RESUMEN
An application of CO2/HCO3--free solution (Zero-CO2) did not increase intracellular pH (pHi) in ciliated human nasal epithelial cells (c-hNECs), leading to no increase in frequency (CBF) or amplitude (CBA) of the ciliary beating. This study demonstrated that the pHi of c-hNECs expressing carbonic anhydrase IV (CAIV) is high (7.64), while the pHi of ciliated human bronchial epithelial cells (c-hBECs) expressing no CAIV is low (7.10). An extremely high pHi of c-hNECs caused pHi, CBF and CBA to decrease upon Zero-CO2 application, while a low pHi of c-hBECs caused them to increase. An extremely high pHi was generated by a high rate of HCO3- influx via interactions between CAIV and Na+/HCO3- cotransport (NBC) in c-hNECs. An NBC inhibitor (S0859) decreased pHi, CBF and CBA and increased CBF and CBA in c-hNECs upon Zero-CO2 application. In conclusion, the interactions of CAIV and NBC maximize HCO3- influx to increase pHi in c-hNECs. This novel mechanism causes pHi to decrease, leading to no increase in CBF and CBA in c-hNECs upon Zero-CO2 application, and appears to play a crucial role in maintaining pHi, CBF and CBA in c-hNECs periodically exposed to air (0.04% CO2) with respiration.
Asunto(s)
Bicarbonatos , Dióxido de Carbono , Anhidrasa Carbónica IV , Cilios , Células Epiteliales , Mucosa Nasal , Humanos , Concentración de Iones de Hidrógeno , Dióxido de Carbono/metabolismo , Cilios/metabolismo , Bicarbonatos/metabolismo , Células Epiteliales/metabolismo , Mucosa Nasal/metabolismo , Mucosa Nasal/citología , Anhidrasa Carbónica IV/metabolismo , Anhidrasa Carbónica IV/genética , Células Cultivadas , Simportadores de Sodio-Bicarbonato/metabolismo , Simportadores de Sodio-Bicarbonato/genéticaRESUMEN
Carbonic anhydrase IV (Car4) is a newly identified receptor that allows adeno-associated virus (AAV) 9P31 to cross the blood-brain barrier and achieve efficient infection in the central nervous system (CNS) in mouse models. However, the molecular mechanism by which engineered AAV capsids with 7-mer insertion in the variable region (VR) VIII recognize these novel cellular receptors is unknown. Here we report the cryo-EM structures of AAV9P31 and its complex with Mus musculus Car4 at atomic resolution by utilizing the block-based reconstruction (BBR) method. The structures demonstrated that Car4 binds to the protrusions at 3-fold axes of the capsid. The inserted 7-mer extends into a hydrophobic region near the catalytic center of Car4 to form stable interactions. Mutagenesis studies also identified the key residues in Car4 responsible for the AAV9P31 interaction. These findings provide new insights into the novel receptor recognition mechanism of AAV generated by directed evolution and highlight the application of the BBR method to studying the virus-receptor molecular mechanism.
