Two optimized combination assays to examine apoptosis pathways in clinical samples.

BACKGROUND: A consequence of a number of diseases is an alteration in apoptosis. Currently, there is no single assay that measures the main stages of apoptosis, requiring that multiple assays be performed. This hinders studies on clinical samples that have limited cell numbers. Our objective was to combine and optimize assays that target specific stages of apoptosis for use in a typical clinical blood sample. METHODS: Two flow cytometric assays were developed for use on peripheral blood mononuclear cells (PBMC) collected in two 8-ml tubes from a single draw. One measures caspase-12 activity, the level of active caspase-3 and DNA fragmentation. The second assesses depolarization of the mitochondria and phosphatidylserine externalization. Cell populations present within the samples were determined by flow cytometry. Apoptosis was validated by ELISA. RESULTS: Each assay was optimized for use with cell numbers and sample volumes typical of clinical blood samples. Each combination assay effectively distinguished apoptotic from nonapoptotic blood cells. CONCLUSIONS: This combined optimized method comprised of two independent assays makes it possible to assay the major pathways of apoptosis in addition to determining the blood cell subsets that are affected.     

Biochemical evidence that berberine bridge enzyme belongs to a novel family of flavoproteins containing a bi-covalently attached FAD cofactor

Berberine bridge enzyme (BBE) is involved in the transformation of (S)-reticuline to (S)-scoulerine in benzophenanthridine alkaloid biosynthesis of plants. In this report, we describe the high level expression of BBE encoded by the gene from Eschscholzia californica (California poppy) in the methylotrophic yeast Pichia pastoris employing the secretory pathway of the host organism. Using a two-step chromatographic purification protocol, 120 mg of BBE could be obtained from 1 liter of fermentation culture. The purified protein exhibits a turnover number for substrate conversion of 8.2 s(-1). The recombinant enzyme is glycosylated and carries a covalently attached FAD cofactor. In addition to the previously known covalent attachment of the 8alpha-position of the flavin ring system to a histidine (His-104), we could also demonstrate that a covalent linkage between the 6-position and a thiol group of a cysteine residue (Cys-166) is present in BBE. The major evidence for the occurrence of a bi-covalently attached FAD cofactor is provided by N-terminal amino acid sequencing and mass spectrometric analysis of the isolated flavin-containing peptide. Furthermore, it could be shown that anaerobic photoirradiation leads to cleavage of the linkage between the 6-cysteinyl group yielding 6-mercaptoflavin and a peptide with the cysteine residue replaced by alanine due to breakage of the C-S bond. Overall, BBE is shown to exhibit typical flavoprotein oxidase properties as exemplified by the occurrence of an anionic flavin semiquinone species and formation of a flavin N(5)-sulfite adduct.     

Optimization of a solid phase extraction procedure for prostaglandin E2, F2 alpha and their tissue metabolites

The primary prostaglandins PGE(2) and PGF(2 alpha) are metabolized in tissues by a series of enzymatic and non-enzymatic reactions. To measure metabolic rates and individual reaction rates it is necessary to extract the parent prostaglandins and metabolites before the separation and quantification of each compound is achieved. Here we have established and optimized a solid phase extraction (SPE) procedure to recover PGE(2), PGF(2 alpha) and their six enzymatic and non-enzymatic tissue metabolites from aqueous solutions including urine, plasma and tissue homogenate. We have used octadecyl-bonded silica gel as the stationary phase and methanol-water mixtures as binary mobile phases. The volumes and concentrations of the washing and elution solutions were optimized individually for each PG. Recoveries of all PG standards were quantitative except for PGEM, which was recovered at 80% efficiency. Biological matrix components interfered with the extraction in a PG- and matrix-specific fashion. Inclusion of 1% formic acid in the loading mixture raised recoveries from urine, plasma and tissue homogenate to >or=90%. This SPE method is the first that has been optimized by systematic elution studies for PGE(2), PGF(2 alpha) and the complement of their tissue metabolites. The procedure is simple, robust and can serve as an effective pre-purification step before downstream separation and quantification of each tissue metabolite of PGE(2) and PGF(2 alpha) from complex biological matrices.     

