Adaptation of Helicoverpa armigera to Soybean Peptidase Inhibitors Is Associated with the Transgenerational Upregulation of Serine Peptidases.

Molecular phenotypes induced by environmental stimuli can be transmitted to offspring through epigenetic inheritance. Using transcriptome profiling, we show that the adaptation of Helicoverpa armigera larvae to soybean peptidase inhibitors (SPIs) is associated with large-scale gene expression changes including the upregulation of genes encoding serine peptidases in the digestive system. Furthermore, approximately 60% of the gene expression changes induced by SPIs persisted in the next generation of larvae fed on SPI-free diets including genes encoding regulatory, oxidoreductase, and protease functions. To investigate the role of epigenetic mechanisms in regulating SPI adaptation, the methylome of the digestive system of first-generation larvae (fed on a diet with and without SPIs) and of the progeny of larvae exposed to SPIs were characterized. A comparative analysis between RNA-seq and Methyl-seq data did not show a direct relationship between differentially methylated and differentially expressed genes, while trypsin and chymotrypsin genes were unmethylated in all treatments. Rather, DNA methylation potential epialleles were associated with transcriptional and translational controls; these may play a regulatory role in the adaptation of H. armigera to SPIs. Altogether, our findings provided insight into the mechanisms of insect adaptation to plant antiherbivore defense proteins and illustrated how large-scale transcriptional reprograming of insect genes can be transmitted across generations.

Diabetes of the Exocrine Pancreas Related to Hereditary Pancreatitis, an Update.

PURPOSE OF REVIEW: The aim was to review evidence about diabetes secondary to hereditary pancreatitis, seeking novel diagnostic and treatment features. RECENT FINDINGS: Hereditary pancreatitis (HP) is an autosomal dominant condition, characterized by recurrent episodes of acute pancreatitis, progression to fibrosis, and chronic pancreatitis. Clinical presentation includes diabetes of the exocrine pancreas (DEP). HP prevalence ranges from 0.3 to 0.57 per 100,000 people, with up to 80% of these develop DEP. This condition often requires specific interventions: with regard to metabolic control, metformin is the first choice for those with mild DEP, and for those in advanced disease, insulin is considered the first-line therapy. Insulin analogues and insulin pump therapy are preferred due to the brittle glycemic pattern and risk of hypoglycemia. In case of exocrine insufficiency, pancreatic enzyme replacement therapy is recommended. Pancreatic polypeptide administration is a promising novel treatment feature. DEP due to HP appears to be a misdiagnosed condition. The requirement of specific management demonstrates the importance of this matter; therefore, appropriate recognition and classification are important.

Early-Onset Acute Recurrent and Chronic Pancreatitis Is Associated with PRSS1 or CTRC Gene Mutations.

OBJECTIVES: To assess whether the age of onset was associated with unique features or disease course in pediatric acute recurrent pancreatitis (ARP) or chronic pancreatitis (CP). STUDY DESIGN: Demographic and clinical information on children with ARP or CP was collected at INSPPIRE (INternational Study Group of Pediatric Pancreatitis: In Search for a CuRE) centers. The Cochran-Armitage trend test and Jonckheere-Terpstra test were used to examine for differences between pediatric age groups (<6, 6-11, and ≥12 years). RESULTS: Between September 2012 and March 2016, 342 children with ARP or CP were enrolled; 129 (38%) were <6 years of age at the time of first diagnosis of acute pancreatitis, 111 (32%) were 6-11 years of age, and 102 (30%) were ≥12 years of age. Early-onset disease was associated with mutations in cationic trypsinogen (PRSS1) (P < .01), chymotrypsin C (CTRC) (P = .01), family history of acute pancreatitis (P = .02), family history of CP (P < .01), biliary cysts (P = .04), or chronic renal failure (P = .02). Later-onset disease was more commonly present with hypertriglyceridemia (P = .04), ulcerative colitis (P = .02), autoimmune diseases (P < .0001), or medication use (P < .01). Children with later-onset disease also were more likely to visit the emergency department (P < .05) or have diabetes (P < .01). CONCLUSIONS: Early-onset pancreatitis is associated strongly with PRSS1 or CTRC mutations and family history of pancreatitis. Children with later-onset disease are more likely to have nongenetic risk factors. Future studies are needed to investigate whether the disease course, response to therapy, or clinical outcomes differ relative to the timing of disease onset.

