Developmental expression profiles of axon guidance signaling and the immune system in the marmoset cortex: Potential molecular mechanisms of pruning of dendritic spines during primate synapse formation in late infancy and prepuberty (I)

The synapse number and the related dendritic spine number in the cerebral cortex of primates shows a rapid increase after birth. Depending on the brain region and species, the number of synapses reaches a peak before adulthood, and pruning takes place after this peak (overshoot-type synaptic formation). Human mental disorders, such as autism and schizophrenia, are hypothesized to be a result of either too weak or excessive pruning after the peak is reached. Thus, it is important to study the molecular mechanisms underlying overshoot-type synaptic formation, particularly the pruning phase.     

A Naturally Occurring Canine Model of Autosomal Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a non-progressive, clinically and genetically heterogeneous disease of impaired night vision. We report a naturally-occurring, stationary, autosomal recessive phenotype in beagle dogs with normal daylight vision but absent night vision. Affected dogs had normal retinas on clinical examination, but showed no detectable rod responses. They had “negative-type” mixed rod and cone responses in full-field ERGs. Their photopic long-flash ERGs had normal OFF-responses associated with severely reduced ON-responses. The phenotype is similar to the Schubert-Bornschein form of complete CSNB in humans. Homozygosity mapping ruled out most known CSNB candidates as well as CACNA2D4 and GNB3. Three remaining genes were excluded based on sequencing the open reading frame and intron-exon boundaries (RHO, NYX), causal to a different form of CSNB (RHO) or X-chromosome (NYX, CACNA1F) location. Among the genes expressed in the photoreceptors and their synaptic terminals, and mGluR6 cascade and modulators, reduced expression of GNAT1, CACNA2D4 and NYX was observed by qRT-PCR in both carrier (n = 2) and affected (n = 2) retinas whereas CACNA1F was down-regulated only in the affecteds. Retinal morphology revealed normal cellular layers and structure, and electron microscopy showed normal rod spherules and synaptic ribbons. No difference from normal was observed by immunohistochemistry (IHC) for antibodies labeling rods, cones and their presynaptic terminals. None of the retinas showed any sign of stress. Selected proteins of mGluR6 cascade and its modulators were examined by IHC and showed that PKCα weakly labeled the rod bipolar somata in the affected, but intensely labeled axonal terminals that appeared thickened and irregular. Dendritic terminals of ON-bipolar cells showed increased Goα labeling. Both PKCα and Goα labeled the more prominent bipolar dendrites that extended into the OPL in affected but not normal retinas. Interestingly, RGS11 showed no labeling in the affected retina. Our results indicate involvement of a yet unknown gene in this canine model of complete CSNB.     

Release of the extracellular matrix from conidia ofBlumeria graminis in relation to germination

The release of extracellular matrix (ECM) and the emergence of germ tubes from conidia ofBlumeria graminis were studied by light microscopy and micromanipulation. More prompt and frequent ECM release was confirmed on an artificial hydrophobic substratum than on an artificial hydrophilic substratum. Conidia initially incubated on the hydrophilic substratum were transferred by micromanipulation to either the hydrophobic or the hydrophilic substrata. Immediately after transfer onto the hydrophobic substratum, 75% of conidia released ECM, whereas only 16% did so upon transfer to the hydrophilic substratum. Conidia transferred onto the hydrophobic substratum produced a primary germ tube (PGT) more promptly and frequently than those transferred to the hydrophilic substratum. Thus, conidia recognize and respond to substratum hydrophobicity perhaps immediately after contact. When inoculated onto either isolated barley cuticle or the hydrophobic artificial substratum, 2/3 of the conidia produced a PGT from their polar regions. By contrast, on the hydrophilic substratum 2/3 of conidia did so from the side region. These results show that substratum hydrophobicity affects the location of PGT emergence from conidia. Furthermore, the study indicates that very rapid recognition of surface hydrophobicity by conidia promotes ECM release and this in turn may influence the location of PGT emergence.     

