Chemicals and Drugs Commons^™

Assessment Of Lipid Extraction From Human Bone On Subsequent Demineralization, Jaeeun Lee

Biological Sciences Theses & Dissertations

The purpose of this study was to assess the effectiveness of various solvents m removal of lipids from human bone, and to determine the effects of lipid extraction on subsequent acid solubilization of the mineral components of bone. Specifically, the following processing steps were studied; 1) ground human bone was de fatted in various ethanol / water mixtures, and / or chloroform / methanol ( 2: 1, v:v ), 2) ground human bone which had lipids extracted ( in absolute ethanol ) and bone which had not had lipids extracted were demineralized using vanous concentrations of hydrochloric acid to assess …

Activation In Vitro Of Transfer Rna-Guanine Ribosyltransferase By Protein Kinase C, Panayota Eriotou

Chemistry & Biochemistry Theses & Dissertations

The purpose of this study was to isolate and purify the enzymes, transfer RNA-guanine ribosyltrasferase and protein kinase C, and to determine whether the phosphorylating enzyme activates the insertion enzyme in vitro. Transfer RNA-guanine ribosyltransferase lost activity within several days after isolation and total, complete reactivation was accomplished in the presence of protein kinase C. This demonstrated that ribosyltransferase's instability is related to the degree of its phosphorylation. It is proposed that modification of tRNA is controlled by protein kinase C. Deactivation of the insertion enzyme leads to hypomodified tRNA which in turn is associated with neoplasia. Potential use …

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

Medical Diagnostics & Translational Sciences Faculty 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 …

Microwell Based Solid Phase Competitive Enzyme Immunoassay For Estradiol And Estriol, Jeng-Ting Yang

Chemistry & Biochemistry Theses & Dissertations

A sensitive and simple microwell based competition enzyme immunoassay for the quantitative determination of estradiol (E₂) and estriol (E₃) was developed. Nicrowells coated with antibody were incubated with antigen followed by adding horseradish peroxidase (HRPO) conjugate. The assay, which can be performed within two hours at room temperature, involved simultaneous incubation of E₂ - or E₃-HRPO conjugate and serum sample in polystyrene microwells coated with anti- E₂ or anti-E₃ gamma globulin fraction.

Gamma globulin was isolated from whole anti-serum by DEAE-cellulose chromatography. A carbodiimide coupling method was utilized to prepare the …

Development Of A Solid Phase Enzyme Immunoassay For Total Serum Testosterone, Li Li

Chemistry & Biochemistry Theses & Dissertations

An enzyme-labeled immunoassay of sufficient range and sensitivity was developed for serum testosterone determination. In this method, the testosterone in the serum sample and testosterone-HRPO conjugate compete with each other for the limited antibody binding sites on the anti-testosterone gamma globulin coated microwell. The assay can be performed in only 2.5 hours.

The gamma globulin fraction was isolated from the anti-testosterone whole serum by DEAE-cellulose column chromatography. Testosterone-peroxidase conjugate was prepared by a carbodiimide coupling method. The interference which arises from the binding of testosterone to steroid hormone binding protein was eliminated by the addition of 17 beta-estradiol. All assay …

The Determination Of Clomiphene Citrate And Tamoxifen In Human Blood Plasma And Semen, Janice Irene Kiss

Chemistry & Biochemistry Theses & Dissertations

Triphenylethylene antiestrogen compounds are used to stimulate ovulation in anovulary women and spermatogenesis in oligozoospermatic males. Clomiphene Citrate (CC) is given therapeuticaly as a mixture of the trans (E) and cis (Z) isomers. Each of these isomers has its own characteristic agonist-antagonist profiles. Only the trans form of Tamoxifen (T) is administered therapeutically.

HPLC methodologies were developed for the determination of CC and Tin human blood and seminal plasma. The methodologies involved extraction of the triphenylethylene compounds using hexane, followed by evaporation of the hexane under N₂. The residue was then taken up in a volume of HPLC …

Regulation Of Queuine Insertion Into Transfer Rna: Effects Of Tumor Promoters, Bonnie Jean Brooks

Chemistry & Biochemistry Theses & Dissertations

The purpose of this study was to treat normal human fibroblasts with known tumor promoters and observe queuine modification of tRNA. The queuine insertion enzyme, tRNAguanine ribosyltransferase, was studied in vivo and in vitro. Tumor promoter-treated human fibroblast cultures exhibited variable queuine-insertion rates with a transient inhibition that correlated with variable levels of queuine modified tRNA over time in culture from passages 3 — 8. In contrast the in vitro studies showed that phorbol cetera and saccharin actually increased insertion with strong evidence indicating phosphorylation as a positive modulating force of the enzyme activity. It is proposed that chronic stimulation …

