Identification of Phenolic Compounds Present in Three Speedwell (Veronica L.) Species and Their Antioxidant Potential.

Extracts from Veronica species (speedwells) are known for the various biological activities they show, such as cytotoxic, antimicrobial, anti-inflammatory, and antioxidant activities. Also, the plants from this genus are known as medicinal plants used in traditional medicine worldwide. Phenolic compounds are specialized metabolites that contribute to biological activity the most. Therefore, the aim of this research is identification and quantification of phenolic compounds present in three Veronica species (Veronica anagallis-aquatica L., Veronica persica Poir., and Veronica polita Fr.) using the liquid chromatography-mass spectrometry (LC-MS/MS) technique. All extracts were tested for antioxidant activity with two methods: DPPH (2,2-diphenyl-1-picrylhydrazyl) and ORAC (oxygen radical absorbance capacity). Also, standards for compounds that were detected in the highest amount in all species were also tested for antioxidant activity. Three different solvents (pure methanol, 80% ethanol, and water) were used for the extraction of phenolic components and their comparison in order to test their antioxidant activity as a final goal. The main compounds present in the tested Veronica extracts were: p-hydroxybenzoic acid, vanillic acid, caffeic acid, gentisic acid, and apigenin. V. anagallis-aquatica contained the highest amount of phenolic components in comparison with the two other tested species, V. persica and V. polita. Caffeic acid showed the highest antioxidant activity in both studied methods with an IC50 value for DPPH activity of 1.99 µg/mL. For the plant extracts, in general, methanolic/ethanolic extracts showed higher activity than water extracts in both methods which was expected, as organic solutions extract more phenolic compounds. This research points to the potential application of extracts of different Veronica species for antioxidant activity.

Substrate-Induced Conformational Changes of the Tyrocidine Synthetase 1 Adenylation Domain Probed by Intrinsic Trp Fluorescence.

Nonribosomal peptide synthetases (NRPS) are multifunctional proteins that catalyze the synthesis of the peptide products with enormous biological potential. The process of biosynthesis starts with the adenylation (A) domain, which during the catalytic cycle undergoes extensive structural rearrangements. In this paper, we present the first study of the tyrocidine synthetase 1 A-domain (TycA-A) fluorescence properties. The TycA-A protein contains five potentially fluorescent Trp residues at positions 227, 301, 323, 376 and 406. The contribution of each Trp to the TycA-A emission was determined using protein variants bearing single Trp to Phe substitutions. The accessibility of the Trp side chains during adenylation showed that only W227 is affected by substrate binding. The protein variant containing solely fluorescent W227 residue was constructed and further used as a probe to explore the binding effect of different non-cognate amino acid substrates. The results indicate a different accessibility of W227 residue in the presence of non-cognate amino acids, which might offer an explanation for the higher aminoacyl-adenenylate leakage. Overall, our results suggest that intrinsic tryptophan fluorescence could be used as a method to probe the effect of substrate binding on the local structure in NRPS adenylation domains.

Anisakis pegreffii (Nematoda: Anisakidae) products modulate oxidative stress and apoptosis-related biomarkers in human cell lines.

BACKGROUND: In countries with elevated prevalence of zoonotic anisakiasis and high awareness of this parasitosis, a considerable number of cases that associate Anisakis sp. (Nematoda, Anisakidae) and different bowel carcinomas have been described. Although neoplasia and embedded larvae were observed sharing the common site affected by chronic inflammation, no association between the nematode and malignancy were directly proved. Similarly, no data are available about the effect of secretory and excretory products of infecting larvae at the host's cellular level, except in respect to allergenic interaction. METHODS: To test the mechanisms by which human non-immune cells respond to the larvae, we exposed the fibroblast cell line HS-68 to two Anisakis products (ES, excretory/secretory products; and EC, crude extract) and evaluated molecular markers related to stress response, oxidative stress, inflammation and apoptosis, such as p53, HSP70, TNF-α, c-jun and c-fos, employing cell viability assay, spectrophotometry, immunoblotting and qPCR. RESULTS: Both Anisakis products led to increased production of reactive oxygen species (ROS), especially in EC-treated cells. While the ES treatment induces activation of kinases suggesting inflammation and cell proliferation (or inhibition of apoptosis), in EC-treated cells, other signaling pathways indicate the inhibition of apoptosis, marked by strong upregulation of Hsp70. Elevated induction of p53 in fibroblasts treated by both Anisakis products, suggests a significantly negative effect on the host DNA. CONCLUSIONS: This study shows that in vitro cell response to Anisakis products can result in at least two different scenarios, which in both cases lead to inflammation and DNA damage. Although these preliminary results are far from proving a relationship between the parasite and cancer, they are the first to support the existence of conditions where such changes are feasible.

