Cell and Developmental Biology Commons^™

Construction Of Rhas And Rhar Overexpression Plasmids And The Effects Of Crp Mutants On E.Coli L-Rhamnose Gene Expression, Amber Bromenshenkel, Susan Egan

Honors Capstones

Capstone submitted as a graduation requirement for the BSU Honors Program.

The Role Of The Arabidopsis Phosphatidylinositol 3-Kinase In Vacuolar Protein Transport, Christopher A. Jones

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

In all eukaryotic organisms vesicle-mediated protein transport is crucial for maintaining cellular homeostasis. Over the past decade significant strides have been made in elucidating the molecular mechanisms governing this complex process. Both genetic and biochemical studies have demonstrated the unequivocal involvement of class III phospatidylinositol 3-kinases (PtdIns 3-kinases) in regulating vesicle transport. While PtdIns 3-kinases have been well studied in both yeast and mammalian systems, the role of these enzymes in plants is poorly understood. In the present study, we demonstrate that the Arabidopsis Ptdlns 3-kinase is localized to the cytoplasm, and perhaps the Golgi apparatus. Moreover, we show that …

Comparison Of Tnf-A Levels Produced By Macrophages Treated With Brucella Abortus And Escherichia Coli Lps, Tajie Harris

Honors Capstones

Capstone submitted as a graduation requirement for the BSU Honors Program.

Determination Of Nuclear Factor-Kappa B Activation In Cultured Renal Epithelial Cells And Cardiac Myocytes Exposed To Cocaine And Morphine, David Angeles Tiangco

Biological Sciences Theses & Dissertations

Nuclear factor-kappa B (NF-kB) is an important transcription factor that participates in the genetic regulation of inflammation in many tissues. Cocaine and heroin are drugs of abuse associated with renal and cardiac pathology. The purpose of this study was to test the hypothesis that exposure to cocaine or morphine (a metabolite of heroin) would activate NF-kB in renal epithelial cells (COS-7) and cardiac myocytes (H9c2) grown in culture. COS-7 and H9c2 cells were co-transfected with an experimental reporter specific for NF-kB activation and a control reporter with constitutive activity. A dual-luciferase assay was used to determine levels of NF-kB activation …

A Cycle Of Vam7p Release From And Ptdins 3-P–Dependent Rebinding To The Yeast Vacuole Is Required For Homotypic Vacuole Fusion, Christine Boeddinghaus, Alexey J. Merz, Rico Laage, Christian Ungermann

Dartmouth Scholarship

Vacuole fusion requires a coordinated cascade of priming, docking, and fusion. SNARE proteins have been implicated in the fusion itself, although their precise role in the cascade remains unclear. We now report that the vacuolar SNAP-23 homologue Vam7p is a mobile element of the SNARE complex, which moves from an initial association with the cis-SNARE complex via a soluble intermediate to the docking site. Soluble Vam7p is specifically recruited to vacuoles and can rescue a fusion reaction poisoned with antibodies to Vam7p. Both the recombinant Vam7p PX domain and a FYVE domain construct of human Hrs block the recruitment of …

Prostate Specific Membrane Antigen (Psma): Immunoassay Development And Characterization Of Transcriptional Regulation, Zhen Xiao

Theses and Dissertations in Biomedical Sciences

Prostate cancer (PCA) is the most common cancer and the second leading cause of death among American men. The high mortality is greatly attributed to the lack of early detection tools and effective treatment for metastasis and relapses. Biomarkers that can discriminate benign from malignant tumor and signal the development of androgen independent and metastatic tumor are needed. A biomarker designated prostate specific membrane antigen (PSMA) has the potential to fulfill this need. The objective of this study is to develop a clinically useful immunoassay for quantitation of serum PSMA and to study the molecular mechanism underlying the upregulation of …

Actin And Myosin Remodeling In The A7r5 Smooth Muscle Cell, Michael E. Fultz

Theses, Dissertations and Capstones

Active remodeling of the cytoskeleton has been proposed to contribute to the low energy cost and maintenance of the sustained contraction in smooth muscle. Using confocal microscopy and standard immunohistochemical techniques, direct observation of actin remodeling was studied in the contracting A7r5 cell in response to the diacylglycerol (DAG) analog phorbol 12, 13 dibutyrate (PDBu). -actin was shown to exist in the resting cell as parallel stress cables that extend across the cell. Stimulation by PDBu resulted in a sustained contraction that occurred in approximately eighty-five percent of the A7r5 cells. The initial contraction was not uniform, but primarily occurred …

