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PubMed2006

Plant fatty acid (ethanol) amide hydrolases.

Fatty acid amide hydrolase (FAAH) plays a central role in modulating endogenous N-acylethanolamine (NAE) levels in vertebrates, and, in part, constitutes an "endocannabinoid" signaling pathway that regulates diverse physiological and behavioral processes in animals. Recently, an Arabidopsis FAAH homologue was identified which catalyzed the hydrolysis of NAEs in vitro suggesting a FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs. Here, we provide evidence to support this concept by identifying candidate FAAH genes in monocots (Oryza sativa) and legumes (Medicago truncatula), which have similar, but not identical, exon-intron organizations. Corresponding M. truncatula and rice cDNAs were isolated and cloned into prokaryotic expression vectors and expressed as recombinant proteins in Escherichia coli. NAE amidohydrolase assays confirmed that these proteins indeed catalyzed the hydrolysis of 14C-labeled NAEs in vitro. Kinetic parameters and inhibition properties of the rice FAAH were similar to those of Arabidopsis and rat FAAH, but not identical. Sequence alignments and motif analysis of plant FAAH enzymes revealed a conserved domain organization for these members of the amidase superfamily. Five amino-acid residues determined to be important for catalysis by rat FAAH were absolutely conserved within the FAAH sequences of six plant species. Homology modeling of the plant FAAH proteins using the rat FAAH crystal structure as a template revealed a conserved protein core that formed the active site of each enzyme. Collectively, these results indicate that plant and mammalian FAAH proteins have similar structure/activity relationships despite limited overall sequence identity. Defining the molecular properties of NAE amidohydrolase enzymes in plants will help to better understand the metabolic regulation of NAE lipid mediators.

Amidohydrolases↗

PubMed2004

Bioactivation of carbamate-based 20(S)-camptothecin prodrugs.

Two new prodrugs of CPT were synthesized, based on carbamate linkages between the 20-hydroxy group of CPT and a linker designed to be enzymatically removed by either Penicillin-G-Amidase or catalytic antibody 38C2. Cell growth inhibition assays showed an up-to-2250-fold difference in toxicity between the prodrugs and the active drug. A significant increase in toxicity was observed upon incubation of the enzyme or the catalytic antibody with the corresponding prodrug. The described derivatives of CPT further our knowledge in the design of prodrugs for use in selective approaches for targeted chemotherapy.

Biotransformation↗

PubMed2004

Aspartame and aspartame derivatives effect human thrombin catalytic activity.

The study of small Asp-Phe analogs was undertaken since this dipeptide sequence is critical in fibrinogen recognition and catalysis. The inhibition of clotting activity by Asp-Phe-methyl ester (aspartame), formyl-Asp-Phe-methyl ester and acetyl-Asp-Phe was biphasic in all cases, indicating the presence of at least two binding sites. The N-terminally blocked derivatives are stronger inhibitors than aspartame. In contrast, tosyl-Gly-Pro-Arg-p'-nitroanilide hydrolysis was inhibited minimally by Asp-Phe-methyl, ester [Ki(app)=98 mM]. Acetyl-Asp-Phe inhibition of thrombin amidase activity was biphasic, tenfold stronger and appeared to be strongly cooperative. These results are discussed with respect to the inhibition of alpha-thrombin by ATP.

Amidohydrolases↗

PubMed2005

Isolation and characterization of trypsin from pyloric caeca of Monterey sardine Sardinops sagax caerulea.

