Fatty acid remodeling in cellular glycerophospholipids following the activation of human T cells

Changes in fatty acid (FA) and glycerophospholipid (GPL) metabolism associated with cell cycle entry are not fully understood. In this study FA-GPL remodeling was investigated in resting and proliferating primary human T cells. Significant changes were measured in the composition and distribution of FAs in GPLs following receptor activation of human T cells. The FA distribution of proliferating T cells was very similar to that of the human Jurkat T cell line and when the stimulus was removed from proliferating T cells, they stopped proliferating and the FA distribution largely reverted back to that of resting T cells. The cellular content of saturated and monounsaturated FAs was significantly increased in proliferating cells, which was associated with an induction of FA synthase and stearoyl-CoA desaturase-1 gene expression. Additionally, cellular arachidonate was redistributed in GPLs in a distinct pattern that was unlike any other FAs. This redistribution was associated with an induction of CoA-dependent and CoA-independent remodeling. Accordingly, significant changes in the expression of several acyl-CoA synthetases, lysophospholipid acyltransferases, and phospholipase A₂ were measured. Overall, these results suggest that metabolic pathways are activated in proliferating T cells that may represent fundamental changes associated with human cell proliferation.     

Cortical Impact Injury in Rats Promotes a Rapid and Sustained Increase in Polyunsaturated Free Fatty Acids and Diacylglycerols

Neurotrauma activates the release of membrane phospholipid-derived second messengers, such as free arachidonic acid (20:4n-6, AA) and diacylglycerols (DAGs). In the present study, we analyze the effect of cortical impact injury of low-grade severity applied to the rat frontal right sensory-motor cortex (FRC) on the accumulation of free fatty acids (FFAs) and DAGs in eight brain areas 30 min and 24 hours after the insult. At these times, accumulation of FFAs and DAGs occurred mainly in the damaged FRC. The cerebellum was the only other brain area that displayed a significant accumulation of DAGs by day one post-injury. By 30 min, accumulation of free AA in the FRC displayed the greatest relative increase (300% over sham value), followed by free docosahexaenoic acid (22:6n-3, DHA, 150%), while both 20:4-DAGs and 22:6-DAGs were increased 100% over sham values. At day one, free 22:6 and 22:6-DAGs showed the greatest increase (590% and 230%, respectively). These results suggest that TBI elicits the hydrolysis of phospholipids enriched in excitable membranes, targeting early on 20:4-phospholipids (by 30 min post-trauma) and followed 24 hours later by preferential hydrolysis of DHA-phospholipids. These lipid metabolic changes may contribute to the initiation and maturation of neuronal and fiber track degeneration observed following cortical impact injury.     

Regulation of prostaglandin E2 production by the superoxide radical and nitric oxide in mouse peritoneal macrophages

The purpose of this study was to elucidate the role of NO and O^-₂ on enzymatic components of cyclooxygenase (COX) pathway in peritoneal macrophages. Activation of murine peritoneal macrophages by lipopolysaccharides (LPS) resulted in time-dependent production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). This stimulation was also accompanied by the production of other reactive oxygen species such as superoxide (O^-₂), and by increased expression of COX-2. Our results provide evidence that O^-₂ may be involved in the pathways that result in arachidonate release and PGE₂ formation by COX-2 in murine peritoneal macrophages stimulated by LPS. However, we were not able to demonstrate that NO participates in the regulation of PG production under our experimental conditions.     

