Biochemistry Commons^™

Association Between Archaeal Prolyl- And Leucyl-Trna Synthetases Enhances TrnaPro Aminoacylation, Mette Praetorius-Ibba, Theresa E. Rogers, Rachel Samson, Zvi Kelman, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacyl-tRNA synthetase-containing complexes have been identified in different eukaryotes, and their existence has also been suggested in some Archaea. To investigate interactions involving aminoacyl-tRNA synthetases in Archaea, we undertook a yeast two-hybrid screen for interactions between Methanothermobacter thermautotrophicus proteins using prolyl-tRNA synthetase (ProRS) as the bait. Interacting proteins identified included components of methanogenesis, protein-modifying factors, and leucyl-tRNA synthetase (LeuRS). The association of ProRS with LeuRS was confirmed in vitro by native gel electrophoresis and size exclusion chromatography. Determination of the steady-state kinetics of tRNA^Pro charging showed that the catalytic efficiency (k_cat/K_m) of ProRS increased 5-fold …

Defensin In The Ticks Dermacentor Variabilis And Ixodes Scapularis, Shane Michael Ceraul

Theses and Dissertations in Biomedical Sciences

This dissertation presents the identification, molecular structure and expression of defensin, an antimicrobial peptide, in the ticks, Dermacentor variabilis and Ixodes scapularis. A 624 bp cDNA molecule for defensin was cloned and sequenced from hemocytes, fat body and midgut of unstimulated D. variabilis ticks. Defensin peptide was detected only in the hemocyte lysates. These results suggest that the defensin from D. variabilis is synthesized and stored in the hemocytes, although the role of other tissues is not excluded.

The ability of the juvenile stage ticks to express the defensin gene was confirmed. Detection of the defensin transcript in all …

Quality Control During Aminoacyl-Trna Synthesis, M. Praetorius-Ibba, S. Ataide, C. Hausmann, J. Levengood, J. Ling, S. Wang, H. Roy, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

The fidelity of translation is determined at two major points: the accuracy of aminoacyl-tRNA selection by the ribosomes and synthesis of cognate amino acid/tRNA pairs by aminoacyl-tRNA synthetases (aaRSs) in the course of the aminoacylation reaction. The most important point in aminoacylation is the accurate recognition of cognate substrates coupled with discrimination of non-cognates. While this is generally accomplished by a single enzyme, we have recently found that discrimination against lysine analogues requires the existence of two unrelated lysyl-tRNA synthetases. For other amino acids, initial recognition is not sufficiently accurate with errors being corrected by an intrinsic editing activity. Recent …

Enterotoxin B Subunit Lectins As Adjuvants For Improvement Of Mucosal Vaccine, Nak-Won Choi

Loma Linda University Electronic Theses, Dissertations & Projects

In comparison with whole organism vaccines, subunit vaccines may be safer for immunization but may lack sufficient immunogenicity to provide complete immunity to the pathogen. To resolve this problem, bacterial and plant enterotoxin B subunit adjuvants containing a variety of receptor-binding properties were used to enhance the immunogenicity of rotavirus subunit vaccines. Enterotoxin B subunit adjuvants were employed to enhance protection against virus infection. Pentameric cholera toxin B subunit (CTB), shiga toxin-1 B subunit (STB) and monomeric ricin toxin B subunit (RTB) molecules were genetically linked to a 90 amino acid peptide from the simian rotavirus (SA11) nonstructural protein NSP4 …

Photoreactive Bicyclic Amino Acids As Substrates For Mutant Escherichia Coli Phenylalanyl-Trna Synthetases, Thomas Bentin, Ramin Hamzavi, Jahan Salomonsson, Hervé Roy, Michael Ibba, Peter E. Nielsen

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Unnatural amino acids carrying reactive groups that can be selectively activated under non-invasive biologically benign conditions are of interest in protein engineering as biological tools for the analysis of protein-protein and protein-nucleic acids interactions. The double ring system phenylalanine analogues benzofuranylalanine and benzotriazolylalanine were synthesized, and their photolability was tested by UV irradiation at 254, 320, and 365 nm. Although both showed photo reactivity, benzofuranylalanine appeared as the most promising compound because this amino acid was activated by UVA (long wavelength) irradiation. These amino acids were also tested for in vitro charging of tRNA^Phe and for protein mutagenesis via …

