Temporal expression of G-protein-coupled receptor 54 (GPR54), gonadotropin-releasing hormones (GnRH), and dopamine receptor D2 (drd2) in pubertal female grey mullet, Mugil cephalus.

The G-protein-coupled receptor 54 (muGPR54) cDNA was cloned from the brain of the grey mullet, and its expression level, as well as those of the gonadotropin-releasing hormones (GnRH1, GnRH2, GnRH3) and dopamine receptor D2 (drd2), in the brain, pituitary and ovary of pubertal fish (early, intermediate, advanced) were determined by real-time quantitative RT-PCR (QPCR). The muGPR54 cDNA has an open reading frame of 1140 bp with a predicted 380 amino acid peptide, containing seven putative transmembrane domains and putative N-glycosylation and protein kinase C phosphorylation sites. QPCR results showed that the early stage of puberty in grey mullet is characterized by significantly high levels of expression of GPR54, GnRH and drd2 in the brain relative to the intermediate and advanced stages, except for GnRH1 that increased at the advanced stage of puberty. In the pituitary, drd2 expression declined significantly at the advanced stage relative to levels at the intermediate stage. Ovarian expression of GPR54 significantly increased from the intermediate stage of puberty relative to the early stage while that of GnRH1 acutely increased at the advanced stage of puberty. The ovarian expression of drd2 decreased as puberty progressed, but the changes were not significant. The results suggest the possible role of GPR54 and GnRH in positively regulating pubertal development in grey mullet and the dopaminergic inhibition of reproductive function mediated by drd2.

Isolation and expression analysis of multiple isoforms of putative farnesoic acid O-methyltransferase in several crustacean species

Farnesoic acid O-methyltransferase (FaMeT) is the enzyme responsible for the conversion of farnesoic acid (FA) to methyl farnesoate (MF) in the final step of MF synthesis. Multiple isoforms of putative FaMeT were isolated from six crustacean species belonging to the families Portunidae, Penaeidae, Scyllaridae and Parastacidae. The portunid crabs Portunus pelagicus and Scylla serrata code for three forms: short, intermediate and long. Two isoforms (short and long) were isolated from the penaeid prawns Penaeus monodon and Fenneropenaeus merguiensis. Two isoforms were also identified in the scyllarid Thenus orientalis and parastacid Cherax quadricarinatus. Putative FaMeT sequences were also amplified from the genomic DNA of P. pelagicus and compared to the putative FaMeT transcripts expressed. Each putative FaMeT cDNA isoform was represented in the genomic DNA, indicative of a multi-gene family. Various tissues from P. pelagicus were individually screened for putative FaMeT expression using PCR and fragment analysis. Each tissue type expressed all three isoforms of putative FaMeT irrespective of sex or moult stage. Protein domain analysis revealed the presence of a deduced casein kinase II phosphorylation site present only in the long isoform of putative FaMeT.

Gene expression profiling of cuticular proteins across the moult cycle of the crab Portunus pelagicus

Background: Crustaceans represent an attractive model to study biomineralization and cuticle matrix formation, as these events are precisely timed to occur at certain stages of the moult cycle. Moulting, the process by which crustaceans shed their exoskeleton, involves the partial breakdown of the old exoskeleton and the synthesis of a new cuticle. This cuticle is subdivided into layers, some of which become calcified while others remain uncalcified. The cuticle matrix consists of many different proteins that confer the physical properties, such as pliability, of the exoskeleton. Results: We have used a custom cDNA microarray chip, developed for the blue swimmer crab Portunus pelagicus, to generate expression profiles of genes involved in exoskeletal formation across the moult cycle. A total of 21 distinct moult-cycle related differentially expressed transcripts representing crustacean cuticular proteins were isolated. Of these, 13 contained copies of the cuticle_1 domain previously isolated from calcified regions of the crustacean exoskeleton, four transcripts contained a chitin_bind_4 domain (RR consensus sequence) associated with both the calcified and un-calcified cuticle of crustaceans, and four transcripts contained an unannotated domain (PfamB_109992) previously isolated from C. pagurus. Additionally, cryptocyanin, a hemolymph protein involved in cuticle synthesis and structural integrity, also displays differential expression related to the moult cycle. Moult stage-specific expression analysis of these transcripts revealed that differential gene expression occurs both among transcripts containing the same domain and among transcripts containing different domains. Conclusion: The large variety of genes associated with cuticle formation, and their differential expression across the crustacean moult cycle, point to the complexity of the processes associated with cuticle formation and hardening. This study provides a molecular entry path into the investigation of the gene networks associated with cuticle formation.

