Organic anion transporting polypeptides (OATP) in zebrafish (Danio rerio): Phylogenetic analysis and tissue distribution

The aim of our study was the initial characterization of Organic anion transporting polypeptides (SLCO gene superfamily) in zebrafish (Danio rerio) as an important model species in biomedical and ecotoxicological research, using phylogenetic analysis, membrane topology prediction and tissue expression profiling. The phylogenetic tree of Oatp superfamily in vertebrates was constructed in Mega 3.1. Software, membrane topology was predicted using HMMTOP algorithm, while qRT-PCR was used to determine tissue-specific gene expression levels. Phylogenetic analysis revealed that Oatp superfamily in zebrafish consists of five families that include 14 SLCO genes. Eight out of 14 transporters do have orthologs or co-orthologs in other vertebrates, while 6 members are found only in fish lineage. Topology analysis showed that all zebrafish Oatps consist of 12 transmembrane domains (TMD) with the large fifth extracellular loop (LP5). Tissue distribution analysis revealed that the expression patterns of Oatp2a1, Oatp2b1 and Oatp3a1 follow tissue distribution patterns of their mammalian (co)orthologs. Expression pattern of a newly identified Oatp1d1 is similar to mouse Oatp1a4, while other new zebrafish Oatps (Oatp1e1, 1f2) do not resemble any of the mammalian Oatps. In summary, the described comprehensive analysis of Oatp superfamily in fish represents a first step towards research on toxicological relevance of uptake transporters in aquatic organisms.     

Differential patterns of large tumor antigen-specific immune responsiveness in patients with BK polyomavirus-positive prostate cancer or benign prostatic hyperplasia

The role of the polyomavirus BK (BKV) large tumor antigen (L-Tag) as a target of immune response in patients with prostate cancer (PCa) has not been investigated thus far. In this study, we comparatively analyzed humoral and cellular L-Tag-specific responsiveness in age-matched patients bearing PCa or benign prostatic hyperplasia, expressing or not expressing BKV L-Tag-specific sequences in their tissue specimens, and in non-age-matched healthy individuals. Furthermore, results from patients with PCa were correlated to 5-year follow-up clinical data focusing on evidence of biochemical recurrence (BR) after surgery (prostate specific antigen level of ≥0.2 ng/ml). In peripheral blood mononuclear cells (PBMC) from patients with PCa with evidence of BR and BKV L-Tag-positive tumors, stimulation with peptides derived from the BKV L-Tag but not those derived from Epstein-Barr virus, influenza virus, or cytomegalovirus induced a peculiar cytokine gene expression profile, characterized by high expression of interleukin-10 (IL-10) and transforming growth factor β1 and low expression of gamma interferon genes. This pattern was confirmed by protein secretion data and correlated with high levels of anti-BKV L-Tag IgG. Furthermore, in PBMC from these PCa-bearing patients, L-Tag-derived peptides significantly expanded an IL-10-secreting CD4(+) CD25(+(high)) CD127(-) FoxP3(+) T cell population with an effector memory phenotype (CD103(+)) capable of inhibiting proliferation of autologous anti-CD3/CD28-triggered CD4(+) CD25(-) T cells. Collectively, our findings indicate that potentially tolerogenic features of L-Tag-specific immune response are significantly associated with tumor progression in patients with BKV(+) PCa.     

Mesostigmatic mites (Acari: Mesostigmata) in nests of the Eurasian griffon vulture (<Emphasis Type="Italic">Gyps fulvus</Emphasis>) in Croatia

We examined the species composition and community structure of mites of the order Mesostigmata (Acari) in nests of the Eurasian griffon vulture (Gyps fulvus Hablizl, 1783) in Croatia. Material collected from 18 nests included 565 mites belonging to seven species. The most abundant species were Leiodinychus orbicularis (C.L. Koch, 1839) (Trematuridae) and Androlaelaps casalis (Berlese, 1887) (Laelapidae). The results were compared with the community structure and frequency of dominant species of Mesostigmata in nests of 32 other bird species. Leiodinychus orbicularis occurred in the nests of 13 species of birds. It is a typical nidicolous species which occurs most frequently in the perennial nests of birds of prey. In contrast, A. casalis rarely occurs in the nests of birds of prey.     

