Structural and functional characterization of <Emphasis Type="Italic">Delphinus delphis</Emphasis> hemoglobin system

Structural analysis of the hemoglobin (Hb) system of Delphinus delphis revealed a high globin multiplicity: HPLC–electrospray ionization-mass spectrometry (ESI-MS) analysis evidenced three major β (β1 16,022 Da, β2 16,036 Da, β3 16,036 Da, labeled according to their progressive elution times) and two major α globins (α1 15,345 Da, α2 15,329 Da). ESI-tandem mass and nucleotide sequence analyses showed that β2 globin differs from β1 for the substitution Val126 → Leu, while β3 globin differs from β2 for the isobaric substitution Lys65 → Gln. The α2 globin differs from the α1 for the substitution Ser15 → Ala. Anion-exchange chromatography allowed the separation of two Hb fractions and HPLC–ESI-MS analysis revealed that the fraction with higher pI (HbI) contained β1, β2 and both the α globins, and the fraction with lower pI (HbII) contained β3 and both the α globins. Both D. delphis Hb fractions displayed a lower intrinsic oxygen affinity, a decreased effect of 2,3-BPG and a reduced cooperativity with respect to human HbA₀, with HbII showing the more pronounced differences. With respect to HbA₀, either the substitution Proβ5 → Gly or the Proβ5 → Ala is present in all the cetacean β globins sequenced so far, and it has been hypothesized that position 5 of β globins may have a role in the interaction with 2,3-BPG. Regarding the particularly lowered cooperativity of HbII, it is interesting to observe that the variant human HbA, characterized by the substitution Lysβ65 → Gln (HbJ-Cairo) has a decreased cooperativity with respect to HbA₀.     

Fatty acid composition of Turbatrix aceti and its use in feeding regimes of Coregonus maraena (Bloch, 1779): is it really a suitable alternative to Artemia nauplii?

By incorporating the free‐swimming nematode Turbatrix aceti into early feeding regimes of the European whitefish Coregonus maraena, the suitability of this nematode species was investigated as an alternative to Artemia nauplii. During a 14‐day feeding trial in a total of 25 aquaria each 1.7 L (each treatment n = 5, 255 larvae/tank) T. aceti was used either as the sole live food or in combination with Artemia nauplii or microdiet to determine the effect of T. aceti on growth performance and survival rate of C. maraena. By analysing the fatty acid composition of T. aceti prior to and after enrichment with INVE spresso^® it was investigated whether the amount of n3‐polyunsaturated fatty acids (n3‐PUFA) in T. aceti could be further enhanced. Supplementation of Artemia nauplii with T. aceti increased growth significantly within the first 5 days of rearing in comparison to the non‐supplemented food treatments (14.39 ± 0.15 mm compared to 13.44 ± 0.18 mm; mean ± SE). However, growth and survival of juvenile C. maraena on nematode‐supplemented Artemia nauplii did not differ significantly from non‐supplemented Artemia nauplii at the end of the 14‐day rearing period (15.22 ± 0.15 mm compared to 14.86 ± 0.24 mm). All feeding treatments containing Artemia nauplii showed significantly higher growth and lower mortality at the end of the experiment in comparison to diets containing only the microdiet or T. aceti or a combination thereof. The overall low performance of T. aceti alone can most likely be explained by an insufficient capacity of C. maraena to digest this nematode species efficiently. Enrichment with INVE spresso^® successfully increased the proportion of DHA in the T. aceti tissue. The results reveal that T. aceti cannot be considered a full alternative to Artemia nauplii, at least not in the rearing of C. maraena, but might be a useful vector of essential fatty acids within the early rearing period of this and potentially other fish species when provided as live food along with Artemia nauplii.     

1‐Acetyl‐3‐[(3R)‐hydroxyfatty acyl]glycerols: Lipid Compounds from Euphrasia rostkoviana Hayne and E. tetraquetra (Bréb.) Arrond.

