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.     

Identification and Characterization of a Novel Galactofuranose-Specific β-D-Galactofuranosidase from Streptomyces Species

β-D-galactofuranose (Galf) is a component of polysaccharides and glycoconjugates and its transferase has been well analyzed. However, no β-D-galactofuranosidase (Galf-ase) gene has been identified in any organism. To search for a Galf-ase gene we screened soil samples and discovered a strain, identified as a Streptomyces species by the 16S ribosomal RNA gene analysis, that exhibits Galf-ase activity for 4-nitrophenyl β-D-galactofuranoside (pNP-β-D-Galf) in culture supernatants. By draft genome sequencing of the strain, named JHA19, we found four candidate genes encoding Galf-ases. Using recombinant proteins expressed in Escherichia coli, we found that three out of four candidates displayed the activity of not only Galf-ase but also α-L-arabinofuranosidase (Araf-ase), whereas the other one showed only the Galf-ase activity. This novel Galf-specific hydrolase is encoded by ORF1110 and has an optimum pH of 5.5 and a Km of 4.4 mM for the substrate pNP-β-D-Galf. In addition, this enzyme was able to release galactose residue from galactomannan prepared from the filamentous fungus Aspergillus fumigatus, suggesting that natural polysaccharides could be also substrates. By the BLAST search using the amino acid sequence of ORF1110 Galf-ase, we found that there are homolog genes in both prokaryotes and eukaryotes, indicating that Galf-specific Galf-ases widely exist in microorganisms.     

Different combinations of Notch ligands and receptors regulate V2 interneuron progenitor proliferation and V2a/V2b cell fate determination

The broad diversity of neurons is vital to neuronal functions. During vertebrate development, the spinal cord is a site of sensory and motor tasks coordinated by interneurons and the ongoing neurogenesis. In the spinal cord, V2-interneuron (V2-IN) progenitors (p2) develop into excitatory V2a-INs and inhibitory V2b-INs. The balance of these two types of interneurons requires precise control in the number and timing of their production. Here, using zebrafish embryos with altered Notch signaling, we show that different combinations of Notch ligands and receptors regulate two functions: the maintenance of p2 progenitor cells and the V2a/V2b cell fate decision in V2-IN development. Two ligands, DeltaA and DeltaD, and three receptors, Notch1a, Notch1b, and Notch3 redundantly contribute to p2 progenitor maintenance. On the other hand, DeltaA, DeltaC, and Notch1a mainly contribute to the V2a/V2b cell fate determination. A ubiquitin ligase Mib, which activates Notch ligands, acts in both functions through its activation of DeltaA, DeltaC, and DeltaD. Moreover, p2 progenitor maintenance and V2a/V2b fate determination are not distinct temporal processes, but occur within the same time frame during development. In conclusion, V2-IN cell progenitor proliferation and V2a/V2b cell fate determination involve signaling through different sets of Notch ligand–receptor combinations that occur concurrently during development in zebrafish.     

Musculoskeletal-see-through mirror: Computational modeling and algorithm for whole-body muscle activity visualization in real time

In this paper, we present a system that estimates and visualizes muscle tensions in real time using optical motion capture and electromyography (EMG). The system overlays rendered musculoskeletal human model on top of a live video image of the subject. The subject therefore has an impression that he/she sees the muscles with tension information through the cloth and skin. The main technical challenge lies in real-time estimation of muscle tension. Since existing algorithms using mathematical optimization to distribute joint torques to muscle tensions are too slow for our purpose, we develop a new algorithm that computes a reasonable approximation of muscle tensions based on the internal connections between muscles known as neuronal binding. The algorithm can estimate the tensions of 274 muscles in only 16 ms, and the whole visualization system runs at about 15 fps. The developed system is applied to assisting sport training, and the user case studies show its usefulness. Possible applications include interfaces for assisting rehabilitation.     

Impact of insulin resistance on enhanced monocyte adhesion to endothelial cells and atherosclerogenesis independent of LDL cholesterol level

Epidemiological studies suggest that insulin resistance is an independent risk factor for cardiovascular disease. However, there is little information on the role of insulin resistance in atherosclerogenesis independent of LDL cholesterol level. The aim of this study was to investigate the impact of systemic insulin resistance on monocyte adhesion to endothelial cells and atherosclerotic lesions independent of LDL cholesterol level. KKAy mice are obese mice with spontaneous diabetes and insulin resistance, and normal levels of LDL cholesterol. In parallel with systemic insulin resistance, decreased insulin signal, and the increased expression of monocyte chemoattractant protein-1 (MCP-1) were noted in macrophages isolated from KKAy mice. These mice showed enhanced monocyte adhesion to the endothelial cells of the thoracic artery. Furthermore, these mice showed expanded atherosclerotic lesions when fed high cholesterol diet. Our data indicate that insulin resistance promotes the atherosclerogenesis independent of LDL cholesterol level. Decreased insulin signaling in macrophages associated with systemic insulin resistance could be involved, at least in part, in this pathological process.     

Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei

The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis-Menten kinetics (K_m of 338 μM and V_max of 601 μmol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1.     