Asunto(s)
Anhidrasa Carbónica IV , Dependovirus , Animales , Ratones , Dependovirus/genética , Anhidrasa Carbónica IV/análisis , Anhidrasa Carbónica IV/metabolismo , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Barrera Hematoencefálica/metabolismo , Vectores GenéticosRESUMEN
An increasing number of studies reveal the deleterious effects of isoflurane (Iso) exposure during pregnancy on offspring cognition. However, no effective therapeutic strategy for Iso-induced deleterious effects has been well developed. Angelicin exerts an anti-inflammatory effect on neurons and glial cells. This study investigated the roles and mechanism of action of angelicin in Iso-induced neurotoxicity in vitro and in vivo. After exposing C57BL/6 J mice on embryonic day 15 (E15) to Iso for 3 and 6 h, respectively, neonatal mice on embryonic day 18 (E18) displayed obvious anesthetic neurotoxicity, which was revealed by the elevation of cerebral inflammatory factors and blood-brain barrier (BBB) permeability and cognitive dysfunction in mice. Angelicin treatment could not only significantly reduce the Iso-induced embryonic inflammation and BBB disruption but also improve the cognitive dysfunction of offspring mice. Iso exposure resulted in an increase of carbonic anhydrase (CA) 4 and aquaporin-4 (AQP4) expression at both mRNA and protein levels in vascular endothelial cells and mouse brain tissue collected from neonatal mice on E18. Remarkably, the Iso-induced upregulation of CA4 and AQP4 expression could be partially reversed by angelicin treatment. Moreover, GSK1016790A, an AQP4 agonist, was used to confirm the role of AQP4 in the protective effect of angelicin. Results showed that GSK1016790A abolished the therapeutic effect of angelicin on Iso-induced inflammation and BBB disruption in the embryonic brain and on the cognitive function of offspring mice. In conclusion, angelicin may serve as a potential therapeutic for Iso-induced neurotoxicity in neonatal mice by regulating the CA4/AQP4 pathway.
Asunto(s)
Disfunción Cognitiva , Furocumarinas , Isoflurano , Leucina/análogos & derivados , Sulfonamidas , Embarazo , Femenino , Ratones , Animales , Isoflurano/toxicidad , Anhidrasa Carbónica IV/metabolismo , Células Endoteliales/metabolismo , Ratones Endogámicos C57BL , Acuaporina 4/genética , Acuaporina 4/metabolismo , Furocumarinas/efectos adversos , Inflamación , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/tratamiento farmacológico , Disfunción Cognitiva/psicología , CogniciónRESUMEN
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.
Asunto(s)
Anhidrasas Carbónicas , Lagartos , Animales , Anhidrasa Carbónica IV , Dióxido de Carbono , CatálisisRESUMEN
Therapeutic irradiation for cancers of the head and neck causes serious and irreversible damage to the salivary glands; the resulting adverse effects on salivary quality and quantity produce detrimental effects on teeth and oral mucosa. The salivary effects have been related mostly to loss of serous acini; damage to the ducts is relatively minor. Other radiation effects include fibrosis, adiposis and vascular damage. Stem cells in the salivary gland ducts have the potential to generate acinar cells in vitro and in vivo. I investigated the ducts and vasculature in irradiated and normal human submandibular glands using immunohistochemical localization of biomarkers of stem cells, duct function and blood vessels. Stem cell markers CK5 and Sca-1 labeled the cytoplasm of the basal and intercalated duct cells and all duct cells, respectively, in both normal and irradiated glands. CA IV, which participates in regulating salivary electrolytes and acid-base balance, labeled the cytoplasm of all ducts. CD34 labeling demonstrated more extensive vasculature in the irradiated glands than in the normal glands. My findings suggest that duct stem cells and at least one duct function persisted, and the vasculature was greater, despite moderate fibrosis, in the irradiated gland.
Asunto(s)
Anhidrasa Carbónica IV , Glándula Submandibular , Humanos , Glándulas Salivales , Citoplasma , Células Madre , Conductos SalivalesRESUMEN
The blood-brain barrier (BBB) presents a major challenge for delivering large molecules to study and treat the central nervous system. This is due in part to the scarcity of targets known to mediate BBB crossing. To identify novel targets, we leverage a panel of adeno-associated viruses (AAVs) previously identified through mechanism-agnostic directed evolution for improved BBB transcytosis. Screening potential cognate receptors for enhanced BBB crossing, we identify two targets: murine-restricted LY6C1 and widely conserved carbonic anhydrase IV (CA-IV). We apply AlphaFold-based in silico methods to generate capsid-receptor binding models to predict the affinity of AAVs for these identified receptors. Demonstrating how these tools can unlock target-focused engineering strategies, we create an enhanced LY6C1-binding vector, AAV-PHP.eC, that, unlike our prior PHP.eB, also works in Ly6a-deficient mouse strains such as BALB/cJ. Combined with structural insights from computational modeling, the identification of primate-conserved CA-IV enables the design of more specific and potent human brain-penetrant chemicals and biologicals, including gene delivery vectors.
