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.

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.

Nrp1 is Activated by Konjac Ceramide Binding-Induced Structural Rigidification of the a1a2 Domain.

Konjac ceramide (kCer) is a plant-type ceramide composed of various long-chain bases and a-hydroxyl fatty acids. The presence of d4t,8t-sphingadienine is essential for semaphorin 3A (Sema3A)-like activity. Herein, we examined the three neuropilin 1 (Nrp1) domains (a1a2, b1b2, or c), and found that a1a2 binds to d4t,8t-kCer and possesses Sema3A-like activity. kCer binds to Nrp1 with a weak affinity of mM dissociation constant (Kd). We wondered whether bovine serum albumin could influence the ligand-receptor interaction that a1a2 has with a single high affinity binding site for kCer (Kd in nM range). In the present study we demonstrated the influence of bovine serum albumin. Thermal denaturation indicates that the a1a2 domain may include intrinsically disordered region (IDR)-like flexibility. A potential interaction site on the a1 module was explored by molecular docking, which revealed a possible Nrp1 activation mechanism, in which kCer binds to Site A close to the Sema3A-binding region of the a1a2 domain. The a1 module then accesses a2 as the IDR-like flexibility becomes ordered via kCer-induced protein rigidity of a1a2. This induces intramolecular interaction between a1 and a2 through a slight change in protein secondary structure.

Development of CVD Silica Membranes Having High Hydrogen Permeance and Steam Durability and a Membrane Reactor for a Water Gas Shift Reaction.

Water gas shift reaction of carbon monoxide (CO) with membrane reactors should be a promising method for hydrogen mass-production because of its high CO conversion, high hydrogen purity and low carbon dioxide emission. For developing such membrane reactors, we need hydrogen permselective membranes with high hydrogen permeance with order of 10-6 mol m-2 s-1 Pa-1 at 573 K and high steam durability. In this study, we have optimized the kind of substrates, precursors, vapor concentration, and chemical vapor deposition (CVD) time using the counter-diffusion CVD method for developing such membranes. The developed membrane prepared from hexamethyldisiloxane has a hydrogen permeance of 1.29 × 10-6 mol m-2 s-1 Pa-1 at 573 K and high steam durability. We also conducted water gas shift reactions with membrane reactors installed the developed silica membranes. The results indicated that reactions proceed efficiently with the conversion around 95-97%, hydrogen purity around 94%, and hydrogen recovery around 60% at space velocity (SV) 7000.

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.

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.

Effectiveness of Magnifying Narrow-Band Imaging Endoscopy for Differential Diagnosis between the High-Risk Mixed-Type and Low-Risk Simple-Type of Low-Grade, Well-Differentiated Gastric Tubular Adenocarcinoma.

Backgrounds. Magnifying endoscopy with narrow-band imaging (NBI-ME) is useful for diagnosing differentiated early gastric cancer (D-EGC). D-EGC is classified as high- or low-grade based on its glandular architectural and cytological atypia. Low-grade, well-differentiated tubular adenocarcinoma (LG-tub1) mixed with high-grade tub1 (HG-tub1) and/or other histological types (M-LG-tub1) may indicate a primitive high-risk malignant lesion compared to histologically simple-type LG-tub1 (S-LG-tub1). Because LG-tub1 is occasionally difficult to diagnose due to its unclear demarcation under conventional white light endoscopy, early precise diagnoses are important. Methods. We compared NBI-ME and postendoscopic submucosal dissection histological findings for 30 S-LG-tub1 and 15 M-LG-tub1 lesions. We classified the NBI-ME findings of S-LG-tub1 (and not D-EGC) into four patterns. The differential diagnosis between M-LG-tub1 and S-LG-tub1 depended on the presence of more than one of these patterns without or with other patterns (referred to as "limited-to-four-pattern [LFP] sign-positive" and "sign-negative", resp.). Result. The sensitivity, specificity, accuracy, positive and negative predictive values, and intraobserver and interobserver agreement, using the "LFP sign" for the differential diagnosis between M-LG-tub1 and S-LG-tub1, were 87.9%, 91.7%, 88.9%, 96.7%, 73.3%, and k = 0.842 and k = 0.737, respectively. Conclusion. NBI-ME may be useful in differentiating between high-risk M-LG-tub1 and low-risk S-LG-tub1.

