[Lucifensin, Chymotrypsin Proteins and Molecular Characterization of Lucilia sericata]. / Lucilia sericata'nin Lusifensin, Kimotripsin Proteinleri ve Moleküler Karakterizasyonlari.

Lucilia sericata, one of the most common species of the Calliphoridae family, is found in large numbers around droppings, garbage and carcasses. This fly species is important in medicine, forensics and veterinary medicine. The larvae of the parasite are important both in veterinary medicine and in combating of the animal diseases, as they cause significant losses in animal production. Since they are one of the first fly colonies to settle on corpses, they can also be used in determining the time of death in the field of forensic medicine. L.sericata larvae used in Maggot debridement treatment (MDT) which is a treatment method with fly larvae, help wound healing by destroying necrotic tissues and infectious agents in wounds. While the larvae protect themselves from polymicrobial flora with the proteins they secrete; at the same time, they make an interesting contribution to wound healing with these molecules secreted. One of the most important molecules discovered in recent years is lucimycin which has an antifungal effect. In addition, lucifensin and chymotrypsin secretions have gained importance in recent years due to their antibacterial effects and especially their effects on resistant gram-negative and positive bacteria. There is a need for the discovery of the molecules that can be alternative in the treatment of non-healing wounds or that can be applied together with existing antibiotics. It is necessary to investigate the antimicrobial characterization of the compounds involved in maggot therapy and their mechanisms. The aim of this study was to clone, molecular characterization and analysis of the antigenic structures of lucifensin and chymotrypsin genes, which are important defensin molecules secreted by L.sericata larvae used in MDT. Primarily, the cultivation of L.sericata colonies to be used in molecular studies were performed. Later, RNA isolation and cDNA synthesis from larvae were carried out. Lucifensin and chymotrypsin genes were individually inserted into the pJet1.2 plasmid by cloning reactions. The presence of the recombinant plasmid was confirmed by PCR screening and DNA sequence analysis methods in all steps. Nucleotide and amino acid based molecular characterizations of these two genes, which are important larval components in wound treatment, have been made. Antigenic regions and three-dimensional structures of the proteins were obtained. The isolate numbered MT495795 of the L.sericata lucifensin gene and the isolate numbered MT495794 of the chymotrypsin gene were registered to GenBank. This data reported for the first time in the Republic of Turkey will contribute to the literature. From the beginning of the 20th century until the discovery of the antibiotics, MDT was applied especially on soldiers but did not find much application area after the discovery of the antibiotics. Drug resistance, which is the most important problem encountered in the treatment of the wounds today, has led to the recall of MDT and its mechanism of action. In this study the data, obtained will constitute a source for the multidisciplinary studies of the scientists from different fields on the discovery and applicability of the important moleculesin the treatment of the wounds.

Colorado potato beetle chymotrypsin genes are differentially regulated in larval midgut in response to the plant defense inducer hexanoic acid or the Bacillus thuringiensis Cry3Aa toxin.

When Colorado potato beetle larvae ingested potato plants treated with the plant defense inducer compound hexanoic acid, midgut chymotrypsin enzyme activity increased, and the corresponding chymotrypsin genes were differentially expressed, evidence of the larval digestive proteolytic system's plasticity. We previously reported increased susceptibility to Cry3Aa toxin in larvae fed hexanoic acid treated plants. Here we show that the most expressed chymotrypsin gene in larvae fed hexanoic acid treated plants, CTR6, was dramatically downregulated in Cry3Aa intoxicated larvae. lde-miR-965-5p and lde-miR-9a-5p microRNAs, predicted to target CTR6, might be involved in regulating the response to hexanoic acid but not to Cry3Aa toxin.

A Jonah-like chymotrypsin from the therapeutic maggot Lucilia sericata plays a role in wound debridement and coagulation.

