Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

In all vertebrate animals, CD8⁺ cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC effectively individualizes the immune response of each member of the species. We have recently developed efficient methods to generate recombinant human MHC-I (also known as human leukocyte antigen class I, HLA-I) molecules, accompanying peptide-binding assays and predictors, and HLA tetramers for specific CTL staining and manipulation. This has enabled a complete mapping of all HLA-I specificities (“the Human MHC Project”). Here, we demonstrate that these approaches can be applied to other species. We systematically transferred domains of the frequently expressed swine MHC-I molecule, SLA-1*0401, onto a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules. A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data indicate that it is possible to extend the biochemical and bioinformatics tools of the Human MHC Project to other vertebrate species.     

The patchwork nature of rolling-circle plasmids: comparison of six plasmids from two distinct Bacillus thuringiensis serotypes

Bacillus thuringiensis, the entomopathogenic bacteria from the Bacillus cereus group, harbors numerous extrachromosomal molecules whose sizes vary from 2 to more than 200kb. Apart from the genes coding for the biopesticide delta-endotoxins located on large plasmids, little information has been obtained on these plasmids and their contribution to the biology of their host. In this paper, we embarked on a detailed comparison of six small rolling-circle replicating (RCR) plasmids originating from two major B. thuringiensis strains. The complete nucleotide sequences of plasmid pGI1, pGI2, pGI3, pTX14-1, pTX14-2, and pTX14-3 have been obtained and compared. Replication functions, comprising, for each plasmid, the gene encoding the Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified and analyzed. Two new families, or homology groups, of RCR plasmids originated from the studies of these plasmids (Group VI based on pGI3 and Group VII based on pTX14-3). On five of the six plasmids, loci involved in conjugative mobilization (Mob-genes and origin of transfer (oriT)) were identified. Plasmids pTX14-1, pTX14-2, and pTX14-3 each harbor an ORF encoding a polypeptide containing a central domain with repetitive elements similar to eukaryotic collagen (Gly-X-Y triplets). These genes were termed bcol for Bacillus-collagen-like genes.     

Demonstration of Neuron-Glia Transfer of Precursors for Gaba Biosynthesis in a Co-Culture System of Dissociated Mouse Cerebral Cortex

Co-cultures of neurons and astrocytes were prepared from dissociated embryonic mouse cerebral cortex and cultured for 7 days. To investigate if these cultures may serve as a functional model system to study neuron-glia interaction with regard to GABA biosynthesis, the cells were incubated either in media containing [U-¹³C]glutamine (0.1, 0.3 and 0.5 mM) or 1 mM acetate plus 2.5 mM glucose plus 1 mM lactate. In the latter case one of the 3 substrates was uniformly ¹³C labeled. Cellular contents and ¹³C labeling of glutamate, GABA, aspartate and glutamine were determined in the cells after an incubation period of 2.5 h. The GABA biosynthetic machinery exhibited the expected complexity with regard to metabolic compartmentation and involvement of TCA cycle activity as seen in other culture systems containing GABAergic neurons. Metabolism of acetate clearly demonstrated glial synthesis of glutamine and its transfer to the neuronal compartment. It is concluded that this co-culture system serves as a reliable model in which functional and pharmacological aspects of GABA biosynthesis can be investigated. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella. An erratum to this article can be found at     

Elevated CO2 increases fungal-based micro-foodwebs in soils of contrasting plant species

Plant and Soil - As different plant species support different soil communities, assessment of climate change impacts on soil communities must consider how these impacts depend on the vegetation. We...     

Velocity and Directionality of the Electrohysterographic Signal Propagation

Objective

The initiation of treatment for women with threatening preterm labor requires effective distinction between true and false labor. The electrohysterogram (EHG) has shown great promise in estimating and classifying uterine activity. However, key issues remain unresolved and no clinically usable method has yet been presented using EHG. Recent studies have focused on the propagation velocity of the EHG signals as a potential discriminator between true and false labor. These studies have estimated the propagation velocity of individual spikes of the EHG signals. We therefore focus on estimating the propagation velocity of the entire EHG burst recorded during a contraction in two dimensions.

Study Design

EHG measurements were performed on six women in active labor at term, and a total of 35 contractions were used for the estimation of propagation velocity. The measurements were performed using a 16-channel two-dimensional electrode grid. The estimates were calculated with a maximum-likelihood approach.

Results

The estimated average propagation velocity was 2.18 (±0.68) cm/s. No single preferred direction of propagation was found.

Conclusion

The propagation velocities estimated in this study are similar to those reported in other studies but with a smaller intra- and inter-patient variation. Thus a potential tool has been established for further studies on true and false labor contractions.     

C-reactive Protein Exists in an NaCl Concentration-dependent Pentamer-Decamer Equilibrium in Physiological Buffer

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca²⁺-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mm Ca²⁺ exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s_20,w⁰ of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca²⁺, CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R_G values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K_D value of 21 μm in solution (140 mm NaCl, 2 mm Ca²⁺) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K_D value of 23 μm (140 mm NaCl, 2 mm Ca²⁺), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mm Ca²⁺ and 140 mm NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.     

Oceanic fronts in the Sargasso Sea control the early life and drift of Atlantic eels

Anguillid freshwater eels show remarkable life histories. In the Atlantic, the European eel (Anguilla anguilla) and American eel (Anguilla rostrata) undertake extensive migrations to spawn in the oceanic Sargasso Sea, and subsequently the offspring drift to foraging areas in Europe and North America, first as leaf-like leptocephali larvae that later metamorphose into glass eels. Since recruitment of European and American glass eels has declined drastically during past decades, there is a strong demand for further understanding of the early, oceanic phase of their life cycle. Consequently, during a field expedition to the eel spawning sites in the Sargasso Sea, we carried out a wide range of dedicated bio-physical studies across areas of eel larval distribution. Our findings suggest a key role of oceanic frontal processes, retaining eel larvae within a zone of enhanced feeding conditions and steering their drift. The majority of the more westerly distributed American eel larvae are likely to follow a westerly/northerly drift route entrained in the Antilles/Florida Currents. European eel larvae are generally believed to initially follow the same route, but their more easterly distribution close to the eastward flowing Subtropical Counter Current indicates that these larvae could follow a shorter, eastward route towards the Azores and Europe. The findings emphasize the significance of oceanic physical–biological linkages in the life-cycle completion of Atlantic eels.