Deoxynucleoside salvage facilitates DNA repair during ribonucleotide reductase blockade in human cervical cancers

Cells generate 2'-deoxyribonucleoside triphosphates (dNTPs) for both replication and repair of damaged DNA predominantly through de novo reduction of intracellular ribonucleotides by ribonucleotide reductase (RNR). Cells can also salvage deoxynucleosides by deoxycytidine kinase/thymidine kinase 1 in the cytosol or by deoxyguanosine kinase/thymidine kinase 2 in mitochondria. In this study we investigated whether the salvage dNTP supply pathway facilitates DNA damage repair, promoting cell survival, when pharmacological inhibition of RNR by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC no.?663249) impairs the de novo pathway. Human cervical cancer cells were subjected to radiation with or without 3-AP under medium deoxynucleoside concentrations of 0, 0.05, 0.5 and 5.0?μM. Efficacy of DNA damage repair was assessed by γ-H2AX flow cytometry and focus counts, by single cell electrophoresis (Comet assay), and by caspase 3 cleavage assay as a marker of treatment-induced apoptosis. Cell survival was assessed by colony formation. We found that deoxyribonucleotide salvage facilitates DNA repair during RNR inhibition by 3-AP and that salvage reduces the radiochemosensitivity of human cervical cancer cells.     

Genome-wide association mapping identifies the genetic basis of discrete and quantitative variation in sexual weaponry in a wild sheep population

Understanding the genetic architecture of phenotypic variation in natural populations is a fundamental goal of evolutionary genetics. Wild Soay sheep (Ovis aries) have an inherited polymorphism for horn morphology in both sexes, controlled by a single autosomal locus, Horns. The majority of males have large normal horns, but a small number have vestigial, deformed horns, known as scurs; females have either normal horns, scurs or no horns (polled). Given that scurred males and polled females have reduced fitness within each sex, it is counterintuitive that the polymorphism persists within the population. Therefore, identifying the genetic basis of horn type will provide a vital foundation for understanding why the different morphs are maintained in the face of natural selection. We conducted a genome-wide association study using ～36000 single nucleotide polymorphisms (SNPs) and determined the main candidate for Horns as RXFP2, an autosomal gene with a known involvement in determining primary sex characters in humans and mice. Evidence from additional SNPs in and around RXFP2 supports a new model of horn-type inheritance in Soay sheep, and for the first time, sheep with the same horn phenotype but different underlying genotypes can be identified. In addition, RXFP2 was shown to be an additive quantitative trait locus (QTL) for horn size in normal-horned males, accounting for up to 76% of additive genetic variation in this trait. This finding contrasts markedly from genome-wide association studies of quantitative traits in humans and some model species, where it is often observed that mapped loci only explain a modest proportion of the overall genetic variation.     

Investigating membrane protein dynamics in living cells.

Live cell imaging is a powerful tool for understanding the function and regulation of membrane proteins. In this review, we briefly discuss 4 fluorescence-microscopy-based techniques for studying the transport dynamics of membrane proteins: fluorescence-correlation spectroscopy, image-correlation spectroscopy, fluorescence recovery after photobleaching, and single-particle and (or) molecule tracking. The advantages and limitations of each approach are illustrated using recent studies of an ion channel and cell adhesion molecules.     

Actin-myosin network reorganization breaks symmetry at the cell rear to spontaneously initiate polarized cell motility

We have analyzed the spontaneous symmetry breaking and initiation of actin-based motility in keratocytes (fish epithelial cells). In stationary keratocytes, the actin network flow was inwards and radially symmetric. Immediately before motility initiation, the actin network flow increased at the prospective cell rear and reoriented in the perinuclear region, aligning with the prospective axis of movement. Changes in actin network flow at the cell front were detectable only after cell polarization. Inhibition of myosin II or Rho kinase disrupted actin network organization and flow in the perinuclear region and decreased the motility initiation frequency, whereas increasing myosin II activity with calyculin A increased the motility initiation frequency. Local stimulation of myosin activity in stationary cells by the local application of calyculin A induced directed motility initiation away from the site of stimulation. Together, these results indicate that large-scale actin-myosin network reorganization and contractility at the cell rear initiate spontaneous symmetry breaking and polarized motility of keratocytes.     

