Lead and copper effects on lipid metabolism in cultured lichen photobionts with different phosphorus status

We evaluated the ability of heavy metals (copper, lead) to alter lipid metabolism in four algal lichen photobionts following short term exposure. Metal concentrations (10 microM) were equivalent to environmentally relevant levels that have been reported to have effects on intact algae. The algae were grown under normal or deficient phosphate conditions to assess any interactions with the heavy metal stress. Given the frequent sensitivity of lichens to copper and lead, there were surprisingly small changes on lipid metabolism, as assessed by radiolabelling from [1-14C]acetate. The main effects, which were seen in a number of cases, were an overall inhibition of total lipid labelling and a relative increase in the labelling of triacylglycerols in the non-polar fraction. Both of these changes can be viewed as reflecting general toxicity of heavy metals. The Coccomyxa photobiont species were more sensitive than Trebouxia species, which fits with the general distribution of the latter in lichens inhabiting harsh environments.     

Lipid metabolism in mucous-dwelling amitochondriate protozoa

Entamoeba, Giardia, and trichomonads are the prominent members of a group known as 'mucosal parasites'. While Entamoeba and Giardia trophozoites colonise the small intestine, trichomonads inhabit the genitourinary tracts of humans and animals. These protozoa lack mitochondria, well-developed Golgi complexes, and other organelles typical of higher eukaryotes. Nonetheless, they have developed unique metabolic pathways that allow them to survive and multiply in the small intestine and reproductive tracts by scavenging nutrients from the host. Various investigators have shown that these protozoa are unable to synthesise the majority of their own lipids and cholesterol de novo; rather, they depend mostly on supplies from outside sources. Therefore, questions of how they transport and utilise exogenous lipids for metabolic purposes are extremely important. There is evidence suggesting that these parasites can take up the lipids and cholesterol they need from lipoprotein particles present in the host and/or in the growth medium. Studies also support the idea that individual lipid and fatty acid molecules can be transported without the help of lipoproteins. Exogenous phospholipids have been shown to undergo fatty acid remodelling (by deacylation/reacylation reactions), which allows these protozoa to alter lipids, bypassing the synthesis of entirely new phospholipid molecules. In addition, many of these amitochondriates are, however, capable of elongating/desaturating long-chain fatty acids, and assembling novel glycophospholipid molecules. In this review, progress in various aspects of lipid research on these organisms is discussed. Attempts are also made to identify steps of lipid metabolic pathways that can be used to develop chemotherapeutic agents against these and other mucosal parasites.     

Three isocoumarins and a benzofuran from the cultured lichen mycobionts of Pyrenula sp

The spore-derived mycobionts of the lichen Pyrenula sp. were cultivated on a malt-yeast extract medium supplemented with 10% sucrose. The investigation of their metabolites resulted in isolation of four compounds, three isocoumarins and a biogenetically related benzofuran; their structures were determined by spectroscopic methods.     

Coccomyxa sp. (Chlorophyta: Chlorococcales), a new pathogen in mussels (Mytilus galloprovincialis) of Vigo estuary (Galicia, NW Spain)

In this work, we describe the occurrence of irregular shaped green aggregations in the mantle, gill filaments, adductor muscle, visceral mass and haemolymph of wild mussels (Mytilus galloprovincialis) collected from the Vigo estuary (Galicia, NW Spain). Microscopic examination of these masses revealed that they consist of intracellular green algae which are spherical to oval in shape, 5 μm in length and 3 μm in width, without flagella and with a smooth surface. The algal cells present a small single nucleus, a mitochondrion, 1-2 parietal chloroplasts and lack pyrenoids. Reproduction is by formation of 2-4 autospores or daughter cells. Pigment analysis reveals the presence of photopigments typical of green algae in addition to alloxanthin, diadinoxanthin and diatoxanthin. These carotenoids are noted for the first time in a parasitic chlorophyte. The signs of infection, together with the morphological observations, suggest that this parasitic algae may be Coccomyxa parasitica. However, further molecular studies are required for confirmation. This is the first report of Coccomyxa algae parasitizing the species M. galloprovincialis.     

