Enzymic Proteolysis byEntamoeba histolytica; biochemical characteristics and relationship with invasiveness

Parasitology ◽  
1960 ◽  
Vol 50 (3-4) ◽  
pp. 531-550 ◽  
Author(s):  
R. A. Neal
Keyword(s):  
Proteolytic Activity ◽  
Culture Media ◽  
Proteolytic Enzymes ◽  
Egg Yolk ◽  
Single Strain ◽  
High Proteolytic Activity ◽  
Serum Inhibitor ◽  
Sulphydryl Groups ◽  
Ph Range

The proteolytic activity of extracts ofEntamoeba histolyticahas been further investigated. Casein and gelatin, but not haemaglobin, were hydrolysed. Activity was observed in the pH range 5·8 to 8·5 with optimal activity at pH 7·5 to 7·9. Activity was optimal at 37° C. and sulphydryl groups were not required. Concomitant bacteria showed no proteolytic activity. Hyaluronidase, collagenase and lecithinase could not be detected.An inhibitor of proteolytic enzyme was present in sera of all animals tested and in egg yolk. All culture media prepared from eggs were inhibitory, but inspissation or dilution of serum inactivated the serum inhibitor. Purified trypsin inhibitors from lima and soy bean were not active against the amoebic enzyme. Proteolytic enzymes were not secreted extracellularlyin vitro.High proteolytic activity was found in two out of five invasive, freshly isolated, strains ofE. histolyticaand after two series of liver passages of a single strain. The significance of these observations is discussed. It is concluded that the present evidence does not convincingly demonstrate that high proteolytic activity is required for tissue invasion by amoebae, but may accompany another factor.

Isolation, Partial Purification and Characterisation of A Plasminogen Activator Produced by Endothelial Cells in Vitro

1979 ◽  
Author(s):  
W.E. Laug
Keyword(s):  
Endothelial Cells ◽  
Proteolytic Activity ◽  
Culture Medium ◽  
Partial Purification ◽  
Diisopropyl Fluorophosphate ◽  
Cell Lysate ◽  
Endothelial Cell Cultures ◽  
Ph Range ◽  
Confluent Endothelial Cell

Cloned endothelial cells obtained from the aorta of 1-2 day old calves produced high fibrinolytic activity, which was 90% dependent upon the presence of plasminogen when grown on 125 I fibrin coated dishes. High plasminogen-dependent proteolytic activity was also demonstrated in the cell lysate and in the culture medium of the cells. The production and secretion of this prtitease were found to increase during the log phase of cell growth and to reach a maximum at con fluency. Thereafter they remained constantly high. This protease, partially purified from the culture medium of confluent endothelial cell cultures, is aiginine specific and activates plasminogen by piOteolytic cleavage to plasmin. Its proteolytic activity which is highest in the pH range of 7.5 to 8.0 is irreversibly inhibited by diisopropyl fluorophosphate, suggesting that it is a serine protease. The molecular weight of this protease is approximately S2000.

284 COMPARATIVE FERTILITY OF FRESHLY-COLLECTED VERSUS FROZEN - THAWED SPERMATOZOA FOR IN VITRO FERTILIZATION IN THE FISHING CAT (PRIONAILURUS VIVERRINUS)

2006 ◽  
Vol 18 (2) ◽  
pp. 249
Author(s):  
G. Magarey ◽  
J. Herrick ◽  
K. Thiangtum ◽  
W. Tunwattana ◽  
W. Swanson
Keyword(s):  
Sperm Motility ◽  
Culture Medium ◽  
Culture Media ◽  
Egg Yolk ◽  
Ovarian Follicles ◽  
Blastocyst Stage ◽  
Motile Sperm ◽  
Vitro Fertilization ◽  
International Transport