Crystallographic and NMR evidence for flexibility in oligosaccharyltransferases and its catalytic significance.

Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of an oligosaccharide to an asparagine residue in glycoproteins. It possesses a binding pocket that recognizes Ser and Thr residues at the +2 position in the N-glycosylation consensus, Asn-X-Ser/Thr. We determined the crystal structures of the C-terminal globular domains of the catalytic subunits of two archaeal OSTs. A comparison with previously determined structures identified a segment with remarkable conformational plasticity, induced by crystal contact effects. We characterized its dynamic properties in solution by (15)N NMR relaxation analyses. Intriguingly, the mobile region contains the +2 Ser/Thr-binding pocket. In agreement, the flexibility restriction forced by an engineered disulfide crosslink abolished the enzymatic activity, and its cleavage fully restored activity. These results suggest the necessity of multiple conformational states in the reaction. The dynamic nature of the Ser/Thr pocket could facilitate the efficient scanning of N-glycosylation sequons along nascent polypeptide chains.

Blockade of inflammatory responses by a small-molecule inhibitor of the Rac activator DOCK2.

Tissue infiltration of activated lymphocytes is a hallmark of transplant rejection and organ-specific autoimmune diseases. Migration and activation of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain Dbl homology domain typically found in guanine nucleotide exchange factors, DOCK2 mediates the GTP-GDP exchange reaction for Rac through its DHR-2 domain. Here, we have identified 4-[3'-(2″-chlorophenyl)-2'-propen-1'-ylidene]-1-phenyl-3,5-pyrazolidinedione (CPYPP) as a small-molecule inhibitor of DOCK2. CPYPP bound to DOCK2 DHR-2 domain in a reversible manner and inhibited its catalytic activity in vitro. When lymphocytes were treated with CPYPP, both chemokine receptor- and antigen receptor-mediated Rac activation were blocked, resulting in marked reduction of chemotactic response and T cell activation. These results provide a rational of and a chemical scaffold for development of the DOCK2-targeting immunosuppressant.

[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.

[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.

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.

Electronic structures of five-coordinate iron(III) porphyrin complexes with highly ruffled porphyrin ring.

The spin states of the iron(III) complexes with a highly ruffled porphyrin ring, [Fe(TEtPrP)X] where X = F-, Cl-, Br-, I-, and ClO4(-), have been examined by 1H NMR, 13C NMR, EPR, and Mössbauer spectroscopy. While the F-, Cl-, and Br- complexes adopt a high-spin (S = 5/2) state, the I- complex exhibits an admixed intermediate-spin (S = 5/2, 3/2) state in CD2Cl2 solution. The I- complex shows, however, a quite pure high-spin state in toluene solution as well as in the solid. The results contrast those of highly saddled [Fe(OETPP)X] where the I- complex exhibits an essentially pure intermediate-spin state both in solution and in the solid. In contrast to the halide-ligated complexes, the ClO4(-) complex shows a quite pure intermediate-spin state. The 13C NMR spectra of [Fe(TEtPrP)ClO4] are characterized by the downfield and upfield shifts of the meso and pyrrole-alpha carbon signals, respectively: delta(meso) = +342 and delta(alpha-py) = -287 ppm at 298 K. The data indicate that the meso carbon atoms of [Fe(TEtPrP)ClO4] have considerable amounts of positive spin, which in turn indicate that the iron has an unpaired electron in the d(xy) orbital; the unpaired electron in the d(xy) orbital is delocalized to the meso positions due to the iron(d(xy))-porphyrin(a(2u)) interaction. Similar results have been obtained in analogous [Fe(TiPrP)X] though the intermediate-spin character of [Fe(TiPrP)X] is much larger than that of the corresponding [Fe(TEtPrP)X]. On the basis of these results, we have concluded that the highly ruffled intermediate-spin complexes such as [Fe(TEtPrP)ClO4] and [Fe(TiPrP)ClO4] adopt a novel (d(xz), d(yz))3(d(xy))1(d(z)(2)1 electron configuration; the electron configuration of the intermediate-spin complexes reported previously is believed to be (d(xy))2(d(xz)), d(yz))2(d(z)(2))1.

[CD25 positive chronic eosinophilic leukemia with myelofibrosis].

