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Bisphenol-A (BPA) is a constituent of polycarbonate plastics and epoxy resins, widely applied on food packaging materials. As BPA exposure results in health hazards, its efficient removal is of crucial importance. In our study five potentially probiotic microorganisms, namely Lactococcus lactis, Bacillus subtilis, Lactobacillus plantarum, Enterococcus faecalis, and Saccharomyces cerevisiae, were tested for their toxicity tolerance to BPA and their BPA removal ability. Although BPA toxicity, evident on all microorganisms, presented a correlation to both BPA addition time and its concentration, all strains exhibited BPA-removal ability with increased removal rate between 0 and 24 h of incubation. BPA degradation resulted in the formation of two dimer products in cells while the compounds Hydroquinone (HQ), 4-Hydroxyacetophenone (HAP), 4-Hydroxybenzoic acid (HBA) and 4-Isopropenylphenol (PP) were identified in the culture medium. In the proposed BPA degradation pathways BPA adducts formation appears as a common pattern, while BPA decomposition as well as the formation, and the levels of its end products present differences among microorganisms. The BPA degradation ability of the tested beneficial microorganisms demonstrates their potential application in the bioremediation of BPA contaminated foods and feeds and provides a means to suppress the adverse effects of BPA on human and animal health.

Five antibacterial peptides produced by Bacillus subtilis NCIB 3610 were purified, quantified, characterized, and identified in the present study. Cell-free extracts were subjected to three purification protocols employing ammonium sulfate or organic solvent precipitation and their combination, followed by ion-exchange chromatography, solid-phase extraction, and preparative high-performance liquid chromatography (HPLC). The combined ammonium sulfate and organic solvent precipitation extraction protocol presented the best results for peptide purification. In the five fractions that presented antimicrobial activity, antibacterial peptides were quantified by the turbidometric method and by HPLC using nisin for external calibration, with the second providing more accurate results. All peptides were pH- and temperature-resistant and their sensitivity to proteases treatment indicated their proteinic nature. The five peptides were subjected to microwave-assisted acid hydrolysis (MAAH) and following derivatization were analyzed using norleucine as the internal standard, to determine their amino acid content. The identification of the isolated peptides using the UniProt and PubChem databases indicated that the four peptides correspond to UniProt entries of the bacteriocins Subtilosin-A (Q1W152) Subtilosin-SbOX (H6D9P4), Ericin B (Q93GH3), Subtilin (P10946), and the fifth to the non-ribosomal antibacterial lipopeptide surfactin (CID:443592). The amino acid content determination and computational analyses, applied in the present work on the antimicrobial peptides of B. subtilis, proved an efficient screening and quantification method of bacteriocins that could potentially be applied in other bacterial strains. The constructed phylogenetic trees heterogeneity observed across the five peptides investigated might be indicative of competitive advantage of the strain.

The production of β-Ν-methylamino-L-alanine (BMAA) in cyanobacteria is triggered by nitrogen-starvation conditions and its biological role, albeit unknown, is associated with nitrogen assimilation. In the present study, the effect of BMAA (773 μg L−1) on nitrogen metabolism and physiology of the non-diazotrophic cyanobacterium and non-BMAA producer, Synechococcus sp. TAU-MAC 0499, was investigated. In order to study the combined effect of nitrogen availability and BMAA, nitrogen-starvation conditions were induced by transferring cells in nitrogen-free medium and subsequently exposing the cultures to BMAA. After short-term treatment (180 min) and in the presence of nitrogen, BMAA inhibited glutamine synthetase, which resulted in low concentration of glutamine. In the absence of nitrogen, although there was no effect on glutamine synthetase, a possible perturbation in nitrogen assimilation is reflected on the significant decrease in glutamate levels. During the long-term exposure (24–96 h), growth, photosynthetic pigments and total protein were not affected by BMAA exposure, except for an increase in protein and phycocyanin levels at 48 h in nitrogen replete conditions. Results suggest that BMAA interferes with nitrogen assimilation, in a different way, depending on the presence or absence of combined nitrogen, providing novel data on the potential biological role of BMAA.

