Optimal assay conditions for aspartate transcarbamylase (ATCase) activity in mesozooplankton

The optimal conditions for storing and assaying mesozooplanktonaspartate transcarb amylase (ATCase) (EC 2.1.3.1 [EC] .) are definedin order to enable the specific activity of this enzyme to beused as an index of mesozooplankton productivity. ATCase activitywas found to be stable for more than 2 weeks when stored inliquid nitrogen, with a negligible loss of activity, whateverthe state of frozen material (cell-free homogenate or wholecells). As an alternative, –20C, –90C freezersand dry ice (–80C) can give more flexibility for storingand transporting material, provided initial freezing has beencarried out at –196C. ATCase specific activity was stableover a large range of total protein concentrations (2–55mg ml^–1) for most mesozooplankton samples, and the enzymeactivity was linear between 0.8 and 80 nmol carbamyl aspartatemin^–1 mg^–1 of total protein. The optimal incubationtime (30 min), temperature (35C) and pH range (9–9.5)for assay of ATCase were similar forCalanus helgolondicus (Crustacea:Copepoda) (G.O.Sars) and three mesozooplankton assemblages sampledoff Plymouth (UK) in May, August and November. However, theKm values for both substrateswere variable: from 3.9 to 79.1for aspartate, and from 0.83 to 3.46 for carbamyl phosphate.Such variations arelikely to be due to the presence of differentATCase catalytic potential, rather than changes in ATCase assemblage.     

Control of the release of digestive enzymes in the larvae of the fall armyworm,Spodoptera frugiperda

There is a basal level of enzyme activity for trypsin, aminopeptidase, amylase, and lipase in the gut of unfed larval (L6) Spodoptera frugiperda. Trypsin activity does not decrease with non‐feeding, possibly because of the low protein levels in plants along with high amino acid requirements for growth and storage (for later reproduction in adults). Therefore, trypsin must always be present so that only a minimal protein loss via egestion occurs. Larvae, however, adjust amylase activity to carbohydrate ingestion, and indeed amylase activity is five‐fold higher in fed larvae compared to unfed larvae. Gut lipase activity is low, typical of insects with a high carbohydrate diet. A flat‐sheet preparation of the ventriculus was used to measure the release of enzymes in response to specific nutrients and known brain/gut hormones in S. frugiperda. Sugars greatly increase (>300%) amylase release, but starch has no effect. Proteins and amino acids have little or no effect on trypsin or aminopeptidase release. The control of enzyme release in response to food is likely mediated through neurohormones. Indeed, an allatostatin (Spofr‐AS A5) inhibits amylase and trypsin, and allatotropin (Manse‐ AT) stimulates amylase and trypsin release. Spofr‐AS A5 also inhibits ileum myoactivity and Manse‐AT stimulates myoactivity. The epithelial secretion rate of amylase and trypsin was about 20% of the amount of enzyme present in the ventricular lumen, which, considering the efficient counter‐current recycling of enzymes, suggests that the secretion rate is adequate to replace egested enzymes. © 2009 Wiley Periodicals, Inc.     

Expression of trypsin-like serine peptidases in pre-imaginal stages of Aedes aegypti (Diptera: Culicidae)

This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate‐SDS‐PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6–8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl‐methyl sulfonyl‐fluoride and N‐α‐Tosyl‐L ‐lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin‐like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin‐like serine proteases occur during the developmental cycle of A. aegypti. © 2011 Wiley Periodicals, Inc.     

