Elastase-like Activity Research Articles

Serine protease inhibition attenuates foreign body giant cell formation

Event Abstract Back to Event Serine protease inhibition attenuates foreign body giant cell formation Aisling N. O'Carroll1, Brendan F. Gilmore1, Colin P. Mccoy1 and Louise Carson1 1 Queen's University Belfast, School of Pharmacy, United Kingdom Introduction: Upon biomaterial implantation, macrophages (MΦ) adhere to the material surface and can fuse to form large, multinucleated Foreign Body Giant Cells (FBGCs) [1]. MΦ adherence and FBGC formation on implanted biomaterial surfaces are pivotal events in the progression of a Foreign Body Response (FBR). The host response is further compounded by protease release from FBGCs, during a process described as ‘frustrated phagocytosis’, causing both biomaterial and tissue component degradation in the peri-implant region [2]. Previous studies highlight roles for matrix metalloproteases (MMP)-9 in MΦ fusion [3]. However, little is known regarding the role of serine proteases in FBGC biology. Work presented details the proteolytic activity of FBGCs and effect of serine protease inhibition on the fusogenic capabilities of primary murine MΦs. Materials and Methods: Fusion assays were performed using primary murine bone marrow derived macrophages (BMM) isolated from WT C57BL/6 mice, and treated with IL-4 (10 ng/ml), GM-CSF (10 ng/ml), and 100 µM affinity-based biotinylated inhibitor probes (or vehicle control) targeting three classes of serine proteases; trypsin-like, chymotrypsin-like and elastase-like proteases [4]. Each inhibitor probe was tested alone, and in combination to highlight possible synergistic activity. Protease temporal expression and activity during FBGC formation was analysed by SDS-PAGE, Zymography and Western Blotting from cell-conditioned media and whole cell lysates. Results: Increased serine protease secretion in FBGCs at Day 2, 5 and 7 is observed, when compared with non-fused mononuclear controls. These proteases range from 38-49 kDa in size. FBGC formation is reduced by almost 50% when subject to protease inhibitor treatment. Statistical analyses show BMM treatment with inhibitors directed towards trypsin-like (P < 0.01), chymotrypsin-like (P < 0.001) and elastase-like (P < 0.05) activity results in a significant decrease in percentage fusion when compared with controls. Combined treatment with all three protease inhibitors dramatically reduces FBGC formation in vitro. Discussion: Western blotting, ultilizing the biotinylated inhibitor probes, suggests an increase in protease activity in fused BMMs overtime when compared with unfused controls, in particular an increase in trypsin-like and chymotrypsin-like actiivity at Day 5 and 7. Simultaneous treatment with all three serine inhibitors targeting chymotrypsin-like, trypsin-like and elastase-like serine proteases results in a significant reduction in FBGC formation, over and above each inhibitor alone, and is indicative of a synergistic effect. Such treatment represents a viable novel strategy to abrogate macrophage fusion. Conclusion: Together, these results denote a function for serine proteases in MΦ fusion and FBGC biology. Serine proteases may represent potential novel therapeutic targets in efforts to prevent the FBR – be it due to their influential role initiating intracellular signalling or indeed orchestrating cytoskeletal reorganisation during the fusion process. Future work will centre on the identification and functional characterisation of these serine proteases, unmasking their mechanistic role in FBGC formation.