A novel, generally applicable method of identifying peptides using HPLC, microwave-assisted acid hydrolysis (MAAH), and bioinformatics is described. Method validation was performed on bacteriocins-antibacterial peptides produced by probiotic bacteria-using nine different bacteriocin isolates secreted by the probiotic Lactococcus lactis. Calibration curves were constructed for 23 amino acid PTH derivatives, and analysis was performed using norleucine as the internal standard. Validation of amino acid analysis performed in the range 2.5-100 nmol/mL indicated excellent method linearity, while the LODs ranged from 0.17 to 2.88 nmol/mL and the LOQs from 0.51 to 8.75 nmol/mL. The MAAH method was developed by irradiating nisaplin for various durations at 700 W, with 7 min providing the best results. The amino acid content of each sample was estimated following the application of MAAH to ten different samples. The bacteriocins in our samples were identified using the UniProt database. Eight of nine peptides were identified as UniProt entries: nisin A (P13068), nisin Z (P29559), I4DSZ9, OB7236, P36499, OB7237, A0A0M7BH60, and T2C9F0. The phylogenetic tree was constructed for nisin A and nisin Z using the multiple sequence aligning tool Clustal Ω. The identified nisin types presented excellent correlation with their ModBase-predicted structures. The present method gives true, precise, and rapid results, and requires only standard technical equipment. Our results suggest that the present approach can facilitate the discovery of novel bacteriocins and provide useful information on not only the amino acid contents of peptides but also the evolution of protein biology. Graphical abstract Identification of eight bacteriocins secreted by the probiotic L. lactis, following microwave assisted acid hydrolysis (MAAH), amino acid content analysis of each sample with HPLC-DAD and bioinformatics analysis using Uniprot, Clustal Ω and ModBase.

The present study describes the development of a simple and efficient screening system that allows identification and quantification of nine bacteriocins produced by Lactococcus lactis. Cell-free L. lactis extracts presented a broad spectrum of antibacterial activity, including Gram-negative bacteria, Gram-positive bacteria, and fungi. The characterization of their sensitivity to pH, and heat, showed that the extracts retained their antibacterial activity at extreme pH values and in a wide temperature range. The loss of antibacterial activity following treatment of the extracts with lipase or protease suggests a lipoproteinaceous nature of the produced antimicrobials. The extracts were subjected to a purification protocol that employs a two phase extraction using ammonium sulfate precipitation and organic solvent precipitation, followed by ion exchange chromatography, solid phase extraction and HPLC. In the nine fractions that presented antimicrobial activity, bacteriocins were quantified by the turbidometric method using a standard curve of nisin and by the HPLC method with nisin as the external standard, with both methods producing comparable results. Turbidometry appears to be unique in the qualitative determination of bacteriocins but the only method suitable to both separate and quantify the bacteriocins providing increased sensitivity, accuracy, and precision is HPLC.

The interactions of the probiotics Bacillus subtilis, Lactococcus lactis and Lactobacillus plantarum with the yeast Saccharomyces cerevisiae were examined in terms of probiotic and biochemical characteristics. Yeast supernatant had a positive effect on the aggregation biofilm formation capacity and hydrophobicity of probiotics, and resulted in increased lactic acid levels, reduced pH values as well as lower RS and FAN levels of probiotics. The effect of probiotics supernatants on yeast was more complex but best results were obtained in the yeast: probiotic CFS ratio of 1:2 for B. subtilis and of 2:1 for the other probiotics. The observed effects depended on the volume ratio of the cell free supernatant to the culture it was applied on. Best results were obtained by the volume ratio probiotic: yeast of (2:1) for B. subtilis and of (1:2) probiotic: yeast for L. plantarum and L. lactis. These ratios were used for further evaluation in vitro against V. anguillarum, resulting in reduced survival and attachment properties of the pathogen. Moreover, the administration of the corresponding combination of bacteria and yeast to Artemia nauplii greatly improved their survival following a challenge with the pathogen. Our results demonstrate that yeast enhances the protective effect of probiotics in a strain specific manner.