Comparative analysis of expression profiling of the trypsin and chymotrypsin genes from Lepidoptera species with different levels of sensitivity to soybean peptidase inhibitors.

Peptidase inhibitors (PIs) are essential proteins involved in plant resistance to herbivorous insects, yet many insect species are able to escape the negative effects of these molecules. We compared the effects of acute and chronic ingestion of soybean peptidase inhibitors (SPIs) on Spodoptera frugiperda and Diatraea saccharalis, two Lepidoptera species with different sensitivities to SPI ingestion. We analyzed the trypsin and chymotrypsin gene expression profiles in both species. Acute exposure of S. frugiperda to the inhibitors activated seven genes (SfChy5, SfChy9, SfChy19, SfChy22, SfTry6, SfTry8, and SfTry10), whereas chronic exposure activated 16 genes (SfChy2, SfChy4, SfChy5, SfChy8, SfChy9, SfChy11, SfChy12, SfChy15, SfChy17, SfChy21, SfChy22, SfTry6, SfTry8, SfTry9, SfTry10, and SfTry12). By contrast, the challenge of D. saccharalis with SPIs did not differentially induce the expression of trypsin- or chymotrypsin-encoding genes, with the exception of DsChy7. Bayesian phylogenetic analysis of S. frugiperda trypsin protein sequences revealed two gene clades: one composed of genes responsive to the SPIs and a second composed of the unresponsive genes. D. saccharalis trypsin proteins were clustered nearest to the S. frugiperda unresponsive genes. Overall, our findings support a hypothesized mechanism of resistance of Noctuidae moths to SPIs, involving gene number expansion of trypsin and chymotrypsin families and regulation of gene expression, which could also explain the variable susceptibility between S. frugiperda and D. saccharalis to these plant inhibitors.

ER stress-associated CTRC mutants decrease stimulated pancreatic zymogen secretion through SIRT2-mediated microtubule dysregulation.

Pancreatitis has been suspected for a long time to have an autodigestive genesis. The main events occurring in the pancreatic acinar cell that initiate acute pancreatitis include inhibition of zymogen secretion and intracellular activation of proteases. Chymotrypsin C (CTRC) is a protective protease that limits trypsin and trypsinogen proteolytic activity. Hereditary pancreatitis-associated CTRC mutants such as p.A73T and p.G61R precipitate within the endoplasmic reticulum (ER) causing ER stress. We found that expression of these mutants reduces amylase secretion from carbachol-stimulated rat pancreatic acinar cells AR42J and isolated mice pancreatic acini. Furthermore, this expression also reduces the levels of acetylated tubulin by increasing both the levels and phosphorylation of the deacetylase SIRT2. Remarkably, inhibition of SIRT2 not only greatly recovers tubulin acetylation, but also amylase secretion in pancreatic acinar cells and isolated acini. However, SIRT2 inhibition does not rescue secretion of the CTRC mutants. These results strongly suggest that CTRC variants associated to ER stress inhibit secretagogue-stimulated pancreatic zymogen secretion by altering microtubule stability. Of note, the extent of this inhibition correlates with the degree of ER stress exhibited by the particular CTRC variant.

Biochemical characterisation of chymotrypsin from the midgut gland of yellowleg shrimp, Penaeus californiensis.

Chymotrypsin from shrimp, Penaeus californiensis, was compared to Bos taurus chymotrypsin, and its structure-function relationship was studied. Catalytic efficiency toward synthetic substrate is lower, but it has a broad specificity and higher activity toward protein substrates, including collagen. It is active at pH 4-10 and fully active up to 50 °C for 2 h and at least nine days at room temperature. The activation peptide is twice as long as bovine chymotrypsinogen, has less disulfide bridges, and is a single polypeptide. Only one activation step is necessary from chymotrypsinogen to the mature enzyme. Postmortem implications in muscle softening and melanisation, resistance to temperature and pH and efficiency with proteinaceous substrates make chymotrypsin useful as a biotechnological tool in food processing. This makes shrimp processing wastes useful as a material for production of fine reagents.

