A putative consensus sequence for the nucleotide-binding site of annexin A6

Reaction-induced infrared difference spectroscopy (RIDS) has been used to investigate the nature of interactions of human annexin A6 (ANXA6) with nucleotides. RIDS results for ANXA6, obtained after the photorelease of GTP-gamma-S, ATP, or P(i) from the respective caged compounds, were identical, suggesting that the interactions between the nucleotide and ANXA6 were dominated by the phosphate groups. Phosphate-induced structural changes in ANXA6 were small and affected only seven or eight amino acid residues. The GTP fluorescent analogue, 2'(3')-O-(2,4,6-trinitrophenyl)guanosine 5'-triphosphate (TNP-GTP), quenched tryptophan fluorescence of ANXA6 when bound to the protein. A binding stoichiometry of 1 mol of nucleotide/mol ANXA6 was established with a K(D) value of 2.8 microM for TNP-GTP. The bands observed on RIDS of ANXA6 halves (e.g., N-terminal half, ANXA6a, and C-terminal half, ANXA6b) were similar to those of the whole molecule. However, their amplitudes were smaller by a factor of 2 compared to those of whole ANXA6. TNP-GTP bound to both fragments of ANXA6 with a stoichiometry of 0.5 mol/mol. However, the binding affinities of ANXA6a and ANXA6b differed from that of ANXA6. Simulated molecular modeling revealed a nucleotide-binding site which was distributed in two distinct domains. Residues K296, Y297, K598, and K644 of ANXA6 were less than 3 A from the bound phosphate groups of either GTP or ATP. The presence of two identical sequences in ANXA6 with the F-X-X-K-Y-D/E-K-S-L motif, located in the middle of ANXA6, at residues 293-301 (within ANXA6a) and at 641-649 (within ANXA6b), suggested that the F-X-X-K-Y-D/E-K-S-L motif was the putative sequence in ANXA6 for nucleotide binding.     

Karyotype and nuclear DNA content of hexa-, octo-, and duodecaploid lines of Bromus subgen. Ceratochloa

The subgenus Ceratochloa of the genus Bromus includes a number of closely related allopolyploid forms or species that present a difficult taxonomic problem. The present work combines data concerning chromosome length, heterochromatin distribution and nuclear genome size of different 6x, 8x and 12x accessions in this subgenus. Special attention is paid to the karyotype structure and genomic constitution of duodecaploid plants recently found in South America. Hexaploid lineages possess six almost indistinguishable genomes and a nuclear DNA content between 12.72 pg and 15.10 pg (mean 1Cx value = 2.32 pg), whereas octoploid lineages contain the same six genomes (AABBCC) plus two that are characterized by longer chromosomes and a greater DNA content (1Cx = 4.47 pg). Two duodecaploid accessions found in South America resemble each other and apparently differ from the North American duodecaploid B. arizonicus as regards chromosome size and nuclear DNA content (40.00 and 40.50 pg vs. 27.59 pg). These observations suggest that the South American duodecaploids represent a separate evolutionary lineage of the B. subgenus Ceratochloa, unrecognized heretofore.     

Bio-oxidation of tripropylene glycol under aerobic conditions

Aerobic biodegradation of tripropylene glycol (PG3) was investigated under the conditions of the OECD screening test 301E and the Continuous Flow Activated Sludge Simulation test (CFAS). A modified two-chamber facility with a denitrification stage was used for the CFAS test. Primary PG3 biodegradation was measured by the HPLC with fluorimetric detection and analyte derivatisation. Metabolites were identified by LC-MS with electrospray ionisation and GC-MS with electron impact ionisation, as well as semiquantitatively determined by the LC-MS technique. PG3 was found to be inherently biodegradable and it exhibits a strong poisonous effect on activated sludge after exceeding the threshold concentration (10 mg l⁻¹). Metabolite accumulation onto the activated sludge is probably responsible for this poisonous effect. Probable biotransformation products of tripropylene glycol under the aerobic conditions include metabolites with a single terminal aldehyde or a ketone group and metabolites with two terminal aldehyde or ketone groups. Their concentration rises at the end of the OECD screening test.     

IL-17 is a critical component of vaccine-induced protection against lung infection by lipopolysaccharide-heterologous strains of Pseudomonas aeruginosa

