Cathepsin L mediates intracellular ileal digestion of gastric intrinsic factor

Although acidic proteases of lysosomal origin are implicated in the degradation of intrinsic factor (IF) during cobalamin (cbl) transport across enterocytes and proximal renal tubule cell lines, the enzyme(s) involved in this process is not known. Recombinant (baculovirus-produced) rat 125I-labeled IF (125I-rIF), 43 kDa, added in vivo to the lumen of rat ileum was converted intracellularly to peptides of 33 and 26 kDa. In vitro rat 125I-rIF was degraded to peptides of 33 and 31 kDa by addition of cathepsin L; this conversion was fully inhibited by leupeptin. Western blot analysis using antiserum against denatured native rat IF identified additional cathepsin L degradation products in the 17- to 23-kDa range. In vitro the binding of cobalamin partially inhibited cathepsin L degradation of IF. Rat rIF produced from either insect (Sf9) or mammalian (CHO) cells and native rat IF were all degraded by cathepsin L, although the prominence of the various products differed in the recombinant preparations, being 33 and 36 kDa, respectively. Native rat IF was most sensitive to proteolysis, and no degradation products were identified. Rat 125I-rIF was taken up by LLC-PK1 cells, and 125I from degraded IF appeared abundantly on the basolateral side of cell monolayers by 1 h. The intracellular products of rat rIF in LLC-PK1 cells were the same size as those produced in vitro by the action of cathepsin L. Antiserum against a human kidney cDNA cathepsin L fusion protein easily demonstrated the protease in rat intestinal mucosa, as well as in all other tissues tested. These data suggest that cathepsin L is the protease responsible for the leupeptin-sensitive intracellular degradation of IF.

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THE EFFICACY AND RELATIVE FIBRIN SELECTIVITY OF PROUROKINASE IN PATIENTS WITH ACUTE MYOCARDIAL INFARCTION

10.1055/s-0038-1643573 ◽

1987 ◽

Author(s):

J Loscalzo

Keyword(s):

Myocardial Infarction ◽

Acute Myocardial Infarction ◽

Degradation Products ◽

Coronary Thrombosis ◽

Therapeutic Option ◽

Human Kidney ◽

Thrombolytic Agents ◽

Hemorrhagic Complications

The use of thrombolytic agents in acute myocardial infarction has gained widespread acceptance as an important early therapeutic option. Acute coronary thrombosis has been found in approximately 80% of patients with acute infarction and the use of standard thrombolytic agents opens these occluded vessels in most cases. Unfortunately, in many individuals standard agents also produce a systemic lytic state with its attendant hemorrhagic complications. Prourokinase (PUK) has been shown to be a relatively fibrin selective thrombolytic agent in vitro owing to its localized conversion to urokinase at the clot surface. Because of this desirable property, we studied the efficacy and selectivity of PUK in vivo in 19 patients with acute myocardial infarction. Each of these patients was documented by angiography to have a totally occluded infarct-related artery. Each patient was treated within six hours (range: 2 to 5.8 hours) of the onset of symptoms with 62.5 mg of PUK derived from the human kidney cell line, TCL598, infused intravenously over 90 minutes. Complete vessel patency was achieved in nine patients within 61 ± 19 minutes of the start of the infusion without apparent hemorrhagic complications. We evaluated the effect of PUK on fibrinogen, on nonspecific fibrinogen degradation products (FDP) the the fibrinogen/fibrin I peptide, Bβ 1-42, as well as on the specific fibrin degradation products, D-dimer (XDP) and the fibrin II peptide, Bβ 15-42. Values for these parameters measured before and at the end of the 90-minute infusion of PUK are given as the mean ± S.E.M.We conclude that PUK is an effective agent with which to achieve coronary thrombolysis and that at the doses used in this study, it aDDears to be relatively fibrin selective.

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Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646468 ◽

1991 ◽

Vol 66 (05) ◽

pp. 609-613 ◽

Cited By ~ 14

Author(s):

I R MacGregor ◽

J M Ferguson ◽

L F McLaughlin ◽

T Burnouf ◽

C V Prowse

Keyword(s):

High Purity ◽

Time Course ◽

Prothrombin Complex Concentrate ◽

Degradation Products ◽

Canine Model ◽

Factor Ix ◽

In Vitro Tests ◽

Albumin Infusion

SummaryA non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.