NEW BIOCHEMICAL MARKERS OF RECURRENCE OF PROSTATE CANCER AFTER HIS TREATMENT

We studied the condition of the blood proteolytic systems (activity of kallikrein (К) and level of prekallikrein (PK), the total proteolytic activity of serine proteinases and elastase-like activity, the leukocytic elastase (LE) activity, the angiotensin-converting enzyme (ACE) activity, and the inhibitor activity of the α1-proteinase inhibitor (α1-PI) and α2-macroglobulin (α2-MG) in 36 patients after radical prostatectomy (RPE). Group I was composed of 28 patients without biochemical recurrence (BR) after RPE. In this group, 18 patients had locally limited cancer, and 10 patients had рТ3. Group II included 8 patients after RPE with development of BR. The prostate cancer stage in Group II was as follows: рT3 in 7 patients, and pT4 in one patient. The average age of the patients in the groups was 61.44±1.39 years. The median PSA before RPE was 8.05 ng/ml (LQ=5.01; UQ=12). The average prostate volume in the groups was 52±2.74 cm3 . The median of observations was 18 months. The control group consisted of 20 healthy males. In Group II patients at 1 month after the RPE, the ACE activity was 136.5% (p1&lt;0.001) higher, and the LE activity was 29.0% (p1&lt;0.05) lower than the corresponding indicators in Group I. The median PSA in Group I at that time was 0.02±0.01 ng/ml, and in Group II it was 0.2±0.04 ng/ml. Thus, increased activity of ACE against the background of the reduced antiproteolytic potential of the blood in patients after RPE is the metabolic basis for development of biochemical recurrence, and can be a marker of its progression. Increased activity of ACE and reduced activity of EA are predictors of BR development as early as at 1 month after RPE, when the PSA level is not yet elevated.

Secretagogue Mechanism of Digestive Enzyme Secretion in the Midgut of Andrallus spinidens Fabricius (Hemiptera: Pentatomidae)

Time for food processing and enzymatic secretion were evaluated in starved and fed adults of Andralus spinidnes a predatory hemipteran serving as biocontrol agent of rice pests. Adults were kept starved for 8 days then they were allowed to feed on Galleria melonella larvae (one larva per adult). They were dissected at time intervals of 1, 3, 6, 12, 24 and 48 h post-feeding to evaluate digestive enzymatic activities. It was observed that gut of starved adult is small and shrunken but gut of fed adults after 12 h was normal and well shape. No digestive enzymatic activity was observed in starved adults. Activity of general protease was the highest in 24 h post-feeding but the highest activity of α-amylase and lipase were noticed 12 h post-feeding. Also, specific protease activities showed different peaks at different time intervals. The highest trypsin-like, chymotrypsin-like and elastase-like activities were observed 12 and 24 h post-feeding although activity of chymotrypsin-like protease was still high even after 48 h. Cathepsin B showed the highest activity after 24 and 48 h but Cathepsin L and D had the highest activity after 12 h of post-feeding. The highest activities of amino- and carboxypeptidase were observed 24 h post-feeding. These results clearly demonstrate secretagogue mechanism of digestion and digestive enzyme secretions in A. spinidens. Also, secretion of digestive enzymes at least 12 h post-feeding could be attributed to extra-oral digestion made by the insects and liquefaction of the food material.

Role of endogenous inhibitors on digestive amylolytic and proteolytic activities of a predatory hemipteran, Andrallus spinidens Fabricius

The role of endogenous inhibitors on digestive amylolytic and proteolytic activities of Andrallus spinidens Fabricius (Hemiptera: Pentatomidae) were studied by using extracted endogenous inhibitors from Pieris brassicae, Naranga aenescens, Chilo suppressalis, Ephestia kuehniella and Hyphantria cunea. The endogenous inhibitors from P. brassicae and N. aenescens had the most inhibitory effects on α-amylase of A. spinidens. General proteolytic activity in the mid-gut of A. spinidens was affected by incubation of the endogenous inhibitors from N. aenescens, H. cunea and P. brassicae, but no inhibitory effects were observed in case of C. suppressalis and E. kuehniella. Extracted inhibitors from C. suppressalis, E. kuehniella and N. aenescens significantly decreased trypsin- and chymotrypsin-like activities in the mid-gut of A. spinidens. All extracted inhibitors significantly decreased elastase-like activity of A. spinidens vs. control, except for C. suppressslis. Extracted inhibitors from all species significantly decreased cathepisns B and L activities but this was not significantly different in case of C. suppressalis. Endogenous inhibitors also affected amino- and carboxypeptidase activities in the mid-gut of A. spinidens. Digestive enzymes of A. spinidens are sensitive to endogenous inhibitors from some preys and this might be the reason that some species are more appropriate than others.

