Production and epitope characterization of mAbs specific for translation factor IF1

Initiation of protein synthesis in bacteria relies on the presence of three translation initiation factors, of which translation initiation factor IF1 is the smallest having a molecular weight of only 8.2 kDa. In addition to its function in this highly dynamic process, the essential IF1 protein also functions as an RNA chaperone. Despite extensive research, the exact function of IF1 in translation initiation has not yet been determined, and the research in the function of the factor has in some areas been impeded by the lack of monoclonal antibodies specific for this protein. Several attempts to induce immune response in mice with wild-type IF1 for the production of antibodies have failed. We have now succeeded in producing monoclonal antibodies specific for IF1 by applying a new immunization strategy involving an antigen combination of IF1 coupled to glutathione S-transferase (GST) and a recombinant dimer of IF1. This resulted in the generation of 6 IgG, 2 IgM, and 1 IgA anti-IF1 antibodies, which can be used in ELISA screening and Western immunoblots. We also provide a mapping of the functional epitopes of the generated anti-IF1 monoclonal antibodies by screening the antibodies for binding to IF1 proteins mutated at single amino acid positions.     

Crystal Structure of the Tetrameric Fibrinogen-like Recognition Domain of Fibrinogen C Domain Containing 1 (FIBCD1) Protein

The high resolution crystal structures of a recombinant fragment of the C-terminal fibrinogen-like recognition domain of FIBCD1, a vertebrate receptor that binds chitin, have been determined. The overall tetrameric structure shows similarity in structure and aggregation to the horseshoe crab innate immune protein tachylectin 5A. The high affinity ligand N-acetylmannosamine (ManNAc) binds in the S1 site, predominantly via the acetyl group with the oxygen and acetamide nitrogen hydrogen-bonded to the protein and the methyl group inserted into a hydrophobic pocket. The binding of the ManNAc pyranose ring differs markedly between the two independent subunits, but in all structures the binding of the N-acetyl group is conserved. In the native structure, a crystal contact results in one of the independent protomers binding the first GlcNAc of the Asn³⁴⁰ N-linked glycan on the other independent protomer. In the ligand-bound structure this GlcNAc is replaced by the higher affinity ligand ManNAc. In addition, a sulfate ion has been modeled into the electron density at a location similar to the S3 binding site in L-ficolin, whereas in the native structure an acetate ion has been placed in the S1 N-acetyl binding site, and a sulfate ion has been placed adjacent to this site. These ion binding sites are ideally placed to receive the N-acetyl and sulfate groups of sulfated GalNAc residues of glycosaminoglycans such as chondroitin and dermatan sulfate. Together, these structures give insight into important determinants of ligand selectivity, demonstrating versatility in recognition and binding while maintaining conservation in N-acetyl and calcium binding.     

The GABAA Antagonist DPP-4-PIOL Selectively Antagonises Tonic over Phasic GABAergic Currents in Dentate Gyrus Granule Cells

GABA_A receptors mediate two different types of inhibitory currents: phasic inhibitory currents when rapid and brief presynaptic GABA release activates postsynaptic GABA_A receptors and tonic inhibitory currents generated by low extrasynaptic GABA levels, persistently activating extrasynaptic GABA_A receptors. The two inhibitory current types are mediated by different subpopulations of GABA_A receptors with diverse pharmacological profiles. Selective antagonism of tonic currents is of special interest as excessive tonic inhibition post-stroke has severe pathological consequences. Here we demonstrate that phasic and tonic GABA_A receptor currents can be selectively inhibited by the antagonists SR 95531 and the 4-PIOL derivative, 4-(3,3-diphenylpropyl)-5-(4-piperidyl)-3-isoxazolol hydrobromide (DPP-4-PIOL), respectively. In dentate gyrus granule cells, SR 95531 was found approximately 4 times as potent inhibiting phasic currents compared to tonic currents (IC₅₀ values: 101 vs. 427 nM). Conversely, DPP-4-PIOL was estimated to be more than 20 times as potent inhibiting tonic current compared to phasic current (IC₅₀ values: 0.87 vs. 21.3 nM). Consequently, we were able to impose a pronounced reduction in tonic GABA mediated current (>70 %) by concentrations of DPP-4-PIOL, at which no significant effect on the phasic current was seen. Our findings demonstrate that selective inhibition of GABA mediated tonic current is possible, when targeting a subpopulation of GABA_A receptors located extrasynaptically using the antagonist, DPP-4-PIOL.     

An easy method for detection of nasopharyngeal carriage of multiple Streptococcus pneumoniae serotypes

In this paper, a simplified method for detection of pneumococcal carriage and for revealing the presence of several serotypes in a nasopharyngeal sample is evaluated. Enrichment broth was used for transportation and for the initial culturing of samples. All specimens were examined directly by the capsular reaction test for the presence of any of the 91 known pneumococcal serotypes. Sub-culturing on blood agar was used for isolation of the pneumococcal strains detected in the primary broth culture. A total of 693 nasopharyngeal swabs were obtained among children, their parents and employees in day care centres. Pneumococci were observed in 363 samples and 36 of these (9.9%) contained more than one serotype (multiple carriages). Two persons carried 3 different serotypes simultaneously. A significant increase in the positive sampling rate (5.8%) was achieved by using the simplified method compared to conventional streaking of the swabs directly on blood agar (p<0.0001).     

