Antioxidative defense organization in retroperitoneal white adipose tissue during acclimation to cold—The involvement of l-arginine/NO pathway

1. Retroperitoneal white adipose tissue (RpWAT) antioxidative defense was investigated in untreated, l-arginine-treated and N^ω-nitro-l-arginine methyl ester (l-NAME)-treated rats kept at 4±1 °C (1, 3, 7, 12, 21 and 45 days) and compared to control rats at 22±1 °C.

2. Cold-acclimation-induced RpWAT weight decrease was accompanied by a decline in glutathione level and increased activity of manganese superoxide dismutase (MnSOD), glutathione S-transferase (GST), catalase, glutathione peroxidase and glutathione reductase at different time-points.

3. l-arginine accelerated RpWAT weight decrease, the increase in MnSOD and GST activities and the prolonged increase of catalase, MnSOD and GST activities. l-NAME delayed cold-induced catalase activity increase and tissue weight decrease. Prolonged l-NAME-treatment had a similar effect on RpWAT as l-arginine.

4. Results suggest the involvement of l-arginine/NO pathway in RpWAT oxidative metabolic augmentation induced by cold-acclimation.

Soy protein hydrolysate ameliorates cardiovascular remodeling in rats with L-NAME-induced hypertension

Pepsin-digested soy protein hydrolysate has been reported to be responsible for many of the physiological benefits associated with soy protein consumption. In the present study, we investigated the effects of soy protein hydrolysate with angiotensin-converting enzyme (ACE) inhibitory potential on the blood pressure and cardiovascular remodeling in rats with N_ω-nitro-l-arginine methyl ester hydrochloride (l-NAME)-induced hypertension. Rats were fed a diet containing l-NAME (50 mg/kg body weight) with or without soy protein hydrolysate (1%, 3% or 5%) for 6 weeks. We found that ingestion of soy protein hydrolysate retarded the development of hypertension during the 6-week experimental period without affecting the amount of food intake. Although there was no difference in plasma ACE activity or tissue nitric oxide levels, ACE activity in the heart of rats consuming soy protein hydrolysate was significantly lower than that of the control group. Moreover, cardiac malonaldehyde and tumor necrosis factor-α concentrations were also lower in the soy protein hydrolysate group. No difference in plasminogen activator inhibitor-1 level was found in plasma or cardiovascular tissue. In the histopathological analysis, we also found that soy protein hydrolysate ameliorated inflammation and left ventricle hypertrophy in the heart. These findings suggest that soy protein hydrolysate might not only improve the balance between circulating nitric oxide and renin–angiotensin system but also show beneficial effects on cardiovascular tissue through its ACE inhibitory activity.     

Gene cloning, purification, and characterization of α-methylserine aldolase from Bosea sp. AJ110407 and its applicability for the enzymatic synthesis of α-methyl-l-serine and α-ethyl-l-serine

The gene encoding α-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysis revealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0% similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. Gel filtration revealed the molecular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer. The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5′-phosphate per mole of the subunit. The V_max value was 1.40 μmol min⁻¹ mg⁻¹, and the K_m value was 1.5 mM for the reaction wherein formaldehyde was released from α-methyl-l-serine. This enzyme could also catalyze the reverse reaction, i.e., the synthesis of α-methyl-l-serine from l-alanine and formaldehyde. This activity was inhibited in the excess of formaldehyde; however, α-methyl-l-serine was efficiently produced from l-alanine in the presence of formaldehyde. This method was also applicable for producing α-ethyl-l-serine from l-2-aminobutyric acid.     

