[Adsorption therapy in sepsis.]

BACKGROUND The activation of multiple pro- and anti-inflammatory mediators is a key feature in the pathophysiology of sepsis. Many of these mediators may directly contribute to organ dysfunction and determine disease severity. So far our ability to modulate these upregulated mediator pathways is very limited. Therefore the adsorption of such mediators via an extracorporeal circuit may be a beneficial intervention during sepsis. OBJECTIVES Recent technical innovations have made this intervention feasible. Both systems for exclusive mediator adsorption and for adsorption beside a conventional renal replacement therapy are now available. Some of the membranes can adsorb a broad range of mediators by rather unspecific binding, whereas others specifically adsorb endotoxin or mediators. DISCUSSION Whilst biochemical efficacy could be demonstrated by some of the systems, controlled and randomized studies demonstrating improved clinical endpoints are still lacking. Therefore the use of such therapies outside clinical studies cannot yet be recommended.

Outbreak investigation identifies a single Listeria monocytogenes strain in sheep with different clinical manifestations, soil and water.

Listeria (L.) monocytogenes causes orally acquired infections and is of major importance in ruminants. Little is known about L. monocytogenes transmission between farm environment and ruminants. In order to determine potential sources of infection, we investigated the distribution of L. monocytogenes genetic subtypes in a sheep farm during a listeriosis outbreak by applying four subtyping methods (MALDI-TOF-MS, MLST, MLVA and PFGE). L. monocytogenes was isolated from a lamb with septicemia and from the brainstem of three sheep with encephalitis. Samples from the farm environment were screened for the presence of L. monocytogenes during the listeriosis outbreak, four weeks and eight months after. L. monocytogenes was found only in soil and water tank swabs during the outbreak. Four weeks later, following thorough cleaning of the barn, as well as eight months later, L. monocytogenes was absent in environmental samples. All environmental and clinical L. monocytogenes isolates were found to be the same strain. Our results show that the outbreak involving two different clinical syndromes was caused by a single L. monocytogenes strain and that soil and water tanks were potential infection sources during this outbreak. However, silage cannot be completely ruled out as the bales fed prior to the outbreak were not available for analysis. Faeces samples were negative, suggesting that sheep did not act as amplification hosts contributing to environmental contamination. In conclusion, farm management appears to be a crucial factor for the limitation of a listeriosis outbreak.

Hygrical shrinkage stresses in tiling systems: Numerical modeling combined with field studies

To track down potential sites of material failure in the tile–mortar–substrate systems, locations and intensities of stress concentrations owing to drying-induced shrinkage are investigated. For this purpose, mechanical properties were measured on real systems and used as input parameters for numerical modeling of the effect of shrinkage of substrate and/or mortar using the finite element code Abaqus. On the base of different geometrical set-ups we demonstrate that stress concentrations in the mortar can become critical when (i) substantial mortar shrinkage occurs, (ii) substrate shrinkage can accumulate over considerable spatial distances, particularly (iii) in situations where the mortar layer is not separated from the substrate by a flexible waterproofing membrane. Hence material failure in the system tile–mortar–substrate can be prevented (or reduced) by (i) an application of the tiles after the major stages of substrate shrinkage, (ii) the use of elasto-plastic deformable tile adhesives which can react elastically on local stress concentrations, (iii) the implementation of flexible membranes, and (iv) a reduction of the field size by the installation of flexible joints.

Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf2.

Exposure of biological membranes to reactive oxygen species creates a complex mixture of distinct oxidized phospholipid (OxPL) species, which contribute to the development of chronic inflammatory diseases and metabolic disorders. While the ability of OxPL to modulate biological processes is increasingly recognized, the nature of the biologically active OxPL species and the molecular mechanisms underlying their signaling remain largely unknown. We have employed a combination of mass spectrometry, synthetic chemistry, and immunobiology approaches to characterize the OxPL generated from the abundant phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and investigated their bioactivities and signaling pathways in vitro and in vivo. Our study defines epoxycyclopentenones as potent anti-inflammatory lipid mediators that mimic the signaling of endogenous, pro-resolving prostanoids by activating the transcription factor nuclear factor E2-related factor 2 (Nrf2). Using a library of OxPL variants, we identified a synthetic OxPL derivative, which alleviated endotoxin-induced lung injury and inhibited development of pro-inflammatory T helper (Th) 1 cells. These findings provide a molecular basis for the negative regulation of inflammation by lipid peroxidation products and propose a novel class of highly bioactive compounds for the treatment of inflammatory diseases.

