MED: Genetics & Genomics Papers

Evidence for a "Wattle and Daub" Model of the Cyst Wall of Entamoeba

2012-01-12T07:01:54Z

Evidence for a "Wattle and Daub" Model of the Cyst Wall of Entamoeba Chatterjee, Anirban; Ghosh, Sudip K.; Jang, Ken; Bullitt, Esther; Moore, Landon; Robbins, Phillips W.; Samuelson, John The cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, is composed of fibrils of chitin and three chitin-binding lectins called Jacob, Jessie3, and chitinase. Here we show chitin, which was detected with wheat germ agglutinin, is made in secretory vesicles prior to its deposition on the surface of encysting Ei. Jacob lectins, which have tandemly arrayed chitin-binding domains (CBDs), and chitinase, which has an N-terminal CBD, were each made early during encystation. These results are consistent with their hypothesized roles in cross-linking chitin fibrils (Jacob lectins) and remodeling the cyst wall (chitinase). Jessie3 lectins likely form the mortar or daub of the cyst wall, because 1) Jessie lectins were made late during encystation; 2) the addition to Jessie lectins to the cyst wall correlated with a marked decrease in the permeability of cysts to nucleic acid stains (DAPI) and actin-binding heptapeptide (phalloidin); and 3) recombinant Jessie lectins, expressed as a maltose-binding proteins in the periplasm of Escherichia coli, caused transformed bacteria to agglutinate in suspension and form a hard pellet that did not dissociate after centrifugation. Jessie3 appeared as linear forms and rosettes by negative staining of secreted recombinant proteins. These findings provide evidence for a "wattle and daub" model of the Entamoeba cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins). Author SummaryParasitic protists, which are spread by the fecal-oral route, have cyst walls that resist environmental insults (e.g. desiccation, stomach acids, bile, etc.). Entamoeba histolytica, the cause of amebic dysentery and liver abscess, is the only protist characterized to date that has chitin in its cyst wall. We have previously characterized Entamoeba chitin synthases, chitinases, and multivalent chitin-binding lectins called Jacob. Here we present evidence that the Entamoeba Jessie3 lectin contributes to the mortar or daub, which makes the cyst wall impenetrable to small molecules. First, the Jessie3 lectin was made after chitin and Jacob lectins had already been deposited onto the surface of encysting Entamoeba. Second, cysts became impenetrable to small molecules at the same time that Jessie3 was deposited into the wall. Third, recombinant Jessie3 lectins self-aggregated and caused transfected bacteria to agglutinate. These results suggest a "wattle and daub" model of the Ei cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins).

Cofilin Activation in Peripheral CD4 T Cells of HIV-1 Infected Patients: A Pilot Study

2012-01-12T07:01:43Z

Cofilin Activation in Peripheral CD4 T Cells of HIV-1 Infected Patients: A Pilot Study Wu, Yuntao; Yoder, Alyson; Yu, Dongyang; Wang, Weifeng; Liu, Juan; Barrett, Tracey; Wheeler, David; Schlauch, Karen Cofilin is an actin-depolymerizing factor that regulates actin dynamics critical for T cell migration and T cell activation. In unstimulated resting CD4 T cells, cofilin exists largely as a phosphorylated inactive form. Previously, we demonstrated that during HIV-1 infection of resting CD4 T cells, the viral envelope-CXCR4 signaling activates cofilin to overcome the static cortical actin restriction. In this pilot study, we have extended this in vitro observation and examined cofilin phosphorylation in resting CD4 T cells purified from the peripheral blood of HIV-1-infected patients. Here, we report that the resting T cells from infected patients carry significantly higher levels of active cofilin, suggesting that these resting cells have been primed in vivo in cofilin activity to facilitate HIV-1 infection. HIV-1-mediated aberrant activation of cofilin may also lead to abnormalities in T cell migration and activation that could contribute to viral pathogenesis.