Magnesium deprivation inhibits a MEK–ERK cascade and cell proliferation in renal epithelial Madin-Darby canine kidney cells

AimsLoss of magnesium (Mg²⁺) inhibits cell proliferation and augments nephrotoxicant-induced renal injury, but the role of Mg²⁺ has not been clarified in detail. We examined the effect of extracellular Mg²⁺ deprivation on a MEK–ERK cascade and cell proliferation using a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells.Main methodsMDCK cells were cultured in Mg²⁺-containing or Mg²⁺-free media. A HA-tagged constitutively active (CA)-MEK1 and a dominant negative (DN)-MEK1 were transfected into MDCK cells. The level of protein was examined by Western blotting. The intracellular free Mg²⁺ concentration ([Mg²⁺]_i) was measured using a fluorescent dye, mag-fura 2. Cell proliferation was determined by WST-1 assay. Dead cells were identified by staining with annexin V-FITC and propidium iodide.Key findingsIn the presence of fetal calf serum (FCS), Mg²⁺ deprivation decreased phosphorylated-ERK1/2 (p-ERK1/2) levels and [Mg²⁺]_i. Re-addition of Mg²⁺ increased p-ERK1/2 levels, which were inhibited by U0126, a specific inhibitor of a MEK–ERK cascade. Glutathione-S-transferase pull-down and coimmunoprecipitation assays showed that CA-MEK1 and DN-MEK1 binds with ERK1/2 in the presence of Mg²⁺. In contrast, neither CA-MEK1 nor DN-MEK1 bound to ERK1/2 in the absence of Mg²⁺. These results indicate that the MEK–ERK cascade is regulated by [Mg²⁺]_i. Cell proliferation was increased by the treatment with FCS or the expression of CA-MEK1 in the presence of Mg²⁺, but was inhibited by Mg²⁺ deprivation. Mg²⁺ deprivation did not increase the number of dead cells.SignificanceMg²⁺ is involved in the regulation of the MEK–ERK cascade and cell proliferation in MDCK cells.     

Vasoactive intestinal peptide stimulates mucus secretion, but nitric oxide has no effect on mucus secretion in the ferret trachea.

Vasoactive intestinal peptide (VIP) and nitric oxide (NO) are neurotransmitters involved in the regulation of bronchial and pulmonary vascular tone. Published studies of the effects of VIP on airway mucus secretion have yielded conflicting results. The purpose of this study was to determine the effect of VIP on mucus secretion in the ferret trachea and if this effect was influenced by NO. We used a sandwich enzyme-linked lectin assay to measure mucin secretion and a turbidimetric assay to measure lysozyme (serous cell) secretion from ferret tracheal segments. VIP (10(-7) M) increased mucin secretion over 2 h. VIP (10(-9) to 10(-5) M) stimulated mucin secretion in a dose-dependent fashion. VIP-induced mucin secretion was partially blocked by a VIP receptor antagonist (a chimeric VIP-pituitary adenylate cyclase-activating peptide analog, VIP receptor antagonist) at a 10-fold excess concentration. At all concentrations tested, neither NG-nitro-L-arginine methyl ester, an inhibitor of NO synthase, nor S-nitroso-N-acetyl-penicillamine, an NO donor, had any significant effect on constitutive or VIP-induced mucus secretion. We conclude that VIP-stimulated mucin and lysozyme secretion was both time dependent and dose dependent and that NO neither stimulates nor inhibits mucus secretion in the ferret trachea.     

Excess tyrosine rescues the reduced activity of proliferation and differentiation of cultured recessive yellow melanocytes derived from neonatal mouse epidermis

The murine recessive yellow (Mc1r(e)) is a loss-of-function mutation in the receptor for alpha-melanocyte-stimulating hormone, melanocortin receptor 1 (Mc1r) and produces yellow coats by inducing pheomelanin synthesis in hair follicular melanocytes. However, it is not known whether the Mc1r(e) mutation affects the proliferation and differentiation of melanocytes. In this study, the proliferation and differentiation of recessive yellow epidermal melanocytes cultured in dibutyryl cyclic AMP-supplemented serum-free medium were investigated in detail. The melanocytes produced mainly eumelanin in this culture system. The proliferation of recessive yellow melanocytes was decreased compared with that of wild-type at the e-locus, black melanocytes. The differentiation of melanocytes was also delayed and inhibited in recessive yellow mice. Tyrosinase (TYR) activity and TYR-related protein 1 (TRP1) and TRP2 (dopachrome tautomerase, DCT) expressions were decreased and, in addition, the maturation of stage IV melanosomes was inhibited. Excess l-tyrosine (l-Tyr) added to the culture media rescued the reduced activity of proliferation of melanocytes. l-Tyr also stimulated TYR activity and TRP1 and TRP2 expressions as well as the maturation of stage IV melanosomes and pigmentation. These results suggest that the Mc1r(e) mutation affects the proliferation and differentiation of melanocytes and l-Tyr rescues the reduced proliferative and differentiative activities by stimulating TYR activity and TRP1 and TRP2 expressions as well as melanosome maturation.