Biochemistry Commons^™

Structural Studies Of Scallop Abductin, A Compressible Elastomer That Translates Hydrophobic Interactions To Elasticity, Felipe Jimenez Jr.

Loma Linda University Electronic Theses, Dissertations & Projects

Abductin is a natural elastomer found as the major component of the elastic hinge ligament in marine scallops. Until now, very little was known about the structure and function of this compressible elastomer. Biochemical studies of a synthetic 25 amino acid peptide of abductin produces fibers that assemble into a gel matrix at room temperature. Further investigation of this phenomenon via temperature-dependent turbidimetry revealed that this peptide self-assembles or aggregates at 23 °C. Secondary structural studies of this peptide at different temperatures revealed that it undergoes an inverse temperature transition much like soluble elastin peptides. Replacing phenylalanine with alanine in …

Plant-Based Multicomponent Vaccines Against Enteric Diseases, Jie Yu

Loma Linda University Electronic Theses, Dissertations & Projects

To generate a plant-based multicomponent oral vaccine against cholera, rotavirus and enterotoxigenic E.coli (ETEC), we have constructed transgenic potato plants that synthesize the cholera toxin B and A2 subunits, the rotavirus enterotoxin NSP4 22 amino acid epitope and the ETEC fimbrial antigen CFA/L To use cholera toxin as a carrier molecule for delivery of multiple antigens to the gut-associated lymphoid tissues, the rotavirus NSP4 epitope was linked to the cholera toxin B subunit (CTB) and the ETEC fimbrial antigen CFA/I was linked to the cholera toxin A2 subunit. Cholera toxin-antigen fusion proteins were detected in transformed tuber tissues by immunoblot …

Glucocorticoid Regulation Of Insulin Like Growth Factor Binding Protein-5 Gene Transcription In Human Osteoblasts, Xiaoying Wang

Loma Linda University Electronic Theses, Dissertations & Projects

Glucocorticoids (GCs) inhibit bone formation in vivo and inhibit osteoblast proliferation and collagen synthesis in vitro. These effects may be mediated by alterations in the insulin-like growth factor (IGF) system. In the present study of normal human osteoblast-like (HOB) cells, we tested the hypothesis that dexamethasone (Dex) inhibits the expression of IGF binding protein-5 (IGFBP-5). Dex decreased IGFBP-5 mRNA levels to 54% of control after 4 hr. Dex did not modify the decay of IGFBP-5 mRNA in transcriptionally arrested osteoblast cells. Dex decreased IGFBP-5 hnRNA levels to 67% of control after 2 hr, and the activity of the human IGFBP- …

Studies On The Purification And Role Of Igfbp-5 Protease In Bone, H. Garrett Rush Thompson

Loma Linda University Electronic Theses, Dissertations & Projects

Osteoporosis is a disease characterized by low bone density and deterioration of mineralized bone leading to enhanced susceptibility to fracture. Several growth factors have been implicated in the coupling of formation to resorption during the bone remodeling process. The net bone forming activity of many growth factors has prompted numerous studies focused on the regulation of osteoblast cell proliferation, differentiation and activity. The current study focuses on the insulin-like growth factor (IGF) system, an important growth factor system involved in the regulation of bone formation and bone resorption.

IGF binding protein -5 (IGFBP-5), the most abundant IGF binding protein in …

Identification And Characterization Of The Cis-Acting Elements Around The Murine Cd4 Cnhancer, Xin Dong

Loma Linda University Electronic Theses, Dissertations & Projects

The cluster determinant 4 (CD4) molecule is a transmembrane glycoprotein. CD4 is essential for normal T helper cell function and plays an important role in T cell development and activation. CD4 is encoded by a single gene located on chromosome 6 in the mouse and chromosome 12 in the human. Both human and murine CD4 genes are divided into ten exons spanning more than 25 kb and have a large non-coding region in the first and third intron. CD4 gene expression is controlled primarily at the transcriptional level during T cell development and activation.

