Arachidonic Acid Production Research Articles

Bioactive Lipids Are Generated to Micromolar Levels during RBC Storage, Even in Leukoreduced Units

Background: The transfusion of stored red blood cells (RBCs) has been linked to negative outcomes, such as Transfusion-Related Acute Lung Injury (TRALI) and Transfusion Related Immunomodulation (TRIM). Bioactive lipids have been implicated as playing an important role in TRALI pathogenesis, both as a "second hit" when anti-HLA antibodies are present and also in anti-HLA independent TRALI. Likewise, bioactive lipids have been suggested to play a role in TRIM.Methods: To further study bioactive lipids in RBC storage, we developed a targeted metabolomics approach, with isotope dilution, to accurately and precisely quantify concentrations of multiple poly-unsaturated fatty acids (PUFAs) and their related oxidation products (oxylipins) in stored RBC units. Analytes in RBC samples were extracted using methanol with isotopically-labeled internal standards and analyzed by liquid chromatography-tandem mass spectrometry using multiple reaction monitoring (LC-MS/MS-MRM). Eight units of leukoreduced (LR) and non-leukoreduced (NLR) RBCs were analyzed longitudinally from 6 to 42 days of storage.Results: LR RBCs had a significant increase in multiple PUFAs over storage, including: arachidonic acid (AA), linoleic acid (LA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), linolenic acid (ALA), dihomo-γ-linolenic acid (DGLA), adrenic acid (22:4), and docosapentaenoic acid (22:5). Only a modest increase was observed over the first 20 days of storage, followed by a rapid increase from day 20 to day 42. AA showed the biggest increase in concentration, from 1.0±0.4 µM at day 6 to 13.4±5.2 µM at day 42 in LR RBC units. Oxidative products of PUFA metabolism were also analyzed. The concentration of hydroxyeicosatetraenoic acids (HETEs), oxidation products of AA, including 5-, 8-, 9-, 11-, 12- and 15-HETEs increased 5 to 13 fold from day 6 to day 42 of storage in LR RBCs. The concentration of other oxylipins including hydroxyoctadecadienoic acids(9- and 13-HODEs)and dihydroxyoctadecenoic acids (, 9, 10- and 12,13-DiHOMEs), hydroxyl-DHA(14-, 16- and 17-HDoHEs) were also significantly increased during storage. If present, prostaglandins (PGE2 and PGD2) and Leukotriene (LTB4), were below the lower limits of detection. Juxta position of LR and NLR RBCs, showed that the concentrations of 12-HETE, 15-HETE, 14-HDoHE, and 17-HDoHE in NLR units were significantly higher at day 42 (p<0.01). Of particular note, the average concentration of 12-HETE reached 1.1±0.8 µM in NLR at day 42, which was 45 fold higher than that in LR units. No differences in LR and NLR RBCs were noted at day 6.Conclusions: We report significant generation of PUFAs and products of lipid metabolism/oxidation over RBC storage, with a particular increase in generation after day 20 of storage. LR decreased the generation of some lipid species (12-HETE in particular); however, accumulation of many bioactive lipids occurs robustly despite LR. Because variation from unit to unit was small at day 6 but substantial at day 42, these findings also support significant donor-to-donor differences in the process of bioactive lipid generation during storage. Exactly how the generation of bioactive lipids effects medical outcomes of transfusion remains undetermined; however, the quantitation allowed by the current methods demonstrates the generation of bioactive lipids up to µM levels, which are known to be sufficient to have significant physiological and/or pathological impacts in other settings. DisclosuresZimring:Immucor Inc.: Research Funding; BloodworksNW: Patents & Royalties: Patent Application filed on technology in this abstract - no royalties.

Metabolic engineering of Mortierella alpina for arachidonic acid production with glycerol as carbon source.

