Colocalization of NO and VIP in neurons of the submucous plexus in the rat intestine

Since very few previous studies have carried out the quantitative analysis for the colocalization of nitric oxide (NO) and vasoactive intestinal peptide (VIP) in the submucous neurons in the rat digestive tract, we applied in vivo treatment of colchicine to enhance the immunoreactivity and examined the colocalization of NO synthase (nNOS) and VIP in neurons of the submucous plexus throughout the rat digestive tract. The density of nNOS-containing neurons in the submucous plexus in the stomach corpus (103±25 cells/cm², n=3) and that in the antrum (157±9 cells/cm², n=3) were significantly lower than those in small and large intestine. However no difference was detected in the cell density among duodenum (1967±188 cells/cm², n=3), jejunum (2640±140 cells/cm², n=3), ileum (2070±42 cells/cm², n=3), proximal colon (2243±138 cells/cm², n=3) and distal colon (2633±376 cells/cm², n=3). The proportion of nNOS-immunoreactive (IR), nNOS/VIP-IR and VIP-IR neurons to the total number of submucous neurons was examined. nNOS/VIP-IR neurons comprised 45–55% of total number of submucous neurons from the duodenum to the proximal colon, however those comprised 66.4±5.1% in the distal colon. The results showed that the dense distribution of nNOS-containing neurons was found in the submucous plexus throughout the small and large intestine, and large population of submucous neurons co-stored nNOS and VIP.     

Structural determinants for the action of grayanotoxin in D1 S4-S5 and D4 S4-S5 intracellular linkers of sodium channel alpha-subunits

We located a novel binding site for grayanotoxin on the cytoplasmic linkers of voltage-dependent cardiac (rH1) or skeletal-muscle (mu 1) Na(+) channel isoforms (segments S4-S5 in domains D1 and D4), using the alanine scanning substitution method. GTX-modification of Na(+) channels, transiently expressed in HEK 293 cells, was evaluated under whole-cell voltage clamp, from the ratio of maximum chord conductance for modified and unmodified Na(+) channels. In mu 1, mutations K237A, L243A, S246A, K248A, K249A, L250A, S251A, or T1463A, caused a moderate, but statistically significant decrease in this ratio. On making corresponding mutations in rH1, only L244A dramatically reduced the ratio. Because in mu 1, the serine at position 251 is the only heterologous residue with respect to rH1 (Ala-252), we made a double mutant L243A&S251A to match the sequence of mu 1 and rH1 in S4-S5 linkers of both domains. This double mutation resulted in a significant decrease in the ratio, to the same extent as L244A substitution in rH1 did, indicating that the site at Leu-244 in rH1 or at Leu-243 in mu 1 is a novel one, exhibiting a synergistic effect of grayanotoxin.     

Immunocytochemical localization of phenylalanine ammonia-lyase in tissues of Populus kitakamiensis

The polypeptide encoded by the partial fragment of cDNA of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), PALcDNAl (Osakabe et al., 1995, Plant Sci. 105: 217–226), isolated from Populus kitakamiensis (P. sieboldii x P. grandidentata), was expressed in Escherichia coli cells. The polypeptide was purified and an antiserum raised against it. The antiserum recognized a protein of 77 kDa on nitrocellulose blots after sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of total protein and the partially purified PAL protein from P. kitakamiensis. Moreover,the antiserum recognized a protein on the blot after non-denaturing polyacrylamide gel electrophoresis of P. kitakamiensis proteins and this protein had PAL activity. Furthermore, the antibody inhibited PAL activity of extracts from stem tissues. These results showed that the antiserum against the partial PAL peptide recognized only the PAL subunits in extracts of P. kitakamiensis. Immunolocalization studies of P. kitakamiensis tissues revealed that the PAL protein was specifically localized in the xylem and the phloem fibers and no immunogold signal was found in the epidermis, the cortex, the pith, or the cambium of either stems or leaves.Abbreviations IgG immunoglobulin G - IPTG isopropylthio--d-galactoside - PAL phenylalanine ammonia-lyase The authors thank Dr. Kunio Hata of Nippon Paper Industries Co., Ltd. (Japan) for supplying P. kitakamiensis. This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 07406008).     

