Short-term responses of soil enzyme activities and stoichiometry to litter input in Castanopsis carlesii and Cunninghamia lanceolata plantations.

Litter input triggers the secretion of soil extracellular enzymes and facilitates the release of carbon (C), nitrogen (N), and phosphorus (P) from decomposing litter. However, how soil extracellular enzyme activities were controlled by litter input with various substrates is not fully understood. We examined the activities and stoichiometry of five enzymes including ß-1,4-glucosidase, ß-D-cellobiosidase, ß-1,4-N-acetyl-glucosaminidase, leucine aminopeptidase and acidic phosphatase (AP) with and without litter input in 10-year-old Castanopsis carlesii and Cunninghamia lanceolata plantations monthly during April to August, in October, and in December 2021 by using an in situ microcosm experiment. The results showed that: 1) There was no significant effect of short-term litter input on soil enzyme activity, stoichiometry, and vector properties in C. carlesii plantation. In contrast, short-term litter input significantly increased the AP activity by 1.7% in May and decreased the enzymatic C/N ratio by 3.8% in August, and decreased enzymatic C/P and N/P ratios by 11.7% and 10.3%, respectively, in October in C. lanceolata plantation. Meanwhile, litter input increased the soil enzymatic vector angle to 53.8° in October in C. lanceolata plantations, suggesting a significant P limitation for soil microorganisms. 2) Results from partial least squares regression analyses showed that soil dissolved organic matter and microbial biomass C and N were the primary factors in explaining the responses of soil enzymatic activity to short-term litter input in both plantations. Overall, input of low-quality (high C/N) litter stimulates the secretion of soil extracellular enzymes and accelerates litter decomposition. There is a P limitation for soil microorganisms in the study area.

Murine cytomegalovirus downregulates ERAAP and induces an unconventional T cell response to self.

The endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) plays a crucial role in shaping the peptide-major histocompatibility complex (MHC) class I repertoire and maintaining immune surveillance. While murine cytomegalovirus (MCMV) has multiple strategies for manipulating the antigen processing pathway to evade immune responses, the host has also developed ways to counter viral immune evasion. In this study, we find that MCMV modulates ERAAP and induces an interferon γ (IFN-γ)-producing CD8+ T cell effector response that targets uninfected ERAAP-deficient cells. We observe that ERAAP downregulation during infection leads to the presentation of the self-peptide FL9 on non-classical Qa-1b, thereby eliciting Qa-1b-restricted QFL T cells to proliferate in the liver and spleen of infected mice. QFL T cells upregulate effector markers upon MCMV infection and are sufficient to reduce viral load after transfer to immunodeficient mice. Our study highlights the consequences of ERAAP dysfunction during viral infection and provides potential targets for anti-viral therapies.

LAP3 contributes to IFN-γ-induced arginine depletion and malignant transformation of bovine mammary epithelial cells.

BACKGROUND: IFN-γ has been traditionally recognized as an inflammatory cytokine that involves in inflammation and autoimmune diseases. Previously we have shown that sustained IFN-γ induced malignant transformation of bovine mammary epithelial cells (BMECs) via arginine depletion. However, the molecular mechanism underlying this is still unknown. METHODS: In this study, the amino acids contents in BMECs were quantified by a targeted metabolomics method. The acquisition of differentially expressed genes was mined from RNA-seq dataset and analyzed bioinformatically. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) assay were performed to detect gene mRNA and protein expression levels. CCK-8 and would healing assays were used to detect cell proliferation and migration abilities, respectively. Cell cycle phase alternations were analyzed by flow cytometry. RESULTS: The targeted metabolomics analysis specifically discovered IFN-γ induced arginine depletion through accelerating arginine catabolism and inhibiting arginine anabolism in BMECs. Transcriptome analysis identified leucine aminopeptidase 3 (LAP3), which was regulated by p38 and ERK MAPKs, to downregulate arginine level through interfering with argininosuccinate synthetase (ASS1) as IFN-γ stimulated. Moreover, LAP3 also contributed to IFN-γ-induced malignant transformation of BMECs by upregulation of HDAC2 (histone deacetylase 2) expression and promotion of cell cycle proteins cyclin A1 and D1 expressions. Arginine supplementation did not affect LAP3 and HDAC2 expressions, but slowed down cell cycle process of malignant BMECs. In clinical samples of patients with breast cancer, LAP3 was confirmed to be upregulated, while ASS1 was downregulated compared with healthy control. CONCLUSIONS: These results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.

