Tighter Control by Chymotrypsin C (CTRC) Explains Lack of Association between Human Anionic Trypsinogen and Hereditary Pancreatitis.

The human pancreas expresses two major trypsinogen isoforms, cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2). Mutations in PRSS1 cause hereditary pancreatitis by altering cleavage of regulatory nick sites by chymotrypsin C (CTRC) resulting in reduced trypsinogen degradation and increased autoactivation. Despite 90% identity with PRSS1 and a strong propensity for autoactivation, mutations in PRSS2 are not found in hereditary pancreatitis suggesting that activation of this isoform is more tightly regulated. Here, we demonstrated that CTRC promoted degradation and thereby markedly suppressed autoactivation of human anionic trypsinogen more effectively than previously observed with cationic trypsinogen. Increased sensitivity of anionic trypsinogen to CTRC-mediated degradation was due to an additional cleavage site at Leu-148 in the autolysis loop and the lack of the conserved Cys-139-Cys-206 disulfide bond. Significant stabilization of anionic trypsinogen against degradation was achieved by simultaneous mutations of CTRC cleavage sites Leu-81 and Leu-148, autolytic cleavage site Arg-122, and restoration of the missing disulfide bridge. This stands in stark contrast to cationic trypsinogen where single mutations of either Leu-81 or Arg-122 resulted in almost complete resistance to CTRC-mediated degradation. Finally, processing of the trypsinogen activation peptide at Phe-18 by CTRC inhibited autoactivation of anionic trypsinogen, although cationic trypsinogen was strongly stimulated. Taken together, the observations indicate that human anionic trypsinogen is controlled by CTRC in a manner that individual natural mutations are unlikely to increase stability enough to promote intra-pancreatic activation. This unique biochemical property of anionic trypsinogen explains the lack of association of PRSS2 mutations with hereditary pancreatitis.

In vitro models for metabolic studies of small peptide hormones in sport drug testing.

Peptide hormones represent an emerging class of potential doping agents. Detection of their misuse is difficult due to their short half-life in plasma and rapid elimination. Therefore, investigating their metabolism can improve detectability. Unfortunately, pharmacokinetic studies with human volunteers are often not allowed because of ethical constraints, and therefore alternative models are needed. This study was performed in order to evaluate in vitro models (human liver microsomes and S9 fraction) for the prediction of the metabolism of peptidic doping agents and to compare them with the established models. The peptides that were investigated include desmopressin, TB-500, GHRP-2, GHRP-6, hexarelin, LHRH and leuprolide. Several metabolites were detected for each peptide after incubation with human liver microsomes, S9 fraction, and serum, which all showed endopeptidase and exopeptidase activity. In vitro models from different organs (liver vs. kidney) were compared, but no significant differences were recorded. Deamidation was not observed in any of the models and was therefore evaluated by incubation with α-chymotrypsin. In conclusion, in vitro models are useful tools for forensic and clinical analysts to detect peptidic metabolic markers in biological fluids.

Comprehensive functional analysis of chymotrypsin C (CTRC) variants reveals distinct loss-of-function mechanisms associated with pancreatitis risk.

OBJECTIVE: The digestive enzyme chymotrypsin C (CTRC) protects against pancreatitis by promoting degradation of trypsinogen, thereby curtailing potentially harmful trypsinogen activation. Loss-of-function variants in CTRC increase the risk for chronic pancreatitis. The aim of the present study was to perform comprehensive functional analysis of all missense CTRC variants identified to date. DESIGN: We investigated secretion, activity and degradation of 27 published and five novel CTRC mutants. We also assessed the effect of five mutants on endoplasmic reticulum (ER) stress. RESULTS: None of the mutants exhibited a gain of function, such as increased secretion or activity. By contrast, 11 mutants showed marked loss of function, three mutants had moderate functional defects, whereas 18 mutants were functionally similar to wild-type CTRC. The functional deficiencies observed were diminished secretion, impaired catalytic activity and degradation by trypsin. Mutants with a secretion defect caused ER stress that was proportional to the loss in secretion. ER stress was not associated with loss-of-function phenotypes related to catalytic defect or proteolytic instability. CONCLUSIONS: Pathogenic CTRC variants cause loss of function by three distinct but mutually non-exclusive mechanisms that affect secretion, activity and proteolytic stability. ER stress may be induced by a subset of CTRC mutants, but does not represent a common pathological mechanism of CTRC variants. This phenotypic dataset should aid in the classification of the clinical relevance of CTRC variants identified in patients with chronic pancreatitis.

