Evaluation of an extended-release formulation of ceftiofur crystalline-free acid in koi (Cyprinus carpio).

The use of an extended release ceftiofur crystalline-free acid formulation (CCFA, Excede For Swine(®) , Pfizer Animal Health) in koi was evaluated after administration of single intramuscular (i.m.) or intracoelomic (i.c.) doses. Twenty koi were divided randomly into a control group and four treatment groups (20 mg/kg i.m., 60 mg/kg i.m., 30 mg/kg i.c., and 60 mg/kg i.c.). Serum ceftiofur-free acid equivalents (CFAE) concentrations were quantified. The pharmacokinetic data were analyzed using a nonlinear mixed-effects approach. Following a CCFA injection of 60 mg/kg i.m., time durations that serum CFAE concentrations were above the target concentration of 4 µg/mL ranged from 0.4 to 2.5 weeks in 3 of 4 fish, while serum CFAE concentrations remained below 4 µg/mL for lower doses evaluated. Substantial inter-individual variations and intra-individual fluctuations of CFAE concentrations were observed for all treatment groups. Histological findings following euthanasia included aseptic granulomatous reactions, but no systemic adverse effects were detected. Given the unpredictable time vs. CFAE concentration profiles for treated koi, the authors would not recommend this product for therapeutic use in koi at this time. Further research would be necessary to correlate serum and tissue concentrations and to better establish MIC data for Aeromonas spp. isolated from naturally infected koi.

The diet of adult psittacids: veterinarian and ethological approaches.

Parrots species are kept as pets because of their colours, their vocal abilities, their longevity and also their behaviours. Nevertheless, many owners do not know how to feed their bird in a healthy way and sometimes veterinarians and ethologists are confronted to dramatic situations. Diet is a key factor to prevent and reduce health and psychogenic disorders. Imbalance can lead to physical, physiological and behavioural modifications that can weaken the owner-bird relationship, cause bird discomfort and sometimes threaten its survival. Psittacids are known for their complex cognitive and communicative abilities. They are social animals and need many interactions. Kept in captivity, they could suffer from boredom because of lack of stimulations and also because of lack of possibility to explore and to forage, which could represent up to 70% of their day time in the wild. Indeed, humans control every parameter in the environment of pet psittacids. They provide a highly digestible diet. In captivity, foraging is not mandatory, and the bird can get bored. Here, we present a review of the literature regarding the quality of the diet and health disorders on one hand and the interaction between foraging opportunities and psychogenic disorders in adult psittacids on the other hand.

Phylogeography of the threatened crayfish (genus Austropotamobius) in Italy: implications for its taxonomy and conservation.

A nucleotide sequence analysis of a portion of the mitochondrial large ribosomal subunit was performed to define the phylogeography of the threatened crayfish Austropotamobius (Decapoda; Astacidae) in Italy. We collected 61 specimens from 31 localities across the Italian peninsula. For the phylogenetic inference, we combined the 61 Austropotamobius spp sequences obtained from this study with 18 sequences deposited in GenBank and corresponding to Italian, French, Irish, Swiss, and Slovenian locations. Among the analysed sequences, 34 distinct haplotypes were detected. Our results confirmed the presence of both A. pallipes and A. italicus in the Italian peninsula and the existence within the latter species of a strong intraspecific genetic variation, due to the occurrence of four subspecies with a well-defined geographic distribution. From a conservation viewpoint, Italy, with its high haplotype variability, may be considered a 'hot spot' for the genetic diversity of the European native crayfish Austropotamobius. We suggest that re-introduction programs should be conducted with extreme caution in Italy, since not only the two Austropotamobius species but also the four A. italicus subspecies are genetically and taxonomically separate units and require independent conservation plans.

Origin and colonization history of the white-clawed crayfish, Austropotamobius pallipes, in Ireland.

