Gain of function by phosphorylation in Presenilin 1-mediated regulation of insulin signaling

During pregnancy, activation of the maternal immune system results in inflammation in the foetal nervous system. The causative agents are pro-inflammatory cytokines like interleukin-1β (IL-1β), produced by the foetus. In this study, we examine the effect of IL-1β on the proliferation and differentiation of neural progenitor cells (NPCs) to better understand its potential effects on the developing brain. We find that the IL-1β receptor (IL-1R1) is expressed in the ventral mesencephalon of the developing brain. Furthermore, IL-1R1 is expressed on Nestin-positive, Sox-2-positive NPCs. IL-1β treatment reduced the numbers of proliferating NPCs, an effect prevented by the IL-1R1 receptor antagonist. LDH and MTT assays, and western blot analysis for cleaved caspase 3 and poly(ADP-ribose) polymerase, confirmed that this was not due to an increase in cell death but rather an induction of differentiation. To further study the effects of IL-1β on cell fate determination, we differentiated NPCs in the presence and absence of IL-1β. Il-1β promoted gliogenesis and inhibited neurogenesis, an effect that required p38-MAPK kinase signalling. In summary, these data show that exposure of NPCs to IL-1β affects their development. This necessitates an examination of the consequences that maternal immune system activation during pregnancy has on the cellular architecture of the developing brain.     

Detection and characterization of a thermophilic biotin biosynthetic enzyme, 7-keto-8-aminopelargonic acid synthase, from various thermophiles

By detailed BLAST searches of the genome database of various thermophiles, five ORFs with similarity to the bioF gene, which encodes 7-keto-8-aminopelargonic acid synthase (BioF) involved in biotin biosynthesis, of Escherichia coli were found: AqbioF, CltbioF, GkbioF, SytbioF, and TsebioF, from Aquifex aeolicus VF5, Clostridium thermocellum ATCC27405, Geobacillus kaustophilus JCM12893, Symbiobacterium thermophilum IAM14863, and Thermosynechococcus elongatus BP-1 respectively. The five purified recombinant bioF gene products, which were overexpressed in E. coli, had the enzyme activity of BioF. The optimum temperature range and thermostability of five BioFs, AqBioF, CltBioF, GkBioF, SytBioF, and TseBioF, were higher than those of E. coli BioF. In particular, AqBioF was found to show the highest thermostability of the α-oxoamine synthase family enzymes reported to date. Substrate specificity experiments revealed that SytBioF was also able to catalyze the reaction of 2-amino-3-ketobutyrate CoA ligase, a member of the α-oxoamine synthase family, and that it used acetyl-CoA and glycine as substrates, like the TTHA1582 protein of Thermus thermophilus. The other purified BioFs, AqBioF and GkBioF, did not show any activity with acyl-CoAs and amino acids other than pimeloyl-CoA and L-alanine as substrates.     

Crystallization strategy for the glycoprotein-receptor complex between measles virus hemagglutinin and its cellular receptor SLAM

Measles virus (MV), one of the most contagious agents, infects immune cells using the signaling lymphocyte activation molecule (SLAM) on the cell surface. A complex of SLAM and the attachment protein, hemagglutinin (MVH), has remained elusive due to the intrinsic handling difficulty including glycosylation. Furthermore, crystals obtained of this complex are either nondiffracting or poorly-diffracting. To solve this problem, we designed a systematic approach using a combination of the following techniques; (1) a transient expression system in HEK293SGnTI(-) cells, (2) lysine methylation, (3) structure-guided mutagenesis directed at better crystal packing, (4) Endo H treatment, (5) single-chain formation for stable complex, and (6) floating-drop vapor diffusion. Using our approach, the receptor-binding head domain of MV-H covalently fused with SLAM was successfully crystallized and diffraction was improved from 4.5 ? to a final resolution of 3.15 ? . These combinational methods would be useful as crystallization strategies for complexes of glycoproteins and their receptors.     

