The effect of relaxed functional constraints on the photosynthetic gene rbcL in photosynthetic and nonphotosynthetic parasitic plants

The photosynthetic gene rbcL has been lost or dramatically altered in some lineages of nonphotosynthetic parasitic plants, but the dynamics of these events following loss of photosynthesis and whether rbcL has sustained functionally significant changes in photosynthetic parasitic plants are unknown. To assess the changes to rbcL associated with the loss of functional constraints for photosynthesis, nucleotide sequences from nonparasitic and parasitic plants of Scrophulariales were used for phylogeny reconstruction and character analysis. Plants in this group display a broad range of parasitic abilities, from photosynthetic ("hemiparasites") to nonphotosynthetic ("holoparasites"). With the exception of Conopholis (Orobanchaceae), the rbcL locus is present in all parasitic plants of Scrophulariales examined. Several holoparasitic genera included in this study, including Boschniakia, Epifagus, Orobanche, and Hyobanche, have rbcL pseudogenes. However, the holoparasites Alectra orobanchoides, Harveya capensis, Harveya purpurea, Lathraea clandestina, Orobanche corymbosa, O. fasciculata, and Striga gesnerioides have intact open reading frames (ORFs) for the rbcL gene. Phylogenetic hypotheses based on rbcL are largely in agreement with those based on sequences of the nonphotosynthetic genes rps2 and matK and show a single origin of parasitism, and loss of photosynthesis and pseudogene formation have been independently derived several times in Scrophulariales. The mutations in rbcL in nonparasitic and hemiparasitic plants would result in largely conservative amino acid substitutions, supporting the hypothesis that functional proteins can experience only a limited range of changes, even in minimally photosynthetic plants. In contrast, ORFs in some holoparasites had many previously unobserved missense substitutions at functionally important amino acid residues, suggesting that rbcL genes in these plants have evolved under relaxed or altered functional constraints.      

Feeding of Lactobacillus sobrius reduces Escherichia coli F4 levels in the gut and promotes growth of infected piglets

The microbial community in the guts of mammals is often seen as an important potential target in therapeutic and preventive interventions. The aim of the present study was to determine whether enterotoxigenic Escherichia coli (ETEC) F4 infection in young animals might be counteracted by a probiotic treatment with Lactobacillus sobrius DSM 16698. The experiment was conducted in three randomized consecutive replications, each consisting of 16 piglets, and including a control group and an L. sobrius fed group, both experimentally challenged with ETEC. During the entire trial, the animals' health status, body weight, and microbial parameters were monitored periodically. Probiotic supplementation containing L. sobrius significantly reduced the levels of ETEC in the ileum when fed directly to piglets after weaning. In contrast, the number of days when the piglets had an increased faecal water content was significantly higher in the probiotic group. Nevertheless, an improved daily weight gain was also observed in the animals that received probiotic L. sobrius relative to the control fed group. The data indicate that L. sobrius may be effective in the reduction of the E. coli F4 colonization and may improve the weight gain of infected piglets.     

Antimicrobial activity of a multispecies probiotic (Ecologic 641) against pathogens isolated from infected pancreatic necrosis

AIMS: Although probiotic prophylaxis has been suggested to prevent small bowel bacterial overgrowth, bacterial translocation and infection of pancreatic necrosis in severe acute pancreatitis, limited data are available on their antimicrobial activity. METHODS AND RESULTS: Using the well-diffusion method, we studied the antimicrobial properties of a multispecies probiotic product (Ecologic 641) against a collection of pathogens cultured from infected pancreatic necrosis. All individual probiotic strains included in the multispecies preparation were able to inhibit the growth of the pathogens to some extent. However, the combination of the individual strains (i.e. the multispecies preparation) was able to inhibit all pathogenic isolates. Probiotic-free supernatants adjusted to pH 7 were not able to inhibit pathogen growth. CONCLUSION: Ecologic 641 is capable of inhibiting growth of a wide variety of pathogens isolated from infected pancreatic necrosis. The antimicrobial properties are to a large extent explained by the production of organic acids. SIGNIFICANCE AND IMPACT OF THE STUDY: Ecologic 641 is currently being used in a Dutch nationwide double-blind, placebo-controlled, randomized multicentre trial in patients with predicted severe acute pancreatitis.     