Inhibition of nitric oxide synthase by cobalamins and cobinamides

Cobalamins are important cofactors for methionine synthase and methylmalonyl-CoA mutase. Certain corrins also bind nitric oxide (NO), quenching its bioactivity. To determine if corrins would inhibit NO synthase (NOS), we measured their effects on -l-[¹⁴C]arginine-to-l-[¹⁴C]citrulline conversion by NOS1, NOS2, and NOS3. Hydroxocobalamin (OH-Cbl), cobinamide, and dicyanocobinamide (CN₂-Cbi) potently inhibited all isoforms, whereas cyanocobalamin, methylcobalamin, and adenosylcobalamin had much less effect. OH-Cbl and CN₂-Cbi prevented binding of the oxygen analog carbon monoxide (CO) to the reduced NOS1 and NOS2 heme active site. CN₂-Cbi did not react directly with NO or CO. Spectral perturbation analysis showed that CN₂-Cbi interacted directly with the purified NOS1 oxygenase domain. NOS inhibition by corrins was rapid and not reversed by dialysis with l-arginine or tetrahydrobiopterin. Molecular modeling indicated that corrins could access the unusually large heme- and substrate-binding pocket of NOS. Best fits were obtained in the “base-off” conformation of the lower axial dimethylbenzimidazole ligand. CN₂-Cbi inhibited interferon-γ-activated Raw264.7 mouse macrophage NO production. We show for the first time that certain corrins directly inhibit NOS, suggesting that these agents (or their derivatives) may have pharmacological utility. Endogenous cobalamins and cobinamides might play important roles in regulating NOS activity under normal and pathological conditions.     

Activity of yeast d-amino acid oxidase on aromatic unnatural amino acids

d-Amino acid oxidase is a FAD-dependent enzyme that catalyses the conversion of the d-enantiomer of amino acids into the corresponding α-keto acid. Substrate specificity of the enzyme from the yeast Rhodotorula gracilis was investigated towards aromatic amino acids, and particularly synthetic α-amino acids.A significant improvement of the activity (V_max,app) and of the specificity constant (the V_max,app/K_m,app ratio) on a number of the substrates tested was obtained using a single-point mutant enzyme designed by a rational approach. With R. gracilis d-amino acid oxidase the complete resolution of d,l-homo-phenylalanine was obtained with the aim to produce the corresponding pure l-isomer and to use the corresponding α-keto acid as a precursor of the amino acid in the l-form.     

Alpha1-adrenoceptors trigger the snake venom production cycle in secretory cells by activating phosphatidylinositol 4,5-bisphosphate hydrolysis and ERK signaling pathway

Loss of venom from the venom gland after biting or manual extraction leads to morphological changes in venom secreting cells and the start of a cycle of production of new venom. We have previously shown that stimulation of both α- and β-adrenoceptors in the secretory cells of the venom gland is essential for the onset of the venom production cycle in Bothrops jararaca. We investigated the signaling pathway by which the α-adrenoceptor initiates the venom production cycle. Our results show that the α₁-adrenoceptor subtype is present in venom gland of the snake. In quiescent cells, stimulation of α₁-adrenoceptor with phenylephrine increased the total inositol phosphate concentration, and this effect was blocked by the phospholipase C inhibitor U73122. Phenylephrine mobilized Ca²⁺ from thapsigargin-sensitive stores and increased protein kinase C activity. In addition, α₁-adrenoceptor stimulation increased the activity of ERK 1/2, partially via protein kinase C. Using RT-PCR approach we obtained a partial sequence of a snake α₁-adrenoceptor (260 bp) with higher identity with α_1D and α_1B-adrenoceptors from different species. These results suggest that α₁-adrenoceptors in the venom secreting cells are probably coupled to a G_q protein and trigger the venom production cycle by activating the phosphatidylinositol 4,5-bisphosphate and ERK signaling pathway.     

A novel mechanism of vascular relaxation induced by sodium nitroprusside in the isolated rat aorta

Sodium nitroprusside (SNP) is an endothelium-independent relaxant agent and its effect is attributed to its direct action on the vascular smooth muscle (VSM). Endothelium modulates the vascular tone through the release of vasoactive agents, such as NO. The aim of this study was to investigate the contribution of the endothelium on SNP vasorelaxation, NO release and Ca²⁺ mobilization. Vascular reactivity experiments showed that endothelium potentiates the SNP-relaxation in rat aortic rings and this effect was abolished by l-NAME. SNP-relaxation in intact endothelium aorta was inhibited by NOS inhibitors for the constitutive isoforms (cNOS). Furthermore, endogenous NO is involved on the SNP-effect and this endogenous NO is released by cNOS. Moreover, Ca²⁺ mobilization study shows that l-NAME inhibited the reduction of Ca²⁺-concentration in VSM cells and reduced the increase in Ca²⁺-concentration in endothelial cells induced by SNP. This enhancement in Ca²⁺-concentration in the endothelial cells is due to a voltage-dependent Ca²⁺ channels activation. The present findings indicate that the relaxation and [Ca²⁺]_i decrease induced by SNP in VSM cells is potentiated by endothelial production of NO by cNOS-activation in rat aorta.     

