A new phylogenetic marker,apolipoprotein B,provides compelling evidence for eutherian relationships

Higher-level relationships within, and the root of Placentalia, remain contentious issues. Resolution of the placental tree is important to the choice of mammalian genome projects and model organisms, as well as for understanding the biogeography of the eutherian radiation. We present phylogenetic analyses of 63 species representing all extant eutherian mammal orders for a new molecular phylogenetic marker, a 1.3kb portion of exon 26 of the apolipoprotein B (APOB) gene. In addition, we analyzed a multigene concatenation that included APOB sequences and a previously published data set (Murphy et al., 2001b) of three mitochondrial and 19 nuclear genes, resulting in an alignment of over 17kb for 42 placentals and two marsupials. Due to computational difficulties, previous maximum likelihood analyses of large, multigene concatenations for placental mammals have used quartet puzzling, less complex models of sequence evolution, or phylogenetic constraints to approximate a full maximum likelihood bootstrap. Here, we utilize a Unix load sharing facility to perform maximum likelihood bootstrap analyses for both the APOB and concatenated data sets with a GTR+Gamma+I model of sequence evolution, tree-bisection and reconnection branch-swapping, and no phylogenetic constraints. Maximum likelihood and Bayesian analyses of both data sets provide support for the superordinal clades Boreoeutheria, Euarchontoglires, Laurasiatheria, Xenarthra, Afrotheria, and Ostentoria (pangolins+carnivores), as well as for the monophyly of the orders Eulipotyphla, Primates, and Rodentia, all of which have recently been questioned. Both data sets recovered an association of Hippopotamidae and Cetacea within Cetartiodactyla, as well as hedgehog and shrew within Eulipotyphla. APOB showed strong support for an association of tarsier and Anthropoidea within Primates. Parsimony, maximum likelihood and Bayesian analyses with both data sets placed Afrotheria at the base of the placental radiation. Statistical tests that employed APOB to examine a priori hypotheses for the root of the placental tree rejected rooting on myomorphs and hedgehog, but did not discriminate between rooting at the base of Afrotheria, at the base of Xenarthra, or between Atlantogenata (Xenarthra+Afrotheria) and Boreoeutheria. An orthologous deletion of 363bp in the aligned APOB sequences proved phylogenetically informative for the grouping of the order Carnivora with the order Pholidota into the superordinal clade Ostentoria. A smaller deletion of 237-246bp was diagnostic of the superordinal clade Afrotheria.     

Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will ‘snap’ Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to ‘lock’ gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules.     

Overexpression of A(3) adenosine receptors decreases heart rate,preserves energetics,and protects ischemic hearts

To determine whether A(3) adenosine receptor (A(3)AR) signaling modulates myocardial function, energetics, and cardioprotection, hearts from wild-type and A(3)AR-overexpressor mice were subjected to 20-min ischemia and 40-min reperfusion while (31)P NMR spectra were acquired. Basal heart rate and left ventricular developed pressure (LVDP) were lower in A(3)AR-overexpressor hearts than wild-type hearts. Ischemic ATP depletion was delayed and postischemic recoveries of contractile function, ATP, and phosphocreatine were greater in A(3)AR-hearts. To determine the role of depressed heart rate and to confirm A(3)AR-specific signaling, hearts were paced at 480 beats/min with or without 60 nmol/l MRS-1220 (A(3)AR-specific inhibitor) and then subjected to ischemia-reperfusion. LVDP was similar in paced A(3)AR-overexpressor and paced wild-type hearts. Differences in ischemic ATP depletion and postischemic contractile and energetic dysfunction remained in paced A(3)AR-overexpressor hearts versus paced wild-type hearts but were abolished by MRS-1220. In summary, A(3)AR overexpression decreased basal heart rate and contractility, preserved ischemic ATP, and decreased postischemic dysfunction. Pacing abolished the decreased contractility but not the ATP preservation or cardioprotection. Therefore, A(3)AR overexpression results in cardioprotection via a specific A(3)AR effect, possibly involving preservation of ATP during ischemia.     

Effect of mutation on helper T-cells and viral population: A computer simulation model for HIV

Summary A Monte Carlo simulation is proposed to study the dynamics of helper T-cells (N _H) and viral (N _V) populations in an immune response model relevant to HIV. Cellular states are binary variables and the interactions are described by logical expressions. Viral population shows a nonmonotonic growth before reaching a constant value while helper T-cells grow to a constant after a relaxation/reaction time. Initially, the population of helper cells grows with time with a power-law, N _H ∼t ^β, before reaching the steady-state; the growth exponent β increases systematically (β ≈ 1 – 2) with the mutation rate (P _mut≈0.1–0.4). The critical recovery time (t _c) increases exponentially with the viral mutation, t _c≈Ae ^αP _mut , with α=4.52±0.29 in low mutation regime and α=15.21±1.41 in high mutation regime. The equilibrium population of helper T-cell declines slowly with P _mut and collapses at ∼ 0.40; the viral population exhibits a reverse trend, i.e., a slow increase before the burst around the same mutation regime.     

