Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 9.686
Filtrar
1.
Cell Death Dis ; 14(1): 7, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36609444

RESUMO

SLC12A5, a neuron-specific potassium-chloride co-transporter, has been reported to promote tumor progression, however, the underlying mechanism remains unclear. Here we report that SLC12A5 functions as an oncogene to promote tumor progression and castration resistance of prostate cancer through the N6-methyladenosine (m6A) reader YTHDC1 and the transcription factor HOXB13. We have shown that the level of SLC12A5 was increased in prostate cancer, in comparison to its normal counterparts, and further elevated in castration-resistant prostate cancer (CRPC). The enhanced expression of SLC12A5 mRNA was associated with neuroendocrine prostate cancer (NEPC) progression and poor survival in prostate cancer. Furthermore, we demonstrated that SLC12A5 promoted the castration resistance development of prostate cancer in addition to the cell proliferation and migration. Interestingly, SLC12A5 was detected in the cell nucleus and formed a complex with nuclear m6A reader YTHDC1, which in turn upregulated HOXB13 to promote the prostate cancer progression. Therefore, our findings reveal a mechanism that how the potassium-chloride cotransporter SLC12A5 promotes the tumor progression and provide a therapeutic opportunity for prostate cancer to apply the neurological disorder drug SLC12A5 inhibitors.


Assuntos
Neoplasias de Próstata Resistentes à Castração , Simportadores , Masculino , Humanos , Neoplasias de Próstata Resistentes à Castração/patologia , Simportadores/genética , Simportadores/metabolismo , Cloretos/metabolismo , Cloretos/uso terapêutico , Castração , Potássio/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Fatores de Processamento de RNA/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo
2.
Sci Adv ; 9(3): eade8346, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36662855

RESUMO

Malfunction of the sialic acid transporter caused by various genetic mutations in the SLC17A5 gene encoding Sialin leads to a spectrum of neurodegenerative conditions called free sialic acid storage disorders. Unfortunately, how Sialin transports sialic acid/proton (H+) and how pathogenic mutations impair its function are poorly defined. Here, we present the structure of human Sialin in an inward-facing partially open conformation determined by cryo-electron microscopy, representing the first high-resolution structure of any human SLC17 member. Our analysis reveals two unique features in Sialin: (i) The H+ coupling/sensing requires two highly conserved Glu residues (E171 and E175) instead of one (E175) as implied in previous studies; and (ii) the normal function of Sialin requires the stabilization of a cytosolic helix, which has not been noticed in the literature. By mapping known pathogenic mutations, we provide mechanistic explanations for corresponding functional defects. We propose a structure-based mechanism for sialic acid transport mediated by Sialin.


Assuntos
Doença do Armazenamento de Ácido Siálico , Simportadores , Humanos , Ácido N-Acetilneuramínico , Microscopia Crioeletrônica , Doença do Armazenamento de Ácido Siálico/genética , Mutação , Simportadores/genética , Simportadores/metabolismo , Transporte de Íons
3.
J Neuroinflammation ; 20(1): 15, 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36691035

RESUMO

The mechanisms by which neonatal inflammation leads to cognitive deficits in adulthood remain poorly understood. Inhibitory GABAergic synaptic transmission plays a vital role in controlling learning, memory and synaptic plasticity. Since early-life inflammation has been reported to adversely affect the GABAergic synaptic transmission, the aim of this study was to investigate whether and how neonatal inflammation affects GABAergic synaptic transmission resulting in cognitive impairment. Neonatal mice received a daily subcutaneous injection of lipopolysaccharide (LPS, 50 µg/kg) or saline on postnatal days 3-5. It was found that blocking GABAergic synaptic transmission reversed the deficit in hippocampus-dependent memory or the induction failure of long-term potentiation in the dorsal CA1 in adult LPS mice. An increase of mIPSCs amplitude was further detected in adult LPS mice indicative of postsynaptic potentiation of GABAergic transmission. Additionally, neonatal LPS resulted in the increased expression and function of K+-Cl--cotransporter 2 (KCC2) and the decreased expression of transforming growth factor-beta 1 (TGF-ß1) in the dorsal CA1 during adulthood. The local TGF-ß1 overexpression improved KCC2 expression and function, synaptic plasticity and memory of adult LPS mice. Adult LPS mice show hypermethylation of TGFb1 promoter and negatively correlate with reduced TGF-ß1 transcripts. 5-Aza-deoxycytidine restored the changes in TGFb1 promoter methylation and TGF-ß1 expression. Altogether, the results suggest that hypermethylation-induced reduction of TGF-ß1 leads to enhanced GABAergic synaptic inhibition through increased KCC2 expression, which is a underlying mechanism of neonatal inflammation-induced hippocampus-dependent memory impairment in adult mice.


