Differential distribution of a SINE element in the Entamoeba histolytica and Entamoeba dispar genomes: Role of the LINE-encoded endonuclease

<p>Abstract</p> <p>Background</p> <p><it>Entamoeba histolytica </it>and <it>Entamoeba dispar </it>are closely related protistan parasites but while <it>E. histolytica </it>can be invasive, <it>E. dispar </it>is completely non pathogenic. Transposable elements constitute a significant portion of the genome in these species; there being three families of LINEs and SINEs. These elements can profoundly influence the expression of neighboring genes. Thus their genomic location can have important phenotypic consequences. A genome-wide comparison of the location of these elements in the <it>E. histolytica </it>and <it>E. dispar </it>genomes has not been carried out. It is also not known whether the retrotransposition machinery works similarly in both species. The present study was undertaken to address these issues.</p> <p>Results</p> <p>Here we extracted all genomic occurrences of full-length copies of EhSINE1 in the <it>E. histolytica </it>genome and matched them with the homologous regions in <it>E. dispar</it>, and vice versa, wherever it was possible to establish synteny. We found that only about 20% of syntenic sites were occupied by SINE1 in both species. We checked whether the different genomic location in the two species was due to differences in the activity of the LINE-encoded endonuclease which is required for nicking the target site. We found that the endonucleases of both species were essentially very similar, both in their kinetic properties and in their substrate sequence specificity. Hence the differential distribution of SINEs in these species is not likely to be influenced by the endonuclease. Further we found that the physical properties of the DNA sequences adjoining the insertion sites were similar in both species.</p> <p>Conclusions</p> <p>Our data shows that the basic retrotransposition machinery is conserved in these sibling species. SINEs may indeed have occupied all of the insertion sites in the genome of the common ancestor of <it>E. histolytica </it>and <it>E. dispar </it>but these may have been subsequently lost from some locations. Alternatively, SINE expansion took place after the divergence of the two species. The absence of SINE1 in 80% of syntenic loci could affect the phenotype of the two species, including their pathogenic properties, which needs to be explored.</p

Interactions of the Human MCM-BP Protein with MCM Complex Components and Dbf4

MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK

Crystal Structure of a Charge Engineered Human Lysozyme Having Enhanced Bactericidal Activity

Human lysozyme is a key component of the innate immune system, and recombinant forms of the enzyme represent promising leads in the search for therapeutic agents able to treat drug-resistant infections. The wild type protein, however, fails to participate effectively in clearance of certain infections due to inherent functional limitations. For example, wild type lysozymes are subject to electrostatic sequestration and inactivation by anionic biopolymers in the infected airway. A charge engineered variant of human lysozyme has recently been shown to possess improved antibacterial activity in the presence of disease associated inhibitory molecules. Here, the 2.04 Å crystal structure of this variant is presented along with an analysis that provides molecular level insights into the origins of the protein's enhanced performance. The charge engineered variant's two mutated amino acids exhibit stabilizing interactions with adjacent native residues, and from a global perspective, the mutations cause no gross structural perturbations or loss of stability. Importantly, the two substitutions dramatically expand the negative electrostatic potential that, in the wild type enzyme, is restricted to a small region near the catalytic residues. The net result is a reduction in the overall strength of the engineered enzyme's electrostatic potential field, and it appears that the specific nature of this remodeled field underlies the variant's reduced susceptibility to inhibition by anionic biopolymers

An Estimate of Avian Mortality at Communication Towers in the United States and Canada

Avian mortality at communication towers in the continental United States and Canada is an issue of pressing conservation concern. Previous estimates of this mortality have been based on limited data and have not included Canada. We compiled a database of communication towers in the continental United States and Canada and estimated avian mortality by tower with a regression relating avian mortality to tower height. This equation was derived from 38 tower studies for which mortality data were available and corrected for sampling effort, search efficiency, and scavenging where appropriate. Although most studies document mortality at guyed towers with steady-burning lights, we accounted for lower mortality at towers without guy wires or steady-burning lights by adjusting estimates based on published studies. The resulting estimate of mortality at towers is 6.8 million birds per year in the United States and Canada. Bootstrapped subsampling indicated that the regression was robust to the choice of studies included and a comparison of multiple regression models showed that incorporating sampling, scavenging, and search efficiency adjustments improved model fit. Estimating total avian mortality is only a first step in developing an assessment of the biological significance of mortality at communication towers for individual species or groups of species. Nevertheless, our estimate can be used to evaluate this source of mortality, develop subsequent per-species mortality estimates, and motivate policy action