Trypsin from pyloric caeca of Monterey sardine was purified by fractionation with ammonium sulfate, gel filtration, affinity and ionic exchange chromatography. Fraction 102, obtained from ionic exchange chromatography, generated one band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass of the isolated trypsin was 25 kDa and showed esterase-specific activity on Nalpha-p-tosyl-L-arginine methyl ester (TAME) that was 4.5 times greater than amidase-specific activity on N-benzoyl-L-arginine-p-nitroanilide. The purified enzyme was partially inhibited by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited by the metallo-protease inactivator EDTA or the chymotrypsin inhibitor tosyl-L-phenylalanine chloromethyl-ketone. The optimum pH for activity was 8.0 and maximum stability was observed between pH 7 and 8. A marked loss in stability was observed below pH 4 and above pH 11. Activity was optimum at 50 degrees C and lost activity at higher temperatures. The kinetic trypsin constants K(m) and k(cat) were 0.051 mM and 2.12 s(-1), respectively, while the catalytic efficiency (k(cat)/K(m)) was 41 s(-1) mM(-1). General characteristics of the Monterey sardine trypsin resemble those of trypsins from other fish, especially trypsins from the anchovy Engraulis japonica and Engraulis encrasicholus and the sardine Sardinops melanostica.

Ammonium Sulfate↗

PubMed2004

Antimicrobial susceptibility patterns, beta-lactamases, and biochemical identification of Yokenella regensburgei strains.

Yokenella regensburgei is an opportunistic human pathogen that phenotypically resembles Hafnia alvei. The susceptibility of 10 Y. regensburgei strains to 75 antimicrobial agents was examined, applying a microdilution procedure in cation-adjusted Mueller-Hinton broth (CAMHB) and IsoSensitest broth (ISB). beta-Lactamases were characterized phenotypically with beta-lactamase activity and induction assays. Genotypically, PCR experiments applying degenerated primer pairs for the detection of AmpC beta-lactamase genes were performed. Examining the phenotypic properties of Yokenella and 76 H. alvei strains with commercial identification systems and conventional tests, a database for an accurate biochemical separation of Y. regensburgei from H. alvei was established. In CAMHB, all tested yokenellae were resistant or at least of intermediate susceptibility to penicillin G, oxacillin, amoxicillin, amoxicillin-clavulanate, cefaclor, cefazoline, loracarbef, cefoxitin, all tested macrolides, lincosamides, streptogramins, ketolides, fusidic acid, glycopeptides, linezolid, and rifampicin. All Yokenella strains were sensitive to several beta-lactams, all tested aminoglycosides, chloramphenicol, folate-pathway inhibitors, fosfomycin, nitrofurantion, quinolones, and tetracyclines. In ISB, the minimum inhibitory concentration (MIC) values of several beta-lactams were one to four MIC doubling dilution steps lower than those found in CAMHB (depending on the beta-lactam). All yokenellae yielded specific amplification products for ampC, and all of these strains expressed beta-lactamases that were strongly inducible. Hydroxyproline amidase, maltosidase, tri-peptidase, proline deaminase, catalase reaction, Voges-Proskauer test, and fermentation of glycerol, melibiose and myo-inositol were suitable parameters to separate Y. regensburgei from H. alvei.

Anti-Bacterial Agents↗

PubMed2005

Natural antimicrobial susceptibility patterns and biochemical identification of Escherichia albertii and Hafnia alvei strains.

