Synthesis, storage and degradation of neutral lipids in yeast

The single cell eukaryote Saccharomyces cerevisiae is an attractive model to study the complex process of neutral lipid (triacylglycerol and steryl ester) synthesis, storage and turnover. In mammals, defects in the metabolism of these lipids are associated with a number of severe diseases such as atherosclerosis, obesity and type II diabetes. Since the yeast harbors many counterparts of mammalian enzymes involved in these pathways, conclusions drawn from research with the microorganism can be readily applied to the higher eukaryotic system. Here, we summarize our current knowledge of yeast neutral lipid metabolism, report about pathways and enzymes contributing to formation and degradation of triacylglycerols and steryl esters, and describe storage of these components in lipid particles. The interplay of different subcellular compartments in neutral lipid metabolism, regulatory aspects of this process and cell biological consequences of dysfunctions will be discussed.     

Anthocyanins: Structural characteristics that result in unique metabolic patterns and biological activities

Interest in anthocyanins has increased immensely during the past decade. From these studies, it is clear that anthocyanins have unique properties: Anthocyanins are absorbed intact and absorption can be saturated; acylation of anthocyanins lowers their apparent absorption; anthocyanidin diglycosides in the form of sambubioside or rutinoside impart increased stability to the anthocyanin molecule; and the quantities excreted in urine are less than 0.1% of intake. However, 60–90% of the anthocyanins may disappear from the gastrointestinal tract within 4 h after a meal. What happens to the bulk of the anthocyanins that disappear is not clear. Degradation accounts for a part of this disappearance, but differs for the various aglycones and may be modified further by the nature of the aglycone glycosylation, which further complicates our understanding of this process. Anthocyanins may play an important role in health promotion in terms of obesity prevention, cardiovascular health, anti-inflammatory and anti-cancer effects.     

Anthocyanins: structural characteristics that result in unique metabolic patterns and biological activities

Interest in anthocyanins has increased immensely during the past decade. From these studies, it is clear that anthocyanins have unique properties: Anthocyanins are absorbed intact and absorption can be saturated; acylation of anthocyanins lowers their apparent absorption; anthocyanidin diglycosides in the form of sambubioside or rutinoside impart increased stability to the anthocyanin molecule; and the quantities excreted in urine are less than 0.1% of intake. However, 60-90% of the anthocyanins may disappear from the gastrointestinal tract within 4 h after a meal. What happens to the bulk of the anthocyanins that disappear is not clear. Degradation accounts for a part of this disappearance, but differs for the various aglycones and may be modified further by the nature of the aglycone glycosylation, which further complicates our understanding of this process. Anthocyanins may play an important role in health promotion in terms of obesity prevention, cardiovascular health, anti-inflammatory and anti-cancer effects.     

Insights into ALS pathomechanisms: from flies to humans

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease causing the death of motor neurons with consequent muscle atrophy and paralysis. Several neurodegenerative diseases have been modeled in Drosophila and genetic studies on this model organism led to the elucidation of crucial aspects of disease mechanisms. ALS, however, has lagged somewhat behind possibly because of the lack of a suitable genetic model. We were the first to develop a fly model for ALS and over the last few years, we have implemented and used this model for a large scale, unbiased modifier screen. We also report an extensive bioinformatic analysis of the genetic modifiers and we show that most of them are associated in a network of interacting genes controlling known as well as novel cellular processes involved in ALS pathogenesis. A similar analysis for the human homologues of the Drosophila modifiers and the validation of a subset of them in human tissues confirm and expand the significance of the data for the human disease. Finally, we analyze a possible application of the model in the process of therapeutic discovery in ALS and we discuss the importance of novel “non-obvious” models for the disease.     