Lipid metabolism during aging of high-alpha-linolenate-phenotype potato tubers

Previous studies demonstrated that high levels of alpha-linolenate in cell membranes of potato tubers (achieved by overexpressing fatty acid desaturases) enhances lipid peroxidation, oxidative stress, and tuber metabolic rate, effectively accelerating the physiological age of tubers. This study details the changes in lipid molecular species of microsomal and mitochondrial membranes from wild-type (WT) and high-alpha-linolenate tubers during aging. The microsomal and mitochondrial polar lipids of high-alpha-linolenate tubers were dominated by 18:3/18:3 and 16:0/18:3 molecular species. Relative to WT tubers, high-alpha-linolenate tubers had a substantially higher 16:0/18:n to 18:n/18:n molecular species ratio in mitochondria and microsomes, potentially reflecting a compensatory response to maintain membrane biophysical properties in the face of increased unsaturation. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) accounted for 53 and 37% of polar lipids, respectively, in mitochondria from younger WT and high-alpha-linolenate tubers. The relative proportions of these phospholipids (PL) did not change during aging of WT tubers. In contrast, PE increased to dominate the PL pool of mitochondria during aging of high-alpha-linolenate tubers. While aging effected an increase in mitochondrial 18:3-bearing PCs and PEs in WT tubers, the concentration of 18:3-bearing PCs fell with a concomitant increase in 18:3-bearing PEs during aging of high-alpha-linolenate tubers. These age- and high-alpha-linolenate-induced changes had no effect on the respiration rate and functional integrity of isolated mitochondria. Differential increases in the respiration rates of WT and high-alpha-linolenate tubers during aging were therefore a consequence of unsaturation-dependent alterations in the microenvironments of cells. Microsomal 18:3-bearing PCs, PEs, digalactosyldiacylglycerols (DGDG), and monogalactosyldiacylglycerols all increased in WT tubers during aging. In contrast, a selective loss of 18:3-bearing PCs and DGDGs from microsomes of high-alpha-linolenate tubers likely reflects a greater susceptibility of membranes to peroxidative catabolism during aging. Aging resulted in an increase in sterol/PL ratio in microsomes from WT tubers, due primarily to a decline in PL. In high-alpha-linolenate tubers, the increase in sterol/PL ratio during aging was due to increases in Delta 5-avenasterol and stigmasterol, indicating membrane rigidification and likely contributing to increased membrane permeability. Age-induced changes in 18:3-bearing lipids in membranes of transformed tubers are discussed relative to the development of oxidative stress and accelerated aging.     

Lipid metabolism in chromoplast membranes from the daffodil: Glycosylation and acylation

The non-photosynthetic chromoplast membranes from the corona ofNarcissus pseudonarcissus L. were investigated for their lipid synthetic capabilities. The following activities were detected: galactosylation of diacylglycerol and galactosydiacylglycerols, glycosylation of sterols, acylation of monogalactosyldiacylglycerol and steryl glycosides from an unknown endogenous donor, acylation of phospholipids from acyl-CoA, and acylation of phosphatidyl inositol from phosphatidyl choline. Furthermore, activities of an acyl thioesterase, a sugar epimerase, and a phospholipase A₂ were measured.Abbreviations MGDG monogalactosyldiacylglycerol - DGDG digalactosyldiacylglycerol - TGDG tri-and tetragalactosyldiacylglycerol - SG steryl glycoside - SL sulfolipid - ACP acyl carrier protein     