Bangladeshi diarrheagenic Hafnia alvei-like strains have been described recently as the new species Escherichia albertii (Int J Syst Evolut Microbiol. 2003;53:807-810). The natural susceptibility of 21 E. albertii and 76 H. alvei strains to 69 antimicrobial agents was examined, applying a microdilution procedure in IsoSensitest broth (for all the strains) and cation-adjusted Mueller-Hinton broth (for some strains). Examining the phenotypic features of both taxa with commercial identification systems and conventional tests, a database for an accurate biochemical separation of E. albertii from H. alvei was also established. Both taxa were naturally sensitive or sensitive and of intermediate susceptibility to aminoglycosides, acylureidopenicillins, ticarcillin, several cephalosporins, carbapenems, aztreonam, quinolones, folate pathway inhibitors, and nitrofurantoin. They were naturally resistant to tetracycline, penicillin G, oxacillin, all macrolides except for azithromycin, lincosamides, streptogramins, glycopeptides, rifampicin, and fusidic acid. Taxon-related differences in natural susceptibility affecting clinical assessment criteria were seen with doxycycline, minocycline, aminopenicillins, some cephalosporins, azithromycin, and fosfomycin. E. albertii was more susceptible than H. alvei to these agents and was naturally sensitive to all beta-lactams (except for penicillin G and oxacillin), azithromycin, and fosfomycin. H. alvei was naturally resistant or of intermediate susceptibility to all tetracyclines, amoxicillin, amoxicillin-clavulanate, ampicillin-sulbactam, narrow-spectrum cephalosporins, azithromycin, and fosfomycin. Motile malonate-negative Hafnia strains (indicating genospecies 2 of the H. alvei complex) were less susceptible to some cephalosporins than nonmotile, malonate-positive hafniae (indicating genospecies 1). Proline deaminase, hydroxyproline amidase, tripeptidase, chitinase, Voges-Proskauer reaction, and assimilation of histidine as well as acid production from glycerol, rhamnose, and xylose were suitable tests to separate strains of E. albertii from those of the H. alvei complex. Although out of the scope of this study, it should be noted that several strains of E. albertii showed acquired resistances to some penicillins and antifolates.

Anti-Bacterial Agents↗

PubMed2005

Plasmid-specified FemABX-like immunity factor in Staphylococcus sciuri DD 4747.

A plasmid from Staphylococcus sciuri DD 4747 had three open reading frames: a replication gene, an N-acetylmuramyl-l-alanine amidase-like gene, and a gene similar to the lysostaphin endopeptidase resistance gene (epr/lif). The epr-like gene was introduced into S. aureus RN4220; the recombinant strain was more resistant to lysostaphin endopeptidase and its cell wall peptidoglycan contained more serines and fewer glycines than the parental strain with the shuttle vector alone. Based on both its function and its similarity to femAB, this gene is a member of the femABX-like immunity gene family. Furthermore, this is the first example of a femABX-like immunity gene that is not linked to the gene for the bacteriolytic enzyme against which it specifies immunity.

Bacterial Proteins↗

PubMed2004

Sequence and transcriptional analysis of a gene cluster of Pseudomonas putida 86 involved in quinoline degradation.

Although quinoline 2-oxidoreductase (Qor) and 1H-2-oxoquinoline 8-monooxygenase (OxoOR), which catalyse the first two steps of quinoline degradation by Pseudomonas putida 86, and their genes have been investigated in some detail, the genetic organization and regulation of the catabolic pathway are not known yet. A gene cluster involved in quinoline degradation was characterized. Upstream of oxoO encoding the oxygenase component of OxoOR, the gene oxoS coding for a XylS-type protein is located. The DNA region downstream of oxoO comprises potential open reading frames (ORFs) that may code for further catabolic enzymes (an alpha/beta-hydrolase fold protein, and an amidase), and for accessory proteins presumably required for the assembly of metal cofactor containing holoenzymes (XdhC-like protein, MoeC- and MobA-like protein(s), IscS and IscU). The potential iscU gene is followed by the genes qorMSL that encode the structural subunits of Qor. Three potential ORFs (ORFs7-9) are located between qorMSL and oxoR, which codes for the reductase component of OxoOR. ORFs7-9 have counterparts in the cox (CO oxidizing system) and nic (nicotine degradation) gene clusters. Transcription of all these genes and ORFs located downstream of oxoS is induced by quinoline or 1H-2-oxoquinoline. Insertional inactivation of oxoS abolished quinoline-induced transcription. However, weak transcription of ORFs7-9 also occurred independent of quinoline and OxoS. The typical tandem recognition site for a XylS-type transcriptional activator was identified in the putative promoter region of qorM, and archetypal XylS indeed was found to activate synthesis of Qor. Motifs corresponding to single half-sites of a XylS-type binding site are located upstream of oxoO, the xdhC-like gene, and oxoR. Putative quinoline-specific transcriptional start sites were identified for these genes, and for qorM. The gene cluster probably is transcribed from several promoters, resulting in multiple overlapping polycistronic mRNAs.