The protective role of verbenalin in rat model of focal cerebral ischemia reperfusion

To investigate the protective mechanism of verbenalin on cerebral ischemia-reperfusion injury. Middle cerebral artery occlusion in the left hemisphere was induced in rats by filament insertion, and rat model of focal cerebral ischemia-reperfusion was established. The high, medium and low dose of verbenalin groups were injected in the tail vein of corresponding drugs 10?min before reperfusion, and submitted for 22?h of reperfusion after the operation. Mortality rate was then calculated, and neurological deficits of rats were scored. The serum of rats was got to determine the S-100β protein level, and the brain tissue was removed to determine the levels of Bax, Bcl-2, Caspase-3 and ATPase. TTC staining was performed on the brain tissue to calculate the percentage of cerebral infarct size. Changes in brain tissue morphology were observed. Rat model of focal cerebral ischemia-reperfusion was successfully replicated. In groups that have taken different doses of verbenalin, the mortality rate, neurological deficit score and the percentage of cerebral infarction size were significantly reduced, and the levels of Bax, Caspase-3, S-100β level of the serum in the brain tissue were also significantly reduced. Increases in the levels of Bcl-2 and ATPase in brain tissue and improvement of pathological damage of hippocampus and cortex were observed. Verbenalin can inhibit the expression of apoptosis genes, promote the expression of anti-apoptosis genes, improve brain microcirculation and energy metabolism, hence reducing cerebral ischemia-reperfusion injury.     

Simultaneous activation of p38 and JNK by arachidonic acid stimulates the cytosolic phospholipase A2-dependent synthesis of lipid droplets in human monocytes

Exposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. This effect appears specific for AA in that it is not mimicked by other fatty acids, whether saturated or unsaturated. LDs are formed by two different routes: (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling, leading to increased triacylglycerol and cholesteryl ester formation utilizing fatty acids coming from the de novo biosynthetic route. Both routes can be dissociated by the arachidonyl-CoA synthetase inhibitor triacsin C, which prevents the former but not the latter. LD formation by AA-induced signaling predominates, accounting for 60–70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A₂α (cPLA₂α), pointing out this enzyme as a key regulator of AA-induced signaling. LD formation in AA-treated monocytes can also be blocked by the combined inhibition of the mitogen-activated protein kinase family members p38 and JNK, which correlates with inhibition of cPLA₂α activation by phosphorylation. Collectively, these results suggest that concomitant activation of p38 and JNK by AA cooperate to activate cPLA₂α, which is in turn required for LD formation possibly by facilitating biogenesis of this organelle, not by regulating neutral lipid synthesis.     

Fatty acid oxidation and myocardial phospholipase A2 activity

Summary Cis-unsaturated fatty acids, but not saturated fatty acids, inhibited phospholipase A₂ activity (PLA₂) in vitro, and may function as endogenous suppressors of lipolysis. To probe the possible role of lipid peroxidation in the regulation of myocardial lipid catabolism, a neutral-active and Ca²⁺-dependent PLA₂ was extracted from rat heart and was partially purified by sulfopropyl cation exchange chromatography. Myocardial PLA, activity was inhibited in a dose-dependent manner by oleic, linoleic, linolenic, and arachidonic acids; the IC₅₀ for arachidonic acid was approx 65 M. Palmitic acid was not inhibitory. When arachidonic acid was incubated at 37°C, exposed to air, there was a time- and pH-dependent peroxidation of the arachidonic acid as monitored by turbidity, thiobarbituric acid reactivity, and thin layer chromatography. Peroxidation was increased as the pH was lowered from 7.5 to 4.5, and was accompanied by a decrease in PLA₂ inhibitory potency. Thus, arachidonate incubated for 24 hours at pH's 4.5, 6.0 and 7.5 lost 84%, 32%, and 20% respectively, of its inhibitory potency. Therefore, in vitro acidosis promotes the oxidation of cis-unsaturated fatty acids and relieves their inhibitory or suppressive activity toward PLA₂s. Increased lipid peroxidation of unesterified unsaturated fatty acids during acidosis may therefore promote lipolysis observed during myocardial ischemia and reperfusion injury.     