Insights into ALS pathomechanisms: from flies to humans

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease causing the death of motor neurons with consequent muscle atrophy and paralysis. Several neurodegenerative diseases have been modeled in Drosophila and genetic studies on this model organism led to the elucidation of crucial aspects of disease mechanisms. ALS, however, has lagged somewhat behind possibly because of the lack of a suitable genetic model. We were the first to develop a fly model for ALS and over the last few years, we have implemented and used this model for a large scale, unbiased modifier screen. We also report an extensive bioinformatic analysis of the genetic modifiers and we show that most of them are associated in a network of interacting genes controlling known as well as novel cellular processes involved in ALS pathogenesis. A similar analysis for the human homologues of the Drosophila modifiers and the validation of a subset of them in human tissues confirm and expand the significance of the data for the human disease. Finally, we analyze a possible application of the model in the process of therapeutic discovery in ALS and we discuss the importance of novel “non-obvious” models for the disease.     

Characterization of the yam tuber storage proteins from Dioscorea batatas exhibiting unique lectin activities

Four major proteins designated DB1, DB2, DB3, and DB4 were isolated and characterized from the yam tuber Dioscorea batatas. The ratios of their yields were 20:50:20:10. DB1 was a mannose-binding lectin (20 kDa) consisting of 10-kDa subunits and was classified as the monocot mannose-binding lectin family. DB2, accounting for 50% of the total protein, was the storage protein, commonly called dioscorins consisting of a 31-kDa subunit. On the basis of amino acid sequence, DB2 was classified to be dioscorin A. DB3 was a maltose-binding lectin, having an apparent molecular mass of 120 kDa and composed of a 66-kDa subunit and two 31-kDa subunits (DB3S). The 66-kDa subunit was further composed of two 31-kDa subunits (DB3L) cross-linked by disulfide bonds. DB3L and DB3S (242 and 241 amino acid residues, respectively) were homologous with each other with 72% sequence identity. They showed a sequence homology to dioscorin B and dioscorin A from Dioscorea alata, with 90 and 93% identity, respectively, and to carbonic anhydrase from Arabidopsis thaliana with about 45% identity. DB3S had one intrachain disulfide bond located at Cys(28)-Cys(187), whereas DB3L had one interchain disulfide bond (Cys(40)-Cys(40)') in addition to the intrachain disulfide bond (Cys(28)-Cys(188)) to form a 66-kDa subunit. DB1 and DB3 agglutinated rabbit erythrocytes at 2.7 and 3.9 microg/ml, respectively. Despite the structural homology between DB2 and DB3, DB2 had no lectin activity. The 66-kDa subunit itself revealed the full hemagglutinating activity of DB3, indicating that DB3L but not DB3S was responsible for the activity. The hemagglutinating activity of DB3 required Ca(2+) ions and was exclusively inhibited by maltose and oligomaltoses (e.g. maltopentaose and maltohexaose) but not by d-glucose. DB3 could not be classified into any known plant lectin family. DB4 was a chitinase, homologous to an acidic chitinase from Dioscorea japonica. DB1, DB2, and DB3 did not show any activity of carbonic anhydrase, amylase, or trypsin inhibitor activity. These results show that two of the four major proteins isolated from the yam tubers D. batatas have unique lectin activities.     

Adenine aminohydrolase from Leishmania donovani: unique enzyme in parasite purine metabolism

