Yoshinori Katakura

DETECTION OF MAGNETIC NANOPARTICLES WITH SUPERCONDUCTING QUANTUM INTERFERENCE DEVICE (SQUID) MAGNETOMETER AND APPLICATION TO IMMUNOASSAYS

A system is developed to magnetically measure biological antigen-antibody reactions with a superconducting quantum interference device (SQUID) magnetometer. In this system, antibodies are labeled with magnetic nanoparticles of γ-Fe2O3, and the antigen-antibody reactions are measured by detecting the magnetic field from the magnetic nanoparticles. A setup of the system is described, and the sensitivity of the system is studied in terms of detectable weight of nanoparticles. Magnetic particles as small as 600 pg can be detected at present. An experiment is also conducted to measure antigen-antibody reaction with the present system. It is shown that the sensitivity of the present system is better than that of the conventional optical method. A one order of magnitude improvement of sensitivity will be realized by the sophistication of the present system.

Enzyme-digested Fucoidan Extracts Derived from Seaweed Mozuku of Cladosiphon novae-caledoniae kylin Inhibit Invasion and Angiogenesis of Tumor Cells.

Fucoidan is a uniquely-structured sulfated polysaccharide found in the cell walls of several types of brown seaweed that has recently, especially as enzyme-digested fucoidan extract, attracted a lot attention due to its anti-tumor potential. In this study, we evaluated the effects of enzyme-digested fucoidan extracts prepared from seaweed Mozuku of Cladosiphon novae-caledoniae kylin on in vitro invasion and angiogenesis abilities of human tumor cells. First, we evaluated the effect of the fucoidan extracts on oxidative stress of tumor cells, and demonstrated that intracellular H2O2 level and released H2O2 from tumor cells were both greatly repressed upon the treatment with the fucoidan extracts, suggesting that fucoidan extracts ameliorate oxidative stress of tumor cells. Next, we tested for the effects of fucoidan extracts on invasion ability of human fibrosarcoma HT1080 cells, showing that fucoidan extracts significantly inhibit their invasion, possibly via suppressing matrix metalloproteinases (MMPs) MMP-2/9 activities. Further, we investigated the effects of the fucoidan extracts on angiogenesis of human uterine carcinoma HeLa cells, and found that fucoidan extracts suppressed expression and secretion of an angiogenesis factor vascular endothelial growth factor (VEGF), resulting in suppressed vascular tubules formation of tumor cells. The results taken together clarified that enzyme-digested fucoidan extracts from Cladosiphon novae-caledoniae kylin possess inhibitory effects on invasion and angiogenesis of tumor cells. These effects might, at least partially, be elicited by the antioxidative potential of enzyme digested fucoidan extracts.

Endostatin inhibits lymph node metastasis by a down-regulation of the vascular endothelial growth factor C expression in tumor cells.

Anti-angiogenic therapy is a newly developed treatment method for malignant tumors. Endostatin has an anti-angiogenetic effect. Endostatin has also been shown to block the growth and metastasis of various cancers through the vascular system. However, there have so far been few reports on the relationship between endostatin and lymph node metastasis. In this study, we investigated the relationship between endostatin and the inhibition of lymph node metastasis. We first made recombinant adenovirus which expressed endostatin gene (Ad-end), and then performed the following experiments. Our findings showed Ad-end to inhibit the proliferation and tube formation of endothelial cells in vitro. In addition, Ad-end inhibited the growth of a human oral squamous cell carcinoma cell line (SQUU-B) implanted subcutaneously in the right flank of nude mice and orthotopically in the tongue of nude mice, and Ad-end also inhibited lymph node metastasis in orthotopic implantation. The number of CD31-positive blood vessels and 5’-nase-positive lymphatic vessels around Ad-end-infected tumors in tongue lesions was significantly lower than that in the control group. The down-regulation of vascular endothelial growth factor C (VEGF-C) in Ad-end-infected SQUU-B cells was recognized by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. These findings suggested that endostatin inhibited lymphangiogenesis and lymph node metastasis by suppressing the production of VEGF-C in tumor cells.