Assuntos
Simportadores , Fator de Crescimento Transformador beta1 , Camundongos , Animais , Fator de Crescimento Transformador beta1/metabolismo , Metilação , Regulação para Baixo , Lipopolissacarídeos/metabolismo , Hipocampo/metabolismo , Plasticidade Neuronal , Transmissão Sináptica/fisiologia , Inflamação/metabolismo , Cognição , Simportadores/metabolismo
4.
Biol Pharm Bull ; 46(1): 86-94, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36596528

RESUMO

From our previous observation that the anesthetic effects of phenobarbital potentiate in rats with a decreased cerebral protein expression of the potassium chloride cotransporter KCC2 (SLC12A5), an in vivo study was conducted to clarify whether the pharmacological effect of phenobarbital alters by stimulating the cerebral tropomyosin receptor kinase B (TrkB) that is known to down-regulate the KCC2 protein expression. The stimulation was performed in rats with repetitious intraperitoneal administration of a TrkB agonist, namely 7,8-dihydroxyflavone (DHF). After that, the rats underwent an intraventricular infusion of phenobarbital using a dwelled cannula, and the onset time of the phenobarbital-induced general anesthesia was determined. In addition, their brain tissues were excised and cerebral cortices were collected. Then, subcellular fractions were prepared and the cerebral expression of various proteins involving the anesthetic effects of phenobarbital was examined. It was demonstrated that phenobarbital induced general anesthesia about 2 times faster in rats receiving the DHF treatment than in control rats, and that the phenobarbital amount in the brain tissue at the onset time of anesthesia was lower in rats with the treatment. Western blotting showed that the cerebral protein expression of KCC2 decreases, and the phosphorylation of the TrkB protein increases with the DHF treatment. These observations indicate that the anesthetic effects of phenobarbital potentiate with the TrkB stimulation and the resultant decrease in the cerebral KCC2 protein expression. The results also suggest that the TrkB protein and its phosphorylation status may be a key modulator of the pharmacological efficacy of phenobarbital.


Assuntos
Flavonas , Simportadores , Ratos , Animais , Tropomiosina/metabolismo , Fenobarbital/farmacologia , Flavonas/farmacologia , Receptor trkB/metabolismo
5.
Neurosci Lett ; 796: 137065, 2023 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-36638954

RESUMO

Focal cortical dysplasia (FCD) represents a group of malformations of cortical development, which are speculated to be related to early developmental defects in the cerebral cortex. According to dysmature cerebral development hypothesis of FCD altered GABAA receptor function is known to contribute to abnormal neuronal network. Here, we studied the possible association between age at seizure onset in FCD with the subunit configuration of GABAA receptors in resected brain specimens obtained from patients with FCD. We observed a significantly higher ratio of α4/α1 subunit-containing GABAA receptors in patients with early onset (EO) FCD as compared to those with late onset (LO) FCD as is seen during the course of development where α4-containing GABAA receptors expression is high as compared to α1-containing GABAA receptors expression. Likewise, the influx to efflux chloride co-transporter expression of NKCC1/KCC2 was also increased in patients with EO FCD as seen during brain development. In addition, we observed that the ratio of GABA/Glutamate neurotransmitters was lower in patients with EO FCD as compared to that in patients with LO FCD. Our findings suggest altered configuration of GABAA receptors in FCD which could be contributing to aberrant depolarizing GABAergic activity. In particular, we observed a correlation of age at seizure onset in FCD with subunit configuration of GABAA receptors, levels of NKCC1/KCC2 and the ratio of GABA/Glutamate neurotransmitters such that the patients with EO FCD exhibited a more critically modulated GABAergic network.


