Cell and Developmental Biology Commons^™

Positive Regulation Of Pka On Human Gonadotropin-Releasing Hormone (Hgnrh) Gene Expression In Human Placental Jeg-3 Cells, Zhaoyang Wen

Chemistry & Biochemistry Theses & Dissertations

Using the human placental choriocarcinoma JEG-3 cell line as an in vitro human placental model, we studied the mechanisms of the PKA positive regulation of the hGnRH gene expression in the human placenta. Studies in JEG3 cells showed that through the PKA catalytic subunit a, human GnRH upstream promoter activity was stimulated by PKA signaling pathway in a cAMP dependent mechanism. The sequence between —202 (Afl II) and —554 (BamH I) base pair in the human GnRH upstream promoter region appeared to be responsible for the PKA positive regulation of the gene expression. Furthermore, Western blot analysis demonstrated the involvement …

Morphine Enhances The Permeability Across Vascular Endothelial Cell Barriers, Jenine Anday

Seton Hall University Dissertations and Theses (ETDs)

Using three types of in vitro endothelial cell barrier models, we investigated the direct effects and mechanisms of morphine on vascular permeability. Our current studies illustrate LPS induces permeability across these VEC barriers, and is significantly enhanced when co-treated with morphine, displayed using the [ 14 CJ-inulin paracellular marker. Incubation with morphine alone induces permeability in a concentration­ depeodent manner, and is not blocked by the addition of naloxone. Morphine enhances the detrimental effects of LPS on cell viability and alone also decreases endothelial cell viability, concentration-dependently, which is also not affected by naloxone, as demonstrated by the trypan blue …

Antioxidation Activity Of Dhea And Its Mechanisms, Sun Yang, Han Rui

Pharmacy Faculty Articles and Research

Objective: The aim of this study was to determine the anti-oxidative activity of a new chemopreventive agent--dehydroepiandrosterone (DHEA), and the mechanisms of action by which DHEA protect the thymocytes and DNA from oxidative damage. Methods: Agarose gel electrophoresis, flow cytometry, single cel! gel electrophoresis, chemiluminescence assay, triazolyl blue tetrazolumbromide (MTT) colorimetry, and three dimensional collagen gel assay were used. Results: In agarose gel electrophoresis, 10 nmol/L DHEA blocked the typical DNA degradation (DNA Ladder) induced by H₂O₂. DHEA 2. 5 nmol/L and 10 nmol/L both significantly decreased the percentage of characteristic apoptotic DNA …

Receptor Number And Caveolar Co-Localization Determine Receptor Coupling Efficiency To Adenylyl Cyclase, Rennolds S. Ostrom, Caroline Gregorian, Ryan M. Drenan, Yang Xiang, John W. Regan, Paul A. Insel

Pharmacy Faculty Articles and Research

Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to β1-adrenergic receptor (β1AR)-selective activation 3.7-fold, to β2AR-selective activation only 1.6-fold and to prostaglandin E2 (PGE2) not at all. Therefore, the rank order of efficacy in …

Cellular Release Of And Response To Atp As Key Determinants Of The Set-Point Of Signal Transduction Pathways, Rennolds S. Ostrom, Caroline Gregorian, Paul A. Insel

Pharmacy Faculty Articles and Research

The determinants of “basal” activity of signaling pathways regulating cellular responses are poorly defined. One possibility is that cells release factors to establish the set-point of such pathways. Here we show that treatment of Madin-Darby canine kidney cells with the nucleotidase apyrase decreases basal arachidonic acid release and cAMP production 30–40% and that inhibitors of P2Y receptor action also affect basal and forskolin-stimulated cAMP accumulation. Changing medium prominently increases extracellular levels of ATP in Madin-Darby canine kidney, COS-7, and HEK-293 cells. Mechanical stimulation of ATP release likely occurs in virtually every experimental protocol with cultured cells, implicating such release and …

Insulinlike Growth Factor 1- And 2-Augmented Collagen Gel Repair Of Facial Osseous Defects, James S. Toung, Roy C. Ogle, Raymond F. Morgan, William H. Lindsey

School of Medical Diagnostics & Translational Sciences Publications

BACKGROUND: Defects of the facial bone structure are common problems for the facial plastic surgeon. Native type 1 collagen gels (T1CGs) have been shown to mediate repair of facial critical-size defects in rat models.

