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1 Telomere length (TL) has recently shown prognostic value in Ewing sarcoma, nonsmall cell lung tumors, gastric cancer, and neuroblastoma. A TL pattern among cancers may provide information to create a better understanding of the molecular or pathogenic characteristics of specific cancer types. Telomeres are hexamer-nucleotide repeat sequences (5’-TTAGGG) at the ends of the chromosomes that serve as cap-like protective mechanisms to maintain the chromosome’s structure after DNA replication. Once TL becomes too short after several cell divisions, a normal cell undergoes senescence or apoptosis. In order to avoid this biological fate in cancer, a tumor cell can activate telomerase to maintain telomere length or elongate their TL by initiating the alternative lengthening of telomeres (ALT) pathway. To measure TLs or telomere repeats, we based our assay on a monochrome, multiplex quantitative PCR. This approach measures the signal generated from the telomere repeats against the signal from a single copy gene in the same DNA sample. The method is unique by measuring 2 fluorescent signals at 2 different temperatures using 1 DNA-intercalating dye within the same reaction tube. Our study included various cancer cell line DNAs including the NCI-60 panel. Preliminary data show statistically significant differences within breast cancer, pancreatic cancer, and osteosarcoma cell lines.
ROLE OF REVERSE TRANSCRIPTASE DOMAIN 0 IN NONLTR RETROTRANSPOSON INTEGRATIONJeremy Cortez, Shawn Christensen.
University of Texas at Arlington, Arlington, TX.
Transposable elements (TEs) are genomic parasites (mobile DNA) with the ability to replicate within the host genome. Of these TEs, nonlong terminal repeat retrotransposons (NLR) make up over 34% of the human genome.
NLRs use a “copy out as RNA, copy in as DNA” replication mechanism. NLR integration occurs through targetprimed reverse transcription (TPRT) where the element encoded DNA endonuclease generates a free chromosomal 3’-OH that is used to prime cDNA synthesis directly at the site of insertion. In addition to DNA endonuclease and reverse transcriptase (RT) activities, NLRs must encode the ability to bind element RNA. The RT of NLR elements has a domain (domain 0) not present in the RT found in long-terminal repeat retrotransposons or retroviruses. It is hypothesized that domain 0 of the NLR RT may be involved in recognizing element RNA. The R2 NLR element from Bombyx Mori is an excellent model system for the molecular and biochemical dissection of TPRT because of R2’s site specificity and the ability to obtain purified components that retain activity in vitro. Site-directed mutagenesis was used to generate a point mutation within domain 0 of the RT. The mutant protein has been purified and is being tested for loss of function using in vitro based DNA and RNA binding reactions that will be analyzed by electrophoretic mobility shift assays and denaturing gel electrophoresis. Characterization of any loss of function when compared to the wildtype protein will determine the role of domain 0 in the R2 integration process.
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Biological Sciences FRI-484
CHARACTERIZATION OF PREVALENT OPHTHALMIC MICROORGANISMS ISOLATED AT AN OPTICAL CLINIC
SERVING ECONOMICALLY DISADVANTAGED AND TRANSIENT POPULATIONSRoquita Garcia, Martha Gonzales, Ricardo Mata, Ana Vallor.
University of the Incarnate Word, San Antonio, TX.
This study seeks to investigate the prevalence of opportunistic microbial species on eyewear. Eyeglasses, when infrequently cleaned or sterilized, may serve as ecological niches for ophthalmic pathogens as compared to eyeglasses which are routinely maintained. If colonization of opportunistic microbes occurs, eyeglasses would serve as a continuous source of reinoculation giving rise to recurrent ocular infections and development of drug resistance.
In addition, eyeglasses may serve as a point of transmission between individuals who share eyeglasses or health care providers handling them during routine exams in an optical setting. A total of 60 subjects will be recruited, 20 from each of the following populations: patients and health care workers recruited from I Care San Antonio optical clinic serving economically disadvantaged and transient populations and control subjects with no clinical association.
