Antigenicity of a proteolytic enzyme of Fasciola hepatica

1988 ◽  
Vol 62 (3) ◽  
pp. 257-260 ◽  
Author(s):  
G. C. Coles ◽  
D. Rubano
Keyword(s):  
Molecular Weight ◽  
Schistosoma Mansoni ◽  
Western Blotting ◽  
Gel Electrophoresis ◽  
Proteolytic Enzyme ◽  
Fasciola Hepatica

ABSTRACTThe possibility was examined of using a haemoglobinase released during in vitro incubation of adult Fasciola hepatica for immunodiagnosis of fascioliasis. By SDS gel electrophoresis the enzyme appeared as two closely migrating bands with a molecular weight of approximately 27 000 daltons. After Western blotting the bands reacted with serum from rats infected with F. hepatica and mice infected with Schistosoma mansoni. The enzyme was therefore not a good antigen for immunodiagnosis.

How anthelmintics help us to understand helminths

Parasitology ◽  
1985 ◽  
Vol 90 (4) ◽  
pp. 675-685 ◽  
Author(s):  
H. Vanden Bossche
Keyword(s):  
Molecular Weight ◽  
Schistosoma Mansoni ◽  
Mode Of Action ◽  
Liver Fluke ◽  
Fasciola Hepatica ◽  
Atp Synthesis ◽  
Aerobic Metabolism ◽  
Unexpected Finding

Study of the mode of action of the anti-parasitic ‘hydrogenophore’, closantel, revealed that mitochondria isolated from livers of untreated rats, 13 weeks after infection with Fasciola hepatica metacercariae, were uncoupled. This uncoupling of mitochondria might be induced by a product(s) excreted by the liver fluke. The lipid extractable product(s) binds to albumin and has a molecular weight between 500 and 1000 Daltons. A second unexpected finding indicates that closantel affects the motility of Schistosoma mansoni in vitro by interferin with mitochondrial ATP synthesis. The results obtained are suggestive of a role for aerobic metabolism in the generation of energy required for motility.

Fibrin-I Degradation Products with a Molecular Weight Higher than That of Fibrinogen

1973 ◽  
Vol 29 (01) ◽  
pp. 122-129 ◽  
Author(s):  
René von Hugo ◽  
Henner Graeff
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Degradation Products ◽  
Gel Filtration ◽  
Agarose Gel ◽  
Two Dimensional ◽  
Plasma Clot ◽  
Two Dimensional Gel Electrophoresis ◽  
Parent Molecule

SummaryThe observation of intravascular lysis of fibrin deposits and of fibrinogen derivatives with a molecular weight higher than the parent molecule in human cases of disseminated intravascular coagulation (DIC) initiated the following in vitro study. Following streptokinase induced plasma clot solubilization fibrinogen derivatives were investigated after ß-alanine precipitation of the plasma samples by polyacrylamide (PAA) gel electrophoresis, intra gel immunoprecipitation, two dimensional gel electrophoresis and by agarose gel filtration. Three fibrin-i degradation products were observed and characterized according to their relative electrophoretic mobility in 5% PAA gel: 0.23, 0.35, 0.46 (fibrinogen: 0.43) x 10-5 cm2/V x sec. They could also be demonstrated after electrophoresis in the presence of 5 M urea. Agarose gel filtration yielded one peak at 180 ml of effluent volume. The 0.23 derivative was eluted in the peak fractions, whilst the 0.35 and 0.46 derivatives were eluted together at approximately 201 ml of the effluent volume (fibrinogen: 225 ml). This indicates, that the three fibrin-i degradation products described are molecular entities with molecular weights higher than fibrinogen and, that the 0.46 derivative has an increased charge/molecular size ratio in comparison with fibrinogen. Corresponding data were obtained by two dimensional gel electrophoresis in gels of different pore size.

scholarly journals The cercarial glycocalyx of Schistosoma mansoni.

