Biochemistry, Biophysics, and Structural Biology Commons^™

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 …

Glucose Mediated Effects On Glucose-6-Phosphate Dehydrogenase Expression, Daryl Arkwright

Masters Theses

Circulating high glucose levels are believed to cause oxidative stress which is implicated in a number of pathological conditions such as cancer, aging, and diabetes. Oxidative stress regulates the expression of glucose-6-phosphate dehydrogenase (G6PDH), the key enzyme in the pentose phosphate pathway which provides reducing equivalents in the form of NADPH. These reducing equivalents are also required by fatty acid synthase (FAS), the enzyme which catalyzes fatty acid biosynthesis. We examined whether glucose, a known inducer of oxidative stress, could regulate either G6PDH or FAS activity in primary rat hepatocytes. We tested the effects of glucose alone or in combination …

Expression, Purification, And Characterization Of Nonstructural Protein 2 (Ns2) Of Bluetongue Virus Serotype 17, Hai Lin

Biology

The NS2 protein of bluetongue virus (BTV) serotype 17 (B17) was expressed in large scale as a Glutathione-S-Transferase (GST) fusion protein in E. coli cells. The size of the expressed NS2/GST fusion protein was about 68 kilodaltons (kDa) when analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This is equal to the sum of the predicted size from the deduced amino acid sequence of the NS2 protein (41 kDa) and the known mass of the GST protein (27 kDa). The NS2/GST fusion protein was purified by absorption to glutathione-conjugated agarose beads (G-beads), and then analyzed on western blots by two …

The Influence Of A Human Repetitive Dna On Genome Stability, Eugenia L. Posey

Electronic Theses and Dissertations

A uniquely human interspersed repetitive DNA sequence family, the L2Hs, are highly polymorphic in human genomes. Several features of interspersed repeated DNA may contribute to the instability observed. Certain motifs (direct repeats, palindromes, and inverted repeats) comprising L2Hs elements may adopt unusual secondary structures such as cruciforms or hairpins. These motifs have been associated with features of genome instability in recombination, insertions and deletions. The L2Hs elements also are AT-rich (76%) compared to the bulk of human DNA (52%). That their dynamic nature (i.e. polymorphisms) may arise from recombination, insertions and deletions has led to the hypothesis that the L2Hs …

The Polarized Sorting Of Membrane Proteins Expressed In Cultured Hippocampal Neurons Using Viral Vectors, Mark Jareb, Gary Banker

Biology Faculty Publications

One model of neuronal polarity (Dotti and Simons, 1990) proposes that neurons and polarized epithelia use similar mechanisms to sort membrane proteins. To explore this hypothesis, we used viral vectors to express proteins in cultured neurons and assessed their distribution using quantitative immunofluorescence microscopy. Basolateral epithelial proteins were polarized to dendrites; more significantly, mutations of sequences required for their basolateral targeting in epithelia also disrupted dendritic targeting. Unexpectedly, apical proteins were not polarized to axons but were expressed at roughly equal amounts in dendrites and axons. These data provide strong evidence that targeting of basolateral and dendritic proteins depends on …

A New Human Selenium-Containing Protein, Vadim N. Gladyshev, Kuan Teh Jeang, John C. Wootton, Dolph L. Hatfield

Vadim Gladyshev Publications

Selenium which occurs in proteins as the amino acid, selenocysteine, is essential for numerous biological processes and for human health. A prominent ⁷⁵Se-labeled protein detected in human T-cells migrated as a 15-kDa band by SDS-polyacrylamide gel electrophoresis. This protein subunit was purified and subjected to tryptic digestion and peptide sequence analyses. Sequences of tryptic peptides derived from the protein corresponded to a human placental gene sequence containing an open reading frame of 162 residues and a readthrough in-frame TGA codon. Three different peptide sequences of the 15-kDa protein corresponded to a nucleotide sequence located downstream of this codon, suggesting …

The Second Messenger Camp Elicits Eating By An Anatomically Specific Action In The Perifornical Hypothalamus, Elizabeth Gillard, Arshad Khan, Rickinder Grewal, Bara Mouradi, Stefany Wolfsohn, B. Stanley

Selected Works Temporary Series

No abstract provided.

