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1
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Mangiacotti M, Fumagalli M, Casali C, Biggiogera M, Forneris F, Sacchi R. Carbonic anhydrase IV in lizard chemical signals. Sci Rep 2023; 13:14164. [PMID: 37644071 PMCID: PMC10465503 DOI: 10.1038/s41598-023-41012-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2023] [Accepted: 08/20/2023] [Indexed: 08/31/2023] Open
Abstract
The evolution of chemical signals is subject to environmental constraints. A multicomponent signal may combine semiochemical molecules with supporting compounds able to enhance communication efficacy. Carbonic anhydrases (CAs) are ubiquitous enzymes catalysing the reversible hydration of carbon dioxide, a reaction involved in a variety of physiological processes as it controls the chemical environment of the different tissues or cellular compartments, thus contributing to the overall system homeostasis. CA-IV isoform has been recently identified by mass spectrometry in the femoral gland secretions (FG) of the marine iguana, where it has been hypothesized to contribute to the chemical stability of the signal, by regulating blend pH. Lizards, indeed, use FG to communicate by delivering the waxy secretion on bare substrate, where it is exposed to environmental stressors. Therefore, we expect that some molecules in the mixture may play supporting functions, enhancing the stability of the chemical environment, or even conferring homeostatic properties to the blend. CA-IV may well represent an important candidate to this hypothesized supporting/homeostatic function, and, therefore, we can expect it to be common in FG secretions of other lizard species. To evaluate this prediction and definitely validate CA identity, we analysed FG secretions of eight species of wall lizards (genus Podarcis), combining mass spectrometry, immunoblotting, immunocytochemistry, and transmission electron microscopy. We demonstrate CA-IV to actually occur in the FG of seven out of the eight considered species, providing an immunochemistry validation of mass-spectrometry identifications, and localizing the enzyme within the secretion mass. The predicted structure of the identified CA is compatible with the known enzymatic activity of CA-IV, supporting the hypothesis that CA play a signal homeostasis function and opening to new perspective about the role of proteins in vertebrate chemical communication.
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Affiliation(s)
- Marco Mangiacotti
- Department of Earth and Environmental Sciences, University of Pavia, Via Taramelli 24, 27100, Pavia, Italy.
| | - Marco Fumagalli
- The Armenise-Harvard Laboratory of Structural Biology, Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, Via Ferrata 9A, 27100, Pavia, Italy
| | - Claudio Casali
- Laboratory of Cell Biology and Neurobiology, Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, Via Ferrata 9, 27100, Pavia, Italy
| | - Marco Biggiogera
- Laboratory of Cell Biology and Neurobiology, Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, Via Ferrata 9, 27100, Pavia, Italy
| | - Federico Forneris
- The Armenise-Harvard Laboratory of Structural Biology, Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, Via Ferrata 9A, 27100, Pavia, Italy
| | - Roberto Sacchi
- Department of Earth and Environmental Sciences, University of Pavia, Via Taramelli 24, 27100, Pavia, Italy
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2
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Yrjänäinen A, Patrikainen MS, Azizi L, Tolvanen MEE, Laitaoja M, Jänis J, Hytönen VP, Nocentini A, Supuran CT, Parkkila S. Biochemical and Biophysical Characterization of Carbonic Anhydrase VI from Human Milk and Saliva. Protein J 2022; 41:489-503. [PMID: 35947329 PMCID: PMC9464147 DOI: 10.1007/s10930-022-10070-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/28/2022] [Indexed: 11/29/2022]
Abstract
Carbonic anhydrases (CA, EC 4.2.1.1) catalyze the hydration of carbon dioxide and take part in many essential physiological processes. In humans, 15 CAs are characterized, including the only secreted isoenzyme CA VI. CA VI has been linked to specific processes in the mouth, namely bitter taste perception, dental caries, and maintenance of enamel pellicle, and implicated in several immunity-related phenomena. However, little is known of the mechanisms of the above. In this study, we characterized human CA VI purified from saliva and milk with biophysical methods and measured their enzyme activities and acetazolamide inhibition. Size-exclusion chromatography showed peaks of salivary and milk CA VI corresponding to hexameric state or larger at pH 7.5. At pH 5.0 the hexamer peaks dominated. SDS- PAGE of milk CA VI protein treated with a bifunctional crosslinker further confirmed that a majority of CA VI is oligomers of similar sizes in solution. Mass spectrometry experiments confirmed that both of the two putative N-glycosylation sites, Asn67 and Asn256, are heterogeneously glycosylated. The attached glycans in milk CA VI were di- and triantennary complex-type glycans, carrying both a core fucose and 1 to 2 additional fucose units, whereas the glycans in salivary CA VI were smaller, seemingly degraded forms of core fucosylated complex- or hybrid-type glycans. Mass spectrometry also verified the predicted signal peptide cleavage site and the terminal residue, Gln 18, being in pyroglutamate form. Thorough characterization of CA VI paves way to better understanding of the biological function of the protein.
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Affiliation(s)
- Alma Yrjänäinen
- Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland
| | - Maarit S Patrikainen
- Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.
| | - Latifeh Azizi
- Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland
| | | | - Mikko Laitaoja
- Department of Chemistry, University of Eastern Finland, Joensuu, Finland
| | - Janne Jänis
- Department of Chemistry, University of Eastern Finland, Joensuu, Finland
| | - Vesa P Hytönen
- Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.,Fimlab Ltd, Tampere University Hospital, Tampere, Finland
| | - Alessio Nocentini
- Neurofarba Department, Sezione di Chimica Farmaceutica, University of Florence, Florence, Italy
| | - Claudiu T Supuran
- Neurofarba Department, Sezione di Chimica Farmaceutica, University of Florence, Florence, Italy
| | - Seppo Parkkila
- Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.,Fimlab Ltd, Tampere University Hospital, Tampere, Finland
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3
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Aspatwar A, Tolvanen MEE, Barker H, Syrjänen L, Valanne S, Purmonen S, Waheed A, Sly WS, Parkkila S. Carbonic Anhydrases in Metazoan Model Organisms: Molecules, Mechanisms, and Physiology. Physiol Rev 2022; 102:1327-1383. [PMID: 35166161 DOI: 10.1152/physrev.00018.2021] [Citation(s) in RCA: 59] [Impact Index Per Article: 19.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
During the past three decades, mice, zebrafish, fruit flies, and Caenorhabditis elegans have been the primary model organisms used for the study of various biological phenomena. These models have also been adopted and developed to investigate the physiological roles of carbonic anhydrases (CAs) and carbonic anhydrase-related proteins (CARPs). These proteins belong to eight CA families and are identified by Greek letters: α, β, γ, δ, ζ, η, θ, and ι. Studies using model organisms have focused on two CA families, α-CAs and β-CAs, which are expressed in both prokaryotic and eukaryotic organisms with species-specific distribution patterns and unique functions. This review covers the biological roles of CAs and CARPs in light of investigations performed in model organisms. Functional studies demonstrate that CAs are not only linked to the regulation of pH homeostasis, the classical role of CAs but also contribute to a plethora of previously undescribed functions.
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Affiliation(s)
- Ashok Aspatwar
- Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland
| | | | - Harlan Barker
- Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.,Fimlab Ltd and TAYS Cancer Centre, Tampere University Hospital, Tampere, Finland
| | - Leo Syrjänen
- Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.,Department of Otorhinolaryngology, Tampere University Hospital, Tampere, Finland
| | - Susanna Valanne
- Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland
| | - Sami Purmonen
- Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland
| | - Abdul Waheed
- Department of Biochemistry and Molecular Biology, Edward A. Doisy Research Center, Saint Louis University School of Medicine, St. Louis, MO, United States
| | - William S Sly
- Department of Biochemistry and Molecular Biology, Edward A. Doisy Research Center, Saint Louis University School of Medicine, St. Louis, MO, United States
| | - Seppo Parkkila
- Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.,Fimlab Ltd and TAYS Cancer Centre, Tampere University Hospital, Tampere, Finland
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4
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Trampert DC, van de Graaf SFJ, Jongejan A, Oude Elferink RPJ, Beuers U. Hepatobiliary acid-base homeostasis: Insights from analogous secretory epithelia. J Hepatol 2021; 74:428-441. [PMID: 33342564 DOI: 10.1016/j.jhep.2020.10.010] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/22/2020] [Revised: 10/03/2020] [Accepted: 10/19/2020] [Indexed: 12/14/2022]
Abstract
Many epithelia secrete bicarbonate-rich fluid to generate flow, alter viscosity, control pH and potentially protect luminal and intracellular structures from chemical stress. Bicarbonate is a key component of human bile and impaired biliary bicarbonate secretion is associated with liver damage. Major efforts have been undertaken to gain insight into acid-base homeostasis in cholangiocytes and more can be learned from analogous secretory epithelia. Extrahepatic examples include salivary and pancreatic duct cells, duodenocytes, airway and renal epithelial cells. The cellular machinery involved in acid-base homeostasis includes carbonic anhydrase enzymes, transporters of the solute carrier family, and intra- and extracellular pH sensors. This pH-regulatory system is orchestrated by protein-protein interactions, the establishment of an electrochemical gradient across the plasma membrane and bicarbonate sensing of the intra- and extracellular compartment. In this review, we discuss conserved principles identified in analogous secretory epithelia in the light of current knowledge on cholangiocyte physiology. We present a framework for cholangiocellular acid-base homeostasis supported by expression analysis of publicly available single-cell RNA sequencing datasets from human cholangiocytes, which provide insights into the molecular basis of pH homeostasis and dysregulation in the biliary system.
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Affiliation(s)
- David C Trampert
- Amsterdam UMC, University of Amsterdam, Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology Endocrinology Metabolism (AGEM), Meibergdreef 9, Amsterdam, the Netherlands
| | - Stan F J van de Graaf
- Amsterdam UMC, University of Amsterdam, Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology Endocrinology Metabolism (AGEM), Meibergdreef 9, Amsterdam, the Netherlands
| | - Aldo Jongejan
- Amsterdam UMC, University of Amsterdam, Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Meibergdreef 9, Amsterdam, the Netherlands
| | - Ronald P J Oude Elferink
- Amsterdam UMC, University of Amsterdam, Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology Endocrinology Metabolism (AGEM), Meibergdreef 9, Amsterdam, the Netherlands
| | - Ulrich Beuers
- Amsterdam UMC, University of Amsterdam, Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology Endocrinology Metabolism (AGEM), Meibergdreef 9, Amsterdam, the Netherlands.