Different pathways of nitrogen and phosphorus regeneration mediated by extracellular enzymes in temperate lakes under various trophic state.

Several Italian and Chinese temperate lakes with soluble reactive phosphorus concentrations < 0.015 mg L-1 were studied to estimate nitrogen and phosphorus regeneration mediated by microbial decomposition and possible different mechanisms driven by prevailing oligo- or eutrophic conditions. Leucine aminopeptidase (LAP), beta-glucosidase (GLU) and alkaline phosphatase (AP), algal, and bacterial biomass were related to trophic and environmental variables. In the eutrophic lakes, high algal and particulate organic carbon concentrations stimulated bacterial respiration (> 20 µg C L-1 h-1) and could favor the release of inorganic phosphorus. High extracellular enzyme activities and phosphorus solubilizing bacteria abundance in sediments accelerated nutrient regeneration. In these conditions, the positive GLU-AP relationship suggested the coupling of carbon and phosphorus regeneration; an efficient phosphorus regeneration and high nitrogen levels (up to 0.067 and 0.059 mg L-1 NH4 and NO3 in Italy; 0.631 and 1.496 mg L-1 NH4 and NO3 in China) led to chlorophyll a peaks of 14.9 and 258.4 µg L-1 in Italy and China, respectively, and a typical algal composition. Conversely, in the oligo-mesotrophic lakes, very low nitrogen levels (in Italy, 0.001 and 0.005 mg L-1 NH4 and NO3, respectively, versus 0.053 and 0.371 mg L-1 in China) induced high LAP, while low phosphorus (33.6 and 46.3 µg L-1 total P in Italy and China, respectively) led to high AP. In these lakes, nitrogen and phosphorus regeneration were coupled, as shown by positive LAP-AP relationship; however, the nutrient demand could not be completely met without the supply from sediments, due to low enzymatic activity and phosphorus solubilizing bacteria found in this compartment.

Molecular cloning and characterization of leucine aminopeptidase gene from Taenia pisiformis.

Leucine aminopeptidase (LAP, EC: 3.4.11.1) is an important metalloexopeptidase that catalyze the hydrolysis of amino-terminal leucine residues from polypeptides and proteins. In this study, a full length of cDNA encoding leucine aminopeptidase of Taenia pisiformis (TpLAP) was cloned by rapid amplification of cDNA-ends using the polymerase chain reaction (RACE-PCR) method. The full-length cDNA of the TpLAP gene is 1823 bp and contains a 1569 bp ORF encoding 533 amino acids with a putative mass of 56.4 kDa. TpLAP contains two characteristic motifs of the M17LAP family in the C-terminal sequence: the metal binding site 265-[VGKG]-271 and the catalytic domain motif 351-[NTDAEGRL]-357. The soluble GST-TpLAP protein was expressed in Escherichia coli Transetta (DE3) and four specific anti-TpLAP monoclonal antibodies (mAbs) were prepared. In enzymatic assays, the optimal activity was observed at pH 9.5 at 45 °C. GST-TpLAP displayed a hydrolyzing activity for the Leu-pNA substrate with a maximum activity of 46 U/ml. The enzymatic activity was significantly enhanced by Mn2+ and completely inhibited by 20 nM bestatin and 0.15 mM EDTA. The native TpLAP was detected specifically in ES components of adult T. pisiformis by western blotting using anti-TpLAP mAb as a probe. Quantitative real-time PCR revealed that the TpLAP gene was expressed at a high level in adult worm tissues, especially in the gravid proglottids (50.71-fold). Immunolocalization analysis showed that TpLAP was located primarily in the subtegumental parenchyma zone and the uterine wall of adult worms. Our results indicate that TpLAP is a new member of the M17LAP family and can be considered as a stage-differentially expressed protein. These findings might provide new insights into the study of the mechanisms of growth, development and survival of T. pisiformis in the final host and have potential value as an attractive target for drug therapy or vaccine intervention.