Exclusive rewards in mutualisms: ant proteases and plant protease inhibitors create a lock–key system to protectAcaciafood bodies from exploitation

Myrmecophytic Acacia species produce food bodies (FBs) to nourish ants of the Pseudomyrmex ferrugineus group, with which they live in an obligate mutualism. We investigated how the FBs are protected from exploiting nonmutualists. Two-dimensional gel electrophoresis of the FB proteomes and consecutive protein sequencing indicated the presence of several Kunitz-type protease inhibitors (PIs). PIs extracted from Acacia FBs were biologically active, as they effectively reduced the trypsin-like and elastase-like proteolytic activity in the guts of seed-feeding beetles (Prostephanus truncatus and Zabrotes subfasciatus), which were used as nonadapted herbivores representing potential exploiters. By contrast, the legitimate mutualistic consumers maintained high proteolytic activity dominated by chymotrypsin 1, which was insensitive to the FB PIs. Larvae of an exploiter ant (Pseudomyrmex gracilis) taken from Acacia hosts exhibited lower overall proteolytic activity than the mutualists. The proteases of this exploiter exhibited mainly elastase-like and to a lower degree chymotrypsin 1-like activity. We conclude that the mutualist ants possess specifically those proteases that are least sensitive to the PIs in their specific food source, whereas the congeneric exploiter ant appears partly, but not completely, adapted to consume Acacia FBs. By contrast, any consumption of the FBs by nonadapted exploiters would effectively inhibit their digestive capacities. We suggest that the term 'exclusive rewards' can be used to describe situations similar to the one that has evolved in myrmecophytic Acacia species, which reward mutualists with FBs but safeguard the reward from exploitation by generalists by making the FBs difficult for the nonadapted consumer to use.

Partial characterization of serine peptidases in larvae of Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae), reveals insensitive peptidases to some plant peptidase inhibitors

Digestive serine peptidases are the major proteolytic enzymes in some coleopterans, including Prostephanus truncatus (Horn). These enzymes have become important targets for the control of insect pests by using plant peptidase inhibitors. Recently, the effect of a trypsin inhibitor from Hyptis suaveolens seeds was tested in artificial diets on P. truncatus with no significant effect on its growth and development (Aguirre et al., 2009) suggesting that the proteolytic system of this insect needs to be studied. In this work, the serine peptidases of this coleopteran were analyzed. Three types of serine peptidase activities were detected: trypsin-like activity with the presence of five peptidases between 23 and 100 kDa; elastase-like activity composed of four peptidases between 42 and 68 kDa, one type of chymotrypsin-like activity which recognized a large substrate (CA) and is composed of eight peptidases between 17 and 95 kDa and another type of chymotrypsin-like activity that recognized a short substrate (CB) with a single activity band of apparent molecular mass of 155 kDa. Most of these activities were composed of anionic peptidases with alkaline optimal pH. In addition, the inhibitory effect of two types of peptidase inhibitors (PI's): synthetic and plant peptidase inhibitors (PPI's) were tested. In general, trypsin-like activity was more sensitive to PPI's whereas CB and elastase-like activities unexpectedly, were unaffected. These results reflect the variety of the P. truncatus serine peptidases, revealing also the presence of two groups of PPI-resistant peptidases that could be playing an important role to circumvent the detrimental effect of plant serine peptidase inhibitors.

Protease Activity Increases in Plasma, Peritoneal Fluid, and Vital Organs after Hemorrhagic Shock in Rats

Hemorrhagic shock (HS) is associated with high mortality. A severe decrease in blood pressure causes the intestine, a major site of digestive enzymes, to become permeable – possibly releasing those enzymes into the circulation and peritoneal space, where they may in turn activate other enzymes, e.g. matrix metalloproteinases (MMPs). If uncontrolled, these enzymes may result in pathophysiologic cleavage of receptors or plasma proteins. Our first objective was to determine, in compartments outside of the intestine (plasma, peritoneal fluid, brain, heart, liver, and lung) protease activities and select protease concentrations after hemorrhagic shock (2 hours ischemia, 2 hours reperfusion). Our second objective was to determine whether inhibition of proteases in the intestinal lumen with a serine protease inhibitor (ANGD), a process that improves survival after shock in rats, reduces the protease activities distant from the intestine. To determine the protease activity, plasma and peritoneal fluid were incubated with small peptide substrates for trypsin-, chymotrypsin-, and elastase-like activities or with casein, a substrate cleaved by multiple proteases. Gelatinase activities were determined by gelatin gel zymography and a specific MMP-9 substrate. Immunoblotting was used to confirm elevated pancreatic trypsin in plasma, peritoneal fluid, and lung and MMP-9 concentrations in all samples after hemorrhagic shock. Caseinolytic, trypsin-, chymotrypsin-, elastase-like, and MMP-9 activities were all significantly (p<0.05) upregulated after hemorrhagic shock regardless of enteral pretreatment with ANGD. Pancreatic trypsin was detected by immunoblot in the plasma, peritoneal space, and lungs after hemorrhagic shock. MMP-9 concentrations and activities were significantly upregulated after hemorrhagic shock in plasma, peritoneal fluid, heart, liver, and lung. These results indicate that protease activities, including that of trypsin, increase in sites distant from the intestine after hemorrhagic shock. Proteases, including pancreatic proteases, may be shock mediators and potential targets for therapy in shock.

