scholarly journals Cloning and Expression of the Major SecretedCathepsin B-Like Protein from Juvenile Fasciola hepatica andAnalysis of Immunogenicity following Liver FlukeInfection

2003 ◽  
Vol 71 (12) ◽  
pp. 6921-6932 ◽  
Author(s):  
Ruby H. P. Law ◽  
Peter M. Smooker ◽  
James A. Irving ◽  
David Piedrafita ◽  
Rebecca Ponting ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cathepsin B ◽  
Fasciola Hepatica ◽  
Cathepsin L ◽  
Cloning And Expression ◽  
Significant Similarity ◽  
Peptide Substrates ◽  
Liver Flukes ◽  
Kinetics Of

ABSTRACT The functions of the cathepsin B-like proteases in liver flukes are unknown and analysis has been hindered by a lack of protein for study, since the protein is produced in small amounts by juvenile flukes. To circumvent this, we isolated and characterized a cDNA encoding the major secreted cathepsin B from Fasciola hepatica. The predicted preproprotein is 339 amino acids in length, with the mature protease predicted to be 254 amino acids long, and shows significant similarity to parasite and mammalian cathepsin B. Only one of the two conserved histidine residues required for cathepsin B exopeptidase activity is predicted to be present. Recombinant preproprotein was produced in yeast, and it was shown that the recombinant proprotein can undergo a degree of self-processing in vitro to the mature form, which is active against gelatin and synthetic peptide substrates. The recombinant protein is antigenic in vaccinated rats, and antibodies to the protein are detected early after infection of rats and sheep with F. hepatica. The kinetics of the response to cathepsin B and cathepsin L after infection of sheep and rats confirm the temporal expression of these proteins during the life cycle of the parasite.

scholarly journals Identification in skeletal muscle of a distinct extracellular pool of amino acids, and its role in protein synthesis

1971 ◽  
Vol 121 (5) ◽  
pp. 817-827 ◽  
Author(s):  
R. C. Hider ◽  
E. B. Fern ◽  
D. R. London
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Incubation Medium ◽  
Extensor Digitorum Longus ◽  
Extensor Digitorum ◽  
Longus Muscle ◽  
Kinetics Of

1. The kinetics of radioactive labelling of extra- and intra-cellular amino acid pools and protein of the extensor digitorum longus muscle were studied after incubations with radioactive amino acids in vitro. 2. The results indicated that an extracellular pool could be defined, the contents of which were different from those of the incubation medium. 3. It was concluded that amino acids from the extracellular pool, as defined in this study, were incorporated directly into protein.

Transport of amino acids by isolated gills of the mussel Mytilus californianus Conrad

1975 ◽  
Vol 62 (2) ◽  
pp. 313-325
Author(s):  
S. H. Wright ◽  
T. L. Johnson ◽  
J. H. Crowe
Keyword(s):  
Amino Acids ◽  
Significant Role ◽  
Transport Mechanism ◽  
Surrounding Medium ◽  
Gill Tissue ◽  
Mytilus Californianus ◽  
Animal Nutrition ◽  
Neutral Amino Acids ◽  
Kinetics Of

The unidirectional influx of cycloleucine into in vitro preparations of gill tissue of the mussel, Mytilus californianus, was determined. Influx was found to be linear for at least an hour, and the kinetics of cycloleucine influx conformed to Michaelis-Menten type kinetics. The transport mechanism(s) for cycloleucine is relatively specific for the L-enantiomorph of neutral amino acids, and is capable of accumulating cycloleucine to intracellular concentrations much higher than those of the surrounding medium. Evedence is presented that the transport of amino acids by gill tissue plays a significant role in whole animal nutrition.

scholarly journals The Zoonotic Helminth Parasite Fasciola hepatica: Virulence-Associated Cathepsin B and Cathepsin L Cysteine Peptidases Secreted by Infective Newly Excysted Juveniles (NEJ)

Animals ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3495
Author(s):  
Tara Barbour ◽  
Krystyna Cwiklinski ◽  
Richard Lalor ◽  
John Pius Dalton ◽  
Carolina De Marco Verissimo
Keyword(s):  
Vaccine Development ◽  
Fasciola Hepatica ◽  
Cathepsin L ◽  
Intestinal Wall ◽  
Mammalian Host ◽  
Parasite Invasion ◽  
Potent Inhibition ◽  
Cysteine Peptidases ◽  
Future Drug

Fasciolosis caused by Fasciola hepatica is a major global disease of livestock and an important neglected helminthiasis of humans. Infection arises when encysted metacercariae are ingested by the mammalian host. Within the intestine, the parasite excysts as a newly excysted juvenile (NEJ) that penetrates the intestinal wall and migrates to the liver. NEJ excystment and tissue penetration are facilitated by the secretion of cysteine peptidases, namely, cathepsin B1 (FhCB1), cathepsin B2 (FhCB2), cathepsin B3 (FhCB3) and cathepsin L3 (FhCL3). While our knowledge of these peptidases is growing, we have yet to understand why multiple enzymes are required for parasite invasion. Here, we produced functional recombinant forms of these four peptidases and compared their physio-biochemical characteristics. Our studies show great variation of their pH optima for activity, substrate specificity and inhibitory profile. Carboxy-dipeptidase activity was exhibited exclusively by FhCB1. Our studies suggest that, combined, these peptidases create a powerful hydrolytic cocktail capable of digesting the various host tissues, cells and macromolecules. Although we found several inhibitors of these enzymes, they did not show potent inhibition of metacercarial excystment or NEJ viability in vitro. However, this does not exclude these peptidases as targets for future drug or vaccine development.

