Cathepsin B is not the processing enzyme for mouse prorenin

2010 ◽  
Vol 298 (5) ◽  
pp. R1212-R1216 ◽  
Author(s):  
Chantal Mercure ◽  
Marie-Josée Lacombe ◽  
Khashayarsha Khazaie ◽  
Timothy L. Reudelhuber
Keyword(s):  
Cathepsin B ◽  
Renin Angiotensin System ◽  
Apparent Molecular Weight ◽  
Culture Cell ◽  
Tissue Culture Cell ◽  
Active Renin ◽  
Processing Enzyme ◽  
Chloroquine Treatment

Renin, an aspartyl protease that catalyzes the rate-limiting step in the renin-angiotensin system (RAS), is proteolytically activated by a second protease [referred to as the prorenin processing enzyme (PPE)] before its secretion from the juxtaglomerular cells of the kidney. Although several enzymes are capable of activating renin in vitro, the leading candidate for the PPE in the kidney is cathepsin B (CTSB) due to is colocalization with the renin precursor (prorenin) in juxtaglomerular cell granules and because of its site-selective activation of human prorenin both in vitro and in transfected tissue culture cell models. To verify the role of CTSB in prorenin processing in vivo, we tested the ability of CTSB-deficient (CTSB−/−) mice to generate active renin. CTSB−/− mice do not exhibit any overt symptoms (renal malformation, preweaning mortality) typical of an RAS deficiency and have normal levels of circulating active renin, which, like those in control animals, rise more than 15-fold in response to pharmacologic inhibition of the RAS. The mature renin enzyme detected in kidney lysates of CTSB−/− mice migrates at the same apparent molecular weight as that in control mice, and the processing to active renin is not affected by chloroquine treatment of the animals. Finally, the distribution and morphology of renin-producing cells in the kidney is normal in CTSB−/− mice. In conclusion, CTSB-deficient mice exhibit no differences compared with controls in their ability to generate active renin, and our results do not support CTSB as the PPE in mice.

scholarly journals Simultaneous immunohistochemical detection of IUdR and BrdU infused intravenously to cancer patients.

1991 ◽  
Vol 39 (4) ◽  
pp. 407-412 ◽  
Author(s):  
M A Miller ◽  
C M Mazewski ◽  
N Yousuf ◽  
Y Sheikh ◽  
L M White ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Solid Tumors ◽  
Bone Marrow Cells ◽  
Tritiated Thymidine ◽  
Culture Cell ◽  
S Phase ◽  
Tissue Culture Cell ◽  
Cell Cycle Time

Cell cycle kinetics of solid tumors in the past have been restricted to an in vitro labeling index (LI) measurement. Two thymidine analogues, bromodeoxyuridine (BrdU) and iododeoxyuridine (IUdR), can be used to label S-phase cells in vivo because they can be detected in situ by use of monoclonal antibodies (MAb) against BrdU (Br-3) or IUdR (3D9). Patients with a variety of solid tumors (lymphoma, brain, colon cancers) received sequential intravenous IUdR and BrdU. Tumor tissue removed at the end of infusion was embedded in plastic and treated with MAb Br-3 and 3D9 sequentially, using a modification of a previously described method. Clearly single and double labeled cells were visible, which enabled us to determine the duration of S-phase (Ts) and the total cell cycle time (Tc), in addition to the LI in these tumors. Detailed control experiments using tissue culture cell lines as well as bone marrow cells from leukemic patients are described, including the comparison of this double label technique with our previously described BrdU-tritiated thymidine technique. We conclude that the two methods are comparable and that the IUdR/BrdU method permits rapid and reliable cell cycle measurements in solid tumors.

