Biochemistry Commons^™

A Kinetic Investigation Of The Mechanism Of Muscle Contraction With A Series Of Nucleotides, Wei Jiang

Theses and Dissertations in Biomedical Sciences

Muscle contraction is thought to be accomplished by sliding of myosin filaments along actin filaments. Although actomyosin naturally uses ATP as an energy source, it can also use other nucleoside triphosphates (NTP) as substrates. In this work, the dependence of rate and equilibrium constants of the various steps in the muscle contraction mechanism upon nucleotide structure was investigated to unravel the dependence of the mechanical properties of the muscle upon changes in different biochemical steps of the mechanism.

The experiments measuring the dissociation of actomyosin by NTP (or mant-NTP) shows that both the rate constant of actomyosin-S1 dissociation and second …

Biotransformation Of Bile Acids, Cholesterol And Steroids. Chapter 13 In: The Ecology And Physiology Of Gastrointestinal Microbes. Vol. 1, New York, Chapman And Hall., Stephen Baron, Phillip B. Hylemon

Biology Faculty Scholarship

No abstract provided.

A Study Of Euplotes Crassus Telomere Proteins And Related Genes, John Scott Perez

Department of Chemistry: Dissertations, Theses, and Student Research

A novel PCR technique used to amplify Euplotes crassus macronuclear chromosomes was developed to provide scientific proof that an Euplotes crassus 1.0 kb gene is not genetically related to an Oxytricha nova 1.8 kb β-telomere protein gene. Theories and experimental procedures associated with the development of this PCR technique were the product of research that will have a profound impact on future studies of telomere protein genes and telomeric DNA in a multitude of eukaryotic cells. The hypothesis that a β-telomere protein does not exist in Euplotes crassus was supported by the result of this study.

Expression of the recombinant …

Determination Of Pancreatic And Salivary Amylase By Enzyme Immunoassay And Their Prevalence In Hyperamylasemic Patients, Sabdra Borgens Ward

Theses and Dissertations in Biomedical Sciences

Currently, amylase determinations are nonspecific for the organ source and are based entirely on the enzymatic properties of amylase to produce a measurable product or byproduct. The determination of pancreatic amylase is important in the diagnosis of acute pancreatitis. Most commercially available tests for amylase employ the measurement of the change in NADH absorbance at 280 nm or of the p-nitrophenol released from a maltotetrose substrate. These are nonspecific measurements of pancreatic amylase and often necessitate other tests to be run such as a serum lipase.

The two predominant isoenzymes of amylase are pancreatic (p-amylase) and salivary (s-amylase); the most …

Signal Transduction In Bacterial Chemotaxis, Edward Heath Rowsell

Loma Linda University Electronic Theses, Dissertations & Projects

This study investigated the site of ATP utilization in the signal transduction pathway of bacterial chemotaxis and localized the point of convergence of a methylation-independent system of chemotaxis with the methylation-dependent system. The identity of the signal originating from the phosphotransferase system was investigated by substituting the fructose-inducible HPr-like protein FPr for HPr in transport and chemotaxis. In addition, a novel chemoattractant, glycerol, was identified for Salmonella typhimurium. Histidine-auxotrophic S. typhimurium strains ST23 (hisF) and ST171 (hisF cheB) were depleted for ATP. The times required for the bacteria to adapt to a step increase in serine, …

Investigation Of Selenium Status In Hypo-, Hyper- And Euthyroid Children, Ping Sun

Chemistry & Biochemistry Theses & Dissertations

Recently, it has been reported that human type I iodothyronine deiodinase, an enzyme important in the conversion of T₄ to T₃ , is a selenoenzyme. Several studies have reported alterations in plasma selenium level in hypo- and hyperthyroid patients.

