Cell and Developmental Biology Commons^™

Solid Phase Extraction Of Mammalian Cell Mitochondrial Dna And Its Electrophoretic Separation In Agarose Gels, Theresa Brick-Miller

Biological Sciences Theses & Dissertations

An improved and efficient electrophoretic procedure for the isolation of mitochondrial DNA (mtDNA) is described. The solid phase extraction procedure for the isolation of mtDNA involves embedding as few as 200,000 Ehrlich ascites tumor cells into a block of agarose, digestion of cellular membranes by detergent action and subsequent electrophoretic separation of nucleic acids in agarose gels. Genomic DNA remains in the original block of agarose while the mtDNA migrates in the separation gel to a position equivalent to that obtained for mtDNA isolated by traditional procedures.

The efficacy of a large number of detergents, agarose concentrations, embedding conditions, embedding …

Acetylcholinesterase In The Neonatal Rat Brain: The Effects Of Prenatal Ethanol Exposure, Linda M. Boland

Biological Sciences Theses & Dissertations

Acetylcholinesterase (AChE) activity in the neonatal rat brain was determined by histochemical localization and quantitative biochemical analysis. AChE-positive perikarya were concentrated in the neostriatum, globus pallidus, medial habenular nucleus, amygdaloid complex, various hypothalamic nuclei, gigantocellular and paragigantocellular reticular nuclei, raphe nuclei, locus coeruleus, and motor nuclei of cranial nerves III-VII and IX-XII. Some areas stained differently or not at all when compared with the adult rat brain. The effects of prenatal ethanol exposure on regional AChE activity were assessed by comparing enzyme levels in offspring exposed via maternal consumption of a liquid diet containing 35 percent ethanol derived calories. This …

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 …

Characterization Of The Dna And Chromosonal Analysis Of Embryonic Dermacentor Variabilis (Say) Cells In Culture, Stanley Nicholas Mason

Biological Sciences Theses & Dissertations

Cultured embryonic Dermacentor variabilis cells were examined by spectrophotometric techniques to determine total cellular DNA concentration and guanine-cytosine content of the DNA and by differential staining procedures to define the karyotype further. The cells were found to have a mean DNA content of 16.98 pg and a DNA guanine-cytosine content of 49%. Chromosome banding attempts resulted in the specific characterization of four chromosomes and differential grouping of the seven remaining chromosomes. A previously unreported submetacentric chromosome was identified.

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 …

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 …

Electron Microscopic Demonstration Of Neural Connections Using Horseradish Peroxidase: A Comparison Of The Tetramethylbenzidine Procedure With Seven Other Histochemical Methods, Keith A. Carson, M-Marsel Mesulam

Biological Sciences Faculty Publications

Eight methods for the electron microscopic demonstration of horseradish peroxidase (HRP) labeling have been compared in adjacent series of vibratome sections of mouse lumbar spinal cord. The tracer, a HRP-wheat germ agglutinin (WGA) conjugate, was injected into the gastrocnemius muscle complex. Following retrograde axonal transport to the lumbar motor neurons and transganglionic anterograde transport of the tracer to the dorsal horn, the HRP activity was demonstrated in eight series of adjacent sections of lumbar spinal cord using eight methods. These included procedures using tetramethylbenzidine (TMB), benzidine dihydrochloride (BDHC), o-tolidine, paraphenylenediamine-pyrocatechol (PPD-PC), and 4 methods using 3,3'-diaminobenzidine (DAB). All eight methods …

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 …

Fine Structure Of The Nucleus And Cytoplasmic Feulgen-Positive Areas In The Developing Oocyte Of Argus (Persicargas) Radiatus, Bonnie J. Harding

Biological Sciences Theses & Dissertations

A transmission electron microscope analysis of the changes that occur in the nuclear chromatin and the Feulgen-positive areas of the oocyte cytoplasm as the female develops, feeds and mates is described. The description includes analysis of oocytes from three types of females: unfed, unmated; fed, unmated; and fed, mated. In all types, dense material passes through the extremely porous nuclear membrane where it accumulates in the cytoplasm.

Nuclei of previtellogenic oocytes appear the same in all females studied. Fibrillar bodies and up to four large nucleoli are seen. No typical chromatin is seen, but structures resembling nucleosomes appear throughout the …

The Analysis Of S-Adenosylmethionine And S-Adenosylhomocysteine In Normal And Neoplastic Cells, Kathryn Elizabeth Godburn

Chemistry & Biochemistry Theses & Dissertations

Altered patterns of methylation have been shown in both ribosomal and transfer RNA isolated from cancer cells. In contrast to the elevated activity of the methyltransferases responsible for this modification, hypomethylation appears as a general characteristic. The endogenous levels of S-adenosylmethionine and S-adenosylhomocysteine are important in relationship to their role as substrate and product of the transmethylation reaction. The levels of S-adenosylmethionine and S-adenosylhomocysteine were determined using phosphocellulose cation exchange or high pressure liquid chromatography. High voltage paper electrophoresis was used in a modified procedure to directly resolve (³⁵s) labeled S-adenosylmethionine and S-adenosylhomocysteine in small quantities of cell …

The Effects Of 712-Dimethylbenz (A) Anthracene On Ovarian Steroidogenesis And Uterine Growth In The Mouse, Jeffrey Lynn Clark

