scholarly journals Fixation of Thy-1 in nervous tissue for immunohistochemistry: a quantitative assessment of the effect of different fixation conditions upon retention of antigenicity and the cross-linking of Thy-1.

1983 ◽  
Vol 31 (2) ◽  
pp. 263-274 ◽  
Author(s):  
R J Morris ◽  
P C Barber
Keyword(s):  
Monoclonal Antibody ◽  
Antigenic Determinant ◽  
Sodium Dodecyl ◽  
Rabbit Antiserum ◽  
Sodium Deoxycholate ◽  
Antigenic Determinants ◽  
Cross Linking ◽  
Cell Surface Antigens ◽  
Vascular Perfusion ◽  
Fixed Tissue

It has proved difficult to obtain good immunohistochemical localization of cell surface antigens in nerve for a number of reasons, prominent among which are problems of fixing this class of molecule without destroying their antigenicity. In the course of developing a fixation procedure suitable for one such antigen. Thy-1, we have quantitatively assessed the effect of different fixation parameters upon the retention of Thy-1 antigenicity and upon the extent of cross-linking of the antigen in the tissue. The former was measured using radioimmunoassays adapted for membrane antigens in fixed tissue, the latter by measuring the proportion of antigen rendered insoluble to the detergent, sodium deoxycholate, and by examining the size of the antigen on sodium dodecyl sulfate--polyacrylamide gels. These approaches demonstrated that minor modifications of the standard vascular perfusion fixation of brain, using both glutaraldehyde and paraformaldehyde, were sufficient to fix the Thy-1 molecule, and at the same time substantially spare its antigenicity. In this study we measured Thy-1 using both a conventional rabbit antiserum and a mouse monoclonal antibody to the Thy-1.1 antigenic determinant. The multiple antigenic determinants recognized by the rabbit antibodies were cumulatively more resistant to fixation than the single antigenic determinant recognized by the monoclonal antibody.

Monoclonal antibody recognizing gp80, a membrane glycoprotein implicated in intercellular adhesion of Dictyostelium discoideum

1983 ◽  
Vol 3 (5) ◽  
pp. 863-870
Author(s):  
B A Murray ◽  
H L Niman ◽  
W F Loomis
Keyword(s):  
Monoclonal Antibody ◽  
Dictyostelium Discoideum ◽  
Posttranslational Modification ◽  
Rabbit Antiserum ◽  
Intercellular Adhesion ◽  
Blocking Effect ◽  
Antigenic Determinants ◽  
Membrane Glycoprotein ◽  
Developmentally Regulated ◽  
Contact Sites

WE have raised a monoclonal antibody, designated E28D8, which reacts with an 80,000-dalton membrane glycoprotein (gp80) of Dictyostelium discoideum. gp80 has been implicated in the formation of the EDTA-resistant adhesions ("contact sites A") which appear during development. The monoclonal antibody reacted with other developmentally regulated proteins of D. discoideum, confirming previous results indicating the presence of common antigenic determinants recognized by polyclonal rabbit antibodies directed to gp80. Periodate sensitivity of the determinants suggests that carbohydrate may be necessary for reactivity. Thus, the determinant recognized by E28D8 may result from a posttranslational modification common to a number of proteins. Some of the proteins that carry the determinant were preferentially localized to posterior cells in slugs. Monoclonal antibody E28D8 did not inhibit contact-sites-A-mediated intercellular adhesion. However, gp80 affinity purified on immobilized monoclonal antibody was able to neutralize the adhesion-blocking effect of rabbit antiserum to gp80. Although gp80 itself may not be essential for cell-cell adhesion, it appears to carry the determinants associated with adhesion.

scholarly journals Towards uterus tissue engineering: a comparative study of sheep uterus decellularisation

2020 ◽  
Vol 26 (3) ◽  
pp. 167-178 ◽  
Author(s):  
T T Tiemann ◽  
A M Padma ◽  
E Sehic ◽  
H Bäckdahl ◽  
M Oltean ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Vascular Perfusion ◽  
Uterus Transplantation ◽  
Live Donors ◽  
Triton X