A 70-year-old man was referred to our hospital in March 2001 for the purpose of evaluation for anemia and thrombocytopenia. Physical examination revealed hepatosplenomegaly, normal skin, and normal neurologic findings. Blood examination showed a white blood cell count of 10,900/microliter, with a differential count of 58.5% eosinophils and 3.5% blast cells. Flow cytometric analysis of eosinophils revealed that they were positive for CD33, CD13, CD25, and HLA-DR. Bone marrow aspiration could not be performed due to dry tap, and bone marrow core biopsy specimen revealed severe myelofibrosis with blastoid cells proliferation. Cytogenetic analysis of bone marrow cells showed isochromosome 17. FISH analysis using a RAR alpha probe (17q21.1) demonstrated 62% of peripheral blood nucleated cells having three signals. BCR/ABL gene rearrangement by FISH analysis was not observed. Allergic disease, infectious disease, parasitic disease, collagen vascular diseases, pulmonary disease, and neoplastic disorders were excluded. Therefore, a diagnosis of chronic eosinophilic leukemia was made. The patient had no symptoms of hypereosinophilia. However, eosinophils with sparse granulation, positivity for CD25, elevated serum levels of soluble IL-2 receptor, and elevated serum levels of eosinophil cationic protein suggested activation of eosinophils. Further analysis is needed regarding the activation of eosinophils in chronic eosinophilic leukemia.

Different clones of t(1;12)/t(12;12) involving the ETV6 gene in a case of acute myeloid leukemia.

We describe here a case of a 77-year-old Japanese man who developed acute myeloid leukemia (AML-M2). Chromosome analysis of the bone marrow blast cells showed 46,XY,del(7q) at onset, and after relapse, two clones, 46,XY,t(1;12) and 46,XY,del(7q),t(12;12), were present. Fluorescence in situ hybridization analysis confirmed that each clone with the 12p abnormality involved the ETV6 gene. These findings suggest that the ETV6 gene rearrangements in this case were apparently independent of contribution to leukemogenesis, because this cytogenetic aberration appeared as a secondary change. To our knowledge, this is the first report of two different clones with ETV6 gene rearrangements in the same patient.

[Human herpesvirus-8 negative primary effusion lymphoma with complete clinical remission after removal of ascites].

A 58-year-old HIV-negative woman was admitted to our hospital with abdominal distension. She had a 5-year history of hypothyroidism and a 4-year history of diabetes mellitus. Physical examination revealed ascites. There was no lymphadenopathy or splenomegaly. Laboratory examination showed elevated levels of serum LDH and Al-p, polyclonal hypergammaglobulinemia, and was positive for anti-nuclear antibody, several autoantibodies and HCV-RNA. A computed tomographic scan of the abdomen and chest showed massive ascites, but there was no evidence of tumor masses or lymph node enlargement. Cytologic examination of the ascitic fluid revealed numerous abnormal lymphocytes which by flow cytometry demonstrated expression of CD5, CD19, CD20, and CD4. Cytogenetical analysis demonstrated a hyperdiploid karyotype, with numerical abnormalities. Southern blot analysis demonstrated rearranged monoclonal bands in JH and c-mycgenes. Polymerase chain reaction (PCR) analysis failed to detect the genomes of EBV and HHV-8 in the abnormal lymphocytes. A diagnosis of primary effusion lymphoma of B cell lineage was made. Following abdominal paracentesis, the patient remained in complete clinical remission for 7 months and died of an unrelated cause (cerebral bleeding). The present case demonstrated an HIV-, HHV-8-, and EBV-negative, and HCV-positive primary effusion lymphoma of B cell lineage, with a unique clinical course.

Spontaneous clinical and cytogenetic remission of aplastic anemia in a patient with del(13q).

We describe the case of a 64-year-old Japanese man with pancytopenia. Bone marrow biopsy findings were consistent with aplastic anemia. The patient was treated by transfusions without immunosuppressive therapy. Chromosome analysis of bone marrow cells at 6 months after onset showed a 46,XY,del(13) (q14q22) karyotype. The pancytopenia resolved gradually over the next 5 years; chromosome analysis of bone marrow cells at that time yielded normal findings. To our knowledge, this is the first report of spontaneous hematologic and cytogenetic remission of aplastic anemia. These findings suggest that the abnormal clone with deletion of the long arm of chromosome 13 was not sufficient for clonal evolution in aplastic anemia in this case.