Effect of chymotrypsin C and related proteins on pancreatic cancer cell migration.

Pancreatic cancer is a malignant cancer with a high mortality rate. The amount of chymotrypsin C in pancreatic cancer cells is only 20% of that found in normal cells. Chymotrypsin C has been reported to be involved in cancer cell apoptosis, but its effect on pancreatic cancer cell migration is unclear. We performed cell migration scratch assays and Transwell experiments, and found that cell migration ability was downregulated in pancreatic cancer Aspc-1 cells that overexpressed chymotrypsin C, whereas the cell migration ability was upregulated in Aspc-1 cells in which chymotrypsin C was suppressed. Two-dimensional fluorescence differential in gel electrophoresis/mass spectrometry method was used to identify the proteins that were differentially expressed in Aspc-1 cells that were transfected with plasmids to induce either overexpression or suppressed expression of chymotrypsin C. Among 26 identified differential proteins, cytokeratin 18 was most obviously correlated with chymotrypsin C expression. Cytokeratin 18 is expressed in developmental tissues in early stages of cancer, and is highly expressed in most carcinomas. We speculated that chymotrypsin C might regulate pancreatic cancer cell migration in relation to cytokeratin 18 expression.

Digestive enzyme activity of two stonefly species (Insecta, Plecoptera) and their feeding habits.

The digestive enzymes of two stoneflies species, Hemimelaena flaviventris and Isoperla morenica, were studied for the first time. These species are temporary water inhabitants and exhibit great feeding plasticity. Although they are traditionally referred to as predators, a previous study revealed that H. flaviventris incorporates some diatoms into its diet in addition to feeding usually on several prey, and I. morenica (in that study under the name of I. curtata) only feeds on animals occasionally. The enzymatic activities of digestive amylase, lipase, protease, trypsin and chymotrypsin were determined for each species at the same developmental stage. The results show that H. flaviventris has a greater digestive enzymatic pool and higher relative and absolute protease, lipase and trypsin activities than I. morenica. The latter has a relative higher amylase activity. As higher amylase activity is typical of phytophagous species and higher protease activity typical of carnivorous species; these results reveal that H. flaviventris is a more efficient zoophagous species than I. morenica. The ecological implications of these findings, including the higher secondary production of H. flaviventris in its habitat, are discussed.

Trypsin and chymotrypsin are involved in the progesterone-induced acrosome reaction in canine spermatozoa.

Acrosomal proteases allow the spermatozoon not only to cross the cumulus cells and penetrate the zona pellucida of the oocyte, but also they are needed for the acrosome reaction process (AR). The present study evaluated in vitro the role of trypsin and chymotrypsin in the acrosome reaction of canine spermatozoa by means of protease inhibitors. Spermatozoa obtained from the second fraction of the ejaculate and devoid of seminal plasma were re-suspended in canine capacitation medium (CCM) and incubated at 38.5 degrees C in 5% CO(2). After 2 h (period of sperm capacitation), aliquots of sperm suspension were incubated separately with trypsin inhibitor NPGB (p-nitrophenyl-p'-guanidino-benzoate); TI (Trypsin inhibitor I-S Type from soybean) and with chymotrypsin inhibitor TPCK (N-tosyl-L-phenylalanine-chloromethyl-ketone) for 30 min. The AR was induced with progesterone and evaluated using the dual fluorescent staining technique 'Hoechst and chlortetracycline'. Acrosomal exocytosis levels were statistically significant higher in the samples treated with progesterone than in the control without inducer. However, the trypsin inhibitors NPGB, TI and the chymotrypsin inhibitor TPCK reduced the percentage of AR when compared with the control with progesterone and without inhibitor (p < 0.001), where the AR values were 45.63 +/- 3.8%, 51.63 +/- 2.8%, 58.38 +/-4.1% and 71.25 +/- 4.9%, respectively. These results show that trypsin and chymotrypsin inhibitors are effective in blocking the acrosome reaction induced by progesterone in canine; in addition, they suggest the participation of respective proteases in the AR process in this species.

Surface protease of Treponema denticola hydrolyzes C3 and influences function of polymorphonuclear leukocytes.