Biochemical characterization of digestive proteases and carbohydrases of the carob moth, Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae)

Digestive proteinases and carbohydrases of Ectomyelois ceratoniae (Zeller) larvae were investigated using appropriate substrates and inhibitors. Midgut pH in larvae was determined to be slightly alkaline. Midgut extracts showed optimum activity for proteolysis of hemoglobin at pH 9–12. Midgut proteinases also hydrolyzed the synthetic substrates of trypsin, chymotrypsin, and elastase at pH 8–11. Maximum digestive α-amylase activity was also observed at pH 8–11. However, optimum activity for α- and β-glucosidase occurred at pH 5–8. Alpha- and β-galactosidases optimum activities occurred at pH 5 and pH 6, respectively. Inhibitors of serine proteases were effective on midgut serine proteases (trypsin and chymotrypsin proteases). Zymogram analyses revealed at least five bands of total proteolytic activity in the larval midgut. Protease-specific zymogram analyses revealed at least four, two, and one isozymes for trypsin-, chymotrypsin-, and elastase-like activities respectively. Two α-amylase isozymes were found in the midgut of fifth instar larvae and in the whole bodies of 1st through 5th instar larvae. Zymogram studies also revealed the presence of one and two bands of activity for β- and α-glucosidase, respectively. Recycling of α-amylase and proteases in the larval midgut was not complete. At least one isozyme of trypsin, chymotrypsin, elastase, and α-amylase were not recycled and were observed in the larval hindgut.

Periodontal disease in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) and periodontal disease (inflammatory diseases of the tissues around teeth) are common disorders associated with diabetes and cardiometabolic risk. Comprehensively examining the periodontal status in PCOS, this study suggests that the susceptibility for periodontal disease may significantly increase in patients with PCOS compared with healthy young women, and that local/periodontal oxidant status appears to be affected in PCOS.

Elastase-like Enzyme Activity in Cultured Human Fibroblast

Elastase-like Enzyme Activity in Cultured Human Fibroblast

Protease Gene Duplication and Proteolytic Activity in Drosophila Female Reproductive Tracts

Secreted proteases play integral roles in sexual reproduction in a broad range of taxa. In the genetic model Drosophila melanogaster, these molecules are thought to process peptides and activate enzymes inside female reproductive tracts, mediating critical postmating responses. A recent study of female reproductive tract proteins in the cactophilic fruit fly Drosophila arizonae, identified pervasive, lineage-specific gene duplication amongst secreted proteases. Here, we compare the evolutionary dynamics, biochemical nature, and physiological significance of secreted female reproductive serine endoproteases between D. arizonae and its congener D. melanogaster. We show that D. arizonae lower female reproductive tract (LFRT) proteins are significantly enriched for recently duplicated secreted proteases, particularly serine endoproteases, relative to D. melanogaster. Isolated lumen from D. arizonae LFRTs, furthermore, exhibits significant trypsin-like and elastase-like serine endoprotease activity, whereas no such activity is seen in D. melanogaster. Finally, trypsin- and elastase-like activity in D. arizonae female reproductive tracts is negatively regulated by mating. We propose that the intense proteolytic environment of the D. arizonae female reproductive tract relates to the extraordinary reproductive physiology of this species and that ongoing gene duplication amongst these proteases is an evolutionary consequence of sexual conflict.

In vivo inhibition of human neutrophil collagenase (MMP-8) activity during long-term combination therapy of doxycycline and non-steroidal anti-inflammatory drugs (NSAID) in acute reactive arthritis.