Zygospore formation between homothallic and heterothallic strains of Closterium

Zygospore formation in different strains of the Closterium peracerosum-strigosum-littorale complex was examined in this unicellular isogamous charophycean alga to shed light on gametic mating strains in this taxon, which is believed to share a close phylogenetic relationship with land plants. Zygospores typically form as a result of conjugation between mating-type plus (mt⁺) and mating-type minus (mt⁻) cells during sexual reproduction in the heterothallic strain, similar to Chlamydomonas. However, within clonal cells, zygospores are formed within homothallic strains, and the majority of these zygospores originate as a result of conjugation of two recently divided sister gametangial cells derived from one vegetative cell. In this study, we analyzed conjugation of homothallic cells in the presence of phylogenetically closely related heterothallic cells to characterize the reproductive function of homothallic sister gametangial cells. The relative ratio of non-sister zygospores to sister zygospores increased in the presence of heterothallic mt⁺ cells, compared with that in the homothallic strain alone and in a coculture with mt⁻ cells. Heterothallic cells were surface labeled with calcofluor white, permitting fusions with homothallic cells to be identified and confirming the formation of hybrid zygospores between the homothallic cells and heterothallic mt⁺ cells. These results show that at least some of the homothallic gametangial cells possess heterothallic mt⁻-like characters. This finding supports speculation that division of one vegetative cell into two sister gametangial cells is a segregative process capable of producing complementary mating types.     

Pyrrolidinyl phenylurea derivatives as novel CCR3 antagonists

Optimization starting with our lead compound 1 (IC₅₀ = 4.9 nM) led to the identification of pyrrolidinyl phenylurea derivatives. Further modification toward improvement of the bioavailability provided (R)-1-(1-((6-fluoronaphthalen-2-yl)methyl)pyrrolidin-3-yl)-3-(2-(2-hydroxyethoxy)phenyl)urea 32 (IC₅₀ = 1.7 nM), a potent and orally active CCR3 antagonist.     

Promotion of Ccn2 expression and osteoblastic differentiation by actin polymerization, which is induced by laminar fluid flow stress

Fluid flow stress (FSS) is a major mechanical stress that induces bone remodeling upon orthodontic tooth movement, whereas CCN family protein 2 (CCN2) is a potent regenerator of bone defects. In this study, we initially evaluated the effect of laminar FSS on Ccn2 expression and investigated its mechanism in osteoblastic MC3T3-E1 cells. The Ccn2 expression was drastically induced by uniform FSS in an intensity dependent manner. Of note, the observed effect was inhibited by a Rho kinase inhibitor Y27632. Moreover, the inhibition of actin polymerization blocked the FSS-induced activation of Ccn2, whereas inducing F-actin formation using cytochalasin D and jasplakinolide enhanced Ccn2 expression in the same cells. Finally, F-actin formation was found to induce osteoblastic differentiation. In addition, activation of cyclic AMP-dependent kinase, which inhibits Rho signaling, abolished the effect of FSS. Collectively, these findings indicate the critical role of actin polymerization and Rho signaling in CCN2 induction and bone remodeling provoked by FSS.     

Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae

ABSTRACT: BACKGROUND: Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital. RESULTS: We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6-7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials. CONCLUSIONS: We demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography.     

Relationship among coronary plaque compliance, coronary risk factors and tissue characteristics evaluated by integrated backscatter intravascular ultrasound

ABSTRACT: BACKGROUND: The purpose of the present study was to evaluate the mechanical properties of coronary plaques and plaque behavior, and to elucidate the relationship among tissue characteristics of coronary plaques, mechanical properties and coronary risk factors using integrated backscatter intravascular ultrasound (IB-IVUS). Methods: Non-targeted plaques with moderate stenosis (plaque burden at the minimal lumen site: 50-70%) located proximal to the site of the percutaneous coronary intervention target lesions were evaluated by IB-IVUS. Thirty-six plaques (less calcified group: an arc of calcification [less than or equal to]10) in 36 patients and 22 plaques (moderately calcified group: 10< an arc of calcification [less than or equal to]60) in 22 patients were evaluated. External elastic membrane volume (EEMV) compliance, lumen volume (LV) compliance, plaque volume (PV) response (difference between PV in systole and diastole), EEM area stiffness index were measured at the minimal lumen site. Relative lipid volume (lipid volume/internal elastic membrane volume) was calculated by IB-IVUS. Results: In the less calcified group, there was a significant correlation between EEMV compliance and the relative lipid volume (r=0.456, p=0.005). There was a significant inverse correlation between EEM area stiffness index and the relative lipid volume (p=0.032, r =-0.358). The LV compliance and EEM area stiffness index were significantly different in the diabetes mellitus (DM) group than in the non-DM group (1.32 +/- 1.49 vs. 2.47 +/- 1.79 %/10 mmHg, p =0.014 and 28.3 +/- 26.0 vs. 15.7 +/- 17.2, p =0.020). The EEMV compliance and EEM area stiffness index were significantly different in the hypertension (HTN) group than in the non-HTN group (0.77 +/- 0.68 vs. 1.57 +/- 0.95 %/10 mmHg, p =0.012 and 26.5 +/- 24.3 vs. 13.0 +/- 16.7, p =0.020). These relationships were not seen in the moderately calcified group. Conclusion: The present study provided new findings that there was a significant correlation between mechanical properties and tissue characteristics of coronary arteries. In addition, our results suggested that the EEMV compliance and the LV compliance were independent and the compliance was significantly impaired in the patients with DM and/or HTN. Assessment of coronary mechanical properties during PCI may provide us with useful information regarding the risk stratification of patients with coronary heart disease.     