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5
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Pemmari T, Laakso J, Patrikainen MS, Parkkila S, Järvinen TAH. Carbonic Anhydrase VI in Skin Wound Healing Study on Car6 Knockout Mice. Int J Mol Sci 2020; 21:ijms21145092. [PMID: 32708518 PMCID: PMC7404312 DOI: 10.3390/ijms21145092] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2020] [Revised: 07/11/2020] [Accepted: 07/15/2020] [Indexed: 01/22/2023] Open
Abstract
Carbonic anhydrases (CAs) contribute to tumor cell migration by generating an acidic environment through the conversion of carbon dioxide to bicarbonate and a proton. CA VI is secreted to milk and saliva, and it could contribute to wound closure, as a potential trophic factor, in animals that typically lick their wounds. Our aim was to investigate whether human CA VI improves skin-wound healing in full-thickness skin-wound models. The effect was studied in Car6 -/- knockout mice and wild type littermates. Half of both mice strains were given topically administered, milk-derived CA VI after wounding and eight hours later. The amount of topically given CA VI exceeded the predicted amount of natural saliva-delivered CA VI. The healing was followed for seven days and studied from photographs and histological sections. Our results showed no significant differences between the treatment groups in wound closure, re-epithelization, or granulation tissue formation, nor did the Car6 genotype affect the healing. Our results demonstrate that CA VI does not play a major role in skin-wound healing and also suggest that saliva-derived CA VI is not responsible for the licking-associated improved wound healing in animals.
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Affiliation(s)
- Toini Pemmari
- Faculty of Medicine and Health Technology, Tampere University, 33520 Tampere, Finland
| | - Jaakko Laakso
- Faculty of Medicine and Health Technology, Tampere University, 33520 Tampere, Finland
| | - Maarit S Patrikainen
- Faculty of Medicine and Health Technology, Tampere University, 33520 Tampere, Finland
| | - Seppo Parkkila
- Faculty of Medicine and Health Technology, Tampere University, 33520 Tampere, Finland
- Fimlab Ltd., Tampere University Hospital, 33520 Tampere, Finland
| | - Tero A H Järvinen
- Faculty of Medicine and Health Technology, Tampere University, 33520 Tampere, Finland
- Department of Orthopedics and Traumatology, Tampere University Hospital, 33520 Tampere, Finland
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6
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Laouar A. Maternal Leukocytes and Infant Immune Programming during Breastfeeding. Trends Immunol 2020; 41:225-239. [PMID: 32057705 DOI: 10.1016/j.it.2020.01.005] [Citation(s) in RCA: 56] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2019] [Revised: 01/10/2020] [Accepted: 01/12/2020] [Indexed: 12/11/2022]
Abstract
The fetal immune system develops in a rather sterile environment relative to the outside world and, therefore, lacks antigenic education. Soon after birth, the newborn is exposed to the hostile environment of pathogens. Recently, animal- and limited human-based studies have indicated that help from the mother, upon transfer of leukocytes and their products via breast milk feeding, greatly assists the newborn's immune system. Here, I discuss the newest advances on how milk leukocytes impact early life immunity, with an emphasis on the development of the infant T cell repertoire and early immune responses in the periphery and gut-associated lymphoid tissue. A deeper understanding of these novel mechanistic insights may inform potential translational approaches to improving immunity in infants.
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Affiliation(s)
- Amale Laouar
- Surgery Department and the Child Health Institute of New Jersey, Robert Wood Johnson Medical School-Rutgers University, 89 French Street, New Brunswick, NJ 08901, USA.
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7
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Towards Understanding Non-Infectious Growth-Rate Retardation in Growing Pigs. Proteomes 2019; 7:proteomes7030031. [PMID: 31514421 PMCID: PMC6789591 DOI: 10.3390/proteomes7030031] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2019] [Revised: 08/30/2019] [Accepted: 09/10/2019] [Indexed: 01/03/2023] Open
Abstract
For growth-rate retardation in commercial growing pigs suffering from non-infectious diseases, no biomarker is available for early detection and prevention of the condition or for the diagnosis of affected animals. The point in question is that the underlying pathological pathway of the condition is still unknown and multiple nutritional or management issues could be the cause of the disease. Common health status markers such as acute phase proteins, adenosine deaminase activity or total antioxidant capacity did not show any alteration in the saliva of animals with growth-rate retardation, so other pathways should be affected. The present study investigates saliva samples from animals with the same commercial crossbreed, sex and age, comparing control pigs and pigs with growth-rate retardation. A proteomics approach based on two-dimensional gel electrophoresis including mass spectrometry together with validation experiments was applied for the search of proteins that could help understand disease mechanisms and be used for early disease detection. Two proteins were detected as possible markers of growth-rate retardation, specifically S100A12 and carbonic anhydrase VI. A decrease in innate immune response was confirmed in pigs with growth-rate retardation, however further studies should be necessary to understand the role of the different CA VI proteoforms observed.
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8
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Engineered Carbonic Anhydrase VI-Mimic Enzyme Switched the Structure and Affinities of Inhibitors. Sci Rep 2019; 9:12710. [PMID: 31481705 PMCID: PMC6722136 DOI: 10.1038/s41598-019-49094-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2019] [Accepted: 08/15/2019] [Indexed: 01/25/2023] Open
Abstract
Secretory human carbonic anhydrase VI (CA VI) has emerged as a potential drug target due to its role in pathological states, such as excess acidity-caused dental caries and injuries of gastric epithelium. Currently, there are no available CA VI-selective inhibitors or crystallographic structures of inhibitors bound to CA VI. The present study focuses on the site-directed CA II mutant mimicking the active site of CA VI for inhibitor screening. The interactions between CA VI-mimic and a series of benzenesulfonamides were evaluated by fluorescent thermal shift assay, stopped-flow CO2 hydration assay, isothermal titration calorimetry, and X-ray crystallography. Kinetic parameters showed that A65T, N67Q, F130Y, V134Q, L203T mutations did not influence catalytic properties of CA II, but inhibitor affinities resembled CA VI, exhibiting up to 0.16 nM intrinsic affinity for CA VI-mimic. Structurally, binding site of CA VI-mimic was found to be similar to CA VI. The ligand interactions with mutated side chains observed in three crystallographic structures allowed to rationalize observed variation of binding modes and experimental binding affinities to CA VI. This integrative set of kinetic, thermodynamic, and structural data revealed CA VI-mimic as a useful model to design CA VI-specific inhibitors which could be beneficial for novel therapeutic applications.
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9
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Sumi KR, Kim SC, Natarajan S, Choi KS, Choi MR, Kim HT, Park JI, Nou IS, Gilmour KM, Kho KH. Molecular cloning and characterization of secretory carbonic anhydrase VI in pufferfish (Takifugu rubripes). Gene 2018; 640:57-65. [PMID: 29030255 DOI: 10.1016/j.gene.2017.10.008] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2017] [Accepted: 10/05/2017] [Indexed: 11/29/2022]
Abstract
Carbonic anhydrase VI (CA VI) has been characterized as a secretory isozyme in mammals. Our present study confirmed the occurrence of CA VI in pufferfish (Takifugu rubripes). In this study, genomic sequence information for the CA VI of pufferfish was used for molecular cloning. We cloned a 1821 bp cDNA sequence, which consisted of a complete coding sequence of 1623bp and a deduced amino acid sequence of 540 amino acids from the open reading frame. A BLAST search indicated that this protein exhibits 53%, 79%, and 67% identity with human, tilapia, and gar CA VI, respectively. It also shows 63%-77% identity with other fish CA VI-like sequences (zebrafish, Asian arowana, salmon, and large yellow croaker). Moreover, alignment of two or more sequences revealed that the protein sequence of pufferfish CA VI has 34%-37% identity with mammalian and fish CA II sequences. An NH2-terminal signal peptide of 18 amino acids in length was predicted in the pufferfish CA VI sequence. Three potential N-linked glycosylation sites and two cysteine residues (Cys-28 and Cys-209) that are likely to form one disulfide bond were present in pufferfish CA VI. In silico and phylogenetic analyses revealed that pufferfish CA VI is an extracellular secretory protein. Active site analysis indicated that this protein is a low-activity CA isozymes due to a characteristic Val/Ile substitution at position 207. Homology modeling of puffer CA VI was performed using the crystal structure of human carbonic anhydrase XIV as a template structure, based on high similarity. Reverse transcription-polymerase chain reaction (PCR), quantitative PCR and in situ hybridization results revealed that, the pufferfish CA VI is highly expressed in liver tissue.
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Affiliation(s)
- Kanij Rukshana Sumi
- Department of Fisheries Science, College of Fisheries and Ocean Sciences, Chonnam National University, 50, Daehak-ro, Yeosu, Jeonnam 59626, Republic of Korea.
| | - Soo Cheol Kim
- Department of Biomedical and Electronic Engineering, College of Engineering, Chonnam National University, Yeosu 59626, Republic of Korea
| | - Sathishkumar Natarajan
- Department of Horticulture, College of Life Science and Natural Resources, Sunchon National University, 255, Jungang-ro, Suncheon-Si, Jeollanam-do 57922, Republic of Korea
| | - Kap Seong Choi
- Department of Food Science and Technology, College of Life Science and Natural Resources, Sunchon National University, 255, Jungang-ro, Suncheon-Si, Jeollanam-do 57922, Republic of Korea.
| | - Myeong Rak Choi
- Department of Biomedical and Electronic Engineering, College of Engineering, Chonnam National University, Yeosu 59626, Republic of Korea.
| | - Hoy-Taek Kim
- Department of Horticulture, College of Life Science and Natural Resources, Sunchon National University, 255, Jungang-ro, Suncheon-Si, Jeollanam-do 57922, Republic of Korea.
| | - Jong-In Park
- Department of Horticulture, College of Life Science and Natural Resources, Sunchon National University, 255, Jungang-ro, Suncheon-Si, Jeollanam-do 57922, Republic of Korea.
| | - Ill-Sup Nou
- Department of Horticulture, College of Life Science and Natural Resources, Sunchon National University, 255, Jungang-ro, Suncheon-Si, Jeollanam-do 57922, Republic of Korea.
| | - Kathleen M Gilmour
- Department of Biology, Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, Canada.
| | - Kang Hee Kho
- Department of Fisheries Science, College of Fisheries and Ocean Sciences, Chonnam National University, 50, Daehak-ro, Yeosu, Jeonnam 59626, Republic of Korea.