Bisphenol-A, a synthetic organic compound with estrogen mimicking properties, may enter bloodstream through either dermal contact or ingestion. Probiotic bacterial uptake of bisphenol can play a major protective role against its adverse health effects. In this paper, a method for the quantification of BPA in bacterial cells of L. lactis and of BPA and its potential metabolites 4-hydroxybenzoic Acid, 4-hydroxyacetophenone and hydroquinone in the culture medium is described. Extraction of BPA from the cells was performed using methanol–H2O/TFA (0.08%) (5:1 v/v) followed by SPE. Culture medium was centrifuged and filtered through a 0.45 μm syringe filter. Analysis was conducted in a Nucleosil column, using a gradient of A (95:5 v/v H2O: ACN) and B (5:95 v/v H2O: ACN, containing TFA, pH 2), with a flow rate of 0.5 mL/min. Calibration curves (0.5–600 μg/mL) were constructed using 4-n-Octylphenol as internal standard (1 > R2 > 0.994). Limit of Detection (LOD) and Limit of Quantification (LOQ) values ranged between 0.23 to 4.99 μg/mL and 0.69 to 15.1 μg/mL respectively. A 24 h administration experiment revealed a decline in BPA concentration in the culture media up to 90.27% while the BPA photodegradation levels were low. Our results demonstrate that uptake and possible metabolism of BPA in L. lactis cells facilitates its removal.

The alterations of immune responses of Artemia franciscana nauplii as a function of culture time and after a challenge with the pathogen Vibrio anguillarum were studied. The effect of the administration of the probiotic bacteria Bacillus subtilis , Lactobacillus plantarum and Lactococcus lactis either alone or in combination with the pathogen was evaluated. The activity of the antioxidant enzymes superoxide dismutase (SOD), Glutathione reductase (GRed), Glutathione transferase (GST) and Phenoloxidase (PO) presented a significant increase as a function of culture time, appeared elevated following probiotic administration and were depleted 48 h following the experimental challenge. Lipid peroxidation reached peak levels at 48 h of culture, when nauplii start feeding and returned to lower values at 144 h, remaining however significantly higher than control ( P 0.05 ). The three probiotics significantly reduced lipid peroxidation in comparison with the corresponding control, while challenge with the pathogen resulted in its threefold increase. Survival of nauplii remained high throughout culture and was either increased or remained at control levels following the administration of the probiotics. The challenge with the pathogen resulted in a significantly decreased survival of 15.3% for the positive control, while in the probiotic treated series survival values were not significantly different from the negative control ( P > 0.05 ). Following a combined administration of each probiotic and the pathogen the activities of all enzymes tested were significantly lower ( P 0.001 ) than the negative control (no treatment), but higher than the positive control (challenge, no probiotic). Lipid peroxidation was significantly lower in the probiotic treated series in comparison to the positive control ( P 0.001 ). The results of the present study provide evidence that major alterations take place as a function of culture time of Artemia nauplii. In addition the pathogen induces an oxidative stress response. The probiotics B. subtilis , L. plantarum and L. lactis protect Artemia against a V. anguillarum challenge by enhancing its immune responses thus contributing to reduced oxidative damage and increased survival.

European sea bass larvae were challenged by bath with Listonella anguillarum strain 332A, 2.5×10(7) CFUmL(-1) for 1h. Fish either received no treatment or oral treatment with Artemia franciscana (Kellog) nauplii enriched with oxolinic acid, or bath treatments with oxolinic acid. Medication commenced 1day following challenge and was performed on days 1, 3 and 5 post-challenge at a dosage of 20mgL(-1) for 2h for bath treatments, while two doses each of 750 nauplii per fish were administered daily for five consecutive days in oral treatments. Cumulative mortality reached 96% for the unmedicated challenged group, 32% in the group receiving bath treatments and 17% in the group receiving medicated nauplii. Pharmacokinetic parameters of oxolinic acid were calculated in sea bass larvae, for both treatments. Steady-state concentrations of oxolinic acid of 48.0 and 75.2μgg(-1) were achieved for bath treatment and oral treatment, respectively, while the elimination half-life was calculated to be 25.1h for bath treatment and 21.7h for oral treatment.

