scholarly journals Evaluation of In Vivo Proteolytic Activity

10.5772/25951 ◽  
2012 ◽  
Author(s):  
Wataru Yoshida ◽  
Akihisa Kamataki ◽  
Miwa Uzuki ◽  
Takashi Sawai
Keyword(s):  
Proteolytic Activity

scholarly journals Demonstration in vivo that stromelysin-3 functions through its proteolytic activity

Oncogene ◽  
2000 ◽  
Vol 19 (12) ◽  
pp. 1605-1612 ◽  
Author(s):  
A Noël ◽  
A Boulay ◽  
F Kebers ◽  
R Kannan ◽  
A Hajitou ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Stromelysin 3

Phenotypic Correction of Adamts13-Deficient Mice by Early Intra-Amniotic Gene Transfer of Lentiviral Vector Encoding ADAMTS13 Genes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 197-197
Author(s):  
Masami Niiya ◽  
Masayuki Endo ◽  
Philip W. Zoltick ◽  
Nidal E. Muvarak ◽  
David G. Motto ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Proteolytic Activity ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Occlusion Time ◽  
Carboxyl Terminal ◽  
Human Wild Type

Abstract ADAMTS13, a member of A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, is mainly synthesized in the hepatic stellate cells, endothelial cells and megakaryocytes or platelets. It controls the sizes of von Willebrand factor (VWF) multimers by cleaving VWF at the Tyr1605-Met1606 bond. Genetic deficiency of plasma ADAMTS13 activity results in hereditary thrombotic thrombocytopenic purpura (TTP), also named Upshaw-Schülman syndrome. To develop a potential gene therapy approach and to determine the domains of ADAMTS13 required for recognition and cleavage of VWF in vivo, a self-inactivating lentiviral vector encoding human wild-type ADAMTS13 or variant truncated after the spacer domain (construct MDTCS) was administrated by intra-amniotic injection on embryonic day 8. Direct stereomicroscopy and immunofluorescent microscopic analysis revealed that the green fluorescent protein (GFP) reporter, ADAMTS13 and MDTCS were predominantly expressed in the heart, kidneys and skin. The synthesized ADAMTS13 and truncated variant were detectable in mouse plasma by immunoprecipitation and Western blot, as well as by proteolytic cleavage of FRETS-VWF73 substrate. The levels of proteolytic activity in plasma of mice expressing ADAMTS13 and MDTCS were 5 ± 7% and 60 ± 70%, respectively using normal human plasma as a standard, and this proteolytic activity persisted for at least 24 weeks in Adamts13−/−mice and 42 weeks in wild-type mice tested (the duration of observation). The mice expressing both recombinant ADAMTS13 and MDTCS showed a significantly decreased ratio of plasma VWF collagen-binding activity to antigen and a reduction in VWF multimer sizes as compared to those in the controls. Moreover, the mice expressing ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time (9.0 ± 0.6 and 25.2 ± 3.2 min, respectively) as compared to the Adamts13−/− mice expressing GFP alone (5.6 ± 0.5 min) (p<0.01). The ferric chloride-induced carotid occlusion time in Adamts13−/− mice expressing ADAMTS13 was almost identical to that in wild type mice with same genetic background (C56BL/6) (8.0 ± 0.2 min) (p>0.05). The data demonstrate the correction of the prothrombotic phenotype in Adamts13−/−mice by gene transfer to the fetus by viral vectors encoding human wild type ADAMTS13 and the carboxyl terminal truncated variant (MDTCS), supporting the feasibility of developing a gene therapy based treatment for hereditary TTP. The discrepancy in the proteolytic activity of MDTCS between in vitro (Zhang P et al. Blood, 2007 in press) and in vivo in the present study suggests the potential cofactors in murine circulation that may rescue the defective proteolytic activity of the carboxyl-terminal truncated ADAMTS13 protease seen in vitro.