An enhancer has been identified approximately …

Identification And Characterization Of Control Elements Within The Murine Cd4 Gene, Zhong Deng

Loma Linda University Electronic Theses, Dissertations & Projects

The control of CD4 gene expression is essential for T lymphocyte development. Since the molecular mechanism for the control of CD4 gene expression during T cell development had not been elucidated, a study of the factors that control CD4 gene expression may lead to further. Toward these goals, we have made a series of recombinant DNA constructs to define the cis-acting transcriptional control elements in the murine CD4 locus that control CD4 gene expression during T cell development. In this study, we have identified multiple cis-acting control elements, which are critical for regulating the expression of the murine …

Telomerase Activity In Prostatic Fluid And Tissue As A Marker For Prostate Cancer, Zhilian Wang

Loma Linda University Electronic Theses, Dissertations & Projects

Telomerase is a ribonucleoprotein enzyme that is able to add telomeric repeats to the ends of eukaryotic DNA to stabilize chromosomes. Telomerase is active in germline cells, stem cells and tumor cells, but not in most human somatic cells. In this study we report the extended use of the Telomeric Repeat Amplification Protocol (TRAP) assay for the detection of telomerase activity in prostate needle biopsy specimens and freshly collected prostatic fluid. Clinical application of a newly developed PCR-ELISA telomerase assay method is also evaluated. Multiple sextant needle biopsy specimens from 56 subjects and prostatic secretion specimens from 72 subjects, with …

Structural Studies Of Recombinant Human Β-Casein Proteins, Yilin Hu

Loma Linda University Electronic Theses, Dissertations & Projects

β-Casein is the major component (~80%) of the human casein system. Interacting with calcium and phosphates through its phosphorylated N-terminus, β-casein participates in the formation of micelles which carry these otherwise insoluble nutrients indispensable for the growth and development of newborns. Human β-casein, or fragments thereof, are also found to have biological effects such as enhancement of calcium absorption. So far, knowledge of micelle formation is limited and structural studies of β-casein, especially its N-terminal portion will provide a better understanding of the structure-function relationship of milk micelles.

To elucidate the role that the N-terminus of the protein plays in …

Regulation Of Connexin43 By Phosphorylation, Maithili M. Shah

Loma Linda University Electronic Theses, Dissertations & Projects

Gap junctions play a crucial role in embryogenesis, especially of the heart where connexin43 is thought to be important for its formation as well as synchronized contraction. Several reports have shown that connexin43 protein is phosphorylated on serine residues in vitro and in vivo.

Studies performed in our laboratory have demonstrated that cell-to-cell communication in cells expressing connexin43 can be controlled rapidly and reversibly by microinjection of active protein kinases or phosphatases that target serine or threonine residues. Phosphorylation by cAMP-dependent protein kinase (PKA) seems to favor channel opening, whereas the effect of protein kinase C (PKC) on channel gating …

Regulation Of Synthesis And Actions Of Bone Growth Factors, Rashmi Malpe

Loma Linda University Electronic Theses, Dissertations & Projects

Among the many bone growth factors, this study focused on the IGF and BMP systems because of some of their unique functions, namely, IGFs function both as local and systemic agents and BMPs are capable of inducing de novo bone formation at non-skeletal sites. There is varied osteoblast differentiation in and load bearing on the skeletal system which led to the first study whose hypothesis was that these skeletal site dependent differences are associated with site-specific differences in IGF system components' production. Results from their quantitation in the CM of normal HBC derived from calvaria, mandibles, ribs, vertebrae, and marrow …

The Effects Of Exposure To Extremely Low-Amplitude, Low-Frequency Electric Fields On Skeletal Tissue, In Vitro, Robert James Fitzsimmons

Loma Linda University Electronic Theses, Dissertations & Projects

In vivo experiments have demonstrated (a) that transient electrical potentials are induced in impact-loaded bone, and (b) that bone formation is increased in response to electrical stimulation. Therefore, it has been hypothesized that, in response to mechanical loading, mechanical energy in bone is transduced into biologically relevant electrical energy. In order to study the mechanism through which electric fields may increase bone formation, a model system was developed were bone cells or bone organ cultures were exposed to a capacitively-coupled electric field of low frequency (8-25 Hz) and extremely low amplitude (estimated 10^-7 V/cm in the culture medium) for …

