Biochemistry, Biophysics, and Structural Biology Commons^™

High Performance Liquid Chromatographic Separation Of Different Types Of Collagen, Yongjoo Chung

Chemistry & Biochemistry Theses & Dissertations

The objective of this research was to develop a method for the separation of different types of collagen using a large pore size (100 nm) reversed-phase C-8 column. Three different types of collagen (type I, III, and V) were sufficiently separated on the column using a mobile phase containing water-acetonitrile-trifluoroacetic acid. Collagens found in each HPLC peak were identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to assay the column effluents.

Trifluoroacetic acid (TFA) was used as an ion pair reagent. This compound improved the chromatographic profile for these proteins on a reversed-phase column. It was observed that with …

The Study Of Natural Product(S) From Opsanus Tau (Linnaeus), The Oyster Toadfish, Miki Taira

Chemistry & Biochemistry Theses & Dissertations

Opsanus tau (Linnaeus), the Oyster toadfish, commonly inhabits the Chesapeake Bay. The Oyster toadfish has been categorized as a venomous and/or ichthyotoxic fish; however, chemical studies to substantiate these claims have not been forthcoming.

The oyster toadfish has dorsal and opercular spines which are believed to secrete a venom(s). Based on our findings, the oyster toadfish does not secrete venom(s) from these spines.

The oyster toadfish secretes large amounts of a mucus when it is irritated. 3-Octanone was believed to be the major ichthyotoxic component of the mucus from the oyster toadfish. Based on our investigation, 3-octanone is present only …

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 …

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 …

Separation Of Transfer Rna Isoacceptors By Reversed Phase W-Porex C-4 High Pressure Liquid Chromatography, Christine D. Witte

Chemistry & Biochemistry Theses & Dissertations

Transfer RNA (tRNA), is a complex class of small molecules which have been proven to play a major role in protein synthesis. Transfer RNA is composed of 20 families of isoacceptors which may recognize only one particular amino acid. This family of isoacceptors may differ from each other by primary sequence or modified nucleosides. Queuine is a modified nucleoside which is found in the anticodon regions of tRNA isoacceptors for asparagine, aspartic acid, tyrosine and histidine. Queuine has gained the interest of many researchers since queuine deficient tRNA was found in many neoplastic and undifferentiated cell-lines. It is often difficult …

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 …

The Determination Of Tamoxifen Citrate In Human Blood Serum And Semen, Karimdad Tahmoresi

Chemistry & Biochemistry Theses & Dissertations

High performance liquid chromatography (HPLC)methodologies for the determination of tamoxifen and its metabolites 4-hydroxytamoxifen and n-desmethyltamoxifen, and using cis-tamoxifen as an internal standard have been developed.

Clinical analyses of tamoxifen and its metabolites in human serum and semen, were performed as follows: The analytes were extracted from serum and semen with hexane, the hexane evaporated, the dried extract then reconstituted with methanol, followed by reverse phase high performance liquid chromatography.

Between-run coefficients of variation (CV'S) for tamoxifen and n-desmethyltamoxifen for serum sample analyses, (n = 15) were, 4% and 13% at mean (SD) concentration of 140.2 (6.2) and 71.8 (9.8) …

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 …

Spectroscopic Study Of Decomposition Products Of Gossypol, David Bernard Hankins

Chemistry & Biochemistry Theses & Dissertations

Basic research into reproductive physiology has begun to identify possible new approaches to controlling fertility. Gossypol, a natural polyphenol compound isolated from cottonseed oil, has been shown to be an effective male antifertility agent. The decomposition products of gossypol which are thought to possess more potent antifertility effects than the parent compound gossypol have been studied extensively in more recent research. The purpose of this research is to develop techniques and methods which could be used to identify decomposition products of gossypol. A model decomposition reaction by which a smaller molecule can be isolated in a pure state has been …

Simultaneous Determination Of 3-Methoxy-4-Hydroxyphenyl-Glycol, Homovanillic Acid, And 5-Hydroxyindoleacetic Acid In Plasma By High Performance Liquid Chromatography, Yung-Te Tseng

