Intracellular localization and induction of a dynamic RNA-editing event of macro-algal V-ATPase subunit A (VHA-A) in response to copper.

A V-ATPase subunit A protein (VHA-A) transcript together with a variant (C793 to U), which introduces a stop codon truncating the subunit immediately downstream of its ATP binding site, was identified within a Fucus vesiculosus cDNA from a heavy metal contaminated site. This is intriguing because the VHA-A subunit is the crucial catalytic subunit responsible for the hydrolysis of ATP that drives ion transport underlying heavy metal detoxification pathways. We employed a chemiluminescent hybridization protection assay to quantify the proportion of both variants directly from mRNA while performing quantification of total transcript using Q-PCR. Polyclonal antisera raised against recombinant VHA-A facilitated simultaneous detection of parent and truncated VHA-A and revealed its cellular and subcellular localization. By exploiting laboratory exposures and samples from an environmental copper gradient, we showed that total VHA-A transcript and protein, together with levels of the truncated variant, were induced by copper. The absence of a genomic sequence representing the truncated variant suggests a RNA editing event causing the production of the truncated VHA-A. Based on these observations, we propose RNA editing as a novel molecular process underpinning VHA trafficking and intracellular sequestration of heavy metals under stress.

Endemic infection reduces transmission potential of an epidemic parasite during co-infection.

Endemic, low-virulence parasitic infections are common in nature. Such infections may deplete host resources, which in turn could affect the reproduction of other parasites during co-infection. We aimed to determine whether the reproduction, and therefore transmission potential, of an epidemic parasite was limited by energy costs imposed on the host by an endemic infection. Total lipids, triacylglycerols (TAG) and polar lipids were measured in cockroaches (Blattella germanica) that were fed ad libitum, starved or infected with an endemic parasite, Gregarina blattarum. Reproductive output of an epidemic parasite, Steinernema carpocapsae, was then assessed by counting the number of infective stages emerging from these three host groups. We found both starvation and gregarine infection reduced cockroach lipids, mainly through depletion of TAG. Further, both starvation and G. blattarum infection resulted in reduced emergence of nematode transmission stages. This is, to our knowledge, the first study to demonstrate directly that host resource depletion caused by endemic infection could affect epidemic disease transmission. In view of the ubiquity of endemic infections in nature, future studies of epidemic transmission should take greater account of endemic co-infections.

Relative efficacies of omega-3 polyunsaturated fatty acids in reducing expression of key proteins in a model system for studying osteoarthritis.

OBJECTIVE: To assess the relative efficacy of three different omega-3 (n-3) polyunsaturated fatty acids (PUFAs) in suppressing the mRNA levels for important proteins involved in the etiology of osteoarthritis (OA). METHODS: A model cell culture system (bovine chondrocytes) was used. Inflammatory factors and enzymes involved in OA were induced by exposure of the chondrocyte cultures to interleukin-1alpha (IL-1alpha). The effect of pre-incubating cultures with various amounts of exogenous fatty acids on subsequent levels of mRNAs was assessed by reverse transcription-polymerase chain reactions (RT-PCR). RESULTS: Exposure of cultures to IL-1alpha induced expression of the cartilage proteinases A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS)-4 and ADAMTS-5, cyclooxygenase (COX)-2, the matrix metalloproteinase (MMP)-3 and the inflammatory cytokines IL-1alpha, interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha). n-3 PUFAs were able to reduce the levels of mRNA for ADAMTS-4, ADAMTS-5, MMP-3, MMP-13, COX-2 (but not COX-1), IL-1alpha, IL-1beta and TNF-alpha. Eicosapentaenoic acid (EPA) was the most effective, followed by docosahexaenoic (DHA) and then alpha-linolenic (ALA) acid. The n-6 PUFA, arachidonic acid (AA) had no effect. CONCLUSION: These results show that omega-3 (n-3) PUFAs cause a reduction in the mRNA levels for various proteins known to be important in the pathology of OA. They provide a molecular explanation, at least in part, for beneficial effects of dietary omega-3 PUFAs for the amelioration of symptoms of the disease. The relative efficacy of EPA suggests that this omega-3 PUFA may be especially useful for dietary supplementation in patients with OA.

Does the membrane's physical state control the expression of heat shock and other genes?

Membranes provide the structural framework that divides cells from their environment and that, in eukaryotic cells, permits compartmentation. They are not simply passive barriers that are liable to be damaged during environmental challenge or pathological states, but are involved in cellular responses and in modulating intracellular signalling. Recent data show that the expression of several genes, particularly those that respond to changes in temperature, ageing or disease, is influenced and/or controlled by the membrane's physical state.

Human rhinovirus-induced inflammatory responses are inhibited by phosphatidylserine containing liposomes.

