Loss Of Phospholipids Research Articles

028 Successful cryopreservation of avian germplasm: Why a multifaceted approach is required

The value of the ability to cryopreserve and store germplasm has long been recognized for indefinite preservation of genetic material, especially for “at-risk” populations. For domestic livestock species, options for preserving genetic material in frozen form range from sperm to oocytes to embryos. More than sixty years ago, the discovery of glycerol’s cryoprotective properties for rooster sperm pioneered the success of modern cryobiology and led to the development of semen cryopreservation for a wide range of species. Despite decades of research, the overall fertility rates of frozen/thawed poultry sperm are highly variable and not reliable enough for preservation of genetic stocks. The relatively low fertilizing ability most likely results from physiological sensitivity to the cryogenic process coupled with the requirement for prolonged sperm functionality in the hen reproductive tract. We have conducted systematic experiments to understand how and why poultry sperm lose functional competence after cryopreservation, and have shown that the glycocalyx, the carbohydrate-rich zone on the sperm surface, is altered by the cryogenic cycle. The amount of sialic acid, a terminal sugar critical for sperm transport in the female reproductive tract, was reduced after cryopreservation; however, we have shown that sperm cells can incorporate exogenous sialic acid, suggesting a strategy for sperm freezing protocols. Second, sperm lipids such as phosphotidylcholine also are altered during cold storage. The ratio of lipids in the sperm membrane determine the overall fluidity of the membrane and impact the ability of sperm to remain viable during the wide temperature fluctuations that are part of the cryogenic cycle. We have shown that supplemental lipids can circumvent the loss of phospholipids during cryopreservation. Finally, we have conducted the first proteomic analysis of poultry sperm and have identified three proteins not previously found in sperm from any species to date (dihydropyrimidinase, mitofilin and mitochondrial tri-functional protein). We currently are evaluating the proteomic profile of frozen/thawed sperm to determine the impact of cryopreservation on sperm proteins. Taken together, these data suggests innovative strategies for developing reliable poultry semen cryopreservation methods. For most species, sperm cryopreservation effectively captures the entire genome; however, in birds, the female is the heterogametic sex. Cryopreservation of avian oocytes and embryos is problematic because of the macrolecithal characteristics of the egg, where the surface-to-volume ratio poses a significant barrier to the movement of solutes/cryoprotectants and impedes effective protection of the cells from freeze/thaw damage. Recent advances for manipulation of poultry primordial germ cells and culture of embryonic stem cells suggests that cryopreservation of these cell types, followed by re-implantation in recipient embryos, could provide a mechanism to preserve the entire genome; albeit the efficiency rates of reconstitution associated with the current technologies are rather low. A novel innovation of cryopreservation and transplantation of ovarian tissue offers an alternative for whole genome preservation in birds (Song and Silversides, 2008); although chemical treatment to reduce the growth of host germ cells does not completely prevent the production of host-derived offspring. Reconstitution of poultry lines will most likely rely on more than one type of cryopreserved material. Source of funding: USDA ARS Project Number: 1245-31000-105-00. Conflict of interest: None declared. [email protected]

Loss of both phospholipid and triglyceride transfer activities of microsomal triglyceride transfer protein in abetalipoproteinemia

Mutations in microsomal triglyceride transfer protein (MTP) cause abetalipoproteinemia (ABL), characterized by the absence of plasma apoB-containing lipoproteins. In this study, we characterized the effects of various MTP missense mutations found in ABL patients with respect to their expression, subcellular location, and interaction with protein disulfide isomerase (PDI). In addition, we characterized functional properties by analyzing phospholipid and triglyceride transfer activities and studied their ability to support apoB secretion. All the mutants colocalized with calnexin and interacted with PDI. We found that R540H and N780Y, known to be deficient in triglyceride transfer activity, also lacked phospholipid transfer activity. Novel mutants S590I and G746E did not transfer triglycerides and phospholipids and did not assist in apoB secretion. In contrast, D384A displayed both triglyceride and phospholipid transfer activities and supported apoB secretion. These studies point out that ABL is associated with the absence of both triglyceride and phospholipid transfer activities in MTP.

