scholarly journals Proteinases in Excretory-Secretory Products ofToxocara canisSecond-Stage Larvae: Zymography and Modeling Insights

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Gonzalo Ernesto González-Páez ◽  
Fernando Alba-Hurtado ◽  
Carlos Gerardo García-Tovar ◽  
Raúl Argüello-García
Keyword(s):  
Serine Proteinase ◽  
Toxocara Canis ◽  
Serine Proteinases ◽  
Bodily Fluids ◽  
Multiple Components ◽  
Proteolytic Activities ◽  
Secretory Products ◽  
Ph Range ◽  
Physiological Substrates

Components released in excretory-secretory products ofToxocara canislarvae (TES) include phosphatidylethanolamine-binding proteins (TES26), mucins (TES120, MUC2-5), and C-type lectins (TES32, TES70) and their biochemical, immunological, and diagnostic properties have been extensively studied albeit proteinase activities towards physiological substrates are almost unknown. Proteolytic activities in TES samples were first analyzed by gel electrophoresis with gelatin as substrate. Major activities of ~400, 120, and 32 kDa in TES were relatively similar over a broad pH range (5.5–9.0) and all these were of the serine-type as leupeptin abolished gelatinolysis. Further, the ~400 kDa component degraded all physiological substrates tested (laminin, fibronectin, albumin, and goat IgG) and the 120 kDa component degraded albumin and goat IgG while proteinases of lower MW (45, 32, and 26 kDa) only degraded laminin and fibronectin, preferentially at alkaline pH (9.0). By protein modeling approaches using the known sequences of TES components, only TES26 and MUC4 displayed folding patterns significantly related to reference serine proteinases. These data suggest that most of serine proteinase activities secretedin vitroby infective larvae ofT. canishave intriguing nature but otherwise help the parasite to affect multiple components of somatic organs and bodily fluids within the infected host.

An initial characterization of the proteolytic enzymes secreted by the adult stage of the human hookworm Necator americanus

Parasitology ◽  
1995 ◽  
Vol 110 (5) ◽  
pp. 555-563 ◽  
Author(s):  
A. Brown ◽  
J. M. Burleigh ◽  
E. E. Billett ◽  
D. I. Pritchard
Keyword(s):  
Serine Proteases ◽  
Metal Ion ◽  
Proteolytic Enzymes ◽  
Maximum Activity ◽  
Biologically Relevant ◽  
Necator Americanus ◽  
Recent Developments ◽  
Proteolytic Activities ◽  
Secretory Products ◽  
Ph Range

SUMMARYThe proteolytic activities present in adult Necator americanus excretory–secretory products have been assessed using biologically relevant, naturally occurring substrates (haemoglobin and fibrinogen) and a number of synthetic fluorogenic and chromogenic substrates. One broad peak of activity was observed against haemoglobin in the pH range 5 to 7, with maximum activity at pH 6·6, while fibrinogenolytic activity was shown to be greater at pH 3·5. Inhibition studies against haemoglobin, fibrinogen and synthetic substrates using a battery of appropriate protease inhibitors indicated the presence of a mixture of aspartyl, cysteinyl and serine proteases. Metal ion (Ca2+, Zn2+ and Fe2+) stimulation was demonstrated, with stimulation by Zn2+ being the most marked. These results are discussed in the context of recent developments in the field of parasite proteolytic enzymes, where they have been suggested as targets for immuno- and chemotherapy.