Two potential probiotics Bacillus subtilis and Lactobacillus plantarum were evaluated for use in aquaculture as preventive measures against vibriosis. In vitro evaluation of the probiotics using co-culture assays with the pathogen Vibrio anguillarum and testing for the production of antibacterial substances showed the presence of antagonism and confirmed the production of antibacterial substances. Both potential probiotics were administered to the live fish feed Artemia franciscana nauplii, offering protection against a subsequent challenge of the nauplii with the fish pathogen V. anguillarum, with best survival rates of the nauplii and the most efficient protection offered by B. subtilis. Nauplii enriched with B. subtilis were further used to evaluate the protection of sea bass larvae against vibriosis. The untreated group of fish challenged with V. anguillarum presented low survival of 36.7 %, while the fish treated with nauplii enriched with the probiotic B. subtilis showed significantly increased survival rates of 86.7 % after challenge with the pathogen. The survival of healthy unchallenged fish treated with the probiotic was not significantly different from control unchallenged fish (90-94 %). Our results indicate that B. subtilis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.

The uptake of flumequine by Artemia nauplii was studied as a function of its concentration in the enrichment medium and of the duration of the enrichment period. An emulsion containing 20, 30, 40 or 50% (w/w) flumequine was administered to nauplii for 4, 8, 12, 24 or 32 h. Increased uptake of flumequine (450.4 ± 15.8 μg/g dry weight) and good survival rates of the nauplii were observed with the 40% emulsion and 24 h enrichment. The concentration data of flumequine in nauplii were best fit to a two phase exponential elimination model with the first phase elimination half-life ( t 1/2α ) and the terminal phase elimination half-life ( t 1/2β ) to be 1 and 19.5 h, respectively. In the efficacy trial 1000 sea bass larvae were challenged by bath with Listonella anguillarum strain 332A, 2.5 × 10 7 CFU/ml for 1 h. Fish either received no treatment or oral treatment with flumequine bioencapsulated in nauplii or bath treatments with flumequine. Medication commenced two days following challenge, for bath treatments performed on day 2, 4 and 6 post challenge at a dosage of 20 mg/l for 2 h. Medication for oral treatments, commenced two days post challenge and two doses each of 700 nauplii per fish daily for five consecutive days. Cumulative mortality reached 96% for the unmedicated challenged group, 43% in the group receiving bath treatments and 29% in the group receiving medicated nauplii. Pharmacokinetic parameters of flumequine were calculated in sea bass larvae, after bath treatment with flumequine and oral treatment with flumequine bioencapsulated in nauplii. Steady state concentrations of flumequine (80.7 μg/g) in sea bass larvae were achieved after a 5-day oral treatment with medicated nauplii and the elimination half-life was found to be 19.5 h.

The glutathione S-transferase enzyme system, which belongs in phase II of detoxification, has been studied in developmental stages of the branchiopod Artemia salina. The highest total activity and specific activity towards 1-chloro-2,4-dinitrobenzene was observed 48 h after hatching. The number of isoenzymes present in A. salina varies between three and four, depending on the developmental stage. The two major isoenzymes, with corresponding isoelectric points of 8.5 and 7.2, were present in all developmental stages, but at a varying rate: as the organism grew older, the alkaline isoenzyme was expressed at a higher rate. The kinetic and molecular characteristics of the enzyme were similar to those of other aquatic organisms. With regards to the kinetic characteristics, significant differences were observed between the two major isoenzymes, and, since the rate of their expression changes as the organism grows, the detoxification potential of the organism varies according to developmental stage.

