Soybean Seed Extracts Preferentially Express Genomic Loci of Bradyrhizobium japonicum in the Initial Interaction with Soybean, Glycine max (L.) Merr

Initial interaction between rhizobia and legumes actually starts via encounters of both partners in the rhizosphere. In this study, the global expression profiles of Bradyrhizobium japonicum USDA 110 in response to soybean (Glycine max) seed extracts (SSE) and genistein, a major soybean-released isoflavone for nod genes induction of B. japonicum, were compared. SSE induced many genomic loci as compared with genistein (5.0 µM), nevertheless SSE-supplemented medium contained 4.7 µM genistein. SSE markedly induced four predominant genomic regions within a large symbiosis island (681 kb), which include tts genes (type III secretion system) and various nod genes. In addition, SSE-treated cells expressed many genomic loci containing genes for polygalacturonase (cell-wall degradation), exopolysaccharide synthesis, 1-aminocyclopropane-1-carboxylate deaminase, ribosome proteins family and energy metabolism even outside symbiosis island. On the other hand, genistein-treated cells exclusively showed one expression cluster including common nod gene operon within symbiosis island and six expression loci including multidrug resistance, which were shared with SSE-treated cells. Twelve putatively regulated genes were indeed validated by quantitative RT-PCR. Several SSE-induced genomic loci likely participate in the initial interaction with legumes. Thus, these results can provide a basic knowledge for screening novel genes relevant to the B. japonicum- soybean symbiosis.Key words: soybean seed extracts, Bradyrhizobium japonicum, expression clusters, genistein, symbiosis     

Conformation-activity relationship on novel 4-pyridylmethylthio derivatives with antiangiogenic activity

We found 4-pyridylmethylthio derivative 1 to be very effective in using antiangiogenesis activity to prevent proliferation of HUVECs (Human Umbilical Vein Endothelial Cells), which was induced by vascular endothelial growth factor (VEGF). Compound 1 was equally effective in inhibiting VEGF receptor2 tyrosine kinase (KDR, IC₅₀ = 26 nM). We deduced that the inhibition was the result of binding the catalytic domain of VEGF receptor2 tyrosine kinase in a similar fashion to both phthalazine derivative PTK787 2 and anthranylamide derivative AAL993 3. In this report, we will describe the conformational analyses, from ab initio MO calculation and X-ray crystallographic analyses, of compound 1 and the analogs, which include non-active 9, all in comparison with 2 and 3. The conformation–activity relationships suggest that a nonbonded intramolecular interaction between the sulfur and the carbonyl oxygen of 1 was very important in inhibiting KDR.     

Neuroinvasiveness of the MR766 strain of Zika virus in IFNAR-/- mice maps to prM residues conserved amongst African genotype viruses

Zika virus (ZIKV) strains are classified into the African and Asian genotypes. The higher virulence of the African MR766 strain, which has been used extensively in ZIKV research, in adult IFNα/β receptor knockout (IFNAR^-/-) mice is widely viewed as an artifact associated with mouse adaptation due to at least 146 passages in wild-type suckling mouse brains. To gain insights into the molecular determinants of MR766’s virulence, a series of genes from MR766 were swapped with those from the Asian genotype PRVABC59 isolate, which is less virulent in IFNAR^-/- mice. MR766 causes 100% lethal infection in IFNAR^-/- mice, but when the prM gene of MR766 was replaced with that of PRVABC59, the chimera MR/PR(prM) showed 0% lethal infection. The reduced virulence was associated with reduced neuroinvasiveness, with MR766 brain titers ≈3 logs higher than those of MR/PR(prM) after subcutaneous infection, but was not significantly different in brain titers of MR766 and MR/PR(prM) after intracranial inoculation. MR/PR(prM) also showed reduced transcytosis when compared with MR766 in vitro. The high neuroinvasiveness of MR766 in IFNAR^-/- mice could be linked to the 10 amino acids that differ between the prM proteins of MR766 and PRVABC59, with 5 of these changes affecting positive charge and hydrophobicity on the exposed surface of the prM protein. These 10 amino acids are highly conserved amongst African ZIKV isolates, irrespective of suckling mouse passage, arguing that the high virulence of MR766 in adult IFNAR^-/- mice is not the result of mouse adaptation.     

