A novel cysteine proteinase inhibitor from seeds of Enterolobium contortisiliquum and its effect on Callosobruchus maculatus larvae

This study focused on the characterization of a novel cysteine proteinase inhibitor from Enterolobium contortisiliquum seeds targeting the inhibition of the growth of Callosobruchus maculatus larvae, an important cosmopolitan pest of the cowpea Vigna unguiculata during storage. The inhibitor was isolated by ion-exchange besides of size exclusion chromatography. EcCI molecular mass is 19,757 Da, composed of two polypeptide chains. It strongly inhibits papain (Ki_app 0.036 nM) and proteinases from the midguts of C. maculatus (80 μg mL^?1, 60% inhibition). The inhibitory activity is reduced by 40% after a heat treatment at 100 °C for 2 h. The protein displayed noxious activity at 0.5% and 1% (w/w) when incorporated in artificial seeds, reducing larval mass in 87% and 92%, respectively. Treatment of C. maculatus larvae with conjugated EcCI-FIT and subsequent biodistribution resulted in high fluorescence intensity in midguts and markedly low intensity in malpighian tubules and fat body. Small amounts of labeled proteins were detected in larvae feces. The detection of high fluorescence in larvae midguts and low fluorescence in their feces indicate the retention of the FITC conjugated EcCI inhibitor in larvae midguts. These results demonstrate the potential of the natural protein from E. contortisiliquum to inhibit the development of C. maculatus.     

基于线粒体DNA控制区的斑翅草螽不同地理种群遗传分化研究

采用PCR扩增结合DNA克隆测序技术,分析了斑翅草螽Conocephalus maculates 9个地理种群mtDNA控制区序列的变异及遗传多样性。切除侧翼RNA基因序列后,最终获得的斑翅草螽mtDNA控制区比对后全长为676 bp,平均碱基组成T(37.8%),C(11.7%),A(41.3%)和G(9.1%)。共检测到98个可变位点,占总位点数的14.5%,其中,9处碱基插入/缺失,74处转换(40个T/C,34个A/G),50处颠换(18个A/T,11个T/G,15个A/C,6个C/G)。共定义46个单倍型,其中,4个为种群间共享单倍型(H02、H05、H08和H10),其余42个为各种群独有单倍型,包括6个种群内共享单倍型(H09、H11、H15、H18、H26和H38)。单倍型总数占实验个体总数的69.7%,除四川峨眉山外,其余种群单倍型百分比均﹥50%。通过两两地理种群间的FST值差异显著性检验,将这些群体分为4组,分别为SC+CQ,GX+FLB+HN+YN,XZ和HB。以长瓣草螽C.gladiatus、峨眉草螽C.emeiensis、悦鸣草螽C.melaenus、竹草螽C.bambusanus为外群,构建的斑翅草螽mtDNA控制区单倍型NJ法系统树形成3个自举支持度较高的分支,其中,分支A由28种单倍体组成,包括本研究中除四川峨眉山(SC)和重庆万州(CQ)以外的7个种群;分支B由12种单倍体组成,包含除菲律宾拉乌尼翁(FLB)和江西南昌(JX)以外的7个种群;分支C由6种单倍型组成,全部来自西藏林芝(XZ)的单倍型。聚类结果表明,斑翅草螽不同地理种群间的遗传分化并不明显,即使是两两群体间FST值差异显著的群体,也未能形成完全独立的分支。     

Effect of a trypsin inhibitor from Dimorphandra mollis seeds on the development of Callosobruchus maculatus

Bruchid larvae cause major losses in grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil, are pests that damage stored seeds. Plants synthesize a variety of molecules, including proteinaceous proteinase inhibitors, to defend themselves against attack by insects. In this work, a trypsin inhibitor (DMTI-II) isolated from Dimorphandra mollis seeds was tested for anti-insect activity against Callosobruchus maculatus larvae. The inhibitor produced ca. 67% mortality to this bruchid when incorporated into an artificial diet at a level of 1%. The doses necessary to cause 50% mortality (LD₅₀) and to reduce weight by 50% (ED₅₀) for DMTI-II were ca. 0.50% and 0.60%, respectively. The action of DMTI-II on C. maculatus larvae may involve the inhibition of trypsin-like activity of larval midgut extracts, the absence of digestion by midgut preparations or with a mixture of pepsin and papain, and its association with a chitin column and chitinous structure in the midgut of this insect.     

