Enhancing ligand–protein binding in affinity thermoprecipitation: Elucidation of spacer effects

Copolymers of N‐isopropylacrylamide and N‐acryloyl amino acid spacers of varying chain length were synthesized. p‐Aminobenzamidine (PABA) was chemically linked to the pendant carboxyl groups of these polymers to obtain thermoprecipitating affinity polymers. The inhibition constant (K_i) of these polymers for trypsin decreased, i.e., the efficiency of PABA–trypsin binding increased with increase in the spacer chain length. The polymer to which PABA was linked through a spacer of five methylene groups exhibited eleven times lower K_i than that of the polymer containing PABA without a spacer. Investigations on model inhibitors N‐acyl‐p‐aminobenzamidines showed that this enhancement in trypsin binding by the polymers was due to the spacer as well as to microenvironmental effects. Recovery and specific activity of the trypsin recovered increased with the spacer chain length. Separation of trypsin from a mixture of trypsin and chymotrypsin was also enhanced with the spacer chain length. The inhibition constants of these affinity polymers were not adversely affected by the crowding effect. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 418–425, 1999.     

Wine phenolic compounds influence the production of volatile phenols by wine-related lactic acid bacteria

Aims: To evaluate the effect of wine phenolic compounds on the production of volatile phenols (4‐vinylphenol [4VP] and 4‐ethylphenol [4EP]) from the metabolism of p‐coumaric acid by lactic acid bacteria (LAB). Methods and Results: Lactobacillus plantarum, Lactobacillus collinoides and Pediococcus pentosaceus were grown in MRS medium supplemented with p‐coumaric acid, in the presence of different phenolic compounds: nonflavonoids (hydroxycinnamic and benzoic acids) and flavonoids (flavonols and flavanols). The inducibility of the enzymes involved in the p‐coumaric acid metabolism was studied in resting cells. The hydroxycinnamic acids tested stimulated the capacity of LAB to synthesize volatile phenols. Growth in the presence of hydroxycinnamic acids, especially caffeic acid, induced the production of 4VP by resting cells. The hydroxybenzoic acids did not significantly affect the behaviour of the studied strains. Some of the flavonoids showed an effect on the production of volatile phenols, although strongly dependent on the bacterial species. Relatively high concentrations (1 g l^?1) of tannins inhibited the synthesis of 4VP by Lact. plantarum. Conclusions: Hydroxycinnamic acids were the main compounds stimulating the production of volatile phenols by LAB. The results suggest that caffeic and ferulic acids induce the synthesis of the cinnamate decarboxylase involved in the metabolism of p‐coumaric acid. On the other hand, tannins exert an inhibitory effect. Significance and Impact of the Study: This study highlights the capacity of LAB to produce volatile phenols and that this activity is markedly influenced by the phenolic composition of the medium.     

Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

The soil bacterium Pseudomonas putida KT2440 has gained increasing biotechnological interest due to its ability to tolerate different types of stress. Here, the tolerance of P. putida KT2440 toward eleven toxic chemical compounds was investigated. P. putida was found to be significantly more tolerant toward three of the eleven compounds when compared to Escherichia coli. Increased tolerance was for example found toward p‐coumaric acid, an interesting precursor for polymerization with a significant industrial relevance. The tolerance mechanism was therefore investigated using the genome‐wide approach, Tn‐seq. Libraries containing a large number of miniTn5‐Km transposon insertion mutants were grown in the presence and absence of p‐coumaric acid, and the enrichment or depletion of mutants was quantified by high‐throughput sequencing. Several genes, including the ABC transporter Ttg2ABC and the cytochrome c maturation system (ccm), were identified to play an important role in the tolerance toward p‐coumaric acid of this bacterium. Most of the identified genes were involved in membrane stability, suggesting that tolerance toward p‐coumaric acid is related to transport and membrane integrity.

