Spectral and polarization sensitivity of juvenile Atlantic salmon (Salmo salar): phylogenetic considerations.

We were interested in comparing the characteristics of polarization sensitivity in Atlantic salmon to those in Pacific salmon. Here we show that the common ancestor to the clade containing Salmo salar, Oncorhynchus mykiss, O. nerka, O. clarkii and Salvelinus fontinalis has the trait of ultraviolet polarization sensitivity. We examined spectral and polarization sensitivity of juvenile Atlantic salmon (Salmo salar) using both optic nerve compound action potential (CAP) and electroretinogram (ERG) recordings. Our experiments employed photic manipulation to adjust the sensitivity of the four cone mechanisms of Atlantic salmon. A spectrally broad background was used to ensure a contribution of all cone mechanisms to both spectral and polarization sensitivity. Chromatic adaptation was used to isolate the sensitivity of each of the four cone mechanisms for both spectral and polarization sensitivity. Under spectrally broad conditions, UV sensitive (UVS), mid wavelength sensitive (MWS) and long wavelength sensitive (LWS) cone mechanisms contributed to polarization sensitivity. CAP recordings produced the typical 'W' shaped polarization sensitivity curve reflecting two active polarization detectors with peaks at e-vector orientations of 0 deg, 90 deg and 180 deg, and troughs at 30 deg and 150 deg. ERG recordings produced a four-peaked polarization sensitivity curve reflecting two active polarization detectors and negative feedback activity, with peaks at e-vectors 0 deg, 45 deg, 90 deg, 135 deg and 180 deg, and troughs at 30 deg, 60 deg, 120 deg and 150 deg. Polarization-sensitivity measurements of isolated cone mechanisms revealed two orthogonal polarization detector mechanisms in Atlantic salmon, identical to that found in rainbow trout and other Pacific salmonid fishes. Moreover, under spectrally broad background conditions, CAP and ERG polarization sensitivity of Atlantic salmon did not differ significantly from that reported in Pacific salmonids.

Development and validation of a capillary electrophoresis assay for the determination of 3,4-diaminopyridine and 4-aminopyridine including related substances.

A capillary electrophoresis (CE) assay has been developed for the quantitation and determination of the impurity profile of the potassium channel blockers 3,4-diaminopyridine and 4-aminopyridine. The compounds were separated from related substances using a capillary of 30 cm effective length, a 50 mM phosphate buffer, pH 2.5 and an applied voltage of 25 kV. The assay was validated with respect to specificity, linearity, range, limits of quantitation and detection, precision and robustness. The method allows the detection and quantitation of impurities at the 0.05% level. The feasibility of the assay was demonstrated by analyzing a commercial sample of 3,4-diaminopyridine. All known related substances could be detected in this sample with the present CE method.

Influence of the structure of cyclodextrins and amino acid sequence of dipeptides and tripeptides on the pH-dependent reversal of the migration order in capillary electrophoresis.

The pH-dependent reversal of the migration order in cyclodextrin (CD)-mediated capillary electrophoresis (CE) enantioseparations of dipeptides and tripeptides has been studied between pH 2.5 and 3.5 using beta-CD and several of its neutral derivatives. The occurrence of the phenomenon depended on both the structure of the CD and the amino acid composition and sequence of the peptides. While an inversion was observed for several peptides when native beta-CD, dimethyl-beta-cyclodextrin or trimethyl-beta-cyclodextrin were added to the run buffer, no alteration of the order occurred in the presence of permethyl-beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin. Most peptides that displayed a change of the migration behavior upon increasing the buffer pH contained Phe at the C-terminus. An ionizable carboxyl group in the peptide structure was a prerequisite. As seen with other uncommon migration effects in CE, the pH-dependent reversal of the migration order occurred in the pH region of the pKa values of the peptide carboxyl functions.

Method development and validation for the chiral separation of peptides in the presence of cyclodextrins using capillary electrophoresis and experimental design.