1985 ◽  
Vol 100 (5) ◽  
pp. 1423-1434 ◽  
Author(s):  
J C Samuelson ◽  
J P Caulfield
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Schistosoma Mansoni ◽  
High Molecular Weight ◽  
Membrane Surface ◽  
Periodate Oxidation ◽  
Apparent Molecular Weight ◽  
Ruthenium Red ◽  
Unit Membrane

Cercariae, the freshwater stage of Schistosoma mansoni infectious to man, are covered by a single unit membrane and an immunogenic glycocalyx. When cercariae penetrate the host skin, they transform to schistosomula by shedding tails, secreting mucous and enzymes, and forming microvilli over their surface. Here the loss of the glycocalyx from cercariae transforming in vitro was studied morphologically and biochemically. By scanning electron microscopy, the glycocalyx was a dense mesh composed of 15-30 nm fibrils that obscured spines on the cercarial surface. The glycocalyx was absent on organisms fixed without osmium and was partially lost when parasites aggregated in their own secretions before fixation. By transmission electron microscopy, a 1-2 microns thick mesh of 8-15-nm fibrils was seen on parasites incubated with anti-schistosomal antibodies or fixed in aldehydes containing tannic acid or ruthenium red. Cercariae transformed to schistosomula when tails were removed mechanically and parasites were incubated in saline. Within 5 min of transformation, organisms synchronously formed microvilli which elongated to 3-5 microns by 20 min and then were shed. However, considerable fibrillar material remained adherent to the double unit membrane surface of schistosomula. For biochemical labeling, parasites were treated with eserine sulfate, which blocked cercarial swimming, secretion, infectivity, and transformation to schistosomula. Material labeled by periodate oxidation and NaB3H4 was on the surface as shown by autoradiography and had an apparent molecular weight of greater than 10(6) by chromatography. Periodate-NaB3H4 glycocalyx had an isoelectric point of 5.0 +/- 0.4 and was precipitable with anti-schistosomal antibodies. More than 60% of the radiolabeled glycocalyx was released into the medium by transforming parasites in 3 h and was recovered as high molecular weight material. Parasites labeled with periodate and fluorescein-thiosemicarbazide and then transformed had a corona of fluorescence containing microvilli, much of which was shed onto the slide. Material on cercariae labeled by lodogen-catalyzed iodination was also of high molecular weight and was antigenic. In conclusion, the cercarial glycocalyx appears to be composed of acidic high molecular weight fibrils which are antigenic and incompletely cleared during transformation.

scholarly journals Dialysis Unmasks the Fungicidal Properties of Glandular Salivary Secretions

2004 ◽  
Vol 72 (5) ◽  
pp. 2703-2709 ◽  
Author(s):  
Eva J. Helmerhorst ◽  
Bianca Flora ◽  
Robert F. Troxler ◽  
Frank G. Oppenheim
Keyword(s):  
Molecular Weight ◽  
Candida Albicans ◽  
Ionic Strength ◽  
Gel Electrophoresis ◽  
Fungicidal Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Inhibitory Effect ◽  
Salivary Proteins ◽  
Dose Dependent

ABSTRACT Several salivary proteins exhibit fungicidal activity against the opportunistic oral pathogen Candida albicans when they are tested as pure proteins in vitro. However, salivary secretions that are examined by the same assays either lack or exhibit very low candidacidal activity. Since ionic strength is known to have an inhibitory effect on the fungicidal activities of some proteins, parotid secretion was subjected to dialysis with membranes having molecular weight cutoffs (MWCOs) of 500, 1,000, 10,000, and 25,000. Dialysis with membranes with MWCOs of ≥1,000 promoted fungicidal activity of parotid secretion, and this activity was dose dependent. The addition of sodium chloride to dialyzed, fungicidal parotid secretion abolished this activity, indicating that the fungicidal component was salt sensitive. Similar results were obtained with submandibular and sublingual secretions. Polyacrylamide gel electrophoresis under native and denaturing conditions was used to analyze the composition of the dialysate. Unexpectedly, proteins with MWs much lower than the nominal MWCOs of the membranes were not lost during dialysis. Among the retained proteins, the two fractions with MWs of approximately 17,000 and 4,000 exhibited fungicidal activity. These results are consistent with the presence of lysozyme and histatins, respectively, which may represent the major candidacidal capacity of dialyzed parotid secretion.

