RESUMO
In this study, a case of Lynch syndrome (LS) family line with a novel mutation site in the MLH1 c.463dupC gene was reported and the clinical and pathogenic genetic features of this family were analyzed. A 40-year-old female patient with colon cancer diagnosed at the First Affiliated Hospital of Kunming Medical University on October 2, 2020 was retrospectively included. The clinical data of the family were collected and the family lineage was drawn. The family tumor history met the Amsterdam Criteria â ¡ and the diagnostic criteria of LS in Chinese, which was a typical LS family lineage. A germline code-shift missense mutation c.463dupC in the MLH1 gene located in exon 6, a possible pathogenic variant, was detected by second-generation sequencing (NGS) in the patient. Subsequently, Sanger sequencing was performed on a total of 20 direct lineage members of the family of the MLH1 gene, 7 cases were found to harbor the mutation and included in the LS high-risk control. Follow-up to October 2023 showed that the patient had endometrial and cervical polyps, one case had colorectal cancer, and two cases had intestinal polyps, all were treated with early intervention and therapy; two cases did not show any clinical symptoms. This study is the first to report a new mutation site for the potentially pathogenic MLH1 c.463dupC, providing a rationale for the pathogenicity of the mutation and standardized health management for familial carriers.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose , Feminino , Humanos , Adulto , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Predisposição Genética para Doença , Estudos Retrospectivos , Proteína 1 Homóloga a MutL/genética , MutaçãoRESUMO
Objective: To analyze the influencing factors of lung function in pneumoconiosis patients, and to provide reference for clinical treatment. Methods: From July 2020 to December 2020, a questionnaire survey was conducted on pneumoconiosis patients in the jurisdiction by using the "Guangdong Province Occupational Disease Prevention and Control Institute" questionnaire, and the relevant items of patients were examined. The rate of counting data is expressed, and the measurement data is expressed by mean and standard deviation. Chi-square test was used for comparison between groups, trend chi-square test was used for trend analysis of ordered classified data. Multivariate analysis was carried out with binary logistic regression model. Results: A total of 1409 pneumoconiosis patients were enrolled. The abnormal rate of lung function in pneumoconiosis patients was 68.77%. The results of trend Chi-square test showed that the abnormal rate of lung function increased with the age of exposure to dust in different age groups (Chi Sqnare Trend=64.12ã8.49ã24.20, P<0.05) . In univariate analysis, there were statistical significance in different dust exposure age, working age, pneumoconiosis stage, complications and occupational pneumoconiosis diseases (P<0.05) . Multiple logistic regression results showed that age of exposure to dust, years of service, stage of pneumoconiosis and complications were the main influencing factors of lung function in pneumoconiosis patients (P<0.05) . Compared with patients aged 0-30 years, patients aged 50-70 years and older had a higher rate of abnormal lung function (OR=2.16, 95%CI: 1.12~4.16; OR=4.82, 95%CI: 2.05~11.35, all P<0.05) ; Compared with patients with 0~20 years of service, patients with 20~30 years of service and more than 30 years of service had a higher rate of abnormal lung function (OR=1.58, 95%CI: 1.10~2.25; OR=1.63, 95%CI: 1.28~2.40, P<0.05) ; Compared with stage â patients, Stage â ¡ and Stage â ¢ patients had a higher rate of abnormal lung function (OR=1.62, 95%CI: 1.20~2.17; OR=2.23, 95%CI: 1.40~3.55, all P<0.05) ; Compared with patients without comorbidities, patients with comorbidities had a higher rate of abnormal lung function (OR=1.68, 95%CI: 1.20~2.38, P<0.05) . Conclusion: The factors such as age of exposure to dust, working age, stage of pneumoconiosis and complications may be the influencing factors of lung function in pneumoconiosis patients.