Availability of tyrosine amide for α-chymotrypsin-catalyzed synthesis of oligo-tyrosine peptides

Oligo-tyrosine peptides such as Tyr-Tyr having angiotensin I-converting enzyme (ACE) inhibitory activity could be synthesized by α-chymotrypsin-catalyzed reaction with l-tyrosine ethyl ester in aqueous media. However, peptide yield in the reaction was below 10%. Since l-tyrosine amide showed highly nucleophilic activity for the deacylation of enzyme through which a new peptide bond was made, its application to the enzymatic peptide synthesis was evaluated in this study. Addition of tyrosine amide into the reaction produced Tyr-Tyr-NH₂, of which yield exceeded 130% on the basis of tyrosine ethyl ester. Although purified Tyr-Tyr-NH₂ did not inhibit ACE activity, α-chymotrypsin could act on the dipeptide amide and convert about 40% of it to Tyr-Tyr. The use of both ester and amide forms of tyrosine is expected to be a potent procedure for α-chymotrypsin-catalyzed synthesis of antihypertensive peptides.     

Mechanisms of catalysis and inhibition operative in the arginine deiminase from the human pathogen Giardia lamblia

Giardia lamblia arginine deiminase (GlAD), the topic of this paper, belongs to the hydrolase branch of the guanidine-modifying enzyme superfamily, whose members employ Cys-mediated nucleophilic catalysis to promote deimination of l-arginine and its naturally occurring derivatives. G. lamblia is the causative agent in the human disease giardiasis. The results of RNAi/antisense RNA gene-silencing studies reported herein indicate that GlAD is essential for G. lamblia trophozoite survival and thus, a potential target for the development of therapeutic agents for the treatment of giardiasis. The homodimeric recombinant protein was prepared in Escherichia coli for in-depth biochemical characterization. The 2-domain GlAD monomer consists of a N-terminal domain that shares an active site structure (depicted by an in silico model) and kinetic properties (determined by steady-state and transient state kinetic analysis) with its bacterial AD counterparts, and a C-terminal domain of unknown fold and function. GlAD was found to be active over a wide pH range and to accept l-arginine, l-arginine ethyl ester, N^α-benzoyl-l-arginine, and N^ω-amino-l-arginine as substrates but not agmatine, l-homoarginine, N^α-benzoyl-l-arginine ethyl ester or a variety of arginine-containing peptides. The intermediacy of a Cys424–alkylthiouronium ion covalent enzyme adduct was demonstrated and the rate constants for formation (k₁ = 80 s⁻¹) and hydrolysis (k₂ = 35 s⁻¹) of the intermediate were determined. The comparatively lower value of the steady-state rate constant (k_cat = 2.6 s⁻¹), suggests that a step following citrulline formation is rate-limiting. Inhibition of GlAD using Cys directed agents was briefly explored. S-Nitroso-l-homocysteine was shown to be an active site directed, irreversible inhibitor whereas N^ω-cyano-l-arginine did not inhibit GlAD but instead proved to be an active site directed, irreversible inhibitor of the Bacillus cereus AD.     

