scholarly journals Inhibitor studies indicate that active cathepsin L is probably essential to its own processing in cultured fibroblasts

1990 ◽  
Vol 272 (1) ◽  
pp. 39-44 ◽  
Author(s):  
A Salminen ◽  
M M Gottesman
Keyword(s):  
Culture Media ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Vitro Assay ◽  
Cultured Fibroblasts ◽  
Low Concentrations ◽  
Procathepsin L ◽  
Nih 3T3 ◽  
Increased Activity

The lysosomal cysteine proteinase cathepsin L is synthesized in cultured mouse NIH 3T3 cells as a 39 kDa precursor and processed intracellularly into active 29 kDa and 20 kDa + 5 kDa lysosomal forms. Addition to culture media of the peptidyl aldehyde leupeptin, a non-covalent inhibitor of cathepsin L, results in the accumulation of the 20 kDa mature form of the enzyme, resulting in increased activity of cathepsin L as measured in an in vitro assay system in the absence of leupeptin. The more potent irreversible cathepsin L inhibitors benzyloxycarbonyl-Phe-Ala-diazomethane and L-transepoxysuccinyl-L-leucylamino-(4-guanidino)butane, when added to living cells at low concentrations, result in accumulation of all partially processed forms of cathepsin L, especially the 29 kDa form, suggesting that cathepsin L is responsible for its own processing. Exogenous procathepsin L introduced into CHO cells by endocytosis via the mannose 6-phosphate receptor is processed in a manner similar to endogenous procathepsin L. We conclude that the major intracellular pathway for processing of procathepsin L, either endogenous or exogenous, probably requires active cathepsin L.

scholarly journals Glycosylation of procathepsin L does not account for species molecular-mass differences and is not required for proteolytic activity

1989 ◽  
Vol 262 (3) ◽  
pp. 931-938 ◽  
Author(s):  
S M Smith ◽  
S E Kane ◽  
S Gal ◽  
R W Mason ◽  
M M Gottesman
Keyword(s):  
Molecular Mass ◽  
Proteolytic Activity ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Enzymic Activity ◽  
A431 Cells ◽  
Molecular Masses ◽  
Procathepsin L ◽  
Endoglycosidase H

Cathepsin L is a major lysosomal cysteine proteinase in mouse and human cells. Despite similar predicted molecular masses, procathepsin L in these two species migrates on SDS/polyacrylamide gels with apparent molecular masses of 39 kDa and 42 kDa respectively. To determine if glycosylation differences account for this discrepancy, and to ascertain whether glycosylation is essential for enzymic activity, mouse and human procathepsins L were expressed at high concentrations in mouse NIH 3T3 cells or in human A431 cells after DNA-mediated transfection of cloned DNAs for these enzymes. In pulse-chase studies, human procathepsin L transfectants synthesized and secreted large amounts of enzymically active 42 kDa proenzyme and processed it into 34 kDa and 26 kDa intracellular peptides, a pattern of secretion and processing similar to that seen with endogenous or transfected mouse procathepsin L. Both translation of cloned procathepsin L cDNAs in vitro and Endoglycosidase H treatment of 39 kDa mouse and 42 kDa human procathepsin L resulted in non-glycosylated proteins 2 kDa lower in molecular mass than the untreated proteins for both species. This suggests that glycosylation differences are not responsible for the molecular-mass disparity between the two species. Moreover, Endoglycosidase H-treated mouse enzyme retained full proteolytic activity, indicating that glycosylation of cathepsin L is not essential for enzymic function.

scholarly journals 3-Pentylcatechol, a Non-Allergenic Urushiol Derivative, Displays Anti-Helicobacter pylori Activity In Vivo

2020 ◽  
Vol 13 (11) ◽  
pp. 384
Author(s):  
Hang Yeon Jeong ◽  
Tae Ho Lee ◽  
Ju Gyeong Kim ◽  
Sueun Lee ◽  
Changjong Moon ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Triple Therapy ◽  
Inflammatory Cells ◽  
Control Group ◽  
Vitro Assay ◽  
Low Concentrations ◽  
H Pylori ◽  
Stomach Mucous Membrane