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10
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Patrikainen MS, Tolvanen MEE, Aspatwar A, Barker HR, Ortutay C, Jänis J, Laitaoja M, Hytönen VP, Azizi L, Manandhar P, Jáger E, Vullo D, Kukkurainen S, Hilvo M, Supuran CT, Parkkila S. Identification and characterization of a novel zebrafish ( Danio rerio) pentraxin-carbonic anhydrase. PeerJ 2017; 5:e4128. [PMID: 29230365 PMCID: PMC5723433 DOI: 10.7717/peerj.4128] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2017] [Accepted: 11/14/2017] [Indexed: 12/29/2022] Open
Abstract
Background Carbonic anhydrases (CAs) are ubiquitous, essential enzymes which catalyze the conversion of carbon dioxide and water to bicarbonate and H+ ions. Vertebrate genomes generally contain gene loci for 15–21 different CA isoforms, three of which are enzymatically inactive. CA VI is the only secretory protein of the enzymatically active isoforms. We discovered that non-mammalian CA VI contains a C-terminal pentraxin (PTX) domain, a novel combination for both CAs and PTXs. Methods We isolated and sequenced zebrafish (Danio rerio) CA VI cDNA, complete with the sequence coding for the PTX domain, and produced the recombinant CA VI–PTX protein. Enzymatic activity and kinetic parameters were measured with a stopped-flow instrument. Mass spectrometry, analytical gel filtration and dynamic light scattering were used for biophysical characterization. Sequence analyses and Bayesian phylogenetics were used in generating hypotheses of protein structure and CA VI gene evolution. A CA VI–PTX antiserum was produced, and the expression of CA VI protein was studied by immunohistochemistry. A knock-down zebrafish model was constructed, and larvae were observed up to five days post-fertilization (dpf). The expression of ca6 mRNA was quantitated by qRT-PCR in different developmental times in morphant and wild-type larvae and in different adult fish tissues. Finally, the swimming behavior of the morphant fish was compared to that of wild-type fish. Results The recombinant enzyme has a very high carbonate dehydratase activity. Sequencing confirms a 530-residue protein identical to one of the predicted proteins in the Ensembl database (ensembl.org). The protein is pentameric in solution, as studied by gel filtration and light scattering, presumably joined by the PTX domains. Mass spectrometry confirms the predicted signal peptide cleavage and disulfides, and N-glycosylation in two of the four observed glycosylation motifs. Molecular modeling of the pentamer is consistent with the modifications observed in mass spectrometry. Phylogenetics and sequence analyses provide a consistent hypothesis of the evolutionary history of domains associated with CA VI in mammals and non-mammals. Briefly, the evidence suggests that ancestral CA VI was a transmembrane protein, the exon coding for the cytoplasmic domain was replaced by one coding for PTX domain, and finally, in the therian lineage, the PTX-coding exon was lost. We knocked down CA VI expression in zebrafish embryos with antisense morpholino oligonucleotides, resulting in phenotype features of decreased buoyancy and swim bladder deflation in 4 dpf larvae. Discussion These findings provide novel insights into the evolution, structure, and function of this unique CA form.
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Affiliation(s)
| | | | - Ashok Aspatwar
- Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland.,Fimlab Ltd., Tampere University Hospital, Tampere, Finland
| | - Harlan R Barker
- Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland
| | | | - Janne Jänis
- Department of Chemistry, University of Eastern Finland, Joensuu, Finland
| | - Mikko Laitaoja
- Department of Chemistry, University of Eastern Finland, Joensuu, Finland
| | - Vesa P Hytönen
- Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland.,Fimlab Ltd., Tampere University Hospital, Tampere, Finland
| | - Latifeh Azizi
- Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland
| | - Prajwol Manandhar
- Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland.,Center for Molecular Dynamics Nepal, Kathmandu, Nepal
| | - Edit Jáger
- Department of Epidemiology, Faculty of Health Sciences, Semmelweis University, Budapest, Hungary
| | - Daniela Vullo
- Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università degli Studi di Firenze, Florence, Italy
| | - Sampo Kukkurainen
- Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland
| | - Mika Hilvo
- Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland.,Zora Biosciences Ltd., Espoo, Finland
| | - Claudiu T Supuran
- Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università degli Studi di Firenze, Florence, Italy
| | - Seppo Parkkila
- Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland.,Fimlab Ltd., Tampere University Hospital, Tampere, Finland
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11
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Variations in the bitterness perception-related genes TAS2R38 and CA6 modify the risk for colorectal cancer in Koreans. Oncotarget 2017; 8:21253-21265. [PMID: 28423504 PMCID: PMC5400581 DOI: 10.18632/oncotarget.15512] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2016] [Accepted: 02/07/2017] [Indexed: 01/29/2023] Open
Abstract
Bitterness perception is known to be an important factor in individuals' dietary behaviors and is also associated with the sensing of nutritious/noxious molecules for subsequent metabolic responses in multiple organs. Therefore, the genetic variation in bitterness sensing may be associated with diet-related diseases, including colorectal cancer (CRC). We investigated the influence of variations in the bitterness-sensing genes taste receptor type 2 member 38 (TAS2R38) and carbonic anhydrase 6 (CA6) on the consumption of food, tobacco and alcohol and the risk of CRC in Koreans. The study population consisted of 681 cases and 1361 controls, and their intake of vegetables, fruits, fiber, fat-food and sweets was analyzed. The genotypes for TAS2R38 A49P, V262A and I296V and CA6 rs2274333 A/G were assessed using the MassArray technique. Our findings suggested that the TAS2R38 diplotype, CA6 rs2274333 and their combined genotype had a negligible influence on dietary and alcohol intake. The combined TAS2R38-CA6 AVI/AVI-AA genotype was associated with higher tobacco consumption than the other genotypes in CRC cases only. However, the genetic variations were a significant risk factor for CRC. The TAS2R38 AVI/AVI diplotype and CA6 G allele were associated with a reduced risk of CRC. Moreover, when the combined genotypes of the subjects were analyzed, possessing both the variant diplotype/variant allele (AVI/AVI+G*) was associated with a greater reduction in the risk of CRC (adjusted OR = 0.49; 95%CI: 0.34-0.74). In summary, variations in the bitterness perception genes TAS2R38 and CA6 did not influence the examined food intake in Koreans. However, those genetic variants were a decisive modifying factor of CRC susceptibility.
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Arabacı T, Çiçek Y, Beydemir Ş, Çanakçı CF, Çanakçı V. Are increased salivary carbonic anhydrase VI levels related to the amount of supragingival dental calculus formation and clinical periodontal scores? J Dent Sci 2015. [DOI: 10.1016/j.jds.2012.12.014] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
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Kazokaitė J, Milinavičiūtė G, Smirnovienė J, Matulienė J, Matulis D. Intrinsic binding of 4-substituted-2,3,5,6-tetrafluorobenezenesulfonamides to native and recombinant human carbonic anhydrase VI. FEBS J 2015; 282:972-83. [PMID: 25586768 DOI: 10.1111/febs.13196] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2014] [Revised: 12/08/2014] [Accepted: 01/12/2015] [Indexed: 11/30/2022]
Abstract
Carbonic anhydrase (CA) VI is a potential drug target for cariogenesis and cancer of the salivary gland. It is the only secreted human CA isozyme which is found in saliva and milk. Here, CA VI was expressed in bacterial and mammalian cell cultures and directly affinity-purified from human saliva. The binding of 4-substituted-2,3,5,6-tetrafluorobenezenesulfonamides to the native and recombinant CA VI from these three sources was compared. Interaction between the enzyme and inhibitors was determined by fluorescent thermal shift assay and isothermal titration calorimetry. The observed dissociation constants were the same within the error margin for all three CA VI preparations. The intrinsic binding parameters for the compounds were obtained by determining and dissecting the binding-linked protonation reactions. Intrinsic thermodynamic parameters of binding arrange the compounds in a buffer- and pH-independent manner. Intrinsic binding constants of nonfluorinated compounds were significantly stronger than those of fluorinated benzenesulfonamides. An opposite result was determined for the observed binding constants. The increase in observed affinity of the fluorinated compounds was due to the fluorine effect on diminishing the pKa of the compounds but not due to direct recognition of the protein. The temperature-stability profiles for recombinant and native CA VI were compared and showed that CA VI is more stable in slightly acidic than neutral conditions.
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Affiliation(s)
- Justina Kazokaitė
- Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Lithuania
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14
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Nishita T, Yatsu J, Watanabe K, Ochiai H, Ichihara N, Orito K, Arishima K. Urinary carbonic anhydrase VI as a biomarker for kidney disease in pigs. Vet J 2014; 202:378-80. [DOI: 10.1016/j.tvjl.2014.07.007] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2013] [Revised: 06/30/2014] [Accepted: 07/14/2014] [Indexed: 11/30/2022]
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McCormick NH, Hennigar SR, Kiselyov K, Kelleher SL. The biology of zinc transport in mammary epithelial cells: implications for mammary gland development, lactation, and involution. J Mammary Gland Biol Neoplasia 2014; 19:59-71. [PMID: 24338187 DOI: 10.1007/s10911-013-9314-4] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/19/2013] [Accepted: 12/04/2013] [Indexed: 02/07/2023] Open
Abstract
Zinc plays a critical role in a vast array of cellular functions including gene transcription, protein translation, cell proliferation, differentiation, bioenergetics, and programmed cell death. The mammary gland depends upon tight coordination of these processes during development and reproduction for optimal expansion, differentiation, and involution. For example, zinc is required for activation of matrix metalloproteinases, intracellular signaling cascades such as MAPK and PKC, and the activation of both mitochondrial-mediated apoptosis and lysosomal-mediated cell death. In addition to functional needs, during lactation the mammary gland must balance providing optimal zinc for cellular requirements with the need to secrete a substantial amount of zinc into milk to meet the requirements of the developing neonate. Finally, the mammary gland exhibits the most profound example of programmed cell death, which is driven by both apoptotic and lysosomal-mediated cell death. Two families of zinc-specific transporters regulate zinc delivery for these diverse functions. Members of the ZIP family of zinc transporters (ZIP1-14) import zinc into the cytoplasm from outside the cell or from subcellular organelles, while members of the ZnT family (ZnT1-10) export zinc from the cytoplasm. Recently, the ion channel transient receptor potential mucolipin 1 (TRPML1) has also been implicated in zinc transport. Herein, we review our current understanding of the molecular mechanisms through which mammary epithelial cells utilize zinc with a focus on the transport of zinc into discrete subcellular organelles for specific cellular functions during mammary gland development, lactation, and involution.