Assuntos
Malformações do Desenvolvimento Cortical , Simportadores , Humanos , Receptores de GABA-A/metabolismo , Simportadores/metabolismo , Cloretos/metabolismo , Ácido gama-Aminobutírico/metabolismo , Convulsões/complicações , Malformações do Desenvolvimento Cortical/metabolismo
6.
Structure ; 31(1): 58-67.e4, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36525976

RESUMO

The melibiose permease MelB is a well-studied Na+-coupled transporter of the major facilitator superfamily. However, the symport mechanism of galactosides and cations is still not fully understood, especially at structural levels. Here, we use single-molecule force spectroscopy to investigate substrate-induced structural changes of MelB from Salmonella typhimurium. In the absence of substrate, MelB equally populates two different states, from which one shows higher mechanical structural stability with additional stabilization of the cytoplasmic middle-loop C3. In the presence of either melibiose or a coupling Na+-cation, however, MelB increasingly populates the mechanically less stable state, which shows a destabilized middle-loop C3. In the presence of both substrate and co-substrate, this mechanically less stable state of MelB is predominant. Our findings describe how both substrates guide MelB transporters to populate two different mechanically stabilized states, and contribute mechanistic insights to the alternating-access action for the galactoside/cation symport catalyzed by MelB.


Assuntos
Melibiose , Simportadores , Melibiose/química , Simportadores/metabolismo , Proteínas de Membrana Transportadoras , Sódio/metabolismo , Transporte de Íons , Cátions
7.
J Med Chem ; 66(1): 657-676, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36584238

RESUMO

The solute carrier (SLC) monocarboxylate transporter 1 (MCT1; SLC16A1) represents a promising target for the treatment of cancer; however, the MCT1 modulator landscape is underexplored with only roughly 100 reported compounds. To expand the knowledge about MCT1 modulation, we synthesized a library of 16 indole-based molecules and subjected these to a comprehensive biological assessment platform. All compounds showed functional inhibitory activities against MCT1 at low nanomolar concentrations and great antiproliferative activities against the MCT1-expressing cancer cell lines A-549 and MCF-7, while the compounds were selective over MCT4 (SLC16A4). Lead compound 24 demonstrated a greater potency than the reference compound, and molecular docking revealed strong binding affinities to MCT1. Compound 24 led to cancer cell cycle arrest as well as apoptosis, and it showed to sensitize these cancer cells toward an antineoplastic agent. Strikingly, compound 24 had also significant inhibitory power against the multidrug transporter ABCB1 and showed to reverse ABCB1-mediated multidrug resistance (MDR).


Assuntos
Antineoplásicos , Simportadores , Simulação de Acoplamento Molecular , Simportadores/metabolismo , Antineoplásicos/farmacologia , Proteínas de Membrana Transportadoras , Indóis/farmacologia , Transportadores de Ácidos Monocarboxílicos
8.
J Comp Neurol ; 531(1): 48-57, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36217249

