Life Sciences Commons^™

Identification Of The Sites Of Action Of Inhibitors Of Mammalian Phospholipase D2 (Pld2) And The Role Of Interacting Protein Partners, Ramya Ganesan

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Phospholipase D (PLD) is a key enzyme for the remodeling of phospholipids in the cell membrane. PLD has been implicated in many physiological functions such as chemotaxis and phagocytosis as well as pathological functions such as ischemia/reperfusion and cancer metastasis. Several small molecule inhibitors of PLD have been recently developed to overcome these pathological effects. We have concentrated on 5-Fluoro-2-Indolyl des-Chlorohalopemide (FIPI) which is an indole derivative of halopemide that inhibits the two mammalian isoforms PLD1 and PLD2. We also concentrate on N-[2-[1-(3-Fluorophenyl)-4-oxo-1,3,-8-triazaspiro[4.5]dec-8-yl]ethyl]-2-naphthalenecarboxamide (NFOT) and N-[2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]dec-8-yl)ethyl]-2-naphthalenecarboxamide (NOPT) that are specific inhibitors of the PLD2 isoform. In spite of knowing their …

Effect Of Herpes Simplex Virus-1 On Macrophage Cd Marker Expression, Hind Obaid Albeshri

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Macrophages are specialized phagocytic cells derived from the peripheral blood mononuclear cells. Macrophages can assume M1 or M2 cellular states upon activation with specific cytokines. Upon activation, macrophages produce either pro- or anti- inflammatory cytokines and thereby perform their functions of antigen engulfment and debris clearance. Macrophages have several cell surface cluster differentiation (CD) markers including CD80, CD163 and CD200R that differentiate M1 macrophages from M2. Downstream signaling and cytokine production varies based on the interaction between the specific CD marker and antigen.

The present study was aimed at analyzing and comparing the effects of HSV-1 infection on un-polarized and …

The Camkkβ Inhibitor Sto-609 Causes Artefacts In Ca2+ Imaging And Selectively Inhibits Bkca In Mouse Carotid Body Type I Cells, Jennifer G. Jurcsisn

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It has previously been reported that AMP-activated protein kinase (AMPK) may be critical for hypoxic chemotransduction in carotid body type I cells (Wyatt et al, 2007). This study sought to determine the importance of the regulatory upstream kinase of AMPK, CamKKβ, in the acute response to hypoxia in isolated mouse type I cells.

Initial studies indicated several previously unreported artefacts associated with using the CamKKβ inhibitor STO-609 and calcium imaging techniques. Strong STO-609 autofluorescence meant that Fura-2 could not be used and X-Rhod-1 imaging revealed that STO-609 quenched fluorescence even in the absence of intracellular Ca2+. The whole-cell configuration of …

Effects Of Rev Protein On Microtubule Arrays In Living Cells, Nayana M. Nikumbh

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The HIV protein Rev regulates the expression of essential viral proteins during the course of infection by a mechanism that is well understood. It promotes nuclear export of viral transcripts, normally retained in the nucleus owing to the presence of introns, by interacting with host cell transport factors. However, over-expression of Rev in cells leads to defects in cell cycle progression, specifically slowing growth and impairing progression through mitosis (43). While it is possible that Rev may be altering the proteins in transport pathways, cell cycle defects may be attributed to Rev's interactions with other proteins.

In vitro experiments show …

Characterization Of Small Conductance Calcium-Activated Potassium Channels In A Human Lens Epithelium Cell Line (B3), Wedad Ali Saqar

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Small conductance calcium-activated potassium (SK) channels belong to the family of K+ channels. They play a significant role in cell volume regulation and in lens homeostasis, and thus are associated with cataract formation. This study was designed to demonstrate the presence of the four different isoforms of SK channels (SK1, SK2, SK3, and SK4) in human lens epithelial (HLE)-B3 cell line using Western blot (WB), immunofluorescence (IF), and reverse transcriptase coupled to the polymerase chain reaction (RT-PCR). Cataract formation involves SK-mediated K+ loss and gain of Na+ ions by lens cells. Further, it has been shown in previous studies that …

Role Of Bumetanide On Insulin Secretion, Mohammed Mashari Almutairi

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Insulin secretion depends on the electrical properties of the ß-cells in pancreatic islets. The consensus mechanism of insulin secretion involves glucose-stimulated metabolic increase in the ATP/ADP ratio. This increase closes ATP-sensitive K+ (KATP) channels resulting in ß-cell plasma membrane depolarization, which in turn activates voltage-dependent Ca2+ channels further resulting in Ca2+ influx and release of insulin stored in granules. In addition to this well-known consensus mechanism, insulin secretion also depends on electrogenic efflux of Cl- ions. The Na+K 2Cl- cotransporters (NKCCs) maintain the intracellular concentration of Cl- ([Cl-]i) at values higher than those predicted at thermodynamic equilibrium. This distribution of …

