Cytological Preservation Using 1,5-Difluro 2, 4-Dinitrobenzene

1967 ◽  
Vol 25 ◽  
pp. 146-147
Author(s):  
Delbert E. Philpott
Keyword(s):  
Hydrogen Atom ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Biological Material ◽  
Free Amino ◽  
Positive Charge ◽  
Cross Linking ◽  
Amino Groups ◽  
Stabilization Of Membranes ◽  
And Cartilage

A protein cross-linking agent, 1,5-difluro 2,4-dinitrobenzene (DFF) has been used for the preservation of red blood cells, liver and cartilage. This difluro compound is known for Its cross-linking ability which results in the stabilization of membranes by reacting with the free amino groups; this removes the positive charge by removal of a hydrogen atom. The double site on the difluro compound can react if the two amino groups are 5Å or more apart. It also reacts with sulfhydryl, tyrosyl or histidyl groups to form stable dinitro phenyl derivatives. The present studies indicate that preservation extends into the ultrastructure level and demonstrates a method for holding biological material for prolonged periods.

Effect of cross-linking agents on insulin associated responses in adipocytes

1982 ◽  
Vol 60 (10) ◽  
pp. 987-1000 ◽  
Author(s):  
H. Joseph Goren ◽  
C. Ronald Kahn
Keyword(s):  
Insulin Receptor ◽  
Glucose Transport ◽  
Free Amino ◽  
Glucose Oxidation ◽  
Insulin Binding ◽  
Cross Linking ◽  
Amino Group ◽  
Amino Groups ◽  
Disuccinimidyl Suberate ◽  
Reactive Groups

The effect of 10 bifunctional cross-linking agents and four monofunctional analogues was studied on isolated adipocytes. [125I]Insulin binding and degradation, basal and insulin-stimulated glucose oxidation, and 3-O-methyl glucose uptake were measured. Two cross-linkers, which possess succinimide ester residues (disuccinimidyl suberate and dithiobis(succinimidyl propionate)) and react selectively with amino groups, appeared to react relatively specifically with the insulin receptor. Both produced a slight stimulation of basal glucose transport and metabolism, a marked inhibition of insulin-stimulated glucose transport and metabolism, and a marked decrease in insulin binding. Pretreatment of cells with unlabelled insulin partially blocked the effect of disuccinimidyl suberate, and as has been previously shown, disuccinimidyl suberate cross-linked insulin to its receptor. A monofunctional analogue of these compounds was 100-fold less active in altering cellular metabolic activity. Bisimidates, such as dimethyl suberimidate, dimethyl adipimidate, and dimethyl dithiobispropionimidate, also react with free amino groups but are more hydrophilic. These agents produced similar effects on glucose oxidation as the succinimide esters, but had little or no effect on insulin binding. The effects of these agents are not blocked by insulin and they do not cross-link insulin to its receptor. Mixed bifunctional reagents containing either a succinimide ester or an imidate and a group which reacts with thiols produced effects similar to the cross-linkers containing two succinimide groups or bisimidates, respectively. The bifunctional arylating agents difluorodinitrobenzene and bis(fluoronitrophenyl)sulfone produce marked effects on insulin binding and glucose oxidation at micromolar concentrations, but the monofunctional analogue fluorodinitrobenzene is almost equally active suggesting that with these compounds chemical modifications and not cross-linking was important. With neither the mixed bifunctional reagents, nor the arylating agents, did insulin pretreatment alter the effect of cross-linker and none of these agents cross-linked [125I]insulin to its receptor. These data suggest that the insulin receptor possesses a free amino group in a hydrophobic environment in its active site. A reactive amino group in a hydrophilic environment as well as other reactive groups are also present in some component of the insulin receptor–effector complex. Chemical modification or cross-linking of these functional groups results in an inhibition or mimicking of insulin action. Further study will be required to identify the exact locus of these sites.

scholarly journals Free amino acids in plasma, red blood cells, polymorphonuclear leukocytes, and muscle in normal and uraemic children

2002 ◽  
Vol 17 (3) ◽  
pp. 413-421 ◽  
Author(s):  
Alberto Canepa ◽  
José Carolino Divino Filho ◽  
Alberto Gutierrez ◽  
Alba Carrea ◽  
Ann‐Marie Forsberg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Free Amino Acids ◽  
Free Amino ◽  
Polymorphonuclear Leukocytes ◽  
Uraemic Children

scholarly journals Variation of Free Amino Acid Concentration in Red Blood Cells of Horse

1984 ◽  
Vol 55 (5) ◽  
pp. 307-314
Author(s):  
Seiki WATANABE ◽  
Kei HANZAWA ◽  
Sachiko KANDA ◽  
Michinari YOKOHAMA ◽  
Kazushige MOGI
Keyword(s):  
Amino Acid ◽  
Red Blood Cells ◽  
Free Amino Acid ◽  
Acid Concentration ◽  
Blood Cells ◽  
Free Amino ◽  
Amino Acid Concentration

