A novel mutation of ALK2, L196P, found in the most benign case of fibrodysplasia ossificans progressiva activates BMP-specific intracellular signaling equivalent to a typical mutation, R206H

Naphthoquinone derivatives have been reported to possess various pharmacological activities, such as antiplatelet, anticancer, antifungal, and antiviral properties. In this study, we investigated the effects of a newly-synthesized naphthoquinone derivative, 2-decylamino-5,8-dimethoxy-1,4-naphthoquinone (2-decylamino-DMNQ), on VSMC proliferation and examined the molecular basis of the underlying mechanism. In a dose-dependent manner, 2-decylamino-DMNQ inhibited PDGF-stimulated VSMC proliferation with no apparent cytotoxic effect. While 2-decylamino-DMNQ did not affect PDGF-Rβ or Akt, it did inhibit the phosphorylation of Erk1/2 and PLCγ1 induced by PDGF. Moreover, 2-decylamino-DMNQ suppressed DNA synthesis through the arrest of cell cycle progression at the G₀/G₁ phase, including the suppression of pRb phosphorylation and a decrease in PCNA expression, which was related to the downregulation of cell cycle regulatory factors, such as cyclin D1/E and CDK 2/4. It was demonstrated that both U0126, an Erk1/2 inhibitor, and U73122, a PLCγ inhibitor, increased the proportion of cells in the G₀/G₁ phase of the cell cycle. Thus, these results suggest that 2-decylamino DMNQ has an inhibitory effect on PDGF-induced VSMC proliferation and the mechanism of this action is through cell cycle arrest at the G₀/G₁ phase. This may be a useful tool for studying interventions for vascular restenosis in coronary revascularization procedures and stent implantation.     

Strongly pH-buffered ringer's solution expands the receptive field size of horizontal cells in the carp retina

By intracellular recordings, we studied the effects of pH buffering on the size of the receptive field and the extent of dye coupling of horizontal cells (HCs) in the light-adapted carp retina. These parameters were compared between data obtained in fortified Ringer's solution and those obtained in control bicarbonate Ringer's of the same pH (7.60). In Ringer's fortified with 10 mM HEPES or 15 mM Tris, the dye-coupling ratio of HCs increased by 71% and 70%, respectively. These fortified Ringer's solutions also depolarized the dark membrane potential and increased the light-evoked response. The HC receptive field profile could be described by the exponential decline in peak response amplitude to a slit of light moved tangentially from the recording electrode. Thus, the receptive field size was determined as a space constant proportional to (gj/gm)(1/2), where gj and gm denote gap and non-gap-junctional conductances. The HEPES- or Tris-fortified Ringer's significantly increased the space constant by 43% and 41%, respectively. Since dye coupling was increased in the fortified Ringer's, it is likely that gj increased more than gm as a result of alkalinization of the cytosol. Since HEPES has an aminosulfonate moiety, it has been assumed to close the hemi-channels of connexin 26, but the pH-buffering effects were essentially the same as those of Tris that has no aminosulfonate moiety. Therefore, it is unlikely that the closure of connexin 26 hemichannels is responsible for the change in the receptive field size of HCs.     

The amino acid sequence of ferredoxin from the alga Mastigocladuslaminosus

Ferredoxin was purified from the thermophilic blue-green alga, Mastigocladuslaminosus. The physicochemical properties of this ferredoxin are similar to those of other [2Fe-2S] plant ferredoxins except for its unusual thermal stability. The primary structure of the protein was determined and consists of 98 amino acid residues, 5 of which are cysteines. The positions of 4 cysteines which bind the iron atoms of the active centre are identical to those in other ferredoxins. The primary structure of the ferredoxin does not reveal any special features to account for its high thermal stability.     

The meiotic recombination checkpoint is regulated by checkpoint rad+ genes in fission yeast

During the course of meiotic prophase, intrinsic double-strand breaks (DSBs) must be repaired before the cell can engage in meiotic nuclear division. Here we investigate the mechanism that controls the meiotic progression in Schizosaccharomyces pombe that have accumulated excess meiotic DSBs. A meiotic recombination-defective mutant, meu13Delta, shows a delay in meiotic progression. This delay is dependent on rec12+, namely on DSB formation. Pulsed-field gel electrophoresis analysis revealed that meiotic DSB repair in meu13Delta was retarded. We also found that the delay in entering nuclear division was dependent on the checkpoint rad+, cds1+ and mek1+ (the meiotic paralog of Cds1/Chk2). This implies that these genes are involved in a checkpoint that provides time to repair DSBs. Consistently, the induction of an excess of extrinsic DSBs by ionizing radiation delayed meiotic progression in a rad17(+)-dependent manner. dmc1Delta also shows meiotic delay, however, this delay is independent of rec12+ and checkpoint rad+. We propose that checkpoint monitoring of the status of meiotic DSB repair exists in fission yeast and that defects other than DSB accumulation can cause delays in meiotic progression.     