Novel features of computer-simulated clonal life of Paramecium caudatum

The clone of the ciliated protozoan Paramecium caudatum has the immaturity period of about 60 fissions and the lifespan of about 600 fissions. These life cycle figures have been depicted through laboratory experiments that allow continuous cell divisions for hundreds, which never occur in nature. We here constructed the nature-mimicking model culture that alternated the log- and stationary phases to allow conjugation, and computer-simulated the age structure modifying parameters such as cell distributions to start the culture, fission rates, death rates, immaturity periods, probabilities of conjugation, proportions of transplantation and so on. The average and maximum ages in the culture after thousands of alternations were converged to 43±2 and 140±5 fissions, respectively, when parameters for the immaturity period and the maximum clonal lifespan were set at 60 and 600 fissions. This result explains why cells collected in nature are usually young and vigorous. The average and maximum ages proportionally prolonged as the immaturity period was prolonged, as reported true for species of the ciliate. These results indicate the validity of our simulation. The average and maximum ages remained unchanged when the initial condition for starting the culture was changed from two complementary mating-type cells to a population with a quadratic-function distribution, and when the fission rate at the log-phase and the death rate at the stationary phase were modified for older ages. The average and maximum ages changed slightly when either the conjugation rate or the proportion of transplantation was somewhat lowered. Although they changed considerably when such parameters as the immaturity period, conjugation rate and death rate were extremely modified, no clones with the age over 230 fissions appeared in any simulations. These results indicate the robustness of the model, which provides us with fresh insight into the structural system of the clonal lifespan of P. caudatum in nature.     

Cigarette smoking and serum soluble Fas levels: Findings from the JACC study

Cigarette smoking enhances low-grade systemic inflammation in the lung and other organs. Activated immune cells play an important role at early and late stages of inflammation, and in recent years, soluble Fas (sFas), an isoform of death molecule Fas, was found to interfere with the apoptotic pathways of these activated immune cells. The aim of this study was to confirm the association between cigarette smoking and sFas levels in healthy male subjects. We measured serum sFas levels of 4415 male subjects selected as controls for a nested case-control study within the large-scale cohort study conducted in Japan, called the JACC Study. Smoking status at baseline was evaluated by a self-administered questionnaire. Least square means of sFas according to smoking status and numbers of cigarettes smoked per day among smokers were calculated and adjusted for possible confounding factors. Mean sFas levels showed an increasing trend across never smokers, past smokers and current smokers, as 2.21 (95% CI: 2.14–2.27) ng/ml, 2.29 (2.22–2.36) ng/ml, and 2.36 (2.30–2.43) ng/ml, respectively. However, no dose-response relationship was observed between the number of cigarettes smoked per day and sFas levels among smokers.     

Glycosphingolipids are not pivotal receptors for Subtilase cytotoxin in vivo: sensitivity analysis with glycosylation-defective mutant mice

Certain glycosphingolipids play important roles as cellular receptor for bacterial toxins with high specificity and strong affinity. In particular AB(5) toxins exhibit typical modes of cell attachment with B5 and invasion and biological effects in cells with A subunit. Subtilase cytotoxin (SubAB) is the prototype of a recently discovered AB(5) cytotoxin family produced by certain strains of Shiga toxigenic Escherichia coli, and shows highly specific serine protease activity toward endoplasmic reticulum chaperone Bip. Since this toxin bound to a mimic of ganglioside GM2, GM2 has been considered to be possible receptor for SubAB. Using six kinds of glycosylation-defective knockout mice lacking certain group of glycosphingolipids, sensitivity to SubAB in vivo was analyzed. Consequently, all mutant mice died at around 70h after intraperitoneal injection of 10 microg (or 7.5 microg) of SubAB as well as wild type mice. These results indicated none of glycolipids are not pivotal receptor for SubAB in the body.