Reversible photochromism of novel silver(I) coordination complexes with 1,2-bis(2′-methyl-5′-(2″-pyridyl)-3′-thienyl)perfluorocyclopentene in crystalline phase

Three novel silver(I) complexes with 1,2-bis(2-methyl-5′-(2″-pyridyl)-3′-thienyl)perfluorocyclopentene (BM-2-PTP) were synthesized by the reaction of Ag(CF₃SO₃) or Ag(CF₃COO) with BM-2-PTP in benzene at different temperatures. The structures of these metal complexes were revealed by X-ray crystallographic analyses and the correlation between crystal structures and photochromic performance was discussed. In complexes 1 and 2, silver(I) is three-coordinated to two nitrogens from distinct ligand molecules as well as one oxygen from anions to form a 1-D polymeric structure. On the other hand, complex 3 contains two crystallographic independent Ag(I) with different coordination environments, and the adjacent BM-2-PTP molecules are connected by Ag-CF₃CO₂-Ag chains to afford a 1-D double chain structure. The difference in structures of three complexes shows the interesting anionic effect on coordination and the subtleness of crystal engineering. It is noted that complex 3 underwent reversible photochromic reaction in crystalline state despite the unfavorable framework to the rotation of thiophene groups.     

Adaptive thresholds to detect differentially expressed genes in microarray data

To detect changes in gene expression data from microarrays, a fixed threshold for fold difference is used widely. However, it is not always guaranteed that a threshold value which is appropriate for highly expressed genes is suitable for lowly expressed genes. In this study, aiming at detecting truly differentially expressed genes from a wide expression range, we proposed an adaptive threshold method (AT). The adaptive thresholds, which have different values for different expression levels, are calculated based on two measurements under the same condition. The sensitivity, specificity and false discovery rate (FDR) of AT were investigated by simulations. The sensitivity and specificity under various noise conditions were greater than 89.7% and 99.32%, respectively. The FDR was smaller than 0.27. These results demonstrated the reliability of the method.     

Pre- and postsynaptic inhibition mediated by GABA(B) receptors in rat ventrolateral periaqueductal gray neurons

The present study examined the actions of a GABA(B)-receptor agonist, baclofen, on synaptic transmission in rat ventrolateral periaqueductal gray (PAG) neurons of brainstem slices by using whole-cell voltage-clamp recordings. Baclofen (10 microM) induced a slow outward current (peak amplitude: 30.1+/-3.1pA, n=13) at -70mV, which persisted in the presence of tetrodotoxin (0.5 microM) and was diminished in the presence of postsynaptic intracellular K(+)-channel blockers (Cs(+) and TEA) and GDP-beta-S, indicating a direct postsynaptic depression mediated by K(+) channels and G proteins. Baclofen (10 microM) also decreased the frequency of both glutamatergic spontaneous EPSC (by 36+/-7%, n=11) and GABAergic spontaneous IPSC (by 37+/-12%, n=6) without changes in their amplitudes, indicating its presynaptic inhibitions. Taken together, the activation of postsynaptic GABA(B) receptors inhibits ventrolateral PAG neurons directly. At the same time, activating presynaptic GABA(B) receptors on glutamatergic and GABAergic nerve terminals inhibits glutamate and GABA release, respectively. The overall effects might influence an output of ventrolateral PAG neurons that build up the descending pain control system to the spinal dorsal horn.     

Derivation of Mesenchymal Stromal Cells from Pluripotent Stem Cells through a Neural Crest Lineage using Small Molecule Compounds with Defined Media

Neural crest cells (NCCs) are an embryonic migratory cell population with the ability to differentiate into a wide variety of cell types that contribute to the craniofacial skeleton, cornea, peripheral nervous system, and skin pigmentation. This ability suggests the promising role of NCCs as a source for cell-based therapy. Although several methods have been used to induce human NCCs (hNCCs) from human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), further modifications are required to improve the robustness, efficacy, and simplicity of these methods. Chemically defined medium (CDM) was used as the basal medium in the induction and maintenance steps. By optimizing the culture conditions, the combination of the GSK3β inhibitor and TGFβ inhibitor with a minimum growth factor (insulin) very efficiently induced hNCCs (70–80%) from hPSCs. The induced hNCCs expressed cranial NCC-related genes and stably proliferated in CDM supplemented with EGF and FGF2 up to at least 10 passages without changes being observed in the major gene expression profiles. Differentiation properties were confirmed for peripheral neurons, glia, melanocytes, and corneal endothelial cells. In addition, cells with differentiation characteristics similar to multipotent mesenchymal stromal cells (MSCs) were induced from hNCCs using CDM specific for human MSCs. Our simple and robust induction protocol using small molecule compounds with defined media enabled the generation of hNCCs as an intermediate material producing terminally differentiated cells for cell-based innovative medicine.     