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Affiliation(s)
- Nicholas H McCormick
- Department of Nutritional Sciences, The Pennsylvania State University, University Park, PA, USA
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16
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Nishita T, Yatsu J, Murakami M, Kamoshida S, Orito K, Ichihara N, Arishima K, Ochiai H. Isolation and sequencing of swine carbonic anhydrase VI, an enzyme expressed in the swine kidney. BMC Res Notes 2014; 7:116. [PMID: 24576305 PMCID: PMC3975938 DOI: 10.1186/1756-0500-7-116] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2013] [Accepted: 02/24/2014] [Indexed: 11/10/2022] Open
Abstract
Background Carbonic anhydrase VI (CA-VI) is produced by the salivary gland and is secreted into the saliva. Although CA-VI is found in the epithelial cells of distal straight tubule of swine kidneys, the exact function of CA-VI in the kidneys remains unclear. Results CA-VI was located in the epithelial cells of distal straight tubule of swine kidneys. A full-length cDNA clone of CA-VI was generated from the swine parotid gland by reverse transcription polymerase chain reaction, using degenerate primers designed based on conserved regions of the same locus in human and bovine tissues. The cDNA sequence was 1348 base pairs long and was predicted to encode a 317 amino acid polypeptide with a putative signal peptide of 17 amino acids. The deduced amino acid sequence of mature CA-VI was most similar (77.4%) to that of human CA-VI. CA-VI expression was confirmed in both normal and nephritic kidneys, as well as parotid. As the primers used in this study spanned two exons, the influence of genomic DNA was not detected. The expression of CA-VI was demonstrated in both normal and nephritic kidneys, and mRNA of CA-VI in the normal kidneys which was the normalised to an endogenous β–actin was 0.098 ± 0.047, while it was significantly lower in the diseased kidneys (0.012 ± 0.007). The level of CA-VI mRNA in normal kidneys was 19-fold lower than that of the parotid gland (1.887). Conclusions The localisation of CA-VI indicates that it may play a specialised role in the kidney.
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Affiliation(s)
- Toshiho Nishita
- Laboratory of Physiology I, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 252-5201, Japan.
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17
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Aidar M, Marques R, Valjakka J, Mononen N, Lehtimäki T, Parkkila S, de Souza AP, Line SRP. Effect of genetic polymorphisms in CA6 gene on the expression and catalytic activity of human salivary carbonic anhydrase VI. Caries Res 2013; 47:414-20. [PMID: 23652931 DOI: 10.1159/000350414] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2012] [Accepted: 03/03/2013] [Indexed: 11/19/2022] Open
Abstract
Carbonic anhydrase isoenzyme VI (CA VI) plays an important role in the homeostasis of oral tissues participating in the processes of taste, protection of dental tissues against the loss of minerals, caries, and possibly in the formation of dental calculus in periodontal disease. This study aimed to verify the correlation between changes in the expression and activity of human salivary carbonic anhydrase VI and genetic polymorphisms in its gene (CA6). The study population consisted of 182 healthy volunteers (female and male, aged 18-22). Samples of total saliva were assayed for CA VI concentrations using a specific time-resolved immunofluorometric assay. CA VI catalytic activity was detected by a modified protocol of Kotwica et al. [J Physiol Pharmacol 2006;57(suppl 8):107-123], adapted to CA VI in saliva. Samples of genomic DNA were genotyped for polymorphisms rs2274327 (C/T), rs2274328 (A/C) and rs2274333 (A/G) by TaqMan® SNP Genotyping Assays. The concentration and catalytic activity of the salivary CA VI obtained for the different genotypes were analyzed using the Kruskal-Wallis nonparametric test and the Dunn test. The results showed that individuals with TT genotype (rs2274327) had significantly lower CA VI concentrations than the individuals with genotypes CT or CC (p < 0.05). There was also an association between polymorphism rs2274333 and salivary CA VI concentrations. There were no associations between the three polymorphisms analyzed and variations in CA VI activity. Our results suggest that polymorphisms in the CA6 gene are associated with the concentrations of secreted CA VI.
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Affiliation(s)
- M Aidar
- Department of Morphology, Piracicaba Dental School, University of Campinas-Unicamp, Sao Paulo, Brazil
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Crystal structure of the secretory isozyme of mammalian carbonic anhydrases CA VI: Implications for biological assembly and inhibitor development. Biochem Biophys Res Commun 2012; 419:485-9. [DOI: 10.1016/j.bbrc.2012.02.038] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2012] [Accepted: 02/07/2012] [Indexed: 11/20/2022]
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Lorenz K, Bader M, Klaus A, Weiss W, Görg A, Hofmann T. Orosensory stimulation effects on human saliva proteome. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2011; 59:10219-10231. [PMID: 21846099 DOI: 10.1021/jf2024352] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/31/2023]
Abstract
Saliva flow induced by 6-gingerol (pungent), hydroxy-α/β-sanshools (tingling), and citric acid (sour) was measured, and the time-dependent changes in the whole saliva proteome were analyzed by means of 2D-PAGE, followed by tryptic in-gel digestion and MALDI-TOF-MS peptide mass fingerprint analysis. The proteins showing significantly decreased abundance after oral 6-gingerol stimulation were identified as glutathione S-transferase P, the heat shock protein β-1, the heat shock 70 kDa protein 1, annexin A1, and cytoplasmic β-actin, whereas prolactin inducible proteins (PIP), short palate, lung and nasal epithelium carcinoma-associated protein 2 (SPLUNC2), zinc-α-2-glycoproteins (Zn-α-GP), and carbonic anhydrase VI (CAVI) were found with increased abundance. As the effects of this study were observed instantaneously upon stimulation, any proteome modulation is very likely to result from the release of proteins from preformed vesicles and not from de novo synthesis. The elevated levels of SPLUNC2, Zn-α-GP, and CAVI might be interpreted to trigger innate protective mechanisms in mucosal immunity and in nonimmune mucosal defense and might play an important role during the initial stage of inflammation.
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Affiliation(s)
- Katharina Lorenz
- Food Chemistry and Molecular Sensory Science, Technische Universität München, Lise-Meitner Strasse 34, D-85354 Freising, Germany
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Culp DJ, Robinson B, Parkkila S, Pan PW, Cash MN, Truong HN, Hussey TW, Gullett SL. Oral colonization by Streptococcus mutans and caries development is reduced upon deletion of carbonic anhydrase VI expression in saliva. Biochim Biophys Acta Mol Basis Dis 2011; 1812:1567-76. [PMID: 21945428 DOI: 10.1016/j.bbadis.2011.09.006] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2011] [Revised: 09/07/2011] [Accepted: 09/09/2011] [Indexed: 12/16/2022]
Abstract
Carbonic anhydrase VI (CA VI), encoded by type A transcripts of the gene Car6, is a secretory product of salivary glands and is found in the enamel pellicle. Because higher caries prevalence is associated with lower salivary concentrations of CA VI in humans, we tested whether CA VI protects enamel surfaces from caries induced by Streptococcus mutans, using Car6(-/-) mice, in which salivary CA VI expression is absent. We detected aberrant Car6 type A transcripts in Car6(-/-) mice, likely targets for nonsense-mediated mRNA decay. Expression of the intracellular stress-induced isoform of CA VI encoded by type B transcripts was restricted to parotid and submandibular glands of wild type mice. The salivary function of Car6(-/-) mice was normal as assessed by the histology and protein/glycoprotein profiles of glands, salivary flow rates and protein/glycoprotein compositions of saliva. Surprisingly, total smooth surface caries and sulcal caries in Car6(-/-) mice were more than 6-fold and 2-fold lower than in wild type mice after infection with S. mutans strain UA159. Recoveries of S. mutans and total microbiota from molars were also lower in Car6(-/-) mice. To explore possible mechanisms for increased caries susceptibility, we found no differences in S. mutans adherence to salivary pellicles, in vitro. Interestingly, higher levels of Lactobacillus murinus and an unidentified Streptococcus species were cultivated from the oral microbiota of Car6(-/-) mice. Collective results suggest salivary CA VI may promote caries by modulating the oral microbiota to favor S. mutans colonization and/or by the enzymatic production of acid within plaque.
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Affiliation(s)
- David J Culp
- Department of Oral Biology, University of Florida, Gainesville, FL 32610, USA.
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NISHITA T, ITOH S, ARAI S, ICHIHARA N, ARISHIMA K. Measurement of carbonic anhydrase isozyme VI (CA-VI) in swine sera, colostrums, saliva, bile, seminal plasma and tissues. Anim Sci J 2011; 82:673-8. [DOI: 10.1111/j.1740-0929.2011.00888.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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Tye CE, Sharma R, Smith CE, Bartlett JD. Altered ion-responsive gene expression in Mmp20 null mice. J Dent Res 2010; 89:1421-6. [PMID: 20929715 DOI: 10.1177/0022034510384625] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
During enamel maturation, hydroxyapatite crystallites expand in volume, releasing protons that acidify the developing enamel. This acidity is neutralized by the buffering activity of carbonic anhydrases and ion transporters. Less hydroxyapatite forms in matrix metalloproteinase-20 null (Mmp20(-/-)) mouse incisors, because enamel thickness is reduced by approximately 50%. We therefore asked if ion regulation was altered in Mmp20(-/-) mouse enamel. Staining of wild-type and Mmp20(-/-) incisors with pH indicators demonstrated that wild-type mice had pronounced changes in enamel pH as development progressed. These pH changes were greatly attenuated in Mmp20(-/-) mice. Expression of 4 ion-regulatory genes (Atp2b4, Slc4a2, Car6, Cftr) was significantly decreased in enamel organs from Mmp20(-/-) mice. Notably, expression of secreted carbonic anhydrase (Car6) was reduced to almost undetectable levels in the null enamel organ. In contrast, Odam and Klk4 expression was unaffected. We concluded that a feedback mechanism regulates ion-responsive gene expression during enamel development.