The presence of the white-clawed crayfish Austropotamobius pallipes complex in Ireland is suspected to be a result of human translocations. Two hypotheses have been formulated about the origin of the crayfish: from British populations or from western French populations. In order to resolve this question, nine Irish crayfish populations (a total of 124 individuals) were sampled along a south-north cline and investigated by combining two molecular markers: mtDNA and RAPDs. The mtDNA marker, analysed by RFLP on the entire molecule, showed an absence of polymorphism within and among Irish populations. The RFLP haplotype found in Irish populations was only recorded in western French populations and was different from those found in English populations. This result may be explained by a human introduction of crayfish to Ireland from western French populations. RAPD analysis showed a clinal reduction of genetic variability within Irish populations from south to north, associated with an increase in their genetic differentiation. A stepwise model of translocation from the south to the north of Ireland is proposed and discussed.

Population genetic structure of the endangered freshwater crayfish Austropotamobius pallipes, assessed using RAPD markers.

Random amplified polymorphic DNA (RAPD) analysis was performed to characterize the genetic diversity of Austropotamobius pallipes, a threatened freshwater crayfish native to Europe. Four decamer primers which generated six unambiguous polymorphic bands were used to analyse crayfish from 21 populations sampled in the major part of its range. Genetic diversity within populations of A. pallipes, estimated by Shannon's diversity index, ranged from 0 to 0.446 with a mean of 0.159. A UPGMA dendrogram constructed from pairwise PhiST values between populations, revealed three clusters corresponding to populations sampled in the southern, northwestern and eastern part of its range. AMOVA analysis revealed a high genetic structure of A. pallipes populations PhiST=0.814, with 73.11% of the genetic variation distributed between these clusters. It suggests a historical geographical separation of these groups into three refugial areas, probably in the Rhine, Mediterranean and Atlantic basins during recent glaciations. The close genetic relationships between English and western French populations are in accordance with a natural postglacial origin of English populations from individuals having survived in an Atlantic refugium. However, the present results suggest that the Irish stock originated from a human translocation of individuals from an Atlantic refugium.

Pyrene degradation by two fungi in a freshwater sediment and evaluation of fungal biomass by ergosterol content.

Mucor racemosus var. sphaerosporus and Phialophora alba were investigated for their abilities to degrade pyrene in a freshwater sediment, with or without glucose supply as nutrient or carbon source, during 90 days. The ergosterol contents in sediment were quantified to estimate fungal biomass and to assess the correlation between fungal activity and biodegradation of pyrene. Results showed that, in an heterogeneous environment, these fungi presented different abilities to degrade pyrene. P. alba increased the degree of pyrene degradation by 9%, compared to the native micro-organisms, but a supply of glucose acted as an inhibitor to pyrene disappearance. M. racemosus var. sphaerosporus was not efficient at sediment bioremediation (with or without glucose added), because it reduced the rate of pyrene degradation by the native microflora. In any case, there was no increase of ergosterol in boxes during bioremediation experiments. In our experimental conditions, ergosterol content could not be correlated to pyrene degradation.

Liquid chromatography study of pyrene degradation by two micromycetes in a freshwater sediment.

Pyrene biodegradation in a freshwater sediment without fungi supply, or inoculated with two sediment micromycetes, Mucor racemosus var. sphaerosporus and Phialophora alba was studied after 0, 5, 13, 28, 60 and 90 days. The influence of glucose addition was estimated, and a liquid chromatographic method for simultaneous quantitative determination of residual anthracene, fluoranthene and pyrene in the sediment was developed. Samples with PAHs were extracted in Soxhlet with ethyl acetate, and LC analysis was performed on a 5 microm Supelcosil column (150 x 4.6 mm I.D.) with gradient elution (2 ml min(-1)) of acetonitrile-water and UV detection at 254 nm. Recoveries of anthracene, fluoranthene and pyrene were 90.3%+/-1.1%, 93.2%+/-0.9% and 90.42%+/-1.9%, respectively, without interference. The native sediment microorganisms (with or without glucose added) have shown 35% pyrene degradation and sediment with glucose inoculated by the strains revealed 40%.

Dansyl amino acid enantiomer separation on a teicoplanin chiral stationary phase: effect of eluent pH.