Thromboxane A2 stimulates mitogen activated protein kinase and arachidonic acid liberation in rabbit platelets

U46619, a thromboxane A₂ mimetic, caused tyrosine phosphorylation of several proteins in rabbit platelets. Among them, 42 kDa protein was identified as a mitogen-activated protein kinase (MAPK). U46619 activated MAPK in a concentration-dependent manner, measured by incorporation of ³²P to a specific substrate for MAPK. U46619 also liberated [³H)arachidonic acid in a concentration-dependent manner. The U46619-induced MAPK activation and [³H]arachidonic acid liberation were inhibited by SQ29548 and by the removal of external Ca²⁺ ions. This is a first demonstration that TXA₂ activates MAPK accompanied with arachidonic acid liberation in rabbit platelets.     

Modulation of the beta-adrenergic response in cultured rat heart cells

Incubation of rocker-cultured neonatal rat heart cells with 3 mM L(+)-lactate led to a sharp increase in the sensitivity of cardiomyocytes to the beta-adrenergic agonist isoprenaline, as measured by their chronotropic response. This effect was accompanied by a reduction in the arachidonic acid content of the total phospholipids. The phospholipase A₂-activator melittin as well as free arachidonic acid induced this supersensitivity to the same degree. On the other hand, the L(+)-lactate-evoked supersensitivity could be blocked by the phospholipase A₂ inhibitors mepacrine and n-bromophenacyl-bromide, suggesting an involvement of phospholipase A₂ in the process of beta-adrenergic sensitization. The sensitizing action of arachidonic acid was blocked by the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid, but not by the cyclooxygenase inhibitor indomethacin. Supersensitivity was likewise evoked by 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), but not by 5-S-HPETE or 5-S-HETE. These findings suggest that the phospholipase A₂-15-lipoxygenase pathway plays a role in the induction of beta-adrenergic supersensitivity in the cultured cardiomyocytes and point to a new physiological role of the lipoxygenase product 15-S-HETE.Abbreviations NDGA nordihydroguaiaretic acid - HETE hydroeicosatetraenoic acid - HPETE hydroperoxyeicosatetraenoic acid     

Modulation of ATPase activities of human erythrocyte membranes by free fatty acids or phospholipase A2

Summary The artificial insertion of increasing amounts of unsaturated fatty acids into human erythrocyte membranes modulated ATPase activities in a biphasic manner, depending on the number and position of double bonds, their configuration, and the chain length. Uncharged long-chain fatty acid derivatives with double bonds and short-chain fatty acids were ineffective. Stearic acid stimulated Na⁺K⁺-ATPase only. Anionic and non-ionic detergents and -lysophosphatidylcholine failed to stimulate ATPase activities at low, and inhibited them at high concentrations.Mg²⁺-ATPase activity was maximally enhanced by a factor of 2 in the presence of monoenoic fatty acids; half-maximal stimulation was achieved at a molar ratio ofcis(trans)-configurated C18 acids/membrane phopholipid of 0.16 (0.26).Na⁺K⁺-ATPase activity was maximally augmented by 20% in the presence of monoenoic C18 fatty acids at 37°C. Half-maximal effects were attained at a molar ratio oleic (elaidic) acid/phospholipid of 0.032 (0.075). Concentrations of free fatty acids which inhibited ATPase activities at 37°C were most stimulatory at reduced temperatures. AT 10°C, oleic acid increased Na⁺K⁺-ATPase activity fivefold (molar ratio 0.22).Unsaturated fatty acids simulated the effect of calmodulin on Ca²⁺-ATPase of native erythrocyte membranes (i.e., increase ofV _max from 1.6 to 5 mol PO ₄ ^3– ·phospholipid^–1·hr^–1, decrease of K _Ca from 6 m to 1.4–1.8 m). Stearic acid decreasedK _Ca (2 m) only, probably due to an increase of negative surface charges.A stimulation of Mg²⁺-ATPase, Na⁺K⁺-ATPase, and Ca²⁺-ATPase could be achieved by incubation of the membranes with phospholipase A₂.An electrostatic segregation of free fatty acids by ATPases with ensuing alterations of surface charge densities and disordering of the hydrophobic environment of the enzymes provides an explanation of the results.     

