In vitro proteolysis of nematode FMRFamide-like peptides (FLPs) by preparations from a free-living nematode (Panagrellus redivivus) and two plant-parasitic nematodes (Heteroderaglycines and Meloidogyne incognita)

2011 ◽  
Vol 86 (1) ◽  
pp. 77-84 ◽  
Author(s):  
E.P. Masler
Keyword(s):  
Cathepsin L ◽  
Cysteine Proteases ◽  
Developmental Differences ◽  
Parasitic Nematodes ◽  
Free Living ◽  
Additional Species ◽  
Substrate Preferences ◽  
Panagrellus Redivivus ◽  
Species Specific

AbstractProteolytic activities in extracts from three nematodes, the plant parasites Heterodera glycines and Meloidogyneincognita, and the free-living Panagrellus redivivus, were surveyed for substrate preferences using a battery of seven FRET-modified peptide substrates, all derived from members of the large FMRF-amide like peptide (FLP) family in nematodes. Overall protease activity in P. redivivus was four- to fivefold greater than in either of the parasites, a result that might reflect developmental differences. Digestion of the M. incognita FLP KHEFVRFa (substrate Abz-KHEFVRF-Y(3-NO2)a) by M. incognita extract was sevenfold greater than with H. glycines extract and twofold greater than P. redivivus, suggesting species-specific preferences. Additional species differences were revealed upon screening 12 different protease inhibitors. Two substrates were used in the screen, Abz-KHEFVRF-Y(3-NO2)a and Abz-KPSFVRF-Y(3-NO2)a), which was digested equally by all three species. The effects of various inhibitor, substrate and extract source combinations on substrate digestion suggest that M. incognita differs significantly from P. redivivus and H. glycines in its complement of cysteine proteases, particularly cathepsin L-type protease.

scholarly journals Anthelmintic tolerance in free-living and facultative parasitic isolates of Halicephalobus (Panagrolaimidae)

Parasitology ◽  
2012 ◽  
Vol 139 (10) ◽  
pp. 1301-1308 ◽  
Author(s):  
P. FONDERIE ◽  
W. BERT ◽  
F. HENDRICKX ◽  
W. HOUTHOOFD ◽  
T. MOENS
Keyword(s):  
Larval Development ◽  
Anthelmintic Resistance ◽  
Positive Control ◽  
Parasitic Nematodes ◽  
Free Living ◽  
Egg Hatching ◽  
Complete Life Cycle ◽  
Panagrellus Redivivus ◽  
Halicephalobus Gingivalis

SUMMARYStudies on anthelmintic resistance in equine parasites do not include facultative parasites. Halicephalobus gingivalis is a free-living bacterivorous nematode and a known facultative parasite of horses with a strong indication of some form of tolerance to common anthelmintic drugs. This research presents the results of an in vitro study on the anthelmintic tolerance of several isolates of Halicephalobus to thiabendazole and ivermectin using an adaptation of the Micro-Agar Larval Development Test hereby focusing on egg hatching and larval development. Panagrellus redivivus and Panagrolaimus superbus were included as a positive control. The results generally show that the anthelmintic tolerance of Halicephalobus to both thiabendazole and ivermectin was considerably higher than that of the closely related Panagrolaimidae and, compared to other studies, than that of obligatory equine parasites. Our results further reveal a remarkable trend of increasing tolerance from fully free-living isolates towards horse-associated isolates. In vitro anthelmintic testing with free-living and facultative parasitic nematodes offers the advantage of observing drug effect on the complete life cycle as opposed to obligatory parasites that can only be followed until the third larval stage. We therefore propose Halicephalobus gingivalis as an experimental tool to deepen our understanding of the biology of anthelmintic tolerance.

scholarly journals Microfluidic deep mutational scanning of the human executioner caspases reveals differences in structure and regulation

2022 ◽  
Vol 8 (1) ◽  
Author(s):  
Hridindu Roychowdury ◽  
Philip A. Romero
Keyword(s):  
Large Scale ◽  
Cysteine Proteases ◽  
Amino Acid Substitutions ◽  
Substrate Preferences ◽  
Functional Differences ◽  
Caspase Family ◽  
Executioner Caspases ◽  
The Impact ◽  
Human Caspase

