Biomedical subjects

J H Crowe

Publications and source records attributed to J H Crowe.

At least 37 records · Page 2Linked to original sources

PubMed1999

The internal calcium concentration of human platelets increases during chilling.

Human platelets must be stored at 22 degreesC in blood banks, because of the well-known phenomenon of cold-induced activation. When human platelets are chilled below room temperature, they undergo shape change and vesicle secretion that resembles physiological agonist-mediated activation. The trigger for the cascade of events leading to platelet activation at hypothermic temperatures is not known, although an increase in the internal calcium concentration ([Ca]i) due to passage of the platelet membranes through their thermotropic phase transition has been proposed. We report here that the fluorescent calcium-sensitive probe, Indo-1, has been used to estimate the internal calcium concentration of human platelets during a reduction in temperature from 20 degreesC to 5 degreesC at a rate of 0.5 degreesC/min. An increase on the order of 100 nM was recorded. Almost all of the increase in [Ca2+]i occurs during the chilling process, as incubation of platelets for 1 h at low temperature did not lead to a continued calcium concentration increase. The increase in [Ca2+]i during chilling is likely to be due to more than a single mechanism, but might include some release of the calcium stores from the dense tubule system. Loading platelets with the calcium chelator BAPTA (1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) dramatically reduced the increase in [Ca2+]i seen during chilling. Antifreeze glycoproteins (AFGPs) isolated from the blood serum of Antarctic fishes, which are known to protect platelets from cold-induced activation, did not eliminate the rise in [Ca2+]i during chilling, suggesting that signaling mechanisms are likely to be involved in cold-induced activation.

Antifreeze Proteins↗

PubMed1999

Are lipid phase transitions responsible for chilling damage in human platelets?

In previous studies we have proposed that the well-known chilling-induced activation of human blood platelets can be ascribed at least in part to a thermotropic phase transition in membrane lipids. The evidence that this is the case is reviewed and amplified in this review, followed by an examination of the available physical data concerning phase transitions in lipid mixtures that mimic the mixture found in platelet membranes. Assuming complete mixing at all temperatures and equal contributions of the members of the mixture to the phase transition, the lipid mixture found in platelets should give values for Tm ranging from about 1 degrees C to about 16 degrees C, depending on the isomers present in the mixture. (The former value is not in agreement with the observed Tm, but the latter is in excellent agreement.) However, examination of the phase diagram for a binary pair of lipids found in platelet membranes shows that ideal mixing almost certainly does not occur; instead of a linear phase diagram, a convex one was obtained. This shape for the phase diagram, which would displace Tm to an unexpectedly elevated temperature, is in agreement with previously published phase diagrams for mixtures of this type. The prediction, based on thermodynamic properties of lipids found in the platelets, is that Tm will be displaced upward in more complex mixtures of the composition found in platelets, a prediction that requires experimental testing.

Blood Platelets↗

PubMed1999

Lipid composition determines the effects of arbutin on the stability of membranes.

Arbutin (hydroquinone-beta-D-glucopyranoside) is an abundant solute in the leaves of many freezing- or desiccation-tolerant plants. Its physiological role in plants, however, is not known. Here we show that arbutin protects isolated spinach (Spinacia oleracea L.) thylakoid membranes from freeze-thaw damage. During freezing of liposomes, the presence of only 20 mM arbutin led to complete leakage of a soluble marker from egg PC (EPC) liposomes. When the nonbilayer-forming chloroplast lipid monogalactosyldiacylglycerol (MGDG) was included in the membranes, this leakage was prevented. Inclusion of more than 15% MGDG into the membranes led to a strong destabilization of liposomes during freezing. Under these conditions arbutin became a cryoprotectant, as only 5 mM arbutin reduced leakage from 75% to 20%. The nonbilayer lipid egg phosphatidylethanolamine (EPE) had an effect similar to that of MGDG, but was much less effective, even at concentrations up to 80% in EPC membranes. Arbutin-induced leakage during freezing was accompanied by massive bilayer fusion in EPC and EPC/EPE membranes. Twenty percent MGDG in EPC bilayers completely inhibited the fusogenic effect of arbutin. The membrane surface probes merocyanine 540 and 2-(6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosph ocholi ne (NBD-C(6)-HPC) revealed that arbutin reduced the ability of both probes to partition into the membranes. Steady-state anisotropy measurements with probes that localize at different positions in the membranes showed that headgroup mobility was increased in the presence of arbutin, whereas the mobility of the fatty acyl chains close to the glycerol backbone was reduced. This reduction, however, was not seen in membranes containing 20% MGDG. The effect of arbutin on lipid order was limited to the interfacial region of the membranes and was not evident in the hydrophobic core region. From these data we were able to derive a physical model of the perturbing or nonperturbing interactions of arbutin with lipid bilayers.