Wild populations of fishing cats (Prionailurus viverrinus) in Southeast Asia are in decline, primarily due to habitat loss. Because the fishing cat population in North American zoos is small (n = 69) and inbred (F = 0.17) with relatively low genetic variation (86%), infusion of new founder genes from Asia is a conservation priority. Importation of cryopreserved semen for use with IVF and ET may offer one alternative to the international transport of living animals. In this study, our objectives were to (1) compare motility longevity of fresh vs. frozen-thawed fishing cat spermatozoa in two culture media, (2) evaluate ovarian responses to exogenous gonadotropins, and (3) assess development of IVF embryos produced with fresh vs. frozen-thawed spermatozoa. Raw semen was collected via electroejaculation from male fishing cats (n = 4), divided into groups, and washed. Two sperm pellets were resuspended in either Ham's F10 medium (HF10; with 5% FBS) or our feline optimized culture medium (FOCM; with 0.4% BSA); another pellet was diluted in TEST egg yolk, cooled to 5�C over 3 h, glycerated (4%), and cryopreserved in straws over LN2 vapor. Frozen sperm samples were thawed, washed, and diluted in either HF10 or FOCM. Fresh and frozen-thawed sperm motility (percent motile, rate of forward progress) in each medium (10 � 106 motile sperm/mL) was assessed (at 0, 1, 3, and 6 h) in microdrops under oil during culture (38�C; 6% CO2 in air). Female fishing cats (n = 10) were treated with exogenous gonadotropins (150 IU eCG, 100 IU hCG, 85-h interval) and ovarian follicles were aspirated laparoscopically. Recovered oocytes were inseminated with fresh (2 � 105 motile sperm/mL) or frozen-thawed (5 � 105 motile sperm/mL) spermatozoa in FOCM microdrops; resulting embryos were either cryopreserved or cultured in FOCM (with 5% FBS added at 72 h post-insemination) for 7 days. Sperm motility over time did not differ (P > 0.05) between media for either fresh or frozen-thawed samples; however, across media, frozen-thawed sperm motility was lower (P < 0.05) and declined faster (P < 0.05) compared to fresh spermatozoa. Females produced an average (�SEM) of 9.8 � 2.9 mature ovarian follicles, allowing recovery of 7.3 � 2.6 high-quality oocytes per female. Oocyte cleavage percentage at 42 h p.i. was lower (P < 0.05) with frozen-thawed spermatozoa (38%, 11/29) compared to freshly collected spermatozoa (68%, 17/25). Overall, 35% (6/17) of cultured embryos developed to blastocysts with no difference (P > 0.05) between embryos produced with frozen-thawed (4/11) vs. fresh (2/6) spermatozoa. Although fishing cat sperm motility and fertility appear compromised after cryopreservation, our results demonstrate the ability of frozen-thawed spermatozoa to produce IVF embryos that are capable of developing to blastocyst stage in vitro. This work was supported by (NIH RR015388).

scholarly journals Optimization of Diet for Post Larvel/Juvenile Sea Bass and Hybrid Stripped Bass Based on Enzymatic Profiles of their Digestive Tracts

1995 ◽  
Author(s):  
Sheenan Harpaz ◽  
Steven G. Hughes ◽  
Pinhas Lindner
Keyword(s):  
Proteolytic Activity ◽  
Proteolytic Enzymes ◽  
Juvenile Fish ◽  
Research Work ◽  
Sea Bass ◽  
Original Research ◽  
Young Fish ◽  
Feed Formulation ◽  
Feed Protein

The overall goal of this research work was to identify the main proteolytic activities which take place in the digestive tracts of young bass fish, and use the knowledge acquired in order to improve feed protein utilization in juvenile fish based on their digestive capacity. The results of the work clearly showed that the young fish possess the entire profile of proteolytic enzymes which is found in adult fish. Yet, in the young fish the level of activity is substantially lower per gram tissue (or gram protein) as compared with the activity found in the digestive tracts of the same fish at an older (larger) age. In addition it was found that the main proteolytic enzyme in these fish is chymotrypsin which accounts for almost 80% of the proteolytic activity. An effort aimed at enhancing this activity has lead to the interesting finding that alcohol substantially enhances the proteolytic activity of fish intestines. Fish intestinal homogenates were used in order to evaluate the suitability of various feeds for the fish. Potential feed proteins were subjected to the proteolytic activity of the fish enzymes in vitro, in a manner simulating the natural process. The proteolytic activity was monitored by the valuation of the products, i.e. amino acid released. This method has proven to be a powerful tool which enables us to predict with a very high degree of accuracy the potential of a feed to promote growth. Selection of feed based on the proteolytic capacity of the fish degestive tracts can now be implemented in feed formulation, as anticipated in the original research proposal.

