[Early determinants of chronic obstructive pulmonary disease: Lung regeneration, a new therapeutic target?] / Déterminants précoces de la bronchopneumopathie chronique obstructive : la régénération pulmonaire, une nouvelle piste thérapeutique ?

Chronic obstructive pulmonary disease, a disease of increasing incidence, is related mainly to smoking. Although symptoms only appear at adulthood, the disease can develop from early life events. For example, bronchopulmonary dysplasia, which occurs in preterm infants, is characterized by airspace enlargement and could lead to late lung consequences. Once the lesions are established, no curative treatment is available. Stimulating lung regeneration from endogenous stem cells is therefore an exciting research domain, particularly through the activation of the mesenchymal contingent located in the lung stem cell niche.

Bacteriology of the Labrador dog gut: a cultural and genotypic approach.

AIMS: To carry out an extensive study of the microflora composition of the Labrador dog gut. METHODS AND RESULTS: Faecal specimens from four Labradors were collected and plated onto growth media designed to recover total anaerobes, bacteroides, bifidobacteria, lactobacilli, clostridia, Gram-positive cocci, total aerobes and coliforms. Morphologically different isolates were collected from all agars inoculated with faeces from one canine individual (repeated four times). A total of 157 out of 171 isolates were identified using 16S rRNA gene sequencing. Sequence analysis showed that agar selectivity was poor, especially when bacteroides and Gram-positive cocci were the targets. Bifidobacteria were not detected in any of the samples analysed, indicating their presence at low or negligible levels. The gene sequences of many of the isolates (n=45, representing 29% of the total) did not correlate with known species in the Ribosomal Database Project and EMBL databases, suggesting the presence of novel gut diversity. CONCLUSIONS: Traditional culture methods fail to reflect the bacterial diversity present in Labrador dog faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown the value of molecular-based methodologies for determining bacterial profiles in the Labrador dog gut microbiota, but has also exposed the limitations of purportedly selective agars.

Development of a technique for the in vivo assessment of flatulence in dogs.

OBJECTIVE: To develop a noninvasive method for the in vivo assessment of flatulence in dogs. ANIMALS: 8 adult dogs. PROCEDURE: Rectal gases were collected via a perforated tube held close to each dog's anus and attached to a monitoring pump fitted with a sensor that recorded hydrogen sulfide concentrations every 20 seconds. Patterns of flatulence were monitored for 14 hours after feeding on 4 days, and within- and between-dog variation was assessed over 4 hours on 4 consecutive days. Rate of hydrogen sulfide production (flatulence index) and frequency and number of emissions were evaluated as potential indicators of flatus characteristics. An odor judge assigned an odor rating to each flatulence episode, and the relationship between that rating and hydrogen sulfide concentration was determined. RESULTS: Flatulence patterns varied within and between dogs. Variation was most pronounced for flatulence index; mean coefficients of variance within dogs over time and between dogs on each day were 75 and 103%, respectively. Flatus with hydrogen sulfide concentrations > 1 parts per million could be detected by the odor judge, and severity of malodor was highly correlated with hydrogen sulfide concentration. Odor ratings were accurately predicted by use of the equation 1.51 X hydrogen sulfide concentration(0.28). CONCLUSIONS AND CLINICAL RELEVANCE: The technique described in this report appears to provide sensitive, reliable, and relevant data and will enable further studies of the factors that influence flatulence in dogs. Use of this technique also has the potential to aid in investigations of colonic physiology and pathology.

Administration of charcoal, Yucca schidigera, and zinc acetate to reduce malodorous flatulence in dogs.

OBJECTIVE: To determine whether feeding activated charcoal, Yucca schidigera, and zinc acetate would ameliorate the frequency and odor characteristics of flatulence in dogs. DESIGN: In vitro screening of active agents followed by a randomized controlled trial. ANIMALS: 8 adult dogs. PROCEDURE: A fecal fermentation system was used to assess the effects of activated charcoal, Yucca schidigera, and zinc acetate alone and in combination on total gas production and production of hydrogen sulfide, the primary determinant of flatus malodor in dogs. All 3 agents were subsequently incorporated into edible treats that were fed 30 minutes after the dogs ate their daily rations, and the number, frequency, and odor characteristics of flatulence were measured for 5 hours, using a device that sampled rectal gases and monitored hydrogen sulfide concentrations. RESULT: Total gas production and number and frequency of flatulence episodes were unaffected by any of the agents. Production of hydrogen sulfide in vitro was significantly reduced by charcoal, Yucca schidigera, and zinc acetate by 71, 38, and 58%, respectively, and was reduced by 86% by the combination of the 3 agents. Consumption of the 3 agents was associated with a significant decrease (86%) in the percentage of flatulence episodes with bad or unbearable odor and a proportional increase in the percentage of episodes of no or only slightly noticeable odor. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that activated charcoal, Yucca schidigera, and zinc acetate reduce malodor of flatus in dogs by altering the production or availability of hydrogen sulfide in the large intestine.

Carnosine-like immunoreactivity in the central nervous system of rats during postnatal development.

In the nervous system of adult rodents, the aminoacylhistidine dipeptides (carnosine and/or homocarnosine) have been shown to be expressed in three main populations of cells: the mature olfactory receptor neurons, a subset of glial cells, and the neuroblasts of the rostral migratory stream. The current study analyzed the distribution of these dipeptides during postnatal development within the rat brain and spinal cord focusing on their pattern of appearance in the glial cells. Double staining methods using antibodies against carnosine and some markers specific for immature (vimentin) and mature (glial fibrillary acidic protein and Rip) glial cell types were used. Glial immunostaining for the aminoacylhistidine dipeptides appears starting from postnatal day 6 and reaches the final distribution in 3-week-old animals. The occurrence of carnosine-like immunoreactivity in astrocytes lags behind that in oligodendrocytes suggesting that, as previously demonstrated by in vitro studies, oligodendrocytes are also able to synthesize carnosine and/or homocarnosine in vivo. Furthermore, the spatiotemporal patterns observed support the hypothesis that the production of these dipeptides coincides with the final stages of glia differentiation. In addition, a strong carnosine-like immunoreactivity is transiently seen in a small population of cells localized in the hypothalamus and in the subfornical organ from birth to postnatal day 21. In these cells, carnosine-like immunoreactivity was not colocalized with any of the glial specific markers used. Moreover, no evidence for colocalization of carnosine and gonadotropin-releasing hormone (GnRH) has been observed.

