Maja Pavela-Vrančić

DSP toxin profile in the coastal waters of the central Adriatic Sea

A monitoring program, carried out in 1996 and 1997, has confirmed that toxic compounds, other than the most frequently detected toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1), are involved in DSP phenomena in the Adriatic Sea. Toxicity was assessed by the mouse bioassay; the content and the nature of the toxic components were established through fluorometric HPLC analysis combined with mass spectrometry. A rare pectenotoxin-2 (PTX-2) derivative, 7-epi-pectenotoxin-2 seco acid (7-epi-PTX-2SA), was the exclusive contaminant of samples collected from the central Adriatic in 1996. Contrary to its marked oral toxicity, intraperitoneally 7-epi-PTX-2SA displayed no toxic effects, hampering its detection by the mouse bioassay. In 1997, its concentration and frequency of appearance were lower than in 1996, with concomitant occurrence of OA, DTX-2, and a new unidentified component related to the DSP toxic group of compounds. This is the first report on the occurrence of DTX-2 in Adriatic mussels. A survey of the phytoplankton community in the surrounding seawater has established the presence of Prorocentrum micans and several potentially toxic species from the Dinophysis genus. A case of unexplained toxicity, associated with the occurrence of Gonyaulax polyedra, suggested possible shellfish contamination with yessotoxin (YTX).

The occurrence of 7-epi-pectenotoxin-2 seco acid in the coastal waters of the central Adriatic (Kaštela Bay)

Okadaic acid (OA) and 7-epi-pectenotoxin-2 seco acid (7-epi-PTX-2SA) were identified as the toxic determinants in mussels from the central Adriatic Sea. The nature of the pectenotoxin-2 derivative was confirmed by chromatographic comparison with toxins present in algae extracts of Dinophysis acuta from Ireland, and by mass spectrometric analysis. The origin of shellfish toxicity has been associated with the occurrence of the Dinophysis species. This is the first report on the incidence of 7-epi-PTX-2SA in the Adriatic region.

Inhibition of alkaline phosphatase activity by okadaic acid, a protein phosphatase inhibitor.

This paper reports on a potential physiological target of okadaic acid (OA), the toxin metabolite responsible for shellfish poisoning and, consequently, human intoxication. OA is a potent promoter of tumor activity, most likely by inhibiting protein phosphatase 1 and 2A (Adv. Cancer. Res. 61 (1993) 143). However, all of its cellular targets have not yet been characterized. The interaction of OA with alkaline phosphatase (ALP) has been investigated in view of its protein phosphatase inhibition activity. Kinetic analysis of ALP from Escherichia coli, human placental and calf intestinal ALP has shown that OA acts as a non-competitive inhibitor of ALP. The bacterial enzyme displays a higher affinity for OA (K(i) 360 nM) than the eukaryotic proteins (human placental ALP, K(i) 2.05 microM; calf intestinal ALP, K(i) 3.15 microM). The inhibition by OA suggests a putative role of ALP in the phosphorylation status, through regulation of the phosphorylation/dephosphorylation equilibrium of proteins with phosphoseryl or phosphothreonyl residues.

Oxidative stability and antioxidant activity of bovine, caprine, ovine and asinine milk

This study compares the oxidative stability of fat isolated from bovine, caprine, ovine and asinine milk as well as the antioxidant activity of casein and whey from these milks. Fat from bovine and asinine milk showed the highest oxidative stability. The antioxidant activity of casein and whey was examined before and after an in vitro digestion process. Whey from asinine milk showed the highest antioxidant activity. The antioxidant activity of whey and casein increased after simulated intestinal digestion. In addition, the results of this study showed that asinine whey exhibited radical scavenging activity comparable with the strong synthetic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).

Dipeptide synthesis by an isolated adenylate-forming domain of non-ribosomal peptide synthetases (NRPS)

A deletion mutant of tyrocidine synthetase 1 (ΔΔTY1), comprising the adenylation domain of TY1 as an independent functional adenylate‐forming unit, was used to investigate the ability of the adenylation domain in non‐ribosomal peptide synthetases to catalyse peptide bond formation from the aminoacyl adenylate intermediate. The results demonstrate that only one substrate amino acid needs to be activated as an aminoacyl adenylate. In view of the potential exploitation of peptide synthetases for enzymatic synthesis of dipeptides of choice, it is important to note that this does not necessarily require a dimodular construct or an intermediate acyl transfer step.

