Influence of HLA genotype on birth weight of patients with Turner syndrome

Summary Growth failure starting before birth is a common characteristic in Turner syndrome, and its pathogenesis is still not completely explained. Experiments performed in mice and rats to test whether a genetic disparity between mothers and offspring and maternal immunological status have any influence on litter size have demonstrated that allogenic litters are significantly larger in size than genetically compatible ones. Studies in humans have given contrasting results, but some authors have found that heterozygosity at enzyme loci and in blood groups is positively correlated with intrauterine growth. HLA class I and II polymorphisms were defined in 53 patients with Turner syndrome and in their parents, and lymphocytotoxic antibody detection was performed in 36 mothers. These data were related to the patients' birth weight. The frequency of the HLA-B16 allele in patients with a birth weight > 10th centile was significantly higher in comparison with those < 10th centile. HLA antigen sharing was present in 43 couples (81.1%). Mean birth weight was 2934 ± 472 g in patients without HLA antigen parental sharing and 2721 ± 529 g in those whose parents shared HLA antigens. The mean birth weight of the 10 patients whose parents do not share HLA antigens was significantly higher than that of the patients with parental HLA–B+DR sharing (P < 0.05) and not significantly higher than in those patients with parental HLA sharing at other HLA loci. Patients whose parents shared B+DR antigens also had significantly smaller birth weights than those with B and A+B+DR sharing (P < 0.025 and P < 0.025). No significant difference in mean birth weight was found in relation to other parameters, such as mother-child histocompatibility, HLA homozygosity and lymphocytotoxic production in the mothers.     

Efficient Translation of Pelargonium line pattern virus RNAs Relies on a TED-Like 3′-Translational Enhancer that Communicates with the Corresponding 5′-Region through a Long-Distance RNA-RNA Interaction

Cap-independent translational enhancers (CITEs) have been identified at the 3´-terminal regions of distinct plant positive-strand RNA viruses belonging to families Tombusviridae and Luteoviridae. On the bases of their structural and/or functional requirements, at least six classes of CITEs have been defined whose distribution does not correlate with taxonomy. The so-called TED class has been relatively under-studied and its functionality only confirmed in the case of Satellite tobacco necrosis virus, a parasitic subviral agent. The 3´-untranslated region of the monopartite genome of Pelargonium line pattern virus (PLPV), the recommended type member of a tentative new genus (Pelarspovirus) in the family Tombusviridae, was predicted to contain a TED-like CITE. Similar CITEs can be anticipated in some other related viruses though none has been experimentally verified. Here, in the first place, we have performed a reassessment of the structure of the putative PLPV-TED through in silico predictions and in vitro SHAPE analysis with the full-length PLPV genome, which has indicated that the presumed TED element is larger than previously proposed. The extended conformation of the TED is strongly supported by the pattern of natural sequence variation, thus providing comparative structural evidence in support of the structural data obtained by in silico and in vitro approaches. Next, we have obtained experimental evidence demonstrating the in vivo activity of the PLPV-TED in the genomic (g) RNA, and also in the subgenomic (sg) RNA that the virus produces to express 3´-proximal genes. Besides other structural features, the results have highlighted the key role of long-distance kissing-loop interactions between the 3´-CITE and 5´-proximal hairpins for gRNA and sgRNA translation. Bioassays of CITE mutants have confirmed the importance of the identified 5´-3´ RNA communication for viral infectivity and, moreover, have underlined the strong evolutionary constraints that may operate on genome stretches with both regulatory and coding functions.     

Albumin binding as a general strategy for improving the pharmacokinetics of proteins