We previously reported that 3-pentylcatechol (PC), a synthetic non-allergenic urushiol derivative, inhibited the growth of Helicobacter pylori in an in vitro assay using nutrient agar and broth. In this study, we aimed to investigate the in vivo antimicrobial activity of PC against H. pylori growing in the stomach mucous membrane. Four-week-old male C57BL/6 mice (n = 4) were orally inoculated with H. pylori Sydney Strain-1 (SS-1) for 8 weeks. Thereafter, the mice received PC (1, 5, and 15 mg/kg) and triple therapy (omeprazole, 0.7 mg/kg; metronidazole, 16.7 mg/kg; clarithromycin, 16.7 mg/kg, reference groups) once daily for 10 days. Infiltration of inflammatory cells in gastric tissue was greater in the H. pylori-infected group compared with the control group and lower in both the triple therapy- and PC-treated groups. In addition, upregulation of cytokine mRNA was reversed after infection, upon administration of triple therapy and PC. Interestingly, PC was more effective than triple therapy at all doses, even at 1/15th the dose of triple therapy. In addition, PC demonstrated synergism with triple therapy, even at low concentrations. The results suggest that PC may be more effective against H. pylori than established antibiotics.

scholarly journals Effects of Temperature and pH on Fusarium oxysporum and Soybean Seedling Disease

Plant Disease ◽  
2019 ◽  
Vol 103 (12) ◽  
pp. 3234-3243
Author(s):  
David R. Cruz ◽  
Leonor F. S. Leandro ◽  
Gary P. Munkvold
Keyword(s):  
Fusarium Oxysporum ◽  
Root Rot ◽  
Radial Growth ◽  
Culture Media ◽  
Fungal Growth ◽  
Artificial Culture ◽  
Vitro Assay ◽  
Seedling Disease ◽  
Soybean Seedlings

Fusarium oxysporum (Fo) is an important pathogen that reduces soybean yield by causing seedling disease and root rot. This study assessed the effects of pH and temperature on Fo fungal growth and seedling disease. In an in vitro assay, 14 Fo isolates collected from symptomatic soybean roots across Iowa in 2007 were grown on artificial culture media at five pH levels (4, 5, 6, 7, and 8) and incubated at four temperatures (15, 20, 25, or 30°C). In a rolled-towel assay, soybean seeds from Fo-susceptible cultivar Jack were inoculated with a suspension of a pathogenic or a nonpathogenic Fo isolate; both isolates were previously designated for their relative aggressiveness in causing root rot at 25°C. The seeds were placed in rolled germination paper, and the rolls were incubated in all combinations of buffer solutions at four pH levels (4, 5, 6, and 7), and four temperatures (15, 20, 25, or 30°C). There was a significant interaction between temperature and pH (P < 0.05) for in vitro radial growth and root rot severity. Isolates showed the most in vitro radial growth after incubation at pH 6 and 25°C. For the rolled-towel assay, the pathogenic isolate caused the most severe root rot at pH 6 and 30°C. Gaussian regression analysis estimates for optimal conditions were pH 6.3 at 27.1°C for maximal fungal growth and pH 5.9 at 30°C for maximal root rot severity. These results indicate that optimal pH and temperature conditions are similar for Fo growth and disease in soybean seedlings and suggest that Fo may be a more important seedling pathogen when soybeans are planted under warm conditions in moderately acidic soils.

scholarly journals Processing of progranulin into granulins involves multiple lysosomal proteases and is affected in frontotemporal lobar degeneration

2020 ◽  
Author(s):  
Swetha Mohan ◽  
Paul J. Sampognaro ◽  
Andrea R. Argouarch ◽  
Jason C. Maynard ◽  
Anand Patwardhan ◽  
...  
Keyword(s):  
Frontotemporal Lobar Degeneration ◽  
Cathepsin L ◽  
Dependent Manner ◽  
Loss Of Function ◽  
New Strategy ◽  
Lysosomal Proteases ◽  
Ph Dependent ◽  
Increased Activity

