Medical Physiology Commons^™

Bitc Sensitizes Pancreatic Adenocarcinomas To Trail-Induced Apoptosis, Christina A. Wicker, Ravi P. Sahu, Kashmira Kulkarni-Datar, Sanjay K. Srivastava, Thomas L. Brown

Neuroscience, Cell Biology & Physiology Faculty Publications

Pancreatic adenocarcinoma is an aggressive cancer with a greater than 95% mortality rate and short survival after diagnosis. Chemotherapeutic resistance hinders successful treatment. This resistance is often associated with mutations in codon 12 of the K-Ras gene (K-Ras 12), which is present in over 90% of all pancreatic adenocarcinomas. Codon 12 mutations maintain Ras in a constitutively active state leading to continuous cellular proliferation. Our study determined if TRAIL resistance in pancreatic adenocarcinomas with K-Ras 12 mutations could be overcome by first sensitizing the cells with Benzyl isothiocyanate (BITC). BITC is a component of cruciferous vegetables and a cell cycle …

Gamma Motor Neurons Express Distinct Genetic Markers At Birth And Require Muscle Spindle-Derived Gdnf For Postnatal Survival, Neil A. Shneider, Meghan N. Brown, Courtney A. Smith, James Pickel, Francisco J. Alvarez

Neuroscience, Cell Biology & Physiology Faculty Publications

Background: Gamma motor neurons (γ-MNs) selectively innervate muscle spindle intrafusal fibers and regulate their sensitivity to stretch. They constitute a distinct subpopulation that differs in morphology, physiology and connectivity from α-MNs, which innervate extrafusal muscle fibers and exert force. The mechanisms that control the differentiation of functionally distinct fusimotor neurons are unknown. Progress on this question has been limited by the absence of molecular markers to specifically distinguish and manipulate γ-MNs. Recently, it was reported that early embryonic γ-MN precursors are dependent on GDNF. Using this knowledge we characterized genetic strategies to label developing γ-MNs based on GDNF receptor expression, …

Neuroendocrine Peptides Neuropeptide-Y (Npy) And Peptide-Yy (Pyy) Suppress Cl- Secretion And K+ Secretion In Guinea Pig Distal Colon Through Action At Y2-Receptors, Susan T. Halm, Jin Zhang, Dan R. Halm

Neuroscience, Cell Biology & Physiology Faculty Publications

Electrogenic Cl^– and K⁺ secretion in isolated mucosa from guinea pig distal colon measured as short-circuit current (I_sc) and transepithelial conductance (G_t) were stimulated by epinephrine (epi), prostaglandin-E₂ (PGE₂) and carbachol (CCh). neuropeptide-Y (NPY) and peptide-YY (PYY) inhibited by 60% Cl^– secretion activated by either PGE₂ or PGE₂+CCh with EC₅₀’s of 16nM and 6nM, respectively. Neither peptide markedly inhibited the transient component of the PGE₂+CCh response. Immunoreactivity (IR) for NPY was present in enteric ganglia and in proximity with crypts. Basolateral membranes of …

Viral Vectors In The Research Laboratory: Just How Safe Are They?, Dawn P. Wooley, Kimberly Kay Morris, Robert Mcrae, John C. Trefry

Neuroscience, Cell Biology & Physiology Faculty Publications

No abstract provided.

Anti-Tumor Effect Of Doxycycline On Glioblastoma Cells, Andrea Wang-Gillam, Eric Siegel, Debra A. Mayes, Laura F. Hutchins, Yi-Hong Zhou

Neuroscience, Cell Biology & Physiology Faculty Publications

AIM: Glioblastoma multiforme (GBM) is the most common primary brain tumor in humans, and it is highly invasive. Doxycycline, first identified as an antimicrobial agent, is a nonspecific inhibitor of matrix metalloproteinases (MMPs). Our objective was to investigate the anti-MMP effect of doxycycline at therapeutically acceptable levels on glioma cells in vitro.