The retention and separation of a series of D,L dansyl amino acids (used as test solutes) on a teicoplanin stationary phase were investigated over a wide range of mobile phase (citrate buffer-methanol, 90:10, v/v) pH. An approach based on the development of various equilibria was carried out in order to describe the retention behavior of the solute in the chromatographic system. The equilibrium constants corresponding to the transfer of the anionic and zwitterionic forms of the dansyl amino acids from the mobile to the stationary phase were determined. These values allowed one to explain the decrease in the retention factor and the associated increase in the separation factor as the eluent pH was increased. Thermodynamic parameter variations were calculated so that the driving forces of the solute association with the teicoplanin phase were derived. This approach indicated that the chiral discrimination was principally controlled by the interaction between the anionic form of the solute and the stationary phase.

Time measurement in the photoperiodic induction of sexual rest in the terrestrial isopod Armadillidium vulgare (Latreille).

The photoperiodic control of sexual rest in Armadillidium vulgare was investigated using various experimental protocols. When reared in conditions of a Nanda-Hamner (i.e. resonance) protocol from their first parturial moult to their post experimental moult, females showed a weak resonance effect in sexual rest incidence. The transfer from a long day cycle to a symmetrical skeleton photoperiod--consisting of two equal light pulses per 24 h of continuous darkness--revealed the involvement of a circadian oscillatory system in the photoperiodic clock of this species. The data, obtained in the whole experiments, suggested that both oscillator and hourglass features are involved in the photoperiodic response controlling the sexual rest in Armadillidium vulgare. Moreover, when non-24-h light-dark cycles (with a long photophase) were applied, a mechanism responsible of arrest of reproduction also implied a photoperiodic counter which accumulated and added up the photoperiodic information within a sensitive period during post parturial intermoult.

Drastic bottlenecks in the endangered crayfish species Austropotamobius pallipes in Spain and implications for its colonization history.

Variation in mitochondrial DNA (mtDNA) was surveyed, using restriction endonucleases, in the white-clawed crayfish, Austropotamobius pallipes lusitanicus, from 14 populations sampled in Spain. Four additional samples from France (1), Slovenia (1) and Italy (2) were also analysed. Among the 11 haplotypes listed, only one was detected from the 154 animals sampled from Spanish populations. This haplotype was also recorded in the Fosso di Ferfereta population (Italy). Estimates of nucleotide sequence divergence among haplotypes ranged from 0.45% to 17.4%. Interpopulational genetic relationships showed that Spanish populations were closely related to those of Fosso di Ferfereta with a small genetic distance (0.0003) found between them. AMOVA revealed that most of the genetic variance (71.97%) was attributed to variation between European regions. These results are in accordance with a drastic bottleneck event during the history of the Spanish populations. Four suggestions, based on human introduction, selection and recent or ancient historical events are discussed in relation to the lack of genetic variation in the Spanish crayfish stock.

Expression of thrombopoietin by umbilical vein endothelial cells.

Current data suggest that the primary source of thrombopoietin (TPO) is the liver. Extra-hepatic sites for TPO production have been demonstrated essentially through the study of the expression of TPO mRNA. In this work, we report that TPO is expressed at low levels by endothelial cells (EC) derived from the umbilical vein (HUVEC). Both TPO mRNA and the protein are expressed and the protein is functional as assessed by biological assay. Expression of TPO by HUVEC may be useful to study the regulation of the production of this cytokine and to understand the apparent specific interactions between mature megakaryocytes and EC in the bone marrow.

Dendritic cells issued in vitro from bone marrow produce PGE(2) that contributes to the immunomodulation induced by antigen-presenting cells.

