The Action of Brinase in Vitro and in Vivo

1975 ◽  
Author(s):  
P. J. Gaffney ◽  
K. Lord ◽  
R. D. Thornes
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Size Range ◽  
Degradation Products ◽  
Molecular Size ◽  
Human Fibrinogen ◽  
Rate Limiting Step ◽  
Rate Limiting

Brinase (an extract of Aspergillus Oryzae) was shown to rapidly digest human fibrinogen in vitro to aggregable degradation products with a molecular size range of 310,000 to 230,000 the latter fragments being more slowly digested to core fragments, Dbr and Ebr. The fibrinogen polypeptide chain susceptibility to Brinase attack was in the order Aα, γ, Bβ, Lysis of the Bβ chain seems to be the rate limiting step in the conversion of the high molecular weight fragments (MW 310,000–230,000) to the core fragments Dbr and Ebr. The conservation of NH2 terminal Tyrosine during fibrinogen digestion and the very transient existence of D dimer fragments during totally crosslinked fibrin lysis suggest that the carboxy end of the γ chain is prone to Brinase attack. The crosslinked α chains of fibrin, while resistant to plasmin, are vigorously digested by Brinase. The plasma of cancer patients being treated with Brinase contained degraded fibrinogen (lacking intact Aα chains) and their aggregates. These aggregates contained some crosslinked γ chains (γ-γ dimers) suggesting that Brinase in vivo exorcises both a lytic and coagulant effect. Thrombin mediated clots in all the plasmas examined contained no crosslinked α chains. Positive plasma ethanol gelation tests can be explained by the presence of the aggregable high molecular weight fragments observed during the in vitro lysis of fibrinogen by Brinase.

The Fibrin Assay Comparison Trial (FACT)

2001 ◽  
Vol 85 (04) ◽  
pp. 671-678 ◽  
Author(s):  
Sybille Zips ◽  
Hanimsah Ergül ◽  
Dieter Heene ◽  
Carl-Erik Dempfle ◽  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Degradation Products ◽  
Plasma Samples ◽  
Fibrin Degradation Products ◽  
Comparison Trial ◽  
Clinical Conditions ◽  
D Dimer

SummaryAlthough D-dimer has gained widespread clinical use as a parameter for detection of in vivo fibrin formation, the issue of standardization of D-dimer assays remains to be resolved. The FACT study was performed to generate basic data for development of calibrators and standard preparations.A set of 86 samples, including plasma samples from patients with DIC, DVT, and other clinical conditions, serial dilutions of pooled plasma samples, and plasma samples containing fibrinogen- and fibrin derivatives, were distributed to 12 manufacturers of D-dimer assays.D-dimer assays differ concerning specificity for crosslinked fibrin, and preference for either high molecular weight fibrin complexes, or low molecular weight fibrin degradation products. Terminal plasmin digests of fibrin clots for calibration produce aberrant results in some assays, especially those with preference for high molecular weight crosslinked fibrin derivatives. The best conformity is achieved by the use of pooled plasma samples from patients with high levels of D-dimer antigen in plasma. In vitro preparations containing a comparable composition of fibrin derivatives to clinical plasma samples may also serve as reference material.

scholarly journals Determining the Rate-Limiting Step for Light-Responsive Redox Regulation in Chloroplasts