The Modulatory Role Of Circulating Microvesicles In Endothelial Progenitor Cell Function Is Altered In T2dm, Hala Mustafa Ammar

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Circulating microvesicles (cMVs) are the extracellular MVs released from the cells in the blood and on the vascular wall. Our previous study demonstrates that cMVs of diabetic mouse are detrimental to endothelial progenitor cells (EPCs), which are known to be very important for maintaining normal endothelial function and structure. In this study, we compared the levels of circulating EPCs and EPC-derived MVs (EPC-MVs) in diabetic and healthy subjects. Also, the migration ability, apoptosis rate and reactive oxygen species (ROS) production of EPCs cultured from diabetic and healthy subjects were determined. More importantly, we evaluated whether cMVs from healthy subjects (ch-MVs) …

Exploring How Middle School Students' Epistemologies In Practice Change Across Time With Varying Content Areas And Knowledge Product Contexts, Mackenzie Evan English

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Recently, the National Research Council developed A Framework for K-12 Science Education (NRC, 2012), to support a new vision of science education. The Framework (2012) focuses on three integrated dimensions--disciplinary core ideas, crosscutting concepts, and scientific and engineering practices which is different than traditional ways of learning science. I focused on students' engagement in scientific practices, attention to epistemological practices (EIP) that guide students' construction, evaluation and revision of knowledge products. I examined how students EIP's changed over time and across contexts with respect to different knowledge product types (models and explanations). I present data from 103, 6th grade students …

Assortative Fertilization In The Elegans-Group Of Caenorhabditis, Sara Rose Seibert

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Assortative fertilization refers to the species-specific interactions between sperm and oocytes that affect the success of fertilization. One type of interaction is chemotaxis of sperm to oocytes. In Caenorhabditis elegans, amoeboid sperm must crawl along the uterine lining towards the spermathecae in response to oocyte-derived prostaglandin signals for fertilization. This chemotactic signaling system likely operates in other species of the Elegans-Group of Caenorhabditis as sperm of C. briggsae and C. remanei do localize to the C. elegans spermathecae. In this project the impacts of species-specific chemotaxis on fertilization and female fecundity were assessed. To accomplish this, the localization of …

Inactivation Of At1a Receptors Attenuate Lactate Accumulation And Improve Cardiac Performance And Acid-Base Homeostasis During Endurance Exercise, Ahmad Mohammed Alhajoj

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Angiotensin Type 1 (AT1) receptors are involved in cardiovascular pathology. Losartan, the AT1 receptor blocker, in combination with exercise has been shown to be effective in improving cardiac performance. The goal of the study was to investigate the role of AT1a receptors on cardiac function, and exercise tolerance in response to aerobic exercise using AT1a receptor knockout (AT1aKO) mice. An exercise wheel system was used for the exercise paradigm. Male (C57BL/6) wild type (WT) and AT1aKO mice were randomly assigned to four groups: WT control (n=6), WT exercise (WTEX, n=8), AT1a KO (KO) control (n=5), and AT1a KO exercise (KOEX, …

Adeno-Associated Virus (Aav) Transduction Of Primary Human Cd4+T Lymphocytes, Sahar Hussein Kamel

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Development of latent HIV-infected CD4+ T lymphocytes is the major cause of HIV treatment failure. Adeno-associated virus (AAV) is an attractive vector for anti-HIV gene therapy due to its lack of pathogenicity, low immunogenicity, and persistent transgene expression. However, a major limitation for AAV gene transfer is the cell-specific tropism of each serotype. Only AAV serotypes 2 and 5 have been investigated in hematopoietic cells, both of these serotypes have shown low transduction efficiency. Since each of the currently described AAV serotypes demonstrate distinct tissue tropism, I hypothesized that other serotypes such as AAV1, 4, 8, or 9 may demonstrate …

Socs1/Socs3 Expression And Virus Replication Of Denv2 And Hsv-1 In Cytokine-Polarized Subsets Of Raw 264.7 Macrophages, Nagarjuna Reddy Cheemarla