Purification,characterization and evaluation of insecticidal potential of trypsin inhibitor from mungbean (<Emphasis Type="Italic">Vigna radiata</Emphasis> L. Wilczek) seeds

Protease inhibitors present in seeds of legumes possess strong inhibitory activity against trypsin and confer resistance against pests. In the present investigation, trypsin inhibitor activity was found in the seed flour extracts of all the eight selected varieties of mungbean under study which was further confirmed by dot blot analysis. All the varieties showed inhibitory activity in vitro against the gut protease of Helicoverpa armigera (HGP). Trypsin inhibitor was purified from mungbean seeds to near homogeneity with 58.1-fold and 22.8% recovery using heat denaturation, NH₄(SO₄)₂ fractionation, ion-exchange chromatography on DEAE-Sephadex A-25 and gel filtration through Sephadex G-75. The molecular mass of the inhibitor was 47 kDa as determined by gel filtration and SDS-PAGE. The inhibitor retained 90% or more activity between pH 4 and 10, however, it was nearly inactive at extreme pH values. The inhibitor was stable up to 80°C but thereafter, the activity decreased gradually retaining nearly 30% of activity when heated at 100°C for 20 min. The inhibitor activity was undetectable at 121°C. Insect bioassay experiment using purified mungbean trypsin inhibitor showed a marked decline in survival (%) of larvae with increase in inhibitor concentration. The larval growth was also extended by the trypsin inhibitor. This study signifies the insecticidal potential of mungbean trypsin inhibitor which might be exploited for raising transgenic plants.     

Polymorphism of the kinase domain of the S-locus receptor kinase gene (SRK) in Brassica oleracea L.

 DNA polymorphism of the S-locus receptor kinase gene (SRK) participating in self-incompatibility in Brassica was analyzed by PCR-RFLP and nucleotide sequencing. In the screening of primers for specific amplification of polymorphic DNA fragments of SRK, the best combination was that of a forward primer (PK1) having the nucleotide sequence of the second exon of S6 SRK and a reverse primer (PK4) having the complementary nucleotide sequence of the fifth exon of S6 SRK. PCR using this primer pair amplified DNA fragments of 0.9–1.0 kb from 36 S haplotypes out of 42 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonuclease(s): 25 types were found in a double digestion with MboI and AfaI. Nucleotide sequencing of the DNA fragments amplified from five S haplotypes showed that the third, fourth, and fifth exons of SRK are highly conserved, and that there are high variations of the second and third introns of SRK, which produced polymorphism of the band pattern in PCR-RFLPs. Another forward primer (PK5) having the nucleotide sequence of the second exon, which is derived from S2 SRK, amplified DNA fragments of almost the same region of SRK from 27 S haplotypes in combination with PK4. Although SRK alleles of the class-II S haplotypes were not amplified, all of the class-I S-haplotypes were amplified with a primer mixture of PK1, PK4 and PK5. The DNA fragments of both SRK alleles in S heterozygotes, or a 1 : 1 mixture of the genomic DNA of different S homozygotes, were amplified without exception, suggesting the usefulness of these primers for the identification of S heterozygotes. The DNA fragment sizes obtained by digestion with restriction endonucleases served as markers for the identification of S haplotypes. Received: 15 December 1996 / Accepted: 14 February 1997     

Effect of jasmonic acid and salicylic acid induced resistance in groundnut on Helicoverpa armigera

Induced resistance in plants affects insect growth and development as a result of the up‐regulation of defence‐related secondary metabolites or enzyme‐binding proteins. In the present study, the effects of jasmonic acid (JA) and salicylic acid (SA) induced resistance in groundnut on Helicoverpa armigera (Hübner) are examined. Larval survival, larval weights and the activities of digestive enzymes (total serine protease and trypsin) and of detoxifying enzymes [glutathione S‐transferase (GST) and esterase (EST)] are studied in insects fed on four groundnut genotypes with moderate levels of resistance to H. armigera (ICGV 86699, ICGV 86031, ICG 2271 and ICG 1697) and a susceptible genotype (JL 24). The plants are pre‐ and/or simultaneously treated with JA and SA, and then infested with H. armigera, which are allowed to feed for 6 days. Significantly lower serine protease and trypsin activities are observed in H. armigera fed on plants treated with JA. Greater GST activity is recorded in insects fed on JA and SA treated plants, whereas EST activity is low in H. armigera larvae fed on plants treated with JA and SA. Serine proteases, trypsin and GST activities and larval weights (r = 0.74–0.95) and larval survival (r = 0.77–0.93) are positively correlated, whereas EST activity and larval weight (r = ?0.55) and larval survival (r = ?0.65) are negatively correlated. The results suggest that midgut digestive and detoxifying enzymes can be used as indicators of the adverse effects of constitutive and/or induced resistance in crop plants on the insect pests and the role of JA and SA in insect pest management.     

