Biochemical characterization of some raptor trypanosomes. I. In vitro cultivation and lectin-binding studies

1986 ◽  
Vol 64 (1) ◽  
pp. 189-194 ◽  
Author(s):  
Carl E. Kirkpatrick ◽  
Cynthia A. Terway-Thompson
Keyword(s):  
Biochemical Characterization ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
The Other ◽  
In Vitro Cultivation ◽  
Type I ◽  
Binding Studies ◽  
Binding Characteristics ◽  
American Kestrels

Nine trypanosome strains from five species of raptors were cultivated in vitro in a monophasic medium. Two morphologically distinct trypanosomes were observed in culture: those from American kestrels (Falco sparverius) were smaller than the other strains. The two kestrel (KT) trypanosome strains showed in vitro growth kinetics that differed from the larger trypanosomes, and the KT strains, unlike the others, required hemin in the medium for growth. The effectiveness of eight plant lectins to induce the agglutination of cultured trypanosomes was studied as a means of differentiating the various strains. It was found that lectins from Lens culinaris and Ricinus communis (type I) were particularly effective in distinguishing the KT strains from the other raptor trypanosome strains. Based on the results of experiments in which lectin-mediated trypanosome agglutination was inhibited by the addition of various monosaccharides, it is concluded that all of the avian trypanosomes studied express surface methyl α-D-mannoside, D(+)-galactose, and (or) α-lactose. Only the relatively large raptor trypanosome isolates expressed N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and α-L(−)-fucose on their surfaces. The differences in lectin-binding characteristics between the two morphologic types of raptor trypanosome were as great as those among each of the avian trypanosomes and the mammalian trypanosomatids Leishmania chagasi and Trypanosoma rhodesiense.

Loss Of Platelet Membrane Glycoproteins Or Terminal Sialic Acid Detected By Fitc-Lectins

1981 ◽  
Author(s):  
L McGregor ◽  
J L McGregor ◽  
K J Clemetson ◽  
M Dechavanne ◽  
E F Lüscher
Keyword(s):  
Sialic Acid ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Binding Studies ◽  
Platelet Surface ◽  
Terminal Sialic Acid

Pre-thrombic conditions in certain individuals resulting from enhanced platelet-vessel wall or platelet-platelet interactions are perhaps characterized by a reduction in certain membrane glycoproteins or loss of terminal sialic acid. In order to investigate if such changes are detectable, the binding of FITC-lectins to human platelets treated under in vitro conditions with certain proteases to mimic possible in vivo changes occuring on the platelet surface, has been examined. Human platelets were isolated, washed and either treated with neuraminidase (10 U) or plasmin (1 CU) before fixing with formaldehyde. Binding studies were performed by the method of Monsigny et al. using FITC labelled wheat germ agglutinin (WGA), Lens culinaris lectin (LCL), Ricinus communis agglutinin (RCA) and concanavalin A (ConA). The number of lectin-binding sites (n) and the dissociation constant (Kd) were obtained by Steck and Wallach reciprocal plots. After neuraminidase or plasmin treatment n was reduced but Kd remained approximately the same with WGA. FITC-RCA-60 gave a slight fluorescence with untreated and very strong fluorescence with neuraminidase treated platelets. Platelet glycoproteins separated by 2-dimensional gel electrophoresis were identified by binding of fluorescent lectins. Plasmin decreased the intensity of GP Ib and IIb and removed Ia completely. Neuraminidase decreased the labelling of Ib by WGA. These techniques show promise as methods of detecting pre-thrombotic conditions.

The Identification and Significance of a Thr→Pro Polymorphism in Kringle IV Type 8 of Apolipoprotein(a)

1997 ◽  
Vol 77 (05) ◽  
pp. 0949-0954 ◽  
Author(s):  
J Prins ◽  
F R Lues ◽  
Y Y van der Hoek ◽  
J J.P Kastelein ◽  
B N Bouma ◽  
...  
Keyword(s):  
Case Control Study ◽  
Amino Acid Position ◽  
Case Control ◽  
Binding Studies ◽  
Binding Characteristics ◽  
Apolipoprotein A ◽  
Significant Independent Risk Factor ◽  
And Gender ◽  
Control Study

