PP2A Inhibition Assay Using Recombinant Enzyme for Rapid Detection of Okadaic Acid and Its Analogs in Shellfish

Okadaic acid and its analogs (OAs) responsible for diarrhetic shellfish poisoning (DSP) strongly inhibit protein phosphatase 2A (PP2A) and thus are quantifiable by measuring the extent of the enzyme inhibition. In this study, we evaluated the suitability of the catalytic subunit of recombinant human PP2A (rhPP2Ac) for use in a microplate OA assay. OA, dinophysistoxin-1(DTX1), and hydrolyzate of 7-O-palmitoyl-OA strongly inhibited rhPP2Ac activity with IC50 values of 0.095, 0.104, and 0.135 nM, respectively. The limits of detection and quantitation for OA in the digestive gland of scallops and mussels were 0.0348 μg/g and 0.0611 μg/g respectively, and, when converted to the whole meat basis, are well below the regulation level proposed by EU (0.16 μg/g whole meat). A good correlation with LC-MS data was demonstrated, the correlation coefficient being 0.996 with the regression slope of 1.097

Connexin 43 Astrocytopathy Linked to Rapidly Progressive Multiple Sclerosis and Neuromyelitis Optica

<div><p>Background</p><p>Multiple sclerosis (MS) and neuromyelitis optica (NMO) occasionally have an extremely aggressive and debilitating disease course; however, its molecular basis is unknown. This study aimed to determine a relationship between connexin (Cx) pathology and disease aggressiveness in Asian patients with MS and NMO.</p> <p>Methods/Principal Findings</p><p>Samples included 11 autopsied cases with NMO and NMO spectrum disorder (NMOSD), six with MS, and 20 with other neurological diseases (OND). Methods of analysis included immunohistochemical expression of astrocytic Cx43/Cx30, oligodendrocytic Cx47/Cx32 relative to AQP4 and other astrocytic and oligodendrocytic proteins, extent of demyelination, the vasculocentric deposition of complement and immunoglobulin, and lesion staging by CD68 staining for macrophages. Lesions were classified as actively demyelinating (n=59), chronic active (n=58) and chronic inactive (n=23). Sera from 120 subjects including 30 MS, 30 NMO, 40 OND and 20 healthy controls were examined for anti-Cx43 antibody by cell-based assay. Six NMO/NMOSD and three MS cases showed preferential loss of astrocytic Cx43 beyond the demyelinated areas in actively demyelinating and chronic active lesions, where heterotypic Cx43/Cx47 astrocyte oligodendrocyte gap junctions were extensively lost. Cx43 loss was significantly associated with a rapidly progressive disease course as six of nine cases with Cx43 loss, but none of eight cases without Cx43 loss regardless of disease phenotype, died within two years after disease onset (66.7% vs. 0%, <i>P</i>=0.0090). Overall, five of nine cases with Cx43 loss and none of eight cases without Cx43 loss had distal oligodendrogliopathy characterized by selective myelin associated glycoprotein loss (55.6% vs. 0.0%, <i>P</i>=0.0296). Loss of oligodendrocytic Cx32 and Cx47 expression was observed in most active and chronic lesions from all MS and NMO/NMOSD cases. Cx43-specific antibodies were absent in NMO/NMOSD and MS patients.</p> <p>Conclusions</p><p>These findings suggest that autoantibody-independent astrocytic Cx43 loss may relate to disease aggressiveness and distal oligodendrogliopathy in both MS and NMO.</p> </div

Loss of oligodendrocytic Cx47 and Cx32 expression in chronic lesions of MS (case MS-5).

<p>KB staining in a chronic inactive lesion of the optic nerve (A). Higher magnification view of the lesion boundary area (B–I, corresponding to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072919#pone-0072919-g005" target="_blank">Figure 5A</a>, square). Immunoreactivities for Cx47 and Cx32 are diminished beyond the demyelinated area (B, C) as revealed by immunostaining for MOG, OSP and MAG (D–F). This lesion is classified as pattern D for Cx43 and pattern A for Cx47/Cx32. In contrast, MLC1, AQP4 and Cx43 expression are up-regulated because of astrogliosis (G–I). MLC1 is localized at perivascular foot processes even in chronic gliotic tissues (G). Scale Bar = 1 mm(A); 200 µm (B–I).</p

Distal oligodendrogliopathy and astrocytopathy in anti-AQP4 antibody-seropositive NMO (case NMO-10).