Actin Dynamics Regulate Myosin Assembly In Muscle Cells, John Dylan Cook

Inquiry: The University of Arkansas Undergraduate Research Journal

During muscle cell (i.e. myocyte )development, organization of actin and myosin into the contractile unit (i.e. sarcomere) is required for proper muscle contraction. By disrupting the processes of muscle cell development, I am able to see which steps play important roles in proper maturation of myocytes. Elucidation of the key pathways in muscle development could lead to a better understanding of human cardiac hypertrophies and muscle myopathies. Since actin filament formation precedes myosin organization, I am using actin assembly inhibitors to determine if actin filaments are a necessary prerequisite for myosin organization. It is hypothesized that disruption of actin will …

Improved Energy Model For Membrane Electroporation In Biological Cells Subjected To Electrical Pulses, R. P. Joshi, Q. Hu, K. H. Schoenbach, H. P. Hjalmarson

Bioelectrics Publications

A self-consistent model analysis of electroporation in biological cells has been carried out based on an improved energy model. The simple energy model used in the literature is somewhat incorrect and unphysical for a variety of reasons. Our model for the pore formation energy E(r) includes a dependence on pore population and density. It also allows for variable surface tension, incorporates the effects of finite conductivity on the electrostatic correction term, and is dynamic in nature. Self-consistent calculations, based on a coupled scheme involving the Smoluchowski equation and the improved energy model, are presented. It is shown that E(r) becomes …

Theoretical Predictions Of Electromechanical Deformation Of Cells Subjected To High Voltages For Membrane Electroporation, R. P. Joshi, Q. Hu, K. H. Schoenbach, H. P. Hjalmarson

Bioelectrics Publications

An electromechanical analysis based on thin-shell theory is presented to analyze cell shape changes in response to external electric fields. This approach can be extended to include osmotic-pressure changes. Our calculations demonstrate that at large fields, the spherical cell geometry can be significantly modified, and even ellipsoidal forms would be inappropriate to account for the deformation. Values of the surface forces obtained from our calculations are in very good agreement with the 1–10 mN/m range for membrane rupture reported in the literature. The results, in keeping with reports in the literature, demonstrate that the final shape depends on membrane thickness. …

Apoptosis Pathways: Presence And Significance In Ejaculated Human Spermatozoa, Steven Lewis Taylor

Theses and Dissertations in Biomedical Sciences

Ejaculated sperm display markers that are indicative of apoptosis in somatic cells. The question remains as to whether sperm have operative apoptosis mechanisms. The aim of this research was to test the hypothesis that apoptosis markers in sperm and somatic cells are different.

Ejaculated human sperm from patients and donors were separated into high and low motility fractions using Percoll™ gradients. Contaminating cells were removed using anti-CD45 conjugated paramagnetic beads. Fractions were divided into groups: staurosporine, anti-Fas antibody, and hydrogen peroxide treated and control. Direct enzymatic measurement of caspase activity, flow cytometric evaluation of phosphatidylserine translocation, immunoblots, and immunocytochemistry were …

Dipyridamole Reduces Oxidative Stress In Human Coronary Artery Endothelial And Hela Cell Models, Matthew E. Giangrante

Seton Hall University Dissertations and Theses (ETDs)

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Functional Requirement Of Aquaporin-5 In Plasma Membranes Of Sweat Glands, Lene N. Nejsum, Tae-Hwan Kwon, Uffe B. Jensen, Ornella Fumagalli, Jørgen Frøkiaer, Carissa M. Krane, Anil G. Menon, Landon S. King, Peter C. Agre, Søren Nielsen

Biology Faculty Publications

The distribution and function of aquaporins (AQPs) have not previously been defined in sweat glands. In this study, AQP1, AQP3, and AQP5 mRNA were demonstrated in rat paw by reverse transcription (RT)–PCR, but AQP2 and AQP4 were not. AQP1, AQP3, and AQP5 protein were confirmed in these tissues by immunoblotting. AQP1 was identified in capillary endothelial cells by immunohistochemical labeling, but not in sweat glands or epidermis. Abundant AQP3 expression was seen in basal levels of epidermis, but not in sweat glands. AQP2 and AQP4 were not observed in either skin or sweat glands. Immunohistochemical labeling revealed abundant AQP5 in …