SummaryElevated plasma levels of lipoprotein(a) [Lp(a)] represent a significant independent risk factor for the development of atherosclerosis. Interindividual levels of apo(a) vary over 1000-fold and are mainly due to inheritance that is linked to the locus of the apolipoprotein(a) [apo(a)] gene. The apo(a) gene encodes multiple repeats of a sequence exhibiting up to 85% DNA sequence homology with plasminogen kringle IV (K.IV), a lysine binding domain. In our search for sequence polymorphisms in the K.IV coding domain, we identified a polymorphism predicting a Thr→Pro substitution located at amino acid position 12 of kringle IV type 8 of apo(a). The functional and clinical significance of this polymorphism was analysed in a case-control study and by comparing the in vitro lysine binding characteristics of the two Lp(a) subtypes.The case-control study (involving 153 subjects having symptomatic atherosclerosis and 153 age and gender matched normolipidemic controls) revealed an overall allele frequency for the Thr12-→Pro substitution in kringle IV type 8 of 14% and a negative association between presence of the Pro12-subtype and symptomatic atherosclerosis (p <0.03). The in vitro lysine binding studies, using Lp(a) isolated from subjects homozygous for either Thr12 or Pro12 in K.IV type 8, revealed comparable lysine-Sepharose binding fractions for the two subtypes. The binding affinity (Kd) for immobilised plasmin degraded des- AA-fibrin (DesafibTM-X) was also comparable for the two subtypes, however a decreased maximal attainable binding (Bmax) for immobilised desafibTM-X was observed for the Pro12-subtype Lp(a).

scholarly journals Different Forms of TFF2, A Lectin of the Human Gastric Mucus Barrier: In Vitro Binding Studies

2019 ◽  
Vol 20 (23) ◽  
pp. 5871 ◽  
Author(s):  
Franziska Heuer ◽  
René Stürmer ◽  
Jörn Heuer ◽  
Thomas Kalinski ◽  
Antje Lemke ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Gastric Mucus ◽  
Binding Studies ◽  
Binding Characteristics ◽  
Duodenal Glands ◽  
Vitro Binding ◽  
Trefoil Factor Family ◽  
Mucus Barrier ◽  
Mass Form

Trefoil factor family 2 (TFF2) and the mucin MUC6 are co-secreted from human gastric and duodenal glands. TFF2 binds MUC6 as a lectin and is a constituent of the gastric mucus. Herein, we investigated human gastric extracts by FPLC and identified mainly high- but also low-molecular-mass forms of TFF2. From the high-molecular-mass forms, TFF2 can be completely released by boiling in SDS or by harsh denaturing extraction. The low-molecular-mass form representing monomeric TFF2 can be washed out in part from gastric mucosa specimens with buffer. Overlay assays with radioactively labeled TFF2 revealed binding to the mucin MUC6 and not MUC5AC. This binding is modulated by Ca2+ and can be blocked by the lectin GSA-II and the monoclonal antibody HIK1083. TFF2 binding was also inhibited by Me-β-Gal, but not the α anomer. Thus, both the α1,4GlcNAc as well as the juxtaperipheral β-galactoside residues of the characteristic GlcNAcα1→4Galβ1→R moiety of human MUC6 are essential for TFF2 binding. Furthermore, there are major differences in the TFF2 binding characteristics when human is compared with the porcine system. Taken together, TFF2 appears to fulfill an important role in stabilizing the inner insoluble gastric mucus barrier layer, particularly by its binding to the mucin MUC6.

scholarly journals Platelet-derived microparticles associate with fibrin during thrombosis

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4651-4663 ◽  
Author(s):  
P Siljander ◽  
O Carpen ◽  
R Lassila
Keyword(s):  
Type I Collagen ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
Type I ◽  
Binding Studies ◽  
Vitro Binding ◽  
Large Platelet ◽  
Platelet Aggregates ◽  
Thrombin Antithrombin Iii Complex

Platelet-derived microparticles (MP) are reported to express both pro- and anticoagulant activities. Nevertheless, their functional significance has remained unresolved. The present study monitored the generation and fate of MP in an experimental model of thrombosis with costimulation of platelets by collagen and thrombin. When minimally anticoagulated (0.5 micromol/L PPACK) blood was perfused over immobilized fibrillar type I collagen in a flow chamber at a low shear rate (300 s(-1)), endogenous thrombin was generated, as evidenced by thrombin-antithrombin III complex. In contrast to full anticoagulation 150 micromol/L PPACK) and the absence of collagen, large platelet aggregates and fibrin ensued during perfusions over collagen in the presence of thrombin. In these thrombi, MP, defined as GPIIbIIIa- and P- selectin-positive vesicles (<1 micron), were found to align fibrin in immunofluorescence and scanning immunoelectron microscopy. Moreover, in sections of embolectomized thromboemboli from patients GPIIbIIIa- and P- selectin-positive material compatible with MP was detected in a fibrin strand-like pattern. In vitro binding studies showed that MP bound to fibrin and acted there as procoagulants. In summary, we show that MP generated during thrombus formation associate with local fibrin. This adhesive function fibrin could imply a sustained modulatory role for MP in evolving thrombi.

