Effects of Temperature and Dietary Lipids on Phospholipid Fatty Acids and Membrane Fluidity in Steinernema carpocapsae

The phospholipid composition of Steinernema carpocapsae was studied in relation to diet and culture temperature. When reared at 18 and 27.5 C on Galleria mellonella or on an artificial diet supplemented with lard, linseed oil, or fish oil as lipid sources, nematode phospholipids contained an abundance of 20-carbon polyunsaturated fatty acids, with eicosapentaenoic acid (20:5(n - 3)) predominant, regardless of the fatty acid composition of the diet. Because the level of linolenic acid (18:3(n - 3)) in nematode phospholipids was very low and because eicosapentaenoic acid was present even when its precursor (linolenic acid) was undetectable in the diet, S. carpocapsae likely produces n - 3 polyunsaturated fatty acids by de novo biosynthesis, a pathway seldom reported in eukaryotic animals. Reduction of growth temperature from 25 to 18 C increased the proportion of 20:5(n - 3) but not other polyunsaturated fatty acids. A fluorescence polarization technique revealed that vesicles produced from phospholipids of nematodes reared at 18 C were less ordered than those from nematodes reared at 27.5 C, especially in the outermost region of the bilayer. Dietary fish oil increased fluidity in the outermost region but increased rigidity in deeper regions. Therefore, S. carpocapsae appears to modify its membrane physical state in response to temperature, and eicosapentaenoic acid may be involved in this response. The results also indicate that nematode membrane physical state can be modified dietarily, possibly to the benefit of host-finding or survival of S. carpocapsae at low temperatures.     

Compositional studies and Biological activities of Perovskia abrotanoides Kar. oils

Background

Current study has been designed to evaluate the chemical composition of essential and fixed oils from stem and leaves of Perovskia abrotanoides and antioxidant and antimicrobial activities of these oils.

Results

GC-MS analysis of essential oil identified 19 compounds with (E)-9-dodecenal being the major component in stem and hexadecanoic acid in leaves. In contrast, GC-MS analysis of fixed oil showed 40 constituents with α-amyrin the major component in stem and α-copaene in leaves. The antioxidant activity showed the highest value of 76.7% in essential oil from leaves in comparison with fixed oil from stem (45.9%) through inhibition of peroxidation in linoleic acid system. The antimicrobial assay tested on different microorganisms (e.g. E. coli, S. aureus, B. cereus, Nitrospira, S. epidermis, A. niger, A. flavus and C. albicans) showed the higher inhibition zone at essential oil from leaves (15.2 mm on B. cereus) as compared to fixed oil from stem (8.34 mm on S. aureus) and leaves (11.2 mm on S. aureus).

Conclusions

The present study revealed the fact that essential oil analyzed from Perovskia abrotanoides stem and leaves could be a promising source of natural products with potential antioxidant and antimicrobial activities, as compared to fixed oil.     

Kocuria marina BS-15 a biosurfactant producing halophilic bacteria isolated from solar salt works in India

Biosurfactant screening was made among the eight halophilic bacterial genera isolated from Kovalam solar salt works in Kanyakumari of India. After initial screening, Kocuria sp. (Km), Kurthia sp. (Ku) and Halococcus sp. (Hc) were found to have positive biosurfactant activity. Biosurfactant derived from Kocuria sp. emulsified more than 50% of the crude oil, coconut oil, sunflower oil, olive oil and kerosene when compared to the other strains. Further, Kocuria marina BS-15 derived biosurfactant was purified and characterized by TLC, FTIR and GC–MS analysis. The TLC analysis revealed that, the purified biosurfactants belong to the lipopeptide group. The IR spectrum results revealed that functional groups are R₂CNN, alkenes and N–H. The GC–MS analysis confirmed the compound as Nonanoic acid and Cyclopropane with the retention time of 12.78 and 24.65, respectively.     

Bicyclosesquiphellandrene and 1-epibicyclosesquiphellandrene: Two new dienes based on the cadalene skeleton

Two new sesquiterpene hydrocarbons, bicyclosesquiphellandrene and 1-epibicyclosesquiphellandrene have been isolated from Piper cubeba oil and Ocimum basilicum oil respectively. Their structures were elucidated spectroscopically and their stereochemistry by acid rearrangement and hydrogenation studies.     

Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants.     

