Variability in gut mucosal secretory IgA in mice along a working day.

OBJECTIVE: To assess the variability of secretory immunoglobulin A (S-IgA) in the lumen and feces of mice along a working day. RESULTS: Mice were maintained under a 12 h light-dark cycle, light period starting at 8 AM. S-IgA was determined in feces and intestinal content (after one or three washes) at three points along the day: at the beginning, in the middle and at the end of the light period (ELP). Significant reduction in the content of S-IgA in the small intestine fluid and in feces was observed at the end of the light cycle, which coincides with the end of a regular working day (8 PM) in any given animal facility. It was also observed that three washes of the small intestine were more effective than one flush to recover a significant higher amount of S-IgA, with the smallest coefficient of variation observed by the ELP. A smaller CV would imply a reduced number of animals needed to achieve the same meaningful results. The results may be useful when designing animal trials for the selection of probiotic candidates based on their capacity of activating S-IgA, since it would imply a more rational use of experimental animals.

Exopolysaccharide from Lactobacillus fermentum Lf2 and its functional characterization as a yogurt additive.

Lactobacillus fermentum Lf2 is a strain which is able to produce high levels (approximately 1 g/l) of crude exopolysaccharide (EPS) when it is grown in optimised conditions. The aim of this work was to characterize the functional aspects of this EPS extract, focusing on its application as a dairy food additive. Our findings are consistent with an EPS extract that acts as moderate immunomodulator, modifying s-IgA and IL-6 levels in the small intestine when added to yogurt and milk, respectively. Furthermore, this EPS extract, in a dose feasible to use as a food additive, provides protection against Salmonella infection in a murine model, thus representing a mode of action to elicit positive health benefits. Besides, it contributes to the rheological characteristics of yogurt, and could function as a food additive with both technological and functional roles, making possible the production of a new functional yogurt with improved texture.

Kinetic analysis of site-specific photoaptamer-protein cross-linking.

ssDNA oligonucleotides containing bromodeoxyuridine, BrdU-photoaptamers, are rapidly emerging as specific protein capture reagents in protein microarray technologies. A mathematical model for the kinetic analysis of photoaptamer-protein photocross-linking reactions is presented. The model is based on specific aptamer/protein binding followed by laser excitation that can lead to either covalent cross-linking of the photoaptamer and protein in the complex or irreversible photodamage to the aptamer. Two distinct kinetic regimes, (1) frozen and (2) rapid equilibrium, are developed analytically to model binding kinetics between laser pulses. The models are used to characterize the photocross-linking between three photoaptamers and their cognate protein targets; photoaptamers 0650 and 0615 cross-link human basic fibroblast growth factor and 0518 cross-links HIV MN envelope glycoprotein. Data for cross-linking reaction yields as a function of both laser energy dose and target protein concentration are analyzed for affinity constants and cross-link reaction rates. The binding dissociation constants derived from the cross-linking data are in good accord with independent measurements; the rapid equilibrium model appears to produce results more consistent with the experimental observations, although there is significant overlap between the two models for most conditions explored here. The rate of photodamage for 0615 and 0518 is 3.5 and 2.5 times that of the specific cross-link, giving low maximum reaction yields of approximately 20% and approximately 30%, whereas 0650 cross-links with a rate over five times higher than its photodamage rate and has a maximum reaction yield exceeding 80%. Quantum yields for the three systems are estimated from the data; photoaptamer 0650 has a reasonably high quantum yield of approximately 0.2 for protein cross-linking, while 0518 and 0615 have quantum yields of 0.07 and 0.02. The work presented here provides a useful set of metrics that allow for refinement of photoaptamer properties.