Surveys were administered addressing hygiene habits and recent episodes of infection and treatment. Samples were taken from subjects and their eyeglasses. Initial microbial characterization entailed identification by microscopy, isolation on differential media, and biochemical tests. Currently, of 40 subjects recruited, 181 microbial samples have been collected and 54 have been isolated from eyeglasses. Preliminary identification of species include Serratia sp., Gram negative and positive rods, Micrococcus luteus, and Staphylococcus spp. Assessment of resistance to antibiotics and germicides routinely used in the clinic is currently being conducted. We predict that there will be a unique microbial populations demonstrating germicide resistance colonizing poorly maintained eyeglasses and transmission from patient to health care worker of these organisms.
THE TWO YEAST GYF PROTEINS SMY2/SYH1 IMPACT CELLULAR PRE-MRNA ABUNDANCERoberto Tapia, Min Chen, Brian C. Rymond.
University of Kentucky, Lexington, KY.
Eukaryotic gene expression is regulated at multiple levels. Two structurally related cytoplasmic yeast proteins Smy2 and Syh1 belong to the phylogenetically conserved GYF protein family, whose members generally function in RNA metabolism. While genetic/physical interaction partners of Smy2 and Syh1 are highly enriched in the splicing and mRNA decay categories, direct evidence linking Smy2 and Syh1 function in cellular RNA processing or stability is still lacking. Here we tested whether the Smy2/Syh1 proteins impact the cellular abundance of unprocessed premRNA exported to the cytoplasm bound with the BBP splicing factor. We predicted that if Smy2/Syh1 help promote the degradation of unprocessed pre-mRNA, then pre-mRNA levels would be elevated in the mutant background.
Yeast with either the smy2::KAN or syh1::KAN single deletions or both deletions grow well. RNA extracted from these cells was assayed by northern blot for the levels of several intron-containing ribosomal protein transcripts (RPS17A, RPS22B, …) and other intron-bearing genes. When cell density increases to the point where growth slows and ribosome demand diminishes, we see a clear increase in unspliced ribosomal protein pre-mRNA in the double deletion mutant. These and related observations support our hypothesis that Smy2 and Syh1 act in pre-mRNA metabolism and suggest that these proteins may promote the turnover of excess pre-mRNA under conditions of growth restricted ribosomal protein demand.
MOLECULAR MECHANISMS OF THE ANTI-BREAST-CANCER EFFECT OF PHYLLOSTACHYS EDULIS
BAMBOO EXTRACT AND COMPOUNDSMatthew Lim, Jason Higa, Jun Panee.
John A. Burns School of Medicine, Honolulu, HI.
Breast cancer (BC) is one of the most common cancers in women in America, and mortality is disproportionately high in some minority groups. Alternative forms of treatment are being considered for those unable to receive or hesitant concerning chemotherapy. This study investigated the molecular mechanisms of the anti-breast-cancer effect of Phyllostachys edulis, which is a widely distributed bamboo. The general hypothesis is that the extract from Phyllostachys edulis may inhibit the development of breast cancer through decreasing cell proliferation and/or inducing cell death. Estrogen receptor positive and negative (ER+ and ER-) human BC cell lines were used as in vitro models, and noncarcinoma mammary cells were used as a control. Fractions and compounds were isolated from the extract through chromatography. Cell proliferation, cell cycle, apoptosis, and ERalpha binding activity was measured
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by DNA-ethidium bromide binding, propidium iodide (PI) staining, and flow cytometry, competitive binding assay, and luciferase reporter assay. The raw extract and compounds from Phyllostachys edulis were more effective in inhibiting the proliferation of ER+ BC cells in comparison to that of the ER- BC cells. This inhibitory effect was associated withweak binding activities to ERalpha; G1 arrest and apoptosis induction; increases of p21(WAF) and BAX protein expression; decrease of cyclin D1 protein expression; increase of total peroxisome proliferator-activated receptor activity; and O-methylation of flavones as active compounds. Further research on the molecular mechanisms and metabolites of bamboo extract may determine whether individual or multiple compounds provide anti-breast-cancer effects.