Five homologous acetylated acylglycerols of 3‐hydroxyfatty acids (chain lengths C(14) – C(18)), named euphrasianins A – E, were characterized for the first time in Euphrasia rostkoviana Hayne (Orobanchaceae) by gas chromatography/mass spectrometry (GC/MS) and high‐performance liquid chromatography/atmospheric pressure chemical ionization‐mass spectrometry (HPLC/APCI‐MSⁿ). In addition to mass spectrometric data, structures of euphrasianins were verified via a three‐step total synthesis of one representative homologue (euphrasianin A). The structure of the latter was confirmed by 1D‐ and 2D‐NMR experiments as well as high‐resolution electrospray ionization‐mass spectrometry (HR‐ESI‐MS). The absolute configuration of the 3‐hydroxyfatty acid moiety at C(3) was found to be R in the natural euphrasianins, which was determined by alkaline hydrolysis and methylation of a purified fraction, followed by chiral GC analysis. Furthermore, in extracts of Euphrasia tetraquetra (Bréb .) Arrond . euphrasianins C and E were detected exclusively, indicating that this subclass of lipid constituents is possibly valuable for fingerprinting methods.     

Identification of metabolites produced from <Emphasis Type="Italic">N</Emphasis>-phenylpiperazine by <Emphasis Type="Italic">Mycobacterium</Emphasis> spp

Mycobacterium sp. 7E1B1W and seven other mycobacterial strains known to degrade hydrocarbons were investigated to determine their ability to metabolize the piperazine ring, a substructure found in many drugs. Cultures were grown at 30°C in tryptic soy broth and dosed with 3.1 mM N-phenylpiperazine hydrochloride; samples were removed at intervals and extracted with ethyl acetate. Two metabolites were purified from each of the extracts by high-performance liquid chromatography; they were identified by mass spectrometry and ¹H nuclear magnetic resonance spectroscopy as N-(2-anilinoethyl)acetamide and N-acetyl-N′-phenylpiperazine. The results show that mycobacteria have the ability to acetylate piperazine rings and cleave carbon-nitrogen bonds.     

Halilectin 1 (H‐1) and Halilectin 2 (H‐2): two new lectins isolated from the marine sponge Haliclona caerulea

Two new lectins named Halilectin 1 (H‐1) and Halilectin 2 (H‐2) were isolated from the marine sponge Haliclona caerulea using a combination of affinity chromatography on stroma fixed onto Sephadex G‐25 and cation and anion exchange chromatography. H‐1 is a monomeric protein with a molecular mass of 40 kDa estimated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and 15 kDa estimated using a TSK gel. Conversely, H‐2 is a homodimeric protein with 15 kDa monomers linked via weak interactions. H‐1 more effectively agglutinates trypsinized rabbit erythrocytes, whereas H‐2 more effectively agglutinates native rabbit erythrocytes. The hemagglutinating activity of H‐1 could be not inhibited by any tested sugars, but H‐2 was inhibited by orosomucoid and porcine stomach mucin. Neither lectin was dependent on divalent ions. H‐1 was stable at basic pH range and temperatures up to 50 °C, whereas H‐2 was stable at acid pH range and temperatures up to 80 °C. The H. caerulea lectins exhibited dose‐dependent toxicity against Artemia nauplii. Additionally, 76% of the primary structure of H‐2 was determined using tandem mass spectrometry to contain a unique amino acid sequence with no similarity to any members of the animal lectin family. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of transgenic zooplankton <Emphasis Type="Italic">Artemia</Emphasis> as a bioreactor to produce exogenous protein