Given that preliminary work has indicated that prostaglandins can play a role in modulating dendritic cell (DC) functions, we addressed the prostaglandin E(2) (PGE(2)) biosynthetic capacity of mouse DC produced in vitro from bone marrow cells. We observed production of significant amounts of PGE(2), which was reduced by at least 80% when cells were incubated in the presence of indomethacin, a COX-1 preferential inhibitor. Indeed, when tested by Western blot analysis with specific COX-1 and COX-2 antibodies, only COX-1 expression could be detected in the bone marrow (BM)-DC. For lipopolysaccharide (LPS)-treated BM-DC, inhibition of PGE(2) production by indomethacin or by NS-398 (a COX-2-selective inhibitor) used alone was less potent. After LPS treatment of BM-DC, COX-1 and COX-2 expression was potent, and inhibition of PGE(2) synthesis needed the presence of both indomethacin and NS-398. We also observed that exogenous PGE(2) diminished the expression of MHC class II molecules by BM-DC and that prostaglandin and indomethacin had antagonistic effects on cell proliferation during the mixed lymphocyte reaction using BM-DC as stimulatory cells. This assessment of PGE(2) suggests that endogenous PGE(2) produced by DC might play a role as an immunomodulating factor during the immune response. This hypothesis is sustained by the fact that IL-12 production by BM-DC is modulated by exogenous PGE(2) as well as endogenous prostaglandin, since either the addition of exogenous PGE(2) or the presence of LPS (which increases endogenous PGE(2) synthesis) decreases IL-12 production, while NS-398 (which decreases LPS-induced PGE(2) synthesis) increases IL-12 synthesis.

Quantitation of ergosterol in river sediment by liquid chromatography.

The ergosterol content of a river sediment can be used as an indicator of fungal activity. A method is developed for the extraction and determination of ergosterol in river sediment as part of a study to assess the correlation between fungal activity and biodegradation of pyrene, which is an environmental pollutant. This method is based on saponification and the liquid-liquid extraction of ergosterol by ethyl acetate. Quantitation and detection are performed isocratically by liquid chromatography on a 5-microm Hypersil C18 column with methanol-acetonitrile (80:20, v/v) as the mobile phase and detection at 282 nm. The detection limit is 50 ng/mL ergosterol, which is equivalent to 0.1 microg/g. The recovery of ergosterol at a concentration level in the range of 2 to 12 microg/mL is 91.7% +/- 3.1% without interferences. This method is applied in order to successfully quantitate the ergosterol content in a river sediment with or without a fungus supply.

DNA migration regimes in hydrodynamic chromatography and slalom chromatography: evidence for a transition.

Recently, we hypothesized that DNA separation mechanisms in slalom chromatography (SC) and packed column hydrodynamic chromatography (HDC) could be connected and simultaneously observed 'J. Chromatogr. A 886 (2000) 1'. The present paper describes the migration of various circular and linear double-stranded DNAs in a chromatographic system using a C1 stationary phase and an acetonitrile-phosphate buffer mixture as a mobile phase. The migration dependence on the flow rate for the different species showed the relative contribution of both the SC and HDC separation mechanisms. A transition between the SC and HDC regimes was demonstrated for the first time. In addition, the data were analyzed in terms of polymer migration in a dense structure. Three different behaviors were distinguished in relation to the size and the compactness of the nucleic acid.

Column efficiency and separation of DNA fragments using slalom chromatography: hydrodynamic study and fractal considerations.

Novel equations (Guillaume Y. C.; et al. Anal. Chem. 2000, 72, 853) were developed to describe the large double-stranded DNA molecule retention in slalom chromatography (SC). These equations were applied for the first time to model both the "apparent selectivity" and the resolution between two eluted DNA fragments on a chromatogram. A study of the column efficiency corroborated the fact that slalom chromatography is not based on an adsorption or equilibrium phenomenon, but can be attributed to a hydrodynamic phenomenon. Using a combination of the dynamics of DNA fragment progression in the column and fractal considerations, it was shown that the apparent selectivity depends both on the DNA fragment sizes and mobile-phase flow rate and therefore a balance between two hydrodynamic regimes. A chromatographic response function was also used to obtain the most efficient separation conditions for a mixture of DNA fragments in a minimum analysis time. The chromatographic data confirmed that in SC the flow rate can increase or maintain the separation efficiency with an associated decrease in the analysis time. This constitutes an attractive outcome in relation to the classical chromatographic separation.