Base Sequence↗

PubMed2006

New model substrates for enzymes hydrolysing polyethyleneterephthalate and polyamide fibres.

Recently the potential of enzymes for surface hydrophilisation and/or functionalisation of polyethyleneterephthalate (PET) and polyamide (PA) has been discovered. However, there was no correlation between enzyme class/activity (e.g. esterase, lipase, cutinase) and surface hydrolysis of these polymers and consequently no simple assay to estimate this capability. Enzymes active on the model substrates bis (benzoyloxyethyl) terephthalate and adipic acid bishexyl-amide, were also capable of increasing the hydrophilicity of PET and PA. When dosed at the identical activity on 4-nitrophenyl butyrate, only enzymes from Thermobifida fusca, Aspergillus sp., Beauveria sp. and commercial enzymes (TEXAZYME PES sp5 and Lipase PS) increased the hydrophilicity of PET fibres while other esterases and lipases did not show any effect. Activity on PET correlated with the activity on the model substrate. Hydrophilicity of fibres was greatly improved based on increases in rising height of up to 4.3 cm and the relative decrease of water absorption time between control and sample of the water was up to 76%. Similarly, enzymes increasing the hydrophilicity of PA fibres such as from Nocardia sp., Beauveria sp. and F. solani hydrolysed the model substrate; however, there was no common enzyme activity (e.g. protease, esterase, amidase) which could be attributed to all these enzymes.

Actinomycetales↗

PubMed2006

Synthesis of tetrapeptide Bz-RGDS-NH2 by a combination of chemical and enzymatic methods.

The tetrapeptide Bz-Arg-Gly-Asp-Ser-NH(2) (Bz-RGDS-NH(2)) was successfully synthesized by a combination of chemical and enzymatic methods in this study. Firstly, the precursor tripeptide Gly-Asp-Ser-NH(2) (GDS-NH(2)) was synthesized by a novel chemical method in four steps including chloroacetylation of l-aspartic acid, synthesis of chloroacetyl l-aspartic acid anhydride, the synthesis of ClCH(2)COAsp-SerOMe and ammonolysis of ClCH(2)COAsp-SerOMe. Secondly, lipase (PPL) was used to catalyze the formation of Bz-RGDS-NH(2) in aqueous water-miscible organic cosolvent systems using Bz-Arg-OEt as the acyl donor and GDS-NH(2) as the nucleophile. The optimum conditions were Bz-Arg-OEt 50 mM; GDS-NH(2) 400 mM; 10 degrees C, 0.1M phosphate buffer, pH 7.5; 60% DMF or 58% DMSO, PPL: 10 mg ml(-1) with the maximum yields of the tetrapeptide of 73.6% for DMF and 70.4% for DMSO, respectively. The secondary hydrolysis of the tetrapeptide product did not take place due to the absence of amidase activity of lipase.

Catalysis↗

PubMed2006

Insights into molecular plasticity of choline binding proteins (pneumococcal surface proteins) by SAXS.

Phosphocholine moieties decorating the pneumococcal surface are used as a docking station for a family of modular proteins, the so-called choline binding proteins or CBPs. Choline recognition is essential for CBPs function and may also be a determinant for their quaternary structure. There is little knowledge about modular arrangement or oligomeric structures in this family. Therefore, we have used the small angle X-ray scattering (SAXS) technique combined with analytical ultracentrifugation in order to model the three-dimensional envelope of two highly different CBPs: the phage encoded Cpl-1 lysozyme and the pneumococcal phosphorylcholine esterase Pce. Both enzymes have an N-terminal catalytic module and a C-terminal choline-binding module (CBM) that attaches them to the bacterial surface and comprises six and ten sequence repeats in Cpl-1 and Pce, respectively. SAXS experiments have shown an inherent conformational plasticity in Cpl-1 that accounts for the different relative position of these regions in the solution and crystal structures. Dimerization of Cpl-1 upon choline binding has been also visualised for the first time, and monomer-monomer interactions take place through the first CBR where a non-canonical choline binding site has now been identified. This mode of association seems to be independent of the absence or presence of the Cpl-1 catalytic module and reveals that the arrangement of the monomers differs from that previously found in the isolated CBM dimer of pneumococcal LytA amidase. In contrast, Pce displays the same modular disposition in the solution and crystal structures, and remains almost invariant upon choline binding. The present results suggest that protein dimerization and duplication of CBRs may be alternative but not equivalent ways of improving cell wall recognition by CBPs, since they provide different interaction geometries for choline residues present in (lipo)teichoic acids.