OBJECTIVE: To evaluate the efficacy of T1CG augmented with insulinlike growth factor (IGF) 1, IGF-2, and a combination of IGF-1 and IGF-2 on the repair of facial critical-size defects in a rodent model.

METHODS: Twenty-four retired male breeder Sprague-Dawley rats were divided into 4 groups of 6 animals. Facial critical-size defects were created by removing the nasalis bones with a bone-cutting drill. Defects were treated with …

Inhibition Of Cpla2-Mediated Arachidonic Acid Release By Cyclic Amp Defines A Negative Feedback Loop For P2y-Receptor Activation In Mdck-D1 Cells, Mingzhao Xing, Steven Post, Rennolds S. Ostrom, Michael Samardzija, Paul A. Insel

Pharmacy Faculty Articles and Research

In Madin-Darby canine kidney D1cells extracellular nucleotides activate P2Y receptors that couple to several signal transduction pathways, including stimulation of multiple phospholipases and adenylyl cyclase. For one class of P2Y receptors, P2Y2 receptors, this stimulation of adenylyl cyclase and increase in cAMP occurs via the conversion of phospholipase A2 (PLA2)-generated arachidonic acid (AA) to prostaglandins (e.g. PGE2). These prostaglandins then stimulate adenylyl cyclase activity, presumably via activation of prostanoid receptors. In the current study we show that agents that increase cellular cAMP levels (including PGE2, forskolin, and the β-adrenergic agonist isoproterenol) can inhibit P2Y receptor-promoted AA release. The protein kinase …

The Isolation And Identification Of Polyphenolics From Grape Seeds And Their Activity Towards Specific Cell Lines, Joseph A. Reddy

Chemistry & Biochemistry Theses & Dissertations

Grape seed extract is being marketed in France for cardiovascular use and skin care. The active constituents of both these drugs are certain polyphenols called procyanidins. A similar seed extract has now been obtained from Virginia Chardonnay grape seeds. Ten of the major components in this extract have been isolated in their pure form by the semi-preparative HPLC separation on a YMC ODS-AQ (Octadecylsilane-AQ) column. The ODS-Aq column has been shown to have a better resolving capacity compared to the Spherisorb ODS (Octadecylsilane) column previously described in the literature. This has been attributed to the ODSAQ support which has both …

The Effects Of Glutathione On Human Keratinocyte, Manisha N. Trivedi

Theses and Dissertations in Biomedical Sciences

Cultured human keratinocytes were grown in vitro to assess the effects of glutathione, a thiol, on human keratinocyte proliferation. Single keratinocytes were harvested from newborn (greater than 5 days old) foreskin and were grown in culture flasks such that experiments could be performed in approximately 2 to 3 weeks. In order to evaluate the effects of glutathione (GSH) on keratinocyte growth, experiments were performed to determine the growth curve, plating efficiency, and incorporation of radiolabelled thymidine. Growth studies, performed with different concentrations of glutathione, revealed that 0.065 μM was the most effective concentration when compared to control cell cultures, and …

Laminin Potentiates Differentiation Of Pcc4uva Embryonal Carcinoma Into Neurons, T. M. Sweeney, Roy C. Ogle, C. D. Little

School of Medical Diagnostics & Translational Sciences Publications

The embryonal carcinoma PCC4uva differentiates into neurons in response to treatment with retinoic acid and dbcAMP. We used this in vitro model system to study the effects of laminin on early neural differentiation. Laminin substrata markedly potentiate neural differentiation of retinoic acid and dbcAMP-treated cultures. Only laminin induced more rapid neural cell body clustering, neurite growth and neurite fasciculation as compared to type IV collagen, type I collagen, and fibronectin substrata. Exogenous laminin substrata promoted greater cell attachment, cellular spreading and growth to confluence than type IV collagen, type I collagen, fibronectin and glass substrata. Laminin-induced effects were inhibited by …

Effect Of Photoperiod On Developmental Morphology And Enolase Isoenzyme Immunohistochemistry In Rat And Djungarian Hamster Superficial Pineal Glands, Chalmer D. Mcclure