Microwell Based Competitive Enzyme Immunoassay For Quantifying Serum Triiodothyronine And Thyroxine Levels, Yiwen Yang

Chemistry & Biochemistry Theses & Dissertations

A microwell based competitive enzyme immunoassay for determination of total thyroxine and triiodothyronine in human serum was developed with use of horseradish peroxidase conjugate. The assay, which can be performed in only 1.5 hours at room temperature, involves simultaneous incubation of T₃- or T₄- HRPO conjugate and serum sample in anti-T₃ or anti-T₄ gamma globulin fraction coated polystyrene microwells.

Isolation of anti-T₃ and anti-T₄ gamma globulin fractions were accomplished from whole antiserum by using DEAE-cellulose chromatograph. A carbodiimide coupling method was described to prepare the T₄- and T₃-HRPO …

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

Medical Diagnostics & Translational Sciences Faculty 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 …

Isolation And Partial Characterization Of Basic, Sperm-Specific, Nuclear Proteins In The Dog And Hampster, Richard William Hall

Biological Sciences Theses & Dissertations

The purpose was to determine whether dogs and hamsters contain the same sperm-specific protamine and testis specific proteins (TP) as other eutherian mammals previously studied. The proteins (protamine and TPs) were isolated from spermatids and mature spermatozoa, and were subjected to electrophoresis in an acid-urea, polyacrylamide gel system according to the procedure of Panyim and Chalkley (1962). The results indicated that, regardless of the anatomical location of sperm acquisition, acid precipitation technique, or denaturing agents used, epididymal and testicular samples from both the dog and hamster contained protamine in elongated spermatids and mature spermatozoa. The protamines from dogs and hamsters …

A Method For The Large Scale Purification Of Hcg (Human Chorionic Gonadotropin) From The Urine Of Pregnant Women, Chun-Shyan Kuo

Chemistry & Biochemistry Theses & Dissertations

A large scale preparation of human chorionic gonadotropin (HCG) from the urine of pregnant women was accomplished by using kaolin adsorption, NH₄OH extraction and DEAE-cellulose ion exchange. A recovery about 81% of HCG and a specific activity in the 2,000-3,000 IU/mg range was achieved using this purification method.

Two HCG activity fractions, HCG I & HCG II, were isolated from DEAE-cellulose chromatography by a stepwise sodium chloride gradient elution. HCG I was eluted at 0.01-0.02 M NaCl while HCG II was eluted at 0.12-0.02 M NaCl.

The differences between HCG I and HCG II have been studied. The …

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

Medical Diagnostics & Translational Sciences Faculty 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 …

Synthesis And Characterization Of N-Alkyl-Adenosine-5'-Diphosphates, Han-Ping Huang

Chemistry & Biochemistry Theses & Dissertations

n-Alkylphosphates and adenosine derivatives have been observed to be competitive inhibitors of coenzyme molecules in many NAD⁺ requiring enzymes. In earlier studies, adenosine derivatives such as AMP, ADP and ADP-ribose were found to be coenzyme competitive inhibitors of the Lactate Dehydrogenase catalyzed reaction. However, n-alkylphosphates, to the contrary, + were found to be noncompetitive inhibitors of NAD⁺.

Furthermore, it was observed previously in Dr. Yuan's laboratory that the effect of n-octylphosphate on the LDH catalyzed reaction was changed from noncompetitive to competitive in the presence of low concentration of AMP. Rossmann et al. reported that the binding …

Investigation Of Complex Formation By Oligomers Of Cytosine And Guanosine, Steven Roberts Davis

Chemistry & Biochemistry Theses & Dissertations

Duplex formation between oligo(C:G) _n where n=3 to 4 was shown not to occur under conditions favorable for duplex formation between poly G and poly C. Instead, a stable guano sine self-structure was found to form which a T_m of 50°C for (Gp)₃ and 80°C for (Gp)₄ at strand concentrations of 10^-5M in 1M NaCl. Neither a duplex nor a self-structure formed in the absence of salt.