     

New insights into the catalytic mechanism of human glycine N‐acyltransferase

Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi‐substrate kinetic parameters of a recombinant human glycine N‐acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colorimetric method and a newly developed HPLC–ESI‐MS/MS method. Previous studies analyzing the kinetic parameters of GLYAT, indicated a random Bi–Bi and/or ping‐pong mechanism. In this study, the hippuric acid concentrations produced by the GLYAT enzyme reaction were analyzed using the allosteric sigmoidal enzyme kinetic module. Analyses of the initial rate (v) against substrate concentration plots, produced a sigmoidal curve (substrate activation) when the benzoyl‐CoA concentrations was kept constant, whereas the plot with glycine concentrations kept constant, passed through a maximum (substrate inhibition). Thus, human GLYAT exhibits mechanistic kinetic cooperativity as described by the Ferdinand enzyme mechanism rather than the previously assumed Michaelis–Menten reaction mechanism.     

Enzymatic formation of a resorcylic acid by creating a structure‐guided single‐point mutation in stilbene synthase

A novel C17 resorcylic acid was synthesized by a structure‐guided Vitis vinifera stilbene synthase (STS) mutant, in which threonine 197 was replaced with glycine (T197G). Altering the architecture of the coumaroyl binding and cyclization pocket of the enzyme led to the attachment of an extra acetyl unit, derived from malonyl‐CoA, to p‐coumaroyl‐CoA. The resulting novel pentaketide can be produced strictly by STS‐like enzymes and not by Chalcone synthase‐like type III polyketide synthases; due to the unique thioesterase like activity of STS‐like enzymes. We utilized a liquid chromatography mass spectrometry‐based data analysis approach to directly compare the reaction products of the mutant and wild type STS. The findings suggest an easy to employ platform for precursor‐directed biosynthesis and identification of unnatural polyketides by structure‐guided mutation of STS‐like enzymes.     

ENZYMES OF FATTY ACID METABOLISM IN OVERWINTERING MATURE LARVAE OF THE PINE NEEDLE GALL MIDGE,THECODIPLOSIS JAPONENSIS*

Abstract In this study, overwintering larvae of pine needle gall midge, Thmodiplosis jaHnensis, were sampled at various dates in the winter of 1997 and profiles of some enzymes of fatty acid metabolism were studied. During overwintering, a decrease in total lipids in T. japonensis larvae suggested the use of fat reserves to maintain basal metabolism. Activities of two enzymes associated with fatty acid synthesis, i. e. malic enzyme and ATP‐dependent citrate lyase, decreased from December to mid‐January, then increased from the end of February, indicating a reduced potential for fatty acid synthesis during the winter. Enzymes for fatty acid oxidation, as indicated by the activities of hydroxyacyl‐CoA dehydrogenase, carnitine‐palmitoyl transferase and acetoacetyl‐CoA thiolase, showed different profiles. The potential for ketone body metabolism, as measured by p‐hydroxybutyrate dehydrogenase activity, decreased in the course of winter, indicating that ketone body as a metabolic fuel during overwintering is not important.     

Relationship between Sulfaguanidine Resistance and Increased Cellulose Production in Acetobacter xylinum BPR3001E

The mechanism of the increased cell growth and cellulose production of Acetobacter xylinum subsp. sucrofermentans BPR3001E, a sulfaguanidine (SG)-resistant mutant, was investigated. We found that adding p-aminobenzoic acid (PABA) to cultures of the parent strain, BPR2001, led to increased levels of intracellular adenosine-related purine compounds and increased cellulose production. Furthermore, adding ATP increased the cellulose production by permeabilized BPR2001 cells. On the other hand, the intracellular levels of PABA and adenosine-related purine compounds in BPR3001E cells were higher than those in BPR2001 cells. These results suggest that SG resistance increases enhance cellulose production through increased levels of intracellular high-energy compounds caused by increased PABA biosynthesis, reflecting the promoted supply of cellulose precursors.     

