Masanari Kitagawa

Proteomic studies of an Antarctic cold-adapted bacterium, Shewanella livingstonensis Ac10, for global identification of cold-inducible proteins.

Proteomic analysis of a cold-adapted bacterium, Shewanella livingstonensis Ac10, isolated from Antarctic seawater was carried out to elucidate its cold-adaptation mechanism. The cells were grown at 4°C and 18°C, and soluble and membrane proteins were analyzed by two-dimensional gel electrophoresis. At 4°C, the relative abundance of 47 soluble proteins and five membrane proteins increased more than twofold, and these proteins were analyzed by peptide mass fingerprinting. Twenty-six soluble proteins and two membrane proteins were identified. These included proteins involved in RNA synthesis and folding (RpoA, GreA, and CspA), protein synthesis and folding (TufB, Efp, LysU, and Tig), membrane transport (OmpA and OmpC), and motility (FlgE and FlgL). Cold-inducible RpoA, GreA, and CspA may be required for efficient and accurate transcription and proper folding of RNA at low temperatures, where base pairing of nucleic acids is stable and undesired secondary structures of RNA tend to form. Tig is supposed to have peptidyl-prolyl cis–trans isomerase activity and facilitate proper folding of proteins at low temperatures. The cold induction of OmpA and OmpC is likely to counteract the low diffusion rate of solutes at low temperatures and enables the efficient uptake of nutrients. These results provided many clues to understand microbial cold-adaptation mechanisms.

Construction of a Low-Temperature Protein Expression System Using a Cold-Adapted Bacterium, Shewanella sp. Strain Ac10, as the Host

ABSTRACT A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4°C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce β-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4°C and 139 mg/liter of culture at 18°C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium, Desulfotalea psychrophila DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system.

Nucleotide Sequence of the Arginine Deiminase Gene of Mycoplasma hominis

The arginine deiminase gene of Mycoplasma hominis was amplified by the polymerase chain reaction, and its entire nucleotide sequence was determined. This gene consists of 1227 base pairs encoding 409 amino acids, and has 35.2% guanine plus cytosine content. Nucleotide sequence homologies of the arginine deiminase gene between M. hominis and M. arginini, and between M. hominis and M. orale were 82.1 and 80.8%, respectively, suggesting that this gene is highly conserved among arginine‐utilizing Mycoplasma species.

Abstract 3137: A novel expansion method for functional natural killer cells and its clinical application

Background: Natural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment of cancer. But, it is difficult to generate the sufficient scale and purity of NK cells, and reliable methods to produce large number of functional NK cells have not been established yet. We have developed novel clinical-grade NK cells expansion method to produce the high purity, large scale and functional NK cells using a combination of recombinant human fibronectin fragment (RetroNectin®) -induced T-cells (RN-T cells), OK-432 and IL-2. We subsequently conducted a Phase 1 clinical study to evaluate the safety and efficacy of this NK cell therapy. In this paper, we address the characteristics of the NK cells elicited by this method and the results of immune monitoring in the clinical trial. Methods: To confirm the significance of RN-T cells as stimulator, we compared the stimulation effects on NK cells between RN-T cells and aCD3-T cells, which stimulated by anti-CD3 mAb only. Next, we analyzed expanded NK cells from PBMCs obtained from 31 cancer patients to verify the efficacy of this method. In the Phase 1 trial, patients with unresectable digestive cancer were enrolled. They received weekly intravenous administration of autologous NK cells elicited by the novel method three times to assess the safety of the number of adoptive cells at 0.5 × 109(cohort1), 1 × 109(cohort 2) and 2 × 109 cells (cohort 3) per dose. The phenotype and cytotoxicity of expanded NK cells were analyzed. For immune monitoring, cytotoxicity of PBMCs and whole blood cytokine levels were examined following NK cell infusion. Results: The stimulation by RN-T cells could induce preferable NK cell proliferation rather than the one by aCD3-T. As results of 31 cancer patients, 688±76-fold expansion was achieved in this system with PBMCs, and the NK cell populations were highly purified (84.7±3.6%) and highly expressed functional markers such as NKG2D (97.3±0.6%) and CD16 (96.8±0.7%). In the Phase 1 clinical trial, no NK cell infusion related severe or unexpected toxicities were observed. The response rate and the disease control rate in 10 per protocol patients were 0% and 50.0%, respectively. Although no clinical responses were observed, the cytotoxicity of PBMCs against K-562 targets increased in most patients (80%). The average of cytotoxic activity of PBMCs increased more than two times after the NK cell infusion. Conclusion: Although no patients receiving the NK cell therapy alone experienced a clinical response, adoptive immunotherapy of the NK cells elicited by the novel method is expected to exert considerable ADCC activity in vivo because of their high expression of CD16. Now, we are conducting clinical trial in which we explore the combination of this novel NK therapy with IgG1 antibodies such as trastuzumab and cetuximab. We also address the ongoing clinical trial in this paper. Citation Format: Takeshi Ishikawa, Naoyuki Sakamoto, Tetsuya Okayama, Kaname Oka, Satoshi Kokura, Mitsuko Ideno, Akiko Kato, Tatsuji Enoki, Masanari Kitagawa, Junichi Mineno, Tomoyo Yasuda, Toshifumi Doi, Yuji Naito, Yoshito Itoh, Toshikazu Yoshikawa. A novel expansion method for functional natural killer cells and its clinical application. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3137. doi:10.1158/1538-7445.AM2015-3137