Structure-function relations of variant and fragment nisins studied with model membrane systems.

Nisin, a 34 residue lantibiotic produced by strains of Lactococcus lactis subsp. lactis, exerts antimicrobial activity against Gram-positive bacteria at the cytoplasmic membrane. The structural aspects of nisin which facilitate membrane interaction and permeabilization have been investigated in planar lipid bilayers and liposomes with proteolytic fragments and site-directed variants. N-Terminal nisin fragments N1-12 and N1-20 had little effect on phospholipid mobility, on macroscopic electrical conductance, or on calcein release from liposomes. By contrast, the I30W nisin A variant induced a time-dependent reduction in lipid mobility, indicative of nisin-membrane surface interactions, as well as a decline in membrane capacitance, rise in conductance, and calcein release from liposomes. In these respects I30W nisin A is similar to native nisin. Charge substitutions were also engineered to generate K12L and H27K nisin A variants, both of which were similar to I30W nisin A with respect to an overall reduction in phospholipid mobility. While the K12L nisin A variant elicited a higher increase in membrane capacitance and electrical conductance than I30W nisin A, the H27K nisin A variant elicited weaker effects. These results point to a substantial role for intramembrane charged residues in controlling ion flow through nisin-doped membranes. Native nisin and variants elicit an enhanced release of calcein from liposomes composed of the negatively-charged phospholipids cardiolipin and phosphatidylserine, compared with phospholipid bearing no net charge, suggesting that an electrostatic attraction encourages the initial nisin-membrane association. The results are discussed in the context of other recently proposed models for nisin action.

Molecular analysis of the regulation of nisin immunity.

The genetic determinants controlling immunity to nisin are coordinately regulated, along with biosynthesis genes, in response to an environmental signal, nisin or a nisin analogue. The nisR gene product, the putative response regulator of nisin biosynthesis, was found to be a vital component of this induction mechanism. This protein forms part of a two-component regulatory system which controls the expression of genes involved in nisin immunity and biosynthesis. Analysis of the structural requirements of the external signal, using nisin fragments and engineered nisin variants, indicated that the 12 amino-terminal residues of the molecule are a minimum requirement for induction, with an intact ring A being an essential component. Changes throughout the molecule also affected its induction capacity. The production of certain variant nisins by engineered lactococcal strains is reduced in parallel with the strains' immunity to nisin. This can be attributed to inefficient induction by the variant molecule. Treating growing cultures with nisin restored full immunity and maximized the yields of nisin variants by the producer strains.

Interaction of the lantibiotic nisin with membranes revealed by fluorescence quenching of an introduced tryptophan.

Nisin is a lantibiotic produced by strains of Lactococcus lactis subsp. lactis. The target for nisin action is the cytoplasmic membrane of gram-positive bacteria. To aid understanding of its mode of action, the interaction of nisin with vesicles of differing phospholipid composition were investigated by fluorescence techniques, using a variant of nisin in which the isoleucine at position 30 was replaced by a tryptophan residue. Activity of the site-directed variant containing tryptophan was established to be similar to that of the wild-type peptide. Fluorescence experiments showed a blue shift of the emission wavelength maximum in the presence of lipid vesicles, indicating that the tryptophan residue enters a more hydrophobic environment. Quenching experiments with aqueous and membrane-restricted quenchers (iodide and spin-labelled lipids, respectively) both confirmed a non-aqueous environment for the Trp30 residue, and implied that the residue resides between 0.36 nm and 0.52 nm from the centre of the membrane, depending on the lipid identity. The results clearly demonstrate that nisin interacts strongly with the hydrophobic phase of lipid vesicles. This interaction is stronger in the presence of negatively charged lipids suggesting their importance in the functional interaction of nisin with membranes.

Interaction of nisin with planar lipid bilayers monitored by fluorescence recovery after photobleaching.

Nisin, a prominent member of the lantibiotic family of antimicrobial agents, has wide application as a food preservative despite poor understanding of its mode of action. Fluorescence recovery after photobleaching has been used with planar lipid bilayers as a model membrane system to examine how nisin might interact with the surface of bacterial cells. Nisin associates with planar lipid bilayers in the absence of an applied membrane potential causing an array of effects consistent with adsorption of nisin onto the membrane surface which involves inhibition of the lateral diffusion and fluorescence of the lipid probe N-(7--1,2,3-benzoxadiazol-4-yl) phosphatidylethanolamine (NBD-PE) and a reduction of the capacitance of the bilayer. Nisin adsorption is dependent on phospholipid composition. In the presence of dioleoylphosphatidylcholine (PC): cardiolipin (CL) 4:1, the rate of lateral mobility of phospholipid is reduced to 61% of the control level which decreases to a value of 46% when CL is replaced by 1-palmitoyl-2-oleoylphosphatidylserine (PS). These effects on bilayer parameters are transient, and with time the values return to near original levels. High electrical conductivity is observed on application of a voltage ramp suggesting that insertion into the membrane follows surface association. Results have been interpreted in terms of a model in which nisin initially binds to the surface of the membrane causing a modulation of bilayer properties.