Effect of removing sialic acids from endothelium on the adherence of circulating platelets in arteries in vivo

1982 ◽  
Vol 214 (1197) ◽  
pp. 471-480 ◽  
Keyword(s):  
Sialic Acid ◽  
Proteolytic Activity ◽  
Negative Charge ◽  
Sialic Acids ◽  
Arterial Walls ◽  
Charge Excess ◽  
Flow Through ◽  
Adhesion Of Platelets ◽  
Prostacyclin Production

The contribution of the net negative charge excess due to sialic acids on endothelium in preventing adhesion of circulating platelets in vivo was investigated in anaesthetized rabbits. Platelets in the rabbit’s circulation were selectively labelled with radioactive 5-hydroxytryptamine in vivo . Segments of carotid arteries temporarily isolated from the circulation were perfused with one or other of two commercial preparations of neuraminidase; the opposite carotid artery was perfused similarly without the enzyme, as control. A neuraminidase preparation from Behringwerke free of proteolytic activity released sialic acid into the perfusate with a peak concentration after 10–15 min which decreased gradually later. A neuraminidase preparation from Sigma that contained demonstrable proteolytic activity released sialic acid similarly during the first hour and thereafter more sialic acid in a second peak. After blood flow through the carotids had been restored the adhesion of labelled platelets in the artery perfused with neuraminidase was compared with that in the artery perfused without the enzyme. The radioactivities were significantly higher in carotids that had been perfused with neuraminidase than in those that had been perfused without the enzyme. Neuraminidase perfusion had no effect on the production of prostacyclin by the carotids. Perfusion with acetylsalicylic acid before neuraminidase increased the adhesion of platelets significantly. It is concluded that diminution in electrostatic repulsion between circulating platelets and vascular endothelium from which the net negative charge excess due to sialic acids has been removed increases the adhesion of circulating platelets, irrespective of the production of prostacyclin by the arterial walls, and that inhibition of prostacyclin production augments this adhesion of platelets.

scholarly journals Influens of mole rats burrow activity to restore the soils proteolytic activity in terms of their man-made pollution

2016 ◽  
Vol 17 (3-4) ◽  
pp. 107-111
Author(s):  
T. A. Zamesova
Keyword(s):  
Heavy Metals ◽  
Metal Concentration ◽  
Proteolytic Activity ◽  
The Body ◽  
Activity Level ◽  
Forming Process ◽  
Vital Functions ◽  
The Impact ◽  
Burrow Activity

Heavy metals, especially their large concentration, is toxic for all living creatures because they are accumulating in the living things tissues. Theirs excessive levels evoke coagulation of proteins that cause immediate death of cells. Heavy metals get into the ground by different ways: directly because of using, precipitation, industrial pollution. Their impacts may be either time limited and have low toxicity or, alternatively, it may be long-termed and expressing the pollutants' ability to accumulate them in the body and supply chains. In the edaphotop block of land biogeocenosis most of the transformation processes begin with soil degradation ones. This leads to disruption of soil-forming process cycle of matter and biological productivity of systems. Optimization of a particular system or a block is possible only by acting on their biological relations, which is responsible for managing the system. Distortion or optimization of relations occurs with the animals environmental-forming, the component of which is animals burrow activity. As a result of it, water and chemicals are being reallocated in soil depths, thermal and aeration modes are changed, formed typical zoogenous micro relief with specific physical and chemical properties of the soil. This improves the living conditions of soil micro-organisms. Last ones are involved in soil enzyme activity forming. Enzymes involved in important biochemical processes: synthesis and humus decomposition, hydrolysis of organic compounds, the schedule remains of plants and microorganisms and converted them available to fixation, fix loose items, are actively involved in the cycling of essential elements for plants, in oxidation renewable reactions, etc. It is possible to determine the extent of microorganism’s activity by means of indexes of soil proteolytic activity. Conducted studies of the impact for animals burrow activity on the soils proteolytic activity for artificial forest under conditions of heavy metal pollution. Control plots were contaminated by lead with concentrations of 32; 160; 320 mg/kg of soil, which correspond to the maximum permissible concentration of 1; 5; 10 MAC. Samples were taken at 1st, 3rd and 15th months after contamination; proteolytic activity was determined by the Mishustin application method. Current proteolytic activity of soil is determined in vivo. It is the result of microorganism’s vital functions. And in turn can be an indicator of microbial activity. Contamination of lead depressing vital functions for soil microorganisms. This effect is more noticeable in the first month after contamination. The characteristic dependence of the soil proteolytic activity on metal concentration was observed also, the higher the MPC, the lower proteolytic activity level. The characteristics' dependence of soil proteolytic activity on metal concentration were also observed: the higher MPC, the lower the activity level. It was shown that on the mole rats burrows-dug with lead contaminated the medium proteolytic activity level exceeded control 1.9; 1.76 and 1.53 times respectively, at 1st; 3rd and 15th months after contamination. Based on this we can say that burrowing activity of animals inhibits inhibitory effect of heavy metals and plays an important role in the self-cleaning environment.