We studied the in vivo effect of long-term doxycycline treatment combined with NSAID on human interstitial collagenases, other matrix metalloproteinases, serine proteinases, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and lactoferrin from saliva and serum during the course of acute reactive arthritis (ReA). Collagenase activity and serine proteases (elastase-like, cathepsin G-like and trypsin-like activities) of saliva (n = 10) and gelatinase, lactoferrin and TIMP-1 of saliva (n = 10) and serum (n = 10) samples before and after 2 months doxycycline treatment, combined with NSAID, were studied by quantitative SDS-PAGE assay, ELISA assay and by spectrophotometric assay. The cellular source and molecular forms of salivary collagenase were characterized by immunoblotting using specific antisera. We found that activities of total and endogenously active interstitial collagenase reduced significantly. The salivary collagenase was found to originate from neutrophils. No fragmentation of either pro 75-kD and active 65-kD MMP-8 was detected after 2 months doxycycline treatment. However, during 2 months doxycycline and NSAID treatment no reduction of salivary and serum gelatinase, lactoferrin and TIMP-1-levels and salivary serine protease activities were detected. The in vivo inhibition of collagenase (MMP-8) activity during long-term doxycycline therapy in human saliva containing inflammatory exudate of ReA patients may contribute to the reduced tissue destruction observed in recent clinical and animal model studies in arthritides during long-term doxycycline/tetracycline treatment.

Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays

Proteolytic activities in soluble protein extracts from Mamestra brassicae (cabbage moth) larval midgut were analysed using specific peptide substrates and proteinase inhibitors. Serine proteinases were the major activities detected, with chymotrypsin-like and trypsin-like activities being responsible for approximately 62% and 19% of the total proteolytic activity towards a non-specific protein substrate. Only small amounts of elastase-like activities could be detected. The serine proteinases were active across the pH range 7–12.5, with both trypsin-like and chymotrypsin-like activities maximal at pH 11.5. The digestive proteinases were stable to the alkaline environment of the lepidopteran gut over the timescale of passage of food through the gut, with 50% of trypsin and 40% of chymotrypsin activity remaining after 6 h at pH 12, 37 °C. Soybean Kunitz trypsin inhibitor (SKTI) ingestion by the larvae had a growth-inhibitory effect, and induced inhibitor-insensitive trypsin-like activity. Qualitative and quantitative changes in proteinase activity bands after gel electrophoresis of gut extracts were evident in SKTI-fed larvae when compared with controls, with increases in levels of most bands, appearance of new bands, and a decrease in the major proteinase band present in extracts from control insects.

COMPARISON OF THE PEPTIDASE ACTIVITY IN THE ONCOSPHERE EXCRETORY/SECRETORY PRODUCTS OF TAENIA SOLIUM AND TAENIA SAGINATA

We compared the peptidase activities of the excretory/secretory (E/S) antigens of oncospheres of Taenia solium and related, but nonpathogenic, Taenia saginata. Taenia solium and T. saginata oncospheres were cultured, and the spent media of 24-, 48-, 72-, and 96-hr fractions were analyzed. Activities for serine peptidases (chymotrypsin-, trypsin-, and elastase-like), cysteine peptidases (cathepsin B-, cathepsin L-, and calpaine-like), and aminopeptidase (B-like peptidases) were tested fluorometrically with peptides coupled to 7-amino-4-methylcoumarin. In both species, the E/S antigens showed cysteine, serine, and aminopeptidase activities. Although no particular peptidase had high activity in T. solium, and was absent in T. saginata, or vice versa, different patterns of activity were found. A chymotrypsin-like peptidase showed the highest activity in both parasites, and it had 10 times higher activity in T. solium than in T. saginata. Trypsin-like and cathepsin B-like activities were significantly higher in T. solium. Minimal levels of cathepsin B were present in both species, and higher levels of elastase-like and cathepsin L-like activity were observed in T. saginata. Taenia solium and T. saginata have different levels and temporal activities of proteolytic enzymes that could play a modulator role in the host specificity for larval invasion through penetration of the intestinal mucosa.

