Plant tissues contain a wide variety of phenolic compounds,
frequently in high concentration. Both non-covalent association of
intact and oxidized phenols and covalent linkage of oxidized phenols
to protein occur. Such interactions can have important biochemical
and nutritional significance.
A model system approach was used to identify and quantitate
amino...
This dissertation describes an investigation of DNA precursor supply for mitochondrial and nuclear DNA synthesis in HeLa cells and Mouse L-cells. HeLa cells were used for the quantitation of cellular and mitochondrial deoxyribonucleoside triphosphate (dNTP) and ribonucleoside triphosphate (rNTP) pools and of changes in pools in response to treatment with...
I have investigated nucleoprotein organization in
chromatin of the yeast, Saccharomyces cerevisiae. My
approach has been to prepare insoluble, minimally per
turbed chromatin from a mitochondrial DNA-free strain of
yeast, using a number of new techniques, and then digest
this substrate with micrococcal nuclease. The soluble
products are electrophoresed on...
The first reported observation of hemoglobin association
with a plasma protein was made by Polonovski and
Jayle in 1938. Since that time, countless investigators
have contributed to the characterization of the hemoglobin-
binding plasma protein, haptoglobin. Haptoglobins
have been found in many mammals, including humans and in
various birds. However,...
The dihydrofolate reductase gene of bacteriophate T4
has been investigated at the structural level to elucide the
unusual regulation of expression of this gene and possible
evolutionary relationships between the ohage and drug resistance
factors-encoded dihydrofolate reductases (DHFR).
A 1.1-kilobase-pair restriction fragment of the T4
multiple mutant dec8 containing frd,...
Investigators have been studying an aggregate
containing enzymes of deoxyribonucleoside triphosphate
biosynthesis in T4-phage infected bacteria. They suggest
that it behaves as an organized complex, efficiently
channeling DNA precursors to the replication apparatus.
Previous work has suggested that about 10 enzyme
activities remain associated through several fractionation
steps. This multi-enzyme...
A rapid nuclear isolation technique was adapted in
order to examine the question of DNA precursor
compartmentation in mammalian cells. By using this method
a reproducible proportion of the cellular nucleotides
remained associated with the isolated nuclei.
Examination, at several different cell densities, of
exponentially growing HeLa cells showed that...
The cytolytic properties of the mosquitocidal 25 Kd protein deltaendotoxin of Bacillus thuringiensis subsp. israelensis (Bti) was studied using the human erythrocyte as a target cell. Both a fixedtime and a kinetic hemolytic bioassay were developed in order to screen a variety of treatments for their influence on the toxinerythrocyte...
Imbalanced deoxyribonucleoside triphosphate (dNTP) pools are mutagenic for DNA synthesis in both intact cells and cell-free replication systems. Almost certainly, such mutagenesis involves competition between correctly and incorrectly base-paired precursors at replication sites. However, there are certain differences between the intact cell and cell-free systems that do not always allow...
Ribosomes are intricate macromolecular complexes which are a major
element of the protein biosynthetic machinery in all life forms. In
Escherichia coli they contain about 50 distinct proteins and 3
ribosomal RNAs. The small 30S ribosomal subunit in E. coli
incorporates 21 proteins and a 16S rRNA. The 16S rRNA...
A simple matrix method for the analysis of protein circular dichroism (CD) spectra is presented and used to compute the secondary structure of globular proteins in their native and denatured states. The method uses singular value decomposition and generalized inverse algorithms, and a basis set of CD spectra for proteins...
Recent studies have demonstrated that perturbations of
intracellular thiol and calcium homeostasis may be important events
in the early development of cell injury by toxic chemicals.
Incubation of isolated rat hepatocytes in a calcium free
medium, severely depleted intracellular Ca²⁺ levels and resulted in
the loss of both cytosolic and...