Insect fauna in nests of the Eurasian griffon vulture (Gyps fulvus) in Croatia

Qualitative, quantitative and trophic structure of insects found in Eurasian griffon vulture nests were analysed. A total of 249 insects belonging to six orders were found in 18 griffon vulture nests, collected in three colonies on the islands Cres and Plavnik (Adriatic Sea). Eudominant orders were beetles (64.26%) and ants (22.49%). Dermestid beetles were present in all examined nests and comprise the permanent nest fauna. Other groups of insects (cockroaches, web spinners, ants, flies, aphids) were found to occur occasionally in nests.     

Haemolymph as compartment for efficient and non-destructive determination of P-glycoprotein (Pgp) mediated MXR activity in bivalves

Measurement of the modulation of accumulation rate of model P-glycoprotein (Pgp) substrates has been a well established methodology for determination of the presence and activity of the multixenobiotic resistance (MXR) defence mechanism in aquatic invertebrates. Most studies have been focused on the gill tissue of various bivalves as a primary compartment for this type of measurements. In this study, we evaluated the potential of measuring the accumulation rate of a fluorescent model Pgp substrate rhodamine B (RB) in haemolymph, plasma and haemocytes of the freshwater painter's mussel (Unio pictorum) as additional potentially useful compartments. The obtained results demonstrated several important advantages of the determination of Pgp mediated MXR transport activity in haemolymph over determinations in gill tissue. The overall MXR response correlated well with the level of Pgp activity simultaneously determined in gills. The method is more sensitive, the procedure is easier and less laborious, and repeated use of same individuals is possible. Finally--the approach is non-destructive, offering a potentially powerful biomarker and research tool for studies directed to the evaluation of ecotoxicological importance of MXR defence and the presence of MXR inhibitors in the environment.     

Meiofauna constitute a considerable portion of invertebrate drift among moss-rich patches within a karst hydrosystem

Aiming to establish the most frequent invertebrate taxa in drift at the small spatial scale within a moss-rich karst tufa-precipitating hydrosystem, we sampled drift among microhabitats differing in substratum type and flow conditions along a tufa barrier-cascading lotic reach. Additionally, we addressed the question of the contribution and the potential significance of meiofauna within the overall invertebrate drift at the small spatial scale. During the study period, a total of 60 invertebrate taxa were recorded in the drift. Six of these taxa belonged to the annelid/arthropod meiofauna and they represented 35% of total drift density. Macroinvertebrates found in drift were represented mainly by larval insects. The composition of the most abundant taxa in total drift was as follows: Alona spp. (Cladocera 26.7%), Riolus spp. (Coleoptera: Elmidae 13.2%), Simulium spp. (Diptera: Simuliidae 12.2%), Enchytraeidae (Oligochaeta 10.4%), Hydrachnidia (6.3%), Orthocladinae (Diptera: Chironomidae 3.9%) and Naididae (Oligochaeta 3.6%). Faunal drift densities and amounts of transported particulate matter (PM) were highest at the fast-flowing sites located at the barriers and lowest at the slow-flowing sites within pools. Similarly to the seasonal amounts of transported PM, faunal drift was lowest in winter, and peaked in autumn and in late spring/early summer. Correlation between flow velocity and PM-faunal drift densities suggested a significant effect of the dislodged PM, though a minor influence of discharge and flow velocity on faunal drift. We suggest that the small-scale habitat heterogeneity and the respective feeding and refugial strategies of the fauna, as well as faunal passive dislodgement initiated by the shear forces of the flow were the most important drivers of observed drift patterns.     

Contrasting Zooplankton Assemblages in Two Oxbow Lakes with Low Transparencies and Narrow Emergent Macrophyte Belts (Krapina River,Croatia)

The aim of this study was to examine the combined effect of water transparency and narrow macrophyte belts on zooplankton assemblages in two oxbow lakes (Krapina River, Croatia). Samples were collected in open water and among helophytes in the littoral zone from April until September 2008. Rotifers were the most abundant group of zooplankton in both lakes, and dominated in the Krapina oxbow lake 1 (KO1). Lake KO1 had significantly lower transparency, lower percentage macrophyte cover and higher chlorophyll a concentration than Krapina oxbow lake 2 (KO2). In lake KO1, variation in the horizontal distribution of cladocerans and rotifers in terms of their abundance seemed to be determined by competition between Bosmina longirostris and Keratella cochlearis, initiated by oscillation in transparency and detritus availability. In lake KO2, with higher transparency and higher percentage macrophyte cover, the abundance of small‐ and large‐bodied cladocerans increased in the littoral zone simultaneously with higher transparency, suggesting fish predation. Results of this study indicated that small differences in transparencies between the two lakes caused significant differences in horizontal distribution of the zooplankton assemblage. Even narrow helophyte belts offered a refuge to zooplankton, although lower transparencies reduced the effectiveness of macrophytes as a refuge from predators. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Stratigraphic framework, discontinuity surfaces, and regional significance of Campanian slope to ramp carbonates from central Dalmatia, Croatia

