Applied Plasma Research

Contains research objectives and reports on three research projects.National Science Foundation (Grant GK-2581)Joint Services Electronics Program under Contract DA 28-043-AMC-02536(E

GTP-dependent structural rearrangement of the eRF1:eRF3 complex and eRF3 sequence motifs essential for PABP binding

Translation termination in eukaryotes is governed by the concerted action of eRF1 and eRF3 factors. eRF1 recognizes the stop codon in the A site of the ribosome and promotes nascent peptide chain release, and the GTPase eRF3 facilitates this peptide release via its interaction with eRF1. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay through its association with cytoplasmic poly(A)-binding protein (PABP) via PAM2-1 and PAM2-2 motifs in the N-terminal domain of eRF3. We have studied complex formation between full-length eRF3 and its ligands (GDP, GTP, eRF1 and PABP) using isothermal titration calorimetry, demonstrating formation of the eRF1:eRF3:PABP:GTP complex. Analysis of the temperature dependence of eRF3 interactions with G nucleotides reveals major structural rearrangements accompanying formation of the eRF1:eRF3:GTP complex. This is in contrast to eRF1:eRF3:GDP complex formation, where no such rearrangements were detected. Thus, our results agree with the established active role of GTP in promoting translation termination. Through point mutagenesis of PAM2-1 and PAM2-2 motifs in eRF3, we demonstrate that PAM2-2, but not PAM2-1 is indispensible for eRF3:PABP complex formation

Nucleotide sequence divergence among DNA fractions of different syngens of Tetrahymena pyriformis

The magnitude of the differences in base sequence of DNA fractions derived from different syngens of the ciliated protozoan Tetrahymena pyriformis was investigated. Each DNA was fractionated into unique and repeated sequences by hydroxylapatite chromatography, and the fractions were tested by in vitro molecular hybridization techniques. The amount of hybrid formed and the thermal stability of the hybrid molecules were examined at different incubation temperatures (50 and 65 C) for unique sequences and at 50 C for repeated sequences. The extent of the reactions involving either unique or repeated sequences was nearly complete when the two DNAs compared were derived from the same syngen. Moreover, intrasyngenic hybrids formed at 50 C (and 65 C for unique sequences) exhibit a high degree of thermal stability. In contrast, the extent of the reactions involving sequences derived from different syngens was low, as expected from the effect of mismatching on rate of reassociation, and intersyngenic hybrids formed at 50 C have low thermal stability. The reaction of unique sequences is further reduced at 65 C and the intersyngenic hybrids formed have a higher thermal stability than those formed at 50 C. The degree to which thermal stability is lowered was then used to estimate the percentage of mispaired bases. The average divergence of unique sequences between syngens is large and of the magnitude found for rodent DNAs from different genera or for Drosophila DNAs from nonsibling species. The repeated sequence fraction may contain more than one component and may be more conserved than the unique sequence fraction.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44120/1/10528_2004_Article_BF00486091.pd

Intersyngenic variations in the esterases and acid phosphatases of Tetrahymena pyriformis

The esterase and acid phosphatase isozymes were surveyed in strains of syngens 2–12 under conditions found to be optimal for syngen 1. Both intersyngenic and intrasyngenic variations were found. Comparisons of the esterases suggest that homologous enzymes are present in certain syngens and that some ordering of the variations with respect to syngen differences is possible. The acid phosphatases are highly polymorphic in different strains even within a syngen, and the variations cannot be ordered with respect to syngen differences. These results are discussed in terms of other types of studies directed at assessing syngen relationships and in terms of the sources of variation. It was concluded that only characters less vulnerable to intra clonal variation will be capable of revealing syngen relationships.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44168/1/10528_2004_Article_BF00485640.pd

Genome amplification and gene expression in the ciliate macronucleus

The focus of this review is on the micronucleus and macronucleus in the ciliated protozoa and the organization and function of the DNA molecules within them. We present (1) some of the structural and functional differences which are known, (2) the genetic evidence for macronuclear units, (3) two hypotheses for the organization of the DNA molecules in the macronucleus to explain these units, and (4) experiments designed to discriminate between these hypotheses. We conclude that the size of the genome is not reduced in the macronucleus and that there are 45 copies of the haploid genome present in the macronucleus of normal strains of Tetrahymena pyriformis and 800 copies in the macronucleus of Paramecium aurelia . The ciliate genome is relatively simple in terms of repeated sequences. However, not all copies of the genes present in the macronucleus may be identical since fractions of differing thermal stability appear after renaturation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44178/1/10528_2004_Article_BF00486122.pd

Information storing by biomagnetites

Since the discovery of the presence of biogenic magnetites in living organisms, there have been speculations on the role that these biomagnetites play in cellular processes. It seems that the formation of biomagnetite crystals is a universal phenomenon and not an exception in living cells. Many experimental facts show that features of organic and inorganic processes could be indistinguishable at nanoscale levels. Living cells are quantum "devices" rather than simple electronic devices utilizing only the charge of conduction electrons. In our opinion, due to their unusual biophysical properties, special biomagnetites must have a biological function in living cells in general and in the brain in particular. In this paper we advance a hypothesis that while biomagnetites are developed jointly with organic molecules and cellular electromagnetic fields in cells, they can record information about the Earth's magnetic vector potential of the entire flight in migratory birds.Comment: 17 pages, 3 figure