Vaccinia virus-infected animal cells have been used to
study the interactions between the replication of
deoxyribonucleic acid (DNA) and the biosynthesis of its
nucleotide precursors. Some antimetabolites that inhibit
DNA replication have as their targets enzymes of nucleotide
biosynthesis. Furthermore, the disruption of nucleotide
metabolism can alter the fidelity of...
Farlowia mollis is a marine red alga that grows in the
intertidal region along the coast of Oregon. We began studying
the chemistry of this alga after a survey of Oregon seaweeds
revealed possibly interesting chemistry and antimicrobial activity
in its crude organic extract.
Over the past three years the...
Ribonucleotide reductase is an important enzyme in the control of
DNA replication within the cell. Ribonucleotide reductase exerts its
control through enzymatic reduction of nucleoside diphosphates. The
bacteriophage T4 enzyme is an example of the class of iron-requiring
reductases which also includes E. coli and mammalian ribonucleotide
reductases. The two...
Bacteriophage T4 gene 42 encodes dCMP hydroxymethylase, an
enzyme unique to the deoxyribonucleotide metabolism of T-even
bacteriophages. To study biochemical and biophysical properties of
the enzyme, as well as the interaction of dCMP hydroxymethylase with
other DNA precursor biosynthetic enzymes in vitro, availability of large
amounts of the enzyme is...
Several levels of eukaryotic chromatin structure have
been observed: the nucleosome, the 10 nm and 30 nm fibers
and loop domains, apparently attached to the nuclear
matrix. In this research, the structure and function of
chromatin at two of these levels was investigated, with
studies on both nucleosome positioning and...
Although the synthesis of DNA precursors is
closely coordinated with DNA replication, it is still
not clear whether deoxyribonucleoside triphosphates
(dNTPs) influence DNA replication independently of
their interactions with DNA polymerase catalytic sites.
In an effort to understand the extent to which rate and
fidelity of DNA replication are regulated...
By using immobilized trypsin, I have been able to
prepare well-defined, stable trypsinized nucleosomes. The
difficulties of lacking of control in the extent of
trypsinization, which were encountered in previous studies
with the use of free trypsin, have been eliminated. The
nucleosomes and oligonucleosomes prepared by immobilized
trypsin are suitable...
For ras gene mutation analysis in the rainbow trout (Oncorhynchus
mykiss) model system, a partial trout ras sequence was identified
using the polymerase chain reaction (PCR). Two synthetic
oligonucleotides based on rat K-ras gene sequence were used as
primers for the PCR procedure. A 90 base pair (bp) sequence,
referred...
The work in this thesis has provided conclusive genetic evidence
that "panhandle" intermediates form during adenovirus replication.
Adenovirus chromosomes lacking 51 by from their left -hand termini
are infectious and capable of regenerating the missing origin
sequence. Yet if an entire inverted terminal repeat is removed, the
adenovirus chromosome is...
Ribosomes are multicomponent macromolecular particles and
are essential for the survival of cells in all organisms. The ribosome's
universal function is to catalyze polypeptide synthesis through
translation of mRNA transcripts. Ribosomes from Escherichia coli,
eubacterial organisms, have a sedimentation coefficient of 70S and are
composed of 30S and 50S ribonucleoprotein...
Vaccinia virus infected monkey kidney cells had been previously shown
to have an increased ribonucleoside diphosphate reductase (RR) activity. DNA
from mutant virus resistant to hydroxyurea were digested with restriction
endonucleases and were shown to have substoichiometric amounts of the Hind
III F fragment. Additional information from Southern blotting experiments...
The ability to faithfully replicate DNA is dependent upon the maintenance
and regulation of its precursors, the deoxyribonucleoside triphosphates.
Enzymes encoded by the bacteriophage T4 have been widely used as models
of biochemical processes. A body of evidence supports the concept that the
bacteriophage T4 enzymes involved in deoxyribonucleotide biosynthesis...
