Maria V. Calvo

High-yield production of mono- and di-oleylglycerol by lipase-catalyzed hydrolysis of triolein

Abstract Lipases from several origins—in soluble form or immobilized on solid supports of different aquaphilicity—have been used for the production of mono- and di-oleylglycerol by hydrolysis of triolein. The porcine pancreatic lipase adsorbed on Celite was found to be the most effective biocatalyst tested. High activity and excellent selectivity toward the formation of intermediate acylglycerols were achieved (the conversion to diolein plus monoolein was 79% after 5 h of reaction). The time course of triolein hydrolysis with free and immobilized lipases was analyzed using a sequential kinetic model, and the apparent first-order rate constants were calculated. The differences found in the rate constants were related to differences in the regioselectivity of the enzymes and/or the aquaphilicity of the supports.

Analysis of Tween 80 as an esterase/ lipase substrate for lipolytic activity assay

The Tween 80 assay to detect lipolytic activities in agar media was evaluated. A spectrophotometric assay for Tween 80 hydrolysis was established. The specific activities with Tween 80, as well as with some conventional lipase-type and esterase-type substrates, were measured using several lipases and esterases. The activity with Tween 80 was similar to that obtained with p-nitrophenyl butyrate; the enzyme activities with both substrates were between the esterase and lipase categories.

Total milk fat extraction and quantification of polar and neutral lipids of cow, goat, and ewe milk by using a pressurized liquid system and chromatographic techniques

Although milk polar lipids such as phospholipids and sphingolipids located in the milk fat globule membrane constitute 0.1 to 1% of the total milk fat, those lipid fractions are gaining increasing interest because of their potential beneficial effects on human health and technological properties. In this context, the accurate quantification of the milk polar lipids is crucial for comparison of different milk species, products, or dairy treatments. Although the official International Organization for Standardization-International Dairy Federation method for milk lipid extraction gives satisfactory results for neutral lipids, it has important disadvantages in terms of polar lipid losses. Other methods using mixtures of solvents such as chloroform:methanol are highly efficient for extracting polar lipids but are also associated with low sample throughput, long time, and large solvent consumption. As an alternative, we have optimized the milk fat extraction yield by using a pressurized liquid extraction (PLE) method at different temperatures and times in comparison with those traditional lipid extraction procedures using 2:1 chloroform:methanol as a mixture of solvents. Comparison of classical extraction methods with the developed PLE procedure were carried out using raw whole milk from different species (cows, ewes, and goats) and considering fat yield, fatty acid methyl ester composition, triacylglyceride species, cholesterol content, and lipid class compositions, with special attention to polar lipids such as phospholipids and sphingolipids. The developed PLE procedure was validated for milk fat extraction and the results show that this method performs a complete or close to complete extraction of all lipid classes and in less time than the official and Folch methods. In conclusion, the PLE method optimized in this study could be an alternative to carry out milk fat extraction as a routine method.

A Novel Role of the Potyviral Helper Component Proteinase Contributes To Enhance the Yield of Viral Particles