STUDIES ON PROTEOLYTIC ACTIVITY AND FUNCTION OF THE THYROID GLAND IN RATS ADMINISTERED EXCESS IODIDE

1976 ◽  
Vol 82 (3) ◽  
pp. 728-736 ◽  
Author(s):  
J. Sinadinović ◽  
K. Liewendahl
Keyword(s):  
Proteolytic Activity ◽  
Proteolytic Enzymes ◽  
Unexpected Finding ◽  
Proteolytic Resistance ◽  
Thyroid Glands ◽  
Excess Iodide ◽  
Rat Thyroid ◽  
And Function ◽  
Free Thyroxine Level

ABSTRACT Thyroid auto-proteolytic activity in rats increased significantly only after several weeks of treatment with 1 mg iodide per day. Iodide added in vitro did not activate thyroid auto-proteolysis, nor was inhibition observed. Thyroid proteolytic activity was also tested using 131I-labelled heat-inactivated thyroid homogenate from control rats as substrate. Significantly increased proteolytic activity was again demonstrated in thyroid glands from rats on excess iodide for several weeks. The increased proteolytic activity is therefore probably not due to a decreased proteolytic resistance of thyroglobulin in the iodide-treated rats. Increased synthesis or decreased degradation of thyroid proteolytic enzymes is a more likely explanation for this phenomenon. These findings are at variance with those of some previous studies showing decreased proteolytic activity in thyroid glands from rats administered excess iodide. Since the rats remained euthyroid the increased auto-proteolysis, which occurred quite late, was considered an unexpected finding and it is at present not certain whether this is related to the successful adaptation. Treatment with iodide did not alter the thyroid and hypophyseal weight or histology. Serum free thyroxine level and thyrotrophin concentration were also unaffected by excess iodide. The adaptation of the intact rat thyroid to prolonged and excess iodide intake seems to occur without the assistance of an altered thyrotrophin secretion.

scholarly journals Proteolytic Enzyme Production by Strains of the Insect Pathogen Xenorhabdus and Characterization of an Early-Log-Phase-Secreted Protease as a Potential Virulence Factor

2010 ◽  
Vol 76 (20) ◽  
pp. 6901-6909 ◽  
Author(s):  
Mustafa K. Massaoud ◽  
Judit Marokh�zi ◽  
Andr�s Fodor ◽  
Istv�n Venekei
Keyword(s):  
Proteolytic Activity ◽  
Proteolytic Enzyme ◽  
Galleria Mellonella ◽  
Proteolytic Enzymes ◽  
Detection Methods ◽  
Logarithmic Phase ◽  
Bacterial Strains ◽  
Native Page ◽  
Protease B

ABSTRACT As a comparison to a similar study on Photorhabdus strains, 15 Xenorhabdus bacterial strains and secondary phenotypic variants of two strains were screened for proteolytic activity by five detection methods. Although the number and intensity of proteolytic activities were different, every strain was positive for proteolytic activity by several tests. Zymography following native PAGE detected two groups of activities with different substrate affinities and a higher and lower electrophoretic mobility that were distinguished as activity 1 and 2, respectively. Zymography following SDS-PAGE resolved three activities, which were provisionally named proteases A, B, and C. Only protease B, an ∼55-kDa enzyme, was produced by every strain. This enzyme exhibited higher affinity to the gelatin substrate than to the casein substrate. Of the chromogenic substrates used, three were hydrolyzed: furylacryloyl-Ala-Leu-Val-Tyr (Fua-ALVY), Fua-LGPA (LGPA is Leu-Gly-Pro-Ala) (a substrate for collagen peptidases), and succinyl-Ala-Ala-Pro-Phe-thiobenzyl (Succ-AAPF-SBzl). All but the Fua-LGPA-ase activity seemed to be from secreted enzymes. According to their substrate preference profiles and inhibitor sensitivities, at least six such proteolytic enzymes could be distinguished in the culture medium of Xenorhabdus strains. The proteolytic enzyme that was secreted the earliest, protease B and the Succ-AAPF-SBzl-hydrolyzing enzyme, appeared from the early logarithmic phase of growth. Protease B could also be detected in the hemolymph of Xenorhabdus-infected Galleria mellonella larvae from 15 h postinfection. The purified protease B hydrolyzed in vitro seven proteins in the hemolymph of Manduca sexta that were also cleaved by PrtA peptidase from Photorhabdus. The N-terminal sequence of protease B showed similarity to a 55-kDa serralysin type metalloprotease in Xenorhabdus nematophila, which had been identified as an orthologue of Photorhabdus PrtA peptidase.