AbstractThe human caspase family comprises 12 cysteine proteases that are centrally involved in cell death and inflammation responses. The members of this family have conserved sequences and structures, highly similar enzymatic activities and substrate preferences, and overlapping physiological roles. In this paper, we present a deep mutational scan of the executioner caspases CASP3 and CASP7 to dissect differences in their structure, function, and regulation. Our approach leverages high-throughput microfluidic screening to analyze hundreds of thousands of caspase variants in tightly controlled in vitro reactions. The resulting data provides a large-scale and unbiased view of the impact of amino acid substitutions on the proteolytic activity of CASP3 and CASP7. We use this data to pinpoint key functional differences between CASP3 and CASP7, including a secondary internal cleavage site, CASP7 Q196 that is not present in CASP3. Our results will open avenues for inquiry in caspase function and regulation that could potentially inform the development of future caspase-specific therapeutics.

A possible alternative mechanism of kinin generation in vivo by cathepsin L

2005 ◽  
Vol 386 (7) ◽  
pp. 699-704 ◽  
Author(s):  
Luciano Puzer ◽  
Juliana Vercesi ◽  
Marcio F.M. Alves ◽  
Nilana M.T. Barros ◽  
Mariana S. Araujo ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Enzyme Inhibitor ◽  
Alternative Pathway ◽  
Cathepsin L ◽  
Specific Inhibitor ◽  
Cysteine Proteases ◽  
Hypotensive Effect ◽  
Alternative Mechanism

Abstract We investigated the ability of cathepsin L to induce a hypotensive effect after intravenous injection in rats and correlated this decrease in blood pressure with kinin generation. Simultaneously with blood pressure decrease, we detected plasma kininogen depletion in the treated rats. The effect observed in vivo was abolished by pre-incubation of cathepsin L with the cysteine peptidase-specific inhibitor E-64 (1 μM) or by previous administration of the bradykinin B2 receptor antagonist JE049 (4 mg/kg). A potentiation of the hypotensive effect caused by cathepsin L was observed by previous administration of the angiotensin I-converting enzyme inhibitor captopril (5 mg/kg). In vitro studies indicated that cathepsin L excised bradykinin from the synthetic fluorogenic peptide Abz-MTSVIRRPPGFSPFRAPRV-NH2, based on the Met375–Val393 sequence of rat kininogen (Abz=o-aminobenzoic acid). In conclusion, our data indicate that in vivo cathepsin L releases a kinin-related peptide, and in vitro experiments suggest that the kinin generated is bradykinin. Although it is well known that cysteine proteases are strongly inhibited by kininogen, cathepsin L could represent an alternative pathway for kinin production in pathological processes.

Biological attributes of Salibro™, a novel sulfonamide nematicide. Part 2: Impact on the fitness of various non-target nematodes

Nematology ◽  
2020 ◽  
pp. 1-17 ◽  
Author(s):  
Tim C. Thoden ◽  
Mariam A. Alkader ◽  
John A. Wiles
Keyword(s):  
Negative Impact ◽  
Soil Health ◽  
Agar Plate ◽  
Parasitic Nematodes ◽  
Published Data ◽  
Plant Parasitic Nematodes ◽  
Free Living ◽  
Plant Parasitic ◽  
Novel Products

