Effect of physicochemical parameters on the stability and activity of garlic alliinase and its use for in-situ allicin synthesis.

Garlic is a well-known example of natural self-defence system consisting of an inactive substrate (alliin) and enzyme (alliinase) which, when combined, produce highly antimicrobial allicin. Increase of alliinase stability and its activity are of paramount importance in various applications relying on its use for in-situ synthesis of allicin or its analogues, e.g., pulmonary drug delivery, treatment of superficial injuries, or urease inhibitors in fertilizers. Here, we discuss the effect of temperature, pH, buffers, salts, and additives, i.e. antioxidants, chelating agents, reducing agents and cosolvents, on the stability and the activity of alliinase extracted from garlic. The effects of the storage temperature and relative humidity on the stability of lyophilized alliinase was demonstrated. A combination of the short half-life, high reactivity and non-specificity to particular proteins are reasons most bacteria cannot deal with allicin's mode of action and develop effective defence mechanism, which could be the key to sustainable drug design addressing serious problems with escalating emergence of multidrug-resistant (MDR) bacterial strains.

α-Glucosidase Inhibitory Activity of Tannat Grape Phenolic Extracts in Relation to Their Ripening Stages.

The present study aimed to screen grape extracts as novel α-glucosidase inhibitors to prevent type-2 diabetes and hyperglycemia. The total polyphenol content (TPC) was measured by Folin-Ciocalteu assay and the stilbene, anthocyanin and flavan-3-ol compounds were measured by Ultra High-Performance Liquid Chromatography coupled to Mass Spectrometry (UHPLC-MS). The α-glucosidase inhibitory of seed and skin Tannat grape extracts at four ripening stages were investigated. The highest TPC values were measured in seeds at the "veraison stage" (65.29 ± 5.33 g of Gallic Acid Equivalent (GAE) per kilogram of Fresh Weight (FW)). This was in accordance with the high flavan-3-ol contents measured for these two extracts (43.22 ± 2.59 and 45.45 ± 6.48 g/kg of seeds FW, respectively). The skin and seed extracts at the first stage of ripening exerted strong α-glucosidase inhibition, exceeding 95% (p < 0.05). A high linear correlation (R = 0.723, p ≤ 0.05) was observed between flavan-3-ol contents and the α-glucosidase inhibitory activity. The stilbene contents and this activity were moderately to strongly anti-correlated (R = -0.828, p ≤ 0.05 for trans-resveratrol). The enzyme kinetic studies revealed a mixed type of inhibition. This study brings promising results for the therapeutic potential of seed and skin Tannat grape extracts as a functional food product with anti-diabetic activity.

Prediction of Ligands Binding Acetylcholinesterase with Potential Antidotal Activity: A Virtual Screening Approach.

Intoxications caused by organophosphorus compounds (OPs) are associated with the reversible, and sometimes irreversible interaction with acetylcholinesterase (AChE). OPs are commonly used as pesticides mainly in developing countries, where the associated poisoning is a major health problem related to suicidal attempts, careless manipulation, and chemical warfare. The current antidotes are oxime-based drugs that can regenerate the AChE catalytic activity. Nevertheless, challenges associated with lack of efficiency and difficulties for crossing blood-brain barrier have motivated the design of novel alternatives. We used a validated molecular docking approach for the virtual screening of 579,890 synthetic ligands and 478 drugs against a human AChE in its apo conformation, and a murine AChE conjugated with the OP tabun. After filtering, 7 hits were selected as potential competitors due to the formation of key interactions within the active site gorge of the AChE structure, and potential reactivators based on interactions with amino acids of the catalytic triad in the presence of organophosphorus compounds. The selected candidates will be further evaluated through in vitro and in vivo assays.

Catalytic activity, structure and stability of proteinase K in the presence of biosynthesized CuO nanoparticles.