METHODS: The MTT assay was used to determine the anti-proliferative effects of doxycycline. MMP2 activity and expression were determined by gelatinase zymography and real-time quantitative RT-PCR, respectively. Cell invasion was assessed by Matrigel invasion assay.

RESULTS: Doxycycline exerted mild anti-proliferative effects on all three glioma cell lines …

Retroviral Vector And Cell-Based Assay For Measuring The Mutation Rate Of Retroviruses Employing Same, Dawn P. Wooley, Kelly Jo Huang

Neuroscience, Cell Biology & Physiology Faculty Publications

Lentiviral-based retrovirus vectors and an in vivo mutation rate assay employing them. More particularly, an assay for directly determining the in vivo mutation rate of HIV-1.

Is Gene Therapy A Good Therapeutic Approach For Hiv-Positive Patients?, Jai G. Marathe, Dawn P. Wooley

Neuroscience, Cell Biology & Physiology Faculty Publications

Despite advances and options available in gene therapy for HIV-1 infection, its application in the clinical setting has been challenging. Although published data from HIV-1 clinical trials show safety and proof of principle for gene therapy, positive clinical outcomes for infected patients have yet to be demonstrated. The cause for this slow progress may arise from the fact that HIV is a complex multi-organ system infection. There is uncertainty regarding the types of cells to target by gene therapy and there are issues regarding insufficient transduction of cells and long-term expression. This paper discusses state-of-the-art molecular approaches against HIV-1 and …

Soluble Amyloid Oligomers Increase Bilayer Conductance By Altering Dielectric Structure, Yuri Sokolov, J. Ashot Kozak, Rakez Kayed, Alexandr Chanturiya, Charles G. Glabe, James E. Hall

Neuroscience, Cell Biology & Physiology Faculty Publications

The amyloid hypothesis of Alzheimer's toxicity has undergone a resurgence with increasing evidence that it is not amyloid fibrils but a smaller oligomeric species that produces the deleterious results. In this paper we address the mechanism of this toxicity. Only oligomers increase the conductance of lipid bilayers and patch-clamped mammalian cells, producing almost identical current-voltage curves in both preparations. Oligomers increase the conductance of the bare bilayer, the cation conductance induced by nonactin, and the anion conductance induced by tetraphenyl borate. Negative charge reduces the sensitivity of the membrane to amyloid, but cholesterol has little effect. In contrast, the area …

Combining An Autologous Peripheral Nervous System “Bridge” And Matrix Modification By Chondroitinase Allows Robust, Functional Regeneration Beyond A Hemisection Lesion Of The Adult Rat Spinal Cord, John D. Houle, Veronica J. Tom, Debra A. Mayes, Gail Wagoner, Napoleon Phillips, Jerry Silver

Neuroscience, Cell Biology & Physiology Faculty Publications

Chondroitinase-ABC (ChABC) was applied to a cervical level 5 (C5) dorsal quadrant aspiration cavity of the adult rat spinal cord to degrade the local accumulation of inhibitory chondroitin sulfate proteoglycans. The intent was to enhance the extension of regenerated axons from the distal end of a peripheral nerve (PN) graft back into the C5 spinal cord, having bypassed a hemisection lesion at C3. ChABC-treated rats showed (1) gradual improvement in the range of forelimb swing during locomotion, with some animals progressing to the point of raising their forelimb above the nose, (2) an enhanced ability to use the forelimb in …

Charge Screening By Internal Ph And Polyvalent Cations As A Mechanism For Activation, Inhibition, And Rundown Of Trpm7/Mic Channels, J. Ashot Kozak, Masayuki Matsushita, Angus C. Nairn, Michael D. Cahalan