OVEREXPRESSION OF THE G PROTEIN-COUPLED ESTROGEN RECEPTOR PROMOTES EARLY MAMMARY
TUMORIGENESIS IN A MOUSE MODELMona Ahmed, Chelin Hu, Helen Hathaway.
University of New Mexico, Albuquerque, NM.
Tumor progression in the mouse mammary epithelium caused by ectopic expression of the polyoma middle T oncoprotein (PyMT) is a reliable model for studying human breast cancer. The G protein-coupled estrogen receptor (GPER) is an intracellular transmembrane estrogen receptor that is localized to the endoplasmic reticulum. GPER plays a role in estrogen pathophysiology by promoting proliferation in breast cancer cells. The goal of our study was to examine the effects of increased GPER activity on early mammary tumorigenesis; therefore, we created mice that overexpressed GPER through transgenic technology and crossbred them to PyMT transgenic mice. The abdominalinguinal mammary glands were collected and fixed from seven-week-old female offspring that were either PyMT transgenic only or doubly transgenic for both GPER and PyMT. One gland was prepared for wholemount, stained with carmine, and photographed. The other gland was embedded in paraffin and sectioned for H&E staining. Morphometric analyses were used to quantify the proportion of mammary gland area containing hyperplasias and tumor-like growths. Our results revealed that mammary glands from mice that were PyMT transgenic and overexpressed GPER had a significantly (p = 0.015) larger tumor-involved area distal to the lymph node when compared to glands from mice that were PyMT transgenic only. Based on these results, it is evident that overexpression of GPER promotes early mammary tumorigenesis.
DEFINING THE CORTICAL DETERMINANTS OF THE DIVISION SITE DURING STARFISH MEIOSISQuincy Billie, Charles Shuster.
New Mexico State University, Las Cruces, NM.
Meiosis in animal cells involves a series of reductive divisions that ultimately give rise to a haploid gamete. In oogenesis, only a single gamete is generated at the end of meiosis, as a result of two highly asymmetric cell divisions that conserve cytoplasm within the oocyte. During prometaphase of meiosis I, the meiotic spindle translocates toward the cell cortex, and eventually docks one pole against the cell surface. Our lab is interested in understanding the cortical cues that enable meiotic spindle migration and docking in starfish oocytes, where meiotic divisions occur at the animal pole. Work in other systems has implicated the PAR complex of proteins in polarized cell divisions, and we wished to test the hypothesis that the PAR complex defines the cortical site of meiotic cell division. Toward these ends, we obtained immature oocytes from the atlantic starfish, Asterias forbesi, and induced meiotic maturation with 1-methyl-adenine. In these oocytes, germinal vesicle breakdown occurred within 20 minutes with a prominent prometaphase MI spindle lying parallel to the animal pole by 45 minutes post-activation. To interfere with PAR complex function, oocytes were pretreated and activated in the absence or presence of a pseudosubstrate inhibitor of atypical PKC (aPKCi). Examination of MI spindles by immunolocalization revealed that inhibition of aPKC had no initial effect on spindle migration to the animal pole. Current efforts are focused on examining spindle rotation in cells with compromised aPKC activity, both by immunofluorescence as well as timelapse live-cell microscopy.
IDENTIFICATION OF MIR-137 TARGETS IN COLON CANCERAlexandria Roy, Amber Smith, Liang Xu.
University of Kansas, Lawrence, KS.
MicroRNAs (miRNAs) are non-protein-coding RNAs that negatively regulate gene expression by inhibiting protein translation. MicroRNAs are frequently dysregulated in many types of cancer, resulting in aberrant gene expression.
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Biological Sciences We recently identified miR-137 as a tumor- suppressive miRNA, and found its expression decreased in approximately 70% of colon cancer tumor samples, as compared to normal tissue. Furthermore, when we reexpressed miR-137 in cancer cell lines, we reduced cell growth, colony formation, and tumorsphere growth. We identified oncogene Musashi-1 (Msi1) as a target of miR-137. However, microRNAs such as miR-137 are known to regulate hundreds of genes, therefore, our goal is to identify additional targets of miR-137 that may be overexpressed in colon cancer.