Conferring the ability to utilize inorganic polyphosphate on ATP-specific NAD kinase

ATP特異性の獲得メカニズムの解明 : 新薬と新しい物質生産系の開発に期待. 京都大学プレスリリース. 2013-09-11.NAD kinase (NADK) is a crucial enzyme for production of NADP(+). ATP-specific NADK prefers ATP to inorganic polyphosphate [poly(P)] as a phosphoryl donor, whereas poly(P)/ATP-NADK utilizes both ATP and poly(P), and is employed in industrial mass production of NADP(+). Poly(P)/ATP-NADKs are distributed throughout Gram-positive bacteria and Archaea, whereas ATP-specific NADKs are found in Gram-negative α- and γ-proteobacteria and eukaryotes. In this study, we succeeded in conferring the ability to utilize poly(P) on γ-proteobacterial ATP-specific NADKs through a single amino-acid substitution; the substituted amino-acid residue is therefore important in determining the phosphoryl-donor specificity of γ-proteobacterial NADKs. We also demonstrate that a poly(P)/ATP-NADK created through this method is suitable for the poly(P)-dependent mass production of NADP(+). Moreover, based on our results, we provide insight into the evolution of bacterial NADKs, in particular, how NADKs evolved from poly(P)/ATP-NADKs into ATP-specific NADKs

Collagen-VI supplementation by cell transplantation improves muscle regeneration in Ullrich congenital muscular dystrophy model mice

6型コラーゲンを補う細胞移植がウルリッヒ型先天性筋ジストロフィーモデルマウスの病態を改善する. 京都大学プレスリリース. 2021-08-24.iPS cells show therapeutic benefits for a rare muscle dystrophy. 京都大学プレスリリース. 2021-08-24.[Background] Mesenchymal stromal cells (MSCs) function as supportive cells on skeletal muscle homeostasis through several secretory factors including type 6 collagen (COL6). Several mutations of COL6A1, 2, and 3 genes cause Ullrich congenital muscular dystrophy (UCMD). Skeletal muscle regeneration deficiency has been reported as a characteristic phenotype in muscle biopsy samples of human UCMD patients and UCMD model mice. However, little is known about the COL6-dependent mechanism for the occurrence and progression of the deficiency. The purpose of this study was to clarify the pathological mechanism of UCMD by supplementing COL6 through cell transplantation. [Methods] To test whether COL6 supplementation has a therapeutic effect for UCMD, in vivo and in vitro experiments were conducted using four types of MSCs: (1) healthy donors derived-primary MSCs (pMSCs), (2) MSCs derived from healthy donor induced pluripotent stem cell (iMSCs), (3) COL6-knockout iMSCs (COL6KO-iMSCs), and (4) UCMD patient-derived iMSCs (UCMD-iMSCs). [Results] All four MSC types could engraft for at least 12 weeks when transplanted into the tibialis anterior muscles of immunodeficient UCMD model (Col6a1KO) mice. COL6 protein was restored by the MSC transplantation if the MSCs were not COL6-deficient (types 1 and 2). Moreover, muscle regeneration and maturation in Col6a1KO mice were promoted with the transplantation of the COL6-producing MSCs only in the region supplemented with COL6. Skeletal muscle satellite cells derived from UCMD model mice (Col6a1KO-MuSCs) co-cultured with type 1 or 2 MSCs showed improved proliferation, differentiation, and maturation, whereas those co-cultured with type 3 or 4 MSCs did not. [Conclusions] These findings indicate that COL6 supplementation improves muscle regeneration and maturation in UCMD model mice

Fundamental physics activities with pulsed neutron at J-PARC(BL05)

"Neutron Optics and Physics (NOP/ BL05)" at MLF in J-PARC is a beamline for studies of fundamental physics. The beamline is divided into three branches so that different experiments can be performed in parallel. These beam branches are being used to develop a variety of new projects. We are developing an experimental project to measure the neutron lifetime with total uncertainty of 1 s (0.1%). The neutron lifetime is an important parameter in elementary particle and astrophysics. Thus far, the neutron lifetime has been measured by several groups; however, different values are obtained from different measurement methods. This experiment is using a method with different sources of systematic uncertainty than measurements conducted to date. We are also developing a source of pulsed ultra-cold neutrons (UCNs) produced from a Doppler shifter are available at the unpolarized beam branch. We are developing a time focusing device for UCNs, a so called "rebuncher", which can increase UCN density from a pulsed UCN source. At the low divergence beam branch, an experiment to search an unknown intermediate force with nanometer range is performed by measuring the angular dependence of neutron scattering by noble gases. Finally the beamline is also used for the research and development of optical elements and detectors. For example, a position sensitive neutron detector that uses emulsion to achieve sub-micrometer resolution is currently under development. We have succeeded in detecting cold and ultra-cold neutrons using the emulsion detector.Comment: 9 pages, 5 figures, Proceedings of International Conference on Neutron Optics (NOP2017

Structural and mutational analysis of amino acid residues involved in ATP specificity of Escherichia coli acetate kinase.