Specific Response of a Novel and Abundant Lactobacillus amylovorus-Like Phylotype to Dietary Prebiotics in the Guts of Weaning Piglets

Using 16S rRNA gene-based approaches, we analyzed the responses of ileal and colonic bacterial communities of weaning piglets to dietary addition of four fermentable carbohydrates (inulin, lactulose, wheat starch, and sugar beet pulp). An enriched diet and a control diet lacking these fermentable carbohydrates were fed to piglets for 4 days (n = 48), and 10 days (n = 48), and the lumen-associated microbiota were compared using denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16S rRNA genes. Bacterial diversities in the ileal and colonic samples were measured by assessing the number of DGGE bands and the Shannon index of diversity. A higher number of DGGE bands in the colon (24.2 ± 5.5) than in the ileum (9.7 ± 4.2) was observed in all samples. In addition, significantly higher diversity, as measured by DGGE fingerprint analysis, was detected in the colonic microbial community of weaning piglets fed the fermentable-carbohydrate-enriched diet for 10 days than in the control. Selected samples from the ileal and colonic lumens were also investigated using fluorescent in situ hybridization (FISH) and cloning and sequencing of the 16S rRNA gene. This revealed a prevalence of Lactobacillus reuteri in the ileum and Lactobacillus amylovorus-like populations in the ileum and the colon in the piglets fed with fermentable carbohydrates. Newly developed oligonucleotide probes targeting these phylotypes allowed their rapid detection and quantification in the ileum and colon by FISH. The results indicate that addition of fermentable carbohydrates supports the growth of specific lactobacilli in the ilea and colons of weaning piglets.     

Interactions of neural glycosaminoglycans and proteoglycans with protein ligands: assessment of selectivity, heterogeneity and the participation of core proteins in binding

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8- fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400- fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.      

Gut flora in health and disease: potential role of probiotics

In a young evolving science, there are always more questions than answers. That is also the situation in the emerging field of Probiotics, and this was made very clear at the International Probiotics Workshop in Amsterdam. In the report of this workshop, we present a selection of the most urgent questions in the field of probiotics. In addition, we propose a few strategies for the future of probiotics research. During the workshop, 120 experts--from disciplines including Human Nutrition, Gastroenterology, Nutritional Therapy, Cell Biology, Microbiology and Immunology--discussed new views on microbe-host interactions and the role of probiotics in prevention and alleviation of gastro-intestinal, atopic and auto-immune diseases. There is a general consensus among the experts that administering defined strains can help in preventing and curing gut flora related diseases: the first clinical trials show a promising role for probiotics. But the system is very complex, and most underlying mechanisms are still unclear. Rapid progress in this field will depend largely on the collaboration between fundamental researchers from different disciplines and medical specialists. Besides, more clinical studies are required to convince authorities and the public of the value of microbial therapies.     

Alternative splicing and protein function

Background  

Alternative splicing is a major mechanism of generating protein diversity in higher eukaryotes. Although at least half, and probably more, of mammalian genes are alternatively spliced, it was not clear, whether the frequency of alternative splicing is the same in different functional categories. The problem is obscured by uneven coverage of genes by ESTs and a large number of artifacts in the EST data.     

High-throughput PCR screening of genes for three-component regulatory system putatively involved in quorum sensing from low-G + C gram-positive bacteria

Quorum sensing of gram-positive bacteria is often regulated by three-component regulatory system composed of autoinducing peptide, sensor kinase and response regulator. We used PCR to study a gene cassette encoding this three-component regulatory system. Degenerate primers were designed from consensus amino acid sequences in the HPK10 subfamily, mostly involved in quorum sensing. Products amplified from genomic DNA of Lactobacillus, Enterococcus, and Clostridium species were cloned and sequenced; their deduced amino acid sequences were similar to those of members of the HPK10 subfamily. Complete genes for the putative gene cassette were cloned by inverse PCR from L. paracasei E93490 and L. plantarum WCFS6. Phylogenetic analysis grouped the cloned putative HPKs into the HPK10 subfamily. These results indicated the usefulness of this high-throughput gene screening and suggested that the three-component regulatory gene cassette are widely present.