Neuroscience, Cell Biology & Physiology Faculty Publications

The Mg²⁺-inhibited cation (MIC) current, believed to represent activity of TRPM7 channels, is found in lymphocytes and mast cells, cardiac and smooth muscle, and several other eukaryotic cell types. MIC current is activated during whole-cell dialysis with divalent-free internal solutions. Millimolar concentrations of intracellular Mg²⁺ (or other divalent metal cations) inhibit the channels in a voltage-independent manner. The nature of divalent inhibition and the mechanism of channel activation in an intact cell remain unknown. We show that the polyamines (spermine, spermidine, and putrescine) inhibit the MIC current, also in a voltage-independent manner, with a potency that parallels …

Resting Potential–Dependent Regulation Of The Voltage Sensitivity Of Sodium Channel Gating In Rat Skeletal Muscle In Vivo, Gregory N. Filatov, Martin J. Pinter, Mark M. Rich

Neuroscience, Cell Biology & Physiology Faculty Publications

Normal muscle has a resting potential of −85 mV, but in a number of situations there is depolarization of the resting potential that alters excitability. To better understand the effect of resting potential on muscle excitability we attempted to accurately simulate excitability at both normal and depolarized resting potentials. To accurately simulate excitability we found that it was necessary to include a resting potential–dependent shift in the voltage dependence of sodium channel activation and fast inactivation. We recorded sodium currents from muscle fibers in vivo and found that prolonged changes in holding potential cause shifts in the voltage dependence of …

Stim1, An Essential And Conserved Component Of Store-Operated Ca2+ Channel Function, Jack Roos, Paul J. Digregorio, Andriy V. Yeromin, Kari Ohlsen, Maria I. Lioudyno, Shenyuan L. Zhang, Olga Safrina, J. Ashot Kozak, Steven L. Wagner, Michael D. Cahalan, Gönül Veliçelebi, Kenneth A. Stauderman

Neuroscience, Cell Biology & Physiology Faculty Publications

Store-operated Ca²⁺ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca²⁺ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca²⁺ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca²⁺ release-activated Ca²⁺(CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity …

A Developmental Switch In The Response Of Drg Neurons To Ets Transcription Factor Signaling, Simon Hippenmeyer, Eline Vrieseling, Markus Sigrist, Thomas Portmann, Celia Laengle, David R. Ladle, Silvia Arber

Neuroscience, Cell Biology & Physiology Faculty Publications

Two ETS transcription factors of the Pea3 subfamily are induced in subpopulations of dorsal root ganglion (DRG) sensory and spinal motor neurons by target-derived factors. Their expression controls late aspects of neuronal differentiation such as target invasion and branching. Here, we show that the late onset of ETS gene expression is an essential requirement for normal sensory neuron differentiation. We provide genetic evidence in the mouse that precocious ETS expression in DRG sensory neurons perturbs axonal projections, the acquisition of terminal differentiation markers, and their dependence on neurotrophic support. Together, our findings indicate that DRG sensory neurons exhibit a temporal …

Distinct Properties Of Crac And Mic Channels In Rbl Cells, J. Ashot Kozak, Hubert H. Kerschbaum, Michael D. Cahalan

Neuroscience, Cell Biology & Physiology Faculty Publications

In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca²⁺ release–activated Ca²⁺ (CRAC) channels open in response to passive Ca²⁺ store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg²⁺ exposes an outwardly rectifying current (Mg²⁺-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg²⁺, spermine, and …

Single Channel Properties And Regulated Expression Of Ca2+ Release-Activated Ca2+ (Crac) Channels In Human T Cells, Alla F. Fomina, Christopher M. Fanger, J. Ashot Kozak, Michael D. Cahalan

Neuroscience, Cell Biology & Physiology Faculty Publications

Although the crucial role of Ca²⁺ influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca²⁺ channels in normal human T lymphocytes. The use of Na⁺ as the permeant ion in divalent-free solution permitted Ca²⁺ release-activated Ca²⁺ (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca²⁺ store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca²⁺ in …