Human rhinovirus (HRV) infections are major contributors to the healthcare burden associated with acute exacerbations of chronic airway disease, such as chronic obstructive pulmonary disease and asthma. Cellular responses to HRV are mediated through pattern recognition receptors that may in part signal from membrane microdomains. We previously found Toll-like receptor signaling is reduced, by targeting membrane microdomains with a specific liposomal phosphatidylserine species, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine (SAPS). Here we explored the ability of this approach to target a clinically important pathogen. We determined the biochemical and biophysical properties and stability of SAPS liposomes and studied their ability to modulate rhinovirus-induced inflammation, measured by cytokine production, and rhinovirus replication in both immortalized and normal primary bronchial epithelial cells. SAPS liposomes rapidly partitioned throughout the plasma membrane and internal cellular membranes of epithelial cells. Uptake of liposomes did not cause cell death, but was associated with markedly reduced inflammatory responses to rhinovirus, at the expense of only modest non-significant increases in viral replication, and without impairment of interferon receptor signaling. Thus using liposomes of phosphatidylserine to target membrane microdomains is a feasible mechanism for modulating rhinovirus-induced signaling, and potentially a prototypic new therapy for viral-mediated inflammation.

Synthesis of sulphoquinovosyl diacylglycerol by higher plants.

The biosynthesis of sulphoquinovosyl diacylglycerol in germinating alfalfa seeds has been examined. Some incorporation of [35S]sulphate into the lipid occurs before chlorophyll production anh this is unaffected by chloramphenicol. Cysteic acid, molybdate, sulphite and sulpholactic acid all reduce incorporation of [35S]sulphate into sulphoquinovosyl diacylgylcerol. Some comparisons are made with other seed types. The results indicate that sulphoquinovosyl diacylgylcerol synthesis in alfalfa probably proceeds by a pathway similar to that in Euglena.

alpha-Hydroxylation of newly synthesised fatty acids by a soluble fraction from germinating pea.

A soluble fraction from germinating pea (Pisum sativum) seeds alpha-hydroxylated newly-synthesised fatty acids to form alpha-hydroxypalmitic and alpha-hydroxystearic acids. In contrast to fatty acid synthesis from [14C] malonyl CoA, alpha-hydroxylation was inhibited by exogenous phospholipids. alpha-Hydroxylation was optimal at pH 8, required reduced pyridine nucleotides and was inhibited by EDTA and imidazole.

Solubilisation, partial purification and properties of acyl-CoA: glycerol-3-phosphate acyltransferase from avocado (Persea americana) fruit mesocarp.

Glycerol-3-phosphate acyltransferase (G3PAT) activity was studied using a microsomal membrane fraction from avocado (Persea americana) mesocarp. G3PAT was shown to be an integral membrane protein, having an active site that appeared to be accessible to the cytoplasmic face of the endoplasmic reticulum, in experiments using limited proteolytic digestion. CHAPS solubilisation (0.25%, w/v) of microsomal G3PAT activity was used as an initial step in purification of this enzyme. Both CHAPS-solubilised and microsomal G3PAT activities were characterised and compared. Affinity chromatography was used to purify microsomal G3PAT for the first time from a plant source. Glycerophosphorylethanolamine coupled to cyanogen bromide-activated Sepharose was used for this purpose. Specific elution of G3PAT by a solution of glycerol-3-phosphate resulted in about 150-fold purification. The significance of the results and the potential usefulness of the purification method for further studies of G3PAT in plants are discussed.

Some characteristics of soluble fatty acid synthesis in germinating pea seeds.

Soluble fractions from germinating pea synthesize palmitic acid de novo and stearic acid by elongation. Malonyl CoA, acyl carrier protein and NADPH are required for both reactions. In contrast to some other plant systems, no requirement was found for divalent cations. On the other hand, the formation of both stearate and palmitate was inhibited by sulphydryl reagents and palmitate elongation was sensitive to arsenite. The products of the reactions were examined and found to be principally acyl-acyl carrier proteins and unesterified fatty acids. Unlike the pea microsomal fractions, the soluble enzymes are stimulated only slightly by the addition of exogenous lipids. The substrate for palmitate elongation is palmitoylacyl carrier protein, which is quantitatively elongated to stearate. Comparisons are made with membrane-localised fatty acid synthesis from the same tissue.

Lipid metabolism in germinating seeds. Purification of ethanolamine kinase from soya bean.

