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1.
J Toxicol Sci ; 45(9): 539-548, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32879253

RESUMO

We investigated the mechanism underlying intestinal cadmium (Cd) uptake based on the mediators (metal transporters) of essential elements, such as Fe, Zn, Cu, and Ca, under normal conditions in female rats. These elements interact with Cd uptake from the intestinal tract. Cd concentration at each site of the small intestine (duodenum, jejunum, and ileum) increased as Cd exposure increased. However, Cd concentration was the highest in the duodenum. The gene expression of ZIP14, DMT1, and ATP7A increased with increase in Cd concentration. Further, Cu concentration decreased as Cd concentration increased. In contrast, Fe concentration displayed a decreasing tendency with the increase in Cd concentration. The gene expression levels of ZIP14, DMT1, and ATP7A were positively correlated with Cd concentration. Immunohistochemical staining revealed the positive sites of ZIP14 and DMT1 scattered in the area adjacent to the goblet cells, resorbable epithelial cells, and lamina propria in the duodenum tissue, according to the increase in Cd concentration. Cd is induced to synthesize and bind to metallothionein (MT-I and -II) and accumulate in the intestinal tissues, mainly in the duodenum. Such findings suggest that Cd, a contaminant element, is taken up from the intestinal tract by multiple metal transporters such as Cu, Fe, and Zn, thereby involving in the intestinal Cd absorption.


Assuntos
Cádmio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Absorção Intestinal/genética , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Cobre , ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , Duodeno/metabolismo , Feminino , Expressão Gênica , Ferro , Metalotioneína/metabolismo , Ratos , Zinco
2.
Nat Commun ; 11(1): 4778, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963241

RESUMO

Obtaining genetic variation information from indica rice hybrid parents and identification of loci associated with heterosis are important for hybrid rice breeding. Here, we resequence 1,143 indica accessions mostly selected from the parents of superior hybrid rice cultivars of China, identify genetic variations, and perform kinship analysis. We find different hybrid rice crossing patterns between 3- and 2-line superior hybrid lines. By calculating frequencies of parental variation differences (FPVDs), a more direct approach for studying rice heterosis, we identify loci that are linked to heterosis, which include 98 in superior 3-line hybrids and 36 in superior 2-line hybrids. As a proof of concept, we find two accessions harboring a deletion in OsNramp5, a previously reported gene functioning in cadmium absorption, which can be used to mitigate rice grain cadmium levels through hybrid breeding. Resource of indica rice genetic variation reported in this study will be valuable to geneticists and breeders.


Assuntos
Variação Genética , Vigor Híbrido/genética , Oryza/genética , Cruzamento , Proteínas de Transporte de Cátions/genética , China , Cruzamentos Genéticos , Deleção de Genes , Genes de Plantas , Hibridização Genética , Oryza/classificação , Filogenia , Proteínas de Plantas/genética , Polimorfismo de Nucleotídeo Único
3.
Proc Natl Acad Sci U S A ; 117(33): 20235-20243, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32753384

RESUMO

All cells require Mg2+ to replicate and proliferate. The macrophage protein Slc11a1 is proposed to protect mice from invading microbes by causing Mg2+ starvation in host tissues. However, the Mg2+ transporter MgtB enables the facultative intracellular pathogen Salmonella enterica serovar Typhimurium to cause disease in mice harboring a functional Slc11a1 protein. Here, we report that, unexpectedly, the Salmonella small protein MgtR promotes MgtB degradation by the protease FtsH, which raises the question: How does Salmonella preserve MgtB to promote survival inside macrophages? We establish that the Salmonella small protein MgtU prevents MgtB proteolysis, even when MgtR is absent. Like MgtB, MgtU is necessary for survival in Slc11a1 +/+ macrophages, resistance to oxidative stress, and growth under Mg2+ limitation conditions. The Salmonella Mg2+ transporter MgtA is not protected by MgtU despite sharing 50% amino acid identity with MgtB and being degraded in an MgtR- and FtsH-dependent manner. Surprisingly, the mgtB, mgtR, and mgtU genes are part of the same transcript, providing a singular example of transcript-specifying proteins that promote and hinder degradation of the same target. Our findings demonstrate that small proteins can confer pathogen survival inside macrophages by altering the abundance of related transporters, thereby furthering homeostasis.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Macrófagos/microbiologia , Magnésio/metabolismo , Salmonella typhimurium/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Macrófagos/fisiologia , Camundongos , Plasmídeos/genética , Salmonella typhimurium/genética , Virulência
4.
Chem Biol Interact ; 329: 109217, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32750324

RESUMO

Developing brain is very sensitive to the influence of environmental factors during gestation and the neonatal period. The aim of the study is to assess cobalt and iron accumulation in the brain as well as changes in the expression of iron-regulatory proteins transferrin receptor 1, hepcidin, and ferroportin in suckling mice. Perinatal exposure to cobalt chloride increased significantly cobalt content in brain tissue homogenates of 18-day-old (d18) and 25-day-old (d25) mice inducing alterations in brain iron homeostasis. Higher degree of transferrin receptor 1 expression was demonstrated in cobalt chloride-exposed mice with no substantial changes between d18 and d25 mice. A weak ferroportin expression was found in 18-day-old control and cobalt-treated mouse brain. Cobalt exposure of d25 mice resulted in increased ferroportin expression in brain compared to the untreated age-matched control group. Hepcidin level in cobalt-exposed groups was decreased in d18 mice and slightly increased in d25 mice. The obtained data contribute for the better understanding of metal toxicity impact on iron homeostasis in the developing brain with further possible implications in neurodegeneration.