Divergence In Non-Cognate Amino Acid Recognition Between Class I And Class Ii Lysyl-Trna Synthetases, Jeffrey D. Levengood, Sandro F. Ataide, Hervé Roy, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Lysine insertion during coded protein synthesis requires lysyl-tRNA^Lys, which is synthesized by lysyl-tRNA synthetase (LysRS). Two unrelated forms of LysRS are known: LysRS2, which is found in eukaryotes, most bacteria, and a few archaea, and LysRS1, which is found in most archaea and a few bacteria. To compare amino acid recognition between the two forms of LysRS, the effects of l-lysine analogues on aminoacylation were investigated. Both enzymes showed stereospecificity toward the l-enantiomer of lysine and discriminated against noncognate amino acids with different R-groups (arginine, ornithine). Lysine analogues containing substitutions at other positions were generally most effective as …

Aminoacyl-Trnas: Setting The Limits Of The Genetic Code, Michael Ibba, Dieter Söll

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacyl-tRNAs (aa-tRNAs) are simple molecules with a single purpose—to serve as substrates for translation. They consist of mature tRNAs to which an amino acid has been esterified at the 3′-end. The 20 different types of aa-tRNA are made by the 20 different aminoacyl-tRNA synthetases (aaRSs, of which there are two classes), one for each amino acid of the genetic code (Ibba and Söll 2000). This would be fine if it were not for the fact that such a straightforward textbook scenario is not true in a single known living organism. aa-tRNAs lie at the heart of gene expression; they interpret …

Positive Regulation Of Pka On Human Gonadotropin-Releasing Hormone (Hgnrh) Gene Expression In Human Placental Jeg-3 Cells, Zhaoyang Wen

Chemistry & Biochemistry Theses & Dissertations

Using the human placental choriocarcinoma JEG-3 cell line as an in vitro human placental model, we studied the mechanisms of the PKA positive regulation of the hGnRH gene expression in the human placenta. Studies in JEG3 cells showed that through the PKA catalytic subunit a, human GnRH upstream promoter activity was stimulated by PKA signaling pathway in a cAMP dependent mechanism. The sequence between —202 (Afl II) and —554 (BamH I) base pair in the human GnRH upstream promoter region appeared to be responsible for the PKA positive regulation of the gene expression. Furthermore, Western blot analysis demonstrated the involvement …

Methods Development For The Identification Of Unknown Proteins, Laura Brumfield Neal

Theses, Dissertations and Capstones

The chemistry of transport, storage, energy production, and reproduction in physiological systems is controlled by the actions and interactions of proteins. A species full complement of these amazing machines is often referred to as its proteome. The new science of proteomics utilizes a set of powerful and evolving tools to characterize the nature of the proteome, and further to fully elucidate the functional attributes of the proteins and their associations.

A methodology was developed by combining several proteomic techniques, such as electrophoresis, RP-HPLC, and mass spectrometry, to correctly identity proteins. Three known proteins (myoglobin, albumin, and actin) were employed for …

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 …

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 …

Generation Of Human Chorionic Gonadotropin Beta Subunit Specific Monoclonal Antibody Using A Synthetic Peptide, Zhe Huang

Chemistry & Biochemistry Theses & Dissertations

Human chorionic gonadotropin (hCG) is well known for early immunological and bioassay tests for early detection of pregnancy. Since the hormone specific beta subunit confers unique biological and immunological specificity to hCG, immunochemical measurement of hCG has achieved greater specificity and sensitivity in recent years by using either the beta subunit as an immunogen or a partially reduced beta subunit polypeptide chain. However, antiserum raised against the entire hCG beta subunit usually cross-reacts to some extent with human luteinizing hormone (hLH). hCG has been found to be present in the extract of tumors and minute quantities of hCG are synthesized …

Archaeal Aminoacyl-Trna Synthesis: Unique Determinants Of A Universal Genetic Code?, Michael Ibba, A. W. Curnow, J. Bono, P. A. Rosa, C. R. Woese, D. Söll

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

No abstract provided.

