Endothelial nitric oxide synthase in the amphibian, Xenopus tropicalis

Nitric oxide (NO) is generated by NO synthase (NOS) of which there are three isoforms: neuronal NOS (nNOS, nos1), inducible NOS (iNOS, nos2), and endothelial NOS (eNOS, nos3). This study utilised the genome of Xenopus tropicalis to sequence a nos3 cDNA and determine if eNOS protein is expressed in blood vessels. A nos3 cDNA was sequenced that encoded a 1177 amino acid protein called XteNOS, which showed closest sequence identity to mammalian eNOS protein. The X. tropicalis nos3 gene and eNOS protein were determined to be an orthologue of mammalian nos3 and eNOS using gene synteny and phylogenetic analyses, respectively. In X. tropicalis, nos3 mRNA expression was highest in lung and skeletal muscle and lower in the liver, gut, kidney, heart and brain. Western analysis of kidney protein using an affinity-purified anti-XteNOS produced a single band at 140kDa. Immunohistochemistry showed XteNOS immunoreactivity in the proximal tubule of the kidney and endocardium of the heart, but not in the endothelium of blood vessels. Thus, X. tropicalis has a nos3 gene that appears not to be expressed in the vascular endothelium.     

Nitric oxide (NO) synthase immunoreactivity in the starfish Marthasterias glacialis

The neuroendocrine system of the starfish Marthasterias glacialis was investigated immunocytochemically using antisera specific for rat neuronal, bovine aortic endothelial, and mouse macrophage, nitric oxide (NO) synthases. Immunoreactivity was detected only with the antibodies specific for the neural enzyme, in the ectoneural and hyponeural tissues of the radial nerve cords and in the basiepithelial plexus and endocrine cells of the digestive tract. The pyloric stomach showed more immunoreactive structures than the other digestive organs, with the rectal caeca showing the least activity. Immunoreactive endocrine cells were located in the cardiac and pyloric stomachs and in the pyloric caeca. Co-localization of the enzyme immunoreactivity, and the staining for NADPH-diaphorase, demonstrate the presence of NO synthase in echinoderms. These results provide further evidence that NO is a neuronal messenger of early phylogenetic origin which has been conserved throughout evolution.     

Vascular distribution of nitric oxide synthase and vasodilation in the Australian lungfish, Neoceratodus forsteri

The presence of nitric oxide synthase (NOS) and role of nitric oxide (NO) in vascular regulation was investigated in the Australian lungfish, Neoceratodus forsteri. No evidence was found for NOS in the endothelium of large and small blood vessels following processing for NADPH-diaphorase histochemistry. However, both NADPH-diaphorase histochemistry and neural NOS immunohistochemistry demonstrated a sparse network of nitrergic nerves in the dorsal aorta, hepatic artery, and branchial arteries, but there were no nitrergic nerves in small blood vessels in tissues. In contrast, nitrergic nerves were found in non-vascular tissues of the lung, gut and kidney. Dual-wire myography was used to determine if NO signalling occurred in the branchial artery of N. forsteri. Both SNP and SIN-1 had no effect on the pre-constricted branchial artery, but the particulate guanylyl cyclase (GC) activator, C-type natriuretic peptide, always caused vasodilation. Nicotine mediated a dilation that was not inhibited by the soluble GC inhibitor, ODQ, or the NOS inhibitor, L-NNA, but was blocked by the cyclooxygenase inhibitor, indomethacin. These data suggest that NO control of the branchial artery is lacking, but that prostaglandins could be endothelial relaxing factors in the vasculature of lungfish.     

Effects of nitric oxide synthase inhibitors and a substrate,L-arginine,on the cardiac function of juvenile salmonid fish

Control of cardiac function was investigated juvenile brown trout (Salmo trutta L.) and rainbow trout (Oncorhynchus mykiss Walbaum) using inhibitors of nitric oxide synthase (NOS), (L-NAME, NG-nitro-L-arginine and L-NMMA, NG-monomethyl-L-arginine) and a substrate of NOS (L-arginine). Salmonid alevins are excellent models for such studies since they are transparent, the beating heart is easily observed, diffusing distances are small, and they respond within a few seconds to exogenously administered chemicals. The response to inhibitors of NOS (L-NAME or L-NMMA) was tachycardia interpreted as vasoconstriction through lowered capacity for synthesis of NO. This could be reversed by addition of L-arginine and the subsequent bradycardia was explained as a vasodilation resulting from increased synthesis of NO. Blood flow into the heart is mainly via the vitelline vein and changes of flow resulting from constriction or dilation of this vessel may be probably major determinants of heart rate. The results provide evidence for the presence NOS in juvenile fish and indicate a physiological role for NO in cardiovascular control.     