Oligomers of guanosine and cytosine were obtained by basic hydrolysis and separated according to chain length using DEAE Sephadex column chromatography. Separation of cytosine oligomers with chain lengths …

Tardive Dyskinesia: A Prevalence Study In An Outpatient Population, Virginia J. Ravizza

Community & Environmental Health Theses & Dissertations

The purpose of this prevalence study was to assess the occurrence of tardive dyskinesia among a chronically mentally ill sample of a suburban mental health clinic. The participants in this study were selected from clients of a suburban mental health clinic who agreed to participate in this prevalence study. There were 45 participants--17 males and 28 females.

The Abnormal Involuntary Movement Scale (AIMS) was selected as the instrument to measure the abnormal involuntary movements exhibited by the participants of the study on the day of testing. The testing occurred during a specific time period, June 8, 1987 to July 2, …

The Synthesis And Evaluation Of The Cardiovascular Activity Of Novel Imidazole Ring Systems, Joseph E. De Los Angeles

Chemistry & Biochemistry Theses & Dissertations

An investigation was undertaken to synthesize and evaluate the cardiovascular activity of two imidazole ring systems. Imidazoles with hypotensive activity would provide novel potential antihypertensive drugs. These novel structures may also find use as chemical probes in the study of the cardiovascular system and as prototypes for other potential cardiovascular agents.

The histamine derivative, 7,7-dipyridyl-4,5,6,7-tetrahydroimidazo-[4,5-c]pyridine (ligand l), was demonstrated to possess significant hypotensive activity in laboratory rats. A 15 mg/Kg dose of ligand 1 produces a statistically significant decrease in blood pressure when injected IP. This hypotensive effect is also associated with marked increase in heart rate. Although compound 1 …

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 …

Negative Cooperatively In The Binding Of 3-Aminopyridine Adenine Dinucleotide To L-Α-Glycero-3-Phosphate Dehydrogenase, David Wheeler

Chemistry & Biochemistry Theses & Dissertations

Glycerol-3-phosphate dehydrogenase catalyzes the reversible reaction:

NAD⁺ + G-3-P ⇌ NADH + DHAP + H⁺

The enzyme is found in organisms from E. coli to man. The primary function of the enzyme varies from organism to organism. Depending on the metabolism of the organism, the enzyme may function primarily to generate G-3-P for lipid (in organisms with a highly aerobic biosynthesis metabolism), or it may function primarily to produce NAD⁺ to support glycolysis. Complex multicellular organisms possess tissue specific isozymes of GPDH whose kinetic parameters reflect the relative importance of G-3-P and NAD⁺ production in various …

Two-Dimensional Electrophoretic Studies Of Normal Spematozoal Proteins, Ying Lin

Chemistry & Biochemistry Theses & Dissertations

Protein profiles of human spermatozoa have been mapped by using high resolution two-dimensional electrophoresis which combines isoelectric focusing as the first dimension and polyacrylamide gradient gels electrophoresis in SDS as the second dimension. The profiles of normal spermatozoa! extraction, acrosomal extraction and seminal plasma showed high resolution in its separation capability. No qualitative or quantitative differences were observed by using Coomassie Brilliant Blue R-250 staining in a much higher concentration of proteins. However, coomassie blue lacks the sensitivity to detect many minor components. Therefore, a silver staining procedure was used, which resulted in greatly increased sensitivity.

Samples from normal fertile …

A Method For The Comparison Of Hdl Containing Apoprotein E And Hdl Cholesterol, Steven A. Knizner

Chemistry & Biochemistry Theses & Dissertations

Human serum high density lipoproteins (HDL) were isolated by preparative salt density gradient ultracentrifugation and analyzed further by heparin-sepharose affinity chromatography. Separation of the major classes of serum lipoproteins was achieved after a single ultracentrifugation on a salt density gradient at 272,000 x g for 36 hours in a swinging bucket rotor. After isolation, the total HDL was subdivided into HDL containing apoprotein E and HDL without this apoprotein by heparin-sepharose affinity chromatography. Relative amounts of these subclasses were then calculated.

HDL-cholesterol was measured after precipitation of LDL and VLDL by phosphotungstic acid and magnesium. The statistical relationship between HDL …

Investigation Of The Coenzyme Binding Site Of Lactate Dehydrogenase From Chicken Muscle, Jia-Hwei Lin

Chemistry & Biochemistry Theses & Dissertations

Investigation of the coenzyme binding site of lactate dehydrogenase was approached by using coenzyme analogs. Inhibition studies of this enzyme using n-alkylphosphates as coenzyme analogs were performed with a fluorometric method. The results showed that all of the n-alkylphosphates were competitive inhibitors with respect to NAD⁺.