Assuntos
Doenças Profissionais , Pneumoconiose , Humanos , Pneumoconiose/epidemiologia , Poeira , Inquéritos e Questionários , PulmãoRESUMO
AIM: To establish an ultrasound-based radiomics model through machine learning methods and then to assess the ability of the model to differentiate infected focal liver lesions from malignant mimickers. MATERIALS AND METHODS: A total of 104 patients with infected focal liver lesions and 485 patients with malignant hepatic tumours were included, consisting of hepatocellular carcinoma (HCC), cholangiocarcinoma (CC), combined hepatocellular-cholangiocarcinoma (cHCC-CC), and liver metastasis. Radiomics features were extracted from grey-scale ultrasound images. Feature selection and predictive modelling were carried out by dimensionality reduction methods and classifiers. The diagnostic effect of the prediction mode was assessed by receiver operating characteristic (ROC) curve analysis. RESULTS: In total, 5,234 radiomics features were extracted from grey-scale ultrasound image of every focal liver lesion. The ultrasound-based radiomics model had a favourable predictive value for differentiating infected focal liver lesions from malignant hepatic tumours, with an area under the curve (AUC) of 0.887 and 0.836 (HCC group), 0.896 and 0.766 (CC group), 0.944 and 0.754 (cHCC-CC group), 0.918 and 0.808 (liver metastasis group), and 0.949 and 0.745 (malignant hepatic tumour group) for the training set and validation set, respectively. CONCLUSIONS: Ultrasound-based radiomics is helpful in differentiating infected focal liver lesions from malignant mimickers and has the potential for use as a supplement to conventional grey-scale ultrasound and contrast-enhanced ultrasound (CEUS).
Assuntos
Neoplasias Hepáticas/diagnóstico por imagem , Ultrassonografia/métodos , Diagnóstico Diferencial , Feminino , Humanos , Fígado/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
1. Fusidic acid (FA) is widely used for the treatment of infections of sensitive osteomyelitis or skin and soft tissue caused by bacteria. However, the role of cytochrome P450s (CYPs) in the metabolism of FA is unclear. In the present study, we screened the main CYPs for the metabolism of FA and studied its interactions with isoform-selective substrates in vitro. 2. The main CYP450s were screened according to the inhibitory effect of specific inhibitors on the metabolism of FA in human liver microsomes (HLMs) or recombinant CYP isoforms. Enzyme kinetic parameters including Ki, Ki', Vmax, and IC50 were calculated to determine the potential of FA to affect CYP-mediated metabolism of isoform-selective substrates. 3. FA metabolism rate was inhibited by 49.8% and 83.1% under CYP2D6, CYP3A4 selective inhibitors in HLMs. In recombinant experiment, the inhibitory effects on FA metabolism were 83.3% for CYP2D6 and 58.9% for CYP3A4, respectively. FA showed inhibition on CYP2D6 and CYP3A4 with Kis of 13.9 and 38.6 µM, respectively. Other CYP isoforms including CYP1A2, CYP2A6, CYP2C9, CYP2E1, and CYP2C19 showed minimal or no effect on the metabolism of FA. 4. FA was primarily metabolized by CYP2D6 and CYP3A4 and showed a noncompetitive inhibition on CYP2D6 and a mixed competitive inhibition on CYP3A4. Drug-drug interactions between FA and other chemicals, especially with substrates of CYP2D6 and CYP3A4, are phenomena that clinicians need to be aware of and cautious about.
