Two cage-type lupin alkaloids from Sophora franchetiana

Two new cage-type lupin alkaloids, (?)-tsukushinamine-B and tsukushinamine-C, have been isolated from the fresh epigeal parts of Sophora franchetiana, along with (?)-cytisine, (?)-N-formylcytisine, (?)-rhombifoline, (?)-anagyrine, (?)-baptifoline and (±)-ammodendrine, as well as (?)-tsukushinamine-A. The structures of these novel tsukushinamine-type lupin alkaloids were determined by spectroscopic data and partly by a chemical reaction. Variations of the alkaloid contents in the seeds, seedlings and various parts of S. franchetiana were also examined.     

Development of AC microelectrophoresis for rapid protein affinity evaluation

We have developed a new method for evaluating the affinity interactions between two different proteins by applying an alternating current (AC) voltage to a micro-flow channel. An AC voltage was applied to the protein-modified microspheres in the micro-flow channel, which resulted in the oscillation of the microspheres owing to their surface charges. The oscillation amplitude showed a linear relationship with the charge density of the microspheres. As an example for protein affinity measurement, the amplitude changes of a profilin-modified microsphere were measured by the addition of actin. In the same electrical condition, the oscillation amplitude of the profilin-modified microsphere increased by ≈175% by binding with actin. Similar results in the principle were obtained for the affinity interaction between biotin and streptavidin. The results showed that the higher the charge density of the microspheres induced by binding with different proteins, the higher the oscillation amplitude of the microspheres, thus, suggesting a possible application of the micro-flow channel and AC voltage on the protein property study, as well as on the biosensor application using the oscillation amplitude changes.     

Replication Study in a Japanese Population of Six Susceptibility Loci for Type 2 Diabetes Originally Identified by a Transethnic Meta-Analysis of Genome-Wide Association Studies

AimWe performed a replication study in a Japanese population to evaluate the association between type 2 diabetes and six susceptibility loci (TMEM154, SSR1, FAF1, POU5F1, ARL15, and MPHOSPH9) originally identified by a transethnic meta-analysis of genome-wide association studies (GWAS) in 2014.MethodsWe genotyped 7,620 Japanese participants (5,817 type 2 diabetes patients and 1,803 controls) for each of the single nucleotide polymorphisms (SNPs) using a multiplex polymerase chain reaction invader assay. The association of each SNP locus with the disease was evaluated using logistic regression analysis.ResultsOf the six SNPs examined in this study, four (rs6813195 near TMEM154, rs17106184 in FAF1, rs3130501 in POU5F1 and rs4275659 near MPHOSPH9) had the same direction of effect as in the original reports, but two (rs9505118 in SSR1 and rs702634 in ARL15) had the opposite direction of effect. Among these loci, rs3130501 and rs4275659 were nominally associated with type 2 diabetes (rs3130501; p = 0.017, odds ratio [OR] = 1.113, 95% confidence interval [CI] 1.019–1.215, rs4275659; p = 0.012, OR = 1.127, 95% CI 1.026–1.238, adjusted for sex, age and body mass index), but we did not observe a significant association with type 2 diabetes for any of the six evaluated SNP loci in our Japanese population.ConclusionsOur results indicate that effects of the six SNP loci identified in the transethnic GWAS meta-analysis are not major among the Japanese, although SNPs in POU5F1 and MPHOSPH9 loci may have some effect on susceptibility to type 2 diabetes in this population.     

Gain-of-function screen identifies a role of the Sec61alpha translocon in Drosophila postmitotic neurotoxicity

To elucidate the intrinsic mechanisms of neurotoxicity induction, including those underlying neural cell death and neurodegeneration, we developed a gain-of-function screen for gene products causing neural cell loss. To identify novel genes with a cell-death-related function in neurons, we screened 4,964 Drosophila GS lines, in which one or two genes from much of the Drosophila genome can be overexpressed. Approximately 0.68% of the GS lines produced phenotypes involving a loss of postmitotic neurons. Of these, we identified and characterized the endd2 gene, which encodes the Drosophila ortholog of Sec61alpha (DSec61alpha), an endoplasmic reticulum protein with protein translocation activity. Ectopic expression of DSec61alpha caused neural cell death accompanied by the accumulation of ubiquitinated proteins, which was mediated by DSec61alpha's translocon activity. This supported our previous observation that the DSec61alpha translocon contributes to expanded polyglutamine-mediated neuronal toxicity, which is also associated with ubiquitinated protein accumulation. These data suggest that the translocon may be a novel component of neural cell death and degeneration pathways. Our approach can be used to identify potential neurotoxic factors within the whole genome, which will increase our understanding of the molecular mechanisms of various types of cell death, including those associated with human neurodegenerative diseases.     

