Comparison of ractopamine residue depletion from internal tissues.

The aim of the study was to compare residue depletion of ractopamine HCl as a ß-adrenergic agonist that promotes muscle growth of animals, from internal tissues on days after its repeat administration to animals. The experiment was carried out in 38 albino guinea pigs. Treated animals (n = 30) were orally administered ractopamine HCl in a dose of 3.5 mg/kg body mass per day for 7 consecutive days. On days 1, 10, 20 and 30 of drug discontinuation, animals were randomly sacrificed and the liver, kidney, lung, heart, muscle, spleen and fat samples were collected. In all matrices, ractopamine concentration was determined using validated enzyme-linked immunosorbent assay (ELISA) as a quantitative screening method. The highest ractopamine concentration was recorded on day 1 in the lungs (55.80 ± 15.62 µg/kg), followed by the kidney (21.85 ± 3.91 µg/kg), spleen (12.59 ± 1.95 µg/kg), fat (10.17 ± 5.02 µg/kg), heart (9.73 ± 0.22 µg/kg), liver (5.58 ± 2.09 µg/kg), and lowest in muscle (2.21 ± 1.02 µg/kg). Ractopamine residues were detected in the lungs in the period of 30 days after withdrawal in significantly higher concentrations in comparison to other investigated matrices, suggesting that depletion of ractopamine from the lungs occurs at a much slower rate than its depletion from other internal tissues.

Gestational diabetes mellitus: Prevention, diagnosis and treatment. A fresh look to a busy corner.

BACKGROUND: Gestational diabetes mellitus (GDM) is a common pregnancy complication characterized by hyperglycaemia with onset or first recognition during pregnancy. Risk factors include family history of diabetes, previous GDM, genetic predisposition for GDM/type 2 diabetes, insulin resistance conditions such as overweight, obesity and ethnicity. Women with GDM are at high risk for fetal macrosomia, small for gestational age, neonatal hypoglycaemia, operative delivery and caesarean delivery. The aim of this narrative review is to summarize the most recent findings of diagnosis and treatment of GDM in order to underline the importance to promote adequate prevention of this disease, especially through lifestyle interventions such as diet and physical activity. METHODS: The research was conducted using the following electronic databases, MEDLINE, EMBASE, Web of Science, Scopus, ClinicalTrial.gov, OVID and Cochrane Library, including all published randomized and non-randomized studies as well as narrative and systematic reviews. RESULTS: The lack of universally accepted criteria makes the definition of diagnosis and prognosis of this condition difficult. Early diagnosis and glucose blood level control may improve maternal and fetal short and long-term outcomes. Treatment strategies include nutritional interventions and exercise. Medical treatment can be necessary if these strategies are not effective. Moreover, novel non-pharmacologic agents such as myo-inositol seem to be effective and safe both in the prevention and the treatment of GDM. CONCLUSIONS: It is important to promote adequate prevention of GDM. Further studies are needed in order to better define the most appropriate strategies for the clinical management of women affected by GDM.

Treatment of a landfill leachate containing compounds of pharmaceutical origin.

Paper reports the results of landfill leachate treatment with membrane bioreactor, nanofiltration and ozonation. Investigated leachate encompasses a number of specific compounds of pharmaceutical origin, including a suite of by-products deriving from the production of vitamin C and propyphenazone. Low biodegradability was observed in MBR (16%) for propyphenazone, while the removal of intermediates from the vitamin C-synthesis was moderate, reaching 30% for diacetone sorbose (DAS) and 69% for diacetone alpha-keto-gulonic acid (DAG). Ozonation almost completely removed propyphenazone but failed to significantly oxidise intermediates from the vitamin C-synthesis. Nanofiltration of the leachate succeeded to remove 99% of DAG and 79% of propyphenazone which made it the most efficient among techniques used.

Effect-directed analysis reveals inhibition of zebrafish uptake transporter Oatp1d1 by caulerpenyne, a major secondary metabolite from the invasive marine alga Caulerpa taxifolia.

