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1.
J Plant Res ; 132(5): 655-665, 2019 Sep.
Article in English | MEDLINE | ID: mdl-31289959

ABSTRACT

XSP25, previously shown to be the most abundant hydrophilic protein in xylem sap of Populus nigra in winter, belongs to a secretory protein family in which the arrangement of basic and acidic amino acids is conserved between dicotyledonous and monocotyledonous species. Its gene expression was observed at the same level in roots and shoots under long-day conditions, but highly induced under short-day conditions and at low temperatures in roots, especially in endodermis and xylem parenchyma in the root hair region of Populus trichocarpa, and its protein level was high in dormant buds, but not in roots or branches. Addition of recombinant PtXSP25 protein mitigated the denaturation of lactate dehydrogenase by drying, but showed only a slight effect on that caused by freeze-thaw cycling. Recombinant PtXSP25 protein also showed ice recrystallization inhibition activity to reduce the size of ice crystals, but had no antifreezing activity. We suggest that PtXSP25 protein produced in shoots and/or in roots under short-day conditions and at non-freezing low temperatures followed by translocation via xylem sap to shoot apoplast may protect the integrity of the plasma membrane and cell wall functions from freezing and drying damage in winter environmental conditions.


Subject(s)
Plant Proteins/genetics , Populus/physiology , Stress, Physiological/genetics , Desiccation , Freezing , Plant Proteins/metabolism , Plant Shoots/physiology , Populus/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Seasons , Xylem/physiology
2.
Langmuir ; 35(23): 7452-7458, 2019 06 11.
Article in English | MEDLINE | ID: mdl-30119611

ABSTRACT

Ice formation remains central to our understanding of the effects of low temperatures on the biological response of cells and tissues. The formation of ice inside of cells and the net increase in crystal size due to recrystallization during thawing is associated with a loss of cell viability during cryopreservation. Because small-molecule ice recrystallization inhibitors (IRIs) can control the growth of extracellular ice, we sought to investigate the ability of two aryl-glycoside-based IRIs to permeate into cells and control intracellular ice recrystallization. An interrupted graded freezing technique was used to evaluate the IRI permeation into human red blood cells (RBCs) and mitigate cell damage during freezing and thawing. The effect of IRIs on the intracellular growth of ice crystals in human umbilical vein endothelial cells (HUVECs) was visualized in real time under different thawing conditions using fluorescence cryomicroscopy. Adding an aryl glycoside to 15% glycerol significantly increased post-thaw RBC integrity by up to 55% during slow cooling compared with the 15%-glycerol-only control group. The characteristics of the cryobiological behavior of the RBCs subjected to the interrupted graded freezing suggest that the aryl-glycoside-based IRI is internalized into the RBCs. HUVECs treated with the IRIs were shown to retain a large number of small ice crystals during warming to high subzero temperatures and demonstrated a significant inhibition of intracellular ice recrystallization. Under slow thawing conditions, the aryl glycoside IRI p-bromophenyl-ß-d-glucoside was shown to be most effective at inhibiting intracellular ice recrystallization. We demonstrate that IRIs are capable of internalizing into cells, altering the cryobiological response of cells to slow and rapid freezing and controlling intracellular ice recrystallization during freezing. We conclude that IRIs have tremendous potential as cryoprotectants for the preservation of cells and tissues at high subzero temperatures.


Subject(s)
Ice , Intracellular Space/drug effects , Intracellular Space/metabolism , Small Molecule Libraries/pharmacology , Crystallization , Human Umbilical Vein Endothelial Cells/cytology , Humans , Permeability
3.
Carbohydr Res ; 439: 1-8, 2017 Feb 01.
Article in English | MEDLINE | ID: mdl-28011438

ABSTRACT

Cryopreservation is an important technique employed for the storage and preservation of biological tissues and cells. The limited effectiveness and significant toxicity of conventionally-used cryoprotectants, such as DMSO, have prompted efforts toward the rational design of less toxic alternatives, including carbohydrate-based surfactants. In this paper, we report the modular synthesis and ice recrystallization inhibition (IRI) activity of a library of variably substituted, carbohydrate-based fluorosurfactants. Carbohydrate-based fluorosurfactants possessed a variable mono- or disaccharide head group appended to a hydrophobic fluoroalkyl-substituted azobenzene tail group. Light-addressable fluorosurfactants displayed weak-to-moderate IRI activity that could be tuned through selection of carbohydrate head group, position of the trifluoroalkyl group on the azobenzene ring, and isomeric state of the azobenzene tail fragment.