Summary Currently a renaissance in chemical nematicides is taking place with novel products like Nimitz® (a.s. fluensulfone), Velum Prime® (a.s. fluopyram) and Salibro™ (a.s. fluazaindolizine – Reklemel™ active) entering the marketplace. Although a considerable amount of published data is already available on their laboratory and field impact on plant-parasitic nematodes, little is understood of their compatibility with the beneficial or free-living nematodes that are part of the soil health network. In a range of laboratory studies, the effects of these nematicides on the vitality and reproduction of several species was tested, including both cosmopolitan free-living nematodes (Acrobeloides, Cruznema, Panagrobelus) as well as commercially applied entomopathogenic nematodes (Steinernema, Heterorhabditis). Within aqueous exposure and agar plate in vitro assays, species sensitivity to those nematicides differed significantly but their fitness (vitality and reproduction; infectivity to insect hosts) was generally not adversely impacted by concentrations of 5-50 ppm (a.s.) of Salibro. Even at 250 ppm (a.s.) of Salibro only some species of the bacterial-feeding species showed some negative impact. By contrast, both Nimitz at 50 ppm (a.s.) and Velum at 5 ppm (a.s.) consistently demonstrated stronger adverse impacts. In second level soil drenching assays, either no, or occasionally slight, adverse effects on the natural community of free-living nematodes were observed if soils were drenched with different volumes of Salibro at 5-50 ppm (a.s.), while relatively stronger reductions were measured within the plant-parasitic species (especially root-knot nematodes). Both Nimitz and Vydate (a.s. oxamyl) showed some degree of compatibility at 5 and 25 ppm (a.s.), respectively, which was generally higher than for Velum Prime at 5 ppm (a.s.). Overall, these data indicate that, when used at common field rates, Salibro will be one of the best options as part of integrated nematode management programmes where the use of chemical nematicides is required.

scholarly journals An Enterotoxin-Like Binary Protein from Pseudomonas protegens with Potent Nematicidal Activity

2017 ◽  
Vol 83 (19) ◽  
Author(s):  
Jun-Zhi Wei ◽  
Daniel L. Siehl ◽  
Zhenglin Hou ◽  
Barbara Rosen ◽  
Jarred Oral ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Caenorhabditis Elegans ◽  
Cholera Toxin ◽  
Nematicidal Activity ◽  
Binary Toxin ◽  
Parasitic Nematodes ◽  
Free Living ◽  
Subunit A ◽  
Content Type ◽  
Panagrellus Redivivus

ABSTRACT Soil microbes are a major food source for free-living soil nematodes. It is known that certain soil bacteria have evolved systems to combat predation. We identified the nematode-antagonistic Pseudomonas protegens strain 15G2 from screening of microbes. Through protein purification we identified a binary protein, designated Pp-ANP, which is responsible for the nematicidal activity. This binary protein inhibits Caenorhabditis elegans growth and development by arresting larvae at the L1 stage and killing older-staged worms. The two subunits, Pp-ANP1a and Pp-ANP2a, are active when reconstituted from separate expression in Escherichia coli. The binary toxin also shows strong nematicidal activity against three other free-living nematodes (Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp.), but we did not find any activity against insects and fungi under test conditions, indicating specificity for nematodes. Pp-ANP1a has no significant identity to any known proteins, while Pp-ANP2a shows ∼30% identity to E. coli heat-labile enterotoxin (LT) subunit A and cholera toxin (CT) subunit A. Protein modeling indicates that Pp-ANP2a is structurally similar to CT/LT and likely acts as an ADP-ribosyltransferase. Despite the similarity, Pp-ANP shows several characteristics distinct from CT/LT toxins. Our results indicate that Pp-ANP is a new enterotoxin-like binary toxin with potent and specific activity to nematodes. The potency and specificity of Pp-ANP suggest applications in controlling parasitic nematodes and open an avenue for further research on its mechanism of action and role in bacterium-nematode interaction. IMPORTANCE This study reports the discovery of a new enterotoxin-like binary protein, Pp-ANP, from a Pseudomonas protegens strain. Pp-ANP shows strong nematicidal activity against Caenorhabditis elegans larvae and older-staged worms. It also shows strong activity on other free-living nematodes (Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp.). The two subunits, Pp-ANP1a and Pp-ANP2a, can be expressed separately and reconstituted to form the active complex. Pp-ANP shows some distinct characteristics compared with other toxins, including Escherichia coli enterotoxin and cholera toxin. The present study indicates that Pp-ANP is a novel binary toxin and that it has potential applications in controlling parasitic nematodes and in studying toxin-host interaction.

scholarly journals IN VITRO EFFECT OF L-CARNITINE ON THE ACTIVITY OF LYSOSOMAL CYSTEINE PROTEASES AND THE STATE OF LYSOSOMAL MEMBRANE