CD4+ T cell-specific deletion of IL-4 receptor alpha prevents ovalbumin-induced anaphylaxis by an IFN-gamma-dependent mechanism

IL-4Ralpha-mediated STAT6 activation serves an essential role in various animal models of allergy and asthma at both the sensitization and effector phases. IL-4 and IL-13 signaling via the IL-4Ralpha chain exacerbates murine anaphylaxis, but the cell-specific requirements for IL-4Ralpha expression are unclear. The purpose of this study was to elucidate the mechanisms of systemic anaphylaxis to OVA in gene-targeted mice with a deletion of the IL-4Ralpha chain in the macrophage/neutrophil or CD4+ T lymphocyte population. Results demonstrated that anaphylaxis in this model was entirely dependent upon the FcgammaRII/III and was associated with mast cell degranulation. Expression of the IL-4Ralpha on CD4+ T cells, but not macrophages or neutrophils, was critical for severe anaphylaxis, characterized by diarrhea, hypothermia, and death. Ab depletion experiments demonstrated that IFN-gamma protected against mortality and severe intestinal pathology despite the presence of Ag and specific Ab. This protection was associated with reduced levels of mast cell protease, a marker of mast cell degranulation, suggesting that IFN-gamma may inhibit mast cell degranulation in vivo. These data suggest that it may be possible to limit the severity of anaphylaxis using rational therapies designed to increase numbers of IFN-gamma-producing cells by targeting IL-4Ralpha signaling in CD4+ T lymphocytes.     

Positive Selection in the N-Terminal Extramembrane Domain of Lung Surfactant Protein C (SP-C) in Marine Mammals

Maximum-likelihood models of codon and amino acid substitution were used to analyze the lung-specific surfactant protein C (SP-C) from terrestrial, semi-aquatic, and diving mammals to identify lineages and amino acid sites under positive selection. Site models used the nonsynonymous/synonymous rate ratio (ω) as an indicator of selection pressure. Mechanistic models used physicochemical distances between amino acid substitutions to specify nonsynonymous substitution rates. Site models strongly identified positive selection at different sites in the polar N-terminal extramembrane domain of SP-C in the three diving lineages: site 2 in the cetaceans (whales and dolphins), sites 7, 9, and 10 in the pinnipeds (seals and sea lions), and sites 2, 9, and 10 in the sirenians (dugongs and manatees). The only semi-aquatic contrast to indicate positive selection at site 10 was that including the polar bear, which had the largest body mass of the semi-aquatic species. Analysis of the biophysical properties that were influential in determining the amino acid substitutions showed that isoelectric point, chemical composition of the side chain, polarity, and hydrophobicity were the crucial determinants. Amino acid substitutions at these sites may lead to stronger binding of the N-terminal domain to the surfactant phospholipid film and to increased adsorption of the protein to the air-liquid interface. Both properties are advantageous for the repeated collapse and reinflation of the lung upon diving and resurfacing and may reflect adaptations to the high hydrostatic pressures experienced during diving. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Reviewing Editor: Dr. Richard Kliman     

Simian immunodeficiency virus SIVmac239 infection of major histocompatibility complex-identical cynomolgus macaques from Mauritius

Nonhuman primates are widely used to study correlates of protective immunity in AIDS research. Successful cellular immune responses have been difficult to identify because heterogeneity within macaque major histocompatibility complex (MHC) genes results in quantitative and qualitative differences in immune responses. Here we use microsatellite analysis to show that simian immunodeficiency virus (SIV)-susceptible cynomolgus macaques (Macaca fascicularis) from the Indian Ocean island of Mauritius have extremely simple MHC genetics, with six common haplotypes accounting for two-thirds of the MHC haplotypes in feral animals. Remarkably, 39% of Mauritian cynomolgus macaques carry at least one complete copy of the most frequent MHC haplotype, and 8% of these animals are homozygous. In stark contrast, entire MHC haplotypes are rarely conserved in unrelated Indian rhesus macaques. After intrarectal infection with highly pathogenic SIVmac239 virus, a pair of MHC-identical Mauritian cynomolgus macaques mounted concordant cellular immune responses comparable to those previously reported for a pair of monozygotic twins infected with the same strain of human immunodeficiency virus. Our identification of relatively abundant SIV-susceptible, MHC-identical macaques will facilitate research into protective cellular immunity.     

