. The initial phase of constriction was nearly absent in peripheral airways of M(3)R-KO mice when SPHK was inhibited by DHS and DMS but was unaffected in M(2)R-KO mice. Quantitative RT-PCR revealed that the disruption of the M(2)R and M(3)R genes had no significant effect on the expression levels of the SPHK1-isoform in peripheral airways. CONCLUSION: These results demonstrate that the SPHK/S1P signaling pathway contributes to cholinergic constriction of murine peripheral airways. In addition, our data strongly suggest that SPHK is activated via the M(2)R. Given the important role of muscarinic mechanisms in pulmonary disease, these findings should be of considerable therapeutic relevance

Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants

BACKGROUND: Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity. METHODOLOGY/PRINCIPAL FINDINGS: We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models. CONCLUSIONS/SIGNIFICANCE: These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity

Circulating immunoglobulin-secreting cells in patients with plasma cell dyscrasia

Abstract Immunoglobulin-secreting cells (ISC) in peripheral blood mononuclear cells (PBM) isolated from patients with plasma cell dyscrasia of various stages were studied using reverse hemolytic plaque assay. Normal healthy individuals contained 35 +/- 16 ISC/10(5) PBM. Aleukemic or subleukemic patients with overt myeloma contained 825 +/- 713 ISC, whereas leukemic patients contained 12,675 +/- 2520 ISC. Patients of premyelomatous stage, however, contained ISC/10(5) PBM of normal range. The relationship between ISC and clinical stage or laboratory data is discussed.</jats:p

Circulating immunoglobulin-secreting cells in patients with plasma cell dyscrasia

Immunoglobulin-secreting cells (ISC) in peripheral blood mononuclear cells (PBM) isolated from patients with plasma cell dyscrasia of various stages were studied using reverse hemolytic plaque assay. Normal healthy individuals contained 35 +/- 16 ISC/10(5) PBM. Aleukemic or subleukemic patients with overt myeloma contained 825 +/- 713 ISC, whereas leukemic patients contained 12,675 +/- 2520 ISC. Patients of premyelomatous stage, however, contained ISC/10(5) PBM of normal range. The relationship between ISC and clinical stage or laboratory data is discussed.</jats:p