Trypanosoma cruzi: Serum levels of nitric oxide and expression of inducible nitric oxide synthase in myocardium and spleen of dogs in the acute stage of infection with metacyclic or blood trypomastigotes

The participation of nitric oxide (NO) in the control of blood parasitemia and parasitism during the acute phase of infection in dogs inoculated with blood trypomastigotes (BT) or metacyclic trypomastigotes (MT group) of Berenice-78 Trypanosoma cruzi strain has been evaluated. Animals of the MT group (n = 4) presented increased levels of serum NO throughout the infection when compared with the BT (n = 4) or control (n = 4) groups, and a delay in parasitemia peak compared with the BT group. In spleen fragments, tissue parasitism was not observed but the MT group presented larger areas associated with inducible NO synthase (iNOS) in relation to BT and control groups. Heart fragments of MT-infected animals exhibited comparatively low tissue parasitism and high iNOS expression, while animals of the BT group presented high inflammatory infiltrate, high tissue parasitism and low iNOS expression. These results indicate that the source of inoculum can interfere with the development of the acute phase of Chagas disease, and may also trigger a distinct parasite-host interaction during this phase.     

Nitric oxide (NO) increase at fertilization in sea urchin eggs upregulates fertilization envelope hardening

Previous studies indicate that the nitric oxide (NO) increase at fertilization in sea urchin eggs is Ca²⁺-dependent and attributed to the late Ca²⁺ rise. However, its role in fertilization still remains unclear. Simultaneous measurements of the activation current, by a single electrode voltage clamp, and NO, using the NO indicator DAF-FM, showed that the NO increase occurred at the time of peak current (t_p) which corresponds to peak [Ca²⁺]_i, suggesting that NO is not related to any other ionic changes besides [Ca²⁺]_i. We measured O₂ consumption by a polarographic method to examine whether NO regulated a respiratory burst for protection as reported in other biological systems. Our results suggested NO increased O₂ consumption. The fluorescence of reduced pyridine nucleotides, NAD(P)H was measured in controls and when the NO increase was eliminated by PTIO, a NO scavenger. Surprisingly, PTIO decreased the rate of the fluorescence change and the late phase of increase in NAD(P)H was eliminated. PTIO also suppressed the production of H₂O₂ and caused weak and high fertilization envelope (FE). Our results suggest that NO increase upregulates NAD(P)H and H₂O₂ production and consolidates FE hardening by H₂O₂.     

Seasonality in expression and distribution of nitric oxide synthase isoforms in the testis of the catfish, Clarias batrachus: Role of nitric oxide in testosterone production

Nitric oxide (NO) is a well-recognized versatile signaling molecule. It is produced by catalytic action of nitric oxide synthase (NOS) on L-arginine in a variety of animal tissues. Existence of different isoforms of NOS has been shown in mammalian testis, but report on their presence in the testis of ectothermic vertebrates is non-existent. This study demonstrates the differential expressions of two isoforms of nitric oxide synthase (neuronal-nNOS and inducible-iNOS) like molecules in different cell types in the testis of seasonally breeding catfish, Clarias batrachus through immunohistochemistry. Positive immunoprecipitation of nNOS and iNOS like molecules were detected in germ cells as well as interstitial cells only in the recrudescing and fully mature fish. The immunoreactions differed in intensity and varied with changing reproductive status. Treatment of adult male fish with NO donor, sodium nitroprusside, and a NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME) increased and decreased the total nitrate and nitrite concentration in the testis, respectively. Sodium nitroprusside and L-NAME also induced simultaneous decline and rise in the testicular testosterone level, respectively. These findings, thus, suggest that NOS isoforms are expressed variedly in different cell types in the testis of reproductively active fish. This investigation also suggests that NO inhibits testosterone production in the testis.     