The Toxicity of a Lipid Transfer Protein (Cc-LTP1) from Coffea canephora Seeds on the Larval Development of Callosobruchus maculatus (Coleoptera: Bruchidae)

In this work, we analyzed the effects of coffee seed proteins, especially Cc-LTP₁ on the larval development of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), a bruchid pest of beans and the most important insect pest of Vigna unguiculata (L.) Walp. Artificial seed assay, which incorporated the F/0-90 fraction from Coffea canephora seeds, resulted in the reduction of oviposition and caused an inhibition of C. maculatus larval development in a dose-dependent manner. The F/0-90 fraction used at a 4 % concentration resulted in the survival of no larvae. The purified Cc-LTP₁, at a concentration of 0.5 %, also demonstrated effective inhibition of larval development, reducing both females oviposition and the weight and number of larvae. Cc-LTP₁ was also able to inhibit the C. maculatus gut α-amylase activity, and immunolabeling by an anti-LTP serum was observed in the midgut tissues of the C. maculatus larvae. Cc-LTP₁ has shown binding affinity towards microvillar cells, endoplasmic reticulum and mitochondria, as demonstrated by micrographic images taken by a transmission electron microscope. The results from this study indicate that Cc-LTP₁ has insecticidal actions toward C. maculatus and exerts anti-nutritional effects with direct actions on intestinal tissues.     

The defensive role of latex in plants: detrimental effects on insects

The defensive role of the latex of Calotropis procera has recently been reported. In this study, latex proteins involved in detrimental effects on insects were evaluated on another important crop pest. The latex was fractionated to obtain its major protein fraction, which was then used to evaluate its insecticidal properties against Callosobruchus maculatus (Coleoptera: Bruchidae) in artificial bioassays. Laticifer proteins (LP) were investigated to characterize their action in such an activity. LP was highly insecticidal at doses as low as 0.1% (W/W). This effect was slightly augmented in F₁ generation reared in artificial seeds containing LP at similar proportions of F₀, but was fully reversed when F₁ developed in LP-free seeds. The insecticidal proteins were not retained in a chitin column, and did not lose their insecticidal activity, even after heat treatment or pronase digestion. However, these samples inhibited papain (EC 3.4.22.2) activity and gut proteases of C. maculatus larvae, and a reverse zymogram showed the presence of protein bands resistant to papain digestion. These activities were not observed in unheated LP as they were probably masked by abundant endogenous cysteine protease (EC 3.4.22.16) activity present in unheated LP. LP was resistant to proteolysis when assayed with C. maculatus gut extract. However, gut proteins of C. maculatus were digested when incubated with LP. These observations and the deleterious effects of LP upon C. maculatus, reinforce the hypothesis that laticifer fluids are involved in plant defense against insects and indicate C. procera latex to be a source of promising insecticidal proteins. The inhibitor of proteolysis present in the latex seems to be resistant to heat and proteolysis and is certainly involved in the detrimental effects observed.     

Acute toxicity of kaolin and essential oils from Mentha pulegium and Zingiber officinale against different stages of Callosobruchus maculatus under laboratory conditions

Naturally derived compounds such as essential oils and natural mineral are relatively cheap, non-toxic to food grains and environmentally friendly and would be suitable alternatives for currently used chemical insecticides if they have high insecticidal effectiveness. In the present study, acute toxicity of kaolin and essential oils from Mentha pulegium and Zingiber officinale were assessed on different stages of Callosobruchus maculatus at 28?±?2?°C, 65?±?5% R. H and dark condition. The calculated LC₅₀ values on the egg, larvae and adult stages of C. maculatus were 1.15, 2.33 and 2.18?μl/ml air for Z. officinale and 0.07, 0.11 and 0.09?μl/ml air for M. pulegium, respectively. The result showed that M. pulegium was more effective essential oil against different stages of C. maculatus compared with the Z. officinale, and also the egg and adult stages of C. maculatus were more susceptible against essential oils compared with larval stage. The LC₅₀ values of kaolin were 0.71 and 0.18?mg/cm² on egg and adult of C. maculatus, respectively. The combination of tested essential oils with kaolin increased mortality of C. maculatus adults compared with their application alone. It was found that tested essential oils and kaolin had high potential in controlling different stages of C. maculatus.     

