Radioactive labelling of the attachment site of C3

Pathology ◽  
1983 ◽  
Vol 15 (3) ◽  
pp. 346
Author(s):  
C.J. Rutherford ◽  
B.M. Babior
Keyword(s):  
Attachment Site ◽  
Radioactive Labelling

Localization of immunolabels by electron microscopy and image averaging

1983 ◽  
Vol 41 ◽  
pp. 282-283
Author(s):  
J. Frank ◽  
P.-Y. Sizaret ◽  
A. Verschoor ◽  
J. Lamy
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Attachment Site ◽  
Uranyl Acetate ◽  
Limulus Polyphemus ◽  
Resolution Limit ◽  
Electron Microscopic ◽  
Image Averaging ◽  
Top View ◽  
Better Than

The accuracy with which the attachment site of immunolabels bound to macromolecules may be localized in electron microscopic images can be considerably improved by using single particle averaging. The example studied in this work showed that the accuracy may be better than the resolution limit imposed by negative staining (∽2nm).The structure used for this demonstration was a halfmolecule of Limulus polyphemus (LP) hemocyanin, consisting of 24 subunits grouped into four hexamers. The top view of this structure was previously studied by image averaging and correspondence analysis. It was found to vary according to the flip or flop position of the molecule, and to the stain imbalance between diagonally opposed hexamers (“rocking effect”). These findings have recently been incorporated into a model of the full 8 × 6 molecule.LP hemocyanin contains eight different polypeptides, and antibodies specific for one, LP II, were used. Uranyl acetate was used as stain. A total of 58 molecule images (29 unlabelled, 29 labelled with antl-LPII Fab) showing the top view were digitized in the microdensitometer with a sampling distance of 50μ corresponding to 6.25nm.

Site-Specific Protein Covalent Attachment to Nanotubes and Its Electronic Impact on Single Molecule Function

2019 ◽  
Author(s):  
Adam Beachey ◽  
Harley Worthy ◽  
William David Jamieson ◽  
Suzanne Thomas ◽  
Benjamin Bowen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Protein ◽  
Functional Integration ◽  
Attachment Site ◽  
Covalent Attachment ◽  
Great Promise ◽  
Functional Coupling ◽  
Phenyl Azide ◽  
Electronic Impact ◽  
Protein Attachment

<p>Functional integration of proteins with carbon-based nanomaterials such as nanotubes holds great promise in emerging electronic and optoelectronic applications. Control over protein attachment poses a major challenge for consistent and useful device fabrication, especially when utilizing single/few molecule properties. Here, we exploit genetically encoded phenyl azide photochemistry to define the direct covalent attachment of three different proteins, including the fluorescent protein GFP, to carbon nanotube side walls. Single molecule fluorescence revealed that on attachment to SWCNTs GFP’s fluorescence changed in terms of intensity and improved resistance to photobleaching; essentially GFP is fluorescent for much longer on attachment. The site of attachment proved important in terms of electronic impact on GFP function, with the attachment site furthest from the functional center having the larger effect on fluorescence. Our approach provides a versatile and general method for generating intimate protein-CNT hybrid bioconjugates. It can be potentially applied easily to any protein of choice; attachment position and thus interface characteristics with the CNT can easily be changed by simply placing the phenyl azide chemistry at different residues by gene mutagenesis. Thus, our approach will allow consistent construction and modulate functional coupling through changing the protein attachment position.</p>

RESULTS OF POLARIZED LIGHT USE FOR STUDY OF STRESS-STRAIN STATE OF CORNEA IN PATHOLOGY OF EXTRAOCULAR MUSCLES

2020 ◽  
pp. 65-69
Author(s):  
Nataliia Mykhailivna Kovtun ◽  
Ihor Volodymyrovych Kaplin
Keyword(s):  
Interference Pattern ◽  
Optical Anisotropy ◽  
Extraocular Muscle ◽  
Strain State ◽  
Polarized Light ◽  
Attachment Site ◽  
Rectus Muscle ◽  
Extraocular Muscles ◽  
Asymmetric Distortion ◽  
Interference Picture

The parameters of interference patterns observed on the cornea in a polarized light in pathology of extraocular muscles were quantitatively evaluated. A study of the shape of 147 interference patterns observed on the cornea in a polarized light showed that the horizontal deviation of an eye in 56 % of cases was caused by the displacement of the attachment of the upper rectus muscle towards the inner rectus muscle. During illumination of the cornea of a live eye with polarized light, a specific interference pattern in the form of a rhombus formed by rainbow stripes (isochromes) is observed. It is established that such an interference pattern is the result of the influence of extraocular muscles on the cornea. The findings showed that the weakening of the force of one of muscles leads to asymmetric distortion of the shape of the interference rhombus, which is manifested with a reduced length of the corresponding section of the diagonal. On the contrary, as the force of the muscle increases, the corresponding section of the diagonal enhances. The displacement of the attachment site of direct extraocular muscle along the line of action is equivalent to a change in effort on its part, indicated appropriately by the shape of the interference rhombus. As the muscle moves away from the line of action, the angle of the interference rhombus is shifted away from the corresponding meridian. Key words: polarized light, optical anisotropy, cornea, extra−ocular muscles, interference picture.