Treponema denticola is a dominant microorganism in human periodontal lesions. One of the major virulence factors of this microorganism is its chymotrypsin-like surface protease, dentilisin. The purpose of this study was to evaluate the effect of dentilisin on human polymorphonuclear leukocytes (PMNs). We used chemiluminescence to assess production of O(-)(2) by PMNs against T. denticola ATCC 35405 and dentilisin-deficient mutant K1. T. denticola ATCC 35405 induced production of O(-)(2), whereas dentilisin-deficient K1 did not. We found that chymostatin, a protease inhibitor, strongly reduced the ability of T. denticola ATCC 35405 to induce production of, O(-)(2), whereas K1 was relatively unaffected. We also used Immunoblot and ELISA to evaluate the activation of complement by this microorganism in relation to PMNs. T. denticola ATCC 35405 hydrolyzed the alpha-chain of C3, producing iC3b. Furthermore, strain ATCC 35405 induced a larger release of MMP-9 from PMNs than strain K1. Dentilisin activated PMNs via complement pathways and may play a role in establishing periodontal lesions.

In silico discovery of enzyme-substrate specificity-determining residue clusters.

The binding between an enzyme and its substrate is highly specific, despite the fact that many different enzymes show significant sequence and structure similarity. There must be, then, substrate specificity-determining residues that enable different enzymes to recognize their unique substrates. We reason that a coordinated, not independent, action of both conserved and non-conserved residues determine enzymatic activity and specificity. Here, we present a surface patch ranking (SPR) method for in silico discovery of substrate specificity-determining residue clusters by exploring both sequence conservation and correlated mutations. As case studies we apply SPR to several highly homologous enzymatic protein pairs, such as guanylyl versus adenylyl cyclases, lactate versus malate dehydrogenases, and trypsin versus chymotrypsin. Without using experimental data, we predict several single and multi-residue clusters that are consistent with previous mutagenesis experimental results. Most single-residue clusters are directly involved in enzyme-substrate interactions, whereas multi-residue clusters are vital for domain-domain and regulator-enzyme interactions, indicating their complementary role in specificity determination. These results demonstrate that SPR may help the selection of target residues for mutagenesis experiments and, thus, focus rational drug design, protein engineering, and functional annotation to the relevant regions of a protein.

Enzyme dehydration using Microglassification™ preserves the protein's structure and function.

Controlled enzyme dehydration using a new processing technique of Microglassification™ has been investigated. Aqueous solution microdroplets of lysozyme, α-chymotrypsin, catalase, and horseradish peroxidase were dehydrated in n-pentanol, n-octanol, n-decanol, triacetin, or butyl lactate, and changes in their structure and function were analyzed upon rehydration. Water solubility and microdroplet dissolution rate in each solvent decreased in the order: butyl lactate > n-pentanol > triacetin > n-octanol > n-decanol. Enzymes Microglassified™ in n-pentanol retained higher activity (93%-98%) than n-octanol (78%-85%) or n-decanol (75%-89%), whereas those Microglassified™ in triacetin (36%-75%) and butyl lactate (48%-79%) retained markedly lower activity. FTIR spectroscopy analyses showed α-helix to ß-sheet transformation for all enzymes upon Microglassification™, reflecting a loss of bound water in the dried state; however, the enzymes reverted to native-like conformation upon rehydration. Accelerated stressed-storage tests using Microglassified™ lysozyme showed a significant (p < 0.01) decrease in enzymatic activity from 46,560 ± 2736 to 31,060 ± 4327 units/mg after 3 months of incubation; however, it was comparable to the activity of the lyophilized formulation throughout the test period. These results establish Microglassification™ as a viable technique for enzyme preservation without affecting its structure or function.

Role of dentilisin in Treponema denticola epithelial cell layer penetration.

Treponema denticola is an oral anaerobic spirochete implicated in periodontal diseases. The chymotrypsin-like protease, dentilisin (PrtP), has been suggested to be an important virulence factor of T. denticola. In this study, we examined the role of dentilisin in T. denticola epithelial monolayer penetration by comparing the wild type and prtP mutant. Wild-type T. denticola can disrupt transepithelial resistance (TER) and substantially penetrate the HEp-2 cell layer. The prtP mutant altered the monolayer only slightly and penetrated the Hep-2 layer in very low numbers. The membrane fraction of wild-type T. denticola is able to complement the prtP mutant in monolayer penetration, while the comparable fraction from the mutant has no such effect. Immunofluorescence studies suggested that wild-type T. denticola altered the TER by likely degrading the tight junctional proteins such as ZO-1. Cytotoxicity was not a major factor in the disruption of TER. The outer membrane vesicles (OMVs) of wild-type T. denticola also disrupted epithelial barrier function and penetrated the epithelial layers. Taken together, these results suggest that T. denticola penetrates the epithelial cell monolayers by altering cellular tight junctions.

