Biochemical and biophysical characterization of the RAS family small GTPase protein DiRAS3.

DiRAS3, also called ARHI, is a RAS (sub)family small GTPase protein that shares 50-60% sequence identity with H-, K-, and N-RAS, with substitutions in key conserved G-box motifs and a unique 34 amino acid extension at its N-terminus. Unlike the RAS proto-oncogenes, DiRAS3 exhibits tumor suppressor properties. DiRAS3 function has been studied through genetics and cell biology, but there has been a lack of understanding of the biochemical and biophysical properties of the protein, likely due to its instability and poor solubility. To overcome this solubility issue, we engineered a DiRAS3 variant (C75S/C80S), which significantly improved soluble protein expression in E. coli. Recombinant DiRAS3 was purified by Ni-NTA and size exclusion chromatography (SEC). Concentration dependence of the SEC chromatogram indicated that DiRAS3 exists in monomer-dimer equilibrium. We then produced truncations of the N-terminal (ΔN) and both (ΔNC) extensions to the GTPase domain. Unlike full-length DiRAS3, the SEC profiles showed that ΔNC is monomeric while ΔN was monomeric with aggregation, suggesting that the N and/or C-terminal tail(s) contribute to dimerization and aggregation. The 1H-15N HSQC NMR spectrum of ΔNC construct displayed well-dispersed peaks similar to spectra of other GTPase domains, which enabled us to demonstrate that DiRAS3 has a GTPase domain that can bind GDP and GTP. Taken together, we conclude that, despite the substitutions in the G-box motifs, DiRAS3 can switch between nucleotide-bound states and that the N- and C-terminal extensions interact transiently with the GTPase domain in intra- and inter-molecular fashions, mediating weak multimerization of this unique small GTPase.

Embryonic staging of bats with special reference to Vespertilio sinensis and its cochlear development.

BACKGROUND: How bats deviate heterochronically from other mammals remains largely unresolved, reflecting the lack of a quantitative staging framework allowing comparison among species. The standard event system (SES) is an embryonic staging system allowing quantitative detection of interspecific developmental variations. Here, the first SES-based staging system for bats, using Asian parti-colored bat (Vespertilio sinensis) is introduced. General aspects of normal embryonic development and the three-dimensional development of the bat cochlea were described for the first time. Recoding the embryonic staging tables of 18 previously reported bat species and Mus musculus into the SES system, quantitative developmental comparisons were performed. RESULTS: It was found that limb bud development of V. sinensis is relatively late among 19 bat species and late limb development is a shared trait of vespertilionid bats. The inner ear cochlear canal forms before the semicircular canal in V. sinensis while the cochlear canal forms after the semicircular canal in non-volant mammals. CONCLUSIONS: The present approach using the SES system provides a powerful framework to detect the peculiarities of bat development. Incorporating the timing of gene expression patterns into the SES framework will further contribute to the understanding of the evolution of specialized features in bats.

Structural and thermodynamic analyses reveal critical features of glycopeptide recognition by the human PILRα immune cell receptor.

Before entering host cells, herpes simplex virus-1 uses its envelope glycoprotein B to bind paired immunoglobulin-like type 2 receptor α (PILRα) on immune cells. PILRα belongs to the Siglec (sialic acid (SA)-binding immunoglobulin-like lectin)-like family, members of which bind SA. PILRα is the only Siglec member to recognize not only the sialylated O-linked sugar T antigen (sTn) but also its attached peptide region. We previously determined the crystal structure of PILRα complexed with the sTn-linked glycopeptide of glycoprotein B, revealing the simultaneous recognition of sTn and peptide by the receptor. However, the contribution of each glycopeptide component to PILRα binding was largely unclear. Here, we chemically synthesized glycopeptide derivatives and determined the thermodynamic parameters of their interaction with PILRα. We show that glycopeptides with different sugar units linking SA and peptides (i.e. "GlcNAc-type" and "deoxy-GlcNAc-type" glycopeptides) have lower affinity and more enthalpy-driven binding than the wild type (i.e. GalNAc-type glycopeptide). The crystal structures of PILRα complexed with these glycopeptides highlighted the importance of stereochemical positioning of the O4 atom of the sugar moiety. These results provide insights both for understanding the unique O-glycosylated peptide recognition by the PILRα and for the rational design of herpes simplex virus-1 entry inhibitors.