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Affiliation(s)
- C E Tye
- Department of Cytokine Biology, Harvard School of Dental Medicine, Boston, MA 02115, USA
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Pan PW, Käyrä K, Leinonen J, Nissinen M, Parkkila S, Rajaniemi H. Gene expression profiling in the submandibular gland, stomach, and duodenum of CAVI-deficient mice. Transgenic Res 2010; 20:675-98. [PMID: 20835760 DOI: 10.1007/s11248-010-9441-2] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2010] [Accepted: 09/01/2010] [Indexed: 12/31/2022]
Abstract
Carbonic anhydrase VI (CAVI) is the only secreted isozyme of the α-carbonic anhydrase family, which catalyzes the reversible reaction [Formula in text]. It appears that CAVI protects teeth and gastrointestinal mucosa by neutralizing excess acidity. However, the evidence for this physiological function is limited, and CAVI may have additional functions that have yet to be discovered. To explore the functions of CAVI more fully, we generated Car6 (-/-) mice and analyzed Car6 (-/-) mutant phenotypes. We also examined transcriptomic responses to CAVI deficiency in the submandibular gland, stomach, and duodenum of Car6 (-/-) mice. Car6 (-/-) mice were viable and fertile and had a normal life span. Histological analyses indicated a greater number of lymphoid follicles in the small intestinal Peyer's patches. A total of 94, 56, and 127 genes were up- or down-regulated in the submandibular gland, stomach, and duodenum of Car6 (-/-) mice, respectively. The functional clustering of differentially expressed genes revealed a number of altered biological processes. In the duodenum, the significantly affected biological pathways included the immune system process and retinol metabolic processes. The response to oxidative stress and brown fat cell differentiation changed remarkably in the submandibular gland. Notably, the submandibular gland, stomach, and duodenum shared one important transcriptional susceptibility pathway: catabolic process. Real-time PCR confirmed an altered expression in 14 of the 16 selected genes. The generation and of Car6 (-/-) mice and examination of the effects of CAVI deficiency on gene transcription have revealed several affected clusters of biological processes, which implicate CAVI in catabolic processes and the immune system response.
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Affiliation(s)
- Pei-wen Pan
- Institute of Medical Technology, University of Tampere, Biokatu 6, 33520, Tampere, Finland.
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Ochiai H, Kanemaki N, Kamoshida S, Murakami M, Ichihara N, Asari M, Nishita T. Determination of full-length cDNA nucleotide sequence of equine carbonic anhydrase VI and its expression in various tissues. J Vet Med Sci 2009; 71:1233-7. [PMID: 19801906 DOI: 10.1292/jvms.71.1233] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
A full-length cDNA clone of an equine carbonic anhydrase (CA)-VI was obtained from the equine parotid gland. The cDNA sequence was 1338 bp long and was predicted to encode a 319 amino acid polypeptide with a putative signal peptide of 18 amino acids. The deduced amino acid sequence of mature CA-VI showed the similarity of 70% to those of other mammalians reported. Westernblot analysis using anti-horse CA-VI peptide detected the single band in parotid gland, and the band reduced its size by treatment with N-glycosidase F. Additionally, CA-VI protein expression was confirmed in submandicular gland and weakly in liver. In contrast, RT-PCR analysis revealed signals in the digestive tract including duodenum, jejunum, ileum, cecum and colon as well as the salivary glands. In addition, certain signals were detected in testis, thyroid gland and liver, but not in nerve tissue, skeletal muscle, spleen or lymph node.
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Affiliation(s)
- Hideharu Ochiai
- Research Institute of Biosciences, Azabu University, Sagamihara, Kanagawa, Japan.
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Ambatipudi KS, Lu B, Hagen FK, Melvin JE, Yates JR. Quantitative analysis of age specific variation in the abundance of human female parotid salivary proteins. J Proteome Res 2009; 8:5093-102. [PMID: 19764810 PMCID: PMC2834885 DOI: 10.1021/pr900478h] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Human saliva is a protein-rich, easily accessible source of potential local and systemic biomarkers to monitor changes that occur under pathological conditions; however, little is known about the changes in abundance associated with normal aging. In this study, we performed a comprehensive proteomic profiling of pooled saliva collected from the parotid glands of healthy female subjects, divided into two age groups 1 and 2 (20-30 and 55-65 years old, respectively). Hydrophobic charge interaction chromatography was used to separate high- from low-abundance proteins prior to characterization of the parotid saliva using multidimensional protein identification technology (MudPIT). Collectively, 532 proteins were identified in the two age groups. Of these proteins, 266 were identified exclusively in one age group, while 266 proteins were common to both groups. The majority of the proteins identified in the two age groups belonged to the defense and immune response category. Of note, several defense related proteins (e.g., lysozyme, lactoferrin and histatin-1) were significantly more abundant in group 2 as determined by G-test. Selected representative mass spectrometric findings were validated by Western blot analysis. Our study reports the first quantitative analysis of differentially regulated proteins in ductal saliva collected from young and older female subjects. This study supports the use of high-throughput proteomics as a robust discovery tool. Such results provide a foundation for future studies to identify specific salivary proteins which may be linked to age-related diseases specific to women.
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Affiliation(s)
- Kiran S. Ambatipudi
- Center for Oral Biology, University of Rochester Medical Center; Rochester, New York 14642
| | - Bingwen Lu
- Department of Chemical Physiology, The Scripps Research Institute; 10550 North Torrey Pines Road, SR-11, La Jolla, CA 92037
| | - Fred K Hagen
- Center for Oral Biology, University of Rochester Medical Center; Rochester, New York 14642
| | - James E. Melvin
- Center for Oral Biology, University of Rochester Medical Center; Rochester, New York 14642
| | - John R. Yates
- Department of Chemical Physiology, The Scripps Research Institute; 10550 North Torrey Pines Road, SR-11, La Jolla, CA 92037
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Sugiura Y, Soeta S, Ichihara N, Nishita T, Murakami M, Amasaki H, Amasaki T, Asari M. Immunohistolocalization and gene expression of the carbonic anhydrase isoenzymes (CA-II and CA-VI) in glands associated with the canine lacrimal apparatus. Anat Histol Embryol 2009; 39:1-6. [PMID: 19874279 DOI: 10.1111/j.1439-0264.2009.00966.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Cytosolic and secretory carbonic anhydrase isoenzymes (CA-II and CA-VI, respectively) were detected by immunohistolocalization using specific canine CA-II and CA-VI antisera. CA-II and CA-VI were identified in glands associated with the canine lacrimal apparatus, such as lacrimal gland, superficial gland of the third eyelid (third eyelid gland) and tarsal gland. CA-II and CA-VI mRNA signals were also detected by reverse-transcriptase polymerase chain reaction in the same tissues. Some serous acinar cells and duct segments in the lacrimal gland and serous acinar cells in the third eyelid gland were immunopositive for anti-CA-II and CA-VI antisera. In particular, some immunopositive acini to CA-II and CA-VI on the edge of the third eyelid gland are histologically similar to sebaceous gland cells. Sebaceous gland cells in the tarsal and ciliary glands also showed immunopositivity to both CA antisera. CA-II and CA-VI gene transcripts were detected in the same regions. These results suggest that secreted CA-VI may form together with cytosolic CA-II, a high-activity isozyme mostly considered as a bicarbonate producer, in a mutually complementary system for the maintenance of bicarbonate levels to regulate pH in tear fluid and protect the corneal epithelia against injuries. In sebaceous gland cells in the lacrimal apparatus, CA-VI may be related to lipogenesis in an unknown function.
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Affiliation(s)
- Y Sugiura
- Azabu University School of Veterinary Medicine, Fuchinobe, Sagamihara, Kanagawa, Japan
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Peres RCR, Camargo G, Mofatto LS, Cortellazzi KL, Santos MCLG, Santos MN, Bergamaschi CC, Line SRP. Association of polymorphisms in the carbonic anhydrase 6 gene with salivary buffer capacity, dental plaque pH, and caries index in children aged 7–9 years. THE PHARMACOGENOMICS JOURNAL 2009; 10:114-9. [DOI: 10.1038/tpj.2009.37] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
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Quintana M, Palicki O, Lucchi G, Ducoroy P, Chambon C, Salles C, Morzel M. Short-Term Modification of Human Salivary Proteome Induced by Two Bitter Tastants, Urea and Quinine. CHEMOSENS PERCEPT 2009. [DOI: 10.1007/s12078-009-9048-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
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Oleksiewicz MB, Kjeldal HO, Klenø TG. Identification of stool proteins in C57BL/6J mice by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. Biomarkers 2008; 10:29-40. [PMID: 16097391 DOI: 10.1080/13547500500038775] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
Gastrointestinal disease is a major cause of mortality in humans and animals, and the detection of disease-associated protein in stool is an established diagnostic method in this context. Yet, no data currently exists about the protein composition of mammalian faeces. Using a newly developed two-dimensional (2D) gel method, 28 of the most abundant proteins in murine faeces were identified. Mammalian faeces contains protein from multiple species (from the individual, from gastrointestinal bacteria, from food, etc.). Yet, it was found that the majority of mouse stool proteins were of mouse origin, with a minority of proteins being derived from food (in particular soybean glycinin and conglycinin) and bacteria (flagellin). Most mouse proteins were proteases and saccharidases derived from the exocrine pancreas. In addition, two unexpected mouse proteins were identified: one was a newly described mucin-like protein from intestinal goblet cells (FcgammaBP); the other was the secreted form of carbonic anhydrase (type VI) from salivary gland. The data suggest that 2D analysis of faecal protein is likely to provide meaningful information about the physiological stage of the gastrointestinal tract. Compared with studies based on biopsies, faecal protein analysis may reduce the number of laboratory animals, and might also allow quicker bridging from animal studies to humans, where biopsy material is more difficult to obtain and is less relevant for general practice use.