(Z)-11-Hexadecenal and (3Z,6Z,9Z)-Tricosatriene: Sex Pheromone Components of the Red Banded Mango Caterpillar Deanolis sublimbalis

The sex pheromone of the red banded mango caterpillar, Deanolis sublimbalis (Lepidoptera: Crambidae), a serious pest of the mango Mangifera indica (Anacardiaceae) in India and Southeast Asia and a recent invader into northern Australia, has been identified. Three candidate compounds were identified from pheromone gland extracts of female moths, using gas chromatography (GC), GC-electroantennographic detection and GC-mass spectrometric analyses, in conjunction with dimethyldisulfide derivatization. Field bioassays established that both (Z)-11-hexadecenal (Z11-16:Ald) and (3Z,6Z,9Z)-tricosatriene (3Z,6Z,9Z-23:Hy) were required for attraction of male D. sublimbalis moths, and 1,000 μg of a 1:1 mix of Z11-16:Ald and 3Z,6Z,9Z-23:Hy was more attractive to male moths than caged virgin females. However, the binary blend was only attractive when the isomeric purity of the monounsaturated aldehyde was >99%, suggesting that the (E)-isomer was inhibitory. Although (Z)-11-hexadecen-1-ol (Z11-16:OH) was tentatively identified in gland extracts, the addition of this compound to the binary blend did not increase the numbers of moths captured. The pheromone can now be used in integrated pest management strategies.

Purification of immunoglobulins from chicken sera by thiophilic gel chromatography

Immunoglobulin Y is different from most of the other immunoglobulins because it does not bind protein A or protein G. Thiophilic gel chromatography has been successfully used to purify IgY from chicken egg yolk, but the technology has not previously been used to purify IgY from serum. In this research note, we describe the optimization of T-gel chromatography for purification of IgY from serum. Data are provided on the recovery and purity of IgY obtained using potassium sulfate buffers of different concentrations. Decreasing the strength of potassium sulfate buffer from 0.5 to 0.3 M did not alter the amount of IgY recovered but increased the purity. Using 0.3 M potassium sulphate, we recovered approximately 63.7% of the serum Ig as almost pure IgY.

An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.

Modeling chickpea growth and development: Nitrogen accumulation and use

Quantitative information regarding nitrogen (N) accumulation and its distribution to leaves, stems and grains under varying environmental and growth conditions are limited for chickpea (Cicer arietinum L.). The information is required for the development of crop growth models and also for assessment of the contribution of chickpea to N balances in cropping systems. Accordingly, these processes were quantified in chickpea under different environmental and growth conditions (still without water or N deficit) using four field experiments and 1325 N measurements. N concentration ([N]) in green leaves was 50 mg g-1 up to beginning of seed growth, and then it declined linearly to 30 mg g-1 at the end of seed growth phase. [N] in senesced leaves was 12 mg g-1. Stem [N] decreased from 30 mg g-1 early in the season to 8 mg g-1 in senesced stems at maturity. Pod [N] was constant (35 mg g-1), but grain [N] decreased from 60 mg g-1 early in seed growth to 43 mg g-1 at maturity. Total N accumulation ranged between 9 and 30 g m-2. N accumulation was closely linked to biomass accumulation until maturity. N accumulation efficiency (N accumulation relative to biomass accumulation) was 0.033 g g-1 where total biomass was -2 and during early growth period, but it decreased to 0.0176 g g-1 during the later growth period when total biomass was >218 g m-2. During vegetative growth (up to first-pod), 58% of N was partitioned to leaves and 42% to stems. Depending on growth conditions, 37-72% of leaf N and 12-56% of stem N was remobilized to the grains. The parameter estimates and functions obtained in this study can be used in chickpea simulation models to simulate N accumulation and distribution.

Non-destructive assessment of Arundo donax (Poaceae) leaf quality

Leaf carbon (C) content, leaf nitrogen (N) content, and C:N ratio are especially useful for understanding plant-herbivore interactions and may be important in developing control methods for the invasive riparian plant Arundo donax L. We measured C content, N content, C:N ratio, and chlorophyll index (SPAD 502 reading) for 768 leaves from A. donax collected over a five year period at several locations in California, Nevada, and Texas. Leaf N was more variable than leaf C, and thus we developed a linear regression equation for estimating A. donax leaf N from the leaf chlorophyll index (SPAD reading). When applied to two independent data sets, the equation (leaf N content % = -0.63 + 0.08 x SPAD) produced realistic estimates that matched seasonal and spatial trends reported from a natural A. donax population. Used in conjunction with the handheld SPAD 502 meter, the equation provides a rapid, non-destructive method for estimating A. donax leaf quality.