The present study describes the application of statistical experimental design to the optimization of enantioselective separations of peptides in capillary electrophoresis in order to obtain optimal operating conditions for routine work. Hydroxypropyl-beta-cyclodextrin was used as chiral selector and Ala-PheOMe as model peptide. The experiments were performed according to a face centered cube response surface experimental design for obtaining information how the factors such as concentration of the chiral selector, pH, buffer concentration and voltage affected the two response goals, resolution and analysis time. In order to achieve the simultaneous optimization of these two major electrophoretic performance goals for efficient and fast separation, the Derringer desirability functions were tested. While in the predefined experiments the analysis time for baseline separation was 25 min the desirability functions proposed a CE method, which diminished the analysis time and permitted the complete separation of the peptide enantiomers within 9 min.

Study of the cis-trans isomerization of enalapril by reversed-phase liquid chromatography.

Enalapril is a dipeptidic angiotensin converting enzyme inhibitor. It exists as a mixture of two conformers in solution with respect to the peptide bond involving the proline amino group. The RPLC of such products may yield peak splitting or multiple peaks as a result of the slow kinetics of the conformation change. In this study, the influence of the flow-rate, pH, temperature, organic modifier and counter ion on the peak shape and the separation of the cis and trans conformers are examined qualitatively by HPLC. It appears that decrease of relaxation time for isomerization with concomitant improvement in peak shape is favoured by a decrease in pH and flow-rate, increase of temperature, choice of organic solvent (nature, amount) and cationic counter ion concentration in the mobile phase. The elution order of the isomers was dependent on the nature of the organic modifier whereas the separation selectivity was improved by an increase of pH or the addition of a negatively charged counter ion. In addition, an NMR investigation on enalapril is described.

Reversed-phase liquid chromatography of lisinopril conformers.

The effect of flow rate, temperature, pH, organic solvent and counter ion on peak shape and separation of the cis and trans conformers of lisinopril are investigated by HPLC. It was demonstrated that complete separation of the two isomers can be achieved at low temperature at either neutral or low pH together with appropriate type and concentration of organic solvent, whereas the elution of lisinopril as a single peak is favored by a decrease of flow rate, elevated temperature, choice of organic solvent (type and amount) and the use of an appropriate counter ion concentration.

Validation of a CE assay for the analysis of isomeric aminopyridines and diaminopyridines.

A capillary electrophoresis assay for the analysis of aminopyridines and diaminopyridines has been developed and validated. The compounds were separated using a 100 mM sodium acetate buffer at pH 5.15, an applied voltage of 20 kV and a capillary of 60 cm effective length, N-(1-naphthyl)ethylenediamine was used as internal standard to compensate for injection errors and minor fluctuations of the migration times. The detection wavelength was set at 240 nm and not optimized for a specific derivative. The assay was validated with respect to specificity, linearity, range, limit of quantitation and detection, precision, and robustness. Within certain limits, the assay also allowed the detection and determination of the other aminopyridine derivatives at the 0.1% level as demonstrated for 3,4-diaminopyridine.

RP-HPLC and NMR study of cis-trans isomerization of enalaprilat.

The angiotensin converting enzyme inhibitor, enalaprilat can exist in solution as cis and trans conformers which interconvert around the amide bond at room temperature. A HPLC with UV detection was performed to study the influence of various chromatographic operational conditions on both rotamers separation and elution of enalaprilat as a single peak. In addition nuclear overhauser enhancement difference was used for the identification of the conformers. The isomer ratio integrated from the obtained 1H NMR result were 71.5:28.5 and 76:24 at 298 and 279 K, respectively where the trans was the major form.

Preliminary study of organochlorine residues in human milk and cord blood.

A large collection of human milk samples and cord blood from various hospitals of Tunis is studied for organochlorine residues. Some results obtained by ECD-GC are given.

Migrating locusts can detect polarized reflections to avoid flying over the sea.