BackgroundAlthough some microorganisms can convert glycerol into valuable products such as polyunsaturated fatty acids, the yields are relative low due primarily to an inefficient assimilation of glycerol. Mortierella alpina is an oleaginous fungus which preferentially uses glucose over glycerol as the carbon source for fatty acid synthesis.ResultsIn the present study, we metabolically engineered M. alpina to increase the utilization of glycerol. Glycerol kinase and glycerol-3-phosphate dehydrogenase control the first two steps of glycerol decomposition. GK overexpression increased the total fatty acid content by 35 %, whereas G3PD1, G3PD2 and G3PD3 had no significant effect. Overexpression of malic enzyme (ME1) but not glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase or isocitrate dehydrogenase significantly increased fatty acid content when glycerol was used as carbon source. Simultaneous overexpression of GK and ME1 enabled M. alpina to accumulate fatty acids efficiently, with a 44 % increase in fatty acid content (% of dry weight), a 57 % increase in glycerol to fatty acid yield (g/g glycerol) and an 81 % increase in fatty acid production (g/L culture). A repeated batch process was applied to relieve the inhibitory effect of raw glycerol on arachidonic acid synthesis, and under these conditions, the yield reached 52.2 ± 1.9 mg/g.ConclusionsThis study suggested that GK is a rate-limiting step in glycerol assimilation in M. alpina. Another restricting factor for fatty acid accumulation was the supply of cytosolic NADPH. We reported a bioengineering strategy by improving the upstream assimilation and NADPH supply, for oleaginous fungi to efficiently accumulate fatty acid with glycerol as carbon source.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0392-4) contains supplementary material, which is available to authorized users.

Identification of Pathways Mediating Growth Differentiation Factor5-Induced Tenogenic Differentiation in Human Bone Marrow Stromal Cells.

To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering.

Identification and characterization of three genes encoding acyl-CoA: diacylglycerol acyltransferase (DGAT) from the microalga Myrmecia incisa Reisigl

Acyl-CoA: diacylglycerol acyltransferase (DGAT) is a key enzyme responsible for triacylglycerol (TAG) synthesis in eukaryotic organisms. The present work reported DGAT genes in the green alga Myrmecia incisa, a promising candidate for arachidonic acid (ArA) production. According to the results of homology search against a transcriptome database, we cloned three cDNAs encoding putative DGAT1 and DGAT2. The 2238-bp, 1056-bp and 1068-bp of open reading frame (ORF) of these three cDNAs, designated as MiDGAT1, MiDGAT2A and MiDGAT2B, were predicted to encode proteins composed of 745, 351 and 355 amino acids. They were separated by 14, 6 and 6 introns, respectively, as revealed by comparing their corresponding cDNA and DNA sequences. Multiple sequence alignment of amino acids indicated that MiDGAT1 had a pleckstrin homology (PH) domain, whilst MiDGAT2s contained a highly conserved HPHG, a characteristic motif of DGAT2 family. To determine the function, they were expressed heterologously in a Saccharomyces cerevisiae mutant strain with impaired TAG metabolism. Results of thin-layer chromatography and BODIPY staining indicated that both MiDGAT1 and MiDGAT2s were able to restore TAG synthesis and lipid body formation. GC-MS analysis indicated that palmitic acid and stearic acid were the major components of TAGs in yeast cells, and their ratio between wild type and the transformed yeasts was not significantly different. Quantitative RT-PCR results showed that the transcript level of MiDGAT2A was regulated by nitrogen starvation, which was consistent with TAG accumulation in M. incisa.

Arachidonic acid‐derived signaling lipids and functions in impaired healing

Very little is known about lipid function during wound healing, and much less during impaired healing. Such understanding will help identify what roles lipid signaling plays in the development of impaired/chronic wounds. We took a lipidomics approach to study the alterations in lipid profile in the LIGHT(-/-) mouse model of impaired healing which has characteristics that resemble those of impaired/chronic wounds in humans, including high levels of oxidative stress, excess inflammation, increased extracellular matrix degradation and blood vessels with fibrin cuffs. The latter suggests excess coagulation and potentially increased platelet aggregation. We show here that in these impaired wounds there is an imbalance in the arachidonic acid (AA) derived eicosonoids that mediate or modulate inflammatory reactions and platelet aggregation. In the LIGHT(-/-) impaired wounds there is a significant increase in enzymatically derived breakdown products of AA. We found that early after injury there was a significant increase in the eicosanoids 11-, 12-, and 15-hydroxyeicosa-tetranoic acid, and the proinflammatory leukotrienes (LTD4 and LTE) and prostaglandins (PGE2 and PGF2α ). Some of these eicosanoids also promote platelet aggregation. This led us to examine the levels of other eicosanoids known to be involved in the latter process. We found that thromboxane (TXA2 /B2 ), and prostacyclins 6kPGF1α are elevated shortly after wounding and in some cases during healing. To determine whether they have an impact in platelet aggregation and hemostasis, we tested LIGHT(-/-) mouse wounds for these two parameters and found that, indeed, platelet aggregation and hemostasis are enhanced in these mice when compared with the control C57BL/6 mice. Understanding lipid signaling in impaired wounds can potentially lead to development of new therapeutics or in using existing nonsteroidal anti-inflammatory agents to help correct the course of healing.

