Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing

Segmental isotopic labeling is a powerful labeling tool to facilitate NMR studies of larger proteins by not only alleviating the signal overlap problem but also retaining features of uniform isotopic labeling. Although two approaches, expressed protein ligation (EPL) and protein trans-splicing (PTS), have been mainly used for segmental isotopic labeling, there has been no single example in which both approaches have been directly used with an identical protein. Here we applied both EPL and PTS methods to a 140 kDa dimeric multi-domain protein E. coli CheA, and successfully produced the ligated CheA dimer by both approaches. In EPL approach, extensive optimization of the ligation sites and the conditions were required to obtain sufficient amount for an NMR sample of CheA, because CheA contains a dimer forming domain and it was not possible to achieve high reactant concentrations (1–5 mM) of CheA fragments for the ideal EPL condition, thereby resulting in the low yield of segmentally labelled CheA dimer. PTS approach sufficiently produced segmentally labeled ligated CheA in vivo as well as in vitro without extensive optimizations. This is presumably because CheA has self-contained domains connected with long linkers, accommodating a seven-residue mutation without loss of the function, which was introduced by PTS to achieve the high yield. PTS approach was less laborious than EPL approach for the routine preparation of segmentally-isotope labeled CheA dimer. Both approaches remain to be further developed for facilitating preparations of segmental isotope-labelled samples without extensive optimizations for ligation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10858-012-9628-3) contains supplementary material, which is available to authorized users

Terrestrial invasion of pomatiopsid gastropods in the heavy-snow region of the Japanese Archipelago

<p>Abstract</p> <p>Background</p> <p>Gastropod mollusks are one of the most successful animals that have diversified in the fully terrestrial habitat. They have evolved terrestrial taxa in more than nine lineages, most of which originated during the Paleozoic or Mesozoic. The rissooidean gastropod family Pomatiopsidae is one of the few groups that have evolved fully terrestrial taxa during the late Cenozoic. The pomatiopsine diversity is particularly high in the Japanese Archipelago and the terrestrial taxa occur only in this region. In this study, we conducted thorough samplings of Japanese pomatiopsid species and performed molecular phylogenetic analyses to explore the patterns of diversification and terrestrial invasion.</p> <p>Results</p> <p>Molecular phylogenetic analyses revealed that Japanese Pomatiopsinae derived from multiple colonization of the Eurasian Continent and that subsequent habitat shifts from aquatic to terrestrial life occurred at least twice within two Japanese endemic lineages. Each lineage comprises amphibious and terrestrial species, both of which are confined to the mountains in heavy-snow regions facing the Japan Sea. The estimated divergence time suggested that diversification of these terrestrial lineages started in the Late Miocene, when active orogenesis of the Japanese landmass and establishment of snowy conditions began.</p> <p>Conclusions</p> <p>The terrestrial invasion of Japanese Pomatiopsinae occurred at least twice beside the mountain streamlets of heavy-snow regions, which is considered the first case of this event in the area. Because snow coverage maintains stable temperatures and high humidity on the ground surface, heavy-snow conditions may have paved the way for these organisms from freshwater to land via mountain streamlets by preventing winter desiccation in mountain valleys. The fact that the terrestrialization of Pomatiopsidae occurred only in year-round wet environments, but not in seasonally dried regions, provides new insight into ancient molluscan terrestrialization.</p

A circulating subset of iNKT cells mediates antitumor and antiviral immunity

新規の循環型iNKT細胞を発見 --抗腫瘍・抗ウイルス感染効果の高い免疫細胞療法の開発への貢献に期待--. 京都大学プレスリリース. 2022-10-24.Invariant natural killer T (iNKT) cells are a group of innate-like T lymphocytes that recognize lipid antigens. They are supposed to be tissue resident and important for systemic and local immune regulation. To investigate the heterogeneity of iNKT cells, we recharacterized iNKT cells in the thymus and peripheral tissues. iNKT cells in the thymus were divided into three subpopulations by the expression of the natural killer cell receptor CD244 and the chemokine receptor CXCR6 and designated as C0 (CD244⁻CXCR6⁻), C1 (CD244⁻CXCR6⁺), or C2 (CD244⁺CXCR6⁺) iNKT cells. The development and maturation of C2 iNKT cells from C0 iNKT cells strictly depended on IL-15 produced by thymic epithelial cells. C2 iNKT cells expressed high levels of IFN-γ and granzymes and exhibited more NK cell–like features, whereas C1 iNKT cells showed more T cell–like characteristics. C2 iNKT cells were influenced by the microbiome and aging and suppressed the expression of the autoimmune regulator AIRE in the thymus. In peripheral tissues, C2 iNKT cells were circulating that were distinct from conventional tissue-resident C1 iNKT cells. Functionally, C2 iNKT cells protected mice from the tumor metastasis of melanoma cells by enhancing antitumor immunity and promoted antiviral immune responses against influenza virus infection. Furthermore, we identified human CD244⁺CXCR6⁺ iNKT cells with high cytotoxic properties as a counterpart of mouse C2 iNKT cells. Thus, this study reveals a circulating subset of iNKT cells with NK cell–like properties distinct from conventional tissue-resident iNKT cells

