Superior protective and therapeutic effects of IL-12 and IL-18 gene-transduced dendritic neuroblastoma fusion cells on liver metastasis of murine neuroblastoma

Fusion vaccine of dendritic cells (DCs) and tumor cells has the advantage of inducing an immune response against multiple tumor Ags, including unknown tumor Ags. Using the liver metastasis model of C1300 neuroblastoma cells, we assessed the protective and therapeutic effects of fusion cells transduced with the IL-12 gene and/or the IL-18 gene. Improving the fusion method by combining polyethylene glycol and electroporation increased loading efficiency. In the A/J mice vaccinated with fusion cells modified with the LacZ gene (fusion/LacZ), IFN-gamma production and CTL activity increased significantly compared with that of DCs/LacZ, C1300/LacZ, or a mixture of the two (mixture/LacZ). With the transduction of IL-12 and IL-18 genes into the fusion cells (fusion/IL-12/IL-18), the level of IFN-gamma increased more than five times that of other fusion groups. In addition, NK cell activity and CTL activity increased significantly compared with that of mixture/LacZ, fusion/LacZ, DC/LacZ, or C1300/LacZ. In the protective and therapeutic studies of fusion cell vaccine, mice vaccinated with fusion/LacZ, fusion/IL-12, fusion/IL-18, or fusion/IL-12/IL-18 showed a significant decrease in liver metastasis and a significant increase in survival compared with mice given a mixture/LacZ, DCs/LacZ, or C1300/LacZ. In particular, the mice receiving fusion/IL-12/IL-18 vaccine showed a complete protective effect and the highest therapeutic effects. The present study investigates the improved loading efficiency of fusion cells and suggests that the introduction of IL-12 and IL-18 genes can induce extremely strong protective and therapeutic effects on liver metastasis of neuroblastoma.     

The Vibrio motor proteins, MotX and MotY, are associated with the basal body of Na-driven flagella and required for stator formation

The four motor proteins PomA, PomB, MotX and MotY, which are believed to be stator proteins, are essential for motility by the Na(+)-driven flagella of Vibrio alginolyticus. When we purified the flagellar basal bodies, MotX and MotY were detected in the basal body, which is the supramolecular complex comprised of the rotor and the bushing, but PomA and PomB were not. By antibody labelling, MotX and MotY were detected around the LP ring. These results indicate that MotX and MotY associate with the basal body. The basal body had a new ring structure beneath the LP ring, which was named the T ring. This structure was changed or lost in the basal body from a DeltamotX or DeltamotY strain. The T ring probably comprises MotX and MotY. In the absence of MotX or MotY, we demonstrated that PomA and PomB were not localized to a cell pole. From the above results, we suggest that MotX and MotY of the T ring are involved in the incorporation and/or stabilization of the PomA/PomB complex in the motor.     

Helical distribution of the bacterial chemoreceptor via colocalization with the Sec protein translocation machinery

In Escherichia coli, chemoreceptor clustering at a cell pole seems critical for signal amplification and adaptation. However, little is known about the mechanism of localization itself. Here we examined whether the aspartate chemoreceptor (Tar) is inserted directly into the polar membrane by using its fusion to green fluorescent protein (GFP). After induction of Tar-GFP, fluorescent spots first appeared in lateral membrane regions, and later cell poles became predominantly fluorescent. Unexpectedly, Tar-GFP showed a helical arrangement in lateral regions, which was more apparent when a Tar-GFP derivative with two cysteine residues in the periplasmic domain was cross-linked to form higher oligomers. Moreover, similar distribution was observed even when the cytoplasmic domain of the double cysteine Tar-GFP mutant was replaced by that of the kinase EnvZ, which does not localize to a pole. Observation of GFP-SecE and a translocation-defective MalE-GFP mutant, as well as indirect immunofluorescence microscopy on SecG, suggested that the general protein translocation machinery (Sec) itself is arranged into a helical array, with which Tar is transiently associated. The Sec coil appeared distinct from the MreB coil, an actin-like cytoskeleton. These findings will shed new light on the mechanisms underlying spatial organization of membrane proteins in E. coli.     

The fern Adiantum capillus-veneris lacks stomatal responses to blue light

We investigated the responses of stomata to light in the fern Adiantum capillus-veneris, a typical species of Leptosporangiopsida. Stomata in the intact leaves of the sporophytes opened in response to red light, but they did not open when blue light was superimposed on the red light. The results were confirmed in the isolated Adiantum epidermis. The red light-induced stomatal response was not affected by the mutation of phy3, a chimeric protein of phytochrome and phototropin in this fern. The lack of a blue light-specific stomatal response was observed in three other fern species of Leptosporangiopsida, i.e. Pteris cretica, Asplenium scolopendrium and Nephrolepis auriculata. Fusicoccin, an activator of the plasma membrane H(+)-ATPase, induced both stomatal opening and H(+) release in the Adiantum epidermis. Adiantum phototropin genes AcPHOT1 and AcPHOT2 were expressed in the fern guard cells. The transformation of an Arabidopsis phot1 phot2 double mutant, which lost blue light-specific stomatal opening, with AcPHOT1 restored the stomatal response to blue light. Taken together, these results suggest that ferns of Leptosporangiopsida lack a blue light-specific stomatal response, although the functional phototropin and plasma membrane H(+)-ATPase are present in this species.     

