Cell and Developmental Biology Commons^™

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 …

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 …

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 …

Ube1l Is A Retinoid Target That Triggers Pml/Rarα Degradation And Apoptosis In Acute Promyelocytic Leukemia, Sutisak Kitareewan, Ian Pitha-Rowe, David Sekula, Christopher H. Lowrey, Michael J. Nemeth, Todd R. Golub, Sarah J. Freemantle, Ethan Dmitrovsky

Dartmouth Scholarship

All-trans-retinoic acid (RA) treatment induces remissions in acute promyelocytic leukemia (APL) cases expressing the t(15;17) product, promyelocytic leukemia (PML)/RA receptor α (RARα). Microarray analyses previously revealed induction of UBE1L (ubiquitin-activating enzyme E1-like) after RA treatment of NB4 APL cells. We report here that this occurs within 3 h in RA-sensitive but not RA-resistant APL cells, implicating UBE1L as a direct retinoid target. A 1.3-kb fragment of the UBE1L promoter was capable of mediating transcriptional response to RA in a retinoid receptor-selective manner. PML/RARα, a repressor of RA target genes, abolished this UBE1L promoter activity. A hallmark of …

Osmosensitive Release Of Neurotransmitter Amino Acids: Relevance And Mechanisms, Herminia Pasantes-Morales, Rodrigo Franco, Lenin Ochoa, Benito Ordaz

School of Veterinary and Biomedical Sciences: Faculty Publications

Hyposmolarity activates amino acid efflux as part of the corrective volume process in a variety of cells. This review discusses the mechanism of amino acid release in brain cells preparations. Results present evidence of substantial differences between the efflux of taurine and that of GABA and glu-tamate, which besides a possible role as osmolytes, have a main function as synaptic transmitters. The differences found concern the efflux time course, the sensitivity to Cl^– channel blockers, the modulation by tyrosine kinases, the influence of PKC and the effect of cytoskeleton disruptive agents. While taurine efflux features fit well with the …

A Mechanistic Study Of Myoglobin Nephrotoxicity, Jennifer L. Minigh

Theses, Dissertations and Capstones

Myoglobin is an endogenous protein that can become nephrotoxic under certain conditions such as crush injuries, drug overdose, and seizures where prolonged contraction of muscle leads to cell death and leakage of myoglobin. The mechanism of myoglobin-induced nephrotoxicity is not fully understood. The purpose of this study was to characterize the sequence and mechanistic events associated with the in vitro toxicity of myoglobin in renal cortical slices. Renal tissue was isolated from Fischer 344 rats. Slices of renal cortex were prepared by freehand. These slices were then incubated for 60-180 minutes with myoglobin (0-12 mg/mL) pretreated with 4 mM ascorbic …

Pkcα Translocation And Actin Remodeling In Contracting A7r5 Smooth Muscle Cells, Chenwei Li

Theses, Dissertations and Capstones

Recent research indicates that protein kinase C (PKC) plays an important role in smooth muscle contraction. Because PKC activation and specificity of substrate phosphorylation is believed to be associated with the relocalization of the enzyme to specific cell sites, we first investigated the subcellular translocation of PKCα in A7r5 smooth muscle cells by confocal microscopy through use of standard immunohistologic staining and PKCα - enhanced green fluorescent protein (PKCα -EGFP) fusion protein expression. PKCα was diffusely distributed throughout the cytosol in the unstimulated A7r5 cell. Upon stimulation with phorbol 12, 13 dibutyrate (PDBu), PKCα was translocated primarily to either the …

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 …

The Osteopetrotic Mutation Toothless (Tl) Is A Loss-Of-Function Frameshift Mutation In The Rat Csf1 Gene: Evidence Of A Crucial Role For Csf-1 In Osteoclastogenesis And Endochondral Ossification, Liesbeth Van Wesenbeeck, Paul R. Odgren, Carole A. Mackay, Marina D'Angelo, Fayez F. Safadi, Steven N. Popoff, Wim Van Hul, Sandy C. Marks Jr.

PCOM Scholarly Works

The toothless (tl) mutation in the rat is a naturally occurring, autosomal recessive mutation resulting in a profound deficiency of bone-resorbing osteoclasts and peritoneal macrophages. The failure to resorb bone produces severe, unrelenting osteopetrosis, with a highly sclerotic skeleton, lack of marrow spaces, failure of tooth eruption, and other pathologies. Injections of CSF-1 improve some, but not all, of these. In this report we have used polymorphism mapping, sequencing, and expression studies to identify the genetic lesion in the tl rat. We found a 10-base insertion near the beginning of the open reading of the Csf1 gene that yields a …

Diverse Roles For The Eph Family Of Receptor Tyrosine Kinases In Carcinogenesis, Masaru Nakamoto, Andrew Bergeman