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Cytokine activation of macrophages leads to "macrophage polarizatio" The M1 subset is polarized by exposure to lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) and M2 polarized cells develop after exposure to interleukin-4 (IL-4) or IL-13. In this study, the RAW 264.7 murine macrophage cell line was used to study the effect herpes simplex virus-Type I (HSV-1) and Dengue virus serotype-2 (DENV2) infections have on cultures of unpolarized (M0) or polarized subsets. The macrophage subsets were characterized using cluster of differentiation markers CD14 and CD86. Uninfected M1 macrophages showed up regulation in expression of the co-stimulatory factor B7.2 (CD86) compared to expression by …

Differentiation Of U-937 Monocytes To Macrophage-Like Cells Polarized Into M1 Or M2 Phenotypes According To Their Specific Environment: A Study Of Morphology, Cell Viability, And Cd Markers Of An In Vitro Model Of Human Macrophages, Fatma Husien S. Abdulhadi

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In this study, the human leukemic monocyte lymphoma cell line U-937 was used as an in vitro model for monitoring monocyte/macrophage differentiation. Phorbol 12- myristate (13) (PMA) was used to activate U-937 cells into macrophage-like cells (M0). After 24 hours of PMA treatment, non- adherent U-937 cells became tightly adherent to the culture plates forming M0 cells. M0 cells were then polarized into the M1 macrophage phenotype by treatment with LPS and IFN-γ for another 24 hours. Each of the cytokines IL-4, IL-13, or IL-10 was applied separately to three M0 cultures for 24 hours to induce the M2 macrophage …

Early Increase Of Cd11c In Human Monocyte-Derived Dendritic Cells In The Presence Of A/California/07/2009 (H1n1pdm), Amber M. Braddock

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Influenza A continues to cause significant morbidity and mortality. Vaccine production time, viral antigenic variability and low vaccine efficacy contribute to the annual pandemics and seasonal epidemics. A better understanding of human immune responses to influenza A could result in more effective vaccines. Dendritic cells (DCs) play an important role in the initiation of immune responses. Populations of B lymphocytes secrete protective antibodies but the role of different populations of B cells in the protective response is unknown. In this thesis we developed assays to determine subclasses of influenza specific antibodies. Mononuclear cells were assessed using flow cytometry to identify …

White Blood Cell Counts, Parasite Prevalence, And Plasma Cortisol Levels Of Dogs In A County Animal Shelter: Changes Over Days And Impact Of A Program Of Repeated Human Interaction, Emily S. Dudley

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Animal shelter housing is highly stressful for a dog, compromising welfare and leading to undesirable behaviors and unknown health consequences. We documented the changes in circulating numbers of white blood cells, plasma cortisol, and fecal parasite shedding of dogs housed for 10 days at a county animal shelter. White blood cell changes were most prominent on Day 10 after arrival to the shelter. Changes included increased total leukocytes, mature neutrophils, and lymphocytes, with less consistent increases in monocytes and neutrophil to lymphocyte ratio (N:L). Fecal parasite shedding was elevated and not affected by day. Cortisol levels of shelter dogs declined …

Development Of A Co-Culture System To Mimic The Transfection Of Hsv-1 From Keratinocytes To Neuronal Cells, David A. Dixon

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To more closely mimic the in vivo progression of HSV-1 a novel in vitro method was created. In this study HEL-30 keratinocytes were infected with a 0.1 MOI of HSV-1 and plated unto a membrane with 8μm diameter pores. The membrane insert was then placed into culture wells that contained Neuro-2a cells (N2A). The neurites from the neuronal cells made cell-to-cell contact with the infected HEL-30 cells and the virus was transmitted into the neuronal cells. To determine if infection occurred in the N2A cells the cells were lysed and the lysate incubated with Vero cells to titrate virus plaque …

Investigating Anatomical And Molecular Aspects Of Proprioceptive Sensory Neuron Diversity Using A Transgenic Mouse Model, Martha Jean Sonner

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Muscle spindles (MS) and Golgi tendon organs (GTOs) are encapsulated, stretch-activated sensory receptors housed within skeletal muscles, yet their functional contributions to proprioceptive monitoring of limb movements are quite different. For example, MS are responsible for detecting stretch of muscle fibers, and GTOs provide feedback regarding muscle tension during contraction. Peripheral axons extending from proprioceptive sensory neurons (PSNs) in the dorsal root ganglia supply MS and GTOs. MS are innervated by Group Ia and II sensory fiber endings, while GTOs are innervated by a single Group Ib sensory fiber. The developmental processes guiding axons from PSNs to their targets and …

Expression, Characterization, Cloning And Functional Impact Of Slc12a5 In The Endocrine Pancreas, Shams Kursan