Ribonucleotide reductase is a remarkable enzyme that catalyzes the rate-limiting
step in the synthesis of the 2'-deoxynucleoside triphosphates. The intent of this project
was to characterize the ribonucleotide reductase encoded by the orthopoxvirus,
vaccinia. The first objective was to study the structural and functional features of the
viral small subunit...
Methods are presented to acquire data from analytical ultracentrifugation experiments
by computer using the absorption optical scanning system of the Beckman Model-E
ultracentrifuge. A computer program was written which analyzes sedimentation velocity
experiments by the van Holde - Weischet method and by the second moment method. The van
Holde -...
Ribonucleoside diphosphate reductase (RR) from vaccinia virus was recently cloned and overexpressed rn Escherichia coli. The amino acid sequence identities of the small and large subunits between the mouse and the vaccinia virus reductase are approximately 80 and 72 percent, respectively. In addition, vaccinia virus RR displays similar complex allosteric...
The first committed step in DNA biosynthesis occurs by direct reduction of
ribonucleotides. This reduction is catalyzed by ribonucleotide reductase (RNR), an
enzyme which uses a unique radical mechanism to facilitate the transformation. All four
DNA precursors are synthesized by a single enzyme. Therefore, an intricate pattern of
regulation has...
Although the Escherichia coli host has almost all of the enzymes necessary to
synthesize nucleotides needed for bacteriophage T4 DNA replication, phage genes
expressed early in infection encode enzymes for de novo DNA precursor biosynthesis
and salvage from degraded host DNA. Eight early enzymes and two host enzymes
comprise the...
The effects of oligoribonucleotides of identical or similar sequences to a
ribosomal protein binding site, specifically S8, on prokaryotic 30S ribosomal assembly
were investigated in Escherichia coli. The oligoribonucleotides were expected to bind to
the target protein S8 and block its incorporation into the 30S subunit, resulting in a nonfunctional...
During catalysis the flavoprotein domain of neuronal nitric-oxide synthase (nNOS) shuttles NADPH-derived reducing equivalents from FAD to FMN and then to the P450-heme enabling heme-based oxygen activation and subsequent NO-synthesis. The binding of Ca²⁺-activated calmodulin (Ca²⁺-CaM) to nNOS alleviates inhibition of flavin mediated electron transfer within the diflavin domain, which...
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LIST OF FIGURES
Figure
1.1 The NOS-catalyzed two-step conversion of L-arginine
toL-citrulline
The vascular system transports components essential to the survival of the individual and acts as a barrier to substances that may injure the organism. Atherosclerosis is a dynamic, lesion producing disease of the arterial system that compromises the functioning of the organ by occlusive and thrombogenic processes. This investigation was...
Deoxyribonucleoside triphosphate (dNTP) concentrations measured in cells are not symmetric. dGTP almost always represents only 5-10% of the total dNTP pools in cells. In an in vitro replication system involving semiconservative replication from an SV 40 origin, the mutation frequency of an M13 phagemid replicated by human cell extracts in...
Butane-grown Pseudomonas butanovora oxidized butane by a soluble butane monooxygenase through the terminal pathway yielding 1 -butanol as the predominant product. Alcohol dehydrogenases (ADHs) involved in butane oxidation in P. butanovora were purified and characterized at the biochemical, genetic and physiological levels. Butane-grown P. butanovora expressed a type I soluble...
Mismatch repair is one of the mechanisms by which cells ensure genomic
stability. Deficiencies in mismatch repair (MMR) increase mutation rates and cancer
risks. In the well-characterized methyl-directed Escherichia co/i system, MMR is
initiated by MutS, Mut L, and MutH proteins. The single MutS protein and the
single MutL protein...
β-catenin is a multi functional protein that is involved in cell-cell adhesion and cell signaling. In non-stimulated cells, β-catenin is tightly down-regulated by GSK-3β-dependent phosphorylation at Ser and Thr residues, followed by rapid ubiquitination and proteasomal degradation. It is well established that mutations within the regulatory GSK-3β region lead to...