4-Chloro-7-nitrobenzofurazan↗

PubMed1998

Interactions of arbutin with dry and hydrated bilayers.

The glycosylated hydroquinone arbutin (4-hydroxyphenyl-beta-D-glucopyranoside) is abundant in certain resurrection plants, which can survive almost complete dehydration for prolonged periods. Little is known about the role of arbutin in vivo, but it is thought to contribute toward survival of the plants in the dry state. We have investigated the interactions of arbutin with model membranes under conditions of high and low hydration, as well as the possible participation of arbutin in carbohydrate glasses formed at low water contents. Retention of a trapped soluble marker inside large unilamellar vesicles and fusion of vesicles was monitored by fluorescence spectroscopy. Effects of arbutin on glass-transition temperatures and hydrated membrane phase-transition temperatures were measured by differential scanning calorimetry. The possible insertion of arbutin into membrane bilayers was estimated by following arbutin auto-fluorescence. Evidence is presented that arbutin does not change the glass-transition temperature of a sucrose/trehalose glass, but that arbutin does interact with hydrated membranes by insertion of the phenol moiety into the lipid bilayer. This interaction causes increased membrane leakage during air-drying by a mechanism other than vesicle-vesicle fusion. Implications of these effects on the dehydrated plant cells, as well as possible methods of obviating the damage, are discussed.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1998

Expression of high-affinity trehalose-H+ symport in Saccharomyces cerevisiae.

The expression of the high-affinity trehalose-H+ symport was investigated in various Saccharomyces cerevisiae strains and culture conditions. Previous kinetic studies of trehalose transport in yeast have revealed the existence of at least two different uptake mechanisms: a high-affinity trehalose-H+ symport activity repressed by glucose, and a constitutive low-affinity transport activity, a putative facilitated diffusion process. Exogenously added trehalose was not an inducer of the high-affinity transport activity, and a correlation between trehalose and maltose uptake by yeast cells was found. Our results indicate that the maltose-H+ symporters encoded by MAL11, MAL21, and MAL41 are not responsible for the trehalose transport activity. The analysis of both trehalose and maltose transport activities in wild-type and in laboratory strains with defined MAL genes showed that the trehalose-H+ symporter was under control of MAL regulatory genes. Our results also suggest that the recently characterized AGT1 gene of S. cerevisiae may encode the high-affinity trehalose-H+ symporter. During diauxic growth on glucose the transport activity was low during the first exponential phase of growth, increased as glucose was exhausted from the medium, and decreased again as the cells reached the late stationary phase. This pattern was coincident with that of the intracellular levels of trehalose. The strong correlation between these two parameters may be of physiological significance during adaptation of yeast cells to stress conditions.

Biological Transport↗

PubMed1998

The effects of chloroplast lipids on the stability of liposomes during freezing and drying.