Haemophilus paragallinarumsecretes metalloproteases

2005 ◽  
Vol 51 (10) ◽  
pp. 893-896 ◽  
Author(s):  
P C Rivero-García ◽  
C Vázquez Cruz ◽  
P Sánchez Alonso ◽  
S Vaca ◽  
E Negrete-Abascal
Keyword(s):  
Molecular Mass ◽  
Proteolytic Activity ◽  
Culture Media ◽  
High Molecular Mass ◽  
Secreted Proteins ◽  
Extracellular Proteases ◽  
Heat Stable ◽  
Infectious Coryza ◽  
Ph Range ◽  
Polyclonal Serum

Haemophilus paragallinarum secretes metalloproteases into different culture media lacking serum. Secreted proteins, concentrated by precipitation with 70% ammonium sulphate ((NH4)2SO4) or methanol, displayed proteolytic activity at >100 kDa molecular mass in 10% polyacrylamide gels co-polymerized with porcine gelatin (0.1%). They were active in a broad pH range (4–9); pH 7.5 being the optimum. Protease activity was inhibited by 20 mmol EDTA/L and reactivated by calcium. The proteolytic activity was heat-stable at 40, 50, and 60 °C, but its activity diminished at 70 °C or higher. Secreted proteins partially degraded chicken immunoglobulin G (IgG) and cross-reacted with a polyclonal serum against a high molecular mass protease secreted by Actinobacillus pleuropneumoniae. Extracellular proteases could play a role in infectious coryza caused by H. paragallinarum.Key words: pathogenicity, secreted protein, infectious coryza.

Hymenolepis diminuta (Cestoda) liberates an inhibitor of proteolytic enzymes during in vitro incubation

Parasitology ◽  
1990 ◽  
Vol 101 (3) ◽  
pp. 455-464 ◽  
Author(s):  
P. W. Pappas ◽  
G. L. Uglem
Keyword(s):  
Proteolytic Activity ◽  
Proteolytic Enzymes ◽  
Hymenolepis Diminuta ◽  
Supernatant Fraction ◽  
Amidase Activity ◽  
Freeze Thaw ◽  
Positive Material ◽  
Bovine Trypsin ◽  
Intestinal Contents

SUMMARYHymenolepis diminuta liberated measurable amounts of ‘Lowry-positive material’ (LPM) and protein during incubation for 2 h in vitro. When tapeworms were incubated in the presence of bovine trypsin (BT), or when BT was added to the medium after removing the tapeworms, the enzyme's proteolytic activity was inhibited significantly. Centrifugation of the medium at 30 000 g yielded a pellet composed of tegumental elements, but this fraction did not inhibit BT. The 30 000 g supernatant fraction contained a chemical(s) that inhibited the proteolytic enzymes of the rodent host's intestinal contents (IC). The inhibitor(s) was stable following repeated freeze-thaw cycles, heat labile, and not degraded by BT or IC, and it inhibited the amidase activity of BT in a non-competitive manner.

scholarly journals Potential Applicability of Chymotrypsin-Susceptible Microcin J25 Derivatives to Food Preservation