Caulerpa taxifolia is a marine alga of tropical and subtropical distribution and a well-known invasive species in several temperate regions. Its invasiveness mainly stems from the production of secondary metabolites, some of which are toxic or repellent substances. In this study we investigated the possible inhibitory effects of C. taxifolia secondary metabolites on the activity of two zebrafish (Danio rerio) uptake transporters that transport organic anions (Oatp1d1) and cations (Oct1). Both transporters were transiently transfected and overexpressed in human embryonic kidney HEK293T cells. Transport activity assays using lucifer yellow (LY) and 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) as model substrates were applied for the determination of Oatp1d1 and Oct1 interactors. A two-step Effect-Directed Analysis (EDA) procedure was applied for the separation and identification of compounds. We identified caulerpenyne (CYN) as the major metabolite in C. taxifolia and reveal its potent inhibitory effect towards zebrafish Oatp1d1 as well as weak effect on zebrafish Oct1 transport. The observed effect was confirmed by testing CYN purified from C. taxifolia, resulting in an IC50 of 17.97 µM, and a weak CYN interaction was also determined for the zebrafish Oct1 transporter. Finally, using Michaelis-Menten kinetics experiments, we identified CYN as a non-competitive inhibitor of the zebrafish Oatp1d1. In conclusion, this study describes a novel mechanism of biological activity in C. taxifolia, shows that CYN was a potent non-competitive inhibitor of zebrafish Oatp1d1, and demonstrates that EDA can be reliably used for characterization of environmentally relevant complex biological samples.

Elimination of aromatic surfactants from municipal wastewaters: comparison of conventional activated sludge treatment and membrane biological reactor.

Behaviour of anionic surfactants of linear alkylbenzene sulphonates (LAS) type and non-ionic surfactants of nonylphenol polyethoxylate (NPnEO) type was studied in the conventional mechanical/biological sewage treatment plant (STP) as well as using a membrane biological reactor (MBR). LAS and NPnEO were determined using high performance liquid chromatography (HPLC) equipped with spectrofluorimetric detection. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) was used for identification and quantification of stable metabolites, including nonylphenol (NP), nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO) and nonylphenoxy carboxylic acids (NPnEC). The study showed that aromatic surfactants belong to the most prominent constituents in the examined municipal wastewaters with typical LAS and NPnEO concentrations of 2-10 mg/L and 0.1-0.5 mg/L, respectively. The removal of aromatic surfactants in conventional STP showed well-known features reported in the literature, including an efficient microbial transformation of the parent molecules and formation of stable metabolic products. The elimination efficiency of aromatic surfactants using the MBR unit was higher than that in the conventional STP, while the composition of recalcitrant nonylphenolic residues in the effluent seems to be ecotoxicologically more favourable due to the lower contributions of the lipophilic metabolites.

Peroral application of water-soluble derivative of propolis (WSDP) and its related polyphenolic compounds and their influence on immunological and antitumour activity.

Polyphenolic compounds are widely distributed in the plant kingdom and display a variety of biological activities, including chemoprevention and growth inhibition of tumours. Propolis contains a conglomerate of polyphenolic compounds. We investigated the effect of propolis and polyphenolic compounds, components of propolis, on the growth and metastatic potential of a transplantable mammary carcinoma (MCa) of the mouse. Metastases in the lung were generated by 2 x 10(5) tumour cells injected intravenously (i.v.). A water-soluble derivative of propolis (WSDP) and the polyphenolic compounds (caffeic acid (CA) and caffeic acid phenethyl ester (CAPE)) were given to mice perorally before or after tumour cell inoculation. WSDP, CA and CAPE reduced the number of metastases in the lung. This implies that the antitumour activities of the compounds used in these studies are mostly related to the immunomodulatory properties of the compounds, their cytotoxicity to tumour cells, and their ability to induce apoptosis and/or necrosis.

Genetic typing of recent classical swine fever virus isolates from Croatia.

During a period of 5 years (1997-2001) several outbreaks of classical swine fever (CSF) were recorded in Croatia. For genetic typing, fragments of 150 nucleotides within the 5'-non-translated region (5'-NTR) and 190 nucleotides within the E2 glycoprotein coding gene of nine field isolates that were derived from domestic pigs and wild boars were used. For better epizootiological understanding, isolates from other European countries were included in the study. The results show that the isolates belong to subgroups 2.1 and 2.3 of CSF virus. Isolates from subgroup 2.1 were collected from domestic pigs during sporadic outbreaks in June 1997 and are genetically closely related. A genomic similarity between these isolates and CSF virus isolates from pigs in other European countries from the same year could also be confirmed. In contrast, the isolate from October 1997 was found to be a member of subgroup 2.3, and is closely related to European CSF virus isolates from outbreaks in the last decade in Western and Central European countries. These results show that two different sources of CSF virus caused outbreaks in Croatia during the same year. Furthermore, a close relationship was found between an isolate from a domestic pig in 1999 and isolates of subgroup 2.3 that originated from Croatian wild boars.

Classical swine fever virus (C strain) distribution in organ samples of inoculated piglets.