ABSTRACT The helper component proteinase (HCPro) is an indispensable, multifunctional protein of members of the genus Potyvirus and other viruses of the family Potyviridae. This viral factor is directly involved in diverse steps of viral infection, such as aphid transmission, polyprotein processing, and suppression of host antiviral RNA silencing. In this paper, we show that although a chimeric virus based on the potyvirus Plum pox virus lacking HCPro, which was replaced by a heterologous silencing suppressor, caused an efficient infection in Nicotiana benthamiana plants, its viral progeny had very reduced infectivity. Making use of different approaches, here, we provide direct evidence of a previously unknown function of HCPro in which the viral factor enhances the stability of its cognate capsid protein (CP), positively affecting the yield of virions and consequently improving the infectivity of the viral progeny. Site-directed mutagenesis revealed that the ability of HCPro to stabilize CP and enhance the yield of infectious viral particles is not linked to any of its previously known activities and helped us to delimit the region of HCPro involved in this function in the central region of the protein. Moreover, the function is highly specific and cannot be fulfilled by the HCPro of a heterologous potyvirus. The importance of this novel requirement in regulating the sorting of the viral genome to be subjected to replication, translation, and encapsidation, thus contributing to the synchronization of these viral processes, is discussed. IMPORTANCE Potyviruses form one of the most numerous groups of plant viruses and are a major cause of crop loss worldwide. It is well known that these pathogens make use of virus-derived multitasking proteins, as well as dedicated host factors, to successfully infect their hosts. Here, we describe a novel requirement for the proper yield and infectivity of potyviral progeny. In this case, such a function is performed by the extensively studied viral factor HCPro, which seems to use an unknown mechanism that is not linked to its previously described activities. To our knowledge, this is the first time that a factor different from capsid protein (CP) has been shown to be directly involved in the yield of potyviral particles. Based on the data presented here, we hypothesize that this capacity of HCPro might be involved in the coordination of mutually exclusive activities of the viral genome by controlling correct assembly of CP in stable virions.

Kinetic and enantioselective behaviour of isoenzymes A and B from Candida rugosa lipase in the hydrolysis of lipids and esters

The isoenzymes A and B from C. rugosa lipase have been kinetically characterized by non-linear regression methods measuring the hydrolysis of tributyrin (lipase activity), p-nitrophenyl acetate (esterase activity) and methyl 2-chloropropionate (enantioselectivity). With tributyrin as substrate, the specificity constant (k::f/K%P) for lipase A is about 2-fold that of the lipase B. This confirms the higher specificity of lipase A for substrates containing butyric acid moieties. However, the kinetic behaviour of both isoenzymes for the hydrolysis of pnitrophenyl acetate is quite similar, with lipase A showing a slight preference for this substrate. Kinetic analysis with the substrates methyl (R)-(+) and methyl (S)-(-) 2-chloropropionates showed that both isoenzymes exhibit stereospecificity for the enantiomer R and display nonMichaelian kinetics of a sigmoidal type. Since this behaviour precludes the usual treatment on the basis of the enantiomeric ratio [(Vmax/Km)&( Vmax/Km)s~ow], enantioselectivity was quantified using the areas under the v-[S] curves. Thus, the ratio between the areas under the curves for the two enantiomers assayed increases from 1.27 for isoenzyme A to 1.68 for isoenzyme B. This result clearly shows that isolation and purification of isoenzymes may enhance the enantioselective properties of an enzymatic system.

Effect of chemical modification of isoenzymes A and B from C. rugosa on their activity and stability

SummaryLipases A and B from C.rugosa have been chemically modified with octanoyl chloride or polyethylene glycol (molecular weight: 750 or 5000). Although the hydrolytic activity was diminished, modification gave rise in some cases to preparations with increased synthetic activity in hexane. Furthermore, PEG5000-lipases A and B were significantly more stable (up to 20-fold) at alkaline pH values than the native enzymes.

Single Amino Acid Changes in the 6K1-CI Region Can Promote the Alternative Adaptation of Prunus- and Nicotiana-Propagated Plum pox virus C Isolates to Either Host

Plum pox virus (PPV) C is one of the less common PPV strains and specifically infects cherry trees in nature. Making use of two PPV-C isolates that display different pathogenicity features, i.e., SwCMp, which had been adapted to Nicotiana species, and BY101, which had been isolated from cherry rootstock L2 (Prunus lannesiana) and propagated only in cherry species, we have generated two infective full-length cDNA clones in order to determine which viral factors are involved in the adaptation to each host. According to our results, the C-P3(PIPO)/6K1/N-CI (cylindrical inclusion) region contains overlapping but not coincident viral determinants involved in symptoms development, local viral amplification, and systemic movement capacity. Amino acid changes in this region promoting the adaptation to N. benthamiana or P. avium have trade-off effects in the alternative host. In both cases, adaptation can be achieved through single amino acid changes in the NIapro protease recognition motif between 6K1 and CI or in nearby sequences. Thus, we hypothesize that the potyvirus polyprotein processing could depend on specific host factors and the adaptation of PPV-C isolates to particular hosts relies on a fine regulation of the proteolytic cleavage of the 6K1-CI junction.