In silico screening of novel inhibitors of M17 Leucine Amino Peptidase (LAP) of Plasmodium vivax as therapeutic candidate.

M17 LAP (Leucine Amino Peptidase) plays an important role in the hydrolysis of amino acids essential for growth and development of Plasmodium vivax (Pv), the pathogen causing malaria. In this paper a homology model of PvLAP was generated using MODELLER v9.15. From different in-silico methods such as structure based, ligand based and de novo drug designing a total of 90 compounds were selected for docking studies. A final list of 10 compounds was prepared. The study reported the identification of 2-[(3-azaniumyl-2-hydroxy-4-phenylbutanoyl) amino]-4-methylpentanoate as the best inhibitor in terms of docking score and pharmacophoric features. The reliability of the binding mode of the inhibitor is confirmed by molecular dynamics (MD) simulation study with GROMACS software for a simulation time of 20ns in water environment. Finally, in silico ADMET analysis of the inhibitors using MedChem Designer v3 evaluated the drug likeness of the best hits to be considered for industrial pharmaceutical research.

Molecular Characterization of Babesia bovis M17 Leucine Aminopeptidase and Inhibition of Babesia Growth by Bestatin.

The M17 leucine aminopeptidase (M17LAP) enzymes of the other apicomplexan parasites have been characterized and shown to be inhibited by bestatin. Though Babesia bovis also belongs to the apicomplexan group, it is not known whether its M17LAP could display similar biochemical properties as well as inhibition profile. To unravel this uncertainty, a B. bovis M17LAP (BbM17LAP) gene was expressed in Escherichia coli , and activity of the recombinant enzyme as well as its inhibition by bestatin were evaluated. The inhibitory effect of the compound on growths of B. bovis and Babesia gibsoni in vitro was also determined. The expression of the gene fused with glutathione S-transferase (GST) yielded approximately 81-kDa recombinant BbM17LAP (rBbM17LAP). On probing with mouse anti-rBbM17LAP serum, a green fluorescence was observed on the parasite cytosol on confocal laser microscopy, and a specific band greater than the predicted molecular mass was seen on Western blotting. The Km and Vmax values of the recombinant enzyme were 139.3 ± 30.25 and 64.83 ± 4.6 µM, respectively, while the Ki was 2210 ± 358 µM after the inhibition. Bestatin was a more potent inhibitor of the growth of B. bovis [IC50 (50% inhibition concentration) = 131.7 ± 51.43 µM] than B. gibsoni [IC50 = 460.8 ± 114.45 µM] in vitro. The modest inhibition of both the rBbM17LAP activity and Babesia parasites' growth in vitro suggests that this inhibition may involve the endogenous enzyme in live parasites. Therefore, BbM17LAP may be a target of bestatin, though more studies with other aminopeptidase inhibitors are required to confirm this.

Purification and characterisation of a glutamic acid-containing peptide with calcium-binding capacity from whey protein hydrolysate.