Dynamic domain arrangement of CheA-CheY complex regulates bacterial thermotaxis, as revealed by NMR

Bacteria utilize thermotaxis signal transduction proteins, including CheA, and CheY, to switch the direction of the cell movement. However, the thermally responsive machinery enabling warm-seeking behavior has not been identified. Here we examined the effects of temperature on the structure and dynamics of the full-length CheA and CheY complex, by NMR. Our studies revealed that the CheA-CheY complex exists in equilibrium between multiple states, including one state that is preferable for the autophosphorylation of CheA, and another state that is preferable for the phosphotransfer from CheA to CheY. With increasing temperature, the equilibrium shifts toward the latter state. The temperature-dependent population shift of the dynamic domain arrangement of the CheA-CheY complex induced changes in the concentrations of phosphorylated CheY that are comparable to those induced by chemical attractants or repellents. Therefore, the dynamic domain arrangement of the CheA-CheY complex functions as the primary thermally responsive machinery in warm-seeking behavior.Peer reviewe

Severity of Menstrual Pain Is Associated with Nutritional Intake and Lifestyle Habits

Recently, the employment rate of women in Japan has steadily increased. Approximately 80% of women experience menstrual pain and premenstrual syndrome (PMS). These symptoms decrease a woman’s quality of life and her work productivity, leading to an economic loss. This cross-sectional study of 321 healthy Japanese women aged 20–39 years aimed to clarify the lifestyle-related factors or nutrient intake that might cause menstrual pain. The participants underwent body composition measurements and completed meal survey sheets and lifestyle questionnaires, including menstrual status, exercise, sleep and breakfast consumption. Based on the questionnaire results, participants were divided into two groups according to the severity of menstrual pain, namely, heavy and light. Chi-square and Wilcoxon signed-rank sum tests were used to compare the severity of menstrual pain in the two groups. In the heavy group, the intake of animal proteins, including fish, vitamin D and vitamin B12, was significantly lower (p p p < 0.05). This study suggests that a lack of animal protein, the accompanying vitamins and fatty acids, and the frequency of breakfast or bathing are associated with the severity of menstrual pain

Imaging of built-in electric field at a p-n junction by scanning transmission electron microscopy

Precise measurement and characterization of electrostatic potential structures and the concomitant electric fields at nanodimensions are essential to understand and control the properties of modern materials and devices. However, directly observing and measuring such local electric field information is still a major challenge in microscopy. Here, differential phase contrast imaging in scanning transmission electron microscopy with segmented type detector is used to image a p-n junction in a GaAs compound semiconductor. Differential phase contrast imaging is able to both clearly visualize and quantify the projected, built-in electric field in the p-n junction. The technique is further shown capable of sensitively detecting the electric field variations due to dopant concentration steps within both p-type and n-type regions. Through live differential phase contrast imaging, this technique can potentially be used to image the electromagnetic field structure of new materials and devices even under working conditions

Anomalous Radioisotope Production for 68ZnO Using Polyethylene by Accelerator Neutrons

We measured the yields of radionuclides after neutron irradiation on an enriched 68ZnO sample covered with polyethylene blocks. The neutrons were generated from the 9Be(d,n) reaction by 40 and 50 MeV deuterons. For 50 MeV deuterons we found anomalously large yields of 67Ga, 66Ga, 69mZn, and 64Cu, more than 20-times larger than the yields for a 68ZnO sample without the polyethylene blocks. At the same time, the yields of the same radioisotopes from an enriched metallic 68Zn sample were almost insensitive to the presence of polyethylene. On the other hand, for 40 MeV deuterons the enhanced production was not observed. This finding would provide a unique capability of accelerator neutrons to simultaneously produce large amounts of several radioisotopes via the neutrons- as well as protons-induced reactions on a single sample. The experimental data were compared with the yields calculated by the simulation code Particle and Heavy Ion Transport code System (PHITS). The calculated yields of these radioisotopes produced from the 68ZnO sample covered with the polyethylene blocks considerably disagree with the measured ones, while for the 68ZnO sample without the blocks they agree with each other