Persistence and fluctuation of lateral dimorphism in fishes

Two morphological types ("righty" and "lefty") have been discovered in several fish species and are referred to as a typical example of antisymmetry. It has been suggested, first, that this dimorphism (called laterality) is inheritable; second, that the frequencies of laterality in each species fluctuate around 0.5; and third, that predators mainly exploit prey of the opposite laterality; that is, lefty and righty predators prey on righties and lefties, respectively. The latter is defined as "cross predation"; the antonym "parallel predation" means predation within the same laterality. We hypothesized that cross predation drives alternation of the survival and reproductive advantages between two morphological types, leading to frequency-dependent selection that maintains the dimorphism. To investigate this, we constructed mathematical models of population dynamics of one prey/one predator systems and three-trophic-level systems with omnivory. Mathematical analysis and computer simulations explained the behavior of the laterality frequency in nature well, insofar as cross predation dominated over parallel predation. Furthermore, the simulations showed that when only one of the morphological types exists in a species, the other type can invade. This suggests that dimorphism is maintained in all interacting species.     

Na+-dependent K+ uptake Ktr system from the cyanobacterium Synechocystis sp. PCC 6803 and its role in the early phases of cell adaptation to hyperosmotic shock

Transmembrane ion transport processes play a key role in the adaptation of cells to hyperosmotic conditions. Previous work has shown that the disruption of a ktrB/ntpJ-like putative Na(+)/K(+) transporter gene in the cyanobacterium Synechocystis sp. PCC 6803 confers increased Na(+) sensitivity, and inhibits HCO(3)(-) uptake. Here, we report on the mechanistic basis of this effect. Heterologous expression experiments in Escherichia coli show that three Synechocystis genes are required for K(+) transport activity. They encode an NAD(+)-binding peripheral membrane protein (ktrA; sll0493), an integral membrane protein, belonging to a superfamily of K(+) transporters (ktrB; formerly ntpJ; slr1509), and a novel type of ktr gene product, not previously found in Ktr systems (ktrE; slr1508). In E. coli, Synechocystis KtrABE-mediated K(+) uptake occurred with a moderately high affinity (K(m) of about 60 microm), and depended on both Na(+) and a high membrane potential, but not on ATP. KtrABE neither mediated Na(+) uptake nor Na(+) efflux. In Synechocystis sp. PCC 6803, KtrB-mediated K(+) uptake required Na(+) and was inhibited by protonophore. A Delta ktrB strain was sensitive to long term hyperosmotic stress elicited by either NaCl or sorbitol. Hyperosmotic shock led initially to loss of net K(+) from the cells. The Delta ktrB cells shocked with sorbitol failed to reaccumulate K(+) up to its original level. These data indicate that in strain PCC 6803 K(+) uptake via KtrABE plays a crucial role in the early phase of cell turgor regulation after hyperosmotic shock.     

Daxx enhances Fas-mediated apoptosis in a murine pro-B cell line,BAF3

Daxx has been shown to play an essential in type I interferon (IFN-/β)-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and nuclear translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify the mechanism of Daxx-mediated apoptosis signaling in B lymphocyte progenitors, here we introduced an efficient suicide switch in a murine pro-B cell line, BAF3, by expressing FK506-binding protein-fused Fas intracellular domain (FKBP-Fas) and Daxx. It allows us to monitor Fas/Daxx-mediated signal by induction of Fas dimerization with the dimerizer drug AP20187. AP20187-mediated Fas dimerization induced not only apoptosis but also Jun N-terminal kinase (JNK) activation. However, AP20187 had no effect on cells expressing either Fas or Daxx only. Furthermore, expression of a JNK inhibitor, the JNK-binding domain of JIP-1, resulted in resistance to AP20187-mediated apoptosis in cells expressing FKBP-Fas and Daxx. These results imply that our novel suicide switch system may provide a powerful tool to delineate or identify the signaling molecules for Daxx-mediated apoptotic machinery in B lymphocyte progenitors through JNK activation.     

