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In our efforts to understand how transcription may be regulated in Entamoeba histolytica , we have examined if this parasite has conserved enzymatic mechanisms for targeted acetylation and deacetylation of histones. Western blotting indicated that basic nuclear proteins in the size range of 16–23 kDa were acetylated in amebic trophozoites, suggesting histone acetylation. Single representatives of the GNAT and MYST family of histone acetyltransferases (HATs) were identified in the E. histolytica genome and their expression in amebic trophozoites was detected by reverse transcription of RNA followed by the polymerase chain reaction (RT-PCR). Full-length recombinant EhMYST protein demonstrated HAT activity with calf thymus histones and showed a preference for histone H4, similar to the yeast MYST protein, Esa1. However, ehMYST did not complement a yeast esa1 mutation. Histone deacetylase (HDAC) activity was detected in nuclear extracts from E. histolytica , and characteristically, was inhibited by trichostatin A (TSA). Consistent with the observation of HDAC activity, RT-PCR analysis demonstrated that an amebic hdac1 homolog ( ehHDAC ) is expressed and appropriately spliced in E. histolytica trophozoites. Our results suggest that mechanisms for histone acetylation and deacetylation are operational in E. histolytica .

The Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) binds to the URE3 sequence of the Gal/GalNAc-inhibitable lectin hgl5 and ferredoxin 1 (fdx) gene promoters. This binding can be inhibited in vitro by addition of calcium. Two EF-hand motifs, which are associated with the ability to bind calcium, are present in the amino acid sequence of URE3-BP. Mutation of the second EF-hand motif in URE3-BP resulted in the loss of calcium inhibition of DNA binding as monitored by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays revealed that URE3-BP was physically bound to the hgl5 and fdx promoters in vivo. Parasite intracellular calcium concentrations were altered by changes in extracellular calcium. Promoter occupancy was lost when intracellular calcium levels were increased by coordinate increases in extracellular calcium. Increased intracellular calcium also resulted in decreased levels of URE3-BP mRNA. Together these results demonstrate that changes in extracellular calcium result in changes in URE3-BP mRNA and in the ability of URE3-BP to bind to URE3-containing promoters. Modulation of URE3-BP by calcium may represent an important mechanism of control of gene expression in E. histolytica.

To study transcriptional regulation in the lower branching eukaryote Entamoeba histolytica, we have identified two sequence-specific DNA-binding proteins that recognize the upstream regulatory element URE4, an enhancer that regulates expression of the Gal/GalNAc lectin heavy subunit gene hgl5. A chromatographic purification of E. histolytica nuclear extracts by gel filtration, cation exchange, and sequence-specific DNA affinity chromatography led to a 700-fold increase in URE4 binding activity and the appearance of two dominant protein species with molecular masses of 28 and 18 kDa. These proteins, termed E. histolytica enhancer-binding proteins 1 and 2 (EhEBP1 and EhEBP2), were sequenced by tandem mass spectroscopy and their corresponding cDNA clones identified. Recombinant EhEBP1 and EhEBP2 were able to bind double-stranded oligonucleotides bearing the URE4 motif in a sequence-specific manner, and antibodies raised against EhEBP1 were able to interfere with the formation of URE4-protein complexes in crude nuclear extracts. Overexpression of EhEBP1 in E. histolytica trophozoites resulted in a 7-fold drop in promoter activity in transiently transfected reporter gene constructs when the URE4 motif was present, confirming its ability to specifically recognize the URE4 motif and suggesting that additional cofactors may be required for transcriptional activation by URE4. Further characterization and identification of binding partners for EhEBP1 and EhEBP2, the first proteins with demonstrated sequence-specific DNA binding activity to be identified in E. histolytica, should provide new insights into transcriptional regulation in this protozoan parasite.

What are the ethical issues or situations Air Force Nurse Practitioners (AFNPs) experience in clinical practice? Which moral or ethical principles are reflected? Little has been written about the ethical issues faced by AFNPs in clinical practice. The description and exploration of these issues was accomplished in this study through the use of qualitative research methodology. Seven active duty AFNPs provided narrative accounts of ethical issues and situations encountered in their clinical practice. The individual narratives were analyzed and findings were presented using four basic principles of biomedical ethics: respect for autonomy, nonmaleficence, beneficence, and justice. Implications of this study illustrate the need for some type of ethics education in advanced practice nursing curricula. Further implications for use in clinical practice include time management, resource utilization, template creation, and training. More research is needed to determine if the experiences of AFNPs are universal to the profession of advanced practice nursing. Patient and healthcare provider satisfaction, and retention of personnel in military service may all be impacted by further research in ethical issues in clinical practice.

Recent studies have increased our knowledge of Entamoeba histolytica cell biology and gene regulation. In the ameba, dominant-negative mutations in the Gal/GalNAc lectin affect adhesion and cytolysis, whereas mutations in meromyosin affect cytoskeletal function. Studying these mutant proteins has improved our understanding of the role of these proteins in E. histolytica virulence. The characterization of the CP5 cysteine protease and the induction of apoptosis in host target cells has led to a better comprehension of the mechanisms by which trophozoites can lyse target cells.