Active and inactive renin in human forearm of hypertensive patients

1991 ◽  
Vol 69 (9) ◽  
pp. 1390-1393 ◽  
Author(s):  
Stefano Taddei ◽  
Stefania Favilla ◽  
Antonio Salvetti
Keyword(s):  
Brachial Artery ◽  
Vascular Tissue ◽  
Physiological Role ◽  
Renin Angiotensin System ◽  
Hypertensive Patients ◽  
Active Renin ◽  
Inactive Renin ◽  
Dose Dependent

Although many in vitro and animal studies indicate the existence of a local renin–angiotensin system, data regarding its physiological role are quite controversial, and moreover, evidence suggesting inactive and active renin release from vascular tissue in vivo is lacking both in animal and humans. The aim of our study was to evaluate whether β-adrenoceptor stimulation, a well-known stimulus to renin production, through isoproterenol might cause local renin production from vessels of the forearm of hypertensive patients. Drugs were infused into the brachial artery at systemically ineffective rates, while forearm blood flow (FBF, venous plethysmography), mean intra-arterial pressure, and heart rate were monitored throughout. Active and inactive vessel renin production was measured by calculating venous-arterial (V-A) differences by simultaneous sampling from brachial artery and an ipsilateral deep vein. Active renin (PRA) and total renin (Sepharose bound trypsin activation) were measured by radioimmunoassay while inactive renin was calculated as the difference between total and active renin. V-A differences were corrected for FBF to calculate renin extraction or production. In a group of 10 patients, isoproterenol, which was infused at increasing cumulative rates (0.03, 0.1, 0.3 μg∙100 mL−1 forearm tissue∙min−1 for 5 min each), caused a dose-dependent increment in FBF that was blunted by intra-arterial propranolol (n = 5) pretreatment (10 μg∙100 mL−1 forearm tissue∙min−1 for 10 min). β-Adrenoceptor stimulation caused a dose-dependent outflow of both active and inactive renin, an effect antagonized by propranolol. In conclusion, our data represent the first evidence in humans of tissue active and inactive renin production in the forearm vascular bed.Key words: tissue renin, active renin, inactive renin, isoproterenol.

Laboratory investigation of virulence among strains of Yersinia enterocolitica and related species isolated from pigs and pork products

1992 ◽  
Vol 38 (2) ◽  
pp. 92-97 ◽  
Author(s):  
Jacob K. P. Kwaga ◽  
John O. Iversen
Keyword(s):  
Yersinia Enterocolitica ◽  
Related Species ◽  
Surface Hydrophobicity ◽  
Culture Cell ◽  
In Vitro Assays ◽  
Tissue Culture Cell ◽  
Good Prediction ◽  
Pork Products

Eighty strains of Yersinia enterocolitica and related species isolated from slaughtered pigs and pork products were tested for possession of virulence-associated phenotypes by employing 12 in vivo and in vitro assays. The isolates could be broadly divided into two groups: (i) strains belonging to pathogenic bioserotypes of Y. enterocolitica that displayed virulence-associated characteristics in most or all assays and (ii) strains belonging to Y. enterocolitica biotype 1A and to related species that were largely negative in these assays. No individual test was found as a single reliable measure of virulence. All strains belonging to Y. enterocolitica serotype O: 1,2,3 were pyrazinamidase positive (indicates avirulence) and autoagglutination negative but were positive in all other virulence assays. Salt aggregation was found to be a better indicator of virulence than latex particle agglutination, both of which measure surface hydrophobicity. Overall, tissue culture cell invasion provided the best selection of a subpopulation of yersiniae that are potentially virulent. However, crystal violet and Congo red binding assays among others provided good prediction of virulence at the time of testing. Our results provide further evidence that swine may constitute an important reservoir of human pathogenic strains. Key words: Yersinia enterocolitica, virulence phenotype, human pathogen.