Differential modification of the phospholipid profile by transient ischemia in rat hippocampal CA1 and CA3 regions

The hippocampal CA1 region is most susceptible to cerebral ischemia in both rodents and humans, whereas CA3 is remarkably resistant. Here, we investigated the possible role of membrane lipids in differential susceptibility in these regions. Transient ischemia was induced in rats via bilateral occlusion of common carotid arteries and membrane lipids were analyzed by mass spectrometry. While lipid profile differences between the intact CA1 and CA3 were rather minor, ischemia caused significant pyramidal cell death with concomittant reduction of phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, plasmalogen and sphingomyelin only in CA1. The phospholipid loss was evenly distributed in most molecular species. Ischemia also significantly increased cell death mediator ceramides only in CA1. Our data suggests that differential susceptibility to ischemia between CA1 and CA3 is not linked to their unique phospholipid profile. Also, selective activation of phospholipase A2, which primarily releases polyunsaturated fatty acids, might not be characteristic to cell death in CA1.

Pathophysiology of Myocardial Ischemia Reperfusion Injury: A Review

Cardiovascular events are one of the leading causes of death in the world. Thrombolysis, percutaneous transluminal coronary angioplasty, and coronary bypass surgery are the general treatment strategies of cardiovascular events. All of these treatment strategies can cause a myocardial ischemia reperfusion (MI/R) injury, which is known to occur on the restoration of coronary blood flow after a period of myocardial infarction (MI). Although there is an only way to save the myocardium from necrotic and apoptotic damages, reperfusion achieved by the restoration of blood flow often aggravates cardiac dysfunction. It is believed that MI/R injury is related to the increased reactive oxygen species (ROS), calcium overloading, and the loss of membrane phospholipids especially during the reperfusion. The harmful effects of ROS on cardiac tissue during the MI/R can be prevented by endogenous antioxidant systems. Also, the complement system plays a crucial role in the inflammatory events of ischemic injury; thereupon it is important in the pathogenesis of the MI/R injury. Polymorphonuclear leukocytes in the reperfusion period are also associated with MI/R injury. Therefore these circumstances can increase the irreversible tissue damage. Although sometimes the reperfusion is provided, blood flow cannot be supplied to the myocardial tissue. This is called a no-reflow phenomenon. A lot of exogenous antioxidant agents can be used to prevent this process of injury. Due to these properties of antioxidants, a number of studies have been carried out and have been reported anywhere in the world. These studies demonstrated that these agents can be used in the MI/R-induced tissue damage and protect the heart against ROS-related myocardial injury.

Depletion of acidic phospholipids influences chromosomal replication in Escherichia coli

In Escherichia coli, coordinated activation and deactivation of DnaA allows for proper timing of the initiation of chromosomal synthesis at the origin of replication (oriC) and assures initiation occurs once per cell cycle. In vitro, acidic phospholipids reactivate DnaA, and in vivo depletion of acidic phospholipids, results in growth arrest. Growth can be restored by the expression of a mutant form of DnaA, DnaA(L366K), or by oriC-independent DNA synthesis, suggesting acidic phospholipids are required for DnaA- and oriC-dependent replication. We observe here that when acidic phospholipids were depleted, replication was inhibited with a concomitant reduction of chromosomal content and cell mass prior to growth arrest. This global shutdown of biosynthetic activity was independent of the stringent response. Restoration of acidic phospholipid synthesis resulted in a resumption of DNA replication prior to restored growth, indicating a possible cell-cycle-specific growth arrest had occurred with the earlier loss of acidic phospholipids. Flow cytometry, thymidine uptake, and quantitative polymerase chain reaction data suggest that a deficiency in acidic phospholipids prolonged the time required to replicate the chromosome. We also observed that regardless of the cellular content of acidic phospholipids, expression of mutant DnaA(L366K) altered the DNA content-to-cell mass ratio.

