Medicine and Health Sciences Commons^™

A Mouse Model For Beta 0-Thalassemia., Baoli Yang, S. Kirby, J. Lewis, P. Detloff, N. Maeda, O. Smithies

Baoli Yang

We have used a "plug and socket" targeting technique to generate a mouse model of beta 0-thalassemia in which both the b1 and b2 adult globin genes have been deleted. Mice homozygous for this deletion (Hbbth-3/Hbbth-3) die perinatally, similar to the most severe form of Cooley anemia in humans. Mice heterozygous for the deletion appear normal, but their hematologic indices show characteristics typical of severe thalassemia, including dramatically decreased hematocrit, hemoglobin, red blood cell counts, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, as well as dramatically increased reticulocyte counts, serum bilirubin concentrations, and red cell distribution …

Appropriate Tissue- And Cell-Specific Expression Of A Single Copy Human Angiotensinogen Transgene Specifically Targeted Upstream Of The Hprt Locus By Homologous Recombination., B. Cvetkovic, Baoli Yang, R. Williamson, C. Sigmund

Baoli Yang

Development of experimental models by genetic manipulation in mice has proven to be very useful in determining the significance of particular genes in the development of or susceptibility to hypertension. Advances in molecular genetics, transgenic mouse technology, and physiological measurements in mice provided an opportunity to go a step further and develop models to analyze the physiological significance of specific gene variants potentially causing hypertension. In this report, we describe the development of a human angiotensinogen transgenic mouse model generated by targeting the human angiotensinogen gene upstream of the mouse HPRT locus by homologous recombination. The main benefit of this …

Identification Of A Tripartite Basal Promoter Which Regulates Human Terminal Deoxynucleotidyl Transferase Gene Expression., D. Bhaumik, Baoli Yang, T. Trangas, J. Bartlett, M. Coleman, D. Sorscher

Baoli Yang

In order to locate the promoter region of the human terminal deoxynucleotidyl transferase gene, serially truncated segments of the 5'-flanking region of the gene were cloned into a chloramphenicol acetyltransferase reporter vector. Transient transfection analyses of the terminal transferase-reporter gene constructs identified the basal promoter region within -34 to +40 base pairs relative to the transcription start site. Three promoter elements were defined in this region. The primary element is within 34 base pairs upstream of the transcription start site. The CAP site is 62 base pairs upstream of the translation start site. The secondary element involves sequences around the …

Regulation Of Terminal Deoxynucleotidyl Transferase Gene Expression In Mice And Men., M. Coleman, Baoli Yang, D. Sorscher

Baoli Yang

A nontemplate directed DNA polymerase, terminal deoxynucleotidyl transferase (terminal transferase) is expressed in a tissue-specific and development stage-specific manner. Its enzymatic properties and tissue localization have implicated the protein in development of normal immune function. Significant progress has been made in understanding the enzymology and important domains of this protein. More recently, studies have focused on regulation of the gene that codes for the protein in mice and humans. The murine gene has yielded to these studies more readily than the human gene. A murine basal promoter element has been identified along with several trans-acting protein factors that may regulate …

Mutational Analysis Of Residues In The Nucleotide Binding Domain Of Human Terminal Deoxynucleotidyl Transferase., Baoli Yang, K. Gathy, M. Coleman

Baoli Yang

Human terminal deoxynucleotidyl transferase (TdT) was overexpressed in a baculovirus system. The pure recombinant enzyme was identical in size, activity, kinetic constants, and metal effects to native enzyme. Three amino acids, within either the putative nucleotide binding domain and part of a DNA polymerase consensus sequence, YGDTDSLF, or a TdT consensus sequence, GGFRRGK, were altered by site-directed mutagenesis. The four mutant forms of terminal transferase were also overexpressed in the baculovirus expression system and purified from Trichoplusia ni larvae by a monoclonal antibody affinity column and compared with wild-type enzyme with respect to thermostabilities, secondary structure, metal effects, and kinetic …

Pharmacogenetic Screening For Susceptibility To Fetal Malformations In Women, D. Van Dyke, V. Ellingrod, M. Berg, Jennifer Niebyl, A. Sherbondy, D. Trembath