RESUMO

The neuronal chloride (Cl-) exporter, KCC2, regulates neuron excitability and development and undergoes a stereotypical pattern of delayed upregulation as neurons mature. KCC2 upregulation favors neural inhibition by establishing a negative Cl- gradient, ensuring GABA-induced Cl- currents are inward and inhibitory. We developed a zebrafish fluorescent reporter line, KCC2b:mCitrine, to track KCC2 expression in vivo during early brain development. KCC2b:mCitrine was first detected at 16 h postfertilization and by day 6 labeled most central and peripheral neurons and processes. At 20 h, expression was greatest in the soma-dense basal neuroepithelium but largely absent in apical and mantle zones where differentiation and migration primarily occur, and time lapse imaging at this stage supports a postmigration upregulation of KCC2b. Central dopamine neurons showed low KCC2b expression as observed in other species. KCC2b:mCitrine fluorescence was stable over minutes in most neurons, but brightness transients observed in single cells fit our expectation for real-time tracking of KCC2b upregulation in new neurons. To further assess whether fluorescence brightness tracks KCC2b expression, zebrafish embryos were exposed to bisphenol-A (BPA), which is known to suppress KCC2 expression. Fluorescence decreased after 6 days of BPA exposure but not after 2 or 4 days, suggesting that it is an accurate but delayed indicator of KCC2b expression. KCC2b:mCitrine zebrafish present a new method for visualizing KCC2b's complex dynamics during brain development, and potentially screening compounds aimed at modulating KCC2 expression.


Assuntos
Simportadores , Animais , Simportadores/metabolismo , Peixe-Zebra/metabolismo , Neurônios/metabolismo , Encéfalo/metabolismo
9.
Biochemistry ; 62(1): 118-133, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36516499

RESUMO

Osmosensing transporter ProP forestalls cellular dehydration by detecting environments with high osmotic pressure and mediating the accumulation of organic osmolytes by bacterial cells. It is composed of 12 transmembrane helices with cytoplasmic N- and C-termini. In Escherichia coli, dimers form when the C-terminal domains of ProP molecules form homodimeric, antiparallel, α-helical coiled coils. No dominant negative effect was detected when inactive and active ProP molecules formed heterodimers in vivo. Purification of ProP in detergent dodecylmaltoside yielded monomers, which were functional after reconstitution in proteoliposomes. With other evidence, this suggests that ProP monomers function independently whether in the monomeric or dimeric state. Amino acid replacements that disrupted or reversed the coiled coil did not prevent in vivo dimerization of ProP detected with a bacterial two-hybrid system. Maleimide labeling detected no osmolality-dependent variation in the reactivities of cysteine residues introduced to transmembrane helix (TM) XII. In contrast, coarse-grained molecular dynamic simulations detected deformation of the lipid around TMs III and VI, on the lipid-exposed protein surface opposite to TM XII. This suggests that the dimer interface of ProP includes the surfaces of TMs III and VI, not of TM XII as previously suggested by crosslinking data. Homology modeling suggested that coiled-coil formation and dimerization via such an interface are not mutually exclusive. In previous work, alterations to the C-terminal coiled coil blocked co-localization of ProP with phospholipid cardiolipin at E. coli cell poles. Thus, dimerization may contribute to ProP targeting, adjust its lipid environment, and hence indirectly modify its osmotic stress response.


Assuntos
Proteínas de Escherichia coli , Simportadores , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Sequência de Aminoácidos , Dimerização , Simportadores/química , Proteínas de Membrana Transportadoras/metabolismo , Fosfolipídeos/metabolismo
10.
J Med Chem ; 66(1): 384-397, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36525250

RESUMO

Due to increased reliance on glycolysis, which produces lactate, monocarboxylate transporters (MCTs) are often upregulated in cancer. MCT4 is associated with the export of lactic acid from cancer cells under hypoxia, so inhibition of MCT4 may lead to cytotoxic levels of intracellular lactate. In addition, tumor-derived lactate is known to be immunosuppressive, so MCT4 inhibition may be of interest for immuno-oncology. At the outset, no potent and selective MCT4 inhibitors had been reported, but a screen identified a triazolopyrimidine hit, with no close structural analogues. Minor modifications to the triazolopyrimidine were made, alongside design of a constrained linker and broad SAR exploration of the biaryl tail to improve potency, physical properties, PK, and hERG. The resulting clinical candidate 15 (AZD0095) has excellent potency (1.3 nM), MCT1 selectivity (>1000×), secondary pharmacology, clean mechanism of action, suitable properties for oral administration in the clinic, and good preclinical efficacy in combination with cediranib.