Although the crustacean Artemia has been commonly used as an experimental organism and served as a live bait feed for aquaculture, gene transfer system on Artemia sp. to generate stable lines is not well developed. In this study, we optimized a condition for cyst-eletroporation and generated stable lines of transgenic A. sinica. Two expression plasmids directed by the hybrid promoters of cytomegalovirus (CMV) and medaka β-actin (Mβ) were co-electroporated on decapsulated cysts: pCMV-Mβ-GFP contained GFP reporter gene and pCMV-Mβ-ypGH contained yellowfin porgy GH (ypGH) cDNA. We examined the GFP shown in the Artemia larvae and found that the expression rate was 13.3% (3,219 out of 24,054 examined). We then chose 200 G0 founders which strongly expressed GFP to generate transgenic lines. Homozygotic strains derived from F4 generation of each transgenic line, A3 and A8, were obtained. We proved that transgenic lines A3 and A8 also harbored pCMV-Mβ-ypGH and produced recombinant ypGH with a concentration of 0.089 and 0.032 μg per 50 homozygotic nauplii, respectively. Ten live Artemia nauplii were fed daily to zebrafish larvae during 25 to 35 days of post-fertilization. The average body length gain rates of zebrafish larvae fed transgenic Artemia were 16–20% greater than those of control group, indicating the exogenous ypGH produced by transgenic Artemia is functional. Therefore, we concluded that the transgenesis on Artemia is developed, and transgenic Artemia might be highly potentially useful as a new bioreactor material for application in aquaculture and biological researches.     

Feeding behaviour,feed selectivity and growth studies of mangrove killifish, <Emphasis Type="Italic">Kryptolebias marmoratus</Emphasis>, larvae using various live and formulated feeds

Nine types of live foods viz. L, S and SS morphotypes of Brachionus plicatilis sp. complex, first instar Artemia franciscana, Fabrea salina, Acartia tsuensis, Tigriopus japonicus, Diaphanosoma celebensis, Moina mongolica and a formulated feed of two sizes (400 and 700 μm) were used to observe feeding behaviour and growth of mangrove killifish, Kryptolebias marmoratus. Behavioural observations were made for one hour on days 0, 1, 5, and 10 after hatching. Focus, unsuccessful and successful attacks and vomit were noted. With rotifers L, S and SS types and newly hatched Artemia nauplii as food, all the larvae showed maximum feeding success throughout the experimental period. Larvae did not consume any of the 700 μm artificial diet. Vomiting was noticed on capturing the ciliate Fabrea and 400 μm artificial diets. Rotifers were ingested in greater numbers. SS-type rotifers were consumed in largest number (209.2/h per individual) on day 10. Significantly greater growth was observed after 10 days rearing with L type rotifer, Artemia nauplii, T. japonicus, A. tsuensis, M. mongolica, D. celebensis, and a mixture of L type rotifer and F. salina (Tukey–Kramer post hoc test, P < 0.05). Feed selectivity experiments on days 0, 1, 5 and 10 revealed that killifish larvae feed preferentially on Artemia nauplii and rotifers from a mixture of Artemia nauplii, rotifers, A. tsuensis, T. japonicus, D. celebensis and M. mongolica. Techniques for culturing various zooplankton at small-scales are also described.     

Characterization of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose-3-epimerase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> that converts <Emphasis Type="SmallCaps">d</Emphasis>-fructose into <Emphasis Type="SmallCaps">d-</Emphasis>psicose

A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn²⁺. At pH 9 and 40°C, 118 g d-psicose l⁻¹ was produced from 700 g d-fructose l⁻¹ after 3 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Repellent efficacy of essential oils and plant extracts against Tribolium castaneum and Plodia interpunctella