In a murine model of acute fatal pneumonia, we previously showed that nasal immunization with a live-attenuated aroA deletant of Pseudomonas aeruginosa strain PAO1 elicited LPS serogroup-specific protection, indicating that opsonic Ab to the LPS O Ag was the most important immune effector. Because P. aeruginosa strain PA14 possesses additional virulence factors, we hypothesized that a live-attenuated vaccine based on PA14 might elicit a broader array of immune effectors. Thus, an aroA deletant of PA14, denoted PA14DeltaaroA, was constructed. PA14DeltaaroA-immunized mice were protected against lethal pneumonia caused not only by the parental strain but also by cytotoxic variants of the O Ag-heterologous P. aeruginosa strains PAO1 and PAO6a,d. Remarkably, serum from PA14DeltaaroA-immunized mice had very low levels of opsonic activity against strain PAO1 and could not passively transfer protection, suggesting that an antibody-independent mechanism was needed for the observed cross-serogroup protection. Compared with control mice, PA14DeltaaroA-immunized mice had more rapid recruitment of neutrophils to the airways early after challenge. T cells isolated from P. aeruginosa DeltaaroA-immunized mice proliferated and produced IL-17 in high quantities after coculture with gentamicin-killed P. aeruginosa. Six hours following challenge, PA14DeltaaroA-immunized mice had significantly higher levels of IL-17 in bronchoalveolar lavage fluid compared with unimmunized, Escherichia coli-immunized, or PAO1DeltaaroA-immunized mice. Antibody-mediated depletion of IL-17 before challenge or absence of the IL-17 receptor abrogated the PA14DeltaaroA vaccine's protection against lethal pneumonia. These data show that IL-17 plays a critical role in antibody-independent vaccine-induced protection against LPS-heterologous strains of P. aeruginosa in the lung.     

High-Throughput Sequencing of Islet-Infiltrating Memory CD4+ T Cells Reveals a Similar Pattern of TCR Vβ Usage in Prediabetic and Diabetic NOD Mice

Autoreactive memory CD4⁺ T cells play a critical role in the development of type 1 diabetes, but it is not yet known how the clonotypic composition and TCRβ repertoire of the memory CD4⁺ T cell compartment changes during the transition from prediabetes to diabetes. In this study, we used high-throughput sequencing to analyze the TCRβ repertoire of sorted islet-infiltrating memory CD4⁺CD44^high T cells in 10-week-old prediabetic and recently diabetic NOD mice. We show that most clonotypes of islet-infiltrating CD4⁺CD44^high T cells were rare, but high-frequency clonotypes were significantly more common in diabetic than in prediabetic mice. Moreover, although the CD4⁺CD44^high TCRβ repertoires were highly diverse at both stages of disease development, dominant use of TRBV1 (Vβ2), TRBV13-3 (Vβ8.1), and TRBV19 (Vβ6) was evident in both prediabetic and diabetic mice. Our findings strongly suggest that therapeutic targeting of cells specifically expressing the dominant TCRβ might reduce pancreatic infiltration in prediabetic mice and attenuate the progression to diabetes.     

Correction to: Lentinula edodes as a Source of Bioelements Released Into Artificial Digestive Juices and Potential Anti-inflammatory Material

Biological Trace Element Research - The original version of this article unfortunately contained a mistake.     

Acclimation of denitrifying activated sludge to a single vs. complex external carbon source during a start-up of sequencing batch reactors treating ammonium-rich anaerobic sludge digester liquors

In this study, denitrification of ammonium-reach anaerobic sludge digester liquor was investigated during start-up periods of two laboratory-scale “fill-and-draw” reactors. One reactor was fed with a single carbon source (ethanol), whereas the other reactor was fed with a complex carbon source (fusel oil). During two acclimation experiments, the structure of microbial community involved in denitrification was analyzed using 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis fingerprints and fluorescent in situ hybridization. The characteristics of the mixed liquor were additionally supported by regular measurements of nitrate uptake rates. The addition of fusel oil and ethanol resulted in a significant enhancement of the denitrification rate and efficiency combined with the increasing volumetric addition of sludge digester liquor up to 15 % of the reactor volume. The microbiological analyses revealed that the addition of sludge digester liquor as well as both external carbon sources (fusel oil and ethanol) did not affect the structure of microbial communities in a severe way. In both reactors, Curvibacter sp. and Azoarcus sp. were found as the most abundant representatives of denitrifiers.     

Identification of (η6-arene)ruthenium(II) protein binding sites in E. coli cells by combined multidimensional liquid chromatography and ESI tandem mass spectrometry: specific binding of [(η6- p -cymene)RuCl2(DMSO)] to stress-regulated proteins and to helicases

An automated multidimensional protein identification technology, which combines biphasic liquid chromatography with electrospray ionisation tandem mass spectrometry (MS/MS), was employed to analyse tryptic peptides from Escherichia coli cells treated with the antiproliferation agent [(η⁶-p-cymene)RuCl₂(DMSO)], where DMSO is dimethyl sulfoxide. MS/MS spectra were recorded for molecular ions generated by neutral loss of p-cymene from intensive peptide ions coordinated by the (η⁶-p-cymene)Ru^II fragment. Matching of the MS/MS spectra of the ruthenated peptides to spectra of proteins in the E. coli database enabled the identification of five protein targets for [(η⁶-p-cymene)RuCl₂(DMSO)]. One of these is the constitutive cold-shock protein cspC, which regulates the expression of genes encoding stress-response proteins, and three of the other targets, ppiD, osmY and sucC, are proteins of the latter type. The DNA damage-inducible helicase dinG was likewise established as a protein target. Aspartate carboxylate functions were identified as the probable Ru binding sites in cspC, ppiD and dinG, and threonine and lysine side chains in osmY and sucC, respectively. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.