Abstract Uterus tissue engineering may dismantle limitations in current uterus transplantation protocols. A uterine biomaterial populated with patient-derived cells could potentially serve as a graft to circumvent complicated surgery of live donors, immunosuppressive medication and rejection episodes. Repeated uterine bioengineering studies on rodents have shown promising results using decellularised scaffolds to restore fertility in a partially impaired uterus and now mandate experiments on larger and more human-like animal models. The aim of the presented studies was therefore to establish adequate protocols for scaffold generation and prepare for future in vivo sheep uterus bioengineering experiments. Three decellularisation protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularisation solutions used were based on 0.5% sodium dodecyl sulphate (Protocol 1) or 2% sodium deoxycholate (Protocol 2) or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100 (Protocol 3). The scaffolds were examined by histology, extracellular matrix quantification, evaluation of mechanical properties and the ability to support foetal sheep stem cells after recellularisation. We showed that a sheep uterus can successfully be decellularised while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (Protocol 2) were the most favourable treatment in our study based on quantifications. However, all scaffolds supported stem cells for 2 weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Hence, this study reports three valuable decellularisation protocols for future in vivo sheep uterus bioengineering experiments.

scholarly journals Monoclonal antibody recognizing gp80, a membrane glycoprotein implicated in intercellular adhesion of Dictyostelium discoideum.

1983 ◽  
Vol 3 (5) ◽  
pp. 863-870 ◽  
Author(s):  
B A Murray ◽  
H L Niman ◽  
W F Loomis
Keyword(s):  
Monoclonal Antibody ◽  
Dictyostelium Discoideum ◽  
Posttranslational Modification ◽  
Rabbit Antiserum ◽  
Intercellular Adhesion ◽  
Blocking Effect ◽  
Antigenic Determinants ◽  
Membrane Glycoprotein ◽  
Developmentally Regulated ◽  
Contact Sites

WE have raised a monoclonal antibody, designated E28D8, which reacts with an 80,000-dalton membrane glycoprotein (gp80) of Dictyostelium discoideum. gp80 has been implicated in the formation of the EDTA-resistant adhesions ("contact sites A") which appear during development. The monoclonal antibody reacted with other developmentally regulated proteins of D. discoideum, confirming previous results indicating the presence of common antigenic determinants recognized by polyclonal rabbit antibodies directed to gp80. Periodate sensitivity of the determinants suggests that carbohydrate may be necessary for reactivity. Thus, the determinant recognized by E28D8 may result from a posttranslational modification common to a number of proteins. Some of the proteins that carry the determinant were preferentially localized to posterior cells in slugs. Monoclonal antibody E28D8 did not inhibit contact-sites-A-mediated intercellular adhesion. However, gp80 affinity purified on immobilized monoclonal antibody was able to neutralize the adhesion-blocking effect of rabbit antiserum to gp80. Although gp80 itself may not be essential for cell-cell adhesion, it appears to carry the determinants associated with adhesion.

scholarly journals Characteristics of a copper-dependent cross-linking reaction between two forms of cytochrome P-450 in rabbit-liver microsomal membranes

1980 ◽  
Vol 187 (1) ◽  
pp. 227-237 ◽  
Author(s):  
P R McIntosh ◽  
R B Freedman
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Molar Ratio ◽  
Cross Linking ◽  
Thiol Compounds ◽  
Microsomal Membranes ◽  
The Cross ◽  
High Concentrations ◽  
Cytochrome P 450

1. In liver microsomal membranes from adult rabbits treated with beta-naphthoflavone, reaction with Cu2+ salts plus 1,10-phenanthroline leads to the cross-linking of the two specifically beta-naphthoflavone-inducible cytochrome P-450 species, form 4 and form 6, to form homo- and hetero-dimer species. 2. The cross-linking is not reversed by treatment with 2-mercaptoethanol, so that it can be observed conveniently and specifically on conventional reducing sodium dodecyl sulphate/polyacrylamide gels. 3. The reaction occurs rapidly, and significant cross-linking is observed after 30s at all temperatures from −10 to 40 degrees C. 4. The cross-linking can be brought about by Cu2+ alone at concentrations greater than 0.5 mM, but not by 1,10-phenanthroline alone; at low Cu2+ concentrations, 1,10-phenanthroline enhances the cross-linking reaction, but high concentrations of 1,10-phenanthroline are inhibitory; the optimal molar ratio of Cu2+ to 1,10-phenanthroline is 4:1.5. The effect of Cu2+ is not mimicked by Mn2+, Fe3+, Fe2+, Co2+, Ni2+, Zn2+ or Ag+; Cu+ is probably also ineffective. 6. The cross-linking reaction is inhibited by the prior addition of high concentrations of EDTA or thiol compounds, by sodium dodecyl sulphate at greater than or equal to 0.1% and by sodium deoxycholate and non-ionic detergents at greater than or equal to 1%; the reaction cannot be reversed by incubation with EDTA or with thiol compounds after reaction with cupric phenanthroline; the cross-linking reaction is not inhibited by prior treatment of microsomal membranes with N-ethylmaleimide. 7. The chemical nature of the cross-linking reaction is unknown, but it is most unlikely that it involves the formation of intermolecular disulphide bonds. 8. The great specificity of the reaction makes it a promising tool for the study of molecular interactions between cytochrome P-450 species in intact microsomal membranes.