Proteinases in the excretory/secretory products (ES) of adult Trichinella spiralis

Parasitology ◽  
1995 ◽  
Vol 111 (2) ◽  
pp. 201-208 ◽  
Author(s):  
V. K. Todorova ◽  
D. P. Knox ◽  
M. W. Kennedy
Keyword(s):  
Size Range ◽  
Proteinase Inhibitors ◽  
Trichinella Spiralis ◽  
Proteinase Activity ◽  
Serine Proteinases ◽  
Protein Substrates ◽  
Defined Media ◽  
Metalloproteinase Inhibitors ◽  
Secretory Products

SUMMARYAdult Trichinella spiralis were maintained in vitro using defined media and the material excreted/secreted (ES) during this time examined for proteolytic enzyme (proteinase) activity using an azocasein assay and gelatin-substrate gels. Several discrete proteinases in the size range 14–100 kDa were observed with optimal activity at pH 7·5. The use of a class-differentiating panel of proteinase inhibitors indicated that serine proteinases were predominant although some inhibition was evident in the presence of cysteine and metalloproteinase inhibitors. Of a panel of potential natural protein substrates tested, ES proteinases only degraded fibrinogen and plasminogen and degradation was, in part, susceptible to the action of serine, cysteine and aspartyl proteinase inhibitors. In addition, antibody harvested from immune but not normal mice inhibited ES proteinase activity, an observation of relevance to the immunobiology of Trichinosis.

scholarly journals Extracellular proteolytic activities expressed by Bacillus pumilus isolated from endodontic and periodontal lesions

2008 ◽  
Vol 57 (5) ◽  
pp. 643-651 ◽  
Author(s):  
Blair T. Johnson ◽  
Lindsey N. Shaw ◽  
Daniel C. Nelson ◽  
John A. Mayo
Keyword(s):  
Proteolytic Activity ◽  
Bacillus Pumilus ◽  
Serine Proteinase ◽  
Glutamyl Endopeptidase ◽  
Rrna Gene ◽  
Root Canals ◽  
Carbohydrate Fermentation ◽  
Proteolytic Activities ◽  
Marginal Periodontitis

The purpose of the present study was to identify 12 Bacillus isolates that had been obtained from root canals of teeth requiring endodontic therapy and from periodontal pockets in severe marginal periodontitis, and to determine whether these isolates exhibited extracellular proteolytic activity and, using in vitro assays, whether any such activity could degrade substrates that would be pathophysiologically relevant with regard to the production of endodontic and periodontal lesions. Biochemical and carbohydrate fermentation patterns were used in the identification of all strains, which was confirmed by determination of the16S rRNA gene sequence for strain BJ0055. Screening for production of extracellular proteolytic activity by all strains was done with a general proteinase substrate. All isolates were identified as representing Bacillus pumilus and all exhibited extracellular proteolytic activity. The putative pathophysiological relevance of extracellular proteinase production in strain BJ0055 was assessed using fluorophore-labelled elastin and collagen and several chromogenic peptides. Probable classes of proteinases acting on each substrate were investigated using class-specific inhibitors. Activity–pH profiles were determined in buffers at different pH values. Extracellular activities that were caseinolytic, elastinolytic, collagenolytic, glutamyl endopeptidase-like, and alanyl tripeptidyl peptidase-like were observed. No trypsin-like activities were detected. Serine- and chymotrypsin-like serine proteinase activities were detected, with activity observed at neutral and alkaline, but not acidic, pH. B. pumilus strains isolated from endodontic and periodontal lesions exhibited extracellular activities that degrade elastin, collagen and other substrates. These activities may be virulence factors that contribute to tissue damage in apical periodontitis and severe marginal periodontitis.

Classes and crossreactivity of proteinases in the excretory–secretory products of Caenorhabditis elegans

2007 ◽  
Vol 81 (1) ◽  
pp. 93-99
Author(s):  
S. Nic an Ultaigh ◽  
M.F. Ryan
Keyword(s):  
Caenorhabditis Elegans ◽  
Ph Value ◽  
Cross Reactivity ◽  
Serine Proteinases ◽  
Cysteine Proteinases ◽  
C Elegans ◽  
Antigenic Relatedness ◽  
Free Living Nematode ◽  
Secretory Products

