A stalk-specific wheat germ agglutinin binding protein, wst34, in Dictyostelium discoideum can be detected with antiserum raised against Dictyostelium mucoroides stalk

1990 ◽  
Vol 68 (4) ◽  
pp. 699-704 ◽  
Author(s):  
Yuzuru Kubohara ◽  
Koji Okamoto
Keyword(s):  
Dictyostelium Discoideum ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyclonal Antiserum ◽  
Stalk Cell ◽  
Affinity Column ◽  
Western Blots

A new stalk-specific wheat germ agglutinin (WGA) binding protein, wst34, has been identified in Dictyostelium discoideum and purified by the use of preparative sodium dodecyl sulfate - polyacrylamide gel electrophoresis and a WGA-affinity column. In normal development, wst34 appears during culmination and is maintained in stalk cells. It has a molecular mass of 34 kilodaltons and a pI value of 5.5–6.5. A polyclonal antiserum raised against stalk cell proteins of Dictyostelium mucoroides recognizes wst34 in western blots of D. discoideum proteins.Key words: Dictyostelium discoideum, Dictyostelium mucoroides, wheat germ agglutinin.

Concanavalin A and wheat germ agglutinin binding glycoproteins associated with cell fusion and zygote differentiation in Dictyostelium discoideum: effects of calcium ions and tunicamycin on glycoprotein profiles

1991 ◽  
Vol 69 (4) ◽  
pp. 282-290 ◽  
Author(s):  
Darren D. Browning ◽  
Danton H. O'Day
Keyword(s):  
Dictyostelium Discoideum ◽  
Cell Fusion ◽  
Concanavalin A ◽  
Calcium Ions ◽  
Sexual Development ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dictyostelium Cell

To determine which glycoproteins may be critical to sexual development in Dictyostelium discoideum, cell samples from different developmental stages were separated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and blotted to nitrocellulose. Concanavalin A (ConA) and wheat germ agglutinin (WGA) binding proteins were visualized on the blots using an immunochemical procedure employing peroxidase–antiperoxidase. ConA labelled at least 28 proteins, but only one band showed calcium-dependent changes in its expression. WGA bound at least 30 proteins and changes in several bands were observed that did not occur in calcium-deficient controls. Two WGA-binding glycoproteins which migrated at 200 and 166 kilodaltons (kDa), respectively, showed developmental changes associated with the time of cell fusion. One WGA-binding and one ConA-binding glycoprotein migrating at 130 and 126 kDa, respectively, appeared later during sexual development, in association with the phase of zygote differentiation. Several WGA- and ConA-binding glycoproteins decreased during sexual development, but were not affected by the absence of calcium ions. Tunicamycin (1 μg/mL) inhibited cell fusion when added to sexual cultures prior to the appearance of the 166-kDa glycoprotein gp166. The effects of this inhibitor on development support the importance of glycoproteins to cell fusion during sexual development in D. discoideum.Key words: Dictyostelium, cell fusion, glycoprotein, tunicamycin, concanavalin A, wheat germ agglutinin, calcium.

scholarly journals Specific glycoprotein changes during development of the chick neural retina.

1978 ◽  
Vol 79 (1) ◽  
pp. 132-137 ◽  
Author(s):  
G Mintz ◽  
L Glaser
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Neural Retina ◽  
Cell Type ◽  
Retinal Antigens ◽  
Qualitative Changes

After separation of whole proteins of chick neural retina by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), a number of glycoproteins can be detected by staining the gels with 125I-labeled wheat germ agglutinin (WGA) and other lectins. The glycoprotein patterns show both quantitative and qualitative changes between days 7 and 13 of development. Some of these glycoproteins can be separated by chromatography on columns of insolubilized lectins. These observations suggest that purification of some of these glycoproteins identified by staining with radioactive lectins would yield retinal antigens which may be specific for developmental stage and cell type.

scholarly journals Trichomonas vaginalis Lipophosphoglycan Mutants Have Reduced Adherence and Cytotoxicity to Human Ectocervical Cells

2005 ◽  
Vol 4 (11) ◽  
pp. 1951-1958 ◽  
Author(s):  
Felix D. Bastida-Corcuera ◽  
Cheryl Y. Okumura ◽  
Angie Colocoussi ◽  
Patricia J. Johnson
Keyword(s):  
Wheat Germ Agglutinin ◽  
Parent Strain ◽  
Trichomonas Vaginalis ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Chemical Mutagenesis ◽  
Reduced Adherence

ABSTRACT The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite.

scholarly journals Preparation and characterization of plasma membrane-enriched fractions from rat pancreatic islets.