Chloroplast thylakoids contain four classes of lipids, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol (cpPG). We have investigated the effects of these lipids on the stability of large unilamellar vesicles made from egg phosphatidylcholine (EPC), by substitution of different fractions of EPC in the membranes by the various chloroplast lipids. Damage to liposomes after freezing to - 18 degrees C was measured as carboxyfluorescein leakage or fusion between vesicles. The presence of all chloroplast lipids increased leakage. However, the maximum amount of leakage and the concentration dependence were dramatically different between the different lipids. Only SQDG induced vesicle fusion, while the non-bilayer lipid MGDG did not. The presence of MGDG in the membranes led to more leakage than the presence of another non-bilayer lipid, egg phosphatidylethanolamine (EPE). In EPE-containing liposomes, leakage was strongly associated with fusion. Combinations of different chloroplast lipids had an additive effect on leakage induced by freezing. Most of the leakage from galactolipid-containing vesicles occurred during the first 15 min of freezing at - 18 degrees C. After a 3 h incubation period, most leakage occurred between 0 degrees C and - 10 degrees C. Lowering the temperature to - 22 degrees C had only a small additional effect. Incubation of liposomes at - 10 degrees C in the presence of 2.5 M NaCl without ice crystallization, approximately the same concentration obtained by freezing to - 10 degrees C, resulted in very little leakage. Air drying of liposomes to low water contents resulted in massive leakage, both from pure EPC vesicles and from vesicles containing galactolipids. The latter vesicles showed more leakage at any given water content than EPC vesicles.

Chloroplasts↗

PubMed1998

Conformational and dynamic properties of a 14 residue antifreeze glycopeptide from Antarctic cod.

The 1H and 13C NMR spectra of a 14-residue antifreeze glycopeptide from Antarctic cod (Tetramatomnus borchgrevinki) containing two proline residues have been assigned. 13C NMR relaxation experiments indicate motional anisotropy of the peptide, with a tumbling time in water at 5 degrees C of 3-4 ns. The relaxation data and lack of long-range NOEs are consistent with a linear peptide undergoing significant segmental motion. However, extreme values of some coupling constants and strong sequential NOEs indicate regions of local order, which are most evident at the two ATPA subsequences. Similar spectroscopic properties were observed in the 16-residue analogue containing an Arg-Ala dipeptide added to the C-terminus. Molecular modeling also showed no evidence of long-range order, but the two ATPA subsequences were relatively well determined by the experimental data. These motifs were quite distinct from helical structures or beta turns commonly found in proteins, but rather resemble sections of an extended polyproline helix. Thus, the NMR data provide a description of the local order, which is of relevance to the mechanism of action of the antifreeze activity of the antifreeze glycopeptides as well as their ability to protect cells during hypothermic storage.

Amino Acid Sequence↗

PubMed1998

Effect of sugars on headgroup mobility in freeze-dried dipalmitoylphosphatidylcholine bilayers: solid-state 31P NMR and FTIR studies.

The effect of the carbohydrates trehalose, glucose, and hydroxyethyl starch (HES) on the motional properties of the phosphate headgroup of freeze-dried dipalmitoylphosphatidylcholine (DPPC) liposomes was studied by means of 31P NMR, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). The results show that trehalose, which is a strong glass former (Tg = 115 degreesC), elevates the onset of the lipid headgroup rotations and preserves some rotational mobility of the phosphate headgroups after cooling from the liquid-crystalline state. Glucose (Tg = 30 degreesC), a very effective depressant of the phase transition temperature of freeze-dried DPPC, markedly elevates the initiation of the temperature of headgroup rotations. On the other hand, the monosaccharide does not preserve the headgroup disordering when cooled from the liquid-crystalline state. These effects are consistent with formation of hydrogen bonds between the OH groups of the sugar and the polar headgroups of DPPC. They show, however, that hydrogen bonding is not sufficient for preservation of the dynamic properties of freeze-dried DPPC. HES, although a very good glass former (Tg > 110 degreesC), does not depress the phase transition temperature and affects only slightly the rotational properties of freeze-dried DPPC. This lack of effect of HES is associated with the absence of direct interactions with the lipid phosphates, as evidenced by the FTIR results. These data show that vitrification of the additive is not sufficient to affect the dynamic properties of dried DPPC.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1998

The role of vitrification in anhydrobiosis.