Lucilia sericata larvae are used in maggot debridement therapy, a traditional wound healing approach that has recently been approved for the treatment of chronic wounds. Maggot excretion products (MEP) contain many different proteases that promote disinfection, debridement and the acceleration of wound healing, e.g. by activating the host contact phase/intrinsic pathway of coagulation. In order to characterise relevant procoagulant proteases, we analysed MEP and identified a chymotrypsin-like serine protease with similarities to Jonah proteases from Drosophila melanogaster and a chymotrypsin from Lucilia cuprina. A recombinant form of the L. sericata Jonah chymotrypsin was produced in Escherichia coli. The activated enzyme (Jonahm) had a pH optimum of 8.0 and a temperature optimum of 37 °C, based on the cleavage of the chromogenic peptide s-7388 and casein. Jonahm reduced the clotting time of human plasma even in the absence of the endogenous protease kallikrein, factor XI or factor XII and digested the extracellular matrix proteins fibronectin, laminin and collagen IV, suggesting a potential mechanism of wound debridement. Based on these characteristics, the novel L. sericata chymotrypsin-like serine protease appears to be an ideal candidate for the development of topical drugs for wound healing applications.

Low-protein diets affect ileal amino acid digestibility and gene expression of digestive enzymes in growing and finishing pigs.

The objective of this study was to evaluate effects of dietary crude protein (CP) intake on ileal amino acid digestibilities and expression of genes for digestive enzymes in growing and finishing pigs. In Experiment 1, 18 growing pigs (average initial BW = 36.5 kg) were assigned randomly into one of three treatments (n = 6/treatment group) representing normal (18 % CP), low (15 % CP), and very low (12 % CP) protein intake. In Experiment 2, 18 finishing pigs (average initial BW = 62.3 kg) were allotted randomly into one of three treatments (n = 6/treatment group), representing normal (16 % CP), low (13 % CP) and very low (10 % CP) protein intake. In both experiments, diets with low and very low CP were supplemented with crystalline amino acids to achieve equal content of standardized ileal digestible Lys, Met, Thr, and Trp, and were provided to pigs ad libitum. Daily feed intake, BW, and feed/gain ratios were determined. At the end of each experiment, all pigs were slaughtered to collect pancreas, small-intestine samples, and terminal ileal chymes. Samples were used for determining expression of genes for digestive enzymes and ileal amino acid digestibilities. Growing pigs fed the 12 % CP and 15 % CP diets had lower final body weight (P < 0.01) and ADG (P < 0.0001) when compared with pigs fed the 18 % dietary CP diet. Growing pigs fed with the 12 % CP diet showed higher digestibilities for CP (P < 0.05), DM (P < 0.05), Lys (P < 0.0001), Met (P < 0.01), Cys (P < 0.01), Thr (P < 0.01), Trp (P < 0.05), Val (P < 0.05), Phe (P < 0.05), Ala (P < 0.05), Cys (P < 0.01), and Gly (P < 0.05) than those fed the 18 % CP diet. Finishing pigs fed the 16 % CP diet had a higher (P < 0.01) final body weight than those fed the 10 % CP diet. mRNA levels for digestive enzymes (trypsinogen, chymotrypsin B, and dipeptidases-II and III) differed among the three groups of pigs (P < 0.05), and no difference was noted in the genes expression between control group and lower CP group. These results indicated that a reduction of dietary CP by a six-percentage value limited the growth performance of growing-finishing pigs and that a low-protein diet supplemented with deficient amino acids could reduce the excretion of nitrogen into the environment without affecting weight gain.

Dietary ß-glucan (MacroGard®) enhances survival of first feeding turbot (Scophthalmus maximus) larvae by altering immunity, metabolism and microbiota.

Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, ß-glucan (MacroGard(®)) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast ß-1,3/1,6-glucan in form of MacroGard(®) at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, microbiota analysis, trypsin activity and size measurements. Along with the feeding of ß-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard(®) fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by ß-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-α and il-1ß was observed. We conclude that the administration of MacroGard(®) induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot.

Identification and characterization of the related immune-enhancing proteins in crab Scylla paramamosain stimulated with rhubarb polysaccharides.