Endoscopic and clinicopathological features of intramucosal, histologically mixed-type, low-grade, well-differentiated gastric tubular adenocarcinoma with the potential for late-onset lymph node metastasis.

BACKGROUND: Intramucosal, histologically mixed-type, low-grade (LG), well-differentiated gastric tubular adenocarcinomas (tub1s; LG-tub1s) have larger mean diameters and exhibit a higher frequency of the gastric mucin phenotype (G-phenotype) than pure LG-tub1s. In proportion to their increases in diameter, G-phenotype differentiated-type early gastric cancer (EGC) tumours reportedly grow to eventually contain (an) undifferentiated-type component(s) and LG-tub1s, which are included in differentiated-type EGCs, reportedly exhibit changes in their glandular architectural and cytological atypia grades from LG to high-grade (HG) and can grow to contain a moderately differentiated tubular adenocarcinoma (tub2) component and undifferentiated components. Because they generally show a higher frequency of malignancy relative to tumours with a higher atypia grade and lower differentiation degree, it is suggested that, among mixed-type LG-tub1s, G-phenotype LG-tub1s containing an HG-tub2 component (LG-tub1s > HG-tub2) with undifferentiated components might lead to late-onset metastasis to lymph nodes even after a successful endoscopic submucosal dissection (ESD). We aimed to clarify the endoscopic and clinicopathological features of these G-phenotype LG-tub1s > HG-tub2. METHODS: Of the 13,217 oesophagogastroduodenoscopies performed at our institutions between September 2008 and March 2016, 185 EGC lesions were evaluated in this retrospective observational study. Among these EGC lesions, 60 intramucosal LG-tub1s were divided into 53 tub1 (44 pure LG-tub1s and nine LG-tub1s containing HG-tub1) lesions and seven LG-tub1 > tub2 (LG-tub1 containing LG- and HG-tub2) lesions. RESULTS: The frequencies of the superficial depressed type (P = 0.026), reddish colour (P = 0.006), HG of contained tub2s (P = 0.006), and G-phenotype (P = 0.028) were significantly higher in the LG-tub1 > tub2 group than those in the tub1 group. However, the largest lesion of the LG-tub1 > tub2 group had a superficial flat appearance, an isochromatic colour, an HG-tub2 and an undifferentiated component, and a large diameter greater than 30 mm, and it exhibited a G-phenotype. CONCLUSIONS: Intramucosal G-phenotype LG-tub1s > HG-tub2 are potential premalignant stomach neoplasms that may have specific endoscopic and clinicopathological features. However, G-phenotype LG-tub1s > HG-tub2 with undifferentiated component, which potentially show higher malignancy than those without undifferentiated components might change from a reddish to isochromatic colour. Accurately diagnosing, treating, and following-up G-phenotype LG-tub1s > HG-tub2 might decrease the number of patients who experience late-onset metastasis after ESD.

Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion.

Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited by inhomogeneity through the formation of dimers, trimers, and larger oligomers. The scFv protein is assumed to be in equilibrium between the closed and open states formed by assembly or disassembly of VH and VL domains. Therefore, the production of an scFv with equilibrium biased to the closed state would be critical to overcome the problem in inhomogeneity of scFv for industrial or therapeutic applications. In this study, we obtained scFv clones stable against GA-pyridine, an advanced glycation end-product (AGE), by using a combination of a phage display system and random mutagenesis. Executing the bio-panning at 37 °C markedly improved the stability of scFvs. We further evaluated the radius of gyration by small-angle X-ray scattering (SAXS), obtained compact clones, and also visualized open.

Population dynamics, synchrony, and environmental quality of Hokkaido voles lead to temporal and spatial Taylor's laws.