Binding Sites↗

PubMed2006

The 1.6 A crystal structure of the catalytic domain of PlyB, a bacteriophage lysin active against Bacillus anthracis.

Lysins are peptidoglycan hydrolases that are produced by bacteriophage and act to lyse the bacterial host cell wall during progeny phage release. Here, we describe the structure and function of a novel bacteriophage-derived lysin, PlyB, which displays potent lytic activity against the Bacillus anthracis-like strain ATCC 4342. This molecule comprises an N-terminal catalytic domain (PlyB(cat)) and a C-terminal bacterial SH3-like domain, SH3b. It is shown that both domains are required for effective catalytic activity against ATCC 4342. Further, PlyB has specific activity comparable to the phage lysin PlyG, an amidase being developed as a therapeutic against anthrax. In contrast to PlyG, however, the 1.6 A X-ray crystal structure of PlyB(cat) reveals that the catalytic domain adopts the glycosyl hydrolase (GH)-25, rather than phage T7 lysozyme-like fold. PlyB therefore represents a new class of anthrax lysin and a new defensive tool in the armament against anthrax-mediated bioterrorism.

Amino Acid Sequence↗

PubMed2006

Evaluation of the role of the arachidonic acid cascade in anandamide's in vivo effects in mice.

The pharmacological profiles of the endocannabinoid anandamide and exogenous cannabinoids (e.g., Delta9-tetrahydrocannabinol) are similar, but not exactly the same. One notable difference is that anandamide's in vivo effects in mice are not blocked by the brain cannabinoid (CB1) receptor antagonist SR141716A. The degree to which the rapid metabolism of anandamide to arachidonic acid might be involved in this unexpected lack of effect was the focus of this study. Mice were tested in a tetrad of tests sensitive to cannabinoids, consisting of spontaneous locomotion, ring immobility, rectal temperature and tail flick nociception. Anandamide and arachidonic acid produced a similar profile of effects, but neither drug was blocked by SR141716A. When hydrolysis of anandamide was inhibited by an amidase inhibitor (phenylmethyl sulfonyl fluoride; PMSF), however, SR141716A significantly attenuated anandamide's effects but did not completely block them. Similarly, the effects of the metabolically stable anandamide analog O-1812 were attenuated by SR141716A. The role of oxidative metabolism in anandamide's effects in the tetrad was also investigated through pharmacological modulation of cyclooxygenase and lipoxygenase, two major classes of enzymes that degrade arachidonic acid. Whereas the non-selective cyclooxygenase inhibitor ibuprofen blocked the in vivo effects of arachidonic acid, it did not alter anandamide's effects. Other modulators of the cyclooxygenase and lipoxygenase pathways also failed to block anandamide's effects. Together, these results offer partial support for a pharmacokinetic explanation of the failure of SR141716A to antagonize the effects of anandamide; however, they also suggest that non-CB1, non-CB2 receptors may be involved in mediation of anandamide's in vivo actions, particularly at higher doses.

Animals↗

PubMed2004

Peptidoglycan recognition proteins (PGRPs).