Juvenile Fasciola hepatica are resistant to killing in vitro by free radicals compared with larvae of Schistosoma mansoni

2000 ◽  
Vol 22 (6) ◽  
pp. 287-295 ◽  
Author(s):  
David Piedrafita ◽  
Terry W. Spithill ◽  
John P. Dalton ◽  
Paul J. Brindley ◽  
Mark R. Sandeman ◽  
...  
Keyword(s):  
Free Radicals ◽  
Schistosoma Mansoni ◽  
Fasciola Hepatica

Low Molecular Weight Branched Polyethylenimine-Graft-Lentinan as a Novel Synthetic Gene Carrier

2013 ◽  
Vol 815 ◽  
pp. 293-298
Author(s):  
Li Jing Min ◽  
Jing Fen Li
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Water Soluble ◽  
Low Molecular Weight ◽  
Dna Migration ◽  
Branched Polyethylenimine ◽  
Soluble Polysaccharide

LMW Branched Polyethylenimine-graft-lentinan was synthesized by performing crosslinker CDI in the presence of water-soluble Polysaccharide (Mw 70,000) and polyethylenimine (PEI, Mw 600). The chemistry of the PEI-g-lentinan obtained were characterized. The results indicated that the amines of lentinan were grafted with PEI. Gel electrophoresis showed that DNA migration was retarded completely at a N/P ratio of 10/1, indicating good DNA condensation capability of PEI-g-lentinan. The cytotoxicity of PEI-g-lentinan was evaluated, and the results reflected that PEI-g-lentinan had a lower cytotoxicity than PEI (25 K). Gene transfection efficiency of PEI-g-chitosan in cos-7 cells was determined. Remarkably,PEI-g-lentinan showed a higher transfection efficiency than that of PEI (25 K) in vitro.

scholarly journals Variability in the Apparent Molecular Weight of Inhibin

1985 ◽  
Vol 38 (3) ◽  
pp. 327 ◽  
Author(s):  
H WG Baker ◽  
LW Eddie ◽  
B Hudson ◽  
HD Niall
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Polyacrylamide Gel ◽  
Pituitary Cells ◽  
Response Curves

An in vitro bioassay based on suppression of GnRH-stimulated FSH secretion by pituitary cells in culture was used to monitor inhibin activity after dialysis, gel filtration or polyacrylamide gel electrophoresis of protein preparations from a variety of gonadal secretions and extracts under native and dissociating conditions. The suggestion that inhibin is a peptide of molecular weight less than 5000 was not confirmed. Although some fractions of low molecular weight suppressed FSH secretion, the amount of activity was low and the dose response curves were not parallel with a standard preparation of inhibin. Under most conditions, inhibin eluted with an apparent molecular weight of about 90 000. However, gel filtration of rete testis fluid protein in I M acetic acid resulted in elution of inhibin activity with a lower apparent molecular weight and with polyacrylamide gel electrophoresis in o� 1% (w/v) sodium dodecylsulfate, the apparent molecular weight was 30 000. It is concluded that inhibin is a protein which tends to aggregate and coelute with larger molecules.

scholarly journals Incorrect Molecular Weights due to inaccurate Prestained Protein Molecular Weight Markers that are used for Gel Electrophoresis and Western Blotting

2020 ◽  
Author(s):  
Rômulo Leão Silva Neris ◽  
Ajuni Kaur ◽  
Aldrin V. Gomes
Keyword(s):  
Molecular Weight ◽  
Molecular Mass ◽  
Western Blotting ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Molecular Weights ◽  
Molecular Masses ◽  
Protein Molecular Weight