Numerous organisms are capable of surviving more or less complete dehydration. A common feature in their biochemistry is that they accumulate large amounts of disaccharides, the most common of which are sucrose and trehalose. Over the past 20 years, we have provided evidence that these sugars stabilize membranes and proteins in the dry state, most likely by hydrogen bonding to polar residues in the dry macromolecular assemblages. This direct interaction results in maintenance of dry proteins and membranes in a physical state similar to that seen in the presence of excess water. An alternative viewpoint has been proposed, based on the fact that both sucrose and trehalose form glasses in the dry state. It has been suggested that glass formation (vitrification) is in itself sufficient to stabilize dry biomaterials. In this review we present evidence that, although vitrification is indeed required, it is not in itself sufficient. Instead, both direct interaction and vitrification are required. Special properties have often been claimed for trehalose in this regard. In fact, trehalose has been shown by many workers to be remarkably (and sometimes uniquely) effective in stabilizing dry or frozen biomolecules, cells, and tissues. Others have not observed any such special properties. We review evidence here showing that trehalose has a remarkably high glass-transition temperature (Tg). It is not anomalous in this regard because it lies at the end of a continuum of sugars with increasing Tg. However, it is unusual in that addition of small amounts of water does not depress Tg, as in other sugars. Instead, a dihydrate crystal of trehalose forms, thereby shielding the remaining glassy trehalose from effects of the added water. Thus under less than ideal conditions such as high humidity and temperature, trehalose does indeed have special properties, which may explain the stability and longevity of anhydrobiotes that contain it. Further, it makes this sugar useful in stabilization of biomolecules of use in human welfare.

Adaptation, Biological↗

PubMed1998

Determination of time-dependent shape changes in red blood cells.

A kinetic method for detecting changes in red blood cell shape with time resolution on the order of seconds is described. The definition of shape index (SI) and the experimental conditions under which the measurements should be conducted are given, and the relationship between SI and morphological index, which is often used to quantitatively assess red blood cell morphology, is established. Further, we show that the method can be used to assess shape changes under a variety of experimental conditions known to cause major perturbations in the morphology of red blood cells (such as addition of lysolecithin or chlorpromazine) and to detect changes in shape during chemical fixation for observation with microscopy. We report here that the ability of glutaraldehyde to fix red blood cells without additional alteration in shape strongly depends on the morphology that is being fixed and the initial state of the cells.

Adult↗

PubMed1997

Stabilization of dry membranes by mixtures of hydroxyethyl starch and glucose: the role of vitrification.

R. P. Goodrich and co-workers (1989, U.S. Patent 4,874,690; 1992, Proc. Natl. Acad. Sci. USA 89,967-971) have reported that red blood cells can be preserved in the dry state by addition of mixtures of hydroxyethyl starch (HES) and glucose. More recently, Spieles and co-workers (1996, Cryo-Lett. 17, 43-52) found that HES alone is insufficient to preserve the dry cells and concluded on this basis that the studies of Goodrich et al. were incorrect. In the present paper we revisit that suggestion, using liposomes as a model to study effects of HES and glucose on membrane stability. In previous studies we and others have established that liposomes can be stabilized in the dry state if they are dried in the presence of disaccharides. Monosaccharides have not been effective. Measurements of effects of glucose on phase transitions in the dry lipids and vibrational frequency of the phosphate headgroup suggest that glucose shows an interaction with dry egg phosphatidylcholine similar to that seen with disaccharides. Nevertheless, glucose does not inhibit fusion in liposomes during drying, and it does not prevent leakage. Hydroxyethyl starch, which has a very high glass transition (Tg), inhibits fusion in the dry liposomes, but it does not depress the liquid crystalline to gel phase transition temperature (Tm) in the dry phospholipids, does not cause a shift in the phosphate vibration indicative of hydrogen bonding of the sugar to the phosphate, and does not stop leakage of trapped carboxyfluorescein. However, if glucose is added to the HES-containing samples, the liposomes are stabilized, so long as the samples are maintained below the Tg of the mixture. If they are heated above that Tg they fuse and leak their contents. We conclude that both glass formation and depression of Tm in the dry lipids are required. The role of glass formation in stabilization during drying of liposomes appears to be inhibition of fusion.