Recently, considerable interest has been focused on immunostimulants to reduce diseases in crab aquaculture. However, information regarding to the related immune-enhancing proteins in crabs is not available yet. In this study, rhubarb polysaccharides were tested for enhancement of the immune activity in crab Scylla paramamosain. Compared with those in the control group, values of, phenoloxidase (PO), alkaline phosphatase (AKP) and alkaline phosphatasein (ACP) activity in the, experimental group were improved significantly 4 d after the treatment. Furthermore, 15 and 17 altered proteins from haemocytes and hepatopancreas, respectively, were found in rhubarb polysaccharide-treated crabs using 2-DE approach. Of these, hemocyanin, chymotrypsin, cryptocyanin, C-type lectin receptor, and ferritin protein were identified by mass spectrometry. In addition, RT-PCR, analysis showed that the mRNA levels of hemocyanin and chymotrypsin increased about 2.4- and 1.4-fold in the experiment group. Moreover, the hemocyanin gene in S. paramamosain (SpHMC) was, cloned and characterized. SpHMC contains one open reading frame of 2022 bp and encodes a polypeptide of 673 amino acids. It is clustered into one branch along with crab hemocyanin in a phylogenetic tree. The mRNA transcripts of SpHMC were detected mainly in the tissues of, hepatopancreas, hemocyte and intestines, and its levels were up-regulated significantly in hemocytes, of S. paramamosain treated with Vibrio parahemolyticus, Beta streptococcus or poly I:C for 6-48 h. Taken together, these studies found 5 related immune-enhancing proteins and a novel heomcyanin homologue with potential pathogen-resistant activities in crab.

The impact of ingested potato type II inhibitors on the production of the major serine proteases in the gut of Helicoverpa armigera.

The flowers of the ornamental tobacco produce high levels of a series of 6 kDa serine protease inhibitors (NaPIs) that are effective inhibitors of trypsins and chymotrypsins from lepidopteran species. These inhibitors have a negative impact on the growth and development of lepidopteran larvae and have a potential role in plant protection. Here we investigate the effect of NaPIs on the activity and levels of serine proteases in the gut of Helicoverpa armigera larvae and explore the adaptive mechanisms larvae employ to overcome the negative effects of NaPIs in the diet. Polyclonal antibodies were raised against a Helicoverpa punctigera trypsin that is a target for NaPIs and two H. punctigera chymotrypsins; one that is resistant and one that is susceptible to inhibition by NaPIs. The antibodies were used to optimize procedures for extraction of proteases for immunoblot analysis and to assess the effect of NaPIs on the relative levels of the proteases in the gut and frass. We discovered that consumption of NaPIs did not lead to over-production of trypsins or chymotrypsins but did result in excessive loss of proteases to the frass.

A complex of genes involved in adaptation of Leptinotarsa decemlineata larvae to induced potato defense.

The Colorado potato beetle (Leptinotarsa decemlineata) is the most important pest of potato in many areas of the world. One of the main reasons for its success lies in the ability of its larvae to counteract plant defense compounds. Larvae adapt to protease inhibitors (PIs) produced in potato leaves through substitution of inhibitor-sensitive digestive cysteine proteases with inhibitor-insensitive cysteine proteases. To get a broader insight into the basis of larval adaptation to plant defenses, we created a "suppression subtractive hybridisation" library using cDNA from the gut of L. decemlineata larvae fed methyl jasmonate-induced or uninduced potato leaves. Four hundred clones, randomly selected from the library, were screened for their relevance to adaptation with DNA microarray hybridizations. Selected enzyme systems of beetle digestion were further inspected for changes in gene expression using quantitative PCR and enzyme activity measurements. We identified two new groups of digestive cysteine proteases, intestains D and intestains E. Intestains D represent a group of structurally distinct digestive cysteine proteases, of which the tested members are strongly upregulated in response to induced plant defenses. Moreover, we found that other digestive enzymes also participate in adaptation, namely, cellulases, serine proteases, and an endopolygalacturonase. In addition, juvenile hormone binding protein-like (JHBP-like) genes were upregulated. All studied genes were expressed specifically in larval guts. In contrast to earlier studies that reported experiments based on PI-enriched artificial diets, our results increase understanding of insect adaptation under natural conditions.

Protein stability: a single recorded mutation aids in predicting the effects of other mutations in the same amino acid site.