Protective mechanism of reduced water against alloxan-induced pancreatic β-cell damage: Scavenging effect against reactive oxygen species

Reactive oxygen species (ROS) cause irreversible damage to biological macromolecules, resulting in many diseases. Reduced water (RW) such as hydrogen-rich electrolyzed reduced water and natural reduced waters like Hita Tenryosui water in Japan and Nordenau water in Germany that are known to improve various diseases, could protect a hamster pancreatic β cell line, HIT-T15 from alloxan-induced cell damage. Alloxan, a diabetogenic compound, is used to induce type 1 diabetes mellitus in animals. Its diabetogenic effect is exerted via the production of ROS. Alloxan-treated HIT-T15 cells exhibited lowered viability, increased intracellular ROS levels, elevated cytosolic free Ca2+ concentration, DNA fragmentation, decreased intracellular ATP levels and lowering of glucose-stimulated release of insulin. RW completely prevented the generation of alloxan-induced ROS, increase of cytosolic Ca2+ concentration, decrease of intracellular ATP level, and lowering of glucose-stimulated insulin release, and strongly blocked DNA fragmentation, partially suppressing the lowering of viability of alloxan-treated cells. Intracellular ATP levels and glucose-stimulated insulin secretion were increased by RW to 2–3.5 times and 2–4 times, respectively, suggesting that RW enhances the glucose-sensitivity and glucose response of β-cells. The protective activity of RW was stable at 4 °C for over a month, but was lost by autoclaving. These results suggest that RW protects pancreatic β-cells from alloxan-induced cell damage by preventing alloxan-derived ROS generation. RW may be useful in preventing alloxan-induced type 1-diabetes mellitus.

Tryptophan-19 of β-lactoglobulin, the only residue completely conserved in the lipocalin superfamily, is not essential for binding retinol, but relevant to stabilizing bound retinol and maintaining its structure

Residue 19 of tryptophan in bovine beta-lactoglobulin (beta-LG) is the only invariant residue throughout the lipocalin superfamily having two characteristic features: binding ability for small hydrophobic molecules and the unique beta-barrel three-dimensional structure. In this study, we investigated whether this strictly conserved Trp-19 of beta-LG would be indispensable for its structure and function such as maintaining the molecular structure and biological activity of beta-LG. Spectroscopic and enzymatic oxidation experiments on retinol bound to W19Y, in which Tyr was substituted for Trp-19, showed that Trp-19 was not critical for this binding, but was important for stably maintaining the environment surrounding retinol and the bound retinol. An using four anti-beta-LG monoclonal antibodies as probes, revealed a structural change in region 20-29, but not in the reverse region of Trp-19. A guanidine hydrochloride-induced unfolding study showed that the conformational stability of W19Y was greatly reduced by 6.9 kcal/mol compared to that of wild-type beta-LG. These facts indicated that Trp-19 is one of the important residues in correctly maintaining the local structure of beta-LG and stably retaining its overall structure, thereby conserving the bound retinol molecule.

SIRT1 prevents replicative senescence of normal human umbilical cord fibroblast through potentiating the transcription of human telomerase reverse transcriptase gene

SIRT1, the mammalian homolog of sirtuins, has emerged as a mediator of the beneficial effects of calorie restriction. Among them, we focused on the SIRT1-induced prevention of cellular senescence, and tried to reveal the molecular mechanisms that define the effects of SIRT1. Firstly in this study, we observed that overexpression of SIRT1 resulted in the prevention of cellular senescence of normal human umbilical cord fibroblast HUC-F2 cells. Here, we focused on the human telomerase reverse transcriptase (hTERT) gene as a target of the SIRT1-induced prevention of cellular senescence. Results showed that SIRT1, SIRT1 activator, resveratrol, and SIRT1 activating condition, starved condition, increased the transcription of hTERT in HUC-F2 cells. Next, we found that SIRT1 increased hTERT transcription in a c-MYC-dependent manner, triggered the transcription of the c-MYC gene and increased the amount of c-MYC recruited to the hTERT promoter. Further, SIRT1 increased the transcriptional activation ability of c-MYC and correspondingly increased the amount of acetylated H4 histone at the hTERT promoter. All of these results indicated that SIRT1 activates hTERT transcription through the involvement of c-MYC, and suggested that this SIRT1-induced augmentation of hTERT transcription resulted in the extension of the cellular life span of HUC-F2 cells.