Negamycin restores dystrophin expression in skeletal and cardiac muscles of mdx mice

The ability of aminoglycoside antibiotics to promote read-through of nonsense mutations has attracted interest in these drugs as potential therapeutic agents in genetic diseases. However, the toxicity of aminoglycoside antibiotics may result in severe side effects during long-term treatment. In this paper, we report that negamycin, a dipeptide antibiotic, also restores dystrophin expression in skeletal and cardiac muscles of the mdx mouse, an animal model of Duchenne muscular dystrophy (DMD) with a nonsense mutation in the dystrophin gene, and in cultured mdx myotubes. Dystrophin expression was confirmed by immunohistochemistry and immunoblotting. We also compared the toxicity of negamycin and gentamicin, and found negamycin to be less toxic. Furthermore, we demonstrate that negamycin binds to a partial sequence of the eukaryotic rRNA-decoding A-site. We conclude that negamycin is a promising new therapeutic candidate for DMD and other genetic diseases caused by nonsense mutations.     

Collagen XVIII,a basement membrane heparan sulfate proteoglycan,interacts with L-selectin and monocyte chemoattractant protein-1

Leukocyte infiltration during inflammation is mediated by the sequential actions of adhesion molecules and chemokines. By using a rat ureteral obstruction model, we showed previously that L-selectin plays an important role in leukocyte infiltration into the kidney. Here we report the purification, identification, and characterization of an L-selectin-binding heparan sulfate proteoglycan (HSPG) expressed in the rat kidney. Partial amino acid sequencing and Western blotting analyses showed that the L-selectin-binding HSPG is collagen XVIII, a basement membrane HSPG. The binding of L-selectin to isolated collagen XVIII was specifically inhibited by an anti-L-selectin monoclonal antibody, EDTA, treatment of the collagen XVIII with heparitinase or heparin but not by chemically desulfated heparin. A cell binding assay showed that the L-selectin-collagen XVIII interaction mediates cell adhesion. Interestingly, collagen XVIII also interacted with a chemokine, monocyte chemoattractant protein-1, and presented it to a monocytic cell line, THP-1, which enhanced the alpha(4)beta(1) integrin-mediated binding of the THP-1 cells to vascular cell adhesion molecule-1. Thus, collagen XVIII may provide a link between selectin-mediated cell adhesion and chemokine-induced cellular activation and accelerate the progression of leukocyte infiltration in renal inflammation.     

Regional concentration and chromatographic characterization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the brain of the bullfrog,Rana catesbeiana

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a regulatory neuropeptide which functions as a hypothalamic factor for pituitary hormone release, and as a neurotransmitter, neuromodulator and neurotrophic factor in both frogs and mammals. This study examined the quantitative distribution and chromatographic characterization of immunoreactive PACAP in the central nervous system (CNS) of the bullfrog, Rana catesbeiana, using an enzyme immunoassay (EIA), named avidin-biotin complex detectable EIA for PACAP, and high-performance liquid chromatographic (HPLC) analysis. The brain of adult bullfrogs contained relatively high levels of immunoreactive PACAP (344.63 pmol/g wet weight of tissue). The average concentrations of immunoreactive PACAP in the regions of the telencephalon, diencephalon, tectum, cerebellum, rhombencephalon, and spinal cord were 213.84, 767.14, 524.94, 192.71, 237.67, and 362.04 pmol/g wet weight of tissue, respectively. The concentrations of immunoreactive PACAP increased with the brain development during metamorphosis, and the concentration of immunoreactive PACAP in the brain of tadpoles at the end of metamorphosis was approximately 200 pmol/g wet weight of tissue. The predominant form of immunoreactive PACAP in the CNS of adult and tadpole was eluted closely with synthetic PACAP38, but another smaller immunoreactivity also appeared in a the fraction, which corresponded to the retention time of synthetic PACAP27, as analyzed by reverse-phase HPLC.     