Structure and functional characterization of a bile acid 7α dehydratase BaiE in secondary bile acid synthesis

Conversion of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) to the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) is performed by a few species of intestinal bacteria in the genus Clostridium through a multistep biochemical pathway that removes a 7α‐hydroxyl group. The rate‐determining enzyme in this pathway is bile acid 7α‐dehydratase (baiE). In this study, crystal structures of apo‐BaiE and its putative product‐bound [3‐oxo‐Δ^4,6‐lithocholyl‐Coenzyme A (CoA)] complex are reported. BaiE is a trimer with a twisted α + β barrel fold with similarity to the Nuclear Transport Factor 2 (NTF2) superfamily. Tyr30, Asp35, and His83 form a catalytic triad that is conserved across this family. Site‐directed mutagenesis of BaiE from Clostridium scindens VPI 12708 confirm that these residues are essential for catalysis and also the importance of other conserved residues, Tyr54 and Arg146, which are involved in substrate binding and affect catalytic turnover. Steady‐state kinetic studies reveal that the BaiE homologs are able to turn over 3‐oxo‐Δ⁴‐bile acid and CoA‐conjugated 3‐oxo‐Δ⁴‐bile acid substrates with comparable efficiency questioning the role of CoA‐conjugation in the bile acid metabolism pathway. Proteins 2016; 84:316–331. © 2016 Wiley Periodicals, Inc.     

Jasmonoyl‐l‐isoleucine hydrolase 1 (JIH1) regulates jasmonoyl‐l‐isoleucine levels and attenuates plant defenses against herbivores

For most plant hormones, biological activity is suppressed by reversible conjugation to sugars, amino acids and other small molecules. In contrast, the conjugation of jasmonic acid (JA) to isoleucine (Ile) is known to enhance the activity of JA. Whereas hydroxylation and carboxylation of JA‐Ile permanently inactivates JA‐Ile‐mediated signaling in plants, the alternative deactivation pathway of JA‐Ile by its direct hydrolysis to JA remains unstudied. We show that Nicotiana attenuata jasmonoyl‐l ‐isoleucine hydrolase 1 (JIH1), a close homologue of previously characterized indoleacetic acid alanine resistant 3 (IAR3) gene in Arabidopsis, hydrolyzes both JA‐Ile and IAA‐Ala in vitro. When the herbivory‐inducible NaJIH1 gene was silenced by RNA interference, JA‐Ile levels increased dramatically after simulated herbivory in irJIH1, compared with wild‐type (WT) plants. When specialist (Manduca sexta) or generalist (Spodoptera littoralis) herbivores fed on irJIH1 plants they gained significantly less mass compared with those feeding on wild‐type (WT) plants. The poor larval performance was strongly correlated with the higher accumulation of several JA‐Ile‐dependent direct defense metabolites in irJIH1 plants. In the field, irJIH1 plants attracted substantially more Geocoris predators to the experimentally attached M. sexta eggs on their leaves, compared with empty vector plants, which correlated with higher herbivory‐elicited emissions of volatiles known to function as indirect defenses. We conclude that NaJIH1 encodes a new homeostatic step in JA metabolism that, together with JA and JA‐Ile‐hydroxylation and carboxylation of JA‐Ile, rapidly attenuates the JA‐Ile burst, allowing plants to tailor the expression of direct and indirect defenses against herbivore attack in nature.     