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Affiliation(s)
- M B Oleksiewicz
- Department of Virology and Molecular Toxicology, Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark.
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30
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[Caries risk estimation in children regarding values of saliva buffer system components and carboanhydrase activity]. VOJNOSANIT PREGL 2008; 65:676-80. [PMID: 18814503 DOI: 10.2298/vsp0809676s] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022] Open
Abstract
BACKGROUND/AIM One of the preconditions for efficacious systematic reduction of caries prevalence and prophylaxis is the determination of risks of this disease appearance. The aim of this study was to prove the significance of salivary carboanhydrase activity determination in estimation of caries risk in children. METHODS The study included 123 children of average age of 13.4+/-0.3 years and permanent dentition. The children were divided into two groups according to caries risk (low and high caries risk groups). Two samples of saliva--unstimulated and stimulated one were taken from each child. Salivary carboanhydrase activity, as well as pH value, bicarbonate and phosphate buffer levels were estimated in both group of saliva samples. RESULTS The investigation showed significantly higher carboanhydrase activity (p < 0.001) in both saliva samples in low caries risk group compared to high caries risk one. In children with low caries risk, both unstimulated and stimulated saliva show significantly higher bicarbonate and phosphate buffer concentrations (p < 0.001), as well as pH values. CONCLUSION The lower caries incidence could be expected in children with high carboanhydrase activity and higher salivary buffer system parameters levels. The presented results suggest that salivary carboanhydrase activity represents the important marker of individual susceptibility for caries appearance in children.
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Krishnamurthy VM, Kaufman GK, Urbach AR, Gitlin I, Gudiksen KL, Weibel DB, Whitesides GM. Carbonic anhydrase as a model for biophysical and physical-organic studies of proteins and protein-ligand binding. Chem Rev 2008; 108:946-1051. [PMID: 18335973 PMCID: PMC2740730 DOI: 10.1021/cr050262p] [Citation(s) in RCA: 569] [Impact Index Per Article: 33.5] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Affiliation(s)
- Vijay M. Krishnamurthy
- Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138
| | - George K. Kaufman
- Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138
| | - Adam R. Urbach
- Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138
| | - Irina Gitlin
- Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138
| | - Katherine L. Gudiksen
- Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138
| | - Douglas B. Weibel
- Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138
| | - George M. Whitesides
- Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138
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32
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Ichihara N, Tsukamoto A, Kasuya T, Shibata S, Nishita T, Murakami M, Amasaki H, Asari M. Gene expression of secretory carbonic anhydrase isozymes in striated ducts of canine salivary glands using laser microdissection system. Anat Histol Embryol 2007; 36:357-60. [PMID: 17845225 DOI: 10.1111/j.1439-0264.2007.00774.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
To clarify whether striated duct cells in canine salivary glands synthesize secretory carbonic anhydrase (CA-VI), as is the case with serous acinar cells, the present study utilized laser microdissection to harvest striated duct cells from canine parotid and submandibular glands, and total RNA extracted from these cells was then amplified by reverse transcription-polymerase chain reaction to assess CA-VI gene expression. The results confirmed the local expression of CA-VI mRNA in striated duct cells. This clarified that, in canine salivary glands, CA-VI is synthesized in not only serous acinar cells, but also striated duct cells.
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Affiliation(s)
- N Ichihara
- Laboratory of Anatomy I, Azabu University School of Veterinary Medicine, Fuchinobe 1-17-71, Sagamihara, Kanagawa 229-8501, Japan.
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Unterkircher CS, Leão MVP, Carvalho YR. Immunization of Balb/c mice with modified auto-antigens for induction of autoimmune sialoadenitis. Braz Dent J 2007; 18:40-4. [PMID: 17639199 DOI: 10.1590/s0103-64402007000100009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2006] [Accepted: 02/02/2006] [Indexed: 11/22/2022] Open
Abstract
Sjögren's syndrome is an autoimmune disease characterized by sialoadenitis and elevated titers of autoantibodies. To assess whether it is possible to induce inflammatory changes in salivary gland tissues, a series of immunizations in Balb/c mice have been undertaken, using salivary gland extract, modified or not, added to several adjuvants. Mice's humoral immune response to salivary gland antigens was monitored by ELISA. Inflammatory cells infiltrating gland tissue were seen 3 months after immunization with salivary gland extract modified with pepsin (AgGp) and metaperiodate (AgGMp). Although pathological progression was not observed, the histopathological picture was similar to the initial phase of Sjögren's syndrome. In addition, a monoclonal antibody reactive with 3 gland polypeptides and anhydrase carbonic II was rescued among B cells from immunized mice. Thus, immunizations with modified autoantigens were able to initiate pathological damage to glandular tissue and stimulate the proliferation of auto-reactive B cells.
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Affiliation(s)
- Carmelinda Schmidt Unterkircher
- Department of Biosciences and Oral Diagnosis, School of Dentistry of São José dos Campos, São Paulo State University, São José dos Campos, SP, Brazil
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34
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Kasuya T, Shibata S, Kaseda M, Ichihara N, Nishita T, Murakami M, Asari M. Immunohistolocalization and Gene Expression of the Secretory Carbonic Anhydrase Isozymes (CA-VI) in Canine Oral Mucosa, Salivary Glands and Oesophagus. Anat Histol Embryol 2007; 36:53-7. [PMID: 17266669 DOI: 10.1111/j.1439-0264.2006.00721.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
The immunohistolocalization of secretory carbonic anhydrase isoenzymes (CA-VI) in canine salivary glands, parotid, submandibular, sublingual and zygomatic glands, oral and oesophageal mucosa was studied using a specific antiserum against a canine CA-VI. In addition, the gene expression of CA-VI from the same tissue was studied using a real-time reverse-transcriptase polymerase chain reaction. In all salivary glands and oesophageal gland, immunostaining intensely localized CA-VI antiserum throughout the cytoplasm of serous acinar cells, including serous demilune and ductal epithelial cells. In contrast, no immunoreaction localized CA-VI in the mucous acinar cells of the gland. CA-VI gene transcripts were also detected in the same areas. The physiological significance of secretory CA-VI in the oral and oesophageal cavity is thought to play a highly specialized role in the maintenance of bicarbonate level in saliva and to protect mucosa from acid injury. It is shown that the major sites of the CA-VI secretion in dogs were in serous (demilune) secretory cells in all four major salivary glands and oesophageal glands in particular.
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Affiliation(s)
- T Kasuya
- Laboratory of Anatomy I, Azabu University School of Veterinary Medicine, Fuchinobe 1-17-71, Sagamihara, Kanagawa, 229-8501, Japan
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35
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Nishita T, Tanaka Y, Wada Y, Murakami M, Kasuya T, Ichihara N, Matsui K, Asari M. Measurement of Carbonic Anhydrase Isozyme VI (CA-VI) in Bovine Sera, Saliva, Milk and Tissues. Vet Res Commun 2006; 31:83-92. [PMID: 17180448 DOI: 10.1007/s11259-006-3423-0] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/14/2005] [Indexed: 11/27/2022]
Abstract
Concentrations of bovine carbonic anhydrase isozyme VI (CA-IV) in bovine serum, saliva, normal milk, colostrum, submandibular gland, liver, and mammary gland were determined. CA-VI was purified from bovine saliva and an antibody to CA-VI was generated. The concentrations of CA-VI in the saliva (7.8 +/- 7.9 microg/ml), serum (2.1+/- 5.7 ng/ml), milk (7.9 +/- 12.1 ng/ml), submandibular gland (284.7 microg/g protein), liver (921.0 +/- 180.7 ng/g protein) and mammary gland (399.6 +/- 191.2 ng/g protein) were determined by ELISA. No seasonal change in CA-VI levels was observed in normal milk. The concentration of CA-VI in colostrum (day 1 post partum) was 119 ng/ml and decreased rapidly by 1 month following birth. Mammary gland contained much smaller amounts than the submandibular gland. CA-VI mRNA was detected in the liver and mammary gland of cow by RT-PCR. The ELISA used in this study proved to be a precise and sensitive method for determining CA-VI concentrations in saliva, serum, milk and tissue specimens from cows. The ELISA may enable the study of changes in CA-VI associated with hereditary or metabolic disorders of the salivary gland, mammary gland and liver using small samples of saliva, serum or milk.
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Affiliation(s)
- T Nishita
- Laboratory of Veterinary Physiology 1, School of Veterinary Medicine, Azabu University, Kanagawa, Japan.
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36
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Kaseda M, Ichihara N, Nishita T, Amasaki H, Asari M. Immunohistochemistry of the Bovine Secretory Carbonic Anhydrase Isozyme (CA-VI) in Bovine Alimentary Canal and Major Salivary Glands. J Vet Med Sci 2006; 68:131-5. [PMID: 16520534 DOI: 10.1292/jvms.68.131] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
In the present study, we firstly demonstrated immunohistochemical expressions of secretory carbonic anhydrase (CA-VI) isozyme in bovine forestomach, large intestine and major salivary glands. CA-VI was detected in basal layer epithelial cells of esophageal and forestomach stratified epithelium, in mucous cells of upper glandular region of large intestine, in serous acinar cells of the parotid gland, in serous demilune cells and some ductal liner cells of mandibular, monostomatic sublingual and esophageal glands. These immunohistolocalizations suggested that bovine CA-VI plays various roles in pH regulation, maintenance of ion and fluid balance, and cell proliferation.