1976 ◽  
Vol 71 (2) ◽  
pp. 606-623 ◽  
Author(s):  
A Lernmark ◽  
A Nathans ◽  
D F Steiner
Keyword(s):  
Plasma Membrane ◽  
Wheat Germ Agglutinin ◽  
Membrane Fraction ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasma Membrane Fraction ◽  
Fresh Plasma ◽  
Rat Islets Of Langerhans ◽  
Membrane Components

Methods have been developed for the isolation on a semi-micro scale of a plasma membrane-enriched fraction from rat islets of Langerhans. An important feature of these experiments is the use of 125I-labeled wheat germ agglutinin as a specific probe for plasma membrane-containing fractions. The partly purified plasma membrane fraction had a density in sucrose of about 1.10 and was enriched in the activities of 5'-nucleotidase, alkaline phosphatase, sodium-potassium, and magnesium-dependent ATPase and adenylate cyclase. It contained only very low levels of acid phosphatase, cytochrome c oxidase, insulin, and RNA. Further purification was hampered by the relatively small amounts of fresh plasma membrane material that could be obtained from 16-24 rats in each experiment. When islets were prelabeled with radioactive fucose, the plasma membrane-enriched fraction contained radioactivity at a four- to fivefold higher specific acivity than the whole islet homogenate. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of plasma membrane-enriched fractions pooled from several experiments revealed a distinctive pattern of protein bands as compared with other less pure fractions. With respect to rapidity, apparent specificity, and easy reversibility of the labeling of the plasma membrane fraction, 125I-wheat germ agglutinin provides a highly useful tool for the detection of microgram quantities of plasma membrane components which should be applicable to many other systems as well.

Purification and characterization of a novel factor which stimulates rat ribosomal gene transcription in vitro by interacting with enhancer and core promoter elements

1990 ◽  
Vol 10 (10) ◽  
pp. 5177-5186
Author(s):  
J Zhang ◽  
S T Jacob
Keyword(s):  
Ribosomal Dna ◽  
Core Promoter ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Affinity Column ◽  
Mobility Shift ◽  
Enhancer Element ◽  
Molecular Weights ◽  
The Core ◽  
Pol I

Previous studies in our laboratory have characterized a 174-base-pair (bp) enhancer sequence in the rat ribosomal DNA spacer region that exhibits all of the characteristics of a polymerase (Pol) II enhancer. Further studies showed that at least half of the enhancer activity resides in a 37-bp motif (E1) within the 174-bp spacer sequence that is located between positions -2.183 and -2.219 kilobase pairs upstream of the initiation site. To identify the factor(s) that binds specifically to the 37-bp enhancer domain, we fractionated whole-cell extract from rat adenocarcinoma ascites cells by chromatography on a series of columns, including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides of molecular weights 79,400 and 89,100 and was completely devoid of RNA Pol I activity. Electrophoretic mobility shift analysis showed that the polypeptides in the purified preparation (designated E1BF) interacted with both the enhancer element and the core promoter. To determine whether each polypeptide can separately bind to the core promoter and the enhancer, the individual components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, renatured, and subjected to gel retardation analysis. This experiment demonstrated that both polypeptides interacted with the two cis-acting sequences. The specificity of the binding was demonstrated by competition with unlabeled 37-bp and core promoter fragments and lack of competition with nonspecific DNAs in the mobility shift assay. The 37-bp enhancer as well as the downstream sequence of the core promoter were protected by E1BF in the DNase I footprinting assay. Addition of E1BF to limiting amounts of fraction DE-B, which contains all factors essential for Pol I-directed transcription, resulted in three- to fourfold stimulation of ribosomal DNA transcription. Comparison of molecular weights and footprinting profiles did not reveal any relationship between E1BF and other Pol I trans-acting factors.

Pig sperm membrane microdomains contain a highly glycosylated 15–25-kDa wheat germ agglutinin-binding protein

2012 ◽  
Vol 426 (3) ◽  
pp. 356-362 ◽  
Author(s):  
Waraporn Kasekarn ◽  
Takeru Kanazawa ◽  
Kazuki Hori ◽  
Tomoyuki Tsuchiyama ◽  
Xue Lian ◽  
...  
Keyword(s):  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Binding Protein ◽  
Membrane Microdomains ◽  
Sperm Membrane

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin

1993 ◽  
Vol 13 (4) ◽  
pp. 2061-2068
Author(s):  
H Shirataki ◽  
K Kaibuchi ◽  
T Sakoda ◽  
S Kishida ◽  
T Yamaguchi ◽  
...  
Keyword(s):  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Brain ◽  
Target Protein ◽  
Dependent Manner ◽  
Gtp Binding Protein ◽  
Reading Frame ◽  
Gtp Binding ◽  
Putative Target

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.