2017 ◽  
Vol 25 (1) ◽  
pp. 14-20 ◽  
Author(s):  
M A. Fomina ◽  
A M. Kudlaeva ◽  
A N. Ryabkov
Keyword(s):  
Acid Phosphatase ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Lysosomal Membrane ◽  
Female Rats ◽  
In Vitro Incubation ◽  
Cathepsin H ◽  
Vitro Effect

The influence of L-carnitine in vitro on the lysosomal cysteine proteinase activity and stability of the lysosomal membrane of the liver homogenates of intact sexually Mature female rats of Wistar line weighing 280-330 g were studied. In the experimental groups isolated lysosomes were incubated in vitro in a solution of L-carnitine during 1, 2 and 4 hours, in the control groups in vitro incubation was carried out in a medium of isolating solution. The activity of ca-thepsins B, L and H was investigated by spectrofluorimetric method of Barrett & Kirschke in two fractions - lysosomal and outside of lysosomes. The activity of acid phosphatase was used as the main marker of a membrane labilization. In vitro incubation of lysosomes showed that carnitine at a concentration of 5 mM increases the total activity of cathepsin B in a one-hour incubation at 73,2% (p=0,008), cathepsin L in a two- and four-hour incubation - at 77,7% (p=0,005) and 42,3% (p=0,013) respectively, and reduces the overall activity of the cathepsin H in a one-hour incubation at 200,0% (p=0,008), in a two-hour - by 67,9% (p=0,05), in a four-hour -27,1% (p=0,02). In addition, incubation in 5 mM L-carnitine solution leads to an increase of unsedimentable activity and fall sedimentaries activity for cathepsin L in a two-hour, and for acid phosphatase - in a two - and four-hour exposure. 5 mM L-carnitine in one - and two-hour incubation stabilizes lysosomal membrane (whereas increase in incubation time up to 4 hours leads to its damage) and increases the selective permeability of the lysosomal membrane for the studied cathepsins, to the greatest extent - for cathepsin H.

scholarly journals Development of a New Antileishmanial Aziridine-2,3-Dicarboxylate-Based Inhibitor with High Selectivity for Parasite Cysteine Proteases

2015 ◽  
Vol 60 (2) ◽  
pp. 797-805 ◽  
Author(s):  
Caroline Schad ◽  
Ulrike Baum ◽  
Benjamin Frank ◽  
Uwe Dietzel ◽  
Felix Mattern ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Neglected Tropical Diseases ◽  
Cathepsin L ◽  
Selective Inhibition ◽  
Cysteine Proteases ◽  
Host Cells ◽  
Th2 Response ◽  
Content Type

ABSTRACTLeishmaniasis is one of the major neglected tropical diseases of the world. Druggable targets are the parasite cysteine proteases (CPs) of clan CA, family C1 (CAC1). In previous studies, we identified two peptidomimetic compounds, the aziridine-2,3-dicarboxylate compounds 13b and 13e, in a series of inhibitors of the cathepsin L (CL) subfamily of the papain clan CAC1. Both displayed antileishmanial activityin vitrowhile not showing cytotoxicity against host cells. In further investigations, the mode of action was characterized inLeishmania major. It was demonstrated that aziridines 13b and 13e mainly inhibited the parasitic cathepsin B (CB)-like CPC enzyme and, additionally, mammalian CL. Although these compounds induced cell death ofLeishmaniapromastigotes and amastigotesin vitro, the induction of a proleishmanial T helper type 2 (Th2) response caused by host CL inhibition was observedin vivo. Therefore, we describe here the synthesis of a new library of more selective peptidomimetic aziridine-2,3-dicarboxylates discriminating between host and parasite CPs. The new compounds are based on 13b and 13e as lead structures. One of the most promising compounds of this series is compound s9, showing selective inhibition of the parasite CPsLmaCatB (a CB-like enzyme ofL. major; also namedL. majorCPC) andLmCPB2.8 (a CL-like enzyme ofLeishmania mexicana) while not affecting mammalian CL and CB. It displayed excellent leishmanicidal activities againstL. majorpromastigotes (50% inhibitory concentration [IC50] = 37.4 μM) and amastigotes (IC50= 2.3 μM). In summary, we demonstrate a new selective aziridine-2,3-dicarboxylate, compound s9, which might be a good candidate for futurein vivostudies.