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The Slc12a5 gene encodes the neuron-specific electroneutral K+Cl- cotransporter-2 (KCC2), a constitutively active Cl- extruder. In mature neurons, KCC2 functionally predominates over other members of the Slc12a family, particularly the Na+K+2Cl- cotransporter-1 (NKCC1), a ubiquitous Cl- loader. Therefore, KCC2 keeps and maintains the intracellular chloride concentration ([Cl-]i) below values predicted by thermodynamic equilibrium and in doing so, KCC2 plays a key role in neuron physiology: it makes possible the inhibitory action of neurotransmitters such as gamma-aminobutyric acid (GABA) and glycine. Neurons and insulin-secreting pancreatic ß-cells share many genes and functions. Indeed, neuron-specific genes have been also found in ß-cells whereas …

Opiliones Biodiversity In Cusuco National Park, Honduras, Brittany N. Damron

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Biodiversity in the tropics is especially diverse and describing species found in the tropics is important. Opiliones species richness is higher in the tropics than the temperate zone, with diversity highest in low latitudes. This study aims to catalog the Opiliones community in Cusuco National Park, Honduras, while estimating species richness and describing their abundance, distribution and community structure. Fifty hours of sampling at 6 sites yielded 18 morphospecies and 264 individual Opiliones were collected. This study added one new described species to the Opiliones fauna of Honduras, and fourteen morphospecies may represent new species. An estimated 43 to 112 …

Expression Of The Alpha, Beta, And Gamma Subunits Of The Interleukin-2 Receptor By Human Vascular Smooth Muscle Cells, Sultan Mohammed Alhayyani

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Interleukin 2 (IL-2) is a member of the cytokine family and contributes to the proliferation, survival, and death of lymphocytes [1]. The interleukin-2 receptor (IL-2) is a tripartite receptor commonly expressed on the surfaces of many lymphoid cells and is composed of three non-covalently associated subunits, alpha (CD25), beta (CD122), and gamma (CD132) [2]. Our laboratory has previously described IL-2 receptor Beta expression by vasculature smooth muscle cells (VSMC) in mice and humans [3]. The current work expands our previous observations by assessing the expression of the alpha and gamma subunits of the IL-2R by VSMCs. Analysis of IL-2R expression …

Co-Evolution Of Mitochondrial And Nuclear Genomes In Caenorhabditis , Emily Elaine Hill

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Nuclear and mitochondrial genomes have co-adapted through evolution. Interactions between these genomes are required for mitochondrial functions. My goal was to determine how divergence of these interactions affected mitochondrial function and organismal fitness. The impact of divergence between Caenorhabditis briggsae populations and between C. briggsae and its sister species, C. nigoni was assessed. To accomplish this, four sets of hybrid lines were constructed. Each hybrid line possessed the nuclear genome of one parental strain and mitochondria from another parental strain. Two sets of hybrid lines were constructed from strains derived from different C. briggsae populations. The other two were constructed …

Effects Of Transcranial Direct Current Stimulation On Expression Of Immediate Early Genes (Ieg's), Jessica Wagner

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Transcranial direct current stimulation (tDCS) has been utilized in human studies to modulate a multitude of psychological, cognitive, and psychiatric disorders. There have been positive behavioral results in human subjects but our knowledge of biological processes occurring during stimulation to elicit behavioral outcomes is limited. Our study utilizes a rodent tDCS (R-tDCS) model in which Sprague Dawley rats receive tDCS in order to examine whether tDCS affects neuronal activation. We examined two immediate early genes (IEG's), cFos and zif268, in order to discern if tDCS affects neuronal activation. Our findings indicate that tDCS does affect neuronal activation by means of …

Direct And Indirect Effects Of White-Tailed Deer (Odocoileus Virginianus) Herbivory On Beetle And Spider Assemblages In Northern Wisconsin, Elizabeth J. Sancomb

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White-tailed deer directly impact vegetation structure and species composition through selective foraging, and indirectly impact other species by altering habitat, food-web interactions, and microclimate. I examined the direct effects of deer exclusion on vegetation communities, and indirect effects on beetle, spider, and web-building spider (WBS) assemblages. Forb and woody plant percent cover were higher in exclosures, while graminoid cover was higher in controls. There were no differences in beetle and spider assemblages between browsed and protected areas. The absence of differences could be attributed to legacy effects, or alternatively high vagility of individuals. WBS assemblages were more abundant and diverse …

Comparison Between Flow Cytometry And Bead Method In Counting Cd4 And Cd8 T Lymphocytes In Mouse Spleen Cells Suspension, Abdulrahman Abdalla Allabidi

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The flow cytometry is gold standard method to count CD4, CD8 human T lymphocytes, and calculate CD8 to CD4 ratio in HIV patients peripheral blood to stage them and monitor their response to treatment. In this study, a comparison was made by counting CD4and CD8 mouse spleen T lymphocytes by flow cytometry, and a method involving antibody coated latex beads. The bead method yielded results comparable to those obtained by flow cytometry. These results indicate that antibody coated beads are suitable to determine CD4 and CD8 + T lymphocytes counts in situation where flow cytometry is not readily available.