The bioavailability of dietary ionised calcium is affected by intestinal basic environment. Calcium-binding peptides can form complexes with calcium to improve its absorption and bioavailability. The aim of this study was focused on isolation and characterisation of a calcium-binding peptide from whey protein hydrolysates. Whey protein was hydrolysed using Flavourzyme and Protamex with substrate to enzyme ratio of 25:1 (w/w) at 49 °C for 7 h. The calcium-binding peptide was isolated by DEAE anion-exchange chromatography, Sephadex G-25 gel filtration and reversed phase high-performance liquid chromatography (RP-HPLC). A purified peptide of molecular mass 204 Da with strong calcium binding ability was identified on chromatography/electrospray ionisation (LC/ESI) tandem mass spectrum to be Glu-Gly (EG) after analysis and alignment in database. The calcium binding capacity of EG reached 67·81 µg/mg, and the amount increased by 95% compared with whey protein hydrolysate complex. The UV and infrared spectrometer analysis demonstrated that the principal sites of calcium-binding corresponded to the carboxyl groups and carbonyl groups of glutamic acid. In addition, the amino group and peptide amino are also the related groups in the interaction between EG and calcium ion. Meanwhile, the sequestered calcium percentage experiment has proved that EG-Ca is significantly more stable than CaCl2 in human gastrointestinal tract in vitro. The findings suggest that the purified dipeptide has the potential to be used as ion-binding ingredient in dietary supplements.

Histochemical distribution of intestinal enzymes of juvenile pacu (Piaractus mesopotamicus) fed lyophilized bovine colostrum.

Enzyme activity was evaluated in the intestine of juvenile pacu, Piaractus mesopotamicus, fed diets containing 0, 10 or 20 % of lyophilized bovine colostrum (LBC) inclusion for either 30 or 60 days. The enzymes intestinal acid and alkaline phosphatase (ACP and ALP, respectively), nonspecific esterase (NSE), lipase (LIP), dipeptidyl aminopeptidase IV (DAP IV) and leucine aminopeptidase (LAP) were studied using histochemistry in four intestinal segments (S1, S2, S3 and rectum). Moderate activity of the DAP IV was detected in the three last intestinal segments, but no differences among the treatments were detected. Enzymes LAP, NSE and LIP were weakly stained in all intestinal segments and the inclusion of 10 or 20 % of LBC in the diet commanded a moderate reaction to NSE in the S3 segment at day 60. ACP activity was detected only in the brush border of the S1 segment of fish fed 0 % LBC for either 30 or 60 days. The activity of ALP was very strong in the first intestinal segment, but a weak reaction was seen in the last segments. The inclusion of 20 % of LBC changed the pattern of staining to the ALP, eliciting moderate staining in S2 at day 30 and S1 at day 60. The consumption of diets containing LBC by juvenile pacu did not have significant implications in intestinal enzymatic activity, which still was not fully stimulated.

[Effects of applying different kind fertilizers on enzyme activities related to carbon, nitrogen, and phosphorus cycles in reddish paddy soil].

Based on the long-term fixed position experimental data from Qianyanzhou Ecological Experiment Station, Chinese Academy of Sciences in 1998, this paper analyzed the effects of applying different kind fertilizers (straw, ST; pig manure, OM; and chemical fertilizer, NPK) on the nutrients (C, N, and P) status and the activities of related enzymes ( beta-1,4-glucosidase, betaG; beta-1,4-N-acetylglucosaminidase, NAG; L-leucine aminopeptidase, LAP; and acid phosphatase, AP) in reddish paddy soil. With the application of OM, the activities of soil betaG, NAG, and LAP increased significantly, as compared with other treatments, and were 1.4, 2. 6, and 1.9 times higher than the control (CK) , respectively. Applying OM also improved the ratio of soil organic carbon to total nitrogen (C/N), but decreased the soil betaG/(NAG+LAP) ratio, suggesting that pig manure could benefit the degradation of soil cellulose and the accumulation of soil organic carbon. Applying NPK increased the activities of soil betaG, NAG, and LAP, but decreased the AP activity, with a decrement of 34% as compared with CK. Under the application of NPK, the soilbetaG/AP and (NAG+ LAP)/AP ratios increased, but the ratios of soil organic carbon to total phosphorus (C/P) and of soil total nitrogen to total phosphorus (N/P) decreased, indicating that chemical fertilizers could induce the accumulation of soil inorganic phosphorus, and inhibit the microbial functions of degrading polysaccharides and phosphate phospholipids.