Recombinant Human Bone Morphogenetic Protein 9 (rhBMP9) Induced Osteoblastic Behaviour on a Collagen Membrane Compared With rhBMP2

BACKGROUND Bone morphogenetic protein 9 (BMP9) has previously been characterized as one of the most osteogenic growth factors of the BMP-family, however, up until now, these experiments have only been demonstrated using adenovirus-transfection experiments (gene therapy). With the recent development of recombinant human (rh)BMP9, the aim of the present study was to investigate its osteopromotive potential versus rhBMP2 when loaded onto a collagen membrane. METHODS ST2 stromal bone marrow cells were seeded onto 1)control; 2)rhBMP2-low(10ng/ml); 3)rhBMP2-high(100ng/ml); 4)rhBMP9-low(10ng/ml); and 5)rhBMP9-high(100ng/ml) porcine collagen membranes. Groups were then compared for cell adhesion at 8 hours, cell proliferation at 1, 3 and 5 days real-time PCR at 3 and 14 days for genes encoding Runx2, alkaline phosphatase(ALP) and bone sialoprotein(BSP) at 3 and 14 days and alizarin red staining at 14 days. RESULTS While rhBMP2 and rhBMP9 demonstrated little effects on cell attachment and proliferation, pronounced increases were observed on osteoblast differentiation. It was found that all groups significantly induced ALP mRNA levels at 3 days and BSP levels at 14 days, however rhBMP9-high demonstrated significantly higher values when compared to all other groups for ALP levels (5-fold increase at 3 days and 2-fold increase at 14 days). Alizarin red staining further revealed that both concentrations of rhBMP9 induced up to 3-fold more staining when compared to rhBMP2. CONCLUSION These results indicate that the combination of collagen membranes with rhBMP9 significantly induced significantly higher ALP mRNA expression and alizarin red staining when compared to rhBMP2. These findings suggest that rhBMP9 may be a suitable growth factor for future regenerative procedures in bone biology.

Poly-N-acetyl glucosamine nanofibers for negative-pressure wound therapies

The wound healing promoting effect of negative wound pressure therapies (NPWT) takes place at the wound interface. The use of bioactive substances at this site represents a major research area for the development of future NPWT therapies. To assess wound healing kinetics in pressure ulcers treated by NPWT with or without the use of a thin interface membrane consisting of poly-N-acetyl glucosamine nanofibers (sNAG) a prospective randomized clinical trial was performed. The safety of the combination of NPWT and sNAG was also assessed in patients treated with antiplatelet drugs. In the performed study, the combination of NPWT and sNAG in 10 patients compared to NPWT alone in 10 patients promoted wound healing due to an improved contraction of the wound margins (p = 0.05) without a change in wound epithelization. In 6 patients treated with antiplatelet drugs no increased wound bleeding was observed in patients treated by NPWT and sNAG. In conclusion, the application of thin membranes of sNAG nanofibers at the wound interface using NPWT was safe and augmented the action of NPWT leading to improved wound healing due to a stimulation of wound contraction.

Lipid topogenesis - 35years on

Glycerophospholipids are the principal fabric of cellular membranes. The pathways by which these lipids are synthesized were elucidated mainly through the work of Kennedy and colleagues in the late 1950s and early 1960s. Subsequently, attention turned to cell biological aspects of lipids: Where in the cell are lipids synthesized? How are lipids integrated into membranes to form a bilayer? How are they sorted and transported from their site of synthesis to other cellular destinations? These topics, collectively termed 'lipid topogenesis', were the subject of a review article in 1981 by Bell, Ballas and Coleman. We now assess what has been learned about early events of lipid topogenesis, i.e. "lipid synthesis, the integration of lipids into membranes, and lipid translocation across membranes", in the 35years since the publication of this important review. We highlight the recent elucidation of the X-ray structures of key membrane enzymes of glycerophospholipid synthesis, progress on identifying lipid scramblase proteins needed to equilibrate lipids across membranes, and new complexities in the subcellular location and membrane topology of phosphatidylinositol synthesis revealed through a comparison of two unicellular model eukaryotes.