A monoclonal antibody that neutralizes poliovirus by cross-linking virions.

1985 ◽  
Vol 54 (1) ◽  
pp. 7-13 ◽  
Author(s):  
A A Thomas ◽  
P Brioen ◽  
A Boeyé
Keyword(s):  
Monoclonal Antibody ◽  
Cross Linking

scholarly journals Vaccination-induced variation in the 140 kD merozoite surface antigen of Plasmodium knowlesi malaria.

1987 ◽  
Vol 165 (2) ◽  
pp. 359-367 ◽  
Author(s):  
F W Klotz ◽  
D E Hudson ◽  
H G Coon ◽  
L H Miller
Keyword(s):  
Monoclonal Antibodies ◽  
Malaria Infection ◽  
Rhesus Monkeys ◽  
Plasmodium Knowlesi ◽  
Rabbit Antiserum ◽  
Antigenic Determinants ◽  
Partial Protection ◽  
Erythrocyte Invasion ◽  
Merozoite Surface Antigen

Immunity to 143/140 kD schizont antigens of a monkey malaria, Plasmodium knowlesi, provides partial protection to lethal malaria infection in rhesus monkeys challenged with uncloned parasites. To determine the capacity of a cloned parasite to generate variants of the 143/140 kD antigens, immunized monkeys were challenged with a clone of P. knowlesi. Parasites recovered 8 d after inoculation with a cloned parasite retained the 143/140 kD antigens. Parasites recovered 30 d after challenge had undergone changes in the 143/140 kD antigens. Antibodies that block erythrocyte invasion in vitro of the inoculum parasites did not inhibit invasion of erythrocytes by two isolates recovered from the immunized monkeys. An isolate from one monkey recovered on day 30 contained clones expressing new 76/72 kD antigens reactive with rabbit antiserum against the 143/140 kD proteins, and other clones expressing no antigens crossreactive with antisera against the 143/140 kD proteins. An isolate from another monkey obtained 59 d after challenge expressed new antigens of 160/155, 115/113, and 87/85 kD. Using monoclonal antibodies, we found that epitopes were lost from the variant proteins, but we were unable to determine whether new epitopes had appeared. We conclude that clones of P. knowlesi can rapidly vary antigenic determinants on the 143/140 kD proteins in animals immunized with these antigens.

scholarly journals Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

1981 ◽  
Vol 197 (3) ◽  
pp. 629-636 ◽  
Author(s):  
J L McKenzie ◽  
A K Allen ◽  
J W Fabre
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Human Brain ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Antibody Affinity ◽  
Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

scholarly journals Mucin biosynthesis. Properties of a bovine tracheal mucin β-6-N-acetylglucosaminyltransferase

1985 ◽  
Vol 227 (2) ◽  
pp. 405-412 ◽  
Author(s):  
P W Cheng ◽  
W E Wingert ◽  
M R Little ◽  
R Wei
Keyword(s):  
Enzyme Activity ◽  
Freezing Point ◽  
Cetylpyridinium Chloride ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Gas Chromatography Mass Spectrometry ◽  
Tween 20 ◽  
Group A ◽  
Michaelis Constants ◽  
Triton X

We have characterized a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-β 1→3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5mM-MnCl2, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba2+, Mg2+ and Ca2+ could partially replace Mn2+, but Co2+, Fe2+, Cd2+ and Zn2+ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl beta-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the 14C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Gal beta 1→3(Glc-NAc beta 1→6)N-acetylgalactosaminitol by beta-hexosaminidase treatment, gas chromatography-mass spectrometry and 1H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis.

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