The desert locust Schistocerca gregaria is a well known migrating insect, travelling long distances in swarms containing millions of individuals. During November 2004, such a locust swarm reached the northern coast of the Gulf of Aqaba, coming from the Sinai desert towards the southeast. Upon reaching the coast, they avoided flying over the water, and instead flew north along the coast. Only after passing the tip of the gulf did they turn east again. Experiments with tethered locusts showed that they avoided flying over a light-reflecting mirror, and when given a choice of a non-polarizing reflecting surface and a surface that reflected linearly polarized light, they preferred to fly over the former. Our results suggest that locusts can detect the polarized reflections of bodies of water and avoid crossing them; at least when flying at low altitudes, they can therefore avoid flying over these dangerous areas.

Separation of dipeptide and tripeptide enantiomers in capillary electrophoresis using carboxymethyl-beta-cyclodextrin and succinyl-beta-cyclodextrin: influence of the amino acid sequence, nature of the cyclodextrin and pH.

The separation of the LL and DD enantiomers of dipeptides and tripeptides using cyclodextrins (CDs) containing carboxyl groups was investigated with respect to the amino acid sequence of the peptides, the nature of the cyclodextrin and the buffer pH. Compared to succinyl-beta-cyclodextrin, carboxymethyl-beta-cyclodextrin was the more universal CD for enantioseparations. Reversal of the enantiomer migration order upon increasing the buffer pH from 2.5 to 3.5 was observed in some cases. As shown for Phe-Phe reversal of the migration order also occurred between pH 3.5 and 5.3. Complexation constants and complex mobilities change with pH as both, the charge of the peptide and the charge of the CD vary depending on the pH. The complexation constants and complex mobilities of the dipeptides Ala-Phe and Phe-Phe were determined in order to explain the enantiomer migration behavior in the pH range 2.5-5.3. While the complexation constants determined the migration order at pH 2.5 and 5.3, complex mobility had a strong influence around pH 3.5-3.8.

Methylation of DNA in NaCl-adapted cells of potato.

Salt-adapted and control cells of the cultivated potato, Solanum tuberosum cultivar Russet Burbank, untreated or treated with 5-azacytidine (an inhibitor of DNA methylation), were compared with respect to: a) % of cytosine methylation in total nuclear DNA, as determined by HPLC; b) fresh and dry weight. Adapted and control cells were compared also with respect to % of cytosine methylation in DNA, which was purified from DNaseI-partially-digested chromatin and size fractionated by electrophoresis in agarose gels. The growth (represented by dry weight) of the NaCl-adapted cells in saline medium lacking 5-azacytidine was similar to that of control cells in standard medium. The adaptation of the cells was correlated with some increase (+16%) of methylation in total DNA and with a much greater increase in the lower molecular weight DNA fractions which were obtained from the presumably more active chromatin. As expected, the treatment of the cells with the methylation inhibitor induced a decrease in the level of methylation. The decrease of methylation, however, was much greater in the adapted cells, whose dry weight, unlike in the control, was not affected by this treatment.

[High resolution gas phase chromatography-mass spectrometry of polychlorinated biphenyl congener residues in samples of biologic origin]. / Chromatographie en phase gazeuse haute résolution-spectrométrie de masse des congénères de biphényles polychlorés dans quelques échantillons d'origine biologique.

A study is performed on polychlorinated biphenyl (PCB) congener residues in samples of human blood and milk as well as in falcon and pigeon eggs. Most of the PCB congeners found in these biological samples were quantified by high-resolution gas chromatography (HRGC). A PCB technical mixture--namely, DP6 (Phenochlor)--was used for the calibration as its composition was previously determined by HRGC-mass spectrometry. The usefulness of such a congener analysis is outlined. It is the first time to the best of our knowledge that a Phenochlor mixture is used for standardization.

pH-dependence of complexion constants and complex mobility in capillary electrophoresis separations of dipeptide enantiomers.

