A solanesyl-diphosphate synthase localizes in glycosomes of Trypanosoma cruzi

We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass approximately 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target.     

Membrane topology of mouse stearoyl-CoA desaturase 1

Stearoyl-CoA desaturase (SCD) is an integral membrane protein anchored in the endoplasmic reticulum. It catalyzes the biosynthesis of monounsaturated fatty acids that are required for the synthesis of triglycerides, cholesteryl esters, and phospholipids. Four mouse isoforms of SCD (SCD1-4) and two human isoforms have been characterized. In the current study, we characterize the topology of the mouse SCD1 isoform. Hydropathy analysis of the 355-amino acid mouse SCD1 protein predicts that the protein contains four transmembrane domains (TMDs) and three loops connecting the membrane-spanning domains. To define the topology of the protein, recombinant SCD1 constructs containing epitope tags were transiently expressed in HeLa cells and analyzed by indirect immunofluorescence and cysteine derivatization. Our data provide evidence that the N and C termini of SCD1 are oriented toward the cytosol with four transmembrane domains separated by two very short hydrophilic loops in the ER lumen and one large hydrophilic loop in the cytosol. In addition, based on the previous observation that SCD is a thiol enzyme, we sought to investigate whether the cysteine residues were essential for enzyme activity through mutagenesis studies, and our data suggest that the cysteines in SCD are not catalytically essential.     

Induction of efficient antitumor immunity using dendritic cells activated by recombinant Sendai virus and its modulation by exogenous IFN-beta gene

Dendritic cell (DC)-based cancer immunotherapy has been paid much attention as a new and cancer cell-specific therapeutic in the last decade; however, little clinical outcome has been reported. Current limitations of DC-based cancer immunotherapy include sparse information about which DC phenotype should be administered. We here report a unique, representative, and powerful method to activate DCs, namely recombinant Sendai virus-modified DCs (SeV/DC), for cancer immunotherapy. In vitro treatment of SeV without any bioactive gene solely led DCs to a mature phenotype. Even though the expression of surface markers for DC activation ex vivo did not always reach the level attained by an optimized amount of LPS, superior antitumor effects to B16F1 melanoma, namely tumor elimination and survival, were obtained with use of SeV-GFP/DC as compared with those seen with LPS/DC in vivo, and the effect was enhanced by SeV/DC-expressing IFN-beta (SeV-murine IFN-beta (mIFN-beta)/DC). In case of the treatment of an established tumor of B16F10 (7-9 mm in diameter), a highly malignant subline of B16 melanoma, SeV-modified DCs (both SeV-GFP/DC and SeV-mIFN-beta/DC), but not immature DC and LPS/DC, dramatically improved the survival of animals. Furthermore, SeV-mIFN-beta/DC but not other DCs could lead B16F10 tumor to the dormancy, associated with strongly enhanced CD8+ CTL responses. These results indicate that rSeV is a new and powerful tool as an immune booster for DC-based cancer immunotherapy that can be significantly modified by IFN-beta, and SeV/DC, therefore, warrants further investigation as a promising alternative for cancer immunotherapy.     

Hydrodynamic-based delivery of an interleukin-22-Ig fusion gene ameliorates experimental autoimmune myocarditis in rats

IL-22 is one of several cytokines with limited homology to IL-10. However, the biological activities of IL-22 are mostly unknown. The purpose of this study was to evaluate the effect of IL-22 on rat experimental autoimmune myocarditis (EAM) and elucidate an aspect of the biological activities of IL-22. Rats were immunized on day 0; IL-22-Ig-treated rats were injected with pCAGGS-IL-22-Ig and control rats with pCAGGS-Ig using hydrodynamics-based gene delivery on day 1 or day 6. IL-22-Ig gene therapy administered on day 1 or day 6 after immunization was effective in controlling EAM as monitored by the heart weight to body weight ratio, and the myocarditis area in rats was sacrificed on day 17. Examination of the expression of IL-22-related genes in purified cells from EAM hearts suggested that IL-22-Ig acting target cells were noncardiomyocytic (NC) noninflammatory cells such as fibroblasts, smooth muscle cells, and endothelial cells. Therefore, we examined the effect of rIL-22 or serum containing IL-22-Ig on the expression of immune-relevant genes in IL-1-stimulated NC cells cultured from EAM hearts. Results showed that the expression of immunologic molecules (PGE synthase, cyclooxygenase-2, MIP-2, MCP-1, IL-6, and cytokine-induced neutrophil chemoattractant-2) in IL-1-stimulated NC cells was significantly decreased by rIL-22 or serum containing IL-22-Ig. EAM was suppressed by hydrodynamics-based delivery of plasmid DNA encoding IL-22-Ig, and the reason for this effectiveness may be that IL-22 suppressed gene expression of PG synthases, IL-6, and chemokines in activated NC noninflammatory cells.     