Innate immune system recognizes microorganisms through a series of pattern recognition receptors that are highly conserved in evolution. Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules that are conserved from insects to mammals and recognize bacteria and their unique cell wall component, peptidoglycan (PGN). Drosophila, mosquito, and mammals have families of 13, 7, and 4 PGRP genes, respectively, and some of these genes are alternatively spliced. PGRPs are differentially expressed in various cells and tissues, their expression is often upregulated by bacteria, and they mediate host responses to bacterial infections. Insect PGRPs have four known effector functions that are unique for insects: activation of prophenoloxidase cascade, activation of Toll receptor, activation of Imd pathway, and induction of phagocytosis. One function, amidase activity, is shared by some insect and mammalian PGRPs, whereas antibacterial activity of some mammalian PGRPs is unique for mammals.

Animals↗

PubMed2005

Purification and characterization of Manduca sexta prophenoloxidase-activating proteinase-1, an enzyme involved in insect immune responses.

Early on, we reported the partial purification of prophenoloxidase-activating proteinase-1 (PAP-1) from the tobacco hornworm, Manduca sexta [Proc. Natl. Acad. Sci. USA 95 (1998) 12220]. PAP-1 requires an auxiliary factor for generating active phenoloxidase (PO) [Insect Biochem. Mol. Biol. 33 (2003) 197; Insect Biochem. Mol. Biol. 34 (2004) 731]. To further characterize their roles in the proteolytic activation of prophenoloxidase (proPO), we purified PAP-1 to near homogeneity by hydroxylapatite, dextran sulfate, gel filtration, and lectin affinity chromatography. With 2.4 x 10(3)-fold purification and 20% yield, we obtained 63 microg PAP-1 from about 120 M. sexta prepupal cuticles (approximately 400 g). The purified glycoprotein (Mr=39,810+/-20; pI=5.6) had the highest amidase activity at pH 8.0 and a low salt concentration. The optimal conditions for proPO activation by PAP-1 and SPHs were: pH 8.0-8.4, PAP:SPH=1.5:1, and 0-10 degrees C for 40-50 min. While PAP-1 and SPHs are reasonably heat stable, PO activity generated after 1h incubation was lower at 20 or 30 degrees C than 0-10 degrees C because activated PO was unstable at a higher temperature. The KMs of PAP-1 toward IEARpNA and proPO were 201+/-18 microM and 16.6+/-3.0 microg/ml, respectively, and the absence of SPHs did not significantly affect KM for the synthetic substrate. PO activity and proPO cleavage were reduced in reaction mixtures containing the same amounts of proPO, PAP-1, and SPHs but increasing concentrations of NaCl. Ionic strength of the reaction buffer may reduce proPO-PAP-SPH interactions, proPO processing, and PO assembly.

Animals↗

PubMed2005

Cloning and expression of Klebsiella phage K11 lysozyme gene.

Previously, the lysozyme gene of the Klebsiella phage K11 was partially sequenced in our lab. Using the sequence information the lysozyme gene of the Klebsiella phage K11 was amplified and cloned using the polymerase chain reaction of the pfu DNA polymerase. The nucleotide sequence of phage K11 lysozyme gene was determined. The open reading frame corresponds to a polypeptide with 151 amino acids and molecular weight of 16,932 Da. The deduced amino acid sequence of this polypeptide shows 74-75% homologies to the T7 and T3 phage lysozymes. Although the gene was efficiently expressed under the control of tac promoter in Escherichia coli XL1-blue cells at 37 degrees C, most of the K11 lysozyme produced was insoluble. When the temperature of cell growth was lowered, however, solubility of the K11 lysozyme was increased gradually. The insoluble protein expressed at 37 degrees C was solubilized in 5 M guanidine-HCl and refolded in the presence of oxido-shuffling agent (GSH/GSSG). Through the refolding process the recombinant lysozyme was solubilized and purified. The purified K11 lysozyme showed transcription inhibition of K11 RNA polymerase as well as amidase activity. These results showed that the lysozyme of bacteriophage K11 is a bifunctional protein that cuts a bond in the bacterial cell wall and selectively inhibits K11 phage RNA polymerase. Also, transcription inhibition ability of K11 lysozyme with T7 or SP6 phage RNA polymerase was measured. T7 RNA polymerase was less inhibited than K11 RNA polymerase by K11 lysozyme. But SP6 RNA polymerase was not nearly inhibited by K11 lysozyme.