Molecular pathogenesis of the cell surface proteins and lipids from Treponema denticola.

Treponema denticola, frequently isolated from the human oral cavity, is thought to be a major pathogen of human periodontal disease. Recent developments in molecular analysis have clarified the surface structure of this microorganism and the characteristics of its pathogenic factors. Structural analysis of the outer sheath showed T. denticola to have a new type of outer membrane lipid. Limited exposure of the major outer sheath protein is suggested by electron-microscopic analysis. A protease-deficient mutant has revealed the roles of the protease in the organization of the outer sheath material and in T. denticola pathogenicity. The surface features that contribute to the pathogenicity of T. denticola in periodontal disease are gradually being elucidated, and are reviewed.

Effect of chymotrypsin on human cholecystokinin release: use of clostripain in the validation of a new radioimmunoassay.

We have developed and validated a new radioimmunoassay for cholecystokinin. In order to establish that the antiserum binds large and small forms of CCK to an equal extent, we used the microbial enzyme clostripain, which cleaves large forms of CCK yielding CCK 8. Cleavage by clostripain of synthetic and purified forms of CCK, and CCK extracted at from human jejunum and CCK in human plasma was found not to affect immunoactivity, indicating that the antiserum reacts similarly with all forms of CCK. There is controversy over whether intraduodenal trypsin inhibits release of CCK in man. We used our radioimmunoassay to investigate whether chymotrypsin, rather than trypsin, could be the major mediator of negative feedback control of CCK release. Six normal subjects received an intraduodenal infusion of L-phenylalanine and L-tryptophan on two occasions, with the addition of either 1 g/l bovine chymotrypsin or 1 g/l albumin. Plasma CCK concentrations rose in response to the amino acid infusion, but were not affected by the addition of chymotrypsin, indicating that this enzyme is not a mediator of CCK feedback regulation in man.

A chymotrypsin-like proteinase that may be involved in desquamation in plantar stratum corneum.

We have recently reported that unipolar cell shedding from plantar stratum corneum incubated in vitro, and the associated degradation of the desmosomal protein desmoglein I, are dependent on the activity of a proteinase that can be inhibited by aprotinin, chymostatin and zinc ion. The aim of this work was to find a proteinase in plantar stratum corneum that fulfils the criteria for being the responsible enzyme. Dissociated plantar corneocytes were incubated with the chymotrypsin substrate 3-carbomethoxypropionyl-L-Arg-L-Pro-L-Tyr-p-nitroanilide hydrochloride (S-2586) and H-D-Ile-Pro-Arg-p-nitroanilide dihydrochloride (S-2288), a substrate for a wide range of serine proteinases with arginine specificity. There was a significant rate of hydrolysis of S-2586, but S-2288 was hydrolysed only very slowly. Extraction of dissociated corneocytes with buffers containing KCl or sodium dodecyl sulphate released one major proteinase that could be detected by electrophoresis in polyacrylamide gels with copolymerized casein and subsequent incubations of the gels. Both the caseinolytic activity and the S-2586-hydrolysing activities were inhibited by aprotinin, chymostatin and zinc ion, but not by leupeptin. The S-2586-hydrolysing activity was also inhibited by soybean trypsin inhibitor and phenylmethylsulphonyl fluoride. Both activities were optimal at pH 7-8 but were also significant at pH 5.5. On gel exclusion chromatography, the S-2586-hydrolysing and caseinolytic activities were eluted with an apparent molecular weight of around 18 kDa. When analyzed by electrophoresis in the presence of sodium dodecyl sulphate under non-reducing conditions the caseinolytic enzyme had an apparent molecular weight of around 25 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

A study on the possible role of chymotrypsin in the aetiology of equine chronic obstructive pulmonary disease (COPD).