This study was conducted to investigate the repellent efficacy of essential oils (Origanum vulgare, Pimpinella anisum, and Tanacetum cinerariifolium) and four plant extracts (Agastache rugosa, Capsicum annuum, Citrus reticulata, and Ginkgo biloba) against Tribolium castaneum (adults and larvae) and Plodia interpunctella (larvae). Gas chromatography/mass spectrometry analysis revealed the presence of carvacrol, anethole, and jasmolin I as the predominant constituent in O. vulgare, P. anisum, and T. cinerariifolium, respectively. Furthermore, ethyl hexopyranoside, 9,12‐octadecadienoic acid, cyclopentanol, and 2‐cresol were identified in A. rugosa, C. annuum, C. reticulata, and G. biloba, respectively. The repellent efficacy of each essential oil, plant extract, and the combination of oils was evaluated using a specially designed cylinder trap for 120 h. Among the three oils, O. vulgare and T. cinerariifolium had greatest repellent efficacy against P. interpunctella larvae. T. cinerariifolium exhibited effective repellence against the adults and larvae of T. castaneum. Therefore, O. vulgare (O) and T. cinerariifolium (T) were selected for further investigation of combined effects. Two essential oils were mixed in three different ratios of OT1 (1:3), OT2 (1:1), and OT3 (3:1). The repellent efficacies of OT1 and OT2 against the adults of T. castaneum were significantly greater than that of OT3. OT1 was effective against the larvae of T. castaneum, whereas OT2 was effective against the larvae of P. interpunctella. OT1 enhanced the repellent efficacy by approximately five times against larvae of T. castaneum, compared with that of T. cinerariifolium. Overall, OT1 was selected as the best repellent substance against all the tested insects.     

Structure and chain conformation of a (1 → 6)-α-d-glucan from the root of Pueraria lobata (Willd.) Ohwi and the antioxidant activity of its sulfated derivative

A water soluble glucan, PLB-2C, was isolated from the water extract of the root of Pueraria lobata (Willd) Ohwi using anion-exchange and gel permeation chromatography. Its structure was investigated by gas chromatography (GC), gas chromatography–mass spectrometry (GC–MS), infrared (IR) spectra, and nuclear magnetic resonance (NMR) spectroscopy of heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) techniques. The results indicated that PLB-2C was a linear glucan composed of (1 → 6)-α-d-Glcp. Chain conformation study showed that the polysaccharide took random coil compact conformation. In vitro cell viability assay by MTT method, its sulfated derivative PLB-2CS which was substituted at 2-O, 3-O, 4-O positions, at 0.1, 1, and 5 mg/ml, could attenuate PC12 cell damage significantly caused by hydrogen peroxide.     

Synergistic roles of acyl‐CoA binding protein (ACBP1) and sterol carrier protein 2 (SCP2) in Toxoplasma lipid metabolism

Toxoplasma gondii relies on apicoplast‐localised FASII pathway and endoplasmic reticulum‐associated fatty acid elongation pathway for the synthesis of fatty acids, which flow through lipid metabolism mainly in the form of long‐chain acyl‐CoA (LCACoAs) esters. Functions of Toxoplasma acyl‐CoA transporters in lipid metabolism remain unclear. Here, we investigated the roles of acyl‐CoA‐binding protein (TgACBP1) and a sterol carrier protein‐2 (TgSCP2) as cytosolic acyl‐CoA transporters in lipid metabolism. The fluormetric binding assay and yeast complementation confirmed the acyl‐CoA binding activities of TgACBP1 and TgSCP2, respectively. Disruption of either TgACBP1 or TgSCP2 caused no obviously phenotypic changes, whereas double disruption resulted in defects in intracellular growth and virulence to mice. Gas chromatography coupled with mass spectrometry (GC–MS) results showed that TgACBP1 or TgSCP2 disruption alone led to decreased abundance of C18:1, whereas double disruption resulted in reduced abundance of C18:1, C22:1, and C24:1. ¹³C labelling assay combined with GC–MS showed that double disruption of TgACBP1 and TgSCP2 led to reduced synthesis rates of C18:0, C22:1, and C24:1. Furthermore, high performance liquid chromatography coupled with high resolution mass spectrometry (HPLC‐HRMS) was used for lipidomic analysis of parasites and indicated that loss of TgACBP1 and TgSCP2 caused serious defects in production of glycerides and phospholipids. Collectively, TgACBP1 and TgSCP2 play synergistic roles in lipid metabolism in T. gondii.     