Antioxidants ◽  
2018 ◽  
Vol 7 (11) ◽  
pp. 153 ◽  
Author(s):  
Keisuke Yoshida ◽  
Toru Hisabori
Keyword(s):  
Redox Regulation ◽  
Reducing Power ◽  
Rubisco Activase ◽  
Power Transfer ◽  
Target Proteins ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Thiol-based redox regulation ensures light-responsive control of chloroplast functions. Light-derived signal is transferred in the form of reducing power from the photosynthetic electron transport chain to several redox-sensitive target proteins. Two types of protein, ferredoxin-thioredoxin reductase (FTR) and thioredoxin (Trx), are well recognized as the mediators of reducing power. However, it remains unclear which step in a series of redox-relay reactions is the critical bottleneck for determining the rate of target protein reduction. To address this, the redox behaviors of FTR, Trx, and target proteins were extensively characterized in vitro and in vivo. The FTR/Trx redox cascade was reconstituted in vitro using recombinant proteins from Arabidopsis. On the basis of this assay, we found that the FTR catalytic subunit and f-type Trx are rapidly reduced after the drive of reducing power transfer, irrespective of the presence or absence of their downstream target proteins. By contrast, three target proteins, fructose 1,6-bisphosphatase (FBPase), sedoheptulose 1,7-bisphosphatase (SBPase), and Rubisco activase (RCA) showed different reduction patterns; in particular, SBPase was reduced at a low rate. The in vivo study using Arabidopsis plants showed that the Trx family is commonly and rapidly reduced upon high light irradiation, whereas FBPase, SBPase, and RCA are differentially and slowly reduced. Both of these biochemical and physiological findings suggest that reducing power transfer from Trx to its target proteins is a rate-limiting step for chloroplast redox regulation, conferring distinct light-responsive redox behaviors on each of the targets.

scholarly journals The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycinlabile and puromycin-non-labile sites on polyribosomes

1971 ◽  
Vol 122 (3) ◽  
pp. 267-276 ◽  
Author(s):  
D. C. N. Earl ◽  
Susan T. Hindley
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Donor Site ◽  
Peptide Bond ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Donor And Acceptor ◽  
Rate Limiting

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0°C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.

scholarly journals Solubility and ADMET profiles of short oligomers of lactic acid

ADMET & DMPK ◽  
2020 ◽  
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>

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

In Vitro and in Vivo Antigen-Induced Release of High-Molecular Weight Neutrophil Chemotactic Activity from Human Nasal Tissue

1988 ◽  
Vol 2 (1) ◽  
pp. 7-11 ◽  
Author(s):  
T. Nagakura ◽  
T. Onda ◽  
Y. likura ◽  
T. Endo ◽  
H. Nagakura ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Nasal Polyps ◽  
Gel Filtration ◽  
Chemotactic Activity ◽  
Nasal Tissue ◽  
Nasal Secretions ◽  
Neutrophil Chemotactic Activity

High molecular weight neutrophil chemotactic activity has been identified in resected human nasal polyps, inferior turbinates, and nasal secretions following antigen challenge. The estimated molecular weight, by gel filtration chromatography, was approximately 600,000. However, a heterogeneity of molecular weight in some patients was recognized. Our results suggest a possible role for high molecular weight-neutrophil chemotactic activity in the pathogenesis of hypersensitivity in the human nasal cavity.

scholarly journals Coassembly of SecYEG and SecA Fully Restores the Properties of the Native Translocon

2018 ◽  
Vol 201 (1) ◽  
Author(s):  
Priya Bariya ◽  
Linda L. Randall
Keyword(s):  
Lipid Bilayers ◽  
Rate Constant ◽  
Apparent Rate Constant ◽  
Membrane Vesicles ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Secretory System ◽  
Rate Limiting

ABSTRACTIn all cells, a highly conserved channel transports proteins across membranes. InEscherichia coli, that channel is SecYEG. Many investigations of this protein complex have used purified SecYEG reconstituted into proteoliposomes. How faithfully do activities of reconstituted systems reflect the properties of SecYEG in the native membrane environment? We investigated by comparing threein vitrosystems: the native membrane environment of inner membrane vesicles and two methods of reconstitution. One method was the widely used reconstitution of SecYEG alone into lipid bilayers. The other was our method of coassembly of SecYEG with SecA, the ATPase of the translocase. For nine different precursor species we assessed parameters that characterize translocation: maximal amplitude of competent precursor translocated, coupling of energy to transfer, and apparent rate constant. In addition, we investigated translocation in the presence and absence of chaperone SecB. For all nine precursors, SecYEG coassembled with SecA was as active as SecYEG in native membrane for each of the parameters studied. Effects of SecB on transport of precursors faithfully mimicked observations madein vivo. From investigation of the nine different precursors, we conclude that the apparent rate constant, which reflects the step that limits the rate of translocation, is dependent on interactions with the translocon of portions of the precursors other than the leader. In addition, in some cases the rate-limiting step is altered by the presence of SecB. Candidates for the rate-limiting step that are consistent with our data are discussed.IMPORTANCEThis work presents a comprehensive quantification of the parameters of transport by the Sec general secretory system in the threein vitrosystems. The standard reconstitution used by most investigators can be enhanced to yield six times as many active translocons simply by adding SecA to SecYEG during reconstitution. This robust system faithfully reflects the properties of translocation in native membrane vesicles. We have expanded the number of precursors studied to nine. This has allowed us to conclude that the rate constant for translocation varies with precursor species.