Localization Of Sodium, Potassium-Adenosine Triphosphatase In The Cerebral Cortex Of Developing Rats, Maki Ujiie

Loma Linda University Electronic Theses, Dissertations & Projects

Active transport via Na⁺, K⁺-ATPase plays an important role in establishing and maintaining proper ionic gradient across all mammalian cell membranes. A strict regulation of Na⁺, and K⁺ ion levels is especially important in excitable cells such as neurons and myocytes. Inhibition of the enzyme is associated with several pathological conditions and has serious detrimental effects on cerebral cortical cells. Yet the enzyme appears to be limited in amount and distribution in newborn rats. In this study, Na_⁺, K⁺- ATPase expression and distribution in early postnatal rats were studied …

A Model Using Radiation And Pws4-Htnf-Α Gene Therapy For Treatment Of Glioblastomas, Angelo G. Baher

Loma Linda University Electronic Theses, Dissertations & Projects

The efficacy of radiotherapy for cancer is limited by the dose that can be safely delivered to the tumor without causing debilitating side effects. In addition, successful treatment of highly malignant tumors such as glioblastomas is likely to require adjunctive therapies to enhance tumor response to radiation. Previous studies have shown immunomodulation and a synergestic reduction in tumor volume of malignant tumors when tumor necrosis factor-α (TNF-α) protein is administered prior to radiation. The major goal of the present investigation was to evaluate the efficacy of pWS4-human TNF-α (pWS4-hTNF-α), a new plasmid construct that expresses human TNF-α protein, together with …

Transcriptional Regulation Of The Bombyx Mori A3 Cytoplasmic Actin Promoter In Insect And Mammalian Cells, Karoly Fatyol

Loma Linda University Electronic Theses, Dissertations & Projects

In this study we have analyzed the activity of the silkworm A3 cytoplasmic actin promoter in homologous insect and heterologous mammalian expression systems. To study the expression of the A3 actin promoter in insect cells we have isolated and characterized a stably transformed Bombyx mori cell line containing a novel selectable marker gene, puromycin N-acetyl transferase under control of transcriptional regulatory signals from the A3 cytoplasmic actin gene. Integration of the transfected plasmids into the host genome was demonstrated by Southern and in situhybridizations. By using this cell line we have identified alternative transcriptional initiation sites for the A3 …

A Search For The Cause Of Excess Thiol Sensitivity In A Mutant Of Escherichia Coli, Hongying Zeng

Loma Linda University Electronic Theses, Dissertations & Projects

The subject of this study was a search for the biochemical basis of hypersensitivity to thiols in a mutant of Escherichia coli (IS16). A 1.3 kb chromosomal fragment from E. coli (introduced into the mutant on a plasmid) successfully abolished the hypersensitivity. This fragment contained only one intact gene, ubiX, which codes for polyprenyl-4-hydroxybenzoatelyase. The decarboxylation of octaprenyl-4- hydroxybenzoate in E. coli is catalyzed not only by the ubiX protein but also by the product of ubiD. The ubiquinone content of IS16 was only 2% of that of its parent strain, while the complementing plasmid increased it …

Salsalate Inhibition Of Thyroid Hormone Transport : A Model Of The Euthyroid Sick Syndrome, Ron Wang

Loma Linda University Electronic Theses, Dissertations & Projects

Multiple alterations of serum thyroid hormone metabolism have been noted in essentially all severe systemic nonthyroidal illnesses (Nil). This spectrum of abnormalities is collectively termed the “euthyroid sick syndrome”. One putative mechanism is the presence of circulating thyroid hormone binding inhibitors. Salsalate and salicylate are known to be such inhibitors. The objectives of this study are: to investigate the thyroid hormone changes induced in healthy people by salsalate, and to investigate the mechanisms of salsalate action, as a model for the euthyroid sick syndrome. This dissertation includes: (1) Study of the effect of salsalate on serum thyroid hormone levels in …