Synergistic effect of conjugated linolenic acid isomers against induced oxidative stress,inflammation and erythrocyte membrane disintegrity in rat model

Background

α-Eleostearic acid and punicic acid, two typical conjugated linolenic acid (CLnA) isomers present in bitter gourd and snake gourd oil respectively, exhibit contrasting cis-trans configuration which made them biologically important.

Methods

Rats were divided into six groups. Group 1 was control and group 2 was treated control. Rats in the groups 3 and 4 were treated with mixture of α-eleostearic acid and punicic acid (1:1) (0.5% and 1.0% respectively) while rats in the groups 5 and 6 were treated with 0.5% of α-eleostearic acid and 0.5% of punicic acid respectively along with sodium arsenite by oral gavage once per day.

Results

Results showed that increase in nitric oxide synthase (NOS) activity, inflammatory markers expression, platelet aggregation, lipid peroxidation, protein oxidation, DNA damage and altered expression of liver X receptor-α (LXR-α) after arsenite treatment were restored with the supplementation of oils containing CLnA isomers. Altered activities of different antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and ferric reducing ability of plasma (FRAP) also restored after oil supplementation. Altered morphology and fluidity of erythrocyte membrane studied by atomic force and scanning electron microscopy, after stress induction were significantly improved due to amelioration in cholesterol/phospholipid ratio and fatty acid profile of membrane. Oils treatment also improved morphology of liver and fatty acid composition of hepatic lipid.

Conclusions

Overall two isomers showed synergistic antioxidant and anti-inflammatory effect against induced perturbations and membrane disintegrity.

General significance

Synergistic antioxidant and anti-inflammatory role of these CLnA isomers were established by this study.     

Orally administered emu oil attenuates disease in a mouse model of Crohn’s-like colitis

Crohn’s disease is a severe, incurable inflammatory bowel disease. Orally administered emu oil has demonstrated anti-inflammatory properties in previous models of gastrointestinal disease. We aimed to determine whether orally administered emu oil could attenuate disease in a mouse model of Crohn’s-like colitis. Female ARC(s) mice (CD-1 equivalent, n = 10/group) were intra-rectally administered water (120 μL) or trinitrobenzene sulfonic acid (TNBS; 3 mg in 50% ethanol; 120 μL bolus) on day 0. Mice were orally administered water (80 μL) or emu oil (80 μL or 160 μL) daily for five days and euthanized on day six. Bodyweight and disease activity were recorded daily. Colonoscopy, burrowing activity, facial grimace, histological parameters (damage severity, small intestinal villus height/crypt depth and colonic crypt depth), myeloperoxidase activity and intestinal permeability were assessed. P < 0.05 was considered statistically significant. TNBS decreased bodyweight (days 1, 2, 4; P < 0.05) and increased disease activity (days 1–6; P < 0.01), compared to normal controls. Emu oil (80 μL) attenuated disease activity on days 5–6 (P < 0.05), although bodyweight loss was not significantly impacted (P > 0.05). Facial grimace and colonoscopy scores were significantly increased in TNBS-control mice; effects attenuated by both volumes of emu oil (P < 0.001). TNBS increased histological damage severity compared to normal controls (P < 0.05); an effect attenuated by 80 μL emu oil (proximal and distal colon; P < 0.05) and 160 μL emu oil (distal colon; P < 0.01). In the ileum, villus height and crypt depth were unaffected by TNBS or emu oil treatment compared to normal (P > 0.05). TNBS-induced distal colonic crypt lengthening was unaffected following emu oil administration (P > 0.05). Remaining parameters, including burrowing, myeloperoxidase activity and intestinal permeability, were unchanged across all treatment groups (P > 0.05). In normal mice, emu oil treatment did not significantly impact any parameter compared to normal controls. In conclusion, emu oil reduced overall disease severity and facial grimace scores in TNBS mice. These results suggest therapeutic potential for orally administered emu oil in the management of Crohn’s disease.     