Η χορήγηση ζωντανής τροφής (ναύπλιοι Artemia) αποτeλeί τυπική διαδικασία και κλασικό τρόπο eκτροφής νeαρών ιχθυδίων τσιπούρας και λαβρακιού. Τα διάφορα, μικροβιακής αιτιολογίας, νοσήματα μe σοβαρότατeς οικονομικές προeκτάσeις συνιστούν ένα από τα σημαντικότeρα προβλήματα κατά το χρονικό τούτο διάστημα. Θeραπeυτικές παρeμβάσeις μe την δια του στόματος, χορήγηση των κατάλληλων αντιμικροβιακών παραγόντων, θeωρούνται και eίναι αφ' eνός μeν πeρισσότeρο eφικτές, αφ' eτέρου δe πeρισσότeρο φιλικές προς το πeριβάλλον. Η χρησιμοποιούμeνη μέθοδος συνίσταται στην τeχνική της βιοeγκύστωσης δηλαδή της eνσωμάτωσης των θeραπeυτικών ουσιών στη ζωντανή τροφή. Σe eιδικά διαμορφωμένeς eγκαταστάσeις του Eργαστηρίου Γeνικής Βιολογίας του Τμήματος Βιολογίας του Αριστοτeλeίου Πανeπιστημίου Θeσσαλονίκης και στα πλαίσια σχeτικού eρeυνητικού προγράμματος χρηματοδοτούμeνου από την Eυρωπαϊκή Ένωση, διeνeργήθηκαν πeιραματισμοί σe ιχθύδια λαβρακιού μe στeλέχη Vibrio anguillarum μe στόχο τη βeλτιστοποίηση των eφαρμοζόμeνων κάθe φορά θeραπeυτικών και προοληπτικών σχημάτων. Στα πλαίσια της eργασίας αυτής παρουσιάζονται συγκριτικά αποτeλέσματα από δύο διαφορeτικές πeιραματικές σeιρές (expermentalset)· κάθe σeιρά πeριλαμβάνeι οκτώ διαφορeτικές πeιραματικές συνθήκeς (experimentalseries), eνώ σe κάθe συνθήκη υπάρχουν πέντe eπαναλήψeις (aliquot). Ο πeιραματισμός θα συνeχιστeί μe τη χρησιμοποίηση και άλλων στeλeχών του ιδίου μικροβιακού eίδους καθώς και άλλων παθογόνων μικροβιακών eιδών. Eπίσης προβλέπeται και η eφαρμογή και άλλων σχeτικών πeιραματικών πρωτοκόλλων.

The pharmacokinetics of trimethoprim (TMP), sulfamethoxazole (SMX) and its metabolite N-acetyl-sulfamethoxazole (N-acetyl-SMX), were studied in Artemia nauplii as a function of the duration and temperature of their storage, following their enrichment with the therapeutics using the bioencapsulation technique. A marked decrease in the therapeutic content of the nauplii was observed upon storage at 18°C and 25°C and it was concluded that medicated nauplii should either be administered fresh to fish larvae or after storage for 8 h at 5°C, at the most. Under the latter conditions, satisfactory levels of TMP and SMX were achieved, high survival rates and dry weight contents of the nauplii were preserved and minimal leakage of the therapeutics to the environment was secured. Treatment of seabass larvae with one, three, six or ten doses of medicated Artemia nauplii, showed that maximum levels of the therapeutics are achieved in fish larvae when 10 doses are used. Following the treatment scheme of oral administration of 10 doses of medicated Artemia nauplii to fish larvae, the residual kinetics of TMP, SMX and N-acetyl-sulfamethoxazole were studied in seabass larvae. TMP and SMX showed different kinetic characteristics. A steady state of SMX concentration is considered to be achieved in fish body tissue during the 5-day medication period. TMP, SMX as well as the metabolite N-acetyl-sulfamethoxazole, were detectable in small amounts in fish body tissue even 100 h-post treatment. These data suggest that oral medication of fish larvae through the use of Artemia nauplii as a carrier of therapeutics, appears to be a quite promising approach to be used as an alternative method of treatment, which could minimize some of the problems arising from the methods currently in use.

The ability of Artemia nauplii to act as carriers of the chemotherapeutics trimethoprim and sulfamethoxazole for the treatment of infectious diseases of fish fry was studied. Both drugs accumulate in the nauplii as a function of time and their doses in the enrichment medium, but in different ratios, indicating different accumulation kinetics of the two drugs. The levels of the therapeutics in the nauplii remain fairly high even 8 hr post enrichment, decreasing thereafter mostly due to their discharge into their surrounding medium. However, the amounts of the discharged therapeutics do not account for their decrease in the nauplii. This, along with the appearance of certain minor peaks observed in HPLC, may well indicate that a metabolic alteration of the therapeutics occurs in the nauplii. A time-course quantification of the two drugs in the gut and the body of the fish larvae showed that they follow different kinetic patterns during their transfer from the digestive tract to the rest of the body. The use of the bioencapsulation technique as means of prevention or therapy was tested on Vibrio anguillarum-challenged sea bass larvae (Dicentrarchus labrax). Highly significant improvement (p < 0·002) in survival was observed when administration of medicated Artemia to fish larvae preceded (S = 81·4% ± 6·2%) or followed (S = 81·2% ± 4·4%) the bacterial challenge, as compared to the challenged-non-treated fish larvae (S = 65·0 ± 7·0%).