131I-induced changes in rat thyroid gland function

Therapeutic doses of (131)I administered to thyrotoxic patients may cause thyroid failure. The present study used a rat model to determine thyroid function after the administration of different doses of (131)I (64-277 microCi). Thirty male Fisher rats in the experimental group and 30 in the control group (untreated) were followed for 6 months. The animals were 4 months old at the beginning of the experiment and were sacrificed at an age of 9 months. Hormone concentration was determined before (131)I administration (4-month-old animals) and three times following (131)I administration, when the animals were 7, 8, and 9 months old. The thyroid glands were removed and weighed, their volume was determined and histopathological examination was performed at the end of the experiment. Significant differences in serum triiodothyronine and thyroid-stimulating hormone concentration, measured at the age of 7, 8, and 9 months, were found in the experimental group. During aging of the animals, the concentration of thyroxin fell from 64.8 +/- 8.16 to 55.0 +/- 6.1 nM in the control group and from 69.4 +/- 6.9 to 25.4 +/- 3.2 nM in the experimental group. Thyroid gland volume and weight were significantly lower in the experimental than in the control group. Thyroid glands from the experimental group showed hyaline thickness of the blood vessel wall, necrotic follicles, a strong inflammatory reaction, and peeling of necrotic cells in the follicles. In conclusion, significant differences in hormone levels and histopathological findings indicated prolonged hypothyroidism after (131)I administration to rats, which was not (131)I dose dependent.

Occurrence and antibiotic susceptibility profiles of Burkholderia cepaciacomplex in coastal marine environment

During an environmental study of bacterial resistance to antibiotics in coastal waters of the Kaštela Bay, Adriatic Sea, Croatia, 47 Burkholderia cepacia complex (Bcc) isolates were recovered from seawater and mussel (Mytilus galloprovincialis) samples. All isolates showed multiple antibiotic resistance. Among the isolates, two Burkholderia cenocepacia isolates produced chromosomally encoded TEM-116 extended-spectrum β-lactamase (ESBL). Analysis of outer membrane proteins revealed that decreased expression of a 36-kDa protein could be associated with a high level of β-lactam resistance in both isolates. Phenotypic study of efflux system also indicated an over-expression of Resistance-Nodulation-Cell Division (RND) efflux-mediated mechanism in one of the isolates. This study demonstrated the presence of Bcc inseawater and M. galloprovincialis, which gives evidence that coastal marine environment, including mussels, could be considered as a reservoir for Bcc species. Detection of ESBL-encoding genes indicates the potential role of these bacteria in the maintenance and dispersion of antibiotic resistance genes.

The A9 Core Sequence from NRPS Adenylation Domain Is Relevant for Thioester Formation

The adenylation (A) domain in nonribosomal peptide synthetases catalyses a two‐step reaction in which an amino acid is activated and then transferred to the neighbouring thiolation (T) domain. In this study, we investigated the role of the conserved A9 core sequence of the A‐domain of tyrocidine synthetase 1, by analysis of single amino acid mutations in the A9 region. Mutation of an absolutely conserved proline (P490G) significantly reduced the conformational stability of the protein, as evidenced by increased susceptibility to proteolytic cleavage and denaturation. All mutant A‐domains were capable of amino acid activation, but the activity in the overall reaction was reduced. Surprisingly, the S491R mutant (mutation at the first residue following the A9 motif) showed elevated overall activity compared to the wild‐type protein. Our results suggest that the A9 core sequence plays a role in the second reaction step, in which it could serve as a “clip” for the proper positioning of residues important for the interaction with the T‐domain, and/or stabilisation of the thioester‐forming conformation.

Evaluation of Olive Fruit Lipoxygenase Extraction Protocols on 9- and 13-Z,E-HPODE Formation

In plant tissues, enzymes implicated in the lipoxygenase (LOX) pathway are responsible for the hydroperoxydation of polyunsaturated fatty acids, ultimately leading to the production of small chemical species involved in several physiological processes. During industrial olive oil production, these enzymes are activated upon crushing and grinding of olive fruit tissue, subsequently leading to the synthesis of volatile compounds responsible for the positive aroma and flavor of the oil. An investigation of LOX activity during olive fruit ripening and malaxation could assist in the production of oils with favorable aroma and taste. Therefore, a reliable method for olive LOX purification is crucial. Here we report a critical review of six LOX extraction protocols, two of which have shown minimum enzyme activity, possibly leading to misconceptions in the interpretation of experimental data. Future research concerning olive LOX should employ extraction methods that preserve enzyme activity.