Drug Stability↗

PubMed1997

Trehalose: a cryoprotectant that enhances recovery and preserves function of human pancreatic islets after long-term storage.

The scarcity of available tissue for transplantation in diabetes and the need for multiple donors make it mandatory to use an optimal cryopreservation method that allows maximal recovery and preservation of beta-cell function. We have developed a method to cryopreserve islets with excellent survival of endocrine cells. Current methods use DMSO as cryoprotectant. Our method involves introducing both DMSO and the disaccharide trehalose into the cells during cooling. Uptake and release of trehalose occurred during the thermotropic lipid-phase transition measured in pancreatic endocrine cells between 5 degrees and 9 degrees C, using [14C]trehalose. Recovery of adult islets after cryopreservation with 300 mmol/l trehalose was 92 vs. 58% using DMSO alone. In vitro function, in terms of insulin content and release in response to secretagogues, was indistinguishable from fresh islets. Grafts from islets cryopreserved with trehalose contained 14-fold more insulin than grafts from islets cryopreserved without trehalose. Results with human fetal islet-like cell clusters (ICCs) were more pronounced: recovery from cryopreservation was 94%, compared with 42% without trehalose. Complete functionality of fetal cells was also restored; tritiated thymidine incorporation and insulin content and release were similar to fresh tissue. After transplantation in nude mice, there was a 15-fold increase in insulin content of grafts from ICCs cryopreserved with trehalose compared with ICCs cryopreserved without trehalose. Thus, the addition of trehalose to cryopreservation protocols leads to previously unobtainable survival rates of human pancreatic endocrine tissue.

Adult↗

PubMed1996

The lytic activity of the bee venom peptide melittin is strongly reduced by the presence of negatively charged phospholipids or chloroplast galactolipids in the membranes of phosphatidylcholine large unilamellar vesicles.

We have investigated the dependence of the lytic activity of the bee venom peptide melittin on the lipid composition of its target membrane. The lysis of large unilamellar liposomes, measured as loss of the fluorescent dye carboxyfluorescein, in the presence of melittin was strongly reduced when the negatively charged lipids phosphatidylglycerol (PG) or phosphatidylserine (PS), or the plant chloroplast lipids monogalactosyldiacylglycerol (MGDG) or digalactosyldiacylglycerol (DGDG) were incorporated into egg phosphatidylcholine (EPC) membranes. This reduction was evident at concentrations below 10 wt% of the additional lipids. It was not due to reduced binding of melittin to the vesicles. It was also not related to a reduced insertion depth of the peptide into the bilayer, as shown by quenching of the intrinsic tryptophan fluorescence of the peptide by the aqueous quencher sodium nitrate. Fourier transform infrared spectroscopy (FTIR) revealed specific interactions of the peptide with the headgroups of the inhibitory lipids. The phosphate peak in PG was shifted by two wavenumbers after the addition of melittin. There was no shift in EPC or PS. Instead, in PS the COO- peak was strongly distorted in the presence of melittin. These data indicate ionic interactions between the basic peptide and the negative charges on the membrane surface. The galactolipids are uncharged. Here the evidence points to hydrogen bonding between melittin and OH-groups of the sugar headgroups. Liposomes containing DGDG were the only case where we found evidence for changes in fatty acyl chain motion due to the presence of melittin, from the CH2-scissoring peaks.

Bee Venoms↗

PubMed1996

Arbutin inhibits PLA2 in partially hydrated model systems.