The sedimentology, microfacies, and stratigraphic age (from planktonic and benthic foraminifera and strontium-isotope stratigraphy) of a 300-m-thick Upper Cretaceous carbonate succession from the Island of ?iovo (central Dalmatia, Croatia) were analyzed in order to determine the lithostratigraphic, depositional, and chronostratigraphic framework. The Cretaceous strata were deposited in the southern part of the long-lasting (Late Triassic to Paleogene) Adriatic-Dinaridic Carbonate Platform (ADCP), one of a few late Mesozoic, intra-Tethyan, peri-Adriatic (sub)tropical archipelagos. The succession is separated by a firmground formational boundary into two lithostratigraphic units: the underlying Middle to Upper Campanian Dol Formation consisting of slope pelagic limestone with intercalated turbidites and debrites, and the overlying Upper Campanian ?iovo Formation composed of outer-ramp bioclastic-lithoclastic and echinoderm-dominated packstone. Age, lithology, and depositional settings of the ?iovo Formation are different from other penecontemporaneous, regionally important inner-platform carbonate successions within the ADCP domain. Therefore, the ?iovo Formation is proposed here as a new lithostratigraphic unit. Regionally important condensed intervals in the form of at least two firmground surfaces, characterized by Thalassinoides burrows (with phosphatic mineralization) that belong to the Glossifungites ichnofacies, occur in the lowermost part of the ?iovo Formation. Abrupt shallowing of depositional environments at the boundary between the Dol and the ?iovo Formation, and the generation of the formational boundary firmground, likely correlate with the regionally recorded Upper Campanian Event that represents a global eustatic sea-level fall. A regionally important subaerial exposure surface with nodular calcrete, rhizoliths, and Microcodium aggregates in the upper part of the ?iovo Formation represents a regional subaerial unconformity that was recorded across the ADCP domain and was interpreted as a consequence of diachronous and differential uplift of various parts of the platform in response to the formation of a forebulge in front of the approaching Dinaridic orogen.     

Molecular Characterization of Zebrafish Oatp1d1 (Slco1d1), a Novel Organic Anion-transporting Polypeptide

The organic anion-transporting polypeptide (OATP/Oatp) superfamily includes a group of polyspecific transporters that mediate transport of large amphipathic, mostly anionic molecules across cell membranes of eukaryotes. OATPs/Oatps are involved in the disposition and elimination of numerous physiological and foreign compounds. However, in non-mammalian species, the functional properties of Oatps remain unknown. We aimed to elucidate the role of Oatp1d1 in zebrafish to gain insights into the functional and structural evolution of the OATP1/Oatp1 superfamily. We show that diversification of the OATP1/Oatp1 family occurs after the emergence of jawed fish and that the OATP1A/Oatp1a and OATP1B/Oatp1b subfamilies appeared at the root of tetrapods. The Oatp1d subfamily emerged in teleosts and is absent in tetrapods. The zebrafish Oatp1d1 is similar to mammalian OATP1A/Oatp1a and OATP1B/Oatp1b members, with the main physiological role in transport and balance of steroid hormones. Oatp1d1 activity is dependent upon pH gradient, which could indicate bicarbonate exchange as a mode of transport. Our analysis of evolutionary conservation and structural properties revealed that (i) His-79 in intracellular loop 3 is conserved within OATP1/Oatp1 family and is crucial for the transport activity; (ii) N-glycosylation impacts membrane targeting and is conserved within the OATP1/Oatp1 family with Asn-122, Asn-133, Asn-499, and Asn-512 residues involved; (iii) the evolutionarily conserved cholesterol recognition interaction amino acid consensus motif is important for membrane localization; and (iv) Oatp1d1 is present in dimeric and possibly oligomeric form in the cell membrane. In conclusion, we describe the first detailed characterization of a new Oatp transporter in zebrafish, offering important insights into the functional evolution of the OATP1/Oatp1 family and the physiological role of Oatp1d1.