Physiology of digestion and the molecular characterization of the major digestive enzymes from Periplaneta americana.

Cockroaches are among the first insects to appear in the fossil record. This work is part of ongoing research on insects at critical points in the evolutionary tree to disclose evolutionary trends in the digestive characteristics of insects. A transcriptome (454 Roche platform) of the midgut of Periplanetaamericana was searched for sequences of digestive enzymes. The selected sequences were manually curated. The complete or nearly complete sequences showing all characteristic motifs and highly expressed (reads counting) had their predicted sequences checked by cloning and Sanger sequencing. There are two chitinases (lacking mucin and chitin-binding domains), one amylase, two α- and three ß-glucosidases, one ß-galactosidase, two aminopeptidases (none of the N-group), one chymotrypsin, 5 trypsins, and none ß-glucanase. Electrophoretic and enzymological data agreed with transcriptome data in showing that there is a single ß-galactosidase, two α-glucosidases, one preferring as substrate maltase and the other aryl α-glucoside, and two ß-glucosidases. Chromatographic and enzymological data identified 4 trypsins, one chymotrypsin (also found in the transcriptome), and one non-identified proteinase. The major digestive trypsin is identifiable to a major P. americana allergen (Per a 10). The lack of ß-glucanase expression in midguts was confirmed, thus lending support to claims that those enzymes are salivary. A salivary amylase was molecularly cloned and shown to be different from the one from the midgut. Enzyme distribution showed that most digestion occurs under the action of salivary and midgut enzymes in the foregut and anterior midgut, except the posterior terminal digestion of proteins. A counter-flux of fluid may be functional in the midgut of the cockroach to explain the low excretory rate of digestive enzymes. Ultrastructural and immunocytochemical localization data showed that amylase and trypsin are released by both merocrine and apocrine secretion mainly from gastric caeca. Finally, a discussion on Polyneoptera digestive physiology is provided.

Properties and secretory mechanism of Musca domestica digestive chymotrypsin and its relation with Drosophila melanogaster homologs.

Musca domestica larvae present two different digestive chymotryptic activities found in the posterior midgut (PMG): one major soluble activity in the lumen and another minor present in cell membrane fractions. Both soluble and membrane-bound chymotryptic activities have different half lives of thermal inactivation (46 °C) in the presence and absence of 10 mM Triton X-100, indicating that they are two different molecular species. Purified soluble chymotryptic activity has pH optimum 7.4 and a molecular mass of 28 kDa in SDS-PAGE. It does not cleave short substrates, such as Suc-F-MCA, preferring longer substrates, such as Suc-AAPF-MCA, with a primary specificity (kcat/Km) for Phe rather than Tyr and Leu residues. In-gel activity revealed a unique band against S-AAPF-MCA with the same migration as purified chymotrypsin. One chymotrypsinogen-like sequence (MdChy1) was sequenced, cloned and recombinantly expressed in Escherichia coli (DE3) Star. MdChy1 is expressed in the proximal posterior midgut (PMG1), as seen by RT-PCR. Expression analysis of other chymotrypsin genes revealed genes expressed at the anterior midgut (AMG) and PMG. Western blot of M. domestica midgut tissues using anti-MdChy1 antiserum showed a single band in samples from AMG and PMG, co-migrating with recombinant and purified enzymes. Immunogold labeling corresponding to Mdchy1 was found in small vesicles (thus indicating exocytosis) and in the lumen of AMG and PMG, corroborating the existence of two similar groups of chymotrypsins. Transcriptomes of M. domestica AMG and whole midgut prepared by pyrosequencing disclosed 41 unique sequences of chymotrypsin-like enzymes (19 probably functional), from which MdChy1 is highly expressed. Phylogenetic reconstruction of Drosophila melanogaster and M. domestica chymotrypsin-like sequences revealed that the chymotrypsin genes expanded before the evolutionary separation of Musca and Drosophila.

General up regulation of Spodoptera frugiperda trypsins and chymotrypsins allows its adaptation to soybean proteinase inhibitor.