Chemical Composition and Insecticidal Activity of Artemisia scoparia Essential Oil against Three Coleopteran Stored-Product Insects

Chemical composition of the essential oil from Artemisia scoparia Waldst et Kit, and its fumigant and repellent activity were investigated against three stored product insects, Callosobruchus maculatus (Fab.), Sitophilus oryzae (L.), and Tribolium castaneum (Herbst). Dry ground leaves were subjected to hydrodistillation using a modified clevenger-type apparatus and the chemical composition of the volatile oil was studied by GC-MS. Nineteen components (99.51% of the total composition) were identified. β-Pinene (19.01%), capillin (17.45%), limonene (15.11%), myrcene (10.95) were found to be the major constituents of the oil. The mortality of 1-7 day old adults of the insect pests increased with concentration from 37 to 926 μL per L air and with exposure time from 3 to 24 h. A concentration of 37 μL per L air and exposure time of 24 h was sufficient to obtain 100% kill of the insects. Callosobruchus maculatus was more susceptible than S. oryzae and T. castaneum. A second more detailed bioassay gave estimates for the LC₅₀ of C. maculatus as 1.46 μL per L air, S. oryzae 1.87 μL per L air and T. castaneum 2.05 μL per L air. Also, the essential oil was significantly more repellent to T. castaneum and S. oryzae than C. maculatus. However, half-life time of the oil for C. maculatus was longer than S. oryzae and T. castaneum. These results show the efficacy of A. scoparia oil for use in organic food protection.     

Angiotensin I-Converting-Enzyme-Inhibitory and Antibacterial Peptides from Lactobacillus helveticus PR4 Proteinase-Hydrolyzed Caseins of Milk from Six Species

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine α_S1-casein (α_S1-CN) 24-47 fragment (f24-47), f169-193, and β-CN f58-76; ovine α_S1-CN f1-6 and α_S2-CN f182-185 and f186-188; caprine β-CN f58-65 and α_S2-CN f182-187; buffalo β-CN f58-66; and a mixture of three tripeptides originating from human β-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 μg/ml (100 μmol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC₅₀) of some of the crude peptide fractions was very low (16 to 100 μg/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC₅₀s were confirmed. An antibacterial peptide corresponding to β-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 μg/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.     

Artificial small RNA-mediated growth inhibition in Escherichia coli and Salmonella enterica serovar Typhimurium

We developed a synthetic RNA approach to identify growth inhibition sequences by cloning random 24-nucleotide (nt) sequences into an arabinose-inducible expression vector. This vector expressed a small RNA (sRNA) of ∼140 nt containing a 24 nt random sequence insert. After transforming Escherichia coli with the vector, 10 out of 954 transformants showed strong growth defect phenotypes and two clones caused cell lysis. We then examined growth inhibition phenotypes in the Salmonella Typhimurium LT2 strain using the twelve sRNAs that exerted an inhibitory effect on E. coli growth. Three of these clones showed strong growth inhibition phenotypes in S. Typhimurium LT2. The most effective sRNA contained the same insert (N1) in both bacteria. The 24 nt random sequence insert of N1 was abundant in guanine residues (ten out of 24 nt), and other random sequences causing growth defects were also highly enriched for guanine (G) nucleotides. We, therefore, generated clones that express sRNAs containing a stretch of 16 to 24 continuous guanine sequences (poly-G₁₆, -G₁₈, -G₂₀, -G₂₂, and -G₂₄). All of these clones induced growth inhibition in both liquid and agar plate media and the poly-G₂₀ clone showed the strongest effect in E. coli. These results demonstrate that our sRNA expression system can be used to identify nucleotide sequences that are potential candidates for oligonucleotide antimicrobial drugs.     

Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies

Bauhinia bauhinoides cruzipain inhibitor (BbCI) and Bauhinia bauhinioides kallikrein inhibitor (BbKI) are cysteine and serine proteinase inhibitors structurally homologous to plant Kunitz-type inhibitors, but are devoid of disulfide bridges. Based on cDNA sequences, we found that BbKI and BbCI are initially synthesized as a prepropeptide comprising an N-terminal signal peptide (19 residues), the mature protein (164 residues) and a C-terminal targeting peptide (10 residues). Partial cDNAs encoding the mature enzymes plus N-terminal His-tags and thrombin cleavage sites were expressed in E. coli and the soluble proteins were purified by one-step nickel affinity chromatography. After thrombin cleavage, both proteins exhibited potent inhibitory activities toward their cognate proteinases like the wild-type proteins. BbCI inhibits human neutrophil elastase ( K i(app) 5.3 nM), porcine pancreatic elastase ( K i(app) 40 nM), cathepsin G ( K i(app) 160 nM) and the cysteine proteinases cruzipain ( K i(app) 1.2 nM), cruzain ( K i(app) 0.3 nM) and cathepsin L ( K i(app) 2.2 nM), while BbKI strongly inhibits plasma kallikrein ( K i(app) 2.4 nM) and plasmin ( K i(app) 33 nM). Circular dichroism spectra of BbCI and BbKI were in agreement with the beta-trefoil fold described for Kunitz inhibitors. The inhibitory potency of both BbCI- and BbKI-type inhibitors suggests that other, non-covalent interactions may compensate for the lack of disulfide bridges.     

Insecticidal Activity and Chemical Composition of Artemisia sieben Besser Essential Oil from Karaj,Iran

Atremisia sieberi Besser is a widely distributed plant that grows in many areas of Iran and has strong insecticidal activity against stored product pests, so an experiment was conducted to investigate fumigant toxicity of the A. sieberi oil collected from Karaj region of Iran. The oil was applied against one to seven day old adults of three major stored product insects including: Callosobruchus maculatus (Fab.), Sitophilus oryzae (L.), and Tribollium castaneum (Herbst). The potency of fumigant toxicity of A. sieberi on C. maculatus was higher (LC₅₀: 1.64 μL per L) than S. oryzae (LC₅₀: 4.41 μL per L) and T. castaneum (LC50: 20.31 μ.L per L). The relationships between the time exposure and oil concentration on mortality show that the mortality was increased as oil concentration and exposure time was increased. The concentration of 185 μL per L and exposure time of 24h was enough to obtain 100% kill of the insects. It was also found that the regions where A. sieberi grows affect essential oil components of the plant and can play an important role in properties of fumigant toxicity.     

The inhibitor profiling of the caspase family of proteases using substrate-derived peptide glyoxals

A series of substrate-based α-keto-β-aldehyde (glyoxal) sequences have been synthesised and evaluated as inhibitors of the caspase family of cysteine proteases. A number of potent inhibitor sequences have been identified. For example, a palmitic acid containing sequence pal-Tyr-Val-Ala-Asp-glyoxal was demonstrated to be an extremely effective inhibitor of caspase-1, inhibiting not only the action of the protease against synthetic fluorogenic substrates (K_i = 0.3 nM) but also blocking its processing of pro-interleukin-1beta (pro-IL-1β). In addition, the peptide Ac-Asp-Glu-Val-Asp-glyoxal, which is based on the consensus cleavage sequence for caspase-3, is a potent inhibitor of this protease (K_i = 0.26 nM) yet only functions as a comparatively modest inhibitor of caspase-1 (K_i = 451 nM). Potent inhibitor sequences were also identified for caspases-6 and -8. However, the degree of discrimination between the family members is limited. The ability of Ac-Asp-Glu-Val-Asp-glyoxal to block caspase-3 like activity in whole cells and to delay the development of apoptosis was assessed. When tested against caspase-3 like activity in cell lysates, Ac-Asp-Glu-Val-Asp-glyoxal displayed effective inhibition similar to that observed against recombinant caspase-3. Treatment of whole cells with this potent caspase-3 inhibitor was however, not sufficient to significantly stall the development of apoptosis in-vitro.     

