Molecular Biology Commons^™

Analysis Of The Phosphorylated Forms Of Protein Kinase R, Christine Quartararo

Honors Scholar Theses

Protein Kinase R (PKR) is induced by interferon and activated by dsRNA. Subsequent autophosphorylation and phosphorylation of eIF2alpha inhibits viral replication. In the latent state PKR exists as an unphosphorylated monomer. Work in the Cole laboratory has shown two additional states, a phosphorylated monomeric state (pPKRm) and a phosphorylated dimeric state (pPKRd). RNA serves as a scaffold bringing two PKRs together allowing dimerization and autophosphorylation to occur. The contribution of each state to the function of PKR remains unclear. Western blots were performed to examine the phosphorylation states of the essential residues,

T446 and T451. Activity assays have shown activation …

Minimotif Miner 2nd Release: A Database And Web System For Motif Search, Sanguthevar Rajasekaran, Sudha Balla, Patrick R. Gradie, Michael R. Gryk, Krishna Kadaveru, Vamsi Kundeti, Mark W. Maciejewski, Tian Mi, Nicholas Rubino, Jay Vyas, Martin R. Schiller

Life Sciences Faculty Research

Minimotif Miner (MnM) consists of a minimotif database and a web-based application that enables prediction of motif-based functions in user-supplied protein queries. We have revised MnM by expanding the database more than 10-fold to approximately 5000 motifs and standardized the motif function definitions. The web-application user interface has been redeveloped with new features including improved navigation, screencast-driven help, support for alias names and expanded SNP analysis. A sample analysis of prion shows how MnM 2 can be used.

Letter From The Dean, Lalit Verma

Discovery, The Student Journal of Dale Bumpers College of Agricultural, Food and Life Sciences

No abstract provided.

Detection Of Biomarkers By Mrs Requires Error Analysis, Jeffrey C. Hoch, Mark W. Maciejewski, Michael R. Gryk

UCHC Articles - Research

Using MRS imaging and singular value decomposition (SVD), Manganas et al. (Science 318, 980,2007) reported a nuclear magnetic resonance diagnostic for neural progenitor cells. Characteristics of MRS experiments are known to be problematic for SVD-based methods. Absent detection using alternate methods of spectrum analysis or controls to quantify the false discovery rate, little significance can be attached to their result.

The Development Of A Gc/Ms Protocol For The Analysis Of Polysaccharides In Echinacea Purpurea, Michaela Knapp

Undergraduate Theses and Capstone Projects

Echinacea purpurea is an herbal supplement used to reduce symptoms of common colds and flu-like illnesses. As of right now, it is not well understood what chemical components in the plant are immunologically active. The four main potentially immunologically active chemical components in E. purpurea are caffeic acid derivatives, alkamides, polyacetylenes, and polysaccharides. In this investigation, a protocol was developed in an attempt to identify polysaccharides present in Puritan’s Pride E. purpurea (aerial) “non-irradiated” herbal supplement First, an estimate of the crude total polysaccharide content in Puritan’s Pride and in a ChromaDex certified botanical standard (E. purpurea -powdered root) was …

Cell Edge Features Affected By Microtubule Inhibitor Combinations, Sonal Uppal, Nancy Boudreau, Elizabeth Wendt, Carol Heckman

Applied Statistics and Operations Research Faculty Publications

The tumor promoter, phorbol 12-myristate 13-acetate (PMA), enhances tumor yield through an epigenetic mechanism. PMA, like another promoter, phosphatase inhibitor okadaic acid, works by maintaining proteins in a phosphorylated state. In order to identify chemicals with promoter and antipromoter effects, this laboratory has developed a standard curve of morphogenetic changes using data from precancerous cell lines that eventually became neoplastic. Using the curve as a basis of comparison, we defined the “signature” phenotype as that adopted when a cell line became neoplastic. The results of solving for signature type disclosed that the microtubule-depolymerizing compound, colchicine, had a promoter-like effect [2]. …

Epichlorohydrin Cross-Linking Of Synthetic Dna Oligomers, Rami W. Zaharan, Julie T. Millard

Undergraduate Research Symposium (UGRS)