Calcium-dependent mitochondrial formation of species promoting strand scission of genomic DNA in U937 cells exposed to tert-butylhydroperoxide: The role of arachidonic acid

Treatment of U937 cells with a sublethal concentration of tert-butylhydroperoxide generates DNA single strand breakage in U937 cells and this response is increased by caffeine, ATP, pyruvate or antimycin A. As we previously reported (Guidarelli, Clementi, Brambilla and Cantoni, (1997) Biochem. J. 328, 801–806), the enhancing effects of antimycin A are mediated by inhibition of complex III and the ensuing formation of superoxides and hydrogen peroxide in a reaction in which ubisemiquinone serves as an electron donor. Active electron transport was required in pyruvate-supplemented cells since the increased genotoxic response occurred as a consequence of enforced mitochondrial Ca²⁺ accumulation, a process driven by the increased electrochemical gradient. The enhancing effects of caffeine or ATP were also the consequence of mitochondrial Ca²⁺ accumulation but these responses were independent on electron transport. The increased formation of DNA lesions resulting from exposure to tert-butylhydroperoxide associated with the Ca²⁺-mobilizing agents or the respiratory substrate was mediated by arachidonic acid generated by Ca²⁺-dependent activation of phospholipase A₂. Melittin, a potent phospholipase A₂ activator, and reagent arachidonic acid mimicked the effects of caffeine, ATP or pyruvate on the tert-butylhydroperoxide-induced DNA single strand breakage.     

Studies on the Cytosolic Phospholipase A2 in Immortalized Astrocytes (DITNC) Revealed New Properties of the Calcium Ionophore,A23187

Besides playing an important role in the maintenance of cell membrane phospholipids, phospholipases A₂ (PLA₂) are responsible for the release of arachidonic acid (AA) which is a precursor for prostaglandin biosynthesis. The cytosolic PLA₂ has been the focus of recent studies, probably due to its ability to respond to protein kinases and changes in intracellular calcium levels. In this study, we examined agents for stimulation of the cytosolic phospholipase A₂ in immortalized astrocytes (DITNC). Incubation of DITNC cells with [¹⁴C]arachidonic acid (AA) resulted in a time-dependent uptake of the label into phospholipids (PL) and neutral glycerides. In prelabeled cells, release of labeled AA could be stimulated by calcium mobilizing agents such as calcium ionophore A23187 (4–20 M) and thimerosal (100 M), and by phorbol myristate acetate (PMA, 100 nM), an agent for activation of protein kinase C. The release of AA could also be stimulated by ATP (200 M), probably through activation of the purinergic receptor but not by glutamate (1 mM). The stimulated release of AA was dependent on extracellular Ca²⁺ and was inhibited by mepacrine (50 M), a non-specific PLA₂ inhibitor. Western blot analysis further confirmed the presence of an 85 kDa cPLA₂ in both membrane and cytosol fractions of these cells and stimulation by A23187 resulted in translocation of this protein to the membrane fraction. Besides labeled fatty acids, A23187 also stimulated the concomitant release of labeled PL into the culture medium and this event was accompanied by the increased release in lactate dehydrogenase (LDH). Results thus revealed that besides activation of cPLA₂, the calcium ionophore A23187 is capable of perturbating cell membrane integrity.     

Effect of pentoxifylline on arachidonic acid metabolism,neutral lipid synthesis and accumulation during induction of the lipocyte phenotype by retinol in murine hepatic stellate cell