Asunto(s)
Barrera Hematoencefálica , Anhidrasa Carbónica IV , Ratones , Humanos , Animales , Barrera Hematoencefálica/metabolismo , Anhidrasa Carbónica IV/genética , Anhidrasa Carbónica IV/metabolismo , Encéfalo/metabolismo , Técnicas de Transferencia de Gen , Primates/genética , Dependovirus/genética , Dependovirus/metabolismoRESUMEN
BACKGROUND: Carbonic anhydrase 4 (CA4) is a member of a large family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide and was found to have low expression in non-small cell lung cancer (NSCLC). However, the specific role of CA4 in NSCLC and the underlying mechanisms remain unknown. METHODS: The bioinformatic analysis on lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) datasets downloaded from The Cancer Genome Atlas (TCGA) database was performed. We found that CA4 expression was lower in tumors than that in normal tissues, which were verified by Real-time PCR. Lower CA4 levels were significantly associated with higher T stages in LUAD and LUSC cohorts. Multivariate analysis showed that CA4 is an independent prognostic factor for NSCLC. Furthermore, the expression of CA4 also correlated with immune infiltration and drug sensitivity. RESULTS: Ectopic expression of CA4 decreased NSCLC cell proliferation in vitro by CCK-8 assay. CA4 caused G0/G1 cell cycle arrest by cell experiments. Mechanistic studies found that CA affects the cell cycle and inhibits cell proliferation by downregulating the expression of CDK2. CONCLUSION: The present findings highlight the role of CA4 in NSCLC and identify CA4 as a potential novel diagnostic and prognostic biomarker for the treatment of NSCLC.
Asunto(s)
Adenocarcinoma del Pulmón , Carcinoma de Pulmón de Células no Pequeñas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Humanos , Adenocarcinoma del Pulmón/genética , Biomarcadores , Anhidrasa Carbónica IV , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , PronósticoRESUMEN
The functional role of membrane-bound carbonic anhydrases (CAs) has been of keen interest in the past decade, and in particular, studies have linked CA in red muscle, heart, and eye to enhanced tissue oxygen extraction in bony fishes (teleosts). However, the number of purported membrane-bound CA isoforms in teleosts, combined with the imperfect system of CA isoform nomenclature, present roadblocks for ascribing physiological functions to particular CA isoforms across different teleost lineages. Here we developed an organizational framework for membrane-bound CAs in teleosts, providing the latest phylogenetic analysis of extant CA4 and CA4-like isoforms. Our data confirm that there are three distinct isoforms of CA4 (a, b, and c) that are conserved across major teleost lineages, with the exception of CA4c gene being lost in salmonids. Tissue distribution analyses suggest CA4a functions in oxygen delivery across teleost lineages, while CA4b may be specialized for renal acid-base balance and ion regulation. This work provides an important foundation for researchers to elucidate the functional significance of CA4 isoforms in fishes.