Glycine supplementation extends lifespan of male and female mice

Diets low in methionine extend lifespan of rodents, though through unknown mechanisms. Glycine can mitigate methionine toxicity, and a small prior study has suggested that supplemental glycine could extend lifespan of Fischer 344 rats. We therefore evaluated the effects of an 8% glycine diet on lifespan and pathology of genetically heterogeneous mice in the context of the Interventions Testing Program. Elevated glycine led to a small (4%–6%) but statistically significant lifespan increase, as well as an increase in maximum lifespan, in both males (p = 0.002) and females (p < 0.001). Pooling across sex, glycine increased lifespan at each of the three independent sites, with significance at p = 0.01, 0.053, and 0.03, respectively. Glycine‐supplemented females were lighter than controls, but there was no effect on weight in males. End‐of‐life necropsies suggested that glycine‐treated mice were less likely than controls to die of pulmonary adenocarcinoma (p = 0.03). Of the 40 varieties of incidental pathology evaluated in these mice, none were increased to a significant degree by the glycine‐supplemented diet. In parallel analyses of the same cohort, we found no benefits from TM5441 (an inhibitor of PAI‐1, the primary inhibitor of tissue and urokinase plasminogen activators), inulin (a source of soluble fiber), or aspirin at either of two doses. Our glycine results strengthen the idea that modulation of dietary amino acid levels can increase healthy lifespan in mice, and provide a foundation for further investigation of dietary effects on aging and late‐life diseases.     

Role of phenolic acids and enzymes of phenylpropanoid pathway in resistance of chayote fruit (Sechium edule) against infestation by melon fly,Bactrocera cucurbitae

Melon fly is a serious pest of cucurbits all over the world causing huge losses to yield. However, the only exception is the chayote fruit (Sechium edule) that shows resistance to melon fly infestation. Studies on culture of melon fly indicated the absence of plant traits resisting oviposition on chayote fruit. However, the melon fly was unable to complete its life cycle successfully on chayote showing that factors inhibiting larval development in melon fly could be attributed to biochemical constituents. Studies were, therefore, carried out to compare the biochemical responses of chayote, a melon fly resistant species and bitter gourd, a susceptible species to melon fly infestation with regard to the levels of phenolic acids and activities of the enzymes of phenylpropanoid pathway (PPP) leading to synthesis of lignin. The resistant chayote exhibited significantly higher accumulation of lignin associated with higher activities of phenylalanine ammonia‐lyase (PAL), tyrosine ammonia‐lyase (TAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD). On the contrary, the susceptible bitter gourd recorded lower activities of PAL, CAD and POD and a decreasing trend of TAL during infestation associated with a lower lignin content. The monomer composition of lignin in the resistant chayote showed twofold higher level of guaiacyl (G) and syringyl (S) units compared to susceptible bitter gourd and the G/S ratio during infestation increased in chayote while decreasing in bitter gourd. The levels of PPP intermediates, p‐coumaric acid was higher in chayote while p‐hydroxy benzoic acid, a chemo‐attractant, was higher in bitter gourd. Incorporation of p‐coumaric acid in the larval diet strongly inhibited larval growth even as p‐hydroxy benzoic acid promoted growth confirming the direct role of p‐coumaric acid in conferring resistance to chayote. The level of salicylic acid, a signal molecule involved in induction of defence response, was higher in chayote compared to bitter gourd. Chayote also exhibited higher level of activity of POD in the phloem exudates compared to bitter gourd. The higher concentration of sugars in exudates of chayote might act like signalling molecules causing activation of plant genes, especially of the phenylpropanoid biosynthesis pathway and possibly produce an osmotic effect inducing resistance against the melon fly. Thus, the study revealed that the resistance in chayote to melon fly infestation is a complex, multi‐layered process in which the activities of PPP enzymes generating phenolic intermediates leading to lignin biosynthesis and the composition of exudates appear to play significant roles. Besides, the study also indicated that different forms of lignin might play a role in the resistance of chayote against melon fly infestation.     