An ultrastructural study of phagocytosis and shrinkage in nutritive phagocytes of the sea urchin <Emphasis Type="Italic">Anthocidaris crassispina</Emphasis>

The ultrastructural mechanisms of waste-sperm phagocytosis and postspawning shrinkage were studied for accessory cells (nutritive phagocytes; NPs) of the sea urchin Anthocidaris crassispina. Sperm cells were phagocytosed by NPs; they penetrated into the cytoplasm of the NPs inside heterophagosomes formed by an invagination of the cell membrane. Single-sperm-containing heterophagosomes aggregated to form large multisperm heterophagosomes that were accompanied by cytoplasmic vesicles and lipids. Two types of vesicle, viz., Golgi-complex-derived electron-dense vesicles (zymogen granules) and smooth-endoplasmic-reticulum-derived electron-lucent vesicles, were incorporated within multisperm heterophagosomes. Completed multisperm heterophagosomes were transformed into electron-dense remnant bodies, the content of which underwent destruction, resulting in empty vacuoles inside the remnant body. The empty vacuoles were then compressed by the surrounding cytoplasm. Shrinkage of NPs occurred upon completion of sperm degeneration in gonad tubules. This process was undertaken by structures termed cell-size-reducing autolysosomes, which performed two types of autolysis, and resulted in the formation of cheese-hole-like vacuoles in the cytoplasm of NPs. Subsequent cytoplasmic compression of these vacuoles was required for the reduction in size of NPs, an essential event for remodeling the cell for the next gametogenetic cycle.We are grateful to the Research Committee, City University of Hong Kong, for the award of a Strategic Grant (no. 7000989). This work also was supported by a grant from Minpromnayka NS 1219.2003.4     

Ras/myc-transformed serum-free mouse embryo cells under simulated inflammatory and infectious conditions increase levels of nitric oxide and matrix metalloproteinase-9 without a direct association between them

Inflammatory and infectious conditions were simulated in cultures of ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells, using interferon-gamma (IFN-γ, 100 units/ml) and lipopolysaccharide (LPS, 0.5 μg/ml) co-treatment for 24 h, to investigate their effects on the expression of inducible nitric oxide synthase (iNOS) mRNA and the production of NO. Aminoguanidine (AG, 1 mM; an NOS inhibitor) along with IFN-γ and LPS, S-nitroso-N-acetyl-DL-penicillamine (SNAP, 100 μM; an NO donor) and/or (±)-N-[(E)-4-Ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4, 100 μM; an NO donor), were also added to analyze the possible association of NO with matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Co-treatment of cells with IFN-γ and LPS increased iNOS mRNA expression, NO production, MMP-9 mRNA expression, and 105 kDa MMP-9 production. Additional treatment with the NOS inhibitor AG inhibited NO production, but did not down-regulate the expression of MMP-9 mRNA or 105 kDa MMP-9. The NO donors SNAP and NOR4 did not affect the expression of MMP-9 mRNA, 105 kDa MMP-9 or TIMP-1 mRNA. These results suggest that ras/myc SFME cells respond to infectious and inflammatory conditions and can enhance malignancy as cancer cells due to their increased levels of NO and MMP-9 production, but that NO is not directly associated with MMP-9 in these cells. H. Yamaguchi and Y. Kidachi contributed equally to this work     

红边龙血树叶绿体基因组特征及其系统发育分析

红边龙血树(Dracaena marginata)是一种在全球广泛种植的龙血树属园艺植物,具有较高的观赏价值和药用价值。本研究首次利用高通量测序技术对红边龙血树叶片进行全基因组测序,组装得到完整的叶绿体基因组序列,并进行注释、序列特征比较和系统发育分析。结果表明,红边龙血树叶绿体基因组包含一个典型的四分体结构,长度为154926 bp,是目前已报道的龙血树属中叶绿体基因组最小的物种;共拥有132个基因,包含86个编码蛋白基因、38个转运RNA基因和8个核糖体RNA基因;密码子偏好性分析发现存在偏好使用A/U碱基结尾的现象,整体上密码子偏好性较低;共鉴定出46个简单重复序列位点和54个长重复序列,分别在大单拷贝区与反向重复区有最大检出率;种间边界分析发现边界区域基因存在相对位置差异,扩张收缩情况总体较为相似;与近缘种进行系统发育分析,红边龙血树与细枝龙血树聚为一类,关系最近,符合形态学分类特征。对红边龙血树叶绿体基因组的解析为龙血树属植物的物种鉴定、遗传多样性和叶绿体转基因工程等提供了重要数据基础。     