Asunto(s)
Anhidrasa Carbónica IV , Anhidrasas Carbónicas , Animales , Anhidrasa Carbónica IV/genética , Filogenia , Anhidrasas Carbónicas/genética , Isoformas de Proteínas/genética , Peces/genética , OxígenoRESUMEN
A library of twenty-two arylthiazolylhydrazono-1,2,3-triazoles incorporating sulfanilamide and metanilamide moieties have been synthesized by utilizing tail-approach and characterized by their IR, 1H NMR, 13C NMR, HRMS and single crystal studies. Further, these newly synthesized compounds were screened in-vitro for their inhibition efficacy against physiologically relevant hCA I, II, IV and IX isoforms. Inhibition data revealed that, in broader sense, sulfanilamide analogues (4a-4k) were comparatively better inhibitors of cytosolic hCA I and II isoforms than metanilamide analogues (5a-5k), whereas exactly opposite trend was observed in case of inhibition of membrane bound hCA IV and transmembrane hCA IX. For hCA I, more than half of the synthesized compounds were found to be moderate inhibitors and three compounds 4b, 5b and 5e (Ki of 40.6, 224.7 and 74.4 nM, respectively) appeared as better inhibitors than reference drug AAZ (Ki = 250 nM). hCA II was potently inhibited by 4e-4g and 5e with Ki of 18.1, 14.1, 14.9 and 17.8 nM, respectively. Interestingly, 4e-4g selectively inhibited hCA II with selectivity of > 15-fold over hCA I, IV and IX isoforms. All the compounds presented moderate to weak inhibition profiles against glaucoma associated hCA IV with Ki of 88 nM-8.87 µM and except 4f, 5k, significant inhibition profiles against tumor associated hCA IX isoform with Ki spanning in range of 0.113 µM-0.318 µM. Moreover, 5e was the only compound among the whole series which effectively inhibited all the tested isoforms.
Asunto(s)
Anhidrasa Carbónica I , Inhibidores de Anhidrasa Carbónica , Antígenos de Neoplasias , Anhidrasa Carbónica I/metabolismo , Anhidrasa Carbónica II , Anhidrasa Carbónica IV , Anhidrasa Carbónica IX , Inhibidores de Anhidrasa Carbónica/química , Humanos , Estructura Molecular , Relación Estructura-Actividad , Sulfanilamida , Sulfonamidas/química , Triazoles/química , Triazoles/farmacologíaRESUMEN
Pulmonary cartilage plays a crucial structural role determining the physiologic airway compressibility and distensibility, necessary for proper mechanical function. This functionality deteriorates with aging due to increased stiffness of both airway muscle and cartilage, as well as, decreased renewal capacity. Altered airway remodeling has been suggested as a pathogenic driver of chronic obstructive pulmonary disease (COPD) through mechanisms still incompletely understood. Using paraffin-embedded lung tissue sections from archived autopsy material from COPD with non-COPD age matched controls a histopathologic analysis focused on inflammation, fibrosis and calcification was performed with special stains (Masson's trichrome and Von Kossa) and immunohistochemistry for carbonic anhydrase IV (CA IV) and Ki-67. COPD lung tissues showed increased peribronchial inflammation compared to the non-COPD. Coarse amphophilic crystalline deposits in bronchial cartilage were more frequently observed in COPD sections, which were compatible with early dystrophic calcification of the extracellular matrix and chondrocytes. Moreover, Von Kossa staining revealed a significant calcium deposition in the cartilages from COPD in comparison to the controls. Interestingly, Ki-67 immunostains demonstrated a higher overall proliferative rate, including epithelial cells, in COPD. Furthermore, Masson's trichrome staining revealed relatively increased peribronchial collagen deposition associated with a fibrotic stromal response, which may be secondary to the inflammatory milieu in COPD. To further characterize the tissue microenvironment associated with dystrophic calcification, immunohistochemistry for CA IV was used, revealing significantly increased expression in chondrocytes and peribronchial tissue in COPD. Our findings demonstrate that dystrophic calcification of the extracellular matrix and chondrocytes can be linked to CA IV expression in COPD and suggest that pH changes in pulmonary tissue associated with inflammation and calcification may play an active role in COPD.