Assuntos
Antineoplásicos , Neoplasias , Simportadores , Humanos , Ácido Láctico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Hipóxia , Transportadores de Ácidos Monocarboxílicos
11.
Dev Psychobiol ; 65(1): e22353, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36567653

RESUMO

In this study, based on the excitatory/inhibitory imbalance theory of autism, the time window of GABA switch, the role of K-Cl co-transporter 2 (KCC2) in adjustment GABA switch, and brain permeability to erythropoietin (EPO), the effects of postnatal -EPO and- nano- erythropoietin (NEPO) have been evaluated in the valproic acid (VPA) rat model of autism. The VPA was administered for animal modeling of autism at gestational day (GD) 12.5 (600 mg/kg). Male offsprings were injected with EPO and NEPO in a clinically proper postnatal dosing regimen on postnatal days (PND) 1-5, and autistic-like behaviors were tested at the end of the first month. Then animals were sacrificed, and neuron morphology and KCC2 expression were examined by Nissl staining and Western blot. According to our findings, high-dose NEPO improved autism-associated phenotypes. Neuroprotective effects of EPO and NEPO have been shown in the hippocampus. Postnatal NEPO treatment reversed KCC2 expression abnormalities induced by prenatal VPA. Our results might support the role of KCC2 in ASD and the excitatory/inhibitory imbalance hypothesis. We suggested Nano- erythropoietin and other KCC2 interventions as a new approach to the early treatment and prevention of autism.


Assuntos
Transtorno Autístico , Eritropoetina , Hipocampo , Simportadores , Animais , Feminino , Humanos , Masculino , Gravidez , Ratos , Transtorno Autístico/induzido quimicamente , Transtorno Autístico/tratamento farmacológico , Transtorno Autístico/metabolismo , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Modelos Animais de Doenças , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologia , Ácido gama-Aminobutírico/uso terapêutico , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Simportadores/metabolismo , Simportadores/farmacologia , Simportadores/uso terapêutico , Ácido Valproico/farmacologia , Eritropoetina/farmacologia , Eritropoetina/uso terapêutico
14.
BMC Prim Care ; 23(1): 317, 2022 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-36476327

RESUMO

BACKGROUND: Sodium-glucose co-transporter 2 inhibitors (SGLT2 I) has cardiorenal protective properties and are recommended for patients with diabetes and established atherosclerotic cardiovascular disease (ASCVD) and/or chronic kidney disease (CKD). Although cardiorenal complications are high in diabetes and pose a significant financial burden on the Hong Kong health care system, the use of SGLT2 I in these populations remains low. And yet this issue has not been explored in Hong Kong primary care. This study aimed to explore factors affecting primary care doctors' prescribing of SGLT2 I in patients with diabetes and established ASCVD/CKD in Hong Kong. METHODS: A phenomenological qualitative research using semi-structured interviews was conducted between January and May 2021 in one Hospital Authority cluster in Hong Kong. Purposive sampling was employed to recruit primary care doctors in the cluster. The Theoretical Domains Framework (TDF) underpinned the study and guided the development of the interview questions. Data was analysed using both inductive and deductive approaches. The Consolidated criteria for reporting qualitative research (COREQ) checklist was used to guide the reporting. RESULTS: Interviews were conducted with 17 primary care doctors. Four overarching themes were inductively identified: knowledge and previous practice patterns influence prescription, balancing risks and benefits, doctors' professional responsibilities, and system barriers. The four themes were then deductively mapped to the nine specific domains of the TDF: knowledge; intention; memory; beliefs about capabilities; beliefs about consequences; goals; role and identity; emotion; and environmental constraints. Most interviewees, to varying extent, were aware of the cardio-renal advantages and safety profile of SGLT2 I but are reluctant to prescribe or change their patients to SGLT2 I because of their knowledge gap that the cardio-renal benefits of SGLT2 I was independent of glyacemic efficacy. Other barriers included their considerations of patients' age and renal impairment, and patients' perceptions and preferences. CONCLUSIONS: Despite evidence-based recommendations of the utilisation of SGLT2 I in patients with established ASCVD/CKD, the prescription behaviour among primary care doctors was affected by various factors, most of which were amendable. Our findings will inform the development of structured interventions to address these factors to improve patients' cardio-renal outcomes.