2009 ◽  
Vol 75 (17) ◽  
pp. 5734-5738 ◽  
Author(s):  
Mar�a Fernanda Pomares ◽  
Ra�l A. Salom�n ◽  
Olga Pavlova ◽  
Konstantin Severinov ◽  
Ricardo Far�as ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proteolytic Enzymes ◽  
Skim Milk ◽  
Egg Yolk ◽  
Food Preservation ◽  
Peptide Antibiotic ◽  
Microcin J25 ◽  
Potential Use

ABSTRACT Microcin J25 (MccJ25) is a 21-residue ribosomally synthesized lariat peptide antibiotic. MccJ25 is active against such food-borne disease-causing pathogens as Salmonella spp., Shigella spp., and Escherichia coli, including E. coli O157:H7 and non-O157 strains. MccJ25 is highly resistant to digestion by proteolytic enzymes present in the stomach and intestinal contents. MccJ25 would therefore remain active in the gastrointestinal tract, affecting normal intestinal microbiota, and this limits the potential use of MccJ25 as a food preservative. In the present paper, we describe a chymotrypsin-susceptible MccJ25 derivative with a mutation of Gly12 to Tyr that retained almost full antibiotic activity and efficiently inhibited the growth of pathogenic Salmonella enterica serovar Newport and Escherichia coli O157:H7 in skim milk and egg yolk. However, unlike the wild-type MccJ25, the MccJ25(G12Y) variant was inactivated by digestive enzymes both in vitro and in vivo. To our knowledge, our results represent the first example of a rational modification of a microcin aimed at increasing its potential use in food preservation.

Some properties of the extracellular proteolytic enzymes of the milk-spoiling organismPseudomonas aeruginosaATCC 10145

1968 ◽  
Vol 35 (3) ◽  
pp. 395-398 ◽  
Author(s):  
H. S. Juffs ◽  
H. W. Doelle
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Proteolytic Activity ◽  
Culture Supernatant ◽  
Proteolytic Enzymes ◽  
Maximum Activity ◽  
Ph Range

SummaryThe proteolytic enzymes present in the culture supernatant ofPseudomonas aeruginosaATCC 10145 were active in the pH range 5·5–9·0 with a maximum activity at pH 7·3. Heating for 15 sec at 72°C resulted in a 36% loss of proteolytic activity whereas heating for 30 min at 63°C resulted, in a 6% loss. Boiling for 2 min completely inactivated the proteolytic enzymes. At 2°C the proteolytic enzymes were stable for at least a month and casein was readily hydrolysed at this temperature.

Proteolysis on the body surface of pyrethroid-sensitive and resistant Varroa destructor

2013 ◽  
Vol 58 (1) ◽  
Author(s):  
Aneta Strachecka ◽  
Grzegorz Borsuk ◽  
Krzysztof Olszewski ◽  
Jerzy Paleolog ◽  
Zbigniew Lipiński
Keyword(s):  
Protease Inhibitor ◽  
Proteolytic Activity ◽  
Body Surface ◽  
Serine Proteases ◽  
Protein Concentration ◽  
Proteolytic Enzymes ◽  
Electrophoretic Analysis ◽  
The Body ◽  
Activity Levels

AbstractThe aim of this work was to determine the activity of proteases and protease inhibitors sampled from the body surface of tau-fluvalinate-sensitive and resistant V. destructor. Proteins were isolated from the tau-fluvalinate-sensitive and resistant mites, while mites untreated with tau-fluvalinate constituted the control. Subsequently, the following methodology was applied: protein concentration assay by the Lowry method — as modified by Schacterle and Pollack; assay of proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; identification of proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee and Lin method; identification of acidic, neutral and basic protease activities by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method and for protease inhibitor detection with the Felicioli method. The highest value of protein concentration was found in the tau-fluvalinate-sensitive V. destructor, while the highest activity levels of acidic, neutral and alkaline proteases were observed in the tau-fluvalinate-resistant mites. Aspartic, serine, thiolic and metallic proteases were found in the drug-resistant and drug-sensitive Varroa mites. The control samples were found to contain aspartic and serine proteases. In an acidic and alkaline environment, the results revealed a complete loss of inhibitor activities in the in vitro analyses and electrophoresis. Serine protease inhibitor activities (at pH 7.0) were high, especially in the group of tau-fluvalinate-resistant mites.

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