Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions

Single and dual amino acid substitution variants were generated in the TCR CDRs of three TCRs that recognize tumor-associated Ags. Substitutions that enhance the reactivity of TCR gene-modified T cells to the cognate Ag complex were identified using a rapid RNA-based transfection system. The screening of a panel of variants of the 1G4 TCR, that recognizes a peptide corresponding to amino acid residues 157-165 of the human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of the HLA-A*02 class I allele, resulted in the identification of single and dual CDR3alpha and CDR2beta amino acid substitutions that dramatically enhanced the specific recognition of NY-ESO-1(+)/HLA-A*02(+) tumor cell lines by TCR gene-modified CD4(+) T cells. Within this group of improved TCRs, a dual substitution in the 1G4 TCR CDR3alpha chain was identified that enhanced Ag-specific reactivity in gene-modified CD4(+) and CD8(+) T cells. Separate experiments on two distinct TCRs that recognize the MART-1 27-35 (AAGIGILTV) peptide/HLA-A*02 Ag complex characterized single amino acid substitutions in both TCRs that enhanced CD4(+) T cell Ag-specific reactivity. These results indicate that simple TCR substitution variants that enhance T cell function can be identified by rapid transfection and assay techniques, providing the means for generating potent Ag complex-specific TCR genes for use in the study of T cell interactions and in T cell adoptive immunotherapy.     

Comparison of the Cecal Microbiota of Domestic and Wild Turkeys

The extent to which production methods alter intestinal microbial communities of livestock is currently unknown. As the intestinal microbiota may affect animal health, nutrition, and food safety, a baseline comparison of the cecal communities of domestic and wild turkeys was performed. Oligonucleotide fingerprinting of ribosomal RNA (rRNA) genes (OFRG) of 2,990 16S rRNA clones and dot blot quantification of dominant populations were used to identify the dominant bacterial taxa. Seventy-three percent of all the clones belonged to as yet uncultured genera. However, at a higher phylogenetic level, the OFRG library was composed of 54% Bacteroidetes clones (52% of the domestic library clones, 56% of the wild library clones), 30% Firmicutes clones (33% of the domestic library clones, 32% of the wild library clones), 3% Proteobacteria clones (5% domestic, 2% wild), and 3% Deferribacteres clones (4% domestic, 1% wild). Seven percent of the clones were unidentifiable (6% domestic, 9% wild). Bacteroidetes clones included the genera Alistipes, Prevotella, Megamonas, and Bacteroides. Of the Clostridiales clones, groups IV, IX, and XIV including genera Faecalibacterium, Megasphaera, Phascolarctobacterium, and Papillibacter were predominant. Lactobacillus, Enterococcus, and Streptococcus bacilli were also identified. beta- delta- and gamma-proteobacterial genera included Acinetobacter, Sutterella, and Escherichia. Deferribacteres clones showed high similarity to Mucispirillum schaedleri. Statistical comparison of the domestic and wild turkey clone libraries indicated similar levels of community richness and evenness despite the fact that the two libraries shared only 30% of the total clone operational taxonomic units. Together these results indicate that although high level taxonomic community structure is similar, high-density turkey production causes considerable divergence of the genera found in the ceca of commercial birds from those of their wild counterparts.     

Small angle X-ray studies reveal that Aspergillus niger glucoamylase has a defined extended conformation and can form dimers in solution

The industrially important glucoamylase 1 is an exo-acting glycosidase with substrate preference for alpha-1,4 and alpha-1,6 linkages at non-reducing ends of starch. It consists of a starch binding and a catalytic domain interspersed by a highly glycosylated polypeptide linker. The linker function is poorly understood and structurally undescribed, and data regarding domain organization and intramolecular functional cooperativity are conflicting or non-comprehensive. Here, we report a combined small angle x-ray scattering and calorimetry study of Aspergillus niger glucoamylase 1, glucoamylase 2, which lacks a starch binding domain, and an engineered low-glycosylated variant of glucoamylase 1 with a short linker. Low resolution solution structures show that the linker adopts a compact structure rendering a well defined extended overall conformation to glucoamylase. We demonstrate that binding of a short heterobidentate inhibitor simultaneously directed toward the catalytic and starch binding domains causes dimerization of glucoamylase and not, as suggested previously, an intramolecular conformational rearrangement mediated by linker flexibility. Our results suggest that glucoamylase functions via transient dimer formation during hydrolysis of insoluble substrates and address the question of the cooperative effect of starch binding and hydrolysis.     

