Positive selection of an MHC class-I restricted TCR in the absence of classical MHC class I molecules

The H-2Ld alloreactive 2C T cell receptor (TCR) is commonly considered as being positively selected on the H-2Kb molecule. Surprisingly, 2C TCR+ CD8+ single-positive T cells emerge in massive numbers in fetal thymic organ culture originating from 2C transgenic, H-2KbDb−/− (2C+KbDb−/−) but not in fetal thymic organ culture from β2-microglobulin−/− 2C transgenic animals. Mature CD8+ T cells are observed in newborn but not in adult 2C+KbDb−/− mice. These CD8+ T cells express the α4β7 integrin, which allows them to populate the intestine, a pattern of migration visualized by intrathymic injection of FITC and subsequent accrual of FITC-labeled lymphocytes in the gut. We conclude that the 2C TCR is reactive not only with H-2Ld and H-2Kb, but also with nonclassical MHC class I products to enable positive selection of 2C+ T cells in the fetal and newborn thymus and to support their maintenance in the intestine.

Silver ion high pressure liquid chromatography provides unprecedented separation of sterols: application to the enzymatic formation of cholesta-5,8-dien-3 beta-ol.

We report that silver ion HPLC provides remarkable separations of C27 sterols differing only in the number or location of olefinic double bonds. This technique has been extended to LC-MS, analysis of purified components by GC, GC-MS, and 1H NMR, and to its use on a semipreparative scale. The application of this methodology for the demonstration of the catalysis, by rat liver microsomes, of the conversion of 7-dehydrocholesterol to cholesta-5,8-dien-3 beta-ol is also presented.

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: cysteine ligation of the [4Fe-4S] cluster with protein rearrangement is preferred over serine ligation.

The [4Fe-4S] cluster of Azotobacter vinelandii ferredoxin I receives three of its four ligands from a Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys sequence at positions 39-45 while the fourth ligand, Cys20, is provided by a distal portion of the sequence. Previously we reported that the site-directed mutation of Cys20 to Ala (C20A protein) resulted in the formation of a new [4Fe-4S] cluster that obtained its fourth ligand from Cys24, a free cysteine in the native structure. That ligand exchange required significant protein rearrangement. Here we report the conversion of Cys20 to Ser (C20S protein), which gives the protein the opportunity either to retain the native structure and use the Ser20 O gamma as a ligand or to rearrange and use Cys24. X-ray crystallography demonstrates that the cluster does not use the Ser20 O gamma as a ligand; rather it rearranges to use Cys24. In the C20S protein the [4Fe-4S] cluster has altered stability and redox properties relative to either C20A or the native protein.

Construction of a 2.8-megabase yeast artificial chromosome contig and cloning of the human methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p21.

Many human malignant cells lack methylthioadenosine phosphorylase (MTAP) enzyme activity. The gene (MTAP) encoding this enzyme was previously mapped to the short arm of chromosome 9, band p21-22, a region that is frequently deleted in multiple tumor types. To clone candidate tumor suppressor genes from the deleted region on 9p21-22, we have constructed a long-range physical map of 2.8 megabases for 9p21 by using overlapping yeast artificial chromosome and cosmid clones. This map includes the type IIFN gene cluster, the recently identified candidate tumor suppressor genes CDKN2 (p16INK4A) and CDKN2B (p15INK4B), and several CpG islands. In addition, we have identified other transcription units within the yeast artificial chromosome contig. Sequence analysis of a 2.5-kb cDNA clone isolated from a CpG island that maps between the IFN genes and CDKN2 reveals a predicted open reading frame of 283 amino acids followed by 1302 nucleotides of 3' untranslated sequence. This gene is evolutionarily conserved and shows significant amino acid homologies to mouse and human purine nucleoside phosphorylases and to a hypothetical 25.8-kDa protein in the pet gene (coding for cytochrome bc1 complex) region of Rhodospirillum rubrum. The location, expression pattern, and nucleotide sequence of this gene suggest that it codes for the MTAP enzyme.

The structure of Ascaris hemoglobin domain I at 2.2 A resolution: molecular features of oxygen avidity.