The Effects Of Zinc On Skeletal Alkaline Phosphatase Activity And Skeletal Tissues, Susan L. Hall

Loma Linda University Electronic Theses, Dissertations & Projects

Zinc is essential for normal growth, however, very little is known about the mechanisms by which zinc influences growth. ALP, an ectoenzyme expressed in high levels in osteoblasts, is a zinc-containing metalloenzyme and is important in bone formation. Preliminary studies showed that low levels of zinc could increase ALP specific activity in human osteosarcoma, SaOS-2 cells. Current studies were designed to characterize this zinc effect, test possible mechanisms, and determine whether the effect is physiologically relevant. Incubation with zinc resulted in a time and dose dependent increase in ALP specific activity in the extracts and conditioned media of SaOS-2 cells, …

Progesterone Regulation Of Insulin Like-Growth Factor : Binding Protein-5 Gene Transcription In Human Osteoblasts, Viroj Boonyaratanakornicit

Loma Linda University Electronic Theses, Dissertations & Projects

Insulin-like growth factor binding protein-5 (IGFBP-5) is produced by osteoblasts and potentiates IGF mitogenic actions in osteoblast cultures. Progesterone (PG) increases osteoblast proliferation and increases osteoblast IGFBP-5 expression. Mechanisms underlying PG induction of IGFI3P-5 expression were investigated in U2 human osteosarcoma cells. In nuclear run-on analyses, PG increased IGF)3P-5 gene transcription to 400% of control. To define the underlying mechanisms, the human IGF13P-5 gene proximal promoter was cloned and sequenced. The proximal 500 bp of this region contains TATA and CAAT boxes, five putative PG response element (PRE) half-sites and two tandem CACCC boxes. Chloramphenicol acetyltransferase (CAT) reporter constructs containing …

Regulation Of Human Osteosarcoma Cell Differentiation By Transforming Growth Factor B1 (Tgfb And 1a,25- Dihydroxyvitamin D3 : The Mechanism Of Tgfb1 Potentiation Of 1,25d3-Induced Alkaline Phosphatase Activity In Human Mg63 Osteosarcoma Cell, Xinde Song

Loma Linda University Electronic Theses, Dissertations & Projects

Transforming growth factor β₁ (TGFβ₁) is a pluripotent cytokine that works in conjunction with other cytokines to modulate their effects on cell function. Our previous work demonstrated that the combination of TGFβ₁ and 1α,25-dihydroxyvitamin D₃ had a synergistic effect on alkaline phosphatase induction (500 to 700%) in human osteoblastic osteosarcoma cells (MG63). The current studies focused on the molecular basis of alkaline phosphatase induction, including the regulation of the signal transduction and modification of 1α,25-dihydroxyvitamin D₃ receptors (VDR). We demonstrated that alkaline phosphatase activity was regulated at the transcription level. TGFβ₁ at 1 …

Mechanism Of Mitogenic Action Of Aluminum Ion On Bone Cells : Potential Involvement Of The Insulin-Like Growth Factor Regulatory System, Aera Yoo

Loma Linda University Electronic Theses, Dissertations & Projects

Micromolar concentrations of aluminum ion (oxidation state of 3 +) consistently stimulated [3H]thymidine incorporation into cell DNA and increased cellular alkaline phosphatase activity (an osteoblastic differentiation marker) in osteoblast-like cells of chicken and human origin. Although biphasic, dose-dependent stimulations were highly reproducible, the maximal stimulatory dose varied among different experiments. Mitogenic doses of aluminum ion also stimulated collagen synthesis in cultured human osteosarcoma TE85 cells. In addition, the same mitogenic concentrations of aluminum ion enhanced the 1,25 dihydroxy vitamin D3-dependent stimulation of osteocalcin secretion. Together, these findings indicate that aluminum at micromolar concentrations, stimulates the proliferation, differentiation, and activity of …

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, …

The Invariant Arginine In Motif 2 Of Escherichia Coli Alanyl-Trna Synthetase : Is Important For Catalysis But Not For Substrate Binding, Ying Lu