34. The effects of biochemical rejuvenation on red blood cell microvesiculation, phosphatidylserine and CD47 expression during hypothermic storage

Red blood cells (RBCs) under blood bank storage conditions undergo biophysical and biochemical alterations, including biochemical depletion of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG), as well as cholesterol and phospholipid loss through microvesiculation. Microvesiculation is the process by which microparticles (MPs), phospholipid vesicles that are less than 1 μm in diameter, are released in response to different stimuli and conditions. RBC microparticles (RMPs) have been shown to be harmful effectors of inflammation and coagulation post transfusion, as well as in vitro indicators of RBC function and viability throughout ex vivo storage. Recent studies suggested that microvesiculation during storage results from biochemical depletion and/or cell senescence (Chernitskii˘ et al. 38 (8) (1993)18–21). Rejuvenation solutions have been shown to be able to restore ATP and 2,3-DPG levels in hypothermically stored RBCs. The purpose of this study was to examine the effects of the biochemical rejuvenation on red blood cell microvesiculation, as well as phosphatidylserine (PS) and CD47 expression during hypothermic storage. Leukoreduced packed RBC units (n = 14) were produced using the standard buffy coat method from whole blood in citrate–phosphate–dextrose (CPD) and stored in saline–adenine–glucose–mannitol (SAGM) at 1–6 °C for up to 49 days. Units were divided into three different experimental groups: untreated control, sham control and rejuvenated group. On days 28, 35, and 42 of hypothermic storage the rejuvenated group was incubated for one hour with 50 mL of rejuvenation solution containing pyruvate, inosine, phosphate and adenine (PIPA), while the sham control group was incubated for 1 h with 50 mL of 1× phosphate buffered saline. RBC units were tested weekly for ATP concentration, quantity of RMPs, and PS and CD47 expression. Microvesiculation was assessed using multicolor flow cytometric (FC) analysis and a spectrophotometric assay was used to determine ATP concentration. The results indicated that ATP significantly decreased throughout the hypothermic storage in untreated and sham RBC units (p

Inhibitory effects of pentachlorophenol on wastewater biofilms as determined by phospholipid analysis and microelectrode

To elucidate the toxic effect of pentachlorophenol (PCP) on the microbial activities of biofilms, we used two methods to evaluate the inhibition caused by PCP. The spatial distribution of phospholipids in biofilms was determined by phospholipid analysis combined with the micro-slicing technique. The inhibition was derived from the reduction in total phospholipid concentrations on the basis of the phospholipid profile in biofilms before and after exposure to PCP. Moreover, using O2 microprofiles as bases, we determined the spatial distributions of the net specific O2 respiration in biofilms with and without exposure to PCP. Furthermore, the inhibition was derived from the reduction in respiratory rates in biofilms. We attribute the discrepancy in results from the two methods to the delay of the loss of phospholipids compared with the decrease in the oxygen uptake rate in response to PCP.

The ratio of phosphatidylcholine to phosphatidylethanolamine does not predict integrity of growing MT58 Chinese hamster ovary cells

Phosphatidylcholine (PC) homeostasis is important for maintaining cellular growth and survival. Cellular growth and apoptosis may also be influenced by the PC to phosphatidylethanolamine (PE) ratio as a reduction in this ratio can result in a loss of membrane integrity. To investigate whether a reduced PC:PE ratio influences cellular growth and apoptosis, we utilized the MT58 cell line, which contains a thermo-sensitive mutation in CTP:phosphocholine cytidylyltransferase-α, the rate-limiting enzyme for PC biosynthesis. Incubation of MT58 cells at the restrictive temperature of 41 °C results in a reduction of cellular PC and induces apoptosis. Furthermore, MT58 cells have a 50% reduction in the PC:PE ratio when incubated at 41 °C. In an attempt to normalize the PC:PE ratio, which may stabilize cellular membranes and rescue MT58 cells from apoptosis, the cells were treated with either silencing RNA to impair PE biosynthesis or lysophosphatidylcholine to increase PC mass. Impairing PE biosynthesis in MT58 cells reduced cellular PE and PC concentrations by 30% and 20%, but did not normalize the PC:PE ratio. Loss of both phospholipids enhanced the onset of apoptosis in MT58 cells. Lysophosphatidylcholine normalized cellular PC, increased PE mass by 10%, restored cellular growth and prevented apoptosis of MT58 cells without normalizing the PC:PE ratio. Furthermore, total amount of cellular PC and PE, but not the PC:PE ratio, correlated with cellular growth ( R 2 = 0.76), and inversely with cellular apoptosis ( R 2 = 0.97). These data suggest the total cellular amount of PC and PE, not the PC:PE ratio, influences growth and membrane integrity of MT58 cells.