Biology Faculty Publications

The Eph family of receptor tyrosine kinases and their cell-presented ligands, the ephrins, are frequently overexpressed in a wide variety of cancers, including breast, small-cell lung and gastrointestinal cancers, melanomas, and neuroblastomas. In particular, one Eph family member, EphA2, is overexpressed in many cancers, including 40% of breast cancers. EphA2 can also transform breast epithelial cells in vitro to display properties commonly associated with the development of metastasis. Remarkably, the oncogenic properties of EphA2 contravene traditional dogma with regard to the oncogenic properties of a growth factor and its receptor tyrosine kinase: while stimulation of EphA2 by its ligand (ephrin-A1) …

Domain-Specific Olivocerebellar Projection Regulated By The Epha-Ephrin-A Interaction, Kazuhiko Nishida, John Flanagan, Masaru Nakamoto

Biology Faculty Publications

Neural maps in the vertebrate central nervous system often show discontinuously segregated, domain-to-domain patterns. However, the molecular mechanism that establishes such maps is not well understood. Here we show that in the chicken olivocerebellar system, EphA receptors and ephrin-As are expressed with distinct levels and combinations in mapping domains. When ephrin-A2 is retrovirally overexpressed in the cerebellum, the olivocerebellar map is disrupted, excluding axons with high receptor activity from ectopic expression domains. Conversely, overexpression of a truncated EphA3 receptor in the cerebellum reduces endogenous ligand activity to undetectable levels and causes aberrant mapping, with high receptor axons invading high ligand …

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. …

Mechanisms Counteracting Swelling In Brain Cells During Hyponatremia, Herminia Pasantes-Morales, Rodrigo Franco, Benito Ordaz, Lenin D. Ochoa

School of Veterinary and Biomedical Sciences: Faculty Publications

Water gain in the brain consequent to hyponatremia is counteracted by mechanisms that initially include a compensatory displacement of liquid from the interstitial space to cerebrospinal fluid and systemic circulation and subsequently an active reduction in cell water accomplished by extrusion of intracellular osmolytes to reach osmotic equilibrium. Potassium (K⁺), chloride (Cl^–), amino acids, polyalcohols, and methylamines all contribute to volume regulation, with a major contribution of ions at the early phase and of organic osmolytes at the late phase of the regulatory process. Experimental models in vitro show that osmolyte fluxes occur via leak pathways …

In Vivo Effect Of Leukemia Inhibitory Factor (Lif) And An Anti-Lif Polyclonal Antibody On Murine Embryo And Fetal Development Following Exposure At The Time Of Transcervical Blastocyst Transfer, Michael H. Mitchell, R. James Swanson, Sergio Oehninger

Biological Sciences Faculty Publications

Leukemia inhibitory factor (LIF) enhances in vitro murine preimplantation development in a time- and dose-dependent fashion. Knockout experiments have demonstrated that endometrial LIF is essential for in vivo murine implantation. We assessed the impact of LIF and an anti-LIF polyclonal antibody (pab) on in vivo development and developed a novel and successful nonsurgical method of embryo transfer for this species, a transcervical blastocyst transfer technique. The objectives of this study were to evaluate the effects of LIF and the anti-LIF pab on 1) implantation, resorption, pregnancy, and viability rates and 2) the overall structural and skeletal development. Two-cell embryos 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 …

16s Ribosomal Dna Sequencing Confirms The Synonymy Of Vibrio Harveyi And V. Carchariae, Eric J. Gauger, Marta Gomez-Chiarri

Fisheries, Animal and Veterinary Sciences Faculty Publications

Seventeen bacterial strains previously identified as Vibrio harveyi (Baumann et al. 1981) or V. carchariae (Grimes et al. 1984) and the type strains of V. harveyi, V. carchariae and V. campbellii were analyzed by 16S ribosomal DNA (rDNA) sequencing. Four clusters were identified in a phylogenetic analysis performed by comparing a 746 base pair fragment of the 16S rDNA and previously published sequences of other closely related Vibrio species. The type strains of V. harveyi and V. carchariae and about half of the strains identified as V. harveyi or V. carchariae formed a single, wellsupported cluster designed as ‘bona …

Preparation Of Novel Hydroxyethyl Amine Isosteres As Potential Cathepsin D Inhibitors, Rose M. Mcconnell, Walter E. Godwin, Amy Stefan, Crystal Newton, Nikki Herring, Crissy Goss

Journal of the Arkansas Academy of Science

Cathepsin D is a lysosomal aspartic protease found in all mammalian cells and is considered to be one of the main catabolic proteinases. Cathepsin D has been suggested to play a role in the metastatic potential of several types of cancer. A high activated cathepsin D level in breast tumor tissue has been associated with an increased incidence of relapse and metastasis. High levels of active cathepsin D have also been found in colon cancer, prostate cancer, uterine cancer, and ovarian cancer. Hydroxyethyl isosteres with cyclic tertiary amine have proven to be clinically useful as inhibitors of aspartyl proteases similar …