Transcriptional induction of Smurf2 ubiquitin ligase by TGF-beta

Smad ubiquitination regulatory factor 2 (Smurf2), a ubiquitin ligase for Smads, plays critical roles in the regulation of transforming growth factor-beta (TGF-beta)-Smad signaling via ubiquitin-dependent degradation of Smad2 and Smad7. We found that TGF-beta stimulates Smurf2 expression. TGF-beta activated the Smurf2 promoter in a TGF-beta responsive cell lines, whereas IL-1alpha, PDGF and epidermal growth factor did not. TGF-beta-mediated Smurf2 promoter activation was inhibited by Smad7 or an activin receptor-like kinase 5 inhibitor but not by dominant negative Smad or disruption of Smad-binding elements in the promoter. Moreover, inhibition of the phosphatidil inositol 3 kinase (PI3K)/Akt pathway suppressed TGF-beta-mediated Smurf2 induction. These results suggest that TGF-beta stimulates Smurf2 expression by Smad-independent pathway such as PI3K/Akt pathway via TGF-beta receptor.     

Nicotine affects mineralized nodule formation by the human osteosarcoma cell line Saos-2

Several in vitro and in vivo studies have indicated that tobacco smoking may be an important risk factor for the development and severity of inflammatory periodontal disease. In the present study, we examined the effect of nicotine on cell proliferation, alkaline phosphatase (ALPase) activity, mineralized nodule formation, and the expression of extracellular matrix proteins in the human osteosarcoma cell line Saos-2. The cells were cultured with Dulbecco's modified Eagle medium containing 10% fetal bovine serum with 0, 10(-4) M, and 10(-3) M nicotine for up to 14 days. Mineralized nodule formation was examined by alizarin red staining, and the calcium content in mineralized nodules was determined using a calcium E-test kit. The expression of extracellular matrix proteins was estimated by determining the levels of their mRNAs using the real-time polymerase chain reaction. Mineralized nodule formation and calcium content in mineralized nodules were remarkably suppressed by nicotine on days 10 and 14 of culture, respectively. ALPase activity as well as type I collagen and osteopontin expression also decreased in the presence of nicotine after 5, 10, and 14 days of culture, respectively. By contrast, the amount of bone sialoprotein increased during 14 days of culture with nicotine. These results suggest that nicotine suppresses osteogenesis through a decrease in ALPase and type I collagen production by osteoblasts.     

Sex Differences in the Default Mode Network with Regard to Autism Spectrum Traits: A Resting State fMRI Study

Autism spectrum traits exist on a continuum and are more common in males than in females, but the basis for this sex difference is unclear. To this end, the present study draws on the extreme male brain theory, investigating the relationship between sex difference and the default mode network (DMN), both known to be associated with autism spectrum traits. Resting-state functional magnetic resonance imaging (MRI) was carried out in 42 females (mean age ± standard deviation, 22.4 ± 4.2 years) and 43 males (mean age ± standard deviation, 23.8 ± 3.9 years) with typical development. Using a combination of different analyses (viz., independent component analysis (ICA), fractional amplitude of low-frequency fluctuation (fALFF), regional homogeneity (ReHo), and seed-based analyses), we examined sex differences in the DMN and the relationship to autism spectrum traits as measured by autism-spectrum quotient (AQ) scores. We found significant differences between female and male subjects in DMN brain regions, with seed-based analysis revealing a significant negative correlation between default-mode resting state functional connectivity of the anterior medial prefrontal cortex seed (aMPFC) and AQ scores in males. However, there were no relationships between DMN sex differences and autism spectrum traits in females. Our findings may provide important insight into the skewed balance of functional connectivity in males compared to females that could serve as a potential biomarker of the degree of autism spectrum traits in line with the extreme male brain theory.