A Ligand Binding Domain Mutation In The Mouse Glucocorticoid Receptor Functionally Links Chromatin Remodeling And Transcription Initiation, Lynn A. Sheldon, Catharine L. Smith, Jack E. Bodwell, Allan U. Munck, Gordon L. Hager

Dartmouth Scholarship

We utilized the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) in vivo to understand how the interaction of the glucocorticoid receptor (GR) with a nucleosome-assembled promoter allows access of factors required for the transition from a repressed promoter to a derepressed, transcriptionally competent promoter. A mutation (C644G) in the ligand binding domain (LBD) of the mouse GR has provided information regarding the steps required in the derepression/activation process and in the functional significance of the two major transcriptional activation domains, AF1 and AF2. The mutant GR activates transcription from a transiently transfected promoter that has a disordered nucleosomal …

Staphylococcal Enterotoxin B Primes Cytokine Secretion And Lytic Activity In Response To Native Bacterial Antigens, K. M. Mason, T. D. Dryden, Nancy J. Bigley, P. S. Fink

Neuroscience, Cell Biology & Physiology Faculty Publications

Superantigens stimulate T-lymphocyte proliferation and cytokine production, but the effects of superantigen exposure on cell function within a complex, highly regulated immune response remain to be determined. In this study, we demonstrate that superantigen exposure significantly alters the murine host response to bacterial antigens in an in vitro coculture system. Two days after exposure to the superantigen staphylococcal enterotoxin B, splenocytes cultured with Streptococcus mutans produced significantly greater amounts of gamma interferon (IFN-gamma) and interleukin-12 than did sham-injected controls. The majority of IFN-gamma production appeared to be CD8(+) T-cell derived since depletion of this cell type dramatically reduced the levels …

Identifying Swelling-Activated Channels From Ion Selectivity Patterns, Dan R. Halm

Neuroscience, Cell Biology & Physiology Faculty Publications

No abstract provided.

Subcellular Localization Of Mineralocorticoid Receptors In Living Cells: Effects Of Receptor Agonists And Antagonists, Géza Fejes-Tóth, David Pearce, Anikó Náray-Fejes-Tóth

Dartmouth Scholarship

Results on the subcellular localization of the mineralocorticoid receptor (MR) have been controversial. To determine the subcellular distribution and trafficking of the MR in living cells after binding of agonists and antagonists, we expressed a MR-green fluorescent protein (GFP) chimera in mammalian cells lacking endogenous MR. The GFP-tagged MR (GFP-MR) remained transcriptionally active, as determined in cotransfection experiments with the MR-responsive reporter, TAT3-LUC. The subcellular localization of GFP-MR was monitored by fluorescence time-lapse microscopy. In the absence of hormone, MR was present both in the cytoplasm and nucleus. Aldosterone induced a rapid nuclear accumulation of the MR. Aldosterone-bound GFP-MR was …

Direct Demonstration Of Retroviral Recombination In A Rhesus Monkey, Dawn P. Wooley, Randall A. Smith, Susan Czajak, Ronald C. Desrosiers

Neuroscience, Cell Biology & Physiology Faculty Publications

Recombination may be an important mechanism for increasing variation in retroviral populations. Retroviral recombination has been demonstrated in tissue culture systems by artificially creating doubly infected cells. Evidence for retroviral recombination in vivo is indirect and is based principally on the identification of apparently mosaic human immunodeficiency virus type 1 genomes from phylogenetic analyses of viral sequences. We infected a rhesus monkey with two different molecularly cloned strains of simian immunodeficiency virus. One strain of virus had a deletion in vpx and vpr, and the other strain had a deletion in nef. Each strain on its own induced low virus …

A Recombinant Inwardly Rectifying Potassium Channel Coupled To Gtp-Binding Proteins, Kim W. Chan, M. Noelle Langan, Jin Liang Sui, J. Ashot Kozak, Amanda Pabon, John A. A. Ladias, Diomedes E. Logothetis

