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.

A promising new target to control fasciolosis: Fasciola hepatica leucine aminopeptidase 2.

Parasite M17 leucine aminopeptidases (LAPs) have been associated with critical roles in different key functions such as the nutrition, migration, and invasion of the natural host. Native or recombinant LAP used as a vaccine antigen has proved effective to elicit protection against Fasciola hepatica infection in sheep, pointing to potential vaccine candidates against fascioliasis in ruminant species. Previously, the FhLAP1, abundantly secreted in vitro by the mature adult parasite was used as a vaccine antigen obtaining promising protection results against F. hepatica challenge in small ruminants. Here, we report the biochemical characterization of a second recombinant LAP (FhLAP2) associated with the juvenile stage of F. hepatica. FhLAP2 showed aminopeptidase activity using different synthetic substrates, including leucine, arginine, and methionine and was increased in the presence of Mn+ 2 and Mg+ 2. The activity was inhibited by bestatin, 1,10-phenanthroline, and EDTA, specific inhibitors of aminopeptidase and/or metalloproteases. Finally, the recombinant FhLAP2 functional form was tested in combination with Freund's incomplete adjuvant in an immunization trial in mice followed by an experimental challenge with F. hepatica metacercariae. The immunization with FhLAP2/FIA resulted in a significant reduction of parasite recovery compared to control groups. The immunized group elicited total specific IgG and subclasses IgG1 and IgG2 antibody responses. This study highlights the potential of a new candidate vaccine formulation with potential applications in natural ruminant hosts, especially those targeting the juvenile stage.

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.

Soil enzyme activity and stoichiometry in secondary grasslands along a climatic gradient of subtropical China.

Soil extracellular enzymes plays key roles in ecosystem carbon (C), nitrogen (N), and phosphorus (P) cycling, and are very sensitive to climatic, plant, and edaphic factors. However, the interactive effects of these factors on soil enzyme activities at large spatial scales remain unclear. Here, we investigated the spatial pattern of the activities of five soil hydrolyzing enzymes [ß-D-cellobiohydrolase (CB), ß-1,4-glucosidase (BG), ß-1,4-N-acetyl-glucosaminidase (NAG), L-leucine aminopeptidase (LAP), and acid phosphatase (AP)], and their C:N:P acquisition ratios in relation to plant inputs and edaphic properties across a 600-km climatic gradient in secondary grasslands of subtropical China. The activities of CB, BG, and NAG decreased while that of LAP increased with the increasing mean annual temperature (MAT). The activities of all enzymes did not significantly vary with the mean annual precipitation (MAP). We found that the activities of BG, NAG, and AP were predominately dependent on plant N contents, while the soil LAP activity was tightly related to soil recalcitrant C and N contents. In contrast, the ecoenzymatic C:nutrient (N and P) acquisition ratios increased with increasing MAP and decreasing MAT, primarily due to the increase in plant input at warmer and wetter sites. In addition to climates, plant C inputs, C use efficiency, soil pH, soil organic C, soil C:P, and N:P ratios explained 79% and 72% of the overall variation in ecoenzymatic C:nutrient and P:N acquisition ratios, respectively. The pattern of ecoenzymatic C:N:P acquisition ratios also revealed unexpected N limitation in subtropical grasslands. Overall, our study highlighted the importance of climate in controlling soil biological C, N, and P acquisition activities through its direct and indirect effects on plant inputs and soil edaphic factors, thereby providing useful information for better understanding and predictions of soil C and nutrient cycling in grassland ecosystems at regional scales.

Effect of fish paste products, fish balls 'tsumire', intake in Sprague-Dawley rats.

The fish paste product, fish balls 'tsumire', is a traditional type of Japanese food made from minced fish as well as imitation crab, kamaboko and hanpen. Although tsumire is known as a high-protein and low-fat food, there is a lack of scientific evidence on its health benefits. Hence, we aimed to investigate the effects of tsumire intake on organ weight and biomarker levels in Sprague-Dawley rats for 84 d as a preliminary study. Six-week-old male Sprague-Dawley rats were divided into two groups: group I, fed normal diets, and group II, fed normal diets with 5 % dried tsumire. Throughout the administration period, we monitored their body weight and food intake; at the end of this period, we measured their organ weight and analysed their blood biochemistry. No significant differences were observed with respect to body weight, food intake, organ weight and many biochemical parameters between the two groups. It was found that inorganic phosphorus and glucose levels were higher in group II rats than in group I rats. On the other hand, sodium, calcium, amylase and cholinesterase levels were significantly lower in group II than in group I. Interestingly, we found that the levels of aspartate aminotransferase, alanine transaminase, lactate dehydrogenase and leucine aminopeptidase in group II were significantly lower than in group I, and that other liver function parameters of group II tended to be lower than in group I. In conclusion, we consider that the Japanese traditional food, 'tsumire,' may be effective as a functional food for human health management worldwide.