Long‐term cattle grazing shifts the ecological state of forest soils

Cattle grazing profoundly affects abiotic and biotic characteristics of ecosystems. While most research has been performed on grasslands, the effect of large managed ungulates on forest ecosystems has largely been neglected. Compared to a baseline seminatural state, we investigated how long‐term cattle grazing of birch forest patches affected the abiotic state and the ecological community (microbes and invertebrates) of the soil subsystem. Grazing strongly modified the soil abiotic environment by increasing phosphorus content, pH, and bulk density, while reducing the C:N ratio. The reduced C:N ratio was strongly associated with a lower microbial biomass, mainly caused by a reduction of fungal biomass. This was linked to a decrease in fungivorous nematode abundance and the nematode channel index, indicating a relative uplift in the importance of the bacterial energy‐channel in the nematode assemblages. Cattle grazing highly modified invertebrate community composition producing distinct assemblages from the seminatural situation. Richness and abundance of microarthropods was consistently reduced by grazing (excepting collembolan richness) and grazing‐associated changes in soil pH, Olsen P, and reduced soil pore volume (bulk density) limiting niche space and refuge from physical disturbance. Anecic earthworm species predominated in grazed patches, but were absent from ungrazed forest, and may benefit from manure inputs, while their deep vertical burrowing behavior protects them from physical disturbance. Perturbation of birch forest habitat by long‐term ungulate grazing profoundly modified soil biodiversity, either directly through increased physical disturbance and manure input or indirectly by modifying soil abiotic conditions. Comparative analyses revealed the ecosystem engineering potential of large ungulate grazers in forest systems through major shifts in the composition and structure of microbial and invertebrate assemblages, including the potential for reduced energy flow through the fungal decomposition pathway. The precise consequences for species trophic interactions and biodiversity–ecosystem function relationships remain to be established, however.     

A study of the interaction of Escherichia coli elongation factor-Tu with aminoacyl-tRNAs by partial digestion with cobra venom ribonuclease

The hydrolysis of several aminoacylated transfer RNAs, by double-strand-specific ribonuclease from Naja oxiana was studied. The sensitivity to this enzyme of Phe-tRNA^Phe, Glu-tRNA^Glu and Met-tRNA_m^Met from Escherichia coli and Phe-tRNA^Phe from yeast was examined, both in the free state and complexed to E. coli elongation factor Tu. The hydrolysis patterns in the isolated state were similar for all aminoacylated tRNAs except Glu-tRNA₂^Glu, which exhibited striking differences probably arising from the existence of several subpopulations of tRNA₂^Glu. When engaged in a ternary complex with EF-Tu and GTP, the aminoacyl-tRNAs were efficiently protected in the amino acid acceptor and TΨC helices, showing that the interaction with EF-Tu primarily takes place at the -C-C-A end and at the amino acid acceptor and TΨC helices. In all cases an increased reactivity of the anticodon stem was observed in the complexed tRNA, possibly resulting from a conformational change in this region of the tRNAs.     

Functional organization of the sortilin Vps10p domain

A Vps10p domain makes up the entire luminal part of Sortilin, and this type of domain is the hallmark of a new family of neuronal receptors that target a variety of ligands, including neurotrophins and neuropeptides. We have shown that two structural features of the Vps10p domain, the N-terminal propeptide and the C-terminal segment of ten conserved cysteines (10CC), are key elements in the function of Sortilin. The propeptide has two functions. (i) It binds the mature part of Sortilin and prevents ligands in the biosynthetic pathway from binding to the uncleaved proreceptor, and (ii) it facilitates receptor transport in early Golgi compartments by a mechanism that does not depend on its ability to prevent ligand binding. In contrast, other Vps10p domain receptors, such as SorLA and SorCS3, do not need their propeptide for normal and swift processing. The 10CC segment constitutes an exchangeable module containing five conserved disulfide bridges, and using module-shuffling and truncations, we have shown that the 10CC segment is a major ligand-binding region in Sortilin.     

Biomass production and water use efficiency in perennial grasses during and after drought stress

Drought is a great challenge to agricultural production, and cultivation of drought‐tolerant or water use‐efficient cultivars is important to ensure high biomass yields for bio‐refining and bioenergy. Here, we evaluated drought tolerance of four C₃ species, Dactylis glomerata cvs. Sevenop and Amba, Festuca arundinacea cvs. Jordane and Kora, Phalaris arundinacea cvs. Bamse and Chieftain and Festulolium pabulare cv. Hykor, and two C₄ species Miscanthus × giganteus and M. lutarioriparius. Control (irrigated) and drought‐treated plants were grown on coarse and loamy sand in 1 m² lysimeter plots where rain was excluded. Drought periods started after harvest and lasted until 80% of available soil water had been used. Drought caused a decrease in dry matter yield (DM; P < 0.001) for all species and cultivars during the drought period. Cultivars Sevenop, Kora and Jordane produced DM at equal levels and higher than the other C₃ cultivars in control and drought‐treated plots both during and after the drought period. Negative correlations were observed between stomatal conductance (g_s) and leaf water potential (P < 0.01) and positive correlations between g_s and DM (P < 0.05) indicating that g_s might be suitable for assessment of drought stress. There were indications of positive associations between plants carbon isotope composition and water use efficiency (WUE) as well as DM under well‐watered conditions. Compared to control, drought‐treated plots showed increased growth in the period after drought stress. Thus, the drought events did not affect total biomass production (DM_total) of the whole growing season. During drought stress and the whole growing season, WUE was higher in drought‐treated compared to control plots, so it seems possible to save water without loss of biomass. Across soil types, M. lutarioriparius had the highest DM_total (15.0 t ha^?1), WUE_total (3.6 g L^?1) and radiation use efficiency (2.3 g MJ^?1) of the evaluated grasses.