Infrequent finding of metallo-ß-lactamase VIM-2 in carbapenem-resistant Pseudomonas aeruginosa strains from Croatia.

One hundred sixty-nine nonreplicate imipenem-resistant Pseudomonas aeruginosa strains isolated in a large hospital on the coastal region of Croatia were studied. The most active antibiotics were colistin and amikacin. Most of the isolates were multiresistant. The most prevalent serotype was O12, followed by O11. Six strains carried the bla(VIM-2) gene located in a novel class 1 integron composed in its variable part of the bla(VIM-2)-bla(oxa-10)-ΔqacF-aacA4 genes. Metallo-ß-lactamase-producing strains belonged to sequence types ST235 and ST111.

Is yessotoxin the main phycotoxin in Croatian waters?

With the aim of investigating whether yessotoxin (YTX) is responsible for diarrhetic shellfish poisoning (DSP) events in Croatian waters, three different methods were combined: a modified mouse bioassay (MBA) that discriminates YTX from other DSP toxins, the enzyme-linked immunosorbent assay method (ELISA) and liquid chromatography-mass spectrometry (LC-MS/MS). Among 453 samples of mussels and seawater analyzed in 2007, 10 samples were DSP positive. Results obtained by the modified MBA method revealed that most of the samples were positive for YTX, with the exception of samples from Lim Bay (LB 1) The ELISA method also identified the presence of YTX in these samples. DSP toxin profiles showed the presence of okadaic acid (OA) in three, and YTX in four out of nine samples that were analyzed by LC-MS/MS. The phytoplankton community structure pattern revealed Lingulodinium polyedrum (Stein) Dodge, which was present in the water prior to and/or during toxicity events at low concentrations (80 to 1440 cells L(-1)), as a potential YTX producing species. It is proposed that L. polyedrum cells accumulated in mussels and the subsequently observed toxicity may be related to metabolism after ingestion, resulting in carboxy YTX as the major analog in the mussel.

Dimer asymmetry and the catalytic cycle of alkaline phosphatase from Escherichia coli.

Although alkaline phosphatase (APase) from Escherichia coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics, supported by a model describing a dimeric enzyme with unequal subunits [Orhanovic S., Pavela-Vrancic M. and Flogel-Mrsic M. (1994) Acta. Pharm.44, 87-95]. The possibility, that the observed asymmetry could be attributed to negative cooperativity in Mg2+ binding, has been examined. The influence of the metal ion content on the catalytic properties of APase from E. coli has been examined by kinetic analyses. An activation study has indicated that Mg2+ enhances APase activity by a mechanism that involves interactions between subunits. The observed deviations from Michaelis-Menten kinetics are independent of saturation with Zn2+ or Mg2+ ions, suggesting that asymmetry is an intrinsic property of the dimeric enzyme. In accordance with the experimental data, a model describing the mechanism of substrate hydrolysis by APase has been proposed. The release of the product is enhanced by a conformational change generating a subunit with lower affinity for both the substrate and the product. In the course of the catalytic cycle the conformation of the subunits alternates between two states in order to enable substrate binding and product release. APase displays higher activity in the presence of Mg2+, as binding of Mg2+ increases the rate of conformational change. A conformationally controlled and Mg2+-assisted dissociation of the reaction product (Pi) could serve as a kinetic switch preventing loss of Pi into the environment.