scholarly journals Myosin at the apical pole of ciliated epithelial cells as revealed by a monoclonal antibody.

1986 ◽  
Vol 103 (2) ◽  
pp. 613-619 ◽  
Author(s):  
C Klotz ◽  
N Bordes ◽  
M C Laine ◽  
D Sandoz ◽  
M Bornens
Keyword(s):  
Monoclonal Antibody ◽  
Striated Muscle ◽  
Ultrastructural Localization ◽  
Basal Bodies ◽  
Myosin Heavy Chains ◽  
Ciliated Cells ◽  
Western Blots ◽  
Apical Pole ◽  
Heavy Chains ◽  
Filamentous Material

A monoclonal antibody (CC-212), obtained in a fusion experiment in which basal bodies from quail oviduct were used as immunogen, has been shown to label the apical pole of ciliated cells and to react with a 200-kD protein. This monoclonal antibody was demonstrated to be an anti-myosin from smooth muscle or from nonmuscular cells using the following criteria: On Western blots it reacted with the myosin heavy chains from gizzard and platelet extracts and from cultured cell line extracts, but did not react with striated muscle myosin heavy chains. By immunofluorescence it decorated the stress fibers of well-spread cells with a characteristic striated pattern, while it did not react with myotubes containing organized myofibrils. On native ciliated cells as well as on Triton-extracted ciliated cortices from quail oviduct, this monoclonal antibody decorated the apical pole with a stronger labeling of the periphery of the apical area. Ultrastructural localization was attempted using the immunogold technique on the same preparation. Myosin was associated with a filamentous material present between striated rootlets and the proximal extremities of the basal bodies. No labeling of the basal body itself or of axoneme was observed.

Identification of proteins associated with microtubule-organizing centres and filaments in the oral apparatus of the ciliate Paramecium tetraurelia

1990 ◽  
Vol 97 (3) ◽  
pp. 553-563
Author(s):  
GUY KERYER ◽  
FRANCINE IFTODE ◽  
MICHEL BORNENS
Keyword(s):  
Monoclonal Antibody ◽  
Spatial Organization ◽  
Buccal Cavity ◽  
Ultrastructural Localization ◽  
Basal Bodies ◽  
Paramecium Tetraurelia ◽  
Single Polypeptide ◽  
Almost All ◽  
Structure And Activity

In an effort to study the assembly of microtubules in ciliates we have isolated the oral apparatus of Paramecium tetraurelia, a major microtubuie-organizing centre (MTOC) and identified several proteins localized in the fibrillar system associated with basal bodies. Using a monoclonal antibody raised against human centrosomes (CTR210) and a polyclonal antibody (OF1) directed against a 87x103Mr protein of the oral apparatus of Tetrahymena, we observed the same decoration of the oral apparatus of Paramecium, both in situ and after isolation. Ultrastructural localization further showed the presence of the antigens in the fibrillar network that forms a layer under almost all the buccal cavity in close apposition to the basal bodies. CTR210 recognized two sets of polypeptides of Mr72 and 80x103, whereas OF1 recognized a single polypeptide of Mr87x103. Only the 80x103Mr polypeptide was also decorated with the monoclonal antibody MPM-2, previously shown to decorate the oral apparatus of Paramecium and known to react with phosphorylated epitopes in a large variety of MTOCs. All the proteins identified with the three antibodies are insoluble at high ionic strength, display several isoforms and apparently belong to the same fibrillar material. The function of this material in the spatial organization, the structure, and activity of the MTOCs is discussed.

Immunocytochemical study of the formation of striated rootlets during ciliogenesis in quail oviduct

1990 ◽  
Vol 95 (3) ◽  
pp. 423-432 ◽  
Author(s):  
M. Lemullois ◽  
M.C. Marty
Keyword(s):  
Monoclonal Antibody ◽  
Polyclonal Antibody ◽  
Ciliated Cell ◽  
Apical Part ◽  
Basal Bodies ◽  
Immunocytochemical Study ◽  
Ciliated Cells ◽  
Immunogold Staining ◽  
Dense Granules ◽  
Basal Pole

In quail oviduct, a 175K (K = 10(3) Mr) protein associated with striated rootlets was previously identified by Klotz and co-workers using monoclonal antibody CC310. As this monoclonal antibody recognizes several proteins on immunoblots of ciliated cells, we prepared a polyclonal antibody monospecific to the 175K protein by intrasplenic immunization of mice. Immunofluorescence study confirmed the distribution of the 175K protein at the apical part of the ciliated cell and its absence in other epithelial cells. Immunogold staining showed that this protein was strongly associated with the fibrillar axis of striated rootlets. The absence of labeling on striation suggested that rootlets were composed of several proteins, with one group forming the fibrillar axis and the second forming the striation. The formation of striated rootlets during ciliogenesis was studied using this polyclonal antibody. The 175K protein appeared at the beginning of centriologenesis in fibrillar material located around dense granules, and then around the generative complex. The formation of rootlets began at the basal pole of migrating basal bodies. The elongation of the rootlet axes took place when basal bodies were anchored to the plasma membrane.