Partial protection with a chimeric tetraspanin-leucine aminopeptidase subunit vaccine against Opisthorchis viverrini infection in hamsters.

Opisthorchiasis is a serious public health problem in East Asia and Europe. The pathology involves hepatobiliary abnormalities such as cholangitis, choledocholithiasis and tissue fibrosis that can develop into cholangiocarcinoma. Prevention of infection is difficult as multiple social and behavioral factors are involved, thus, progress on a prophylactic vaccine against opisthorchiasis is urgently needed. Opisthorchis viverrini tetraspanin-2 (Ov-TSP-2) was previously described as a potential vaccine candidate conferring partial protection against O. viverrini infections in hamsters. In this study, we generated a recombinant chimeric form of the large extracellular loop of Ov-TSP-2 and O. viverrini leucine aminopeptidase, designated rOv-TSP-2-LAP. Hamsters were vaccinated with 100 and 200 µg of rOv-TSP-2-LAP formulated with alum-CpG adjuvant via intraperitoneal injection and evaluated the level of protection against O. viverrini infection. Our results demonstrated that the number of worms recovered from hamsters vaccinated with either 100 or 200 µg of rOv-TSP-2-LAP were significantly reduced by 27% compared to the adjuvant control group. Furthermore, the average length of worms recovered from animals vaccinated with 200 µg of rOv-TSP-2-LAP was significantly shorter than those from the control adjuvant group. Immunized hamsters showed significantly increased serum levels of anti-rOv-TSP-2 IgG and IgG1 compared to adjuvant control group, suggesting that rOv-TSP-2-LAP vaccination induces a mixed Th1/Th2 immune response in hamsters. Therefore, the development of a suitable vaccine against opisthorchiasis requires further work involving new vaccine technologies to improve immunogenicity and protective efficacy.

Polymorphisms of LAP3 gene and their association with the growth traits in the razor clam Sinonovacula constricta.

Leucine aminopeptidase 3 (LAP3) is an important proteolytic enzyme that catalyzes the hydrolysis of leucine residues from the amino termini of protein or peptide substrates and plays a critical role in protein metabolism and growth. In the present study, we investigated the full-length cDNA sequence of the LAP3 gene in Sinonovacula constricta (ScLAP3) using expressed sequence tags and rapid amplification of cDNA ends. The full-length ScLAP3 cDNA was 2885 bp, with a 1560 bp open reading frame encoding 519 amino acids. Sequence analysis revealed that ScLAP3 shared 70.9% identity with LAP3 from the blood clam Tegillarca granosa and 62.0-68.0% with other species. ScLAP3 was expressed in all six tested tissues, with significantly higher expression levels in the foot compared with mantle, adductor muscle, liver, gills, and siphon tissues in adults (P < 0.01). In the eight developmental stages, ScLAP3 expression gradually increased, with significantly higher levels in D-shaped larvae compared with other developmental stages (P < 0.01), suggesting that it may be involved in the formation of certain organs during early development. Association analysis identified three shared single nucleotide polymorphisms (SNPs), c.1073A > G, c.1139C > T and c.1154A > G in exons of ScLAP3 gene from 177 individuals of two groups, one selective strain and one wild population, which had significant effects on growth traits of S. constricta. The results provided candidate genetic markers to assist selective breeding of razor clams toward improved growth.

Moderate protection is induced by a chimeric protein composed of leucine aminopeptidase and cathepsin L1 against Fasciola hepatica challenge in sheep.