Here, CuO nanoparticles were synthesized using Sambucus nigra (elderberry) fruit extract. Further, the binding of proteinase K, as a model enzyme with green synthesized nanoparticles was investigated. The results demonstrated that the structural changes in enzyme were induced by the binding of nanoparticles. These changes were accompanied by the decrease in the Michaelis-Menten constant at 298 K. This means that the enzyme affinity for the substrate was increased. Thermodynamic parameters of protein stability and protein-ligand binding were estimated from the spectroscopic measurements at 298-333 K. Depending on the temperature, CuO nanoparticles showed a dual effect on the thermodynamic stability and binding affinity of enzyme. Nanoparticles increase the stability of the native state of enzyme at room temperature. On the other hand, nanoparticles stabilize the unfolded state of enzyme at 310-333 K. An overall favorable Gibbs energy change was observed for the binding process at 298-333 K. The enzyme-nanoparticle binding is enthalpically driven at room temperature. It was concluded that hydrogen bonding plays a key role in the interaction of enzyme with nanoparticles at 298-310 K. At higher temperatures, the protein-ligand binding is entropically driven. This means that hydrophobic association plays a major role in the proteinase K-CuO binding at 310-333 K.

Novel urushiol derivatives as HDAC8 inhibitors: rational design, virtual screening, molecular docking and molecular dynamics studies.

Three series of novel urushiol derivatives were designed by introducing a hydroxamic acid moiety into the tail of an alkyl side chain and substituents with differing electronic properties or steric bulk onto the benzene ring and alkyl side chain. The compounds' binding affinity toward HDAC8 was screened by Glide docking. The highest-scoring compounds were processed further with molecular docking, MD simulations, and binding free energy studies to analyze the binding modes and mechanisms. Ten compounds had Glide scores of -8.2 to -10.2, which revealed that introducing hydroxy, carbonyl, amino, or methyl ether groups into the alkyl side chain or addition of -F, -Cl, sulfonamide, benzamido, amino, or hydroxy substituents on the benzene ring could significantly increase binding affinity. Molecular docking studies revealed that zinc ion coordination, hydrogen bonding, and hydrophobic interactions contributed to the high calculated binding affinities of these compounds toward HDAC8. MD simulations and binding free energy studies showed that all complexes possessed good stability, as characterized by low RMSDs, low RMSFs of residues, moderate hydrogen bonding and zinc ion coordination and low values of binding free energies. Hie147, Tyr121, Phe175, Hip110, Phe119, Tyr273, Lys21, Gly118, Gln230, Leu122, Gly269, and Gly107 contributed favorably to the binding; and Van der Waals and electrostatic interactions provided major contributions to the stability of these complexes. These results show the potential of urushiol derivatives as HDAC8 binding lead compounds, which have great therapeutic potential in the treatment of various malignancies, neurological disorders, and human parasitic diseases.

Novel Class IIa-Selective Histone Deacetylase Inhibitors Discovered Using an in Silico Virtual Screening Approach.

Histone deacetylases (HDAC) contain eighteen isoforms that can be divided into four classes. Of these isoform enzymes, class IIa (containing HDAC4, 5, 7 and 9) target unique substrates, some of which are client proteins associated with epigenetic control. Class IIa HDACs are reportedly associated with some neuronal disorders, making HDACs therapeutic targets for treating neurodegenerative diseases. Additionally, some reported HDAC inhibitors contain hydroxamate moiety that chelates with zinc ion to become the cofactor of HDAC enzymes. However, the hydroxamate functional group is shown to cause undesirable effects and has poor pharmacokinetic profile. This study used in silico virtual screening methodology to identify several nonhydroxamate compounds, obtained from National Cancer Institute database, which potentially inhibited HDAC4. Comparisons of the enzyme inhibitory activity against a panel of HDAC isoforms revealed these compounds had strong inhibitory activity against class IIa HDACs, but weak inhibitory activity against class I HDACs. Further analysis revealed that a single residue affects the cavity size between class I and class IIa HDACs, thus contributing to the selectivity of HDAC inhibitors discovered in this study. The discovery of these inhibitors presents the possibility of developing new therapeutic treatments that can circumvent the problems seen in traditional hydroxamate-based drugs.

Zn2+-dependent autocatalytic activity of the Bordetella pertussis CyaA-hemolysin.