Assuntos
Doenças Cardiovasculares , Diabetes Mellitus Tipo 2 , Insuficiência Renal Crônica , Inibidores do Transportador 2 de Sódio-Glicose , Simportadores , Humanos , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Diabetes Mellitus Tipo 2/complicações , Doenças Cardiovasculares/tratamento farmacológico , Pesquisa Qualitativa , Insuficiência Renal Crônica/complicações , Prescrições , Glucose , Sódio
15.
Cells ; 11(23)2022 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-36497071

RESUMO

The liver is the most common site for colorectal cancer (CRC)-associated metastasis. There remain unsatisfactory medications in liver metastasis given the incomplete understanding of pathogenic mechanisms. Herein, with an orthotopic implantation model fed either regular or high-fat diets (HFD), more liver metastases were associated with an expansion of conjugated bile acids (BAs), particularly taurocholic acid (TCA) in the liver, and an increased gene expression of Na+-taurocholate cotransporting polypeptide (NTCP). Such hepatic BA change was more apparently shown in the HFD group. In the same model, TCA was proven to promote liver metastases and induce a tumor-favorable microenvironment in the liver, characterizing a high level of fibroblast activation and increased proportions of myeloid-derived immune cells. Hepatic stellate cells, a liver-residing source of fibroblasts, were dose-dependently activated by TCA, and their conditioned medium significantly enhanced the migration capability of CRC cells. Blocking hepatic BA uptake with NTCP neutralized antibody can effectively repress TCA-triggered liver metastases, with an evident suppression of tumor microenvironment niche formation. This study points to a new BA-driven mechanism of CRC-associated liver metastases, suggesting that a reduction of TCA overexposure by limiting liver uptake is a potential therapeutic option for CRC-associated liver metastasis.


Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Simportadores , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/genética , Simportadores/metabolismo , Ácido Taurocólico/farmacologia , Ácido Taurocólico/metabolismo , Microambiente Tumoral
16.
Front Endocrinol (Lausanne) ; 13: 882788, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36568087

RESUMO

Introduction: A mathematical model of the pituitary-thyroid feedback loop is extended to deepen the understanding of the Allan-Herndon-Dudley syndrome (AHDS). The AHDS is characterized by unusual thyroid hormone concentrations and a mutation in the SLC16A2 gene encoding for the monocarboxylate transporter 8 (MCT8). This mutation leads to a loss of thyroid hormone transport activity. One hypothesis to explain the unusual hormone concentrations of AHDS patients is that due to the loss of thyroid hormone transport activity, thyroxine (T 4) is partially retained in thyroid cells. Methods: This hypothesis is investigated by extending a mathematical model of the pituitary-thyroid feedback loop to include a model of the net effects of membrane transporters such that the thyroid hormone transport activity can be considered. A nonlinear modeling approach based on the Michaelis-Menten kinetics and its linear approximation are employed to consider the membrane transporters. The unknown parameters are estimated through a constrained parameter optimization. Results: In dynamic simulations, damaged membrane transporters result in a retention of T 4 in thyroid cells and ultimately in the unusual hormone concentrations of AHDS patients. The Michaelis-Menten modeling approach and its linear approximation lead to similar results. Discussion: The results support the hypothesis that a partial retention of T 4 in thyroid cells represents one mechanism responsible for the unusual hormone concentrations of AHDS patients. Moreover, our results suggest that the retention of T 4 in thyroid cells could be the main reason for the unusual hormone concentrations of AHDS patients.