The chymotrypsin activity of seven batches of Micropolyspora faeni and of five batches of Aspergillus fumigatus culture extracts, prepared for inhalation challenge in horses, was assayed and was found to range between 0.29 and 1.45 units/mg protein and 0.02 and 0.20 units/mg protein respectively. Horses affected with chronic obstructive pulmonary disease (COPD) were challenged with two batches of each antigen which had different chymotrypsin activities and no significant correlations were found between the degree of response to challenge and the chymotrypsin activity of the antigens. Inhalation of two doses of nebulised, purified chymotrypsin over 4 days did not induce signs of respiratory disease in COPD-affected horses. However, repeated chymotrypsin inhalations after an interval of 3 weeks caused an exacerbation of signs of COPD in one horse. These studies suggest that, although repeated inhalation of purified chymotrypsin may induce respiratory hypersensitivity in horses, the chymotrypsin-like enzymes of M. faeni and A. fumigatus do not play a major role in the precipitation of clinical signs of equine COPD.

The involvement of cytosolic chymotrypsin-like, trypsin-like, and cucumsin-like activities in degradation of insulin and insulin-like growth factor I by epithelial tissues.

Using specific substrates, benzyloxycarbonyl-Gly-Gly-Leu-p-nitroanilide, benzyloxycarbonyl-Gly-Gly-Arg-2-naphthylamide and benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide, cytosolic chymotrypsin-like, trypsin-like and cucumsin-like activities were determined, respectively, in rat epithelial tissues and differentiated human Caco-2 cells. The cytosolic fractions of rat colonic, rectal, nasal, and alveolar epithelial cells and differentiated human Caco-2 cells contained these three distinct enzyme activities. However, effects of enzyme inhibitors revealed that these three distinctive activities were not extensively involved in cytosolic or homogenate degradation of insulin and insulin-like growth factor I (IGF-I). It is concluded that proteasome-like activities may not significantly limit nonparenteral absorption of peptide and protein drugs such as insulin and IGF-I.

Characterization of the proteolytic activity of commercial proteases and strained ruminal fluid.

The objective of this research was to formulate a mixture of commercial proteases that would mimic the rate and extent of protein degradation obtained using strained ruminal fluid. The proteolytic activity of strained ruminal fluid and several commercial proteases was characterized using 13 L-amino acid p-nitroanilides as artificial substrates. A mixture of Streptomyces griseus protease, chymotrypsin, and proteinase K at .042, 2.5, and .5 enzyme units/mL, respectively, was similar to the activity of strained ruminal fluid against the same artificial substrates. However, degradative activities were different in incubations with feed proteins as substrates. The rates of degradation of expeller soybean meal, solvent soybean meal, and casein were .08, .05, and .08/h, respectively, using the enzyme mixture and .03, .15, and .24/h using strained ruminal fluid. A second experiment compared degradative activity of S. griseus protease at .066 enzyme units/mL, ficin at .5 enzyme units/mL, and a mixture of trypsin, carboxypeptidase B, chymotrypsin, and carboxypeptidase A at 116.6, .5, 2.5, and .5 enzyme units/mL, respectively. Protein degradation rates obtained with strained ruminal fluid were two to six times faster than those obtained with the enzyme mixtures. A third experiment compared the degradability of 15 feed proteins with the mixture of trypsin, carboxypeptidase B, chymotrypsin and carboxypeptidase A to that with strained ruminal fluid. Degradation rates obtained using strained ruminal fluid ranged from .007 to .217/h; degradation rates using the enzyme mixture ranged from .010 to .079/h and were lower (P = .004) than with strained ruminal fluid. Overall, the experiments indicated that the commercial enzymes tested did not mimic the protein degradative activity of strained ruminal fluid.

Development of digestive enzymes in pigs with emphasis on lipolytic activity in the stomach and pancreas.

