Hydrogen peroxide formation of polymorphonuclear leukocytes stimulated with cytochalasin D

Possible cellular sites to form superoxide (O₂⁻) and H₂O₂ were studied in polymorphonuclear leukocytes stimulated with either cytochalasin D (CD) or bacteria. When the cells were stimulated with cytochalasin, the ratio of O₂⁻ to H₂O₂ appearing in the medium was about 2 (determined separately) and the appearance of H₂O₂ in the medium was inhibited by the addition of cytochrome c, a scavenger of O₂⁻. The inhibition was reversed by the addition of superoxide dismutase. The cells phagocytosing bacteria released O₂⁻ at the beginning but the release subsided corresponding to the increase in the direct release of H₂O₂. p-Chloromercuribenzenesulfonate, a non-permeant reagent, inhibited the H₂O₂ formation by the cells stimulated with CD, whereas it did not inhibit the formation by the phagocytosing cells. These observations supported a hypothesis that the cells stimulated with the cytochalasin release O₂⁻ from the cell surface, whereas the phagocytosing cells release H₂O₂ from an intracellular site, probably the phagosomes formed by the invagination of the plasma membrane. An electron microscopic finding that the vacuole-like structures found in the cytochalasin-treated cells have openings to outside, also supported the hypothesis. The particle-bound NADPH-oxidase activity of the cytochalasin-treated cells was several times higher than that of the resting cells and the activation was apparently parallel with the O₂⁻-releasing activity of the cells. No activation of the NADH-oxidase was observed.     

MEGA11: Molecular Evolutionary Genetics Analysis Version 11

The Molecular Evolutionary Genetics Analysis (MEGA) software has matured to contain a large collection of methods and tools of computational molecular evolution. Here, we describe new additions that make MEGA a more comprehensive tool for building timetrees of species, pathogens, and gene families using rapid relaxed-clock methods. Methods for estimating divergence times and confidence intervals are implemented to use probability densities for calibration constraints for node-dating and sequence sampling dates for tip-dating analyses. They are supported by new options for tagging sequences with spatiotemporal sampling information, an expanded interactive Node Calibrations Editor, and an extended Tree Explorer to display timetrees. Also added is a Bayesian method for estimating neutral evolutionary probabilities of alleles in a species using multispecies sequence alignments and a machine learning method to test for the autocorrelation of evolutionary rates in phylogenies. The computer memory requirements for the maximum likelihood analysis are reduced significantly through reprogramming, and the graphical user interface has been made more responsive and interactive for very big data sets. These enhancements will improve the user experience, quality of results, and the pace of biological discovery. Natively compiled graphical user interface and command-line versions of MEGA11 are available for Microsoft Windows, Linux, and macOS from www.megasoftware.net.     

Inhibition of alpha-globin gene expression by RNAi

RNA interference (RNAi), a process by which target messenger RNA (mRNA) is cleaved by small interfering complementary RNA (siRNA), is widely used for investigations of regulation of gene expression in various cells. In this study, siRNA complementary to 5′ region of exon II of α-globin mRNA was examined for its role in erythroid colony forming cells (ECFCs) isolated from normal peripheral blood donor. On day 6 of cell culture, 1 × 10⁶ ECFCs were transfected with lipofectamine-containing α-globin specific siRNA. After 48 h of transfection, α-globin specific siRNA produced significantly reduction of α-globin mRNA level in a dose-dependent manner, but it did not affect the level of β-globin mRNA. Significantly, decreased numbers of hemoglobinized erythroid cells relative to the control were observed supporting the inhibitory effect of this α-globin mRNA specific siRNA.     

B7-DC induced by IL-13 works as a feedback regulator in the effector phase of allergic asthma

B7-DC is a costimulatory molecule belonging to the B7 family. We previously found that treatment with anti-B7-DC mAb during the effector phase enhances asthma phenotypes in mice. We investigated the mechanisms of B7-DC induction and how B7-DC regulates asthma phenotypes. In allergen-challenged IFN-γ-deficient mice, anti-B7-DC mAb failed to enhance the asthma phenotypes although the induction of B7-DC on dendritic cells of the mice was comparable with that on dendritic cells of wild-type mice. B7-DC on dendritic cells was up-regulated by IL-13 in vitro. The induction of B7-DC on dendritic cells after allergen challenge was attenuated by blockade of IL-13 in vivo. The asthma phenotypes were enhanced in B7-DC-deficient mice, more than in wild-type mice. The enhancement was concurrent with the down-regulation of IFN-γ and up-regulation of IL-13. These results suggest that B7-DC induced by IL-13 works as a feedback regulator by up-regulating IFN-γ production during the effector phase of allergic asthma.     