Bioactive Milk Lipids

Current information about the nutritional composition of milk fat is required for the consumer and therefore essential for the successful development of dairy industries as well as marketing their products. The progress in the knowledge concerning some milk fat components that possess biological properties and health benefits beyond their nutritional significance, has a growing interest in the dairy industry to design and formulate products that incorporate specific bioactive components derived from milk. In the last two decades, special attention has been paid to the fatty acid (FA) composition on all short, medium chain and branched fatty acid as well as linoleic conjugated acid (CLA) in milk and dairy products. Trans monounsaturated fatty acids profiles from dairy fat have gained increasing relevance because they may have metabolic properties distinct from those of other origins, hydrogenation reaction for instance. Other minor lipid compounds with biological activity, phospholipids and cholesterol are part of the fat globule membrane. This review summarizes the current knowledge in milk fat research with a brief overview of the importance of dairy lipids as biological molecules with emphasis on the different bioactive compounds present in this fraction.

Susceptibility to excitotoxicity in aged hippocampal cultures and neuroprotection by non-steroidal anti-inflammatory drugs: role of mitochondrial calcium.

Brain damage after insult and cognitive decline are related to excitotoxicity and strongly influenced by aging, yet mechanisms of aging‐dependent susceptibility to excitotoxicity are poorly known. Several non‐steroidal anti‐inflammatory drugs (NSAIDs) may prevent excitotoxicity and cognitive decline in the elderly by an unknown mechanism. Interestingly, after several weeks in vitro, hippocampal neurons display important hallmarks of neuronal aging in vivo. Accordingly, rat hippocampal neurons cultured for several weeks were used to investigate mechanisms of aging‐related susceptibility to excitotoxicity and neuroprotection by NSAIDs. We found that NMDA increased cytosolic Ca2+ concentration in young, mature and aged neurons but only promoted apoptosis in aged neurons. Resting Ca2+ levels and responses to NMDA increased with time in culture which correlated with changes in expression of NMDA receptor subunits. In addition, NMDA promoted mitochondrial Ca2+ uptake only in aged cultures. Consistently, specific inhibition of mitochondrial Ca2+ uptake decreased apoptosis. Finally, we found that a series of NSAIDs depolarized mitochondria and inhibited mitochondrial Ca2+ overload, thus preventing NMDA‐induced apoptosis in aged cultures. We conclude that mitochondrial Ca2+ uptake is critical for age‐related susceptibility to excitotoxicity and neuroprotection by NSAIDs.

A temperature-controlled amplicon system derived from Plum pox potyvirus.

The control of replication can facilitate a viral amplicon to reach high expression levels by enabling the virus to escape host defence mechanisms and reducing the deleterious effects of viral infection. We have developed a novel system to regulate amplicon expression by controlling the temperature of plant growth. Nicotiana benthamiana plants were transformed at two different temperatures with a cDNA copy of the Plum pox potyvirus genome harbouring the open reading frame 2 of Porcine circovirus 2 between the nuclear inclusion protein b and coat protein coding sequences. Although transformation at 27 degrees C mainly yielded nonexpressing amplicons, lines with a tight control of amplicon expression were obtained. Viral replication was not detected in these plants when germinated at 28 degrees C, but was observed when the plants were shifted to 20 degrees C. In lines transformed at 24 degrees C, although the amplicon was expressed at 28 degrees C, viral accumulation was low and caused only minor growing defects. Viral replication was enhanced in these plants by shifting the temperature to 20 degrees C; under such conditions, the amplicon reached higher and more persistent expression levels than in plants transformed at 27 degrees C. These results demonstrate the utility of temperature regulation to control viral amplicon expression.