Anti-tumor effect of a recombinant Bifidobacterium strain secreting a claudin-targeting molecule in a mouse breast cancer model.

Bifidobacterium is a nonpathogenic strain of anaerobic bacteria that selectively localizes and proliferates in tumors. It has emerged as a specific carrier of anticancer proteins against malignant tumors. Claudins are tetraspanin transmembrane proteins that form tight junctions. Claudin-4 is overexpressed in certain epithelial malignant cancers. The C-terminal fragment of the Clostridium perfringens enterotoxin (C-CPE), an exotoxin without the cytotoxic domain, strongly binds to claudin-4. The C-CPE fusion toxin (C-CPE-PE23), which targets claudin-4, strongly suppresses tumor growth; however, C-CPE fusion toxins exhibit hepatic toxicity. In this study, we successfully generated a strain of Bifidobacterium longum that secreted C-CPE-PE23 (B. longum-C-CPE-PE23) and was specific to and cross reactive with human and mouse claudin-4. We evaluated the therapeutic potential of this strain against triple-negative breast cancer using a mouse model. C-CPE-PE23 decreased cell viability in a dose-dependent manner in human and mouse breast cancer cell lines. After intravenous injection, Bifidobacterium was specifically distributed in the tumors of mice bearing breast cancer tumors. Moreover, B. longum-C-CPE-PE23 significantly suppressed tumor growth in mice with breast cancer without serious side effects, such as weight loss or hepatic and renal damage. We suggest that B. longum-C-CPE-PE23 is a good candidate for breast cancer treatment. Bifidobacterium could also be used as a drug delivery system for hepatotoxic agents.

Toxicity of Gold Nanoparticles in Mice due to Nanoparticle/Drug Interaction Induces Acute Kidney Damage.

Nanomaterials are innovative materials with many useful properties, but there is concern regarding their many unknown effects on living organisms. Gold nanoparticles are widely used as industrial materials because of their excellent properties. The potential biological hazards of gold nanoparticles are unknown, and thus, here we examined the in vivo effects of gold nanoparticles 10, 50, and 100 nm in diameter (GnP10, GnP50, and GnP100, respectively) and their interactions with drugs in mice to clarify their safety in mammals. Cisplatin, paraquat, and 5-aminosalicylic acid cause side-effect damage to the liver and kidney in mice. No hepatotoxicity or nephrotoxicity was observed when any of the gold nanoparticles alone were administered via the tail vein. In contrast, co-administration of GnP-10 with cisplatin, paraquat, or 5-aminosalicylic acid caused side-effect damage to the kidney. This suggests that gold nanoparticles with a particle size of 10 nm are potentially nephrotoxic due to their interaction with drugs.

[Development of an Anticancer Therapy Using Recombinant Bifidobacterium as a New Drug Delivery System (DDS)].

　Intravenously administered obligate anaerobic bacteria, such as bifidobacteria, grow specifically in tumor tissues. This specificity is attributed to the following: (1) Vascular walls in tumor tissues have nanometer- to micrometer-wide cracks, which allow the bacteria to pass through; (2) the intratumoral environment is hypoxic, due to poor vascularization, and therefore bifidobacteria can survive and proliferate in this anaerobic environment; (3) bifidobacteria cannot survive in well-oxygenated normal tissues. Moreover, unlike gram-negative bacteria, the gram-positive bifidobacteria do not produce endotoxins; therefore, there is no risk of endotoxin shock associated with their intravenous administration. Recently, the utility of bifidobacteria for specific drug delivery to tumor tissues has been highlighted. We have established a novel anti-cancer drug-delivery system using Bifidobacterium longum for the specific release of anti-tumor antibodies (e.g., antibody-drug complexes or single-chain antibodies) to targeted tumor tissues. Here, we introduce the results of our investigation.

Hepatotoxicity and Drug/Chemical Interaction Toxicity of Nanoclay Particles in Mice.

