MITOPLD is a mitochondrial protein essential for nuage formation and piRNA biogenesis in the mouse germline

MITOPLD is a member of the phospholipase D superfamily proteins conserved among diverse species. Zucchini (Zuc), the Drosophila homolog of MITOPLD, has been implicated in primary biogenesis of Piwi-interacting RNAs (piRNAs). By contrast, MITOPLD has been shown to hydrolyze cardiolipin in the outer membrane of mitochondria to generate phosphatidic acid, which is a signaling molecule. To assess whether the mammalian MITOPLD is involved in piRNA biogenesis, we generated Mitopld mutant mice. The mice display meiotic arrest during spermatogenesis, demethylation and derepression of retrotransposons, and defects in primary piRNA biogenesis. Furthermore, in mutant germ cells, mitochondria and the components of the nuage, a perinuclear structure involved in piRNA biogenesis/function, are mislocalized to regions around the centrosome, suggesting that MITOPLD may be involved in microtubule-dependent localization of mitochondria and these proteins. Our results indicate a conserved role for MITOPLD/Zuc in the piRNA pathway and link mitochondrial membrane metabolism/signaling to small RNA biogenesis.     

Changes in the microbial community structure during thermophilic composting of manure as detected by the quinone profile method

Quinone profiles and physico-chemical properties were measured to characterize the microbial community structure during a 14-day thermophilic composting of cattle manure mixed with rice straw as a bulking agent. The change in total quinone content (TQ) and the divergence of quinone (DQ) indicated that the microbial biomass reached a peak followed by a decrease, whereas the microbial community diversity increased continuously during the composting process. The high mole fraction of ubiquinones in the raw materials, and at the beginning of the composting period suggested that fungi and/or Proteobacteria were present. The predominance of MK-7 from days 3 to 7 suggested that Bacillus spp. were the main microbial species. An increase in partially saturated and long-chain menaquinones during the latter composting period indicated that the proliferation of various species of Actinobacteria was occurring. The microbial community structure, as expressed by TQ and DQ, corresponded well to physico-chemical properties such as the C/N ratio, pH, O₂ consumption and compost mass reduction.     

Early bone marrow hematopoietic defect in simian/human immunodeficiency virus C2/1-infected macaques and relevance to advance of disease

To clarify hematological abnormalities following infection with human immunodeficiency virus (HIV), we examined the hematopoietic capability of bone marrow by using cynomolgus monkeys infected with pathogenic simian/human immunodeficiency virus (SHIV) strain C2/1, an animal model of HIV infection. The relationship between the progress of the infection and the CD4/CD8 ratio of T lymphocytes or the amount of SHIV C2/1 viral load in the peripheral blood was also investigated. A colony assay was performed to assess the hematopoietic capability of bone marrow stem cells during the early and advanced phases of the infection. Colonies of granulocytes-macrophages (GM) were examined by PCR for the presence of the SIVmac239 gag region to reveal direct viral infection. There was a remarkable decrease in the CFU-GM growth on days 1 and 3 postinoculation, followed by recovery on day 56. During the more advanced stage, the CFU-GM growth decreased again. There was minimal evidence of direct viral infection of pooled cultured CFU-GM despite the continuously low CD4/CD8 ratios. These results indicate that the decrease in colony formation by bone marrow stem cells is reversible and fluctuates with the advance of the disease. This decrease was not due to direct viral infection of CFU-GM. Our data may support the concept that, in the early phase, production of inhibitory factors or deficiency of a stimulatory cytokine is responsible for some of the bone marrow defects described in the SHIV C2/1 model.     

A new method for the modification of fibroin heavy chain protein in the transgenic silkworm

We constructed a new plasmid vector for the production of a modified silk fibroin heavy chain protein (H-chain) in the transgenic silkworm. The plasmid (pHC-null) contained the promoter and the 3' region of a gene encoding the H-chain and the coding regions for the N-terminal domain and the C-terminal domain of the H-chain. For the model protein, we cloned a foreign gene that encoded EGFP between the N-terminal domain and the C-terminal domain in pHC-null and generated transgenic silkworms that produced a modified H-chain, HC-EGFP. Transgenic silkworms produced HC-EGFP in the posterior part of silk gland cells, secreted it into the lumen of the gland, and produced a cocoon with HC-EGFP as part of the fibroin proteins. N-terminal sequencing of HC-EGFP localized the signal sequence cleavage site to between positions A((21)) and N((22)). These results indicate that our new plasmid successfully produced the modified H-chain in a transgenic silkworm.     