Taylor's law (TL) asserts that the variance in a species' population density is a power-law function of its mean population density: log(variance) = a + b × log(mean). TL is widely verified. We show here that empirical time series of density of the Hokkaido gray-sided vole, Myodes rufocanus, sampled 1962-1992 at 85 locations, satisfied temporal and spatial forms of TL. The slopes (b ± standard error) of the temporal and spatial TL were estimated to be 1.613 ± 0.141 and 1.430 ± 0.132, respectively. A previously verified autoregressive Gompertz model of the dynamics of these populations generated time series of density which reproduced the form of temporal and spatial TLs, but with slopes that were significantly steeper than the slopes estimated from data. The density-dependent components of the Gompertz model were essential for the temporal TL. Adding to the Gompertz model assumptions that populations with higher mean density have reduced variance of density-independent perturbations and that density-independent perturbations are spatially correlated among populations yielded simulated time series that satisfactorily reproduced the slopes from data. The slopes (b ± standard error) of the enhanced simulations were 1.619 ± 0.199 for temporal TL and 1.575 ± 0.204 for spatial TL.

Flood disturbance and predator-prey effects on regional gradients in species diversity.

The effects of both abiotic factors and biotic interactions among guilds (i.e., inter-guild effects) have been suggested to be important for understanding spatial variation in species diversity; however, compared to the abiotic effects, the processes by which the inter-guild effects are mediated have been little described. Hence, we investigated stream invertebrate assemblages on Hokkaido Island, Japan, and assessed how the processes of determining regional patterns in species diversity differed among guilds (collector-filterers, collector-gatherers/shredders, scrapers, and predators) by taking both inter-guild and abiotic effects into consideration using Bayesian networks. Collector-gatherers/shredders, collector-filterers, and predators exhibited significant regional gradients in taxonomic richness. Gradients in the former two guilds can be generated by variation in flood disturbance regardless of interactions with other guilds. The gradient in predator taxonomic richness was indirectly related to the disturbance and was directly generated by bottom-up effects through their prey (collector-gatherers/shredders and collector-filterers). We found that not only environmental factors, but also inter-guild effects may be essential for forming the regional gradient in predators, unlike those for collector-gatherers/shredders and collector-filterers. The processes underlying the regional variation in taxonomic richness of the three guilds are interpreted in terms of the "more individuals" hypothesis, facilitation, and predator-prey relationships.

Protocol to identify the ligand binding site of Mincle using NMR spectroscopy.

Mincle (macrophage-inducible C-type lectin, CLEC4E) is a C-type lectin immune-stimulatory receptor that can be targeted for inducing potent adjuvant effects. Mincle can recognize trehalose dimycolate and related glycolipids. Here, we present a protocol to identify the ligand binding mode of Mincle. We describe steps for preparing labeled Mincle ectodomain, data acquisition, and analysis of nuclear magnetic resonance experiments using non-detergent sulfobetaine-195. This protocol can be applied to other protein-ligand interactions that have aggregation problems for complex formation. For complete details on the use and execution of this protocol, please refer to Furukawa et al.1.

Refugia in glacial ages led to the current discontinuous distribution patterns of the dark red-backed vole Myodes rex on Hokkaido, Japan.

The terrestrial mammalian fauna of the North Japanese island, Hokkaido, is more similar to that of Southern Siberia than to the main island of Japan, Honshu. Three species of the genus Myodes (Muridae, Rodentia) are found on Hokkaido, but not on Honshu. While Myodes rufocanus and M. rutilus are widely distributed across Hokkaido as well as the Eurasian continent, M. rex, which is endemic to Hokkaido and its adjacent islands, shows a discontinuous distribution pattern. We analyzed the phylogeographic history of M. rex using the mitochondrial DNA control region in order to interpret their discontinuous distribution pattern. Phylogenetic relationships among 54 distinct haplotypes showed that M. rex can be divided into four clades that occur on the northern, central, and southern regions of the Hokkaido mainland and on Rishiri Island, respectively. The phylogroups in the northern and central regions were largely separated in space, although several areas of sympatry were found. The phylogroup in the southern region, which was clearly separated from other phylogroups, showed markedly low genetic variability. All analyzed individuals from the population on Rishiri belonged to a separate lineage. Across a range of divergence rate estimates, we dated the basal divergence of all phylogroups to the mid to late Pleistocene, with subsequent signals of population expansion within lineages. We conclude that current phylogeographic structure in M. rex likely reflects Pleistocene survival in several separate refugia in situ. Past glacial ages have thus played an important role in shaping the current distribution patterns of mammalian species on Hokkaido.

