Removal of lipopolysaccharide from protein solution using nanostructured porous supports bearing lipid membranes.

Polymeric lipid membranes of N-octadecylchitosan, which consists of 70 mol% of 2-(octadecylamino)-2-deoxy-d-glucopyranose, 17 mol% of 2-amino-2-deoxy-d-glucopyranose, and 13 mol% of 2-acetamido-2-deoxy-d-glucopyranose, were covalently immobilized to carboxylated porous supports composed of chitosan and used for the adsorption of pyrogenic lipopolysaccharide. When human serum albumin solution, including 5 mg mL-1 of albumin and 5.6 ng mL-1 of lipopolysaccharide, was passed through a column packed with the resulting porous supports bearing lipid membranes assembled in nanoscale, lipopolysaccharide was removed to as low as a detection limit of 0.020 ng mL-1 with a quantitative recovery of protein. On the other hand, in the case of directly N-octadecylated porous supports having cationic and hydrophobic ligands which are not assembled as lipid membranes, lipopolysaccharide could not be removed to the detection limit and protein recovery was lower than the porous supports bearing lipid membranes. The difference above as well as difference from conventional adsorbents suggested that the selectivity was attributable to an interaction between the cationic lipid membranes of N-octadecylchitosan and lipopolysaccharide as well as protein. The porous supports bearing lipid membranes were stable in 0.5 M NaOH and 0.1 M HCl at ambient temperature. Considering the confirmed excellent selectivity and chemical stability, their practical use as separation media in the pharmaceutical manufacturing can be expected.

Diversity of the formyltetrahydrofolate synthetase (FTHFS) gene in the proximal and mid ostrich colon.

We analysed fragments of the formyltetrahydrofolate synthetase (FTHFS) gene, which encodes a key enzyme in reductive acetogenesis, from the bacterial flora in the proximal (PC) and mid (MC) colon of three ostriches to assess and compare bacterial diversity in this organ. Two clone libraries of FTHFS fragments were constructed from DNA extracted from digesta of the PC and MC, and a total of 46 cloned sequences were analysed from each library. A wide variety of FTHFS sequences were recovered. The coverage of the PC and MC libraries was 90.0% and 83.3%, respectively. Shannon-Wiener index (H') and Chao1 of the MC library were higher than those of PC library. The sequences from each library were classified into 15 operational taxonomic units (OTUs) and clusters. Only four OTUs in cluster I were distantly related to known acetogens from human feces and rumen, suggesting the presence of the novel acetogens. Phylogenetic analysis suggests that composition of FTHFS sequences differs for the PC and MC.

Microbial diversity in ostrich ceca as revealed by 16S ribosomal RNA gene clone library and detection of novel Fibrobacter species.

The ostrich (Struthio camelus) is a herbivorous bird and although the hindgut is known as the site for fiber digestion, little is known about the microbial diversity in the ostrich hindgut. Our aim was to analyze the microbial diversity in ostrich ceca using a 16S ribosomal RNA gene (rDNA) clone library approach. A total of 310 clones were sequenced and phylogenetically analyzed and were classified into 110 operational taxonomy units (OTUs) based on a 98% similarity criterion. The similarity of the sequences ranged from 86 to 99% and 95 OTUs had less than 98% similarity to the sequences in the public databases. Coverage and the Shannon-Wiener index (H') of the library were 83.9% and 4.29, respectively. The sequences were assigned to the following 6 phyla: Firmicutes (50.9% of the total number of sequences), Bacteroidetes (39.4%), Fibrobacteres (6.5%), Euryarchaeota (1.9%), Spirochaetes (1.0%), and Verrucomicrobia (0.3%); approximately 90% of the sequences were affiliated with Firmicutes and Bacteroidetes. The only OTU of Fibrobacteres (OTU 107), had 93 and 90% similarity to Fibrobacter succinogenes and F. intestinalis, respectively, suggesting a new species of Fibrobacter in ostrich ceca. Clostridium coccoides and C. leptum formed major groups within the Firmicutes. There was no OTU with high similarity (> or =98%) to the 16S rDNA of cultivated fibrolytic bacteria in our library. Although two OTUs were affiliated with Euryarchaeota, no sequence was affiliated with methanogenic Archaea. This study presents the very complex ostrich cecal microbial community, in which the majority of the bacterial species have not yet been cultivated.

Detection of fiber-digesting bacteria in the ceca of ostrich using specific primer sets.

The purpose of this study was to detect three fibrolytic bacteria, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus, in the cecal digesta of the ostrich (Struthio camelus) by PCR using a species-specific primer set for each 16S ribosomal RNA gene (16S rDNA). Although amplified DNA fragments obtained from each primer set had the expected size, the clone library derived from the amplimer contained non-specific sequences. The F. succinogenes-specific primer set recovered a partial 16S rDNA sequence of an uncultivated Fibrobacter with low similarity (<95%) and distantly related phylogenetic positioning to Fibrobacter sequences deposited in the databases, indicating a novel species of Fibrobacter. The sequence was considered to be identical to a clone detected in our previous experiment. Thus, we confirm that the gastrointestinal tract of the ostrich is one of the habitats of Fibrobacter species. The clone library derived from the R. flavefaciens-specific primer set contained a 16S rDNA sequence with 97% similarity to R. flavefaciens, indicating it could be one of a major fibrolytic bacterium in the ostrich ceca. No R. albus 16S rDNA sequence was found in the clone library of the R. albus-specific primer set.

Molecular characterization of chicken growth hormone secretagogue receptor gene.

Synthetic growth hormone secretagogues stimulate growth hormone secretion by binding to a specific receptor, growth hormone secretagogue receptor (GHS-R). In this study, we investigated the cDNA and the genomic structure of chicken GHS-R. Chicken GHS-R gene is composed of two exons separated by an intron. Two GHS-R mRNA species, cGHS-R1a and cGHS-R1a-variant (cGHS-R1aV) are generated by alternative splicing of a primary transcript. cGHS-R1a protein is predicted to have seven transmembrane domains by a high degree of amino acid sequence identity with mammalian and teleost homologs. cGHS-R1aV lacks the transmembrane-6 domain due to a 48 bp deletion. RT-PCR analysis showed widespread tissue distributions of cGHS-R1a and cGHS-R1aV mRNAs with much higher amounts of cGHS-R1a in all the tissues.