Cell and Developmental Biology Commons^™

Induction Of Apoptosis In Human Prostate Cancer Cells By Resveratrol, Gary Zulfikar Morris

Chemistry & Biochemistry Theses & Dissertations

Recently attention has been brought to trans-resveratrol's {TR) anticancer activity, as determined through a number of cultured cancer cell models. This activity was attributed to TR behaving as an estrogen, and the orientation of TR' s hydroxyl groups. Based on this work it was of interest to determine whether TR would also be toxic in prostate cancer cells; if toxic, did TR induce necrosis or apoptosis in the cells; was it toxic through hormone mediated pathways; and were TR's hydroxyl groups responsible for its biological activity. To this end, cellular viability was assessed in two different prostate cancer cell …

An Ultrastructural Survey Of The Nuchal Glands Of Rhabdophis Tigrinus (Serpentes: Colubridae), Kathleen A. Roberts

Biological Sciences Theses & Dissertations

Rhabdophis tigrinus, an Asian natricine snake, possesses a series of integumentary glands located in the nuchal skin. Previous studies indicated that the nuchal glands are of mesodermal origin, contain cardiac steroids analogous to bufotoxins found in the parotoid glands of toads, which are included in the snake's diet. The nuchal glands are defensive in function. Preliminary histological studies revealed differences in quality of the gland tissue compared to previous studies, and difference in fixation techniques were suspected. Seven specimens of R. tigrinus were fixed by perfusion using a conservative fixation process standard for electron microscopy. The nuchal gland series …

The Occurrence Of The Freshwater Clams, Musculium Partumeium (Say) And Pisidium Casertanum (Poli) (Bivalvia: Sphaeriidae), In The Hawaiian Islands, Albert J. Burky, Carl M. Way, Skippy Hau, M. Eric Benbow

Biology Faculty Publications

Sphaeriid clams were collected from an ancient, continuously cultivated taro pond complex at Ke‘anae Peninsula, Maui. Both species are known for adaptations for temporary pond habitats and could have been initially introduced by the earliest Hawaiian settlers transported with moist taro root stock from other areas of Oceania.

Intracellular Transport, Assembly, And Degradation Of Wild-Type And Disease-Linked Mutant Gap Junction Proteins, Judy K. Vanslyke, Suzanne M. Deschênes, Linda S. Musil

Biology Faculty Publications

More than 130 different mutations in the gap junction integral plasma membrane protein connexin32 (Cx32) have been linked to the human peripheral neuropathy X-linked Charcot–Marie–Tooth disease (CMTX). How these various mutants are processed by the cell and the mechanism(s) by which they cause CMTX are unknown. To address these issues, we have studied the intracellular transport, assembly, and degradation of three CMTX-linked Cx32 mutants stably expressed in PC12 cells. Each mutant had a distinct fate: E208K Cx32 appeared to be retained in the endoplasmic reticulum (ER), whereas both the E186K and R142W mutants were transported to perinuclear compartments from which …

The Docking Stage Of Yeast Vacuole Fusion Requires The Transfer Of Proteins From A Cis-Snare Complex To A Rab/Ypt Protein, Albert Price, Darren Seals, William Wickner, Christian Ungermann

Dartmouth Scholarship

The homotypic fusion of yeast vacuoles requires Sec18p (NSF)-driven priming to allow vacuole docking, but the mechanism that links priming and docking is unknown. We find that a large multisubunit protein called the Vam2/6p complex is bound to cis-paired SNAP receptors (SNAREs) on isolated vacuoles. This association of the Vam2/6p complex with the cis-SNARE complex is disrupted during priming. The Vam2/6p complex then binds to Ypt7p, a guanosine triphosphate binding protein of the Rab family, to initiate productive contact between vacuoles. Thus, cis-SNARE complexes can contain Rab/Ypt effectors, and these effectors can be mobilized by NSF/Sec18p-driven priming, allowing their direct …

Roles Of Hof1p, Bni1p, Bnr1p, And Myo1p In Cytokinesis In Saccharomyces Cerevisiae, Elizabeth Ann Vallen, J. Caviston, E. Bi

Biology Faculty Works

Cytokinesis in Saccharomyces cerevisiae occurs by the concerted action of the actomyosin system and septum formation. Here we report on the roles of HOF1,BNI1, and BNR1 in cytokinesis, focusing on Hof1p. Deletion of HOF1 causes a temperature-sensitive defect in septum formation. A Hof1p ring forms on the mother side of the bud neck in G2/M, followed by the formation of a daughter-side ring. Around telophase, Hof1p is phosphorylated and the double rings merge into a single ring that contracts slightly and may colocalize with the actomyosin structure. Upon septum formation, Hof1p splits into two rings, disappearing upon cell separation. Hof1p …