scholarly journals Transformation-associated alterations in interactions between pre-B cells and fibronectin

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2311-2320 ◽  
Author(s):  
FM Lemoine ◽  
S Dedhar ◽  
GM Lima ◽  
CJ Eaves
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Attachment ◽  
Attachment Site ◽  
Receptor Antibodies

Abstract Marrow stromal elements produce as yet uncharacterized soluble growth factors that can stimulate the proliferation of murine pre-B cells, although close contact between these two cell types appears to ensure a better pre-B cell response. We have now shown that freshly isolated normal pre-B cells (ie, the B220+, surface mu- fraction of adult mouse bone marrow) adhere to fibronectin (FN) via an RGD cell-attachment site, as shown in a serum-free adherence assay, and they lose this functional ability on differentiation in vivo into B cells (ie, the B220+, surface mu+ fraction). Similarly, cells from an immortalized but stromal cell-dependent and nontumorigenic murine pre-B cell line originally derived from a Whitlock-Witte culture were also found to adhere to fibronectin (FN) via an RGD cell-attachment site. Moreover, in the presence of anti-FN receptor antibodies, the ability of this immortalized pre-B cell line to proliferate when co-cultured with a supportive stromal cell line (M2–10B4 cells) was markedly reduced (down to 30% of control). This suggests that pre-B cell attachment to FN on stromal cells may be an important component of the mechanism by which stromal cells stimulate normal pre-B cell proliferation and one that is no longer operative to control their more differentiated progeny. Two differently transformed pre-B cell lines, both of which are autocrine, stromal-independent, tumorigenic in vivo, and partially or completely differentiation-arrested at a very early stage of pre-B cell development, did not bind to FN. In addition, anti-FN receptor antibodies were much less effective in diminishing the ability of these tumorigenic pre-B cells to respond to M2–10B4 cell stimulation, which could still be demonstrated when the tumorigenic pre-B cells were co- cultured with M2–10B4 cells at a sufficiently low cell density. Analysis of cell surface molecules immunoprecipitated from both the nontumorigenic and tumorigenic pre-B cell lines by an anti-FN receptor antibody showed an increase in very late antigen (VLA) alpha chain(s) in both tumorigenic pre-B cell lines and a decrease in the beta 1 chain in one. Interestingly, all of the pre-B cell lines expressed similar amounts of messenger RNA for the beta 1 chain of the FN receptor. These results suggest that alteration of FN receptor expression on pre-B cells may represent a mechanism contributing to the outgrowth of leukemic pre-B cells with an autocrine phenotype and capable of stromal cell-independent, autonomous growth.

INTERACTION OF S PROTEIN/VITRONECTIN WITH CULTURED ENDOTHELIAL CELLS: PROMOTION OF ATTACHMENT AND SPECIFIC BINDING

1987 ◽  
Author(s):  
K T Preissner ◽  
E Anders ◽  
G Müller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Cell Attachment ◽  
Specific Binding ◽  
Umbilical Vein ◽  
Attachment Site ◽  
Cell Types ◽  
S Protein ◽  
Specific Association ◽  
Umbilical Vein Endothelial Cells ◽  
Adhesive Proteins

The interaction of the complement inhibitor S protein, which is identical to the serum spreading factor, vitronectin, with cultured human endothelial cells of macro- and microvas- cular origin was investigated. Purified S protein, coated for 2 h on polystyrene petri dishes, induced concentration- and time-dependent attachment and spreading of human umbilical vein endothelial cells (HUVEC) as well as human omental tissqe microvasular endothelial cells (HOTMEC) at 37°C. With 3 × 105 cells/ml (final concentration) more than 50% of the cells attached within 2 h incubation at 0.3 - 3 μg/ml S protein. The effect of S protein was specific, since only monospecific antibodies against S protein prevented attachment of cells, while antibodies against fibronectin, fibrinogen or von Wille-brand factor were uneffective. The pentapeptide Gly-Arg-Gly-Asp-Ser, which contains the cell-attachment site of these adhesive proteins including S protein, inhibited the activity of S protein to promote attachment of endothelial cells in a concentration-dependent fashion; at 200 μM peptide, less than 10% of the cells became attached. Direct binding of S protein to HUVEC and HOTMEC was studied with cells in suspension at a concentration of 1 × 106 cells/ml in the presence of 1% (w/v) human serum albumin and 1 mM CaCl2 and was maximal after 120 min. Both cell types bound S protein in a concentration-dependent fashion with an estimated dissociation constant KD=0.2pM. More than 80% of bound radiolabelled S protein was displaced by unlabelled S protein, whereas binding was reduced to about 50% by the addition in excess of either fibronectin, fibrinogen, von Willebrand factor or the pentapeptide. These findings provide evidence for the specific association of S protein with endothelial cells, ultimately leading to attachment and spreading of cells. Although the promotion of attachment was highly specific for S protein, other adhesive proteins than S protein, also known to associate with endothelial cells, may in part compete with direct S protein binding.