Plasma protein binding can be an effective means of improving the pharmacokinetic properties of otherwise short lived molecules. Using peptide phage display, we identified a series of peptides having the core sequence DICLPRWGCLW that specifically bind serum albumin from multiple species with high affinity. These peptides bind to albumin with 1:1 stoichiometry at a site distinct from known small molecule binding sites. Using surface plasmon resonance, the dissociation equilibrium constant of peptide SA21 (Ac-RLIEDICLPRWGCLWEDD-NH(2)) was determined to be 266 +/- 8, 320 +/- 22, and 467 +/- 47 nm for rat, rabbit, and human albumin, respectively. SA21 has an unusually long half-life of 2.3 h when injected by intravenous bolus into rabbits. A related sequence, fused to the anti-tissue factor Fab of D3H44 (Presta, L., Sims, P., Meng, Y. G., Moran, P., Bullens, S., Bunting, S., Schoenfeld, J., Lowe, D., Lai, J., Rancatore, P., Iverson, M., Lim, A., Chisholm, V., Kelley, R. F., Riederer, M., and Kirchhofer, D. (2001) Thromb. Haemost. 85, 379-389), enabled the Fab to bind albumin with similar affinity to that of SA21 while retaining the ability of the Fab to bind tissue factor. This interaction with albumin resulted in reduced in vivo clearance of 25- and 58-fold in mice and rabbits, respectively, when compared with the wild-type D3H44 Fab. The half-life was extended 37-fold to 32.4 h in rabbits and 26-fold to 10.4 h in mice, achieving 25-43% of the albumin half-life in these animals. These half-lives exceed those of a Fab'(2) and are comparable with those seen for polyethylene glycol-conjugated Fab molecules, immunoadhesins, and albumin fusions, suggesting a novel and generic method for improving the pharmacokinetic properties of rapidly cleared proteins.     

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or “scarring” is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first “organ” culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.     

The stratigraphy of the Middle Stone Age sediments at Pinnacle Point Cave 13B (Mossel Bay, Western Cape Province, South Africa)

Pinnacle Point Cave 13B (PP13B) has provided the earliest archaeological evidence for the exploitation of marine shellfish, along with very early evidence for use and modification of pigments and the production of bladelets, all dated to approximately 164?ka (Marean et?al., 2007). This makes PP13B a key site in studies of the origins of modern humans, one of a handful of sites in Africa dating to Marine Isotope Stage 6 (MIS 6), and the only site on the coast of South Africa with human occupation confidently dated to MIS 6. Along with this MIS 6 occupation there are rich archaeological sediments dated to MIS 5, and together these sediments are differentially preserved in three different areas of the cave. The sediments represent a complex palimpsest of geogenic, biogenic, and anthropogenic input and alteration that are described and interpreted through the use of a variety of macrostratigraphic, micromorphologic, and geochemical techniques. Three independent dating techniques allow us to constrain the age range of these sediments and together provide the stratigraphic context for the analyses of the material that follow in this special issue.     

Key Importance of Small RNA Binding for the Activity of a Glycine-Tryptophan (GW) Motif-containing Viral Suppressor of RNA Silencing

Viruses express viral suppressors of RNA silencing (VSRs) to counteract RNA silencing-based host defenses. Although virtually all stages of the antiviral silencing pathway can be inhibited by VSRs, small RNAs (sRNAs) and Argonaute (AGO) proteins seem to be the most frequent targets. Recently, GW/WG motifs of some VSRs have been proposed to dictate their suppressor function by mediating interaction with AGO(s). Here we have studied the VSR encoded by Pelargonium line pattern virus (family Tombusviridae). The results show that p37, the viral coat protein, blocks RNA silencing. Site-directed mutagenesis of some p37 sequence traits, including a conserved GW motif, allowed generation of suppressor-competent and -incompetent molecules and uncoupling of the VSR and particle assembly capacities. The engineered mutants were used to assess the importance of p37 functions for viral infection and the relative contribution of diverse molecular interactions to suppressor activity. Two main conclusions can be drawn: (i) the silencing suppression and encapsidation functions of p37 are both required for systemic Pelargonium line pattern virus infection, and (ii) the suppressor activity of p37 relies on the ability to bind sRNAs rather than on interaction with AGOs. The data also caution against potential misinterpretations of results due to overlap of sequence signals related to distinct protein properties. This is well illustrated by mutation of the GW motif in p37 that concurrently affects nucleolar localization, efficient interaction with AGO1, and sRNA binding capability. These concomitant effects could have been overlooked in other GW motif-containing suppressors, as we exemplify with the orthologous p38 of turnip crinkle virus.     