Assuntos
Encéfalo/metabolismo , Cobalto/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas Reguladoras do Ferro/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cobalto/metabolismo , Feminino , Hepcidinas/genética , Hepcidinas/metabolismo , Ferro/metabolismo , Proteínas Reguladoras do Ferro/genética , Camundongos , Camundongos Endogâmicos ICR , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo
5.
Aquat Toxicol ; 225: 105524, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32610223

RESUMO

In the present research, the effects of exposure to a sublethal concentration of zinc (Zn) on metal and ion homeostasis, and the regulation and the localization of various Zn transporters (i.e., the Zrt-Irt Like Protein (ZIP) family of Zn transporters), were investigated in zebrafish (Danio rerio) during early development. Exposure to an elevated level of Zn [4 µM (high) vs. 0.25 µM (control)] from 0 day post-fertilization (dpf) resulted in a significant increase in the whole body content of Zn at 5 dpf. A transient decrease in the whole body calcium (Ca) level was observed in 3 dpf larvae exposed to high Zn. Similarly, whole body nickel (Ni) and copper (Cu) contents were also reduced in 3 dpf larvae exposed to high Zn. Importantly, the magnitude of reduction in whole body Ni and Cu contents following Zn exposure was markedly higher than that in Ca content, suggesting that internal Ni and Cu balance were likely more sensitive to Zn exposure in developing zebrafish. Exposure to high Zn altered the mRNA expression levels of specific zip transporters, with an increase in zip1 (at 3 dpf) and zip8 (at 5 dpf), and a decrease in zip4 (at 5 dpf). The expression levels of most zip transporters tended to decrease from 3 dpf to 5 dpf with the exception of zip4 and zip8. Results from in situ hybridization revealed that several zip transporters exhibited distinct spatial distribution (e.g., zip8 in the intestinal tract, zip14 in the pronephric tubules). Overall, our findings suggested that exposure to sublethal concentrations of Zn disrupts the homeostasis of essential metals during early development and that different ZIP transporters may play unique roles in regulating Zn homeostasis in various organs in developing zebrafish.


Assuntos
Proteínas de Transporte de Cátions/genética , Homeostase/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Proteínas de Peixe-Zebra/genética , Peixe-Zebra , Zinco/toxicidade , Animais , Cálcio/metabolismo , Cobre/metabolismo , Relação Dose-Resposta a Droga , Larva/efeitos dos fármacos , Larva/metabolismo , Nível de Efeito Adverso não Observado , Poluentes Químicos da Água/metabolismo , Peixe-Zebra/fisiologia , Zinco/metabolismo
6.
Gene ; 756: 144914, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32574759

RESUMO

The life history of the Chinese mitten crab (Eriocheir japonica sinensis) includes two migrations: a feeding migration and a reproductive migration. Ambient salinity is one of the most critical factors during migration. In this study, the salinity adaptation mechanism of Chinese mitten crabs was simulated using continuous salinity changes. The expression of six key genes [Na+/K+-ATPase α subunit (NAK-α), V-type H+-ATPase subunit A (VHA-A), Zinc transporter (ZnT), Cl- channel protein 2 (CLCN2), ubiquitin/ribosomal S27 fusionprotein (S27), and glutathione S-transferase (GST)] and the activities of three enzymes [Na+/K+-ATPase (NAK), V-type H+-ATPase (VHA), and glutathione S-transferase (GST)] were evaluated in ten groups exposed to a range of salinity changes during mariculture based on the transcriptome data obtained from short term salinity-induced crabs (ES) compared to control group in freshwater crabs (EF). The results revealed that different genes exhibited different roles in physiological regulation. In total, 3,599 unigenes were significantly and differentially expressed in a comparison between the EF and ES treatments. A novel modulation of gene expression and the corresponding enzyme activity of NAK and VHA exhibited similar patterns. As genes related to osmoregulation, NAK and VHA showed similar patterns of both gene expression and enzyme activity in mariculture. During the gradual change in salinity from 0‰ to 25‰ and back to 0‰, the gene expression and enzyme activities of NAK and VHA initially increased (0‰ â†’ 10‰), weakened (10‰ â†’ 20‰) and then increased again (20‰ â†’ 25‰ â†’ 0‰). S27 could serve as a reference gene in the expression analysis of Chinese mitten crabs under salinity stress. ZnT and CLCN2 were involved in osmoregulation as functional proteins. Our findings provide insights into the regulation mechanisms employed during the migration of the Chinese mitten crab.