Assuntos
Ácido Fusídico/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Humanos , Inativação Metabólica/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Isoformas de Proteínas/metabolismo , Relação Estrutura-AtividadeRESUMO
1.Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of tanshinone IIA, a famous Chinese medicine used for many years to treat cardiovascular disorders. However, the role of cytochrome P450 (CYP) enzymes in the metabolism of STS was unclear. In this study, we screened the main CYPs for the metabolism of STS and studied their interactions in vitro. 2.Seven CYPs were screened for the metabolism of STS by human liver microsomes (HLMs) or recombinant CYP isoforms. To determine the potential of STS to affect CYP-mediated phase I metabolism in humans, phenacetin (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), metoprolol (CYP2D6), chlorzoxazone (CYP2E1), S-Mephenytoin (CYP2C19), and midazolam (CYP3A4) were used as the respective probe substrates. Enzyme kinetic studies were performed to investigate the mode of inhibition of the enzyme-substrate interactions. 3.STS inhibited the activity of CYP3A4 in a dose-dependent manner in the HLMs and CYP3A4 isoform. Other CYP isoforms, including CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on the metabolism of STS. 4.The results suggested that STS primarily inhibits the activities of CYP3A4 in vitro, and STS has the potential to perpetrate drug-drug interactions with other CYP3A4 substrates.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Fenantrenos/farmacologia , Interações Medicamentosas , Humanos , Microssomos Hepáticos/metabolismoRESUMO
Recent evidence indicates that second messengers and protein kinases regulate the activity and expression of glutamate transporters. The aim of the present study was to determine if direct activation of protein kinases C or A modulates the activity of the sodium-dependent glutamate transporter EAAC1. EAAC1 modulation was studied in cRNA-injected Xenopus oocytes by measuring [3H]L-glutamate uptake or glutamate-evoked uptake currents. We found that activation of PKA was ineffective, whereas treatment with the PKC agonist phorbol 12-myristate 13-acetate (PMA) caused a significant decrease in EAAC1 transport activity (IC(50)=44.7+/-12 nM). PMA-induced EAAC1 inhibition was PKC-mediated because the inhibition could be blocked by specific PKC inhibitors and incubation with the inactive 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) did not affect EAAC1. Saturation studies of glutamate-evoked uptake currents showed that PMA-mediated inhibition was due to a decrease in I(max) with no change in K(m). PMA simultaneously decreased membrane capacitance (C(m)) and transport-associated current and increased cytosolic accumulation of EAAC1 protein, compared to control. These results suggest that PKC activation inhibits EAAC1 by promoting its retrieval from the plasma membrane. PMA also significantly decreased glutamate uptake in a Madin-Darby canine kidney (MDCK) cell line stably transfected with EAAC1 but enhanced EAAC1-mediated glutamate uptake in the rat C6 glioma cells, consistent with previous observations. Because activation of PKC by phorbol esters leads to opposite effects on EAAC1 activity in different culture models, we conclude that the PKC-mediated regulation of EAAC1 is cell-type specific.
Assuntos
Sistema X-AG de Transporte de Aminoácidos , Carcinógenos/farmacologia , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ácido Glutâmico/metabolismo , Proteína Quinase C/metabolismo , Simportadores , Acetato de Tetradecanoilforbol/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Membrana Celular/efeitos dos fármacos , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Transportador 3 de Aminoácido Excitatório , Proteínas de Transporte de Glutamato da Membrana Plasmática , Ácido Glutâmico/farmacocinética , Humanos , Indóis/farmacologia , Maleimidas/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Forbóis/farmacologia , Proteína Quinase C/efeitos dos fármacos , RNA Complementar/farmacologia , Trítio/farmacocinética , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Xenopus laevisRESUMO
Recent advances in several experimental techniques have enabled detailed structural information to be obtained for floating (Langmuir) monolayers and Langmuir-Blodgett films. These techniques are described briefly and their application to the study of films of fatty acids and their salts is discussed. Floating monolayers on aqueous subphases have been shown to possess a complex polymorphism with phases whose structures may be compared to those of smectic mesophases. However, only those phases that exist at high surface pressures are normally used in Langmuir-Blodgett (LB) deposition. In single LB monolayers of fatty acids and fatty acid salts the acyl chains are in the all-trans conformation with their long axes normal to the substrate. The in-plane molecular packing is hexagonal with long-range bond orientational order and short-range positional order: known as the hexatic-B structure. This structure is found irrespective of the phase of the parent floating monolayer. The structures of multilayer LB films are similar to the structures of their bulk crystals, consisting of stacked bilayer lamellae. Each lamella is formed from two monolayers of fatty acid molecules or ions arranged head to head and held together by hydrogen bonding between pairs of acids or ionic bonding through the divalent cations. With acids the acyl chains are tilted with respect to the substrate normal and have a monoclinic structure, whereas the salts with divalent cations may have the chains normal to the substrate or tilted. The in-plane structures are usually centred rectangular with the chains in the trans conformation and packed in a herringbone pattern. Multilayer films of the acids show only a single-step order-disorder transition at the melting point. This temperature tends to rise as the number of layers increases. Complex changes occur when multilayer films of the salts are heated. Disorder of the chains begins at low temperatures but the arrangement of the head groups does not alter until the melting temperature is reached. Slow heating to a temperature just below the melting temperature gives, with some salts, a radical change in phase. The lamellar structure disappears and a new phase consisting of cylindrical rods lying parallel to the substrate surface and stacked in a hexagonal pattern is formed. In each rod the cations are aligned along the central axis surrounded by the disordered acyl chains.
