Partial characterization of proteolytic enzymes in different developmental stages of Ostertagia ostertagi

1993 ◽  
Vol 67 (4) ◽  
pp. 271-278 ◽  
Author(s):  
H. de Cock ◽  
D. P. Knox ◽  
E. Claerebout ◽  
D. C. de Graaf
Keyword(s):  
Proteinase Inhibitor ◽  
Developmental Stages ◽  
Proteolytic Enzymes ◽  
Ostertagia Ostertagi ◽  
Protein Substrates ◽  
Stage Specificity ◽  
Specific Manner ◽  
Secretory Products

AbstractProteolytic enzymes present in extracts of third (L3) and fourth (L4) stage larvae and adults of the cattle nematode Ostertagia ostertagi were defined on the basis of pH optima and proteinase inhibitor sensitivity in spectrophotometric assays using azocasein and elastin-orcein as protein substrates. Evidence that different classes of proteinases are expressed in a stage specific manner was provided by the contrasting pH optima and inhibitor sensitivities shown by the enzymes in the different parasite stages. Stage specificity was confirmed by gelatin-substrate analysis. In addition, proteolytic activity was sought in the excretory/secretory products (ES) of the L4 following simple in vitro culture. Contrasting pH and inhibitor sensitivities as well as gelatin-substrate analysis showed that different proteinases were present in somatic L4 extracts and L4 ES products. The secreted proteinases may be useful targets for serodiagnosis or vaccination.

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.

A somatic cell-derived system for studying both early and late mitotic events in vitro

1989 ◽  
Vol 94 (3) ◽  
pp. 449-462
Author(s):  
J. Nakagawa ◽  
G.T. Kitten ◽  
E.A. Nigg
Keyword(s):  
Chromatin Condensation ◽  
Nuclear Lamina ◽  
Free System ◽  
Cell Free System ◽  
Protein Substrates ◽  
Nuclear Envelope Breakdown ◽  
Biochemical Fractionation ◽  
A Cell

We describe a cell-free system for studying mitotic reorganization of nuclear structure. The system utilizes soluble extracts prepared from metaphase-arrested somatic chicken cells and supports both the disassembly and subsequent partial reassembly of exogenous nuclei. By fluorescence microscopy, biochemical fractionation, protein phosphorylation assays and electron microscopy, we show that chicken embryonic nuclei incubated in extracts prepared from metaphase-arrested chicken hepatoma cells undergo nuclear envelope breakdown, lamina depolymerization and chromatin condensation. These prophase-like events are strictly dependent on ATP and do not occur when nuclei are incubated in interphase extracts. Compared to interphase extracts, metaphase extracts show increased kinase activities toward a number of nuclear protein substrates, including lamins and histone H1; moreover, they specifically contain four soluble phosphoproteins of Mr 38,000, 75,000, 95,000 and 165,000. Following disassembly of exogenous nuclei in metaphase extracts, telophase-like reassembly of a nuclear lamina and re-formation of nuclear membranes around condensed chromatin can be induced by depletion of ATP from the extract. We anticipate that this reversible cell-free system will contribute to the identification and characterization of factors involved in regulatory and mechanistic aspects of mitosis.

SUSCEPTIBILITY OF PROTEINS USED IN CALF MILK REPLACERS TO HYDROLYSIS BY VARIOUS PROTEOLYTIC ENZYMES

1980 ◽  
Vol 60 (4) ◽  
pp. 907-914 ◽  
Author(s):  
K. J. JENKINS ◽  
S. MAHADEVAN ◽  
D. B. EMMONS
Keyword(s):  
Trichloroacetic Acid ◽  
Proteolytic Enzymes ◽  
Milk Proteins ◽  
In Vitro Study ◽  
Enzyme Treatment ◽  
Protein Hydrolysis ◽  
Protein Substrates ◽  
Optimum Ph ◽  
Milk Replacers