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), …

Small Glutamine-Rich Protein/Viral Protein U–Binding Protein Is A Novel Cochaperone That Affects Heat Shock Protein 70 Activity, Peter C. Angeletti, Doriann Walker, Antonito T. Panganiban

Nebraska Center for Virology: Faculty Publications

Molecular chaperone complexes containing heat shock protein (Hsp) 70 and Hsp90 are regulated by cochaperones, including a subclass of regulators, such as Hsp70 interacting protein (Hip), C-terminus of Hsp70 interacting protein (CHIP), and Hsp70-Hsp90 organizing factor (Hop), that contain tetratricopeptide repeats (TPRs), where Hsp70 refers to Hsp70 and its nearly identical constitutive counterpart, Hsc70, together. These proteins interact with the Hsp70 to regulate adenosine triphosphatase (ATPase) and folding activities or to generate the chaperone complex. Here we provide evidence that small glutamine-rich protein/viral protein U–binding protein (SGT/UBP) is a cochaperone that negatively regulates Hsp70. By “Far-Western” and pull-down assays, SGT/UBP …

Influence Of Protein Tyrosine Kinases On Cell Volume Change-Induced Taurine Release, Herminia Pasantes-Morales, Rodrigo Franco

School of Veterinary and Biomedical Sciences: Faculty Publications

Taurine efflux occurs in association with cell swelling in both hyposmotic and isosmotic conditions and during cell shrinkage in apoptotic death. Release occurs through a leak pathway, is largely Ca²⁺-independent and is sensitive to Cl^– channel blockers. Taurine efflux elicited by hyposmolarity is reduced or suppressed by tyrosine kinase blockers and increased by tyrosine phosphatase inhibitors. The specific kinases involved are still unknown and may be different in the various cell types. Non-receptor and scr-related protein kinases have been identified in some cells as elements that directly phosphorylate the taurine efflux pathway. Possible tyrosine kinase targets are …

Methods And Compositions For Protection Against Bovine Viral Diseases, Subramaniam Srikumaran

School of Veterinary and Biomedical Sciences: Faculty Publications

The present invention relates to methods and compositions for eliciting an immune response against bovine viral epitopes. The methods comprise combining at least one heat shock protein with at least one bovine viral epitope to form a purified epitope/heat shock protein complex and administration of an immune system stimulating amount of the purified epitope/heat shock protein complex. The compositions comprise, a purified epitope/heat shock protein complex comprising at least one bovine viral epitope complexed with at least one heat shock protein, and a pharmaceutically acceptable carrier, diluent or excipient.

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 …

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 …

Manipulation Of Prenatal Hormones And Dietary Phytoestrogens During Adulthood Alter The Sexually Dimorphic Expression Of Visual Spatial Memory, Edwin D. Lephart, Trent D. Lund

Faculty Publications

In learning and memory tasks, requiring visual spatial memory (VSM), males exhibit higher performance levels compared to females (a difference attributed to sex steroid hormonal influences). Based upon the results from our companion investigation, this study examined the influence of prenatal sex steroid hormone manipulations on VSM in adulthood, as assessed in the radial arm maze. Additionally, the influence of dietary soy phytoestrogens (i.e., the presence of high or low estrogen-like compounds present in the animal's diet) on VSM was examined in combination with the prenatal hormonal manipulations. Results: Radial arm maze performance on a phytoestrogen-rich diet: 1) females treated …

Visual Spatial Memory Is Enhanced In Female Rats (But Inhibited In Males) By Dietary Soy Phytoestrogens, Edwin D. Lephart, Trent D. Lund, Timothy W. West, Lilyan Y. Tian, Lihong H. Bu, Daniel L. Simmons, Kenneth Setchell, Herman Adlercreutz

Faculty Publications

In learning and memory tasks, requiring visual spatial memory (VSM), males exhibit superior performance to females (a difference attributed to the hormonal influence of estrogen). This study examined the influence of phytoestrogens (estrogen-like plant compounds) on VSM, utilizing radial arm-maze methods to examine varying aspects of memory. Additionally, brain phytoestrogen, calbindin (CALB), and cyclooxygenase-2 (COX-2) levels were determined. Results: Female rats receiving lifelong exposure to a high-phytoestrogen containing diet (Phyto-600) acquired the maze faster than females fed a phytoestrogen-free diet (Phyto-free); in males the opposite diet effect was identified. In a separate experiment, at 80 days-of-age, animals fed the Phyto-600 …

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 …