AbstractProteinases released during the in vitro maintenance of asynchronous cultures of the free-living nematode Caenorhabditis elegans were characterized on the basis of subunit composition, fluorogenic substrate specificity, inhibitor sensitivity and pH optima. Cysteine proteinases are present in the excretory–secretory products (ESP) as indicated by the hydrolysis of cathepsin fluorogenic substrates and confirmed by immunoblotting. Serine proteinases were predominant as indicated by substrate gel analysis and inhibitor studies. The presence of metallo-proteinases was also indicated by inhibitor studies. The optimal pH value for cysteine proteinases was 5.5, while serine proteinases were optimal at pH 8.0. As a control, cultures of Escherichia coli, the diet of C. elegans, were extracted separately and gave no evidence of overlap with C. elegans ESP. Cross reactivity between the ESP of C. elegans and antibodies raised against the ESP of the equine parasite Strongylus vulgaris indicated antigenic relatedness of a proteic epitope. This is the first study to characterize the ESP of C. elegans and to display its relatedness with that of S. vulgaris.

The use of 35S-labelled Toxocara canis to determine larval numbers in tissues

1992 ◽  
Vol 66 (4) ◽  
pp. 279-287 ◽  
Author(s):  
K. C. Carter
Keyword(s):  
Post Infection ◽  
Half Life ◽  
Toxocara Canis ◽  
Second Stage ◽  
Infective Larvae ◽  
Secretory Products ◽  
Formed Part ◽  
Antibody Levels ◽  
The Brain

ABSTRACTThe potential of using 35S-labelled larvae to determine the number of second-stage Toxocara canis larvae present in the tissues of infected animals was assessed. Infective larvae were labelled by in vitro culture with medium containing 35S-methionine. The amount of radiolabel attached to larvae decayed exponentially with time and had an in vitro mean half life of 3·54±0·65 days. The ‘lost’ radiolabel was incorporated into proteins which formed part of the worm's excretory/secretory products. The levels of radioactivity present in different organs of BALB/c mice, infected with 35S-labelled T. canis larvae, varied over the course of infection. Initially most of the radioactivity was present in liver, but over the course of infection 35S liver levels gradually decreased and brain levels increased. By day 14 post-infection the majority of the isotope was present in the brain (p<0·01). Assessment of antibody levels on day 14 post-infection showed that infection with 35S labelled T. canis larvae induced the production of parasite-specific IgM, IgG and IgG1 antibodies.

scholarly journals A rapid, parasite-dependent cellular response to Dirofilaria immitis in the Mongolian jird (Meriones unguiculatus)

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Christopher C. Evans ◽  
Katherine M. Day ◽  
Yi Chu ◽  
Bridget Garner ◽  
Kaori Sakamoto ◽  
...  
Keyword(s):  
Cellular Response ◽  
Dirofilaria Immitis ◽  
Cell Attachment ◽  
Early Response ◽  
Meriones Unguiculatus ◽  
Filarial Parasite ◽  
Immune Mediated ◽  
Secretory Products ◽  
Species Specific

Abstract Background The Mongolian jird (Meriones unguiculatus) has long been recognized as a permissive host for the filarial parasite Brugia malayi; however, it is nonpermissive to another filarial parasite, canine heartworm (Dirofilaria immitis). By elucidating differences in the early response to infection, we sought to identify mechanisms involved in the species-specific clearance of these parasites. We hypothesized that the early clearance of D. immitis in intraperitoneal infection of the jird is immune mediated and parasite species dependent. Methods Jird peritoneal exudate cells (PECs) were isolated and their attachment to parasite larvae assessed in vitro under various conditions: D. immitis and B. malayi cultured separately, co-culture of both parasites, incubation before addition of cells, culture of heat-killed parasites, and culture with PECs isolated from jirds with mature B. malayi infection. The cells attaching to larvae were identified by immunohistochemistry. Results In vitro cell attachment to live D. immitis was high (mean = 99.6%) while much lower for B. malayi (mean = 5.56%). This species-specific attachment was also observed when both filarial species were co-cultured, with no significant change from controls (U(9, 14) = 58.5, p = 0.999). When we replicated these experiments with PECs derived from jirds subcutaneously infected with B. malayi, the results were similar (99.4% and 4.72% of D. immitis and B. malayi, respectively, exhibited cell attachment). Heat-killing the parasites significantly reduced cell attachment to D. immitis (mean = 71.9%; U(11, 14) = 7.5, p < 0.001) while increasing attachment to B. malayi (mean = 16.7%; U(9, 15) = 20, p = 0.002). Cell attachment to both species was reduced when larvae were allowed a 24-h pre-incubation period prior to the addition of cells. The attaching cells were identified as macrophages by immunohistochemistry. Conclusions These results suggest a strongly species-dependent response from which B. malayi could not confer protection by proxy in co-culture. The changes in cell attachment following heat-killing and pre-incubation suggest a role for excretory/secretory products in host immune evasion and/or antigenicity. The nature of this attachment is the subject of ongoing study and may provide insight into filarial host specificity.