Ethanolamine kinase has been purified to homogeneity from germinating soya bean (Glycine max L.) seeds. The purified enzyme had a molecular weight of 17--19 000 as estimated by gel filtration and sodium dodecyl suphate-polyacrylamide gel electrophoresis. It would not phosphorylate choline, had a Km for ethanolamine of 8 microM and utilised Mg-ATP. The kinase could be purified in a 37 000 molecular weight form (dimer) which would easily dissociate on storage. In contrast to ethanolamine kinase whose activity was unaffected by the presence of choline in the assay system, soya bean choline kinase, although not phosphorylating ethanolamine, was competitively inhibited by the latter. The purification of specific choline and ethanolamine kinases from germinating soya bean confirmed in vivo observations which had indicated separate enzymes.

Acetyl-CoA carboxylase exerts strong flux control over lipid synthesis in plants.

The importance of acetyl-CoA carboxylase in regulation of lipid synthesis for barley and maize leaves has been quantitatively assessed using, as specific inhibitors, the herbicides fluazifop and sethoxydim. Apparent flux control coefficients of about 0.58 and 0.52 were determined for acetyl-CoA carboxylase in barley and maize leaves, respectively. These results show that acetyl-CoA carboxylase is the major flux controlling enzyme for light-stimulated lipid synthesis in these tissues.

Mitochondria Increase Three-Fold and Mitochondrial Proteins and Lipid Change Dramatically in Postmeristematic Cells in Young Wheat Leaves Grown in Elevated CO2.

A dramatic stimulation in mitochondrial biogenesis during the very early stages of leaf development was observed in young wheat plants (Triticum aestivum cv Hereward) grown in elevated CO2 (650 [mu]L L-1). An almost 3-fold increase in the number of mitochondria was observed in the very young leaf cells at the base of the first leaf of a 7-d-old wheat plant. In the same cells large increases in the accumulation of a mitochondrial chaperonin protein and the mitochondrial 2-oxoglutarate dehydrogenase complex and pyruvate dehydrogenase complex were detected by immunolabeling. Furthermore, the basal segment also shows a large increase in the rate of radiolabeling of diphosphatidylglycerol, a lipid confined to the inner mitochondrial membrane. This dramatic response in very young leaf cells to elevated CO2 suggests that the numerous documented positive effects of elevated CO2 on wheat leaf development are initiated as early as 12 h postmitosis.

Daphnia magna can tolerate short-term starvation without major changes in lipid metabolism.

Daphnia magna is a common crustacean that is adapted to brief spells of fasting. Lipids are naturally a major component of their diet and are stored as energy reserves. However, there has been some controversy in the literature on the extent to which dietary lipids are used directly for complex lipid formation in Daphnia. We examined lipid metabolism in D. magna by labeling the animals using [1-14C]acetate and then followed the turnover of radiolabeled lipids during a pulse chase. Daphnia were either fed or maintained without food during the chase period. The decrease in radioactivity during the chase was relatively unaffected by feeding, although there were some differences in the distribution of radioactivity between lipid classes or individual FA. The polar lipids, which were four times better labeled than nonpolar lipids, contained the most radioactivity in the zwitterionic phosphoglycerides, PE and PC. Under the experimental conditions, the turnover of the polar membrane lipids was unaffected by feeding. Within nonpolar lipids, TAG accounted for up to about 80% of the label, followed by DAG. Overall, our data show that D. magna is capable of high rates of lipid radiolabeling de novo and, in addition, is able to use--and indeed may be dependent on--some dietary components such as the PUFA linoleate and alpha-linolenate. The results also clearly show that Daphnia is able to tolerate brief spells of fasting (24 h) with very little change to its lipid metabolism.

A plant metallothionein produced in E. coli.

A metallothionein cDNA was generated from pea (Pisum sativum L.) roots, amplified by PCR and inserted into a plasmid for expression in E. coli. Purification of the resultant product generated 3 pools of cadmium-containing material after DEAE-cellulose chromatography. The amino acid composition of each was in excellent agreement with that predicted for pea metallothionein. A cadmium content of approximately 6 g.atoms per mole of protein was estimated. N-terminal sequence analysis revealed that the recombinant molecule had been proteolysed within the extended region linking the 2 cysteine-rich (putative) metal-binding regions. The significance of these findings in terms of the protein folding/targeting of the molecule are considered.

Direct identification and quantification of the cofactor in glutamate semialdehyde aminotransferase from pea leaves.

Glutamate semialdehyde aminotransferase, a key enzyme in the synthetic pathway leading to chlorophyll was purified from pea (Pisum sativum) leaves. Although the preparation contained a single contaminant the enzyme could be unambiguously identified as a dimer of subunit molar mass 45 kDa having an absorption spectrum consistent with the presence of pyridoxamine phosphate as cofactor. The cofactor was released by treatment with strong phosphate at low pH and was identified and quantified fluorimetrically. The specific activity of the enzyme (1.4 mumol.min-1.mg-1; 23 nkatal.mg-1) is very much higher than previously reported.