Enzyme linked immunosorbent assays (ELISAs) and a nested polymerase chain reaction after reverse transcription (RT-PCR) were used for the detection of the Chinese strain (C strain) of classical swine fever virus (CSFV) in blood and tissue samples of experimentally inoculated piglets. One group of 10 piglets was inoculated with C strain material from rabbits and a second one with material from infected minipig kidney (MPK) cell culture. Tested blood samples were taken on the day of inoculation as well as on days 2, 4, 6, 8, 10, 13 and 16. Samples of spleen, tonsil and brain tissue were collected from piglets on days 6, 8, 10, 13 and 16 and tested for glycoprotein E(RNS) and protein NS2-3 using commercially available ELISA kits. E(RNS) and NS2-3 were detected earlier in blood samples of piglets inoculated with the C strain propagated in a cell culture. Regardless of propagation the presence of the viral E(RNS) and NS2-3 was detected in spleen and tonsil samples simultaneously. The C strain propagated in a cell culture was found in only one brain sample, whereas, the virus propagated in rabbits was detected in 70% of the brain samples. For the detection of the CSFV RNA in blood samples, a part within the 5' non-coding region was amplified. The differences in the results gained by antigen detection in blood samples decreased when nested RT-PCR was used.

Detection and genetic characterization of porcine circovirus type 2 (PCV2) in pigs from Croatia.

Porcine circovirus type 2 (PCV2) from the Circoviridae family has recently been associated with two serious diseases of swine, post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). During 2002, several outbreaks of clinical disease in pigs with weights ranging from 10 to 70 kg occurred on four farms in different locations in Croatia. The signs were consistent with PMWS and PDNS. Apart from progressive weight loss, pneumonia and/or diarrhoea, multifocal erythematous skin lesions and dermal necrosis were also observed. The PCR results obtained from PCV2 specific oligonucleotide primers confirmed a PCV2 infection. In addition, archive samples that were classical swine fever virus positive and derived from domestic pigs during an outbreak in 1997 were included in this study and one out of the three isolates was found to be positive for PCV2. For a better epizootiological understanding, genetic typing of representative isolates was carried out and compared with available isolates reported in the GenBank databases.

Comparison of antibody values in sera of pigs vaccinated with a subunit or an attenuated vaccine against classical swine fever.

Ten pigs, aged 85 days, were vaccinated with a subunit vaccine containing 32 microg of classical swine fever virus glycoprotein E2 (gp E2) (group 1), and a further 10 pigs were vaccinated with a C strain vaccine (10(4+/-0.15) TCID50/ml), produced by amplification in minipig kidney (MPK) cell culture (group 2). Nine non-vaccinated pigs served as a control group (group 3). Serum samples were collected before (day 0) and at 4, 10, 21 and 28 days after vaccination and were analysed by two commercially available enzyme immunoassays and by a neutralizing peroxidase-linked assay (NPLA). At the same times, peripheral blood was taken for determining the total leukocyte count and the body temperature was taken daily. Antibodies were not detected in serum samples collected before vaccination (day 0), and no side-effects that could be connected with vaccination were observed during the trial. Ten days after vaccination 6/10 pigs vaccinated with the subunit vaccine were seropositive. On days 21 and 28, the ratios of serologically positive to vaccinated pigs were 9/10 and 10/10, respectively. Four of the ten pigs that were vaccinated with the C strain vaccine were positive on day 21 and 9/10 on day 28. However, the results of the NPLA showed that only 4/10 pigs had an antibody titre > 1:32 at the end of the trial in both the vaccinated groups, even though the subunit vaccine initiated an earlier and higher level of neutralizing antibodies than the vaccine produced from the C strain. Challenge was performed 28 days after vaccination on four randomly selected pigs from both vaccinated groups. The pigs survived the challenge without showing any clinical signs of classical swine fever (CSF), while two nonvaccinated control pigs died on the 10th and 12th days after infection.

Immunophenotyping of leukocyte subsets in peripheral blood and palatine tonsils of prefattening pigs.