Three New Records of Mortierella Species Isolated from Crop Field Soil in Korea

Three new fungal species of the genus Mortierella, Mortierella zychae, Mortierella ambigua, and Mortierella indohii, have been reported in Korea. The fungi were encountered during a study on the fungal community of soil samples collected from different locations in Korea. The species were identified based on molecular and morphological analyses. This study presents detailed descriptions of the morphological observations and molecular phylogenetic analysis of these three fungi. All three species were found to be sensitive to triphenyltetrazolium chloride staining. M. zychae demonstrated the highest intensity of mycelial staining, indicating that this species has the highest potential to produce arachidonic acid of the three species. The staining results indicated that the newly recorded species could potentially be useful for arachidonic acid production.

Orai3 Surface Accumulation and Calcium Entry Evoked by Vascular Endothelial Growth Factor.

Vascular endothelial growth factor (VEGF) acts, in part, by triggering calcium ion (Ca(2+)) entry. Here, we sought understanding of a Synta66-resistant Ca(2+) entry pathway activated by VEGF. Measurement of intracellular Ca(2+) in human umbilical vein endothelial cells detected a Synta66-resistant component of VEGF-activated Ca(2+) entry that occurred within 2 minutes after VEGF exposure. Knockdown of the channel-forming protein Orai3 suppressed this Ca(2+) entry. Similar effects occurred in 3 further types of human endothelial cell. Orai3 knockdown was inhibitory for VEGF-dependent endothelial tube formation in Matrigel in vitro and in vivo in the mouse. Unexpectedly, immunofluorescence and biotinylation experiments showed that Orai3 was not at the surface membrane unless VEGF was applied, after which it accumulated in the membrane within 2 minutes. The signaling pathway coupling VEGF to the effect on Orai3 involved activation of phospholipase Cγ1, Ca(2+) release, cytosolic group IV phospholipase A2α, arachidonic acid production, and, in part, microsomal glutathione S-transferase 2, an enzyme which catalyses the formation of leukotriene C4 from arachidonic acid. Shear stress reduced microsomal glutathione S-transferase 2 expression while inducing expression of leukotriene C4 synthase, suggesting reciprocal regulation of leukotriene C4-synthesizing enzymes and greater role of microsomal glutathione S-transferase 2 in low shear stress. VEGF signaling via arachidonic acid and arachidonic acid metabolism causes Orai3 to accumulate at the cell surface to mediate Ca(2+) entry and downstream endothelial cell remodeling.

Arachidonic acid production by Mortierella alpina using raw crop materials [pdf

Arachidonic acid production by Mortierella alpina using raw crop materials [pdf

Arachidonic acid production by Mortierella alpina using raw crop materials

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, China

ROLE OF THROMBOXANE AND LEUKOTRIENES IN MECHANISMS OF CONTRACTILE REACTIONS OF PORTAL VEIN, INDUCED BY ACETYLCHLINE AND PHENYLEPHRINE

Effects of picotamide and zileuton on tonic contractile activity of the rat portal vein preparations, induced by acetylcholine (2.10(-5) mol/1) and phenylephrine (5.10(-7) mol/1) were investigated. Conversion of arachidonic acid products (prostaglandins, leukotrienes) synthesized by endothelial cells, plays an important role in the local regulation of vascular tone. The compounds formed in a cascade of enzymatic transformations can modulate the effect of other vasoactive factors. Picotamide (6,5.10(-5) mol/1) - thromboxane receptor and thromboxane -synthase blocker - depress acetylcholine-induction tonic contraction of isolated segments of portal vein with intact endothelium by 29% and norepinephrine-induction reduction of 45% relative to the control values. The obtained results indicate a participation of thromboxane and/or endoperoxide H2 in this reaction. Partial inhibition of the contractions by 5-lipoxygenase blocker zileuton(4,2.10(-5) mol/1) at 23% relative to control values suggests, that products of lipoxigenase pathways of arachidonic acid conversion are involved in mechanisms of specified reactions. These data indicate complex mechanisms of regulation of vascular tone of the portal vein, which play an important role eicosanoids. Further study of these mechanisms is necessary for the formation of basic knowledge, as well as to elucidate the mechanisms of occurrence and development of pathological conditions of vessels and the development of methods of their correction.