This dissertation examines the interaction of the linker histone with DNA and with nucleosomes. The first goal of the project was to characterize the interaction of the linker histone with DNA. Three factors previously reported to influence the linker histone's interaction with DNA were examined: ratio of linker histone to...
Oxidative stress is the major driving force behind the aging process and many age-related diseases. However, direct experimental evidence of whether antioxidants, such as ascorbate (AA) and lipoic acid (LA) can slow the progression of aging process and/or reduce risks of developing degenerative disease is largely absent. This suggests a...
Exposure to polycyclic aromatic hydrocarbons such as benzo[a]pyrene (B[a]P) has been determined to be a risk factor for various forms of human cancer. PAH DNA adducts have been shown to cause mutations, but carcinogenesis is also accompanied by alterations in gene expression. Inhibiting individual cytochrome P450s could clarify the interaction...
With respect to a multienzyme complex of deoxyribonucleoside triphosphate (dNTP) synthesis somehow juxtaposed with DNA replication sites, our laboratory has demonstrated the existence of a multienzyme complex in T4-infected E. coli, named the T4 dNTP synthetase complex, but the idea of direct linkage of dNTP synthesis to DNA replication and...
This thesis involves analyses of 3₂-helices, a-helices and of the change in residual
entropy of a protein upon chemical modification. Part of the long-term goal of
understanding the formation, function and stability of proteins requires that all
conformations be accurately assigned and structurally understood. By a statistical
analysis of 258...
The enzymes of dNTP synthesis in T4 infection associate to form a multienzyme complex, the T4 dNTP synthetase complex, facilitating the flow of metabolites en route to dNTPs, and their subsequent flow into DNA. Study of protein-protein interactions helps one to understand how the enzymes in the complex are organized...
Oxygen derived radicals are involved in many aspects of life from aging and cell signaling to disease states as diverse as heart disease, diabetes, neurodegeneration and inflammation. Therefore, understanding the function of antioxidant defense proteins and the effects of oxygen derived radicals on protein function is essential to elucidate the...
The endoplasmic reticulum (ER) is a crucial organelle in the cell where protein folding and processing occurs, and conditions that negatively affect ER functions are highly detrimental to the cell. The accumulation of unfolded/misfolded proteins in the ER leads to a condition known as ER Stress. The ER has developed...
Aerobic organisms have evolved many sensory mechanisms that allow
response to oxidants in the environment. One area of interest is the relationship
between the activity of the tumor suppressor protein p53 and the redox state of
thioredoxin. Human p53 activity is severely compromised in budding yeast lacking
thioredoxin reductase. Evidence...
Vitamin C is an essential component of the human diet. Uptake of vitamin C in the cell is regulated by the function of sodium-dependent vitamin C transporters (SVCTs). SVCT1 (encoded by the SLC23A1 gene) is expressed in the colon, liver and kidney, where it plays a vital role in determining...
One of the distinctive features of mitochondria is that they have their own DNA. When compared to nuclear DNA, it is known that mitochondrial DNA (mtDNA) not only has a much higher rate of spontaneous mutation, but also arises from metabolically distinct precursor pools. Others from our laboratory have demonstrated...
Eukaryotic cells have developed elaborate biochemical processes to counteract oxidative stress, electrophiles, or carcinogens due to environmental insults. One of these important cellular defense mechanisms is mediated by the transcription factor Nrf2 that have been shown to have decrease activity with age. While the process of which Nrf2 is involved...
Cytoplasmic dynein is an ATP-dependent, microtubule-based molecular motor involved in the positioning and trafficking of cellular cargo. The cargo binding sub-domain of dynein contains the natively disordered intermediate chain (IC) and the homodimeric light chains (Tctex1, LC8 and LC7). The structure and stoichiometry of this complex, how the light chains...