Abstract Background: Progranulin loss-of-function mutations are linked to frontotemporal lobar degeneration with TDP-43 positive inclusions (FTLD-TDP-Pgrn). Progranulin (PGRN) is an intracellular and secreted pro-protein that is proteolytically cleaved into individual granulin peptides, which are increasingly thought to contribute to FTLD-TDP-Pgrn disease pathophysiology. Intracellular PGRN is processed into granulins in the endo-lysosomal compartments. Therefore, to better understand the conversion of intracellular PGRN into granulins, we systematically tested the ability of different classes of endo-lysosomal proteases at a range of pH setpoints.Results: In vitro cleavage assays identified multiple enzymes that can process human PGRN into multi- and single-granulin fragments in a pH-dependent manner. We confirmed the role of cathepsin B and cathepsin L in PGRN processing and showed that these and several previously unidentified lysosomal proteases (cathepsins E, G, K, S and V) are able to process PGRN in variable, pH-dependent manners. In addition, we have demonstrated a new role for asparagine endopeptidase (AEP) in processing PGRN, with AEP having the unique ability to liberate granulin F from the pro-protein. Brain tissue from individuals with FTLD-TDP-Pgrn show increased PGRN processing to granulin F, correlating with increased activity of AEP, in a region-specific manner. Conclusions: This study demonstrates that multiple lysosomal proteases may work in concert to liberate granulins and implicates both AEP and granulin F in the neurobiology of FTLD-TDP-Pgrn. Modulating progranulin cleavage may represent a new strategy to modulate PGRN and granulin levels in disease.

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 Processing of progranulin into granulins involves multiple lysosomal proteases and is affected in frontotemporal lobar degeneration

2020 ◽  
Author(s):  
Swetha Mohan ◽  
Paul J. Sampognaro ◽  
Andrea R. Argouarch ◽  
Jason C. Maynard ◽  
Anand Patwardhan ◽  
...  
Keyword(s):  
Frontotemporal Lobar Degeneration ◽  
Cathepsin L ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Specific Manner ◽  
Lysosomal Proteases ◽  
Ph Dependent ◽  
Increased Activity

Abstract Background - Progranulin loss-of-function mutations are linked to frontotemporal lobar degeneration with TDP-43 positive inclusions (FTLD-TDP-Pgrn). Progranulin (PGRN) is an intracellular and secreted pro-protein that is proteolytically cleaved into individual granulin peptides, which are increasingly thought to contribute to FTLD-TDP-Pgrn disease pathophysiology. Intracellular PGRN is processed into granulins in the endo-lysosomal compartments. Therefore, to better understand the conversion of intracellular PGRN into granulins, we systematically tested the ability of different classes of endo-lysosomal proteases to process PGRN at a range of pH setpoints. Results - In vitro cleavage assays identified multiple enzymes that can process human PGRN into multi- and single-granulin fragments in a pH-dependent manner. We confirmed the role of cathepsin B and cathepsin L in PGRN processing and showed that these and several previously unidentified lysosomal proteases (cathepsins E, G, K, S and V) are able to process PGRN in distinctive, pH-dependent manners. In addition, we have demonstrated a new role for asparagine endopeptidase (AEP) in processing PGRN, with AEP having the unique ability to liberate granulin F from the pro-protein. Brain tissue from individuals with FTLD-TDP-Pgrn show increased PGRN processing to granulin F and an increased activity of AEP, in a region-specific manner. Conclusions - This study demonstrates that multiple lysosomal proteases may work in concert to liberate multi-granulin fragments and granulins. It also implicates both AEP and granulin F in the neurobiology of FTLD-TDP-Pgrn. Modulating progranulin cleavage and granulin production may represent therapeutic strategies for FTLD-Pgrn and other progranulin-related diseases.

scholarly journals Processing of progranulin into granulins involves multiple lysosomal proteases and is affected in frontotemporal lobar degeneration

2020 ◽  
Author(s):  
Swetha Mohan ◽  
Paul J. Sampognaro ◽  
Andrea R. Argouarch ◽  
Jason C. Maynard ◽  
Anand Patwardhan ◽  
...  
Keyword(s):  
Frontotemporal Lobar Degeneration ◽  
Cathepsin L ◽  
Dependent Manner ◽  
Loss Of Function ◽  
New Strategy ◽  
Lysosomal Proteases ◽  
Ph Dependent ◽  
Increased Activity

