Spectrophotometric determination of inorganic phosphate in the presence of thiol compounds

A spectrophotometric method for inorganic phosphate determination in the presence of thiol compounds is described. Thiol compounds, which interfere with the measurement of inorganic phosphate by a modification of the method of Gomori, are removed by carboxymethylation by iodoacetate prior to the formation and reduction of phosphomolybdate complex. A linear standard curve is obtained by this method, and the method is suitable for the assay of a phosphate-releasing enzyme when the measurement must be performed in the presence of thiol compounds.     

Reproduction of Japanese encephalitis virus in human glioma cells, 118MGC: inhibitory effect of host factor(s)

Japanese encephalitis viruses (JEV) were well propagated in human glioma cells, 118MGC until the first 24 hrs after virus infection. However, after 24 hrs, virus growth rate was quickly reduced. This unusual pattern of virus growth was different from the cases in others cells, e.g. IMR-32, Vero and C6/36 cells. The fact that actinomycin-D retained the high yields of JEV in 118MGC cells suggests that some suppressing factors against JEV replication are produced in MGC cells. Interestingly, culture fluids of 118MGC cells indicated inhibitory effect to JEV reproduction, but other culture fluids from several cell lines had no effect. This inhibitory effect of the MGC-culture fluids was lost by heat-treatment at 60 C. In addition, the infectivity of JEV was rapidly decreased by the incubation with MGC-culture fluids. These findings suggest that 118MGC cells produce and secret some inhibitory factors against JEV replication.     

Morphological changes induced by the calcium ionophore A23187 in rat basophilic leukemia (2H3) cells.

RBL-2H3 cells have been widely used to study histamine release in vitro. It was previously shown that these cells undergo striking morphological changes after IgE-mediated secretion. The present study was undertaken to examine if the morphological changes were dependent on activation of the Fc epsilon receptor. Therefore, the cells were stimulated to release histamine by two different mechanisms: activation of the Fc epsilon receptor by antigen and treatment with the calcium ionophore A23187. Cell surface and cytoskeletal changes were examined by fluorescence microscopy and scanning electron microscopy after either IgE- or ionophore-mediated histamine release. After exposure of the cells to either secretagogue, the cells spread over the surface of the culture dish and underwent rearrangement of the cytoskeleton. In addition, scanning electron microscopy revealed that deep ruffles developed on the surface of the cells undergoing IgE-mediated release. The surface changes were not as pronounced with the ionophore. The distribution of the cytoskeletal elements was examined by immunofluorescence using FITC-phalloidin and antibodies against vimentin and tubulin. In unstimulated cells actin was localized at the cell periphery, just under the plasma membrane. In the stimulated cells it was associated with the cell periphery and concentrated in the surface ruffles. As the stimulated cells spread, intermediate filaments and microtubules became distributed throughout the cell body, but there was no obvious association with the membrane ruffles. These morphological changes were dependent on the presence of extracellular calcium and on the concentration of ionophore or antigen, and were also correlated with the amount of histamine released. Additionally, IgE-mediated stimulation led to increased uptake of the soluble-phase tracer Lucifer yellow, whereas stimulation with the ionophore A23187 showed no increase in Lucifer yellow internalization. Ionophore A23187 produced changes similar but not identical to those seen in the RBL-2H3 cells after IgE-mediated histamine release. The differences may be owing to the involvement of the Fc epsilon receptor in IgE-mediated secretion.     

XX/XY System of Sex Determination in the Geophilomorph Centipede Strigamia maritima

We show that the geophilomorph centipede Strigamia maritima possesses an XX/XY system of sex chromosomes, with males being the heterogametic sex. This is, to our knowledge, the first report of sex chromosomes in any geophilomorph centipede. Using the recently assembled Strigamia genome sequence, we identified a set of scaffolds differentially represented in male and female DNA sequence. Using quantitative real-time PCR, we confirmed that three candidate X chromosome-derived scaffolds are present at approximately twice the copy number in females as in males. Furthermore, we confirmed that six candidate Y chromosome-derived scaffolds contain male-specific sequences. Finally, using this molecular information, we designed an X chromosome-specific DNA probe and performed fluorescent in situ hybridization against mitotic and meiotic chromosome spreads to identify the Strigamia XY sex-chromosome pair cytologically. We found that the X and Y chromosomes are recognizably different in size during the early pachytene stage of meiosis, and exhibit incomplete and delayed pairing.     

Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1⁺ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1⁺ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1⁺ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.     

Population and Single-Cell Analysis of Human Cardiogenesis Reveals Unique LGR5 Ventricular Progenitors in Embryonic Outflow Tract

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Structural basis of the strict specificity of a bacterial GH31 α-1,3-glucosidase for nigerooligosaccharides

Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is one of the most diverse families of carbohydrate-active enzymes, containing various enzymes that act on α-glycosides. However, the function of some GH31 groups remains unknown, as their enzymatic activity is difficult to estimate due to the low amino acid sequence similarity between characterized and uncharacterized members. Here, we performed a phylogenetic analysis and discovered a protein cluster (GH31_u1) sharing low sequence similarity with the reported GH31 enzymes. Within this cluster, we showed that a GH31_u1 protein from Lactococcus lactis (LlGH31_u1) and its fungal homolog demonstrated hydrolytic activities against nigerose [α-D-Glcp-(1→3)-D-Glc]. The k_cat/K_m values of LlGH31_u1 against kojibiose and maltose were 13% and 2.1% of that against nigerose, indicating that LlGH31_u1 has a higher specificity to the α-1,3 linkage of nigerose than other characterized GH31 enzymes, including eukaryotic enzymes. Furthermore, the three-dimensional structures of LlGH31_u1 determined using X-ray crystallography and cryogenic electron microscopy revealed that LlGH31_u1 forms a hexamer and has a C-terminal domain comprising four α-helices, suggesting that it contributes to hexamerization. Finally, crystal structures in complex with nigerooligosaccharides and kojibiose along with mutational analysis revealed the active site residues involved in substrate recognition in this enzyme. This study reports the first structure of a bacterial GH31 α-1,3-glucosidase and provides new insight into the substrate specificity of GH31 enzymes and the physiological functions of bacterial and fungal GH31_u1 members.     

Genetic relationship and distribution of the Japanese wild boar (Sus scrofa leucomystax) and Ryukyu wild boar (Sus scrofa riukiuanus) analysed by mitochondrial DNA

Mitochondrial genetic variations were used to investigate the relationships between two Japanese wild boars, Japanese wild boar (Sus scrofa leucomystax) and Ryukyu wild boar (S.s. riukiuanus). Nucleotide sequences of the control (27 haplotypes) and cytochrome b (cyt-b) regions (19 haplotypes) were determined from 59 Japanese wild boars, 13 Ryukyu wild boars and 22 other boars and pigs. From phylogenetic analyses, the mtDNA of Ryukyu wild boar has a distinct lineage from that of Japanese wild boar, which was classified into the Asian pig lineage. This result suggests that the Ryukyu wild boar has a separate origin from the Japanese wild boar.     

Series of vectors to evaluate the position and the order of two different affinity tags for purification of protein complexes

A series of protein expression vectors with dual-affinity tags has been developed. With these constructed vectors, FLAG and hexahistidine tags were fused to a given protein at either the N- or the C-terminal ends or both, for a total of six combinations. Three auxotrophy markers were introduced into each construct, thus yielding 18 different vectors. These vectors allow evaluation of different positions and orders of two different tags. To confirm the efficacy of these vectors, we purified a histone acetyltransferase (Esa1p)-containing complex. First, an appropriate position of the tags was selected through small-scale purification. Next, large-scale purification was done for the selected construct, yielding an Esa1p-containing complex that was comparable to an Esa1p-containing complex (NuA4) obtained by a conventional activity-based purification. These vectors provide a convenient way to select the best position of tags for efficient purification of protein complexes also applicable in proteomics studies.     

Synthesis of sulfoquinovosylacylglycerols, inhibitors of eukaryotic DNA polymerase alpha and beta

Sulfoquinovosyldiacylglycerols (SQDGs) and sulfoquinovosylmonoacylglycerols (SQMGs), bearing diverse fatty acids, were synthesized from D-glucose, and were examined for enzymatic inhibitions of DNA polymerase alpha and beta. These results indicated that the carbon numbers of the fatty acids were highly related to the activities, at least in vitro, of eukaryotic DNA polymerase inhibition.