Blade guard for rotary lawn mowers

A blade guard for rotary lawn mowers comprising a plurality of spaced apart elongated members which extend longitudinally in the intended direction of travel of the mower device. The space between the forward ends of the elongated members and the forward end of the mower cowling is open and free from obstructions to permit the grass to be sucked upwardly for cutting by the mower blade. The elongated members have varying lengths to define an effective area substantially the same as the area defined by the unsharpened center portion of the mower blade when rotating so that the sharpened ends of the blade extend outwardly beyond the effective area of the guard means. The guard means is hinged to the mower cowling to permit convenient cleaning of the mower. An ignition interlock means is also connected to the guard.

Algoritmos de distribución de cargas de proceso en computación con membranas

Con el surgir de los problemas irresolubles de forma eficiente en tiempo polinomial en base al dato de entrada, surge la Computación Natural como alternativa a la computación clásica. En esta disciplina se trata de o bien utilizar la naturaleza como base de cómputo o bien, simular su comportamiento para obtener mejores soluciones a los problemas que los encontrados por la computación clásica. Dentro de la computación natural, y como una representación a nivel celular, surge la Computación con Membranas. La primera abstracción de las membranas que se encuentran en las células, da como resultado los P sistemas de transición. Estos sistemas, que podrían ser implementados en medios biológicos o electrónicos, son la base de estudio de esta Tesis. En primer lugar, se estudian las implementaciones que se han realizado, con el fin de centrarse en las implementaciones distribuidas, que son las que pueden aprovechar las características intrínsecas de paralelismo y no determinismo. Tras un correcto estudio del estado actual de las distintas etapas que engloban a la evolución del sistema, se concluye con que las distribuciones que buscan un equilibrio entre las dos etapas (aplicación y comunicación), son las que mejores resultados presentan. Para definir estas distribuciones, es necesario definir completamente el sistema, y cada una de las partes que influyen en su transición. Además de los trabajos de otros investigadores, y junto a ellos, se realizan variaciones a los proxies y arquitecturas de distribución, para tener completamente definidos el comportamiento dinámico de los P sistemas. A partir del conocimiento estático –configuración inicial– del P sistema, se pueden realizar distribuciones de membranas en los procesadores de un clúster para obtener buenos tiempos de evolución, con el fin de que la computación del P sistema sea realizada en el menor tiempo posible. Para realizar estas distribuciones, hay que tener presente las arquitecturas –o forma de conexión– de los procesadores del clúster. La existencia de 4 arquitecturas, hace que el proceso de distribución sea dependiente de la arquitectura a utilizar, y por tanto, aunque con significativas semejanzas, los algoritmos de distribución deben ser realizados también 4 veces. Aunque los propulsores de las arquitecturas han estudiado el tiempo óptimo de cada arquitectura, la inexistencia de distribuciones para estas arquitecturas ha llevado a que en esta Tesis se probaran las 4, hasta que sea posible determinar que en la práctica, ocurre lo mismo que en los estudios teóricos. Para realizar la distribución, no existe ningún algoritmo determinista que consiga una distribución que satisfaga las necesidades de la arquitectura para cualquier P sistema. Por ello, debido a la complejidad de dicho problema, se propone el uso de metaheurísticas de Computación Natural. En primer lugar, se propone utilizar Algoritmos Genéticos, ya que es posible realizar alguna distribución, y basada en la premisa de que con la evolución, los individuos mejoran, con la evolución de dichos algoritmos, las distribuciones también mejorarán obteniéndose tiempos cercanos al óptimo teórico. Para las arquitecturas que preservan la topología arbórea del P sistema, han sido necesarias realizar nuevas representaciones, y nuevos algoritmos de cruzamiento y mutación. A partir de un estudio más detallado de las membranas y las comunicaciones entre procesadores, se ha comprobado que los tiempos totales que se han utilizado para la distribución pueden ser mejorados e individualizados para cada membrana. Así, se han probado los mismos algoritmos, obteniendo otras distribuciones que mejoran los tiempos. De igual forma, se han planteado el uso de Optimización por Enjambres de Partículas y Evolución Gramatical con reescritura de gramáticas (variante de Evolución Gramatical que se presenta en esta Tesis), para resolver el mismo cometido, obteniendo otro tipo de distribuciones, y pudiendo realizar una comparativa de las arquitecturas. Por último, el