The quantitative and distribution patterns of porcine peripheral blood and tonsillar lymphoid/myeloid cell subsets were assessed in order to establish the immune status of farm pigs prior to their transfer to fattening units. Peripheral blood and tonsillar samples were taken from clinically healthy, nonvaccinated, 12-week-old pigs, either ex vivo or following euthanasia. Single-colour flow cytometry, using monoclonal antibodies (mAbs) reactive with the swine leukocyte cluster of differentiation (CD) antigens, gave the proportions of lymphoid (9.7% CD4+, 8.0% CD8+, 36.9% CD5a+, 20.3% CD16+, 6.9% CD21+, 86.3% CD45+, 41.8% CD45RA+, 48.3% CD45RC+), null cells (6.9%) and myeloid cells (23.7% CD11b+ and 5.4% SWC3a+) in peripheral blood. In situ identification and distribution of lymphoid cells in the tonsils (CD3a+, CD21+, CD45RA+, CD45RC+) was performed with anti-CD mAbs using the avidin-biotin complex method. Most CD3a+ cells were in the parafollicular areas, with many cells in the follicles. CD21+ cells were scattered throughout the parafollicular area, with only a few cells inside lymphoid follicles. CD45RA+ cells were mostly concentrated in the follicles but many positive cells were present in the parafollicular area. Many CD45RC+ cells were visible in the parafollicular area, a few positive cells were in the crypt epithelium, and single cells were inside the follicles.

Rapid immunoassay method for the determination of clenbuterol and salbutamol in blood.

The aim of the study was to evaluate the adequacy of enzyme-linked immunosorbent assay (ELISA) in the post-exposure determination of the ß-agonists clenbuterol and salbutamol in animal plasma and serum. Experimental guinea pigs (n = 20) were treated with two doses (0.25 and 2.5 mg/kg) of clenbuterol (n = 10) and salbutamol (n = 10) for seven days, whereas the control animal group (n = 10) was left untreated. Validation of the applied method yielded acceptable recovery (mean > 70%) and repeatability rates, showing ELISA to be applicable for the semi-quantitative determination of both analytes in both matrices, preferably in plasma. In both matrices, clenbuterol concentrations were proven to be significantly (14-fold) higher than those of salbutamol. Concentrations of both analytes were higher in plasma than in serum. The application of a 10-fold higher clenbuterol and salbutamol dose (2.5 mg/kg) resulted in concentrations 3- to 4-fold higher for clenbuterol and 2- to 3-fold higher for salbutamol, indicating a different release rate of these two ß-agonists.

Chemical indicators of anthropogenic impacts in sediments of the pristine karst lakes.

The anthropogenic impact on the pristine karst lakes was investigated using combination of specific parameters, including multielemental analysis of major inorganic constituents (Al, K, Fe) and trace metals (Li, Ag, Cd, Sn, Pb, Bi, Cr, Co, Ni, Cu, Zn and Sb), polycyclic aromatic hydrocarbons (PAHs) and anionic surfactants of linear alkylbenzene sulfonate (LAS) type. The study was performed in the Plitvice Lakes National Park, situated in a sparsely populated area of the northwestern Dinarides, central Croatia. Dated cores of recent sediments from the two biggest lakes, Lake Prosce and Lake Kozjak, were analysed for the selected contaminants using highly specific methods, involving inductively coupled plasma mass spectrometry (ICP/MS), gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The concentration of inorganic constituents reflected primarily the geological background of the area as well as geomorphological and geochemical characteristics of the Plitvice Lakes. Due to the higher terrigenous input, the concentration of all elements was significantly higher in the Lake Prosce. The concentration of toxic metals was relatively low in both lakes, except for Cd (>1 mg kg(-1)) and Pb (up to 40 mg kg(-1)). The vertical profiles of these metals suggested that elevated concentrations of Cd were of natural origin, derived from the erosion of the Jurassic dolomite bedrock, while Pb was predominately of recent anthropogenic origin. A similar distribution pattern, suggesting the same prevailing mechanism of input, was observed for pyrolytic PAHs. The characteristic diagnostic PAH ratios revealed that higher PAHs prevailingly originated from the combustion of biomass and fossil fuels. LAS, which represent highly specific indicators of untreated wastewaters, were found in rather high concentrations in the recent sediment layers (up to 4.7 mg kg(-1)), suggesting that contaminated household and hotel wastewaters reach the Lakes, very probably by leaking through the porous karst rocks.

Phytoplankton abundance and pigment biomarkers in the oligotrophic, eastern Adriatic estuary.