Chemistry & Biochemistry Theses & Dissertations

A rapid, reliable, and sensitive method for simultaneous determination of 3-methoxy-4-hydroxyphenylglycol (MHPG), Homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5- HIAA) in plasma is described. The method is based on high performance liquid chromatography with electrochemical detection. Samples were filtered with Amicon Centrifree micropartition systems and extracted with organic solvent before chromatography. Chromatographic separation of sample components was achieved isocratically in a C18 reverse-phase column. Quantitation was performed by measuring the peak height ratios of the sample components to an internal standard. Analytical recoveries ranged from 50.78 (SD 4.2%) to 67.94 (SD 3.28). Within-run coefficient of variations (CVs) ranged from 3.78 …

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 …

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 …

The Synthesis And Evaluation Of 5-Phenyloxazolidines As Potential Cardiovascular Drugs, Tammy C. Wang

Chemistry & Biochemistry Theses & Dissertations

Drugs such as adrenoceptor antagonists and certain centrally acting agents are known to lower blood pressure and thus serve as clinically useful antihypertensive agents. Norepinephrine 4, a natural ∝-adrenoceptor agonist, is the major agent associated with increased blood pressure in the periphery. Based on a careful structure-activity comparison, a series of substituted oxazolidines, 20, 21, 22 and 23, have been designed as possible antihypertensive candidates. Specifically, 22 and 23 have been synthesized to serve as potential norepinephrine antagonists.

Preliminary evaluation of these compounds indicated that they were not antihypertensive in nature. The more active compound, 23, …

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 …

Modulation Of Queuine Uptake In Cultured Human Fibroblasts By Phorbol Esters And Interferons, Debra L. Crane

Chemistry & Biochemistry Theses & Dissertations

Queuosine, a nucleoside found exclusively in the first position of the anticodon of transfer RNA (tRNA), is generated post-transcriptionally in an exchange of guanine for queuine by the modifying enzyme tRNA-guanine ribosyltransferase. Phorbol ester tumor promoters were shown to induce queuine hypomodified tRNA, and that phorbol ester action was due, in part, to inhibition of queuine transport across the cell membrane. An unidentified protein designated conditioned medium factor (CMF) that potentiated a phorbol-induced inhibition of queuine uptake was also documented. We suggest here that phorbol ester-induced inhibition of queuine uptake is not a significant factor in inducing queuine-deficient tRNA although …

Reactions Of Aqueous Chlorine In The Gastric Fluid Of The Rat, Michael Gary Nickelsen

Chemistry & Biochemistry Theses & Dissertations

Gastric fluid was recovered from Sprague-Dawley rats that had been fasted for 24 hr or 48 hr and administered 4 mL deionized water. The protein, carbohydrate and chloride ion content of the gastric fluid was determined.

When gastric fluid is chlorinated with radiolabeled chlorine to concentrations between 323 and 375 mg/L, analyzed by high performance chromatography followed by liquid scintillation counting, as much as 59% of the radiolabeled chlorine was associated with organic components of the gastric fluid.

Model studies with the aromatic amino acids and the protein pepsin showed the formation of chlorinated analogs. Two chlorinated amino acid derivatives …

Use Of 52Cr For The Quantitative Determination Of Red Cell Volume, Claude Michael Hanbury

Chemistry & Biochemistry Theses & Dissertations

Chromium-52 has recently been suggested for use as a new agent to label red cells for the in vivo quantitation of red cell volume (1). In this paper, the development and validation of a routine ⁵²cr labeling procedure is described.

The accuracy, precision, and detection limits of chromium analysis by Zeeman effect atomic absorption spectroscopy was evaluated in the concentration range of 1 - 10 ug Cr/L.

Red cell chromium uptake was evaluated as a function of time, temperature, and concentration. Red cells labeled with a 2.5 mg/L chromium solution for 30 minutes at room temperature exhibited optimal label …

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 …

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 …

Synthesis Of Potential Long Acting Calcium Channel Antagonists: A Study Of 1,4-Dihydropyridine Analogs, Thomas E. Christos

Chemistry & Biochemistry Theses & Dissertations

An investigation was undertaken to synthesize and evaluate longer acting analogs to the antihypertensive agent nifedipine. Analogs that are capable of covalent bonding to the nifedipine receptor were considered. These compounds may show increased activity as they will remain at the receptor for a longer period of time.