TLR-dependent induction of IFN-beta mediates host defense against Trypanosoma cruzi

Host resistance to the intracellular protozoan parasite Trypanosoma cruzi depends on IFN-gamma production by T cells and NK cells. However, the involvement of innate immunity in host resistance to T. cruzi remains unclear. In the present study, we investigated host defense against T. cruzi by focusing on innate immunity. Macrophages and dendritic cells (DCs) from MyD88(-/-)TRIF(-/-) mice, in which TLR-dependent activation of innate immunity was abolished, were defective in the clearance of T. cruzi and showed impaired induction of IFN-beta during T. cruzi infection. Neutralization of IFN-beta in MyD88(-/-) macrophages led to enhanced T. cruzi growth. Cells from MyD88(-/-)IFNAR1(-/-) mice also showed impaired T. cruzi clearance. Furthermore, both MyD88(-/-)TRIF(-/-) and MyD88(-/-)IFNAR1(-/-) mice were highly susceptible to in vivo T. cruzi infection, highlighting the involvement of innate immune responses in T. cruzi infection. We further analyzed the molecular mechanisms for the IFN-beta-mediated antitrypanosomal innate immune responses. MyD88(-/-)TRIF(-/-) and MyD88(-/-)IFNAR1(-/-) macrophages and DCs exhibited defective induction of the GTPase IFN-inducible p47 (IRG47) after T. cruzi infection. RNA interference-mediated reduction of IRG47 expression in MyD88(-/-) macrophages resulted in increased intracellular growth of T. cruzi. These findings suggest that TLR-dependent expression of IFN-beta is involved in resistance to T. cruzi infection through the induction of IRG47.     

Cutting Edge: Lentiviral short hairpin RNA silencing of PTEN in human mast cells reveals constitutive signals that promote cytokine secretion and cell survival

Engagement of the FcepsilonRI expressed on mast cells induces the production of phosphatidylinositol 3, 4, 5-trisphosphate by PI3K, which is essential for the functions of the cells. PTEN (phosphatase and tensin homologue deleted on chromosome ten) directly opposes PI3K by dephosphorylating phosphatidylinositol 3, 4, 5-trisphosphate at the 3' position. In this work we used a lentivirus-mediated short hairpin RNA gene knockdown method to study the role of PTEN in CD34(+) peripheral blood-derived human mast cells. Loss of PTEN caused constitutive phosphorylation of Akt, p38 MAPK, and JNK, as well as cytokine production and enhancement in cell survival, but not degranulation. FcepsilonRI engagement of PTEN-deficient cells augmented signaling downstream of Src kinases and increased calcium flux, degranulation, and further enhanced cytokine production. PTEN-deficient cells, but not control cells, were resistant to inhibition of cytokine production by wortmannin, a PI3K inhibitor. The findings demonstrate that PTEN functions as a key regulator of mast cell homeostasis and FcepsilonRI-responsiveness.     

Recombination of the GFP gene to the BFP gene using a man-made site-selective DNA cutter

By using the recently developed man-made DNA cutter [a combination of Ce(IV)/EDTA and two DNA additives], green fluorescent protein (GFP) was converted to closely related blue fluorescent protein (BFP). The phosphodiester linkages at T196-A200 in the sense strand of GFP were hydrolyzed by the cutter, and the A1-T196 fragment in the product was selectively connected with the downstream fragment (C197-A720) of BFP by T4 DNA ligase. This recombination changed three codons in the GFP gene (TGC at 196–198, TAT at 199–201, and ACC at 502–504) to TCT, CAT, and ATC in BFP, and accordingly three amino acids in GFP (Cys65, Tyr66, and Thr167) were altered to Ser65, His66, and Ile167. The recombinant gene was successfully expressed in Escherichia coli and emitted blue fluorescence, confirming the absence of undesired side reactions (mutation, deletion, insertion, depurination, etc.) in the DNA manipulation. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Role of VAMP-2, VAMP-7, and VAMP-8 in constitutive exocytosis from HSY cells