Identification of hepatitis C virus (HCV) 2a-derived epitope peptides having the capacity to induce cytotoxic T lymphocytes in human leukocyte antigen-A24+ and HCV2a-infected patients

Since virus-specific cytotoxic T lymphocytes (CTLs) play a critical role in preventing the spread of hepatitis C virus (HCV), vaccine-based HCV-specific CTL induction could be a promising strategy to treat HCV-infected patients. In this study, we tried to identify HCV2a-derived epitopes, which can induce human leukocyte antigen (HLA)-A24-restricted and peptide-specific CTLs. Peripheral blood mononuclear cells of HCV2a-infected patients or healthy donors were stimulated in vitro with HCV2a-derived peptides, which were prepared based on the HLA-A24 binding motif. As a result, three peptides (HCV2a 576-584, HCV2a 627-635, and HCV2a 1085-1094) efficiently induced peptide-specific CTLs from HLA-A24(+) HCV2a-infected patients as well as healthy donors. The cytotoxicity was exhibited by peptide-specific CD8(+) T cells in an HLA-A24-restricted manner. In addition, the HCV2a 627-635 peptide was frequently recognized by immunoglobulin G of HCV2a-infected patients. These results indicate that the identified three HCV2a peptides might be applicable to peptide-based immunotherapy for HLA-A24(+) HCV2a-infected patients.     

G protein-coupled receptor 30 is an estrogen receptor in the plasma membrane

Recently, GPR30 was reported to be a novel estrogen receptor; however, its intracellular localization has remained controversial. To investigate the intracellular localization of GPR30 in vivo, we produced four kinds of polyclonal antibodies for distinct epitopes on GPR30. Immunocytochemical observations using anti-GPR30 antibody and anti-FLAG antibody show that FLAG-GPR30 localizes to the plasma membrane 24 h after transfection. Treatment with estrogen (17beta-estradiol or E2) causes an elevation in the intracellular Ca2+ concentration ([Ca2+]i) within 10 s in HeLa cells expressing FLAG-GPR30. In addition, E2 induces the translocation of GPR30 from the plasma membrane to the cytoplasm by 1 h after stimulation. Immunohistochemical analysis shows that GPR30 exists on the cell surface of CA2 pyramidal neuronal cells. The images on transmission electron microscopy show that GPR30 is localized to a particular region associated with the plasma membranes of the pyramidal cells. These data indicate that GPR30, a transmembrane receptor for estrogen, is localized to the plasma membrane of CA2 pyramidal neuronal cells of the hippocampus in rat brain.     

Nuclear markers reveal that inter-lake cichlids' similar morphologies do not reflect similar genealogy

The apparent inter-lake morphological similarity among East African Great Lakes' cichlid species/genera has left evolutionary biologists asking whether such similarity is due to sharing of common ancestor or mere convergent evolution. In order to answer such question, we first used Geometric Morphometrics, GM, to quantify morphological similarity and then subsequently used Amplified Fragment Length Polymorphism, AFLP, to determine if similar morphologies imply shared ancestry or convergent evolution. GM revealed that not all presumed morphological similar pairs were indeed similar, and the dendrogram generated from AFLP data indicated distinct clusters corresponding to each lake and not inter-lake morphological similar pairs. Such results imply that the morphological similarity is due to convergent evolution and not shared ancestry. The congruency of GM and AFLP generated dendrograms imply that GM is capable of picking up phylogenetic signal, and thus GM can be potential tool in phylogenetic systematics.     

CoQ10 deficiencies and MNGIE: Two treatable mitochondrial disorders

Background

Although causative mutations have been identified for numerous mitochondrial disorders, few disease-modifying treatments are available. Two examples of treatable mitochondrial disorders are coenzyme Q₁₀ (CoQ₁₀ or ubiquinone) deficiency and mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).

Scope of review

Here, we describe clinical and molecular features of CoQ₁₀ deficiencies and MNGIE and explain how understanding their pathomechanisms have led to rationale therapies. Primary CoQ₁₀ deficiencies, due to mutations in genes required for ubiquinone biosynthesis, and secondary deficiencies, caused by genetic defects not directly related to CoQ₁₀ biosynthesis, often improve with CoQ₁₀ supplementation. In vitro and in vivo studies of CoQ₁₀ deficiencies have revealed biochemical alterations that may account for phenotypic differences among patients and variable responses to therapy. In contrast to the heterogeneous CoQ₁₀ deficiencies, MNGIE is a single autosomal recessive disease due to mutations in the TYMP gene encoding thymidine phosphorylase (TP). In MNGIE, loss of TP activity causes toxic accumulations of the nucleosides thymidine and deoxyuridine that are incorporated by the mitochondrial pyrimidine salvage pathway and cause deoxynucleoside triphosphate pool imbalances, which, in turn cause mtDNA instability. Allogeneic hematopoetic stem cell transplantation to restore TP activity and eliminate toxic metabolites is a promising therapy for MNGIE.

Major conclusions

CoQ₁₀ deficiencies and MNGIE demonstrate the feasibility of treating specific mitochondrial disorders through replacement of deficient metabolites or via elimination of excessive toxic molecules.

General significance

Studies of CoQ₁₀ deficiencies and MNGIE illustrate how understanding the pathogenic mechanisms of mitochondrial diseases can lead to meaningful therapies. This article is part of a Special Issue entitled: Biochemistry of Mitochondria, Life and Intervention 2010.