Dietary flaxseed oil supplementation ameliorates the effect of cisplatin on brush border membrane enzymes and antioxidant system in rat intestine.

Cisplatin (CP; cis-diamminedichloroplatinum II) is a drug widely used against different types of solid tumors. Patients receiving CP, however, experience very profound and long lasting gastrointestinal symptoms. Recently, ω-3 polyunsaturated fatty acid-enriched flaxseed/flaxseed oil (FXO) has shown numerous health benefits. The present study was undertaken to investigate whether FXO can prevent CP-induced adverse biochemical changes in the small intestine of rats. A single intraperitoneal dose of CP (6 mg/kg body weight) was administered to male Wistar rats fed with control diet (CP group) and FXO diet (CPFXO group). Administration of CP led to a significant decline in the specific activities of brush border membrane enzymes both in the mucosal homogenates and in the isolated membrane vesicles. Lipid peroxidation and total sulfhydryl groups were altered upon CP treatment, indicating the generation of oxidative stress. The activities of SOD, catalase and glutathione peroxidase also decreased in CP-treated rats. In contrast, dietary supplementation of FXO prior to and following CP treatment significantly attenuated the CP-induced changes in all these parameters. FXO feeding markedly enhanced resistance to CP-elicited adverse gastrointestinal effects. The results suggest that FXO owing to its intrinsic biochemical/antioxidant properties is an effective agent in reducing the adverse effects of CP on intestine.

Monitoring stepwise proteolytic degradation of peptides by supramolecular domino tandem assays and mass spectrometry for trypsin and leucine aminopeptidase.

A label-free optical detection method has been designed that allows direct monitoring of enzymatic peptide digestion in vitro. The method is based on the addition of a reporter pair, composed of the macrocyclic host cucurbit[7]uril (CB7) and the fluorescent dye acridine orange (AO), to detect the proteolytic degradation of peptides. The enzymatic activity of trypsin and leucine aminopeptidase (LAP) was investigated using H-LSRFSWGA-OH as a substrate. The substrate as well as the intermediary and final products (i.e., H-FSWGA-OH and phenylalanine) formed during its enzymatic hydrolysis differ in their binding affinity to the receptor CB7, which results in varying degrees of dye displacement and, therefore, different fluorescence intensities. CB7 showed a relatively weak binding constant of K approximately 10(4) M(-1) with the substrate, a relatively strong binding constant of K > or = 10(6) M(-1) with H-FSWGA-OH (which is a final product formed by trypsin digestion and the intermediary product formed during the enzymatic activity of LAP), and a moderate binding constant of K < or = 10(5) M(-1) with phenylalanine. Owing to this differential binding affinity of CB7 with the substrate and the corresponding products, the digestion of a peptide by trypsin was followed as a decrease in fluorescence signal, while the complete degradation of the peptide by LAP was monitored as a decrease and a subsequent increase in fluorescence signal. The k(cat)/K(M) value for trypsin (2.0 x 10(7) min(-1) M(-1)) was derived from the change in fluorescence signal with time. Additionally, the complete degradation of the peptide by LAP was also followed by mass spectrometry. The use of a supramolecular sensing ensemble (macrocyclic host and dye) as a fluorescent reporter pair gives this method the flexibility to adapt for monitoring the stepwise degradation of different biologically relevant peptides by other proteases.

Interaction of soyasaponins with plant ingredients in diets for Atlantic salmon, Salmo salar L.