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Affiliation(s)
- Misato Kaseda
- Department of Anatomy, School of Veterinary Medicine, Azabu University, 1-17-71 fuchinobe, Sagamihara-shi, Kanagawa 229-8501, Japan
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37
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Kimoto M, Kishino M, Yura Y, Ogawa Y. A role of salivary carbonic anhydrase VI in dental plaque. Arch Oral Biol 2005; 51:117-22. [PMID: 15961059 DOI: 10.1016/j.archoralbio.2005.04.007] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2005] [Accepted: 04/26/2005] [Indexed: 12/25/2022]
Abstract
OBJECTIVE Carbonic anhydrase (CA) VI is a unique secreted isozyme of CA, which catalyzes the reversible reaction CO2 +H2O<-->H+ +HCO3-. CA VI has been thought to provide a greater buffering capacity to fluids into which it is secreted. This study was performed to confirm this in saliva. DESIGN Nine healthy subjects participated in the study. The pH of the dental plaque from each subject was monitored after a mouth rinse with 10% sucrose with or without 10(-5)M acetazolamide, a specific inhibitor of CA. Also CA was examined in plaque by enzyme histochemistry, immunohistochemistry and Western blot analysis. RESULTS Though sucrose and sucrose plus inhibitor yielded Stephan curves with a similar temporal pattern, the pH values of the latter were significantly lower than those of the former. Plaque exhibited CA activity by enzyme histochemistry. Immunohistochemistry and Western analysis demonstrated that the activity was due to CA VI but not to CA I or CA II. CONCLUSIONS The results indicate that CA VI in saliva penetrates plaque and facilitates acid neutralization by salivary bicarbonate. Therefore, CA VI may be considered an anti-caries protein in saliva.
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Affiliation(s)
- Masaya Kimoto
- Department of Oral Pathology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan
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Kleinke T, Wagner S, John H, Hewett-Emmett D, Parkkila S, Forssmann WG, Gros G. A distinct carbonic anhydrase in the mucus of the colon of humans and other mammals. J Exp Biol 2005; 208:487-96. [PMID: 15671337 DOI: 10.1242/jeb.01398] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
SUMMARY
We have collected gastrointestinal, mainly colonic, mucus from humans,guinea pigs, rats, and normal and carbonic anhydrase II (CAII)-deficient mice. In the mucus of all species, substantial CA activity was present. Using antibodies against human CA isoforms we found that the human mucus CA differs from cytosolic CAI and CAII, membrane-bound CAIV, and the secreted CAVI of saliva. The high sensitivity of mucus CA to acetazolamide rules out its identity with cytosolic CAIII. Partial sequences obtained from the purified human mucus CA show similarity, but not identity, with human CAI, and clear differences from the other known CAs. Additional evidence concerning the CA isoform present in mucus was obtained for the mucus CA of other species and was derived from: (1) the mucus of CAII-deficient mice, whose high CA activity confirms that it is not CAII that is responsible; (2) the inhibitory effect of iodide, which shows that mucus CA from mice, guinea pig and humans does not have the high anion sensitivity of CAI; (3) the inactivating effect of 0.2%SDS on guinea pig, mouse and human mucus CA, ruling out the SDS-resistant CAIV; and (4) the partitioning of guinea-pig mucus CA into the water phase in Triton X114 phase separation experiments, which also argues against its identity with membrane-bound CAs, such as CAIV. A comparison of colonic mucus CA activity in normal and germ-free rats indicates that the mucus CA is not of bacterial origin but is produced by the gastrointestinal tissues. We conclude(from its immunoreactivity, from iodide inhibition and from partial amino acid sequences) that mucus CA of human origin probably represents an isozyme, which is specific for mucus and is not identical with the known CA isozymes. The results obtained for mucus CA of other species collectively point in the same direction.
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Affiliation(s)
- Tanja Kleinke
- Zentrum Physiologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany
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39
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Kivelä AJ, Kivelä J, Saarnio J, Parkkila S. Carbonic anhydrases in normal gastrointestinal tract and gastrointestinal tumours. World J Gastroenterol 2005; 11:155-63. [PMID: 15633208 PMCID: PMC4205394 DOI: 10.3748/wjg.v11.i2.155] [Citation(s) in RCA: 115] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Carbonic anhydrases (CAs) catalyse the hydration of CO2 to bicarbonate at physiological pH. This chemical interconversion is crucial since HCO3- is the substrate for several biosynthetic reactions. This review is focused on the distribution and role of CA isoenzymes in both normal and pathological gastrointestinal (GI) tract tissues. It has been known for many years that CAs are widely present in the GI tract and play important roles in several physiological functions such as production of saliva, gastric acid, bile, and pancreatic juice as well as in absorption of salt and water in intestine. New information suggests that these enzymes participate in several processes that were not envisioned earlier. Especially, the recent reports on plasma membrane-bound isoenzymes IX and XII have raised considerable interest since they were reported to participate in cancer invasion and spread. They are induced by tumour hypoxia and may also play a role in von Hippel-Lindau (VHL)-mediated carcinogenesis.
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Affiliation(s)
- Antti-J Kivelä
- Department of Anatomy and Cell Biology, University of Oulu, Finland.
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40
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Leinonen JS, Saari KA, Seppänen JM, Myllylä HM, Rajaniemi HJ. Immunohistochemical demonstration of carbonic anhydrase isoenzyme VI (CA VI) expression in rat lower airways and lung. J Histochem Cytochem 2004; 52:1107-12. [PMID: 15258187 DOI: 10.1369/jhc.4a6282.2004] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Carbonic anhydrase isoenzyme VI (CA VI), which is transported in high concentrations in saliva and milk into the alimentary tract, is an important element of mucosal protection in the upper alimentary tract. Like alimentary tract mucosa, the respiratory tract mucosa is also exposed to heavy microbial, physical, and chemical stress. The protective and renewal-promoting factors present in the surface mucus of the respiratory tract are mainly produced by the seromucous tracheobronchial glands. Here we studied the secretion of CA VI by these glands in adult and developing rats using immunohistochemical techniques. The serous acinar and duct cells of the tracheobronchial glands stained for CA VI. The presence of the enzyme also in the duct content indicates its active secretion into the surface mucus. CA VI was also visible in the secretory cells and at the base of the ciliated cells of the tracheobronchial surface epithelium. Moreover, the Clara cells of the bronchiolar surface epithelium stained for CA VI. These findings are consistent with the hypothesis that CA VI has a mucosa-protective role not only in the gastrointestinal tract but also in the respiratory tract, where CA VI may act as a pivotal pH neutralizer and growth factor.
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Affiliation(s)
- Jukka S Leinonen
- Department of Anatomy and Cell Biology, PO Box 5000, 90014 University of Oulu, Oulu, Finland.
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41
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Murakami M, Kasuya T, Matsuba C, Ichihara N, Nishita T, Fujitani H, Asari M. Nucleotide sequence and expression of a cDNA encoding canine carbonic anhydrase VI (CA-VI). ACTA ACUST UNITED AC 2004; 14:195-8. [PMID: 14509831 DOI: 10.1080/1042517031000112633] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
A full-length cDNA clone of a canine carbonic anhydrase VI (CA-VI) was generated from the canine parotid gland by using a reverse transcription-polymerase chain reaction (RT-PCR) technique with degenerate primers designed from conserved regions of the same locus in humans and bovines employing RACE (rapid amplification of cDNA ends) techniques. The cDNA sequence was 1351 base pairs (bp) long and was predicted to encode a 320-amino-acid polypeptide containing a putative signal peptide of 17 amino acids. The deduced amino acid sequence of mature CA-VI showed the highest similarity of 74% to that of human CA-VI. RT-PCR analysis with primers specific to the canine CA-VI demonstrated strong signals in the major salivary glands and weak signals in the minor salivary glands and esophagus of a healthy dog. No CA-VI mRNA was detected in the pancreas, liver or the digestive tract except the esophagus.
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Affiliation(s)
- Masaru Murakami
- Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 229-8501, Japan.
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Kitade K, Nishita T, Yamato M, Sakamoto K, Hagino A, Katoh K, Obara Y. Expression and localization of carbonic anhydrase in bovine mammary gland and secretion in milk. Comp Biochem Physiol A Mol Integr Physiol 2003; 134:349-54. [PMID: 12547264 DOI: 10.1016/s1095-6433(02)00268-4] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Little attention has been paid to carbonic anhydrase VI (CA VI), a secretory type isozyme, in the bovine mammary gland, although the gland is an important exocrine gland and CA VI is known to localize in exocrine glands such as salivary and lacrimal glands in various animal species. In the present study mRNA expression and protein localization of CA VI in isolated gland tissues and in cloned epithelial cells from the mammary gland of Holstein cows (Bos taurus) were observed by reverse transcript polymerase chain reaction and immunocytochemistry. Also, changes of CA VI concentrations in milk were measured for 2 months postpartum by an enzyme-linked immunosorbent assay. CA VI gene expression was detected in the gland tissues and epithelial cells, and CA VI protein was localized in the cytoplasm of the epithelial cells. Colostrum contained the highest concentration of CA VI protein (100 ng/ml), decreasing in an exponential manner (P<0.001). We conclude that bovine mammary epithelial cells synthesize and secrete CA VI in colostrum at higher concentration than in normal milk, implying its role to compensate for low CA VI secretion in neonatal calves.
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Affiliation(s)
- Keitaro Kitade
- Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, 981-8555, Sendai, Japan.
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Axford SE, Sharp N, Ross PE, Pearson JP, Dettmar PW, Panetti M, Koufman JA. Cell biology of laryngeal epithelial defenses in health and disease: preliminary studies. Ann Otol Rhinol Laryngol 2001; 110:1099-108. [PMID: 11768697 DOI: 10.1177/000348940111001203] [Citation(s) in RCA: 101] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Esophageal epithelium has intrinsic antireflux defenses, including carbonic anhydrases (CAs I to IV) that appear to be protective against gastric reflux. This study aimed to investigate the expression and distribution of CA isoenzymes in laryngeal epithelium. Laryngeal biopsy specimens collected from the vocal fold and interarytenoid regions were analyzed by Western blotting and immunofluorescence. Carbonic anhydrases I and II were expressed by the majority of samples analyzed. In contrast, CA III was differentially expressed in the interarytenoid samples and was not detected in any vocal fold samples. The expression of CA III was increased in esophagitis as compared to normal esophageal tissue. Carbonic anhydrase I and III isoenzymes were distributed cytoplasmically in the basal and lower prickle cell layers. The laryngeal epithelium expresses some CA isoenzymes and has the potential to protect itself against laryngopharyngeal reflux. Laryngeal tissue may be more sensitive to injury due to reflux damage than the esophageal mucosa because of different responses of CA isoenzymes.