Myosin VI: a multifunctional motor

2004 ◽  
Vol 32 (5) ◽  
pp. 685-688 ◽  
Author(s):  
I. Lister ◽  
R. Roberts ◽  
S. Schmitz ◽  
M. Walker ◽  
J. Trinick ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Actin Filaments ◽  
Biochemical Characterization ◽  
Force Transducer ◽  
The Other ◽  
Myosin Vi ◽  
Coated Pits ◽  
Binding Partners ◽  
Alternative Approach

Myosin VI moves towards the minus end of actin filaments unlike all the other myosins so far studied, suggesting that it has unique properties and functions. Myosin VI is present in clathrin-coated pits and vesicles, in membrane ruffles and in the Golgi complex, indicating that it has a wide variety of functions in the cell. To investigate the cellular roles of myosin VI, we have identified a variety of myosin VI-binding partners and characterized their interactions. As an alternative approach, we have studied the in vitro properties of intact myosin VI. Previous studies assumed that myosin VI existed as a dimer but our biochemical characterization and electron microscopy studies reveal that myosin VI is a monomer. Using an optical tweezers force transducer, we showed that monomeric myosin VI is a non-processive motor with a large working stroke of 18 nm. Potential roles for myosin VI in cells are discussed.

Changing surface antigen and carbohydrate patterns during the development of Oesophagostomum dentatum

Parasitology ◽  
1999 ◽  
Vol 119 (5) ◽  
pp. 491-501 ◽  
Author(s):  
A. JOACHIM ◽  
B. RUTTKOWSKI ◽  
A. DAUGSCHIES
Keyword(s):  
Concanavalin A ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Soybean Agglutinin ◽  
Peanut Agglutinin ◽  
Oesophagostomum Dentatum ◽  
Periodate Treatment

Living and fixed specimen of Oesophagostomum dentatum were labelled in situ with serum antibodies or a panel of biotin- labelled lectins. Specific binding of antibodies was observed in all parasitic stages – freshly exsheathed 3rd-stage larvae (L3), 3rd- and 4th-stage (L4) larvae cultured in vitro and L3 and L4 and adults isolated from pig intestines. The shedding of the stained layer by motile larvae was inhibited by levamisole-induced paralysis. Larvae cultured in vitro exposed serum-derived proteins on their surface which could be labelled with secondary antibody directed against the respective serum donor species. While freshly exsheathed larvae were recognized by O. dentatum-positive serum only, older larvae and adults cross-reacted with serum from pigs infected with O. quadrispinulatum, a closely related species. Lectin binding varied considerably between stages. While binding was not observed in pre-parasitic stages, Concanavalin A, Soybean Agglutinin, Wheat Germ Agglutinin, Ricinus communis Agglutinin and Peanut Agglutinin bound to developing larvae in varying degrees. Dolichos biflorus Agglutinin only bound to advanced (luminal) larval stages, while adults generally displayed only weak or partial lectin binding (except with Concanavalin A and Wheat Germ Agglutinin). Ulex europaeus Agglutinin only labelled larvae derived from cultures containing 10% pig serum. Cleavage of the carbohydrate residues by sodium periodate treatment resulted in reduction of antibody binding to cultured larvae, but not to freshly exsheathed L3. Concanavalin A, Soybean Agglutinin, and Peanut Agglutinin binding was also reduced by periodate treatment, while binding of Wheat Germ Agglutinin and Ricinus communis Agglutinin was inhibited only in early L3, but not in older stages. The different lectin labelling patterns are related to the different stages of the nematode – infective, invasive, histotropic, and luminal – and may serve as a mode of adaptation for the parasite against the host's immune attack by surface glycoprotein variation, together with antigen shedding (as demonstrated by labelling of motile larvae) and a possible acquisition of host molecules at the parasite's surface. Furthermore, a possible role of this developmental variation in surface carbohydrates in parasite–parasite interactions is discussed.