The Second Messenger Camp Elicits Eating By An Anatomically Specific Action In The Perifornical Hypothalamus, Gillard, Arshad Khan, Rickinder Grewal, Bara Mouradi, Stefany Wolfsohn, B. Stanley

Departmental Papers (Biology)

No abstract provided.

The Establishment Of Standard Methods And Experimental Procedures For The Prototypical Icp/Tof-Ms Using An Icp-Aes, Joseph Riley

Mahurin Honors College Capstone Experience/Thesis Projects

The ICP-AES was used to develop a standard test method for the preparation of samples for analysis purposes, a method that could also be transposed onto the new prototypical ICP/TOF-MS. Whereas both of these instruments are analytical spectroscopic instruments and use the ICP's plasma as an energy source, the ICP/TOF-MS also has a mass spectrometer attached to it. This addition gives the instrument better reliability, accuracy, and precision in analysis as well as faster analysis time. Since the ICP-AES is a precursor of the ICP/TOF-MS, it can be used to develop a standard development method. The experiment involved using a …

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 …

Molecular Staging Of Colorectal Cancer: K-Ras Mutation Analysis Of Lymph Nodes Upstages Dukes’ B2 Patients, Jennifer S. Thebo

Dissertations

The successful treatment of colorectal cancer depends upon the ability of staging systems to identify patients who are at risk for recurrence. The currently used systems fail in a significant number of patients. Multiple attempts have been made to improve upon these systems in order to provide better treatment for at-risk patients.

A variety of oncogenes and tumor suppressor genes have been implicated in colorectal cancer. Mutations in one of these, K-ras, occur in 40-60% of colorectal carcinomas. However, the relationship between specific mutations and clinical outcome is unclear.

The purpose of this study was to determine whether or not …

Enhancement Of Atp Levels And Glucose Metabolism During An Infection By Chlamydia: Nmr Studies Of Living Cells, David M. Ojcius, H. Degani, Joel Mispelter, Alice Dautry-Varsat

All Dugoni School of Dentistry Faculty Articles

The Chlamydia species are obligate intracellular bacteria that proliferate only within the infected cell. Since the extracellular bacteria are metabolically inert and there are no cell-free systems for characterizingChlamydia metabolism, we studied metabolic changes related to ATP synthesis and glycolysis in HeLa cells infected withChlamydia psittaci during the course of the 2-day infection cycle using noninvasive 31P and 13C NMR methods. We find that the infection stimulates ATP synthesis in the infected cell, with a peak of ATP levels occurring midway through the infection cycle, when most of the metabolically active bacteria are proliferating. The infection also stimulates synthesis of …

Fission Yeast Mitotic Regulator Dsk1 Is An Sr Protein-Specific Kinase, Zhaohua Irene Tang, Mitsuhiro Yanagida, Ren-Jang Lin

WM Keck Science Faculty Papers

Intricate interplay may exist between pre-mRNA splicing and the cell division cycle, and fission yeast Dsk1 appears to play a role in such a connection. Previous genetic analyses have implicated Dsk1 in the regulation of chromosome segregation at the metaphase/anaphase transition. Yet, its protein sequence suggests that Dsk1 may function as a kinase specific for SR proteins, a family of pre-mRNA splicing factors containing arginine-serine repeats. Using an in vitro system with purified components, we showed that Dsk1 phosphorylated human and yeast SR proteins with high specificity. The Dsk1-phosphorylated SF2/ASF protein was recognized strongly by a monoclonal antibody (mAb104) known …

Land Use Patterns In Relation To Lake Water Quality In The Great Pond Watershed, Problems In Environmental Science Course (Biology 493), Colby College, Colby Environmental Assessment Team, Colby College

Colby College Watershed Study: Great Pond (2012, 2010, 1998)

Lakes are natural resources, which have many effects on the land surrounding them, They support adjacent communities by providing water and regulating temperatures, helping to define the surrounding ecosystem, and serving as sources of drinking water as well as recreation. The prolonged presence of human activity in a watershed can disturb the physical and chemical cycles of the lake and its surrounding ecosystems (Henderson-Sellers and Markland 1987). Over time, lakes undergo a process called eutrophication, a natural aging process during which the nutrient levels increase and dissolved oxygen levels decrease (Smith and Smith 1998). As the lake ages or becomes …