Phytoplankton distribution and environmental characteristics were determined in a shallow, highly stratified and oligotrophic estuary (Zrmanja, eastern Adriatic). Samples were collected in two contrasting seasons; winter (February 2000), when river discharge was high, and in summer (July 2003), a period of drought. Phytoplankton distribution was closely related to salinity gradients, nutrient levels, and water residence time. Microscopic analysis revealed that phytoplankton was composed mainly of marine diatoms, dinoflagellates, cryptophytes, green flagellates, and coccolithophorids. The dominant biomarker pigments were fucoxanthin, alloxanthin and 19'-hexanoyloxyfucoxanthin, while lower, but indicative contributions of peridinin and chlorophyll b were also noted. Maximum abundance and biomass were found in the middle estuary in winter and in the upper estuary in summer. The estuary is mostly P-limited. Development of chain-forming marine diatoms was evident in winter. Due to the reduced nutrient input in summer, the biomass accumulated in the upper estuary (1,000 ng chlorophyll a l(-1)) was composed mostly of nanoplanktonic unicellular diatoms, nanoplanktonic marine dinoflagellates, cryptophytes, and chlorophytes. The concentrations of about 200 ng l(-1) hex-fuco, suggested that the contribution of prymnesiophytes to total biomass was comparable to that of diatoms and dinoflagellates. In the middle estuary and coastal sea, PO(4) and TIN were 3.5 times lower, resulting in a fivefold decrease in biomass (<100 ng chlorophyll a l(-1)). The oligotrophic Zrmanja and other karstic rivers discharging in the eastern Adriatic Sea, provide insufficient source of nutrients and low productivity of the eastern Adriatic Sea.

Influence of honey bee products on transplantable murine tumours.

The effect of propolis [it is a water-soluble derivative (WSDP)] and related polyphenolic compounds of propolis (caffeic acid, caffeic acid phenethyl ester and quercetin), honey, royal jelly and bee venom on tumour growth, metastasizing ability and induction of apoptosis and necrosis in murine tumour models (mammary carcinoma and colon carcinoma) was investigated. WSDP and related polyphenolic compounds showed significant anti-metastatic effect (P < 0.01 and P < 0.001) given either before or after tumour-cell inoculation. Oral or systemic application of WSDP or caffeic acid significantly reduced subcutaneous tumour growth and prolonged the survival of mice. Honey also exerted pronounced anti-metastatic effect (P < 0.05) when applied before tumour-cell inoculation (peroral 2 g kg(-1) for mice or 1 g kg(-1) for rats, once a day for 10 consecutive days). Royal jelly did not affect metastasis formation when given intraperitoneally or subcutaneously. However, intravenous administration of royal jelly before tumour-cell inoculation significantly (P < 0.05) inhibited metastasis formation. When mice were given 10(5) tumour cells intravenously immediately after bee venom injection, the number of tumour nodules in the lung was significantly lower (P < 0.001) than in untreated mice or mice treated with bee venom subcutaneously. Local presence of bee venom in the tissue caused significant delay in subcutaneous tumour formation. These findings clearly demonstrate that anti-tumour and anti-metastatic effects of bee venom are highly dependent on the route of injection and on close contact between components of the bee venom and tumour cells. These data show that honey bee products given orally or systemically may have an important role in the control of tumour growth and tumour metastasizing ability.

Proportions and phenotypic expression of peripheral blood leucocytes in pigs vaccinated with an attenuated C strain and a subunit E2 vaccine against classical swine fever.

The influence of an attenuated classical swine fever virus C strain vaccine and a subunit E2 vaccine against classical swine fever on the peripheral blood leucocyte proportion and phenotypic expression in 12-week-old pigs was studied. The C strain was amplified in minipig kidney cell culture and final product contained 10(4 +/- 0.15) TCID50/ml, while the subunit vaccine contained 32 microg per dose of gp E2. Haematological findings showed that the vaccines did not cause leucopenia or lymphocytopenia and the number of neutrophils and eosinophils during the observation period was within physiological range. The results of the proportion of CD4a+, CD5a+, CD8a+, wCD21+, CD45RA+, CD45RC+ , non-T non-B, SWC3a+ and CD11b+ cells were gained by single-colour flow cytometry. At the end of the trial a significantly increase of percentage of CD4+, CD5a+, CD8+, wCD21+ cells has been found in pigs that received the subunit vaccine and the percentage of CD4+, CD5a+, CD8+, CD45RA+ and CD45RC+ cells was higher in pigs that received the attenuated vaccine. Twenty-eight days after vaccination the percentage of CD4+, CD45RA+ and CD45RC+ was significantly higher in pigs vaccinated with the C strain than in pigs vaccinated with the subunit vaccine. In contrary, the percentage of the wCD21- cells was higher in pigs that received the subunit vaccine. Statistically higher values of SWC3a+ and lower values of CD11b+ cells was observed in pigs that received the attenuated vaccine than in pigs vaccinated with the subunit vaccine. Taken altogether, our results showed that the subunit vaccine produced a better stimulation of B cells and CD11b+ monocytes/macrophages /granulocytes/NK cells, whereas the attenuated vaccine induced a higher response of Th cells, naive/memory cells and macrophages/neutrophils. Thus, both vaccines were able to influence the porcine immune system, by activating different subsets of the immune effector/accessory cells.