Leucine aminopeptidase (FhLAP) and cathepsin L1 (FhCL1) of Fasciola hepatica play a critical role in parasite feeding, migration through host tissue, and immune evasion. These antigens have been tested for immune protection as single components with variable degrees of success. The chimeric-protein approach could improve protection levels against fasciolosis. Previously, we reported the design and construction of a chimeric protein composed of antigenic sequences of FhLAP and FhCL1 of F. hepatica. The goal of the present study was to express and evaluate the immune-protective capacity of this chimeric protein (rFhLAP-CL1) in sheep. Animals were randomly allocated into five groups with five animals in each group. Groups 1, 2 and 3 were immunized twice with 100 µg, 200 µg and 400 µg of rFhLAP-CL1 emulsified with Quil A adjuvant, whereas groups 4 and 5 were the adjuvant control and infection control groups, respectively. The animals were then challenged with 200 metacercariae two weeks after the rFhLAP-CL1 booster. The fluke burden was reduced by 25.5%, 30.7% (p < 0.05) and 46.5% (p < 0.01) in sheep immunized with 100 µg, 200 µg and 400 µg of chimeric protein, respectively, in comparison to the infection control group. There was a reduction of 22.7% (p < 0.05) and 24.4% (p < 0.01) in fecal egg count in groups 2 and 3, respectively, compared to the infection control group. Sheep immunized with chimeric protein produced F. hepatica excretion-secretion product-specific total IgG antibody, which were increased after challenge. Moreover, the levels of rFhLAP-CL1-specific IgG1 and IgG2 isotypes in immunized sheep increased rapidly two weeks after the first immunization and were significantly more elevated than those of the control groups, indicating a mixed Th1/Th2 response. This is a preliminary evaluation of the chimeric protein rFhLAP-CL1 as a possible immunogen against F. hepatica infection in sheep.

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.

Effects of a novel method for enteral nutrition infusion involving a viscosity-regulating pectin solution: A multicenter randomized controlled trial.

BACKGROUND & AIMS: The initial complications associated with infusion of enteral nutrition (EN) for clinical and nutritional care are vomiting, aspiration pneumonia, and diarrhea. There are many recommendations to prevent these complications. A novel method involving a viscosity-regulating pectin solution has been demonstrated. In Japan, this method along with the other so-called "semi-solid EN" approaches has been widely used in practice. However, there has been no randomized clinical trial to prove the efficiency and safety of a viscosity-regulating pectin solution in EN management. Therefore, we planned and initiated a multicenter randomized controlled trial to determine the efficiency and safety. METHODS: This study included 34 patients from 7 medical institutions who participated. Institutional review board (IRB) approval was obtained from all participating institutions. Patients who required EN management were enrolled and randomly assigned to the viscosity regulation of enteral feeding (VREF) group and control group. The VREF group (n = 15) was managed with the addition of a viscosity-regulating pectin solution. The control group (n = 12) was managed with conventional EN administration, usually in a gradual step-up method. Daily clinical symptoms of pneumonia, fever, vomiting, and diarrhea; defecation frequency; and stool form were observed in the 2 week trial period. The dose of EN and duration of infusion were also examined. RESULTS: A favorable trend for clinical symptoms was noticed in the VREF group. No significant differences were observed in episodes of pneumonia, fever, vomiting, and diarrhea between the 2 groups. An apparent reduction in infusion duration and hardening of stool form were noted in the VREF group. CONCLUSIONS: The novel method involving a viscosity-regulating pectin solution with EN administration can be clinically performed safely and efficiently, similar to the conventional method. Moreover, there were benefits, such as improvement in stool form, a short time for EN infusion, and a reduction in vomiting episodes, with the use of the novel method. This indicates some potential advantages in the quality of life among patients receiving this novel method.

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.

Diversity evaluation based on morphological, physiological and isozyme variation in genetic resources of garlic (Allium sativum L.) collected worldwide.

The aim of this study was to obtain primary information about the global diversity of garlic (Allium sativum L.) by evaluating morphological, physiological and isozyme variation. A total of 107 garlic accessions collected worldwide were grown in Yamaguchi, Japan. Five morphological traits (bulb weight, bulb diameter, number of cloves per bulb, number of bulbils and scape length) and one physiological trait (bolting period) of the collected garlic showed wide variation. Meanwhile, a total of 140 garlic accessions, including the 107 mentioned above, were characterized by leucine aminopeptidase (LAP) and phosphoglucoisomerase (PGI) isozyme analyses; they clearly showed polymorphisms in putative isozyme loci (Lap-1, Lap-2 and Pgi-1). Allelic frequencies were estimated in each group of accessions categorized by their geographical origin, and the observed (Ho) and expected (He) heterozygosities were calculated. The allelic frequencies differed between groups. A principal component analysis based on morpho-physiological data indicated a grouping of the garlic accessions into Central Asian and Northern Mediterranean groups as well as others. We discuss the roles of artificial and natural selection that may have caused differentiation in these traits, on the assumption that ancestral domesticated garlic populations have adapted in various regions using standing variation or mutations that accumulated during expansion, and have evolved along with human-preferred traits over a long history of cultivation.