Role of human immunodeficiency virus (HIV)-specific T-cell immunity in control of dual HIV-1 and HIV-2 infection

Progressive immune dysfunction and AIDS develop in most cases of human immunodeficiency virus type 1 (HIV-1) infection but in only 25 to 30% of persons with HIV-2 infection. However, the natural history and immunologic responses of individuals with dual HIV-1 and HIV-2 infection are largely undefined. Based on our previous findings, we hypothesized that among patients with dual infection the control of HIV-1 is associated with the ability to respond to HIV-2 Gag epitopes and to maintain HIV-specific CD4⁺ T-cell responses. To test this, we compared the HIV-specific ex vivo IFN-γ enzyme-linked immunospot (ELISPOT) assay responses of 19 dually infected individuals to those of persons infected with HIV-1 or HIV-2 only. Further, we assessed the functional profile of HIV Gag-specific CD4⁺ and CD8⁺ T cells from nine HIV dually infected patients by using a multicolor intracellular cytokine staining assay. As determined by ELISPOT assay, the magnitude and frequency of IFN-γ-secreting T-cell responses to gene products of HIV-1 were higher than those to gene products of HIV-2 (2.64 versus 1.53 log₁₀ IFN-γ spot-forming cells/10⁶ cells [90% versus 63%, respectively].) Further, HIV-1 Env-, Gag-, and Nef- and HIV-2 Gag-specific responses were common; HIV-2 Nef-specific responses were rare. HIV-specific CD4⁺ T helper responses were detected in nine of nine dually infected subjects, with the majority of these T cells producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and, to a lesser extent, interleukin-2. The HIV-1 plasma viral load was inversely correlated with HIV-2 Gag-specific IFN-γ-/TNF-α-secreting CD4⁺ and HIV-2 Gag-specific IFN-γ-secreting CD8⁺ T cells. In conclusion, the T-cell memory responses associated with containment of single HIV-1 and HIV-2 infection play a similar significant role in the immune control of dual HIV-1 and HIV-2 infection.     

Dimerization of Hsp90 is required for in vivo function. Design and analysis of monomers and dimers

Heat shock protein 90 (Hsp90) plays a central role in signal transduction and has emerged as a promising target for anti-cancer therapeutics, but its molecular mechanism is poorly understood. At physiological concentration, Hsp90 predominantly forms dimers, but the function of full-length monomers in cells is not clear. Hsp90 contains three domains: the N-terminal and middle domains contribute directly to ATP binding and hydrolysis and the C domain mediates dimerization. To study the function of Hsp90 monomers, we used a single-chain strategy that duplicated the C-terminal dimerization domain. This novel monomerization strategy had the dual effect of stabilizing the C domain to denaturation and hindering intermolecular association of the ATPase domain. The resulting construct was predominantly monomeric at physiological concentration and did not function to support yeast viability as the sole Hsp90. The monomeric construct was also defective at ATP hydrolysis and the activation of a kinase and steroid receptor substrate in yeast cells. The ability to support yeast growth was rescued by the addition of a coiled-coil dimerization domain, indicating that the parental single-chain construct is functionally defective because it is monomeric.     

Comprehensive characterization of MHC class II haplotypes in Mauritian cynomolgus macaques

There are currently no nonhuman primate models with fully defined major histocompatibility complex (MHC) class II genetics. We recently showed that six common MHC haplotypes account for essentially all MHC diversity in cynomolgus macaques (Macaca fascicularis) from the island of Mauritius. In this study, we employ complementary DNA cloning and sequencing to comprehensively characterize full length MHC class II alleles expressed at the Mafa-DPA, -DPB, -DQA, -DQB, -DRA, and -DRB loci on the six common haplotypes. We describe 34 full-length MHC class II alleles, 12 of which are completely novel. Polymorphism was evident at all six loci including DPA, a locus thought to be monomorphic in rhesus macaques. Similar to other Old World monkeys, Mauritian cynomolgus macaques (MCM) share MHC class II allelic lineages with humans at the DQ and DR loci, but not at the DP loci. Additionally, we identified extensive sharing of MHC class II alleles between MCM and other nonhuman primates. The characterization of these full-length-expressed MHC class II alleles will enable researchers to generate MHC class II transferent cell lines, tetramers, and other molecular reagents that can be used to explore CD4+ T lymphocyte responses in MCM.