A mechanism for translationally coupled mRNA turnover: interaction between the poly(A) tail and a c-fos RNA coding determinant via a protein complex.

mRNA turnover mediated by the major protein-coding-region determinant of instability (mCRD) of the c-fos proto-oncogene transcript illustrates a functional interplay between mRNA turnover and translation. We show that the function of mCRD depends on its distance from the poly(A) tail. Five mCRD-associated proteins were identified: Unr, a purine-rich RNA binding protein; PABP, a poly(A) binding protein; PAIP-1, a poly(A) binding protein interacting protein; hnRNP D, an AU-rich element binding protein; and NSAP1, an hnRNP R-like protein. These proteins form a multiprotein complex. Overexpression of these proteins stabilized mCRD-containing mRNA by impeding deadenylation. We propose that a bridging complex forms between the poly(A) tail and the mCRD and ribosome transit disrupts or reorganizes the complex, leading to rapid RNA deadenylation and decay.

Mobile-phase-viscosity dependence on DNA separation in slalom chromatography.

Slalom chromatography (SC) is an alternative chromatographic procedure for the separation of relatively large double-stranded DNA molecules and is based on a new principle. The retardation of the DNA fragments from the cleavage of the Lambda DNA by the KpnI restriction enzyme was studied using an acetonitrile-phosphate buffer as a mobile phase with various concentrations of viscosity modifier (i.e. glycerol) and a C1 column as a stationary phase. The DNA molecule retention was accurately described over the glycerol concentration range using a model previously established. It was shown that the eluent viscosity increase enhanced the slalom chromatographic capacity to separate the DNA fragments. A connection between SC and 'hydrodynamic chromatography' processes was predicted to link the two processes in a global separation mechanism based on a non-equilibrium principle.

Differential effect of interferon alpha on chronic myelogenous leukaemia and normal haematopoietic progenitors in a stromal cell co-culture context: role of the flt3 ligand.

Interferon alpha (IFN-alpha) is used to treat chronic myelogenous leukaemia (CML) patients. However, its target(s) remain(s) unknown. One possibility is that there is a differing sensitivity of the leukaemic from the normal colony-forming cell (CFC) compartments to IFN-alpha. Co-cultures of progenitors with stromal cells provide a valuable tool to dissect direct and indirect activities of IFN-alpha. In this study, we have used endothelial cells (EC) as a source of stromal cells. In co-cultures of normal progenitors with EC, IFN-alpha increased the generation of clonogenic cells, mainly via an increased production of flt3 ligand (FL) by EC. In contrast, in co-cultures of CML progenitors with EC, IFN-alpha inhibited the generation of clonogenic cells, mainly by direct inhibition on the progenitors, the up-regulation of FL production by stromal cells being unable to compensate for the direct inhibitory effects of IFN-alpha. These data provide evidence for a differential effect of IFN-alpha on the growth of CML and normal CFC cells in a stromal context and suggest that an alteration in the response of CML progenitor cells to FL is important in the explanation of this differential effect.

C60 and C70 HPLC retention reversal study using organic modifiers

A novel equation (Guillaume Y. C. et al. Anal. Chem. 1998, 70, 608) modeling the weak polar solute retention in reversed-phase liquid chromatography (RPLC) was applied to fullerene molecules C60 and C70. In RPLC, with an organic modifier (OM)/water mobile phase, the fullerene cluster solvation energies were calculated for OM = methanol, ethanol, propanol, butanol, and pentanol. An enthalpy-entropy compensation revealed that the type of interactions between fullerenes and the stationary phase was independent of both the fullerene and organic modifier structures. The energetics of OM and OM-water cluster exchange processes in the mobile phase were investigated in relation to the carbon atom number of the hydrophobic chain of the OM. Two linear correlations were found between the Gibbs free energy changes in the solvent exchange processes which confirmed that (i) a reversal elution order existed for C60 and C70 when methanol was changed into ethanol, propanol, butanol, pentanol and that (ii) the mobile phase was dominant in governing selectivity changes in nonpolar solutes.