Male-biased dispersal causes intersexual differences in the subpopulation structure of the gray-sided vole.

The genetic structure of gray-sided voles was investigated at a spatial scale of 2 km using mtDNA sequences. The control region (674bp) of 162 voles was sequenced and 18 haplotypes were identified. Within 0.5-ha trapping plots (n = 8), the number of haplotypes and gene diversity was significantly greater in males than in females. The fixation index among plots for females (F GP = 0.241) was 3 times as large as that for males (0.075), implying male-biased dispersal. A simulation analysis showed that the observed genetic structure in males could be generated by modifying the observed haplotype distribution of females by adding the effects of local male dispersal. Half of the pairwise F GP (15/28) showed significant differentiation in females, whereas almost none (1/28) were significant in males. Isolation by distance was observed in females, whereas no clear spatial pattern was observed in males. Most pairwise F GP for females were not significant in the short- and intermediate-distance classes (≤1.0 km) as with those for males, whereas all showed significant differentiation in the long-distance class (>1.0 km) for females, but not for males. These findings indicate that the extent of subpopulations within which individuals interact differs between sexes.

Interactive effects of two rodent species on the seed dispersal of Japanese walnut.

The effects of seed dispersers on plant fitness (seed dispersal effectiveness, SDE) have been evaluated based on the number (quantity) and recruitment probability (quality) of dispersed seeds. Although seeds of most zoochorous species are dispersed by two or more animal species, which may interact with each other, SDE has often been studied assuming a one-plant and one-animal species system. We compared the SDE of Japanese walnut (Juglans ailanthifolia) between squirrel-only and squirrel-mouse sites in natural forests of Hokkaido, Japan, and found that the SDE from the red squirrel (Sciurus vulgaris), considered a primary seed disperser, was altered by an alternative seed disperser species, the Japanese wood mouse (Apodemus speciosus). Seed removal rates at the squirrel-mouse site were significantly higher than those at the squirrel-only site, and both dispersed seeds and seedlings were less aggregated, with a strongly repulsive relationship with adult conspecific trees at the squirrel-mouse site. Seedlings established themselves at a location with fewer medium-sized trees (< 10 cm DBH) at the squirrel-mouse site. These results suggest that the interactive effect of the rodent species affects the SDE of Japanese walnut.

Structural basis for plastic glycolipid recognition of the C-type lectin Mincle.

Mincle (macrophage-inducible C-type lectin, CLEC4E) is a C-type lectin immune-stimulatory receptor for cord factor, trehalose dimycolate (TDM), which serves as a potent component of adjuvants. The recognition of glycolipids by Mincle, especially their lipid parts, is poorly understood. Here, we performed nuclear magnetic resonance analysis, revealing that titration of trehalose harboring a linear short acyl chain showed a chemical shift perturbation of hydrophobic residues next to the Ca-binding site. Notably, there were split signals for Tyr201 upon complex formation, indicating two binding modes for the acyl chain. In addition, most Mincle residues close to the Ca-binding site showed no observable signals, suggesting their mobility on an ∼ ms scale even after complex formation. Mutagenesis study supported two putative lipid-binding modes for branched acyl-chain TDM binding. These results provide novel insights into the plastic-binding modes of Mincle toward a wide range of glycol- and glycerol-lipids, important for rational adjuvant development.

Ecological correlates and determinants in the geographical variation of deer morphology.