Arbutin is a glycosylated hydroquinone found at high concentrations in certain plants capable of surviving extreme and sustained dehydration. In this paper, we examine a potential role of this molecule in anhydrobiosis. We have studied its effects on the physical properties of phospholipids and on preservation of liposomes during drying. Arbutin depresses the gel to liquid crystalline phase transition temperature of dry phospholipids, as measured by differential scanning calorimetry, with a pattern similar to that seen in phospholipids dried with the disaccharide trehalose. Unlike trehalose, however, arbutin does not protect dry liposomes from leaking their contents. Also, using Fourier transform infrared spectroscopy, we found an increase in the vibrational frequency of the phosphate asymmetric stretch in partially hydrated phospholipids in the presence of arbutin. Trehalose, by contrast, depresses the frequency of the phosphate in dry phospholipids, indicating that the modes of interaction of trehalose and arbutin with the bilayer are different. Previously, we have shown that phospholipases can be active in liposomes with surprisingly low water contents. Based on the structural similarity of arbutin to a known inhibitor of phospholipase A2 (PLA2), it appeared possible that arbutin might serve as an inhibitor of phospholipases. Liposomes of varying composition were lyophilized in the presence and absence of phospholipases. When the liposomes were partially rehydrated at 76% relative humidity, arbutin inhibited PLA2, but did not inhibit phospholipases B or C. Accumulation of enzyme product in the liposome membranes was measured by analytical thin layer chromatography, and was taken as a measure of enzyme activity. Arbutin did not inhibit any of the enzymes in the presence of excess water. Based on these data, hypotheses are presented concerning the mechanism of PLA2 inhibition by arbutin in the mostly dehydrated state.

Arbutin↗

PubMed1996

Antifreeze glycoproteins inhibit leakage from liposomes during thermotropic phase transitions.

Antifreeze glycoproteins (AFGPs), found in the blood of polar fish at concentrations as high as 35 g/liter, are known to prevent ice crystal growth and depress the freezing temperature of the blood. Previously, Rubinsky et al. [Rubinsky, B., Mattioli, M., Arav, A., Barboni, B. & Fletcher, G. L. (1992) Am. J. Physiol. 262, R542-R545] provided evidence that AFGPs block ion fluxes across membranes during cooling, an effect that they ascribed to interactions with ion channels. We investigated the effects of AFGPs on the leakage of a trapped marker from liposomes during chilling. As these liposomes are cooled through the transition temperature, they leak approximately 50% of their contents. Addition of less than 1 mg/ml of AFGP prevents up to 100% of this leakage, both during chilling and warming through the phase transition. This is a general effect that we show here applies to liposomes composed of phospholipids with transition temperatures ranging from 12 degrees C to 41 degrees C. Because these results were obtained with liposomes composed of phospholipids alone, we conclude that the stabilizing effects of AFGPs on intact cells during chilling reported by Rubinsky et al. may be due to a nonspecific effect on the lipid components of native membranes. There are other proteins that prevent leakage, but only under specialized conditions. For instance, antifreeze proteins, bovine serum albumin, and ovomucoid all either have no effect or actually induce leakage. Following precipitation with acetone, all three proteins inhibited leakage, although not to the extent seen with AFGPs. Alternatively, there are proteins such as ovotransferrin that have no effect on leakage, either before or after acetone precipitation.

Animals↗

PubMed1996

Is vitrification involved in depression of the phase transition temperature in dry phospholipids?

Recent literature has suggested that the depression of the phase transition temperature (Tm) in dry phospholipids by sugars may be ascribed to vitrification of the stabilizing solute, rather than by the direct interaction between sugar and phospholipid we have proposed. Koster et al. ((1994) Biochim. Biophys. Acta 1193, 143-150) claim that the only necessity is that the glass transition (Tg) for the sugar exceed Tm for the lipid. Evidence is presented in the present paper that this is not sufficient. Based on the vitrification hypothesis of Koster et al., the predicted order of effectiveness in depressing Tm in dry dipalmitoylphosphatidylcholine (DPPC) is dextran > or = hydroxyethyl starch > stachyose > raffinose > trehalose > sucrose > glucose. In fact, the opposite order was seen. The effect of raffinose, sucrose, or trehalose on Tm in dry DPPC depends on the thermal history of the sample, as we have reported previously. When DPPC dried with trehalose is heated for the first time, Tm is about 55 degrees C, but on the second and subsequent heating scans Tm falls to about 25 degrees C. Koster et al. suggest that this effect is due to heating the sample above Tg rather than to melting the hydrocarbon chains. We present evidence here that all that is required is for the chains to be melted. Further, we show that retention of residual water by DPPC dried with trehalose depends on the drying temperature, but is independent of drying temperature with glucose, a finding that is consistent with direct interaction. We conclude that vitrification is not in itself sufficient to depress Tm in dry phospholipids.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1996

Membrane phase transition of intact human platelets: correlation with cold-induced activation.