Nanomaterials are relatively new and unconventional materials with many useful properties, but their effects on biological systems are poorly understood. Nanoclay is a general term for layered mineral silicate nanoparticles that are ideally suited for use in clay-based nanocomposites. The potential biological hazards of nanoclays have not been addressed, however. Therefore, we investigated the in vivo effects and drug interactions of nanoclays. In mice, administration of nanoclay particles via the tail vein led to acute liver injury. Co-administration of nanoclay and carbon tetrachloride, paraquat, or cisplatin resulted in both liver and kidney injury. Our findings thus indicate that nanoclay particles are potentially hepato- and nephrotoxic.

Potential role of voltage-sensing phosphatases in regulation of cell structure through the production of PI(3,4)P2.

Voltage-sensing phosphatase, VSP, consists of the transmembrane domain, operating as the voltage sensor, and the cytoplasmic domain with phosphoinositide-phosphatase activities. The voltage sensor tightly couples with the cytoplasmic phosphatase and membrane depolarization induces dephosphorylation of several species of phosphoinositides. VSP gene is conserved from urochordate to human. There are some diversities among VSP ortholog proteins; range of voltage of voltage sensor motions as well as substrate selectivity. In contrast with recent understandings of biophysical mechanisms of VSPs, little is known about its physiological roles. Here we report that chick ortholog of VSP (designated as Gg-VSP) induces morphological feature of cell process outgrowths with round cell body in DF-1 fibroblasts upon its forced expression. Expression of the voltage sensor mutant, Gg-VSPR153Q with shifted voltage dependence to a lower voltage led to more frequent changes of cell morphology than the wild-type protein. Coexpression of PTEN that dephosphorylates PI(3,4)P2 suppressed this effect by Gg-VSP, indicating that the increase of PI(3,4)P2 leads to changes of cell shape. In addition, visualization of PI(3,4)P2 with the fluorescent protein fused with the TAPP1-derived pleckstrin homology (PH) domain suggested that Gg-VSP influenced the distribution of PI(3,4)P2 . These findings raise a possibility that one of the VSP's functions could be to regulate cell morphology through voltage-sensitive tuning of phosphoinositide profile.

The composition of volatile aroma components, flavanones, and polymethoxylated flavones in Shiikuwasha (Citrus depressa Hayata) peels of different cultivation lines.

Citrus peels are important sources of various pleasant aroma compounds and valuable bioactive substances. To investigate differences in the composition and content of Shiikuwasha (Citrus depressa Hayata) peels from different cultivation lines, the composition of volatile aroma components, flavanones, and polymethoxylated flavones (PMFs) in four Shiikuwasha cultivation lines was examined. The composition of volatile aroma components in cold-pressed extracts of Shiikuwasha peels was analyzed using gas chromatography-flame ionization detection and gas chromatography-mass spectrophotometry. The extracts contained mainly monoterpene hydrocarbons (93.40-97.25%), including limonene (46.52-68.26%) and γ-terpinene (21.48-30.52%). Differences in the composition of volatile aroma compounds in the Shiikuwasha cultivation lines were revealed using principal component analysis. Additionally, the composition of flavanones and PMFs was determined using high-performance liquid chromatography methods. Neohesperidin (96.58%) was the predominant flavanone in 'Izumi kugani' peel, while the other peels had high hesperidin contents (89.26-98.66%). Moreover, the PMFs of Shiikuwasha peels were composed of nobiletin (56.74-64.77%) and tangeretin (23.17-34.70%).

Volatile aroma components and antioxidant activities of the flavedo peel extract of unripe Shiikuwasha (Citrus depressa Hayata).