Whole Animal And Cellular Strategies In The Regulation Of Bone Mass In The Rat, Weidong Geng

Theses, Dissertations and Capstones

In order to determine the strategies in regulation of bone mass in the rat and better understand the pathophysiological mechanism of the postmenopausal osteoporosis, a series of studies were conducted at the whole animal and cellular level. At the whole animal level, female Sprague-Dawley rats were ovariectomized (Ovx) or sham-operated (control) at 18 weeks and the entire skeleton obtained at 24 weeks (baseline) or after an additional 31 day (28 week) interval on a normal (1.0%) or deficient (0.02%) calcium diet. Ovx rats on the normal calcium diet showed a 42% increase in whole body bone resorption and a 70% …

The Skn7 Response Regulator Of Saccharomyces Cerevisiae Interacts With Hsf1 In Vivo And Is Required For The Induction Of Heat Shock Genes By Oxidative Stress, Desmond C. Raitt, Anthony L. Johnson, Alexander M. Erkine, Kozo Makino, Brian Morgan, David S. Gross, Leland H. Johnston

Scholarship and Professional Work – COPHS

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein–protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and …

Cell Cycle-Dependent Binding Of Yeast Heat Shock Factor To Nucleosomes, Christina Bourgeois Venturi, Alexander M. Erkine, David S. Gross

Scholarship and Professional Work – COPHS

In the nucleus, transcription factors must contend with the presence of chromatin in order to gain access to their cognate regulatory sequences. As most nuclear DNA is assembled into nucleosomes, activators must either invade a stable, preassembled nucleosome or preempt the formation of nucleosomes on newly replicated DNA, which is transiently free of histones. We have investigated the mechanism by which heat shock factor (HSF) binds to target nucleosomal heat shock elements (HSEs), using as our model a dinucleosomal heat shock promoter (hsp82-ΔHSE1). We find that activated HSF cannot bind a stable, sequence-positioned nucleosome in G₁-arrested …

Cellular Release Of And Response To Atp As Key Determinants Of The Set-Point Of Signal Transduction Pathways, Rennolds S. Ostrom, Caroline Gregorian, Paul A. Insel

Pharmacy Faculty Articles and Research

The determinants of “basal” activity of signaling pathways regulating cellular responses are poorly defined. One possibility is that cells release factors to establish the set-point of such pathways. Here we show that treatment of Madin-Darby canine kidney cells with the nucleotidase apyrase decreases basal arachidonic acid release and cAMP production 30–40% and that inhibitors of P2Y receptor action also affect basal and forskolin-stimulated cAMP accumulation. Changing medium prominently increases extracellular levels of ATP in Madin-Darby canine kidney, COS-7, and HEK-293 cells. Mechanical stimulation of ATP release likely occurs in virtually every experimental protocol with cultured cells, implicating such release and …

Human Gonadotropin-Releasing Hormone (Hgnrh) Gene Expression And Hormone Regulation In Human Placental Jeg-3 Cells, Heming Zheng

Theses and Dissertations in Biomedical Sciences

Using the human placental choriocarcinoma JEG-3 cell line as an in vitro human placental model, I studied the mechanisms of the tissue-specific expression and steroid hormone regulation of the hGnRH gene in the human placenta. The results showed that all of the previously identified four elements are required for the full activity of the hGnRH upstream promoter in JEG-3 cells, while the element 4 (FP4, −987/−968) is the most important. Studies performed with 5′ end deletion of this region confirmed these observations. Further, supershift assay using Oct-1 antibody demonstrated the involvement of Oct-1 in the FP4 DNA-protein interaction in JEG-3 …

Telomerase Activity In Human Preimplantation Embryos, Diane Lynne Wright

Theses and Dissertations in Biomedical Sciences

Telomerase, a ribonucleoprotein, has been described as an essential component of highly proliferative cells, which stabilizes the telomeres and avoids cellular senescence. Telomerase has been identified in various embryonic cell stages, hematopoietic cells, and in >85% of tumor tissue biopsies analyzed. The ability to measure the potential to proliferate successfully could provide an objective measure of an embryo's quality. The objectives of this study were to modify the telomeric repeat amplification protocol (TRAP) assay system for increased sensitivity to allow detection of telomerase activity in the single cell of an oocyte and embryo, obtain telomerase activity levels for the oocyte …

The Kinesin-Related Protein, Hset, Opposes The Activity Of Eg5 And Cross-Links Microtubules In The Mammalian Mitotic Spindle, Vicki Mountain, Calvin Simerly, Louisa Howard, Asako Ando, Gerald Schatten, Duane A. Compton