Proteolytic degradation of the ∼100-kDa isolated RTX (Repeat-in-ToXin) subdomain (CyaA-RTX) of the Bordetella pertussis CyaA-hemolysin (CyaA-Hly) was evidently detected upon solely-prolonged incubation. Here, a truncated CyaA-Hly fragment (CyaA-HP/BI) containing hydrophobic and acylation regions connected with the first RTX block (BI1015-1088) was constructed as a putative precursor for investigating its potential autocatalysis. The 70-kDa His-tagged CyaA-HP/BI fragment which was over-expressed in Escherichia coli as insoluble aggregate was entirely solubilized with 4 M urea. After re-naturation in a Ni2+-NTA affinity column, the purified-refolded CyaA-HP/BI fragment in HEPES buffer (pH 7.4) supplemented with 2 mM CaCl2 was completely degraded upon incubation at 37 °C for 3 h. Addition of 1,10-phenanthroline‒an inhibitor of Zn2+-dependent metalloproteases markedly reduced the extent of degradation for CyaA-HP/BI and CyaA-RTX, but the degradative effect was clearly enhanced by addition of 100 mM ZnCl2. Structural analysis of a plausible CyaA-HP/BI model revealed a potential Zn2+-binding His-Asp cluster located between the acylation region and RTX-BI1015-1088. Moreover, Arg997‒one of the identified cleavage sites of the CyaA-RTX fragment was located in close proximity to the Zn2+-binding catalytic site. Overall results demonstrated for the first time that the observed proteolysis of CyaA-HP/BI and CyaA-RTX fragments is conceivably due to their Zn2+-dependent autocatalytic activity.

Noncoupled Fluorescent Assay for Direct Real-Time Monitoring of Nicotinamide N-Methyltransferase Activity.

Nicotinamide N-methyltransferase (NNMT) is an important biotransforming enzyme that catalyzes the transfer of a labile methyl group from the ubiquitous cofactor S-5'-adenosyl-l-methionine (SAM) to endogenous and exogenous small molecules to form methylated end products. NNMT has been implicated in a number of chronic disease conditions, including metabolic disorders, cardiovascular disease, cancer, osteoarthritis, kidney disease, and Parkinson's disease. We have developed a novel noncoupled fluorescence-based methyltransferase assay that allows direct ultrasensitive real-time detection of the NNMT reaction product 1-methylquinolinium. This is the first assay reported to date to utilize fluorescence spectroscopy to directly monitor NNMT product formation and activity in real time. This assay provided accurate kinetic data that allowed detailed comparative analysis of the NNMT reaction mechanism and kinetic parameters. A reaction model based on a random bireactant mechanism produced global curve fits that were most consistent with steady-state initial velocity data collected across an array of substrate concentrations. On the basis of the reaction mechanism, each substrate could independently bind to the NNMT apoenzyme; however, both substrates bound to the complementary binary complexes with an affinity ∼20-fold stronger compared to their binding to the apoenzyme. This reaction mechanism implies either substrate-induced conformational changes or bireactant intermolecular interactions may stabilize the binding of the substrate to the binary complex and formation of the ternary complex. Importantly, this assay could rapidly generate concentration response curves for known NNMT inhibitors, suggesting its applicability for high-throughput screening of chemical libraries to identify novel NNMT inhibitors. Furthermore, our novel assay potentially offers a robust detection technology for use in SAM substrate competition assays for the discovery and development of SAM-dependent methyltransferase inhibitors.

Arginase Inhibitors: A Rational Approach Over One Century.