Structural characterization of the acid-degraded secondary cell wall polymer of Geobacillus stearothermophilus PV72/p2

The secondary cell wall polymer (SCWP) from Geobacillus stearothermophilus PV72/p2, which is involved in the anchoring of the surface-layer protein to the bacterial cell wall layer, is composed of 2-amino-2-deoxy- and 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-mannose, and 2-acetamido-2-deoxy-D-mannuronic acid. The primary structure of the acid-degraded polysaccharide--liberated by HF-treatment from the cell wall--was determined by high-field NMR spectroscopy and mass spectrometry using N-acetylated and hydrolyzed polysaccharide derivatives as well as Smith-degradation. The polysaccharide was shown to consist of a tetrasaccharide repeating unit containing a pyruvic acid acetal at a side-chain 2-acetamido-2-deoxy-alpha-D-mannopyranosyl residue. Substoichiometric substitutions of the repeating unit were observed concerning the degree of N-acetylation of glucosamine residues and the presence of side-chain linked 2-acetamido-2-deoxy-beta-D-glucopyranosyl units: [Formula: see text].     

Structural characterization of homogalacturonan by NMR spectroscopy-assignment of reference compounds

Complete assignment of ¹H and ¹³C NMR of six hexagalactopyranuronic acids with varying degree and pattern of methyl esterification is reported. The NMR experiments were run at room temperature using approximately 2 mg of sample making this method convenient for studying the structure of homogalacturonan oligosaccharides.     

Polymorphic microsatellite loci for eusocial wasps (Hymenoptera: Vespidae)

Hymenopterans are an important model for studying the evolution of cooperation in animal societies. Here, we characterize 19 microsatellite loci, isolated from the common wasp Vespula vulgaris, that can be used to study genetic variation in three genera (seven species) within the Vespidae. The number of alleles in V. vulgaris was moderate, varying from 2 to 14, with expected heterozygosity ranging between 0.04 and 0.93. Eleven loci amplified DNA in V. austriaca and Dolichovespula sylvestris, nine in V. germanica, eight in Vespa crabro and V. rufa, seven in D. media and only five loci could be used for D. norwegica.     

Avian richness and abundance in temperate Danish forests: tree variables important to birds and their conservation

Many studies on avian diversity and forest structure have focused onfiner scale forest variables such as foliage height diversity, foliagediversity, foliage density, vertical distribution of vegetation and horizontalvegetation density. From a conservation and forestry operational point of viewit would be of great interest if tree variables influenced directly by forestrymanagement decisions also had significant influence on avian richness andabundance. The species, age and size of a tree are examples of such treevariables. A great number of studies also have focused on avian diversityindices to reveal relationships with vegetation variables. However, it may bemore appropriate for foresters and conservation officers to operate withrichness and abundance measures directly, because indices complicateinterpretations on the relative importance of the two variables (richness andabundance) constituting the index. Fourteen managed temperate forests in Denmarkwere investigated for avian species richness and abundance and related tomeasures on different tree variables influenced directly by forestry managementdecisions. A rapid assessment method of avian richness and abundance wasemployed. It consisted of point-counts of bird richness and abundance within 1km² of forest. General linear models were tested byanalyses of variance statistics to reveal the tree variables most important toavian richness and abundance. It was found that more old trees, more treespecies and more tree size-classes correlated with more bird species andindividuals. However, some variation in bird richness and abundance was alsorelated to site quality and/or chance colonization. Moreover, it was shown thatthe guild of cavity-nesting birds correlated positively to age of tree stand.The potential number of bird species in Danish forest is similar to that innearly pristine forest in Poland, and much larger than that recorded in any ofthe forests investigated. Together with the results above, this indicates a highpotential for squeezing in more avian species in a higher quality forest from abiodiversity point of view. Modern Danish forestry affects tree variablesinfluenced directly by forestry management decisions. Such tree variables havegreat influence on avian richness and abundance, but simple measures in forestrypractices can be taken to enhance the conservation of bird richness andabundance.     

On the bovine F blood group system

The F system of three Danish cattle breeds as determined by four specific anti-sera is described. In the Jersey breed three alleles are recognised. In the Danish cattle breeds there was no indication of a null allele. However, the phenotypes observed in zebu cattle by means of four reagents suggest the presence of at least six alleles in the bovine F system. Furthermore, the data show that the factors V₁ and V₂ do not form a linear subtype system in all cattle breeds.