Metastatic Recurrence in a Pancreatic Cancer Patient Derived Orthotopic Xenograft (PDOX) Nude Mouse Model Is Inhibited by Neoadjuvant Chemotherapy in Combination with Fluorescence-Guided Surgery with an Anti-CA 19-9-Conjugated Fluorophore

The aim of this study is to determine the efficacy of neoadjuvant chemotherapy (NAC) with gemcitabine (GEM) in combination with fluorescence-guided surgery (FGS) on a pancreatic cancer patient derived orthotopic xenograft (PDOX) model. A PDOX model was established from a CA19-9-positive, CEA-negative tumor from a patient who had undergone a pancreaticoduodenectomy for pancreatic adenocarcinoma. Mice were randomized to 4 groups: bright light surgery (BLS) only; BLS+NAC; FGS only; and FGS+NAC. An anti-CA19-9 or anti-CEA antibody conjugated to DyLight 650 was administered intravenously via the tail vein of mice with the pancreatic cancer PDOX 24 hours before surgery. The PDOX was brightly labeled with fluorophore-conjugated anti-CA19-9, but not with a fluorophore-conjugated anti-CEA antibody. FGS was performed using the fluorophore-conjugated anti-CA19-9 antibody. FGS had no benefit over BLS to prevent metastatic recurrence. NAC in combination with BLS did not convey an advantage over BLS to prevent metastatic recurrence. However, FGS+NAC significantly reduced the metastatic recurrence frequency to one of 8 mice, compared to FGS only after which metastasis recurred in 6 out of 8 mice, and BLS+NAC with metastatic recurrence in 7 out of 8 mice (p = 0.041). Thus NAC in combination with FGS can reduce or even eliminate metastatic recurrence of pancreatic cancer sensitive to NAC. The present study further emphasizes the power of the PDOX model which enables metastasis to occur and thereby identify the efficacy of NAC in combination with FGS on metastatic recurrence.     

Effective repetitive dystrophin gene transfer into skeletal muscle of adult mdx mice using a helper-dependent adenovirus vector expressing the coxsackievirus and adenovirus receptor (CAR) and dystrophin

BACKGROUND: The helper-dependent adenovirus (HDAd) vector is less immunogenic and has a larger cloning capacity of up to 37 kb enough to carry the full-length dystrophin cDNA. However, high and long-term expression of dystrophin transduced to mature muscle still remains difficult. One of the main reasons for this is that the expression of the coxsackievirus and adenovirus receptor (CAR) is very low in mature muscle. METHODS: We have constructed two different HDAd vectors. One contains the LacZ and the murine full-length dystrophin expression cassette (HDAdLacZ-dys), and the other is a new, improved vector containing the CAR and the dystrophin expression cassette (HDAdCAR-dys). RESULTS: We initially demonstrated high dystrophin expression and prevention of the dystrophic pathology in mdx muscle injected during the neonatal phase with HDAdLacZ-dys. Furthermore, we demonstrated that repeated injections of HDAdCAR-dys into mature muscle led to approximately nine times greater dystrophin-positive fibers in number than a single injection, thereby recovering the expression of dystrophin-associated proteins. This data has also shown that HDAdCAR-dys enabled administration of adenovirus (Ad) vector to the host with pre-existing immunity to the same serotype of Ad. CONCLUSIONS: Repetitive injections of the HDAd vector containing the CAR and the dystrophin expression cassette could improve the efficiency of subsequent dystrophin gene transfer to mature mdx muscle. This result suggests that our new HDAd vector will provide a novel gene therapy strategy for Duchenne muscular dystrophy, raising the prospects for gene therapy of other hereditary myopathies.     

Role of isozyme group-specific sequence 4 in the isozyme-specific properties of human aldolase C

To assess which regions of the aldolase C molecule are required for exhibiting isozyme-specific kinetic properties, we have constructed nine chimeric enzymes of human aldolases A and C. Kinetic studies of these chimeric enzymes revealed that aldolase C absolutely required its own isozyme group-specific sequences (IGS), particularly IGS-4, for exhibiting the characteristics of aldolase C which differ significantly from those of isozymes A and B (Kusakabe T, Motoki K, Hori K. Human aldolase C: characterization of the recombinant enzyme expressed in Escherichia coli. J Biochem (Tokyo) 1994;115:1172–7). Whereas human aldolases A and B required their own isozyme group-specific sequences-1 and -4 (IGS-1 and -4) as the main determinants of isozyme-specific kinetic properties (Motoki K, Kitajima Y, Hori K. Isozyme-specific modules on human aldolase A molecule. J Biol Chem 1993;268:1677–83; Kusakabe T, Motoki K, Sugimoto Y, Takasaki Y, Hori K. Human aldolase B: liver-specific properties of the isoenzyme depend on type B isozyme group-specific sequence. Prot. Eng. 1994;7:1387–93), the present studies indicate that the IGS-1 is principally substitutable between aldolases A and C. The kinetic data also suggests that the connector-2 (amino acid residues 243–306) may modulate the interaction of IGS units with the α/β barrel of the aldolase molecule.     

