Naoka Komori

Phosrestin I undergoes the earliest light-induced phosphorylation by a calcium/calmodulin-dependent protein kinase in drosophila photoreceptors

Activation of PI-PLC initiates two independent branches of protein phosphorylation cascades catalyzed by either PKC or Ca2+/calmodulin-dependent protein kinase (CaMK). We find that phosrestin I (PRI), a Drosophila homolog of vertebrate photoreceptor arrestin, undergoes light-induced phosphorylation on a subsecond time scale which is faster than that of any other protein in vivo. We determine that a CaMK activity is responsible for in vitro PRI phosphorylation at Ser366 in the C-terminal tryptic segment, MetLysSer(P)IleGluGlnHisArg, in which Ser(P) represents phosphoserine366. We also demonstrate that Ser366 is the phosphorylation site of PRI in vivo by identifying the molecular species resulting from in-gel tryptic digestion of purified phospho-PRI using HPLC-electrospray ionization tandem quadrupole mass spectroscopy. From these data, we conclude that the CaMK pathway, not the PKC pathway, is responsible for the earliest protein phosphorylation event following activation of PI-PLC in living Drosophila photoreceptors.

Mechanisms of sustained cutaneous vasodilation induced by spinal cord stimulation

This study was performed to investigate whether spinal cord stimulation (SCS) at intensities below motor threshold prolongs cutaneous vasodilation and whether sustained vasodilation by SCS is mediated through sympathetic inhibition and/or antidromic activation of sensory fibers. SCS was applied to the dorsal surface of the L2-L3 spinal cord of anesthesized rats with stimulus parameters used clinically (i.e., 50 Hz, 0.2 ms duration, and stimulus intensity at 30%, 60%, or 90% of motor threshold). Peripheral vasodilation induced by 5-min SCS was not attenuated by hexamethonium, an autonomic ganglion-blocking agent, but was abolished by dorsal rhizotomy. SCS at < or = 60% of motor threshold increased cutaneous blood flow to the level similar to that obtained at 90% of motor threshold, but the vasodilation did not last for 5 min. SCS-induced vasodilation at 90% of motor threshold persisted for the entire stimulation period up to 30 min, and the vasodilation was not attenuated by hexamethonium. It is concluded that sustained vasodilation, which is induced by SCS at only 90% of motor threshold, in this study was mediated via antidromic activation of sensory fibers.

Sensory fibers containing vanilloid receptor-1 (VR-1) mediate spinal cord stimulation-induced vasodilation

BACKGROUND AND AIMS Spinal cord stimulation (SCS) is used to improve peripheral blood flow in selected populations of patients with ischemia of the extremities. Previous studies show that antidromic activation of sensory fibers is an important mechanism that contributes to SCS-induced vasodilation. However, the characteristics of sensory fibers involved in vasodilation are not fully known. This study investigated the contribution of vanilloid receptor type 1 (VR-1) containing fibers to SCS-induced vasodilation. METHODS A unipolar ball electrode was placed on the left dorsal column at the lumbar 2-3 spinal cord segments (L2-L3) in sodium pentobarbital anesthetized, paralyzed and ventilated rats. Cutaneous blood flows from both ipsilateral (left) and contralateral (right) hind foot pads were recorded with laser Doppler flow perfusion monitors. SCS (50 Hz; 0.2 ms) was applied through the ball electrode at 30%, 60%, 90% and 300% of motor threshold (MT). Resiniferatoxin (RTX), an ultra potent analog of capsaicin and VR-1 receptor agonist, was used to suppress the activities of VR-1 containing sensory fibers. RESULTS SCS at 30%, 60%, 90% and also at 300% of MT significantly increased cutaneous blood flow in the ipsilateral foot pad compared to that in the contralateral side. RTX (2 microg/kg, i.v.) significantly attenuated SCS-induced vasodilation of the ipsilateral side (P<0.05, n=7) compared with responses prior to RTX administration. A pledget of cotton soaked with RTX (2 microg/ml) placed on L2-L3 spinal cord significantly decreased SCS-induced vasodilation of the ipsilateral side at 30%, 60%, 90% and 300% of MT (P<0.05, n=7) compared with responses prior to RTX administration. Additionally, topical application of a pledget of cotton soaked with RTX (2 microg/ml) on the sciatic nerve at the middle level of the thigh or on the tibial nerve at the lower level of the lower hindlimb also decreased SCS-induced vasodilation (n=5). CONCLUSION SCS-induced vasodilation is predominantly mediated via VR-1 containing sensory fibers.

Ocular proteomics: cataloging photoreceptor proteins by two-dimensional gel electrophoresis and mass spectrometry.

Publisher Summary Two-dimensional (2-D) gel electrophoresis, invented more than two decades ago, is a powerful technique for isolating proteins from a complex biological mixture. Important developments have taken place in the initiation and maturation of “genome projects” in which all the genome information in human and in some model animals will be registered. Technological development in automated DNA sequencing has led to the completion of some genome projects for microbes and lower eukaryotes. Another development took place in in modern mass spectrometry initiated by inventions of two new ionization methods, that is, electrospray ionization and matrix-assisted laser desorption ionization. This chapter describes a set of protocols to perform 2-D gel electrophoresis of proteins extracted from bovine retina, in-gel digestion of protein spots, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) to obtain peptide mass fingerprints, a search of genome databases (through the Internet) for peptide fingerprints, and analysis of the resulting data.

Extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) pathways involved in spinal cord stimulation (SCS)-induced vasodilation

BACKGROUND AND AIMS SCS is used to improve peripheral circulation in selected patients with ischemia of the extremities. However the mechanisms are not fully understood. The present study investigated whether blockade of ERK and AKT activation modulated SCS-induced vasodilation. METHODS A unipolar ball electrode was placed on the left dorsal column at the lumbar 2-3 spinal segments in rats. Cutaneous blood flows from left and right hind foot pads were recorded with laser Doppler flow perfusion monitors. SCS was applied through a ball electrode at 60% or 90% of MT. U0126, an inhibitor of ERK kinase, or LY294002, an inhibitor of PI3K upstream of AKT, was applied to the lumbar 3-5 spinal segments (n=7, each group). RESULTS U0126 (100 nM, 5 microM and 250 microM) significantly attenuated SCS-induced vasodilation at 60% (100 nM: P<0.05; 5 microM and 250 microM: P<0.01, respectively) and 90% of MT (100 nM and 5 microM: P<0.05; 250 microM: P<0.01, respectively). LY294002 at 100 microM also attenuated SCS-induced vasodilation at 60% and 90% of MT (P<0.05). CONCLUSIONS These data suggest that ERK and AKT pathways are involved in SCS-induced vasodilation.

Metabolic enrichment of omega-3 polyunsaturated fatty acids does not reduce the onset of idiopathic knee osteoarthritis in mice

OBJECTIVE We evaluated the effect of a reduction in the systemic ratio of n-6:n-3 polyunsaturated fatty acids (PUFAs) on changes in inflammation, glucose metabolism, and the idiopathic development of knee osteoarthritis (OA) in mice. We hypothesized that a lower ratio of n-6:n-3 PUFAs would protect against OA markers in cartilage and synovium, but not bone. DESIGN Male and female fat-1 transgenic mice (Fat-1), which convert dietary n-6 to n-3 PUFAs endogenously, and their wild-type (WT) littermates were fed an n-6 PUFA enriched diet for 9-14 months. The effect of gender and genotype on serum PUFAs, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and glucose tolerance was tested by 2-factor analysis of variance (ANOVA). Cortical and trabecular subchondral bone changes were documented by micro-focal computed tomography (CT), and knee OA was assessed by semi-quantitative histomorphometry grading. RESULTS The n-6:n-3 ratio was reduced 12-fold and 7-fold in male and female Fat-1 mice, respectively, compared to WT littermates. IL-6 and TNF-α levels were reduced modestly in Fat-1 mice. However, these systemic changes did not reduce osteophyte development, synovial hyperplasia, or cartilage degeneration. Also the fat-1 transgene did not alter subchondral cortical or trabecular bone morphology or bone mineral density. CONCLUSIONS Reducing the systemic n-6:n-3 ratio does not slow idiopathic changes in cartilage, synovium, or bone associated with early-stage knee OA in mice. The anti-inflammatory and anti-catabolic effects of n-3 PUFAs previously reported for cartilage may be more evident at later stages of disease or in post-traumatic and other inflammatory models of OA.

Biochemical Characterization of Cone Cyclic Nucleotide-Gated (CNG) Channel Using the Infrared Fluorescence Detection System

Cone vision mediated by photoreceptor cyclic nucleotide-gated (CNG) channel is essential for central and color vision and visual acuity. Cone CNG channel is composed of two structurally related subunit types, CNGA3 and CNGB3. Naturally occurring mutations in cone CNG channel are associated with a variety of cone diseases including achromatopsia, progressive cone dystrophy, and some maculopathies. Nevertheless, our understanding of the structure of cone CNG channel is quite limited. This is, in part, due to the challenge of studying cones in a rod-dominant mammalian retina. We have demonstrated a robust expression of cone CNG channel and a lack of rod CNG channel in the cone-dominant Nrl−/− retina and shown that the Nrl−/− mouse line is a valuable model to study cone CNG channel. This work examined the complex structure of cone CNG channel using infrared fluorescence Western detection combined with chemical cross-linking and blue native-PAGE. Our results suggest that the native cone CNG channel is a heterotetrameric complex likely at a stoichiometry of three CNGA3 and one CNGB3.