The existence of a diverse serine proteinase gene family in lepidopteran insects suggests they play a significant role in the insect adaptation to plant proteinase inhibitors. These proteinases have been shown to be involved in the process of proteolytic digestion in insect larvae. We carried out a selective transcriptome study of midguts from Spodoptera frugiperda larvae fed on a diet supplemented with soybean proteinase inhibitor (SPI). Using subtracted cDNA libraries made of gut-expressed transcripts, a total of 2100 partial sequences were obtained, of those 38% were related to digestive process. Two large and diverse groups of chymotrypsins and trypsins were obtained, and some of these proteinase-encoding genes were further characterized by quantitative RT-PCR. The transcription analyses revealed two groups: one group of genes constitutively expressed in the control larvae that is up regulated by introducing SPI to the diet, and a second group that is absent in the control but is induced by the SPI-rich diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis and also up regulation of existing enzymes. Proteases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteases were also insensitive to a broad-spectrum potato proteinase inhibitor preparation. We propose that adaptation of S. frugiperda to SPI follows a "shotgun" approach, based on a general up regulation of a large set of endoproteinases.

Chymotrypsin genes in the malaria mosquitoes Anopheles aquasalis and Anopheles darlingi.

Four closely related chymotrypsin genes were identified in Anopheles aquasalis and Anopheles darlingi (Anachy1, Anachy2, Andchy1 and Andchy2). The deduced amino-acid sequences were compared to other chymotrypsin sequences. These sequences were used to infer phylogenetic relationships among the different species. Genomic cloning revealed that, in contrast to An. aquasalis and A. gambiae, the chymotrypsin genomic locus in An. darlingi had a short intergenic region that accompanied the inverted position of the genes, suggesting inversion mechanisms in this species related to transposable elements. Alignments of the sequences upstream of the transcription start sites of Anachy1, Anachy2, Andchy1 and Andchy2 revealed areas with high similarity containing palindromic sequences. Northern analysis from An. aquasalis indicated that the transcription of chy 1 and 2 are induced by blood feeding.

Molecular cloning and characterization of a midgut chymotrypsin-like enzyme from the lesser grain borer, Rhyzopertha dominica.

A cDNA encoding a chymotrypsinogen-like protein in midguts of the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) was cloned and sequenced. The 901 bp cDNA contains an 816-nucleotide open reading frame encoding 272-amino acids. The predicted molecular mass and pI of the mature enzyme are 23.7 kDa and 4.64, respectively. The encoded protein includes amino acid sequence motifs that are conserved with 5 homologous chymotrypsinogen proteins from other insects. Features of the putative chymotrypsin-like protein from R. dominica include the serine proteinase active site (His(90), Asp(133), Ser(226)), conserved cysteine residues for disulfide bridges, the residues (Gly(220), Gly(243), Asp(252)) that determine chymotrypsin specificity, and both zymogen activation and signal peptides. A TPCK-sensitive caseinolytic protein (P6) with an estimated molecular mass of 24 kDa is present in midgut extracts of R. dominica and can be resolved by electrophoresis on 4-16% polyacrylamide gels. The molecular mass of this caseinolytic enzyme is similar to that of the chymotrypsin deduced from cDNA. Midgut extracts of R. dominica readily hydrolyzed azocasein and N-succinyl-alanine-alanine-proline-phenylalanine-p- nitroanilide (SAAPFpNA), a chymotrypsin-specific substrate. Properties of the enzymes responsible for these activities were partially characterized with respect to distribution in the gut, optimum pH, and sensitivity toward selected proteinase inhibitors. Optimal activity against both azocasein and SAAPFpNA occurs in a broad pH range from about 7 to 10. Both azocasein and SAAPFpNA activities, located primarily in the anterior midgut region, are inhibited by aprotinin, phenylmethyl sulphonylfluoride (PMSF), and soybean trypsin inhibitor (STI). TPCK (N-alpha-tosyl-L-phenylalanine chloromethyl ketone) and chymostatin inhibited more than 60% of SAAPFpNA but only about 10-20% of azocasein activity. These results provide additional evidence for the presence of serine proteinases, including chymotrypsin, in midguts of R. dominica. Arch. Insect Biochem. Physiol. 43:173-184, 2000.Published 2000 Wiley-Liss, Inc.