Regulation of lipocalin-type prostaglandin D synthase gene expression by Hes-1 through E-box and interleukin-1 beta via two NF-kappa B elements in rat leptomeningeal cells

The promoter function of the rat lipocalin-type prostaglandin D synthase (L-PGDS) gene was characterized in primary cultures of leptomeningeal cells prepared from the neonatal rat brain. Luciferase reporter assays with deletion and site-directed mutation of the promoter region (-1250 to +77) showed that an AP-2 element at -109 was required for activation and an E-box at +57, for repression. Binding of nuclear factors to each of these cis-elements was demonstrated by an electrophoretic mobility shift assay. Several components of the Notch-Hes signaling pathway, Jagged, Notch1, Notch3, and Hes-1, were expressed in the leptomeningeal cells. Human Hes-1 co-expressed in the leptomeningeal cells bound to the E-box of the rat L-PGDS gene, and repressed the promoter activity of the rat L-PGDS gene in a dose-dependent manner. The L-PGDS gene expression was up-regulated slowly by interleukin-1 beta to the maximum level at 24 h. The reporter assay with deletion and mutation revealed that two NF-kappa B elements at -1106 and -291 were essential for this up-regulation. Binding of two NF-kappa B subunits, p65 and c-Rel, to these two NF-kappa B elements occurred after the interleukin-1 beta treatment. Therefore, the L-PGDS gene is the first gene identified as the target for the Notch-Hes signal through the E-box among a variety of genes involved in the prostanoid biosynthesis, classified to the lipocalin family, and expressed in the leptomeninges. Moreover, the L-PGDS gene is a unique gene that is activated slowly by the NF-kappa B system.     

Studies on Lipase in Rat Brain

Lipase activity was measured in homogenates of rat cerebral hemispheres using radioactive glycerol trioleate emulsified with Triton X-100 as substrate. The labeled oleic acid was separated from the ester with a methanol-chloroform-heptane mixture. Under these assay conditions, the activity showed pH optima at about 5.5 and 7.5. The final products of these lipase activities were suggested to be free fatty acid and glycerol.     

Successful tumor eradication was achieved by collaboration of augmented cytotoxic activity and anti-angiogenic effects following therapeutic vaccines containing helper-activating analog-loaded dendritic cells and tumor antigen DNA

We reported previously that pigeon cytochrome c-derived peptides (Pan-IA), which bind broad ranges of MHC class II molecules efficiently, activate T helper (Th) function in mice. In an experimental model, Pan-IA DNA vaccines augmented antitumor immunity in tumor antigen-immunized mice. To elicit more potent antitumor immunity and to eradicate tumors in a therapeutic setting, Pan-IA-loaded dendritic cells (DCs) were inoculated in combination with vaccines including ovalbumin (OVA) antigen DNA in tumor-bearing mice. Seventy percent of the immunized mice survived tumor-free for at least 4 months after treatment. In contrast, mice vaccinated with OVA DNA, either with or without naïve DCs, did not eliminate the tumors and died within 5 weeks. Only in mice vaccinated with OVA DNA and Pan-IA-loaded DCs were both cytotoxic and helper responses specific for OVA induced at the spleen and tumor sites as well as at the vaccination sites. Furthermore, accumulation of OVA-specific CD4⁺ and CD8⁺ T lymphocytes and interferon-gamma-mediated anti-angiogenesis were observed in the tumors of these mice. Thus, the combined vaccination primed both tumor-specific cytotoxicity and helper immunity resulting in augmented tumor lysis ability and anti-angiogenic effects. This is the first report to show that most established tumors were successfully eradicated by collaboration of potent antitumor immunity and anti-angiogenic effects by vaccination with tumor antigens and helper-activating analogs. This novel vaccination strategy is broadly applicable, regardless of identifying helper epitopes in target molecules, and contributes to the development of therapeutic cancer vaccines.