Cultivar affects browning susceptibility of freshly cut star fruit slices

Consumption of freshly-cut horticultural products has increased in the last few years. The principal restraint to using freshly-cut carambola is its susceptibility to tissue-browning, due to polyphenol oxidase-mediated oxidation of phenolic compounds present in the tissue. The current study investigated the susceptibility to browning of star fruit slices (Averrhoa carambola L.) of seven genotypes (Hart, Golden Star, Taen-ma, Nota-10, Malasia, Arkin, and Fwang Tung). Cultivar susceptibility to browning as measured by luminosity (L*) varied significantly among genotypes. Without catechol 0.05 M, little changes occurred on cut surface of any cultivars during 6 hour at 25 degrees C, 67% RH. Addition of catechol led to rapid browning, which was more intense in cvs. Taen-ma, Fwang Tung, and Golden Star, with reduction in L* value of 28.60%, 27.68%, and 23.29%, respectively. Browning was more intense in the center of the slices, particularly when treated with catechol, indicating highest polyphenol oxidase (PPO) concentration. Epidermal browning, even in absence of catechol, is a limitation to visual acceptability and indicates a necessity for its control during carambola processing. Care must be given to appropriate selection of cultivars for fresh-cut processing, since cultivar varied in browning susceptibility in the presence of catechol.

Response of minimally processed carambola to chemical treatments and low-oxygen atmospheres

Fresh-cut carambola (Averrhoa carambola L.) has limited marketability due to cut-surface browning. The effect of chemical treatments (ascorbic acid, citric acid and Ca-EDTA), controlled atmosphere (0.4-20.3% O2) and the association of these processes was investigated. Post-cutting dip and low-oxygen atmospheres did not prevent discoloration or improve sensory and physicochemical parameters. However, ascorbic acid (0.5% and 1%) dips reduced polyphenol oxidase (PPO) activity during storage at 4.5 °C, with 1% ascorbic acid inducing the lowest activity. Although cut-surface browning of 'Maha' slices was not relevant, carambola slices treated with 1% ascorbic acid in association with 0.4% oxygen did not present significant browning or loss of visual quality for up to 12 days, 3 days longer than low oxygen alone (0.4% O2), thus, their quality can be significantly improved by combining both treatments.

The effects of salivary gland extracts from Boophilus microplus ticks on mitogen-stimulated bovine lymphocytes

The effect of salivary gland extract (SGE) from the tick Boophilus microplus was examined in mitogen-stimulated lymphocytes in vitro. SGE was added to lymphocytes of seven cattle together with the mitogens concanavalin A (ConA), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Semi-purified B cells from another seven cattle were stimulated with the mitogen lipopolysaccharide (LPS). PHA and ConA stimulated proliferation of lymphocytes to the same extent, but the inhibition due to SGE of Boophilus microplus on the proliferative response stimulated by PHA (39.0% ± 9.3%) was less than the inhibition of proliferative response stimulated by ConA (75.4% ± 6.9%). In contrast, SGE of B. microplus stimulated the proliferation of B cells in the presence of LPS in a dose-dependent manner. Enhanced stimulation of B cells by SGE at >4 μg in culture was greater than twice that observed when B cells were stimulated by LPS alone. SGE does not have a direct suppressive effect on bovine B cell proliferation; however, in vivo the effectiveness of B cell responses might be influenced by other immune factors, such as cytokine profiles.

Alkaloid production by isolates of the sorghum ergot pathogen (Claviceps africana) from Australia and other countries

