scholarly journals Decoration of Fibrin with Extracellular Chaperones

2019 ◽  
Vol 119 (10) ◽  
pp. 1624-1631
Author(s):  
Simone Talens ◽  
Frank W. G. Leebeek ◽  
Robert Veerhuis ◽  
Dingeman C. Rijken
Keyword(s):  
Heat Stress ◽  
Protein Unfolding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Thioflavin T ◽  
Fibrin Formation ◽  
Α2 Macroglobulin ◽  
Apolipoprotein J ◽  
Fibrin Clots

Background Many proteins bind to fibrin during clot formation in plasma. We previously identified by mass spectrometry the most abundant proteins that noncovalently bind to fibrin clots. Several of these proteins (e.g., apolipoprotein J/clusterin, haptoglobin, α2-macroglobulin, α1-antitrypsin) can act as extracellular chaperones. Objective We hypothesize that clot-binding proteins may interact with fibrin as chaperones. The goal of this study is to test this hypothesis and to investigate the origin of the cross-β or amyloid structures in fibrin clots, which are associated with protein unfolding. Methods and Results A thioflavin T assay was used to detect cross-β structures. A steadily increasing amount was measured in the fibrinogen fraction of plasma during heat stress, a standard treatment to induce unfolding of proteins. Heat-stressed plasma was clotted and clot-bound proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results showed that the amounts of the clot-bound proteins were related to the duration of the heat stress. This indicates that cross-β structures in unfolded fibrin(ogen) are involved in clot binding of the proteins, which supports our chaperone hypothesis. A contributing role of fibrin formation itself was studied by clotting purified fibrinogen with thrombin in the presence of thioflavin T. The fluorescence intensity increased in time in the presence of thrombin, but did not increase in its absence. This provides evidence for the generation of cross-β structures during fibrin formation. Conclusion Fibrin clots generated in plasma are decorated with extracellular chaperones. The binding of these chaperones involves cross-β structures originating both from unfolded fibrinogen and from fibrin formation.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

scholarly journals Accelerated hepatic haem catabolism in the selenium-deficient rat

1977 ◽  
Vol 168 (1) ◽  
pp. 105-111 ◽  
Author(s):  
R F Burk ◽  
M A Correia
Keyword(s):  
Urinary Excretion ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Microsomal Cytochrome ◽  
Haem Oxygenase ◽  
Cytochrome P 450

1. Hepatic microsomal cytochrome P-450 concentrations are lower in selenium-deficient rats treated with phenobarbital for 4 days than in similarly treated control rats. 2. No defect in haem synthesis was found on the basis of measurements of delta-aminolaevulinate synthase (EC 2.3.1.37), delta-aminolaevulinate dehydratase (EC 4.2.1.24) and ferrochelatase (EC 4.99.1.1) activities, and urinary excretion of delta-aminolaevulinate, porphobilinogen, uroporphyrin and coproporphyrin. 3. No defect in apo-(cytochrome P-450) separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. An increase in haem catabolism was found. An 8-fold increase in hepatic microsomal haem oxygenase (EC 1.14.99.3) activity occurred in selenium-deficient rats after phenobarbital treatment, compared with a less than 2-fold increase in control rats. Also excretion of 14CO in the breath after administration of delta-amino[5-14C]laevulinate was greater by phenobarbital-treated selenium-deficient rats than by similarly treated controls. 5. These studies demonstrate that the defective induction of cytochrome P-450 by phenobarbital in selenium-deficient rats is accompanied by increased haem catabolism. This could be due to increased breakdown of cytochrome P-450 or to catabolism of haem before it attaches to the apo-cytochrome. The role of selenium in stabilizing cytochrome P-450 and/or in protecting haem from breakdown remains to be determined.

Substitution of the γ-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking

Blood ◽  
2004 ◽  
Vol 103 (11) ◽  
pp. 4157-4163 ◽  
Author(s):  
Nobuo Okumura ◽  
Oleg V. Gorkun ◽  
Fumiko Terasawa ◽  
Susan T. Lord
Keyword(s):  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Dimer Formation ◽  
Polymer Formation ◽  
Crystallographic Structures ◽  
Link Formation