The perienteric hemoglobin of the parasitic nematode Ascaris has an exceptionally high affinity for oxygen. It is an octameric protein containing two similar heme-binding domains per subunit, but recombinant constructs expressing a single, monomeric heme-binding domain (domain 1; D1) retain full oxygen avidity. We have solved the crystal structure of D1 at 2.2 A resolution. Analysis of the structure reveals a characteristic globin fold and illuminates molecular features involved in oxygen avidity of Ascaris perienteric hemoglobin. A strong hydrogen bond between tyrosine at position 10 in the B helix (tyrosine-B10) and the distal oxygen of the ligand, combined with a weak hydrogen bond between glutamine-E7 and the proximal oxygen, grips the ligand in the binding pocket. A third hydrogen bond between these two amino acids appears to stabilize the structure. The B helix of D1 is displaced laterally by 2.5 A when compared with sperm whale myoglobin. This shifts the tyrosine-B10 hydroxyl far enough from liganded oxygen to form a strong hydrogen bond without steric hindrance. Changes in the F helix compared with myoglobin contribute to a tilted heme that may also be important for oxygen affinity.

Payment order for Daniel Parker on behalf of his son, 1783 July 10

Handwritten order to John Sale to pay scholarship funds to Daniel Parker for use by his son, signed by Charles Chauncey, John Clarke, Jonathan Williams, James Thwing, and Jacob Williams.

Lost in Translation? Russian Media Portrayals and Laypersons’ Interpretations of Angelina Jolie’s Breast Cancer Discourse

In May 2013, Angelina Jolie revealed that because she had a family history of breast and ovarian cancer and carried a rare BRCA gene mutation, she had undergone a preventive double mastectomy. Media coverage has been extensive around the world, including in Russia, not an English-language country, where all global news is inevitably filtered by translation. After examining the reactions of Russian mass media and members of the public to Jolie’s disclosure, I consider what transformations have occurred with Jolie’s message in the process of cross-cultural transfer. I explore the mass media portrayal of Jolie’s announcement, laypersons’ immediate and prolonged reactions, and the reflections of patients involved directly in the field of hereditary breast cancer. To my knowledge, this multifaceted and bilingual project is the first conceptualization of Jolie’s story as it has been translated in a different sociocultural environment. I start with examination of offline and online publications that appeared in Russia within two months after Jolie’s announcement. In this part of my analysis, I conceptualize the representation of Jolie’s case in Russian mass media and grasp what sociocultural waves were generated by this case among general lay audiences. Another part of my study contains the results of qualitative in-depth interviews. Eight women with a family history of hereditary breast cancer were recruited to participate in the research. The findings represent Jolie’s case through the eyes of Russian women with the same gene mutation as Jolie. Consolidating my findings, I argue that Jolie’s announcement was misinterpreted and misrepresented by Russian mass media, as well as misunderstood by a considerable part of the media audience. Jolie’s perspective on hereditary breast cancer mostly remained unheard among members of the Russian public. I make suggestions about the reasons for such a phenomenon, and demonstrate how Jolie’s case is implicated in politics, economics, and the culture of contemporary Russia.

British Balance of Competence Reviews, Part I: ‘Competences about right, so far’. EPIN Working Paper No. 35, October 2013

The first in a series for a CEPS-EPIN project entitled “The British Question and the Search for a Fresh European Narrative” this paper is pegged on an ambitious ongoing exercise by the British government to review all the competences of the European Union. The intention is that this should provide a basis for informed debate before the referendum on the UK remaining in the EU or not, which is scheduled for 2017. This paper summarises the first six reviews, each of which runs to around 80 pages, covering foreign policy, development policy, taxation, the single market, food safety, and public health. The present authors then add their own assessments of these materials. While understandably giving due place to British interests, they are of general European relevance. The substantive conclusions of this first set of reviews are that the competences of the EU are judged by respondents to be ‘about right’ on the whole, which came as a surprise to eurosceptic MPs and the tabloid media. Our own view is that the reviews are objective and impressively researched, and these populist complaints are illustrating the huge gap between the views of informed stakeholders and general public opinion, and therefore also the hazard of subjecting the ‘in or out’ choice for decision by referendum. If the referendum is to endorse the UK’s continuing membership there will have to emerge some fresh popular narratives about the EU. The paper therefore concludes with some thoughts along these lines, both for the UK and the EU as a whole.