Influences Of Acid Mine Drainage On Water Chemistry Of Blackwater River Watershed, Carla C. Morato-Lara

Theses, Dissertations and Capstones

Acidic mine drainage flow from active and abandoned coal mines has been a major source of water pollution in the Appalachian Mountain region of the eastern USA. Water samples were collected and analyzed from Blackwater River watershed during 2 years (September 1996 - May 1998). The water quality measurements determined that the Beaver Creek and North Fork tributaries were affected by acid mine drainage but not Yellow Creek. Beaver Creek and North Fork stations had high sulfate and manganese markers of acid mine drainage, whereas Yellow Creek was slightly acidic with low sulfate and manganese. North Fork was the primary …

The Role Of Domain I In Laminin Chain Assembly, George D. Kamphaus

Theses, Dissertations and Capstones

Laminin, a major component of basement membrane, is a trimeric glycoprotein comprised of three chains - α, β and γ (Burgeson et al., 1994). An order for trimer assembly has been deduced: first, the β and γ chains bind to form a dimer and subsequently α is added to complete the trimer (I. Hunter et al., 1990 & 1992; Utani et al., 1994 & 1995). The C-terminal portions, found within the protein structural domain I of the p and y chains, are implicated in dimer and trimer formation by biochemical studies performed extracellularly (Utani, et al., 1994 & 1995; Nomizu …

Crystal Structures Of Copper(Ii) Complexes Of Some 2-Methyl-8-Quinolinols And Implications For Their Antifungal Activity / Massud Shoja, Herman Gershon, Diana Bray, And Donald D. Clarke Department Of Chemistry, Fordham University, Bronx, Ny 10458, Usa New York Botanical Garden, Bronx, Ny 10458, Usa, Massud Shoja Phd, Herman Gershon, Diana Bray, Donald Dudley Clarke Phd

Chemistry Faculty Publications

A hypothesis that the geometry of a potential fungicide must be consistent with that of the pores of the fungal spore wall in order to penetrate it and be toxic has been developed. Certain bis(8-quinolinolato)copper(II) complexes seemed to contradict this. To resolve this issue, structures of bis(7-fluoro-8-quinolinolato )copper(II) (1), bis(2-methyl-8-quinolinolato )copper(II) (2), and bis(2-methyl-7-nitro-8-quinolinolato)copper(II) (3) were solved. The ligands of 1 are square planar with copper at the center of symmetry. In 2 and 3 the methyl group at C2 interacts with the other 8- quinolinol ligand, producing a significant distortion of the square planar geometry which causes a rotation …

Revision Of The Assigned Structures Of 5- And 7-Iodo-8-Quinolinols And 5- And 7-Iodo-2-Methyl-8-Quinolinols / Donald D. Clarke, Herman Gershon, Massud Shoja, And Mei-Wen Yen Department Of Chemistry, Fordham University, Bronx, Ny 10458, Usa, Donald Dudley Clarke Phd, Herman Gershon, Massud Shoja Phd, Mei-Wen Yen

Chemistry Faculty Publications

Revised structures are presented for 5- and 7-iodo-8-quinolinols and for 5- and 7-iodo-2-methyl-8-quinolinols based on NMR studies. UV spectroscopic characterization of the compounds was also carried out

The Use Of Emergent Rocks As Refugia For The Cheat Mountain Salamander, Plethodon Nettingi Green, Beth Anne Pauley

Theses, Dissertations and Capstones

Plethodon nettinqi was listed as a threatened species in 1989 by the United States Fish and Wildlife Service (Pauley, 1991). Its total range is within 5 counties in eastern West Virginia. There are fewer then 60 disjunct populations known and most populations are above 3,000 ft. and are associated with emergent rocks or narrow ravines with Rhododendron. It is hypothesized the P. netting! survived lumbering practices at the turn of the century by taking refuge beneath large emergent rocks and narrow ravines with Rhododendron. This study examined environmental factors associated with emergent rocks that might regulate the distribution of P. …

Eating Induced By Perifornical Camp Is Behaviorally Selective And Involves Protein Kinase Activity, Elizabeth Gillard, Arshad Khan, Bara Mouradi, Omkar Nalamwar, B. Stanley

Selected Works Temporary Series

No abstract provided.