Loma Linda University Electronic Theses, Dissertations & Projects

The best understood functional activity of the pineal gland is its diurnal production of melatonin in response to environmental lighting cues. Several enzymes of the melatonin pathway respond to daily photoperiod changes, for example hydroxyindole-O-methyltransferase (HIOMT) and serotonin N-acetyltransferase (SNAT). Increased levels of the glycolytic enzyme neuron-specific enolase (NSE) are thought to reflect increased physiological demands placed on neurons and neuroendocrine tissues. Homodimer non-neuronal enolase isoenzyme (NNE) is immunolocalized to cells, and the hybrid enolase (consisting of subunits from NSE and NNE) has been seen in cerebellar stellate and basket cells. Although not rate limiting, concentrations of both NSE and …

Collagen Binding Proteins Derived From The Embryonic Fibroblast Cell Surface Recognize Arginine-Glycine-Aspartic Acid, Roy C. Ogle, Charles D. Little

School of Medical Diagnostics & Translational Sciences Publications

Several cell surface proteins (Mr = 120,000, 90,000, 63,000 and 47,000) apparently integral to embryonic fibroblast plasma membranes were extracted with detergent and isolated by collagen affinity chromatography. Certain of these proteins (Mr = 120,000, 90,000, and 47,000) were specifically eluted from collagen affinity columns by synthetic peptides containing the amino acid sequence arginyl-glycyl-aspartic acid (RGD). These data show that a number of collagen binding proteins exist on the embryonic fibroblast cell surface. Some of the proteins may be collagen receptors binding to RGD sequences in the collagen molecule while at least one of the proteins (Mr = 63,000) recognizes …

Hypoxanthine-Induced Differentiation Of Cultured Human Leukemia Cells, Gayle Jennette Singleton

Chemistry & Biochemistry Theses & Dissertations

Human cultured leukemia cells appear to have a decreased amount of inosine in their tRNA. When cells with inosine deficient tRNA are placed in a hypoxanthine fortified media, they incorporate hypoxanthine into their tRNA by the action of the enzyme tRNA-hypoxanthine ribosyl transferase. This generates the nucleoside inosine in the tRNA. The cultured human leukemia cell lines, CCRF-CEM, HL-60, and HGPRT(-) HL- 60, incorporate hypoxanthine into their tRNA, as determined by tRNA isolation, hydrolysis, and HPLC analysis. Hypoxanthine treatment dramatically inhibited cell growth in conjunction with partial induction of differentiation in the CCRF-CEM, HL-60, and HGPRT ( - ) HL-60 …

Policy For Identifying And Assessing The Health Risks Of Toxic Substances, Maine Bureau Of Health

Maine Collection

Policy for Identifying and Assessing the Health Risks of Toxic Substances

by Norman T. Anderson, Environmental Toxicology Program, Division of Disease Control, Bureau of Health (February, 1988).

Contents: Section I: Introduction / Section II: Methods / Section III: Exposure Assessment / Section IV: Hazard Identification / Section V: Hazard Assessment / Section VI: Risk Characterization / List of Figures / List of Tables

Laminin Receptors For Neurite Formation, H. K. Kleinman, Roy C. Ogle, F. B. Cannon, C. D. Little, T. M. Sweeney, L. Luckenbill-Edds

School of Medical Diagnostics & Translational Sciences Publications

Laminin, a basement membrane glycoprotein promotes both cell attachment and neurite outgrowth. Separate domains on laminin elicit these responses, suggesting that distinct receptors occur on the surface of cells. NG108-15 neuroblastoma-glioma cells rapidly extend long processes in the presence of laminin. We report here that 125I-labeled laminin specifically binds to these cells and to three membrane proteins of 67, 110, and 180 kDa. These proteins were isolated by affinity chromatography on laminin-Sepharose. The 67-kDa protein reacted with antibody to the previously characterized receptor for cell attachment to laminin. Antibodies to the 110-kDa and 180-kDa bands demonstrated that the 110-kDa protein …

Determination Of Human Sperm Cell Acrosin Activity From Normal And Subfertile Men, Dilrowshan H. Haque

Chemistry & Biochemistry Theses & Dissertations

Acrosin is an acrosomal enzyme which is reported to be involved in sperm penetration through cumulus, zona, and ooplasma. This enzyme has been reported to be significantly lower in sperm of infertile men compared to sperm of fertile men. The acrosin assay which has been developed can be used for studies in patients with suspected infertility and may be used on a fraction of most semen specimens being evaluated in a clinical andrology laboratory.