Novel pyridazinone derivatives as butyrylcholinesterase inhibitors

In the current study, forty-four new [3-(2/3/4-methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl carbamate derivatives were synthesized and evaluated for their ability to inhibit electric eel acetylcholinesterase (EeAChE) and equine butyrylcholinesterase (eqBuChE) enzymes. According to the inhibitory activity results, [3-(2-methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl heptylcarbamate (16c, eqBuChE, IC₅₀ = 12.8 μM; EeAChE, no inhibition at 100 μM) was the most potent eqBuChE inhibitor among the synthesized compounds and was found to be a moderate inhibitor compared to donepezil (eqBuChE, IC₅₀ = 3.25 μM; EeAChE, IC₅₀ = 0.11 μM). Kinetic and molecular docking studies indicated that compounds 16c and 14c (hexylcarbamate derivative, eqBuChE, IC₅₀ = 35 μM; EeAChE, no inhibition at 100 μM) were mixed-type inhibitors which accommodated within the catalytic active site (CAS) and peripheral anionic site (PAS) of hBuChE through stable hydrogen bonding and π-π stacking. Furthermore, it was determined that [3-(2-methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl (4-methylphenyl)carbamate 7c (eqBuChE, IC₅₀ = 34.5 μM; EeAChE, 38.9% inhibition at 100 μM) was the most active derivative against EeAChE and a competitive inhibitor binding to the CAS of hBuChE. As a result, 6-(2-methoxyphenyl)pyridazin-3(2H)-one scaffold is important for the inhibitory activity and compounds 7c, 14c and 16c might be considered as promising lead candidates for the design and development of selective BuChE inhibitors for Alzheimer’s disease treatment.     

Does conjugation of antioxidants improve their antioxidative/anti-inflammatory potential?

A series of symmetric and asymmetric spermine (SPM) conjugates with all-trans-retinoic acid (ATRA), acitretin (ACI), (E)-3-(trioxsalen-4′-yl)acrylic acid (TRAA) and l-DOPA, amides of ACI, l-DOPA and TRAA with 1-aminobutane, benzylamine, dopamine and 1,12-diaminobutane as well as hybrid conjugates of O,O′-dimethylcaffeic acid (DMCA) with TRAA or N-fumaroyl-indole-3-carboxanilide (FICA) and 2-(2-aminoethoxy)ethanol were synthesized and their antioxidant properties were studied. The reducing activity (RA)% of the compounds were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging assay and found to be in the range 0–92(20 min)%/96(60 min)% at 100 μM, the most powerful being the conjugates l-DOPA-SPM-l-DOPA (8, RA = 89%/96%) and l-DOPA-dopamine (13, RA = 92%/92%). Conjugate DMCA-NH(CH₂CH₂O)₂-FICA (14) was the most powerful LOX inhibitor with IC₅₀ 33.5 μM, followed by the conjugates ACI-NHCH₂Ph (10, IC₅₀ 40.5 μM), ACI-SPM-TRAA (7, IC₅₀ 41.5 μM), DMCA-NH(CH₂CH₂O)₂-TRAA (15, IC₅₀ 65 μM), 13 (IC₅₀ 81.5 μM) and ACI-dopamine (11, IC₅₀ 87 μM). The most potent inhibitors of lipid peroxidation at 100 μM were the conjugates 15 (98%) and ACI-SPM-ACI (4, 97%) whereas all other compounds showed activities comparable or lower than trolox. The most interesting compounds, namely ATRA-SPM-ATRA (3), 4, 10, 11 and 15, as well as unconjugated compounds such as ATRA and dopamine, were studied for their anti-inflammatory activity in vivo on rat paw oedema induced by Carrageenan and found to exhibit, for doses of 0.01 mmol/mL of conjugates per Kg of rat body weight, weaker anti-inflammatory activities (3.6–40%) than indomethacin (47%) with conjugate 3 being the most potent (40%) in this series of compounds. The cytocompatibility of selected compounds was evaluated by the viability of RAMEC cells in the presence of different concentrations (0.5–50 μM) of the compounds. Conjugates 3 (IC₅₀ 2.6 μM) and 4 (IC₅₀ 4.7 μM) were more cytotoxic than the corresponding unconjugated retinoids ATRA (IC₅₀ 18.3 μM) and ACI (IC₅₀ 14.6 μM), whereas conjugate 15 (IC₅₀ 12.9 μM) was less cytotoxic than either DCSP (IC₅₀ 11.3 μM) or the tert-butyl ester of TRAA (IC₅₀ 2.9 μM).     