Asunto(s)
Anhidrasa Carbónica IV/genética , Anhidrasas Carbónicas , Enfermedad Pulmonar Obstructiva Crónica , Bronquios/metabolismo , Anhidrasa Carbónica IV/metabolismo , Cartílago/metabolismo , Cartílago/patología , Fibrosis , Humanos , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patologíaRESUMEN
Primary sulfonamide derivatives with various heterocycles represent the most widespread group of potential human carbonic anhydrase (hCA) inhibitors with high affinity and selectivity towards specific isozymes from the hCA family. In this work, new 4-aminomethyl- and aminoethyl-benzenesulfonamide derivatives with 1,3,5-triazine disubstituted with a pair of identical amino acids, possessing a polar (Ser, Thr, Asn, Gln) and non-polar (Ala, Tyr, Trp) side chain, have been synthesized. The optimized synthetic, purification, and isolation procedures provided several pronounced benefits such as a short reaction time (in sodium bicarbonate aqueous medium), satisfactory yields for the majority of new products (20.6-91.8%, average 60.4%), an effective, well defined semi-preparative RP-C18 liquid chromatography (LC) isolation of desired products with a high purity (>97%), as well as preservation of green chemistry principles. These newly synthesized conjugates, plus their 4-aminobenzenesulfonamide analogues prepared previously, have been investigated in in vitro inhibition studies towards hCA I, II, IV and tumor-associated isozymes IX and XII. The experimental results revealed the strongest inhibition of hCA XII with low nanomolar inhibitory constants (Kis) for the derivatives with amino acids possessing non-polar side chains (7.5-9.6 nM). Various derivatives were also promising for some other isozymes.
Asunto(s)
Aminoácidos/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/farmacología , Sulfonamidas/química , Triazinas/química , Antígenos de Neoplasias , Anhidrasa Carbónica I/antagonistas & inhibidores , Anhidrasa Carbónica II/antagonistas & inhibidores , Anhidrasa Carbónica IV/antagonistas & inhibidores , Anhidrasa Carbónica IX/antagonistas & inhibidores , Anhidrasas Carbónicas/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , BencenosulfonamidasRESUMEN
Persistent pain affecting patients with inflammatory bowel diseases (IBDs) is still very difficult to treat. Carbonic anhydrase (CA) represents an intriguing pharmacological target considering the anti-hyperalgesic efficacy displayed by CA inhibitors in both inflammatory and neuropathic pain models. The aim of this work was to evaluate the effect of inhibiting CA IV, particularly when expressed in the gut, on visceral pain associated with colitis induced by 2,4-di-nitrobenzene sulfonic acid (DNBS) in rats. Visceral sensitivity was assessed by measuring animals' abdominal responses to colorectal distension. Repeated treatment with the selective CA IV inhibitors AB-118 and NIK-67 effectively counteracted the development of visceral pain induced by DNBS. In addition to pain relief, AB-118 showed a protective effect against colon damage. By contrast, the anti-hyperalgesic activity of NIK-67 was independent of colon healing, suggesting a direct protective effect of NIK-67 on visceral sensitivity. The enzymatic activity and the expression of CA IV resulted significantly increased after DNBS injection. NIK-67 normalised CA IV activity in DNBS animals, while AB-118 was partially effective. None of these compounds influenced CA IV expression through the colon. Although further investigations are needed to study the underlying mechanisms, CA IV inhibitors are promising candidates in the search for therapies to relieve visceral pain in IBDs.
Asunto(s)
Anhidrasa Carbónica IV/efectos de los fármacos , Inhibidores de Anhidrasa Carbónica/farmacología , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Dolor Visceral/tratamiento farmacológico , Animales , Anhidrasa Carbónica IV/metabolismo , Inhibidores de Anhidrasa Carbónica/metabolismo , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/uso terapéutico , Colon/efectos de los fármacos , Colon/metabolismo , Modelos Animales de Enfermedad , Enfermedades Inflamatorias del Intestino/metabolismo , Ratas Sprague-Dawley , Dolor Visceral/metabolismoRESUMEN
"Multi-action" Pt(IV) derivatives of cisplatin with combretastatin A4 (CA4) bioactive ligands that are conjugated to Pt(IV) by carbonate are unique because the ligand (IC50 < 10 nM) is dramatically 1000-folds more cytotoxic than cisplatin in vitro. The Pt(IV)-CA4 prodrugs were as cytotoxic as CA4 itself, indicating that the platinum moiety probably plays an insignificant role in triggering cytotoxicity, suggesting that the Pt(IV)-CA4 complexes act as prodrugs for CA4 rather than as true multi-action prodrugs. In vivo tests (Lewis lung carcinoma) show that ctc-[Pt(NH3)2(PhB)(CA4)Cl2] inhibited tumor growth by 93% compared to CA4 (67%), cisplatin (84%), and 1:1:1 cisplatin/CA4/PhB (85%) while displaying <5% body weight loss compared to cisplatin (20%) or CA4 (10%). In this case, and perhaps with other extremely potent bioactive ligands, platinum(IV) acts merely as a self-immolative carrier triggered by reduction in the cancer cell with only a minor contribution to cytotoxicity.