ABSTRACTThe most widely used Western blotting protein standards are prestained proteins of known molecular mass (kDa). They are also utilized for sodium dodecyl sulphate (SDS) Polyacrylamide Gel Electrophoresis (PAGE) to determine the molecular mass of proteins separated by electrophoresis. The objective of this study was to assess the reliability of different commercially available protein standards in predicting accurate protein molecular weights. We performed this experiment by running Criterion TGX gels with five prestained protein standards (Thermo Fisher SeeBlue Plus 2, Bio-Rad Precision Plus Protein Dual-color, Thermo Fisher Spectra Multi-color, Novex-Sharp Pre-stained, and Invitrogen iBright Pre-Stained). To evaluate their accuracy, we utilized highly purified Bovine Serum Albumin (BSA, 66.44 kDa) and Cytochrome C (Cyto C, 11.62 kDa). We also made use of the dimers of BSA (132.88 kDa) and Cyt C (23.24 kDa) that are present on SDS-PAGE gels. Our results suggest that three of the standards were less accurate at higher molecular masses with the iBright marker having the highest error in determining the expected 132.88 kDa molecular weight. The SeeBlue Plus 2 was accurate at identifying the 132.88 kDa molecular weight protein band but was less reliable for the three other lower molecular weight proteins. These findings have significant implications for the determination of protein masses because researchers rely on these standards to evaluate the molecular masses of their protein(s). We suggest that at least two different protein standards should be initially used in electrophoresis gels and for Western blotting in order to get accurate protein molecular weight results.

scholarly journals Plasma crosslinked fibrin polymers: quantitation based on tissue plasminogen activator conversion to D-dimer and measurement in normal and patients with acute thrombotic disorders

Blood ◽  
1992 ◽  
Vol 80 (3) ◽  
pp. 709-717 ◽  
Author(s):  
A Kornberg ◽  
CW Francis ◽  
VJ Marder
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Western Blotting ◽  
Gel Electrophoresis ◽  
Degradation Products ◽  
Thrombotic Disease ◽  
Tissue Plasminogen ◽  
D Dimer

Abstract Plasma crosslinked fibrin polymers (XLFP) are formed as a result of in vivo hemostatic activation and are elevated in thrombotic disease. We have investigated the plasmic degradation of plasma XLFP in vitro to provide information regarding the pattern of crosslinking and the composition of degradation products. Plasma XLFP were identified by sodium dodecyl sulfate (SDS)-agarose electrophoresis and Western blotting and quantitated by gel scanning. D-dimer was measured by enzyme-linked immunosorbent assay and the results were verified by SDS- polyacrylamide gel electrophoresis and Western blotting of the digests. Complete degradation of XLFP occurred only after supplementation of plasma with plasminogen (5 U/mL) and incubation with recombinant tissue plasminogen activator (rt-PA), indicating that the normal plasma plasminogen concentration limits plasmic degradation in vitro. Gel electrophoresis showed that the principal terminal degradation products of XLDP were fragments D, DD, and E, indicating that crosslinking occurred primarily through gamma chain dimers. After adding a low concentration of thrombin to plasma in vitro, XLFP increased progressively before clotting, and the concentration correlated with the increase in the D-dimer concentration after degradation (r = .98). Plasma XLFP and D-dimer concentrations in plasmic digests were significantly elevated in patients with stroke (150 +/- 83 micrograms/mL and 88 +/- 32 micrograms/mL), myocardial infarction (217 +/- 110 micrograms/mL and 84 +/- 30 micrograms/mL), and venous thrombosis (187 +/- 80 micrograms/mL and 86 +/- 19 micrograms/mL) compared with normals (28 +/- 12 micrograms/mL and 25 +/- 7 micrograms/mL). There was a strong correlation between the plasma concentration of XLFP and the D-dimer immunoreactivity of plasma after plasmic degradation (r = .87). The results indicate that XLFP in plasma are crosslinked primarily through gamma chains and degrade to fragment DD with plasminogen activation. Also, the immunoreactivity of in vitro plasmic digests of plasma reflects the concentration of XLFP and may provide a useful indirect measure of in vivo hemostatic activation in patients with thrombotic disease.

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