Preliminary Experiments with a Defined Culture Medium for Larval Fasciola hepatica

1973 ◽  
Vol 47 (2) ◽  
pp. 181-189 ◽  
Author(s):  
R. S. V. Pullin
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Fasciola Hepatica ◽  
Dry Weight ◽  
Defined Medium ◽  
Inorganic Salts ◽  
Final Maturation ◽  
Defined Culture ◽  
Stock Solutions

A defined medium is described as a basis for in vitro culture work with larval Fasciola hepatica. This medium, termed BCM, can be quickly made up by using a system of stock solutions. BCM contains inorganic salts, glucose, amino acids, vitamins and antibiotics, but no lipid or proteins. Rediae can be dissected from infected snails for culture, but many appear to be contaminated with bacteria. Large rediae cannot survive in BCM but free immature cercariae can complete their final maturation in vitro. This final maturation, from the 30th to the 35th day after miracidial penetration of donor snails, includes tail growth and appearance of body pigmentation. Cercariae matured in vitro encyst successfully when transferred from BCM to water. Small rediae survive in BCM for 5 days, but show no growth or development measured as dry weight and total nitrogen.

Toward understanding recurrent meningioma: the potential role of lysosomal cysteine proteases and their inhibitors

2010 ◽  
Vol 112 (5) ◽  
pp. 940-950 ◽  
Author(s):  
Tamara T. Lah ◽  
Isabelle Nanni ◽  
Miha Trinkaus ◽  
Philipe Metellus ◽  
Christophe Dussert ◽  
...  
Keyword(s):  
Cystatin C ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Who Grade ◽  
Simpson Grade ◽  
Cysteine Cathepsins ◽  
Chick Heart ◽  
Embryonic Chick Heart ◽  
Meningioma Recurrence

Object The first aim of this study was to diagnose more aggressive and potentially recurrent meningiomas using an in vitro embryonic chick heart invasiveness assay in which lysosomal enzyme cathepsin B was used as the invasiveness marker. The second aim was to confirm if cathepsin B and/or cathepsin L and their endogenous inhibitors were also prognostic parameters in the clinical study of 119 patients with meningioma. Methods Primary meningioma cultured spheroids were “confronted” with embryonic chick heart spheroids in vitro, and cathepsin B was used as molecular marker to immunolabel the invasive tumor cells. In vitro invasion assays of the malignant meningioma cells were used to assess the invasive potential related to the cysteine cathepsins. As to the second aim, the possible association of cathepsin B along with selected molecular markers, cathepsin L, and endogenous cysteine protease inhibitors (stefins A and B and cystatin C) with meningioma malignancy was determined using enzyme-linked immunosorbent assays in tumor homogenates. Univariate and multivariate analyses were used to compare these parameters with established biological markers of meningioma recurrence in 119 patients with meningiomas. Results The more invasive tumors, which characteristically overgrew the normal tissue, were identified even within a group of histologically benign meningiomas. More intensive staining of cathepsin B in these tumors was not only found at the tumor front, but also in the invading pseudopodia of a single migrating tumor cells. Matrigel invasion of malignant meningioma cells was significantly altered by modulating cathepsin B activity and by stefin B silencing. In the clinical samples of meningioma, the levels of cathepsins B and L, stefin B, and cystatin C were highest in the tumors of higher histological grades, whereas stefin A and progesterone receptor were the only markers that were significantly increased and decreased, respectively, in WHO Grade III lesions. With respect to the prognosis of relapse, cathepsin L (p = 0.035), stefin B (p = 0.007), cystatin C (p = 0.008), and progesterone receptor (p = 0.049) levels were significant, whereas cathepsin B was not a prognosticator. As expected, WHO grade, age, and Simpson grade (complete tumor resection) were prognostic, with Simpson grade only relevant in the short term (up to 90 months) but not in longer-term follow-up. Of note, the impact of all these parameters was lost in multivariate analysis, due to overwhelming prognostic impact of stefin B (p = 0.039). Conclusions The data indicate that the cysteine cathepsins and their inhibitors are involved in a process related to early meningioma recurrence, regardless of their histological classification. Of note, the known tumor invasiveness marker cathepsin B, measured in whole-tumor homogenates, was not prognostic, in contrast to its endogenous inhibitor stefin B, which was highly significant and the only independent prognostic factor to predict meningioma relapse in multivariate analysis and reported herein for the first time. Stefin B inhibition of local invasion was confirmed by in vitro invasion assay, although its other functions cannot be excluded.