Isolation,identification and characterization of soil microbes which degrade phenolic allelochemicals

Aims: To isolate and characterize microbes in the soils containing high contents of phenolics and to dissolve the allelopathic inhibition of plants through microbial degradation. Methods and Results: Four microbes were isolated from plant soils using a screening medium containing p‐coumaric acid as sole carbon source. The isolates were identified by biochemical analysis and sequences of their 16S or 18S rDNA, and designated as Pseudomonas putida 4CD1 from rice (Oryza sativa) soil, Ps. putida 4CD3 from pine (Pinus massoniana) soil, Pseudomonas nitroreducens 4CD2 and Rhodotorula glutinis 4CD4 from bamboo (Bambusa chungii) soil. All isolates degraded 1 g l^?1 of p‐coumaric acid by 70–93% in inorganic and by 99% in Luria‐Bertani solutions within 48 h. They also effectively degraded ferulic acid, p‐hydroxybenzoic acid and p‐hydroxybenzaldehyde. The microbes can degrade p‐coumaric acid and reverse its inhibition on seed germination and seedling growth in culture solutions and soils. Low pHs inhibited the growth and phenolic degradation of the three bacteria. High temperature inhibited the R. glutinis. Co²⁺ completely inhibited the three bacteria, but not the R. glutinis. Cu²⁺, Al³⁺, Zn²⁺, Fe³⁺, Mn²⁺, Mg²⁺ and Ca²⁺ had varying degrees of inhibition for each of the bacteria. Conclusions: Phenolics in plant culture solutions and soils can be decomposed through application of soil microbes in laboratory or controlled conditions. However, modification of growth conditions is more important for acidic and ions‐contaminated media. Significance and Impact of the Study: The four microbes were first isolated and characterized from the soils of bamboo, rice or pine. This study provides some evidence and methods for microbial control of phenolic allelochemicals.     

The effect of ovariectomy of serum amino acids and cholesterol in the rat

Summary As steroid hormones are known to influence amino acid metabolism we tested the hypothesis that ovariectomy should lead to significant changes in this system.We found that after ovariectomy serum alanine was significantly decreased (p = 0.0006) in contrast to serum glycine and branched chain amino acids (BCAA). The ratio of glycine/BCAA, a parameter for anabolism or catabolism was not changed after ovariectomy. If, however, the amino acid alanine as the link to carbohydrate and lipid metabolism was introduced the alanine/BCAA ratio was significantly altered (p = 0.01).Although serum cholesterol was altered as well (increased,p = 0.03), no significant correlation with alanine was found. We can therefore assume that there are two independent mechanisms for lipid and amino acid changes after ovariectomy.The most prominent finding was that estradiol replacement after ovariectomy restored increased cholesterol levels but did not restore alanine levels. Other ovarial hormones must be incriminated for the regulation of alanine metabolism. The anabolic effects of estradiol as decreasing glycine and BCAA were noticed which rules out insufficient estradiol replacement.     

Structure-function relationships of the antibacterial activity of phenolic acids and their metabolism by lactic acid bacteria

Aims: To determine structure–function relationships of antibacterial phenolic acids and their metabolites produced by lactic acid bacteria (LAB). Methods and Results: Minimum inhibitory concentrations (MICs) of 6 hydroxybenzoic and 6 hydroxycinnamic acids were determined with Lactobacillus plantarum, Lactobacillus hammesii, Escherichia coli and Bacillus subtilis as indicator strains. The antibacterial activity of phenolic acids increased at lower pH. A decreasing number of hydroxyl groups enhanced the activity of hydroxybenzoic acids, but had minor effects on hydroxycinnamic acids. Substitution of hydroxyl groups with methoxy groups increased the activity of hydroxybenzoic, but not of hydroxycinnamic, acid. Metabolism of chlorogenic, caffeic, p‐coumaric, ferulic, protocatechuic or p‐hydroxybenzoic acids by L. plantarum, L. hammesii, Lactobacillus fermentum and Lactobacillus reuteri was analysed by LC‐DAD‐MS. Furthermore, MICs of substrates and metabolites were compared. Decarboxylated and/or reduced metabolites of phenolic acids had a lower activity than the substrates. Strain‐specific metabolism of phenolic acids generally corresponded to resistance. Conclusions: The influence of lipophilicity on the antibacterial activity of hydroxybenzoic acids is stronger than that of hydroxycinnamic acids. Metabolism of phenolic acids by LAB detoxifies phenolic acids. Significance and Impact of the Study: Results allow the targeted selection of plant extracts for food preservation, and selection of starter cultures for fermented products.     