An in vitro study was conducted to assess the hydrolytic susceptibility of various milk and non-milk proteins (soybean, rapeseed, fish) used in calf milk replacers to endogenous and commercial proteolytic enzymes. Extent of protein hydrolysis (%) was calculated from the reduced amount of protein precipitated by 10% trichloroacetic acid following enzyme treatment. All of the enzymes tested hydrolyzed the milk proteins more extensively than the non-milk proteins both at their optimum pH, and at the pH (6.1) of calf abomasal contents immediately after feeding. At both optimum pH and pH 6.1, the highest average hydrolysis value for all protein substrates was obtained with pronase followed by papain, trypsin, pancreatin, chymotrypsin, Mucor miehei rennet andchymosin (calf rennet). All substrates were hydrolyzed extensively by pepsin at pH 2.0 but, as expected, very little hydrolysis occurred with this enzyme atpH6.1.

In vitro cultivation of Hematodinium sp. isolated from Atlantic snow crab, Chionoecetes opilio: partial characterization of late developmental stages

Parasitology ◽  
2014 ◽  
Vol 142 (04) ◽  
pp. 598-611 ◽  
Author(s):  
PETER H. GAUDET ◽  
RICHARD J. CAWTHORN ◽  
MELANIE A. BUOTE ◽  
J. FRANK MORADO ◽  
GLENDA M. WRIGHT ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Partial Characterization ◽  
Snow Crab ◽  
In Vitro Cultivation ◽  
Chionoecetes Opilio

Characterization of Ostertagia ostertagi annexin-like proteins at different developmental stages

2017 ◽  
Vol 116 (5) ◽  
pp. 1515-1522
Author(s):  
Pooja Sharma ◽  
Mark Jenkins ◽  
Dante Zarlenga ◽  
Ray Fetterer ◽  
Zhengguo Xiao ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Ostertagia Ostertagi

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.

Characterization of an extracellular protease and its cDNA from the nematode-trapping fungus Monacrosporium microscaphoides

2006 ◽  
Vol 52 (2) ◽  
pp. 130-139 ◽  
Author(s):  
Miao Wang ◽  
Jinkui Yang ◽  
Ke-Qin Zhang
Keyword(s):  
Serine Protease ◽  
Catalytic Triad ◽  
Infection Process ◽  
Gene Encoding ◽  
Protein Substrates ◽  
Nematocidal Activity ◽  
Infection Mechanisms ◽  
Biocontrol Potential

To better exploit the biocontrol potential of nematophagous fungi, it is important to fully understand the molecular background of the infection process. In this paper, several nematode-trapping fungi were surveyed for nematocidal activity. From the culture filtrate of Monacrosporium microscaphoides, a neutral serine protease (designated Mlx) was purified by chromatography. This protease could immobilize the nematode Penagrellus redivivus in vitro and degrade its purified cuticle, suggesting that Mlx could serve as a virulence factor during infection. Characterization of the purified protease revealed a molecular mass of approximately 39 kDa, an isoelectric point of 6.8, and optimum activity at pH 9 at 65 °C. Mlx has broad substrate specificity, and it hydrolyzes protein substrates, including casein, skimmed milk, collagen, and bovine serum albumin. The gene encoding Mlx was also cloned and the nucleotide sequence was determined. The deduced amino acid sequence contained the conserved catalytic triad of aspartic acid – histidine – serine and showed high similarity with two cuticle-degrading proteases (PII and Aoz1), which were purified from the nematode-trapping fungus Arthrobotrys oligospora. Research on infection mechanisms of nematode-trapping fungi has thus far only focused on A. oligospora. However, little is known about other nematode-trapping fungi. Our report is among the first to describe the purification and cloning of an infectious protease from a different nematode-trapping fungus.Key words: extracellular serine protease, Monacrosporium microscaphoides, nematode-trapping fungus, nematocidal activity.

scholarly journals Characterization of Putative Effectors from the Cereal Cyst Nematode Heterodera avenae