UNLABELLED: The flavedo peel extracts of unripe Shiikuwasha (Citrus depressa Hayata) fruits were extracted using steam distillation (SD) or a cold-press (CP) system. Volatile aroma content and composition were determined using gas chromatography (GC) and each compound was identified using gas chromatography-mass spectrophotometry (GC-MS). The major constituents of the extracts were monoterpene hydrocarbons (91.75-93.75%[709.32-809.05 mg/100 g of fresh flavedo peel]) including limonene (43.08-45.13%[341.46-379.81 mg/100 g of fresh flavedo peel]), γ-terpinene (27.88-29.06%[219.90-245.86 mg/100 g of fresh flavedo peel]), and p-cymene (8.13-11.02%[61.47-97.22 mg/100 g of fresh flavedo peel]). The extraction process used was determined to be a decisive factor that affects the composition of key citrus aroma components, as well as the antioxidant activities of the Shiikuwasha fruit. Antioxidant capabilities of the extracts were examined by assay of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity and ß-carotene bleaching inhibition. The cold-press extraction system may better retain the total phenolic content of the flavedo peel and display superior antioxidant activities, compared to the steam distillation extraction method. PRACTICAL APPLICATION: Shiikuwasha (Citrus depressa Hayata) is a type of small citrus fruit, and has been used as raw material for beverage and food additive productions in Japan. It had a unique aroma composition in which the limonene content of its peels is lower than that of other commonly known citrus peels. The present study detailed the volatile aroma composition, as well as antioxidant capabilities of Shiikuwasha peel extracts of different extraction methods, that are cold-press and steam distillation methods. The results of this study may provide a basis for selection of Shiikuwasha peel extracts in food industry for citrus flavor production.

Bioluminescence imaging of c-fos gene expression accompanying filial imprinting in the newly hatched chick brain.

Bioluminescence imaging is a powerful tool for examining gene expression in living animals. Previously, we reported that exogenous DNA could be successfully delivered into neurons in the newly hatched chick brain using electroporation. Here, we show the in vivo bioluminescence imaging of c-fos promoter activity and its upregulation, which is associated with filial imprinting. The upregulation of c-fos gene expression correlated with both the strength of the chicks' approach activity to the training object and the acquisition of memory. The present technique should be a powerful tool for analyzing the time changes in neural activity of certain brain areas in real-time during memory formation, using brains of living animals.

Suppression of the ecdysteroid-triggered growth arrest by a novel Drosophila membrane steroid binding protein.

Ecdysteroid is a crucial steroid hormone in insects, especially during metamorphosis. Here, we show that the Drosophila membrane steroid binding protein (Dm_MSBP) is a novel structural homolog of the vertebrate membrane-bound receptor component for progesterone. Dm_MSBP exhibited binding affinity to ecdysone when expressed on the cell surface of Drosophila S2 cells. In S2 cells, the stable overexpression of Dm_MSBP suppressed the growth arrest triggered by 20-hydroxyecdysone and prevented the temporal activation of extracellular signal-regulated kinase proteins. These results suggest that Dm_MSBP is a membranous suppressor to ecdysteroid and blocks the signaling by binding it in extracellular fluid.

Gene expression profile in cerebrum in the filial imprinting of domestic chicks (Gallus gallus domesticus).

In newly hatched chicks, gene expression in the brain has previously been shown to be up-regulated following filial imprinting. By applying cDNA microarrays containing 13,007 expressed sequence tags, we examined the comprehensive gene expression profiling of the intermediate medial mesopallium in the chick cerebrum, which has been shown to play a key role in filial imprinting. We found 52 up-regulated genes and 6 down-regulated genes of at least 2.0-fold changes 3h after the training of filial imprinting, compared to the gene expression of the dark-reared chick brain. The up-regulated genes are known to be involved in a variety of pathways, including signal transduction, cytoskeletal organization, nuclear function, cell metabolism, RNA binding, endoplasmic reticulum or Golgi function, synaptic function, ion channel, and transporter. In contrast, fewer genes were down-regulated in the imprinting, coinciding with the previous data that the total RNA synthesis increased associated with filial imprinting. Our data suggests that the filial imprinting involves the modulation of multiple signaling pathways.

Up-regulation of microtubule-associated protein 2 accompanying the filial imprinting of domestic chicks (Gallus gallus domesticus).

Using cDNA microarrays, we have identified elsewhere the genes of microtubule-associated proteins as a group up-regulated in newly hatched chick brains after filial imprinting training. Here we show by in situ hybridization that the mRNA for the microtubule-associated protein 2 (MAP2) gene was enriched in the mesopallium and the hippocampus in the trained chick brain. The regionally specific enrichments of MAP2 mRNA were not observed in the brain of dark-reared or light-exposed chick as controls, implying an association between the degree of expression and the strength of the learned preference. In agreement with the gene expression, MAP2 protein was accumulated in the mesopallium of the trained chick brain, but not in the brains of the controls. The accumulation of MAP2 was found in the cytosol of neurons and co-localized with beta-tubulin, suggesting a change in microtubule assembly. Our results suggest a postnatal reorganization of cytoskeleton following filial imprinting.