In liver fibrosis, the quiescent hepatic stellate cells (HSC) are activated to proliferate and express the activated myofibroblast phenotype, losing fat droplets and the stored vitamin A, and depositing more extracellular matrix. Therapeutic strategies for liver fibrosis are focused on HSC. Pentoxifylline (PTF), an analog of the methylxanthine, prevents the biochemical and histological changes associated with animal liver fibrosis. The aim of the present study was to investigate the phenotypic change of myofibroblasts into quiescent lipocytes by PTF and/or retinol, using a permanent cell line GRX that represents murine HSC. We studied the action of both drugs on the synthesis of neutral lipids, activity of phospholipase A₂ (PLA₂), release of arachidonic acid (AA) and prostaglandins synthesis. Accumulation and synthesis of neutral lipids was dependent upon association of retinol with PTF. PTF (0.5 mg/mL) alone did not induce lipid accumulation and synthesis, but in cells induced by physiologic concentration of retinol (1–2.5 M), it increased the quantity of stored lipids. Retinol and PTF (5 M and 0.1 mg/mL, respectively) had a synergistic effect on neutral lipid synthesis and accumulation. In higher PTF concentrations (0.5 and 0.7 mg/ml), the synthesis was stimulated but accumulation decreased. Membrane-associated PLA₂ activity decreased after PTF treatment, which increased the AA release 8 fold, and significantly increased the production of PGE₂, but not of PGF₂. However, when in presence of retinol, we observed a slightly higher increase in PGE₂ and PGF₂ production. In conclusion, PTF treatment generated an excess of free AA. We propose that retinol counteracts the action of PTF on the AA release and PGs production, even though both drugs stimulated the lipocyte induction in the HSC.     

Studies on the status of tyrosyl residues in phospholipases A2 fromNaja naja atra andNaja nigricollis snake venoms

Two phospholipases A₂ (PLA₂) fromNaja naja atra andNaja nigricollis snake venoms were subjected to tyrosine modification withp-nitrobenzenesulfonyl fluoride (NBSF) atpH 8.0. Three major NBS derivatives from each PLA₂ were separated by high-performance liquid chromatography. The results of amino acid analysis showed that only two Tyr residues out of nine were modified, and the modified residues were identified to be Tyr-3 and Tyr-63 (or Tyr-62) in the sequence. Spectrophotometric titration indicated that the phenolic group of Tyr-3 and Tyr-63 (or Tyr-62) had apK of 10.1 and 11.0, respectively. The reactivity of Tyr-3 toward NBSF was not affected in the presence or absence of Ca ²⁺; however, the reactivity of Tyr-63 (or Tyr-62) toward NBSF was greatly enhanced by Ca²⁺. Modification of Tyr-63 (or Tyr-62) resulted in a marked decrease in both lethality and enzymatic activity. Conversely, modification of Tyr-3 inN. naja atra PLA₂ could cause more than a sixfold increase in lethal potency, in sharp contrast to the loss of enzymatic activity.Tyrosine-63-modifiedN. naja atra PLA₂ exhibited the same Ca²⁺-induced difference spectra as that of native PLA₂, indicating that the Ca²⁺-binding ability of Tyr-63-modifiedN. naja atra PLA₂ was not impaired. However, Tyr-3-modified PLA₂ and all Tyr-modifiedN. nigricollis CMS-9 were not perturbed by Ca²⁺, revealing that the Ca²⁺-binding ability have been lost after tyrosine modification. These results suggest that Tyr-62 inN. nigricollis CMS-9 and Tyr-3 in both enzymes are involved in Ca²⁺ binding. AtpH 8.0, both native PLA₂ enzymes enhance the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, while all of the Tyr-modified derivatives did not enhance the emission intensity at all either in the presence or absence of Ca²⁺, suggesting that the hydrophobic pocket that interacts with ANS might be the substrate binding site, in which Tyr-3 and Tyr-63 (or Tyr-62) are involved.     

Phospholipase A2 in auxin and elicitor signal transduction in cultured parsley cells (Petroselinum crispum L.)