STAT3 modulates CD4+ T mitochondrial dynamics and function in aging

Aging promotes numerous intracellular changes in T cells that impact their effector function. Our data show that aging promotes an increase in the localization of STAT3 to the mitochondria (mitoSTAT3), which promotes changes in mitochondrial dynamics and function and T-cell cytokine production. Mechanistically, mitoSTAT3 increased the activity of aging T-cell mitochondria by increasing complex II. Limiting mitoSTAT3 using a mitochondria-targeted STAT3 inhibitor, Mtcur-1 lowered complex II activity, prevented age-induced changes in mitochondrial dynamics and function, and reduced Th17 inflammation. Exogenous expression of a constitutively phosphorylated form of STAT3 in T cells from young adults mimicked changes in mitochondrial dynamics and function in T cells from older adults and partially recapitulated aging-related cytokine profiles. Our data show the mechanistic link among mitoSTAT3, mitochondrial dynamics, function, and T-cell cytokine production.     

Multiple modes of ligand recognition: Crystal structures of cyclin-dependent protein kinase 2 in complex with ATP and two inhibitors,olomoucine and isopentenyladenine

Cyclin-dependent kinases (CDKs) are conserved regulators of the eukaryotic cell cycle with different isoforms controlling specific phases of the cell cycle. Mitogenic or growth inhibitory signals are mediated, respectively, by activation or inhibition of CDKs which phosphorylate proteins associated with the cell cycle. The central role of CDKs in cell cycle regulation makes them a potential new target for inhibitory molecules with anti-proliferative and/or anti-neoplastic effects. We describe the crystal structures of the complexes of CDK2 with a weakly specific CDK inhibitor, N6-(δ2-isopentenyl)adenine, and a strongly specific inhibitor, olomoucine. Both inhibitors are adenine derivatives and bind in the adenine binding pocket of CDK2, but in an unexpected and different orientation from the adenine of the authentic ligand ATP. The N6-benzyl substituent in olomoucine binds outside the conserved binding pocket and is most likely responsible for its specificity. The structural information from the CDK2-olomoucine complex will be useful in directing the search for the next generation inhibitors with improved properties. © 1995 Wiley-Liss, Inc.     

SNPs in the neural cell adhesion molecule 1 gene (NCAM1) may be associated with human neural tube defects

Neural tube defects (NTDs) are common birth defects, occurring in approximately 1/1,000 births; both genetic and environmental factors are implicated. To date, no major genetic risk factors have been identified. Throughout development, cell adhesion molecules are strongly implicated in cell–cell interactions, and may play a role in the formation and closure of the neural tube. To evaluate the role of neural cell adhesion molecule 1 (NCAM1) in risk of human NTDs, we screened for novel single-nucleotide polymorphisms (SNPs) within the gene. Eleven SNPs across NCAM1 were genotyped using TaqMan. We utilized a family-based approach to evaluate evidence for association and/or linkage disequilibrium. We evaluated American Caucasian simplex lumbosacral myelomeningocele families (n=132 families) using the family based association test (FBAT) and the pedigree disequilibrium test (PDT). Association analysis revealed a significant association between risk for NTDs and intronic SNP rs2298526 using both the FBAT test (P=0.0018) and the PDT (P=0.0025). Using the HBAT version of the FBAT to look for haplotype association, all pairwise comparisons with SNP rs2298526 were also significant. A replication study set, consisting of 72 additional families showed no significant association; however, the overall trend for overtransmission of the less common allele of SNP rs2298526 remained significant in the combined sample set. In addition, we analyzed the expression pattern of the NCAM1 protein in human embryos, and while NCAM1 is not expressed within the neural tube at the time of closure, it is expressed in the surrounding and later in differentiated neurons of the CNS. These results suggest variations in NCAM1 may influence risk for human NTDs.Other members of NTD Collaborative Group involved in this study are listed in the appendix     

Characterization of in vitro models of SLC30A10 deficiency

BioMetals - Manganese (Mn), an essential metal, can be toxic at elevated levels. In 2012, the first inherited cause of Mn excess was reported in patients with mutations in SLC30A10, a Mn efflux...     

Preconditioning of Caenorhabditis elegans to anoxic insult by inactivation of cholinergic,GABAergic and muscle activity

For most metazoans, oxygen deprivation leads to cell dysfunction and if severe, death. Sublethal stress prior to a hypoxic or anoxic insult (“preconditioning”) can protect cells from subsequent oxygen deprivation. The molecular mechanisms by which sublethal stress can buffer against a subsequent toxic insult and the role of the nervous system in the response are not well understood. We studied the role of neuronal activity preconditioning to oxygen deprivation in Caenorhabditis elegans. Animals expressing the histamine gated chloride channels (HisCl1) in select cell populations were used to temporally and spatially inactivate the nervous system or tissue prior to an anoxic insult. We find that inactivation of the nervous system for 3 h prior to the insult confers resistance to a 48-h anoxic insult in 4th-stage larval animals. Experiments show that this resistance can be attributed to loss of activity in cholinergic and GABAergic neurons as well as in body wall muscles. These observations indicate that the nervous system activity can mediate the organism's response to anoxia.