In vitro phagocytosis of carrier mouse red blood cells is increased by Band 3 cross-linking or diamide treatment

2001 ◽  
Vol 34 (3) ◽  
pp. 143 ◽  
Author(s):  
José A. Jordán ◽  
F. Javier Alvarez ◽  
L. Alfredo Lotero ◽  
Angel Herráez ◽  
José C. Díez ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Band 3 ◽  
Cross Linking

scholarly journals THE EFFECT OF FIXATION WITH FORMALDEHYDE AND GLUTARALDEHYDE ON THE COMPOSITION OF PHOSPHOLIPIDS EXTRACTABLE FROM RAT HYPOTHALAMUS

1969 ◽  
Vol 17 (7) ◽  
pp. 482-486 ◽  
Author(s):  
R. C. ROOZEMOND
Keyword(s):  
Vinyl Ether ◽  
Free Amino ◽  
Tissue Extract ◽  
Cross Linking ◽  
Hydrolytic Cleavage ◽  
Amino Groups ◽  
Ether Bond ◽  
Rat Hypothalamus ◽  
The Cross

Fixation of rat hypothalamus in 4% formaldehyde + 1% CaCl2 for 24 hr at 0°C reduced the amount of extractable ethanolamine phospholipids considerably. This decrease may be caused by hydrolytic cleavage of the vinyl ether bond in phosphatidalethanolamine and by reaction of formaldehyde with the free amino groups in ethanolamine phospholipids. Evidence is presented that the reaction with free amino groups may be the main cause for the decrease of extractable phospholipids when dealing with a fixative that contains glutaraldehyde and is buffered at pH 7. In this case no phosphatidylserine and hardly any phosphatidylethanolamine could be detected in the tissue extract. It is presumed that these phospholipids are fixed to proteins by the cross-linking action of glutaraldehyde involving free amino groups of proteins and phospholipids.

scholarly journals Glutaraldehyde fixation of sodium transport in dog red blood cells.

1984 ◽  
Vol 84 (5) ◽  
pp. 789-803 ◽  
Author(s):  
J C Parker
Keyword(s):  
Red Blood Cells ◽  
Cell Volume ◽  
Blood Cells ◽  
Cross Linking ◽  
Glutaraldehyde Fixation ◽  
Cell Shrinkage ◽  
Transport Pathway ◽  
Turn On ◽  
Two Phases ◽  
Chemical Cross Linking

The large increase in passive Na flux that occurs when dog red blood cells are caused to shrink is amiloride sensitive and inhibited when Cl is replaced by nitrate or thiocyanate. Activation and deactivation of this transport pathway by manipulation of cell volume is reversible. Brief treatment of the cells with 0.01-0.03% glutaraldehyde can cause the shrinkage-activated transporter to become irreversibly activated or inactivated, depending on the volume of the cells at the time of glutaraldehyde exposure. Thus, if glutaraldehyde is applied when the cells are shrunken, the amiloride-sensitive Na transporter is activated and remains so regardless of subsequent alterations in cell volume. If the fixative is applied to swollen cells, no amount of subsequent shrinkage will turn on the Na pathway. In its fixed state, the activated transporter is fully amiloride sensitive, but it is no longer inhibited when Cl is replaced by thiocyanate. The action of glutaraldehyde thus allows one to dissect the response to cell shrinkage into two phases. Activation of the pathway is affected by anions and is not prevented by amiloride. Once activated and fixed, the anion requirement disappears. Amiloride inhibits movement of Na through the activated transporter. These experiments demonstrate how a chemical cross-linking agent may be used to study the functional properties of a regulable transport pathway.

scholarly journals Avidin attachment to biotinylated erythrocytes induces homologous lysis via the alternative pathway of complement

Blood ◽  
1991 ◽  
Vol 78 (10) ◽  
pp. 2611-2618
Author(s):  
VR Muzykantov ◽  
MD Smirnov ◽  
GP Samokhin
Keyword(s):  
Blood Cells ◽  
Surface Density ◽  
Positive Charge ◽  
Alternative Pathway ◽  
Cross Linking ◽  
Restriction Factor ◽  
Agglutination Titer ◽  
Alternative Pathway Of Complement ◽  
The Cross