Ultrastructural Localization of Acid Mucosubstance in Skeletal Muscle

1967 ◽  
Vol 25 ◽  
pp. 52-53 ◽  
Author(s):  
William J. Dougherty ◽  
Samuel S. Spicer
Keyword(s):  
Striated Muscle ◽  
Divalent Cations ◽  
Ultrastructural Localization ◽  
Major Elements ◽  
Frozen Sections ◽  
Thorium Dioxide ◽  
Iron Solution ◽  
Coupling System ◽  
Psoas Muscles ◽  
Formalin Fixed

In recent years, considerable attention has focused on the morphological nature of the excitation-contraction coupling system of striated muscle. Since the study of Porter and Palade, it has become evident that the sarcoplastic reticulum (SR) and transverse tubules constitute the major elements of this system. The problem still exists, however, of determining the mechamisms by which the signal to interdigitate is presented to the thick and thin myofilaments. This problem appears to center on the movement of Ca++ions between myofilaments and SR. Recently, Philpott and Goldstein reported acid mucosubstance associated with the SR of fish branchial muscle using the colloidal thorium dioxide technique, and suggested that this material may serve to bind or release divalent cations such as Ca++. In the present study, Hale's iron solution adapted to electron microscopy was applied to formalin-fixed myofibrils isolated from glycerol-extracted rabbit psoas muscles and to frozen sections of formalin-fixed rat psoas muscles.

Ciliary coordination in newt lung cells requires microtubule-based linkages between basal bodies: A correlative light and EM study

1992 ◽  
Vol 50 (1) ◽  
pp. 570-571
Author(s):  
Robert Hard ◽  
Gerald Rupp ◽  
Matthew L. Withiam-Leitch ◽  
Lisa Cardamone
Keyword(s):  
Basal Body ◽  
Untreated Control ◽  
Beat Frequency ◽  
Primary Cultures ◽  
Living Cells ◽  
Power Stroke ◽  
Ultrastructural Changes ◽  
Lung Cells ◽  
Basal Bodies ◽  
Ciliated Cells

In a coordinated field of beating cilia, the direction of the power stroke is correlated with the orientation of basal body appendages, called basal feet. In newt lung ciliated cells, adjacent basal feet are interconnected by cold-stable microtubules (basal MTs). In the present study, we investigate the hypothesis that these basal MTs stabilize ciliary distribution and alignment. To accomplish this, newt lung primary cultures were treated with the microtubule disrupting agent, Colcemid. In newt lung cultures, cilia normally disperse in a characteristic fashion as the mucociliary epithelium migrates from the tissue explant. Four arbitrary, but progressive stages of dispersion were defined and used to monitor this redistribution process. Ciliaiy beat frequency, coordination, and dispersion were assessed for 91 hrs in untreated (control) and treated cultures. When compared to controls, cilia dispersed more rapidly and ciliary coordination decreased markedly in cultures treated with Colcemid (2 mM). Correlative LM/EM was used to assess whether these effects of Colcemid were coupled to ultrastructural changes. Living cells were defined as having coordinated or uncoordinated cilia and then were processed for transmission EM.

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Ashia Siddiqua ◽  
Michael Wilkinson ◽  
Vijay Kakkar ◽  
Yatin Patel ◽  
Salman Rahman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Functional Characterization ◽  
Human Platelets ◽  
Western Blots ◽  
Activated Platelets ◽  
Reducing Conditions ◽  
Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

scholarly journals Monoclonal antibody studies of creatine kinase. The ART epitope: evidence for an intermediate in protein folding

1989 ◽  
Vol 257 (2) ◽  
pp. 461-469 ◽  
Author(s):  
G E Morris
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Creatine Kinase ◽  
Specific Binding ◽  
Trypsin Digestion ◽  
Proteinase K ◽  
Enzyme Linked Immunosorbent Assay ◽  
Muscle Creatine Kinase ◽  
Western Blots ◽  
Methionine Residues

Chemical cleavage at cysteine residues with nitrothiocyanobenzoic acid shows that the last 98 amino acids of the 380-amino-acid sequence of chick muscle creatine kinase are sufficient for binding of the monoclonal antibody CK-ART. Removal of the last 30 amino acids by cleavage at methionine residues with CNBr results in loss of CK-ART binding. CK-ART binding is also lost when these C-terminal methionine residues are oxidized to sulphoxide, but binding is regained on reduction. Proteinase K ‘nicks’ native CK at a single site near the C-terminus and two fragments of 327 amino acides and 53 amino acids can be separated by subsequent SDS or urea treatment. CK-ART still binds normally to ‘nicked’ CK, which is enzymically inactive. After treatment with either urea (in a competition enzyme-linked immunosorbent assay) or SDS (on Western blots), however, CK-ART binds to neither of the two fragments, although these treatments do not affect binding to intact CK. This suggests that parts of both CK fragments contribute to the CK-ART epitope. CK-ART is both species- and isoenzyme-specific, binding only to chick M-CK. The only C-terminal regions containing chick-specific sequences are residues 300-312 and residues 368-371, the latter group being close to the essential methionine residues. We suggest that one, or possibly both, of these regions is involved in forming the conformational epitope on the surface of the CK molecule which CK-ART recognizes. Native CK is resistant to trypsin digestion. The C-terminal half of urea-treated and partly-refolded CK is also resistant to trypsin digestion, whereas the N-terminal half is readily digested. The results suggest a C-terminal region which can refold more rapidly than the rest of the CK molecule and provide evidence for an intermediate in CK refolding.

Monospecific Antibodies against the Three Mammalian Fast Limb Myosin Heavy Chains

2000 ◽  
Vol 272 (1) ◽  
pp. 303-308 ◽  
Author(s):  
Christine A. Lucas ◽  
Lucia H.D. Kang ◽  
Joseph F.Y. Hoh
Keyword(s):  
Myosin Heavy Chains ◽  
Heavy Chains ◽  
Monospecific Antibodies
Sign in / Sign up

Export Citation Format

Share Document