Signal-activated phospholipase A₂ cleavesphosphatidylcholine (PC) into free fatty acids and LPC, respectively.Using bis-BODIPY-PC as an indicator substrate for phospholipaseA₂ which is taken up by parsley cells, active auxins atconcentrations as low as 1 M and a fungal elicitor induced fattyacid-accumulation. Nordihydroguajaretic acid inhibited the accumulationof fatty acid induced by the elicitor. In addition to this, theelicitor, but not auxin, decreased the pool size of diacylglycerol,which seemed to originate from a PC-splitting phospholipase C, whichwould be a new enzyme in plant signal transduction. However, thiselicitor is known to rapidly increase cytosolic calcium in parsley cellsand this activates phospholipase C. Thus, activation of phospholipase Cshould lead to an increase of diacylglycerol and not to a decrease whichmight indicate a discrepancy between animal and plant phospholipidsignal transduction.     

Identification of novel phospholipase A2 group IX members in metazoans

Sequence homologues of the bacterium Streptomyces violaceoruber and sea anemone Nematostella vectensis PLA₂ pfam09056 members were identified in several bacteria, fungi and metazoans illustrating the evolution of this PLA₂ sub-family. Comparison of their molecular structures revealed that bacteria and fungi members are part of the GXIV of PLA₂s while metazoan representatives are similar with GIX PLA₂ of the marine snail Conus magus. Members of GXIV and GIX PLA₂s show modest overall sequence similarity (21–35%) but considerable motif conservation within the putative Ca²⁺-binding, catalytic sites and cysteine residue positions which are essential for enzyme function. GXIV PLA₂s of bacteria and fungi typically contain four cysteine residues composing two intramolecular disulphide bonds. GIX PLA₂ homologues were identified in cnidarians and molluscs and in a single tunicate but appear to be absent from other metazoan genomes. The mature GIX PLA₂ deduced peptides contain up to ten cysteine residues capable of forming five putative disulphide bonds. Three disulphide bonds were identified in GIX PLA₂s, two of which correspond to those localized in GXIV PLA₂s. Phylogenetic analysis demonstrates that metazoan GIX PLA₂s cluster separate from the bacterial and fungal GXIV PLA₂s and both pfam09056 members form a group separate from the prokaryote and eukaryote GXIIA PLA₂ pfam06951. Duplicate PLA₂ pfam09056 genes were identified in the genomes of sea anemone N. vectensis and oyster Crassostrea gigas suggest that members of this family evolved via species-specific duplication events. These observations indicate that the newly identified metazoan pfam09056 members may be classified as GIX PLA₂s and support the idea of the common evolutionary origin of GXIV and GIX PLA₂ pfam09056 members, which emerged early in bacteria and were maintained in the genomes of fungi and selected extant metazoan taxa.     

Enzymatic Esterification of Long Polyunsaturated Fatty Acids and Lyso-Phosphatidylcholine in Isooctane and Ethanol

Phosphatidylcholine (PC) was synthesized from lyso-PC and long poly-unsaturated (n-3) fatty acids (PUFA) using immobilized phospholipase A₂. The esterification was performed using the fatty acids as the main solvent and isooctane or ethanol (99.5%) at low concentrations (7–45%) as additional solvents. The temperature was kept constant at 45d`C and the water concentrations were carefully controlled.

The best yield of PC (22%) was found in the isooctane system at a low water content (22% of the dry immobilized enzyme). In the ethanol system, the yield of PC was only half. The best yields were attained when the concentrations of both isooctane and ethanol were below 7% and the reaction time was very long (9 days). Improved contact between the enzyme and the substrates would probably increase the reaction rate.     

Secondary messengers and phospholipase A2 in auxin signal transduction

Despite recent progress auxin signal transduction remains largely scetchy and enigmatic. A good body of evidence supports the notion that the ABP1 could be a functional receptor or part of a receptor, respectively, but this is not generally accepted. Evidence for other functional receptors is lacking, as is any clearcut evidence for a function of G proteins. Protons may serve as second messengers in guard cells but the existing evidence for a role of calcium remains to be clearified. Phospholipases C and D seem not to have a function in auxin signal transduction whereas the indications for a role of phospholipase A₂ in auxin signal transduction accumulated recently. Mitogen-activated protein kinase (MAPK) is modulated by auxin and the protein kinase PINOID has a role in auxin transport modulation even though their functional linkage to other signalling molecules is ill-defined. It is hypothesized that signal transduction precedes activation of early genes such as IAA genes and that ubiquitination and the proteasome are a mechanism to integrate signal duration and signal strength in plants and act as major regulators of hormone sensitivity.     