Novel amides of 1,1‐bis‐(carboxymethylthio)‐1‐arylethanes: Synthesis,characterization, acetylcholinesterase,butyrylcholinesterase, and carbonic anhydrase inhibitory properties

The thiolation reaction was carried out in a benzene solution at 80°C and p‐substituted ketones and mercaptoacetic acid in a molar ratio (1:4) of in the presence of a catalytic amount of toluene sulfonic acids. The enzyme inhibition activities of the novel amides of 1,1‐bis‐(carboxymethylthio)‐1‐arylethanes derivatives were investigated. These novel amides of 1,1‐bis‐(carboxymethylthio)‐1‐arylethanes derivatives showed good inhibitory action against acetylcholinesterase (AChE) butyrylcholinesterase (BChE), and human carbonic anhydrase I and II isoforms (hCA I and II). AChE inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs and toxins. Many clinically established drugs are carbonic anhydrase inhibitors, and it is highly anticipated that many more will eventually find their way into the market. The novel synthesized compounds inhibited AChE and BChE with K_i values in the range of 0.64–1.47 nM and 9.11–48.12 nM, respectively. On the other hand, hCA I and II were effectively inhibited by these compounds, with K_i values between 63.27–132.34 and of 29.63–127.31 nM, respectively.     

Involvement of glutathione metabolism in Eichhornia crassipes tolerance to arsenic

• Aquatic macrophytes are potentially useful for phytoremediation programmes in environments contaminated by arsenic (As). Biochemical and physiological modification analyses in different plant parts are important to understand As tolerance mechanisms.
• The objective was to evaluate glutathione metabolism in leaves and roots of Eichhornia crassipes (Mart.) Solms treated to As. Specimens of E. crassipes were cultured for 3 days in Clark's nutrient solution containing 7 μm As. The enzymes ATP sulphurylase (ATPS), glutathione reductase (GR), glutathione peroxidase (GSH‐Px), glutathione sulphotransferase (GST) and γ‐glutamylcysteine synthetase (γ‐ECS) activity, glutathione content, total protein and non‐protein thiols were evaluated.
• The ATPS activity increased in roots. GR activity in leaves and GSH‐Px in roots were lower. GST activity was higher in roots and lower in leaves, and γ‐ECS activity was higher in leaves. Glutathione levels were lower, total thiol levels were higher and non‐protein levels did not change in E. crassipes leaves and roots. Exposure to As increased enzyme activity involved with sulphur metabolism, such as ATPS. Higher GR activity and lower GSH‐Px indicate increased glutathione conjugation to As due to increased GSH availability. The higher GST activity indicates its participation in As detoxification and accumulation through As GSH conjugation. Changes in glutathione and thiol levels suggest high phytochelatin synthesis.
• In conclusion, the increments in ATPS, GR, GST and γ‐ECS activity indicate that these enzymes are involved in GSH metabolism and are part of the E. crassipes As detoxification mechanism.

     

Functional analysis of genes for benzoate metabolism in the albicidin biosynthetic region of Xanthomonas albilineans