Growth factor array fabrication using a color ink jet printer

We have developed a novel method for growth factor analysis using a commercial color ink jet printer to fabricate substrata patterned with growth factors. We prepared substrata with insulin printed in a simple pattern or containing multiple areas of varying quantities of printed insulin. When we cultured the mouse myoblast cell line, C2C12, on the insulin-patterned substrata, the cells were grown in the same pattern with the insulin-printed pattern. Cell culture with the latter substrata demonstrated that quantity control of insulin deposition by a color ink jet printer is possible. For further applications, we developed substrata with insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF) spotted in 16 different areas in varying combinations and concentrations (growth factor array). With this growth factor array, C2C12 cells were cultured, and the onset of muscle cell differentiation was monitored for the expression of the myogenic regulator myogenin. The ratio of cells expressing myogenin varied with the doses of IGF-I and bFGF in the sections, demonstrating a feasibility of growth factor array fabrication by a color ink jet printer. Since a printer manipulates several colors, this method can be easily applied to multivariate analyses of growth factors and attachment factors affecting cell growth and differentiation. This method may provide a powerful tool for cell biology and tissue engineering, especially for stem cell research in investigating unknown conditions for differentiation.     

Distribution of antigenic oligomannosyl side chains in the cell wall mannans of several strains of<Emphasis Type="Italic"> Candida tropicalis</Emphasis>

In order to clarify the distribution of antigenic oligomannosyl side chains in the cell wall mannans of the pathogenic yeast Candida tropicalis, the chemical structure of mannans isolated from four C. tropicalis strains was investigated using nuclear magnetic resonance, two-dimensional homonuclear Hartmann-Hahn (2D-HOHAHA) spectroscopy. Two-dimensional maps of the 2D-HOHAHA clearly showed the distribution of oligomannosyl side chains in the mannans. The linear side chain Manalpha1-3Manalpha1-(2Manalpha1-)(n)2Man [n> or =2] is present in the mannans from C. tropicalis IFO 0589 and IFO 1400, but not in the mannans from IFO 0199 and IFO 1647. The mannan of IFO 0589 is the only mannan with the branched side chains, Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man and Manalpha1-2Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man [n> or =2]. However, this mannan lacked the phosphate group and the beta-1,2-linked oligomannosyl side chain which are features of this group. The mannans of the C. tropicalis strains IFO 0589 and IFO 1400 possessed the side chains containing an alpha-1,3-linked mannose residue previously observed in Candida albicans.     

Synthesis,thermal stability and resistance to enzymatic hydrolysis of the oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2'-O-methyluridines

The synthesis of oligonucleotides (ODNs) containing 5-(N-aminohexyl)carbamoyl-2′-O-methyluridine (D) is described, and thermal stability and resistance to enzymatic hydrolysis of the ODNs are compared with ODNs containing 5-(N-aminohexyl)carbamoyl-2′-deoxyuridine (H). The ODNs containing D and the complementary RNA demonstrated a duplex thermal stabilization of 0.4–3.9°C per modification depending on the position and the number, while the ODNs containing H with the RNA showed slightly less effective thermal stabilization. Further more, the ODNs containing D were found to be more resistant to nucleolytic hydrolysis, not only by snake venom phosphodiesterase (SVPD; a 3′-exonuclease) but also by DNase I (an endonuclease). The half-life of the 17mer containing five molecules of D against nucleolytic hydrolysis by SVPD was 240 times greater than the unmodified 17mer ODN, which is 1.8 times greater than the ODN containing 5Hs in the same sequence. Against DNase I, the same ODN containing 5Ds was 24 times greater stable than the unmodified 17mer and 15 times more stable than the ODN containing 5Hs. We also examined whether the duplexes formed by the ODNs containing D and the complementary RNAs could be a substrate of Escherichia coli RNase H. It was revealed that a minimum of five contiguous unmodified 2′-deoxyribonucleosides between Ds was required to constitute a substrate of E.coli RNase H. Thus, the ODN with Ds and at least five contiguous unmodified 2′-deoxyribonucleosides between Ds was found to be a candidate for a novel antisense molecule.