The microbiology of butyrate formation in the human colon

A physical map of the Enterococcus faecium ATCC19434 chromosome was constructed by NotI, I-CeuI and Sse8387I. The chromosome was a circular DNA of 2600 kb in size, and contained six rRNA operons (rrn). The locations and orientations of the six rrn operons and 24 different determinants were mapped. Genomes of three additional E. faecium strains were also analyzed by I-CeuI digestion, and the genome sizes were found to vary from 2550 to 2995 kb. We further investigated the genome sizes and number of rrn operons in four E. faecalis, one E. avium, and one E. durans strains. The genome sizes were larger than E. faecium: 3000-3250 kb in E. faecalis, 3445 kb in E. avium, and 3070 kb in E. durans. E. avium and E. durans contained six rrn operons as in E. faecium, but all the E. faecalis strains possessed four rrn operons.     

Heterologous expression of bacteriocin E-760 in Chlamydomonas reinhardtii and functional analysis

The use of antimicrobial peptides (AMPs) synthesized by bacteria (bacteriocins) is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production. The bacteriocin E-760 isolated from the genus Enterococcus sp. has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria. In this study, the expression of a chimeric protein coding for E-760 in the nucleus of C. reinhardtii was evaluated, as well as, its antibacterial activity. The synthetic gene E-760S was inserted into the genome of C. reinhardtii using Agrobacterium tumefaciens. A transgenic line was identified in TAP medium with hygromycin and also by PCR. The increment in the culture medium temperature of the transgenic strain at 35 °C for 10 minutes, increased the production level of the recombinant protein from 0.14 (Noninduced culture, NIC) to 0.36% (Induced culture, IC) of total soluble proteins (TSP); this was quantified by an ELISA assay. Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log, Streptococcus agalactiae in 0.48 U log, Enterococcus faecium in 0.36 U log, Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae, the activity was 0.07 U log. These results demonstrate that the nucleus transformation of C. reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.     

Phylogenetic Relationships of Butyrate-Producing Bacteria from the Human Gut

Butyrate is a preferred energy source for colonic epithelial cells and is thought to play an important role in maintaining colonic health in humans. In order to investigate the diversity and stability of butyrate-producing organisms of the colonic flora, anaerobic butyrate-producing bacteria were isolated from freshly voided human fecal samples from three healthy individuals: an infant, an adult omnivore, and an adult vegetarian. A second isolation was performed on the same three individuals 1 year later. Of a total of 313 bacterial isolates, 74 produced more than 2 mM butyrate in vitro. Butyrate-producing isolates were grouped by 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism analysis. The results indicate very little overlap between the predominant ribotypes of the three subjects; furthermore, the flora of each individual changed significantly between the two isolations. Complete sequences of 16S rDNAs were determined for 24 representative strains and subjected to phylogenetic analysis. Eighty percent of the butyrate-producing isolates fell within the XIVa cluster of gram-positive bacteria as defined by M. D. Collins et al. (Int. J. Syst. Bacteriol. 44:812–826, 1994) and A. Willems et al. (Int. J. Syst. Bacteriol. 46:195–199, 1996), with the most abundant group (10 of 24 or 42%) clustering with Eubacterium rectale, Eubacterium ramulus, and Roseburia cecicola. Fifty percent of the butyrate-producing isolates were net acetate consumers during growth, suggesting that they employ the butyryl coenzyme A-acetyl coenzyme A transferase pathway for butyrate production. In contrast, only 1% of the 239 non-butyrate-producing isolates consumed acetate.     