scholarly journals The Responses of Bioactive Betanin Pigment and Its Derivatives from a Red Beetroot (Beta vulgaris L.) Betalain-Rich Extract to Hypochlorous Acid

2021 ◽  
Vol 22 (3) ◽  
pp. 1155
Author(s):  
Karolina Starzak ◽  
Katarzyna Sutor ◽  
Tomasz Świergosz ◽  
Boris Nemzer ◽  
Zbigniew Pietrzkowski ◽  
...  
Keyword(s):  
Beta Vulgaris ◽  
Hypochlorous Acid ◽  
Innate Immune ◽  
Inflammatory Factors ◽  
Oxygen Species ◽  
Plant Pigments ◽  
Important Food ◽  
Inflammatory Conditions ◽  
Ph Range

Neutrophils produce hypochlorous acid (HOCl) as well as other reactive oxygen species as part of a natural innate immune response in the human body; however, excessive levels of HOCl can ultimately be detrimental to health. Recent reports suggest that betacyanin plant pigments can act as potent scavengers of inflammatory factors and are notably effective against HOCl. Comparison of the in vitro anti-hypochlorite activities of a novel betalain-rich red beetroot (Beta vulgaris L.) extract with its pure betalainic pigments revealed that the extract had the highest anti-hypochlorite activity, far exceeding the activity of all of the betalainic derivatives and selected reference antioxidants. This suggests that it may be an important food-based candidate for management of inflammatory conditions induced by excessive HOCl production. Among all pigments studied, betanidin exhibited the highest activity across the pH range.

scholarly journals Newly Emerging Airborne Pollutants: Current Knowledge of Health Impact of Micro and Nanoplastics

2021 ◽  
Vol 18 (6) ◽  
pp. 2997
Author(s):  
Alessio Facciolà ◽  
Giuseppa Visalli ◽  
Marianna Pruiti Ciarello ◽  
Angela Di Pietro
Keyword(s):  
Surface Area ◽  
Current Knowledge ◽  
Oxidative Degradation ◽  
Health Impact ◽  
Outdoor Exposure ◽  
Environmental Matrices ◽  
Potential Health ◽  
Bodily Fluids

Plastics are ubiquitous persistent pollutants, forming the most representative material of the Anthropocene. In the environment, they undergo wear and tear (i.e., mechanical fragmentation, and slow photo and thermo-oxidative degradation) forming secondary microplastics (MPs). Further fragmentation of primary and secondary MPs results in nanoplastics (NPs). To assess potential health damage due to human exposure to airborne MPs and NPs, we summarize the evidence collected to date that, however, has almost completely focused on monitoring and the effects of airborne MPs. Only in vivo and in vitro studies have assessed the toxicity of NPs, and a standardized method for their analysis in environmental matrices is still missing. The main sources of indoor and outdoor exposure to these pollutants include synthetic textile fibers, rubber tires, upholstery and household furniture, and landfills. Although both MPs and NPs can reach the alveolar surface, the latter can pass into the bloodstream, overcoming the pulmonary epithelial barrier. Despite the low reactivity, the number of surface area atoms per unit mass is high in MPs and NPs, greatly enhancing the surface area for chemical reactions with bodily fluids and tissue in direct contact. This is proven in polyvinyl chloride (PVC) and flock workers, who are prone to persistent inflammatory stimulation, leading to pulmonary fibrosis or even carcinogenesis.

