Tarantula toxins interact with voltage sensors within lipid membranes

Voltage-activated ion channels are essential for electrical signaling, yet the mechanism of voltage sensing remains under intense investigation. The voltage-sensor paddle is a crucial structural motif in voltage-activated potassium (K(v)) channels that has been proposed to move at the protein-lipid interface in response to changes in membrane voltage. Here we explore whether tarantula toxins like hanatoxin and SGTx1 inhibit K(v) channels by interacting with paddle motifs within the membrane. We find that these toxins can partition into membranes under physiologically relevant conditions, but that the toxin-membrane interaction is not sufficient to inhibit K(v) channels. From mutagenesis studies we identify regions of the toxin involved in binding to the paddle motif, and those important for interacting with membranes. Modification of membranes with sphingomyelinase D dramatically alters the stability of the toxin-channel complex, suggesting that tarantula toxins interact with paddle motifs within the membrane and that they are sensitive detectors of lipid-channel interactions.     

MSC-DC interactions: MSC inhibit maturation and migration of BM-derived DC

BACKGROUND: Mesenchymal stromal cells (MSC) comprise one of the BM stromal cells that are known to support hematopoiesis. It has also been suggested recently that MSC display immunosuppressive capacities through inhibiting the differentiation of monocyte-derived DC. DC travel to the lymph nodes (LN) to present Ag to T cells, and CCL21 is the chemokine that plays an important role in DC migration into the T-cell area of LN. We addressed the effect of MSC on this chemotactic activity of DC, one of the typical characteristics upon maturation. METHODS: BM cells were isolated and then cultured for generation of myeloid DC in the presence of GM-CSF and/or lipopolysaccharide with or without MSC. MSC were identified by flow cytometry of the immunologic markers and by performing colony-forming unit fibroblast assay. Migration of DC was observed with a newly developed time-lapse video microscopic technique. RESULTS: MSC co-culture inhibited the initial differentiation of DC, as well as their maturation. The matured DC actively migrated directionally in response to CCL21, a powerful DC-attracting chemokine, whereas the MSC co-cultured DC did not. DISCUSSION: Collectively, the findings of these experiments raise the possibility that MSC suppress the migratory function of DC and so they may serve immunoregulatory activities through the modulation of the Ag-presenting function of DC.     

Structural requirement of phenylthiourea analogs for their inhibitory activity of melanogenesis and tyrosinase

Effect of a series of 1-phenylthioureas 1a-k and 1,3-disubstituted thioureas 2a-k were evaluated against melanin formation in melanoma B16 cell line and mushroom tyrosinase. Inhibitory activity of tyrosinase of 1-phenylthioureas 1a-k is parallel to their melanogenic inhibition. Thus, the melanogenic inhibition in melanoma B16 cells of 1-phenylthioureas could be the result of inhibition of tyrosinase. However, 1,3-diaryl or 1-phenyl-3-alkylthioureas, 2a-k, appears as melanogenic inhibitor without inhibition of tyrosinase. The molecular docking study of 1e and 2b to binding pocket of tyrosinase provided convincing explanation regarding the necessity of direct connection of planar phenyl to thiourea unit without N'-substitution of phenylthioureas 1 as tyrosinase inhibitor and 2 as non-tyrosinase inhibitor.     

Characterization and expression of the pseudorabies virus (NYJ strain) glycoproteins in Bombyx mori cells and larvae

To characterize the NYJ strain of pseudorabies virus (PRV; Alphaherpesvirus of swine) isolated from the serum of an infected swine in Korea, the nucleotide sequence of three major glycoproteins (gB, gC, and gD) was analyzed. The expression of most potent immunogenic glycoprotein (gD) was also investigated using a Bombyx mori nucleopolyhedrovirus (BmNPV) expression system. The length of the glycoprotein genes corresponding to gB, gC, and gD of the NYJ strain were 2751 bp, 1443 bp, and 1203, respectively, and their identity ranged from 94.2% to 99.8% when compared with other strains. Phylogenetic analyses using these sequences showed that the NYJ strain forms a distinct branch with high bootstrap support. A novel transfer vector (pBmKSK4) was engineered with the polyhedrin promoter of BmNPV and a 6xHis tag to express glycoprotein gD in Bm5 cells and silkworm, B. mori, larvae. The immunogenicity of recombinant gD was demonstrated by its specific detection in both Bm5 cells and silkworm larvae by porcine anti-PRV antibody. The results of this study have implications both for the taxonomy of Korean PRV strains and vaccine development.     

Comparative characteristics of three human embryonic stem cell lines

Human embryonic stem (hES) cells have unique features including unlimited growth capacity, expression of specific markers, normal karyotypes and an ability to differentiate. Many investigators have tried to use hES cells for cell-based therapy, but there is little information about the properties of available hES cell lines. We compared the characteristics of three hES cell lines. The expression of SSEA-1, -3, -4, and APase, was examined by immunocytochemistry, and Oct-4 expression was analyzed by RT-PCR. Differentiation of the hES cells in vitro and in vivo led to the formation of embryoid bodies (EBs) or teratomas. We examined the expression of tissue-specific markers in the differentiated cells by semiquantitative RT-PCR, and the ability of each hES cell line to proliferate was measured by flow cytometry of DNA content and ELISA. The three hES cell lines were similar in morphology, marker expression, and teratoma formation. However there were significant differences (P < 0.05) between the differentiated cells formed by the different cell lines in levels of expression of tissue-specific markers such as renin, kallikrein, Glut-2, beta- and delta-globin, albumin, and alpha1-antitrypsin (alpha1-AT). The hES cell lines also differed in proliferative activity. Our observations should be useful in basic and clinical hES cell research.     

Ornithinibacillus scapharcae sp. nov., isolated from a dead ark clam

A novel Gram-positive, aerobic, motile, hemolytic, endospore-forming and rod-shaped bacterium TW25^T was isolated from a dead ark clam during a mass mortality event on the South coast of Korea. The strain grew optimally at 30°C, at pH 8–9, and with 1% (w/v) NaCl. The 16S rRNA gene sequence analysis indicated that strain TW25^T was associated with the genus Ornithinibacillus and that it was most closely related to the type strain of Ornithinibacillus californiensis (98.5% similarity). The dominant cellular fatty acids were iso-C15:0, anteiso-C15:0 and C16:0. The peptidoglycan amino acid type was A4β, containing l-ornithine and d-aspartic acid. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, four unidentified phospholipids, two unidentified aminolipids and two unidentified lipids. The major respiratory quinone was menaquinone-7 (MK-7). The G + C content of genomic DNA was 36.7 mol%. DNA–DNA hybridization experiments with related strains revealed lower than 11 ± 3% relatedness. Based on this polyphasic taxonomic study, strain TW25^T represents a novel species in the genus Ornithinibacillus, for which the name Ornithinibacillus scapharcae sp. nov. is proposed. The type strain is TW25^T (=KACC 15116^T = JCM 17314^T).