Mitochondrial ATP synthase. Interaction of a synthetic 50-amino acid, beta-subunit peptide with ATP

A 50-amino acid peptide predicted by chemical modification studies of F1 and by comparison with adenylate kinase to comprise part of an ATP-binding domain within the beta-subunit of mitochondrial ATP synthase has been synthesized and purified. In the numbering system used for bovine heart beta, the peptide consists of amino acid residues from aspartate 141 at the N-terminal end to threonine 190 at the carboxyl end. In Tris-Cl buffer, pH 7.4, the peptide undergoes a dramatic reaction with ATP resulting in precipitate formation. Analysis of the precipitate shows it to contain both peptide and ATP. Similar to the ATPase activity of F1 and the binding of nucleotide to the enzyme, the capacity of ATP to induce precipitation of the peptide is decreased markedly by lowering pH. Interaction of the peptide with the fluorescent ATP analog, TNP-ATP (2'(3')-O-(2,4-6-trinitrophenyl)-adenosine 5'-triphosphate), can be demonstrated in solution at low concentrations. A 7-fold enhancement in fluorescence is observed when 2.5 microM TNP-ATP interacts with 2.5 microM peptide. Divalent cation is neither required for ATP-induced precipitation of the peptide nor for demonstrating interaction between TNP-ATP and peptide, just as Mg2+ is not required for nucleotide binding to F1. These results indicate that the beta-subunit peptide studied here comprises at least part of a nucleotide-binding domain within the mitochondrial ATP synthase complex.     

The sub‐nanomolar binding of DNA–RNA hybrids by the single‐chain Fv fragment of antibody S9.6

The monoclonal antibody S9.6 binds DNA–RNA hybrids with high affinity, making it useful in research and diagnostic applications, such as in microarrays and in the detection of R‐loops. A single‐chain variable fragment (scFv) of S9.6 was produced, and its affinities for various synthetic nucleic acid hybrids were measured by surface plasmon resonance (SPR). S9.6 exhibits dissociation constants of approximately 0.6 nM for DNA–RNA and, surprisingly, 2.7 nM for RNA–RNA hybrids that are AU‐rich. The affinity of the S9.6 scFv did not appear to be strongly influenced by various buffer conditions or by ionic strength below 500 mM NaCl. The smallest epitope that was strongly bound by the S9.6 scFv contained six base pairs of DNA–RNA hybrid. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.     

The essential mosquito-stage P25 and P28 proteins from Plasmodium form tile-like triangular prisms

P25 and P28 proteins are essential for Plasmodium parasites to infect mosquitoes and are leading candidates for a transmission-blocking malaria vaccine. The Plasmodium vivax P25 is a triangular prism that could tile the parasite surface. The residues forming the triangle are conserved in P25 and P28 from all Plasmodium species. A cocrystal structure shows that a transmission-blocking antibody uses only its heavy chain to bind Pvs25 at a vertex of the triangle.     

Subdomain 3 of Plasmodium falciparum VAR2CSA DBL3x Is Identified as a Minimal Chondroitin Sulfate A-binding Region

Molecular interactions between the VAR2CSA protein, expressed on the surface of Plasmodium falciparum-infected erythrocytes, and placental chondroitin sulfate A (CSA) are primarily responsible for pregnancy-associated malaria (PAM). Interrupting these interactions may prevent or ameliorate the severity of PAM. Several of the Duffy binding-like (DBL) domains of VAR2CSA, including the DBL3x domain, have been shown to bind CSA in vitro, but a more detailed understanding of how DBL domains bind CSA is needed. In this study, we demonstrate that subdomain 3 (S3), one of the three subdomains of VAR2CSA DBL3x by itself, is the major contributor toward CSA binding. NMR spectroscopy and flow cytometry analyses show that S3 and the intact DBL3x domain bind CSA similarly. Mutations within the S3 portion of DBL3x markedly affect CSA binding. Both recombinant molecules, S3 and DBL3x, are recognized by antibodies in the plasma of previously pregnant women living in malaria-endemic regions of Mali, but much less so by plasma from men of the same regions. As the S3 sequence is highly conserved in all known VAR2CSA proteins expressed by different parasite isolates obtained from various malaria endemic areas of the world, the identification of S3 as an independent CSA-binding region provides a compelling molecular basis for designing interventions against PAM.     

Redundancy and plasticity of neutralizing antibody responses are cornerstone attributes of the human immune response to the smallpox vaccine

The smallpox vaccine is widely considered the gold standard for human vaccines, yet the key antibody targets in humans remain unclear. We endeavored to identify a stereotypic, dominant, mature virion (MV) neutralizing antibody target in humans which could be used as a diagnostic serological marker of protective humoral immunity induced by the smallpox vaccine (vaccinia virus [VACV]). We have instead found that diversity is a defining characteristic of the human antibody response to the smallpox vaccine. We show that H3 is the most immunodominant VACV neutralizing antibody target, as determined by correlation analysis of immunoglobulin G (IgG) specificities to MV neutralizing antibody titers. It was determined that purified human anti-H3 IgG is sufficient for neutralization of VACV; however, depletion or blockade of anti-H3 antibodies revealed no significant reduction in neutralization activity, showing anti-H3 IgG is not required in vaccinated humans (or mice) for neutralization of MV. Comparable results were obtained for human (and mouse) anti-L1 IgG and even for anti-H3 and anti-L1 IgG in combination. In addition to H3 and L1, human antibody responses to D8, A27, D13, and A14 exhibited statistically significant correlations with virus neutralization. Altogether, these data indicate the smallpox vaccine succeeds in generating strong neutralizing antibody responses not by eliciting a stereotypic response to a single key antigen but instead by driving development of neutralizing antibodies to multiple viral proteins, resulting in a "safety net" of highly redundant neutralizing antibody responses, the specificities of which can vary from individual to individual. We propose that this is a fundamental attribute of the smallpox vaccine.     