Evaluation of extra capsular lymph node involvement in patients with extra-hepatic bile duct cancer

ABSTRACT: BACKGROUND: Lymph node metastasis (LNM) is one of the most important prognostic factors for extra-hepatic bile duct carcinoma (ExHBDC). Extra capsular lymph node involvement (ExCLNI) is the extension of cancer cells through the nodal capsule into the perinodal fatty tissue. The prognostic impact of ExCLNI has been shown to be mainly significant in head and neck malignancies. Recently, the prognostic impacts of ExCLNI have been evaluated in gastrointestinal malignancies. However, no data are available regarding the incidence and prognostic significance of extra-capsular lymph node involvement (ExCLNI) in resectable ExHBDCs. The aim of the present study is first to evaluate the incidence of ExCLNI in surgically-treated ExHBDCs and, second, to determine the prognostic impact of ExCLNI in patients with surgically-treated ExHBDCs. METHODS: A total of 228 patients, (110 cases of hilar cholangiocarcinoma and 118 cases of distal cholangiocarcinoma), with surgically-treated ExHBDCs were included in this retrospective study. ExCLNI was defined as the extension of cancer cells through the nodal capsule into the perinodal fatty tissue. The existence of ExCLNI and its prognostic value were analyzed as a subgroup of lymph node metastasis. RESULTS: ExCLNI was detected in only 22% of patients with lymph node metastasis of surgically-treated ExHBDC. The presence of ExCLNI correlated with distal cholangiocarcinoma (P = 0.002). On univariate analysis for survival, perineural invasion, vascular invasion, histological grade, and lymph node metastasis were statistically significant factors. On multivariate analysis, only lymph node metastasis was identified as a significant independent prognostic factor in patients with resectable ExHBDC. Subgroups of lymph node metastasis including the presence of ExCLNI, location of lymph node metastasis, and the number of lymph node metastasis had no statistically significant impact on survival. CONCLUSION: ExCLNI was present in only 22% of the LNM (7% of overall patients) in patients with surgically-treated ExHBDCs. And ExCLNI would have no impact on the survival of patients with surgically-treated ExHBDCs.     

Recognition of Non-Harmonic Natural Sounds by Small Mammals Using Competitive Training

Animals recognize biologically relevant sounds, such as the non-harmonic sounds made by some predators, and respond with adaptive behaviors, such as escaping. To clarify which acoustic parameters are used for identifying non-harmonic, noise-like, broadband sounds, guinea pigs were conditioned to a natural target sound by introducing a novel training procedure in which 2 or 3 guinea pigs in a group competed for food. A set of distinct behavioral reactions was reliably induced almost exclusively to the target sound in a 2-week operant training. When fully conditioned, individual animals were separately tested for recognition of a set of target-like sounds that had been modified from the target sound, with spectral ranges eliminated or with fine or coarse temporal structures altered. The results show that guinea pigs are able to identify the noise-like non-harmonic natural sounds by relying on gross spectral compositions and/or fine temporal structures, just as birds are thought to do in the recognition of harmonic birdsongs. These findings are discussed with regard to similarities and dissimilarities to harmonic sound recognition. The results suggest that similar but not identical processing that requires different time scales might be used to recognize harmonic and non-harmonic sounds, at least in small mammals.