15N2 Incorporation and metabolism in the lichen Peltigera aphthosa Willd

The Nostoc in the cephalodia of the lichen Peltigera aphthosa Willd. fixed ¹⁵N₂ and the bulk of the nitrogen fixed was continuously transferred from it to its eukaryotic partners (a fungus and a green alga, Coccomyxa sp.). Kinetic studies carried out over the first 30 min, after exposure of isolated cephalodia to ¹⁵N₂, showed that highest initial ¹⁵N₂-labelling was into NH ₄ ⁺ . After 12 min little further increase in the NH ₄ ⁺ label occurred while that in the amide group of glutamine and in glutamate continued to increase. The ¹⁵N-labelling of the amino group of glutamine and of aspartate increased more slowly, followed by an increase in the labelling of alanine. When total incorporation of ¹⁵N-label was calculated, the overall pattern was found to be rather similar except that, throughout the experiment, the total ¹⁵N incorporated into glutamate was about six times greater than that into the amide group of glutamine. Pulse chase experiments, in which ¹⁴N₂ was added to cephalodia previously exposed to ¹⁵N₂, showed that the NH ₄ ⁺ pool rapidly became depleted of ¹⁵N-label, followed by decreases in the labelling of glutamate, the amide group of glutamine and aspartate. The ¹⁵N-labelling of alanine, however, continued to increase for a period. When isolated cephalodia were treated with L-methionine-SR-sulphoximine, an inhibitor of glutamine synthetase (EC 6.3.1.2), and azaserine, an inhibitor of glutamate synthase (EC 2.6.1.53), there was no detectable labelling in glutamine although the ¹⁵N-labelling of glutamate increased unimpaired. On treating the cephalodia with amino-oxyacetate, an inhibitor of aminotransferase activity, the alanine pool decreased. Evidence was obtained that glutamine synthetase and glutamate synthase were located in the Nostoc, and that glutamate dehydrogenase (EC 1.4.1.4) and various amino-transferases were located in the cephalodial fungus. Possible implications of these findings are discussed.Abbreviations MSX L-methionine-SR-sulphoximine - AOA amino-oxyacetate - HEPES N-2-hydroxymethylpiperazine-N-2-ethane sulphonic acid - Tris tris-(hydroxymethyl) methylamine - GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - GPT glutamate-pyruvate aminotransferase - APT aspartate-pyruvate aminotransferase - ADH alanine dehydrogenase - GOT glutamate-oxaloacetate aminotransferase     

A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to gamma-glutamylcysteine and lacks phytochelatin synthase activity

Phytochelatins are glutathione-derived, non-translationally synthesized peptides essential for cadmium and arsenic detoxification in plant, fungal and nematode model systems. Recent sequencing programs have revealed the existence of phytochelatin synthase-related genes in a wide range of organisms that have not been reported yet to produce phytochelatins. Among those are several cyanobacteria. We have studied one of the encoded proteins (alr0975 from Nostoc sp. strain PCC 7120) and demonstrate here that it does not possess phytochelatin synthase activity. Instead, this protein catalyzes the conversion of glutathione to gamma-glutamylcysteine. The thiol spectrum of yeast cells expressing alr0975 shows the disappearance of glutathione and the formation of a compound that by LC-MSMS analysis was unequivocally identified as gamma-glutamylcysteine. Purified recombinant protein catalyzes the respective reaction. Unlike phytochelatin synthesis, the conversion of glutathione to gamma-glutamylcysteine is not dependent on activation by metal cations. No evidence was found for the accumulation of phytochelatins in cyanobacteria even after prolonged exposure to toxic Cd2+ concentrations. Expression of alr0975 was detected in Nostoc sp. cells with an antiserum raised against the protein. No indication for a responsiveness of expression to toxic metal exposure was found. Taken together, these data provide further evidence for possible additional functions of phytochelatin synthase-related proteins in glutathione metabolism and provide a lead as to the evolutionary history of phytochelatin synthesis.     

Oxidative stress induces distinct physiological responses in the two Trebouxia phycobionts of the lichen Ramalina farinacea

Background and Aims

Most lichens form associations with Trebouxia phycobionts and some of them simultaneously include genetically different algal lineages. In other symbiotic systems involving algae (e.g. reef corals), the relative abundances of different endosymbiotic algal clades may change over time. This process seems to provide a mechanism allowing the organism to respond to environmental stress. A similar mechanism may operate in lichens with more than one algal lineage, likewise protecting them against environmental stresses. Here, the physiological responses to oxidative stress of two distinct Trebouxia phycobionts (provisionally named TR1 and TR9) that coexist within the lichen Ramalina farinacea were analysed.

Methods

Isolated phycobionts were exposed to oxidative stress through the reactive oxygen species propagator cumene hydroperoxide (CuHP). Photosynthetic pigments and proteins, photosynthesis (through modulated chlorophyll fluorescence), the antioxidant enzymes superoxide dismutase (SOD) and glutathione reductase (GR), and the stress-related protein HSP70 were analysed.