Abstract Crystallographic structures indicate that γ-chain residue Asn308 participates in D:D interactions and indeed substitutions of γAsn308 with lysine or isoleucine have been identified in dysfibrinogens with impaired polymerization. To probe the role of Asn308 in polymerization, we synthesized 3 variant fibrinogens: γAsn308 changed to lysine (γN308K), isoleucine (γN308I), and alanine (γN308A). We measured thrombin-catalyzed polymerization by turbidity, fibrinopeptide release by high-performance liquid chromatography, and factor XIIIa–catalyzed cross-linking by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. In the absence of added calcium, polymerization was clearly impaired with all 3 variants. In contrast, at 0.1 mM calcium, only polymerization of γN308K remained markedly abnormal. The release of thrombin-catalyzed fibrinopeptide B (FpB) was delayed in the absence of calcium, whereas at 1 mM calcium FpB release was delayed only with γN308K. Factor XIIIa–catalyzed γ-γ dimer formation was delayed with fibrinogen (in absence of thrombin), whereas with fibrin (in presence of thrombin) γ-γ dimer formation of only γN308K was delayed. These data corroborate the recognized link between FpB release and polymerization. They show fibrin cross-link formation likely depends on the structure of protofibrils. Together, our results show substitution of Asn308 with a hydrophobic residue altered neither polymer formation nor polymer structure at physiologic calcium concentrations, whereas substitution with lysine altered both.

Purification and Characterization of Kallikrein from Plasma of Patients with Acute Pancreatitis

1981 ◽  
Vol 60 (2) ◽  
pp. 199-205 ◽  
Author(s):  
Naotika Toki ◽  
Hiroyuki Sumi ◽  
Sumiyoshi Takasugi
Keyword(s):  
Acute Pancreatitis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Purification And Characterization ◽  
Α2 Macroglobulin ◽  
Sepharose 4B

1. A kallikrein-like enzyme in plasma of patients with acute pancreatitis was further purified by successive hydroxyapatite/cellulose and Sepharose-4B column chromatography. 2. By these procedures 0.26 mg of purified enzyme with a specific activity of 215 S-2266 chromozyme units/mg of protein was obtained from 10 ml of original plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had an apparent molecular weight of 31 000 as measured by gel filtration on Sephadex G-200. 4. It was confirmed immunologically that this enzyme was pancreatic kallikrein, which is distinct from plasma kallikrein, and that it could combine with α2-macroglobulin only in the presence of trypsin.

Serotypes of Neisseria meningitidis associated with an increased incidence of meningitis cases in the Hamilton area, Ontario, during 1978 and 1979

1983 ◽  
Vol 29 (1) ◽  
pp. 129-136 ◽  
Author(s):  
Fraser E. Ashton ◽  
J. Alan Ryan ◽  
Colina Jones ◽  
Bernard R. Brodeur ◽  
Benito B. Diena
Keyword(s):  
Neisseria Meningitidis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Age Groups ◽  
Double Diffusion ◽  
Sds Page ◽  
Class 1 ◽  
Group B

The distribution of serotypes among strains of Neisseria meningitidis responsible for a marked increase of meningitis cases in the Hamilton area, Ontario, in 1978 and 1979 was determined. Twenty-six serogroup B and two serogroup W135 strains isolated from cerebrospinal fluid, blood, and skin of 28 patients were serotyped by agar gel double diffusion. Twenty-one (81 %) of the group B strains were serotype 2b as judged by the formation of characteristic serotype precipitin bands with the specific anti-2996 (type 2b) serum. Fourteen of the serotype 2b strains also reacted with anti-77252 serum, which suggested that one strain or several closely related strains were mainly responsible for the increase in meningitis during the 2-year period. Examination of the outer membrane complexes (OMC) of the strains by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS–PAGE) revealed that all 21 of the serotype 2b strains contained the class 2 protein (molecular weight 41 500) which is known to be the site of the serotype 2b determinant. Further characterization of the serotype 2b,77252 strains by enzyme-linked immunosorbent assays (ELISA) and SDS–PAGE suggested that the 77252 determinant was present in the class 1 proteins of these strains. The serotype 2b containing strains were isolated from 77.7 and 70% of males and females, respectively, from 81.8% of children less than 5 years of age, and from 75.0% of patients of all age groups. The study indicates the important role of serotype 2b meningococci in causing the increased incidence of meningitis and further substantiates the important association of the serotype 2b determinant with group B serotype 2 meningococcal disease in Canada.

scholarly journals Effects of Tetrahydrobiopterin on Endothelial Dysfunction in Rats with Ischemic Acute Renal Failure

2000 ◽  
Vol 11 (2) ◽  
pp. 301-309
Author(s):  
MASAO KAKOKI ◽  
YASUNOBU HIRATA ◽  
HIROSHI HAYAKAWA ◽  
ETSU SUZUKI ◽  
DAISUKE NAGATA ◽  
...  
Keyword(s):  
Perfusion Pressure ◽  
Tissue Injury ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Excretory Function ◽  
Calcium Dependent ◽  
Ischemic Tissue ◽  
No Release ◽  
Dimeric Form