Effects of Chloride Ion Binding on the Photochemical Properties of Salinibacter Sensory Rhodopsin I

Microbial organisms utilize light not only as energy sources but also as signals by which rhodopsins (containing retinal as a chromophore) work as photoreceptors. Sensory rhodopsin I (SRI) is a dual photoreceptor that regulates both negative and positive phototaxis in microbial organisms, such as the archaeon Halobacterium salinarum and the eubacterium Salinibacter ruber. These organisms live in highly halophilic environments, suggesting the possibility of the effects of salts on the function of SRI. However, such effects remain unclear because SRI proteins from H. salinarum (HsSRI) are unstable in dilute salt solutions. Recently, we characterized a new SRI protein (SrSRI) that is stable even in the absence of salts, thus allowing us to investigate the effects of salts on the photochemical properties of SRI. In this study, we report that the absorption maximum of SrSRI is shifted from 542 to 556 nm in a Cl⁻-dependent manner with a K_m of 307 ± 56 mM, showing that Cl⁻-binding sites exist in SRI. The bathochromic shift was caused not only by NaCl but also by other salts (NaI, NaBr, and NaNO₃), implying that I⁻, Br⁻, and NO₃⁻ can also bind to SrSRI. In addition, the photochemical properties during the photocycle are also affected by chloride ion binding. Mutagenesis studies strongly suggested that a conserved residue, His131, is involved in the Cl⁻-binding site. In light of these results, we discuss the effects of the Cl⁻ binding to SRI and the roles of Cl⁻ binding in its function.     

The diversity of mycorrhizal fungi in Japanese Cephalanthera species

The diversity of mycorrhizal fungi among four Japanese Cephalanthera (Orchidaceae) species was examined at a total of seven sites, based on sequence variation in nuclear ribosomal DNA. This is the first report to detect mycorrhizal fungi in C. subaphylla. Two patterns of mycorrhizal associations were confirmed from our results. C. falcata has lower fungal specificity, associating with Russulaceae, Sebacinales and Thelephoraceae. By contrast, the three Cephalanthera species C. erecta, C. subaphylla, and C. longifolia were associated mainly with Thelephoraceae fungi. There is no large difference found in mycobionts between green and albino individuals of C. falcata and no distinct preference of Japanese Cephalanthera species for a specific fungal group of Thelephoraceae.     

Eighteen Coral Genomes Reveal the Evolutionary Origin of Acropora Strategies to Accommodate Environmental Changes

The genus Acropora comprises the most diverse and abundant scleractinian corals (Anthozoa, Cnidaria) in coral reefs, the most diverse marine ecosystems on Earth. However, the genetic basis for the success and wide distribution of Acropora are unknown. Here, we sequenced complete genomes of 15 Acropora species and 3 other acroporid taxa belonging to the genera Montipora and Astreopora to examine genomic novelties that explain their evolutionary success. We successfully obtained reasonable draft genomes of all 18 species. Molecular dating indicates that the Acropora ancestor survived warm periods without sea ice from the mid or late Cretaceous to the Early Eocene and that diversification of Acropora may have been enhanced by subsequent cooling periods. In general, the scleractinian gene repertoire is highly conserved; however, coral- or cnidarian-specific possible stress response genes are tandemly duplicated in Acropora. Enzymes that cleave dimethlysulfonioproprionate into dimethyl sulfide, which promotes cloud formation and combats greenhouse gasses, are the most duplicated genes in the Acropora ancestor. These may have been acquired by horizontal gene transfer from algal symbionts belonging to the family Symbiodiniaceae, or from coccolithophores, suggesting that although functions of this enzyme in Acropora are unclear, Acropora may have survived warmer marine environments in the past by enhancing cloud formation. In addition, possible antimicrobial peptides and symbiosis-related genes are under positive selection in Acropora, perhaps enabling adaptation to diverse environments. Our results suggest unique Acropora adaptations to ancient, warm marine environments and provide insights into its capacity to adjust to rising seawater temperatures.     

A case of fibrosarcoma on the perivertebral surface of a ferret with hind limb paralysis

A 2.5 year-old female ferret had a stiff palpable mass arising from the dorsal surface of the thoracic (T) to lumbar (L) vertebrae with paralysis of the hind limbs. By myelography the dorsal and ventral lines of contrast were not observed in the area forwarded of L3. Grossly, the tumor encircled the dorsal vertebrae. Microscopically, tumor cells were proliferated intimately and were attached to the vertebrae surface involving surrounding fatty and connective tissues. The tumor consisted of fibroblastic cells with prominent cellular atypia. The bromodeoxyuridine (BrdU) labeling index to examine cellular kinetics was high (11.8%). Based on macro and micropathological features, the present tumor was diagnosed as periosteal fibrosarcoma arising from perivertebral connective tissue.     

Analyses of group III secreted phospholipase A2 transgenic mice reveal potential participation of this enzyme in plasma lipoprotein modification, macrophage foam cell formation, and atherosclerosis

Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.