Jennifer R Niebyl

OBJECTIVE: To present a review of the literature and research on the pharmacogenetics of congenital defects, with a focus on the need for predictive maternal genotype assays. DATA SOURCE: MEDLINE searches (January 1985-January 1999), past reference reviews, and unpublished research. STUDY SELECTION: Review of relevant human, animal, and basic science studies. DATA EXTRACTION: Data on research on polymorphisms, genotyping, cytochrome P450 enzyme systems, epoxide hydrolase, folate metabolism, metabolism of anticonvulsant medications, molecular genetics of neural tube defects, variations in drug metabolism, and environmental exposures were evaluated. DATA SYNTHESIS: Data synthesis includes not only a review of the literature but suggests …

Quantitative Interpretation Of A Genetic Model Of Carcinogenesis Using Computer Simulations, Donghai Dai, B. Beck, X. Wang, C. Howk, Y. Li

Donghai Dai

The genetic model of tumorigenesis by Vogelstein et al. (V theory) and the molecular definition of cancer hallmarks by Hanahan and Weinberg (W theory) represent two of the most comprehensive and systemic understandings of cancer. Here, we develop a mathematical model that quantitatively interprets these seminal cancer theories, starting from a set of equations describing the short life cycle of an individual cell in uterine epithelium during tissue regeneration. The process of malignant transformation of an individual cell is followed and the tissue (or tumor) is described as a composite of individual cells in order to quantitatively account for intra-tumor …

Selective Down-Regulation Of Progesterone Receptor Isoform B In Poorly Differentiated Human Endometrial Cancer Cells: Implications For Unopposed Estrogen Action., N. Kumar, J. Richer, G. Owen, E. Litman, K. Horwitz, Kimberly Leslie

Kimberly K. Leslie

The uterine endometrium responds to unopposed estrogen stimulation with rapid cell proliferation. Progesterone protects the endometrium against the hyperplastic effects of estradiol (E2) through progesterone receptors (PRs), of which two isoforms are expressed: human (h) PRA and PRB. hPRB has a longer NH2 terminus and may function differently from hPRA. Thus, the relative expression of hPRA:hPRB is likely to be important for the action of progesterone. We hypothesized that the hPRA:hPRB ratios may be abnormal in endometrial cancer, leading to a lack of normal progesterone protection against the growth-promoting effects of E2. To test this hypothesis, well-differentiated Ishikawa endometrial cancer …

Estrogen Receptors Are Present In Human Granulosa Cells., B. Hurst, M. Zilberstein, J. Chou, B. Litman, J. Stephens, Kimberly Leslie

Kimberly K. Leslie

Recent studies failed to detect estrogen receptors in primate follicles. This study was initiated to determine whether estrogen receptor (ER) messenger ribonucleic acid (mRNA) is present in human granulosa cells and, further, if functional ER proteins are present. To evaluate the presence of ER, RNA from human granulosa cells obtained at the time of oocyte retrieval for assisted reproduction was extracted, and complementary DNA synthesis was performed by the reverse transcriptase-polymerase chain reaction. Oligonucleotide primers were used to amplify basepairs 570-852 in the B- and C- domains of the ER mRNA. Southern blotting was performed and confirmed that the amplified …

The Role Of Oestrogen In Follicular Development., B. Hurst, Kimberly Leslie

Kimberly K. Leslie

Most of our knowledge of ovarian physiology is based upon studies that have demonstrated functional oestrogen receptors in the ovaries of lower animal species. The presence of oestrogen receptors in primate granulosa cells has been questioned by some investigators. However, we have found oestrogen receptor messenger RNA in human granulosa cells by reverse transcriptase-PCR assay. Furthermore, using immortalized granulosa cell lines transfected with a plasmid containing an oestrogen response element, a functional oestrogen receptor was confirmed. These experiments strongly support the hypothesis that human granulosa cells express biologically active oestrogen receptor.