Biological Sciences Theses & Dissertations

It has been previously demonstrated that 7,12-dimethylbenz(a) anthracene (DMBA) is an initiator of ovarian granulosa cell tumors. It is also known that DMBA leads to destruction of primordial oocytes, and that tumor induction may involve the interaction of pituitary gonadotropins. However, the effects of DMBA on ovarian steroidogenesis and uterine growth in relation to the tumorigenic process have not been investigated. Therefore, the present study was conducted to examine these parameters using adult C57Bl/6N mice treated in proestrus and diestrus II with 0.1 ml corn oil with or without 1.5 mg DMBA and killed 44 hours later. The uteri and …

Structural Studies Of Preimplantation Rabbit Embryos Using Ftir Atr, Amy R. Moler-Booth

Chemistry & Biochemistry Theses & Dissertations

FTIR was used in conjunction with a surface selective technique, known as attenuated total reflectance (ATR), to study the infrared spectra of preimplantation rabbit embryos. The rabbit embryos ranged in aged from 3 to 6 days. Since an embryo is mostly water, deuteration of the embryo was necessary to eliminate the intense water background present in the mid-infrared region. A set of spectral standards were tested, in order to identify the bands seen in embryo spectra. By analyzing and comparing the spectra from 3, 4, 5 and 6 day embryos, differences could be noted. In particular, bands defined as Amide …

Multiple Methods Of Analysis Of 2'-O-Methylation In 5.8s Rrna, Sharon H. Ryan

Chemistry & Biochemistry Theses & Dissertations

5.8S rRNA is a low molecular weight ribosomal RNA which is found hydrogen bonded to 28S rRNA in the eucaryotic cell. There are two nucleotides which have 2'-0-methylations; a guanine residue at position 77 is fully methylated and a uridine residue at position 14 which is partially methylated. This partial 2'-0-methylation of the uridine residue has been found to vary with the tissue source, with the highest level in normal tissue and the lowest level in neoplastic tissue. In order to study the significance of this site-specific methylation, a reliable and convenient method of analysis was needed.

Early studies on …

Detection Of Unique Tumor-Associated Proteins In The Urine Of Patients With Transitional Cell Carcinoma By High Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis, Robert David Lehman

Biological Sciences Theses & Dissertations

A two-dimensional polyacrylamide gel electrophoretic technique was developed for the detection of unique tumor-associated proteins in urine of patients with transitional cell carcinoma (TCC) of the urinary bladder. This technique yielded high resolution of polypeptides in the urine of cancer patients. Urine was chosen as a source of tumor-associated components because the location of the tum or should release already solublized tum or substances in to the urine.

Urinary proteins were concentrated and then analyzed by the two - dimensional electrophoretic technique using isoelectric focusing in the first dimension and sodium dodecylsulfate - polyacrylamide gel electrophoresis in the second dimension. …

A Potential Macroregulatory Mechanism For Growth Regulation In Neurospora Crassa, Martha Mooney

Biological Sciences Theses & Dissertations

Neurospora produces a phase specific, cationic mucopolysaccharide composed primarily of galactosamine (GAG-MP) which becomes part of the cell wall and is later secreted into the media. Its appearance coincides with the onset of the restricted phase of growth and causes efflux of small molecular weight metabolites when incubated with conidial cells. This activity may be a product of electrostatic interactions of the GAG-MP with the conidial plasma membrane. The activity is blocked by acetylation of the primary amines on the molecule, digestion by enzymes that hydrolyze carbohydrate linkages and the inclusion of NaCl (lM) in the GAG-MP/conidia reaction mixture. The …

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoretic Studies Demonstrating Cytoplasmic Protein Components Unique To The Mycelial Phase And To The Yeast Phase Of Candida Albicans, Theresa Catherine Burgess

Biological Sciences Theses & Dissertations

Blastospore and mycelial cytoplasmic extracts from four strains of Candida albicans, varying in virulence, were compared by sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis Cytoplasmic extracts were made by disrupting the cultures in a Braun homogenizer followed by ultracentrifugation. The supernatant fractions were then resolved on polyacrylamide gels containing SDS. Each of the preparations yielded a distinct pattern. There were mycelial specific and blastospore specific protein bands. There were more components resolved from the blastospore phase cytoplasmic extracts than from the mycelial phase cytoplasmic extracts. There were bands common to both phases. These results demonstrate protein components unique …

Comparison Of Interferon Produced In Vitro By Monkey Kidney Cells Demonstrating Interference Against Polio Ii, Coxsackie A-9, Influenza A2/Hong Kong/68, And Echo-9 Viruses, Archer Duncan Farmer Jr.

Biological Sciences Theses & Dissertations

Abstract unavailable

Cytogenic Studies Of A Chesapeake Bay Population Of Molgula Manhattensis (Dekay) (Ascidiacea: Stolidobranchia), Elizabeth A. Crawford

Biological Sciences Theses & Dissertations

Abstract unavailable.

The Effects Of The Cyclic 3'5'-Monophosphate Of Adenosine And Its Dibutyryl Derivative On The In-Vitro Immunization Of Mouse Spleen Cell Suspensions, James A. Blanks

Biological Sciences Theses & Dissertations

Abstract unavailable.