Previous studies on patterns in ungulate size variations have emphasized the effect of a particular environmental factor such as Bergmann's rule and the island rule. However, although multiple environmental factors may influence the body size, these studies focused on a single factor, and various measurements that may be influenced by different environmental factors (at least partly) were used as indices of body size. In this study, we used several skull and limb measurements to examine size variations among island populations of sika deer (Cervus nippon) in southern Japan considering the effects of multiple environmental factors. We found that all measurements differed markedly between populations. We focused on the skull and limb condylobasal length (CBL) and metacarpal length because they had the most important variations among the populations and the largest sample sizes. The common environmental factors influencing CBL and metacarpal length were island area and precipitation. Since these environmental factors reflect the availability of food resources, the causal factor of body size variation may be food resources. Interpopulation variation in metacarpal length was greater than that of CBL, indicating that metacarpal length may be affected by additional factors besides the common factors shared with CBL. Specific environmental factors influencing relative (CBL adjusted) metacarpal length were precipitation and slope. A common direct cause of those environmental factors was discussed in relation to topography. Analyses of phenotypic variation using multiple measurements with multiple environmental factors are useful to gain insight into underlying causes and can lead to identification of a measurement-specific variation with a specific driving force.

[Fungemia caused by Scedosporium prolificans in myelodysplastic syndrome].

We report a case of fungemia caused by Scedosporium prolificans, an emerging pathogen. An 83-year-old man with myelodysplastic syndrome (MDS) and agranulocytosis was admitted for pneumonia in January 2009. He was treated with meropenem, minocycline, and gamma-globulin for pneumonia and G-CSF and platelet transfusion for MDS. Although he recovered from pneumonia as neutrophil count increased, intermittent fever continued. On hospital day 17, blood culture yielded fungal colonies indicating S. prolificans. Voriconazole was started immediately, but the man's general condition deteriorated with cerebral infarction and he died of cerebral hemorrhage on hospital day 65. Attention must therefore be paid to the increasing scedosporiosis incidence in Japan.

A Case Report of Radiotherapy for Skull Lesions of Langerhans Cell Histiocytosis With Dural Invasion.

Background: Langerhans cell histiocytosis (LCH) is a rare disease, especially in adults. It is often associated with non-fatal bone and skin lesions and has relatively good radiosensitivity. In contrast, brain and lymph node metastases from LCH lesions are considered to be less sensitive to radiotherapy. Case Report: At our institution, 30 Gy radiotherapy was used to treat bone lesions with dural invasion in a patient with adult-onset LCH. The patient was treated with chemotherapy and radiotherapy for 21 years since the initial diagnosis. After radiotherapy, the tumor shrank rapidly, and a complete response was achieved 1 year after treatment. The patient survived without local recurrence. Conclusion: Here, we report the details of this case, along with a review of the literature. We suggest that even with invasion of the tissues around the bone lesions in LCH, local recurrence can be prevented by middle radiation doses.

Crystallographic snapshots of Tom20-mitochondrial presequence interactions with disulfide-stabilized peptides.

Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The Tom20 protein, residing on the mitochondrial surface, recognizes the N-terminal presequences of precursor proteins. We previously determined the crystal structures of the Tom20-presequence complex. The successful crystallization involved tethering the presequence to Tom20 through an intermolecular disulfide bond with an optimized linker. In this work, we assessed the tethering method. The intermolecular disulfide bond was cleaved in crystal with a reducing agent. The pose (i.e., conformation and position) of the presequence was identical to the previously determined pose. In another experiment, a longer linker than the optimized length was used for the tethering. The perturbation of the tether changed the pose slightly, but the interaction mode was preserved. These results argue against the forced interaction of the presequence by its covalent attachment to Tom20. Second, as an alternative method referred to as "molecular stiffening", we introduced a disulfide bond within the presequence peptide to restrict the freedom of the peptide in the unbound states. One presequence analogue exhibited over 100-fold higher affinity than its linear counterpart and generated cocrystals with Tom20. One of the two crystallographic snapshots revealed a known pose previously determined by the tethering method, and the other snapshot depicted a new pose. These results confirmed and extended the dynamic, multiple bound state model of the Tom20-presequence interactions and also demonstrated the validity of the molecular tethering and stiffening techniques in studies of transient protein-peptide interactions.

Tom20 recognizes mitochondrial presequences through dynamic equilibrium among multiple bound states.

Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The N-terminal presequences of mitochondrial-precursor proteins contain a diverse consensus motif (phi chi chi phi phi, phi is hydrophobic and chi is any amino acid), which is recognized by the Tom20 protein on the mitochondrial surface. To reveal the structural basis of the broad selectivity of Tom20, the Tom20-presequence complex was crystallized. Tethering a presequence peptide to Tom20 through a disulfide bond was essential for crystallization. Unexpectedly, the two crystals with different linker designs provided unique relative orientations of the presequence with respect to Tom20, and neither configuration could fully account for the hydrophobic preference at the three hydrophobic positions of the consensus motif. We propose the existence of a dynamic equilibrium in solution among multiple states including the two bound states. In accordance, NMR 15N relaxation analyses suggested motion on a sub-millisecond timescale at the Tom20-presequence interface. We suggest that the dynamic, multiple-mode interaction is the molecular mechanism facilitating the broadly selective specificity of the Tom20 receptor toward diverse mitochondrial presequences.

Interspecific differences in tannin intakes of forest-dwelling rodents in the wild revealed by a new method using fecal proline content.

Mammalian herbivores adopt various countermeasures against dietary tannins, which are among the most widespread plant secondary metabolites. The large Japanese wood mouse Apodemus speciosus produces proline-rich salivary tannin-binding proteins in response to tannins. Proline-rich proteins (PRPs) react with tannins to form stable complexes that are excreted in the feces. Here, we developed a new method for estimating the tannin intake of free-living small rodents, by measuring fecal proline content, and applied the method to a field investigation. A feeding experiment with artificial diets containing various levels of tannic acid revealed that fecal proline content was clearly related to dietary tannin content in three species (A. speciosus, Apodemus argenteus, and Myodes rufocanus). We then used fecal proline content to estimate the tannin intakes of these three forest-dwelling species in a forest in Hokkaido. In the autumn, estimated tannin intakes increased significantly in the Apodemus species, but not in M. rufocanus. We speculated that an increase in tannin intake during autumn may result from consumption of tannin-rich acorns. This hypothesis was consistent with population fluctuation patterns of the three species, which were well-synchronized with acorn abundance for the Apodemus species but not for M. rufocanus.

Canonical versus non-canonical transsynaptic signaling of neuroligin 3 tunes development of sociality in mice.

Neuroligin 3 (NLGN3) and neurexins (NRXNs) constitute a canonical transsynaptic cell-adhesion pair, which has been implicated in autism. In autism spectrum disorder (ASD) development of sociality can be impaired. However, the molecular mechanism underlying NLGN3-mediated social development is unclear. Here, we identify non-canonical interactions between NLGN3 and protein tyrosine phosphatase δ (PTPδ) splice variants, competing with NRXN binding. NLGN3-PTPδ complex structure revealed a splicing-dependent interaction mode and competition mechanism between PTPδ and NRXNs. Mice carrying a NLGN3 mutation that selectively impairs NLGN3-NRXN interaction show increased sociability, whereas mice where the NLGN3-PTPδ interaction is impaired exhibit impaired social behavior and enhanced motor learning, with imbalance in excitatory/inhibitory synaptic protein expressions, as reported in the Nlgn3 R451C autism model. At neuronal level, the autism-related Nlgn3 R451C mutation causes selective impairment in the non-canonical pathway. Our findings suggest that canonical and non-canonical NLGN3 pathways compete and regulate the development of sociality.

[Accurate, preoperative diagnosis of duodenal duplication based on its characteristic endoscopic findings and radiographic presentation].

An 18-year-old woman was admitted to our hospital with pain in the right upper quadrant of the abdomen and fever. Diagnostic imaging studies, namely, upper gastrointestinal roentogenography and endoscopy, were performed, and an oval cystic tumor was detected in the second part of the duodenum. On the basis of the characteristic findings of the imaging studies and blood tests, we established a definitive diagnosis of duodenal duplication complicated with acute pancreatitis, even before surgical exploration. On surgical examination, the main pancreatic duct appeared to communicate internally with the cystic lesion. The histopathological examination of the surgical specimen confirmed the accuracy of the preoperative diagnosis.