Key Results

Photosynthetic performance was severely impaired by CuHP in phycobionts, as indicated by decreases in the maximal PSII photochemical efficiency (F_v/F_m), the quantum efficiency of PSII (Φ_PSII) and the non-photochemical dissipation of energy (NPQ). However, the CuHP-dependent decay in photosynthesis was significantly more severe in TR1, which also showed a lower NPQ and a reduced ability to preserve chlorophyll a, carotenoids and D1 protein. Additionally, differences were observed in the capacities of the two phycobionts to modulate antioxidant activities and HPS70 levels when exposed to oxidative stress. In TR1, CuHP significantly diminished HSP70 and GR but did not change SOD activities. In contrast, in TR9 the levels of both antioxidant enzymes and those of HSP70 increased in response to CuHP.

Conclusions

The better physiological performance of TR9 under oxidative conditions may reflect its greater capacity to undertake key metabolic adjustments, including increased non-photochemical quenching, higher antioxidant protection and the induction of repair mechanisms.     

Infection status of dragonflies with Plagiorchis muris metacercariae in Korea.

Plagiorchis muris has been found in both house and field rats as well as in humans. The infection status of the second intermediate hosts of P. muris is prerequisite in understanding their biological features in an ecosystem. Six species of dragonflies were caught in a wide range of areas in Korea; and they were Sympetrum darwinianum, S. eroticum, S. pedomontanum, S. infuscatum, Pantala flavoscens, Calopteryx atrata, and Orthetrum albistylum speciosum. The occurrence of P. muris metacercariae in dragonflies was nationwide with various infection rates. The metacercarial burden of P. muris in the surveyed areas was the highest in S. eroticum followed by S. darwinianum, S. pedomontanum, and C. atrata. The highest infection rate by P. muris metacercariae was found in S. darwinianum followed by S. eroticum. The metacercarial burden was particularly heavy in the dragonflies found in Hamyang-gun and Kosong-gun, Kyongsangnam-do. It is, therefore, likely that dragonflies play a significant role as the second intermediate host in the life cycle of P. muris in Korea.     

饥饿胁迫对瓦氏黄颡鱼脂肪代谢的影响

在水温(252)℃条件下, 对初始体重为(23.652.82) g的瓦氏黄颡鱼进行30d饥饿处理, 于饥饿第0、第7、第15和第30天取样, 分析了饥饿胁迫对瓦氏黄颡鱼的生长、体成分、脂肪酸组成和脂肪代谢相关基因表达的影响. 结果表明: 饥饿胁迫显著降低了瓦氏黄颡鱼肥满度、脂体比及肝体指数(p0.05). 肌肉脂肪含量也随着饥饿时间的延长而下降(p0.05). 肝脏中的饱和脂肪酸和肌肉中的单不饱和脂肪酸显著下降, 而肝脏中多不饱和脂肪酸和单不饱和脂肪酸及肌肉中的多不饱和脂肪酸显著上升(p0.05). 此外, 肌肉中的n-6和n-3及肝脏中的n-6多不饱和脂肪酸显著上升, 而肝脏中的n-3多不饱和脂肪酸显著下降(p0.05), 表明瓦氏黄颡鱼饥饿期间主要消耗饱和脂肪酸及单不饱和脂肪酸供能, 保留多不饱和脂肪酸. 饥饿胁迫15-30d, 瓦氏黄颡鱼肝脏脂蛋白酯酶、肝酯酶及肉碱酰基转运酶mRNA表达显著高于对照组(0d)(p0.05), 而脂肪酸结合蛋白及脂肪酸合成酶mRNA的表达显著低于对照组(p0.05), 表明饥饿胁迫可能会促进肝脏脂肪分解供能, 降低脂肪的生物合成.       