Microwave‐assisted solid‐phase peptide synthesis of neurosecretory protein GL composed of 80 amino acid residues

We recently identified a novel cDNA encoding a small secretory protein of 80 amino acid residues, termed neurosecretory protein GL (NPGL), from the chicken hypothalamus. Homologs of NPGL have been reported to be present in mammals, such as human and rat. NPGL is amidated at its C‐terminus, contains an intramolecular disulfide bond, and is hydrophobic in nature. In this study, we have optimized the synthesis of the entire 80‐amino acid peptide sequence of rat NPGL by microwave‐assisted solid‐phase peptide synthesis. NPGL was obtained with a 10% yield when the coupling reactions were performed using 1‐[Bis(dimethylamino)methylene]‐1H‐1,2,3‐triazolo[4,5‐b]pyridinium‐3‐oxid hexafluorophosphate (HATU) at 50 °C for 5 min, and Fmoc deprotections were performed using 40% piperidine containing 0.1 M HOBt. Furthermore, the disulfide bond of NPGL was formed with 20% yield with the use of glutathione‐containing redox buffer and 50% acetonitrile. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Root endophytes enhance stress‐tolerance of Cicuta virosa L. growing in a mining pond of eastern Japan

Cicuta virosa L. plants can grow in a pond subjected to heavy‐metal inputs at the Hitachi mine, eastern Japan. They accumulate heavy‐metal elements, especially high concentrations of zinc (Zn), in their roots. We focused on the role that root bacterial endophytes play in the heavy‐metal uptake of plants and the provision of heavy‐metal tolerance within plants. Our purpose was to clarify the effects of endophytes on: (i) Zn accumulation in C. virosa roots; (ii) growth of C. virosa seedlings; and (iii) heavy‐metal tolerance of C. virosa plants. Root endophytic Pseudomonas putida and Rhodopseudomonas sp., which induced the high production of Zn‐chelating compounds, were selected for the seedling inoculation test. The results of the inoculation test demonstrated that both strains of endophytes increased Zn accumulation in C. virosa roots by solubilizing Zn in the sediment. Both strains also increased the growth of seedlings by possible production of indole‐3‐acetic acid in the plant. The heavy‐metal tolerance of C. virosa seedlings was likely promoted by producing metal‐chelating compounds that detoxify the metals in the plant tissues, and by decreasing the heavy‐metal contents in the tissues via rapid seedling growth. Thus, such mutualistic interactions between plants and bacteria contribute to the persistence of C. virosa in this severe environment.     

Design and synthesis of novel DFG-out RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors: 2. Synthesis and characterization of a novel imide-type prodrug for improving oral absorption

As an alternative to the previously reported solid dispersion formulation for enhancing the oral absorption of thiazolo[5,4-b]pyridine 1, we investigated novel N-acyl imide prodrugs of 1 as RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors. Introducing N-acyl promoieties at the benzanilide position gave chemically stable imides. N-tert-Butoxycarbonyl (Boc) introduced imide 6 was a promising prodrug, which was converted to the active compound 1 after its oral administration in mice. Cocrystals of 6 with AcOH (6b) possessed good physicochemical properties with moderate thermodynamic solubility (19μg/mL). This crystalline prodrug 6b was rapidly and enzymatically converted into 1 after its oral absorption in mice, rats, dogs, and monkeys. Prodrug 6b showed in vivo antitumor regressive efficacy (T/C=-6.4%) in an A375 melanoma xenograft model in rats. Hence, we selected 6b as a promising candidate and are performing further studies. Herein, we report the design, synthesis, and characterization of novel imide-type prodrugs.     

Effects of temperature on the development and survival of an endangered butterfly,Lycaeides argyrognomon (Lepidoptera: Lycaenidae) with estimation of optimal and threshold temperatures using linear and nonlinear models

The effects of temperature on the development and survival of Lycaeides argyrognomon were examined in the laboratory. The eggs, larvae and pupae were reared at temperatures of 15, 17.5, 20, 25, 30 and 33°C under a long‐day photoperiod of 16‐h light and 8‐h darkness. The survival rates of the first–third instars ranged from 40.0 to 82.4%. The mortalities of the fourth instar were lower than those of the first–third instars. The development time of the overall immature stage decreased from 78.33 days at 15°C to 21.07 days at 30°C, and then increased to 24.33 days at 33°C. The common linear model and the Ikemoto–Takai model were used to estimate the thermal constant (K) and the developmental zero (T₀). The values of T₀ and K for the overall immature stages were 10.50°C and 418.83 degree‐days, and 9.71°C and 451.68 degree‐days by the common model and the Ikemoto–Takai model, respectively. The upper temperature thresholds (T_max) and the optimal temperatures (T_opt) of the egg, the first–third instars and the overall immature stages were estimated by the three nonlinear models. The ranges of T_opt estimated were from 30.33°C to 32.46°C in the overall immature stages and the estimates of T_max of the overall immature stages by the Briere‐1 and the Briere‐2 models were 37.18°C and 33.00°C, respectively. The method to predict the developmental period of L. argyrognomon using the nonlinear models was discussed based on the data of the average temperature per hour.     

Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition

The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2alpha kinase family identifies helix alphaG as critical for the specific recognition of eIF2alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2alpha substrate recognition.