Structure of the oligosaccharides isolated from Dalbergia sissoo Roxb. leaf polysaccharide

A water-soluble polysaccharide isolated from Dalbergia sissoo Roxb. leaves was purified and major homogeneous fraction obtained by GPC. Complete hydrolysis of the polysaccharide followed by paper chromatography and GLC analysis indicated the presence of l-rhamnose, d-glucuronic acid, d-galactose and d-glucose in molar ratio of 1:1:2:2.33, respectively. Partial hydrolysis of the polysaccharide furnished one tri-[I], one hepta-[II] and one nona-[III] saccharides. Hydrolysis of the oligosaccharide I, II and III followed by GLC analysis furnished d-glucose and l-rhamnose (2:1); l-rhamnose, d-galactose and d-glucuronic acid (1:3:3); and l-rhamnose, d-galactose and d-glucose (1:3:5), respectively. Methylation analysis and periodate oxidation of the oligosaccharide I indicated the presence of two non reducing glucose units linked to rhamnose by 1→2 and 1→4 linkages, respectively. Oligosaccharide II is a branched molecule with a main chain consisting of 1,3-linked β-d-galactopyranosyl (2 mol), 1,3,4 linked α-l-rhamnopyranosyl (1 mol) and 1,4,6 linked β-d-galactopyranosyl unit (1 mol) and non reducing β-d-glucuronic acid at the end along with side chains of β-d-glucouronopyranosyl units (2 mol). Oligosaccharide III is also a branched molecule with a main chain consisting of 1,3,4 linked α-l-rhamnopyranosyl (1 mol), 1,2,4 linked β-d-glucopyranosyl (1 mol), 1,3 and 1,4 linked β-d-galactopyranosyl (2 and 1 mol, respectively) having β-d-glucopyranosyl as a non reducing end.     

Glycosylated nervogenic acid derivatives from Liparis condylobulbon (Reichb.f.) leaves

Three new nervogenic acid glycosides, 1-O-α-l-rhamnopyranosyl 3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoate, 3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoic acid, and bis{3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoyl} 1,2-O-β-d-glucopyranose, which we named condobulbosides A–C, were isolated from a methanol extract of the leaves of Liparis condylobulbon together with an apigenin C-glycoside, schaftoside. Their structures were established on the basis of spectral techniques, namely, UV, IR, HR-MS spectroscopy, both 1D and 2D NMR experiments, and chemical reactions.     

Purification and characterization of a novel imidazole dipeptide synthase from the muscle of the Japanese eel Anguilla japonica

We have purified a novel enzyme from eel white muscle which catalyzes the syntheses of imidazole dipeptides, such as carnosine (β-alanyl-l-histidine), anserine (β-alanyl-π-methyl-l-histidine), and balenine (ophidine; β-alanyl-τ-methyl-l-histidine), directly from their precursors. The enzyme was purified 1130-fold from eel muscle by a series of column chromatographies. Although eel muscle contains a large amount of carnosine and only trace amounts of anserine and balenine, the anserine synthesizing activity was by far the highest. From gel permeation chromatography, the molecular mass of the enzyme was calculated to be 275 kDa. SDS-PAGE of the purified enzyme represented a band around 43 kDa, suggesting that the native enzyme is a hexamer or heptamer. The optimal pH and temperature were around 9.5 and 60 °C, respectively. K_m values for β-alanine and π-methyl-l-histidine were 44 and 89 mM, respectively. The enzyme was greatly activated by Zn²⁺ and inhibited by EDTA. The N-terminal amino acid sequence of 25 residues of the purified enzyme showed 52% amino acid identity to 38–62 residues of zebrafish haptoglobin precursor. The purified enzyme also exhibited hydrolytic activity against these imidazole dipeptides.     

Oligochitosan induced Brassica napus L. production of NO and H2O2 and their physiological function

NO (nitric oxide) and H₂O₂ (hydrogen peroxide) are important signaling molecule in plants. Brassica napus L. was used to understand oligochitosan inducing production of NO (nitric oxide) and H₂O₂ (hydrogen peroxide) and their physiological function. The result showed that the production of NO and H₂O₂ in epidermal cells of B. napus L. was induced with oligochitosan by fluorescence microscope. And it was proved that there was an interaction between NO and H₂O₂ with L-NAME (N^G-nitro-l-arg-methyl eater), which is an inhibitor of NOS (NO synthase) in mammalian cells that also inhibits plant NO synthesis, and CAT (catalase), which is an important H₂O₂ scavenger, respectively. It was found that NO and H₂O₂ induced by oligochitosan took part in inducing reduction in stomatal aperture and LEA protein gene expression of leaves of B. napus L. All these results showed that oligochitosan have potential activities of improving resistance to water stress.     