Arginase (EC 3.5.3.1) is the bimanganese enzyme that converts L-arginine into ornithine and urea. This enzyme was discovered more than a century ago and early α-amino acids were identified as weak inhibitors. It was only during the 90s, after nitric oxide (NO) was reported as one of the most important biological mediators and when tight interrelation of arginase and NO synthase was found, that the development of arginase inhibitors was accelerated. The regulation of arginase activity by the N-hydroxy-L-arginine (3, NOHA) intermediate of the NO synthesis was the starting point of the N-hydroxy-nor-arginine (21, nor-NOHA) that proved to be the first micromolar inhibitor. The previously known manganese and arginase binding by borate inspired the 2(S)-amino-6-boronohexanoic acid (39, ABH) and S-(2-boronoethyl)-L-cysteine (40, BEC) now both considered as reference compounds in arginase inhibition. The high-resolution crystal structure of arginase and molecular modeling has rendered possible the recent design of (53) the strongest α,α-disubstituted derivatives of ABH. Simultaneously, traditional medicinal plants have contributed as a source of molecular diversity to the discovery of arginase inhibitors. This rational, step-by-step approach serves as guide in the present review where emphasis is placed on structure activity relationships. Highlights exhaustive review on arginase inhibitors highlight is made on rational approach to conception and structure activity relationships evaluation model is systematically mentioned with results.

Dimethyl sulfide protects against oxidative stress and extends lifespan via a methionine sulfoxide reductase A-dependent catalytic mechanism.

Methionine (Met) sulfoxide reductase A (MsrA) is a key endogenous antioxidative enzyme with longevity benefits in animals. Only very few approaches have been reported to enhance MsrA function. Recent reports have indicated that the antioxidant capability of MsrA may involve a Met oxidase activity that facilities the reaction of Met with reactive oxygen species (ROS). Herein, we used a homology modeling approach to search the substrates for the oxidase activity of MsrA. We found that dimethyl sulfide (DMS), a main metabolite that produced by marine algae, emerged as a good substrate for MsrA-catalytic antioxidation. MsrA bounds to DMS and promoted its antioxidant capacity via facilitating the reaction of DMS with ROS through a sulfonium intermediate at residues Cys72, Tyr103, and Glu115, followed by the release of dimethyl sulfoxide (DMSO). DMS reduced the antimycin A-induced ROS generation in cultured PC12 cells and alleviated oxidative stress. Supplement of DMS exhibited cytoprotection and extended longevity in both Caenorhabditis elegans and Drosophila. MsrA knockdown abolished the cytoprotective effect and the longevity benefits of DMS. Furthermore, we found that the level of physiologic DMS was at the low micromolar range in different tissues of mammals and its level decreased after aging. This study opened a new window to elucidate the biological role of DMS and other low-molecular sulfides in the cytoprotection and aging.

Transcriptome-guided gene isolation and functional characterization of UDP-xylose synthase and UDP-D-apiose/UDP-D-xylose synthase families from Ornithogalum caudatum Ait.

KEY MESSAGE: The present study first identified the involvement of OcUAXS2 and OcUXS1-3 in anticancer polysaccharides biosynthesis in O. caudatum. UDP-xylose synthase (UXS) and UDP-D-apiose/UDP-D-xylose synthase (UAXS), both capable of converting UDP-D-glucuronic acid to UDP-D-xylose, are believed to transfer xylosyl residue to anticancer polysaccharides biosynthesis in Ornithogalum caudatum Ait. However, the cDNA isolation and functional characterization of genes encoding the two enzymes from O. caudatum has never been documented. Previously, the transcriptome sequencing of O. caudatum was performed in our laboratory. In this study, a total of six and two unigenes encoding UXS and UAXS were first retrieved based on RNA-Seq data. The eight putative genes were then successfully isolated from transcriptome of O. caudatum by reverse transcription polymerase chain reaction (RT-PCR). Phylogenetic analysis revealed the six putative UXS isoforms can be classified into three types, one soluble and two distinct putative membrane-bound. Moreover, the two UAXS isoenzymes were predicted to be soluble forms. Subsequently, these candidate cDNAs were characterized to be bona fide genes by functional expression in Escherichia coli individually. Although UXS and UAXS catalyzed the same reaction, their biochemical properties varied significantly. It is worth noting that a ratio switch of UDP-D-xylose/UDP-D-apiose for UAXS was established, which is assumed to be helpful for its biotechnological application. Furthermore, a series of mutants were generated to test the function of NAD+ binding motif GxxGxxG. Most importantly, the present study determined the involvement of OcUAXS2 and OcUXS1-3 in xylose-containing polysaccharides biosynthesis in O. caudatum. These data provide a comprehensive knowledge for UXS and UAXS families in plants.