Assuntos
Ácidos Graxos/química , Sais/química , Ácidos Graxos não Esterificados/química , Cinética , Conformação Molecular , TermodinâmicaRESUMO
Energy-dependent intestinal calcium absorption is important for the maintenance of calcium and bone homeostasis, especially when dietary calcium supply is restricted. The active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is a crucial regulator of this process and increases the expression of the transient receptor potential vanilloid 6 (Trpv6) calcium channel that mediates calcium transfer across the intestinal apical membrane. Genetic inactivation of Trpv6 in mice (Trpv6(-/-)) showed, however, that TRPV6 is redundant for intestinal calcium absorption when dietary calcium content is normal/high and passive diffusion likely contributes to maintain normal serum calcium levels. On the other hand, Trpv6 inactivation impaired the increase in intestinal calcium transport following calcium restriction, however without resulting in hypocalcemia. A possible explanation is that normocalcemia is maintained at the expense of bone homeostasis, a hypothesis investigated in this study. In this study, we thoroughly analyzed the bone phenotype of Trpv6(-/-) mice receiving a normal (approximately 1%) or low (approximately 0.02%) calcium diet from weaning onwards using micro-computed tomography, histomorphometry and serum parameters. When dietary supply of calcium is normal, Trpv6 inactivation did not affect growth plate morphology, bone mass and remodeling parameters in young adult or aging mice. Restricting dietary calcium had no effect on serum calcium levels and resulted in a comparable reduction in bone mass accrual in Trpv6(+/+) and Trpv6(-/-) mice (-35% and 45% respectively). This decrease in bone mass was associated with a similar increase in bone resorption, whereas serum osteocalcin levels and the amount of unmineralized bone matrix were only significantly increased in Trpv6(-/-) mice. Taken together, our findings indicate that TRPV6 contributes to intestinal calcium transport when dietary calcium supply is limited and in this condition indirectly regulates bone formation and/or mineralization.
Assuntos
Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Canais de Cálcio/metabolismo , Cálcio da Dieta/farmacologia , Cálcio/metabolismo , Homeostase/efeitos dos fármacos , Absorção Intestinal/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Envelhecimento/efeitos dos fármacos , Envelhecimento/patologia , Animais , Remodelação Óssea/efeitos dos fármacos , Cálcio/sangue , Canais de Cálcio/deficiência , Canais de Cálcio/genética , Duodeno/efeitos dos fármacos , Duodeno/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fosfatos/sangue , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genéticaRESUMO
Gene mutations in WNK4 kinase cause a genetic form of hypertension by affecting multiple ion transport pathways through different mechanisms. Cai et al. report that the inhibitory effect of WNK4 on the thiazide-sensitive sodium-chloride cotransporter occurs through the lysosomal degradation pathway in mammalian cells.