Isolation of a buprofezin co-metabolizing strain of <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. DFS35-4 and identification of the buprofezin transformation pathway

Buprofezin is a widely used insecticide that has caused environmental pollution in many areas. However, biodegradation of buprofezin by pure cultures has not been extensively studied, and the transformation pathway of buprofezin remains unclear. In this paper, a buprofezin co-metabolizing strain of DFS35-4 was isolated from a buprofezin-polluted soil in China. Strain DFS35-4 was preliminarily identified as Pseudomonas sp. based on its morphological, physiological, and biochemical properties, as well as 16S rRNA gene analysis. In the presence of 2.0 g l⁻¹ sodium citrate, strain DFS35-4 degraded over 70% of 50 mg l⁻¹ buprofezin in 3 days. Strain DFS35-4 efficiently degraded buprofezin in the pH range of 5.0–10.0 and at temperatures between 20 and 30°C. Three metabolites, 2-imino-5-phenyl-3-(propan-2-yl)-1,3,5-thiadiazinan-4-one, 2-imino-5-phenyl-1,3,5-thiadiazinan-4-one, and methyl(phenyl) carbamic acid, were identified during the degradation of buprofezin using gas chromatography–mass spectrometry (GC–MS) and tandem mass spectrometry (MS/MS). A partial transformation pathway of buprofezin in Pseudomonas sp. DFS35-4 was proposed based on these metabolites.     

Life span,growth and mortality in the western Pacific goby <Emphasis Type="Italic">Trimma benjamini</Emphasis>, and comparisons with <Emphasis Type="Italic">T. nasa</Emphasis>

Examination of daily increment rings in the saccular otoliths of 91 specimens of the small goby, Trimma benjamini, reveal a maximum age of 140 days with an average pelagic larval duration of 33.9 ± 4.3 days (SD), or 24.2% of the maximum lifespan. Estimates of daily mortality rate ranged from 2.9% to 6.3%. Comparisons of these results with those for T. nasa suggest that 1) the growth rate of T. benjamini males does not decrease with age as it does for T. nasa; 2) T. benjamini has a longer lifespan and lower daily mortality rate than T. nasa; and 3) T. nasa has a faster growth rate than T. benjamini. These results reinforce the potentially important role of small, planktivorous, outer reef fishes in reef trophodynamics, as well as highlight the need for further research on small reef fishes.     

Mass‐trapping trials for the control of pine processionary moth in a pine woodland recreational area

The pine processionary moth, Thaumetopoea pityocampa, causes serious defoliation to Cedrus, Pinus and Pseudotsuga trees, as well as health problems in humans, pets and farm animals due to their urticating hairs. Environmentally friendly strategies for the management of T. pityocampa include: removal of egg batches, removal of nests, trapping of migrant larvae, spraying microbial or Insect Growth Regulator (IGR) insecticides and biocontrol, as well as pheromone‐based adult trapping and mating‐disruption. In the present paper, results on innovative technology for the control of T. pityocampa infestation using pheromone mass‐trapping are reported. Two 1‐ha plots were identified in the study area (central‐south Italy), a pine woodland recreational site growing Pinus halepensis. In the experimental plot (MT‐plot), 10 G‐traps (funnel trap type) baited with (Z)‐13‐hexadecen‐11‐ynyl acetate sex pheromone component were placed for mass‐trapping of adults; the other plot was used as a control‐plot (C‐plot). The T. pityocampa population was monitored using the two central traps in the MT‐plot and two traps positioned in the C‐plot. In addition, the winter nests made by T. pityocampa larvae overwintering on pine trees were counted. After 2 years of mass‐trapping, the number of adults trapped by the monitoring pheromone traps decreased in the MT‐plot, but not in the C‐plot, whereas the number of nests decreased in both plots. Statistical results highlighted significant differences in trap catches between the two plots but not between years. In the case of nests, differences among plots were not significant before the mass‐trapping, but significant after 1‐year treatment. According to our results, the mass‐trapping technique is able to reduce T. pityocampa infestations. This pheromone method can be applied in combination with other control systems in the context of integrated pest management in recreational areas.     

