Suppression of high pacing-induced ANP secretion by antioxidants in isolated rat atria

Reactive oxygen species (ROS) are formed as a natural by-product of the normal metabolism of oxygen and have important roles in cell signaling. The aim of this study was to investigate direct effects of ROS on atrial hemodynamics and ANP secretion in isolated perfused beating rat atria with antioxidants. When atria were paced at 1.2 Hz, N-acetyl cystein (antioxidant, NAC), α-lipoic acid (antioxidant), tempol (superoxide dismutase mimic), and apocynin (NADPH oxidase inhibitor; NOX inhibitor) did not affect ANP secretion and atrial contractility. When pacing frequency was increased from 1.2 Hz to 4 Hz, the ANP secretion increased and atrial contractility decreased. H₂O₂ level was increased in perfusate obtained from atria stimulated by high pacing frequency. NAC, α-lipoic acid and tempol attenuated high pacing frequency-induced ANP secretion but apocynin did not. In contrast, pyrogallol (a superoxide generator) augmented high pacing frequency-induced ANP secretion. NOX-4 protein was increased by high pacing stimulation and in diabetic rat atria. In diabetic rat atria, high pacing frequency caused an increased ANP secretion and a decreased atrial contractility, that were markedly attenuated as compared to control rats. NAC and apocynin reduced high pacing frequency-induced ANP secretion in diabetic rat atria. These results suggest that intracellular ROS formation partly through an increasing NOX activity in response to high pacing frequency is associated with an increased ANP secretion in rat atria.     

The effects of the perinatal treatment with 5-hydroxytryptophan or tranylcypromine on the peripheral and central serotonin homeostasis in adult rats

Serotonin (5HT) is a biologically active amine present in mammals in the brain and the peripheral tissues. Autism is a neurodevelopmental disorder in which 5HT homeostasis is disturbed both centrally and peripherally, but the relationship between the 5HT disturbances in the two compartments is not understood. In an attempt to explore the relationship between the disturbed peripheral 5HT homeostasis and central 5HT functioning, we exposed the developing rat brain to increased 5HT concentrations, by treatment of rats with subcutaneous injections of the immediate 5HT precursor 5-hydroxy-l-tryptophan (5HTP, 25 mg/kg), or the non-selective MAO inhibitor tranylcypromine (TCP, 2 mg/kg), during the period of the most intensive development of 5HT neurons - from gestational day 13 to post-natal day 21. The effects of the mentioned treatments on peripheral and central 5HT levels were then studied in adult rats. Platelet and plasma 5HT concentrations (measured by ELISA), as well as cortical and midbrain 5HT, tryptophan and 5-hydroxyindoleacetic acid levels (measured by HPLC) were determined in twelve 5HTP treated and eight TCP treated rats, and compared with the values measured in 10 control, saline treated rats. Treatment with 5HTP significantly raised peripheral but not central 5HT concentrations. At adult age, peripheral 5HT homeostasis was re-established, while modest decrease in 5HT concentration was observed in frontal cortex, presumably due to hyperserotonemia-induced loss of 5HT terminals during brain development. Treatment with TCP induced significant 5HT elevations in both compartments. At adult age, permanent changes in 5HT homeostasis were observed, both peripherally (as hyperserotonemia) and centrally (as altered 5HT metabolism with decreased 5HT concentrations). Further studies are planned in order to explore the nature of the different disturbances of 5HT homeostasis induced by the two compounds, and their results are expected to shed some light on the role of hyperserotonemia in autism.     

黄河裸裂尻鱼细胞色素C氧化酶Ⅰ、Ⅱ和Ⅲ亚基基因的克隆及序列特征分析

采用RT-PCR技术克隆获得了黄河裸裂尻鱼(Schizopygopsis pylzovi)CO Ⅰ、Ⅱ、Ⅲ基因的编码序列,并对此进行了初步分析.结果表明,黄河裸裂尻鱼CO I基因全长为1 551 bp,开放阅读框(ORF)由基因全长组成,编码516个氨基酸;COⅡ基因全长为691 bp,开放阅读框为690 bp,编码2...     