Intersyngenic variations in the esterases of axenic stocks of Paramecium aurelia

The esterase isozymes were surveyed in axenic stocks of syngens 1, 2, 4, 5, 6, and 8 of Paramecium aurelia by starch gel electrophoresis. In paramecia there appear to be four types of esterases which are clearer in axenic than in bacterized stocks. Each type differs in its substrate specificity and/or its response to the inhibitor eserine sulfate. Minor variations in type D esterases sometimes occur in different extracts of the same stock and may result from changes in the temperature of growth of the cells or growth cycle differences. Differences in the mobility of the A, B, or C (cathodal) types of esterases may occur in different syngens. They also occur for the A and B types among stocks within a syngen, but the frequency is low, except in the case of syngen 2. Since each of the types of esterases varies independently, at least four and possibly more genes appear to specify the esterases in the species complex. Some pairs of syngens vary in their electrophoretic positions for all types of esterases. Other pairs have identical zymograms. This observation suggests that some syngens may differ from each other by as many as four esterase genes, while others may not differ at all. The difference between P. aurelia and Tetrahymena pyriformis in the degree of intrasyngenic variation observed for enzymes is discussed in relation to other types of characters, the organization of the genetic material in the macronucleus, the presence of symbionts, and their breeding systems. It is suggested that enzyme variation is achieved by the action of different selective forces in these two groups of ciliated protozoa.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44170/1/10528_2004_Article_BF00485643.pd

Double blind, randomized, placebo controlled clinical trial for the treatment of diabetic foot ulcers, using a nitric oxide releasing patch: PATHON

<p>Abstract</p> <p>Background</p> <p>Diabetes Mellitus constitutes one of the most important public health problems due to its high prevalence and enormous social and economic consequences. Diabetic foot ulcers are one of the chronic complications of diabetes mellitus and constitute the most important cause of non-traumatic amputation of inferior limbs. It is estimated that 15% of the diabetic population will develop an ulcer sometime in their lives. Although novel therapies have been proposed, there is no effective treatment for this pathology. Naturally produced nitric oxide participates in the wound healing process by stimulating the synthesis of collagen, triggering the release of chemotactic cytokines, increasing blood vessels permeability, promoting angiogenic activity, stimulating the release of epidermical growth factors, and by interfering with the bacterial mitochondrial respiratory chain. Topically administered nitric oxide has demonstrated to be effective and safe for the treatment of chronic ulcers secondary to cutaneous leishmaniasis. However, due to their unstable nitric oxide release, the topical donors needed to be applied frequently, diminishing the adherence to the treatment. This difficulty has led to the development of a multilayer polymeric transdermal patch produced by electrospinning technique that guarantees a constant nitric oxide release. The main objective of this study is to evaluate the effectiveness and safety of this novel nitric oxide releasing wound dressing for the treatment of diabetic foot ulcers.</p> <p>Methods and design</p> <p>A double-blind, placebo-controlled clinical trial, including 100 diabetic patients was designed. At the time of enrollment, a complete medical evaluation and laboratory tests will be performed, and those patients who meet the inclusion criteria randomly assigned to one of two groups. Over the course of 90 days group 1 will receive active patches and group 2 placebo patches. The patients will be seen by the research group at least every two weeks until the healing of the ulcer or the end of the treatment. During each visit the healing process of the ulcer, the patient's health status and the presence of adverse events will be assessed. Should the effectiveness of the patches be demonstrated an alternative treatment would then be available to patients.</p> <p>Trial registration</p> <p>NCT00428727.</p

R497K polymorphism in epidermal growth factor receptor gene is associated with the risk of acute coronary syndrome

<p>Abstract</p> <p>Background</p> <p>Previous studies suggested that genetic polymorphisms in the epidermal growth factor receptor (EGFR) gene had been implicated in the susceptibility to some tumors and inflammatory diseases. EGFR has been recently implicated in vascular pathophysiological processes associated with excessive remodeling and atherosclerosis. Acute coronary syndrome (ACS) is a clinical manifestation of preceding atherosclerosis. Our purpose was to investigate the association of the EGFR polymorphism with the risk of ACS. In this context, we analyzed the HER-1 R497K and EGFR intron 1 (CA)_nrepeat polymorphisms in 191 patients with ACS and 210 age- and sex-matched controls in a Chinese population, using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) strategy and direct sequencing.</p> <p>Results</p> <p>There were significant differences in the genotype and allele distribution of R497K polymorphism of the EGFR gene between cases and controls. The <it>Lys </it>allele had a significantly increased risk of ACS compared with the <it>Arg </it>allele (adjusted OR = 1.49, 95% CI: 1.12–1.98, adjusted <it>P </it>= 0.006). However, no significant relationship between the number of (CA)_nrepeats of EGFR intron 1 (both alleles < 20 or any allele ≥ 20) and the risk of ACS was observed (adjusted OR = 0.97, 95% CI: 0.58–1.64, adjusted <it>P </it>= 0.911). Considering these two polymorphisms together, there was no statistically significant difference between the two groups.</p> <p>Conclusion</p> <p>R497K polymorphism of the EGFR gene is significantly associated with the risk of ACS. Our data suggests that R497K polymorphism may be used as a genetic susceptibility marker of the ACS.</p

Genomics and proteomics approaches to the study of cancer-stroma interactions

<p>Abstract</p> <p>Background</p> <p>The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression.</p> <p>Methods</p> <p>The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells.</p> <p>Results</p> <p>We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (<it>ARID4A</it>, <it>CALR</it>, <it>GNB2L1</it>, <it>RNF10</it>, <it>SQSTM1</it>, <it>USP9X</it>) were validated by real time PCR.</p> <p>Conclusions</p> <p>A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.</p