Comparison of faecal Lactobacillus populations in experimental animals from different breeding facilities and possible consequences for probiotic studies

AIMS: The effect of probiotic lactobacilli is likely dependent on the indigenous Lactobacillus strains in the intestinal tract. Since a substantial number of probiotic studies is performed in rodents, we compared the Lactobacillus strains of different rat and mouse populations in three animal facilities. METHODS AND RESULTS: SDS-PAGE and 16S rDNA analysis of cultured faecal lactobacilli revealed that different Lactobacillus strains were detected in genetically similar Wistar rats bred at different locations. Further, within the same animal facility host genetics did not affect the types of the predominant lactobacilli strains. CONCLUSIONS: Our results show that the environmental background of laboratory animals rather than host genetics determines the indigenous Lactobacillus strains that are found. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings underline the importance of microflora analysis in probiotic studies.     

Purification and characterization of thermophilin T, a novel bacteriocin produced by Streptococcus thermophilus ACA-DC 0040

ACA-DC 0040 produced an antimicrobial agent, which was named thermophilin T, active against several lactic acid bacteria strains of different species and food spoilage bacteria, such as Clostridium sporogenes C22/10 and Cl. tyrobutyricum NCDO-1754. The crude antimicrobial compound is sensitive to proteolytic enzymes and α-amylase, heat-stable (100 °C for 30 min), resistant to pH exposure at pH 1–12 and demonstrates a bactericidal mode of action against the sensitive strain Lactococcus cremoris CNRZ-117. The production of bacteriocin was optimized approximately 10-fold in an aerobic fermenter held at constant pH 5·8 and 6·2. Ultrafiltration experiments with culture supernatant fluids containing the bacteriocin, and further estimation of molecular weight with gel filtration chromatography, revealed that bacteriocin in the native form has a molecular weight in excess of 300 kDa. SDS-gel electrophoresis of partially purified thermophilin T showed that bacteriocin activity was associated with a protein band of approximately 2·5 kDa molecular mass.     

Development of enterobacterium-specific oligonucleotide probes based on the surface-exposed regions of outer membrane proteins.

Outer membrane proteins of members of the family Enterobacteriaceae consist of conserved membrane-spanning segments and hypervariable, surface-exposed regions. We demonstrate that the hypervariable DNA segments corresponding to the surface-exposed regions of these proteins can be used to develop specific DNA probes for the identification of members of the family Enterobacteriaceae.     

One hundred years ago : Fashion and medicine

Objective: To determine which attributes of clinical practice guidelines influence the use of guidelines in decision making in clinical practice. Design: Observational study relating the use of 47 different recommendations from 10 national clinical guidelines to 12 different attributes of clinical guidelines—for example, evidence based, controversial, concrete. Setting: General practice in the Netherlands. Subjects: 61 general practitioners who made 12 880 decisions in their contacts with patients. Main outcome measures: Compliance of decisions with clinical guidelines according to the attribute of the guideline. Results: Recommendations were followed in, on average, 61% (7915/12 880) of the decisions. Controversial recommendations were followed in 35% (886/2497) of decisions and non-controversial recommendations in 68% (7029/10 383) of decisions. Vague and non-specific recommendations were followed in 36% (826/2280) of decisions and clear recommendations in 67% (7089/10 600) of decisions. Recommendations that demanded a change in existing practice routines were followed in 44% (1278/2912) of decisions and those that did not in 67% (6637/9968) of decisions. Evidence based recommendations were used more than recommendations for practice that were not based on research evidence (71% (2745/3841) v 57% (5170/9039)). Conclusions: People and organisations setting evidence based clinical practice guidelines should take into account some of the other important attributes of effective recommendations for clinical practice.

Key messages

• Specific attributes of clinical practice guidelines determine whether they are used in practice
• Evidence based recommendations are better followed in practice than recommendations not based on scientific evidence
• Precise definitions of recommended performance improve the use of guidelines
• Testing the feasibility and acceptance of clinical guidelines among the target group is important for effective implementation
• People setting evidence based guidelines need to understand the attributes of effective guidelines

     

Identification of the domain which determines the g,m serotype of the flagellin of Salmonella enteritidis.

Clones expressing fragments of the flagellin protein of Salmonella enteritidis were constructed and screened with a g,m-specific monoclonal antibody. Results showed that the g,m epitope is localized between amino acids 258 and 348 of the flagellin. The fliC gene, encoding the flagellin of S. enteritidis, was proven to be the only flagellin gene present in S. enteritidis.     