Assuntos
Proteínas de Transporte/fisiologia , Transporte de Íons/fisiologia , Túbulos Renais Distais/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Proteínas de Transporte/análise , Humanos , Hipertensão/etiologia , Hipertensão/genética , Hipertensão/fisiopatologia , Túbulos Renais Distais/química , Lisossomos/efeitos dos fármacos , Lisossomos/fisiologia , Mutação , Pseudo-Hipoaldosteronismo/etiologia , Pseudo-Hipoaldosteronismo/genética , Pseudo-Hipoaldosteronismo/fisiopatologia , Transdução de Sinais/fisiologia , Simportadores de Cloreto de Sódio/análise , Simportadores de Cloreto de Sódio/efeitos dos fármacos , Simportadores de Cloreto de Sódio/fisiologiaRESUMO
Grazing incidence x-ray-diffraction investigations of the structures of Langmuir-Blodgett films of cadmium behenate with 1, 2, 3, 5, and 21 monolayers are reported. The single monolayer film, deposited on a hydrophilic substrate, showed a hexagonal structure, whereas the bilayer film, deposited on a hydrophobic substrate, had a rectangular structure with herringbone orientation of the acyl chains. With multilayer films formed on a hydrophilic substrate, it was possible to detect that the hexagonal structure of the first layer was retained when additional layers were deposited and that the additional layers had the same rectangular structure as the bilayer.
Assuntos
Ácidos Graxos/química , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas , Estrutura Molecular , Difração de Raios XRESUMO
Two arsenic-resistant plasmids were constructed and introduced into Thiobacillus ferrooxidans strains by conjugation. The plasmids with the replicon of wide-host-range plasmid RSF1010 were stable in T. ferrooxidans. The arsenic resistance genes originating from the heterotroph were expressed in this obligately autotrophic bacterium, but the promoter derived from T. ferrooxidans showed no special function in its original host.
RESUMO
We report here the genomic structures of the genes encoding human calcium transport proteins CaT1 and CaT2, which belong to a recently identified class of highly selective calcium entry channels. The mRNA for CaT1 was expressed more abundantly than that for CaT2 in three major tissues involved in transcellular calcium transport, namely intestine, kidney, and placenta, as determined by quantitative PCR. The genes encoding CaT1 and CaT2, ECAC2 and ECAC1, respectively, are completely conserved in terms of exon size in the coding regions. They also share similar intron-exon structures with the genes encoding the closely related, nonselective cation channels VR1, VRL-1, OTRPC4 (also known as VR-OAC, Trp12, and VRL-2), and a hypothetical protein, VRL-3. We conclude that ECAC2 and ECAC1, which encode calcium selective channels, share a common ancestral gene with the genes encoding the related nonselective cation channels.
Assuntos
Canais de Cálcio/química , Sequência Conservada , Sequência de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos Básicos , Animais , Canais de Cálcio/biossíntese , Canais de Cálcio/genética , Canais de Cálcio/fisiologia , Clonagem Molecular , Sequência Conservada/genética , Éxons/genética , Feminino , Biblioteca Gênica , Humanos , Intestino Delgado/química , Rim/química , Rim/metabolismo , Dados de Sequência Molecular , Oócitos/química , Oócitos/metabolismo , Placenta/química , Gravidez , Canais de Cátion TRPV , Xenopus laevis/genéticaRESUMO
The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and pUB307 were transferred to acidophilic, obligately chemolithotrophic Thiobacillus ferrooxidans from Escherichia coli by conjugation. A genetic marker of kanamycin resistance was expressed in T. ferrooxidans. Plasmid RP4 was transferred back to E. coli from T. ferrooxidans. The broad-host-range IncQ vector pJRD215 was mobilized to T. ferrooxidans with the aid of plasmid RP4 integrated in the chromosome of E. coli SM10. pJRD215 was stable, and all genetic markers (kanamycin/neomycin and streptomycin resistance) were expressed in T. ferrooxidans. By the use of suicide vector pSUP1011, transposon Tn5 was introduced into T. ferrooxidans. The influence of some factors on plasmid transfer from E. coli to T. ferrooxidans was investigated. Results showed that the physiological state of donor cells might be important to the mobilization of plasmids. The transfer of plasmids from E. coli to T. ferrooxidans occurred in the absence of energy sources for both donor and recipient.