Noncovalent attachment of avidin to the membrane of prebiotinylated red blood cells (RBCs) induces lysis via the alternative pathway of complement (APC). Lysis is not species-dependent; RBCs from humans, rabbits, rats, and sheep were lysed with both autologous and all heterologous sera. Both biotinylated and native cells were not lysed. Lysis was observed at an avidin surface density of about 10(5) molecules per cell. Acylation of avidin prevents lysis and decreases the positive charge of the avidin. Lysis depends on the length of the cross-linking agent used for the biotin attachment to the membrane. An increase in the length of the cross-linking agent was accompanied by an enhancement of the lysis and the agglutination titer of biotinylated RBCs in a solution of avidin. It is suggested that avidin attachment induces some transformations of the cell membrane that lead to the conversion from “APC nonactivator” cells to “APC activator” cells. The interaction of avidin with membrane APC-restrictors (decay-accelerating factors, type 1 receptor for complement, homologous restriction factor, and others), the charge of avidin, and its cross-linking ability in lysis are discussed. It is proposed that membrane rearrangement induced by multipoint avidin attachment to biotinylated membrane is the main reason for avidin-induced elimination of APC restriction.

scholarly journals Schistosomula of Schistosoma mansoni use lysophosphatidylcholine to lyse adherent human red blood cells and immobilize red cell membrane components.

1986 ◽  
Vol 103 (3) ◽  
pp. 819-828 ◽  
Author(s):  
D E Golan ◽  
C S Brown ◽  
C M Cianci ◽  
S T Furlong ◽  
J P Caulfield
Keyword(s):  
Membrane Proteins ◽  
Schistosoma Mansoni ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Transmembrane Protein ◽  
Band 3 ◽  
Cross Linking ◽  
Lateral Mobility ◽  
Human Red Blood Cells ◽  
Lipid Probe

Human red blood cells (RBCs) adhere to and are lysed by schistosomula of Schistosoma mansoni. We have investigated the mechanism of RBC lysis by comparing the dynamic properties of transmembrane protein and lipid probes in adherent ghost membranes with those in control RBCs and in RBCs treated with various membrane perturbants. Fluorescence photobleaching recovery was used to measure the lateral mobility of two integral membrane proteins, glycophorin and band 3, and two lipid analogues, fluorescein phosphatidylethanolamine (Fl-PE) and carbocyanine dyes, in RBCs and ghosts adherent to schistosomula. Adherent ghosts manifested 95-100% immobilization of both membrane proteins and 45-55% immobilization of both lipid probes. In separate experiments, diamide-induced cross-linking of RBC cytoskeletal proteins slowed transmembrane protein diffusion by 30-40%, without affecting either transmembrane protein fractional mobility or lipid probe lateral mobility. Wheat germ agglutinin- and polylysine-induced cross-linking of glycophorin at the extracellular surface caused 80-95% immobilization of the transmembrane proteins, without affecting the fractional mobility of the lipid probe. Egg lysophosphatidylcholine (lysoPC) induced both lysis of RBCs and a concentration-dependent decrease in the lateral mobility of glycophorin, band 3, and Fl-PE in ghost membranes. At a concentration of 8.4 micrograms/ml, lysoPC caused a pattern of protein and lipid immobilization in RBC ghosts identical to that in ghosts adherent to schistosomula. Schistosomula incubated with labeled palmitate released lysoPC into the culture medium at a rate of 1.5 fmol/h per 10(3) organisms. These data suggest that lysoPC is transferred from schistosomula to adherent RBCs, causing their lysis.

scholarly journals Avidin attachment to biotinylated erythrocytes induces homologous lysis via the alternative pathway of complement

Blood ◽  
1991 ◽  
Vol 78 (10) ◽  
pp. 2611-2618 ◽  
Author(s):  
VR Muzykantov ◽  
MD Smirnov ◽  
GP Samokhin
Keyword(s):  
Blood Cells ◽  
Surface Density ◽  
Positive Charge ◽  
Alternative Pathway ◽  
Cross Linking ◽  
Restriction Factor ◽  
Agglutination Titer ◽  
Alternative Pathway Of Complement ◽  
The Cross

Abstract Noncovalent attachment of avidin to the membrane of prebiotinylated red blood cells (RBCs) induces lysis via the alternative pathway of complement (APC). Lysis is not species-dependent; RBCs from humans, rabbits, rats, and sheep were lysed with both autologous and all heterologous sera. Both biotinylated and native cells were not lysed. Lysis was observed at an avidin surface density of about 10(5) molecules per cell. Acylation of avidin prevents lysis and decreases the positive charge of the avidin. Lysis depends on the length of the cross-linking agent used for the biotin attachment to the membrane. An increase in the length of the cross-linking agent was accompanied by an enhancement of the lysis and the agglutination titer of biotinylated RBCs in a solution of avidin. It is suggested that avidin attachment induces some transformations of the cell membrane that lead to the conversion from “APC nonactivator” cells to “APC activator” cells. The interaction of avidin with membrane APC-restrictors (decay-accelerating factors, type 1 receptor for complement, homologous restriction factor, and others), the charge of avidin, and its cross-linking ability in lysis are discussed. It is proposed that membrane rearrangement induced by multipoint avidin attachment to biotinylated membrane is the main reason for avidin-induced elimination of APC restriction.

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