Dartmouth Scholarship

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without …

Identification And Characterization Of Determinants Of Head And Neck Tumor Cell Invasion, Yangguan Wu

Theses and Dissertations in Biomedical Sciences

Head and neck squamous cell carcinoma (HNSCC) is a common malignant disease with poor prognosis. The majority of patients die from local invasion or lymphatic metastasis. The mechanism(s) underlining the invasiveness of HNSCC are poorly understood. Utilizing a panel of HNSCC cell lines previously established in our laboratory, we tested the application and relevance of the three-step hypothesis of tumor invasion to HNSCC and investigated the mechanism(s) pertaining to the regulation of each step in the invasive process. Data presented in this thesis demonstrated that tumor cell invasion in HNSCC is a complex process involving three repeated sequential steps: adhesion, …

Tissue-Specific Expression And Steroid Hormone Regulation Of Human Gonadotropin -Releasing Hormone (Hgnrh) Gene In Placental Cells (Jeg -3 Cells), Zhengguang Chen

Theses and Dissertations in Biomedical Sciences

Using JEG-3 cells as an in vitro model, I investigated the mechanisms behind the tissue-specific expression and steroid hormone regulation of the hGnRH gene in the human placenta. The hGnRH upstream promoter was found to be functionally active in JEG-3 cells. The DNA sequence responsible for functioning of the upstream promoter in JEG-3 cells is narrowed to a region between –1048 bp and –730 bp. This DNA fragment contains four elements, which can bind with nuclear extract from JEG-3 cells (but not from GT1-7 cells).

Estradiol (E2) represses the hGnRH upstream promoter activity in JEG-3 cells. This inhibition is receptor-mediated, …

The Timing And Pattern Of Myogenesis In Hymenochirus Boettgeri, Matthew T. Smetanick, Rafael O. De Sá

Biology Faculty Publications

Differences in the relative timing of homologous developmental events among closely related species, known as heterochronies, may provide valuable clues in understanding evolutionary relationships (McKinney, 1988; McNamara, 1995). Examining the timing of myogenic events is a relatively easy and effective method for finding heterochronic events. For example, whether muscle proteins and myofibrils appear before or after multinucleation can be determined through histological techniques (Kielbowna, 1981). Simple observations of live specimens can pinpoint functional landmarks such as first twitch (spontaneous or due to external stimuli) and first heartbeat.

Development Of The Suprarostral Plate Of Pipoid Frogs, Rafael O. De Sá, Charles C. Swart

Biology Faculty Publications

The rostral region of nonpipoid tadpoles has two sets of cartilages, the cornua trabeculae and the suprarostral cartilages, whereas the rostral region in pipoid larvae is occupied by a single and continuous cartilage, the suprarostral plate. The homology of this region in pipoid and nonpipoids tadpoles has been controversial. We examined the early formation and development of the suprarostral plate using serially cross-sectioned specimens of Rhinophrynus, Xenopus, and Hymenochirus. We conclude that the cartilaginous structures present in the rostral area of pipoid and nonpipoid larvae are homologous. Furthermore, we found two different developmental patterns among pipoid larvae. The chondrocranium …

Cell Cycle-Dependent Sequencing Of Cell Fate Decisions In Caenorhabditis Elegans Vulva Precursor Cells, Victor Ambros

Dartmouth Scholarship

In Caenorhabditis elegans, the fates of the six multipotent vulva precursor cells (VPCs) are specified by extracellular signals. One VPC expresses the primary (1°) fate in response to a Ras-mediated inductive signal from the gonad. The two VPCs flanking the 1° cell each express secondary (2°) fates in response to lin-12-mediated lateral signaling. The remaining three VPCs each adopt the non- vulval tertiary (3°) fate. Here I describe experiments examining how the selection of these vulval fates is affected by cell cycle arrest and cell cycle-restricted lin-12 activity. The results suggest that lin-12 participates in two

INTRODUCTION

Cell-cell signaling is …

Insulinlike Growth Factor 1- And 2-Augmented Collagen Gel Repair Of Facial Osseous Defects, James S. Toung, Roy C. Ogle, Raymond F. Morgan, William H. Lindsey

School of Medical Diagnostics & Translational Sciences Publications

BACKGROUND: Defects of the facial bone structure are common problems for the facial plastic surgeon. Native type 1 collagen gels (T1CGs) have been shown to mediate repair of facial critical-size defects in rat models.