Nonhuman primate intestinal villous M-like cells: an effective poliovirus entry site

Humans and some Old World monkeys, chimpanzees, and cynomolgus macaques, are susceptible to oral poliovirus (PV) infection. Interestingly, rhesus macaques, although sensitive to injected PV, are not susceptible to gut infection. Not much is known about the initial event of gut infection by PV in rhesus macaques so far. Here, we show that PV can efficiently enter the lamina propria (LP) by penetrating across intestinal villous M-like cells in rhesus macaques. We found by immunofluorescence analysis that PV effectively invades LP rather than germinal centers (GCs) in rhesus macaques despite expressing PV receptor CD155 on cells within GCs and LP. Furthermore, energy dispersive X-ray spectroscopy demonstrated that gold-labeled PV is spatiotemporally internalized into villous M-like cells and engulfed by macrophage-like cells in LP. These results suggest that rhesus macaques may be resistant to productive gut PV infection owing to a defective translocation of PV to GCs.     

Characterization of Xylanolytic Enzymes in Clostridium cellulovorans: Expression of Xylanase Activity Dependent on Growth Substrates

Xylanase activity of Clostridium cellulovorans, an anaerobic, mesophilic, cellulolytic bacterium, was characterized. Most of the activity was secreted into the growth medium when the bacterium was grown on xylan. Furthermore, when the extracellular material was separated into cellulosomal and noncellulosomal fractions, the activity was present in both fractions. Each of these fractions contained at least two major and three minor xylanase activities. In both fractions, the pattern of xylan hydrolysis products was almost identical based on thin-layer chromatography analysis. The major xylanase activities in both fractions were associated with proteins with molecular weights of about 57,000 and 47,000 according to zymogram analyses, and the minor xylanases had molecular weights ranging from 45,000 to 28,000. High alpha-arabinofuranosidase activity was detected exclusively in the noncellulosomal fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan-, cellobiose-, and cellulose-grown cultures had different subunit compositions. Also, when xylanase activity in the cellulosomes from the xylan-grown cultures was compared with that of cellobiose- and cellulose-grown cultures, the two major xylanases were dramatically increased in the presence of xylan. These results strongly indicated that C. cellulovorans is able to regulate the expression of xylanase activity and to vary the cellulosome composition depending on the growth substrate.     

Cryopreservation of Hyoscyamus niger adventitious roots by vitrification

An auxin-independent adventitious root culture of Hyoscyamus niger was established, and the roots were successfully cryopreserved with a high regeneration rate of 93.3 percnt; by vitrification method. The root tips were cultured for 12 to 14 days in phytohormone-free Murashige and Skoog (MS) liquid medium, and were excised and precultured on Woody Plant (WP) solid medium supplemented with 0.3 mol/L sucrose at 25 °C in the dark. After 1 day, they were treated with MS-based loading solution for 10 min, followed by soaking in MS-based PVS2 for 10 min at 0 °C. The treated root tips were immersed in liquid nitrogen (-196 °C). For recovery, the root tips were thawed rapidly at 40 °C and washed with MS medium containing 1 mol/L sucrose prior to plating onto WP solid medium. The regenerated roots were evaluated by their growth and tropane alkaloid production. The growth and alkaloid content of regenerated roots analyzed using HPLC were found to be almost the same as those of non-treated roots.     

Synthesizing Oligomers from Monomeric Nucleotides in Simulated Hydrothermal Environments

Dimers and trimers of adenosine monophosphate(AMP) were synthesized from AMP in environmentssimulating hot vents on the sea floor of the primitiveEarth. The simulated environments were made in the flowreactor, in which an aqueous solution of reactants wascirculated from the hot to the cold region repeatedly. Theoligomerization proceeded most significantly when the hotreaction solution at about 110 °C was abruptly ejected into the cold environment maintained at about 0 °C.     

Hydrothermal Circulation of Seawater through Hot Vents and Contribution of Interface Chemistry to Prebiotic Synthesis

Synthesizing oligopeptides from glycine andalanine in a flow reactor, which stimulates constanthydrothermal circulation of seawater through hot vents onthe primitive Earth, demonstrated that an exponential growthof the products is possible. The initial rapid growth of the product is a consequence of using the products formed inone cycle as the starting materials for the cycle of synthesis.     

Isolation and characterization of Xenopus laevis homologs of the mouse inv gene and functional analysis of the conserved calmodulin binding sites

The homozygous inv （inversion of embryonic turning） mouse mutant shows situs inversus and polycystic kidney disease, both of which result from the lack of the inv gene. Previously, we suggested that inv may be important for the left-right axis formation, not only in mice but also in Xenopus, and that calmodulin regulates this inv protein function. Here, we isolated and characterized two Xenopus laevis homologs （Xinv-1 and Xinv-2） of the mouse inv gene, and performed functional analysis of the conserved IQ motifs that interact with calmodulin. Xinv-1 expresses early in development in the same manner as mouse inv does. Unexpectedly, a full-length Xenopus inv mRNA did not randomize cardiac orientation when injected into Xenopus embryos, which is different from mouse inv mRNA. Contrary to mouse inv mRNA, Xenopus inv mRNA with mutated IQ randomized cardiac orientation. The present study indicates that calmodulin binding sites （IQ motifs） are crucial in controlling the biological activity of both mouse and Xenopus inv proteins. Although mouse and Xenopus inv genes have a quite similar structure, the interaction with calmodulin and IQ motifs ofXenopus inv and mouse inv proteins may regulate their function in different ways.