Sources of seasonal variability in mesozooplankton aspartate transcarbamylase activity in coastal waters off Plymouth, UK

Some investigators have proposed aspartate transcarbamylase(ATCase) activity as an overall index of mesozooplankton productivity.However, seasonal changes in mesozooplankton species compositionhave never been investigated as a possible source of variationin ATCase activity. In this study, we investigate mesozooplanktoncomposition in terms of (i) developmental stages, (ii) speciesand developmental stages body mass and (iii) species composition,and their relationship to ATCase activity. In controlled laboratoryconditions, ATCase activity variability was closely relatedto changes in somatic growth rate of the copepod Calanus helgolandicus,but was not related to changes in nucleic acid concentrations.It can be argued, however, that the activity of this enzymeis partially involved in copepod somatic productivity, and shouldbe a good index of embryogenesis. In addition, changes in ATCaseactivity were not significantly influenced by variability inmesozooplankton biomass, when investigated both on inter- andintraspecific levels. Finally, when a complete seasonal cyclewas investigated at a fixed station off Plymouth (English Channel),ATCase activity was not correlated with the abundance of anymesozooplankton species apart from copepodites and adult C.helgolandicus.Furthermore, ATCase activity measured both on mesozooplanktonand female C.helgolandicus was significantly correlated (R²= 0.72, n = 33, P < 0.001) throughout the year, apart fromApril. At that particular time of the year, ATCase activitywas in phase with the peak of abundance of copepod eggs andnauplii. It is suggested that mesozooplankton peaks of ATCaseactivity reflect two periods in the life history of the copepod:embryogenesis and terminal moult. We propose further experimentsto test this hypothesis and to promote the development of molecularbiomarkers in order to characterize specific zooplankton metabolicprocesses.     

Proteolytic profile in the larval midgut of Chilo suppressalis Walker (Lepidoptera: Crambidae)

Chilo suppressalis is a key constraint on production of rice. The current research was conducted to study the types of digestive proteases in the larval midgut of C. suppressalis. It was found that activity of total digestive proteases increased from the first to the fifth larval instars, which showed different nutritional requirements. Four types of proteinases and two types of exopeptidase were identified so that their activities from the highest to the lowest activities is trypsin‐like, chymotrypsin‐like and elastase for proteinases, and amino and carboxypeptidases for exopeptidases. Meanwhile, just one type of cysteine protease, cathepsin D, was determined in the fourth and fifth instar larvae. The optimal pH for activity of total protease was found to be pH 9–10 and optimal temperature was observed to be 35–40°C, where there was the highest proteolytic activity. Some specific inhibitors of proteases including PMSF, TLCK, TPCK, DTT, E‐64, cystatin, phenanthroline and EDTA were used to confirm the types of proteases in the midgut of C. suppressalis.     

Programmed Cell Death in Cortical Chick Embryo Astrocytes Is Associated with Activation of Protein Kinase PK60 and Ceramide Formation