OBJECTIVE: To evaluate the efficacy of T1CG augmented with insulinlike growth factor (IGF) 1, IGF-2, and a combination of IGF-1 and IGF-2 on the repair of facial critical-size defects in a rodent model.

METHODS: Twenty-four retired male breeder Sprague-Dawley rats were divided into 4 groups of 6 animals. Facial critical-size defects were created by removing the nasalis bones with a bone-cutting drill. Defects were treated with …

Interaction Of Laminin Beta2 With Eps8 And Desmin In Muscle Cells, Luke Xueliang Cui

Theses, Dissertations and Capstones

Laminin β2 is localized to the synaptic basal lamina (BL) in muscle and glomerular BL in kidney. To find interacting proteins, a mouse kidney cDNA library was screened with domain I of rat laminin β2 using the yeast two-hybrid system. Fifteen positive clones were found. The DNA sequence of three each of these clones corresponded to the phosphoprotein Eps8 (epidermal growth factor receptor pathway substrate clone 8, which is tyrosine phosphorylated after EGF stimulation) and the intermediate filament (IF) protein keratin. To determine if this interaction occurred in muscle cells, C2 cells transfected with the rat …

Cloning And Sequencing Of A Cellobiohydrolase Gene From Trichoderma Harzianum Fp108, Patrick Guilfoile, Ron Burns, Zu-Yi Gu, Matt Amundson, Fu-Hsian Chang

Journal of the Minnesota Academy of Science

A cbh1 cellobiohydrolase gene was cloned and sequenced from the fungus Trichoderma harzianum FP108. The cloning was performed by PCR amplification of T. harzianum genomic DNA, using PCR primers whose sequence was based on the cbh1 gene from Trichoderma reesei. The 3' end of the gene was isolated by inverse PCR; attempts to clone regions upstream of the 5' end of the gene were unsuccessful. Sequence comparisons suggest that this gene is closely related to cbb1 genes from other Trichoderma species. In particular, all catalytically important amino acids in the protein sequence deduced from the T harzianum cbb1 gene are …

Cooperative Binding Of Heat Shock Factor To The Yeast Hsp82 Promoter In Vivo And In Vitro, Alexander M. Erkine, Serena F. Magrogan, Edward A. Sekinger, David S. Gross

Scholarship and Professional Work – COPHS

revious work has shown that heat shock factor (HSF) plays a central role in remodeling the chromatin structure of the yeastHSP82 promoter via constitutive interactions with its high-affinity binding site, heat shock element 1 (HSE1). The HSF-HSE1 interaction is also critical for stimulating both basal (noninduced) and induced transcription. By contrast, the function of the adjacent, inducibly occupied HSE2 and -3 is unknown. In this study, we examined the consequences of mutations in HSE1, HSE2, and HSE3 on HSF binding and transactivation. We provide evidence that in vivo, HSF binds to these three sites cooperatively. This cooperativity is seen …

Antibodies To Surface Igm Can Accelerate Apoptosis Of Mature B-Lymphocytes At Sub - Stimulatory Concentrations, Erica Anderson-Nissen, Robert F. Ashman

Journal of the Minnesota Academy of Science

Antibody to B-cell surface immunoglobulin D (IgD) or surface IgM results in crosslinking of Ig molecules and signal transduction. The function of these surface immunoglobulins has traditionally been investigated by extensive crosslinking experiments and interest has been focused on activation assays. We investigated the effects on apoptosis of culture with anti-(mathematical symbol) antibody (anti-(mathematical symbol)) concentrations ranging from 0.001 (mathematical symbol) mL-1 to 50 (mathematical symbol)g mL-1. Previous experiments have shown that weak dose anti-(mathematical symbol) antibody (anti-(mathematical symbol)) increases mature B-cell apoptosis at both 16- and 64-hour time points, while greater dose anti-(mathematical symbol) results in cell cycle entry …

Inhibition Of Cpla2-Mediated Arachidonic Acid Release By Cyclic Amp Defines A Negative Feedback Loop For P2y-Receptor Activation In Mdck-D1 Cells, Mingzhao Xing, Steven Post, Rennolds S. Ostrom, Michael Samardzija, Paul A. Insel

Pharmacy Faculty Articles and Research

In Madin-Darby canine kidney D1cells extracellular nucleotides activate P2Y receptors that couple to several signal transduction pathways, including stimulation of multiple phospholipases and adenylyl cyclase. For one class of P2Y receptors, P2Y2 receptors, this stimulation of adenylyl cyclase and increase in cAMP occurs via the conversion of phospholipase A2 (PLA2)-generated arachidonic acid (AA) to prostaglandins (e.g. PGE2). These prostaglandins then stimulate adenylyl cyclase activity, presumably via activation of prostanoid receptors. In the current study we show that agents that increase cellular cAMP levels (including PGE2, forskolin, and the β-adrenergic agonist isoproterenol) can inhibit P2Y receptor-promoted AA release. The protein kinase …