Comparison of the bacterial Enhancin-like proteins from Yersinia and Bacillus spp. with a baculovirus Enhancin

Enhancins are a class of metalloproteases found in some baculoviruses that enhance viral infection by degrading the peritrophic membrane (PM) of the insect midgut. However, sequencing has revealed enhancin-like genes with 24-25% homology to viral enhancins, in the genomes of Yersinia pestis and Bacillus anthracis. AcMNPV does not encode enhancin therefore recombinant AcMNPV budded viruses (BVs) and polyhedra inclusion bodies (PIBs) were generated expressing the bacterial Enhancins. Bacterial Enhancins were found to be cytotoxic when compared to viral enhancin, however, larval bioassays suggested that the bacterial Enhancins did not enhance infection in the same way as viral Enhancin. This suggests that the bacterial Enhancins may have evolved a distinct biochemical function.     

Water status related photosynthesis and carbon isotope discrimination in species of the lichen genusPseudocyphellaria with green or blue-green photobionts and in photosymbiodemes

Summary Green lichens have been shown to attain positive net photosynthesis in the presence of water vapour while blue-green lichens require liquid water (Lange et al. 1986). This behaviour is confirmed not only for species with differing photobionts in the genusPseudocyphellaria but for green and blue-green photobionts in a single joined thallus (photosymbiodeme), with a single mycobiont, and also when adjacent as co-primary photobionts. The different response is therefore a property of the photobiont. The results are consistent with published photosynthesis/water content response curves. The minimum thallus water content for positive net photosynthesis appears to be much lower in green lichens (15% to 30%, related to dry weight) compared to blue-greens (85% to 100%). Since both types of lichen rehydrate to about 50% water content by water vapour uptake only green lichens will show positive net photosynthesis. It is proposed that the presence of sugar alcohols in green algae allow them to retain a liquid pool (concentrated solution) in their chloroplasts at low water potentials and even to reform it by water vapour uptake after being dried. The previously shown difference in δ¹³C values between blue-green and green lichens is also retained in a photosymbiodeme and must be photobiont determined. The wide range of δ¹³C values in lichens can be explained by a C₃ carboxylation system and the various effects of different limiting processes for photosynthetic CO₂ fixation. If carboxylation is rate limiting, there will be a strong discrimination of¹³CO₂, at high internal CO₂ partial pressure. The resulting very low δ¹³C values (-31 to-35‰) have been found only in green lichens which are able to photosynthesize at low thallus water content by equilibraiton with water vapour. When the liquid phase diffusion of CO₂ becomes more and more rate limiting and the internal CO₂ pressure decreases, the¹³C content of the photosynthates increases and less negative δ¹³C values results, as are found for blue-green lichens.     

Extracellular polysaccharides of Erwinia futululu,a bacterium associated with a fungal canker disease of Eucalyptus spp

Extracellular polysaccharides (EPSs) produced by an Erwinia spp. associated with a fungal canker disease of Eucalyptus were fractionated into two polysaccharides, one that was identified with that produced by Erwinia stewartii. The other has a similar structure, but with one terminal Glc residue replaced by pyruvic acid to give 4,6-O-[(R)-1-carboxyethylidene)-Galp. Their structures were determined using a combination of chemical and physical techniques including methylation analysis, periodate oxidation, low-pressure gel filtration and anion-exchange chromatographies, high-pH anion-exchange chromatography, mass spectrometry and 1D and 2D 1H NMR spectroscopy. The new polysaccharides, identified as EPS Futululu FF-1 and FF-2, have the following structures:The molecular weights of the polysaccharides range from 1.3-2.1x10(6) and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions.     

Vibrio gallaecicus sp. nov. isolated from cultured clams in north-western Spain

A group of three motile facultative anaerobic marine bacteria were isolated from cultured Manila clams (Ruditapes philippinarum) in Galicia, north-western Spain. The strains were characterized phenotypically and genotypically. Phylogenetic analysis of the 16S rRNA gene and four housekeeping genes, RNA polymerase α-chain (rpoA), RecA protein (recA), the α-subunit of bacterial ATP synthase (atpA) and the uridine monophosphate (UMP) kinase (pyrH), indicated that these strains were closely related to the Vibrio splendidus clade. The amplified fragment length polymorphism (AFLP) fingerprints, DNA–DNA hybridizations and phylogenies of the housekeeping and 16S rRNA gene sequences showed that the three strains represented a different species from all currently described vibrios. The new species could be differentiated from its nearest neighbours on the basis of several phenotypic features. The three strains are therefore a novel species within the genus Vibrio, for which the name Vibrio gallaecicus is proposed, with the type strain being VB 8.9^T(=CECT 7244^T=LMG 24045^T).     