Abstract: Embryonic astrocytes respond readily to serine/threonine kinase regulation in terms of cytoskeleton assembly, mitotic activity, and cell fate. We now present evidence that these responses include apoptosis. Staurosporine induced apoptosis in astrocyte cultures derived from chick embryo cerebral hemispheres, as assayed both by immunocytochemical detection of new 3-hydroxy DNA ends and production of 200-bp DNA fragment laddering. Staurosporine treatment also resulted in the prolonged (>24 h) activation of a 60-kDa serine/threonine protein kinase (PK60), increased ceramide formation (fourfold after 24 h), increased glutamine synthetase activity, and significant apoptosis (40%) after 24 h. PK60 was shown to be cytoskeleton associated and its activity, as measured by phosphorylation of myelin basic protein, was rapid, increased for up to 3 h, and was stable for at least 24 h. Other protein kinase C inhibitors, H8, sphingosine, calphostin C, or the protein kinase A inhibitor KT5720 did not induce either PK60 activation or apoptosis. The dose-dependent increase in [³H]palmitate labeling of ceramide and a specific decrease in labeling of its precursor sphingomyelin were not blocked by the biosynthetic inhibitor fumonisin β1 but were increased (in a dose-dependent manner) by the coaddition of the ceramidase inhibitor oleoylethanolamine. Exogenous addition of C2-ceramide induced apoptosis but did not activate PK60. These results suggest that apoptosis in embryonic astrocytes involves pathways similar to those described in other cell types and that the activation of PK60 and formation of ceramide are early events in the pathway.     

Changes in digestive enzymes through developmental and molt stages in the spiny lobster, Panulirus argus

Changes in major digestive enzymes through developmental and molt stages were studied for the spiny lobster Panulirus argus. There were significant positive relationships between specific activity of trypsin and amylase enzymes and lobster size, whereas esterase and lipase specific activities decreased as lobsters aged. No relationship was found between amylase/trypsin ratio and lobster size. Positive trends were found, however, for trypsin/lipase and amylase/lipase ratios. Results suggest that changes in enzyme activity respond to the lobsters' physiological needs for particular dietary components although multivariate analysis suggested that enzyme activities could be not totally independent of diet. On the other hand, the pattern of changes of major enzyme activities through molt cycle was similar for most enzymes studied. Following molt, trypsin, chymotrypsin, amylase, and lipase activities gradually increased to maximal levels at late intermolt (C4) and premolt (D). There were no variations in the electrophoretic pattern of digestive enzymes through developmental and molt stages and thus, it is demonstrated that regulation is exerted quantitatively rather than qualitatively. Further studies on the effect of other intrinsic and extrinsic factors on digestive enzyme activities are needed to fully understand digestive abilities and regulation mechanisms in spiny lobsters.     

First estimates of metabolic rate in Atlantic bluefin tuna larvae

Atlantic bluefin tuna is an iconic scombrid species with a high commercial and ecological value. Despite their importance, many physiological aspects, especially during the larval stages, are still unknown. Metabolic rates are one of the understudied aspects in scombrid larvae, likely due to challenges associated to larval handling before and during respirometry trials. Gaining reliable estimates of metabolic rates is essential to understand how larvae balance their high growth needs and activity and other physiological functions, which can be very useful for fisheries ecology and aquaculture. This is the first study to (a) estimate the relationship between routine metabolic rate (RMR) and larval dry weight (DW) (mass scaling exponent) at a constant temperature of 26°C, (b) measure the RMR under light and darkness and (c) test whether the interindividual differences in the RMR are related to larval nutritional status (RNA/DNA and DNA/DW). The RMR scaled nearly isometrically with body size (b = 0.99, 0.60–31.56 mg DW) in contrast to the allometric relationship observed in most fish larvae (average b = 0.87). The results show no significant differences in larval RMR under light and darkness, suggesting similar larval activity levels in both conditions. The size explained most of the variability in RMR (97%), and nutritional condition was unrelated to the interindividual differences in routine metabolism. This is the first study to report the metabolic rates of Atlantic bluefin tuna larvae and discuss the challenges of performing bioenergetic studies with early life stages of scombrids.     