TAK1 represses transcription of the human telomerase reverse transcriptase gene

In human cells, telomerase activity is tightly regulated by the expression of its catalytic subunit, namely, the human telomerase reverse transcriptase (hTERT). However, the molecular mechanisms involved in the regulation of hTERT expression have not been completely clarified. We have previously reported that transforming growth factor β (TGF-β) represses the expression of the hTERT gene. In the present study, we demonstrated that TGF-β-activated kinase 1 (TAK1), originally identified as a mitogen-activated kinase kinase kinase, represses the hTERT core promoter activity in an E-box-independent manner, and it also represses the transcription of the hTERT gene in human lung adenocarcinoma cell line, A549 cells. This TAK1-induced repression was found to be caused by the recruitment of histone deacetylase to Sp1 at the hTERT promoter and a consequent reduction in the amount of acetylated histone H4 at the hTERT promoter. Finally, we demonstrated that TAK1 induces cellular senescence programs in normal human diploid cells. Thus, we assume that TAK1 triggers the repression mechanisms of the hTERT gene as a result of evoking cellular senescence programs. Considered together, TAK1 is thought to play a causative role in the determination of a finite replicative lifespan of normal and cancer cells.

Immortalization by gene transfection.

Cultured cell lines that maintain specific differentiated phenotypes have been indispensable tools in cell biology. Progress in understanding the function of differentiated cells in vivo can be facilitated by creating cell lines via immortalizing gene transduction, if they retain the essential differentiated features of the same cells in vivo. Rodent cells immortalize spontaneously with a frequency of 10(-5) to 10(-6). Thus, it is easy to isolate immortal cells from rodent cell populations even without the transfer of immortalizing genes. Immortalizing genes can be used to increase this frequency to approximately 100%. In contrast, the spontaneous immortalization of human cells is a very rare event; the frequency is thought to be < 10(-12). Immortalizing genes can also be used to increase this frequency. Several genes that promise efficient immortalization of cultured cells have been identified. Immortalizing genes include simian virus 40 large T antigen, papillomaviruses E6 and E7, adenovirus E1A, Epstein-Barr virus, human T-cell leukemia virus, herpesvirus saimiri, oncogenes, and mutant p53 gene. Equally important, innovative means of gene delivery have been developed as well. These immortalizing genes, together with gene transfer methodologies, have provided the means to generate cell lines from cell types that are not abundant or are difficult to obtain in pure form in primary culture, are in short supply as human cells, and/or have brief lifetimes in culture. This chapter focuses primarily on the immortalization method by gene transfection. The chapter is not meant to be comprehensive, but rather to provide an account of the power and usefulness of immortalization methodology.

In vitro immunization of human peripheral blood lymphocytes: Establishment of B cell lines secreting IgM specific for cholera toxin B subunit from lymphocytes stimulated with IL-2 and IL-4

In vitro immunization (IVI) techniques have a great potential in the production of human monoclonal antibodies (MAbs) against various antigens. An IVI method of human peripheral blood lymphocytes (PBL) has been developed with a human lung adenocarcinoma cell line in our laboratory. Although several cancer specific human MAbs were successfully generated by using this IVI method, it was not available for soluble antigens, which prompted us to improve the method for generation of human MAbs against soluble antigens. IVI with soluble antigens was effectively caused by the addition of muramyl dipeptides, interleukin-2 and interleukin-4. It was found that the difference of sensitivity of lymphocytes depending upon donors could be overcome by finding the optimal concentrations of IL-2 and IL-4. IVI of human PBL was performed with cholera toxin B subunit (CTB) and the immunized B cells were transformed by Epstein-Barr virus. Anti-CTB antibody was detected using an indirect ELISA. B cells producing anti-CTB antibodies were directly cloned by a soft agar cloning method.

IL-10 augments antibody production in in Vitro immunized lymphocytes by inducing a Th2-type response and B cell maturation

An in vitro immunization (IVI) protocol enables antigen specific antibody production from L-Leucyl-L-Leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBL) upon antigen stimulation in the presence of IL-2, IL-4, and muramyl dipeptide. In the course of our studies, we have found that IL-10 added at the antigen sensitization significantly augmented antibody production level from the LLME-treated PBL. In the present study, we tried to demonstrate the role of IL-10 in the augmentation of antibody production in an IVI protocol by clarifying the cytokine expression profiles in CD4+ and CD8+ T cells. The results showed that IL-10 skewed the Th1/Th2 balance to Th2-type responses by suppressing Th1-type cytokine production and augmenting Th2-type cytokine production in CD4+ and CD8+ T cells, as well as in CD19+ B cells. Furthermore, IL-10 augmented the expression of CD38, an antigen marker of plasma cells, on B cells, which clearly indicates that IL-10 promoted differentiation and maturation of B cells in an IVI protocol. These results indicate that IL-10 plays an important role in setting the cellular milieu to produce antibodies in an IVI protocol.