TRAIL-transduced dendritic cells protect mice from acute graft-versus-host disease and leukemia relapse

TRAIL preferentially induces apoptotic cell death in a wide variety of transformed cells, whereas it induces no apoptosis, but inhibits activation of Ag-specific T cells via blockade of cell cycle progression. Although accumulating results suggest that TRAIL is involved in the maintenance of immunological homeostasis under steady state conditions as well as in the initiation and progression of immunopathologies, the potential regulatory effect of TRAIL on immune responses and its therapeutic potential in immunological diseases remains unclear. We report in this study the potential usefulness of TRAIL-transduced dendritic cells (DCs) for the treatment of lethal acute graft-vs-host disease (GVHD) and leukemia relapse. DCs genetically modified to express TRAIL showed potent cytotoxicity against both alloreactive T cells and leukemic cells through the induction of apoptosis. In addition, treatment with genetically modified DCs expressing TRAIL of allogeneic BM transplants recipients with leukemia was effective for protection against acute GVHD and leukemia relapse. Thus, gene transfer of TRAIL to DCs is a novel modality for the treatment of acute GVHD and leukemia relapse by selective targeting of pathogenic T cells and leukemic cells.     

Characterization of Eag1 Channel Lateral Mobility in Rat Hippocampal Cultures by Single-Particle-Tracking with Quantum Dots

Voltage-gated ion channels are main players involved in fast synaptic events. However, only slow intracellular mechanisms have so far been described for controlling their localization as real-time visualization of endogenous voltage-gated channels at high temporal and spatial resolution has not been achieved yet. Using a specific extracellular antibody and quantum dots we reveal and characterize lateral mobility as a faster mechanism to dynamically control the number of endogenous ether-a-go-go (Eag)1 ion channels inside synapses. We visualize Eag1 entering and leaving synapses by lateral diffusion in the plasma membrane of rat hippocampal neurons. Mathematical analysis of their trajectories revealed how the motion of Eag1 gets restricted when the channels diffuse into the synapse, suggesting molecular interactions between Eag1 and synaptic components. In contrast, Eag1 channels switch to Brownian movement when they exit synapses and diffuse into extrasynaptic membranes. Furthermore, we demonstrate that the mobility of Eag1 channels is specifically regulated inside synapses by actin filaments, microtubules and electrical activity. In summary, using single-particle-tracking techniques with quantum dots nanocrystals, our study shows for the first time the lateral diffusion of an endogenous voltage-gated ion channel in neurons. The location-dependent constraints imposed by cytoskeletal elements together with the regulatory role of electrical activity strongly suggest a pivotal role for the mobility of voltage-gated ion channels in synaptic activity.     

A single amino acid in human APOBEC3F alters susceptibility to HIV-1 Vif

Human APOBEC3F (huA3F) potently restricts the infectivity of HIV-1 in the absence of the viral accessory protein virion infectivity factor (Vif). Vif functions to preserve viral infectivity by triggering the degradation of huA3F but not rhesus macaque A3F (rhA3F). Here, we use a combination of deletions, chimeras, and systematic mutagenesis between huA3F and rhA3F to identify Glu(324) as a critical determinant of huA3F susceptibility to HIV-1 Vif-mediated degradation. A structural model of the C-terminal deaminase domain of huA3F indicates that Glu(324) is a surface residue within the α4 helix adjacent to residues corresponding to other known Vif susceptibility determinants in APOBEC3G and APOBEC3H. This structural clustering suggests that Vif may bind a conserved surface present in multiple APOBEC3 proteins.     

Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and Agrobacterium tumefaciens-mediated gene transfer

The hemibiotrophic ascomycete Colletotrichum higginsianum is the casual agent of anthracnose disease of cruciferous plants. High efficiency transformation by Agrobacterium tumefaciens-mediated gene transfer has been established for this fungus. However, targeted gene mutagenesis through homologous recombination rarely occurs in C. higginsianum. We have identified and disrupted the C. higginsianum homologue of the human Ku70 gene, ChKU70, which encodes a protein that plays a role in non-homologous end-joining for repair of DNA breaks. chku70 mutants showed a dramatic increase in the frequency of integration of introduced exogenous DNA fragments by homologous recombination without any detectable phenotypic defects. This result demonstrates that the chku70 mutant is an efficient recipient for targeted gene mutagenesis in C. higginsianum. We have also developed a novel approach [named direct repeat recombination-mediated gene targeting (DRGT)] for targeted gene disruption through Agrobacterium tumefaciens-mediated gene transfer. DRGT utilizes homologous recombination between repeated sequences on the T-DNA flanking a partial fragment of the target gene. Our results suggest that DRGT in the chku70 mutant background could be a useful tool for rapid isolation of targeted gene disruptants in C. higginsianum.