Ceramides are a group of lipids in the sphingolipid family that are potent cell signaling molecules. Elevations in ceramide levels above the norm generally lead to apoptosis. Evidence from in vitro studies suggests that accumulation of ceramides within mitochondria leads to dysfunction of the mitochondria. This includes inhibition of the...
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Tory M. Hagen
Ceramides are a group of lipids in the sphingolipid family that are potent
Polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (B[a]P), are environmental pollutants linked to increased disease susceptibilities. Alpha-Tocopherol (αT) supplementation decreases B[a]P-DNA adducts in smokers, particularly women; but the mechanism is unknown. To test the hypothesis that αT protection from B[a]P exposure is gender-dependent, male and female rats received 7 daily subcutaneous...
Decline in cardiac pump function is a hallmark of aging where mitochondrial decay is an important underlying cause. Although certainly multifactorial in nature, both dysfunction of the machinery involved in the chemiosmotic process of energy transduction and lower capacity to maintain fatty acid-driven respiration are identified as intrinsic factors of...
Human cathelicidin antimicrobial peptide (CAMP/LL-37) is a cationic antimicrobial peptide that is widely expressed by myeloid and epithelial cells at the human-environment interface. It possesses broad spectrum antimicrobial capacity against bacteria, fungi and viruses. In addition to its direct antimicrobial activity, CAMP/LL-37 also attracts and recruits monocytes, neutrophils and other...
A unique, permanent root-growth arrest phenotype was observed in UV-B irradiated Arabidopsis thaliana roots lacking the DNA damage response kinase ATR. Segregation analysis suggests the dependence of this phenotype on another, unidentified gene, termed ursu. High-throughput sequencing technologies were used to map and identify the causal ursu mutation. Illumina HiSeq...
DNA segments in the fungus Fusarium graminearum were identified as possible genes encoding for chromodomain proteins (CDPs), and targeted for genetic manipulation. Targeted single-gene deletion strains, as wells as GFP-S tag fusion strains, were generated from wildtype F. graminearum protoplasts, and verified strains were screened for phenotypic differences, such as...
The preconditioning agent poly-ICLC has recently been shown to protect against cerebral injury in the mouse model, when administered systemically prior to ischemic insult. However, the mechanism by which poly-ICLC provides neuroprotection is currently unknown, but it has been shown to protect against ischemia-induced bloodbrain barrier (BBB) disruption. Inflammation contributes...
Nuclear Factor, Erythroid Derived 2, Like 2 (NFE2L2 or Nrf2) is the primary transcription factor in cellular defense against oxidative and xenobiotic stresses in higher eukaryotes. This basic leucine zipper transcription factor regulates over 200 antioxidant, detoxification, and lipid metabolizing genes by binding to the Antioxidant Response Element (ARE; a...
As organisms age they lose the ability to respond to both exogenous and endogenous stresses, therefore placing them at a greater risk for the development of many diseases. The transcription factor Nrf2 is responsible for the induction of many oxidative stress response genes and therefore 3proteins. Additionally, Nrf2-mediated stress response...
A study was conducted to evaluate the developmental toxicity of three commonly detected mycotoxins in food (deoxynivalenol, patulin, and zearalenone) using the zebrafish (Danio rerio) model. Mycotoxin exposures were carried out at five concentrations (0.0064, 0.064, 0.64, 6.4, and 64 μM). Morphological and photomotor screenings were conducted at 24 hours...
Exploring and quantifying the parameters regarding contact guidance may provide valuable insight to the precise mechanisms controlling cell movement, especially in a three-dimensional environment. The objective of this project was to quantify the relationship between MDA-MB-231 cell morphogenesis and the geometry of the cell’s local microenvironment, which was modeled by...