Generation of Inactive Renin in Vitro and in Vivo: Reversible Inactivation of the Active Plasma Enzyme in Rats

1982 ◽  
Vol 63 (s8) ◽  
pp. 171s-174s ◽  
Author(s):  
Jack D. Barrett ◽  
Peter Eggena ◽  
Mohinder P. Sambhi
Keyword(s):  
Active Form ◽  
Renin Angiotensin System ◽  
Active Plasma ◽  
Reversible Inactivation ◽  
Active Renin ◽  
Plasma Enzyme ◽  
Inactive Renin ◽  
Rate Of Decline

1. Active and total (trypsin treatment) plasma renins were measured in normal Wistar rats and in rats in which the renin-angiotensin system was stimulated by ether anaesthesia. 2. After incubation of normal plasma in vitro in the absence of angiotensinase inhibitors, active renin declined. This decline was shown to be due to the conversion of active renin into an inactive form, which could be re-activated by trypsin. 3. In plasma from renin-stimulated rats, the rate of decline of active renin in vitro was accelerated; however, the relative amount of inactive renin generated was decreased. 4. Ligation of the kidneys of the ether-anaesthetized animal resulted in a build-up in vivo of inactive renin concomitant with the decline of active renin. 5. These data demonstrate the conversion of active into inactive renin in vitro and indicate that inactive renin can also be generated in vivo from the active form of the enzyme. 6. Multiple forms of inactive renin may exist; some may be true ‘prorenins’ (renin zymogens) produced in the kidney, and others may result from post-biosynthetic modifications of the active plasma enzyme.

scholarly journals Adipose and Muscle Cell Co-Culture System: A Novel In Vitro Tool to Mimic the In Vivo Cellular Environment

Biology ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 6
Author(s):  
Palaniselvam Kuppusamy ◽  
Dahye Kim ◽  
Ilavenil Soundharrajan ◽  
Inho Hwang ◽  
Ki Choon Choi
Keyword(s):  
Drug Development ◽  
Culture System ◽  
Cell Types ◽  
Culture Cell ◽  
In Vitro Techniques ◽  
Culture Systems ◽  
Complex Interactions ◽  
Cellular Environment

A co-culture system allows researchers to investigate the complex interactions between two cell types under various environments, such as those that promote differentiation and growth as well as those that mimic healthy and diseased states, in vitro. In this paper, we review the most common co-culture systems for myocytes and adipocytes. The in vitro techniques mimic the in vivo environment and are used to investigate the causal relationships between different cell lines. Here, we briefly discuss mono-culture and co-culture cell systems and their applicability to the study of communication between two or more cell types, including adipocytes and myocytes. Also, we provide details about the different types of co-culture systems and their applicability to the study of metabolic disease, drug development, and the role of secretory factors in cell signaling cascades. Therefore, this review provides details about the co-culture systems used to study the complex interactions between adipose and muscle cells in various environments, such as those that promote cell differentiation and growth and those used for drug development.

scholarly journals Cathepsin B Localizes in the Caveolae and Participates in the Proteolytic Cascade in Trabecular Meshwork Cells. Potential New Drug Target for the Treatment of Glaucoma

2020 ◽  
Vol 10 (1) ◽  
pp. 78
Author(s):  
April Nettesheim ◽  
Myoung Sup Shim ◽  
Angela Dixon ◽  
Urmimala Raychaudhuri ◽  
Haiyan Gong ◽  
...  
Keyword(s):  
Trabecular Meshwork ◽  
Cathepsin B ◽  
Molecular Mechanisms ◽  
Culture Media ◽  
Ecm Remodeling ◽  
Trabecular Meshwork Cells ◽  
Proteolytic Cascade

Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTBko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFβ/Smad signaling in TM cells vitro and in vivo in CTSBko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.

scholarly journals Accelerated Repurposing and Drug Development of Pulmonary Hypertension Therapies for COVID-19 Treatment Using an AI-Integrated Biosimulation Platform

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 1912
Author(s):  
Kaushik Chakravarty ◽  
Victor G. Antontsev ◽  
Maksim Khotimchenko ◽  
Nilesh Gupta ◽  
Aditya Jagarapu ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Renin Angiotensin System ◽  
Mechanistic Modeling ◽  
Channel Blockers ◽  
Angiotensin System ◽  
In Silico Modeling ◽  
Site Of Action ◽  
Line Of Therapy