Infection status with trematode metacercariae in pond smelts, Hypomesus olidus

Many Koreans usually eat raw pond smelts, Hypomesus olidus, in the winter. This study was performed to evaluate the infection status with trematode metacercariae in pond smelts from January 1998 through February 1999. Among 1,305 fish collected, 459 were purchased from wholesale dealers in Chinchon-gun, Chungchongbuk-do, and the rest of them were caught with a casting net in Soyangho (Lake), Taehoman (Bay) and Paekkokchosuchi (Pond). Seven species of trematode metacercariae including two unidentified ones were detected from 1,305 pond smelts. The number of detected trematode metacercariae according to the species are as follow: Clonorchis sinensis 8, Holostephanus nipponicus 7, Cyathocotyle orientalis 24, Diplostomum sp. 14, and Metorchis orientalis 7. From the above results, it was confirmed that H. olidus plays a role as the second intermediate host of some kinds of trematode including C. sinensis in Korea. Our report shows possible clonorchiasis caused by eating raw pond smelts.     

Trouble with lichen: the re-evaluation and re-interpretation of thallus form and fruit body types in the molecular era

Following discussions of the definition of the terms ‘lichen’ and ‘thallus’, the role of lichenization in the evolution of asco- and basidiomycetes, and divergence and convergence in fruit body types, the morphogenetic interpretation of types of thallus form in lichens is reviewed. Attention is drawn to the various morphogenetic hypotheses proposed to explain the lichen thallus, but it is concluded that it is best interpreted as a novel phenotype with no exact homologue. Similar ascomatal and thallus types are found in lichen-forming fungi of different orders and families, as now revealed by molecular phylogenetic studies. These are interpreted as examples of convergent evolution, strategies by which unrelated fungi either display captured algae to maximize photosynthetic opportunities, or to attach themselves to a substratum. Phenotypic evolution of fruit body and thallus types in the major orders and clades is summarized, and the thallus types known in each order are tabulated. An hypothesis relating the evolution of these structures to hygroscopic movements is proposed, and the critical position of lichens in developing an integrated approach to ascomycete evolution is emphasized.     

Cytochemical location of urease in the cell wall of two different lichen phycobionts

The enzyme urease has been located in the cell wall of recently isolated phycobionts from Evernia prunastri and Xanthoria parietina lichens. Cytochemical detection is achieved by producing a black, electron-dense precipitate of cobalt sulfide proceeding from CO(2) evolved from urea in the presence of cobalt chloride. Cellular fractionation reveals that about 80% of total urease activity was associated to the cell wall on both phycobionts whereas only 20% was recovered as soluble protein.     

Meta-analysis in assessment of the prevalence and annual incidence of Giardia spp. and Cryptosporidium spp. infections in humans in the Nordic countries

We aimed to apply the meta-analysis in the studies of protozoan pathogens in order to obtain a general overview of the prevalence and annual incidence of Giardia spp. and Cryptosporidium spp. infections in asymptomatic and symptomatic human populations in the Nordic countries of Denmark, Finland, Norway and Sweden. In combining the data of 13 clinically and methodologically non-heterogeneous studies published before 2004 using the random effects model with DerSimonian-Laird estimator, we estimated the prevalence (% prevalence: 95% confidence limits) of Giardia cases in the asymptomatic (i.e. no gastroenteric symptoms) general population to be 2.97% (2.64; 3.31) and in the symptomatic population 5.81% (5.34; 6.30). For Cryptosporidium the prevalences were 0.99% (0.81; 1.19) and 2.91% (2.71; 3.12), respectively. In analyzing the data, we estimated that there will be 4670 (4300; 5060) symptomatic cases of Giardia and 3340 (3110; 3580) symptomatic cases of Cryptosporidium annually per 100,000 general population in the Nordic countries. The vast majority of cases will remain unregistered in the national registers of infectious diseases, since for single registered cases there will be 254-867 cases of Giardia undetected/unregistered and 4072 to 15,181 cases of Cryptosporidium, respectively.