Rna Bulge Entropies Correlate With Peptide Binding Strengths For Hiv-1 And Biv Tar Rna Because Of Improved Conformational Access, Brooke Lustig, I Baharand, R L. Jernigan

Faculty Publications, Chemistry

For the binding of peptides to wild-type HIV-1 and BIV TAR RNA and to mutants with bulges of various sizes, changes in the ΔΔG values of binding were determined from experimental Kd values. The corresponding entropies of these bulges are estimated by enumerating all possible RNA bulge conformations on a lattice and then applying the Boltzmann relationship. Independent calculations of entropies from fluctuations are also carried out using the Gaussian network model (GNM) recently introduced for analyzing folded structures. Strong correlations are seen between the changes in free energy determined for binding and the two different unbound entropy calculations. The …

Vibrational Sum Frequency Spectroscopy Of Surfactants And Phospholipids Monolayers At Liquid-Liquid Interfaces, B. L. Smiley, R. A. Walker, D. E. Gragson, T. E. Hannon, G. L. Richmond

Chemistry and Biochemistry

Work from our laboratory on vibrational sum frequency spectroscopic investigations of molecular ordering at the carbon tetrachloride-water interface is reviewed. Simple charged surfactants adsorbed at the liquid-liquid interface are seen to induce alignment of interfacial water molecules to a degree which is dependent on the induced surface potential. Saturation of water molecule alignment occurs at a surfactant surface concentration corresponding to a calculated surface potential of approximately 160 mV. In complementary studies, the relative degree of hydrocarbon chain ordering within monolayers of symmetric phosphatidylcholines of different chain lengths is inferred by the relative signal contributions of the methyl and methylene …

Sequence Profile Of The Parallel Β Helix In The Pectate Lyase Superfamily, Susan Heffron, Gregory R. Moe, Volker Sieber, Jerome Mengaud, Pascale Cossart, Jacqueline Vitali, Frances Jumak

Physics Faculty Publications

The parallel β helix structure found in the pectatelyasesuperfamily has been analyzed in detail. A comparative analysis of known structures has revealed a unique sequenceprofile, with a strong positional preference for specific amino acids oriented toward the interior of the parallel β helix. Using the unique sequenceprofile, search patterns have been constructed and applied to the sequence databases to identify a subset of proteins that are likely to fold into the parallel β helix. Of the 19 families identified, 39% are known to be carbohydrate-binding proteins, and 50% belong to a broad category of proteins with sequences containing leucine-rich repeats …

Evaluation Of The Role Of Fructosamine As An Indicator Of Diabetic Control, Katherine Hooley

Theses

Diabetes mellitus, a common metabolic disease, is characterised by excess circulating glucose concentrations. Such a hyperglycaemic status provides the stimulus for irreversible glycation (non-enzymatically post-translational) of structural and functional proteins. Protein glycation, although a cause of complication (neuropathy, micro/macrovascular disease), provides a time-averaged index of diabetes status and control. In particular, glycated plasma proteins analysis, i.e., the fructosamine assay, has received favourable response due to its speed, simplicity, low cost, and ease of automation. However, lack of standardisation and a universally-accepted calibrant has limited technical and subsequent clinical confidence in fructosamine analysis. In response, a comprehensive technical evaluation (including novel …

Chemical Synthesis, Bacterial Expression, And Characterization Of Pro-Gnrh/Gap, A Precursor Protein Of Two Biologically Active Peptide Hormones, Louis E. Mcadory

Theses and Dissertations

Two biologically active peptides, gonadotropin releasing hormone (GnRH) and GnRH associated peptide (GAP) are both derived from a common prohormone precursor protein, pro-GnRH/GAP. Both peptides are cosecreted from hypothalamic neurosecretory granules and are involved in the regulation of mammalian reproduction. A calcium dependent, neutral pH serine protease discovered in this laboratory, GAP-releasing enzyme, is the most likely processing enzyme of pro-GnRH/GAP. GAP-releasing enzyme is immunologically related to PC1/3, a member of the prohormone convertase (PC) class of processing endoproteinases.