Estrogen Receptors Are Identified In The Glioblastoma Cell Line U138mg., Kimberly Leslie, D. Keefe, S. Powell, F. Naftolin

Kimberly K. Leslie

OBJECTIVE: The antiestrogen tamoxifen has been found to be effective in decreasing glioblastoma cell proliferation, but the mechanism underlying this effect and whether it is through the estrogen receptor (ER) is controversial. The objective of this study was to determine whether ERs are present in three human glioblastoma cell lines--HS683, U138MG, and JHN J889H--using the most sensitive techniques available. METHODS: Ligand binding and flow cytometry were employed to identify estrogen and progesterone receptors. The reverse transcriptase-polymerase chain reaction was used to identify ER mRNA, and a novel reporter gene transfection assay demonstrated that the ER was capable of activating gene …

A Phase Ii Evaluation Of Lapatinib In The Treatment Of Persistent Or Recurrent Epithelial Ovarian Or Primary Peritoneal Carcinoma: A Gynecologic Oncology Group Study, A. Garcia, M. Sill, H. Lankes, A. Godwin, R. Mannel, D. Armstrong, R. Carolla, M. Liepman, N. Spirtos, E. Fischer, Kimberly Leslie

Kimberly K. Leslie

OBJECTIVE: Activation and dimerization of the ERBB family play a role in the pathogenesis and progression of ovarian cancer. We conducted a phase II trial to evaluate the activity and tolerability of lapatinib in patients with recurrent or persistent epithelial ovarian cancer (EOC) and to explore the clinical value of expression levels of epidermal growth factor receptors (EGFR), phosphorylated EGFR, HER-2/neu, and Ki-67, and the presence of EGFR mutations. METHODS: Eligible patients had recurrent or persistent EOC or primary peritoneal carcinoma, measurable disease, and up to 2 prior chemotherapy regimens for recurrent disease. Patients were treated with lapatinib 1500 mg/day. …

Progesterone Regulation Of Activating Protein-1 Transcriptional Activity: A Possible Mechanism Of Progesterone Inhibition Of Endometrial Cancer Cell Growth, Donghai Dai, E. Litman, E. Schonteich, Kimberly Leslie

Kimberly K. Leslie

The uterine endometrium and cancers derived from it are classic models of hormone action: estrogen promotes growth and progesterone inhibits proliferation and results in differentiation. We have now identified a major pathway through which progesterone causes these growth-limiting effects. Ligand-bound progesterone receptors modulate the composition and transcriptional activity of members of the activating protein-1 (AP-1) family, and in particular, c-Jun. First, a dominant negative form of c-Jun inhibits the constitutive growth of Hec50co cells in a manner similar to the effects of progesterone through progesterone B receptors. Second, progesterone inhibits the transcriptional activity of the AP-1 complex in reporter gene …

Hormonal Induction Of Polo-Like Kinases (Plks) And Impact Of Plk2 On Cell Cycle Progression In The Rat Ovary, Feixue Li, Misung Jo, Thomas E. Curry, Jing Liu

Obstetrics and Gynecology Faculty Publications

The highly conserved polo-like kinases (Plks) are potent regulators of multiple functions in the cell cycle before and during mitotic cell division. We investigated the expression pattern of Plk genes and their potential role(s) in the rat ovary during the periovulatory period. Plk2 and Plk3 were highly induced both in intact ovaries and granulosa cells in vivo after treatment with the luteinizing hormone (LH) agonist, human chorionic gonadotropin (hCG). In vitro, hCG stimulated the expression of Plk2 in granulosa cells, but not Plk3. This induction of Plk2 expression was mimicked by both forskolin and phorbol 12 myristate 13-acetate (PMA). Moreover, …

Preimplantation Embryo Programming: Transcription, Epigenetics, And Culture Environment., Veronique Duranthon, Andrew J Watson, Patrick Lonergan