Piceatannol and resveratrol share inhibitory effects on hydrogen peroxide release, monoamine oxidase and lipogenic activities in adipose tissue, but differ in their antilipolytic properties.

Piceatannol is a hydroxylated derivative of resveratrol. While both dietary polyphenols coexist in edible plants and fruits, and share equivalent concentrations in several wines, the influence of piceatannol on adiposity has been less studied than that of resveratrol. Though resveratrol is now recognized to limit fat deposition in various obesity models, the benefit of its dietary supplementation remains under debate regarding human obesity treatment or prevention. The research for more potent resveratrol analogs is therefore still undergoing. This prompted us to compare various effects of piceatannol and resveratrol directly on human adipose tissue (hAT). Hydrogen peroxide release was measured by Amplex Red-based fluorescence in subcutaneous hAT samples from obese patients. Interactions of stilbenes with human amine oxidases and quinone reductase were assessed by radiometric methods, computational docking and electron paramagnetic resonance. Influences on lipogenic and lipolytic activities were compared in mouse adipocytes. Resveratrol and piceatannol inhibited monoamine oxidase (MAO) with respective IC50 of 18.5 and 133.7 µM, but not semicarbazide-sensitive amine oxidase (SSAO) in hAT. For both stilbenes, the docking scores were better for MAO than for SSAO. Piceatannol and resveratrol similarly hampered hydrogen peroxide detection in assays with and without hAT, while they shared pro-oxidant activities when incubated with purified quinone reductase. They exhibited similar dose-dependent inhibition of adipocyte lipogenic activity. Only piceatannol inhibited basal and stimulated lipolysis when incubated at a dose ≥100 µM. Thus, piceatannol exerted on fat cells dose-dependent effects similar to those of resveratrol, except for a stronger antilipolytic action. In this regard, piceatannol should be useful in limiting the lipotoxicity related to obesity when ingested or administered alone - or might hamper the fat mobilization induced by resveratrol when simultaneously administered with it.

Treated calcium pectinate beads for the covalent immobilization of ß-d-galactosidase.

The calcium pectinate (CP) gel beads were treated with polyethyleneimine (PEI) and glutaraldehyde (GA). This treatment greatly enhanced the beads' mechanical strength. Moreover, it enabled the CP beads to covalently immobilize enzymes, such as ß-d-galactosidase (ß-gal). The central composite design (CCD) was applied to optimize the PEI/GA treatment while employing the observed activity of the immobilized ß-gal as a response. The CCD predicted that treating the CP beads with a 3.49% PEI solution of pH 10.55, followed by a 5.66% GA solution in 0.1M phosphate buffer pH 7.02 would allow for the immobilization of 6.25U/g gel of the ß-gal. The verification experiment run at these optimum conditions offered 6.285±0.22U/g gel immobilized ß-gal which was in close agreement with the predicted value. The reusability study revealed that the ß-gal immobilized onto the treated CP beads retained 79.34% of its initial activity after being used for fourteen times.

Chinese herb medicine against Sortase A catalyzed transformations, a key role in gram-positive bacterial infection progress.

Many Gram-positive bacteria can anchor their surface proteins to the cell wall peptidoglycan covalently by a common mechanism with Sortase A (SrtA), thus escaping from the host's identification of immune cells. SrtA can complete this anchoring process by cleaving LPXTG motif conserved among these surface proteins and thus these proteins anchor on the cell wall. Moreover, those SrtA mutants lose this capability to anchor these relative proteins, with these bacteria no longer infectious. Therefore, SrtA inhibitors can be promising anti-infective agents to cure bacterial infections. Chinese herb medicines (CHMs) (chosen from Science Citation Index) have exhibited inhibition on SrtA of Gram-positive pathogens irreversibly or reversibly. In general, CHMs are likely to have important long-term impact as new antibacterial compounds and sought after by academia and the pharmaceutical industry. This review mainly focuses on SrtA inhibitors from CHMs and the potential inhibiting mechanism related to chemical structures of compounds in CHMs.