GAP-releasing enzyme recognizes the eight residue processing site within pro-GnRH/GAP, G⁶LRPGGKR¹³, and correctly cleaves the R^{13 …}

Development Of An Atomic Force Microscopic Immunosensor For The Ferritin Antigen, Brendan O'Connell

Theses

The adsorption conditions for a range of different immunoreactants to both polystyrene and ultrasmooth silanised silicon surfaces are optimised. The conditions studied were concentration of protein, time and temperature of incubation, pH, molarity and ionic strength of the buffer. The anti-ferritin surface was selected as our model system. This immunosensor was used to develop and evaluate a millispot ELISA for ferritin. The millispot ferritin ELISA developed utilises a sample volume of 6pl and a sample incubation time of 10 minutes. Precision and accuracy studies show the assay to have a dynamic assay range of 7.8-lOOng/ml ferritin. The millispot ELISA detected …

Development Of A Differential Assay To Selectively Measure Α-Amylase Isoenzymes., Miriam O'Connor

Theses

α-Amylase is a heterogeneous polypeptide, primarily found in the Salivary and Pancreatic glands, giving rise to two isoenzymes, namely Salivary- and Pancreatic-type Amylases. Total α-Amylase activity measurements are routinely carried out in hospital laboratories to aid in the diagnosis of diseases of the pancreas and in the investigation of pancreatic function in patients with severe abdominal pains. With the development and wide availability of modem chromogenic oligosaccharide substrates specific for Amylase, assays have become more sensitive, rapid and easier to use.

Differential Amylase Isoenzyme assays ware developed to distinguish isoenzyme levels. Several methods were developed for isoenzyme determination. Of these. …

Development Of An Elisa Method For The Measurement Of Cd44 Expression In Human Serum, Ciara Hurley

Theses

CD44 is a cell surface glycoprotein (mol. wt. =85-95 kDa) which has been implicated in lymphocyte-HEV interactions and the process of lymphocyte homing. CD44 is a complex glycoprotein which undergoes extensive alternative splicing to generate various CD44 isoforms. CD44 functions include the delivery of an activation signal to T-cells, binding to hyaluronate, mediation of lymphocyte homing, and the spread of metastatic cells. CD44 has been particularly implicated in the dissemination of non- Hodgkin's lymphoma and as such may serve as a useful prognostic marker of disease progression. CD44 measurement in serum may provide a rapid, inexpensive and relatively non-invasive prognostic …

Development And Validation Of An Improved Ultrasensitive Assay For Cardiac Troponin I (Ctni), William G. Blake

Theses

Cardiac troponin I (cTnl) has been reported to be a highly specific marker for myocardial injury. In patients suffering from acute coronary syndromes, a sensitive serum marker which is closely related to the extent of myocardial damage which provides prognostic data and which can be measured rapidly is desirable. In this thesis a “sandwich” enzyme immunoassay was developed to measure cTnI. The developed assay was reproducible with the inter assay precision ranging from 4.8-14% and the intra assay precision ranging from 3.1-9.5%. The limit of detection of the assay was calculated as 0.385 ng/ml. The upper reference range (non-parametric 95% …

Type I Camp-Dependent Protein Kinase Delays Apoptosis In Human Neutrophils At A Site Upstream Of Caspase-3, Lav K. Parvathenani, E. Stephen Buescher, Enrique Chacon-Cruz, Stephen J. Beebe

Bioelectrics Publications

Current data suggest that apoptosis controls neutrophil numbers in tissues. We analyzed roles for and the sites of action for the cAMP-dependent protein kinases (cAPKs) in apoptosis induced in human neutrophils by in vitro storage, cycloheximide (CHX) exposure, and anti-Fas exposure. Treatment with 8-chlorophenylthio-cAMP (8-CPT-cAMP) prolonged the time required for 50% of the cells to exhibit apoptotic morphology (t 50) from 16.3 to 41.8 h (in vitro culture), from 2.4 to 7.8 h (CHX), and from 4.8 to 6.5 h (anti-Fas). CHX ± 8-CPT-cAMP did not significantly alter resting intracellular calcium levels and H-89, a selective inhibitor of cAPK, had …