Nature of extracellular signal that triggers RhoA/ROCK activation for the basal internal anal sphincter tone in humans

The extracellular signal that triggers activation of rho-associated kinase (RhoA/ROCK), the major molecular determinant of basal internal anal sphincter (IAS) smooth muscle tone, is not known. Using human IAS tissues, we identified the presence of the biosynthetic machineries for angiotensin II (ANG II), thromboxane A2 (TXA2), and prostaglandin F2α (PGF2α). These end products of the renin-angiotensin system (RAS) (ANG II) and arachidonic acid (TXA2 and PGF2α) pathways and their effects in human IAS vs. rectal smooth muscle (RSM) were studied. A multipronged approach utilizing immunocytochemistry, Western blot analyses, and force measurements was implemented. Additionally, in a systematic analysis of the effects of respective inhibitors along different steps of biosynthesis and those of antagonists, their end products were evaluated either individually or in combination. To further describe the molecular mechanism for the IAS tone via these pathways, we monitored RhoA/ROCK activation and its signal transduction cascade. Data showed characteristically higher expression of biosynthetic machineries of RAS and AA pathways in the IAS compared with the RSM. Additionally, specific inhibition of the arachidonic acid (AA) pathway caused ~80% decrease in the IAS tone, whereas that of RAS lead to ~20% decrease. Signal transduction studies revealed that the end products of both AA and RAS pathways cause increase in the IAS tone via activation of RhoA/ROCK. Both AA and RAS (via the release of their end products TXA2, PGF2α, and ANG II, respectively), provide extracellular signals which activate RhoA/ROCK for the maintenance of the basal tone in human IAS.

English

This research focuses on isolation and identification of polyunsaturated fatty acid (PUFA) producing fungi from natural sources of Thailand, followed by experimental designs for carbon and nitrogen utilizations of the potential strains. The results show that 8 and 16 isolates of lower fungi from freshwater and wet-fallen leaves, respectively, could produce PUFAs. Among these isolates, the NR06 that was isolated from wet-fallen leaves at Tad Ta Phu waterfall, Nakhon Ratchasrima province. They showed the highest level productions of both biomass (15.49±0.24 g×l-1) and total fatty acids (16.44±0.30%). The major essential fatty acid composition was found to be arachidonic acid (ARA; C20:4n6) (32.24±0.35%). The minors were linoleic acid (C18:2n6) (8.26±0.59%) and γ-linoleic acid (GLA; C18:3n6) (5.48±0.08%). Besides the morphological characterization, taxonomic identification by the 636 bp-ITS region sequencing and phylogenetic analysis were performed. It was demonstrated that the fungal isolate NR06 was classified in the closest species of Mortierella elongata with 99% similarity (GenBank accession no. KF181625). Statistically based experimental designs (Design Expert 7.1 software) were applied to the optimization of biomass and fatty acid production in the NR06 culture. Using the glucose as a carbon source, there was a significant effect on both biomass and ARA production (p-values < 0.0001), whereas using yeast extract as a nitrogen source affected only ARA production (p-value = 0.02). It was suggested that the NR06 isolate could be further optimized for an improvement as a potential ARA producing strain. Keywords: Polyunsaturated fatty acids (PUFAs), arachidonic acid (ARA), lower fungi, Mortierella elongata. Abbreviation: PUFAs, Polyunsaturated fatty acids; ARA, arachidonic acid.