Citrate synthase and pyruvate kinase activities during early life stages of the shrimp Farfantepenaeus paulensis (Crustacea,Decapoda, Penaeidae): effects of development and temperature

Energy metabolism in early life stages of the shrimp Farfantepenaeus paulensis subjected to temperature reduction (26 and 20 °C) was determined using the activities of citrate synthase (CS) and pyruvate kinase (PK). At both temperatures, weight-specific activity of CS decreased throughout the ontogenetic development from protozoea II (PZ II) to postlarva XII–XIV (PL XII–XIV). PK activity reached a pronounced peak in PL V–VI, followed by a further decrease in PL XII–XIV. Temperature reduction produced variation in oxygen consumption rates (QO₂), ammonia–N excretion and in enzyme activities. Ammonia–N excretion was higher at 20 °C in mysis III (M III), PL V–VI and PL XII–XIV, resulting in substantially lower O:N ratios in these stages. QO₂ was increased in protozoea II (PZ II) and mysis I (M I) at 26 °C, while no difference in QO₂ was detected in the subsequent stages at either temperature. This fact coincided with higher CS and PK activities in M III, PL V–VI and PL XII–XIV at 20 °C compared with 26 °C. Regressions between individual enzyme activities and dry weight exhibited slope values of 0.85–0.92 for CS and 1.1–1.2 for PK and temperature reduction was reflected by higher slope values at 20 than at 26 °C for both enzymes. Weight-specific CS activity was positively correlated with QO₂ at 20 and 26 °C, and may thus be used as an indicator of aerobic metabolic rate throughout the early stages of F. paulensis. The variation in enzyme activities is discussed in relation to possible metabolic adaptations during specific ontogenetic events of the F. paulensis life cycle. Here, the catalytic efficiency of energy-metabolism enzymes was reflected in ontogenetic shifts in behaviour such as larval settlement and the adoption of a benthic existence in early postlarvae. In most cases, enhanced enzyme activities appeared to counteract negative effects of reduced temperature.     

Metabolism and foraging strategies of mid‐latitude mesozooplankton during cyanobacterial blooms as revealed by fatty acids,amino acids,and their stable carbon isotopes

Increasing sea surface temperatures (SST) and blooms of lipid‐poor, filamentous cyanobacteria can change mesozooplankton metabolism and foraging strategies in marine systems. Lipid shortage and imbalanced diet may challenge the build‐up of energy pools of lipids and proteins, and access to essential fatty acids (FAs) and amino acids (AAs) by copepods. The impact of cyanobacterial blooms on individual energy pools was assessed for key species temperate Temora longicornis and boreal Pseudo‐/Paracalanus spp. that dominated field mesozooplankton communities isolated by seasonal stratification in the central Baltic Sea during the hot and the cold summer. We looked at (a) total lipid and protein levels, (b) FA trophic markers and AA composition, and (c) compound‐specific stable carbon isotopes (δ¹³C) in bulk mesozooplankton and in a subset of parameters in particulate organic matter. Despite lipid‐poor cyanobacterial blooms, the key species were largely able to cover both energy pools, yet a tendency of lipid reduction was observed in surface animals. Omni‐ and carnivory feeding modes, FA trophic makers, and δ¹³C patterns in essential compounds emphasized that cyanobacterial FAs and AAs have been incorporated into mesozooplankton mainly via feeding on mixo‐ and heterotrophic (dino‐) flagellates and detrital complexes during summer. Foraging for essential highly unsaturated FAs from (dino‐) flagellates may have caused night migration of Pseudo‐/Paracalanus spp. from the deep subhalocline waters into the upper waters. Only in the hot summer (SST>19.0°C) was T. longicornis submerged in the colder subthermocline water (~4°C). Thus, the continuous warming trend and simultaneous feeding can eventually lead to competition on the preferred diet by key copepod species below the thermocline in stratified systems. A comparison of δ¹³C patterns of essential AAs in surface mesozooplankton across sub‐basins of low and high cyanobacterial biomasses revealed the potential of δ¹³C‐AA isoscapes for studies of commercial fish feeding trails across the Baltic Sea food webs.     