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Accumulation of Macromolecular Particles in the Reticuloendothelial System (RES) Mediated by Platelet Aggregation and Disaggregation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651386 ◽

1969 ◽

Vol 22 (03) ◽

pp. 496-507 ◽

Cited By ~ 17

Author(s):

W.G van Aken ◽

J Vreeken

Keyword(s):

Platelet Aggregation ◽

Degradation Products ◽

Reticuloendothelial System ◽

Caproic Acid ◽

Carbon Particles ◽

Carbon Clearance ◽

Aggregation And Disaggregation ◽

Platelet Aggregates

SummaryCarbon particles cause platelet aggregation in vitro and in vivo. Prior studies established that substances which modify thrombocyte aggregation also influence the rate at which carbon is cleared from the blood.This study was performed in order to elucidate the mechanism by which the carbon-platelet aggregates specifically accumulate in the RES.Activation of fibrinolysis by urokinase or streptokinase reduced the carbon clearance rate, probably due to generated fibrinogen degradation products (FDP). Isolated FDP decreased the carbon clearance and caused disaggregation of platelets and particles in vitro. Inhibition of fibrinolysis by epsilon-amino-caproic acid (EACA), initially accelerated the disappearance of carbon and caused particle accumulation outside the RES, predominantly in the lungs. It is supposed that platelet aggregation and locally activated fibrinolysis act together in the clearance of particles. In the normal situation the RES with its well known low fibrinolytic activity, becomes the receptor of the particles.

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The Effects of Brinolase (Fibrinolytic Enzyme from Aspergillus Oryzae) on Platelet Aggregation of Dog and Man

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649039 ◽

1972 ◽

Vol 28 (01) ◽

pp. 031-048 ◽

Cited By ~ 4

Author(s):

W. H. E Roschlau ◽

R Gage

Keyword(s):

Platelet Aggregation ◽

Aspergillus Oryzae ◽

Degradation Products ◽

Blood Platelet ◽

Human Platelets ◽

Fibrinolytic Enzyme ◽

Inhibitory Effects ◽

Blood Platelet Aggregation

SummaryInhibition of blood platelet aggregation by brinolase (fibrinolytic enzyme from Aspergillus oryzae) has been demonstrated with human platelets in vitro and with dog platelets in vivo and in vitro, using both ADP and collagen as aggregating stimuli. It is suggested that the optimal inhibitory effects of brinolase occur indirectly through the generation of plasma fibrinogen degradation products, without compromising platelet viability, rather than by direct proteolysis of platelet structures.

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Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-021-00558-8 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Miao-Miao Zhao ◽

Wei-Li Yang ◽

Fang-Yuan Yang ◽

Li Zhang ◽

Wei-Jin Huang ◽

...

Keyword(s):

Drug Development ◽

Cathepsin L ◽

Human Cells ◽

New Drugs ◽

Disease Course ◽

Promising Target ◽

First Time

AbstractTo discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

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Secreted KIAA1199 promotes the progression of rheumatoid arthritis by mediating hyaluronic acid degradation in an ANXA1-dependent manner

Cell Death and Disease ◽

10.1038/s41419-021-03393-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Wei Zhang ◽

Guoyu Yin ◽

Heping Zhao ◽

Hanzhi Ling ◽

Zhen Xie ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Hyaluronic Acid ◽

Dependent Manner ◽

Collagen Induced Arthritis ◽

Intracellular Degradation ◽

Main Pathway ◽

First Time

AbstractIn inflamed joints, enhanced hyaluronic acid (HA) degradation is closely related to the pathogenesis of rheumatoid arthritis (RA). KIAA1199 has been identified as a hyaladherin that mediates the intracellular degradation of HA, but its extracellular function remains unclear. In this study, we found that the serum and synovial levels of secreted KIAA1199 (sKIAA1199) and low-molecular-weight HA (LMW-HA, MW < 100 kDa) in RA patients were significantly increased, and the positive correlation between them was shown for the first time. Of note, treatment with anti-KIAA1199 mAb effectively alleviated the severity of arthritis and reduced serum LMW-HA levels and cytokine secretion in collagen-induced arthritis (CIA) mice. In vitro, sKIAA1199 was shown to mediate exogenous HA degradation by attaching to the cell membrane of RA fibroblast-like synoviosytes (RA FLS). Furthermore, the HA-degrading activity of sKIAA1199 depended largely on its adhesion to the membrane, which was achieved by its G8 domain binding to ANXA1. In vivo, kiaa1199-KO mice exhibited greater resistance to collagen-induced arthritis. Interestingly, this resistance could be partially reversed by intra-articular injection of vectors encoding full-length KIAA1199 instead of G8-deleted KIAA119 mutant, which further confirmed the indispensable role of G8 domain in KIAA1199 involvement in RA pathological processes. Mechanically, the activation of NF-κB by interleukin-6 (IL-6) through PI3K/Akt signaling is suggested to be the main pathway to induce KIAA1199 expression in RA FLS. In conclusion, our study supported the contribution of sKIAA1199 to RA pathogenesis, providing a new therapeutic target for RA by blocking sKIAA1199-mediated HA degradation.