2018 ◽  
Vol 108 (2) ◽  
pp. 264-274 ◽  
Author(s):  
Jiang-Kuan Cui ◽  
Huan Peng ◽  
Fen Qiao ◽  
Gao-Feng Wang ◽  
Wen-Kun Huang ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Target Genes ◽  
Developmental Stages ◽  
Heterodera Avenae ◽  
Parasitic Nematodes ◽  
Sequencing Analysis ◽  
Rnai Technology

Few molecular details of effectors of Heterodera avenae parasitism are known. We performed a high-throughput sequencing analysis of the H. avenae transcriptome at five developmental stages. A total of 82,549 unigenes were ultimately obtained, and 747 transcripts showed best hits to genes putatively encoding carbohydrate-active enzymes in plant-parasitic nematodes that play an important role in the invasion process. A total of 1,480 unigenes were homologous to known phytonematode effectors, and 63 putative novel effectors were identified in the H. avenae transcriptomes. Twenty-three unigenes were analyzed by qRT-PCR and confirmed to be highly expressed during at least one developmental stage. For in situ hybridization, 17 of the 22 tested putative effectors were specifically expressed and located in the subventral gland cells, and five putative novel effectors were specifically expressed in the dorsal gland. Furthermore, 115 transcripts were found to have putative lethal RNA interference (RNAi) phenotypes. Three target genes with lethal RNAi phenotypes and two of the four tested putative effectors were associated with a decrease in the number of cysts through in vitro RNAi technology. These transcriptomic data lay a foundation for further studies of interactions of H. avenae with cereal and H. avenae parasitic control.

Characterization of phosphoglycan-containing secretory products ofLeishmania

Parasitology ◽  
1994 ◽  
Vol 108 (S1) ◽  
pp. S63-S71 ◽  
Author(s):  
T. Ilg ◽  
Y.-D. Stierhof ◽  
M. Wiese ◽  
M. J. McConville ◽  
P. Overath
Keyword(s):  
Parasitophorous Vacuole ◽  
Secretory Product ◽  
Flagellar Pocket ◽  
Lesion Development ◽  
Causative Agents ◽  
Secretory Products ◽  
Species Specific ◽  
Modify Protein

SUMMARYThis article presents an overview on phosphoglycan-containing components secreted by the insect and mammalian stages of several species ofLeishmania, the causative agents of leishmaniasis in the Old and New World. Firstly, promastigotes of all three species considered,L. mexicana, L. donovaniandL. major, shed lipophosphoglycan (LPG) into the culture medium possibly by release of micelles from the cell surface. Like the cell-associated LPG, culture supernatant LPG is arhphiphilic and composed of a lysoalkylphosphatidylinositol-phosphosaccharide core connected to species-specific phosphosaccharide repeats and oligosaccharide caps. Secondly, all three species release hydrophilic phosphoglycan. Thirdly, all three species appear to secrete proteins covalently modified by phosphosaccharide repeats and oligosaccharide caps. In the case of promastigotes ofL. mexicana, these components are organized as two filamentous polymers released from the flagellar pocket: the secreted acid phosphatase (sAP) composed of a 100 kDa phosphoglycoprotein and a protein- containing high-molecular-weight-phosphoglycan (proteo-HMWPG) and fibrous networks likewise composed of phosphoglycan possibly linked to protein. Structural analyses and gene cloning suggest that the parasites can covalently modify protein regions rich in serine and threonine residues by the attachment of phosphosaccharide repeats capped by oligosaccharides. We propose that the networks formedin vitrocorrespond to fibrous material previously demonstrated in the digestive tract of infected sandflies. In the case ofL. donovani, the sAP is also modified by phosphoglycans but contains neither proteo-HMWPG nor does it aggregate to filaments. Finally,L. mexicanaamastigotes release proteo-HMWPG via the flagellar pocket into the parasitophorous vacuole of infected macrophages. This material appears to be released into the tissue of the infected mammal upon rupture of infected macrophages during lesion development. This secretory product may contribute to the pathology of lesion development.

Sign in / Sign up

Export Citation Format

Share Document