MOTIVATION: Accurate prediction of protein stability is important for understanding the molecular underpinnings of diseases and for the design of new proteins. We introduce a novel approach for the prediction of changes in protein stability that arise from a single-site amino acid substitution; the approach uses available data on mutations occurring in the same position and in other positions. Our algorithm, named Pro-Maya (Protein Mutant stAbilitY Analyzer), combines a collaborative filtering baseline model, Random Forests regression and a diverse set of features. Pro-Maya predicts the stability free energy difference of mutant versus wild type, denoted as ΔΔG. RESULTS: We evaluated our algorithm extensively using cross-validation on two previously utilized datasets of single amino acid mutations and a (third) validation set. The results indicate that using known ΔΔG values of mutations at the query position improves the accuracy of ΔΔG predictions for other mutations in that position. The accuracy of our predictions in such cases significantly surpasses that of similar methods, achieving, e.g. a Pearson's correlation coefficient of 0.79 and a root mean square error of 0.96 on the validation set. Because Pro-Maya uses a diverse set of features, including predictions using two other methods, it also performs slightly better than other methods in the absence of additional experimental data on the query positions. AVAILABILITY: Pro-Maya is freely available via web server at http://bental.tau.ac.il/ProMaya. CONTACT: nirb@tauex.tau.ac.il; wolf@cs.tau.ac.il SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Purification, cDNA cloning, and recombinant expression of chymotrypsin C from porcine pancreas.

Chymotrypsin C is a bifunctional secretory-type serine protease in pancreas; besides proteolytical activity, it also exhibits a calcium-decreasing activity in serum. In this study, we purified activated chymotrypsin C from porcine pancreas, and identified its three active forms. Active chymotrypsin C was found to be different in the length of its 13-residue activation peptide due to carboxydipeptidase (present in the pancreas) degradation or autolysis of the activated chymotrypsin C itself, resulting in the removal of several C-terminus residues from the activation peptide. After limited chymotrypsin C cleavage with endopeptidase Lys C, several purified peptides were partially sequenced, and the entire cDNA sequence for porcine chymotrypsin C was cloned. Recombinant chymotrypsinogen C was successfully expressed in Escherichia coli cells as inclusion bodies. After refolding and activation with trypsin, the comparison of the recombinant chymotrypsin C with the natural form showed that their proteolytic and calcium-decreasing activities were at the same level. The successful expression of chymotrypsin C gene paves the way to further mutagenic structure-function studies.

Molecular basis for the resistance of an insect chymotrypsin to a potato type II proteinase inhibitor.

Plants produce a variety of proteinase inhibitors (PIs) that have a major function in defense against insect herbivores. In turn, insects have developed strategies to minimize the effect of dietary PIs on digestion. We have discovered that Helicoverpa larvae that survive consumption of a multidomain serine PI from Nicotiana alata (NaPI) contain high levels of a chymotrypsin that is not inhibited by NaPI. Here we describe the isolation of this NaPI-resistant chymotrypsin and an NaPI-susceptible chymotrypsin from Helicoverpa larvae, together with their corresponding cDNAs. We investigated the mechanism of resistance by mutating selected positions of the NaPI-susceptible chymotrypsin using the corresponding amino acids of the NaPI-resistant chymotrypsin. Four critical residues that conferred resistance to NaPI were identified. Molecular modeling revealed that a Phe-->Leu substitution at position 37 in the chymotrypsin results in the loss of important binding contacts with NaPI. Identification of the molecular mechanisms that contribute to PI resistance in insect digestive proteases will enable us to develop better inhibitors for the control of lepidopteran species that are major agricultural pests worldwide.

The RY sequence is necessary but not sufficient for the transcription activation of a winged bean chymotrypsin inhibitor gene in developing seeds.

A winged bean Kunitz-type chymotrypsin inhibitor (WCI) is expressed in seeds and tuberous roots. In seeds, the expression of WCI is restricted to the period between the mid- and late-maturation stage. To understand the mechanisms that regulate the expression of WCI genes, we analyzed the promoter activity of the upstream region of the WCI-3b gene, which encodes a major WCI protein, in transgenic tobacco plants. By using a series of constructs with 5' deletions in the upstream sequences, the region between -882 and -623, relative to the transcription start site, was shown to contain multiple sequences which are responsible for high level expression in mid-maturation stage seeds. However, when this region was fused to the cauliflower mosaic virus 35S core promoter in both orientations, the chimeric promoters showed only a weak transcription activity in transgenic tobacco plants. Further analyses using internal deletion constructs revealed that the region between -882 and -174 is required for the transcription activation. Disruption of the RY sequence at -517, which is conserved in many seed protein genes, resulted in a drastic reduction of the transcription activity in seeds. These results suggest that sequences necessary for high level induction of the WCI-3b gene transcription in developing seeds are dispersed in the region between -882 and -174, and that the RY sequence is one of these sequences.