Pyruvate kinase activity as index of carbohydrate assimilation by mesozooplankton: an early field implementation in the Bay of Biscay,NE Atlantic

Estimates of carbohydrate assimilation within the mesozooplanktoncompartment are a fundamental concern in oceanography becausethey give a general expression of the biologically-regulatedincorporation of metabolic fuel produced from inorganic nutrientsby phytoplankton in the sea. A new tool is proposed for theassessment of such processes. It is based on the measurementof the activity of an enzyme, pyruvate kinase (PK), on samplesof the whole mesozooplankton community. Here, the results arepresented of an early attempt at such an implementation in theBay of Biscay (north-east Atlantic) in springtime. A main areasituated in front of an estuary (Gironde) has been extensivelystudied—the same sampling grid was covered on three occasions(two 20 day time lags between each). Strong mesozooplanktonPK activities were related to high values of nitrates or chlorophylla; the inverse was also true. A significant correlation hasbeen found between thermal stratification and enzyme activity.On a brooder scale, in the southern part of the Bay of Biscaycontinental shelf, the overall significant influence of de-saltedwater coming from rivers was revealed by spatial variationsin mesozooplankton PK activities. The whole dataset suggeststhat this new tool should be potentially very useful in certainfuture studies, especially in fisheries oceanography which oftenrequires characterization of mesoscale features.     

Partial characterization of the proteolytic enzymes in the gut of the banana weevil, Cosmopolites sordidus, and effects of soybean Kunitz trypsin inhibitor on larval performance

The proteolytic enzymes in the gut of the banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae), have been characterized. Both larvae and adults rely on a complex proteolytic system based on at least cathepsin D‐, cathepsin B‐, trypsin‐, chymotrypsin‐, leucine aminopeptidase‐, carboxypeptidase A‐, and carboxypeptidase B‐like activities. All endoproteolytic activities were higher in the anterior section of the gut, whereas the exopeptidases were evenly distributed in the anterior and middle sections, and almost no activity was detected in the posterior section. Gelatin‐containing gels confirmed the spatial organization of the proteolytic digestive process. According to this proteolytic profile, the STI (soybean Kunitz trypsin inhibitor) was tested in vivo to establish its potential as a resistance factor against C. sordidus. Newly hatched larvae fed on diets containing 0.2% (w/w) STI experience lower survival rates and display significant reductions in larval growth. Biochemical analysis carried out on guts of larvae reared on STI‐treated diet showed a reduction of trypsin‐like activity compared to that from larvae fed on control diet. This decrease was compensated with an induction of cathepsin B, whereas cathepsin D, chymotrypsin, and leucine aminopeptidase were not affected. These results are discussed as a basis for selecting appropriate inhibitors to obtain transgenic banana and plantain plants with enhanced resistance to this pest.     

Partial characterization of a major gut thiol proteinase from larvae of Callosobruchus maculatus F.

Much of the proteolytic activity in the digestive tract of Callosobruchus maculatus larvae can be attributed to a thiol proteinase(s) that hydrolyzes [3H]methemoglobin optimally at pH 5.0. Maximal hydrolysis of [3H]methemoglobin, [3H]alpha-casein, and N-benzoyl-DL-arginine napthylamide-(BANA) required the presence of thiol reducing agents. Larval gut proteinase activity was strongly inhibited by p-hydroxymercuribenzoic acid (pHMB), Nethylmaleimide (NEM), and iodoacetic acid (IAA) but was unaffected by the Bowman-Birk and Kunitz proteinase inhibitors from soybeans or by lima bean trypsin inhibitor. L-Trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E-64), a specific inhibitor of thiol proteinases, potently inhibited proteolysis of [3H]methemoglobin by larval gut homogenates. Proteolytic activity in the larval gut was located in the lumen contents and thus appears to play a major role in extracellular digestion. The pH of the larval midgut is slightly acidic, and midgut contents exhibit a negative redox potential, conditions supporting the activity of a thiol proteinase. The significance of these findings is discussed with reference to the vulnerability of this digestive proteinase as a target for existing or genetically engineered plant chemical defenses.     