Effect of Flavourzyme(®) on Angiotensin-Converting Enzyme Inhibitory Peptides Formed in Skim Milk and Whey Protein Concentrate during Fermentation by Lactobacillus helveticus.

Angiotensin-converting enzyme inhibitory (ACE-I) activity as affected by Lactobacillus helveticus strains (881315, 881188, 880474, and 880953), and supplementation with a proteolytic enzyme was studied. Reconstituted skim milk (12% RSM) or whey protein concentrate (4% WPC), with and without Flavourzyme(®) (0.14% w/w), were fermented with 4 different L. helveticus strains at 37 °C for 0, 4, 8, and 12 h. Proteolytic and in vitro ACE-I activities, and growth were significantly affected (P < 0.05) by strains, media, and with enzyme supplementation. RSM supported higher growth and produced higher proteolysis and ACE-I compared to WPC without enzyme supplementation. The strains L. helveticus 881315 and 881188 were able to increase ACE-I to >80% after 8 h of fermentation when combined with Flavourzyme(®) in RSM compared to the same strains without enzyme supplementation. Supplementation of media by Flavourzyme(®) was beneficial in increasing ACE-I peptides in both media. The best media to release more ACE-I peptides was RSM with enzyme supplementation. The L. helveticus 881315 outperformed all strains as indicated by highest proteolytic and ACE-I activities.

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.

Estimation of leucine aminopeptidase and 5-nucleotidase increases alpha-fetoprotein sensitivity in human hepatocellular carcinoma cases.

PURPOSE: To find parameters that can increase alpha-fetoprotein (AFP) sensitivity and so help in accurate diagnosis and rapid management of hepatocullular carcinoma (HCC), as AFP has limited utility of distinguishing HCC from benign hepatic disorders for its high false-positive and false negative rates. MATERIALS AND METHODS: Serum levels of AFP, 5'-nucleotidase enzyme activity (5-NU) and leucine aminopeptidase enzyme (LAP) activity were measured in 40 individuals. RESULTS: LAP and 5'NU were elevated in HCC at p<0.001. Pearson correlation coefficients showed that changes in AFP exhibited positive correlation with both 5'-NU and LAP at (p<0.001). The complementary use of LAP only with AFP resulted in an increase in sensitivity of AFP from 75% to 90% in detecting HCC. The complementary use of both LAP and 5-NU with AFP resulted in an increased sensitivity of AFP in detecting HCC from 75% to 95%. CONCLUSIONS: LAP and 5-FU can be determined in HCC patients in combination with AFP to improve its sensitivity and decrease false negative results.

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.

Alterations in the Helicoverpa armigera midgut digestive physiology after ingestion of pigeon pea inducible leucine aminopeptidase.

Jasmonate inducible plant leucine aminopeptidase (LAP) is proposed to serve as direct defense in the insect midgut. However, exact functions of inducible plant LAPs in the insect midgut remain to be estimated. In the present investigation, we report the direct defensive role of pigeon pea inducible LAP in the midgut of Helicoverpa armigera (Lepidoptera: Noctuidae) and responses of midgut soluble aminopeptidases and serine proteinases upon LAP ingestion. Larval growth and survival was significantly reduced on the diets supplemented with pigeon pea LAP. Aminopeptidase activities in larvae remain unaltered in presence or absence of inducible LAP in the diet. On the contrary, serine proteinase activities were significantly decreased in the larvae reared on pigeon pea LAP containing diet as compared to larvae fed on diet without LAP. Our data suggest that pigeon pea inducible LAP is responsible for the degradation of midgut serine proteinases upon ingestion. Reduction in the aminopeptidase activity with LpNA in the H. armigera larvae was compensated with an induction of aminopeptidase activity with ApNA. Our findings could be helpful to further dissect the roles of plant inducible LAPs in the direct plant defense against herbivory.