Albicidins are potent DNA-gyrase-inhibiting antibiotics and phytotoxins synthesised by Xanthomonas albilineans. Functions have been deduced for some clustered biosynthetic genes, including a PKS-NRPS megasynthase, methyltransferases and regulatory genes, and resistance genes including a transporter and a gyrase-binding protein. More puzzling is the presence in this cluster of apparent aromatic metabolism genes. Here, we describe functional analysis of several such genes and propose a model for their role. An apparent benzoate CoA ligase (xabE) proved essential for albicidin production and pathogenicity. A neighbouring operon includes genes for p-aminobenzoate (PABA) metabolism. A PABA synthase fusion (pabAB) restored prototrophy in pabA and pabB mutants of Escherichia coli, proving functionality. Inactivation of pabAB increased susceptibility to sulphanilamide but did not block albicidin production. X. albilineans contains a remote pabB gene which evidently supplies enough PABA for albicidin biosynthesis in culture. Additional capacity from pabAB may be advantageous in more demanding environments such as infected plants. Downstream from pabAB are a known resistance gene (albG) and ubiC which encodes a p-hydroxybenzoate (PHBA) synthase. PHBA protects X. albilineans from inhibition by PABA. Therefore, coordinated expression may protect X. albilineans against toxicity of both the PABA intermediate and the albicidin product, under conditions that induce high-level antibiotic biosynthesis.     

Properties of Recombinant Staphylococcus haemolyticus Cystathionine β‐Lyase (metC) and Its Potential Role in the Generation of Volatile Thiols in Axillary Malodor

Enzymes implicated in cysteine and methionine metabolism such as cystathionine β‐lyase (CBL; EC 4.4.1.8), a pyridoxal‐5′‐phosphate (PLP)‐dependent carbon–sulfur lyase, have been shown to play a central role in the generation of sulfur compounds. This work describes the unprecedented cloning and characterization of the metC‐cystathionine β‐lyase from the axillary‐isolated strain Staphylococcus haemolyticus AX3, in order to determine its activity and its involvement in amino acid biosynthesis, and in the generation of sulfur compounds in human sweat. The gene contains a cysteine/methionine metabolism enzyme pattern, and also a sequence capable to effect β‐elimination. The recombinant enzyme was shown to cleave cystathionine into homocysteine and to convert methionine into methanethiol at low levels. No odor was generated after incubation of the recombinant enzyme with sterile human axillary secretions; sweat components were found to have an inhibitory effect. These results suggest that the generation of sulfur compounds by Staphylococci and the β‐lyase activity in human sweat are mediated by enzymes other than the metC gene or by the concerted activities of more than one enzyme.     

Arachidonic acid alleviates the detrimental effects of acetylsalicylic acid on human granulosa cells performance in vitro

Here, we investigated the biological effects of arachidonic acid (AA) in human cumulus granulosa cells (CGCs) after exposure to ASA. Cells were isolated from the follicular fluid and incubated with 0.5 mM acetylsalicylic acid (ASA) and 50 µM AA. Cell viability was analyzed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. E₂ and P₄ levels were measured by chemiluminescence assay. Expression of genes including CYP19A1, FACN, and SCD1 was measured by real‐time polymerase chain reaction assay. Oxidative status was analyzed by monitoring glutathione peroxidase activity. The fatty acid profile was analyzed by the gas chromatography technique. Enzyme‐linked immunosorbent assay was used to measure prostaglandin E₂ (PGE₂) in CGCs after exposure to ASA and AA. Protein levels of the estrogen receptor were studied by immunofluorescence staining. Ultrastructural changes were evaluated by transmission electron microscopy imaging. ASA treatment reduced E₂ production, Cyp19a1 expression, glutathione peroxidase (GPx) activity, and estradiol receptor expression in CGCs. The addition of AA prevented the ASA‐induced E₂ reduction (p < .05) and expression of Cyp19a1. Moreover, AA increased the antioxidant capacity of CGCs exposed to ASA by promoting GPx activity (p < .05). AA increased monounsaturated fatty acid/saturated fatty acid ratio compared with the ASA group (p < .05). AA supplementation triggered the synthesis and secretion of PGE₂ in ASA‐treated CGCS (p < .05). Cytoplasmic vacuolation observed in the ASA group and treatment with AA intensified vacuolation rate. The expression of the estrogen receptor was increased after AA supplementation. Data demonstrated that AA decreased the detrimental effects of ASA on human CGCs after 72 hr.