THE INFLUENCE OF A 21 kDa KUNITZ‐TYPE TRYPSIN INHIBITOR FROM NONHOST MADRAS THORN,Pithecellobium dulce,SEEDS ON H. armigera (HÜBNER) (LEPIDOPTERA: NOCTUIDAE)

A trypsin inhibitor purified from the seeds of the Manila tamarind, Pithecellobium dulce (PDTI), was studied for its effects on growth parameters and developmental stages of  Helicoverpa armigera. PDTI exhibited inhibitory activity against bovine trypsin (～86%; ～1.33 ug/ml IC₅₀). The inhibitory activity of PDTI was unaltered over a wide range of temperature, pH, and in the presence of dithiothreitol. Larval midgut proteases were unable to digest PDTI for up to 12 h of incubation. Dixon and Lineweaver–Burk double reciprocal plots analysis revealed a competitive inhibition mechanism and a K_i of ～3.9 × 10^?8 M. Lethal dose (0.50% w/w) and dosage for weight reduction by 50% (0.25% w/w) were determined. PDTI showed a dose‐dependent effect on mean larval weight and a series of nutritional disturbances. In artificial diet at 0.25% w/w PDTI, the efficiency of conversion of ingested food, of digested food, relative growth rate, and growth index declined, whereas approximate digestibility, relative consumption rate, metabolic cost, consumption index, and total developmental period were increased in larvae. This is the first report of antifeedant and antimetabolic activities of PDTI on midgut proteases of  H. armigera.     

Changes in activity of the regulatory glycolytic enzymes and of the pyruvate-dehydrogenase complex during the development of Xenopus laevis

In this study we investigated the variations of the maximal activities of the rate-controlling glycolytic enzymes (i.e., hexokinase, HK; phosphofructokinase, PFK; pyruvate kinase, PK) and of the pyruvate-dehydrogenase complex (PDHc) during the early embryogenesis of Xenopus laevis (from cleavage through hatching). All the enzymatic assays, using different coupled reactions, were performed spectrophotometrically on cytosolic and mitochondrial fractions. The maximal HK activity increases markedly from neurulation onwards, PFK activity presents a peak around gastrulation, PK activity remains relatively constant throughout the period studied and the highest PDHc activity is observed during cleavage. The specific activities display the same temporal pattern. Furthermore, in the sequence of reactions by which glucose is degraded to form acetyl-CoA, the maximal activities of PFK and PK are not limiting while those of HK and PDHc could be rate-limiting at relatively late developmental stages (hatching).     

A Thermostable phytase from <Emphasis Type="Italic">Neosartorya spinosa</Emphasis> BCC 41923 and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C. Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its K _m and V _max for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested, including Fe²⁺, Fe³⁺, and Al³⁺. When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained. However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin at a trypsin/phytase ratio of 0.01 (U/U).     

Annual variations in the specific activity of fructose 1,6-bisphosphatase, alanine aminotransferase and pyruvate kinase in the Sparus aurata liver

We determined the annual change in the intermediary metabolism of glucose through the variations of specific activity of fructose 1,6-bisphosphatase (FBPase), alanine aminotransferase (AAT) and pyruvate kinase (PK). Fish (average mass 330 g) were kept in cages under natural conditions of temperature and photoperiod and fed with a commercial diet. FBPase, AAT and PK increased their activity in June in different ways: AAT and PK increased V(max), and FBPase increased the velocity at subsaturating substrate concentrations, changing K(m). The reproduction period modified the annual tendency of changes in the enzyme activities in both parameters, K(m) and V(max), except for K(m) of PK which shows a circa-annual rhythm. No relation between the changes of enzymes activity and photoperiod or temperature has been found in this study, except for K(m) of AAT which presents a positive correlation with photoperiod and a negative correlation with temperature.