Neuroscience, Cell Biology & Physiology Faculty Publications

GTP-binding (G) proteins have been shown to mediate activation of inwardly rectifying potassium (K+) channels in cardiac, neuronal and neuroendocrine cells. Here, we report functional expression of a recombinant inwardly rectifying channel which we call KGP (or hpKir3.4), to signify that it is K+ selective, G-protein-gated and isolated from human pancreas. KGP expression in Xenopus oocytes resulted in sizeable basal (or agonist-independent) currents while coexpression with a G-protein-linked receptor, yielded additional agonist-induced currents. Coexpression of KGP and hGIRK1 (a human brain homolog of GIRK1/Kir3.1) produced much larger basal currents than those observed with KGP or hGIRK1 alone, and upon coexpression …

Antisense Oligodeoxynucleotide Inhibition Of A Swelling-Activated Cation Channel In Osteoblast-Like Osteosarcoma Cells., Randall L. Duncan, Neil Kizer, Elizabeth L. Barry, Peter P A Friedman, Keith A. Hruska

Dartmouth Scholarship

By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following …

Effects Of Natural Sequence Variation On Recognition By Monoclonal Antibodies Neutralize Simian Immunodeficiency Virus Infectivity, Weon Sang Choi, Catherine Collignon, Clotilde Thiriart, Dawn P. Wooley, E. J. Scott, Karen A. Kent, Ronald C. Desrosiers

Neuroscience, Cell Biology & Physiology Faculty Publications

The determinants of immune recognition by five monoclonal antibodies (KK5, KK9, KK17, Senv7.1, and Senv101.1) that neutralize simian immunodeficiency virus infectivity were analyzed. These five neutralizing monoclonal antibodies were generated to native SIVmac251 envelope glycoprotein expressed by a vaccinia virus recombinant vector. All five recognize conformational or discontinuous epitopes and require native antigen for optimal recognition. These monoclonal antibodies also recognize SIVmac239 gp120, but they do not recognize gp120 of two natural variants of SIVmac239, 1-12 and 8-22, which evolved during the course of persistent infection in vivo (D.P.W. Burns and R.C. Desrosiers, J. Virol. 65:1843-1854, 1991). Recombinant viruses which …

High Rates Of Frameshift Mutations Within Homo-Oligomeric Runs During A Single Cycle Of Retroviral Replication, Dawn P. Wooley, H. M. Temin

Neuroscience, Cell Biology & Physiology Faculty Publications

Homo-oligomeric runs were inserted into a spleen necrosis virus-based retrovirus vector to determine the nature and rate of mutations within runs of 10 to 12 identical nucleotides during a single replication cycle. Clones of helper cells containing integrated copies of retroviral vectors were used to produce virus for infection of target (nonhelper) cells. Proviral sequences from target cell clones were compared with proviral sequences from helper cell clones to study mutations that occurred during a single cycle of replication. In addition to the internal region spanning the homo-oligomeric inserts, a naturally occurring run of 10 T's in the long terminal …

Simian Immunodeficiency Virus Mutants Resistant To Serum Neutralization Arise During Persistent Infection Of Rhesus Monkeys, Dawn P. Wooley, Catherine Collignon, Ronald C. Desrosiers

Neuroscience, Cell Biology & Physiology Faculty Publications

We previously described the pattern of sequence variation in gp120 following persistent infection of rhesus monkeys with the pathogenic simian immunodeficiency virus SIVmac239 molecular clone (D.P.W. Burns and R.C. Desrosiers, J. Virol. 65:1843, 1991). Sequence changes were confined largely to five variable regions (V1 to V5), four of which correspond to human immunodeficiency virus type 1 (HIV-1) gp120 variable regions. Remarkably, 182 of 186 nucleotide substitutions that were documented in these variable regions resulted in amino acid changes. This is an extremely nonrandom pattern, which suggests selective pressure driving amino acid changes in discrete variable domains. In the present study, …

Gelatinase Activity In The Mouse Uterus During Early Pregnancy, Carol A. Brenner, Richard R. Adler, Gary L. Nieder

Neuroscience, Cell Biology & Physiology Faculty Publications

No abstract provided.