Sa1362 Rectal Hyposensitivity and Slow Colonic Transit Are More Common in Patients With Outlet Obstruction by Balloon Expulsion Testing: A Retrospective Cohort Analysis

G A A b st ra ct s along different steps of biosynthesis of these end products, and their antagonists on the basal IAS tone. These agents were given either individually or in certain combinations. Molecular mechanisms for the IAS tone via these pathways were tracked down by monitoring RhoA/ROCK signal transduction cascade. Activation of RhoA/ROCK was monitored using translocation of these molecular proteins in particulate fractions. Results: U46619, a stable analog of TXA2, PGF2α and Ang II produced concentration (10-9 to 10-5 M)-dependent increase in the IAS tone. Interestingly, maximal increase in the IAS tone by U46619 and PGF2α was 98.5 ± 1.5% and 65.0 ± 6.0% maximal respectively, while increase in the IAS tone by Ang II was limited to 19.1 ± 2.8%. These increases in IAS tone were significantly higher than those in the RSM (p < 0.05; n = 6 to 8). Data further showed characteristically higher expression of biosynthetic enzymatic machineries of RAS and AA pathways in the IAS as compared with the RSM. Additionally, specific inhibition of AA pathway caused ~80% decrease in the IAS tone, while that of RAS produced only ~20% decrease. Signal transduction studies revealed that the end products of both AA and RAS pathways cause increase in IAS tone via activation of RhoA/ROCK. Conclusion: Activation of arachidonic acid (AA) and renin-angiotensin system (RAS) pathways both increase basal tone in the internal anal sphincter (IAS). The AA pathway is more potent activator of the IAS tone than the RAS pathway. Both the RAS and AA pathways lead to synthesis and release the end products (TXA2, PGF2α, and Ang II, respectively), which in turn activate their respective G-protein-coupled receptors TPR, FPR, and AT1-R. This activation provides extracellular signals which activate RhoA/ROCK for the maintenance of the basal tone in human IAS. These studies were supported by the NIH grant (RO1DK035385).

Organochlorine insecticides induce NADPH oxidase-dependent reactive oxygen species in human monocytic cells via phospholipase A2/arachidonic acid.

Bioaccumulative organohalogen chemicals, such as organochlorine (OC) insecticides, have been increasingly associated with disease etiology; however, the mechanistic link between chemical exposure and diseases, such as atherosclerosis, cancer, and diabetes, is complex and poorly defined. Systemic oxidative stress stemming from OC exposure might play a vital role in the development of these pathologies. Monocytes are important surveillance cells of the innate immune system that respond to extracellular signals possessing danger-associated molecular patterns by synthesizing oxyradicals, such as superoxide, for the purpose of combating infectious pathogens. We hypothesized that OC chemicals can be toxic to monocytes because of an inappropriate elevation in superoxide-derived reactive oxygen species (ROS) capable of causing cellular oxidative damage. Reactive oxyradicals are generated in monocytes in large part by NADPH oxidase (Nox). The present study was conducted to examine the ability of two chlorinated cyclodiene compounds, trans-nonachlor and dieldrin, as well as p,p'-DDE, a chlorinated alicyclic metabolite of DDT, to stimulate Nox activity in a human monocytic cell line and to elucidate the mechanisms for this activation. Human THP-1 monocytes treated with either trans-nonachlor or dieldrin (0.1-10 μM in the culture medium) exhibited elevated levels of intracellular ROS, as evidenced by complementary methods, including flow cytometry analysis using the probe DCFH-DA and hydroethidine-based fluorometric and UPLC-MS assays. In addition, the induced reactive oxygen flux caused by trans-nonachlor was also observed in two other cell lines, murine J774 macrophages and human HL-60 cells. The central role of Nox in OC-mediated oxidative stress was demonstrated by the attenuated superoxide production in OC-exposed monocytes treated with the Nox inhibitors diphenyleneiodonium and VAS-2870. Moreover, monocytes challenged with OCs exhibited increased phospho-p47(phox) levels and enhanced p47(phox) membrane localization compared to that in vehicle-treated cells. p47(phox) is a cytosolic regulatory subunit of Nox, and its phosphorylation and translocation to the NOX2 catalytic subunit in membranes is a requisite step for Nox assembly and activation. Dieldrin and trans-nonachlor treatments of monocytes also resulted in marked increases in arachidonic acid (AA) and eicosanoid production, which could be abrogated by the phospholipase A2 (PLA2) inhibitor arachidonoyltrifluoromethyl ketone (ATK) but not by calcium-independent PLA2 inhibitor bromoenol lactone. This suggested that cytosolic PLA2 plays a crucial role in the induction of Nox activity by increasing the intracellular pool of AA that activates protein kinase C, which phosphorylates p47(phox). In addition, ATK also blocked OC-induced p47(phox) serine phosphorylation and attenuated ROS levels, which further supports the notion that the AA pool liberated by cytosolic PLA2 is responsible for Nox activation. Together, the results suggest that trans-nonachlor and dieldrin are capable of increasing intracellular superoxide levels via a Nox-dependent mechanism that relies on elevated intracellular AA levels. These findings are significant because chronic activation of monocytes by environmental toxicants might contribute to pathogenic oxidative stress and inflammation.