The effect of antioxidants on the response of the rabbit urinary bladder to in vitro ischemia/reperfusion

To evaluate the protective effects of two naturally occurring antioxidants, α-Lipoic acid and coenzyme Q10 on the response to in vitro ischemia of the rabbit urinary bladder. We measured free fatty acid (FFA) content, phospholipid (PL) content, malondialdehyde (MDA) levels, and phospholipase A(2) activity (PLA) of subcellular compartments. Twenty New Zealand White male rabbits were separated into four groups of five rabbits each. The in vitro whole bladders from Groups 1 and 2 received a 3 h incubation under normal oxygenated physiological conditions. The bladders were stimulated by field stimulation at 1 and 3 h. The bladders from groups 3 and 4 underwent 1 h incubation time under normal oxygenated physiological conditions. After 1 h, the bladders were stimulated with field stimulation. After a maximal pressure response was recorded, the stimulation was turned off and the bath medium changed to one equilibrated with 95% nitrogen, 5% oxygen without glucose (ischemic medium) and incubated for 1 h with field stimulations occurring at 5 min intervals during this time. At the end of this hour of ischemia with repetitive stimulation, the bath was changed to an oxygenated medium with glucose for a 1-h reperfusion period after which the stimulation was repeated. The rabbits from groups 2 and 4 received α-Lipoic acid (10 mg/kg/day) + Coenzyme Q10 (3 mg/kg/day) by gavage for 4 weeks before the experiment. At the end of the experimental period, each bladder was opened longitudinally, and the muscle and mucosa separated by blunt dissection, frozen under liquid nitrogen, and stored at -80°C for biochemical analyses. Each tissue was fractionated by differential centrifugation into nuclear, mitochondrial, synaptosomal, and cytosol (supernatant) components. PL, FFA, MDA, and PLA were analyzed using standard biochemical techniques. Post-ischemic contractility only returned to 30% of control of the untreated group. However, post-ischemic contractility of the treated group returned to approximately 70% of control. PL loss in the muscle mitochondria and synaptosomes was prevented by antioxidant treatment, while the mucosal layer showed a significant drop in PL with antioxidants treatment. Administration of CoQ + LA significantly decreased MDA levels in both control and ischemic tissues in both the muscle and mucosal bladder layers, especially substantial in the microsomal and mitochondrial components. Treatment had variable effects on PLA(2) activity. Treatment of bladder dysfunction with antioxidants daily can be beneficial in man to prevent or delay the onset of progressive loss of bladder function especially that due to ischemic damage to mitochondrial and microsomal lipids. CoQ10 + LA can provide similar protection of the bladder muscle and mucosa against lipid oxidative stress as they have been shown to protect against protein oxidative damage.

<em>Arabidopsis thaliana</em> Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

The effect of in vitro ischemia/reperfusion on contraction, free fatty acid content, phospholipid content, and malondialdehyde levels of the rabbit urinary bladder