Amidohydrolases↗

PubMed2006

Antimicrobial activity of lysostaphin and a Listeria monocytogenes bacteriophage endolysin produced and secreted by lactic acid bacteria.

The expression and secretion signals of the Sep protein from Lactobacillus fermentum BR11 were used to direct export of two peptidoglycan hydrolases by Lb. fermentum BR11, Lactobacillus rhamnosus GG, Lactobacillus plantarum ATCC 14917 and Lactococcus lactis MG1363. The production levels, hydrolytic and bacteriocidal activities of the Listeria monocytogenes bacteriophage N-acetylmuramoyl-l-alanine amidase endolysin Ply511 and the glycylglycine endopeptidase lysostaphin were examined. Buffering of the growth media to a neutral pH allowed detection of Ply511 and lysostaphin peptidoglycan hydrolytic activity from all lactic acid bacteria. It was found that purified Ply511 has a pH activity range similar to that of lysostaphin with both enzymes functioning optimally under alkaline conditions. Supernatants from lactobacilli expressing lysostaphin reduced viability of methicillin resistant Staphylococcus aureus (MRSA) by approximately 8 log(10) CFU/ml compared to controls. However, supernatants containing Ply511 were unable to control L. monocytogenes growth. In coculture experiments, both Lb. plantarum and Lb. fermentum synthesizing lysostaphin were able to effectively reduce MRSA cell numbers by >7.4 and 1.7 log(10)CFU/ml, respectively, while lactic acid bacteria secreting Ply511 were unable to significantly inhibit the growth of L. monocytogenes. Our results demonstrate that lysostaphin and Ply511 can be expressed in an active form from different lactic acid bacteria and lysostaphin showed superior killing activity. Lactobacilli producing lysostaphin may have potential for in situ biopreservation in foodstuffs or for prevention of S. aureus infections.

Anti-Bacterial Agents↗

PubMed2005

Inhibition of leukocyte function and interleukin-2 gene expression by 2-methylarachidonyl-(2'-fluoroethyl)amide, a stable congener of the endogenous cannabinoid receptor ligand anandamide.

Arachidonylethanolamide (anandamide, AEA) has been identified as an endogenous ligand for cannabinoid receptors CB1 and CB2. Characterization of the direct cannabimimetic actions of anandamide has been hampered by its short duration of action and rapid degradation in in vivo and in vitro systems to arachidonic acid, a precursor in the biosynthesis of a broad range of biologically active molecules. In the present studies, we utilized 2-methylarachidonyl-(2'-fluoroethyl)amide (F-Me-AEA), an analog of anandamide resistant to enzymatic degradation, to determine whether F-Me-AEA modulated T cell function similar to that of plant-derived cannabinoids. Indeed, F-Me-AEA at low micromolar concentrations exhibited a marked inhibition of phorbol ester plus calcium ionophore (PMA/Io)-induced IL-2 protein secretion and steady state mRNA expression. Likewise, a modest suppression of the mixed lymphocyte response was observed in the presence of F-Me-AEA indicating an alteration in T cell responsiveness to allogeneic MHC class II antigens. F-Me-AEA was also found to modestly inhibit forskolin-stimulated adenylate cyclase activity in thymocytes and splenocytes, a hallmark of cannabinoid receptor agonists. Further characterization of the influence of F-Me-AEA on the cAMP signaling cascade revealed an inhibition of CREB-1/ATF-1 phosphorylation and subsequently, an inhibition of CRE DNA binding activity. Characterization of nuclear binding proteins further revealed that NF-AT and, to a lesser extent, NF-kappaB DNA binding activities were also suppressed. These studies demonstrate that F-Me-AEA modulates T cell function in a similar manner to plant-derived and endogenous cannabinoids and therefore can be utilized as an amidase- and hydrolysis-resistant endogenous cannabinoid.

Animals↗