Elucidating The Effects Of Tcdd On The Polymorphic Human Hs1,2 Enhancer, Abdullah Freiwan

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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental toxin known to inhibit immunoglobulin (Ig) gene expression in various animal studies. We have identified the mouse 3'Ig heavy chain regulatory region (3'IgH RR) as a sensitive transcriptional target of TCDD, which may mediate the inhibitory effect of TCDD on Ig expression. Interestingly, the human hs1,2 enhancer is polymorphic and has been associated with a number of autoimmune diseases.

Suggesting a species difference, TCDD inhibited mouse hs1,2 enhancer activation and activated basal human hs1,2 (hs-hs1,2) enhancer activity in the mouse B-cell line. The objective of this study was to elucidate the effects of TCDD …

Binding And Entry Mechanisms Of Adenovirus In Polarized Epithelial Cells, Trisha Lynn Brockman

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Adenovirus's primary receptor, the Coxsackievirus and adenovirus receptor (CAR), has two transmembrane isoforms with differential localization within polarized epithelial cells (CAREx7 (basolateral), CAREx8 (apical)). I hypothesized that each isoform of CAR is degraded at different rates and is regulated by Src-family kinases. Increasing or decreasing the concentration of CAR is predicted to directly alter adenoviral entry. To test this hypothesis, inducible Madin Darby Canine Kidney (MDCK) cell lines either expressing CAREx7, CAREx8, or mCherry, under a doxycycline-inducible promoter were used in pulse-chase structured experiments to calculate the half-lives of these proteins. Alternatively, a polarized model lung epithelium (CaLu-3) treated with …

Effect Of The Broad-Spectrum Caspase Inhibitor Q-Vd-Oph On Neurodegeneration And Neuroinflammation Of Sarin Exposed Mice, Ekta J. Shah

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Organophosphates (OP) cause CNS hyperstimulation resulting in seizures, convulsions and brain damage. Sarin is a toxic nerve agent used in Syria, the Gulf War and in terrorist attacks in Japan. Exposure to sarin is associated with presence of dead neurons, activation of astrocytes, microglia and neuroinflammation. Since, cell death and neuroinflammation are found in sarin exposed animals, we evaluated the efficacy of Q-VD-OPh, a broad spectrum caspase inhibitor that prevents cell death and has anti-inflammatory properties. Currently, treatment for sarin exposures is effective only if administered within 30-40 minutes of exposure. In this study, adult female C57BL/6 mice were subjected …

Investigating The Undefined Role Of Subunit Iiiin Cytochrome C Oxidase Functioning Using Dicyclohexylcarbodiimide Chemical Modification; Insight Into Enzyme Structure And Molecular Mechanism, Kelli Nicole Fisher

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In the cell metabolic cycle, cytochrome c oxidase (COX) is the final electron acceptor of the respiratory chain which reduces molecular oxygen into water. It is bound in the inner mitochondrial membrane and on the plasma membrane of bacterial species. Energy produced through electron transfer is coupled to the pumping of protons against the electrochemical gradient in order to fuel the proton motive force for the synthesis of most of the ATP in the cell. The three mitochondrial encoded subunits of COX, I, II, and III, are conserved across species and the complete function of subunit III remains unknown. Dicyclohexylcarbodiimide …

Increased Urinary Angiotensin Converting Enzyme 2 (Ace2) And Neprilysin (Nep) In Type 2 Diabetic Patients, Sridevi Gutta

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Chronic Kidney Disease (CKD) has reached epidemic proportions affecting more than 20 million adults in the US. CKD is routinely diagnosed and defined as reduced glomerular filtration rate (GFR), increased urinary albumin creatinine ratio (UACR) or both. Diabetes, hypertension and obesity share a major part in causing CKD. Angiotensin (1-7) (Ang (1-7)) is a vasodilator that plays an important renoprotective role in the renin angiotensin system, counteracting the vasoconstrictor and proliferative effects of Ang II. ACE2 and NEP which form Ang (1-7) by degrading Ang II and Ang I, respectively. A Disintegrin and Metalloproteinase (ADAM) 17, which is responsible for …