Using Fourier transform infrared spectroscopy (FTIR), we have determined the phase transition temperature (Tm) of lipids in intact human platelets and have shown that it occurs between 15 and 18 degrees C, the temperature at which cold activation of platelets has previously been reported (Zucker and Borrelli, 1954, Blood, 28:602-608; White and Krivit, 1967, Blood, 30:625-635). The temperature at which the platelets pass through Tm is highly correlated with initial platelet shape change. However, shape change continues after the cells have passed through the phase transition. Cold-induced activation has previously prevented long-term storage of platelets at 4 degrees C. Antifreeze glycoproteins (AFGPs) isolated from polar fishes previously have been used to prevent ice crystal growth during freezing of tissues as well as leakage of solutes from liposomes as they were chilled through their Tm. We sought to determine if these AFGPs were able to stabilize platelets for long-term storage at 4 degrees C. Incubating platelets with antifreeze glycoproteins during long-term storage and rapid rewarming to 37 degrees C abrogated granule secretion associated with cold activation in a dose-dependent manner. This work suggests that AFGPs may be a possible solute for use in long-term low temperature storage of platelets.

Antifreeze Proteins↗

PubMed1996

Phase transition temperature and chilling sensitivity of bovine oocytes.

A limiting factor for achieving cryopreservation of oocytes is direct chilling injury (DCI), which occurs during cooling. DCI, or cold shock, is defined as an irreversible damage expressed shortly after exposure to low, but not freezing, temperatures. The primary target of DCI is thought to be the plasma membrane. Recently, an association between DCI in sperm and the thermotropic phase transition of their membrane lipids was demonstrated. In the present study, we examined the phase transition of the membrane lipids of immature and in vitro-matured bovine oocytes during cooling, using Fourier transform infrared spectroscopy (FTIR). The phase transition of the membrane lipids of oocytes at the germinal vesicle (GV) stage occurred between 13 and 20 degrees C, while a very broad phase transition, which centered around 10 degrees C, was observed for mature oocytes (MII) stage. Thermotropic phase transitions were demonstrated to be related to the temperature at which DCI affected the integrity of the oocyte membranes. When immature oocytes were cooled to 13 degrees C, fewer oocytes (40%) retained their membrane integrity than after exposure to 4 degrees C (51%) or holding them at 38 degrees C (78%), (as determined by the Fluorescein Diacetate-FDA test). This finding might suggest that holding immature oocytes at the phase transition temperature is more damaging to their membranes than exposure to lower temperatures. By contrast, no significant differences in membrane integrity were observed when in vitro-matured oocytes were cooled to the same temperatures. Subsequently, GV oocytes were cooled to 4 degrees C, and 26% underwent maturation and 19% underwent fertilization in vitro. In vitromatured oocytes that were cooled to 4 degrees C displayed a slightly decreased rate of fertilization; the overall fertilization was 60% with 24% polyspermy, rather than the 76% fertilization rate with 12% polyspermy obtained with those not subjected to cooling. The high rate of polyspermy indicates that a site(s) other than the plasma membrane is affected during cooling of bovine oocytes. Nucleated bovine GV oocytes were electrofused with in vitro-matured and enucleated oocytes, and then cooled to 4 degrees C. Evaluation of the membrane integrity of the fused oocytes showed that these oocytes are chilling resistant, which strongly suggests that alteration of the membrane composition of an oocyte can change the cell's susceptibility to low temperatures. This finding led to an improvement in the survival of oocytes after cryopreservation.

Animals↗