Okadaic Acid Induces Selective Arrest of Protein Transport in the Rough Endoplasmic Reticulum and Prevents Export into COPII-Coated Structures

Quantitative immunoelectron microscopy and subcellular fractionation established the site of endoplasmic reticulum (ER)-Golgi transport arrest induced by the phosphatase inhibitor okadaic acid (OA). OA induced the disappearance of transitional element tubules and accumulation of the anterograde-transported Chandipura (CHP) virus G protein only in the rough ER (RER) and not at more distal sites. The block was specific to the early part of the anterograde pathway, because CHP virus G protein that accumulated in the intermediate compartment (IC) at 15°C could gain access to Golgi stack enzymes. OA also induced RER accumulation of the IC protein p53/p58 via an IC-RER recycling pathway which was resistant to OA and inhibited by the G protein activator aluminium fluoride. The role of COPII coats in OA transport block was investigated by using immunofluorescence and cell fractionation. In untreated cells the COPII coat protein sec 13p colocalized with p53/p58 in Golgi-IC structures of the juxtanuclear region and peripheral cytoplasm. During OA treatment, p53/p58 accumulated in the RER but was excluded from sec 13p-containing membrane structures. Taken together our data indicate that OA induces an early defect in RER export which acts to prevent entry into COPII-coated structures of the IC region.     

Small, non-peptide C5a receptor antagonists: part 2

Starting from 2, several highly potent C5a receptor antagonists were synthesised through alpha-amide substitution. Attempts to increase the polarity of these compounds through the introduction of basic centres or incorporation into weakly basic heterocycles is described.     

Acetate utilization and butyryl coenzyme A (CoA):acetate-CoA transferase in butyrate-producing bacteria from the human large intestine

Seven strains of Roseburia sp., Faecalibacterium prausnitzii, and Coprococcus sp. from the human gut that produce high levels of butyric acid in vitro were studied with respect to key butyrate pathway enzymes and fermentation patterns. Strains of Roseburia sp. and F. prausnitzii possessed butyryl coenzyme A (CoA):acetate-CoA transferase and acetate kinase activities, but butyrate kinase activity was not detectable either in growing or in stationary-phase cultures. Although unable to use acetate as a sole source of energy, these strains showed net utilization of acetate during growth on glucose. In contrast, Coprococcus sp. strain L2-50 is a net producer of acetate and possessed detectable butyrate kinase, acetate kinase, and butyryl-CoA:acetate-CoA transferase activities. These results demonstrate that different functionally distinct groups of butyrate-producing bacteria are present in the human large intestine.     

Golgi fragmentation during Fas-mediated apoptosis is associated with the rapid loss of GM130

During apoptosis, the Golgi complex becomes fragmented and key proteins (e.g., GRASP65 and p115) are targets for caspase cleavage. GM130, an integral membrane protein, contributes to the maintenance of Golgi structure and facilitates membrane fusion with secretory vesicles. We show that GM130 levels decrease during Fas-induced apoptosis but not during staurosporine-induced apoptosis while in both models p115 levels remain unaffected. We conclude that GM130 is rapidly diminished during Fas-mediated apoptosis associated with Golgi fragmentation in contrast to previous studies which have suggested that loss of GM130 during apoptosis is a late event.     

Identification of a novel antigen on the apical surface of rat alveolar epithelial type II and Clara cells

Here we describe a monoclonal antibody (MMC4) that recognizes a novel antigen on the apical surface of rat alveolar epithelial type II and Clara cells in the lung, proximal tubule epithelial cells in the kidney, and villus epithelial cells in the small intestine. Biochemical analysis showed that the MMC4 antigen was sensitive to heating and proteinase K digestion and that it is distributed in the detergent-rich phase after Triton X-114 phase separation. These data suggest that the MMC4 antigen is an integral membrane protein. Glycerol gradient sedimentation identified two forms of the MMC4 antigen: one with a sedimentation coefficient of 10.1 and one with a sedimentation coefficient of 1.66, suggesting that the antigen may be part of a multiprotein complex. During rat development (fetal day 16 to adult), the MMC4 antigen increased 12-fold in the lung and 200-fold in the kidney. In the intestine, the MMC4 antigen increased 150-fold by neonatal day 1 and then decreased to adult values. Our data demonstrate that the MMC4 antigen is unlike known type II cell- and Clara cell-associated proteins. The MMC4 monoclonal antibody will be useful as a marker of epithelial cell phenotype in development and injury studies.