Microbial biodegradation of aromatic alkanoic naphthenic acids is affected by the degree of alkyl side chain branching

Naphthenic acids (NAs) occur naturally in oil sands and enter the environment through natural and anthropogenic processes. NAs comprise toxic carboxylic acids that are difficult to degrade. Information on NA biodegradation mechanisms is limited, and there are no studies on alkyl branched aromatic alkanoic acid biodegradation, despite their contribution to NA toxicity and recalcitrance. Increased alkyl side chain branching has been proposed to explain NA recalcitrance. Using soil enrichments, we examined the biodegradation of four aromatic alkanoic acid isomers that differed in alkyl side chain branching: (4′-n-butylphenyl)-4-butanoic acid (n-BPBA, least branched); (4′-iso-butylphenyl)-4-butanoic acid (iso-BPBA); (4′-sec-butylphenyl)-4-butanoic acid (sec-BPBA) and (4′-tert-butylphenyl)-4-butanoic acid (tert-BPBA, most branched). n-BPBA was completely metabolized within 49 days. Mass spectral analysis confirmed that the more branched isomers iso-, sec- and tert-BPBA were transformed to their butylphenylethanoic acid (BPEA) counterparts at 14 days. The BPEA metabolites were generally less toxic than BPBAs as determined by Microtox assay. n-BPEA was further transformed to a diacid, showing that carboxylation of the alkyl side chain occurred. In each case, biodegradation of the carboxyl side chain proceeded through beta-oxidation, which depended on the degree of alkyl side chain branching, and a BPBA degradation pathway is proposed. Comparison of 16S rRNA gene sequences at days 0 and 49 showed an increase and high abundance at day 49 of Pseudomonas (sec-BPBA), Burkholderia (n-, iso-, tert-BPBA) and Sphingomonas (n-, sec-BPBA).     

Genome-Wide Association Study Dissects the Genetic Architecture of Seed Weight and Seed Quality in Rapeseed (Brassica napus L.)

Association mapping can quickly and efficiently dissect complex agronomic traits. Rapeseed is one of the most economically important polyploid oil crops, although its genome sequence is not yet published. In this study, a recently developed 60K Brassica Infinium^® SNP array was used to analyse an association panel with 472 accessions. The single-nucleotide polymorphisms (SNPs) of the array were in silico mapped using ‘pseudomolecules’ representative of the genome of rapeseed to establish their hypothetical order and to perform association mapping of seed weight and seed quality. As a result, two significant associations on A8 and C3 of Brassica napus were detected for erucic acid content, and the peak SNPs were found to be only 233 and 128 kb away from the key genes BnaA.FAE1 and BnaC.FAE1. BnaA.FAE1 was also identified to be significantly associated with the oil content. Orthologues of Arabidopsis thaliana HAG1 were identified close to four clusters of SNPs associated with glucosinolate content on A9, C2, C7 and C9. For seed weight, we detected two association signals on A7 and A9, which were consistent with previous studies of quantitative trait loci mapping. The results indicate that our association mapping approach is suitable for fine mapping of the complex traits in rapeseed.     

Changes in growth,hormones levels and essential oil content of Ammi visnaga L. plants treated with some bioregulators

The effects of foliar application of different concentrations of amino acids (tyrosine and phenylalanine) and phenolic acids (trans-cinnamic acid, benzoic acid and salicylic acid) on growth, pigment content, hormones levels and essential oil content of Ammi visnaga L were carried out during two successive seasons. It is clear that foliar application of either amino acids or phenolics significantly promoted the growth parameters in terms of shoot height, fresh and dry biomass, number of branches and number of umbels per plant. The increment of growth parameter was associated with elevated levels of growth promoters (IAA, GA3, total cytokinins) and low level of ABA. The greatest increase in the previously mentioned parameters was measured in plants exposed to different concentrations of phenols particularly in benzoic acid-treated plants. Such effect was concentration dependent. All treatments led to significant increments in yield seeds and oil content. Moreover, gas liquid chromatographic analysis revealed that the main identified components of essential oil were 2,2-dimethyl butanoic acid, isobutyl isobutyrate, α-isophorone, thymol, fenchyl acetate and linalool. Phenolics and amino acid treatments resulted in qualitative differences in these components of essential oil.     

Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP

The massive influx of crude oil into the Gulf of Mexico during the Deepwater Horizon (DWH) disaster triggered dramatic microbial community shifts in surface oil slick and deep plume waters. Previous work had shown several taxa, notably DWH Oceanospirillales, Cycloclasticus and Colwellia, were found to be enriched in these waters based on their dominance in conventional clone and pyrosequencing libraries and were thought to have had a significant role in the degradation of the oil. However, this type of community analysis data failed to provide direct evidence on the functional properties, such as hydrocarbon degradation of organisms. Using DNA-based stable-isotope probing with uniformly ¹³C-labelled hydrocarbons, we identified several aliphatic (Alcanivorax, Marinobacter)- and polycyclic aromatic hydrocarbon (Alteromonas, Cycloclasticus, Colwellia)-degrading bacteria. We also isolated several strains (Alcanivorax, Alteromonas, Cycloclasticus, Halomonas, Marinobacter and Pseudoalteromonas) with demonstrable hydrocarbon-degrading qualities from surface slick and plume water samples collected during the active phase of the spill. Some of these organisms accounted for the majority of sequence reads representing their respective taxa in a pyrosequencing data set constructed from the same and additional water column samples. Hitherto, Alcanivorax was not identified in any of the previous water column studies analysing the microbial response to the spill and we discuss its failure to respond to the oil. Collectively, our data provide unequivocal evidence on the hydrocarbon-degrading qualities for some of the dominant taxa enriched in surface and plume waters during the DWH oil spill, and a more complete understanding of their role in the fate of the oil.     

AtDSEL, an Arabidopsis cytosolic DAD1-like acylhydrolase, is involved in negative regulation of storage oil mobilization during seedling establishment

Mobilization of seed storage reserves is essential for seed germination and seedling establishment. Here, we report that AtDSEL, an Arabidopsis thalianaDAD1-like Seedling Establishment-related Lipase, is involved in the mobilization of storage oils for early seedling establishment. AtDSEL is a cytosolic member of the DAD1-like acylhydrolase family encoded by At4g18550. Bacterially expressed AtDSEL preferentially hydrolyzed 1,3-diacylglycerol and 1-monoacylglycerol, suggesting that AtDSEL is an sn-1-specific lipase. AtDSEL-overexpressing transgenic Arabidopsis plants (35S:AtDSEL) were defective in post-germinative seedling growth in medium without an exogenous carbon source. This phenotype was rescued by the addition of sucrose to the growth medium. In contrast, loss-of-function mutant plants (atdsel-1 and atdsel-2) had a mildly fast-growing phenotype regardless of the presence of an exogenous carbon source. Electron microscopy revealed that 5-day-old 35S:AtDSEL cotyledons retained numerous peroxisomes and oil bodies, which were exhausted in wild-type and mutant cotyledons. The impaired seedling establishment of 35S:AtDSEL was not rescued by the addition of an exogenous fatty acid source, and 35S:AtDSEL seedling growth was insensitive to 2,4-dichlorophenoxybutyric acid, indicating that β-oxidation was blocked in AtDSEL-overexpressers. These results suggest that AtDSEL is involved in the negative regulation of seedling establishment by inhibiting the breakdown of storage oils.     

Cloning,characterization, and expression analysis of acyl–acyl carrier protein (ACP)-thioesterase B from seeds of Chinese Spicehush (Lindera communis)

Acyl–acyl carrier protein (ACP) thioesterases (TE EC 3.1.2.14) are fatty acid biosynthesis key enzymes that determine fatty acid carbon chain length in most plant tissues. A full-length cDNA corresponding to one of the fatty acyl–ACP thioesterase (Fat) genes, designated LcFatB, was isolated from developing Lindera communis seeds using PCR and RACE with degenerate primers based on conserved sequences of multiple TE gene sequences obtained from GenBank. The 1788 bp cDNA had an open reading frame (ORF) of 1260 bp encoding a protein of 419 amino acids. The deduced amino acid sequence showed 61–73% identity to proteins in the FatB class of plant thioesterases. Real-time quantitative PCR analysis revealed that LcFatB was expressed in all tissues of L. communis, with the highest expression in the developing seeds 75 days after flowering. Recombinant pET-MLcFatB was constructed using the pET-30a vector and transformed into Escherichia coli BL21(DE3)△ FadE, a strain that deleted the acyl-CoA dehydrogenase (FadE). SDS-PAGE analysis of proteins isolated from pET-MLcFatB E. coli cells after induction with IPTG revealed a protein band at ~ 40.5 kDa, corresponding to the predicted size of LcFatB mature protein. The decanoic acid and lauric acid contents of the pET-MLcFatB transformant were increased significantly. These findings suggest that an LcFatB gene from a non-traditional oil-seed tree could be used to function as a saturated acyl–ACP thioesterase and could potentially be used to modify the fatty acid composition of seed oil from L. communis or other species through transgenic approaches.     