Heterogeneity of Human Biliary Mucin: Functional Implications

1994 ◽  
Vol 86 (1) ◽  
pp. 75-82 ◽  
Author(s):  
J. Henriëtte Klinkspoor ◽  
Michel J. A. van Wijland ◽  
Carolien A. M. Koeleman ◽  
Willem van Dijk ◽  
Guido N. J. Tytgat ◽  
...  
Keyword(s):  
Cholesterol Gallstone ◽  
Lectin Binding ◽  
Gallstone Disease ◽  
Functional Characterization ◽  
The Other ◽  
Binding Studies ◽  
Human Gallbladder ◽  
Patient Heterogeneity ◽  
Model Bile

1. Human gallbladder mucin has been implicated as playing a role in the pathogenesis of gallstones. In previous studies no differences have been found in the content or composition of mucins derived from control bile or cholesterol gallstone bile. Until now, no differences were also found between these two groups of mucins with regard to their ability to cause cholesterol nucleation. In the accompanying paper we have reported that there is a strong heterogeneity of gallbladder mucins derived from individual patients (M. J. A. van Wijland, J. H. Klinkspoor, L. Th. de Wit, R. P. J. Oude Elferink, G. N. J. Tytgat and A. K. Groen, Clin Sci 1994; 86: 67–74). In the present study we further investigated a possible patient to patient heterogeneity of mucin by means of immunological and functional characterization of mucins isolated from hepatic bile of six different patients with gallstones. 2. Considerable heterogeneity was found. Two of the mucins barely reacted with a polyclonal anti-mucin antibody, whereas the other four mucins reacted very strongly. Lectin-binding studies indicated that the glycans of these two mucins expressed less D-N-acetylgalactosamine residues than the other four mucins. This was confirmed by analyses of the glycan compositions. These studies furthermore indicated that the glycans were of the O-linked type, contained α-D-N-acetylglucosamine and were fucosylated, sialy-lated and sulphated to different extents. Except for a strong heterogeneity in the sugar composition of the mucins, heterogeneity was also found in the biological activity of the mucins. The two immunologically diverging mucins nucleated cholesterol from model bile 1–2 days earlier and also caused an almost threefold more rapid rupture of cholesterol/phospholipid vesicles than the other mucins. 3. We conclude that considerable differences exist between mucins derived from individual patients and that the heterogeneity in glycan composition might play a role in the pathogenesis of gallstone disease.

scholarly journals Distribution of cell surface saccharides on pancreatic cells. II. Lectin-labeling patterns on mature guinea pig and rat pancreatic cells.

1979 ◽  
Vol 80 (1) ◽  
pp. 77-95 ◽  
Author(s):  
M F Maylié-Pfenninger ◽  
J D Jamieson
Keyword(s):  
Guinea Pig ◽  
Endocrine Cells ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Acinar Cells ◽  
Binding Studies ◽  
Lectin Receptors ◽  
Pancreatic Cells ◽  
Receptor Sites ◽  
Centroacinar Cells

The surface saccharide composition of collagenase-dispersed pancreatic cells from adult guinea pig and rat glands was examined by using eight lectins and their ferritin conjugates: Concanavalin A (ConA); Lens culinaris (LCL); Lotus tetragonolobus (LTL); Ricinus communis agglutinins I and II (RCA I, RCA II); Soybean agglutinin (SBA); Ulex europeus lectin (UEL); and wheat germ agglutinin (WGA). Binding studies of iodinated lectins and lectin-ferritin conjugates both revealed one population of saturable, high-affinity receptor sites on the total cell population (approximately 95% acinar cells). Electron microscopy, however, revealed differences in lectin-ferritin binding to the plasmalemma of acinar, centroacinar, and endocrine cells. Whereas acinar cells bound heavily all lectin conjugates, endocrine and centroacinar cells were densely labeled only by ConA, LCL, WGA, and RCA I, and possessed few receptors for LTL, UEL, and SBA. Endocrine and centroacinar cells could be differentiated from each other by using RCA II, which binds to centroacinar cells but not to endocrine cells. Some RCA II receptors appeared to be glycolipids because they were extracted by ethanol and chloroform-methanol in contrast to WGA receptors which resisted solvent treatment but were partly removed by papain digestion. RCA I receptors were affected by neither treatment. The apparent absence of receptors for SBA on endocrine and centroacinar cells, and for RCA II on endocrine cells, was reversed by neuraminidase digestion, which suggested masking of lectin receptors by sialic acid. The absence of LTL and UEL receptors on endocrine and centroacinar cells was not reversed by neuraminidase. We suggest that the differential lectin-binding patterns observed on acinar, centroacinar, and endocrine cells from the adult pancreas surface-carbohydrate-developmental programs expressed during morphogenesis and cytodifferentiation of the gland.