scholarly journals The RgpB C-Terminal Domain Has a Role in Attachment of RgpB to the Outer Membrane and Belongs to a Novel C-Terminal-Domain Family Found in Porphyromonas gingivalis

2006 ◽  
Vol 188 (17) ◽  
pp. 6376-6386 ◽  
Author(s):  
Christine A. Seers ◽  
Nada Slakeski ◽  
Paul D. Veith ◽  
Todd Nikolof ◽  
Yu-Yen Chen ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Proteolytic Activity ◽  
Porphyromonas Gingivalis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteolytic Processing ◽  
Terminal Domain ◽  
Catalytically Active ◽  
Membrane Attachment

ABSTRACT Porphyromonas gingivalis produces outer membrane-attached proteins that include the virulence-associated proteinases RgpA and RgpB (Arg-gingipains) and Kgp (Lys-gingipain). We analyzed the P. gingivalis outer membrane proteome and identified numerous proteins with C-terminal domains similar in sequence to those of RgpB, RgpA, and Kgp, indicating that these domains may have a common function. Using RgpB as a model to investigate the role of the C-terminal domain, we expressed RgpB as a full-length zymogen (recombinant RgpB [rRgpB]), with a catalytic Cys244Ala mutation [rRgpB(C244A)], or with the C-terminal 72 amino acids deleted (rRgpB435) in an Arg-gingipain P. gingivalis mutant (YH522AB) and an Arg- and Lys-gingipain mutant (YH522KAB). rRgpB was catalytically active and located predominantly attached to the outer membrane of both background strains. rRgpB(C244A) was inactive and outer membrane attached, with a typical attachment profile for both background strains according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in YH522KAB, the prodomain was not removed. Thus, in vivo, RgpB export and membrane attachment are independent of the proteolytic activity of RgpA, RgpB, or Kgp. However, for maturation involving proteolytic processing of RgpB, the proteolytic activity of RgpB, RgpA, or Kgp is required. The C-terminally-truncated rRgpB435 was not attached to the outer membrane and was located as largely inactive, discrete 71-kDa and 48-kDa isoforms in the culture supernatant and the periplasm. These results suggest that the C-terminal domain is essential for outer membrane attachment and may be involved in a coordinated process of export and attachment to the cell surface.

Inhibition of Fas-mediated Apoptosis in Yac-1 Cell via Anti-Fas Ribozyme

2004 ◽  
Vol 36 (3) ◽  
pp. 199-205
Author(s):  
Min Zhang ◽  
Fang Liu ◽  
Wei He ◽  
Yong You ◽  
Ping Zou ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Proteolytic Activity ◽  
Caspase 3 ◽  
Hammerhead Ribozyme ◽  
Annexin V ◽  
In Vitro Transcription ◽  
Rt Pcr ◽  
Fas Mrna