A Note On Cascade Climbing Of Migrating Goby And Shrimp Postlarvae In Two Maui Streams, M. Eric Benbow, Leslie Luchar Orzetti, Mollie D. Mcintosh, Albert J. Burky

Biology Faculty Publications

In this study, we documented cascade climbing rates of 133 and 230 postlarvae of Lentipes concolor (O‘opu alamo‘o) and Atyoida bisulcata (Opae kahaole), respectively, from two streams on the island of Maui, Hawaii. Climbing measurements and observations were made of postlarvae at the water-substrate interface in cascade habitats of constricted water flow. Both species were observed to move in short bursts of forward progression within or above the pulsing water-substrate interface. Goby postlarval climbing rates ranged from 0.04 – 1.50 cm s–1 and were slower than shrimp rates which ranged from 0.30 – 3.06 cm s–1. The high variability is …

Mechanism For Membrane Electroporation Irreversibility Under High-Intensity, Ultrashort Electrical Pulse Conditions, R. P. Joshi, K. H. Schoenbach

Bioelectrics Publications

An improved electroporation model is used to address membrane irreversibility under ultrashort electric pulse conditions. It is shown that membranes can survive a strong electric pulse and recover provided the pore distribution has a relatively large spread. If, however, the population consists predominantly of larger radii pores, then irreversibility can result. Physically, such a distribution could arise if pores at adjacent sites coalesce. The requirement of close proximity among the pore sites is more easily satisfied in smaller organelles than in outer cell membranes. Model predictions are in keeping with recent observations of cell damage to intracellular organelles (e.g., mitochondria), …

Analysis Of Protein Localization And Secretory Pathway Function Using The Yeast Saccharomyces Cerevisiae, Elizabeth Ann Vallen

Biology Faculty Works

The isolation and characterization of mutants has been crucial in understanding a number of processes in the field of cell biology. In this exercise, students examine the effects of mutations in the secretory pathway on protein localization. Yeast strains deficient for synthesis of histidinol dehydrogenase are transformed with a plasmid encoding a chimeric protein. The chimera contains a signal sequence fused to histidinol dehydrogenase. A strain with a defect in the translocation of secretory proteins into the endoplasmic reticulum (ER) accumulates sufficient histidinol dehydrogenase in the cytoplasm to grow on media lacking histidine. In contrast, yeast proficient for secretion, or …

Mutations In Sid2, A Novel Gene In Saccharomyces Cerevisiae, Cause Synthetic Lethality With Sic1 Deletion And May Cause A Defect During S Phase, Matthew D. Jacobson , '97, Claudia C. Muñoz , '99, Kirstin S. Knox , '99, Beth E. Williams , '01, Lenette L. Lu , '02, Frederick Cross , '78, Elizabeth Ann Vallen

Biology Faculty Works

SIC1 encodes a nonessential B-type cyclin/CDK inhibitor that functions at the G1/S transition and the exit from mitosis. To understand more completely the regulation of these transitions, Mutations causing synthetic lethality with sic1 Delta were isolated. In this screen, we identified a novel gene, SID2, which encodes an essential protein that appear., to be required for DNA replication or repair. sid2-1 sic1 Delta strains and sid2-21 temperature-sensitive strains arrest preanaphase as large-budded cells with a single nucleus, a short spindle, and an similar to 2C DNA content. RAD9, which is necessary for the DNA damage checkpoint, is required for the …

The Wings Of Bombyx Mori Develop From Larval Discs Exhibiting An Early Differentiated State: A Preliminary Report, Madhuri Kango-Singh, Amit Singh, K. P. Gopinathan

Biology Faculty Publications

Lepidopteran insects present a complex organization of appendages which develop by various mechanisms. In the mulberry silkworm, Bombyx mori, a pair of meso- and meta-thoracic discs located on either side in the larvae gives rise to the corresponding fore- and hind-wings of the adult. These discs do not experience massive cell rearrangements during metamorphosis and display the adult wing vein pattern. We have analysed wing development in B. mori by two approaches, viz., expression of patterning genes in larval wing discs, and regulatory capacities of larval discs following explantation or perturbation. Expression of Nubbin is seen all over the presumptive …

Salivary Acinar Cells From Aquaporin 5-Deficient Mice Have Decreased Membrane Water Permeability And Altered Cell Volume Regulation, Carissa M. Krane, James E. Melvin, Ha-Van Nguyen, Linda Richardson, Jennifer E. Towne, Thomas Doetschman, Anil G. Menon