In Vitro Platelet Binding Compared with In Vivo Thrombus Imaging Using αIIbβ3-Targeted Radioligands

1998 ◽  
Vol 80 (11) ◽  
pp. 845-851 ◽  
Author(s):  
Jan Romano ◽  
Alan Maurer ◽  
Linda Knight
Keyword(s):  
Animal Model ◽  
The Other ◽  
Organ Distribution ◽  
Pulmonary Emboli ◽  
Binding Studies ◽  
Platelet Binding ◽  
Αiibβ3 Integrin ◽  
Surprising Finding

SummaryRadioligands for the αIIbβ3 integrin on platelets are being studied for their ability to image venous thrombi and pulmonary emboli. One such radioligand, 123I-bitistatin, was previously shown to have higher thrombotic uptake in an animal model than other disintegrins, but the reason for this difference was not clear. The purpose of this study was to evaluate three labeled disintegrins, bitistatin, kistrin and barbourin, to look for in vitro differences in platelet binding which could explain the in vivo behavior.Disintegrins labeled with 125I were compared in vitro for extent of binding to platelets and rates of binding and dissociation. These findings were related to organ distribution and image quality for imaging thrombotic lesions, following administration of 123I-disintegrins in an animal model. Fibrinogen at 8.8 μmol/l was able to displace 125Ibarbourin and 125I-kistrin more rapidly from ADP-stimulated platelets, with half-times of 3.5 and 10.7 min, compared with 125I-bitistatin (31.6 min). At equivalent concentrations in whole blood, a higher percentage of bitistatin bound to platelets compared with the other two. In vivo, kistrin and barbourin had significantly lower thrombus:muscle and pulmonary embolus:lung ratios in images compared with bitistatin. There was evidence of more metabolic deiodination of labeled kistrin and barbourin in vivo compared with bitistatin. A surprising finding was that conventional in vitro platelet binding studies did not predict the relative in vivo behavior of labeled disintegrins.The results suggest that labeled bitistatin has improved targeting of thrombi because it is less easily displaced from stimulated platelets, permitting longer lesion retention. It also appears to have a greater association with resting platelets in the blood, which may increase bioavailability and delay metabolic breakdown.

Relative occupation of type-I and type-II corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications

1987 ◽  
Vol 115 (3) ◽  
pp. 459-467 ◽  
Author(s):  
J. M. H. M. Reul ◽  
F. R. van den Bosch ◽  
E. R. de Kloet
Keyword(s):  
Rat Brain ◽  
Plasma Concentrations ◽  
Striking Difference ◽  
Classical Type ◽  
Type I ◽  
Type Ii ◽  
Binding Studies ◽  
Receptor Sites ◽  
Basal Plasma

ABSTRACT The rat brain contains two receptor systems for corticosterone: the type-I corticosterone-preferring receptor and the classical type-II glucocorticoid receptor. The two receptor populations can be distinguished in binding studies with the 'pure' synthetic glucocorticoid 11β,17β-dihydroxy-6-methyl-17α (1-propynyl)-androsta-1,4,6-trione-3-one (RU 28362). In-vitro autoradiography and quantitative image analysis showed that the type-I receptor was localized almost exclusively in the hippocampus, whereas the type-II receptor extended throughout the brain, with the highest levels in the nucleus paraventricularis, nucleus supraopticus and in the thalamic, amygdaloid, hippocampal and septal regions. Unoccupied type-I and type-II receptor sites, as measured in vitro by cytosol binding of 3H-labelled steroids, displayed a large difference in the rate of appearance after adrenalectomy. The availability of type-I receptors exhibited a marked increase, reaching maximal levels within 4–7 h, and then remained constant until 2 weeks after adrenalectomy. The availability of type-II receptors did not change considerably during the first 24 h after adrenalectomy, but displayed a large increase in capacity during the subsequent 2 weeks. After adrenocortical activation as a consequence of exposure to a novel environment, plasma concentrations of corticosterone increased to reach a peak of 811 nmol/l after 30 min and attained the basal concentration (43 nmol/l) after 240 min. During this time, occupation of type-I receptors increased from 77·8% at 0 min to 97% at 30–60 min and then declined to 84·8% after 240 min. Occupation of the type-II receptors was 28·1% at 0 min, 74·5% after 30 min and 32·8% after 240 min. Injection of dexamethasone (25 μg/100 g body wt) at 08.00 h resulted in suppression of basal plasma concentrations of corticosterone and prevented the circadian-driven rise in circulating corticosterone. Occupation of type-I receptors did not change considerably as a result of injection of dexamethasone, but occupation of type-II receptors was markedly increased till 16.00 h compared with that after injection of vehicle. It was concluded that the type-I and type-II receptors are not only localized differently in the rat brain, but also exhibit a striking difference in occupation after manipulation of the pituitary-adrenocortical system. The data further support the concept of a type-I receptor-mediated tonic activating influence and a type-II receptor-mediated feedback action of corticosterone on brain function. J. Endocr. (1987) 115, 459–467

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