Epichlorohydrin (ECH), an important chemical in the synthetic polymer industry, is a bifunctional alkylating agent with the potential to form DNA interstrand crosslinks. Occupational exposure to this suspect carcinogen leads to chromosomal aberrations, and ECH has been shown to undergo reaction with DNA in vivo and in vitro. We are using denaturing polyacrylamide gel electrophoresis to assess cross-linking of synthetic DNA oligomers by both ECH and the related compound, epibromohydrin (EBH). Both epihalohydrins produce a low-mobility band on denaturing gels consistent with an interstrand cross-link. Moreover, the efficiencies, sequence preferences, reaction kinetics, and pH dependence differ for the two compounds, …

A Critical Role For Kalirin In Ngf Signaling Through Trka, Kausik Chakrabarti, Rong Lin, Noraisha I. Schiller, Yanping Wang, David Koubi, Ying-Xin Fan, Brian B. Rudkin, Gibbes R. Johnson, Martin R. Schiller

Life Sciences Faculty Research

Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low …

The Role Of Domain I In Laminin Chain Assembly, George D. Kamphaus

Theses, Dissertations and Capstones

Laminin, a major component of basement membrane, is a trimeric glycoprotein comprised of three chains - α, β and γ (Burgeson et al., 1994). An order for trimer assembly has been deduced: first, the β and γ chains bind to form a dimer and subsequently α is added to complete the trimer (I. Hunter et al., 1990 & 1992; Utani et al., 1994 & 1995). The C-terminal portions, found within the protein structural domain I of the p and y chains, are implicated in dimer and trimer formation by biochemical studies performed extracellularly (Utani, et al., 1994 & 1995; Nomizu …

Type I Camp-Dependent Protein Kinase Delays Apoptosis In Human Neutrophils At A Site Upstream Of Caspase-3, Lav K. Parvathenani, E. Stephen Buescher, Enrique Chacon-Cruz, Stephen J. Beebe

Bioelectrics Publications

Current data suggest that apoptosis controls neutrophil numbers in tissues. We analyzed roles for and the sites of action for the cAMP-dependent protein kinases (cAPKs) in apoptosis induced in human neutrophils by in vitro storage, cycloheximide (CHX) exposure, and anti-Fas exposure. Treatment with 8-chlorophenylthio-cAMP (8-CPT-cAMP) prolonged the time required for 50% of the cells to exhibit apoptotic morphology (t 50) from 16.3 to 41.8 h (in vitro culture), from 2.4 to 7.8 h (CHX), and from 4.8 to 6.5 h (anti-Fas). CHX ± 8-CPT-cAMP did not significantly alter resting intracellular calcium levels and H-89, a selective inhibitor of cAPK, had …

Effects Of Creatine Supplementation On Muscular Strength And Power Development In Well-Trained Football Players, Josef D. Mcquain

Theses, Dissertations and Capstones

The purpose of this study was to determine the effects of creatine supplementation on strength and power performance in well-trained football players. Twenty-one subjects were randomly assigned to three treatment groups: 1) creatine monohydrate, 2) creatine monohydrate+dextrose, or 3) placebo utilizing a double-blind design. Prior to assignment, subjects were matched according to lean body mass. After a five day loading dose, a maintenance dose was administered for 12 days. Test protocols included three anaerobic work bouts: l) two 30 s Wingate bike tests interspersed with five minutes recovery were used to ascertain relative and absolute power, 2) two 100 yard …

An Immunomodulating Mycotoxin Interferes With The Development Of Autoimmune Diabetes In Diabetes-Prone Bb/Wor Rats, Honggang Liu

Theses, Dissertations and Capstones

Various fungal products have immunomodulating activity and some have been studied regarding prevention of transplantation rejection. Prior to this investigation, the mycotoxin, gliotoxin (GT), has never been investigated as an immunotherapeutic drug for autoimmune disease. GT is a fungal secondary metabolite and a member of the epipolythiodioxopiperazine (ETP) family which has been shown to inhibit phagocytosis, induction of cytolytic T cells and the proliferation of T cells following mitogen stimulation. GT also induces in vitro apoptosis in certain immune cell types. More importantly, GT exhibits selective activity towards cells of hemopoietic origin.