Biology Faculty Publications

Aquaporins (AQPs) are channel proteins that regulate the movement of water through the plasma membrane of secretory and absorptive cells in response to osmotic gradients. In the salivary gland, AQP5 is the major aquaporin expressed on the apical membrane of acinar cells. Previous studies have shown that the volume of saliva secreted by AQP5-deficient mice is decreased, indicating a role for AQP5 in saliva secretion; however, the mechanism by which AQP5 regulates water transport in salivary acinar cells remains to be determined. Here we show that the decreased salivary flow rate and increased tonicity of the saliva secreted byAqp5 …

Characterization And Regulation Of Skn-Directed Autoimmune Skin Lesions, Pamela Jane Staton

Theses, Dissertations and Capstones

In the Skn model of autoimmunity, pathogenic T cells sensitized to Skn epidermal antigens (Skn-immune spleen cells or SIS cells) elicited disease in the form of skin lesions when adoptively transferred into shaved immunoincompetent syngeneic adult recipients (‘lesion-forming animals’) while concomitant injection of immunoregulatory normal spleen cells at twice the concentration (2 x normal spleen cells or 2XNS cells) of SIS cells significantly lessened lesion development (‘lesion-controlling animals’) by Day 7 post cell transfer. In a time-course RT-PCR cytokine analysis of skin from lesion-forming and lesion-controlling animals, IL-7 mRNA was significantly elevated in lesion-controlling skin on Day 7 while no …

Concentration Of Specific Biogenic Amines In Ventricular Csf Of Type A And B Parkinson's Disease Patients On Sinemet, Justin Robert Ahlman

Theses Digitization Project

No abstract provided.

A Role For Actin, Cdc1p, And Myo2p In The Inheritance Of Late Golgi Elements In Saccharomyces Cerevisiae, Olivia W. Rossanese, Catherine A. Reinke, Brooke J. Bevis, Adam T. Hammond, Irina B. Sears, James O'Connor, Benjamin S. Glick

Faculty Publications

In Saccharomyces cerevisiae, Golgi elements are present in the bud very early in the cell cycle. We have analyzed this Golgi inheritance process using fluorescence microscopy and genetics. In rapidly growing cells, late Golgi elements show an actin-dependent concentration at sites of polarized growth. Late Golgi elements are apparently transported into the bud along actin cables and are also retained in the bud by a mechanism that may involve actin. A visual screen for mutants defective in the inheritance of late Golgi elements yielded multiple alleles of CDC1. Mutations in CDC1 severely depolarize the actin cytoskeleton, and these …

Self-Consistent Simulations Of Electroporation Dynamics In Biological Cells Subjected To Ultrashort Electrical Pulses, R. P. Joshi, Q. Hu, R. Aly, K. H. Schoenbach, H. P. Hjalmarson

Bioelectrics Publications

The temporal dynamics of electroporation of cells subjected to ultrashort voltage pulses are studied based on a coupled scheme involving the Laplace, Nernst-Plank, and Smoluchowski equations. A pore radius dependent energy barrier for ionic transport, accounts for cellular variations. It is shown that a finite time delay exists in pore formation, and leads to a transient overshoot of the transmembrane potential V_mem beyond 1.0 V. Pore resealing is shown to consist of an initial fast process, a 10⁻⁴s delay, followed by a much slower closing at a time constant of about 10 ⁻¹s. This establishes a …

Receptor Number And Caveolar Co-Localization Determine Receptor Coupling Efficiency To Adenylyl Cyclase, Rennolds S. Ostrom, Caroline Gregorian, Ryan M. Drenan, Yang Xiang, John W. Regan, Paul A. Insel

Pharmacy Faculty Articles and Research

Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to β1-adrenergic receptor (β1AR)-selective activation 3.7-fold, to β2AR-selective activation only 1.6-fold and to prostaglandin E2 (PGE2) not at all. Therefore, the rank order of efficacy in …

Aquaporin 5-Deficient Mouse Lungs Are Hyperresponsive To Cholinergic Stimulation, Carissa M. Krane, Christopher N. Fortner, Arthur R. Hand, Dennis W. Mcgraw, John N. Lorenz, Susan E. Wert, Jennifer E. Towne, Richard J. Paul, Jeffrey A. Whitsett, Anil G. Menon