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 …

Development Of Fluorescent Techniques In The Kinetic Characterization Of Reverse Transcriptase, Kevin Kevin Howerton

Chemistry & Biochemistry Theses & Dissertations

Reverse transcriptase (RT) is an RNA-directed DNA polymerase isolated from human immunodeficiency virus (HIV) and other retroviruses. It has been the primary target for anti-HIV research since the discovery of its vital role in the retroviral life-cycle. The conventional method for carrying out kinetic characterization and inhibition studies of reverse transcriptase involves the use of radioactive materials. Although this method is effective, it is both time consuming, due to a necessary repeated washing step, and hazardous to the worker and environment. Furthermore, strict laws limit the use of radioactive materials to licensed workers and laboratories. In response to these concerns, …

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 …

Developmental And Hormonal Regulation Of The Expression Of Cytosolic Phospholipase A2 Protein Levels In Baboon Placenta, Kathleen Elizabeth Dunstan

Chemistry & Biochemistry Theses & Dissertations

Cytosolic phospholipase A₂ ( cPLA₂) specifically catalyzes the release of arachidonate from membrane phospholipids, thus initiating synthesis of prostaglandins, thromboxanes, and other eicosanoids. The human and nonhuman primate placenta actively synthesize high levels of eicosanoids which play both an autocrine and paracrine role in placental function. Previous studies in Dr. Miriam D. Rosenthal's laboratory have shown that cPLA₂ mRNA levels are developmentally regulated. The present study was designed to determine whether placental cPLA₂ protein levels increased during gestation. Since the marked increases in estrogen levels during normal pregnancy have been shown to enhance morphological and …

The Effect Of Growth Hormone Release On Plasma And Erythrocyte Selenium And Zinc Levels In Children, Maria Estela Legal De Reid

Chemistry & Biochemistry Theses & Dissertations

Research studies have suggested that selenium may be important in the release of growth hormone, GH. A selenium-containing amino-acid residue, selenocysteine, has been identified as part of the active site of the three isoenzymes (Type I, II and III iodothyronine 5' -deiodinase) responsible for the synthesis of active thyroid hormone, T_3, which is reported to stimulate GH secretion. The importance of zinc in growth is well- documented because of its involvement in transcription and expression of GH and its intermediary, insulin growth factor I or IGF- I. Thus, selenium and zinc levels in plasma may be altered in response …

An Enzyme-Linked Immunosorbent Assay For The Detection Of Cardiac Myoglobin Using Monoclonal Antibodies, Deborah F. Kelly

Chemistry & Biochemistry Theses & Dissertations

The most prevalent life-threatening disease worldwide, secondary to coronary artery thrombosis is Acute Myocardial Infarction (AMI). Increases in three clinical markers have been explored as an early means of AMI detection: Cardiac myoglobin, and the two specific cardiac isoforms of Creatine Kinase, CK-MM and CK-MB. However, myoglobin levels have shown to give a more accurate correlation (R = 0.89) than does Creatine Kinase evaluation. Therefore, we have developed a two-site Enzyme-Linked Immunosorbent Assay (ELISA) using monoclonal antibodies against myoglobin isolated from human heart tissue. This assay is based on specific anti-genantibody interactions and is sensitive at the nanogram level. We …

Competitive Enzyme Immunoassay Of Catechin In Human Blood Serum, Jay Fedorowicz

Chemistry & Biochemistry Theses & Dissertations

( +)-Catechin is a type of polyphenolic compound, and it is enriched in fruits, plants, and various beverages. Polyphenolic compounds also play an important role in the human diet. Many scientists, especially wine researchers, have proposed that(+)catechin and its polymers could have many positive health effects. Thus, it is essential to learn more about (+)-catechin and other polyphenolics. Presently, high performance liquid chromatography is the only method to detect ( + )-catechin levels in human blood serum, but this method has the high cost of instrumentation and does not provide good sensitivity. The objective of this project is to develop …

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, …

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 …

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 …

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 …