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Cathepsin L inhibition suppresses drug resistance in vitro and in vivo: a putative mechanism

AJP Cell Physiology ◽

10.1152/ajpcell.00082.2008 ◽

2009 ◽

Vol 296 (1) ◽

pp. C65-C74 ◽

Cited By ~ 35

Author(s):

Xin Zheng ◽

Fei Chu ◽

Pauline M. Chou ◽

Christine Gallati ◽

Usawadee Dier ◽

...

Keyword(s):

Drug Resistance ◽

Cancer Cell ◽

Trichostatin A ◽

Cathepsin L ◽

Active Form ◽

Cancer Resistance ◽

Protease Inhibition ◽

Resistance To Chemotherapy

Cathepsin L is a lysosomal enzyme thought to play a key role in malignant transformation. Recent work from our laboratory has demonstrated that this enzyme may also regulate cancer cell resistance to chemotherapy. The present study was undertaken to define the relevance of targeting cathepsin L in the suppression of drug resistance in vitro and in vivo and also to understand the mechanism(s) of its action. In vitro experiments indicated that cancer cell adaptation to increased amounts of doxorubicin over time was prevented in the presence of a cathepsin L inhibitor, suggesting that inhibition of this enzyme not only reverses but also prevents the development of drug resistance. The combination of the cathepsin L inhibitor with doxorubicin also strongly suppressed the proliferation of drug-resistant tumors in nude mice. An investigation of the underlying mechanism(s) led to the finding that the active form of this enzyme shuttles between the cytoplasm and nucleus. As a result, its inhibition stabilizes and enhances the availability of cytoplasmic and nuclear protein drug targets including estrogen receptor-α, Bcr-Abl, topoisomerase-IIα, histone deacetylase 1, and the androgen receptor. In support of this, the cellular response to doxorubicin, tamoxifen, imatinib, trichostatin A, and flutamide increased in the presence of the cathepsin L inhibitor. Together, these findings provided evidence for the potential role of cathepsin L as a target to suppress cancer resistance to chemotherapy and uncovered a novel mechanism by which protease inhibition-mediated drug target stabilization may enhance cellular visibility and, thus, susceptibility to anticancer agents.

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Solubility and ADMET profiles of short oligomers of lactic acid

ADMET & DMPK ◽

10.5599/admet.843 ◽

2020 ◽

Cited By ~ 1

Author(s):

Daniela Dascălu ◽

Diana Larisa Roman ◽

Madalina Filip ◽

Alecu Aurel Ciorsac ◽

Vasile Ostafe ◽

...

Keyword(s):

Molecular Weight ◽

Glucocorticoid Receptors ◽

Degradation Products ◽

Organic Anion ◽

Toxicity Tests ◽

Medical Applications ◽

Eye Injuries ◽

Toxicological Effects

<p class="ADMETkeywordsheading">Polylactic acid (PLA) is a polymer with an increased potential to be used in different medical applications, including tissue engineering and drug-carries. The use of PLA in medical applications implies the evaluation of the human organism's response to the polymer inserting and to its degradation products. Consequently, within this study, we have investigated the solubility and ADMET profiles of the short oligomers (having the molecular weight lower than 3000 Da) resulting in degradation products of PLA. There is a linear decrease of the molar solubility of investigated oligomers with molecular weight. The results that are obtained also reveal that short oligomers of PLA have promising pharmacological profiles and limited toxicological effects on humans. These oligomers are predicted as potential inhibitors of the organic anion transporting peptides OATP1B1 and OATP1B3, they present minor probability to affect the androgen and glucocorticoid receptors, have a weak potential of hepatotoxicity, and may produce eye injuries. These outcomes may be used to guide or to supplement in vitro and/or in vivo toxicity tests such as to enhance the biodegradation properties of the biopolymer.</p>

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In Vivo Entombment of Bacteria and Fungi during Calcium Oxalate, Brushite and Struvite Urolithiasis

Kidney360 ◽

10.34067/kid.0006942020 ◽

2020 ◽

pp. 10.34067/KID.0006942020

Author(s):

Jessica J. Saw ◽

Mayandi Sivaguru ◽

Elena M. Wilson ◽

Yiran Dong ◽

Robert A. Sanford ◽

...