Using polarized Zeeman-effect atomic absorption spectroscopy, we measured selenium, zinc, copper and manganese concentrations in the plasma and/or red blood cells in children with or without thyroid disease being seen at the outpatient clinic at Children's Hospital of the King's Daughters. Children with thyroid disease were subdivided into untreated, treated and nonresponsive groups. Data were analyzed using Student's t-test …

Separate Metabolic Pathways Leading To Dna Fragmentation And Apoptotic Nuclear Chromatin Condensation, D. Sun, Shibo Jiang, Li-Mou Zheng, David M. Ojcius, John Ding-E. Young

All Dugoni School of Dentistry Faculty Articles

Apoptosis is the predominant form of cell death observed in a variety of physiological and pathological conditions such as cancer involution, insect metamorphosis, the development of the immune and nervous systems, and embryogenesis. The typical nuclear changes taking place in apoptotic cells include extensive condensation of chromatin and internucleosomal DNA fragmentation into units of 200 base pairs. However, the mechanisms responsible for both chromatin condensation and DNA fragmentation have yet to be elucidated. In this study, micrococcal nuclease and the divalent cations, Ca2+ and Mg2+, were applied to isolated nuclei in an attempt to reconstitute in vitro the digestion of …

Severe Bone Marrow Suppression In A Patient With Rheumatoid Arthritis On Methotrexate, M P. Iqbal, A A. Ali, A A. Alvi

Department of Biological & Biomedical Sciences

No abstract provided.

Sds Non-Acrylamide Polymeric Gel-Filled Capillary Electrophoresis For Molecular Size Separation Of Protein, Devon Andres

Honors Theses

Sodium dodecyl sulfide (SDS) non-acrylamide gel-filled capillary columns are a new technology being used for analysis and separation of biotechnology-derived proteins. This research was to compare this new technology to the current methods of SDS polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance size-exclusion chromatography (HPSEC). The molecular mass of four different recombinant proteins were determined by two commercialized SDS non-acrylamide gel-filled capillary columns, SDS-PAGE, and HPSEC. The data obtained showed that the SDS non-acrylamide gel-filled capillary columns were compatible with the SDS-PAGE technique for molecular mass determination. HPSEC was shown to be unreliable for molecular weight determination. SDS non-acrylamide gel-filled capillary …

Aerotaxis In Bacillus Subtilis, Laurence Samuel Wong

Loma Linda University Electronic Theses, Dissertations & Projects

B. subtilis, like Escherichia coli and Salmonella typhimurium, responded to a step increase in oxygen concentration by swimming smoothly (~ 35 s duration) and to a decrease in oxygen by tumbling. In a spatial gradient of oxygen in liquid media, a band of cells congregated near the air interface. Aerotaxis required a functioning respiratory chain. Adaptation of B. subtilis, E. coli and S. typhimurium to media containing amino acids requires methylation of receptors/methyl-accepting chemotaxis proteins. Adaptation to oxygen and phosphotransferase sugar substrates in B. subtilis was dependent on methylation, unlike similar adaptation in E. coli and S. typhimurium. …

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 …

Longitudinal Study Of Plasma And Erythrocyte Selenium Levels In Type I Diabetic Children During Development And Treatment Of Ketoacidosis, Pi-Hsia Fan

Chemistry & Biochemistry Theses & Dissertations

Most studies on the biological activity of selenium have focused on its role at the active site in the ubiquitous enzyme, glutathione peroxidase (GSH-Px) which catalyzes the removal of hydrogen peroxide and organic hydroperoxides (1). A connection between selenium deficiency and a wide range of diseases in animals and humans has been reviewed (2). Development of various forms of human cancer (3), failure of the immune system (4), as well as some cardiovascular diseases (5) have been reported or suspected to correlate with selenium deficiency. However, the physiological function of selenium in these different diseases is not fully understood, and …

Separation And Quantification Of Queuine-Modified And Unmodified Transfer Rna Isoacceptors, Jorge Miguel Pereira De Oliveira Da Silva Santos

Chemistry & Biochemistry Theses & Dissertations

The discovery of a base-exchange modification in the first position of the anticodon of four transfer RNA (tRNA) isoacceptor families has been shown to be correlated with a decrease in the rate of cell division in several tumorigenic tissues. This fact led to the hypothesis that wobbling in the anticodon might be a control point wich mediates the rate of translation. A suitable method for separation of different tRNA isoacceptor families had to be created in order to generate a means for quantitatively measuring degrees of this base modification in tRNA. Radio labelled amino acids were used to charge tRNA …

Purification And Ligand Binding Of A Soluble Class I Mhc Molecule Consisting Of The First Three Domains Of H-2kd Fused To B2-Microglobulin Expressed In The Baculovirus/Insect Cell System, Francois Godeau, Immanuel F. Luescher, David M. Ojcius, Cecile Saucier, Estelle Mottez, Lucien Cabanie, Philippe Kourilsky

All Dugoni School of Dentistry Faculty Articles

A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative …

Appearance Of A Methotrexate Binding Plasma Protein During High Dose Methotrexate Therapy, M P. Iqbal, I Malik, N Mehboobali

Department of Biological & Biomedical Sciences

No abstract provided.