scholarly journals An Assessment of the Bioactivity of Coffee Silverskin Melanoidins

Foods ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 68 ◽  
Author(s):  
Silvia Tores de la Cruz ◽  
Amaia Iriondo-DeHond ◽  
Teresa Herrera ◽  
Yolanda Lopez-Tofiño ◽  
Carlos Galvez-Robleño ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Dietary Fiber ◽  
High Molecular Weight ◽  
Research Work ◽  
Weight Fraction ◽  
High Molecular Weight Fraction ◽  
Total Dietary Fiber ◽  
Coffee Silverskin

Melanoidins present in coffee silverskin, the only by-product of the roasting process, are formed via the Maillard reaction. The exact structure, biological properties, and mechanism of action of coffee silverskin melanoidins, remain unknown. This research work aimed to contribute to this novel knowledge. To achieve this goal, melanoidins were obtained from an aqueous extract of Arabica coffee silverskin (WO2013004873A1) and was isolated through ultrafiltration (>10 kDa). The isolation protocol was optimized and the chemical composition of the high molecular weight fraction (>10 kDa) was evaluated, by analyzing the content of protein, caffeine, chlorogenic acid, and the total dietary fiber. In addition, the structural analysis was performed by infrared spectroscopy. Antioxidant properties were studied in vitro and the fiber effect was studied in vivo, in healthy male Wistar rats. Melanoidins were administered to animals in the drinking water at a dose of 1 g/kg. At the fourth week of treatment, gastrointestinal motility was evaluated through non-invasive radiographic means. In conclusion, the isolation process was effective in obtaining a high molecular weight fraction, composed mainly of dietary fiber, including melanoidins, with in vitro antioxidant capacity and in vivo dietary fiber effects.

scholarly journals The Immunosuppressive Activity of Plasmic Degradation Products of Human Fibrinogen

1977 ◽  
Author(s):  
T.S. Edgington ◽  
L.K. Curtiss ◽  
E.F. Plow
Keyword(s):  
Cell Response ◽  
Degradation Products ◽  
Thymidine Uptake ◽  
Immunosuppressive Activity ◽  
Human Fibrinogen ◽  
Peptide Fraction ◽  
Intact Molecule ◽  
Stimulation Of

Plasmic cleavage of human fibrinogen leads to generation of immunosuppressive activity not expressed by the intact molecule, and which is demonstrable in vitro and in vivo. This activity is not associated with the high molecular weight derivatives X, Y, D and E, but is present in the small dialyzable peptide fraction obtained from plasmic digestion. The peptides inhibit in a non-toxic fashion, the stimulation of 3H-thymidine uptake and blastogenesis of lymphocytes by phytohemagglutinin (PHA) and allogeneic cells (MLC) under conditions of both macrophage dependence and macrophage independence. The peptides also suppress the plaque-forming cell response of mice to sheep red blood cells in vivo. Approximately 30 μg peptides/culture leads to a 50% inhibition of the PHA and MLC systems, and approximately 400 μg/mouse produces a 50% suppression of the plaque-forming cell response. Intact fibrinogen chains exhibit negligible activity, but plasmic digests of Aα chain are suppressive. Consistent with derivation from the Aa chain was the demonstration that the activity was generated from limited plasmic digest of fibrinogen which produced fragment X, and this activity was soluble at 80°C for 10 minutes. The release of the active peptide by limited plasmic degradation, and the activity of these peptides at physiologic concentrations suggests that this system may be of importance in vivo in association with local fibrinogenolysis or fibrinolysis at sites of thrombosis. This has been in part substantiated by the experimental initiation of fibrinogenolysis in vivo with streptokinase.

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