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Affiliation(s)
- S E Axford
- Department of Molecular and Cellular Pathology, Ninewells Hospital and Medical School, University of Dundee, Scotland
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Saarnio J, Parkkila S, Parkkila AK, Pastoreková S, Haukipuro K, Pastorek J, Juvonen T, Karttunen TJ. Transmembrane carbonic anhydrase, MN/CA IX, is a potential biomarker for biliary tumours. J Hepatol 2001; 35:643-9. [PMID: 11690711 DOI: 10.1016/s0168-8278(01)00193-3] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
BACKGROUND/AIMS Carbonic anhydrase isoenzyme IX (MN/CA IX) is a transmembrane protein with a suggested function in maintaining the acid-base balance and intercellular communication. Previous studies have demonstrated that MN/CA IX is expressed in the basolateral plasma membrane of normal biliary epithelial cells, but not in hepatocytes. This study was designed to examine the expression of MN/CA IX in hepatobiliary neoplasms and to elucidate its value as a marker for biliary differentiation. METHODS Fifty-seven hepatobiliary lesions were immunostained for MN/CA IX using biotin-streptavidin complex method. Twenty samples containing normal biliary epithelium and five containing normal liver tissue were used as controls. RESULTS In the biliary epithelial tumours, immunostaining for MN/CA IX was mainly localized at the basolateral surface of the epithelial cells, like in normal mucosa. All non-invasive dysplastic lesions and 57% of invasive lesions of gall-bladder expressed MN/CA IX. In liver, 78% of cholangiocellular malignant lesions showed a positive reaction for MN/CA IX, whereas only 33% of hepatocellular carcinomas showed a weak immunoreaction. CONCLUSIONS Our results suggest that abnormal expression of MN/CA IX may be linked to malignant transformation of hepatobiliary cells. In addition, it seems to be a promising marker for biliary differentiation in hepatobiliary neoplasms.
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Affiliation(s)
- J Saarnio
- Department of Surgery, University of Oulu, Kajaanintie 52A, FIN-90220 Oulu, Finland.
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Nishita T, Sakomoto M, Ikeda T, Amasaki H, Shino M. Purification of carbonic anhydrase isozyme VI (CA-VI) from swine saliva. J Vet Med Sci 2001; 63:1147-9. [PMID: 11714035 DOI: 10.1292/jvms.63.1147] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Salivary or secreted carbonic anhydrase (CA), which constitutes a new class of CA, designated CA-VI, was isolated. Swine CA-VI purified from swine saliva by inhibitor-affinity chromatography and ion exchange chromatography had a specific activity of 5,468 units/mg. The molecular weight was 250,000, as determined by gel filtration under non-denaturing conditions, and the subunit molecular weight was found to be 37,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that swine CA-VI consists of 7 subunits. The treatment of the enzyme with endo-N-acetylglucosaminidase F reduced its subunit molecular weight from 37,000 to 35,000 and 32,000. We raised a rabbit antibody against purfied swine CA-VI. Double immunodiffusion showed that anti-swine CA-VI serum reacted with swine CA-VI and swine saliva, but not with hemolysate (containing CA-I and CA-Il) or muscle extracts (containing CA-III). The concentration of CA-VI in swine saliva, measured using single radial immunodiffusion, was 0.027 +/- 0.017 mg/mg total protein.
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Affiliation(s)
- T Nishita
- Laboratories of Veterinary Physiology 1, Azabu University, Sagamihara, Kanagawa, Japan
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Karhumaa P, Leinonen J, Parkkila S, Kaunisto K, Tapanainen J, Rajaniemi H. The identification of secreted carbonic anhydrase VI as a constitutive glycoprotein of human and rat milk. Proc Natl Acad Sci U S A 2001; 98:11604-8. [PMID: 11553764 PMCID: PMC58776 DOI: 10.1073/pnas.121172598] [Citation(s) in RCA: 83] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2000] [Accepted: 04/09/2001] [Indexed: 01/11/2023] Open
Abstract
In addition to essential nutrients, human milk contains several classes of bioactive factors such as enzymes, hormones, and growth factors, many of which are implicated in infantile growth and development. Secretory carbonic anhydrase isoenzyme VI (CA VI) has been identified earlier as an essential component of mammalian saliva, and we demonstrate here by using biochemical and immunohistochemical techniques that it is also an elementary component of milk. The 42-kDa glycopolypeptide purified from human milk in CA inhibitor affinity chromatography shared 100% homology with salivary CA VI in the protein sequence analysis (40% coverage), and its digestion with PNGase F resulted in a polypeptide backbone similar in size to salivary CA VI. Quantification of CA VI in milk by using a time-resolved immunofluorometric assay revealed an approximately eight-times-higher concentration in human colostrum than in mature milk, the latter corresponding to the levels previously detected in human saliva. The high concentration in the colostrum, in particular its functional and structural stability in an acidic milieu, and its growth-supporting role in the taste buds suggest that milk CA VI is an essential factor in normal growth and development of the infant alimentary tract.
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Affiliation(s)
- P Karhumaa
- Department of Anatomy and Cell Biology, University of Oulu, FIN-90014 Oulu, Finland.
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Affiliation(s)
- W S Sly
- Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104, USA
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Abstract
Recently, an increasing number of carbonic anhydrase (CA) isozymes have been discovered in the human pancreas. These isozymes are classified as the CA family with various molecular structures and different subcellular localizations: cytoplasmic CA II, mitochondrial CA VB, secretory CA VI, membrane-bound CA IV, and transmembrane CA IX and XII. However, there is little evidence concerning their pathophysiological roles. Here, we reviewed the expression of CA isozymes in the human pancreas and proposed hypotheses related to their physiological and pathological roles.
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Affiliation(s)
- I Nishimori
- First Department of Internal Medicine, Kochi Medical School, Japan.
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Christie KN, Thomson C. The distribution of carbonic anhydrase II in human, pig and rat oesophageal epithelium. THE HISTOCHEMICAL JOURNAL 2000; 32:753-7. [PMID: 11254091 DOI: 10.1023/a:1004105228370] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Carbonic anhydrase II (CA II) is present in human oesophageal epithelial cells and probably involved in protecting the mucosa against acidic gastric refluxate. If this is the case, then it is likely that the enzyme will be more concentrated at or near the gastro-oesophageal junction. To answer this question, and determine whether CA II is present and similarly distributed in other species, we also examined the oesophageal epithelium of the rat and pig. In the rat, CA II was largely absent from the oesophageal epithelium, but present in the stratified squamous epithelium of the gastric forestomach as an approximately 2 mm-long collar around the entrance to the corpus, a site that roughly corresponds to the gastro-oesophageal junction in other animals. The enzyme was present mainly in basal and prickle cells. In upper and middle pig oesophagus, CA II was largely confined to basal cells and isolated groups of stratified superficial prickle cells. CA II-containing epithelial cells were highly concentrated in the thickened epithelium at the gastro-oesophageal junction (about four-times thicker than upper or middle). Reactive cells were present throughout the depth of the epithelium, but noticeably more concentrated in the basal and superficial prickle cell layers. CA II was also prominent in the most superficial cell layers in islands of the oesophageal mucosa within the gastric cardia. In man, CA II was confined largely to the basal half of the epithelium in the upper and middle regions of oesophagus. The distribution of CA II at the gastro-oesophageal junction took different forms. In general, there were more CA II-reactive cells at or closer to the lumen. The superficial prickle cell layers tended to exhibit more CA II than the deeper layers, with basal and epibasal cells containing little or no enzyme. In other regions of the same specimens, CA II-containing cells were present from the basal to the most luminal layers. If CA II in oesophageal epithelial cells in the region of the gastro-oesophageal junction (or in the case of the rat the forestomach/corpus junction) is important in the defence against refluxate, then it is in a vulnerable site, since bile salts are potent inhibitors of the enzyme. The action of bile salts on CA II may be an important factor in the initiation of oesophageal disease.
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Affiliation(s)
- K N Christie
- Department of Anatomy & Physiology, University of Dundee, United Kingdom
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Henkin RI, Martin BM, Agarwal RP. Decreased parotid saliva gustin/carbonic anhydrase VI secretion: an enzyme disorder manifested by gustatory and olfactory dysfunction. Am J Med Sci 1999; 318:380-91. [PMID: 10616163 DOI: 10.1097/00000441-199912000-00005] [Citation(s) in RCA: 70] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
BACKGROUND Taste and smell dysfunction has been reported to occur in patients with a variety of clinical problems. We wanted to investigate a specific group of patients in whom taste and smell dysfunction occurred putatively related to a specific biochemical abnormality in a salivary growth factor [gustin/carbonic anhydrase (CA) VI] considered responsible for maintenance of taste bud function. METHODS Eighteen patients developed loss and/or distortion of taste and smell after an acute influenza-type illness. They were evaluated clinically, by psychophysical tests of taste and smell function, by measurement of parotid salivary gustin/CAVI by a radioimmunoassay and by measurement of serum, urine, and salivary zinc. Biopsies of circumvallate papillae were obtained in 6 patients and examined by transmission electron microscopy. Similar studies were performed in 55 asymptomatic volunteers with biopsies of circumvallate papillae performed in 4. RESULTS Taste and smell acuity were impaired in patients compared with healthy volunteers and parotid gustin/CAVI, salivary, and serum zinc concentrations were lower in patients than in healthy volunteers. Taste buds in circumvallate papillae of patients exhibited severe vacuolization, cellular degeneration, and absence of dense extracellular material. CONCLUSIONS These results describe a clinical disorder formulated as a syndrome of hyposmia (decreased smell acuity), hypogeusia (decreased taste acuity), dysosmia (distorted smell function), dysgeusia (distorted taste function), and decreased secretion of parotid saliva gustin/CAVI with associated pathological changes in taste bud anatomy. Because gustin/CAVI is found in humans only in parotid saliva and has been associated with taste bud growth and development these results suggest that inhibition of synthesis of gustin/CAVI is associated with development of taste bud abnormalities and thereby loss of taste function.
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Affiliation(s)
- R I Henkin
- The Taste and Smell Clinic, Washington, DC 20016, USA
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