Cells represent microcosms of spatial and temporal structural organization, with the achievement of internal spatial organization relying upon a collection of macromolecular motor complexes to transport and localize components throughout the cell. Cytoplasmic dynein is one such motor complex, and is the principal ATP-dependent motor for retrograde transport along microtubules...
Many people die yearly worldwide due to diseases associated with aging, and if the
process of aging in humans was more understood, the health of these patients could be
improved. While many biochemical actions are involved with the aging process, loss of
proteostasis has been called one of the most...
An ideal labeling reaction between protein and surface should generate a protein-patterned surface with control over the orientation of the desired protein. Controlling orientation would generate a homogeneous, un-fouled protein surface and allow for maximum efficiency in regards to the activity, reactivity, and stability of the protein. In this research...
Atherosclerosis is the underlying cause of ischemic heart disease and stroke and is the leading cause of death worldwide, especially in developed countries. As an inflammatory disease of arteries in a hyperlipidemic milieu, expression of adhesion molecules, such as endothelial-leukocyte adhesion molecule-1 (E-selectin) and intercellular adhesion molecule-1 (ICAM-1), on endothelial...
The aging process is a biological phenomenon in mammals that heavily contributes to the onset of chronic diseases. While there are many theories that attempt to explain the biological onset of aging, one emerging theory involves chronic inflammation. Chronic inflammation has been previously linked to several age-related diseases and is...
Amyotrophic lateral sclerosis (ALS) is a devastating neurological disorder characterized by neuromuscular junction decay, motor neuron death, progressive paralysis, and eventually death of the individual, usually by respiratory failure. Oxidative stress is a prominent hallmark of the disease and is often accompanied and exacerbated by mitochondrial dysfunction and neuroinflammation. This...
Over the past century, life expectancy in the United States has dramatically increased leading to an increasingly aging population with people reaching, and spending more years in ‘old age’. While this unprecedented shift in population demographics represents great strides for humanity, it is not without cost. One consequence of longer...
The inverse electron demand Diels Alder reaction between tetrazines and strained alkenes is an exceptionally useful tool in functionalizing to biomolecules since it is orthogonal to the chemistry of most living systems and have exceptionally high rate constants. In particular reactions between strained trans-cyclooctenes (sTCO) and tetrazines can achieve second...
Xanthohumol, a prenylated flavonoid found in hops and beer, possesses a long-standing reputation to improve health. Xanthohumol’s cytotoxicity to a wide variety of cancer cell lines is well-established. Herein we provide further insight into the cytotoxicity of not only xanthohumol, but also its non-estrogenic derivatives, to a number of colonic...
Dysferlin is a ∼230 kDa terminally anchored membrane protein that is ubiquitously expressed, but is particularly enriched in skeletal and cardiac muscle tissue. Mutations covering the length of the protein have been linked to muscle wasting diseases including limb-girdle muscular dystrophy and Myoshi myopathy. Dysferlin has been shown to play...
Systems biology is a powerful approach which considers and sheds light on all of the puzzle pieces which make up complex biological processes, and is an effective alternative to unraveling these processes using traditional molecular approaches alone. It is a natural companion approach for computational biology, which leverages the power...
Alzheimer’s Disease (AD) is a neurodegenerative pathology currently affecting nearly 44 million individuals worldwide, yet there are not currently any effective treatments or preventions for AD despite the rapid development in our understanding of the disease over the last four decades. The medical and sanitary innovations of the last century...
Hop (Humulus lupulus L. var. lupulus) is a diploid, dioecious plant with an extensive history of cultivation and use in brewing, as a textile, and for its therapeutic properties. Hop is prized for its ability to produce a variety of aromatic and flavor compounds, as well as compounds with anti-microbial...
Cell signaling is often mediated by protein-protein interactions, which must be specific, tunable, and transient to allow agile responsiveness to cellular messages. Due to their unique properties, multivalent, intrinsically disordered proteins make ideal candidates to accomplish these vital tasks. A single protein with multiple binding sites may bind numerous partners,...