uso de estimadores para el tiempo de aplicación y comunicación, y las variaciones en la topología de árbol de membranas que pueden producirse de forma no determinista con la evolución del P sistema, hace que se deba de monitorizar el mismo, y en caso necesario, realizar redistribuciones de membranas en procesadores, para seguir obteniendo tiempos de evolución razonables. Se explica, cómo, cuándo y dónde se deben realizar estas modificaciones y redistribuciones; y cómo es posible realizar este recálculo. Abstract Natural Computing is becoming a useful alternative to classical computational models since it its able to solve, in an efficient way, hard problems in polynomial time. This discipline is based on biological behaviour of living organisms, using nature as a basis of computation or simulating nature behaviour to obtain better solutions to problems solved by the classical computational models. Membrane Computing is a sub discipline of Natural Computing in which only the cellular representation and behaviour of nature is taken into account. Transition P Systems are the first abstract representation of membranes belonging to cells. These systems, which can be implemented in biological organisms or in electronic devices, are the main topic studied in this thesis. Implementations developed in this field so far have been studied, just to focus on distributed implementations. Such distributions are really important since they can exploit the intrinsic parallelism and non-determinism behaviour of living cells, only membranes in this case study. After a detailed survey of the current state of the art of membranes evolution and proposed algorithms, this work concludes that best results are obtained using an equal assignment of communication and rules application inside the Transition P System architecture. In order to define such optimal distribution, it is necessary to fully define the system, and each one of the elements that influence in its transition. Some changes have been made in the work of other authors: load distribution architectures, proxies definition, etc., in order to completely define the dynamic behaviour of the Transition P System. Starting from the static representation –initial configuration– of the Transition P System, distributions of membranes in several physical processors of a cluster is algorithmically done in order to get a better performance of evolution so that the computational complexity of the Transition P System is done in less time as possible. To build these distributions, the cluster architecture –or connection links– must be considered. The existence of 4 architectures, makes that the process of distribution depends on the chosen architecture, and therefore, although with significant similarities, the distribution algorithms must be implemented 4 times. Authors who proposed such architectures have studied the optimal time of each one. The non existence of membrane distributions for these architectures has led us to implement a dynamic distribution for the 4. Simulations performed in this work fix with the theoretical studies. There is not any deterministic algorithm that gets a distribution that meets the needs of the architecture for any Transition P System. Therefore, due to the complexity of the problem, the use of meta-heuristics of Natural Computing is proposed. First, Genetic Algorithm heuristic is proposed since it is possible to make a distribution based on the premise that along with evolution the individuals improve, and with the improvement of these individuals, also distributions enhance, obtaining complexity times close to theoretical optimum time. For architectures that preserve the tree topology of the Transition P System, it has been necessary to make new representations of individuals and new algorithms of crossover and mutation operations. From a more detailed study of the membranes and the communications among processors, it has been proof that the total time used for the distribution can be improved and individualized for each membrane. Thus, the same algorithms have been tested, obtaining other distributions that improve the complexity time. In the same way, using Particle Swarm Optimization and Grammatical Evolution by rewriting grammars (Grammatical Evolution variant presented in this thesis), to solve the same distribution task. New types of distributions have been obtained, and a comparison of such genetic and particle architectures has been done. Finally, the use of estimators for the time of rules application and communication, and variations in tree topology of membranes that can occur in a non-deterministic way with evolution of the Transition P System, has been done to monitor the system, and if necessary, perform a membrane redistribution on processors to obtain reasonable evolution time. How, when and where to make these changes and redistributions, and how it can perform this recalculation, is explained.