Adenine aminohydrolase (AAH) is an enzyme that is not present in mammalian cells and is found exclusively in Leishmania among the protozoan parasites that infect humans. AAH plays a paramount role in purine metabolism in this genus by steering 6-aminopurines into 6-oxypurines. Leishmania donovani AAH is 38 and 23% identical to Saccharomyces cerevisiae AAH and human adenosine deaminase enzymes, respectively, catalyzes adenine deamination to hypoxanthine with an apparent K(m) of 15.4 μM, and does not recognize adenosine as a substrate. Western blot analysis established that AAH is expressed in both life cycle stages of L. donovani, whereas subcellular fractionation and immunofluorescence studies confirmed that AAH is localized to the parasite cytosol. Deletion of the AAH locus in intact parasites established that AAH is not an essential gene and that Δaah cells are capable of salvaging the same range of purine nucleobases and nucleosides as wild type L. donovani. The Δaah null mutant was able to infect murine macrophages in vitro and in mice, although the parasite loads in both model systems were modestly reduced compared with wild type infections. The Δaah lesion was also introduced into a conditionally lethal Δhgprt/Δxprt mutant in which viability was dependent on pharmacologic ablation of AAH by 2'-deoxycoformycin. The Δaah/Δhgprt/Δxprt triple knock-out no longer required 2'-deoxycoformycin for growth and was avirulent in mice with no persistence after a 4-week infection. These genetic studies underscore the paramount importance of AAH to purine salvage by L. donovani.     

Evolution of metabolic diversity: Insights from microbial polyketide synthases

Polyketides are a family of complex natural products that are built from simple carboxylic acid building blocks. In microorganisms, the majority of these secondary metabolites are produced by exceptionally large, multifunctional proteins termed polyketide synthases (PKSs). Each unit of a type I PKS assembly line resembles a mammalian type fatty acid synthase (FAS), although certain domains are optionally missing. The evolutionary analysis of microbial PKS has revealed a long joint evolution process of PKSs and FASs. The phylogenomic analysis of modular type I PKSs as the most widespread PKS type in bacteria showed a large impact of gene duplications and gene losses on the evolution of type I PKS in different bacterial groups. The majority of type I PKSs in actinobacteria and cyanobacteria may have evolved from a common ancestor, whereas in proteobacteria most type I PKSs were acquired from other bacterial groups. The modularization of type I PKSs almost unexceptionally started with multiple duplications of a single ancestor module. The repeating modules represent ideal platforms for recombination events that can lead to corresponding changes in the actual chemistry of the products. The analysis of these “natural reprogramming” events of PKSs may assist in the development of concepts for the biocombinatorial design of bioactive compounds.     

Insights into Maize LEA proteins: from proteomics to functional approaches

LEA (late embryogenesis abundant) proteins participate in plant stress tolerance responses, but the mechanisms by which protection occurs are not fully understood. In the present work the unfolded proteins from maize dry embryos were analyzed by mass spectrometry. Twenty embryo proteins were identified, and among them 13 corresponded to LEA-type proteins. We selected three major LEA proteins, Emb564, Rab17 and Mlg3, belonging to groups 1, 2 and 3, respectively, and we undertook a comparative study in order to highlight differences among them. The post-translational modifications of native proteins were analyzed and the anti-aggregation properties of recombinant Emb564, Rab17 and Mgl3 proteins were evaluated in vitro. In addition, the protective effects of the LEA proteins were assessed in living cells under stress in Escherichia coli cells and in Nicotiana bentamiana leaves agroinfiltrated with fluorescent LEA-green fluorescent protein (GFP) fusions. Protein visualization by confocal microscopy indicated that cells expressing Mg3-GFP showed reduced cell shrinkage effects during dehydration and that Rab17-GFP co-localized to leaf oil bodies after heat shock. Overall, the results highlight differences and suggest functional diversity among maize LEA groups.     

Regulation of K+ channel activities in plants: from physiological to molecular aspects

Plant voltage-gated channels belonging to the Shaker family participate in sustained K+ transport processes at the cell and whole plant levels, such as K+ uptake from the soil solution, long-distance K+ transport in the xylem and phloem, and K+ fluxes in guard cells during stomatal movements. The attention here is focused on the regulation of these transport systems by protein-protein interactions. Clues to the identity of the regulatory mechanisms have been provided by electrophysiological approaches in planta or in heterologous systems, and through analogies with their animal counterparts. It has been shown that, like their animal homologues, plant voltage-gated channels can assemble as homo- or heterotetramers associating polypeptides encoded by different Shaker genes, and that they can bind auxiliary subunits homologous to those identified in mammals. Furthermore, several regulatory processes (involving, for example, protein kinases and phosphatases, G proteins, 14-3-3s, or syntaxins) might be common to plant and animal Shakers. However, the molecular identification of plant channel partners is still at its beginning. This paper reviews current knowledge on plant K+ channel regulation at the physiological and molecular levels, in the light of the corresponding knowledge in animal cells, and discusses perspectives for the deciphering of regulatory networks in the future.     