To evaluate the effects of in vitro ischemia/reperfusion on contractile response to field stimulation (FS), free fatty acid (FFA) content, phospholipid (PL) content, and malondialdehyde (MDA) levels of the rabbit urinary bladder. There is significant evidence that ischemia/reperfusion injury is linked to obstructive bladder dysfunction secondary to men with benign prostatic hyperplasia (BPH). Twelve New Zealand White male rabbits were separated into two groups of six rabbits each. Each rabbit was euthanized, and the bladder was surgically removed intact for whole bladder incubation. The bladders in Group 1 received a 3-h incubation under normal oxygenated physiological conditions. These bladders received electrical field stimulation (32Hz) after 1 and 3h. The bladders associated with Group 2 received a 1-h incubation under normal oxygenated physiological conditions. At the end of this 1-h period, the bladders were subjected to FS. After a maximal pressure response was recorded, the stimulation was turned off and the bath medium was changed to one equilibrated with 95% nitrogen, 5% oxygen without glucose (ischemic medium) and incubated for 1h with field stimulations (32Hz) occurring at 5-min intervals to represent overactive bladder dysfunction. At the end of this hour of ischemia with repetitive stimulation, the bath was changed to an oxygenated medium with glucose for a 1-h period after which the stimulation was repeated. At the end of the experimental period, each bladder was opened longitudinally and the muscle and mucosa separated by blunt dissection, frozen under liquid nitrogen, and stored at -80°C for biochemical analyses. Each tissue was fractionated by differential centrifugation into nuclear, mitochondrial, synaptosomal, and supernatant (cytosol) components. PL, FFA, and MDA content were analyzed for each fraction using standard biochemical techniques. The bladder contractile responses decreased during the period of in vitro ischemia and returned to only 30% of control after reperfusion. In vitro ischemia/reperfusion showed the following: (1) There was a modest but significant decrease in the FFA content of the microsomes of the muscle and significant increases in the FFA content of the nuclei and mitochondria of the mucosa. (2) There were decreases in the PL content of the homogenate and microsomes of the muscle and decreases in the PL content of the homogenate, microsomes, and supernatant of the mucosa. (3) Significant increases were observed in the MDA levels of the homogenate, mitochondria, and microsomes of both the muscle and mucosa. The significant increases in the lipid peroxidation of the bladder smooth muscle are consistent with the marked decrease in the contractile ability of the bladder following ischemia/reperfusion. The specific increased lipid peroxidation of the mitochondrial and microsomal components is consistent with the specific dysfunctions of the mitochondria and innervations observed following I/R in earlier published studies. The marked increases in lipid peroxidation in the mucosa associated with the loss of PL and FFA from this component are consistent with the significant dysfunction in both the antiadherence and antipermeability properties of the mucosa and may play a major role in the symptomatic nature of I/R-linked diseases of the bladder.

Differential involvement of rat sperm choline glycerophospholipids and sphingomyelin in capacitation and the acrosomal reaction

Rat spermatozoa main lipid classes and their fatty acids were studied to assess their possible changes in capacitation and the acrosomal reaction (AR), induced in vitro. Capacitation-associated protein tyrosine phosphorylation, and the efflux of 30% of the total cholesterol from gametes to the medium, took place concomitantly with the release of a similar percentage, i.e., a larger amount, of the total phospholipid, mostly after hydrolysis of the major choline glycerophospholipids (CGP). Main medium lipid metabolites after capacitation were lyso-CGP and polyenoic fatty acids typical of CGP (22:4n-9, 22:5n-6), as free fatty acids (FFA). The AR, induced by a calcium ionophore, resulted in further phospholipid loss, but the produced metabolites remained in the gametes. CGP decrease in AR accounted for some additional FFA and lyso-CGP, but mostly for (22:5n-6-rich) diglycerides. Hydrolysis of sphingomyelins (SM) to ceramides also occurred, mostly affecting species with very long chain polyenoic fatty acids. Quantitatively, CGP and SM were the lipid classes decreasing the most after capacitation and AR, respectively. The massive cholesterol and phospholipid loss from the gametes during capacitation is thus associated with protein phosphorylation, a function that has been located to the sperm tail. The lipid metabolites produced during AR, by accumulating in the gamete heads, could be implicated in sperm–oocyte interactions.

The ultra-low friction of the articular surface is pH-dependent and is built on a hydrophobic underlay including a hypothesis on joint lubrication mechanism

In this study, the influence of pH on interfacial energy distributed over the phospholipids-bilayer surface model and the effect of hydrophobicity on coefficient of friction ( f) were investigated by using microelectrophoresis. An important clinical implication of deficiency in hydrophobicity is the loss of phospholipids that is readily observed in osteoarthritis joints. This paper establishes the influence of pH on interfacial energy upon an increase f, which might be associated with a decrease of hydrophobicity of the articular surface.