Drosophila Fascin Mutants Are Rescued By Overexpression Of The Villin-Like Protein, Quail, Kelly Cant, Brenda A. Knowles, Shalina Mahajan-Miklos, Matthew Heintzelman, Lynn Cooley

Dartmouth Scholarship

Actin bundle assembly in specialized structures such as microvilli on intestinal epithelia and Drosophila bristles requires two actin bundling proteins. In these systems, the distinct biochemical properties and temporal localization of actin bundling proteins suggest that these proteins are not redundant. During Drosophila oogenesis, the formation of cytoplasmic actin bundles in nurse cells requires two actin bundling proteins, fascin encoded by the singed gene and a villin-like protein encoded by the quail gene. singed and quail mutations are fully recessive and each mutation disrupts nurse cell cytoplasmic actin bundle formation. We used P-element mediated germline transformation to overexpress quail in …

Transport Of Er Vesicles On Actin Filaments In Neurons By Myosin V, George M. Langford, Joel S. Tabb, Bradley J. Molyneaux, Darien L. Cohen, Sergei A. Kuznetsov

Biology - All Scholarship

Axoplasmic organelles in the giant axon of the squid have been shown to move on both actin filaments and microtubules and to switch between actin filaments and microtubules during fast axonal transport. The objectives of this investigation were to identify the specific classes of axoplasmic organelles that move on actin filaments and the myosin motors involved. We developed a procedure to isolate endoplasmic reticulum (ER) from extruded axoplasm and to reconstitute its movement in vitro. The isolated ER vesicles moved on exogenous actin filaments adsorbed to coverslips in an ATP-dependent manner without the addition of soluble factors. Therefore myosin was …

Transport Of Er Vesicles On Actin Filaments In Neurons By Myosin V, Joel S. Tabb, Bradley J. Molyneaux, Darien L. Cohen, Sergei A. Kuznetsov, George M. Langford

Dartmouth Scholarship

Axoplasmic organelles in the giant axon of the squid have been shown to move on both actin filaments and microtubules and to switch between actin filaments and microtubules during fast axonal transport. The objectives of this investigation were to identify the specific classes of axoplasmic organelles that move on actin filaments and the myosin motors involved. We developed a procedure to isolate endoplasmic reticulum (ER) from extruded axoplasm and to reconstitute its movement in vitro. The isolated ER vesicles moved on exogenous actin filaments adsorbed to coverslips in an ATP-dependent manner without the addition of soluble factors. Therefore myosin was …

Transport Of Axl2p Depends On Erv14p, An Er–Vesicle Protein Related To The Drosophila Cornichon Gene Product, Jacqueline Powers, Charles Barlowe

Dartmouth Scholarship

COPII-coated ER-derived transport vesicles from Saccharomyces cerevisiae contain a distinct set of membrane-bound polypeptides. One of these polypeptides, termed Erv14p (ER-vesicle protein of 14 kD), corresponds to an open reading frame on yeast chromosome VII that is predicted to encode an integral membrane protein and shares sequence identity with the Drosophila cornichon gene product. Experiments with an epitope-tagged version of Erv14p indicate that this protein localizes to the ER and is selectively packaged into COPII-coated vesicles. Haploid cells that lack Erv14p are viable but display a modest defect in bud site selection because a transmembrane secretory protein, Axl2p, is not …

Efficacy Of AllowashTm Solution In Solubilization Of Bone Marrow, Alyce Marie Linthurst

Biological Sciences Theses & Dissertations

LifeNet currently cleans allograft bone utilizing Allowash^TM Technology, which encompasses the use of a closed continuous circulation chamber to remove bone marrow by flushing it with Allowash^TM Solution for a prescribed time and temperature after which, it is rinsed with various solutions to remove residual detergent that could be toxic to the recipient. A portion of this process employs Allowash^TM Solution to improve the solubilization and removal of protein and cellular elements in the red bone marrow. This study examines the effectiveness of Allowash^TM Solution in comparison to its component detergents Nonidet P40, Nonoxynol 9 and …

Sec35p, A Novel Peripheral Membrane Protein, Is Required For Er To Golgi Vesicle Docking, Susan M. Vanrheenen, Xiaochun Cao, Vladimir V. Lupashin, Charles Barlowe, M. Gerard Waters

Dartmouth Scholarship

SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (Wuestehube et al., 1996. Genetics. 142:393–406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target …