Antileishmanial and Cytotoxic Effects of Essential Oil and Methanolic Extract of Myrtus communis L.

Plants used for traditional medicine contain a wide range of substances that can be used to treat various diseases such as infectious diseases. The present study was designed to evaluate the antileishmanial effects of the essential oil and methanolic extract of Myrtus communis against Leishmania tropica on an in vitro model. Antileishmanial effects of essential oil and methanolic extract of M. communis on promastigote forms and their cytotoxic activities against J774 cells were evaluated using MTT assay for 72 hr. In addition, their leishmanicidal activity against amastigote forms was determined in a macrophage model, for 72 hr. Findings showed that the main components of essential oil were α-pinene (24.7%), 1,8-cineole (19.6%), and linalool (12.6%). Findings demonstrated that M. communis, particularly its essential oil, significantly (P<0.05) inhibited the growth rate of promastigote and amastigote forms of L. tropica based on a dose-dependent response. The IC₅₀ values for essential oil and methanolic extract was 8.4 and 28.9 μg/ml against promastigotes, respectively. These values were 11.6 and 40.8 μg/ml against amastigote forms, respectively. Glucantime as control drug also revealed IC₅₀ values of 88.3 and 44.6 μg/ml for promastigotes and amastigotes of L. tropica, respectively. The in vitro assay demonstrated no significant cytotoxicity in J774 cells. However, essential oil indicated a more cytotoxic effect as compared with the methanolic extract of M. communis. The findings of the present study demonstrated that M. communis might be a natural source for production of a new leishmanicidal agent.     

Role of methylotrophs in the degradation of hydrocarbons during the Deepwater Horizon oil spill

The role of methylotrophic bacteria in the fate of the oil and gas released into the Gulf of Mexico during the Deepwater Horizon oil spill has been controversial, particularly in relation to whether organisms such as Methylophaga had contributed to the consumption of methane. Whereas methanotrophy remains unqualified in these organisms, recent work by our group using DNA-based stable-isotope probing coupled with cultivation-based methods has uncovered hydrocarbon-degrading Methylophaga. Recent findings have also shown that methylotrophs, including Methylophaga, were in a heightened state of metabolic activity within oil plume waters during the active phase of the spill. Taken collectively, these findings suggest that members of this group may have participated in the degradation of high-molecular-weight hydrocarbons in plume waters. The discovery of hydrocarbon-degrading Methylophaga also highlights the importance of considering these organisms in playing a role to the fate of oil hydrocarbons at oil-impacted sites.     

Identification of a Pair of Phospholipid:Diacylglycerol Acyltransferases from Developing Flax (Linum usitatissimum L.) Seed Catalyzing the Selective Production of Trilinolenin

The oil from flax (Linum usitatissimum L.) has high amounts of α-linolenic acid (ALA; 18:3^cisΔ9,12,15) and is one of the richest sources of omega-3 polyunsaturated fatty acids (ω-3-PUFAs). To produce ∼57% ALA in triacylglycerol (TAG), it is likely that flax contains enzymes that can efficiently transfer ALA to TAG. To test this hypothesis, we conducted a systematic characterization of TAG-synthesizing enzymes from flax. We identified several genes encoding acyl-CoA:diacylglycerol acyltransferases (DGATs) and phospholipid:diacylglycerol acyltransferases (PDATs) from the flax genome database. Due to recent genome duplication, duplicated gene pairs have been identified for all genes except DGAT2-2. Analysis of gene expression indicated that two DGAT1, two DGAT2, and four PDAT genes were preferentially expressed in flax embryos. Yeast functional analysis showed that DGAT1, DGAT2, and two PDAT enzymes restored TAG synthesis when produced recombinantly in yeast H1246 strain. The activity of particular PDAT enzymes (LuPDAT1 and LuPDAT2) was stimulated by the presence of ALA. Further seed-specific expression of flax genes in Arabidopsis thaliana indicated that DGAT1, PDAT1, and PDAT2 had significant effects on seed oil phenotype. Overall, this study indicated the existence of unique PDAT enzymes from flax that are able to preferentially catalyze the synthesis of TAG containing ALA acyl moieties. The identified LuPDATs may have practical applications for increasing the accumulation of ALA and other polyunsaturated fatty acids in oilseeds for food and industrial applications.