Synthesis of biomolecules from N2, CO,and H2O by electric discharge

A model primitive gas containing a mixture of N₂, CO and water vapor over a water pool (300 mL, 37 °C) was subjected to electric discharges. The discharge vessel (7 L in volume) was equipped with a CO₂ absorber (The CO₂ being formed during the discharge), thus simulating possible absorption of CO₂ in the primitive ocean. The vessel also has a cold trap ( –15 °C), which protects the primary products against the further decomposition in the discharge phase by enabling these products to adhere to the trap. Since the partial pressures of CO and N₂ decreased at rates of 1.5–1.7 cmHg day^–1 and 0.1–0.2 cmHg day^–1, respectively, the gases were added at regular intervals. The solution was analyzed at regular intervals for HCN, HCHO and urea, and maximum concentrations of about 50, 2, and 140 mM were observed. The discharge phase was continued for 6 months. In the solution, glycine (5.6% yield based on the carbon), glycylglycine (0.64%), orotic acid (0.004%) and small amounts of the other amino acids were found.     

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.     

Nicotine induces dendritic spine remodeling in cultured hippocampal neurons

Cholinergic neurons in the CNS are involved in synaptic plasticity and cognition. Both muscarinic and nicotinic acetylcholine receptors (nAChRs) influence plasticity and cognitive function. The mechanism underlying nAChR‐induced plasticity, however, has remained elusive. Here, we demonstrate morphological changes in dendritic spines following activation of α4β2* nAChRs, which are expressed on glutamatergic pre‐synaptic termini of cultured hippocampal neurons. Exposure of the neurons to nicotine resulted in a lateral enlargement of spine heads. This was abolished by dihydro‐β‐erythroidine, an antagonist of α4β2* nAChRs, but not by α‐bungarotoxin, an antagonist of α7 nAChRs. Tetanus toxin or a mixture of 2‐amino‐5‐phosphonovaleric acid and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, antagonists of NMDA‐ and AMPA‐type glutamate receptors, blocked the nicotine‐induced spine remodeling. In addition, nicotine exerted full spine‐enlarging response in the post‐synaptic neuron whose β2 nAChR expression was knocked down. Finally, pre‐treatment with nicotine enhanced the Ca²⁺‐response of the neurons to glutamate. These data suggest that nicotine influences the activity of glutamatergic neurotransmission through the activation of pre‐synaptic α4β2 nAChRs, resulting in the modulation of spinal architecture and responsiveness. The present findings may represent one of the cellular mechanisms underlying cholinergic tuning of brain function.

     

Continuous culture of novel mitochondrial cells lacking nuclei

We isolated stable cell lines, designated as mitochondrial cells, from cybrids obtained by fusing mitochondria-less HeLa cells with platelets from patients with Leigh syndrome, a subtype of mitochondrial encephalomyopathy. The cells contain a pathogenic point mutation, T9176C, in the mitochondrial DNA. Hematoxylin-eosin staining, confocal fluorescent microscopy and flow cytometry in fixed or living cells showed that the majority of these mitochondrial cells lack nuclear DNA and nuclei, but contain active mitochondria. Despite the absence of nuclear DNA, these cells can be continuously generated in culture. Therefore, it is likely that they arise from the minority of cells which possess a nucleus.     

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.     

Xylose (glucose) isomerase gene from the thermophile Thermus thermophilus: cloning, sequencing, and comparison with other thermostable xylose isomerases.

The xylose isomerase gene from the thermophile Thermus thermophilus was cloned by using a fragment of the Streptomyces griseofuscus gene as a probe. The complete nucleotide sequence of the gene was determined. T. thermophilus is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized. The gene codes for a polypeptide of 387 amino acids with a molecular weight of 44,000. The Thermus xylose isomerase is considerably more thermostable than other described xylose isomerases. Production of the enzyme in Escherichia coli, by using the tac promoter, increases the xylose isomerase yield 45-fold compared with production in T. thermophilus. Moreover, the enzyme from E. coli can be purified 20-fold by simply heating the cell extract at 85 degrees C for 10 min. The characteristics of the enzyme made in E. coli are the same as those of enzyme made in T. thermophilus. Comparison of the Thermus xylose isomerase amino acid sequence with xylose isomerase sequences from other organisms showed that amino acids involved in substrate binding and isomerization are well conserved. Analysis of amino acid substitutions that distinguish the Thermus xylose isomerase from other thermostable xylose isomerases suggests that the further increase in thermostability in T. thermophilus is due to substitution of amino acids which react during irreversible inactivation and results also from increased hydrophobicity.     

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.