Biochemical Investigation Of Gout And Its Familial Incidences, Chin-Ling Hsu

Chemistry & Biochemistry Theses & Dissertations

Gout is a chronic metabolic disorder caused by deposition of uric acid on the joint. It is categorized into two types: primary and secondary. Primary gout is uric acid overproduction, caused by excessive synthesis of the purine precursors. Secondary gout occurs also as the result of overproduction or decreased renal excretion of uric acid resulting from other disease processes or medication.

The two purine bases, hypoxanthine and xanthine, liberated from ribosides by the action of nucleoside phosphorylase, are degraded to uric acid as the final excretory product in the purine degradation pathway. Hypoxanthine and xanthine are the physiological substrates of …

Kinetic Characterization Of A Recombinant C-Terminal Mutant Of Reverse Transcriptase From The Human Immunodeficiency Virus, Thomas S. Heard

Chemistry & Biochemistry Theses & Dissertations

The human immunodeficiency virus (HIV) reverse transcriptase (RT) (EC 2.7.7.49) is the central replication enzyme for HIV. In general, the kinetic mechanism for this and all other polymerases involves the ordered binding of two substrates: a primer-template (PT) followed by a deoxyribonucleoside triphosphate (dNTP). Previous investigations prompted this research when it was discovered that the substrate dNTP, in absence of PT, could protect a recombinant c-terminal mutant HIV-1 RT from inhibition by pyridoxal-5'-monophosphate (PLP), an active-site dNTP inhibitor. In contrast, the non-mutant recombinant HIV-1 RT required both substrates for protection from PLP inhibition. This investigation sought to determine if this …

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 …

Heterogeneity Of Methotrexate Binding In Human Colon Tumor Cells, M P. Iqbal, N Mehboobali, M A. Waqar, M Ahmed

Department of Biological & Biomedical Sciences

[3H]-methotrexate binding at pH 5.0 and pH 7.2 by the cytosol of tumor tissues and the surrounding normal areas of the gastrointestinal tract of patients suffering from colon or gastric cancer has been used to identify in these cells the presence of a binder of methotrexate having low-affinity for this drug in addition to the enzyme dihydrofolate reductase. Scatchard analysis of the [3H]-methotrexate binding data by a colon tumor sample also reveals that there are two binders of this drug present in the cytosol of these cells. The association constant (Kass) for one binder of methotrexate is = 5.6 x …

Cytolytic And Ion Channel Forming Properties Of The N-Terminus Of Lymphocyte Perforin, David M. Ojcius, Pedro M. Persechini, Li-Mou Zheng, Paulo C. Notaroberto, Sandro C. Adeodato, John Ding-E. Young

All Dugoni School of Dentistry Faculty Articles

Perforin lyses cells by binding to the target cell membrane, where it polymerizes into large nonspecific pores. It is shown here that the first 34 amino acids of the N-terminal region of either human or murine perforin are soluble in aqueous medium and spontaneously insert into membranes. The N-terminal peptides lyse liposomes and nucleated cells, and they form ion channels in planar bilayers, some of which are comparable to those previously described for perforin. The lytic activity of the N-terminal domains does not require calcium, is independent of the lipid headgroup composition, and can be inhibited by heparin. Tumor cells …

Molecular Mechanism Of The Stimulation Of Alkaline Phosphatase Activity In Human Bone Cells By 1,25(Oh)2 D3, Eru Kyeyune-Nyombi

Loma Linda University Electronic Theses, Dissertations & Projects

To facilitate this study an in vitro human model system was established that exhibited many aspects of normal osteoblasts. The human osteosarcoma cell line (TE85 cells) expressed a skeletal alkaline phosphatase activity (an accepted bone cell differentiation marker) which was stimulated by 1,25(OH)₂D₃ (a potent differentiating agent), under serum-free conditions in a dose-dependent, time-dependent, and cell density-dependent manner. Cytochemical analysis of the stimulation of ALP activity by 1,25(OH)₂D₃ showed that 1,25(OH)₂D₃ increased the number of TE85 cells that expressed detectable ALP activity, suggesting that 1,25(OH)₂D₃ promoted the process …