Reconstruction and analysis of a genome-scale metabolic model of the oleaginous fungus Mortierella alpina.

BackgroundMortierella alpina is an oleaginous fungus used in the industrial scale production of arachidonic acid (ARA). In order to investigate the metabolic characteristics at a systems level and to explore potential strategies for enhanced lipid production, a genome-scale metabolic model of M. alpina was reconstructed.ResultsThis model included 1106 genes, 1854 reactions and 1732 metabolites. On minimal growth medium, 86 genes were identified as essential, whereas 49 essential genes were identified on yeast extract medium. A series of sequential desaturase and elongase catalysed steps are involved in the synthesis of polyunsaturated fatty acids (PUFAs) from acetyl-CoA precursors, with concomitant NADPH consumption, and these steps were investigated in this study. Oxygen is known to affect the degree of unsaturation of PUFAs, and robustness analysis determined that an oxygen uptake rate of 2.0 mmol gDW−1 h−1 was optimal for ARA accumulation. The flux of 53 reactions involving NADPH was significantly altered at different ARA levels. Of these, malic enzyme (ME) was confirmed as a key component in ARA production and NADPH generation. When using minimization of metabolic adjustment, a knock-out of ME led to a 38.28% decrease in ARA production.ConclusionsThe simulation results confirmed the model as a useful tool for future research on the metabolism of PUFAs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-014-0137-8) contains supplementary material, which is available to authorized users.

Efficient production of arachidonic acid by Mortierella alpina through integrating fed-batch culture with a two-stage pH control strategy

Arachidonic acid (ARA) yield and productivity of Mortierella alpina mutant D20 were enhanced by integrating a fed-batch culture combined with a two-stage pH control strategy. Following a kinetic analysis of the whole fermentation process, a two-stage pH control strategy was developed in which the pH was maintained at 5.5 for the first 48h and then shifted to 6.5 till the end of fermentation. Using this strategy, a maximum ARA production of 8.12g/L was achieved. On the basis of pH control, the effects of fed-batch cultures on ARA productivity were further investigated. A maximum ARA productivity of 1.40g/L/d was obtained with a two-stage constant-speed glucose feeding strategy, starting with a glucose concentration of 50g/L. This strategy was simple and economical to operate, and it may be possible to apply this approach for large-scale industrial production of ARA.

The effect of pH, aeration, and temperature on arachidonic acid synthesis by Mortierella alpina

The effects of pH, aeration, and temperature on the growth of fungal strain Mortierella alpina LPM-301 and the synthesis of lipids and arachidonic acid in glycerol-containing medium were studied. Arachidonic acid production in the stationary growth phase was found to depend considerably on the pH value; it reached the optimum at pH 6.0 and was irreversibly inhibited at a pH of 3.0. The PO2 values in a range from 10 to 50% showed no marked effect on mycelium growth or the synthesis of lipids and arachidonic acid. The temperature optimum for arachidonic acid production was 20-22 degrees C. Under continuous cultivation, the amount of arachidonic acid reached 29.8% of lipids and 7.4% of biomass. The arachidonic acid yield from the glycerol consumed was 4.1% by mass and 8.8% by energy. It is suggested that arachidonic acid synthesis at an unfavorable pH and elevated temperatures was limited by the activity of A-12-desaturase and by the conversion of linoleic to arachidonic acid, respectively.