长江口中国花鲈食性分析

中国花鲈(Lateolabrax maculatus)是长江口重要的经济鱼类之一。根据2010年7月至10月在长江口崇明东滩团结沙和东旺沙水域采集到的胃含物样品,对中国花鲈的摄食习性进行了研究。结果表明,从中国花鲈胃含物中共鉴定出6类27种饵料生物,其多样性指数H’值为2.484,均匀度指数J’为0.7535,优势指数D为0.1262。采用百分比相对重要性指数(IRI%)和综合指标优势指数(Ip)分析的结果较一致,鱼类是长江口中国花鲈夏季主要食物,其百分比相对重要性指数(IRI%)和综合指标优势指数(Ip)分别为82.63%和94.48,鮻(Liza haematocheila)为优势饵料生物(IRI%=41.89,Ip=53.20)。团结沙和东旺沙中国花鲈食性差异较大,主成分分析(PCA)表明鲚属(Coilia spp.)、舌虾虎鱼(Glossogobius giuris)、脊尾白虾(Exopalaemoncarinicauda)和雷伊著名团水虱(Gnorimosphaeroma rayi)是造成这种差异的主要饵料生物种类。     

A plant Kunitz-type inhibitor mimics bradykinin-induced cytosolic calcium increase and intestinal smooth muscle contraction

Abstract: BbKI is a kallikrein inhibitor with a reactive site sequence similar to that of kinins, the vasoactive peptides inserted in kininogen moieties. This structural similarity probably contributes to the strong interaction with plasma kallikrein, the enzyme that releases, from high-molecular weight kininogen (HMWK), the proinflammatory peptide bradykinin, which acts on B2 receptors (B2R). BbKI was examined on smooth muscle contraction and Ca2+ mobilization, in which the kallikrein-kinin system is involved. Contrary to expectations, BbKI (1.8 μm) increased [Ca2+]cand contraction, as observed with BK (2.0 μm). Not blocked by B1 receptors (B1R), the BbKI agonistic effect was blocked by the B2R antagonist, HOE-140 (6 μm), and the involvement of B2R was confirmed in B2R-knockout mice intestine. The same tissue response was obtained using a synthetic peptide derived from the BbKI reactive site structure, more resistant than BK to angiotensin I-converting enzyme (ACE) hydrolysis. Depending on the concentration, BbKI has a dual effect. At a low concentration, BbKI acts as a potent kallikrein inhibitor; however, due to the similarity to BK, in high concentrations, BbKI greatly increases Ca2+ release from internal storages, as a consequence of its interaction with B2R. Therefore, the antagonistic and agonistic effects of BbKI may be considered in conditions of B2R involvement.     

Bacillus xiamenensis sp. nov., isolated from intestinal tract contents of a flathead mullet (Mugil cephalus)

A taxonomic study was carried out on strain HYC-10^T, which was isolated from the intestinal tract contents of a flathead mullet, Mugil cephalus, captured from the sea off Xiamen Island, China. The bacterium was observed to be Gram positive, oxidase and catalase positive, rod shaped, and motile by subpolar flagella. The bacterium was found to grow at salinities of 0–12 % and at temperatures of 8–45 °C. The isolate was found to hydrolyze aesculin and gelatin, but was unable to reduce nitrate to nitrite. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain HYC-10^T belongs to the genus Bacillus, with highest sequence similarity (99.3 %) to Bacillus aerophilus 28K^T, Bacillus stratosphericus 41KF2a^T and Bacillus altitudinis DSM 21631^T, followed by Bacillus safensis DSM 19292^T (99.5 %) and Bacillus pumilus DSM 27^T (99.5 %), while the sequence similarities to others were all below 97.6 %. The genomic ANIm values between strain HYC-10^T and three type strains (B. altitudinis DSM 21631^T, B. safensis DSM 19292^T and B. pumilus DSM 27^T) were determined to range from 89.11 to 91.53 %. The DNA–DNA hybridization estimate values between strain HYC-10^T and the three type strains were from 36.60 to 44.00 %. The principal fatty acids identified were iso-C_15:0 (39.1 %), anteiso-C_15:0 (22.7 %), iso-C_17:0 (13.1 %), C_16:0 (6.1 %), anteiso-C_17:0 (5.8 %) and iso-C_16:0 (5.1 %). The G+C content of the chromosomal DNA was determined from the draft genome sequence to be 41.3 mol%. The respiratory quinone was determined to be MK-7 (100 %). Phosphatidylglycerol, diphosphatidylglycerol, aminoglycolipid, two glycolipids and two unknown phospholipids were found to be present. The combined genotypic and phenotypic data show that strain HYC-10^T represents a novel species of the genus Bacillus, for which the name Bacillus xiamenensis sp. nov. is proposed, with the type strain HYC-10^T (=CGMCC NO.1.12326^T = LMG 27143^T = MCCC 1A00008^T).     