高山被孢霉中二酰甘油酰基转移酶2同源基因的克隆、表达和活性分析

[背景] 高山被孢霉（Mortierella alpina）是一种可积累大量花生四烯酸（Arachidonic Acid,AA）的产油丝状真菌,其所产脂肪酸主要被组装到甘油骨架上以三酰甘油（Triacylglycerol,TAG）形式存在。二酰甘油酰基转移酶（Diacylglycerol Acyltransferase,DGAT）是TAG生物合成途径的关键酶,对于高山被孢霉TAG的生产具有重要意义。[目的] 通过探究高山被孢霉DGAT2在TAG生物合成方面的功能特点,以期为提高产油真菌的TAG产量及改善TAG的脂肪酸组成提供参考。[方法] 利用序列比对在高山被孢霉ATCC32222基因组中筛选出2个编码DGAT2的候选基因MaDGAT2A/2B,在酿酒酵母（Saccharomyces cerevisiae）中异源表达后进行功能分析,并在外源添加AA条件下通过检测TAG产量进一步分析MaDGAT2A/2B的活性,最后在高山被孢霉中同源过表达MaDGAT2A/2B,通过检测重组菌总脂肪酸产量及组分以分析MaDGAT2A/2B的体内活性。[结果] MaDGAT2A在S. cerevisiae中异源表达时,重组酵母菌TAG的产量达到细胞干重的3.06%,为对照组的4.91倍;而MaDGAT2B未明显提高重组酵母菌TAG的产量。在外源添加AA时,MaDGAT2A/2B均可显著促进重组酵母菌中TAG合成,表达MaDGAT2A的重组酵母菌TAG含量为对照组的3.67倍,表达MaDGAT2B的重组酵母菌TAG含量为对照组的2.61倍。MaDGAT2A/2B在高山被孢霉中过表达对其总脂肪酸产量无显著影响,但可显著提高总脂肪酸中AA的含量,AA占总脂肪酸比例最高达到39.15%,相比对照组提高16.14%。[结论] MaDGAT2A/2B可以参与TAG的生物合成,表明2个候选基因编码的蛋白具有DGAT活性,并且可提高高山被孢霉脂肪酸中AA的含量,对于改善产油真菌的脂肪酸组成从而提高其应用价值具有重要意义。     

Antibacterial properties of recombinant human non-pancreatic secretory phospholipase A2

Human non-pancreatic secretory phospholipase A₂ (hnpsPLA₂) is a group IIA phospholipase A₂ which plays an important role in the innate immune response. This enzyme was found to exhibit bactericidal activity toward Gram-positive bacteria, but not Gram-negative ones. Though native hnpsPLA₂ is active over a broad pH range, it is only highly active at alkaline conditions with the optimum activity pH of about 8.5. In order to make it highly active at neutral pH, we have obtained two hnpsPLA₂ mutants, Glu89Lys and Arg100Glu that work better at neutral pH in a previous study. In the present study, we tested the bactericidal effects of the native hnpsPLA₂ and the two mutants. Both native hnpsPLA₂ and the two mutants exhibit bactericidal activity toward Gram-positive bacteria. Furthermore, they can also kill Escherichia coli, a Gram-negative bacterium. The two mutants showed better bactericidal activity for E. coli at neutral pH than the native enzyme, which is consistent with the enzyme activities. As hnpsPLA₂ is highly stable and biocompatible, it may provide a promising therapy for bacteria infection treatment or other bactericidal applications.