Acetate kinase (AK) generally utilizes ATP as a phosphoryl donor, but AK from Entamoeba histolytica (PPi-ehiAK) uses pyrophosphate (PPi), not ATP, and is PPi-specific. The determinants of the phosphoryl donor specificity are unknown. Here, we inferred 5 candidate amino acid residues associated with this specificity, based on structural information. Each candidate residue in Escherichia coli ATP-specific AK (ATP-ecoAK), which is unable to use PPi, was substituted with the respective PPi-ehiAK amino acid residue. Each variant ATP-ecoAK had an increased Km for ATP, indicating that the 5 residues are the determinants for the specificity to ATP in ATP-ecoAK. Moreover, Asn-337 of ATP-ecoAK was shown to be particularly significant for the specificity to ATP. The 5 residues are highly conserved in 2625 PPi-ehiAK homologs, implying that almost all organisms have ATP-dependent, rather than PPi-dependent, AK

Synthesis and properties of tetrathiafulvalenes bearing 6-aryl-1,4-dithiafulvenes

Novel multistage redox tetrathiafulvalenes (TTFs) bearing 6-aryl-1, 4-dithiafulvene moieties were synthesized by palladium-catalyzed direct C–H arylation. In the presence of a catalytic amount of Pd(OAc)2, P(t-Bu3)·HBF4, and an excess of Cs2CO3, the C–H arylation of TTF with several aryl bromides bearing 1, 3-dithiol-2-ylidenes took place efficiently to produce the corresponding π-conjugated molecules. We also succeeded in the estimation of the oxidation potentials and number of electrons involved in each oxidation step of the obtained compounds by digital simulations

Circatidal activity rhythm in the mangrove cricket Apteronemobius asahinai

Mangrove forests are influenced by tidal flooding and ebbing for a period of approximately 12.4 hours (tidal cycle). Mangrove crickets (Apteronemobius asahinai) forage on mangrove forest floors only during low tide. Under constant darkness, most crickets showed a clear bimodal daily pattern in their locomotor activity for at least 24 days; the active phases of approximately 10 hours alternated with inactive phases of approximately 2 hours, which coincided with the time of high tide in the field. The free-running period was 12.56±0.13 hours (mean±s.d., n=11). This endogenous rhythm was not entrained by the subsequent 24 hours light–dark cycle, although it was suppressed in the photophase; the active phase in the scotophase continued from the active phase in the previous constant darkness, with no phase shift. The endogenous rhythm was assumed to be a circatidal rhythm. On the other hand, the activity under constant darkness subsequent to a light–dark cycle was more intense in the active phase continuing from the scotophase than from the photophase of the preceding light–dark cycle; this indicates the presence of circadian components. These results suggest that two clock systems are involved in controlling locomotor activity in mangrove crickets

Development of the Multidisciplinary Educational Materials on Locally-Caught Fish "Skipjack Tuna"

We developed the multidisciplinary educational materials on locally-caught fish "Skipjack tuna" and worked with 13-hour unit focusing on fisheries industry. Skipjack tuna is one of the most important fish as a food and fishery resource in Kochi. We focused on biology, distribution, and cuisine of Skipjack tuna, and translated multidisciplinary complex knowledge into appropriate levels for our target audience (fifth grader). Major element of the class practices was the learning cycle, which articulated three sequential instructional phases: introduction, cooperative learning, and overview. In introduction, students work with the materials to ask questions on Skipjack tuna (called "Katsuo Quiz"). In cooperative learning, students work in small groups to help one another and write what they have learnt in "Hidensho". In overview, teacher presents the overview to students to promote understandings. After one cycle is completed, the three phases are repeated with new material to revisit and encourage deep understandings of Skipjack tuna and fisheries industry. Collaborative and interactive relationships among university, industry, and learner (consumer) were also the central components in our study. It was suggested that the materials and practices in this study would promote deep understanding of Skipjack tuna. That might lead to promote fish consumption and revitalize local economy