We evaluated the role of VAMP-2/synaptobrevin, VAMP-7/TI-VAMP, and VAMP-8/endobrevin in exocytic pathways of HSY cells, a human parotid epithelial cell line, by coexpressing these VAMP proteins tagged with green fluorescent protein (GFP) and human growth hormone (hGH) as a secretory cargo. Exocytosis of hGH was constitutive and the fluorescent signal of hGH–GFP was observed in the Golgi area and small vesicles quickly moving throughout the cytoplasm. The cytoplasmic vesicles containing hGH overlapped well with VAMP-7-GFP, but did so scarcely with VAMP-2-GFP or VAMP-8-GFP. However, when the vesicle transport from the trans-Golgi network to the plasma membrane was arrested by incubation at 20°C for 2 h and then released by warming up to 37°C; VAMP-2-GFP and hGH were clearly colocalized together in small cytoplasmic vesicles. Neither VAMP-7-GFP nor hGH–GFP was colocalized with LAMP-1, a marker for lysosomes and late endosomes. These results suggest that (1) VAMP-2 can be one of the v-SNAREs for constitutive exocytosis; (2) VAMP-7 is involved in the constitutive exocytosis as a slow, minor v-SNARE, but not in the lysosomal transport; and (3) VAMP-8 is unlikely to be a v-SNARE for constitutive exocytosis in HSY cells.     

Identification of multipotent progenitors in the embryonic mouse kidney by a novel colony-forming assay

Renal stem or progenitor cells with a multilineage differentiation potential remain to be isolated, and the differentiation mechanism of these cell types in kidney development or regeneration processes is unknown. In an attempt to resolve this issue, we set up an in vitro culture system using NIH3T3 cells stably expressing Wnt4 (3T3Wnt4) as a feeder layer, in which a single renal progenitor in the metanephric mesenchyme forms colonies consisting of several types of epithelial cells that exist in glomeruli and renal tubules. We found that only cells strongly expressing Sall1 (Sall1-GFP(high) cells), a zinc-finger nuclear factor essential for kidney development, form colonies, and that they reconstitute a three-dimensional kidney structure in an organ culture setting. We also found that Rac- and JNK-dependent planar cell polarity (PCP) pathways downstream of Wnt4 positively regulate the colony size, and that the JNK pathway is also involved in mesenchymal-to-epithelial transformation of colony-forming progenitors. Thus our colony-forming assay, which identifies multipotent progenitors in the embryonic mouse kidney, can be used for examining mechanisms of renal progenitor differentiation.     

Multiple roles of mesenchymal beta-catenin during murine limb patterning

Recently canonical Wnt signaling in the ectoderm has been shown to be required for maintenance of the apical ectodermal ridge (AER) and for dorsoventral signaling. Using conditional gain- and loss-of-function beta-catenin alleles, we have studied the role of mesenchymal beta-catenin activity during limb development. Here, we show that loss of beta-catenin results in limb truncations due to a defect in AER maintenance. Stabilization of beta-catenin also results in truncated limbs, caused by a premature regression of the AER. Concomitantly, in these limbs, the expression of Bmp2, Bmp4 and Bmp7, and of the Bmp target genes Msx1, Msx2 and gremlin, is expanded in the mesenchyme. Furthermore, we found that the expression of Lmx1b, a gene exclusively expressed in the dorsal limb mesenchyme and involved in dorsoventral patterning, is reduced upon loss of beta-catenin activity and is expanded ventrally in gain-of-function limbs. However, the known ectodermal regulators Wnt7a and engrailed 1 are expressed normally. This suggests that Lmx1b is also regulated, in part, by a beta-catenin-mediated Wnt signal, independent of the non-canoncial Wnt7a signaling pathway. In addition, loss of beta-catenin results in a severe agenesis of the scapula. Concurrently, the expression of two genes, Pax1 and Emx2, which have been implicated in scapula development, is lost in beta-catenin loss-of-function limbs; however, only Emx2 is upregulated in gain-of-function limbs. Mesenchymal beta-catenin activity is therefore required for AER maintenance, and for normal expression of Lmx1b and Emx2.