Abstract To detect a new and more effective way against apoptosis mouse lymphomatic cell line-Yac-1 in which fas gene was expressed highly was used as a model for studying the effects of anti-Fas ribozyme on Fas-mediated apoptosis. A hammerhead ribozyme gene targeting the fas mRNA was synthesized and its in vitro transcription vector was constructed, which was transfected into Yac-1 cells using electroporation. Rz596 expression was detected using RT-PCR, and Fas expression in Yac-1 cells was detected using RT-PCR, Western blot and flow cytometry. After treated with anti-Fas antibody (JO2), Yac-1 cell viability was measured with MTT assay, caspase-3 proteolytic activity was detected, and cell apoptosis was measured according to annexin V apoptosis detecting kit. Anti-Fas ribozyme could cleave fas mRNA efficiently in vivo and in vitro. Fas expression in Yac-1 cells transfected with anti-Fas ribozyme was decreased remarkably and correlated with resistance to Fas-mediated apoptosis as determined by flow cytometry and caspase-3 proteolytic activity. Anti-Fas ribozyme was detected in cells transfected with pU6-RZ596 and pU6-dRZ596 and could remarkably decrease the Fas expression in Yac-1 cells, which made Yac-1 cells get rid of Fas-mediated apoptosis. Because of wide expression of fas in organs and tissues, our research was very useful for studying the inhibition of apoptosis of many organs and tissues in the future.

Activation of C1

1984 ◽  
Vol 306 (1129) ◽  
pp. 283-292 ◽  
Keyword(s):  
Electron Microscopy ◽  
Neutron Diffraction ◽  
Proteolytic Activity ◽  
Functional Properties ◽  
Immune Complexes ◽  
Negative Control ◽  
C1 Inhibitor ◽  
Calcium Dependent ◽  
Catalytic Domains

The first component of complement, C1, is a calcium-dependent complex of two loosely interacting subunits: C1q, responsible for the binding of activators to C1; C1r2—C1s2, which supports the autoactivation potential of C1, together with the proteolytic activity of activated C1 on its two substrates, C4 and C2. Isolated dimeric C1r2 is able to autoactivate through an intradimer crossproteolysis; this capacity is lost when C1r2 is associated with two molecules of C1s inside the calcium-dependent C1r2-C1s2 subunit; this capacity is again observed in reconstituted C1. A model for reconstituted soluble C1 is proposed, based on electron microscopy, neutron diffraction, ultra-centrifugation, various biochemical findings, as well as functional properties of C1 or of its subcomponents. The flexible rod-like structure of C1r2-C1s2 is folded around two arms of C1q, with the catalytic domains of C1r and C1s inserted inside the cone defined by the C1q stalks. Activation of C1 which, in vivo , is controlled by C1 inhibitor, can be achieved by various activators, such as immune complexes; it appears to result from the suppression of a negative control and resides in a positive modulation of the intrinsic autocatalytic potential of C1r inside C1.

Experimental studies of the pathogenesis of infections due to Pseudomonas aeruginosa: extracellular protease and elastase as in vivo virulence factors

1979 ◽  
Vol 25 (5) ◽  
pp. 593-599 ◽  
Author(s):  
Ian Alan Holder ◽  
Constantine G. Haidaris
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mouse Model ◽  
Proteolytic Activity ◽  
Virulence Factors ◽  
Proteolytic Enzyme ◽  
Experimental Studies ◽  
Toxin Production ◽  
Significant Delay ◽  
Lethal Injection

The effects on mortality of supplemental injections of protease and elastase were determined in burned mice infected with non-lethal inocula of a toxin-producing but non-proteolytic- enzyme-producing strain of Pseudomonas aeruginosa. When a variety of solutions containing proteolytic enzyme were injected under these conditions, the mortality increased significantly. This did not occur when organisms other than P. aeruginosa were used. Injections of the enzyme solutions alone were non-lethal. Injection of a solution of α2-macroglobulin, which was shown to inhibit proteolytic activity, together with a proteolytic enzyme – toxin producing strain of P. aeruginosa caused a significant delay in mortality when compared with controls. It was concluded that protease, elastase, and toxin production were necessary for P. aeruginosa to express full virulence in the burned mouse model.

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