Molecular tools have the potential to differentiate microscopically similar gut micro-eukaryotes that may have significantly different relationships with the human host. Using broad range Entamoeba primers to amplify a section of the eukaryotic 18S small subunit ribosomal RNA gene a novel member of the Entamoeba family (Entamoeba bangladeshi) has recently been identified. The goal of this review is to place this species in the context of what is already known about this genus and to discuss the tools and data needed to elucidate the host-microbe relationship.

We have successfully transfected Entamoeba histolytica trophozoites with constructs containing ferredoxin sequences fused to the reporter gene luciferase. We have determined the conditions and parameters necessary to maximise transient luciferase expression in our system. Our optimal construct gave values of 268 x 10(3) relative light units per second (RLU s-1) when assayed representing a stimulation of 18,000-fold over the control vector. Comparison of differing constructs allowed us to conclude that the 5' and 3' ferredoxin sequences are both necessary for optimal luciferase expression from our vectors. Transcriptional initiation occurs within the consensus sequence ATTCA in both construct and chromosomal ferredoxin promoters.

The Entamoeba histolytica transcription factor Upstream Regulatory Element 3-Binding Protein (URE3-BP) is a calcium-responsive regulator of two E. histolytica virulence genes, hgl5 and fdx1. URE3-BP was previously identified by a yeast one-hybrid screen of E. histolytica proteins capable of binding to the sequence TATTCTATT (Upstream Regulatory Element 3 (URE3)) in the promoter regions of hgl5 and fdx1. In this work, precise definition of the consensus URE3 element was performed by electrophoretic mobility shift assays (EMSA) using base-substituted oligonucleotides, and the consensus motif validated using episomal reporter constructs. Transcriptome profiling of a strain induced to produce a dominant-positive URE3-BP was then used to identify additional genes regulated by URE3-BP. Fifty modulated transcripts were identified, and of these the EMSA defined motif T[atg]T[tc][cg]T[at][tgc][tg] was found in over half of the promoters (54% p, Author Summary Most infections with Entamoeba histolytica are asymptomatic. However, in a minority of cases, they develop into invasive and even life-threatening amebiasis. We suspect, based on prior studies of invasive amebae, that changes in amebic gene expression enable the transition from asymptomatic to invasive infection. Our long-term goal is to identify the genetic program required to cause amebic colitis. Here, we studied a transcription factor named URE3-BP that controls the expression of two virulence genes, the Galactose and Galactose N- acetyl- galactosamine inhibitable lectin (Gal/GalNAc lectin) and ferredoxin. We suspected that this factor might coordinate invasiveness by co-regulating additional virulence factors. The consensus DNA motif that is recognized by URE3-BP was identified by reporter gene assays and by electromobility shift assays. We then inducibly expressed a constitutively active form of the transcription factor, and measured the changes in total amebic gene expression mediated by overexpression of this dominant-positive version of URE3-BP. This analysis allowed for a further definition of the functional URE3 motif. Inducible expression of URE3-BP led to changes in the transcript levels of several novel amebic membrane proteins. In conclusion, this genome-wide analysis of a transcription factor and its cis-acting regulatory sequence in Entamoeba histolytica has identified new transcripts regulated by URE3-BP that may play a role in trophozoite motility within a coordinated virulence-specific gene regulatory network.

The sequence of a 2.67-kilobase section of the Escherichia coli chromosome that contains the rep gene has been determined. This gene codes for a protein of predicted Mr 72,800, a DNA helicase, which is also a single-stranded DNA-dependent ATPase. The sequenced region contains an open reading frame of the correct length and orientation to encode the Rep protein. A secondary structure for the protein can be formulated from the amino acid sequence. We have compared both the primary and the secondary structures of Rep with other proteins and find the greatest homology between Rep and E. coli helicase II, the product of the uvrD gene.

Entamoeba histolytica possesses a family of approximately 100 putative transmembrane kinases (TMKs), indicating that the parasite has an extensive means of environmental sensing. The TMKs have been divided into nine sub-groups based on the sequence composition of their intracellular kinase as well as extracellular cysteine-rich domains. EhTMKB1-9 has been recently shown to be expressed in proliferating trophozoites and induced by serum. Interference with EhTMKB1-9 by antisense RNA knockdown or expression of a truncated protein diminished proliferation, adhesion and cytotoxicity. Here we report the involvement of EhTMKB1-9 in phagocytosis and its virulence function in the formation of amebic colitis. Trophozoites induced to express higher levels of wild type EhTMKB1-9 showed increased capacity for endocytosis. In contrast, cells compromised for the EhTMKB1-9 expression through antisense inhibition showed significantly lower levels of phagocytosis and endocytosis under the experimental conditions. The role of EhTMKB1-9 as a virulence factor was studied using animal models of amebiasis. Trophozoites expressing high levels of mutant protein lacking the kinase domain showed a competitive disadvantage with regard to survival as well as invasive phenotype in the murine model of amebic colitis. The same parasites however, were not compromised in their ability to generate abscess in the gerbil model of invasive liver amebiasis. EhTMKB1-9 is the second member from the “B” group of EhTMKs which seems to be deployed by the parasite during intestinal infection. TMKs are attractive targets for drug development because of their requirement in virulence and proliferation.