Effect of Systemically Administered Naproxen Sodium on Clinical Parameters and Myeloperoxidase and Elastase‐Like Activity Levels in Gingival Crevicular Fluid

The present study was conducted to determine the possible effect of naproxen sodium on clinical status and the enzymatic profile of gingival crevicular fluid (GCF) when given as adjunct to periodontal treatment. A total of 34 subjects with chronic periodontitis were selected and divided into two groups to receive either naproxen sodium or placebo. At baseline, GCF samples were obtained and probing depths (PD), gingival index (GI), plaque index (PI), and gingival bleeding index (GBI) scores were recorded. In the non-steroidal anti-inflammatory drug (NSAID) group, patients were treated with a protocol consisting of baseline periodontal treatment (scaling, root planing) and naproxen sodium (275 mg) administration daily for 6 weeks. In the placebo group, patients received the same treatment except placebo was given instead of naproxen sodium. At the end of the experimental period, clinical recordings and GCF sampling were repeated. Myeloperoxidase (MPO) and elastase-like enzyme activity (ELA) levels were determined in GCF samples by a spectrophotometric method. GCF enzymatic content was calculated both as total enzyme activity and enzyme concentration. All of the clinical parameters, except mean GBI, were significantly lower in the experimental group (P <0.05). At baseline and at the end of the experimental period, there were no significant differences between the NSAID and placebo groups regarding GCF MPO and ELA levels in either mode of data presentation (P <0.05). However, in the NSAID group, mean ELA concentration (P = 0.002) and mean total ELA (P = 0.003) presented significant decreases with treatment. Also, with treatment, a general reduction in MPO levels was seen; however, this difference was not significant. Although constant and stable correlations between GCF enzyme levels and clinical parameters could not be found, positive and strong correlations were observed between total enzyme activity and enzyme concentrations. Based on the positive clinical effect and the ELA profile of GCF, it can be suggested that NSAIDs given as an adjunct to baseline periodontal treatment could be beneficial in the outcome of treatment.

Digestive proteolytic and amylolytic activities in Trogoderma granarium Everts (Dermestidae: Coleoptera)

The Khapra beetle, Trogoderma granarium is a serious post-harvest pest of wheat. Digestive proteinase and amylase activities of larvae and adult insects were investigated using general and specific substrates and inhibitors. Gut pH in larvae was determined to be slightly acidic in the midgut and more acidic in the hindgut. However, midgut extracts from larvae showed an optimum activity for proteolysis against azocasein and haemoglobin at alkaline pH (broad optimum in the range 8–11). Proteinase extracted from larval guts hydrolyzed the synthetic substrates BApNA(N α–benzoyl–L–arginine– p–nitroanilide) and SAAPPpNA (N–succinyl–alanine–alanine–proline–phenylalanine– p–nitroanilide) for trypsin- and chymotrypsin-like activities, respectively. The specific substrate for elastase-like activity SAAApNA (N–succinyl–alanine–alanine–alanine– p–nitroanilide) was not hydrolyzed by midgut extract of larvae. The most effective inhibitors of azocaseinolytic hydrolysis were trypsin and chymotrypsin-like serine proteinase inhibitors. Casein zymogram analysis revealed at least six proteinase bands in the larval midgut extract and showed the presence of trypsin-like serine proteinases. No cysteine proteinases were detected in larval midgut extract using specific cysteine proteinase substrates, inhibitors and activators. Carboxypeptidase A activity also was detected in larval midgut extract using FAPP(N-(3-(2-furyl) acryloyl)- l-phenylalanyl- l-phenylalanine) and showed optimal activity at alkaline pH. Activity of α-amylase using starch as substrate, was determined to be maximal around neutral pH (pH 6–8), which is correlated with the pH prevailing in the midgut. No proteolytic and amylolytic activities were detected using conventional substrates in the digestive system of the adult suggesting no significant protein and carbohydrate digestion occurs in the adult beetle which does not need to feed.