The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa

Enzyme histochemistry and immunocytochemistry were used to determine the distribution of neurons in the snail Helix aspersa which exhibited nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and/or immunoreactivity to nitric oxide synthase (NOS). NADPH diaphorase-positive cells and fibres were distributed extensively throughout the central and peripheral nervous system. NADPH diaphorase-positive fibres were present in all neuropil regions of the central and peripheral ganglia, in the major interganglionic connectives and in peripheral nerve roots. NADPH diaphorase-positive cell bodies were found consistently in the eyes, the lips, the tentacular ganglia and the procerebral lobes of the cerebral ganglia; staining of cell bodies elsewhere in the nervous system was capricious. The distribution of NOS-like immunoreactivity differed markedly from that of NADPH diaphorase activity. Small clusters of cells which exhibited NOS-like immunoreactivity were present in the cerebral and pedal ganglia; fibres which exhibited NOS-like immunoreactivity were present in restricted regions of the neuropil of the central ganglia. The disjunct distributions of NADPH diaphorase activity and NOS-like immunoreactivity in the neurvous system of Helix suggest that the properties of neuronal NOS in molluscs may differ sigificantly from those described previously for vertebrate animals.     

Hypoxia enhances gene expression of inducible nitric oxide synthase and matrix metalloproteinase-9 in ras/myc-transformed serum-free mouse embryo cells under simulated inflammatory and infectious conditions

Ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells were treated with interferon-gamma (IFN-gamma; 100 U/ml) and/or lipopolysaccharide (LPS; 0.5 microg/ml) for 24 h to simulate inflammatory and infectious conditions and investigate their effects on the expression of inducible nitric oxide synthase (iNOS) mRNA, nitric oxide (NO) and matrix metalloproteinase-9 (MMP-9). In addition, aminoguanidine (AG; 1 mM), a NOS inhibitor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 10-200 microM), an NO donor or (+/-)-N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4; 10-200 microM), an NO donor, were added to analyze possible associations of NO with MMP-9. Tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 were also measured to analyze possible relationships of NO with the MMP-9/TIMP balance. Furthermore, the cells were treated with 1% O2 under the simulated inflammatory and infectious conditions and the mRNA expressions of iNOS and MMP-9 were analyzed to investigate the possible effects of hypoxia on the expression of genes involved in tumor malignant progression and distant metastasis. Co-treatment with IFN-gamma and LPS increased the expression levels of iNOS mRNA, NO and MMP-9, but NO may not be directly associated with MMP-9 or the MMP-9/TIMP balance. Treatment with 1% O2 markedly increased the gene expression levels of iNOS and MMP-9, indicating that ras/myc SFME cells alter the expression levels of tumor-associated genes and possibly enhance their malignancy as cancer cells under inflammatory, infectious and hypoxic conditions.     

Effects of experimental infection with Eimeria bovis on the balance of sodium, potassium and water in calves

Balance trials were performed to investigate the effects of experimental Eimeria bovis coccidiosis on the metabolism of water, sodium and potassium in calves. Non-infected pair-fed controls and controls fed according to plan were included in the study to allow differentiation between the effects due to infection and due to changes in feed intake. Primary infection with 5 × 10⁴ (group A) or 1 × 10⁵ (group B) oocysts caused mild diarrhoea in three out of four group A calves and mild to severe haemorrhagic diarrhoea in all five group B calves. Losses of sodium and potassium via faeces tended to increase in the infected calves during patency and apparent digestibility (AD) of these minerals was comparably low. In the urine of the infected calves the Na/K-ratio decreased due to a reduced urinary excretion of sodium. The retention (RT) of sodium was particularly high in the calves that had received the higher oocyst dose. Potassium RT did not underlie significant changes during the course of coccidiosis. In the infected calves the plasma level of sodium was reduced transiently while the level of potassium remained fairly stable. Infections with the higher oocyst dose caused a distinct reduction of fluid excretion via urine which compensated for the increased faecal water losses during severe diarrhoea. Reinfection of the group A calves with 1 × 10⁵ oocysts did not cause any significant metabolic impairment. The results of this study indicate that although acute sublethal bovine coccidiosis alters electrolyte and water metabolism the overall balance of electrolytes and water is largely maintained by physiologic adaptation.     