Aged PROP1 Deficient Dwarf Mice Maintain ACTH Production

Humans with PROP1 mutations have multiple pituitary hormone deficiencies (MPHD) that typically advance from growth insufficiency diagnosed in infancy to include more severe growth hormone (GH) deficiency and progressive reduction in other anterior pituitary hormones, eventually including adrenocorticotropic hormone (ACTH) deficiency and hypocortisolism. Congenital deficiencies of GH, prolactin, and thyroid stimulating hormone have been reported in the Prop1null (Prop1-/-) and the Ames dwarf (Prop1df/df) mouse models, but corticotroph and pituitary adrenal axis function have not been thoroughly investigated. Here we report that the C57BL6 background sensitizes mutants to a wasting phenotype that causes approximately one third to die precipitously between weaning and adulthood, while remaining homozygotes live with no signs of illness. The wasting phenotype is associated with severe hypoglycemia. Circulating ACTH and corticosterone levels are elevated in juvenile and aged Prop1 mutants, indicating activation of the pituitary-adrenal axis. Despite this, young adult Prop1 deficient mice are capable of responding to restraint stress with further elevation of ACTH and corticosterone. Low blood glucose, an expected side effect of GH deficiency, is likely responsible for the elevated corticosterone level. These studies suggest that the mouse model differs from the human patients who display progressive hormone loss and hypocortisolism

Zinc Coordination Is Required for and Regulates Transcription Activation by Epstein-Barr Nuclear Antigen 1

Epstein-Barr Nuclear Antigen 1 (EBNA1) is essential for Epstein-Barr virus to immortalize naïve B-cells. Upon binding a cluster of 20 cognate binding-sites termed the family of repeats, EBNA1 transactivates promoters for EBV genes that are required for immortalization. A small domain, termed UR1, that is 25 amino-acids in length, has been identified previously as essential for EBNA1 to activate transcription. In this study, we have elucidated how UR1 contributes to EBNA1's ability to transactivate. We show that zinc is necessary for EBNA1 to activate transcription, and that UR1 coordinates zinc through a pair of essential cysteines contained within it. UR1 dimerizes upon coordinating zinc, indicating that EBNA1 contains a second dimerization interface in its amino-terminus. There is a strong correlation between UR1-mediated dimerization and EBNA1's ability to transactivate cooperatively. Point mutants of EBNA1 that disrupt zinc coordination also prevent self-association, and do not activate transcription cooperatively. Further, we demonstrate that UR1 acts as a molecular sensor that regulates the ability of EBNA1 to activate transcription in response to changes in redox and oxygen partial pressure (pO2). Mild oxidative stress mimicking such environmental changes decreases EBNA1-dependent transcription in a lymphoblastoid cell-line. Coincident with a reduction in EBNA1-dependent transcription, reductions are observed in EBNA2 and LMP1 protein levels. Although these changes do not affect LCL survival, treated cells accumulate in G0/G1. These findings are discussed in the context of EBV latency in body compartments that differ strikingly in their pO2 and redox potential

The mediating effects of first call resolution on call centers’ performance

This article aims to examine and validate the prepositions of the mediating impacts of first call resolution (FCR) on caller satisfaction within the contact center industry.A survey of 168 call center managers was analyzed through structural equation modeling, constituting an overall 43.3 per cent response rate for this study.The results show that FCR positively mediates the relationship between knowledge management, technology-based CRM and caller satisfaction within the inbound customer contact centers.We have empirically assessed call centers/contact centers’ success through caller satisfaction (an observed variable through their 2009 customer survey in Malaysia).Consequently, this study cannot generalize its findings in all other countries.Our strong argument is that within the operational variables, FCR is statistically significant and positively mediates knowledge management applications. But very important to note is that the customer contact centers are first touch points to a company's goods or services, and that many other factors such as product quality, company policy, target markets, decision-making processes and so on are also determinants of caller satisfaction, but fall outside the operational control of contact center activities.This research has empirically established that a company's capability in effectively acquiring a valid understanding of its current and potential customers’ information through CRM technologies will positively impact its acquisitions, customization, management and retention of customers.It also avails both the academic and contact center management the benefits that are inherent in measuring the impact of knowledge management and technology-based CRM on inbound FCR and caller satisfaction.This study finally recommends alternative areas for future research