Loss of Mitochondrial DNA in the Yeast Cardiolipin Synthase crd1 Mutant Leads to Up-regulation of the Protein Kinase Swe1p That Regulates the G2/M Transition

The anionic phospholipid cardiolipin and its precursor phosphatidylglycerol are synthesized and localized in the mitochondrial inner membrane of eukaryotes. They are required for structural integrity and optimal activities of a large number of mitochondrial proteins and complexes. Previous studies showed that loss of anionic phospholipids leads to cell inviability in the absence of mitochondrial DNA. However, the mechanism linking loss of anionic phospholipids to petite lethality was unclear. To elucidate the mechanism, we constructed a crd1Deltarho degrees mutant, which is viable and mimics phenotypes of pgs1Delta in the petite background. We found that loss of cardiolipin in rho degrees cells leads to elevated expression of Swe1p, a morphogenesis checkpoint protein. Moreover, the retrograde pathway is activated in crd1Deltarho degrees cells, most likely due to the exacerbation of mitochondrial dysfunction. Interestingly, the expression of SWE1 is dependent on retrograde regulation as elevated expression of SWE1 is suppressed by deletion of RTG2 or RTG3. Taken together, these findings indicate that activation of the retrograde pathway leads to up-regulation of SWE1 in crd1Deltarho degrees cells. These results suggest that anionic phospholipids are required for processes that are essential for normal cell division in rho degrees cells.

Using Confocal Laser Scanning Microscopy To Probe the Milk Fat Globule Membrane and Associated Proteins

The bovine milk fat globule membrane (MFGM) is an important, biologically relevant membrane due to its functional and health properties. Its composition has been thoroughly studied, but its structure, especially the lateral organization of its components, still remains unclear. We have used confocal laser scanning microscopy (CLSM) to investigate the surface structure of the MFGM in globules with different degrees of processing using two types of fluorescently labeled phospholipid probes and a protein dye. Using this technique, we have observed heterogeneities in the distribution of MFGM lipids and proteins relating to the processing and size of the globules. The effect of pretreating the milk (centrifugation, pasteurization-homogenization and churning) was studied by double-staining the surface of the milk fat globules, followed by observation using CLSM, and by determining the phospholipid profile of raw milk, raw cream, processed milk and buttermilk powder. Our findings agree with other techniques by showing that the composition of the MFGM changes with processing through the loss of phospholipids and the adsorption of caseins and whey proteins onto the surface.

Loss of adipose tissue and plasma phospholipids: Relationship to survival in advanced cancer patients

Extensive loss of adipose tissue is a key feature of cancer cachexia. Advanced cancer patients also exhibit low plasma phospholipids. It is not known whether these processes coincide across the cancer trajectory nor has their relationship with survival been defined. Changes in adipose tissue mass and plasma phospholipids were characterized within 500days prior to death and prognostic significance assessed. Adipose tissue rate of change was determined in a retrospective cohort of patients who died of colorectal and lung cancers (n=108) and who underwent >2 computed tomography scans in the last 500days of life. Plasma phospholipid fatty acids were measured prospectively in a similar cohort of patients with metastatic cancer (n=72). Accelerated loss of adipose tissue begins at 7months from death reaching an average loss of 29% of total AT 2months from death. Plasma phospholipid fatty acids were 35% lower in patients closest to death versus those surviving >8months. Losses of phospholipid fatty acids and adipose tissue occur in tandem and are predictive of survival. Depletion of plasma phospholipids likely indicates a deficit of essential fatty acids in the periphery which may contribute to loss of adipose tissue.

NO inhibits platelet apoptosis by cGMP-dependent and-independent pathways

Background Platelets are specialized anucleate cells that play key roles in hemostasis through their ability to rapidly adhere to subendothelial matrix proteins and endothelial cells (platelet adhesion) and to other activated platelets (platelet aggregation). Platelet activation is an irreversible process resulting ultimately in platelet apoptosis and death. The NO-cGMP-PKG pathway plays an essential role in platelet inhibition, however the role of this pathway in regulation of platelet apoptosis has not been investigated in detail so far. Surface exposure of negatively charged phospholipid phosphatidylserine (Annexin V-binding) and loss of mitochondrial membrane potential (ΔΨm) of activated platelets were used as markers of platelet apoptosis induced by thrombin (up to 0.5 U/ml), convulxin (Cvx 5 ng/ml) or by a mixture of thrombin/convulxin (Thr/Cvx 0.005 U/ml/5 ng/ml).

Changes in Band 3 oligomeric state precede cell membrane phospholipid loss during blood bank storage of red blood cells.