Pharmacokinetics, Immune Response, And Biodistribution Of Iodine-131-Labeled Chimeric Mouse/Human Igg1, K 17-1a Monoclonal Antibody, Ruby Meredith, Albert Lobuglio, Walter Plott, Roger Orr, Ivan Brezovich, Charles Russell, Elizabeth Harvey, Michael Yester, Mansoor Saleh

Haematology and Oncology, East Africa

Pharmacokinetics,immunogenicity,and biodistributionof a 131I-Iabeledmouse/human chimenc monoclonal antibody (C17-1A) was studiedin six metastaticcoloncancerpatients. Pharmacokinetics obtalned from serum radioactivity or chi mera concentration were identical after 5 mCi of 131I(>17-1A withmeanalphahalf-livesof 17.6±2.3 and19.7±2.9 and meanbeta half-livesof 100.9±16.1 and 106.4±14.1 hr, respectively. HPLC analysis documented the monomeric chi rneiic17-lA withoutevidenceof immunecomplexesorfree 1311 None of the patients developed antibody after ‘31I-chi merle17-1A exposure.Radiolocalizationoccurredin known areas of disease >4 cm in all patients. The half-life of total body radioactivity was 58 ±7 hr by whole-body counts and 64 ±13 hr by urine measurements. Whole-body and bone marrow dose estimates ranged from 0.75-1 .03 and …

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 …

Development Of Catalytic Rnas As Potential Anti-Hiv-1 Agents, Pairoj Sae Chang

Loma Linda University Electronic Theses, Dissertations & Projects

RNA molecules with enzymatic activity (ribozymes) targeted to the genome of HIV-1 were developed and demonstrated to mediate site-specific cleavage of the HIV-1 RNA in vitro and to inhibit viral proliferation intracellularly. Two classes of ribozymes that incorporate a "hammerhead" catalytic center were utilized, eotide elements in the viral sequence to provide half of the The first class used nuclcatalytic center. A complementary strand harboring the remaining active site nucleotides was supplied in trans and directed the site-specific cleavage of the HIV-1 RNA target. A second type of ribozyme incorporated the entire catalytic center, minus the site of cleavage, within …

Proto-Oncogene Regulation By Growth Factors In Bone Cells, Harold Lyndon Merriman

Loma Linda University Electronic Theses, Dissertations & Projects

Previous studies have shown that bone cells in culture produce a number of growth factors that are important in bone regulation. Chick and mouse primary calvarial cultures consist of a mixture of cell types that make it difficult to interpret the results from these model systems. In contrast, MC3T3-E1 cells are a clonal mouse osteoblast-like cell line. Since MC3T3-E1 cells consist of a single population of calvarial cells, they make an ideal system in which to study the autocrine effects of bone growth factors.

Based on this work, MC3T3-E1 cells are now known to produce IGF-I, TGF-beta and IGF-II in …

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 …

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 …

Maternal Immunomodulation Of Neonatal Alloantigen Response, Leh Chang

Loma Linda University Electronic Theses, Dissertations & Projects

Remarkable success has been achieved in the transplantation of allogeneic cardiac grafts into newborn infants at Loma Linda University Medical Center. The superior graft survival rate documented in these patients has not correlated with the degree of immunosuppression rendered, or the selection of genetically matched donors. However, the clinical success has correlated with the age of the recipient at the time of receiving a transplant. Patients receiving an allograft within the first few weeks of life are unique in that they seem to accept the alloantigens of their cardiac graft while responding aggressively to antigens in their environment. These observations …

The Role Of Small Peptides In Cancer Physiology And Chemotherapy, Bao-Ling Tsay

Theses and Dissertations in Biomedical Sciences

The targeting of proven anticancer drugs specifically to cancer cells would provide a unique opportunity to restrict neoplasms without damaging the cancer patient. The present research utilizes the phenomenon of illicit transport, i.e. the coupling of normally impermeant metabolites to permeant metabolites, in targeting the drug melphalan to mouse Ehrlich ascites tumor cells. The dipeptide beta-alanyl-melphalan was synthesized and tested in vitro for toxicity towards mouse Ehrlich ascites tumor cells, mouse liver cells, and mouse 3T3 embryonic cells. The parent compound, melphalan, was used as a control treatment. In addition, both melphalan and beta-alanyl-melphalan were utilized in in vivo chemotherapeutic …