Molecular investigations of cytochrome c oxidase subunit I (COI) and the internal transcribed spacer (ITS) in the poultry red mite, Dermanyssus gallinae, in northern Europe and implications for its transmission between laying poultry farms

Samples of Dermanyssus gallinae (DeGeer) (Acari: Dermanyssidae) from more than 49 Norwegian and Swedish laying poultry farms, and additional samples collected from Scottish, Finnish, Danish and Dutch layer farms, were compared genetically. Analysis of partial mitochondrial gene cytochrome c oxidase subunit I (COI) sequences of mites from Norway and Sweden revealed 32 haplotypes. Only single haplotypes were found on most farms, which suggests that infections are recycled within farms and that transmission routes are few. Both Norwegian and Swedish isolates were found in the two major haplogroups, but no haplotypes were shared between Norway and Sweden, indicating little or no recent exchange of mites between these countries. There appears to be no link between haplotypes and geographical location as identical haplotypes were found in both the northern and southern Swedish locations, and haplotypes were scattered in locations between these extremes. The current data suggest that wild birds in Sweden are not a reservoir for D. gallinae infection of layer farms as their mites were genetically distinct from D. gallinae of farm layer birds. Transmission of the poultry red mite in Scandinavia is thus likely to depend on synantropic factors such as the exchange of contaminated material or infested birds between farms or facilities.     

Further characterization and mode of action of dactylomelin-P, an antibacterial protein isolated from the ink of the sea hare Aplysia dactylomela (Rang, 1828)

Sea hares are well known, nearly shell-less, marine opisthobranchs that use a complex repertoire of chemicals for defense and communication instead of a conventional gastropod shell. The most conspicuous characteristic of these invertebrates is the secretion of ink, which is rich in bioactive proteins. Many of these proteins belong to a family of L-amino acid oxidases (L-AAOs). In the current study, we aimed to determine whether dactylomelin-P, an antibacterial protein isolated from the ink of Aplysia dactylomela, could act as an L-AAO. We also investigated its biochemical properties and antibacterial mechanism of action. We found that dactylomelin-P is an acidic protein (pI = 5.0), rich in glutamic acid/glutamine, aspartic acid/asparagine, tyrosine, serine, and proline. It was stable under a broad pH range (3.0-12.0), after heating to 55 °C for 30 min, and after treating with protease. Its N-terminal amino acid sequence was DGVCSNRRQCNKEVCGSSYDVAIVGA and showed high similarity to other sea hare proteins previously identified as L-AAOs. The L-AAO activity was confirmed in an enzymatic assay, which showed that dactylomelin-P could oxidize L-lysine and L-arginine. We also demonstrated that the bacteriostatic activity of dactylomelin-P was mediated by hydrogen peroxide generated in the enzymatic reaction, but it acted as a bactericide in the presence of L-lysine and L-arginine. Transmission electron microscopy analyses showed that dactylomelin-P bound to growth-phase bacteria without causing morphological alterations to the cells. The bactericidal effect seems to involve H₂O₂ and other reactive components since it was not counteracted by H₂O₂ scavengers. Our findings showed biochemical, functional, and phylogenetic similarities among L-AAOs isolated from sea hares; this offers new insight into the evolution of these proteins and their roles in chemical defense.     

Pollinator sharing in dioecious figs (Ficus: Moraceae)

As one of the most specialized pollination syndromes, the fig (Ficus)–fig wasp (Agaonidae) mutualism can shed light on how pollinator behaviour and specificity affect plant diversification through processes such as reproductive isolation and hybridization. Pollinator sharing among species has important implications for Ficus species delimitation and the evolutionary history of the mutualism. Although agaonid wasp pollinators are known to visit more than one host species in monoecious figs, pollinator sharing has yet to be documented in dioecious figs. The present study investigated the frequency of pollinator sharing among sympatric, closely‐related dioecious figs in Ficus sections Sycocarpus and Sycidium. Molecular and morphological species identification established the associations between pollinating agaonid wasp species and host fig species. Cytochrome oxidase I was sequenced from 372 Ceratosolen pollinators of Ficus section Sycocarpus and 210 Kradibia pollinators of Ficus section Sycidium. The association between fig species and morphologically distinct clades of pollinator haplotypes was predominantly one‐to‐one. In Ceratosolen, six of 372 pollinators (1.5%) visited fig species other than the predominant host. No pollinator sharing was detected between the two Sycidium host species, although a rare hybrid shared Kradibia pollinators with both parental species. These findings point to low rates of pollinator sharing among closely‐related dioecious fig species in sympatry, and perhaps lower rates than among monoecious figs. Such rare events could be evolutionarily important as mechanisms for gene flow among fig species. Differences in rates of pollinator sharing among fig lineages might explain the conflicting phylogenetic patterns inferred among monoecious figs, dioecious figs, and their respective pollinators. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 546–558.     