The effects of combining soyasaponins with plant ingredients on intestinal function and fish health were investigated in an 80 d study with Atlantic salmon (270 g) distributed thirty each into twenty-four tanks with seawater. Soyasaponins were supplemented (2 g/kg) to diets with maize gluten (MG), pea protein concentrate (PPC) and sunflower (SFM), rapeseed (RSM) or horsebean meals. A diet with soyabean meal (SBM) and another with wheat gluten and soyasaponins served as reference diets. Marked soyasaponin effects were observed when combined with PPC. This combination induced inflammation in the distal intestine (DI) similar to SBM, reduced feed intake, apparent digestibility of lipid, most amino acids and ash, decreased bile salt levels in intestinal chyme and decreased leucine aminopeptidase (LAP) activity but increased trypsin activity in the DI. No enteritis was observed in other diet groups, but small consistent negative soyasaponin effects were seen on lipid and fatty acid digestibility, faecal DM and LAP activity of the DI. Soyasaponin combination with RSM reduced digestibility of all nutrients including minerals. The mineral effect was also seen for SFM, whereas with MG and SFM a positive soyasaponin effect on feed intake was observed. Caution should be exercised to avoid ingredient combinations giving high saponin levels, a condition that appears to be a key factor in diet-induced enteritis together with certain plant ingredients.

Up-regulation of leucine aminopeptidase-A in cadmium-treated tomato roots.

The effects of cadmium (Cd) on aminopeptidase (AP) activities and Leucine-AP (LAP) expression were investigated in the roots of tomato (Solanum lycopersicum L., var Ibiza) plants. Three-week-old plants were grown for 10 days in the presence of 0.3-300 µM Cd and compared to control plants grown in the absence of Cd. AP activities were measured using six different p-nitroanilide (p-NA) substrates. Leu, Met, Arg, Pro and Lys hydrolyzing activities increased in roots of Cd-treated plants, while Phe-pNA cleavage was not enhanced after Cd treatments. The use of peptidase inhibitors showed that most of the Leu-pNA hydrolyzing activity was related to one or several metallo-APs. Changes in Lap transcripts, protein and activities were measured in the roots of 0 and 30-µM Cd-treated plants. LapA transcript levels increased in Cd-treated roots, whereas LapN RNAs levels were not modified. To assess amount of Leu-pNA hydrolyzing activity associated with the hexameric LAPs, LAP activity was measured following immunoprecipitation with a LAP polyclonal antiserum. LAP activity increased in Cd-treated roots. There was a corresponding increase in LAP-A protein levels detected in 2D-immunoblots. The role of LAP-A in the proteolytic response to Cd stress is discussed.

Evaluation of the marine alga Ulva rigida as a food supplement: effect of intake on intestinal, hepatic, and renal enzyme activities in rats.

The use of seaweeds as a food is more widespread in Eastern than in Western countries, although demand for these plants has increased in the West because their possible usefulness as dietary supplements. However, very little is known about the effects of regular consumption of algae. The aim of the present study was to determine the composition of Ulva rigida and to evaluate the effects of dietary supplementation of the diet with 10% alga for 4 weeks on dietary intake, growth, protein efficiency ratio, diet conversion ratio, and some organ weights in growing male rats. We also studied the effect of inclusion of the alga in the diet on intestinal, hepatic, and renal enzymatic activities. U. rigida was found to be a good source of protein and carbohydrates. Food intake was higher in the U. rigida group than in the control group, but ingestion of alga did not have any effect on the other trophic parameters. The intestinal disaccharidase and leucine aminopeptidase activities were lower in rats fed with alga than in control rats, but γ-glutamyl transpeptidase activity was higher in the kidneys of alga-fed rats than in control rats. U. rigida contains high amounts of protein, carbohydrates, vitamins, and minerals and low amounts of lipids. Analysis of the amino acid composition revealed good-quality protein. The addition of alga to the diet inhibited disaccharidase activities, which suggested that alga consumption could be useful in some chronic disorders associated with pertubations of glucose homeostasis caused by carbohydrate absorption.

Leucine aminopeptidase, beta-glucosidase and alkaline phosphatase activity rates and their significance in nutrient cycles in some coastal Mediterranean sites.