Isolates of Claviceps africana from Australia, Africa, Asia, and America were tested for the production of dihydroergosine (DHES), and its biogenic precursors dihydroelymoclavine (DHEL) and festuclavine (FEST), in culture. Several growth media were evaluated to optimise alkaloid production with little success. The best of these involved 2-stage culturing on high-sucrose substrate. Australian C. africana isolates varied widely and inconsistently in alkaloid production, with DHES concentrations in mycelium ranging from: <0.1 to 9 mg DHES/kg; <0.1 to 1.6 mg DHEL/kg; and <0.1 to 0.4 mg FEST/kg. In a separate experiment using similar culturing techniques, DHES was produced by 2 of 3 Australian isolates, 1 of 3 USA isolates, 1 of 4 Indian isolates, the sole Puerto Rican isolate, the sole Japanese isolate, but not the sole South African isolate. In this experiment, DHES concentrations detected in mycelium of Australian isolates (0.1-1.0 mg DHES/kg) were of similar magnitude to isolates from other countries (0.2-1.8 mg DHES/kg). Three C. africana isolates, including one that produced only traces of alkaloid in culture after 8 weeks, were inoculated onto panicles of sterile male sorghum plants. After 8 weeks, all 3 isolates produced 10-19 mg DHES/kg in the panicles, demonstrating that the growing plant favoured more consistent alkaloid production than culture medium.

Effect of feeding sorghum ergot (Claviceps africana) to sows during mid-lactation on plasma prolactin and litter performance

Diets containing 3% sorghum ergot (16 mg alkaloids/kg, including 14 mg dihydroergosine/kg) were fed to 12 sows from 14 days post-farrowing until weaning 14 days later, and their performance was compared with that of 10 control sows. Ergot-fed sows displayed a smaller weight loss during lactation of 24 kg/head vs. 29 kg/head in control sows (p > 0.05) despite feed consumption being less (61 kg/head total feed intake vs. 73 kg/head by control sows; p < 0.05). Ergot-fed sows had poorer weight gain of litters over the 14-day period (16.6 kg/litter vs. 28.3 kg/litter for controls; p < 0.05) despite an increase in consumption of creep feed by the piglets from the ergot-fed sows (1.9 kg/litter compared with 1.1 kg/litter by the control; p > 0.05). Sow plasma prolactin was reduced with ergot feeding after 7 days to 4.8 μg/l compared with 15.1 μg/l in the control sows (p < 0.01) and then at weaning was 4.9 μg/l compared with 8.0 μg/l (p < 0.01) in the control sows. Two sows fed ergot ceased lactation early, and the above sow feed intakes, body weight losses with litter weight gains and creep consumption indirectly indicate an ergot effect on milk production.

Spiroacetal biosynthesis in insects from diptera to hymenoptera: The Giant Ichneumon wasp Megarhyssa nortoni nortoni Cresson

The volatile components of the mandibular gland secretion generated by the Giant Ichneumon parasitoid wasp Megarhyssa nortoni nortoni Cresson are mainly spiroacetals and methyl ketones, and all have an odd number of carbon atoms. A biosynthetic scheme rationalizing the formation of these diverse components is presented. This scheme is based on the results of incorporation studies using 2H-labeled precursors and [18O]dioxygen. The key steps are postulated to be decarboxylation of β-ketoacid equivalents, β-oxidation (chain shortening), and monooxygenase-mediated hydroxylation leading to a putative ketodiol that cyclizes to spiroacetals. The generality of the role of monooxygenases in spiroacetal formation in insects is considered, and overall, a cohesive, internally consistent theory of spiroacetal generation by insects is presented, against which future hypotheses will have to be compared.

Epithiospecifier protein activity in broccoli: The link between terminal alkenyl glucosinolates and sulphoraphane nitrile

The chemical nature of the hydrolysis products from the glucosinolate-myrosinase system depends on the presence or absence of supplementary proteins, such as epithiospecifier proteins (ESPs). ESPs (non-catalytic cofactors of myrosinase) promote the formation of epithionitriles from terminal alkenyl glucosinolates and as recent evidence suggests, simple nitriles at the expense of isothiocyanates. The ratio of ESP activity to myrosinase activity is crucial in determining the proportion of these nitriles produced on hydrolysis. Sulphoraphane, a major isothiocyanate produced in broccoli seedlings, has been found to be a potent inducer of phase 2 detoxification enzymes. However, ESP may also support the formation of the non-inductive sulphoraphane nitrile. Our objective was to monitor changes in ESP activity during the development of broccoli seedlings and link these activity changes with myrosinase activity, the level of terminal alkenyl glucosinolates and sulphoraphane nitrile formed. Here, for the first time, we show ESP activity increases up to day 2 after germination before decreasing again to seed activity levels at day 5. These activity changes paralleled changes in myrosinase activity and terminal alkenyl glucosinolate content. There is a significant relationship between ESP activity and the formation of sulforaphane nitrile in broccoli seedlings. The significance of these findings for the health benefits conferred by eating broccoli seedlings is briefly discussed.