Alternative antigens reduce cross-reactions in an ELISA for the detection of Salmonella enteritidis in poultry

Two alternative antigens for the use in detection of antibodies to salmonellas were investigated: firstly, lipopolysaccharide (LPS) from members of the D2 group, having antigens O : 9, 46, and flagella antigens. Whereas LPS from the D2 group did not discriminate sufficiently with control sera, flagella antigens reacted specifically with antibodies directed to serotype specific H antigens. When flagella antigens were used to screen sera from birds of commercial flocks, however, cross-reactivity between flagella antigens was observed. When both LPS and flagella antigens were used to screen sera from chickens infected with Salmonella, enteritidis, the sera gave higher titres with flagella antigens during the early stages of infection, and titres with flagella antigens dropped earlier after infection had ended than titres with lipopolysaccharide.     

The relation between gaze aversion and cortisol reactivity in middle childhood

The present study sought to investigate the relation between ethological observations of children's gaze aversion during a psychosocial stress task and their cortisol reactivity to the task, and how this relation might be moderated by how stressful the children perceived the stress task to be.     

Membrane protein-ligand interactions in Escherichia coli vesicles and living cells monitored via a biosynthetically incorporated tryptophan analogue.

This paper presents a deceptively straightforward experimental approach to monitoring membrane protein-ligand interactions in vesicles and in living Escherichia coli cells. This is achieved via the biosynthetic incorporation of 7-azatryptophan, a tryptophan analogue with a red-shifted absorption spectrum, allowing collection of the emission signal of the target protein in a high tryptophan background via red-edge excitation. The approach is demonstrated for the mannitol permease of E. coli (EII(mtl)), an integral membrane protein of 637 amino acids, including four tryptophans, and single-tryptophan mutants of EII(mtl). By using a tryptophan auxotroph, a high level of 7-azatryptophan incorporation in EII(mtl) was achieved. The change in emission signal of the purified enzyme upon mannitol binding (-28%) was 4-fold larger than with EII(mtl) containing tryptophan, demonstrating the known higher sensitivity of this analogue for changes in the microenvironment [Schlesinger, R. (1968) J. Biol. Chem. 243, 3877-3883]. Changes in emission signal could also be monitored (-5%) when the enzyme was situated in vesicles, although it constituted only 10-15% of the total cytoplasmic membrane fraction. Of the five single-tryptophan mutants, the emission signal of the mutant with 7-azatryptophan at position 198 was the most sensitive for mannitol binding. Changes in emission signal not only were observed in vesicles (-18%) but also could be monitored in viable cells (-5%). The fact that only modest expression levels and no protein purification are needed makes this a useful approach for the characterization of numerous protein systems under in vitro and in vivo conditions.     

Accumulation of a 50-kDa protein during leaf senescence of alstroemeria cut flowering stems

Leaf senescence of alstroemeria cut flowering stems in the dark was accompanied by a decrease in the soluble protein and chlorophyll content. Two-dimensional (2D) electrophoresis revealed that a polypeptide with an apparent molecular mass of 50 (± 2) kDa and isoelectric point of 5.0 (± 0.1) accumulated during the senescence process. Treatments which delayed leaf senescence (light and gibberellic acid) diminished the accumulation of this polypeptide. This polypeptide was purified and one of the peptides, resulting from digestion with trypsin, was sequenced. The se quence shows a high degree of homology to that of 3-isopropylmalate dehydrogenase. This enzyme plays a role in the biosynthesis of leucine. Possibly, leucine plays a role in carbon partitioning between sources and sinks in this plant.     

Asynchrony between pronuclear development and protein synthetic changes in zygotes of the mouse derived from oocytes aged postovulation in vivo and fertilized in vivo

The pattern of proteins synthesized by one-cell embryos derived from unaged oocytes and oocytes aged postovulation in vivo was analyzed by means of 35S-methionine labeling and gel electrophoresis. The oocytes were obtained after ovulation induction by an injection of luteinizing hormone-releasing hormone (LHRH) at proestrus or after a superovulation procedure. The analysis was performed in unfertilized aged and unaged secondary oocytes and in zygotes derived from them. The patterns of proteins synthesized by secondary oocytes from all experimental groups were very similar: The oocytes showed a predominant synthesis of 35 kDa proteins. Zygotes from aged as well as unaged LHRH-induced oocytes also showed a predominant synthesis of one group of polypeptides with a relative molecular weight of about 35 kDa. The proteins of the 35 kDa protein complex migrated in an upper (u), middle (m), or lower (l) band in 10% polyacrylamide sodium dodecyl sulfate (SDS) gels. The u- and m-band 35 kDa proteins were shown to be synthesized by secondary oocytes. Early pronuclear zygotes from unaged LHRH-induced oocytes synthesized u- and m- but no l-band 35 kDa proteins. In contrast, part (38%, n = 47) of the early pronuclear zygotes from aged LHRH-induced oocytes did synthesize the l-band 35 kDa proteins. Late pronuclear zygotes (LPZ) from aged as well as unaged oocytes synthesized predominantly the l-band 35 kDa proteins. However, although only 5.8% (n = 51) of LPZ from unaged oocytes synthesize m- and l-band 35 kDa proteins, these bands of proteins are present in 25% (n = 24) of the LPZ from aged oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)