In aquatic microbial ecology, knowledge of the processes involved in the turnover of organic matter is of utmost importance to understand ecosystem functioning. Microorganisms are major players in the cycling of nutrients (nitrogen, phosphorus) and carbon, thanks to their enzymatic activities (leucine aminopeptidase, LAP, alkaline phosphatase, AP, and beta-glucosidase, beta-GLU) on organic polymers (proteins, organic phosphates and polysaccharides, respectively). Estimates of the decomposition rates of organic polymers are performed using fluorogenic compounds, whose hydrolysis rate allow us to obtain information on the "potential" metabolic activity of the prokaryotic community. This paper refers the enzyme patterns measured during recent oceanographic cruises performed in some coastal Mediterranean sites, not yet fully investigated in terms of microbial biogeochemical processes. Mean enzyme activity rates ranged from 5.24 to 5558.1 nM/h, from 12.68 to 244.73 nM/h and from 0.006 to 9.51 nM/h for LAP, AP and beta-GLU, respectively. The highest LAP and AP activity rates were measured in the Gulf of Milazzo (Tyrrhenian Sea) and in the Straits of Messina, in association with the lowest bacterioplankton abundance; in contrast, the lowest ones were found in the northern Adriatic Sea. beta-GLU was more active in the Straits of Messina. Activity rates were analysed in relation to the main environmental variables. Along the northern Adriatic coastal side affected by the Po river, significant inverse relationships linked LAP and AP with salinity, pointing out that fluvial inputs provided organic substrates for microbial metabolism. Both in the Gulf of Manfredonia and in the Straits of Messina, LAP and AP levels were inversely related with the concentration of nitrate and inorganic phosphorus, respectively. In the Gulf of Milazzo, high cell-specific AP measured in spite of phosphorus availability suggested the role of this enzyme not only in phosphorus, but also in carbon release.

Extracellular proteases and possible disease related virulence mechanisms of two marine bacteria implicated in an opportunistic bacterial infection of Nephrops norvegicus.

The extracellular products (ECP) secreted by two strains of gram-negative bacteria isolated from Nephrops norvegicus exhibiting signs of an opportunistic bacterial infection were investigated with the objective of understanding their role in the spoilage of host muscle tissue and identifying disease related virulence mechanisms. ECP from Vibrio sp. demonstrated no proteolytic activity. ECP from Pseudoalteromonas sp. (isolate N10) degraded several substrates, including azocasein and host muscle tissue. Proteolytic activity increased with temperature. Substrate-impregnated sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the effect of the isolates' ECP on the molecular weight of proteins derived from abdominal muscle tissue revealed that the ECP of Pseudoalteromonas sp. selectively degraded the myosin heavy chain, troponin-T, troponin-I, paramyosin and several unidentified muscle proteins approximately 110 kDa in size. Topomyosin was also reduced in quantity. Degradation of SDS-PAGE gels impregnated with host muscle proteins, by the ECP of Pseudoalteromonas sp. revealed 3 zones of proteolysis, with estimated molecular weights between 100 and 30 kDa, indicating multiple proteases in the ECP. Through the API ZYM system, both isolates demonstrated strong leucine arylamidase activity, with the Vibrio sp. showing strong acid phosphatase activity. These enzymes have been identified as disease related virulence mechanisms in other bacterial pathogens. There is likely a complex pathway to the final condition, involving virulence factors of other species and the stresses involved in capture and transport.

Identification and characterization of Paragonimus westermani leucine aminopeptidase.