The Role of Natural Progesterone Administration on the Levels of Progesterone-induced Blocking Factor, Interleukin-10 and the Prolongation of Gestational Length in Impending Preterm Delivery

ABSTRACTProgesterone plays an important role in maintaining pregnancy. The success of pregnancy depends on the balance of immune response between the mother and the fetus. Progesterone regulates the immunological response through progesteroneinduced blocking factor (PIBF) which is synthesized by lymphocytes. Progesterone-induced blocking factor inhibits the production of arachidonic acid, reduced NK cell activity, and modulates the cytokine balance by increasing interleukin-10 (IL-10) level that is produced by Th2. This study aims to determine the roles of natural progesterone administration on PIBF and IL-10 levels and the increase in gestational length.This is an experimental research with pre- and post-test design. Subjects were pregnant women who met the criteria (n = 22). The treatment was done for 7 days. Progesteroneinduced blocking factor and IL-10 levels were measured before and after the administration of natural progesterone and the length of gestation were noted. The study was conducted at Dr Hasan Sadikin Hospital within July to December 2013.Results showed that after the administration of natural progesterone, there were increases on PIBF (p = 0.039) and IL-10 levels (p = 0.031). A positive correlation was noted between PIBF and IL-10 levels (r = 0.549; Spearman rank correlation test). There was also positive correlation between PIBF serum level (moderate strength correlation) and the length of gestation (r = 0.428; Point biserial correlation test). Progesterone-induced blocking factor serum level had more influence on the prolongation of the pregnancy compared to IL-10 level (OR: 1.016, 95% CI: 1.001 to 1.03; p = 0.033).Increased levels of PIBF and IL-10 after the administration of natural progesterone on the impending preterm delivery may prolong the gestation. Progesterone-induced blocking factor serum level have more dominant role in prolongation of gestation compared to IL-10.How to cite this articleThipasary Y, Effendi JS, Anwar AD. The Role of Natural Progesterone Administration on the Levels of Progesterone-induced Blocking Factor, Interleukin-10 and the Prolongation of Gestational Length in Impending Preterm Delivery. J South Asian Feder Obst Gynae 2015;7(3):118-125.

Comparative biochemical characterization of the monoacylglycerol lipase inhibitor KML29 in brain, spinal cord, liver, spleen, fat and muscle tissue

Monoacylglycerol lipase (MAGL) is part of the endocannabinoid and the prostaglandin signaling system. MAGL degrades the endocannabinoid 2-arachidonoylglycerol (2-AG) into glycerol and arachidonic acid. MAGL-induced arachidonic acid is the primary source for prostaglandin synthesis in the brain. 2-AG mainly induces neuroprotective and anti-inflammatory effects, whereas prostaglandins are related to pro-inflammatory effects inducing neurotoxicity. Therefore, inhibition of MAGL represents a promising target for neurological diseases characterized by inflammation. However, as 2-AG is an agonist for the cannabinoid receptor 1 (CB1), inhibition of MAGL might be associated with unwanted cannabimimetic effects. Here, we show that oral administration of KML29, a highly selective inhibitor of MAGL, induced large and dose-dependent changes in 2-AG levels in vivo in brain and spinal cord of mice. Of note, MAGL inhibition by KML29 induced a decrease in prostaglandin levels in brain and most peripheral tissues but not in the spinal cord. MAGL expression was highest in fat, liver and brain, whereas the cytosolic phospholipase A2 (cPLA2), a further enzyme responsible for arachidonic acid production, was highly expressed in spinal cord, muscle and spleen. In addition, high doses (10 mg/kg) of KML29 induced some cannabimimetic effects in vivo in the tetrad test, including hypothermia, analgesia and hypomotility without induction of cataleptic behavior. In summary, inhibition of MAGL by KML29 represents a promising strategy for targeting the cannabinoid and prostaglandin system of the brain with only a moderate induction of cannabimimetic effects.

Enhanced arachidonic acid production from Mortierella alpina combining atmospheric and room temperature plasma (ARTP) and diethyl sulfate treatments

To obtain mutant strains with higher arachidonic acid (ARA) yields, the oleaginous fungus Mortierella alpina was mutated using atmospheric and room temperature plasma (ARTP) coupled with diethyl sulfate (DES). A visual compound filter operation was used in which a screening medium was supplemented with cerulenin, an inhibitor of fatty acid synthase (FAS), and triphenyltetrazolium chloride (TTC). The mutant strain D20 with an ARA production of 5.09g/L, a 40.61% increase over the original strain (3.62g/L), was isolated. The relative ARA content increased from 38.99% to 45.64% of total fatty acids. After optimizing fermentation conditions, the maximum ARA yield (6.82g/L) for strain D20 was obtained in shake flasks. This work provides an appropriate strategy for obtaining high ARA-yield strains by conventional random mutation methods with an efficient screening assay.