Lipocalin 2 alleviates iron toxicity by facilitating hypoferremia of inflammation and limiting catalytic iron generation.

Iron is an essential transition metal ion for virtually all aerobic organisms, yet its dysregulation (iron overload or anemia) is a harbinger of many pathologic conditions. Hence, iron homeostasis is tightly regulated to prevent the generation of catalytic iron (CI) which can damage cellular biomolecules. In this study, we investigated the role of iron-binding/trafficking innate immune protein, lipocalin 2 (Lcn2, aka siderocalin) on iron and CI homeostasis using Lcn2 knockout (KO) mice and their WT littermates. Administration of iron either systemically or via dietary intake strikingly upregulated Lcn2 in the serum, urine, feces, and liver of WT mice. However, similarly-treated Lcn2KO mice displayed elevated CI, augmented lipid peroxidation and other indices of organ damage markers, implicating that Lcn2 responses may be protective against iron-induced toxicity. Herein, we also show a negative association between serum Lcn2 and CI in the murine model of dextran sodium sulfate (DSS)-induced colitis. The inability of DSS-treated Lcn2KO mice to elicit hypoferremic response to acute colitis, implicates the involvement of Lcn2 in iron homeostasis during inflammation. Using bone marrow chimeras, we further show that Lcn2 derived from both immune and non-immune cells participates in CI regulation. Remarkably, exogenous rec-Lcn2 supplementation suppressed CI levels in Lcn2KO serum and urine. Collectively, our results suggest that Lcn2 may facilitate hypoferremia, suppress CI generation and prevent iron-mediated adverse effects.

Nanobioconjugates of Candida antarctica lipase B and single-walled carbon nanotubes in biodiesel production.

Carboxylated single-walled carbon nanotubes (SWCNTCOOH) were used as support for covalent immobilization of Candida antarctica lipase B (CaL-B) using linkers with different lengths. The obtained nanostructured biocatalysts with low diffusional limitation were tested in batch mode in the ethanolysis of the sunflower oil. SWCNTCOOH-CaL-B proved to be a highly efficient and stable biocatalyst in acetonitrile (83.4% conversion after 4h at 35°C, retaining >90% of original activity after 10 cycles).

Characteristics and Applicability of Phytase of the Yeast Pichia anomala in Synthesizing Haloperoxidase.

The phytase of the yeast Pichia anomala is a histidine acid phosphatase based on signature sequences and catalytic amino acids identified by site-directed mutagenesis. Among modulators, N-bromosuccinimide and butanedione inhibit phytase, while Ca(2+) and Ni(2+) stimulate slightly. Vanadate exhibits competitive inhibition of phytase, making it bifunctional to act as haloperoxidase. Molecular docking supports vanadate to share its binding site with phytate. The T 1/2, activation energy (E a ), temperature quotient (Q 10), activation energy of thermal inactivation (Ed), and enthalpy (ΔH d (0) ) of the enzyme are 4.0 min (80 °C), 27.72 kJ mol(-1), 2.1, 410.62 kJ mol(-1), and ∼407.8 kJ mol(-1) (65-80 °C), respectively. The free energy of the process (ΔG d (o) ) increases from 49.56 to 71.58 kJ mol(-1) with rise in temperature, while entropy of inactivation (ΔS d (0) ) remains constant at ∼1.36 kJ mol(-1) K(-1). The supplementation of whole wheat dough with rPPHY resulted in 72.5 % reduction in phytic acid content of bread. These characteristics confirm that the phytase has adequate thermostability for its applicability as a food and feed additive.

Inhibitory effect of ephedrannins A and B from roots of Ephedra sinica STAPF on melanogenesis.