Paragonimus westermani is a tissue-invading trematode parasite that causes inflammatory lung disease as well as systemic infections including cerebral invasion in carnivorous mammals. While aminopeptidases play important roles in trematodes in the catabolism of host hemoglobin, an essential source of nutrient for the parasite, little is known about aminopeptidase in Paragonimus. Presently, we isolated a cDNA encoding a 58 kDa P. westermani leucine aminopeptidase (PwLAP). Deduced amino acid sequence of PwLAP exhibited significant sequence homology with LAP from Schistosoma spp. and Fasciola hepatica. Biochemical analysis of the recombinant PwLAP protein demonstrated preferential substrate specificity for Leu-NHMec and inhibition by EDTA, 1,10-phenanthroline, and bestatin, which are conserved characteristics of the M17 family of leucine aminopeptidase. PwLAP exhibited relatively higher enzyme activity in the presence of Mn2+ compared to Schistosoma mansoni LAP. Based on the biochemical properties and immunohistochemical analysis, PwLAP is concluded to represent a leucine aminopeptidase. The enzyme is most likely responsible for the catabolism of host hemoglobin, and, hence, represents a potential target of Paragonimus chemotherapy.

Effects of different dietary levels of fish protein hydrolysates on growth, digestive enzymes, gut microbiota, and resistance to Vibrio anguillarum in European sea bass (Dicentrarchus labrax) larvae.

Two fish protein hydrolysates (FPH) were incorporated into four diets prepared for start-feeding sea bass larvae, at two different levels (10% and 19% of total ingredients): a commercial FPH, CPSP, in which the molecular mass of the main fraction of soluble peptides (51%) was between 500-2500 Da, and an experimental FPH obtained by acidic silage of sardine offal, SH, with a main portion of soluble peptides (54%) ranging from 200 to 500 Da. The diet with 10% of the commercial FPH gave the best results in terms of growth, survival and intestinal development, as evaluated by the early activity of digestive enzymes in the brush border membrane (alkaline phosphatase and aminopeptidase N). This was related to the low level of Vibrio spp. counted in the larvae of group C10. The high dose of FPH, especially in the experimental preparation rich in short peptides, seemed to favour the dominance of Vibrio sp. TYH3, which behaved opportunistically. The effect of the experimental FPH was ambiguous, since early larvae challenged with Vibrio anguillarum were more resistant to the pathogen, especially at high FPH dose (group S19). This might be due either to direct antagonism between V. anguillarum and Vibrio sp. TYH3, or to the stimulation of the immune response in the larvae. These results indicate that different molecular weight fractions and concentrations of feed-soluble peptides may affect the growth performance and immunological status of sea bass larvae. Consequently, a low dose of commercial FPH seems advisable, both for larval development and for the bacterial environment, although further research is required to determine and characterize peptide fractions that may have a beneficial effect on growth and immune response, and to determine their optimal inclusion levels in diets for sea bass larvae.

Microbial community dynamics during assays of harbour oil spill bioremediation: a microscale simulation study.

AIMS: Microcosm experiments simulating an oil spill event were performed to evaluate the response of the natural microbial community structure of Messina harbour seawater following the accidental load of petroleum. METHODS AND RESULTS: An experimental harbour seawater microcosm, supplemented with nutrients and crude oil, was monitored above 15 days in comparison with unpolluted ones (control microcosms). Bacterial cells were counted with a Live/Dead BacLight viability kit; leucine aminopeptidase, beta-glucosidase, alkaline phosphatase, lipase and esterase enzymes were measured using fluorogenic substrates. The microbial community dynamic was monitored by isolation of total RNA, RT-PCR amplification of 16S rRNA, cloning and sequencing. Oil addition stimulated an increase of the total bacterial abundance, leucine aminopeptidase and phosphatase activity rates, as well as a change in the community structure. This suggested a prompt response of micro-organisms to the load of petroleum hydrocarbons. CONCLUSIONS: The present study on the viability, specific composition and metabolic characteristics of the microbial community allows a more precise assessment of oil pollution. Both structural and functional parameters offer interesting perspectives as indicators to monitor changes caused by petroleum hydrocarbons. SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of microbial structural successions at oil-polluted sites is essential for environmental bioremediation. Data obtained in microcosm studies improve our understanding of natural processes occurring during oil spills.