Production and structural analysis of the polysaccharide secreted by <Emphasis Type="Italic">Trametes (Coriolus) versicolor</Emphasis> ATCC 200801

Trametes versicolor ATCC 200801 secretes 4.1 g L⁻¹ of exopolysaccharide (EPS) when synthetic minimal medium and low-shear bioreactor cultivation technique are used. Structural and compositional analyses by thin layer chromatography, gas chromatography–mass spectrometry, electrospray ionization tandem mass spectrometry, and nuclear magnetic resonance spectroscopy yielded predominantly glucose and small amounts of galactose, mannose, arabinose, and xylose. The main EPS is composed of β-1,3/β-1,6-linked d-glucose molecules which is identical with Schizophyllan but does not possess a triple helical arrangement as secondary structure. Two molar mass fractions were detected by size exclusion chromatography yielding weight-average molecular weights of 4,100 and 2.6 kDa. Protein content varies between 2–3.6% (w/w). The exopolysaccharide is different in the nature of the glycosidic linkage, composition of monosaccharides, protein content, and weight-average molecular weight compared to the well-known polysaccharopeptide (PSP) and polysaccharopeptide Krestin (PSK).     

Rubrivivaxin,a new cytotoxic and cyclooxygenase-I inhibitory metabolite from <Emphasis Type="Italic">Rubrivivax benzoatilyticus</Emphasis> JA2

A new phenol terpenoid ester metabolite was isolated from the culture supernatant of Rubrivivax benzoatilyticus JA2 when the organism grown on L-phenylalanine as sole source of nitrogen under photoheterotrophic condition. The compound was characterized by HPLC, LCMS, IR, NMR (¹H, ¹³C) and Mass spectrometry, as a 3, 4-dihydroxybenzoyl-terpenoid ester [IUPAC: 3, 4-dihydroxy, 5-carboxy, 3-methylpentyl ester], which has a molecular mass (m/z) of 298 and we designated as rubrivivaxin. The significance of cyclooxygenase-I (COX-I) inhibitory, antimicrobial, cytotoxic activities against U937 (Human leukemic monocyte lymphoma) and Jurkat (T lymphocyte) cell lines conferred by rubrivivaxin.     

Purification and characterization of a high molecular mass serine carboxypeptidase from <Emphasis Type="Italic">Monascus pilosus</Emphasis>

Two serine carboxypeptidases, MpiCP-1 and MpiCP-2, were purified to homogeneity from Monascus pilosus IFO 4480. MpiCP-1 is a homodimer with a native molecular mass of 125 kDa composed of two identical subunits of 61 kDa, while MpiCP-2 is a high mass homooligomer with a native molecular mass of 2,263 kDa composed of about 38 identical subunits of 59 kDa. This is unique among carboxypeptidases and distinguishes MpiCP-2 as the largest known carboxypeptidase. The two purified enzymes were both acidic glycoproteins. MpiCP-1 has an isoelectric point of 3.7 and a carbohydrate content of 11%, while for MpiCP-2 these values were 4.0 and 33%, respectively. The optimum pH and temperature were around 4.0 and 50°C for MpiCP-1, and 3.5 and 50°C for MpiCP-2. MpiCP-1 was stable over a broad range of pH between 2.0 and 8.0 at 37°C for 1 h, and up to 55°C for 15 min at pH 6.0, but MpiCP-2 was stable in a narrow range of pH between 5.5 and 6.5, and up to 50°C for 15 min at pH 6.0. Phenylmethylsulfonylfluoride strongly inhibited MpiCP-1 and completely inhibited MpiCP-2, suggesting that they are both serine carboxypeptidases. Of the substrates tested, benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu) was the best for both enzymes. The K_m, V_max, K_cat and K_cat/K_m values of MpiCP-1 for Z-Tyr-Glu at pH 4.0 and 37°C were 1.33 mM, 1.49 mM min^–1, 723 s^–1 and 545 mM^–1 s^–1, and those of MpiCP-2 at pH 3.5 and 37°C were 1.55 mM, 1.54 mM min^–1, 2,039 s^–1 and 1,318 mM^–1 s^–1, respectively.