Keyword(s):

Calcium Oxalate ◽

Stone Formation ◽

Human Kidney ◽

Rrna Gene ◽

Bacterial Cells ◽

Stone Formers ◽

Bacteria And Fungi ◽

Struvite Stones

Background: Human kidney stones form via repeated events of mineral precipitation, partial dissolution and reprecipitation, which are directly analogous to similar processes in other natural and man-made environments where resident microbiomes strongly influence biomineralization. High-resolution microscopy and high-fidelity metagenomic (microscopy-to-omics) analyses, applicable to all forms of biomineralization, have been applied to assemble definitive evidence of in vivo microbiome entombment during urolithiasis. Methods: Stone fragments were collected from a randomly chosen cohort of 20 patients using standard percutaneous nephrolithotomy (PCNL). Fourier transform infrared (FTIR) spectroscopy indicated that 18 of these patients were calcium oxalate (CaOx) stone formers, while one patient each formed brushite and struvite stones. This apportionment is consistent with global stone mineralogy distributions. Stone fragments from 7 of these 20 patients (5 CaOx, 1 brushite and 1 struvite) were thin sectioned and analyzed using brightfield (BF), polarization (POL), confocal, superresolution autofluorescence (SRAF) and Raman techniques. DNA from remaining fragments, grouped according to each of the 20 patients, were analyzed with amplicon sequencing of 16S rRNA gene sequences (V1-V3, V3-V5) and internal transcribed spacer (ITS1, ITS2) regions. Results: Bulk entombed DNA was sequenced from stone fragments in 11 of the 18 CaOx patients, as well as the brushite and struvite patients. These analyses confirmed the presence of an entombed low-diversity community of bacteria and fungi, including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Aspergillus niger. Bacterial cells ~1 µm in diameter were also optically observed to be entombed and well-preserved in amorphous hydroxyapatite spherules and fans of needle-like crystals of brushite and struvite. Conclusions: These results indicate a microbiome is entombed during in vivo CaOx stone formation. Similar processes are implied for brushite and struvite stones. This evidence lays the groundwork for future in vitro and in vivo experimentation to determine how the microbiome may actively and/or passively influence kidney stone biomineralization.

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A Validated HPLC-PDA-HRMS Method to Investigate the Biological Stability and Metabolism of Antiparasitic Triterpenic Esters

Molecules ◽

10.3390/molecules26237154 ◽

2021 ◽

Vol 26 (23) ◽

pp. 7154

Author(s):

Laura Schioppa ◽

Fanta Fall ◽

Sergio Ortiz ◽

Jacques H. Poupaert ◽

Joelle Quetin-Leclercq

Keyword(s):

Medicinal Plants ◽

Degradation Products ◽

Biological Stability ◽

Acidic Media ◽

Microsomal Metabolism ◽

Plasma Enzymes ◽

Absorption Studies ◽

H 30

Pentacyclic triterpenes (PTs) are commonly found in medicinal plants with well-known antiparasitic effects. Previous research on C-3 and C-27 triterpenic esters showed effective and selective in vitro antiparasitic activities and in vivo effectiveness by parenteral routes. The aim of this study was to determine triterpenic esters’ stability in different biological-like media and the main microsomal degradation products. An HPLC-PDA method was developed and validated to simultaneously analyze and quantify bioactive triterpenic esters in methanol (LOQ: 2.5 and 1.25–100 µg/mL) and plasma (LOQ: 5–125 µg/mL). Overall, both triterpenic esters showed a stable profile in aqueous and buffered solutions as well as in entire plasma, suggesting gaining access to the ester function is difficult for plasma enzymes. Conversely, after 1 h, 30% esters degradation in acidic media was observed with potential different hydrolysis mechanisms. C-3 (15 and 150 µM) and C-27 esters (150 µM) showed a relatively low hepatic microsomal metabolism (<23%) after 1 h, which was significantly higher in the lowest concentration of C-27 esters (15 µM) (>40% degradation). Metabolic HPLC-PDA-HRMS studies suggested hydrolysis, hydroxylation, dehydration, O-methylation, hydroxylation and/or the reduction of hydrolyzed derivatives, depending on the concentration and the position of the ester link. Further permeability and absorption studies are required to better define triterpenic esters pharmacokinetic and specific formulations designed to increase their oral bioavailability.

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