Development of cell-expressed and virion-incorporated CCR5-targeted vaccine

Our previous study demonstrated that the immunization with a cycloimmunogen derived from extracellular loop-2 (ECL-2) of CCR5 (cDDR5) attenuated acute phase of CCR5-tropic simian-human immunodeficiency virus (SHIV)_SF162P3 replication in vivo. Although the study showed that the antisera raised against cDDR5 reacted with cell-expressed CCR5, we have not yet demonstrated whether the antisera can react with virion-incorporated CCR5. Here, we show that rhesus cDDR5 (rcDDR5)-specific antibodies react with not only cell-expressed but also virion-incorporated simian CCR5s (siCCR5s), but may predominantly exert their inhibitory effects on simian immunodeficiency virus (SIV) infection by the binding of cell-expressed rather than virion-incorporated CCR5s. These results suggest that the virion-incorporated CCR5 may contribute to the reactivation of the anti-rcDDR5 antibody-producing B-cells by SIV particles after rcDDR5 immunization, although the binding of anti-rcDDR5 antibody to virion-incorporated CCR5 results in a partial inhibitory effect on SIV infection.     

Superoxide scavenging activity of pirfenidone-iron complex

Pirfenidone (PFD) is focused on a new anti-fibrotic drug, which can minimize lung fibrosis etc. We evaluated the superoxide () scavenging activities of PFD and the PFD-iron complex by electron spin resonance (ESR) spectroscopy, luminol-dependent chemiluminescence assay, and cytochrome c reduction assay. Firstly, we confirmed that the PFD-iron complex was formed by mixing iron chloride with threefold molar PFD, and the complex was stable in distillated water and ethanol. Secondary, the PFD-iron complex reduced the amount of produced by xanthine oxidase/hypoxanthine without inhibiting the enzyme activity. Thirdly, it also reduced the amount of released from phorbor ester-stimulated human neutrophils. PFD alone showed few such effects. These results suggest the possibility that the scavenging effect of the PFD-iron complex contributes to the anti-fibrotic action of PFD used for treating idiopathic pulmonary fibrosis.     

Applicability of new spin trap agent, 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide, in biological system

Electron spin resonance using spin-trapping is a useful technique for detecting direct reactive oxygen species, such as superoxide (). However, the widely used spin trap 2,2-dimethyl-3,4-dihydro-2H-pyrrole N-oxide (DMPO) has several fundamental limitations in terms of half-life and stability. Recently, the new spin trap 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (DPhPMPO) was developed by us. We evaluated the biological applicability of DPhPMPO to analyze in both cell-free and cellular systems. DPhPMPO had a larger rate constant for and formed more stable spin adducts for than DMPO in the xanthine/xanthine oxidase (X/XO) system. In the phorbol myristate acetate-activated neutrophil system, the detection potential of DPhPMPO for was significantly higher than that of DMPO (k_DMPO = 13.95 M⁻¹ s⁻¹, k_DPhPMPO = 42.4 M⁻¹ s⁻¹). These results indicated that DPhPMPO is a potentially good candidate for trapping in a biological system.     

Uptake and recovery of gold ions from electroplating wastes using eggshell membrane.

The animal byproduct, hen eggshell membrane (ESM), was evaluated for its ability to sorb gold ions (dicyanoaurate(I) and tetrachloroaurate(III)) from solutions and electroplating wastewater. The gold uptake was dependent on pH, temperature and co-ions present in the solutions, with pH 3.0 being the optimum value. The equilibrium data followed the Langmuir isotherm model with maximum capacities of 147 mg Au(I)/g dry weight and 618 mg Au(III)/g, respectively. Desorption of sorbed gold(I) with 0.1 mol/l NaOH resulted in no changes of the biosorbent gold uptake capacity through five consecutive sorption/desorption cycles. In column experiments, selective recovery of gold from electroplating wastewater containing various metal ions was noted. The affinity of metal sorption was in the order Au > Ag > Co > Cu > Pb > Ni > Zn.     

Effects of Chloroplast DNA Content on the Cell Proliferation and Aging in Chlamydomonas reinhardtii

Chlamydomonas is an unicellular green alga that contains one cup-shaped chloroplast with about 60 copies of cpDNA. Chloroplasts (cp) multiply in the cytoplasm of the plant cell by binary division, with multiple copies of cpDNA transmitted and maintained in successive generations. The effect of cpDNA copy number on cell proliferation and aging was investigated using a C. reinhardtii moc mutant, which has an undispersed cp-nucleoid and unequal segregation of cpDNA during cell division. When the mother cell divided into four daughters, one moc daughter cell chloroplast contained about 60 copies of cpDNA, and the chloroplasts in the three other daughter cells contained the 4–7 copies of cpDNA. In liquid medium, the number of moc cells at the period of stationary phase was about one-third that of the wild type. To observe the process of proliferation and aging in the mother cell, we used solid medium. Three out of four moc cell spores were preferentially degenerated 60 days after cell transfer. To confirm this, wild-type and moc mother cells containing four daughter cells were treated with novobiocin to inhibit cpDNA replication. Cell degeneration increased only in the moc strain following novobiocin introduction. In total, our results suggest that cells possessing smaller amounts of cpDNA degenerate and age more rapidly. Received 7 September 2000/ Accepted in revised form 14 February 2001     

Effect of polymyxin B on liposomal membranes derived from Escherichia coli lipids

The specificity of the action of polymyxin B was studied using liposomes as a model membrane system. Liposomes prepared from total lipids of Gram-negative bacteria Escherichia coli, a mixture of purified E. coli phosphatidylethanolamine and cardiolipin and a mixture of phosphatidylethanolamine and phosphatidylglycerol, were extremely sensitive to polymyxin while those prepared from lipids of Gram-positive bacteria Streptococcus sanguis, lipids of sheep erythrocyte membranes, mixtures of egg lecithin and negatively charged amphiphatic molecules, were less sensitive to the action of the antibiotic. Chlolesterol was shown to suppress the polymyxin-induced response in liposomes.     