The COVID-19 pandemic has reached over 100 million worldwide. Due to the multi-targeted nature of the virus, it is clear that drugs providing anti-COVID-19 effects need to be developed at an accelerated rate, and a combinatorial approach may stand to be more successful than a single drug therapy. Among several targets and pathways that are under investigation, the renin-angiotensin system (RAS) and specifically angiotensin-converting enzyme (ACE), and Ca2+-mediated SARS-CoV-2 cellular entry and replication are noteworthy. A combination of ACE inhibitors and calcium channel blockers (CCBs), a critical line of therapy for pulmonary hypertension, has shown therapeutic relevance in COVID-19 when investigated independently. To that end, we conducted in silico modeling using BIOiSIM, an AI-integrated mechanistic modeling platform by utilizing known preclinical in vitro and in vivo datasets to accurately simulate systemic therapy disposition and site-of-action penetration of the CCBs and ACEi compounds to tissues implicated in COVID-19 pathogenesis.

Processing of the precursor to a chloroplast ribosomal protein made in the cytosol occurs in two steps, one of which depends on a protein made in the chloroplast

1985 ◽  
Vol 5 (5) ◽  
pp. 1093-1099
Author(s):  
R J Schmidt ◽  
N W Gillham ◽  
J E Boynton
Keyword(s):  
Molecular Weight ◽  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Apparent Molecular Weight ◽  
Protein L ◽  
Mature Form ◽  
Chloroplast Protein Synthesis ◽  
Made In

In pulse-chase experiments in which log-phase cells of Chlamydomonas reinhardtii were labeled in vivo for 5 min with H2(35)SO4, fluorographs of immunoprecipitates from whole cell extracts revealed that chloroplast ribosomal proteins L-2, L-6, L-21, and L-29, which are made in the cytosol and imported, appeared in their mature forms. However, in the case of chloroplast ribosomal protein L-18, which is also made in the cytoplasm and imported, a prominent precursor with an apparent molecular weight of 17,000 was found at the end of a 5-min pulse. This precursor was processed to its mature size (apparent molecular weight of 15,500) within the first 5 min of the subsequent chase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the precursor to L-18 formed in vivo was 1.5 kilodaltons smaller than the primary product detected in translations of Chlamydomonas polyadenylated RNA in vitro. Upon a 10-min incubation with a postribosomal supernatant from Chlamydomonas, the 18,500-dalton precursor detected in vitro could be partially converted into a polypeptide that comigrated with the 17,000-dalton precursor detected in extracts of cells labeled in vivo. Under conditions in which the total amounts of chloroplast proteins had been reduced and cells were made to synthesize ribosomes rapidly, the apparent half-life of the 17,000-dalton precursor was extended over that seen in log-phase cells. When chloroplast protein synthesis was inhibited with lincomycin for 3 h before labeling under these conditions, the 17,000-dalton L-18 precursor but not the mature form was found, and the precursor was slowly degraded during a 60-min chase. When cells were placed in the dark for 3 h before labeling, processing of this precursor to the mature form appeared unaffected, but the chloroplast-synthesized ribosomal protein L-26 was detected, indicating that chloroplast protein synthesis was still occurring. We interpret these results to indicate that the maturation of protein L-18 in vivo involves at least two processing steps, one of which depends on a protein made on chloroplast ribosomes.

scholarly journals Cathepsin B inhibition interferes with metastatic potential of human melanoma: an in vitro and in vivo study

2010 ◽  
Vol 9 (1) ◽  
pp. 207 ◽  
Author(s):  
Paola Matarrese ◽  
Barbara Ascione ◽  
Laura Ciarlo ◽  
Rosa Vona ◽  
Carlo Leonetti ◽  
...  
Keyword(s):  
Cathepsin B ◽  
Metastatic Potential ◽  
Human Melanoma ◽  
In Vivo Study
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