Arginine-induced conformational change in the c-ring/a-subunit interface of ATP synthase

The rotational mechanism of ATP synthases requires a unique interface between the stator a subunit and the rotating c-ring to accommodate stability and smooth rotation simultaneously. The recently published c-ring crystal structure of the ATP synthase of Ilyobacter tartaricus represents the conformation in the absence of subunit a. However, in order to understand the dynamic structural processes during ion translocation, studies in the presence of subunit a are required. Here, by intersubunit Cys-Cys cross-linking, the relative topography of the interacting helical faces of subunits a and c from the I. tartaricus ATP synthase has been mapped. According to these data, the essential stator arginine (aR226) is located between the c-ring binding pocket and the cytoplasm. Furthermore, the spatially vicinal residues cT67C and cG68C in the isolated c-ring structure yielded largely asymmetric cross-linking products with aN230C of subunit a, suggesting a small, but significant conformational change of binding-site residues upon contact with subunit a. The conformational change was dependent on the positive charge of the stator arginine or the aR226H substitution. Energy-minimization calculations revealed possible modes for the interaction between the stator arginine and the c-ring. These biochemical results and structural restraints support a model in which the stator arginine operates as a pendulum, moving in and out of the binding pocket as the c-ring rotates along the interface with subunit a. This mechanism allows efficient interaction between subunit a and the c-ring and simultaneously allows almost frictionless movement against each other.     

Genetic characterization of captive Cuban crocodiles (Crocodylus rhombifer) and evidence of hybridization with the American crocodile (Crocodylus acutus)

There is a surprising lack of genetic data for the Cuban crocodile (Crocodylus rhombifer), especially given its status as a critically endangered species. Samples from captive individuals were used to genetically characterize this species in comparison with other New World crocodilians. Partial mitochondrial sequence data were generated from cyt-b (843 bp) and the tRNA(Pro)- tRNA(Phe)-D-loop region (442 bp). Phylogenetic analyses were performed by generating maximum parsimony, maximum likelihood, and Bayesian-based topologies. In addition, in an effort to identify species-specific alleles, ten polymorphic microsatellite loci were genotyped. Distance and model-based clustering analyses were performed on microsatellite data, in addition to a model-based assignment of hybrid types. Both mitochondrial and nuclear markers identified two distinct C. rhombifer genetic sub-clades (alpha and beta); and microsatellite analyses revealed that most admixed individuals were F(2) hybrids between C. rhombifer-alpha and the American crocodile (C. acutus). All individuals in the C. rhombifer-beta group were morphologically identified as C. acutus and formed a distinct genetic assemblage. J. Exp. Zool. 309A:649-660, 2008. (c) 2008 Wiley-Liss, Inc.     

The structure of 3-methylaspartase from Clostridium tetanomorphum functions via the common enolase chemical step.

Methylaspartate ammonia-lyase (3-methylaspartase, MAL; EC ) catalyzes the reversible anti elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to give mesaconic acid. This reaction lies on the main catabolic pathway for glutamate in Clostridium tetanomorphum. MAL requires monovalent and divalent cation cofactors for full catalytic activity. The enzyme has attracted interest because of its potential use as a biocatalyst. The structure of C. tetanomorphum MAL has been solved to 1.9-A resolution by the single-wavelength anomalous diffraction method. A divalent metal ion complex of the protein has also been determined. MAL is a homodimer with each monomer consisting of two domains. One is an alpha/beta-barrel, and the other smaller domain is mainly beta-strands. The smaller domain partially occludes the C terminus of the barrel and forms a large cleft. The structure identifies MAL as belonging to the enolase superfamily of enzymes. The metal ion site is located in a large cleft between the domains. Potential active site residues have been identified based on a combination of their proximity to a metal ion site, molecular modeling, and sequence homology. In common with all members of the enolase superfamily, the carboxylic acid of the substrate is co-ordinated by the metal ions, and a proton adjacent to a carboxylic acid group of the substrate is abstracted by a base. In MAL, it appears that Lys(331) removes the alpha-proton of methylaspartic acid. This motif is the defining mechanistic characteristic of the enolase superfamily of which all have a common fold. The degree of structural conservation is remarkable given only four residues are absolutely conserved.