Asunto(s)
Antineoplásicos/farmacología , Anhidrasa Carbónica IV/metabolismo , Compuestos Organoplatinos/farmacología , Profármacos/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Anhidrasa Carbónica IV/química , Línea Celular , Proliferación Celular/efectos de los fármacos , Cricetulus , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ligandos , Estructura Molecular , Compuestos Organoplatinos/síntesis química , Compuestos Organoplatinos/química , Profármacos/síntesis química , Profármacos/química , Prohibitinas , Relación Estructura-ActividadRESUMEN
Carbonic anhydrases (CAs) catalyze the hydration and dehydration of carbon dioxide. They are important for regulating ions, fluid and acid-base balance in many tissues. The location of CAs by cell type is important for understanding their roles in these functions. CAs II and VI have been demonstrated using immunohistochemistry (IHC) in the serous acinar cells of human salivary glands and ducts of rat salivary glands. CA IV has been localized by IHC to the ducts of rat salivary glands. CA IV also is present in human parotid glands as shown by real time-polymerase chain reaction (RT-PCR), but this method does not show the distribution of the CA isozymes by cell type. We investigated the cell-specific distribution of CA IV in the human parotid gland. Sections from five formalin fixed, paraffin embedded specimens of human parotid gland were subjected to IHC for CA IV using a commercial antibody. Moderate to strong reactions were found in the cell membranes and cytoplasm of the intercalated, striated and excretory ducts and capillaries, and reactions in the acini were limited to faint areas in some cells. These results indicate that CA IV participates in the regulation of bicarbonate/carbon dioxide fluxes in the ductal system of the human parotid gland.
Asunto(s)
Anhidrasas Carbónicas , Glándula Parótida , Animales , Anhidrasa Carbónica IV , Humanos , Inmunohistoquímica , Ratas , Glándulas Salivales , Glándula SubmandibularRESUMEN
Various sulfonamide derivatives are intensively studied as anticancer agents owing to their inhibitory activity against human tumor-associated carbonic anhydrase isoforms. In this work, different synthetic procedures for the series of 1,3,5-triazinyl-aminobenzenesulfonamide conjugates with amino acids, possessing polar uncharged, negatively charged, and hydrophobic side chain, were studied and optimized with respect to the yield/purity of the synthesis/product as well as the time of synthetic reaction. These procedures were compared to each other via characteristic HPLC-ESI-DAD/QTOF/MS analytical product profiles, and their benefits as well as limitations were discussed. For new sulfonamide derivatives, incorporating s-triazine with a symmetric pair of polar and some less-polar proteinogenic amino acids, inhibition constants (KIs) against four human carboanhydrases (hCAs), namely cytosolic hCA I, II, transmembrane hCA IV, and the tumor-associated, membrane-bound hCA IX isoforms, were computationally predicted applying various methods of the advanced statistical analysis. Quantitative structure-activity relationship (QSAR) analysis indicated an impressive KI ratio (hCA II/hCA IX) 139.1 and hCA IX inhibition constant very similar to acetazolamide (KI = 29.6 nM) for the sulfonamide derivative disubstituted with Gln. The derivatives disubstituted with Ser, Thr, and Ala showed even lower KIs (8.7, 13.1, and 8.4 nM, respectively).