Purification,cloning and expression of spinach leaf sucrose-phosphate synthase in Escherichia coli

Sucrose-phosphate synthase (SPS) from leaves of spinach (Spinacia oleracea L.) has been purified to homogeneity by a procedure involving precipitation with polyethylenglycol and chromatography over diethylaminoethylcellulose, Ω-aminohexylagarose, Mono Q and Blue Affinity columns. The purification factor was 838 and the final specific activity was 1.3 nkat · (mg protein)^?1. On denaturing gels the major polypeptide was 120 kDa but there was also a variable amount of smaller polypeptides in the range of 90 to 110 kDa. A new activity stain was developed to allow visualization of SPS in gels. The holoenzyme had a molecular weight of about 240 and 480 kDa in native gels and Sepharose, respectively. A high-titre polyclonal antibody was obtained which reacted with SPS from other species including wheat, potato, banana and maize. Screening of a spinach-leaf cDNA-expression library with the antibody allowed the isolation of a full-length clone. Sequencing revealed a predicted molecular weight of 117649 Da, and considerable homology with the recently published sequence for maize leaf (Worrell et al. 1991, Plant Cell 3, 1121–1130). Expression of the spinach-leaf SPS gene in Escherichia coli resulted in biological activity, revealed by the presence of SPS activity in extracts and the accumulation of sucrose-6-phosphate and sucrose in the bacteria.     

Effects of nitric oxide in 5-hydroxytryptamine-, cholera toxin-, enterotoxigenic Escherichia coli- and Salmonella Typhimurium-induced secretion in the porcine small intestine

The effects of nitric oxide (NO) in the secretory response to the endogenous secretagogue 5-hydroxytryptamine (5-HT), the enterotoxins heat-labile enterotoxigenic Escherichia coli (ETEC) toxin (LT) and cholera toxin (CT), and various cultures of ETEC and Salmonella serotype Typhimurium in the porcine small intestine (Sus scrofa) were investigated. In anaesthetized pigs, jejunal tied-off loops were instilled with 5-HT, LT, CT, various cultures of ETEC or S. Typhimurium. Pigs were given intravenously isotonic saline or isotonic saline containing the NO synthase inhibitor, N^ω-nitro-l-arginine methyl ester (L-NAME). L-NAME significantly induced an increased fluid accumulation in loops induced by 5-HT, ETEC and stn-mutated S. Typhimurium. Fluid accumulation in loops instilled with wild-type S. Typhimurium was increased by L-NAME, although not significantly, while there was no effect on fluid accumulation induced by an invH-mutated isogenic strain. No significant effect of L-NAME was observed on the fluid accumulation induced by the purified enterotoxins LT and CT. The results also demonstrated a relatively large difference in the ability to induce fluid accumulation between the bacteria strains. Diastolic, systolic and mean blood pressures were significantly increased and the body temperature was significantly decreased in groups of pigs treated with L-NAME. In conclusion, the results suggest that NO has a proabsorptive effect in the intact porcine jejunum and is involved in the systemic vascular tone.     

Involvement of nitric oxide in elicitor-induced defense responses and secondary metabolism of Taxus chinensis cells

This work was to characterize the generation of nitric oxide (NO) in Taxus chinensis cells induced by a fungal elicitor extracted from Fusarium oxysporum mycelium and the signal role of NO in the elicitation of plant defense responses and secondary metabolite accumulation. The fungal elicitor at 10-100 microg/ml (carbohydrate equivalent) induced a rapid and dose-dependent NO production in the Taxus cell culture, which exhibited a biphasic time course, reaching the first plateau within 1 h and the second within 12 h of elicitor treatment. The NO donor sodium nitroprusside potentiated elicitor-induced H2O2 production and cell death but had little influence on elicitor-induced membrane K+ efflux and H+ influx (medium alkalinization). NO inhibitors Nomega-nitro-L-arginine and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide partially blocked the elicitor-induced H2O2 production and membrane ion fluxes. Moreover, the NO inhibitors suppressed elicitor-induced activation of phenylalanine ammonium-lyase and accumulation of diterpenoid taxanes (paclitaxel and baccatin III). These results suggest that NO plays a signal role in the elicitor-induced responses and secondary metabolism activities in the Taxus cells.     

Species-independent expression of nitric oxide synthase in the sarcolemma region of visceral and somatic striated muscle fibers

The expression and distribution of nitric oxide synthase (NOS) was studied by use of the newly designed specific histochemical NADPH diaphorase staining method and the indirect immunofluorescence technique employing an antiserum to brain NOS in visceral and somatic striated muscles of several mammalian species. Histochemical activity and immunoreactivity were located in the sarcolemma region of type I and II fibers of all muscles investigated. Visceral muscles were more strongly stained than somatic muscles. Furthermore, type II fibers, identified by staining of myosin adenosine triphosphatase activity after pre-incubation at alkaline pH, were more intensely labeled than type I fibers. In addition, NOS activity was detected in the area of the sarcolemma of intrafusal fibers. No obvious differences between species were observed. It was concluded that NOS of striated muscles probably makes up the richest and most important nitric oxide source in mammals.