Identification of hepatic niche harboring human acute lymphoblastic leukemic cells via the SDF-1/CXCR4 axis

In acute lymphoblastic leukemia (ALL) patients, the bone marrow niche is widely known to be an important element of treatment response and relapse. Furthermore, a characteristic liver pathology observed in ALL patients implies that the hepatic microenvironment provides an extramedullary niche for leukemic cells. However, it remains unclear whether the liver actually provides a specific niche. The mechanism underlying this pathology is also poorly understood. Here, to answer these questions, we reconstituted the histopathology of leukemic liver by using patients-derived primary ALL cells into NOD/SCID/Yc (null) mice. The liver pathology in this model was similar to that observed in the patients. By using this model, we clearly demonstrated that bile duct epithelial cells form a hepatic niche that supports infiltration and proliferation of ALL cells in the liver. Furthermore, we showed that functions of the niche are maintained by the SDF-1/CXCR4 axis, proposing a novel therapeutic approach targeting the extramedullary niche by inhibition of the SDF-1/CXCR4 axis. In conclusion, we demonstrated that the liver dissemination of leukemia is not due to nonselective infiltration, but rather systematic invasion and proliferation of leukemic cells in hepatic niche. Although the contribution of SDF-1/CXCR4 axis is reported in some cancer cells or leukemic niches such as bone marrow, we demonstrated that this axis works even in the extramedullary niche of leukemic cells. Our findings form the basis for therapeutic approaches that target the extramedullary niche by inhibiting the SDF-1/CXCR4 axis.     

High-Efficiency and Robust Expression System for Stable Isotope-Labeled Peptides

Ubiquitin–peptide fusion protein system enables preparation of stable isotope labeled peptides through the expression of the protein in E. coli cells in labeled media (Kohno et al. (1998) J Biomol NMR 12:109–121). Advantages of the system over others include: very specific cleavage of the bond between ubiquitin and target peptide moieties by yeast ubiquitin hydrolase and low cost for the protease which can also be expressed in E. coli cells. The former point is particularly important since other frequently used proteases, such as factor Xa and thrombin, often show non-specific cleavages at sites unexpected from their nominal specificities. We improved the yield of the peptide by adapting the codon usage of ubiquitin gene for the expression in E. coli cells, by using RNase E-deficient host strains, and by modifying purification procedure. The yield of mastoparan-X was increased threefold by these modifications. We also succeeded in the preparation of labeled magainin 2, an antimicrobial peptide that could not be expressed at all by the previous method, by choosing host strains and culture media. The HSQC signals of the ¹⁵N-labeled magainin 2 in an aqueous solution were completely resolved in spite of the severe overlap of the 1D proton signals, confirming that the stable isotope labeling is quite useful for analysis of peptides.     

Effects of Escherichia coli ribosomal protein S12 mutations on cell-free protein synthesis.

We examined the effects of Escherichia coli ribosomal protein S12 mutations on the efficiency of cell-free protein synthesis. By screening 150 spontaneous streptomycin-resistant isolates from E. coli BL21, we successfully obtained seven mutants of the S12 protein, including two streptomycin-dependent mutants. The mutations occurred at Lys42, Lys87, Pro90 and Gly91 of the 30S ribosomal protein S12. We prepared S30 extracts from mutant cells harvested in the mid-log phase. Their protein synthesis activities were compared by measuring the yields of the active chloramphenicol acetyltransferase. Higher protein production (1.3-fold) than the wild-type was observed with the mutant that replaced Lys42 with Thr (K42T). The K42R, K42N, and K42I strains showed lower activities, while the other mutant strains with Lys87, Pro90 and Pro91 did not show any significant difference from the wild-type. We also assessed the frequency of Leu misincorporation in poly(U)-dependent poly(Phe) synthesis. In this assay system, almost all mutants showed higher accuracy and lower activity than the wild-type. However, K42T offered higher activity, in addition to high accuracy. Furthermore, when 14 mouse cDNA sequences were used as test templates, the protein yields of nine templates in the K42T system were 1.2-2 times higher than that of the wild-type.