The role of nitric oxide (NO) in ischemic renal injury is still controversial. NO release was measured in rat kidneys subjected to ischemia and reperfusion to determine whether (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4), a cofactor of NO synthase (NOS), reduces ischemic injury. Twenty-four hours after bilateral renal arterial clamp for 45 min, acetylcholine-induced vasorelaxation and NO release were reduced and renal excretory function was impaired in Wistar rats. Administration of BH4 (20 mg/kg, by mouth) before clamping resulted in a marked improvement of those parameters (10-8 M acetylcholine, Δrenal perfusion pressure: sham-operated control -45 ± 5, ischemia -30 ± 2, ischemia + BH4 -43 ± 4%; ΔNO: control +30 ± 6, ischemia +10 ± 2, ischemia + BH4 +23 ± 4 fmol/min per g kidney; serum creatinine: control 23 ± 2, ischemia 150 ± 27, ischemia + BH4 48 ± 6 μM; mean ± SEM). Most of renal NOS activity was calcium-dependent, and its activity decreased in the ischemic kidney. However, it was restored by BH4 (control 5.0 ± 0.9, ischemia 2.2 ± 0.4, ischemia + BH4 4.3 ± 1.2 pmol/min per mg protein). Immunoblot after low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the dimeric form of endothelial NOS decreased in the ischemic kidney and that it was restored by BH4. These results suggest that the decreased activity of endothelium-derived NO may worsen the ischemic tissue injury, in which depletion of BH4 may be involved.

scholarly journals Role of Immunoglobulin A Monoclonal Antibodies against P23 in Controlling Murine Cryptosporidium parvum Infection

1998 ◽  
Vol 66 (9) ◽  
pp. 4469-4473 ◽  
Author(s):  
F. Javier Enriquez ◽  
Michael W. Riggs
Keyword(s):  
Monoclonal Antibodies ◽  
Cryptosporidium Parvum ◽  
Matched Control ◽  
Intestinal Parasites ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoglobulin A ◽  
Passive Immunization ◽  
Immunofluorescence Assay

ABSTRACT Cryptosporidium parvum is an important diarrhea-causing protozoan parasite of immunocompetent and immunocompromised hosts. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections with bacteria, viruses, and parasites, but little is known about the role of IgA in the control of C. parvuminfection. We assessed the role of IgA during C. parvum infection in neonatal mice. IgA-secreting hybridomas were developed by using Peyer’s patch lymphocytes from BALB/c mice which had been orally inoculated with viable C. parvumoocysts. Six monoclonal antibodies (MAbs) were selected for further study based on indirect immunofluorescence assay reactivity with sporozoite and merozoite pellicles and the antigen (Ag) deposited on glass substrate by gliding sporozoites. Each MAb was secreted in dimeric form and recognized a 23-kDa sporozoite Ag in Western immunoblots. The Ag recognized comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with P23, a previously defined neutralization-sensitive zoite pellicle Ag. MAbs were evaluated for prophylactic or therapeutic efficacy against C. parvum, singly and in combinations, in neonatal BALB/c mice. A combination of two MAbs given prophylactically prior to and 12 h following oocyst challenge reduced the number of intestinal parasites scored histologically by 21.1% compared to the numbers in mice given an isotype-matched control MAb (P < 0.01). Individual MAbs given therapeutically in nine doses over a 96-h period following oocyst challenge increased efficacy against C. parvuminfection. Four MAbs given therapeutically each reduced intestinal infection 34.4 to 42.2% compared to isotype-matched control MAb-treated mice (P < 0.05). One MAb reduced infection 63.3 and 72.7% in replicate experiments compared to isotype-matched control MAb-treated mice (P < 0.0001). We conclude that IgA MAbs directed to neutralization-sensitive P23 epitopes may have utility in passive immunization against murineC. parvum infection.

scholarly journals Role of glycoprotein IIa (beta 1 subunit of very late activation antigens) in platelet functions

Blood ◽  
1991 ◽  
Vol 78 (8) ◽  
pp. 2021-2026 ◽  
Author(s):  
S Parmentier ◽  
B Catimel ◽  
L McGregor ◽  
LL Leung ◽  
JL McGregor
Keyword(s):  
Platelet Adhesion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Lag Phase ◽  
Type Iii Collagen ◽  
Type I ◽  
Western Blots ◽  
Activation Antigens ◽  
Platelet Functions