Anion Permeation In An Apical Membrane Chloride Channel Of A Secretory Epithelial Cell, Dan R. Halm, Raymond A. Frizzell

Neuroscience, Cell Biology & Physiology Faculty Publications

Single channel currents though apical membrane Cl channels of the secretory epithelial cell line T84 were measured to determine the anionic selectivity and concentration dependence of permeation. The current-voltage relation was rectified with single channel conductance increasing at positive potentials. At 0 mV the single channel conductance was 41 ± 2 pS. Permeability, determined from reversal potentials, was optimal for anions with diameters between 0.4 and 0.5 nm. Anions of larger diameter had low permeability, consistent with a minimum pore diameter of 0.55 nm. Permeability for anions of similar size was largest for those ions with a more symmetrical charge …

Strain-Specific Neutralizing Determinant In The Transmembrane Protein Of Simian Immunodeficiency Virus, Toshiaki Kodama, Dawn P. Wooley, Daniel P. Silva, Fulvia Dimarzo Veronese, Ronald C. Desrosiers

Neuroscience, Cell Biology & Physiology Faculty Publications

Monoclonal antibody SF8/5E11, which recognizes the transmembrane protein (TMP) of simian immunodeficiency virus of macaque monkeys (SIVmac), displayed strict strain specificity. It reacted with cloned and uncloned SIVmac251 but not with cloned SIVmac142 and SIVmac239 on immunoblots. This monoclonal antibody neutralized infection by cloned, cell-free SIVmac251 and inhibited formation of syncytia by cloned SIVmac251-infected cells; these activities were specific to cloned SIVmac251 and did not occur with the other viruses. Site-specific mutagenesis was used to show that TMP amino acids 106 to 110 (Asp-Trp-Asn-Asn-Asp) determined the strain specificity of the monoclonal antibody. This strain-specific neutralizing determinant is located within a …

Selection Of Genetic Variants Of Simian Immunodeficiency Virus In Persistently Infected Rhesus Monkeys, Dawn P. Wooley, Ronald C. Desrosiers

Neuroscience, Cell Biology & Physiology Faculty Publications

Genetic and antigenic variation may be one means by which lentiviruses that cause AIDS avoid elimination by host immune responses. Genetic variation in the envelope gene (env) was studied by comparing the nucleotide sequences of 27 clones obtained from two rhesus monkeys infected with molecularly cloned simian immunodeficiency virus. All 27 clones differed from each other and differed from the input clone in the gp120 (SU) portion of the envelope gene. Nucleotide substitutions were shown to accumulate with time at an average rate of 8.5 per 1,000 per year in SU. Surprisingly, the majority of nucleotide substitutions (81%) resulted in …

Significance Of Premature Stop Codons In Env Of Simian Immunodeficiency Virus, Toshiaki Kodama, Dawn P. Wooley, Yathirajulu M. Naidu, Harry W. Kestler Iii, Muthiah D. Daniel, Yen Li, Ronald C. Desrosiers

Neuroscience, Cell Biology & Physiology Faculty Publications

The location of the translational termination codon for the transmembrane protein (TMP) varies in three infectious molecular clones of simian immunodeficiency virus from macaques (SIV_mac). The SIV_mac251 and SIV_mac142 infectious clones have premature stop signals that differ in location by one codon; transfection of these DNAs into human HUT-78 cells yielded virus with a truncated TMP (28 to 30 kilodaltons [kDa]). The SIV_mac239 infectious clone does not have a premature stop codon in its TMP-coding region. Transfection of HUT-78 cells with this clone initially yielded virus with a full-length TMP (41 kDa). …