Assuntos
Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Técnicas de Transferência de Genes , Plasmídeos/genética , Thiobacillus/genética , Conjugação Genética , Marcadores Genéticos , Resistência a Canamicina/genéticaRESUMO
The calcium-release-activated Ca2+channel, ICRAC, is a highly Ca2+-selective ion channel that is activated on depletion of either intracellular Ca2+ levels or intracellular Ca2+ stores. The unique gating of ICRAC has made it a favourite target of investigation for new signal transduction mechanisms; however, without molecular identification of the channel protein, such studies have been inconclusive. Here we show that the protein CaT1 (ref. 4), which has six membrane-spanning domains, exhibits the unique biophysical properties of ICRAC when expressed in mammalian cells. Like ICRAC, expressed CaT1 protein is Ca2+ selective, activated by a reduction in intracellular Ca2+ concentration, and inactivated by higher intracellular concentrations of Ca2+. The channel is indistinguishable from ICRAC in the following features: sequence of selectivity to divalent cations; an anomalous mole fraction effect; whole-cell current kinetics; block by lanthanum; loss of selectivity in the absence of divalent cations; and single-channel conductance to Na+ in divalent-ion-free conditions. CaT1 is activated by both passive and active depletion of calcium stores. We propose that CaT1 comprises all or part of the ICRAC pore.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Animais , Células CHO , Canais de Cálcio/genética , Cricetinae , Cricetulus , Eletrofisiologia , Ratos , Canais de Cátion TRPV , Transfecção , Células Tumorais CultivadasRESUMO
A newly cloned, human epithelial Ca2+ transport protein (CaT1) was expressed in Xenopus laevis oocytes, and its single channel characteristics were examined. The CaT1 channel shows a strong dependence upon hyperpolarizing voltages, being activated by very negative voltages. The probability of channel opening and mean open times increase substantially at more negative voltages in the range of -90 to -160 mV. In addition, CaT1 channel activity was markedly inhibited by micromolar levels of a noncompetitive antagonist of the IP3 receptor originally isolated from a marine sponge, Xestospongin C. This inhibitory effect could be mediated indirectly via the binding of Xestospongin C to the inositol-trisphosphate (IP3) receptor or, alternatively, by a direct action on the CaT1 channel itself. Independent of its mechanism of action in inhibiting CaT1, Xestospongin C will provide a useful tool for elucidating the physiological role(s) of this novel epithelial Ca2+ channel.