Subject(s)
Cryopreservation , Cryoprotective Agents/chemistry , Ice/analysis , Small Molecule Libraries/chemistry , Surface-Active Agents/chemistry , Azo Compounds/chemistry , Cryoprotective Agents/chemical synthesis , Crystallization , Disaccharides/chemistry , Halogenation , Light , Molecular Structure , Monosaccharides/chemistry , Photochemical Processes , Small Molecule Libraries/chemical synthesis , Structure-Activity Relationship , Surface-Active Agents/chemical synthesis
4.
Sci Rep ; 6: 23619, 2016 Mar 29.
Article in English | MEDLINE | ID: mdl-27021850

ABSTRACT

During cryopreservation, ice recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures. In North America, cryopreservation of human red blood cells (RBCs) utilizes high concentrations of glycerol. RBC units frozen under these conditions must be subjected to a time-consuming deglycerolization process after thawing in order to remove the glycerol to <1% prior to transfusion thus limiting the use of frozen RBC units in emergency situations. We have identified several low molecular mass ice recrystallization inhibitors (IRIs) that are effective cryoprotectants for human RBCs, resulting in 70-80% intact RBCs using only 15% glycerol and slow freezing rates. These compounds are capable of reducing the average ice crystal size of extracellular ice relative to a 15% glycerol control validating the positive correlation between a reduction in ice crystal size and increased post-thaw recovery of RBCs. The most potent IRI from this study is also capable of protecting frozen RBCs against the large temperature fluctuations associated with transient warming.


Subject(s)
Blood Preservation/methods , Cryopreservation/methods , Cryoprotective Agents/pharmacology , Erythrocytes/drug effects , Hemolysis/drug effects , Small Molecule Libraries/pharmacology , Cell Survival/drug effects , Cold Temperature , Cryoprotective Agents/chemistry , Crystallization , Erythrocytes/metabolism , Freezing , Glycerol/pharmacology , Humans , Ice , Microscopy, Fluorescence/methods , Microscopy, Video/methods , Molecular Structure , Small Molecule Libraries/chemistry
5.
Cryobiology ; 70(2): 79-89, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25595636

ABSTRACT

Most antifreeze proteins (AFPs) exhibit two types of "antifreeze activity" - thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity. The mechanism of TH activity has been studied in depth and is the result of an adsorption of AFPs to the surface of ice with an ice-binding face (IBF). In contrast, the mechanism of ice recrystallization and its inhibition is considerably less understood. In this paper, we examine several different antifreeze proteins, glycoproteins and mutants of the Lolium perenne AFP (LpAFP) to understand how IRI activity is modulated independently of TH activity. This study also examines the ability of the various AF(G)Ps to protect HepG2 cells from cryoinjury. Post-thaw cell viabilities are correlated to TH, IRI activity as well as dynamic ice shaping ability and single ice crystal growth progressions. While these results demonstrate that AF(G)Ps are ineffective as cryoprotectants, they emphasize how ice crystal habit and most importantly, ice growth progression affect HepG2 cell survival during cryopreservation.


Subject(s)
Antifreeze Proteins/chemistry , Cell Survival/physiology , Cryopreservation , Cryoprotective Agents/chemistry , Glycoproteins/chemistry , Adsorption , Animals , Crystallization , Fish Proteins/chemistry , Hep G2 Cells , Humans , Ice , Lolium/chemistry , Protein Binding
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