Asunto(s)
Aminoácidos/química , Inhibidores de Anhidrasa Carbónica/química , Anhidrasas Carbónicas/química , Sulfonamidas/química , Triazinas/química , Acetazolamida/farmacología , Antígenos de Neoplasias/química , Anhidrasa Carbónica I/química , Anhidrasa Carbónica II/química , Anhidrasa Carbónica IV/química , Anhidrasa Carbónica IX/química , Inhibidores de Anhidrasa Carbónica/farmacología , Cromatografía Líquida de Alta Presión , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas , Modelos Moleculares , Redes Neurales de la Computación , Isoformas de Proteínas/metabolismo , Programas Informáticos , Relación Estructura-Actividad , BencenosulfonamidasRESUMEN
In most vertebrates, red blood cell carbonic anhydrase (RBC CA) plays a critical role in carbon dioxide (CO2) transport and excretion across epithelial tissues. Many early-diverging fishes (e.g., hagfish and chondrichthyans) are unique in possessing plasma-accessible membrane-bound CA-IV in the gills, allowing some CO2 excretion to occur without involvement from the RBCs. However, implications of this on RBC CA function are unclear. Through homology cloning techniques, we identified the putative protein sequences for RBC CA from nine early-diverging species. In all cases, these sequences contained a modification of the proton shuttle residue His-64, and activity measurements from three early-diverging fish demonstrated significantly reduced CA activity. Site-directed mutagenesis was used to restore the His-64 proton shuttle, which significantly increased RBC CA activity, clearly illustrating the functional significance of His-64 in fish red blood cell CA activity. Bayesian analyses of 55 vertebrate cytoplasmic CA isozymes suggested that independent evolutionary events led to the modification of His-64 and thus reduced CA activity in hagfish and chondrichthyans. Additionally, in early-diverging fish that possess branchial CA-IV, there is an absence of His-64 in RBC CAs and the absence of the Root effect [where a reduction in pH reduces hemoglobin's capacity to bind with oxygen (O2)]. Taken together, these data indicate that low-activity RBC CA may be present in all fish with branchial CA-IV, and that the high-activity RBC CA seen in most teleosts may have evolved in conjunction with enhanced hemoglobin pH sensitivity.
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Anhidrasa Carbónica IV/sangre , Anhidrasa Carbónica IV/genética , Eritrocitos/enzimología , Proteínas de Peces/sangre , Proteínas de Peces/genética , Peces/sangre , Peces/genética , Sustitución de Aminoácidos , Animales , FilogeniaRESUMEN
Colon adenocarcinoma (COAD) is a common noncutaneous carcinoma worldwide with high morbidity and mortality. Effective prevention methods are far from being met. Both diagnostic and prognostic models that can precisely and accurately predict the status and survival time of COAD are urgently needed. In the field of COAD, there have been limited studies on molecular biomarkers that can predict disease status and prognosis. Hence, an important task is to identify these biomarkers. We aimed to identify important risk genes that have the ability not only to diagnose tumors but also to predict overall survival. A comprehensive analysis was performed in this study. Finally, carbonic anhydrase 1 (CA1) and CA4 were identified as potential biomarkers due to their predictive roles in diagnosis and prognosis, and the results were further confirmed by a series of analyses. Overall, these findings are of great importance and may facilitate individualized treatment in diagnosis and prognosis.