The chiral separation of the LL- and DD-enantiomers of the dipeptides Ala-Tyr, Phe-Phe, and Asp-PheOMe has been investigated at pH 2.5 and pH 3.5 using beta-cyclodextrin (beta-CD), heptakis-(2,6-di-O-methyl)-beta-cyclodextrin, and heptakis-(2,3,6-tri-O-methyl)-beta-cyclodextrin as chiral selectors. According to electrospray mass spectrometry, heptakis-(2,6-di-O-methyl)-beta-cyclodextrin was a mixture of six isomers. Reversal of the enantiomer migration order upon increasing the buffer pH from 2.5 to 3.5 was observed for all peptides with beta-cyclodextrin, for Ala-Tyr and Phe-Phe in the presence of heptakis-(2,3,6-tri-O-methyl)-beta-cyclodextrin, and for Ala-Tyr using heptakis-(2,6-di-O-methyl)-beta-cyclodextrin. The migration behavior could be explained on the basis of the complexation constants and the mobilities of the peptide-cyclodextrin complexes. Both, the binding constants and complex mobilities decreased with increasing pH as the overall-charge of the peptides decreased. While the complexation constants primarily determined the migration order at pH 2.5, complex mobility dominated in most cases at pH 3.5.

Interleukin-2 activity of colonic lamina propria mononuclear cells in a rat model of experimental colitis.

BACKGROUND: Altered interleukin 2 (IL-2) production has been implicated in the pathogenesis of inflammatory bowel diseases. METHODS: The temporal relationship between IL-2, prostaglandin E2 (PGE2) production, and mucosal injury was evaluated by isolated colonic lamina propria mononuclear cells (LPMC), using the trinitrobenzene sulfonic acid model of rat colitis. RESULTS: Spontaneous LPMC IL-2 activity was significantly increased in chronic (5 weeks) but not acute (5 days) or resolved colitis groups. IL-2 activity after concanavalin A activation was highest in the groups with resolved and chronic colitis. PGE2 production was significantly increased in LPMC cultures in acute or chronic colitis as well as the ethanol control groups but not the resolved colitis group. The addition of indomethacin to LPMC cultures decreased PGE2 levels in all groups, whereas IL-2 activity increased only for the chronic and resolved colitis groups. No correlation was found between PGE2 and IL-2 production by LPMC. CONCLUSIONS: In this experimental model, LPMC IL-2 production varied according to the severity and duration of the inflammation. Increased PGE2 production does not appear to be responsible for the IL-2 alterations in colitis.

Hexachlorobenzene in Tunisian mothers' milk, cord blood and foodstuffs.

This paper reports some preliminary results concerning hexachlorobenzene (HCB) contamination of Tunisian human milk, human cord and adult blood and foodstuffs (cows' milk, meat, eggs). The analyses were performed by gas chromatography with electron-capture detection.

Interleukin-2 production in pediatric inflammatory bowel disease: evidence for dissimilar mononuclear cell function in Crohn's disease and ulcerative colitis.

Crohn's disease (CD) and ulcerative colitis (UC) represent clinically distinct chronic inflammatory bowel diseases (IBD) of unknown etiology. Although the mucosal immune system is implicated in their pathogenesis, immunological differences between the two disorders are not well defined. The aim of this study was to compare in vitro mucosal T-lymphocyte function in CD and UC. Peripheral blood mononuclear cell interleukin-2 (IL-2) production was similar in pediatric IBD and control patients under unstimulated conditions, but was significantly increased in response to phytohemagglutinin (PHA) stimulation for the UC group. Lamina propria mononuclear cells (LPMNC) isolated from colonic resections in IBD patients had significantly lower spontaneous proliferation and IL-2 production in vitro than did LPMNC of control patients. In contrast, significantly greater IL-2 production was detected when the LPMNC of CD patients were cultured with PHA, in comparison with those of UC or control patients. When indomethacin, a prostaglandin synthetase inhibitor, was added to the cultures, significantly increased IL-2 secretion was observed for CD LPMNC, but not for UC cultures, under both stimulated and unstimulated conditions. These findings demonstrate abnormal LPMNC IL-2 production in IBD. Furthermore, our data suggest that inhibition of the prostaglandin synthetase pathway upregulates IL-2 production by LPMNC in CD. These results support the hypothesis that immunoregulatory mechanisms controlling IL-2 production differ between CD and UC.