Within-run precision studies yielded coefficients of variation of 11% for a low-activity pool and 9% for normalactivity pool specimens. Between-run precision studies using a single donor …

The Effects Of Luteinizing Hormone And Adenosine 3',5'-Cyclic Monophosphate On Phospholipid Metabolism By Luteal Mitochondria, Jim John Sadighian

Chemistry & Biochemistry Theses & Dissertations

Luteinizing hormone (LH) increases intracellular concentrations of adenosine 3', 5 '-cyclic monophosphate and the phosphoinositides, phosphatidylinositol (PI), PI 4'-phosphate (PIP) and PI 4' .5 1 - bispbosphate (PIP₂). It is believed that cAMP and the phosphoinositides act concertedly to regulate mitochondrial conversion of cholesterol to pregnenolone. This study examined the effects of LH and N⁶ ,O² -dibutyryl cAMP (dbcAMP) on phospholipids metabolism by luteal mitochondria and the influence of dbcAMP and the phosphoinosi tides on mitochondrial steroid production. Mitochondria were isolated from unincubated and incubated luteal tissue by differential centrifugation. Phospholipids were extracted from the mitochondria …

A Quantitative Study Of The Distinctive Proteins Of The Eosinophil: A Comparison Of Normals And Eosinophilic Patients, James M. Olson

Honors Theses

Eosinophils have long been associated with parasitic and allergic diseases. Methods of procurement and purification of eosinophils in the last ten years have allowed intensive investigation into their components, properties, functions, and mechanisms (Gleich et al, 1982). The goals of this project are to separate eosinophils in blood samples taken from patients with eosinophilia and from normals based on density over distontinuous Metrizamide gradients; to determine quantitatively the levels of the three distinctive proteins of the eosinophil, specifically MBP, EDN, and ECP; and to compare the levels of these proteins found in eosinophils of patients with eosinophilia to the amounts …

Evaluation Of Peptide Assimilation In Ehrlich Ascites Tumor Cells, John M. Castellano

Biological Sciences Theses & Dissertations

The dipeptides glycyl-L-(³H)-leucine and L-phenylalanyl-L-(³H)-leucine were synthesized in radioactive form and utilized in the analysis of peptide transport and hydrolysis by Ehrlich ascites tumor cells. The tumor cells were cultured in the abdominal cavity of CF-1 albino mice and harvested into sterile isotonic buffer for all experimental analyses.

The results of this study reveal that the tumor cells rapidly accumulate radiolabel when incubated with the above mentioned peptides, but that the peptide is most probably hydrolyzed extracellularly and free L-(³)-leucine transported. This hypothesis is based on the observations of reduction in accumulation of radiolabel …

In Vitro Enzymatic Assay Of Rna Methylation, Pamela Jean Eubanks Gallup

Chemistry & Biochemistry Theses & Dissertations

An assay procedure for in vitro enzymatic methylation of mannnalian ribosomal RNA has been developed in this study. The assay procedure, utilized for the comparison of normal and neoplastic methylase activities (using mouse liver and Ehrlich ascites cells as sources of enzyme), is a modification of previously published methods (52,53). A 100,000 x g supernatant (SlOO) enzyme preparation was incubated with 28S-5.8S rRNA and tritium-labeled S-adenosyl-L-methionine. The RNA was extracted, applied to DEAE cellulose paper, washed, and the radioactivity counted. The neoplastic cell methylase preparation was more active in methylating both exogenous neoplastic and normal 28S-5.8S rRNA than the normal …

A Comparative Consideration Of Lipoprotein Distribution And Stress Response In The Mallard Duck And Man, Janice Mackay

Honors Theses

Lipid is associated with all of the major serum protein fractions of both the mallard duck and man. however, whereas the ρ lipoproteins predominate in man with little lipo-albumin evident, according to available literature, in ducks the lipo-albumin constitutes more than half of the serum lipoproteins, and a substantial lipo-prealbumin fraction is evident. Most studies of lipo-proteins are carried out on human serum primarily for the purpose of diagnosis of abnormalities. The stress response in many animals includes a rise in serum α-globulin levels, but its physiological basis is still speculative. While a consideration of the changes in lipoprotein distribution …