Rapid Immunochromatographic Detection of Serum Anti-α-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy

We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy.     

Infection by Toxoplasma gondii Induces Amoeboid-Like Migration of Dendritic Cells in a Three-Dimensional Collagen Matrix

Toxoplasma gondii, an obligate intracellular parasite of humans and other warm-blooded vertebrates, invades a variety of cell types in the organism, including immune cells. Notably, dendritic cells (DCs) infected by T. gondii acquire a hypermigratory phenotype that potentiates parasite dissemination by a ‘Trojan horse’ type of mechanism in mice. Previous studies have demonstrated that, shortly after parasite invasion, infected DCs exhibit hypermotility in 2-dimensional confinements in vitro and enhanced transmigration in transwell systems. However, interstitial migration in vivo involves interactions with the extracellular matrix in a 3-dimensional (3D) space. We have developed a collagen matrix-based assay in a 96-well plate format that allows quantitative locomotion analyses of infected DCs in a 3D confinement over time. We report that active invasion of DCs by T. gondii tachyzoites induces enhanced migration of infected DCs in the collagen matrix. Parasites of genotype II induced superior DC migratory distances than type I parasites. Moreover, Toxoplasma-induced hypermigration of DCs was further potentiated in the presence of the CCR7 chemotactic cue CCL19. Blocking antibodies to integrins (CD11a, CD11b, CD18, CD29, CD49b) insignificantly affected migration of infected DCs in the 3D matrix, contrasting with their inhibitory effects on adhesion in 2D assays. Morphological analyses of infected DCs in the matrix were consistent with the acquisition of an amoeboid-like migratory phenotype. Altogether, the present data show that the Toxoplasma-induced hypermigratory phenotype in a 3D matrix is consistent with integrin-independent amoeboid DC migration with maintained responsiveness to chemotactic and chemokinetic cues. The data support the hypothesis that induction of amoeboid hypermigration and chemotaxis/chemokinesis in infected DCs potentiates the dissemination of T. gondii.     

Genetic abolishment of hepatocyte proliferation activates hepatic stem cells

Quiescent hepatic stem cells (HSCs) can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1), an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs) by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.     

Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

Tumor associated macrophages are known to be closely linked with tumor progression and metastasis. On the other hand, clusterin is overexpressed in several tumor types and regarded as a putative tumor-promoting factor due to this overexpression and the subsequent induction of chemoresistance. In our previous study, clusterin was found to induce the expression of matrix metalloproteinase-9 (MMP-9) in macrophages, and MMP-9 is known to be essential for tumor cell migration and invasion via basement membrane breakdown. Because paracrine interactions between tumor cells and surrounding macrophages regulate metastasis, these findings raise the possibility that clusterin promotes the secretion of cytokines in macrophages in addition to MMP-9. Here, we demonstrate that clusterin upregulates the expressions of chemotactic cytokines, that is, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1β (MIP-1β), regulated upon activation, normal T cell expressed and secreted (RANTES), and tumor necrosis factor-α (TNF-α) in Raw264.7 macrophages. In particular, clusterin stimulated TNF-α secretion via the activations of ERK, JNK, and PI3K/Akt pathways in a time and dose-dependent manner. Furthermore, clusterin-induced TNF-α secretion was found to play a critical role in the chemotactic migration of Raw264.7 macrophages. It was also found that clusterin acts directly as a chemoattractant for macrophages. Together, these results suggest that clusterin stimulates the expression and secretion of TNF-α, which plays a critical role in promoting macrophage chemotaxis, via ERK, JNK, and PI3K/Akt pathways. Collectively, these findings describe a novel function for clusterin as an inducer of TNF-α in macrophages and their chemotactic migration, and suggest that clusterin has a tumor-promoting effect.     

Protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Arabidopsis NADPH oxidases and may function as a trigger for the positive feedback regulation of Ca2+ and reactive oxygen species

Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in signalling and development. Given the high toxicity of ROS, their production is tightly regulated. In Arabidopsis, respiratory burst oxidase homologue F (AtrbohF) encodes NADPH oxidase. Here we characterised the activation of AtRbohF using a heterologous expression system. AtRbohF exhibited ROS-producing activity that was synergistically activated by protein phosphorylation and Ca2+. The two EF-hand motifs of AtRbohF in the N-terminal cytosolic region were crucial for its Ca2+-dependent activation. AtrbohD and AtrbohF are involved in stress responses. Although the activation mechanisms for AtRbohD and AtRbohF were similar, AtRbohD had significantly greater ROS-producing activity than AtRbohF, which may reflect their functional diversity, at least in part. We further characterised the interrelationship between Ca2+ and phosphorylation regarding activation and found that protein phosphorylation-induced activation was independent of Ca2+. In contrast, K-252a, a protein kinase inhibitor, inhibited the Ca2+-dependent ROS-producing activity of AtRbohD and AtRbohF in a dose-dependent manner, suggesting that protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Rboh. Positive feedback regulation of Ca2+ and ROS through AtRbohC has been proposed to play a critical role in root hair tip growth. Our findings suggest that Rboh phosphorylation is the initial trigger for the plant Ca2+-ROS signalling network.     

Tryptophan dimer produced by water-stressed bahia grass is an attractant for <Emphasis Type="Italic">Gigaspora margarita</Emphasis> and <Emphasis Type="Italic">Glomus caledonium</Emphasis>

We isolated and elucidated the structure of several stimulants for arbuscular mycorrhizal fungi (AMF) in water-stressed bahia grass roots. We could isolate some compounds that promoted the growth of Gigaspora margarita Becker and Hall and Glomus caledonium (Nicol. and Gerd.) Trappe and Gerd. In these compounds, tryptophan dimer (Trp–Trp) was elucidated the structure. Trp–Trp was abundantly produced in water-stressed bahia grass roots and exuded to the soil, although it was scarcely detected in non-stressed root exudates. Interestingly, this peptide strongly attracted the hyphae of Gi. margarita and G. caledonium and promoted their hyphal growth in vitro (1.8 × longer than the control). Tryptophan, however, had no effect on hyphal growth and attraction. Thus, Trp–Trp exuded from water-stressed roots would play an important role as a major signal for AMF. An erratum to this article can be found at     

An alloplasmic male-sterile line of Brassica oleracea harboring the mitochondria from Diplotaxis muralis expresses a novel chimeric open reading frame, orf72

Nuclear so-called fertility-restorer genes reverse the pollen sterility of cytoplasmic male-sterile (CMS) plants caused by disturbed mitochondrial-nuclear interactions. We identified a CMS-associated chimeric mitochondrial gene in an alloplasmic CMS line of Brassica oleracea in the 'mur' system. This novel chimeric gene, orf72, was found in the mitochondrial genome of donor cytoplasm. It was located downstream of normal rps7 and contained part of atp9 (atp9-b). It was expressed specifically on the nuclear background of CMS B. oleracea, partially suppressed in the fertility-restored line and entirely suppressed in the cytoplasmic donor.