Autoimmune diseases are disorders caused by immune responses …

The Regulation Of Glucose Transport In Cultured Vascular Smooth Muscle Cells By Angiotensin Ii And Glucose, Leslie Ann Quinn

Theses, Dissertations and Capstones

Glucose transport was assessed in vascular smooth muscle (VSM) cells by measuring the uptake of a radiolabeled non-metabolizable glucose analog, [3H]-2-deoxglucose. VSM cells, isolated from rat aortae by enzymatic digestion, were maintained in culture in Dulbecco's Modified Eagle Medium supplemented with 10% newborn calf serum at 37°C with 5% CO2 and air. Angiotensin II (AU) increased glucose transport by 84%. Significant stimulation occurred by two hours of exposure with the maximum effect being observed between six and eight hours. All effects were concentration dependent with a threshold response being detected at 0.1 nM. All-stimulated transport was blocked by an AU …

Effect Of Retinoic Acid On The Expression And Function Of Ap-1 Transcription Factor In B16 Mouse Melanoma Cells: Role Of Protein Kinase, Sejal H. Desai

Theses, Dissertations and Capstones

Retinoic acid (RA) induces differentiation of B16 mouse melanoma cells. This differentiation is accompanied by an increase in protein kinase Ca (PKCα) protein level and selective enrichment in nuclear-associated PKCα. PKC is thought to regulate gene expression through the TPA response element (TRE). This element is specifically recognized by the AP-1 transcription factor composed of jun and fos family members. In this study, I have analyzed the effect of RA on the expression and function of AP-1 in B16 mouse melanoma cells. Transient transfection analysis of B16 cells using leuciferase reporter gene constructs with or without AP-1 elements indicated that …

Regulation Of Melanogenesis In B16 Mouse Melanoma Cells By Protein Kinase C (Pkc), Harish Mahalingam

Theses, Dissertations and Capstones

The pigment cell-specific expression of tyrosinase and TRP1 has been shown to be important for the production of melanin in pigmented cells. Using a pigmented cell line, B16 mouse melanoma, we obtained evidence that PKC plays a major role in regulating melanogenesis. Chronic treatment with phorbol dibutyrate (PDBu) leads to downregulation of PKC activity and protein levels. This is accompanied by a loss of pigmentation which is correlated with a 50% reduction and a complete loss of TRP1 and tyrosinase respectively. Similar results were obtained with Northern and Western blotting indicating that PKC may regulate the steady state levels of …

Morfología Y Moléculas: Bases Complementarias De La Sistemática Moderna, Rafael O. De Sá, David M. Hillis

Biology Faculty Publications

RESUMEN

El auge de las técnicas moleculares en las últimas dos décadas ha traído nuevos enfoques en los estudios sistemáticos. Aquí presentaremos una revisión del uso, las aplicaciones, y los problemas de las principales metodologías de trabajo. Cada una de estas técnicas es aplicable a algunos problemas e inapropiada para otros, y es importante entender las suposiciones y limitaciones de cada técnica. Concluimos que el uso combinado de los enfoques morfológicos y moleculares otorga mejor resolución a los problemas sistemáticos.

ABSTRACT

The development of molecular techniques during the last two decades has brought new approaches to the analysis of systematic …

The Cγ Subunit Is A Unique Isozyme Of The Camp-Dependent Protein Kinase, Stephen J. Beebe, Paul Salomonsky, Tore Jahnsen, Yixin Li

Bioelectrics Publications

There are at least three isozymes (Cα, Cβ, and Cγ) of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase (PKA) (Beebe, S., Oyen, O., Sandberg, M., Froysa, A., Hansson, V., and Jahnsen, T. (1990) Mol. Endocrinol. 4, 465-475). To compare the Cγ and Cα isozymes, the respective cDNAs were expressed in permanently transformed Kin-8 PKA-deficient Y1 adrenal cells using the mouse metallothionein promoter. The recombinant C subunits were characterized as immunoreactive, zinc-inducible, cAMP-dependent kinase activities. In contrast to Cα, histone was a better substrate than Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) for Cγ. Furthermore, Cγ histone kinase activity was not inhibited by the …

Cell Surface-Binding Sites For Progesterone Mediate Calcium Uptake In Human Sperm, Peter F. Blackmore, Joseph Neulan, Frank Lattanzio, Stephen J. Beebe

Bioelectrics Publications

Recent studies (e.g. Blackmore, P. F., Beebe, S. J., Danforth, D. R., and Alexander, N.) (1990) J. Biol. Chem. 265, 1376-1380) have shown that in human sperm, progesterone produces a rapid increase in intracellular free calcium ([Ca2+]i) and an induction of the acrosome reaction (e.g. Osman, R. A., Andria, M. L., Jones, A. D., and Meizel, S. (1989) Biochem. Biophys. Res. Commun. 160, 828-833). In this study, the location of progesterone receptors on the cell surface of human sperm was identified using progesterone immobilized on bovine serum albumin (BSA) (progesterone 3-(O-carboxymethyl)oxime:BSA) as well as progesterone and its 3-O-carboxymethyloxime derivative. Using …