Biology Faculty Publications

Although aquaporin 5 (AQP5) is the major water channel expressed in alveolar type I cells in the lung, its actual role in the lung is a matter of considerable speculation. By using immunohistochemical staining, we show that AQP5 expression in mouse lung is not restricted to type I cells, but is also detected in alveolar type II cells, and in tracheal and bronchial epithelium. Aqp5 knockout (Aqp5−/−) mice were used to analyze AQP5 function in pulmonary physiology. Compared with Aqp5+/+ mice, Aqp5−/− mice show a significantly increased concentration-dependent bronchoconstriction to intravenously administered Ach, as shown by …

Hawaiian Freshwater Polychaeta: A Potentially Substantial Trophic Component Of Stream Depositional Habitats, M. Eric Benbow, Albert J. Burky, Carl M. Way

Biology Faculty Publications

In this paper we report the widespread occurrence of large annelids (Polychaeta: Nereididae) in Hawaiian stream depositional habitats, drawing attention to the lack of knowledge of Hawaiian stream energetics. Specimens of Namalycastis sp. were collected from five Hawaiian Islands from May–July 1995 and from the island of Maui from July–December 1999 and April–July 2000. Most specimens collected were N. hawaiiensis, while two specimens of N. abiuma were collected from a single site on the island of Molokai. Specimens were collected from elevations ranging from 3–240 m; several collections were from sites above at least one major (> 15 m …

Chondrocranium And Visceral Skeleton Of Atelopus Tricolor And Atelophryniscus Chrysophorus, E O. Lavilla, Rafael O. De Sá

Biology Faculty Publications

Atelopus tricolor and Atelophryniscus chrysophorus have gastromyzophorous tadpoles. Aside from specific differences, the larval chondrocrania and visceral skeletons show several shared character states, including an almost quadrangular, open chondrocranium , cornua trabeculae with expanded tips, cartilago suprarostralis as a single element, a posteriorly projected arcus subocularis quadrati, a closed muscular tunnel, and a simplified branchial basket, with only three pairs of ceratobranchiales. They are compared with diverse character states of suctorial larvae considering them as causally related to the stream life adaptations.

Characterization Of The Biological Activities Of Recombinant Fusion Protein Green Fluorescent Protein/Human Zona Pellucida Protein 3 (Gfp/Hzp3), Zhiyong Lin

Theses and Dissertations in Biomedical Sciences

Despite numerous reports indicating the successful production of bioactive recombinant ZP3, no report has shown the rhZP3 having direct binding activity with human sperm. Recombinant ZP3 generated from our previous study displayed binding activity with human sperm through indirect evidence from hemizona assay (HZA).

This present study focused on the production of recombinant ZP3 with direct binding activity with human sperm. Through the application of a pEGFP expression vector, fusion protein GFP/ZP3 was successfully generated and expressed. The expression of GFP/ZP3 was evidenced by RT-PCR and western blot. The fusion protein was partially purified by Ni-NTA affinity column from cell …

Do Timing And Pattern Of Myogenesis Correlate With Life History Mode In Anurans?, Matthew T. Smetanick, Rafael O. De Sá, Gary P. Radice

Biology Faculty Publications

The timing and pattern of myogenesis varies among anurans that have been studied and the different pat- terns may provide useful phylogenetic information. Specific myogenic markers have been described (Muntz, 1975; Kielbowna, 1981; Boudjelida & Muntz, 1987; Radice et al., 1989) and they can provide infor- mation on evolutionary changes for closely related lineages within a clade. For example, we previously com- pared first appearance of a muscle-specific protein, first twitch of axial muscle, onset of multinucleation within axial myotome, and first heartbeat in two pipid genera (Smetanick et al., 1999). We found that although the timing of myogenesis differed, …

Fast Transport Of Neurofilament Protein Along Microtubules In Squid Axoplasm, Veena Prahlad, Brian T. Helfand, George M. Langford, Ron D. Vale, Robert D. Goldman

Dartmouth Scholarship

Using squid axoplasm as a model system, we have visualized the fast transport of non-filamentous neurofilament protein particles along axonal microtubules. This transport occurs at speeds of 0.5-1.0 microm/second and the majority of neurofilament particles stain with kinesin antibody. These observations demonstrate, for the first time, that fast (0.5-1.0 microm/second) transport of neurofilament proteins occurs along microtubules. In addition, our studies suggest that neurofilament protein can be transported as non-membrane bound, nonfilamentous subunits along axons, and that the transport is kinesin-dependent. Microtubule-based fast transport might therefore provide a mechanism for the distribution and turnover of neurofilament, and perhaps other cytoskeletal …