Characterization of fatty acid elongase enzymes from germinating pea seeds.

In vitro biosynthesis of radioactive arachidate and behenate was observed when microsomal fractions of germinating pea seeds were incubated with exogenous stearoyl-CoA (18:0-CoA) or arachidoyl-CA (20:0-CoA) in the presence of NADPH, [2-14C]malonyl-CoA and ATP. Characterization of parameters required for optimal stearoyl- and arachidoyl-CoA elongation revealed that, at least, two chain-length-specific elongases are necessary for very-long-chain fatty acid synthesis. Both enzymes were found to be sensitive to the group-selective reagents, p-CMB, NEM, iodoacetate, arsenite and phenylglyoxal. Subcellular fractionation studies indicated that both of these elongases were localized mainly in the endoplasmic reticulum.

Lipoxygenase pathway in olive callus cultures (Olea europaea).

Stimulation of the lipoxygenase pathway in olive fruit initiates a cascade of reactions that begins with the regio- and stereospecific di-oxygenation of polyunsaturated fatty acids containing a cis, cis-1,4 pentadiene moiety. Later products of the pathway include volatiles that influence the organoleptic properties of harvested olive oil. In this study, we have investigated lipoxygenase activity in olive callus cultures, and found that there is evidence of several isoforms of the enzyme with different pH optima and substrate specificities. Endogenous lipoxygenase activity was detected throughout the growth cycle of olive callus, particularly during the log phase of growth, suggesting that olive lipoxygenases are intimately involved in growth. The most prominent lipoxygenase activity in tissue cultures was found to be soluble but significant activities were detected in the plastid fraction. In addition, hydroperoxide lyase (HPL) activity was measured in the calli; both 13- and 9-HPL activities were found which were particulate.

Lipid composition of subcellular membranes from larvae and prepupae of Drosophila melanogaster.

Subcellular membranes were analyzed for their lipid composition and protein content at two developmental points representing the third instar wandering larvae and prepupal stages of Drosophila. At both stages, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were the major constituents with phosphatidylinositol (PI), phosphatidylserine (PS), diphosphatidylglycerol (DPG) and phosphatidic acid (PA) being relatively minor components. In total homogenates and in the nuclear-enriched fraction there was no significant difference in the phospholipid composition of the wandering larvae and prepupae. In mitochondria only a significant increase in the minor component PS was observed in the prepupae. In lysosomal membranes on the other hand, the relative abundance of the major components PE and PC increased in the prepupae although the molar ratios of the two lipids remained almost constant. The fatty acid composition of the phospholipids remained virtually unchanged in all of the fractions examined, including the lysosomes, and there was no evidence of lipid peroxidation. With regard to cellular degeneration and the involvement of lysosomes, we conclude that mechanisms other than gross modification of the lipid and/or lipid/protein ratio of their membranes are involved in the liberation of the acid phosphatase contents.

Synthesis of phospholipids by human peritoneal mesothelial cells.

OBJECTIVE: To assess the capacity of cultured human peritoneal mesothelial cells to synthesize choline-containing phospholipids. The study compares the phospholipids secreted from cultured cells with those which we, and others, have identified in the dialysate of patients treated by continuous ambulatory peritoneal dialysis (CAPD). PATIENTS: CAPD effluent was collected from 8 patients who had been receiving CAPD treatment for at least 11 months and who had normal ultrafiltration. CELL CULTURES: Using human omental tissue, homogeneous cultures of mesothelial cells were established. METHODS: Synthesis of phospholipids by mesothelial cells was assessed following incubation with [methyl-14C] choline chloride--a precursor capable of being incorporated into phosphatidylcholine (PtdCho) and sphingomyelin. Lipids from CAPD effluent, cultured cells, and cell medium were extracted in chloroform/methanol. Phospholipids were separated and identified by thin layer chromatography. Synthesis and secretion of PtdCho and other choline-containing lipids by the mesothelial cells were determined by beta scintillation counting of the appropriate bands, while the fatty acid composition of the phospholipids was ascertained by gas liquid chromatography. RESULTS: Synthesis and secretion of PtdCho by mesothelial cells were observed during a 96-hour period. When maintained in medium replete with essential fatty acids, the fatty acid composition of the PtdCho synthesized by cultured mesothelial cells closely resembled that isolated from the peritoneal cavity. CONCLUSION: The demonstration of phospholipid secretion from mesothelial cells, with a fatty acid composition similar to the phospholipids isolated from peritoneal dialysate, lends added support to the hypothesis that the mesothelial cells are the source of the peritoneal phospholipids. As such they offer a useful experimental system in which to study peritoneal phospholipid synthesis.