Loma Linda University Electronic Theses, Dissertations & Projects

Structural motifs 2 and 3, located in the active site of class II aminoacyl-tRNA synthetases, each contain an invariant arginine thought to participate in interactions with ATP. For Escherichia coli alanyl-tRNA synthetase (AlaRS), sequence comparisons indicate that Arg69 should be aligned with the invariant arginine in motif 2 of other class II synthetases. Site-directed random mutagenesis has been employed to generate a set of proteins containing amino acid substitutions in a portion of motif 2 of AlaRS. In this set, only mutations at position 69 caused the enzyme to lose ability to complement growth of an alaS deletion strain, …

Tyrosyl Protein Phosphorylation In Human Bone Cells : Its Potential Role In Mediating Fluoride Induced Osteoblast Proliferation, Alan Blair Thomas

Loma Linda University Electronic Theses, Dissertations & Projects

This dissertation was designed to test the hypothesis that fluoride-stimulated osteoblast proliferation is mediated by specific inhibition of a phosphotyrosyl protein phosphatase, resulting in increased steady state levels of tyrosyl phosphorylation of key cellular proteins. Cellular levels of tyrosyl phosphorylation were determined by an immunoblot detection assay in which cellular proteins were separated by SDS-PAGE, transblotted to nitrocellulose membranes and the phosphotyrosyl proteins were identified by anti-phosphotyrosine monoclonal antibodies. The relative amount of tyrosyl phosphorylation was quantitated by laser densitometric analysis of the autoradiograms. This method showed that fluoride induced dose- and time-dependent changes in the level of tyrosyl phosphorylation …

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. …

Molecular Genetics Of Endocrine Induced Regression Of Mouse Leydig Cell Tumors, Milton Rolando Drachenberg

Loma Linda University Electronic Theses, Dissertations & Projects

Chronic estrogen treatment results in malignant Leydig cell tumor formation in genetically susceptible strains of mice. The hypothesis that the mechanism of estrogen induced carcinogenesis includes a gene amplification event was tested using this in vivo endocrine carcinogenesis model. High molecular weight DNA isolated from liver, hyperplastic chryptorchid testes, primary Leydig cell tumors and transplanted tumor lines progressing to estrogen nondependence for growth, was studied using Southern blot analysis and the in-gel renaturation technique to identify amplification of characterized and anonymous sequences. No consistent gene amplification event could be demonstrated either in carcinogenesis or tumor progression to an estrogen nondependent …

Aerotaxis In Halobacterium Halobium, Jemima Clara Lindbeck

Loma Linda University Electronic Theses, Dissertations & Projects

Aerotaxis, previously studied in eubacteria was investigated in the archaebacterium Halobacterium halobium. A quantitative temporal assay for aerotaxis of H. halobium was developed using computer assisted motion analysis. Aerotaxis was most pronounced in early log phase cultures. Peak aerotaxis was found to coincide with peak respiration consistent with a model in which respiration is required for aerotaxis. In a spatial assay a mutant strain lacking the proton motive force (pmf)-generating pigments, bacteriorhodopsin (bR) and halorhodopsin (hR), exhibited enhanced aerotaxis bands relative to the bR⁺hR⁺ parent. The enhanced aerotaxis in the mutant cells was not due solely to …

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 …

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 …

K-Casein And Its Interaction In Human Milk Micelles, Barbara C. Dev

Loma Linda University Electronic Theses, Dissertations & Projects

Human milk K-casein was isolated from the acid-precipitated casein fraction on Sephadex G-200 and Mono Q HPLC. Samples of K-casein purified from different donors were found similar in both amino acid and carbohydrate composition. A molar extinction coefficient of 11.2 was determined on the basis of amino acid analysis with a norleucine internal standard. Tracer K-casein for human micelle studies was ³H-labeled in the sialic acid moiety of its carbohydrate. The human micelle system was investigated in its native form at 37°C. Micelle reformation by re-equilibration in skimmed milk at 4°C (overnight) followed by 3 h at 37°C was …

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 …