The system of sulfated galactans from the red seaweed Gymnogongrus torulosus (Phyllophoraceae, Rhodophyta): Location and structural analysis

Sulfated polysaccharides were localized in the cuticle, cortex and medulla of the gametophyte thallus, being more concentrated in the intercellular matrix than in the cell walls. During the water extraction sequence, a small percentage of galactan sulfates (5.1% of dry seaweed) with average low M_r (6–11.4 kDa) were extracted at room temperature without disturbing the cellular arrangement, while sulfated galactans of average medium M_r (18–45 kDa) were obtained by further hot-water extractions (52.4% of dry seaweed), with diorganization of the tissue. The residue (40.0% of dry seaweed) still contained carrageenan-type (major) and agaran-type (minor) galactans. Part of these galactans was extracted with 8.4% LiCl solution in DMSO, from which “pure” κ/ι-carrageenans were isolated.Carrageenans and agarans were extracted in a ratio 1:0.5, showing the highest amount of agaran-structures for a carrageenophyte. The galactans comprise alternating 4-sulfated (major) and non-sulfated (minor) 3-linked β-d-galactopyranose units, and 4-linked α-galactopyranose units with the following substitutions: (i) non-sulfated and 2-sulfated 3,6-anhydro-α-d-galactopyranose residues in the carrageenan-structures, which belong to the κ-family (κ/ι-carrageenans); (ii) 3-sulfated α-l-galactopyranose units and 2-sulfated 3,6-anhydro-α-l-galactopyranose residues in the agaran-structures.Alkaline treatment and alkaline dialysis of the main extracts gave “pure” κ/ι-carrageenans, showing that carrageenan molecules are extracted together with low M_r agarans or agaran-dl-hybrids.     

Flesinoxan challenge suggests that chronic treatment with paroxetine in rats does not desensitize receptors controlling 5-HT synthesis

It has been proposed that the desensitization of 5-HT_1A (5-hydroxytryptamine; serotonin) receptors following chronic therapy with selective serotonin reuptake inhibitors (SSRIs) is necessary for their therapeutic efficacy. Stimulation of the 5-HT_1A receptors decreases serotonin (5-HT) synthesis and release, but it is not clear if the receptors are fully desensitized following chronic SSRI treatment. The main objective of this study was evaluation of ability of 5-HT_1A receptors to modulate 5-HT synthesis after 14-day paroxetine treatment. 5-HT_1A receptor sensitivity following chronic administration of the SSRI paroxetine was assessed by the ability of an acute challenge with the 5-HT_1A agonist, flesinoxan, to modulate 5-HT synthesis in the rat brain. The rates of 5-HT synthesis were measured using the α-[¹⁴C]methyl-l-tryptophan autoradiographic method. The rats were treated for 2 weeks with paroxetine (10 mg/(kg day), s.c., delivered by osmotic minipump). After this treatment, the rats received an acute challenge with flesinoxan (5 mg/kg, i.p.), while the control rats were injected with the vehicle. Forty minutes following the flesinoxan injection, the tracer, α-[¹⁴C]methyl-l-tryptophan, was injected over 2 min. 5-HT synthesis rates were calculated from autoradiographically measured tissue tracer concentrations and plasma time–activity curves. The results demonstrated that the acute flesinoxan challenge produced a significant decrease in 5-HT synthesis rates throughout the rat brain. The greatest decrease was observed in the ventral hippocampus, somatosensory cortex and the ascending serotonergic cell bodies. In comparison with data reported on an acute challenge with flesinoxan in naïve rats (rats without any other treatment), the results presented here suggest a greater effect of flesinoxan on synthesis reduction in rats chronically treated with paroxetine. The results also suggest that the 5-HT receptors were not fully desensitized by paroxetine treatment, and that the stimulation of 5-HT_1A receptors with an agonist is still capable of reducing 5-HT synthesis.     