scholarly journals Glochidial infection by the endangered Margaritifera margaritifera (Mollusca) increased survival of salmonid host (Pisces) during experimental Flavobacterium disease outbreak

2021 ◽  
Author(s):  
M. Motiur R. Chowdhury ◽  
Amitav Roy ◽  
Kalle Auvinen ◽  
Katja Pulkkinen ◽  
Hanna Suonia ◽  
...  
Keyword(s):  
Post Infection ◽  
Current Knowledge ◽  
Virulent Strain ◽  
Freshwater Mussels ◽  
Attachment Site ◽  
Disease Outbreak ◽  
Host Susceptibility ◽  
Control Fish ◽  
Margaritifera Margaritifera ◽  
Past Infection

AbstractCo-infections are common in host-parasite interactions, but studies about their impact on the virulence of parasites/diseases are still scarce. The present study compared mortality induced by a fatal bacterial pathogen, Flavobacterium columnare between brown trout infected with glochidia from the endangered freshwater pearl mussel, Margaritifera margaritifera, and uninfected control fish during the parasitic period and after the parasitic period (i.e. glochidia detached) in a laboratory experiment. We hypothesised that glochidial infection would increase host susceptibility to and/or pathogenicity of the bacterial infection. We found that the highly virulent strain of F. columnare caused an intense disease outbreak, with mortality reaching 100% within 29 h. Opposite to the study hypothesis, both fresh ongoing and past infection (14 months post-infection) with glochidia prolonged the fish host’s survival statistically significantly by 1 h compared to the control fish (two-way ANOVA: fresh-infection, F1, 82 = 7.144, p = 0.009 and post-infection, F1, 51 = 4.227, p = 0.044). Furthermore, fish survival time increased with glochidia abundance (MLR: post-infection, t = 2.103, p = 0.045). The mechanism could be connected to an enhanced non-specific immunity or changed gill structure of the fish, as F. columnare enters the fish body mainly via the gills, which is also the glochidia’s attachment site. The results increase current knowledge about the interactions between freshwater mussels and their (commercially important) fish hosts and fish pathogens and also emphasise the importance of (unknown) ecosystem services (e.g., protection against pathogens) potentially associated with imperilled freshwater mussels.

scholarly journals Host-Selected Amino Acid Changes at the Sialic Acid Binding Pocket of the Parvovirus Capsid Modulate Cell Binding Affinity and Determine Virulence

2006 ◽  
Vol 80 (3) ◽  
pp. 1563-1573 ◽  
Author(s):  
Alberto López-Bueno ◽  
Mari-Paz Rubio ◽  
Nathan Bryant ◽  
Robert McKenna ◽  
Mavis Agbandje-McKenna ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Sialic Acid ◽  
Parallel Evolution ◽  
Attachment Site ◽  
Fibroblast Cell ◽  
Scid Mice ◽  
Wild Type ◽  
Large Plaque ◽  
Capsid Sequence

ABSTRACT The role of receptor recognition in the emergence of virulent viruses was investigated in the infection of severe combined immunodeficient (SCID) mice by the apathogenic prototype strain of the parvovirus minute virus of mice (MVMp). Genetic analysis of isolated MVMp viral clones (n = 48) emerging in mice, including lethal variants, showed only one of three single changes (V325M, I362S, or K368R) in the common sequence of the two capsid proteins. As was found for the parental isolates, the constructed recombinant viruses harboring the I362S or the K368R single substitutions in the capsid sequence, or mutations at both sites, showed a large-plaque phenotype and lower avidity than the wild type for cells in the cytotoxic interaction with two permissive fibroblast cell lines in vitro and caused a lethal disease in SCID mice when inoculated by the natural oronasal route. Significantly, the productive adsorption of MVMp variants carrying any of the three mutations selected through parallel evolution in mice showed higher sensitivity to the treatment of cells by neuraminidase than that of the wild type, indicating a lower affinity of the viral particle for the sialic acid component of the receptor. Consistent with this, the X-ray crystal structure of the MVMp capsids soaked with sialic acid (N-acetyl neuraminic acid) showed the sugar allocated in the depression at the twofold axis of symmetry (termed the dimple), immediately adjacent to residues I362 and K368, which are located on the wall of the dimple, and approximately 22 Å away from V325 in a threefold-related monomer. This is the first reported crystal structure identifying an infectious receptor attachment site on a parvovirus capsid. We conclude that the affinity of the interactions of sialic-acid-containing receptors with residues at or surrounding the dimple can evolutionarily regulate parvovirus pathogenicity and adaptation to new hosts.

scholarly journals Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase

1994 ◽  
Vol 301 (2) ◽  
pp. 577-583 ◽  
Author(s):  
K Oda ◽  
J Cheng ◽  
T Saku ◽  
N Takami ◽  
M Sohda ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Endoplasmic Reticulum ◽  
Aspartic Acid ◽  
Chimeric Protein ◽  
Attachment Site ◽  
In Vitro Translation ◽  
Placental Alkaline Phosphatase ◽  
Wild Type ◽  
Aspartic Acid Residue

Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.

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