<p>In active lesions of the cerebral white matter, KB staining and MOG immunostaining show remaining myelin (A, B). Patterns of preferential MAG loss and marked loss of GFAP immunoreactivity are seen (C, D). Higher magnification reveals sharply demarcated, prominent MAG loss in this lesion with infiltration of numerous CD68-positive macrophages, whereas immunoreactivity for MOG, MBP and OSP is preserved in the lesion (E–I). Immunoreactivity for Cx47 is diminished compared with non-affected white matter (J). Complete loss of AQP4 and Cx43 in degenerative, GFAP-positive astrocytes (K–M) and complement deposition are observed around blood vessels with perivascular cell cuffing (N, O). Complement components are present within foamy macrophages in this lesion (P). Nogo-A-positive oligodendrocytes are markedly decreased in this lesion and some remaining oligodendrocytes show nuclear condensation, suggesting apoptotic changes (Q). This lesion is classified as pattern A for Cx43 and pattern B for Cx47/Cx32. Scale Bar = 4 mm (A–D); 200 µm (E–M); 50 µm (N, O); 20 µm (P, Q).</p

Coexistence of distal oligodendrogliopathy in active NMO lesions in case NMO-4

<p>(A–L) CD68 immunostaining demonstrates massive infiltration of macrophages in the cerebral peduncle (A, arrows). Immunoreactivity for MOG is relatively preserved but is completely lost for MAG (B, C, arrows). Higher magnification of the lesion (D–L). Immunoreactivity for MOG is relatively preserved in contrast to complete loss of MAG in this lesion (D, E). Cx47 expression is diminished compared with non-affected white matter (F). Lesion boundary areas (G–I). A dotted line indicates the boundary. Immunostainings for Cx47 and Cx32 are slightly diminished but preserved inside the lesion (G, H). Immunostaining of Nogo-A is markedly decreased and apoptotic nuclear condensation of oligodendrocytes is present (I, insert). Immunoreactivities for AQP4 and Cx43 within highly degenerative GFAP-positive astrocytes are completely lost (J–L). This lesion is classified as pattern B for Cx43 and pattern B for Cx47/Cx32. Scale Bar = 4 mm(A-C); 0.5 mm (D–F); 50 µm (G, H); 100 µm (I); 200 µm (J–L).</p

Loss of Cx47 and Cx32 in chronic active NMO lesions in case NMO-6

<p>(A–M) Low magnification view of KB staining (A) and immunostainings for MOG (B), Cx47 (C) and Cx43 (D) in the cerebrum. Immunoreactivity for Cx47 is extensively diminished in the demyelinating lesion with cavitation in the cerebral white matter, whereas immunoreactivity for MOG is relatively preserved (A–C). This lesion is classified as pattern D for Cx43 and pattern A for Cx47/Cx32. Up-regulation of Cx43 immunoreactivity is observed in the corresponding demyelinating area (D). Arrows indicates cavitation and arrowheads show the lesion boundary (C, D). CD68-positive foamy macrophages are observed at the periphery and perivascular areas of the lesion. No foamy macrophages contain KB-positive granules within their cytoplasm (E, F, insert). Immunoreactivities for MAG and OSP are decreased in these lesions (G, H). Expression levels of Cx47 and Cx32 are extensively reduced in the lesion (I, J) compared with the non-affected white matter (K, L). Numerous Cx43- positive astrocytes are present in this lesion suggesting astrogliosis. Cx43 immunoreactivity is also preserved in the perivascular areas (M). Scale Bar = 2 mm (A–D); 20 µm (E); 100 µm (F–J, M); 50 µm (K, L).</p

Cx43 and AQP4 astrocytopathy in active lesions of MS (case MS-3).

<p>Low magnification view of GFAP (A) and Cx43 (B) immunostaining in the pons. Immunoreactivity for Cx43 is markedly diminished in multiple lesions covered by GFAP-positive astrocytes (A, B, arrows). Massive perivascular cuffing, mostly consisting of lymphocytes, is observed in these lesions (C). Immunoreactivities for Cx32 and Cx47 are decreased in these lesions (D, E) compared with non-affected white matter (D, E, insert). Loss of MAG compared with MOG and OSP is prominent (F–H). Infiltration of macrophages phagocytosing myelin debris, which are immunopositive for myelin proteins (F, H, insert). Numerous GFAP-positive hypertrophic astrocytes exist in perivascular areas and parenchyma of these lesions (I). Patchy loss of AQP4 and diffuse loss of Cx43 in the center and periphery of lesions (J, K). Some hypertrophic astrocytes demonstrate membranous staining for AQP4 (J, insert). This lesion is classified as pattern A for Cx43 and pattern B for Cx47/Cx32. Scale Bar = 4 mm (A, B); 50 µm (C); 100 µm (D–K).</p

Relationship of inflammatory components with patterns of Cx43 loss in MS and NMO lesions.

<p>(A, B) Positivity rates of perivascular lymphocytic cuffing or perivascular deposition of complement and immunoglobulin in active (A) or chronic active (B) lesions of MS cases according to Cx43 patterns. (C, D) Positivity rates of perivascular lymphocytic cuffing or perivascular deposition of complement and immunoglobulin in active (C) or chronic active (D) lesions of NMO/NMOSD cases according to Cx43 patterns. Patterns N lesions are excluded in this figure.</p