Large phospholipid vesicles loaded with the water soluble antibiotic oxytetracycline were formed using the reverse phase evaporation technique. Addition of cholesterol in the lipid phase and suspension of the liposomes in low pH solutions, greatly improved stability of the liposomes upon storage. Liposomised oxytetracycline was administered to Anemia franciscana nauplii using the bioencapsulation technique. Survival and larval development of the nauplii was not affected by the presence of liposomes in the enrichment medium. Determination of oxytetracycline levels in the liposomal preparations and in the nauplii, using reverse phase high performance liquid chromatography, showed significant amounts of antibiotic in the nauplii. The uptake of the liposomised antibiotic by the nauplii depended on the amount of the administered antibiotic and the duration of the enrichment period.

The effect of adenosine on the calcium paradox in the isolated frog heart was studied. Addition of adenosine during calcium depletion protected the frog heart against a calcium paradox. This protective effect was indicated by reduced protein and creatine kinase release, maintenance of electrical activity, and recovery of mechanical activity during reperfusion. Tissue calcium determination results showed that adenosine protected frog myocardial cells by reducing the massive calcium influx during reperfusion possibly through an action on calcium channels. Adenosine exerted its action in a dose-dependent manner; a concentration of 10 μM adenosine provided maximum protection of myocardial cells against the calcium paradox damage. Higher concentrations of adenosine produced side effects on both electrical and mechanical activity. These results are discussed in terms of the possible mechanism involved in the protective effect of adenosine.Key words: calcium paradox, adenosine, frog heart.

The effect of inorganic slow channel blockers on the calcium paradox in the frog heart was examined. Addition of the divalent cations of manganese, cobalt, nickel, or barium during calicium depletion protected the frog heart against a calcium paradox. This protective effect was indicated by reduced protein release, maintenance of electrical activity, and recovery of mechanical activity during reperfusion. Tissue calcium determination results showed that in the control paradox in the absence of divalent cations, there is an efflux of calcium from myocardial cells during calcium depletion and a massive influx of calcium during the following reperfusion, leading to a calcium overload. Divalent cations protected frog myocardial cells, when present in the calcium-free perfusion medium, by reducing both calcium efflux during calcium depletion and the massive calcium influx during reperfusion. The effectiveness of the added divalent cations showed a strong dependence upon their ionic radius. The most potent inhibitors of the calcium paradox in the frog heart were the divalent cations having an ionic radius closer to the ionic radius of calcium. These results are discussed in terms of the possible mechanism involved in the protective effect of manganese, cobalt, nickel, and barium.

The changes in myocardial energy metabolism of isolated perfused Rana ridibunda hearts subjected to prolonged calcium depletion and reperfusion with calcium-containing medium were studied. Calcium-free perfusion resulted in an increase in the concentrations of glucose, glucose-6-phosphate, alpha-ketoglutarate and malate. The myocardial contents of high-energy phosphates were maintained while concentrations of key amino acids were significantly altered. During the reperfusion period the tissue high-energy phosphate content fell abruptly. A marked increase in glycolytic flux and lactate production was observed. The tissue contents of citric acid cycle intermediates and key amino acids decreased. Examination of the activities of marker enzymes during the calcium-free and reperfusion periods showed that only cytoplasmic enzymes are lost during reperfusion, while the activities of other enzymes remained unchanged. The results suggest that the fluxes of both glycolysis and the citric acid cycle are significantly altered during calcium depletion and following repletion in the amphibian heart. The major characteristics of calcium paradox-induced damage in Rana ridibunda heart are the depletion of high-energy stores, the impairment of mitochondrial oxidative metabolism, and a significant increase in anaerobic metabolism.