Assuntos
Canais de Cálcio/fisiologia , Quelantes/farmacologia , Ácido Edético/análogos & derivados , Oxazóis/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Animais , Canais de Cálcio/efeitos dos fármacos , Ácido Edético/farmacologia , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Compostos Macrocíclicos , Potenciais da Membrana/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Canais de Cátion TRPV , Xenopus laevisRESUMO
The human epithelial channels, CaT1 and CaT2, were expressed in oocytes, and their single-channel characteristics were compared. In the presence of Na+ and K+ as charge carriers in the pipette solutions, channel activities were observed only when the the extracellular sides of the patches were exposed to nominally Ca2+- and Mg2+-free solutions. In patches of both CaT1- and CaT2-expressing oocytes, multiple channel openings were observed, but the current levels were higher in CaT2-expressing oocytes, particularly at more negative voltages. With K+ as a charge carrier in patches of CaT1-expressing oocytes, the channel activity was low at -10 to -60 mV, but increased dramatically at more negative potentials. This voltage dependence was observed in the presence of both Na+ and K+. The channel activity with Na+, however, was higher at all potentials. Differences between the voltage dependencies for the two cations were also observed in CaT2-expressing oocytes, but the channel activities were higher than those in CaT1-expressing oocytes, particularly in the presence of Na+. We also found that low concentrations of extracellular Mg2+ (5-50 microm) elicited a strong inhibitory action on the CaT channels. Activation of the CaT1 and CaT2 channels by hyperpolarization and other factors may promote increased Ca2+ entry that participates in stimulation of intestinal absorption and renal reabsorption and/or other Ca2+ transport mechanisms in epithelial cells.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Sistemas de Transporte de Aminoácidos Básicos , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Células Epiteliais/fisiologia , Humanos , Magnésio/farmacologia , Potenciais da Membrana/fisiologia , Oócitos/metabolismo , Potássio/metabolismo , RNA Complementar/genética , Ratos , Sódio/metabolismo , Canais de Cátion TRPV , Transfecção , Xenopus laevisRESUMO
Excitatory amino acid transporter family (EAAT) contains several structure-related membrane proteins. They are essential for the removal of glutamate released from pre-synaptic terminal to terminate its action of synaptic transduction and maintaining the normal concentration of neurotransmitters in nerve system. To study these proteins in single animal model, we cloned several members of EAAT family, named mGLAST-1, mGLT-1, mEAAC1 and mASCT1, from a neonatal mouse brain cDNA library. The cDNA sequence of mASCT1 was firstly reported in mouse, it is composed of 3787 bp which has an open reading frame (ORF) encoding a protein of 532 amino acid residues. The mASCT1 protein was expressed in Xenopus oocyte and the function was characterized by 3H-Ser uptaking. The homology between human ASCT1 and mouse ASCT1 is 89.3%. The DNA sequence data shows the variance in length and composition exists in the sequence of 5'UTR and 3'UTR of mRNA in the family members of EAAT. This phenomenon may indicate a post-transcription regulation mechanism might exist in the gene expression of mouse EAAT family members.
Assuntos
Sistema ASC de Transporte de Aminoácidos/genética , Sistema X-AG de Transporte de Aminoácidos/genética , Encéfalo/metabolismo , DNA Complementar/genética , Transportador 1 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/genética , Simportadores/genética , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , Proteínas de Transporte de Glutamato da Membrana Plasmática , Camundongos , Dados de Sequência MolecularRESUMO
Active absorption of calcium from the intestine and reabsorption of calcium from the kidney are major determinants of whole body calcium homeostasis. Two recently cloned proteins, CaT1 and ECaC, have been postulated to mediate apical calcium uptake by rat intestine and rabbit kidney, respectively. By screening a rat kidney cortex library with a CaT1 probe, we isolated a cDNA encoding a protein (CaT2) with 84.2 and 73.4% amino acid identities to ECaC and CaT1, respectively. Unlike ECaC, CaT2 is kidney-specific in the rat and was not detected in intestine, brain, adrenal gland, heart, skeletal muscle, liver, lung, spleen, thymus, and testis by Northern analysis or reverse transcription polymerase chain reaction. The expression pattern of CaT2 in kidney was similar to that of calbindin D(28K) and the sodium calcium exchanger 1, NCX1, by in situ hybridization of adjacent sections. Furthermore, the mRNAs for CaT2 and calbindin D(28K) were colocalized in the same cells. CaT2 mediated saturable calcium uptake with a Michaelis constant (K(m)) of 0.66 mm when expressed in Xenopus laevis oocytes. Under voltage clamp condition, CaT2 promoted inward currents in X. laevis oocytes upon external application of Ca(2+). Sr(2+) and Ba(2+) but not Mg(2+) also evoked inward currents in CaT2-expressing oocytes. Similar to the alkaline earth metal ions, application of Cd(2+) elicited inward current in CaT2-expressing oocytes with a K(m) of 1.3 mm. Cd(2+), however, also potently inhibited CaT2-mediated Ca(2+) uptake with an IC(50) of 5.4 micrometer. Ca(2+) evoked currents were reduced at low pH and increased at high pH and were only slightly affected by the L-type voltage-dependent calcium channel antagonists, nifedipine, verapamil, diltiazem, and the agonist, Bay K 8644, even at relatively high concentrations. In conclusion, CaT2 may participate in calcium entry into the cells of the distal convoluted tubule and connecting segment of the nephron, where active reabsorption of calcium takes place via the transcellular route. The high sensitivity of CaT2 to Cd(2+) also provides a potential explanation for Cd(2+)-induced hypercalciuria and resultant renal stone formation.