Characterization of excretory–secretory products from protoscoleces of Echinococcus granulosus and evaluation of their potential for immunodiagnosis of human cystic echinococcosis

Parasitology ◽  
2004 ◽  
Vol 129 (3) ◽  
pp. 371-378 ◽  
Author(s):  
D. CARMENA ◽  
J. MARTÍNEZ ◽  
A. BENITO ◽  
J. A. GUISANTES
Keyword(s):  
Echinococcus Granulosus ◽  
Cystic Echinococcosis ◽  
Secretory Protein ◽  
Major Protein ◽  
Healthy Donors ◽  
Secretory Products ◽  
Protein Components ◽  
Human Cystic Echinococcosis

This study describes, for the first time, the characterization of excretory–secretory antigens (ES-Ag) from Echinococcus granulosus protoscoleces, evaluating their usefulness in the immunodiagnosis of human cystic echinococcosis. ES-Ag were obtained from the first 50 h maintenance of protoscoleces in vitro. This preparation contained over 20 major protein components which could be distinguished by 1-dimensional SDS–PAGE with apparent masses between 9 and 300 kDa. The culture of of protoscoleces from liver produced a greater variety of excretory–secretory protein components than those from lung. Determination of enzymatic activities of secreted proteins revealed the presence of phosphatases, lipases and glucosidases, but no proteases. These findings were compared to those obtained from somatic extracts of protoscoleces and hydatid cyst fluid products. Immunochemical characterization was performed by immunoblotting with sera from individuals infected by cystic echinococcosis (n=15), non-hydatidic parasitoses (n=19), various liver diseases (n=24), lung neoplasia (n=16), and healthy donors (n=18). Antigens with apparent masses of 89, 74, 47/50, 32, and 20 kDa showed specificity for immunodiagnosis of human hydatidosis. The 89 and 74 kDa components corresponded to antigens not yet described in E. granulosus, whereas proteins of 41–43 kDa and 91–95 kDa were recognized by the majority of the non-hydatid sera studied.

Metalloproteases and egg-hatching in Pediculus humanus, the body (clothes) louse of humans (Phthiraptera: Insecta)

Parasitology ◽  
2007 ◽  
Vol 135 (1) ◽  
pp. 125-130 ◽  
Author(s):  
V. M. BOWLES ◽  
A. R. YOUNG ◽  
S. C. BARKER
Keyword(s):  
Protease Activity ◽  
The Body ◽  
Egg Hatching ◽  
Egg Hatch ◽  
Pediculus Humanus ◽  
Sds Page ◽  
Metalloprotease Inhibitors ◽  
Egg Shell ◽  
Ph Range

SUMMARYTo investigate the biochemical components of egg-hatch in the body louse, Pediculus humanus, egg-shell-washings (ESW) were collected during the first 2 h post-hatching and analysed by gelatin SDS-PAGE. These ESW contained proteases with molecular mass in the range of 25–100 kDa; the most abundant proteases were ~25 kDa. The 3 main regions of protease activity in the one-dimensional gelatin SDS-PAGE gels resolved to at least 23 distinct regions of protease activity when analysed by two-dimensional gelatin SDS-PAGE, with iso-electric points spread over the entire 3 to 10 pH range. Mechanistic characterization indicated that the ESW contained proteases of the metallo-class, inhibited by both 1,10-phenanthroline and EDTA. Several protease inhibitors were tested for their ability to inhibit louse egg-hatch in vitro. The metalloprotease inhibitor 1,10-phenanthroline and the aminopeptidase inhibitor bestatin significantly inhibited (P<0·05) louse egg-hatch (100% and 58%, respectively). The presence of metalloproteases at the time of egg-hatch and the inhibition of egg-hatch in P. humanus by metalloprotease inhibitors suggests a crucial role for these proteases in the hatching of this medically important parasite.

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