Telomere Disruption Results in Non-Random Formation of De Novo Dicentric Chromosomes Involving Acrocentric Human Chromosomes

2012-01-12T07:01:41Z

Telomere Disruption Results in Non-Random Formation of De Novo Dicentric Chromosomes Involving Acrocentric Human Chromosomes Stimpson, Kaitlin M.; Song, Ihn Young; Jauch, Anna; Holtgreve-Grez, Heidi; Hayden, Karen E.; Bridger, Joanna M.; Sullivan, Beth A. Genome rearrangement often produces chromosomes with two centromeres (dicentrics) that are inherently unstable because of bridge formation and breakage during cell division. However, mammalian dicentrics, and particularly those in humans, can be quite stable, usually because one centromere is functionally silenced. Molecular mechanisms of centromere inactivation are poorly understood since there are few systems to experimentally create dicentric human chromosomes. Here, we describe a human cell culture model that enriches for de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. The induced dicentrics vary in structure near fusion breakpoints and like naturally-occurring dicentrics, exhibit various inter-centromeric distances. Many functional dicentrics persist for months after formation. Even those with distantly spaced centromeres remain functionally dicentric for 20 cell generations. Other dicentrics within the population reflect centromere inactivation. In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the α-satellite DNA array associated with CENP-A is reduced compared to the same array before dicentric formation. Extra-chromosomal fragments that contained CENP-A often appear in the same cells as dicentrics. Some of these fragments are derived from the same α-satellite DNA array as inactivated centromeres. Our results indicate that dicentric human chromosomes undergo alternative fates after formation. Many retain two active centromeres and are stable through multiple cell divisions. Others undergo centromere inactivation. This event occurs within a broad temporal window and can involve deletion of chromatin that marks the locus as a site for CENP-A maintenance/replenishment. Author Summary Endogenous human centromeres are defined by large arrays of α-satellite DNA. A portion of each α-satellite array is assembled into CENP-A chromatin, the structural and functional platform for kinetochore formation. Most chromosomes are monocentric, meaning they have a single centromere. However, genome rearrangement can produce chromosomes with two centromeres (dicentrics). In most organisms, dicentrics typically break during cell division; however, dicentric human chromosomes can be stable in mitosis and meiosis. This stability reflects centromere inactivation, a poorly understood phenomenon in which one centromere is functionally silenced. To explore molecular and genomic events that occur at the time of dicentric formation, we describe a cell-based system to create dicentric human chromosomes and monitor their behavior after formation. Such dicentrics can experience several fates, including centromere inactivation, breakage, or maintaining two functional centromeres. Unexpectedly, we also find that dicentrics with large (<20Mb) inter-centromeric distances are stable through at least 20 cell divisions. Our results highlight similarities and differences in dicentric behavior between humans and model organisms, and they provide evidence for one mechanism of centromere inactivation by centromeric deletion in some dicentrics. The ability to create dicentric human chromosomes provides a system to test other mechanisms of centromere disassembly and dicentric chromosome stability.

Transferability and Fine-Mapping of Genome-Wide Associated Loci for Adult Height across Human Populations

2012-01-12T07:01:41Z

Transferability and Fine-Mapping of Genome-Wide Associated Loci for Adult Height across Human Populations Shriner, Daniel; Adeyemo, Adebowale; Gerry, Norman P.; Herbert, Alan; Chen, Guanjie; Doumatey, Ayo; Huang, Hanxia; Zhou, Jie; Christman, Michael F.; Rotimi, Charles N. Human height is the prototypical polygenic quantitative trait. Recently, several genetic variants influencing adult height were identified, primarily in individuals of East Asian (Chinese Han or Korean) or European ancestry. Here, we examined 152 genetic variants representing 107 independent loci previously associated with adult height for transferability in a well-powered sample of 1,016 unrelated African Americans. When we tested just the reported variants originally identified as associated with adult height in individuals of East Asian or European ancestry, only 8.3% of these loci transferred (p-values=0.05 under an additive genetic model with directionally consistent effects) to our African American sample. However, when we comprehensively evaluated all HapMap variants in linkage disequilibrium (r2=0.3) with the reported variants, the transferability rate increased to 54.1%. The transferability rate was 70.8% for associations originally reported as genome-wide significant and 38.0% for associations originally reported as suggestive. An additional 23 loci were significantly associated but failed to transfer because of directionally inconsistent effects. Six loci were associated with adult height in all three groups. Using differences in linkage disequilibrium patterns between HapMap CEU or CHB reference data and our African American sample, we fine-mapped these six loci, improving both the localization and the annotation of these transferable associations.