Biochemistry Commons^™

Biochemistry Of Hemolysin Toxin Activation By Fatty Acylation: Characterization Of An Internal Protein Acyltransferase, Michael S. Trent

Electronic Theses and Dissertations

Hemolysin toxin produced and secreted by pathogenic Escherichia coli is one of a family of cytolytic, structurally homologous protein toxins known as RTX (repeats in toxin) toxins. RTX toxins are products of a gene cluster, CABD . The A gene product, nontoxic hemolysin (proHlyA) is made toxic by post-translational fatty acylation of two internal lysine residues. HlyC, C gene product, is essential for acylation, and acyl-acyl carrier protein (ACP) is the acyl donor. HlyB and HlyD are involved in secretion of the toxin. HlyC was thought to serve as an internal protein acyltransferase and remained uncharacterized until now. ProHlyA and …

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 …

Expression, Isolation And Purification Of Human Zona Pellucida Protein 3, Ting-Fung Chi

Theses and Dissertations in Biomedical Sciences

Mammalian fertilization involves interactions of sperm surface receptors with the ligands of the zona pellucida, an extracellular matrix surrounding the ovulated oocytes. In humans, the zona pellucida is composed of three major glycoproteins. One of them, ZP3, participates in the primary sperm binding and in the subsequent triggering of the spermatozoa's acrosome reaction. Studies on the role of this specific protein in the human fertilization process are hampered by the limited amount of available biologically functional proteins.

By use of a pcDNA 3.1(+) expression vector, a transfecting-vector was constructed containing a 1.3 kb histidine tagged hZP3 cDNA. This histidine tagged …

Characterization Of The Biological Functions Of Human Recombinant Zona Pellucida Protein 3, Yu Wen Juan

Theses and Dissertations in Biomedical Sciences

Recombinant human zona pellucida protein 3 (rhZP3), expressed. isolated and purified from PA-1 cells, is characterized for its biological activity and the role in the signal transduction pathway. Characterization of the biological activity of rhZP3 was detected by hemizona assay and immunofluorescence staining of acrosome reaction The results indicated that rhZP3 exhibited an inhibition in the binding assay (HZI 43.6 +/-3.3; n = 9; 30 ng/mL rhZP3) and induction of acrosome reaction (198.6% +/- 77.2% increase from baseline; n = 29; 30 ng/mL rhZP3). It was further confirmed by the transmission electron microscopy that there was no difference in morphology …

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, …

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 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 …

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 …

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 …

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 …

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 …

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 …

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 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% …

Development And Validation Of Flow-Injection (Continuous-Flow) Elisa Techniques., Declan Gerard Spillane

Theses

A flow-through enzyme-linked immunosorbent assay was developed based on affinity chromatography using the determination of ferritin in serum as a model system. In this method, samples and standards are introduced to separate columns containing immobilised anti-ferritin antibody, and antigen bound by solid-phase antibody is subsequently detected using an anti-ferritin-alkaline phosphatase conjugate. To detect immobilised label, p-nitrophenyl phosphate is added and product is developed in the column at room temperature. Following elution of product from the column, the absorbance is measured and the columns are regenerated using a low pH elution. The final developed system requires approximately 1.5 h for the …

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 …

Determination Of Trace Levels Of Lead In Whole Blood By Graphite Furnace Atomic Absorption Spectroscopy, Leslie A. Sombers

Dissertations, Theses, and Masters Projects

No abstract provided.

Identification Of An Amylase-Like Gene In Chironomus Riparius, Loriann Mahurter

Seton Hall University Dissertations and Theses (ETDs)

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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 …}