Isolation, Cultivation, and Characterization of Borrelia burgdorferi from Rodents and Ticks in the Charleston Area of South Carolina

2000 ◽  
Vol 38 (1) ◽  
pp. 120-124
Author(s):  
J. H. Oliver ◽  
K. L. Clark ◽  
F. W. Chandler ◽  
L. Tao ◽  
A. M. James ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Borrelia Burgdorferi ◽  
Reference Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Western Blots ◽  
Cotton Rat ◽  
Cotton Mouse ◽  
B 31

ABSTRACT Twenty-eight Borrelia burgdorferi isolates from the Charleston, S.C., area are described. This represents the first report and characterization of the Lyme disease spirochete from that state. The isolates were obtained from December 1994 through December 1995 from the tick Ixodes scapularis , collected from vegetation, and from the rodents Peromyscus gossypinus (cotton mouse), Neotoma floridana (eastern wood rat), and Sigmodon hispidus (cotton rat). All isolates were screened immunologically by indirect immunofluorescence with monoclonal antibodies to B. burgdorferi -specific outer surface protein A (OspA) (antibodies H5332 and H3TS) and B. burgdorferi -specific OspB (antibodies H6831 and H614), a Borrelia (genus)-specific antiflagellin antibody (H9724), Borrelia hermsii -specific antibodies (H9826 and H4825), and two polyclonal antibodies (one to Borrelia species and another to B. burgdorferi ). Six of the isolates were analyzed by exposing Western blots to monoclonal antibodies H5332, H3TS, H6831, and H9724. All isolates were also analyzed by PCR with five pairs of primers known to amplify selected DNA target sequences specifically reported to be present in the reference strain, B. burgdorferi B-31. The protein profiles of six of the isolates (two from ticks, one from a cotton mouse, two from wood rats, and one from a cotton rat) also were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the 28 Charleston isolates are B. burgdorferi sensu stricto based on their similarities to the B. burgdorferi B-31 reference strain.

scholarly journals Mechanism of neutrophil chemiluminescence induced by wheat germ agglutinin: partial characterization of the antigens recognized by wheat germ agglutinin

Blood ◽  
1984 ◽  
Vol 64 (5) ◽  
pp. 1094-1102 ◽  
Author(s):  
Y Ozaki ◽  
J Iwata ◽  
T Ohashi
Keyword(s):  
Membrane Proteins ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Sodium Dodecyl ◽  
Submaxillary Gland ◽  
Binding Property ◽  
Molecular Weights ◽  
Acetylneuraminic Acid ◽  
Cell Extracts ◽  
High Concentrations

Abstract Wheat germ agglutinin (WGA) stimulated neutrophils to produce significant levels of luminol-dependent chemiluminescence (CL). Since WGA is known to bind N-acetylglucosamine (GlcNAc) oligomers and N- acetylneuraminic acid (NANA), we attempted to determine which binding property of WGA is essential for induction of CL. The succinylated form of WGA (SuWGA), which is no longer able to bind NANA, was still able to induce CL. N-Acetylglucosamine at a concentration of 20 mmol/L almost completely inhibited WGA-induced CL production by neutrophils, whereas bovine submaxillary gland mucin, a potent blocker of NANA binding of WGA, failed to inhibit CL production. Lectins with the GlcNAc-binding property were examined for their ability to induce CL. Those that have higher valences and have a tendency to bind GlcNAc oligomers in the internal portion of glycoconjugates were able to induce CL, whereas those that have low valences and bind terminal GlcNAc of glycoconjugates failed to induce CL even at high concentrations. Attempts were made to characterize the neutrophil membrane proteins recognized by WGA. Glycoproteins with a molecular weight of 25,000 daltons were identified by a 50 mmol/L GlcNAc elution of WGA gels loaded with 125I-labeled neutrophil membrane proteins. Elution with 500 mumol/L GlcNAc trimer produced several glycoproteins of different molecular weights in addition to the glycoproteins of 25,000 daltons. 125I-labeled WGA and SuWGA were used for autoradiographic analysis of cell extracts of the neutrophils separated on sodium dodecyl sulfate polyacrylamide gels. WGA recognized multiple glycoproteins of different molecular weights, whereas SuWGA bound only a few of them. Glycoproteins of 25,000 daltons, probably corresponding to those identified by 50 mmol/L GlcNAc elution, were also recognized.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500 ◽  
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

Abstract A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

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