Mouthparts in Leptotrombidium larvae (Acariformes: Trombiculidae)

Mouthparts of Leptotrombidium larvae (Acariformes: Trombiculidae), potential vectors of tsutsugamushi disease agents, were studied in detail using light microscopy, scanning electron microscopy, and transmission electron microscopy. The mouthparts incorporated within the pseudotagma gnathosoma are composed of the infracapitulum ventrally and the chelicerae dorsally. The ventral wall of the infracapitulum is formed by a wide mentum posteriorly and a narrowed malapophysis anteriorly. The malapophysis firmly envelops the distal cheliceral portions by its lateral walls. The lateral lips of the malapophysis are flexible structures hiding the cheliceral blades in inactive condition and turning back forming a type of temporary sucker closely applied to the host skin during feeding. The roof of the infracapitulum is formed by a weakly sclerotized labrum anteriorly and a cervix with the capitular apodemes extending posteriorly. The labral muscles are lacking. The capitular apodemes serve as origin for pharyngeal dilators running to the dorsal wall of the pharynx fused with the bottom of the infracapitulum. The basal cheliceral segments are separated from each other besides the very posterior portions where they are movably joined by the inner walls. The sigmoid pieces serve for insertion of the cheliceral elevators originating at the posterior portions of the basal segments. The movable digits reveal the solid basal sclerite and the cheliceral blade curved upward with a tricuspid cap on its tip. Dendrites of nerve cells run along the digits to their tips. The ganglia are placed within the basal segments just behind the movable digits. The chelicerae also reveal well developed flexible fixed digits overhanging the basal portions of the blades. The gnathosoma possesses several sets of extrinsic muscles originating at the scutum and at the soft cuticle behind it. Laterally, the gnathosoma bears five‐segmented palps with a trifurcate palpal claw. J. Morphol. 277:424–444, 2016. © 2016 Wiley Periodicals, Inc.     

Crystal Structure of the cGMP-dependent Protein Kinase II Leucine Zipper and Rab11b Protein Complex Reveals Molecular Details of G-kinase-specific Interactions

cGMP-dependent protein kinase (PKG)-interacting proteins (GKIPs) mediate cellular targeting of PKG isoforms by interacting with their leucine zipper (LZ) domains. These interactions prevent aberrant signaling cross-talk between different PKG isotypes. To gain detailed insight into isotype-specific GKIP recognition by PKG, we analyzed the type II PKG leucine zipper domain and found that residues 40–83 dimerized and specifically interacted with Rab11b. Next, we determined a crystal structure of the PKG II LZ-Rab11b complex. The PKG II LZ domain presents a mostly nonpolar surface onto which Rab11b docks, through van der Waals interactions. Contact surfaces in Rab11b are found in switch I and II, interswitch, and the β1/N-terminal regions. This binding surface dramatically differs from that seen in the Rab11 family of interacting protein complex structures. Structural comparison with PKG Iα and Iβ LZs combined with mutagenic analysis reveals that GKIP recognition is mediated through surface charge interactions.     

Evolutionarily Conserved Interaction between the Phosphoproteins and X Proteins of Bornaviruses from Different Vertebrate Species

Bornavirus, a non-segmented, negative-strand RNA viruses, is currently classified into several genetically distinct genotypes, such as Borna disease virus (BDV) and avian bornaviruses (ABVs). Recent studies revealed that bornavirus genotypes show unique sequence variability in the putative 5′ untranslated region (5′ UTR) of X/P mRNA, a bicistronic mRNA for the X protein and phosphoprotein (P). In this study, to understand the evolutionary relationship among the bornavirus genotypes, we investigated the functional interaction between the X and P proteins of four bornavirus genotypes, BDV, ABV genotype 4 and 5 and reptile bornavirus (RBV), the putative 5′ UTRs of which exhibit variation in the length. Immunofluorescence and immunoprecipitation analyses using mammalian and avian cell lines revealed that the X proteins of bornaviruses conserve the ability to facilitate the export of P from the nucleus to the cytoplasm via interaction with P. Furthermore, we showed that inter-genotypic interactions may occur between X and P among the genotypes, except for X of RBV. In addition, a BDV minireplicon assay demonstrated that the X and P proteins of ABVs, but not RBV, can affect the polymerase activity of BDV. This study demonstrates that bornaviruses may have conserved the fundamental function of a regulatory protein during their evolution, whereas RBV has evolved distinctly from the other bornavirus genotypes.     

Prolonged motility of fowl spermatozoa in vitro due to a low molecular weight factor(s) released from cultured embryonic cells

Fowl spermatozoa were incubated at 41°C in a supernatant removed from a 4-day culture medium of embryonic chick skeletal muscle cells. Their motility, as assessed at room temperature (20–25°C), was maintained for 48 h. Fertilizing ability was also retained for at least 24 h. In contrast, spermatozoa incubated in the fresh culture medium lost their motility and fertilizing ability rapidly. A filtrate of the 4-day culture medium, obtained by passing the fluid through an Amicon PM-10 ultrafiltration membrane, prolonged the motility of spermatozoa. These results suggested that a low molecular weight factor(s) (mol. wt. < 10 000) supplied by the cultured cells effectively prolonged the motility of spermatozoa.