A high-performance liquid chromatography (HPLC) analytical method for the determination of oxolinic acid and flumequine in Artemia nauplii is described. The samples were extracted and cleaned up by a solid-phase extraction (SPE) procedure using SPE C18 cartridges. Oxolinic acid and flumequine were determined by reversed-phase HPLC using a mobile phase of methanol-0.1 M phosphate buffer, pH 3 (45:55, v/v) and a UV detection wavelength of 254 nm. Calibration curves were linear for oxolinic acid in the range of 0.2-50 microg/g (r2=0.9998) and for flumequine in the range of 0.3-50 microg/g (r2=0.9994). Mean recoveries amounted to 100.8% and 98.4% for oxolinic acid and flumequine, respectively. The quantification limit was 0.2 microg/g for oxolinic acid and 0.3 microg/g for flumequine. Quantitative data from an in vivo feeding study indicated excellent uptake of both drugs by Artemia nauplii.

A high-performance liquid chromatographic analytical method was developed for the determination of oxytetracycline in Artemia nauplii. A solid-phase extraction protocol was used to recover oxytetracycline and the internal standard tetracycline, from the Artemia samples. Oxytetracycline was analyzed using a 150 x 4.6 mm I.D. Hypersil-ODS column, a mobile phase of acetonitrile-tetrahydrofuran-0.01 M oxalic acid buffer (pH 3.0) (15:3:82, v/v), and an ultraviolet detection wavelength of 365 nm. The calibration curve of oxytetracycline in Artemia was linear (r2 = 0.9998) from 0.1 to 6.4 micrograms/g of tissue. Using a signal-to-noise ratio of 4:1 the oxytetracycline detection limit was 10 ng/g of tissue. Mean recovery of oxytetracycline amounted to 97%, while intra-assay variability was 1.5%. Quantitative data from an in-vivo feeding study indicated an excellent uptake of oxytetracycline by Artemia, as its levels reached 25.6 micrograms per g of nauplii.

The effect of perfusion temperature and duration of calcium deprivation on the occurrence of the calcium paradox was studied in the isolated frog heart. Loss of electrical and mechanical activity, ion fluxes, creatine kinase and protein release were used to define cell damage. Perfusion was performed at 22, 27, 32, and 37 degrees C, and calcium deprivation lasted 10, 20, 30, or 40 min. At 22 degrees C and 27 degrees C even a prolonged calcium-free perfusion failed to induce a calcium paradox. After 30 min of calcium-free perfusion at 37 degrees C ventricular activity ceased and a major contraction occurred followed by an increase in resting tension. During the 15-min re-perfusion period the release of creatine kinase was 158.24 +/- 2.49 IU.g dry wt-1, and the total amount of protein lost was 70.37 +/- 0.73 mg.g dry wt-1, while lower perfusion temperatures resulted in a decreased loss of protein and creatine kinase. Ion fluxes in the perfusion effluent indicate that during re-perfusion a massive calcium influx accompanied by a potassium and a magnesium efflux, and an apparent sodium efflux, occur at a perfusion temperature of 37 degrees C after 30 min of calcium deprivation. The results suggest that the basic principles and damaging effects of calcium overloading are common to both mammalian and frog hearts.

Glutathione (GSH) transferase isoenzymes have been partially resolved from the cytosol of Schistocephalus solidus (plerocercoid) by GSH affinity chromatography and chromatofocusing at pH 7-5. The presence of isomeric forms was also suggested by analytical isoelectric focusing and high-performance liquid chromatography (HPLC). Gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that GSH transferase forms were dimers with a subunit size of approximately 24 kDa. The major GSH transferase form in S. solidus (plerocercoid) showed greater biochemical relationship to the Mu family of mammalian GSH transferase compared to the mammalian Alpha or Pi families. The major subunit purified by GSH affinity chromatography and reversed-phase HPLC also showed high N-terminal homology with the Mu family. A minor GSH transferase form appeared more biochemically related to the Alpha family with respect to substrate specificity and inhibitor sensitivity. The major GSH transferase was inhibited by haematin-related compounds, bile acids and a number of anthelmintics including members of the benzimidazole and phenol-based class of compounds. The major GSH transferase had conjugating activity with members of the trans, trans-2,4-alkadienal and trans-2-alkenal series, secondary products of lipid peroxidation.