Progesterone And 17 Α-Hydroxyprogesterone: Novel Stimulators Of Calcium Influx In Human Sperm, Peter F. Blackmore, Stephen J. Beebe, Douglas R. Danforth, Nancy Alexander

Bioelectrics Publications

Progesterone and 17 alpha-hydroxyprogesterone (but not other steroids such as testosterone, corticosterone, beta-estradiol, estrone, dehydroepiandrosterone, 20 alpha-hydroxypregnen-3-one, androstenedione, and pregnenolone) were shown to cause an immediate increase, in free cytosolic calcium ([Ca2+]i) in both capacitated and noncapacitated human sperm, using the fluorescent indicator fura 2. Significant increases in [Ca2+]i were observed with 10 ng/ml progesterone, while maximum effects were seen with 1 microgram/ml progesterone. Two other steroids 11 beta-hydroxyprogesterone and 5 alpha-pregnane-3,20-dione exhibited significant activity to increase [Ca2+]i. This increase in [Ca2+]i elicited by progesterone was entirely due to Ca2+ influx from the extracellular medium since the increase in [Ca2+]i …

The Characterization Of Ribosomal Rna Gene Chromatin From Physarum Polycephalum, Sally A. Amero, Vicky L. Montoya, Wendy L. Murdoch, Roy C. Ogle, John L. Keating, Robert M. Grainger

School of Medical Diagnostics & Translational Sciences Publications

We have isolated ribosomal RNA gene (rDNA) chromatin from Physarum polycephalum using a nucleolar isolation procedure that minimizes protein loss from chromatin and, subsequently, either agarose gel electrophoresis or metrizamide gradient centrifugation to purify this chromatin fraction (Amero, S. A., Ogle, R. C., Keating, J. L., Montoya, V. L., Murdoch, W. L., and Grainger, R. M. (1988) J. Biol. Chem. 263, 10725-10733). Metrizamide-purified rDNA chromatin obtained from nucleoli isolated according to the new procedure has a core histone/DNA ratio of 0.77:1. The major core histone classes comigrate electrophoretically with their nuclear counterparts on Triton-acid-urea/sodium dodecyl sulfate two-dimensional gels, although they …

The Purification Of Ribosomal Rna Gene Chromatin From Physarum Polycephalum, Sally A. Amero, Roy C. Ogle, John L. Keating, Vicky L. Montoya, Wendy L. Murdoch, Robert M. Grainger

School of Medical Diagnostics & Translational Sciences Publications

We have undertaken the purification of ribosomal RNA gene (rDNA) chromatin from the slime mold Physarum polycephalum, in order to study its chromatin structure. In this organism rDNA exists in nucleoli as highly repeated minichromosomes, and one can obtain crude chromatin fractions highly enriched in rDNA from isolated nucleoli. We first developed a nucleolar isolation method utilizing polyamines as stabilization agents that results in a chromatin fraction containing far more protein than is obtained by the more commonly used divalent cation isolation methods. The latter method appears to result in extensive histone loss during chromatin isolations. Two methods were then …

Short-Term Feedback Regulation Of Camp By Accelerated Degradation In Rat Tissues, Tom W. Gettys, Peter F. Blackmore, J. Bruce Redmon, Stephen J. Beebe, Jackie D. Corbin

Bioelectrics Publications

A recent study showed that cAMP analogs lowered cAMP levels in rat hepatocytes. The present work demonstrates that cAMP analogs also lowered cAMP in a rapid, concentration-dependent manner in heart and fat cells. In order to determine if the cAMP-dependent protein kinase mediated this effect, techniques were developed to assay the protein kinase activity ratio in hepatocytes treated with cAMP analogs. The activation of protein kinase and phosphorylase in hepatocytes by 8-pClΦS-cAMP (where 8-pClΦS- indicates 8-parachlorothiophenyl-) was concentration-dependent and occurred in parallel to proportionate decreases in cAMP. More than 20% of the cAMP binding sites on the protein kinase were …

Camp-Dependent Protein Kinase Activation Lowers Hepatocyte Camp, Jackie D. Corbin, Stephen J. Beebe, Peter F. Blackmore