Structural characterization of an O-linked tetrasaccharide from Pseudomonas syringae pv. tabaci flagellin

The flagellin of Pseudomonas syringae pv. tabaci is a glycoprotein that contains O-linked oligosaccharides composed of rhamnosyl and 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methylglucosyl residues. These O-linked glycans are released by hydrazinolysis and then labeled at their reducing ends with 2-aminopyridine (PA). A PA-labeled trisaccharide and a PA-labeled tetrasaccharide are isolated by normal-phase high-performance liquid chromatography. These oligosaccharides are structurally characterized using mass spectrometry and NMR spectroscopy. Our data show that P. syringae pv. tabaci flagellin is glycosylated with a tetrasaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1→3)-α-l-Rhap-(1→2)-α-l-Rhap-(1→2)-α-l-Rha-(1→, as well a trisaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1→3)-α-l-Rhap-(1→2)-α-l-Rha-(1→, which was identified in a previous study.     

Inhibitors of bacterial N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) and demonstration of in vitro antimicrobial activity

The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) is a critical bacterial enzyme for the construction of the bacterial cell wall. A screen biased toward compounds containing zinc-binding groups (ZBG’s) including thiols, carboxylic acids, boronic acids, phosphonates and hydroxamates has delivered a number of micromolar inhibitors of DapE from Haemophilus influenzae, including the low micromolar inhibitor l-captopril (IC₅₀ = 3.3 μM, K_i = 1.8 μM). In vitro antimicrobial activity was demonstrated for l-captopril against Escherichia coli.     

Kinetic characterization of recombinant human cystathionine β-synthase purified from E. coli

Cystathionine β-synthase (CBS) catalyzes the pyridoxal-5′-phosphate-dependent condensation of l-serine and l-homocysteine to form l-cystathionine in the first step of the transsulfuration pathway. Although effective expression systems for recombinant human CBS (hCBS) have been developed, they require multiple chromatographic steps as well as proteolytic cleavage to remove the fusion partner. Therefore, a series of five expression constructs, each incorporating a 6-His tag, were developed to enable the efficient purification of hCBS via immobilized metal ion affinity chromatography. Two of the constructs express hCBS in fusion with a protein partner, while the others bear only the affinity tag. The addition of an amino-terminal, 6-His tag, in the absence of a protein fusion partner and in the absence or presence of a protease-cleavable linker, was found to be sufficient for the purification of soluble hCBS and resulted in enzyme with 86–91% heme saturation and with activity similar to that reported for other hCBS expression constructs. The continuous assay for l-Cth production, employing cystathionine β-lyase and l-lactate dehydrogenase as coupling enzymes, was employed here for the first time to determine the steady-state kinetic parameters of hCBS, via global analysis, and revealed previously unreported substrate inhibition by l-Hcys (K_i^l-Hcys = 2.1 ± 0.2 mM). The kinetic parameters for the hCBS-catalyzed hydrolysis of l-Cth to l-Ser and l-Hcys were also determined and the k_cat/K_m^l-Cth of this reaction is only 2-fold lower than the k_cat/K_m^l-SER of the physiological, condensation reaction.     

L-Amino acid oxidase from Naja naja oxiana venom

A new l-amino acid oxidase (LAAO) was isolated from the Central Asian cobra Naja naja oxiana venom by size exclusion, ion exchange and hydrophobic chromatography. The N-terminal sequence and the internal peptide sequences share high similarity with other snake venom l-amino acid oxidases, especially with those isolated from elapid venoms. The enzyme is stable at low temperatures (− 20 °C, − 70 °C) and loses its activity by heating at 70 °C. Specific substrates for the isolated protein are l-phenylalanine, l-tryptophan, l-methionine and l-leucine. The enzyme has antibacterial activity inhibiting the growth of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. N. naja oxiana LAAO dose-dependently inhibited ADP- or collagen-induced platelet aggregation with IC₅₀ of 0.094 μM and 0.036 μM, respectively. The antibacterial and anti-aggregating activity was abolished by catalase.