Patterns of DNA Barcode Variation in Canadian Marine Molluscs

Background

Molluscs are the most diverse marine phylum and this high diversity has resulted in considerable taxonomic problems. Because the number of species in Canadian oceans remains uncertain, there is a need to incorporate molecular methods into species identifications. A 648 base pair segment of the cytochrome c oxidase subunit I gene has proven useful for the identification and discovery of species in many animal lineages. While the utility of DNA barcoding in molluscs has been demonstrated in other studies, this is the first effort to construct a DNA barcode registry for marine molluscs across such a large geographic area.

Methodology/Principal Findings

This study examines patterns of DNA barcode variation in 227 species of Canadian marine molluscs. Intraspecific sequence divergences ranged from 0–26.4% and a barcode gap existed for most taxa. Eleven cases of relatively deep (>2%) intraspecific divergence were detected, suggesting the possible presence of overlooked species. Structural variation was detected in COI with indels found in 37 species, mostly bivalves. Some indels were present in divergent lineages, primarily in the region of the first external loop, suggesting certain areas are hotspots for change. Lastly, mean GC content varied substantially among orders (24.5%–46.5%), and showed a significant positive correlation with nearest neighbour distances.

Conclusions/Significance

DNA barcoding is an effective tool for the identification of Canadian marine molluscs and for revealing possible cases of overlooked species. Some species with deep intraspecific divergence showed a biogeographic partition between lineages on the Atlantic, Arctic and Pacific coasts, suggesting the role of Pleistocene glaciations in the subdivision of their populations. Indels were prevalent in the barcode region of the COI gene in bivalves and gastropods. This study highlights the efficacy of DNA barcoding for providing insights into sequence variation across a broad taxonomic group on a large geographic scale.     

Expression of miRNAs in ovine fetal gonads: potential role in gonadal differentiation

Background  

Gonadal differentiation in the mammalian fetus involves a complex dose-dependent genetic network. Initiation and progression of fetal ovarian and testicular pathways are accompanied by dynamic expression patterns of thousands of genes. We postulate these expression patterns are regulated by small non-coding RNAs called microRNAs (miRNAs). The aim of this study was to identify the expression of miRNAs in mammalian fetal gonads using sheep as a model.     

Mitochondrial ATP synthase. cDNA cloning, amino acid sequence, overexpression, and properties of the rat liver alpha subunit

The predicted amino acid sequence of the alpha subunit of the rat liver mitochondrial ATP synthase has been obtained by sequencing a cDNA for the alpha subunit. Analysis of the sequence shows that it contains the A and B consensus sequences found in many nucleotide-binding proteins. Twelve amino acids of the rat liver alpha subunit differ from the sequence of the bovine heart alpha subunit; four of these involve differences in charge. The rat liver alpha subunit, from arginine 15 to the C-terminal proline 510, has been overexpressed in Escherichia coli using the alkaline phosphatase promoter (phoA) and leader peptide to direct the export of the expressed protein to the bacterial periplasm. By treating the cells with lysozyme, osmotic shock, and alkaline pH washes, the alpha subunit can be extracted in high yield (greater than 25 mg/liter) and in a high state of purity. The expressed alpha subunit remains soluble at pH 9.5 or greater and precipitates when treated with Mg2+ ions at low millimolar concentration. The bacterially expressed alpha subunit interacts with 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), resulting in a marked fluorescence enhancement upon binding. An enhancement of fluorescence is also observed upon the interaction of the alpha subunit with TNP-ADP. Preincubating the alpha subunit with 1.5 mM ATP significantly reduces the fluorescence enhancement seen with TNP-ATP. The alpha subunit binds TNP-ATP with an apparent Kd in the low micromolar range (1-5 microM) and binds TNP-ADP with an affinity at least 10-fold lower. This work shows that the rat liver alpha subunit can be overexpressed in E. coli to yield a large amount of functional protein. With the acquisition of the overexpressed alpha subunit, it is now possible to test the reconstitution of ATPase activity from a mixture of recombinant and rat liver-derived subunits and to test the formation of complexes by the overexpressed alpha and beta subunits of the rat liver F1-ATPase.     

Molecular evolution of mitochondrial 12S RNA and cytochrome b sequences in the pantherine lineage of Felidae

DNA sequence comparisons of two mitochondrial DNA genes were used to infer phylogenetic relationships among 17 Felidae species, notably 15 in the previously described pantherine lineage. The polymerase chain reaction (PCR) was used to generate sequences of 358 base pairs of the mitochondrial 12S RNA gene and 289 base pairs of the cytochrome b protein coding gene. DNA sequences were compared within and between 17 felid and five nonfelid carnivore species. Evolutionary trees were constructed using phenetic, cladistic, and maximum likelihood algorithms. The combined results suggested several phylogenetic relationships including (1) the recognition of a recently evolved monophyletic genus Panthera consisting of Panthera leo, P. pardus, P. onca, P. uncia, P. tigris, and Neofelis nebulosa; (2) the recent common ancestry of Acinonyx jubatus, the African cheetah, and Puma concolor, the American puma; and (3) two golden cat species, Profelis temmincki and Profelis aurata, are not sister species, and the latter is strongly associated with Caracal caracal. These data add to the growing database of vertebrate mtDNA sequences and, given the relatively recent divergence among the felids represented here (1-10 Myr), allow 12S and cytochrome b sequence evolution to be addressed over a time scale different from those addressed in most work on vertebrate mtDNA.