Bioelectrics Publications

Rat hepatocyte protein kinase was activated by incubating the cells with various cAMP analogs. Boiled extracts were then prepared and Sephadex G-25 chromatography was carried out. The G-25 procedure separated the analogs from cAMP since the resin had the unexpected property of binding cyclic nucleotides with differing affinities. Separation was necessary because the analogs would otherwise interfere with the sensitive protein kinase activation method developed for assay of cAMP. The cAMP analogs, but not 5'-AMP, lowered basal cAMP by 50-70%. The effect was rapid, analog concentration-dependent, and occurred parallel with phosphorylase activation, suggesting that the cAMP analogs act through cAMP-dependent …

Discriminative Insulin Antagonism Of Stimulatory Effects Of Various Camp Analogs On Adipocyte Lipolysis And Hepatocyte Glycogenolysis, Stephen J. Beebe, J. Bruce Redmon, Peter F. Blackmore, Jackie D. Corbin

Bioelectrics Publications

Although insulin effectively blocked hormone-stimulated glycerol output in adipocytes or phosphorylase activation in hepatocytes, the inhibitory effect of insulin on cAMP analog-stimulated cells depended on the cAMP analog used. Of the 20 analogs tested in adipocytes and 13 tested in hepatocytes, the effects of about half of them were effectively blocked by insulin, whereas the effects of many of them were not inhibited at all. In order to approach the explanation for this discriminative insulin action, the inhibitory effects of insulin on the responses to the analogs in the intact cells were correlated with the in vitro cAMP analog specificity …

Purification And Characterization Of A Camp- And Ca2+-Calmodulin-Independent Glycogen Synthase Kinase From Porcine Renal Cortex, Stephen J. Beebe, Erwin M. Reimann, Keith K. Schlender

Bioelectrics Publications

We recently reported the partial purification of a cAMP-independent and Ca²⁺-calmodulin-independent glycogen synthase kinase from porcine renal cortex (Schlender, K. K., Beebe, S. J., and Reimann, E. M. (1981) Cold Spring Harbor Conf. Cell Proliferation, 389-400). Subsequent purification indicated that the enzyme preparation consisted of at least three forms of glycogen synthase kinase which could be resolved by ATP gradient elution from aminoethylphosphate-agarose (AEP-agarose). The predominant form of glycogen synthase kinase, which eluted from AEP-agarose between 2 and 6 mM ATP, was purified approximately 800-fold and is designated GSK-A1. It had a molecular weight of 45,000-50,000 as determined …

Microheterogeneity Of Type Ii Camp-Dependent Protein Kinase In Various Mammalian Species And Tissues, Alison M. Robinson-Steiner, Stephen J. Beebe, Stephen R. Rannels, Jackie D. Corbin

Bioelectrics Publications

Excluding autophosphorylated species, at least six forms of the regulatory subunit of type II cAMP-dependent protein kinase (R(II)) from various mammalian tissues were identified by sodium dodecyl sulfate (SDS) gel electrophoresis of purified samples and of crude preparations photoaffinity labeled with 8-azido[32P] cAMP and by gel filtration. After autophosphorylation some heart R(II) forms termed type IIA (bovine, porcine, equine, and dog) shifted to a more slowly migrating band on SDS gels while others termed type IIB (rat, guinea pig, rabbit, and monkey) did not detectably shift. Both subclasses of R(II) exhibited variation in apparent M(r) on SDS gels. Bovine and …

Two Classes Of Camp Analogs Which Are Selective For The Two Different Camp-Binding Sites Of Type Ii Protein Kinase Demonstrate Synergism When Added Together To Intact Adipocytes, Stephen J. Beebe, Rob Holloway, Stephen R. Rannels, Jackie D. Corbin

Bioelectrics Publications

Twenty-five cyclic nucleotide analogs were tested individually to act as lipolytic agents and to activate adipocyte protein kinase. The lipolytic potency of individual analogs correlated better with their K(a) for protein kinase and their lipophilicity rather than with either parameters alone. Some of the most potent lipolytic analogs had high I50 values for the particulate low K(m) cAMP phosphodiesterase suggesting that their effect was not due to raising endogenous cAMP levels through inhibition of phosphodiesterase. The most potent lipolytic analogs contained a thio moiety at the C-8 or C-6 position. These analogs exhibited concave upward dose-response curves. At high concentrations …