Expression of Matrix Metalloproteinases Subsequent to UrogenitalChlamydia muridarumInfection of Mice

The central hypothesis of this study was that matrix metalloproteinases (MMPs) would be enhanced following murine chlamydial infection and that their expression would vary in mouse strains that differ in their susceptibility to chronic chlamydia-induced disease. To address this hypothesis, female C3H/HeN and C57BL/6 mice were infected intravaginally with Chlamydia muridarum. Uterine and oviduct tissues were assessed for transcription of MMP genes and their tissue inhibitors. An increased activity of MMP genes relative to preinfection tissues was observed in the C3H/HeN mice when compared to C57BL/6 mice. Using gelatin zymography, we detected constitutive MMP-2 activity in both strains of mice but an increase in MMP-9. Casein zymography indicated the presence of two elastase-like activities consistent with MMP-12 and possibly MMP-7. Western blotting and antigen capture enzyme-linked immunoassay also confirmed an increase in MMP-9 but constitutive MMP-2 expression subsequent to the infection in both strains of mice. In C57BL/6 mice, MMP-9 was present in monomer and dimer form throughout the 56-day monitoring period. C3H/HeN mice produced dimeric MMP-9, but increases in the monomer form were also observed through day 14. Post-translational modification of MMP-9 between the two strains also differed. Immunohistochemistry revealed neutrophils as a prominent source for MMP-9 in both strains of mice. We conclude that differences in the relative expression and activity of MMPs, particularly MMP-9, occur in mice differing in their susceptibility to the development of chronic chlamydial disease. These differences may account for disparate outcomes with regard to chronic sequelae of the disease.

Interplay between antibacterial effectors: a macrophage antimicrobial peptide impairs intracellular Salmonella replication.

Antimicrobial peptides have established an important role in the defense against extracellular infections, but the expression of cationic peptides within macrophages as an antibacterial effector mechanism against intracellular pathogens has not been demonstrated. Macrophage expression of the murine cathelicidin-related antimicrobial peptide (CRAMP) was increased after infection by the intracellular pathogen Salmonella typhimurium, and this increase required reactive oxygen intermediates. By using CRAMP-deficient mice or synthetic CRAMP peptide, we found that CRAMP impaired Salmonella cell division in vivo and in vitro, resulting in long filamentous bacteria. This impaired bacterial cell division also depended on intracellular elastase-like serine protease activity, which can proteolytically activate cathelicidins. Macrophage serine protease activity induced filamentation and enhanced the activity of CRAMP in vitro. A peptide-sensitive Salmonella mutant showed enhanced survival within macrophages derived from CRAMP-deficient mice, indicating that Salmonella can sense and respond to cationic peptides in the intracellular environment. Although cationic peptides have been hypothesized to have activity against pathogens within macrophages, this work provides experimental evidence that the antimicrobial arsenal of macrophages includes cathelicidins. These results show that intracellular reactive oxygen intermediates and proteases regulate macrophage CRAMP expression and activity to impair the replication of an intracellular bacterial pathogen, and they highlight the cooperativity between macrophage antibacterial effectors.

Digestive Enzymes and Stylet Morphology of &lt;I&gt;Deraeocoris nigritulus&lt;/I&gt; (Uhler) (Hemiptera: Miridae) Reflect Adaptations for Predatory Habits

Abstract Deraeocoris nigritulus (Uhler) is often collected from the cones of Virginia pine, Pinus virginiana. Most species of Deraeocoris are predacious, but whether this plant bug is phytophagous or predacious is not known. To better understand the feeding adaptations of this mirid, the digestive enzymes from the salivary glands and anterior midgut were analyzed, and the mouthpart stylets were investigated with scanning electron microscopy. Evidence of a trypsin-like enzyme, a chymotrypsin-like enzyme, and pectinase were found in the salivary glands. Low levels of trypsin-like, chymotrypsin-like, elastase-like, pectinase, and α-amylase activities, as well as high levels of α-glucosidase activity, were found in the anterior midgut. The right maxillary stylet has two rows of at least seven strongly recurved teeth in front of at least three weakly recurved teeth on the inner surface, all pointing away from the head. Therefore, this insect is equipped with digestive enzymes and mouthparts mainly adapted for ...

Structure and function of an isozyme of earthworm proteases as a new biocatalyst

The amino acid sequence of the earthworm-serine protease, isozyme C, which shows not only elastase-like activity but also trypsin-like activity, was determined. The catalytic triad of the trypsin family, His, Asp, Ser, was conserved in isozyme C, but the primary substrate determinant of trypsin, Asp, was missing in isozyme C, the same as in elastase. One of the two Gly at the entrance of the substrate-binding pocket of trypsin was replaced by Val as in elastase, however, the other was replaced by Ser whereas Thr is present in elastase. Furthermore, isozyme C also showed esterase-like activity, which was applicable for the synthesis of useful substances.