Abstract Very late activation antigens (VLAs) are glycoproteins (GPs) that play a major role in platelet adhesion to extracellular matrix. These GPs, members of the integrin family, are heterodimer complexes with different alpha subunits noncovalently associated with a common beta 1 subunit known as GPIIa. GPIa-IIa (also known as VLA2), GPIc-IIa (VLA5), and GPIc*-IIa (VLA6) are involved, respectively, in platelet adhesion to collagen, fibronectin, and laminin. At this stage, very little is known about the role of GPIIa in platelet adhesive functions. In this study, we have generated a monoclonal antibody (MoAb) (LYP22) directed against GPIIa. Immunoaffinity chromatography using LYP22 combined with two-dimensional nonreduced-reduced sodium dodecyl sulfate- polyacrylamide gel electrophoresis shows that the antibody brings down all VLA subunits. Western blots indicate that the binding site of LYP22 on GPIIa is disulfide bridge-dependent. The number of LYP22 binding sites is not increased on stimulation with thrombin and is in the range of what is observed with another anti-GPIIa MoAb (A-1A5). LYP22 is the first anti-GPIIa MoAb to inhibit aggregation and secretion of washed platelets stimulated with collagen, thrombin, or arachidonic acid. Moreover, the lag-phase usually observed on collagen stimulation is significantly prolonged (by 60 seconds) in the presence of LYP22. This lag-phase, mediated by LYP22, is also observed in the presence of plasma proteins and is coupled with a reduced effect on collagen- induced platelet aggregation. In addition, LYP22 affects the adhesion of resting platelets to type III collagen, but not to fibronectin, laminin, or type I collagen. These results strongly indicate that the site on GPIIa, bearing the LYP22 epitope, is an active participant in signal transduction controlling platelet functions.

scholarly journals Role of interleukin-15 and interleukin-18 in the secretion of sIL-6R and sgp130 by human neutrophils

2003 ◽  
Vol 12 (3) ◽  
pp. 179-183 ◽  
Author(s):  
E. Jablonska ◽  
M. Marcinczyk
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Neutrophils ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cytoplasmic Proteins ◽  
Wide Range ◽  
Interleukin 15 ◽  
Culture Supernatants

Background:Available data indicate that neutrophils (PMN) produce a wide range of cytokines with the potential to modulate immune response. Recent investigation have shown that interleukin (IL)-15 and IL-18 potentiated several functions of normal neutrophils. It has been reported that IL-18-induced cytokine production may be significantly enhanced by coincident addition of IL-15.Aims:In the present study we compared the effect of recombinant human (rh)IL-15 and rhIL-18 as well as effect of a rhIL-15 and rhIL-18 combination on the induction secretion of sIL-6Rα and sgp130 by human neutrophils. Methods: PMN were isolated from heparinized whole blood of healthy persons. The PMN were cultured for 18 h at 37°C in a humidified incubator with 5% CO2. rhIL-15 and/or rhIL-18 and lipopolysaccharide were tested to PMN stimulation. The culture supernatants of PMN were removed and examined for the presence of sIL-6R and sgp130 by human enzyme-linked immunosorbent assay kits. Cytoplasmic protein fractions of PMN were analysed for the presence of sIL-6R and sgp130 by western blotting using monoclonal antibodies capable of detecting these proteins. Cells were lysed and cytoplasmic proteins were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred onto nitrocellulose and incubated with the primary monoclonal antibodies anti-sIL-6R and anti-sgp130. The membranes were incubated at room temperature with alkaline phosphatase anti-mouse immunoglobulin G. Immunoreactive protein bans were visualized by an AP Conjugate Substrate Kit.Results and conclusion:The results of our investigation revealed that IL-15 alone, similarly to IL-18, has no significant ability for the regulation of both soluble IL-6 receptors, sIL-6R and sgp130, released by human neutrophils. It is interesting to note that the secretion of sgp130 was changed after PMN stimulation with rhIL-15 in the presence of rhIL-18. The combination of rhIL-15 and rhIL-18 was shown to induce PMN to secretion relatively higher amounts of sgp130 compared with the stimulation of PMN with rhIL-15 alone and rhIL-18 alone. The results obtained suggest that IL-15 and IL-18, belonging to the inflammatory cytokines, through the regulation of sgp130 secretion must be also considered as anti-inflammatory mediators that may influence the balance reactions mediated by the IL-6 cytokine family.

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