Abstract Background - Progranulin loss-of-function mutations are linked to frontotemporal lobar degeneration with TDP-43 positive inclusions (FTLD-TDP-Pgrn). Progranulin (PGRN) is an intracellular and secreted pro-protein that is proteolytically cleaved into individual granulin peptides, which are increasingly thought to contribute to FTLD-TDP-Pgrn disease pathophysiology. Intracellular PGRN is processed into granulins in the endo-lysosomal compartments. Therefore, to better understand the conversion of intracellular PGRN into granulins, we systematically tested the ability of different classes of endo-lysosomal proteases at a range of pH setpoints. Results - In vitro cleavage assays identified multiple enzymes that can process human PGRN into multi- and single-granulin fragments in a pH-dependent manner. We confirmed the role of cathepsin B and cathepsin L in PGRN processing and showed that these and several previously unidentified lysosomal proteases (cathepsins E, G, K, S and V) are able to process PGRN in variable, pH-dependent manners. In addition, we have demonstrated a new role for asparagine endopeptidase (AEP) in processing PGRN, with AEP having the unique ability to liberate granulin F from the pro-protein. Brain tissue from individuals with FTLD-TDP-Pgrn show increased PGRN processing to granulin F, correlating with increased activity of AEP, in a region-specific manner. Conclusions - This study demonstrates that multiple lysosomal proteases may work in concert to liberate granulins and implicates both AEP and granulin F in the neurobiology of FTLD-TDP-Pgrn. Modulating progranulin cleavage may represent a new strategy to modulate PGRN and granulin levels in disease.

Immunodiagnostic potency of homologous antigens for natural Haemonchus contortus infection in small ruminants in plate and paperenzyme linked immunosorbent assay

2016 ◽  
Author(s):  
Niranjan Kumar ◽  
Bhupamani Das ◽  
Mehul M. Jadav ◽  
Jayesh B. Solanki
Keyword(s):  
Molecular Weight ◽  
Haemonchus Contortus ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Small Ruminants ◽  
Somatic Antigen ◽  
Predictive Values ◽  
Dot Elisa ◽  
Sensitivity Specificity

The objective of this study was to characterize Haemonchus contortus antigens, and to standardize and evaluate indirect plate and dot-ELISA using homologous antigens in the small ruminants. Electrophoretic separation of somatic antigen in reducing condition on 15% polyacrylamide gel resolved into 16 proteins of molecular weight ranging from 14-100 kDa. Two step ethanolic extraction of the supernatant of in-vitro culture of H. contortus yielded excretory-secretory (E-S) antigen/ cathepsin L cysteine proteinase of molecular weight 28 kDa. The animals (Goats=103; Sheep=91) were broadly kept into post-mortem (PM) and faecal examined groups and further sub-grouped based on mono or multiply helminths infection. At many occasion, the somatic antigen found to cross reacts with other helminths parasites thus minimizing the specificity of the selected tests and antigens. There was no any direct correlation between the parasites load and ELISA reactivity pattern. The prevalence rate of haemonchosis was 55.7 (34/61) in goats/ 47.6 (40/84) % in sheep as per PM examination while it was 45.63 (47/103) in goats/ 41.76% (38/91) in sheep and 36.89 (38/103) in goats/ 35.16% (32/91) in sheep using E-S antigen based plate and dot-ELISA, respectively. With E-S antigen, the overall % sensitivity, specificity, positive and negative predictive values of plate-ELISA was 89.74 (goats)/ 80.95 (sheep), 81.25 (goats)/ 91.84 (sheep), 74.47 (goats)/ 89.47 (sheep), 92.86 (goats)/ 84.91 (sheep), respectively while for dot-ELISA it was 66.67 (goats)/ 61.9 (sheep), 81.25 (goats)/ 87.76 (sheep), 68.42 (goats)/ 81.25 (sheep), 80 (goats)/ 72.88 (sheep), respectively, so the tests and E-S antigen can be recommended for the detection haemonchosis in the small ruminants.

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