Denitrification Activity of a Remarkably Diverse Fen Denitrifier Community in Finnish Lapland Is N-Oxide Limited

Peatlands cover more than 30% of the Finnish land area and impact N₂O fluxes. Denitrifiers release N₂O as an intermediate or end product. In situ N₂O emissions of a near pH neutral pristine fen soil in Finnish Lapland were marginal during gas chamber measurements. However, nitrate and ammonium fertilization significantly stimulated in situ N₂O emissions. Stimulation with nitrate was stronger than with ammonium. N₂O was produced and subsequently consumed in gas chambers. In unsupplemented anoxic microcosms, fen soil produced N₂O only when acetylene was added to block nitrous oxide reductase, suggesting complete denitrification. Nitrate and nitrite stimulated denitrification in fen soil, and maximal reaction velocities (v_max) of nitrate or nitrite dependent denitrification where 18 and 52 nmol N₂O h^-1 g_DW ^-1, respectively. N₂O was below 30% of total produced N gases in fen soil when concentrations of nitrate and nitrite were <500 μM. v_max for N₂O consumption was up to 36 nmol N₂O h^-1 g_DW ^-1. Denitrifier diversity was assessed by analyses of narG, nirK/nirS, and nosZ (encoding nitrate-, nitrite-, and nitrous oxide reductases, respectively) by barcoded amplicon pyrosequencing. Analyses of ~14,000 quality filtered sequences indicated up to 25 species-level operational taxonomic units (OTUs), and up to 359 OTUs at 97% sequence similarity, suggesting diverse denitrifiers. Phylogenetic analyses revealed clusters distantly related to publicly available sequences, suggesting hitherto unknown denitrifiers. Representatives of species-level OTUs were affiliated with sequences of unknown soil bacteria and Actinobacterial, Alpha-, Beta-, Gamma-, and Delta-Proteobacterial sequences. Comparison of the 4 gene markers at 97% similarity indicated a higher diversity of narG than for the other gene markers based on Shannon indices and observed number of OTUs. The collective data indicate (i) a high denitrification and N₂O consumption potential, and (ii) a highly diverse, nitrate limited denitrifier community associated with potential N₂O fluxes in a pH-neutral fen soil.     

Diel patterns in space use, food and metabolic activity of Galaxias maculatus (Pisces: Galaxiidae) in the littoral zone of a shallow Patagonian lake

We detected that Galaxias maculatus exhibits a pattern where metabolic activity increases after sunrise and peaks between noon and sunset, but this species feeds in the afternoon, until several hours after sunset. Moreover, we showed that G. maculatus is observed in the littoral zone during the day, disappears completely from this zone after sunset and returns at sunrise. Littoral prey species are common in the diet of G. maculatus, but this study showed that pelagic prey is also present during twilight and night hours in smaller individuals (<50 mm), which is related to habitat use. These behavioural rhythms are especially important for G. maculatus, which runs a high predation risk when consuming prey that is widely available outside the littoral zone. This risk is ameliorated under the protection of low light intensity. Thus, G. maculatus is a key species linking lower trophic levels, such as the plankton community, to higher levels of native and exotic piscivores. These displacements of G. maculatus generate an active flow of energy and matter between habitats, with a potentially profound effect on the entire food network and energy dynamics of the lake.