The extracellular adenosine deaminase growth factor, ADGF/CECR1, plays a role in Xenopus embryogenesis via the adenosine/P1 receptor.

Adenosine deaminase-related growth factors (ADGF), also known as CECR1 in vertebrates, are a novel family of growth factors with sequence similarity to classical cellular adenosine deaminase. Although genes for ADGF/CECR1 have been identified in both invertebrates as well as vertebrates, their in vivo functions in vertebrates remain unknown. We isolated cDNA clones for two cerc 1s from Xenopus laevis. Both recombinant Xenopus CECR1s exhibited adenosine deaminase and growth factor activity, and the adenosine deaminase activity was found to be indispensable for growth factor activity. The Xenopus cerc 1s are expressed in the somites, pronephros, eyes, cement gland, neural tube, and neural floor plate of the embryos. Knock-down of these two genes using morpholino oligonucleotides caused a reduction in the body size and abnormalities of the body axis in the Xenopus embryos, accompanied by selective changes in the expression of developmental marker genes. Injection of adenosine, agonists for adenosine/P1 receptors, or adenosine deaminase inhibitor into late gastrula archenteron embryos resulted in developmental defects similar to those caused by morpholino oligonucleotide injection. These results show, for the first time, the involvement of CECR1s via the adenosine/P1 receptors in vertebrate embryogenesis via regulation of extracellular adenosine concentrations.

In-vivo gene transfer into newly hatched chick brain by electroporation.

Newly hatched domestic chicks serve as ideal models for studies of the neural basis of behavioral plasticity, particularly for understanding the mechanisms of learning such as filial imprinting. To elucidate the molecular basis and gene functions involved in learning, we developed an in-vivo gene-transfer system in the brain of a living chick using electroporation. When green fluorescent protein-encoding plasmids were transfected to a chick brain, green fluorescence was clearly observed, and expression at the protein level was confirmed by immunoblotting. Most of the transfected brain cells were neuronal cells with dendrites. This neuron-selective electroporation system will facilitate the analysis of gene functions in the living chick brain and provide further clues as to the molecular mechanisms of avian learning.

Involvement of insect-derived growth factor (IDGF) in the cell growth of an embryonic cell line of flesh fly.

Insect-derived growth factor (IDGF) is the first adenosine deaminase-related growth factor (ADGF) purified from the conditioned medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly). Here we show the requirement of IDGF for the growth of NIH-Sape-4 cells. Growth factor activity was abolished by adsorption of IDGF from the conditioned medium of NIH-Sape-4 cells. In addition, knockdown of IDGF gene expression by RNA interference (RNAi) significantly reduced IDGF secretion from the cells following cell growth inhibition. The IDGF gene was strongly expressed in the hemocytes, and IDGF increased the viability of the larval hemocytes. These data provide evidence that IDGF is required for the growth of NIH-Sape-4 cells and possibly for hemocyte viability.

ATP-dependent rotation of mutant ATP synthases defective in proton transport.

During ATP hydrolysis, the gammaepsilon c10 complex (gamma and epsilon subunits and a c subunit ring formed from 10 monomers) of F0F1 ATPase (ATP synthase) rotates relative to the alpha3beta3delta ab2 complex, leading to proton transport through the interface between the a subunit and the c subunit ring. In this study, we replaced the two pertinent residues for proton transport, cAsp-61 and aArg-210 of the c and a subunits, respectively. The mutant enzymes exhibited lower ATPase activities than that of the wild type but exhibited ATP-dependent rotation in planar membranes, in which their original assemblies are maintained. The mutant enzymes were defective in proton transport, as shown previously. These results suggest that proton transport can be separated from rotation in ATP hydrolysis, although rotation ensures continuous proton transport by bringing the cAsp-61 and aArg-210 residues into the correct interacting positions.