scholarly journals Impact of exocrine pancreatic adaptation on in vitro protein digestibility

1993 ◽  
Vol 69 (2) ◽  
pp. 359-369 ◽  
Author(s):  
P. Valette ◽  
H. Malouin ◽  
T. Corring ◽  
L. Savoie
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Pancreatic Enzyme ◽  
Protein Digestibility ◽  
Low Molecular Weight ◽  
Rapeseed Proteins ◽  
Total Capacity ◽  
Kinetics Of

An in vitro enzymic method was used to study the kinetics of digestion of casein and rapeseed proteins. After a predigestion step with pepsin (EC 3.4.23.1), the protein substrates were submitted to a 24 h hydrolysis either with pancreatin or pancreatic juices of pigs adapted either to casein or rapeseed diets and whose enzyme activities were different. After 3, 6 and 24 h of in vitro digestion, dialysates were collected and analysed for content of nitrogen, amino acids and low-molecular-weight peptides. For a long-term hydrolysis (24 h), overall digestibility of both substrates was not affected by the composition of pancreatic enzyme mixtures. However, at the beginning of hydrolysis a significant effect of pancreatic juices was observed, i.e. individual amino acid digestibility was generally higher when casein pancreatic juice was used for hydrolysis and their relative pattern of release was modified. For both substrates the proportion of amino acids released as low-molecular-weight peptides was not affected by the enzyme mixture used and made up about two-thirds of the total digested material. It is concluded that exocrine pancreatic adaptation to protein sources does not affect the total capacity of protein digestion. However, the changes in initial kinetics of release of amino acids are more dependent on the nature of the protein tested than on the composition of pancreatic enzyme mixtures.

scholarly journals Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2′ and P3′ variations in extended substrates

2000 ◽  
Vol 347 (1) ◽  
pp. 123-129 ◽  
Author(s):  
Fernada C. Vieira PORTARO ◽  
Ana Beatriz F. SANTOS ◽  
Maria Helena S. CEZARI ◽  
Maria Aparecida JULIANO ◽  
Luiz JULIANO ◽  
...  
Keyword(s):  
Amino Acids ◽  
Significant Influence ◽  
Active Site ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Cysteine Proteinases ◽  
Aminobenzoic Acid ◽  
Site Analysis ◽  
Hydrophobic Amino Acids ◽  
Human Cathepsin

We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S4 and S3 subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S2ʹ and S3ʹ. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P4, P3, P2ʹ and P3ʹ were made. The S4 to S2ʹ subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S3ʹ, indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S4, S3, S2ʹ and S3ʹ of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P2ʹ respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P3ʹ. Basic residues at P3 and P4 were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P2ʹ (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His111 (H111A) and His110 (H110A) of cathepsin B led to an increase in kcat values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L.

Uptake of a Series of Neutral Dipeptides Including l-Alanyl-l-Alanine, Glycylglycine and Glycylsarcosine by Hamster Jejunum in Vitro

1984 ◽  
Vol 67 (5) ◽  
pp. 541-549 ◽  
Author(s):  
D. M. Matthews ◽  
D. Burston
Keyword(s):  
Amino Acids ◽  
Free Amino ◽  
Inhibitory Effect ◽  
Progressive Increase ◽  
The Other ◽  
Uptake System ◽  
Apparent Affinity ◽  
Inhibitory Ability ◽  
Kinetics Of

1. This paper is the last of a set reporting an investigation of the kinetics of jejunal uptake and inhibitory ability of a series of neutral dipeptides, glycylglycine, l-ananyl-l-alanine, l-valyl-l-valine and l-leucyl-l-leucine, with progressively longer and more lipophilic side chains. 2. The results suggested that at pH 5, uptake of l-alanyl-l-alanine, like that of l-valyl-l-valine and l-leucyl-l-leucine, was the result of two processes, uptake of intact peptide and uptake of free amino acid released extracellularly. On the other hand, uptake of glycylglycine was entirely in the form of intact peptide. In contrast to uptake of l-valyl-l-valine and l-leucyl-l-leucine, the proportion of intact l-alanyl-l-alanine taken up by mediated transport was greatest at the lowest concentration studied and smallest at the highest concentration. 3. Taking the series of results as a whole, whereas the corresponding series of amino acids, glycine, l-alanine, l-valine and l-leucine, showed a progressive increase in apparent affinity for uptake and a decrease in Vmax., we could find no such regular progression with the peptides. 4. The results of work on inhibition of uptake of one dipeptide by another were unexpectedly complex. Examples were the very powerful inhibitory effect of l-valyl-l-valine on uptake of glycylsarcosine, not suggested by the Kt of the former peptide, and the failure of glycylsarcosine to cause complete inhibition of uptake of l-alanyl-l-alanine and l-leucyl-l-leucine, though it could completely inhibit uptake of l-valyl-l-valine. There may be more than one uptake system for intact peptides, but we cannot yet suggest an explanation for all the results on inhibitions of uptake.

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