BACKGROUND: Melanogenesis, a process producing the pigment melanin in human skin, eyes and hair, is a major physiological response against various environmental stresses, in particular exposure to ultraviolet radiation, and its pathway is regulated by a key enzyme, tyrosinase. In this study, we evaluated the effects of ephedrannins A and B, which are polyphenols from the roots of Ephedra sinica, commonly used in herbalism in oriental countries, on mushroom tyrosinase and melanogenesis in B16F10 melanoma cells. METHODS: Their effects on mushroom tyrosinase were determined via kinetic studies using a spectrophotometric analysis and those on melanin and tyrosinase production in melanoma cells treated with α-MSH (melanin stimulating hormone) were examined using PCR and ELISA. RESULTS: Both ephedrannins A and B exhibited concentration-dependent inhibitory effects on L-tyrosine oxidation by mushroom tyrosinase, and the inhibition mechanism was competitive and reversible with L-tyrosine as the substrate. In addition, melanin production in melanoma cells was also suppressed in a concentration-dependent manner by ephedrannins A and B without significant effects on cell proliferation at the concentrations tested. Both compounds showed inhibitory effects on melanin production by suppressing the transcription of tyrosinase in the cells. CONCLUSION: Both compounds exhibited significant inhibitory effects, but the inhibition by ephedrannin B was much more effective than that by ephedrannin A. Both ephedrannins A and B may be good candidates for a whitening agent for skin. GENERAL SIGNIFICANCE: This is the first report that describes effective inhibition of melanin production by ephedrannins A and B isolated from Ephedra roots.

Cloning and characterization of a farnesyl pyrophosphate synthase from Matricaria recutita L. and its upregulation by methyl jasmonate.

Matricaria recutita (L.), commonly known as chamomile, is one of the most valuable medicinal plants because it synthesizes a large number of pharmacologically active secondary metabolites known as α-bisabolol and chamazulene. Although the plant has been well characterized in terms of chemical constituents of essential oil as well as pharmacological properties, little is known about the genes responsible for biosynthesis of these compounds. In this study, we report a new full-length cDNA encoding farnesyl diphosphate synthase (FPS), a key enzyme in the pathway of biosynthesis of isoprenoids, from M. recutita. The cDNA of MrFPS comprises 1032 bp and encodes 343 amino acid residues with a calculated molecular mass of 39.4 kDa. The amino acid sequence homology and phylogenetic analysis indicated that MrFPS belongs to the plant FPS super-family and is closely related to FPS from the Asteraceae family. Expression of the MrFPS gene in Escherichia coli yielded FPS activity. Using real-time quantitative PCR, the expression pattern of the MrFPS gene was analyzed in different tissues of M. recutita as well as in response to methyl jasmonate. The expression analysis demonstrated that MrFPS expression varies in different tissues (with maximal expression in flowers and stems) and was significantly elevated in response to methyl jasmonate. This study will certainly enhance our understanding of the role of MrFPS in the biosynthesis and regulation of valuable secondary metabolites in M. recutita at a molecular level.

Glutathione-garlic sulfur conjugates: slow hydrogen sulfide releasing agents for therapeutic applications.

Natural organosulfur compounds (OSCs) from Allium sativum L. display antioxidant and chemo-sensitization properties, including the in vitro inhibition of tumor cell proliferation through the induction of apoptosis. Garlic water- and oil-soluble allyl sulfur compounds show distinct properties and the capability to inhibit the proliferation of tumor cells. In the present study, we optimized a new protocol for the extraction of water-soluble compounds from garlic at low temperatures and the production of glutathionyl-OSC conjugates during the extraction. Spontaneously, Cys/GSH-mixed-disulfide conjugates are produced by in vivo metabolism of OSCs and represent active molecules able to affect cellular metabolism. Water-soluble extracts, with (GSGaWS) or without (GaWS) glutathione conjugates, were here produced and tested for their ability to release hydrogen sulfide (H2S), also in the presence of reductants and of thiosulfate:cyanide sulfurtransferase (TST) enzyme. Thus, the TST catalysis of the H2S-release from garlic OSCs and their conjugates has been investigated by molecular in vitro experiments. The antiproliferative properties of these extracts on the human T-cell lymphoma cell line, HuT 78, were observed and related to histone hyperacetylation and downregulation of GAPDH expression. Altogether, the results presented here pave the way for the production of a GSGaWS as new, slowly-releasing hydrogen sulfide extract for potential therapeutic applications.