Assuntos
Simportadores , Glândula Tireoide , Humanos , Hormônios Tireóideos , Proteínas de Membrana Transportadoras , Modelos Teóricos , Homeostase , Transportadores de Ácidos Monocarboxílicos/genética , Simportadores/genética
17.
J Vis Exp ; (190)2022 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-36571412

RESUMO

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition. Failure in proper regulation of KCC2 is deleterious and has been associated with the prevalence of several neurological diseases, including epilepsy. There has been considerable progress with regard to understanding the mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enable researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. Here, the protocol highlights how to investigate KCC2 phosphorylation at kinase regulatory sites - Thr906 and Thr1007- using western blotting technique. There are other classic methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis. A few of these additional techniques will be briefly discussed in this manuscript.


Assuntos
Epilepsia , Simportadores , Humanos , Simportadores/genética , Cloretos , Neurônios/metabolismo , Western Blotting , Ácido gama-Aminobutírico
18.
Cells ; 11(23)2022 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-36497119

RESUMO

KCC2 mediates extrusion of K+ and Cl- and assuresthe developmental "switch" in GABA function during neuronal maturation. However, the molecular mechanisms underlying KCC2 regulation are not fully elucidated. We investigated the impact of transforming growth factor beta 2 (TGF-ß2) on KCC2 during neuronal maturation using quantitative RT-PCR, immunoblotting, immunofluorescence and chromatin immunoprecipitation in primary mouse hippocampal neurons and brain tissue from Tgf-ß2-deficient mice. Inhibition of TGF-ß/activin signaling downregulates Kcc2 transcript in immature neurons. In the forebrain of Tgf-ß2-/- mice, expression of Kcc2, transcription factor Ap2ß and KCC2 protein is downregulated. AP2ß binds to Kcc2 promoter, a binding absent in Tgf-ß2-/-. In hindbrain/brainstem tissue of Tgf-ß2-/- mice, KCC2 phosphorylation at T1007 is increased and approximately half of pre-Bötzinger-complex neurons lack membrane KCC2 phenotypes rescued through exogenous TGF-ß2. These results demonstrate that TGF-ß2 regulates KCC2 transcription in immature neurons, possibly acting upstream of AP2ß, and contributes to the developmental dephosphorylation of KCC2 at T1007. The present work suggests multiple and divergent roles for TGF-ß2 on KCC2 during neuronal maturation and provides novel mechanistic insights for TGF-ß2-mediated regulation of KCC2 gene expression, posttranslational modification and surface expression. We propose TGF-ß2 as a major regulator of KCC2 with putative implications for pathophysiological conditions.


Assuntos
Células-Tronco Neurais , Simportadores , Fator de Crescimento Transformador beta2 , Animais , Camundongos , Hipocampo/citologia , Hipocampo/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Fosforilação , Fator de Crescimento Transformador beta2/metabolismo , Simportadores/metabolismo
20.
Int J Mol Sci ; 23(24)2022 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-36555151

RESUMO

This study confirmed the effect of sodium/iodine symporter (NIS) expression on existing drugs by in vitro and in vivo tests using cultured cell lines. The tumor growth inhibitory effect of sodium astatide ([211At]NaAt) was evaluated by in vitro and in vivo tests using human thyroid cancer cells (K1, K1/NIS and K1/NIS-DOX). NIS expression in cancer cells was controlled using the Tet-On system. [131I]NaI was used as control existing drug. From the results of the in vitro studies, the mechanism of [211At]NaAt uptake into thyroid cancer cells is mediated by NIS, analogous to [131I]NaI, and the cellular uptake rate correlates with the expression level of NIS. [211At]NaAt's ability to inhibit colony formation was more than 10 times that of [131I]NaI per becquerel (Bq), and [211At]NaAt's DNA double-strand breaking (DSB) induction was more than ten times that of [131I]NaI per Bq, and [211At]NaAt was more than three times more cytotoxic than [131I]NaI (at 1000 kBq each). In vivo studies also showed that the tumor growth inhibitory effect of [211At]NaAt depended on NIS expression and was more than six times that of [131I]NaI per Bq.


Assuntos
Compostos de Iodo , Simportadores , Neoplasias da Glândula Tireoide , Humanos , Simportadores/genética , Simportadores/metabolismo , Radioisótopos do Iodo/uso terapêutico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...