Lipid loss in the form of vesicles contributes to the red blood cell (RBC) storage lesion, and this loss of lipid is correlated with changes in membrane protein function. Sensitive spectroscopic techniques were used to measure changes in Band 3 oligomeric state during storage of RBCs, compared to metabolic changes and phospholipid loss. The aim of the study was to determine whether changes in the macromolecular organization of membrane proteins occur before, coincident with, or after lipid loss during RBC storage. Five RBC units were collected from normal volunteers and stored under standard blood bank conditions, and both metabolic changes and lipid loss were measured by multiple assays. Band 3 oligomeric state was assessed by time-resolved phosphorescence anisotropy and fluorescence resonance energy transfer of eosin-5-maleimide-labeled RBC ghosts. Extracellular pH decreased and extracellular potassium increased rapidly during cold storage of blood. Band 3 on the RBC membrane exhibited a shift from small to large oligomers early in the storage period and before detectable loss of phospholipid from the RBC membrane. The immobilized fraction of Band 3, that which is tethered to the cytoskeletal network via spectrin and ankyrin, did not change during cold storage. Our results demonstrate that changes in the macromolecular organization of membrane proteins on the RBC occur early in storage, and these changes may induce phospholipid loss, irreversible morphologic changes, and loss of function during RBC storage.

Cryopreservation of Canine Semen – New Challenges

Egg yolk (EY) protects cell membranes against cold shock, and it prevents or restores the loss of phospholipids from the membrane. EY has been widely used in semen extenders. It has been added to Tris-Glucose buffer and has been widely used for cooling and cryopreservation of canine semen. EY is not a defined entity, but a complex biological compound containing proteins, vitamins, phospholipids, glucose and antioxidants which are all potentially useful for cell membrane integrity. Unfortunately, it also is a biologically hazardous compound. Hence, whole EY needs to be replaced by other chemically defined components for semen processing in dogs. Freezing poor semen does not improve its quality, so attention must be focused on how to cope with dogs whose semen does not freeze well, and on designing individual freezing extenders for semen from such males. Furthermore, differences have been found among canid species in the ability of their spermatozoa to withstand freezing. There are differences in sperm membrane fatty acid composition among species, which may explain part of these differences. If the presence of long-chained polyunsaturated fatty acids contributes to increased membrane fluidity, this relationship may be biphasic, i.e. either too much membrane fluidity, or too little, could compromise successful sperm cryopreservation. An increase in fluidity of the outer leaflet of the plasma membrane has been shown in frozen thawed dog spermatozoa. The protective effect of exogenous lipids may lie in close association with the membrane rather than in modification or rearrangement of the membrane. This also points at lipids as an important, if not entirely new group of substances, which may substitute standard EY-based diluents in preserving sperm survival during freezing. EY-derived phospholipids or lecithin could be used to replace whole EY. Vegetable lecithin is currently investigated to avoid using substances of animal origin. EY also contains antioxidants which prevent cells from oxidative damage due to the generation of reactive oxygen species. An increasing number of publications now recognize the significance of protecting sperm from this damage during processing by using dietary or diluent supplemented antioxidants. This paper aims at looking at some of the new challenges in freezing of dog semen.

Dirty-appearing white matter in multiple sclerosis

"Dirty-appearing white matter" (DAWM) in multiple sclerosis (MS) is defined as a region(s) with ill-defined borders of intermediate signal intensity between that of normal-appearing white matter (NAWM) and that of plaque on T(2)-weighted and proton density imaging. To delineate the histopathology of DAWM, four formalin-fixed cerebral hemisphere slices of three MS patients with DAWM were scanned with T(2)- weighted and proton density sequences. The myelin water fraction (MWF) was obtained by expressing the short T(2) component as a fraction of the total T(2) distribution. Hemispheric sections were then stained with Luxol fast blue (LFB) for myelin phospholipids, for myelin basic protein (MBP) and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) for myelin; Bielschowsky silver impregnation for axons; and for glial fibrillary acidic protein (GFAP) for astrocytes. Compared to NAWM, DAWM showed reduction in MWF, corresponding to a reduction of LFB staining. DAWM also showed reduced Bielschowsky staining. Quantitatively, the change in MWF in DAWM most consistently correlated with the change in LFB staining. The findings of this preliminary study suggest that DAWM is characterized by loss of myelin phospholipids, detected by the short T(2) component, and axonal reduction.