Obstetrics & Gynaecology Publications

Preimplantation development directs the formation of an implantation- or attachment-competent embryo so that metabolic interactions with the uterus can occur, pregnancy can be initiated, and fetal development can be sustained. The preimplantation embryo exhibits a form of autonomous development fueled by products provided by the oocyte and also from activation of the embryo's genome. Despite this autonomy, the preimplantation embryo is highly influenced by factors in the external environment and in extreme situations, such as those presented by embryo culture or nuclear transfer, the ability of the embryo to adapt to the changing environmental conditions or chromatin to become reprogrammed …

Oocyte Cytoplasmic Maturation: A Key Mediator Of Oocyte And Embryo Developmental Competence., A J Watson

Obstetrics & Gynaecology Publications

Efforts have intensified to successfully mature and inseminate oocytes in vitro and then culture ensuing embryos to transferable stages from a large number of mammalian species. Success varies, but generally even for the most successful species it is only possible to obtain a maximum of a 40 to 50% development of zygotes to the blastocyst stage. Reduced oocyte developmental competence is suggested as a primary reason for the reduced potential of in vitro-produced embryos. The vast majority of in vitro-matured oocytes are meiotically competent; however, many do not attain an optimal oocyte diameter before insemination. Variations in oocyte in vitro …

Effect Of Serum And Cumulus Cell Expansion On Marker Gene Transcripts In Bovine Cumulus-Oocyte Complexes During Maturation In Vitro., Michele D Calder, Anita N Caveney, Marc-Andre Sirard, Andrew J Watson

Obstetrics & Gynaecology Publications

OBJECTIVE: To determine the distribution of transcripts encoding the FSH receptor (FSHr), LH receptor (LHr), connexin 43 (Cx43), cyclooxygenase-2 (COX-2), and prostaglandin E(2) receptors 2 and 3 (EP2 and EP3) within bovine cumulus-oocyte complexes (COCs) and denuded oocytes and investigate the influence of gonadotropins, serum, and cumulus cell expansion on the abundance of transcripts encoding these genes.

DESIGN: Prospective controlled animal study.

SETTING: University research laboratory.

PATIENT(S): Animal models for human studies.

INTERVENTION(S): Cumulus-oocyte complexes were treated in culture with serum and gonadotropin-supplemented media to examine the effects to mRNA transcript levels.

MAIN OUTCOME MEASURE(S): Variation in mRNA transcript levels. …

The Gamma-Subunit Of The Na-K-Atpase As A Potential Regulator Of Apical And Basolateral Na+-Pump Isozymes During Development Of Bovine Pre-Attachment Embryos., L C Barcroft, S E Gill, A J Watson

Obstetrics & Gynaecology Publications

Expression and activity of the Na-K-ATPase within the basolateral membrane domains of the trophectoderm epithelium provide the driving force for accumulation of Na(+) and Cl(-) across the nascent epithelium, mediating fluid movement into the forming blastocoel. Within the trophectoderm of the bovine blastocyst, multiple isozymes of the Na-K-ATPase are expressed. Immunolocalization has demonstrated that the alpha1-isozyme localizes within the basolateral membrane, whereas the alpha 3-isozyme localizes to the apical cell margins. Gene-specific RT-PCR and wholemount indirect immunofluorescence confocal laser scanning microscopy were used to examine expression of the Na-K-ATPase gamma-subunit (a regulatory subunit of the Na-K-ATPase) throughout development of bovine …

Differential Involvement Of Na(+),K(+)-Atpase Isozymes In Preimplantation Development Of The Mouse., D J Macphee, D H Jones, K J Barr, D H Betts, A J Watson, G M Kidder

Obstetrics & Gynaecology Publications

Na(+),K(+)-ATPase plays an essential role in mammalian blastocoel formation (cavitation) by driving trans-epithelial sodium transport. Previously, the alpha1 and beta1 subunit isoforms of this enzyme were identified in preimplantation mouse embryos and were assumed to be responsible for this function. Here we show that mRNAs encoding an additional alpha subunit isoform (alpha3) and the remaining two beta subunit isoforms are also present in preimplantation embryos. Whereas alpha3 mRNA accumulates between the four-cell and the blastocyst stages and thus results from embryonic transcription, the same could not be demonstrated for beta2 and beta3 mRNAs. Immunoblot analyses confirmed that these subunits are …