Effect of cryopreservation on lipids and some physiological features of spermatozoa from rams pastured in highlands and in valleys

The effect of low temperature preservation on the motility and morphology of acrosomes, acrosomal proteolytic activity, phospholipid and fatty acid composition of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE), and the cholesterol/phospholipid molar ratio in sperm from rams housed in the highlands or in the valleys, were studied. The indices of motility and morphological integrity of sperm from highland rams were much greater compared with those of valley rams. Phosphatidyl choline (PC) of the highland rams was more unsaturated, while PE was more saturated compared with those of valley rams. Cryopreservation of the sperm from highland rams significantly increased the content of choline plasmalogen, accompanied by a slight rise in the levels of lysophosphatidyl choline (LPC) and phosphatidyl inositol (PI) in their sperm. The fatty acid composition altered following cryopreservation. These variations were mainly due to a decrease in the amount of docosahexaenic acid and an increase in the amounts of linoleic and palmitic fatty acids. The results may be indicative of the fact that the alterations in the sperm of the valley rams were more pronounced and they may be attributed to the structural features of the sperm, as well as a reduced concentration of oxygen in the organs and tissues of the highland rams.     

Insights into physiological responses of mosses Physcomitrella patens and Pohlia drummondii to lichen secondary metabolites

Protoplasma - It is widely accepted that allelopathy among mosses and lichens do exist due to its similar ecological needs, though it is rarely documented. With an aim to test whether there is an...     

Insights into stereochemical features of sulphated carbohydrates: X-ray crystallographic and modelling investigations

The three-dimensional structures of the 2-, 3-, 4- and 6-monosulphatesof methyl -D-galactopyranoside have been determined by X-raycrystallography; the first two as the sodium salt, the thirdas both the sodium and potassium salts, and the fourth as apotassium salt. These represent the principal sulphated monomersof the carrageenan polysaccharides. The results extend our knowledgeof the stereochemical features, such as ring conformation, sulphategeometry, hydrogen bonding and cation co-ordination, which characterizesulphated monosaccharides. The stereochemical data have beenused to derive a mean geometry of the O-sulphate group and aset of force constants for use in molecular mechanics calculationson sulphated monosaccharides. These may be used in an extrapolationof the populations of stable conformers of related oligo- andpolysaccharides. crystal structures molecular mechanics O-sulphated carbohydrates X-ray diffraction     

Microplate assay for quantitation of neutral lipids in extracts from microalgae

Lipid quantitation is widespread in the algae literature, but popular methods such as gravimetry, gas chromatography and mass spectrometry (GC–MS), and Nile red cell staining suffer drawbacks, including poor quantitation of neutral lipids, expensive equipment, and variable results among algae species, respectively. A high-throughput microplate assay was developed that uses Nile red dye to quantify neutral lipids that have been extracted from algae cells. Because the algal extracts contained pigments that quenched Nile red fluorescence, a mild bleach solution was used to destroy pigments, resulting in a nearly linear response for lipid quantities in the range of 0.75 to 40 μg. Corn oil was used as a standard for quantitation, although other vegetable oils displayed a similar response. The assay was tested on lipids extracted from three species of Chlorella and resulted in close agreement with triacylglycerol (TAG) levels determined by thin layer chromatography. The assay was found to more accurately measure algal lipids conducive to biodiesel production and nutrition applications than the widely used gravimetric assay. Assay response was also consistent among different species, in contrast to Nile red cell staining procedures.