scholarly journals The Intestinal Protozoan Parasite Entamoeba histolytica Contains 20 Cysteine Protease Genes, of Which Only a Small Subset Is Expressed during In Vitro Cultivation

2003 ◽  
Vol 2 (3) ◽  
pp. 501-509 ◽  
Author(s):  
Iris Bruchhaus ◽  
Brendan J. Loftus ◽  
Neil Hall ◽  
Egbert Tannich
Keyword(s):  
Entamoeba Histolytica ◽  
Cysteine Protease ◽  
Cathepsin L ◽  
Protozoan Parasite ◽  
Cysteine Proteases ◽  
Small Subset ◽  
In Vitro Cultivation ◽  
Intestinal Protozoan ◽  
Parasite Life Cycle

ABSTRACT Cysteine proteases are known to be important pathogenicity factors of the protozoan parasite Entamoeba histolytica. So far, a total of eight genes coding for cysteine proteases have been identified in E. histolytica, two of which are absent in the closely related nonpathogenic species E. dispar. However, present knowledge is restricted to enzymes expressed during in vitro cultivation of the parasite, which might represent only a subset of the entire repertoire. Taking advantage of the current E. histolytica genome-sequencing efforts, we analyzed databases containing more than 99% of all ameba gene sequences for the presence of cysteine protease genes. A total of 20 full-length genes was identified (including all eight genes previously reported), which show 10 to 86% sequence identity. The various genes obviously originated from two separate ancestors since they form two distinct clades. Despite cathepsin B-like substrate specificities, all of the ameba polypeptides are structurally related to cathepsin L-like enzymes. None of the previously described enzymes but 7 of the 12 newly identified proteins are unique compared to cathepsins of higher eukaryotes in that they are predicted to have transmembrane or glycosylphosphatidylinositol anchor attachment domains. Southern blot analysis revealed that orthologous sequences for all of the newly identified proteases are present in E. dispar. Interestingly, the majority of the various cysteine protease genes are not expressed in E. histolytica or E. dispar trophozoites during in vitro cultivation. Therefore, it is likely that at least some of these enzymes are required for infection of the human host and/or for completion of the parasite life cycle.

scholarly journals Curtisia dentata (Cornaceae) leaf extracts and isolated compounds inhibit motility of parasitic and free-living nematodes

2009 ◽  
Vol 76 (2) ◽  
Author(s):  
L.J. Shai ◽  
E.S. Bizimenyera ◽  
V. Bagla ◽  
L.J. McGaw ◽  
J.N. Eloff
Keyword(s):  
Medicinal Plant ◽  
Betulinic Acid ◽  
Small Ruminants ◽  
Anthelmintic Activity ◽  
Parasitic Nematodes ◽  
Leaf Extracts ◽  
Free Living ◽  
C Elegans

Haemonchus contortus and Trichostrongylus colubriformis are among the most important parasitic nematodes of small ruminants. Caenorhabditis elegans, a free-living nematode, is used as a model for evaluating anthelmintic activity of a variety of test substances. Extracts of several medicinal plants are useful in vitro and in vivo against nematode development. Extracts of Curtisia dentata, a South African medicinal plant, and compounds isolated from leaves of this plant were investigated for anthelmintic activity against T. colubriformis, H. contortus and C. elegans. The acetone and dichloromethane extracts were active against all nematodes at concentrations as low as 160 μg/mℓ. Betulinic acid and lupeol were active against the parasitic nematodes only at the high concentrations of 1 000 and 200 μg/mℓ, respectively. All compounds were effective against C. elegans with active concentrations as low as 8 μg/mℓ. Betulinic acid was less active than lupeol and ursolic acid against C. elegans. The acetone and dichloromethane extracts were also active against C. elegans with a concentration of 0.31 mg/mℓ resulting in almost 80 % inhibition of larval motility. The use of free-living nematodes may provide information on the activity of potential anthelmintics against parasitic nematodes. Extracts of various medicinal plant species may provide solutions to ill-health of small ruminants caused by parasitic nematodes in poor communities of southern Africa.

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