The effect of age and weaning on the activities of digestive enzymes with emphasis on the lipolytic enzymes before and after weaning was investigated. The activities of amylase, chymotrypsin, trypsin, carboxyl ester hydrolase, pancreatic lipase, and colipase in pancreatic tissue and the activity of gastric lipase in the cardiac mucosa of the stomach in 45 pigs were response variables. The activity of trypsin was not affected by weaning and the rate of increase was similar during the whole experiment. The activities of chymotrypsin and amylase decreased at weaning (P < .05). After weaning the activity of chymotrypsin increased more slowly than before weaning (P < .001), whereas the rate of increase of amylase activity remained unchanged. Lipase, colipase, and carboxyl ester hydrolase activities decreased at weaning (P < .001), whereas gastric lipase activity increased at weaning (P < .01). The development of lipase, colipase, and carboxyl ester hydrolase activity decreased postweaning (P < .01), whereas gastric lipase activity increased before weaning and remained constant after weaning. Pancreatic lipase had a considerably higher capacity for hydrolyzing tributyrin, and the total activity of pancreatic lipase was up to 600 times higher than that of gastric lipase. The lipolytic enzymes displayed a non-parallel pattern of development, and we suggest that this reflects the importance of these enzymes during the suckling and postweaning phases, respectively. However, the significance of gastric lipase for the digestion of fat in pigs remains to be elucidated.

Stunting syndrome in broilers: effect of age and exogenous amylase and protease on performance, development of the digestive tract, digestive enzyme activity, and apparent digestibility.

Day-old male, meat-type chicks raised in brooder batteries were infected by orally administering an inoculum prepared from intestines of broiler chicks infected with stunting syndrome (SS). Naive controls were kept in a parallel room. The chicks were fed a commercial starter diet supplemented with two levels of enzyme preparations to 14 d of age. The experiment was continued to the age of 6 wk in order to estimate compensatory feed intake and growth. In a parallel study, digestibility of the feed was determined from 1 to 3 wk of age with control or inoculated chicks. The enzymes amylase and proteases were produced by Bacillus subtilis and Penicillium emersonii. Enzyme supplementation had no effect on feed intake, growth, or feed utilization, or on digestibility of fat, starch, protein, or energy. Because enzyme supplementation did not consistently affect performance of chicks and no interactions were observed between enzyme supplementation and infection status, data are presented for effects of infection only. Inoculation of SS-infective material reduced performance to 4 wk. Compensatory growth and feed intake were observed from the age of 4 wk onward. At the age of 6 wk the slight retardation of the inoculated chicks was not significant. On Week 1, retention of fat, starch, protein, and energy was significantly depressed in the inoculated chicks. At the age of 2 wk, retention of starch was not depressed, and at the age of 3 wk, the only consistent depression was that observed for fat. The proventriculus weight and content were consistently higher in inoculated chicks, as were the small intestine and intestinal content. The pH of the gizzard content was higher, and that of the small intestine content was lower, in the inoculated birds than in their control counterparts. Stunting syndrome infection was accompanied by a significant depression of trypsin activity in the pancreas at the age of 1 and 2 wk. At these periods, amylase and chymotrypsin were not affected. At 6 wk of age, the activities of amylase, trypsin, and chymotrypsin in the pancreas were higher in the inoculated than in the control birds. In the intestinal chime, amylase, trypsin, and chymotrypsin activities were lower in the inoculated birds on Week 1 and 2 (NS for amylase on Week 1). On Week 6, the activity of all enzymes assayed was higher in the inoculated birds (NS for amylase). It is suggested that the main factors depressing feed intake and growth in SS-infected birds are most probably beyond those of digestion.

[Relation between acute phase proteins and body defense: application to the monitoring of infectious states]. / Relations entre protéines de la phase aiguë et défenses de l'organisme: application à la surveillance des états infectieux.

All acute and/or chronic pathological processes resulting in tissue damage and destruction lead to an inflammatory response. The purpose of this response is homeostatic and is made up of local and systemic events, the signs of which, taken as a whole, constitute the acute phase syndrome. Among the metabolic changes occurring in this syndrome, the rise in the plasma concentration of a group of heterogenous proteins known as the "Acute Phase Reactant Proteins" ( APRP ) is a very reliable and sensitive indicator of the presence of pathology. Most of these proteins are involved in the mechanisms initiating and controlling the inflammatory response. Some of them seem to establish a link between the body's specific and non-specific defence mechanisms. APRP are indicators of the inflammatory response without being specific to a particular etiology. The degree of variation in APRP levels is not generally speaking a good indicator of the severity or spread of the tissue lesions. On the other hand, they are very useful for detecting and monitoring infectious and/or inflammatory states, whether these are being treated or not. Thus during anti-infectious or anti-inflammatory treatment the return of APRP levels to their physiological baseline is a very good argument in favour of the efficacy of such treatment.

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.