High glucose concentration affects the oxidant-antioxidant balance in cultured mouse podocytes

Hyperglycemia is well-recognized and has long-term complications in diabetes mellitus and diabetic nephropathy. In podocytes, the main component of the glomerular barrier, overproduction of reactive oxygen species (ROS) in the presence of high glucose induces dysfunction and increases excretion of albumin in urine. This suggests an impaired antioxidant defense system has a role in the pathogenesis of diabetic nephropathy. We studied expression of NAD(P)H oxidase subunits by Western blotting and immunofluorescence and the activities of the oxidant enzyme, NAD(P)H, and antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), in mouse podocytes cultured in a high glucose concentration (30 mM). We found long-term (3 and 5 days) exposure of mouse podocytes to high glucose concentrations caused oxidative stress, as evidenced by increased expression of Nox4 and activities of NAD(P)H oxidase (Δ 182%) and SOD (Δ 39%) and decreased activities of GPx (Δ -40%) and CAT (Δ -35%). These biochemical changes were accompanied by a rise in intracellular ROS production and accumulation of hydrogen peroxide in extracellular space. The role of Nox4 in ROS generation was confirmed with Nox4 siRNA. In conclusion, high glucose concentration affects the oxidant-antioxidant balance in mouse podocytes, resulting in enhanced generation of superoxide anions and its attenuated metabolism. These observations suggest free radicals may play an important role in the pathogenesis of diabetic nephropathy.     

Expression and characterization of the terminal heme synthetic enzymes from the hyperthermophile Aquifex aeolicus

The terminal two heme biosynthetic pathway enzymes, protoporphyrinogen oxidase and ferrochelatase, of the hyperthermophilic bacterium Aquifex aeolicus have been expressed in Escherichia coli, purified to homogeneity, and biochemically characterized. Ferrochelatase and protoporphyrinogen oxidase of this organism are both monomeric, as was found for the corresponding enzymes of Bacillus subtilis. However, unlike the B. subtilis proteins, both A. aeolicus enzymes are membrane-associated. Both proteins have temperature optima over 60 degrees C. This is the first demonstration of functional heme biosynthetic enzymes in an extreme thermophilic bacterium.     

基于融合双亲短肽提高葡萄糖氧化酶的热稳定性

葡萄糖氧化酶(Glucose oxidase,简称GOD)可氧化葡萄糖生产葡萄糖酸及其衍生物,在新型无抗饲料添加剂的开发中具有良好的应用潜力,但其生产加工过程中尚存在热稳定差的瓶颈问题。本研究采用融合双亲短肽技术提高葡萄糖氧化酶的热稳定性,分别选取8种不同氨基酸长度、不同Linker连接的双亲短肽(Self-assembling peptides,SAPs)融合至黑曲霉来源的葡萄糖氧化酶的N端,构建了8种融合型酶SAP1-GS-GOD、SAP1-PT-GOD、SAP2-PT-GOD、SAP3-PT-GOD、SAP4-PT-GOD、SAP5-PT-GOD、SAP6-PT-GOD、SAP7-PT-GOD,并在毕赤酵母GS115中异源表达。获得的连接PT Linker的融合酶在60℃下孵育60 min后的相对酶活均高于初始酶。其中,融合酶SAP5-PT-GOD在60℃下孵育30 min的相对酶活为67%,是相同处理条件下初始酶相对酶活的10.9倍。同时,融合酶SAP1-PT-GOD、SAP2-PT-GOD、SAP3-PT-GOD、SAP5-PT-GOD的K_(cat)/K_m值较初始酶均有进一步的提高。研究表明,在葡萄糖氧化酶的N端融合以PT为Linker的目标双亲短肽能有效提高葡萄糖氧化酶的热稳定性,通过对融合酶分子内作用力进行分析,酶分子内氢键的增加对融合酶热稳定性的提高效果最为显著。上述研究获得的热稳定性提高的融合葡萄糖氧化酶及其效果机制分析对提高酶在加工和应用过程中的活性具有重要的研究意义和应用价值。