A review of dietary stilbenes: sources and bioavailability

Stilbenes are a class of phenolic metabolites found in various edible plants, such as grapevine, berries and peanuts. Their bioactivitiy and their potential benefits for human health have been the subject of several studies. Among all identified stilbenes, resveratrol has been particularly studied and results from literature showed that it presents several biological activities, including antioxidant, anti-inflammatory and antiproliferative effects. Likewise, some researches focused on other stilbenes and highlighted similar biological activity for those compounds. However, stilbenes present a high diversity in their phenolic structures (various chemical substituents and polymerization) which is a determining factor of their absorption and metabolism rates. Consequently, this could affect the effectiveness of stilbenes in vivo. In this context, an evaluation of the bioavailability and metabolism of stilbenes is necessary to move forward with pharmacological and clinical studies. Hence, this review aims to present recently obtained data and results concerning stilbenes sources and bioavailability, as a contribution to the valorization of the role of dietary stilbenes in the human diet.     

Stress “Deafness” Reveals Absence of Lexical Marking of Stress or Tone in the Adult Grammar

A Sequence Recall Task with disyllabic stimuli contrasting either for the location of prosodic prominence or for the medial consonant was administered to 150 subjects equally divided over five language groups. Scores showed a significant interaction between type of contrast and language group, such that groups did not differ on their performance on the consonant contrast, while two language groups, Dutch and Japanese, significantly outperformed the three other language groups (French, Indonesian and Persian) on the prosodic contrast. Since only Dutch and Japanese words have unpredictable stress or accent locations, the results are interpreted to mean that stress “deafness” is a property of speakers of languages without lexical stress or tone markings, as opposed to the presence of stress or accent contrasts in phrasal (post-lexical) constructions. Moreover, the degree of transparency between the locations of stress/tone and word boundaries did not appear to affect our results, despite earlier claims that this should have an effect. This finding is of significance for speech processing, language acquisition and phonological theory.     

Variation in eggshell traits between geographically distant populations of pied flycatchers Ficedula hypoleuca

The expression and impact of maternal effects may vary greatly between populations and environments. However, little is known about large‐scale geographical patterns of variation in maternal deposition to eggs. In birds, as in other oviparous animals, the outermost maternal component of an egg is the shell, which protects the embryo, provides essential mineral resources and allows its interaction with the environment in the form of gas exchange. In this study, we explored variation of eggshell traits (mass, thickness, pore density and pigmentation) across 15 pied flycatcher populations at a large geographic scale. We found significant between‐population variation in all eggshell traits, except in pore density, suggesting spatial variation in their adaptive benefits or in the females’ physiological limitations during egg laying. Between‐ population variation in shell structure was not due to geographic location (latitude and longitude) or habitat type. However, eggshells were thicker in populations that experienced higher ambient temperature during egg laying. This could be a result of maternal resource allocation to the shell being constrained under low temperatures or of an adaptation to reduce egg water loss under high temperatures. We also found that eggshell colour intensity was positively associated with biliverdin pigment concentration, shell thickness and pore density. To conclude, our findings reveal large‐ scale between‐population variation of eggshell traits, although we found little environmental dependency in their expression. Our findings call for further studies that explore other environmental factors (e.g. calcium availability and pollution levels) and social factors like sexual selection intensity that may account for differences in shell structure between populations.     

Inter-locality variation in reproductive success of the citril finch Serinus citrinella

Habitat quality is generally thought to affect breeding success. We tested this effect comparing differences in clutch size and reproductive success between citril finch Serinus citrinella sub-populations closely located (<5 km) but differing in habitat quality, within the Port del Comte mountain, in the Catalonian Pre-Pyrenees. We found that birds in the low quality area (Bofia) showed significantly lower hatching, breeding and nesting success than finches in the high quality area (Vansa). These differences in reproductive success fit well with recently found differences in citril finch body mass, fat score, diet, survival rate and speed of moult between these two localities.