Asunto(s)
Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Anhidrasa Carbónica IV/genética , Anhidrasa Carbónica I/genética , Neoplasias del Colon/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/patología , Adulto , Anciano , Colon/patología , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/patología , Regulación Neoplásica de la Expresión Génica/genética , Humanos , PronósticoRESUMEN
Taste information is detected by taste cells and then transmitted to the brain through the taste nerve fibers. According to our previous data, there may be specific coding of taste quality between taste cells and nerve fibers. However, the molecular mechanisms underlying this coding specificity remain unclear. The purpose of this study was to identify candidate molecules that may regulate the specific coding. GeneChip analysis of mRNA isolated from the mice taste papillae and taste ganglia revealed that 14 members of the cadherin superfamily, which are important regulators of synapse formation and plasticity, were expressed in both tissues. Among them, protocadherin-20 (Pcdh20) was highly expressed in a subset of taste bud cells, and co-expressed with taste receptor type 1 member 3 (T1R3, a marker of sweet- or umami-sensitive taste cells) but not gustducin or carbonic anhydrase-4 (markers of bitter/sweet- and sour-sensitive taste cells, respectively) in circumvallate papillae. Furthermore, Pcdh20 expression in taste cells occurred later than T1R3 expression during the morphogenesis of taste papillae. Thus, Pcdh20 may be involved in taste quality-specific connections between differentiated taste cells and their partner neurons, thereby acting as a molecular tag for the coding of sweet and/or umami taste.
Asunto(s)
Cadherinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Papilas Gustativas/metabolismo , Gusto/fisiología , Animales , Anhidrasa Carbónica IV/metabolismo , Femenino , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Transgénicos , Plasticidad Neuronal/fisiología , Protocadherinas , Receptores Acoplados a Proteínas G/genética , Sinapsis/metabolismo , Transducina/metabolismo , Ganglio del Trigémino/metabolismoRESUMEN
The lung microvasculature is essential for gas exchange and commonly considered homogeneous. We show that VEGFA from the epithelium is required for a distinct endothelial cell (EC) population in the mouse lung. Vegfa is predominantly expressed by alveolar type 1 (AT1) cells and locally required to specify a subset of ECs. Single-cell RNA sequencing (scRNA-seq) reveals that â¼15% of lung ECs are transcriptionally distinct-marked by Carbonic anhydrase 4 (Car4)-and arise from bulk ECs, as suggested by trajectory analysis. Car4 ECs have extensive cellular projections and are separated from AT1 cells by a limited basement membrane without intervening pericytes. Car4 ECs are specifically lost upon epithelial Vegfa deletion; without Car4 ECs, the alveolar space is aberrantly enlarged despite the normal appearance of myofibroblasts. Lung Car4 ECs and retina tip ECs have common and distinct features. These findings support a signaling role of AT1 cells and shed light on alveologenesis.
Asunto(s)
Células Epiteliales Alveolares/metabolismo , Células Endoteliales/citología , Endotelio Vascular/citología , Pulmón/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Células Epiteliales Alveolares/citología , Animales , Anhidrasa Carbónica IV/genética , Anhidrasa Carbónica IV/metabolismo , Células Cultivadas , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Pulmón/citología , Pulmón/crecimiento & desarrollo , Ratones , Morfogénesis , Miofibroblastos/citología , Neovascularización Fisiológica , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
With the aim to obtain novel compounds possessing both strong affinity against human carbonic anhydrases and low toxicity, we synthesised novel thiourea and sulphonamide derivatives 3, 4 and 10, and studied their in vitro inhibitory properties against human CA I, CA II and CA IX. We also evaluated the toxicity of these compounds using zebrafish larvae. Among the three compounds, derivative 4 showed efficient inhibition against hCA II (KI = 58.6 nM). Compound 10 showed moderate inhibition against hCA II (KI = 199.2 nM) and hCA IX (KI = 147.3 nM), whereas it inhibited hCA I less weakly at micromolar concentrations (KI = 6428.4 nM). All other inhibition constants for these compounds were in the submicromolar range. The toxicity evaluation studies showed no adverse effects on the zebrafish larvae. Our study suggests that these compounds are suitable for further preclinical characterisation as potential inhibitors of hCA I, II and IX.