During the course of this study, previously unreported and unexpected products were encountered and lead to a more intense examination of the Hantzsch synthesis. Of particular interest was the oxazolidine 25 which was a major component of the Hantzsch synthesis when ethanolamine was used instead of ammonium hydroxide. Also isolated during this …

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 …

Magnesium, Copper And Zinc Concentrations In Blood From Children With Diabetes Mellitus, Linda Lowers Jenkins

Chemistry & Biochemistry Theses & Dissertations

Trace element levels in blood from diabetic children were investigated and compared to control children. Plasma, erythrocyte, mononuclear and polymorphonuclear cells were analyzed for magnesium, copper, and zinc content. Leukocyte separation was performed by density gradient centrifugation on Ficoll-Hypaque, followed by washing steps to remove platelet contamination and hypotonic lysis to remove red cell contamination. Polarized Zeeman-effect atomic absorption spectroscopy was used for trace metal analysis.

Decreased plasma magnesium levels were observed in the diabetic children. This finding is well documented and probably due to increased urinary magnesium loss from glycosuria. Lymphocyte magnesium levels in the diabetic children were also …

Investigation Of Nickle As A Matrix Modifier In The Atomic Absorption Analysis Of Tin, Charles William Saunders

Chemistry & Biochemistry Theses & Dissertations

Nickel has been widely used as a matrix modifier in the flameless atomic absorption analysis of arsenic and selenium. Its utility as a modifier for tin has been investigated using Polarized Zeeman effect Graphite Furnace Atomic Absorption Spectroscopy. Peak area, peak shape, and appearance times were compared in modified and nonmodified standards using the system computer's internal integration and a detector/readout system with 18 msec resolution.

The addition of nickel at 600 mg/L increased the tin absorbance signal five-fold. The improved signal to noise ratio resulted in a two-fold increase in the sensitivity and a lower limit of detection. The …

Determination Of Total And Unfilterable Plasma Aluminum In Uremic Patients, Theodore R. Delbridge

Chemistry & Biochemistry Theses & Dissertations

Aluminum accumulation in the soft tissues and bone of end stage renal failure patients has been described in the literature. In addition to aluminum normally encountered through ubiquitous sources, these patients are subject to exposures through the dialysate used for hemodialysis and through aluminum-containing phosphate binders. These increased exposures, associated with a decreased ability to excrete aluminum through the kidneys, lead to aluminum accumulation which may result in the onset of an encephalopathy syndrome and/or osteomalacic osteodystrophy. These complications may be very devastating.

Blood serum or plasma aluminum levels do not reflect the total body burden of aluminum. However, they …

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 …

Correlation Studies Between Human Seminal Plasma Trace Metal Concentrations And Semen Quality, De-Shen Mei

Chemistry & Biochemistry Theses & Dissertations

Methodologies for the determination of cadmium, copper, selenium and zinc concentration in human seminal plasma have been developed using polarized Zeeman-effect atomic absorption spectroscopy. Within-run coefficients of variation (CY's) for cadmium, copper, selenium and zinc from pooled seminal plasma were found to be 8%, 5% , 4% and 0.5% at a mean (SD) concentration of 0.48 (0.04), 147 (7 .6), 53 (2. 1) and 122,900 (700) μg/ℓ, (n = 10), respectively.

Between-run CV's were determined to be 4% , 7%, 5% and 7% at a mean (SD) concentration of 2.4 (0.1), 147 (10. 1), 67.7 (3.4) and 115,800 (8300) …

The Effects Of Tri-O-Cresyl Phosphate On 2',3'-Cyclicnucleotide-3'-Phosphohydrolase Activity In Neural And Non-Neural Tissue Of The Rat, Mark K. Schlager

Chemistry & Biochemistry Theses & Dissertations

A total of 44 Long-Evans rats received 5.7 g/kg tri-O-cresyl phosphate (TOCP) orally every 3 days for 6 days. A second group of 13 rats received 4.6 g/kg orally every 3 days for 6 days. Animals were administered atropine sulfate (10 mg/kg) intraperitoneally every 6 hours or as needed to alleviate cholinergic symptoms. Ten control animals were given corn oil orally. Eight rats (18%) survived the 5.7 g/kg dose regimen and 5 rats (38%) survived the 4.6 g/kg dose regimen. Surviving rats were killed at 30-33 days post-dose. Signs of delayed neuropathy included ataxia and motor impairment.

The CNP activity …