Strain rate effect on semi‐crystalline plla mechanical properties measured by instrumented indentation tests

Poly(L‐lactide) is a widely studied biomaterial, currently approved for use in a range of medical devices. Its mechanical properties can be tailored giving the material different crystallinity degrees. PLLA presents a complex non‐linear behaviour that depends not only on structural parameters such as crystallinity degree but also on external parameters such as strain rate and temperature. Failure of polymeric implants is attributed to their intrinsic time‐dependent performance under static loading conditions.

Corrosión inducida por contaminantes hidrosolubles en la intercara metal/pintura

El objetivo principal de este trabajo es profundizar en el conocimiento del fenómeno de la corrosión subpelicular inducida por contaminantes hidrosolubles en la intercara metal/pintura. La contaminación salina del substrato es una situación común en la práctica: la superficie metálica suele estar expuesta a atmósferas contaminadas antes de ser recubierta, limpieza previa del metal con abrasivos contaminados, etc. La eliminación total de estos contaminantes resulta muy difícil de conseguir incluso con las técnicas más sofisticadas de limpieza. Esta investigación se centra en la determinación del efecto de la naturaleza del contaminante y la naturaleza y espesor del recubrimiento en el proceso de corrosión subpelicular del acero. En la investigación se utilizaron dos barnices de naturaleza diferente: poliuretano y vinílico; y se aplicaron a tres espesores diferentes. Los contaminantes empleados en este trabajo fueron: NaCl, NH4C1, CaCl2, Na2S04, (NH4)2S04, NaN03, NH4N03, Ca(N03)2. Los ensayos se realizaron en una cámara de condensación de humedad permanente. Los tiempos de exposición fueron 100, 300 y 600 horas. La velocidad de corrosión se evaluó gravimétricamente, mediante la técnica de pérdida de peso. Se realizaron estudios de permeabilidad al oxígeno y al agua de películas libres de substrato, evaluación de la velocidad de corrosión de probetas sin pintar inmersas en soluciones salinas de los contaminantes seleccionados, conductividad de dichas soluciones salinas, solubilidad del oxígeno en las soluciones salinas, adherencia en seco y en húmedo a diferentes tiempos de exposición. Se aporta evidencia respecto al control ejercido en el proceso corrosivo por el oxígeno que permea a través de la película, mientras que la permeación de agua controla la pérdida de adherencia del recubrimiento. Ambas permeabilidades dependen de la naturaleza del recubrimiento y de su espesor. Se ha investigado la influencia de la naturaleza del contaminante en la intercara metal/pintura. La naturaleza del catión parece quedar enmascarada por el efecto definitivo del anión. La concentración salina ejerce asimismo un efecto importante en la corrosión subpelicular. ABSTRACT The main aim of this work is to study in depth the knowledge of underfilm corrosión induced by hydrosoluble contaminants at the metal/paint Ínterface. The saline contamination of the substrate is a common situation in practice: metallic surfaces use to be exposed to polluted atmospheres, previous cleaning of the metal with contaminated abrasives, etc. Total elimination of these contaminants is hard to obtain even with modern cleaning techniques. This research is focused in determining the effect of contaminant nature, coating nature and its thickness on the steel underfilm corrosión process. In this work we used two varnishes with different nature: polyurethane and vinyl; they were applied in three different thicknesses. The saline contaminants employed were: NaCl, NH4C1, CaCl2, Na2S04, (NH4)2S04, NaN03, NH4N03/ Ca(N03)2. The tests were carried out in a condensation humidity chamber. The period of exposure were 100, 300 and 600 hours. Corrosión rate was assessed by weight loss. Simultaneously, studies on oxygen and water permeability of free films, assessing on corrosión rate of uncoated samples immersed in saline solutions of the selected contaminants, conductivity of these solutions, oxygen solubility in saline solutions, wet and dry adhesión of the polyurethane varnish at different periods of exposure, were carried out. There is clear evidence about control on corrosión process of oxygen that passes through the coating, while the passing of water controls the loss of adhesión of the coating. Both, water and oxygen permeation, depend on the nature and thickness of the coating. It has been researched the inf luence of the nature of contaminant at the metal/paint interface. The nature of the catión seems to be "masked" by the definitive effect of the nature of anión. The saline concentration also exerts an important effect on underfilm corrosión.

NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron–sulfur cluster N2 to quinone

The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron–sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[3H]pyridaben ([3H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron–sulfur cluster N2 to quinone.

The Golgi and Endoplasmic Reticulum Remain Independent during Mitosis in HeLa Cells

Partitioning of the mammalian Golgi apparatus during cell division involves disassembly at M-phase. Despite the importance of the disassembly/reassembly pathway in Golgi biogenesis, it remains unclear whether mitotic Golgi breakdown in vivo proceeds by direct vesiculation or involves fusion with the endoplasmic reticulum (ER). To test whether mitotic Golgi is fused with the ER, we compared the distribution of ER and Golgi proteins in interphase and mitotic HeLa cells by immunofluorescence microscopy, velocity gradient fractionation, and density gradient fractionation. While mitotic ER appeared to be a fine reticulum excluded from the region containing the spindle-pole body, mitotic Golgi appeared to be dispersed small vesicles that penetrated the area containing spindle microtubules. After cell disruption, M-phase Golgi was recovered in two size classes. The major breakdown product, accounting for at least 75% of the Golgi, was a population of 60-nm vesicles that were completely separated from the ER using velocity gradient separation. The minor breakdown product was a larger, more heterogenously sized, membrane population. Double-label fluorescence analysis of these membranes indicated that this portion of mitotic Golgi also lacked detectable ER marker proteins. Therefore we conclude that the ER and Golgi remain distinct at M-phase in HeLa cells. To test whether the 60-nm vesicles might form from the ER at M-phase as the result of a two-step vesiculation pathway involving ER–Golgi fusion followed by Golgi vesicle budding, mitotic cells were generated with fused ER and Golgi by brefeldin A treatment. Upon brefeldin A removal, Golgi vesicles did not emerge from the ER. In contrast, the Golgi readily reformed from similarly treated interphase cells. We conclude that Golgi-derived vesicles remain distinct from the ER in mitotic HeLa cells, and that mitotic cells lack the capacity of interphase cells for Golgi reemergence from the ER. These experiments suggest that mitotic Golgi breakdown proceeds by direct vesiculation independent of the ER.

Phosphatidylcholine Synthesis Influences the Diacylglycerol Homeostasis Required for Sec14p-dependent Golgi Function and Cell Growth

Phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids in eukaryotic cells and thus have major roles in the formation and maintenance of vesicular membranes. In yeast, diacylglycerol accepts a phosphocholine moiety through a CPT1-derived cholinephosphotransferase activity to directly synthesize phosphatidylcholine. EPT1-derived activity can transfer either phosphocholine or phosphoethanolamine to diacylglcyerol in vitro, but is currently believed to primarily synthesize phosphatidylethanolamine in vivo. In this study we report that CPT1- and EPT1-derived cholinephosphotransferase activities can significantly overlap in vivo such that EPT1 can contribute to 60% of net phosphatidylcholine synthesis via the Kennedy pathway. Alterations in the level of diacylglycerol consumption through alterations in phosphatidylcholine synthesis directly correlated with the level of SEC14-dependent invertase secretion and affected cell viability. Administration of synthetic di8:0 diacylglycerol resulted in a partial rescue of cells from SEC14-mediated cell death. The addition of di8:0 diacylglycerol increased di8:0 diacylglycerol levels 20–40-fold over endogenous long-chain diacylglycerol levels. Di8:0 diacylglcyerol did not alter endogenous phospholipid metabolic pathways, nor was it converted to di8:0 phosphatidic acid.

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.