Assuntos
Canais de Cálcio/fisiologia , Córtex Renal/metabolismo , Néfrons/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos Básicos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/química , Canais de Cálcio/genética , DNA Complementar , Biblioteca Gênica , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/fisiologia , Estrutura Secundária de Proteína , Coelhos , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Canais de Cátion TRPV , Xenopus laevisRESUMO
Yeast membrane proteins SMF1, SMF2, and SMF3 are homologues of the DCT1 metal ion transporter family. Their functional characteristics and the implications of these characteristics in vivo have not yet been reported. Here we show that SMF1 expressed in Xenopus oocytes mediates H(+)-dependent Fe(2+) transport and uncoupled Na(+) flux. SMF1-mediated Fe(2+) transport exhibited saturation kinetics (K(m) = 2.2 microM), whereas the Na(+) flux did not, although both processes were electrogenic. SMF1 is also permeable to Li(+), Rb(+), K(+), and Ca(2+), which likely share the same uncoupled pathway. SMF2 (but not SMF3) mediated significant increases in both Fe(2+) and Na(+) transport compared with control oocytes. These data are consistent with the concept that uptake of divalent metal ions by SMF1 and SMF2 is essential to yeast cell growth. Na(+) inhibited metal ion uptake mediated by SMF1 and SMF2 expressed in oocytes. Consistent with this, we found that increased sensitivity of yeast to EGTA in the high Na(+) medium is due to inhibition of SMF1- and SMF2-mediated metal ion transport by uncoupled Na(+) pathway. Interestingly, DCT1 also mediates Fe(2+)-activated uncoupled currents. We propose that uncoupled ion permeabilities in metal ion transporters protect cells from metal ion overload.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions , Hidrogênio/metabolismo , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Animais , Cálcio/farmacocinética , Cátions/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Ácido Egtázico/farmacologia , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Lantânio/farmacologia , Mutagênese , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Ratos , Saccharomyces cerevisiae/metabolismo , Cloreto de Sódio/farmacologia , Água/metabolismo , XenopusRESUMO
A multiple-imaging-plate detector system and focusing monochromator have been developed and successfully applied to the time-resolved study of phase transitions in Langmuir-Blodgett films by grazing-incidence X-ray diffraction (GIXD). The monochromator described here combines fixed-exit-beam height with sagittal focusing of the second crystal. The design is similar to that of Matsushita et al. [Matsushita, Ishikawa & Oyanagi (1986). Nucl. Instrum. Methods, A246, 377-379], with the exception that the motion of the first crystal is achieved via a computer-controlled X-Y translation table rather than a set of cams. The second crystal is a ribbed Si(111) wafer mounted in a four-point bending mechanism. The first reported application of imaging plates to a GIXD study was carried out by our group and proved to be very successful in the determination of thin-film structure [Foran, Peng, Steitz, Barnes & Gentle (1996). Langmuir, 12, 774-777]. To extend the capabilities of this system, an imaging-plate camera was designed and built which can accommodate up to 13 imaging plates (40 x 20 cm) inside the vacuum chamber of the main diffractometer at the Australian Beamline at the Photon Factory.