Differential expression of alternative splice variants of β-arrestin-1 and -2 in rat central nervous system and peripheral tissues

Members of arrestin/β‐arrestin protein family are thought to participate in agonist‐mediated desensitization of G‐protein‐coupled receptors, including rhodopsin and β2‐adrenergic receptor. Unlike in human and cow, splice variants of this protein family in rat have not been studied extensively, and there has been no report on their existence at protein level. Hence, a previous report by others on the localization of both β‐arrestin‐1 and ‐2 in a wide range of innervated rat tissues could imply their broad receptor specificity. In this report we show the presence of two alternatively spliced forms of β‐arrestin‐1 in several rat tissues using both reverse transcription–polymerase chain reaction and Western immunoblot. Splicing of β‐arrestin‐1 pre‐mRNA appears to be subject to differential regulation between the rat CNS and peripheral tissues. In contrast, we detected no splice variants of β‐arrestin‐2 in rat. A comparison of the genomic DNA sequences of bovine and rat β‐arrestin‐2, where the splicing of bovine β‐arrestin‐2 mRNA has been reported, revealed a high degree of homology in their organization of exons and introns as well as certain differences that might be responsible for the different processing of β‐arrestin‐2 mRNA in the two species. Our two‐dimensional isoelectric focusing gels using rat spinal cord and heart tissues demonstrate isoelectric heterogeneity of rat β‐arrestin‐1, suggesting that β‐arrestin‐1 is subject to post‐translational modification unlike β‐arrestin‐2.

Effects of immunization with natural and recombinant lysine decarboxylase on canine gingivitis development

Periodontal disease, gingival inflammation (gingivitis) and periodontal attachment loss (periodontitis), causes tooth loss and susceptibility to chronic inflammation. Professionally scaling and cleaning the teeth regularly controls the disease, but is expensive in companion animals. Eikenella corrodens is common in canine oral cavities where it is a source of lysine decarboxylase (LDC). In human dental biofilms (plaques), LDC converts lysine to cadaverine and impairs the gingival epithelial barrier to bacteria. LDC vaccination may therefore retard gingivitis development. Year-old beagle dogs provided blood samples, and had weight and clinical measurements (biofilm and gingivitis) recorded. After scaling and cleaning, two dogs were immunized subcutaneously with 0.2mg native LDC from E. corrodens and 2 sets of four dogs with 0.2mg recombinant LDC purified from Escherichia coli. A third set of 4 dogs was immunized intranasally. Rehydragel(®), Emulsigen(®), Polygen™ or Carbigen™ were used as adjuvant. Four additional pairs of dogs were sham-immunized with each adjuvant alone (controls). Immunizations were repeated twice, 3 weeks apart, and clinical measurements were obtained after another 2 weeks, when the teeth were scaled and cleaned again. Tooth brushing was then stopped and the diet was changed from hard to soft chow. Clinical measurements were repeated after 1, 2, 3, 4, 6 and 8 weeks. Compared with sham-immunized dogs, gingivitis was reduced over all 8 weeks of soft diet after subcutaneous immunization with native LDC, or after intranasal immunization with recombinant LDC in Carbigen™, but for only 6 of the 8 weeks after subcutaneous immunization with recombinant LDC in Emulsigen(®) (repeated measures ANOVA). Subcutaneous vaccination induced a strong serum IgG antibody response that decreased during the soft diet period, whereas intranasal immunization induced a weak serum IgA antibody response that did not decrease. Immunization with recombinant LDC may provide protection from gingivitis if procedures are optimized.

Mechanism for the regulation of mammalian cGMP phosphodiesterase6. 1: Identification of its inhibitory subunit complexes and their roles

Cyclic GMP phosphodiesterase (PDE) in bovine rod photoreceptor outer segments (OS) comprises a catalytic subunit complex (Pαβ) and two inhibitory subunits (Pγ) and is regulated by the α subunit of transducin (Tα). Here, we show an overall mechanism for PDE regulation by identifying Pγ complexes in OS homogenates prepared with an isotonic buffer. Before Tα activation, three Pγ complexes exist in the soluble fraction. Complex a, a minor complex, contains Pαβ, Tα, and a protein named Pδ. Complex b, Pαβγγb, has a PDE activity similar to that of membranous Pαβγγ, PαβγγM, and its level, although its large portion is Pδ-free, is estimated to be 20–30% of the total Pαβγγ. Complex c, (Pγ·GDP-Tα)2c, appears to be a dimer of Pγ·GDP-Tα. Upon Tα activation, (1) complex a stays unchanged, (2) Pαβγγb binds to membranes, (3) the level of (Pγ·GDP-Tα)2c is reduced as its GTP-form is produced, (4) complex d, Pγ·GTP-Tαd, is formed on membranes and its substantial amount is released to the soluble fraction, and (5) membranous Pαβγγ, PαβγγM and/or Pαβγγb, becomes Pγ-depleted. These observations indicate that Pγ as a complex with GTP-Tα dissociates from Pαβγγ on membranes and is released to the soluble fraction and that Pγ-depleted PDE is the GTP-Tα-activated PDE. After GTP hydrolysis, both (Pγ·GDP-Tα)2c and Pγ·GDP-Tαd, without liberating Pγ, deactivate Pγ-depleted PDE. The preferential order to be used for the deactivation is membranous Pγ·GDP-Tαd, solubilized Pγ·GDP-Tαd and (Pγ·GDP-Tα)2c. Release of Pγ·GTP-Tα complexes to the soluble fraction is relevant to light adaptation.