The n-alkyl-ADPs were synthesized from AMP and dicycloheaylammonium salts of n-alkylphosphates. The synthetic compounds were subjected to NMR spectroscopic studies, thin layer chromatography and determination of the ratio of adenosine moiety to phosphate groups. The results indicated that the synthetic compounds were n-alkyl-ADPs.

These synthetic compounds were used as coenzyme analogs in …

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 …

High Resolution Two Dimensional Electrophoresis Of Soluble Proteins In Human Spermatozoa, Hsing-Ying Liu

Chemistry & Biochemistry Theses & Dissertations

The evaluation of human semen is very important for diagnosis of male infertility. Combined with the analysis of seminal plasma, a comprehensive biochemical screening of human spermatozoan proteins may be of assistance in providing qualitative and quantitative information to the physician to assist with evaluation.

In this study, human spermatozoa were ruptured by sonication. The soluble spermatozoan proteins were separated from the cellular debris by centrifugation and then analyzed in dissociated forms by two dimensional electrophoresis. The separated proteins were visualized by a silver stain method.

It has been shown that these combined techniques can resolve- four to five hundred …

Development Of A Procedure For Analysis Of High Density Lipoprotein Subclasses, Chan Chin

Chemistry & Biochemistry Theses & Dissertations

Human serum high density lipoprotein subclasses, HDL₂ and HDL₃ , were isolated by preparative salt density gradient ultracentrifugation and further analyzed by electrophoresis on a 4 to 15% concentration gradient polyacrylamide gel.

The separation of the major classes of serum lipoproteins was achieved after a single ultracentrifugation for 272,000 g at 15°c in a swinging bucket rotor. High resolution concentration gradient gel electrophoresis was found to be particularly suitable for the separation of lipoproteins. The isolation of HDL by precipitation methods, heparin-manganese and dextran-magnesium have also been evaluated in this study, and were found not to completely separate …

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 …

The Synthesis And Biochemical Evaluation Of Potential Acetylcholinesterase Reactivators, Andrea Mcdearmid Lunsford

Chemistry & Biochemistry Theses & Dissertations

Objectives of this research were to (1) synthesize, and (2) evaluate new compounds which would be potential reactivators of organophosphate-inhibited acetylcholinesterase.

A series of 1,8-diazafluorenone and 2,2'bipyridylketone oximes and oxime methiodides were synthesized (11, 13, 14, 22, 24). These compounds were developed as structural analogs of 2-PAM (2-Pyridinealdoxime methiodide) and TMB-4 (1,1Trimethylene-Bis(4-formylpyridinium bromide)dioxime), the current drugs of choice for treatment of organophosphorous poisoning.

Acetylcholinesterase from electric eel was inhibited with DFP (diisopropylfluorophosphate), an irreversible-type inhibitor, and the inhibited enzyme was treated with the various reactivator compounds. The reactivation assays were monitored with pH stat apparatus, and the data were evaluated …

Multiple Forms Of Nicotinamide Adenine Dinucleotide Glycohydrolase From Bull Semen, Stanley D. Yeatts Ii

Chemistry & Biochemistry Theses & Dissertations

Nicotinamide adenine dinucleotide glycohydrolase (NADase) of bull semen is an extracellular, soluble enzyme capable of catalyzing the hydrolysis of the nicotinamide N-ribosidic linkage of NAD⁺. In search for a rapid, simple procedure for the preparation of this enzyme, it was found that Matrex Gel Red A (Amicon) column packing served very well as an affinity gel for NADase. The enzyme was applied to the Matrex Red A column (21 cm x 1.5 cm) in 10 mM sodium phosphate buffer, pH 6.0. Surprisingly, two fractions of NADase activity were obtained when the column was eluted with a sodium chloride …

High Resolution Two-Dimensional Electrophoretic Study Of Human Seminal Plasma Proteins, Edward E. Gaunt

Chemistry & Biochemistry Theses & Dissertations

A comprehensive biochemical screening procedure for the evaluation of seminal plasma constituents is needed which, when used in conjunction with semen analysis, can provide the physician with both qualitative and quantitative information regarding the fertility status of an individual.

High resolution two-dimensional electrophoresis is a technique whereby proteins in a complex mixture such as a biological fluid are separated in one dimension by molecular charge and in the second dimension by molecular mass. It has been the purpose of this investigation to adapt and evaluate this technique for the analysis of human seminal plasma.

Our efforts have shown that this …