Assuntos
Braquiúros/genética , Braquiúros/fisiologia , Osmorregulação , Adaptação Fisiológica , Migração Animal , Animais , Proteínas de Artrópodes/genética , Proteínas de Transporte de Cátions/genética , Água Doce , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Brânquias/fisiologia , Masculino , Salinidade , Análise de Sequência de RNA
7.
mSphere ; 5(3)2020 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-32461278

RESUMO

Zinc supplementation in cell culture has been shown to inhibit various viruses, like herpes simplex virus, rotavirus, severe acute respiratory syndrome (SARS) coronavirus, rhinovirus, and respiratory syncytial virus (RSV). However, whether zinc plays a direct antiviral role in viral infections and whether viruses have adopted strategies to modulate zinc homeostasis have not been investigated. Results from clinical trials of zinc supplementation in infections indicate that zinc supplementation may be beneficial in a pathogen- or disease-specific manner, further underscoring the importance of understanding the interaction between zinc homeostasis and virus infections at the molecular level. We investigated the effect of RSV infection on zinc homeostasis and show that RSV infection in lung epithelial cells leads to modulation of zinc homeostasis. The intracellular labile zinc pool increases upon RSV infection in a multiplicity of infection (MOI)-dependent fashion. Small interfering RNA (siRNA)-mediated knockdown of the ubiquitous zinc uptake transporter ZIP1 suggests that labile zinc levels are increased due to the increased uptake by RSV-infected cells as an antiviral response. Adding zinc to culture medium after RSV infection led to significant inhibition of RSV titers, whereas depletion of zinc by a zinc chelator, N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) led to an increase in RSV titers. The inhibitory effect of zinc was specific, as other divalent cations had no effect on RSV titers. Both RSV infection and zinc chelation by TPEN led to reactive oxygen species (ROS) induction, whereas addition of zinc blocked ROS induction. These results suggest a molecular link between RSV infection, zinc homeostasis, and oxidative-stress pathways and provide new insights for developing strategies to counter RSV infection.IMPORTANCE Zinc deficiency rates in developing countries range from 20 to 30%, and zinc supplementation trials have been shown to correct clinical manifestations attributed to zinc deficiency, but the outcomes in the case of respiratory infections have been inconsistent. We aimed at understanding the role of zinc homeostasis in respiratory syncytial virus (RSV) infection. Infection of lung epithelial cell lines or primary small-airway epithelial cells led to an increase in labile zinc pools, which was due to increased uptake of zinc. Zinc supplementation inhibited RSV replication, whereas zinc chelation had an opposing effect, leading to increases in RSV titers. Increases in labile zinc in RSV-infected cells coincided with induction of reactive oxygen species (ROS). Both zinc depletion and addition of exogenous ROS led to enhanced RSV infection, whereas addition of the antioxidant inhibited RSV, suggesting that zinc is part of an interplay between RSV-induced oxidative stress and the host response to maintain redox balance.


Assuntos
Infecções por Vírus Respiratório Sincicial/patologia , Vírus Sincicial Respiratório Humano/metabolismo , Replicação Viral/efeitos dos fármacos , Zinco/metabolismo , Zinco/farmacologia , Células A549 , Adolescente , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Criança , Pré-Escolar , Células Epiteliais/metabolismo , Etilenodiaminas/farmacologia , Feminino , Interações Hospedeiro-Patógeno , Humanos , Pulmão/citologia , Pulmão/metabolismo , Masculino , Estresse Oxidativo/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia
8.
Proc Natl Acad Sci U S A ; 117(23): 12931-12942, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32457148

RESUMO

Retinal disease and loss of vision can result from any disruption of the complex pathways controlling retinal development and homeostasis. Forward genetics provides an excellent tool to find, in an unbiased manner, genes that are essential to these processes. Using N-ethyl-N-nitrosourea mutagenesis in mice in combination with a screening protocol using optical coherence tomography (OCT) and automated meiotic mapping, we identified 11 mutations presumably causative of retinal phenotypes in genes previously known to be essential for retinal integrity. In addition, we found multiple statistically significant gene-phenotype associations that have not been reported previously and decided to target one of these genes, Sfxn3 (encoding sideroflexin-3), using CRISPR/Cas9 technology. We demonstrate, using OCT, light microscopy, and electroretinography, that two Sfxn3 -/- mouse lines developed progressive and severe outer retinal degeneration. Electron microscopy showed thinning of the retinal pigment epithelium and disruption of the external limiting membrane. Using single-cell RNA sequencing of retinal cells isolated from C57BL/6J mice, we demonstrate that Sfxn3 is expressed in several bipolar cell subtypes, retinal ganglion cells, and some amacrine cell subtypes but not significantly in Müller cells or photoreceptors. In situ hybridization confirmed these findings. Furthermore, pathway analysis suggests that Sfxn3 may be associated with synaptic homeostasis. Importantly, electron microscopy analysis showed disruption of synapses and synaptic ribbons in the outer plexiform layer of Sfxn3 -/- mice. Our work describes a previously unknown requirement for Sfxn3 in retinal function.


Assuntos
Proteínas de Transporte de Cátions/genética , Degeneração Retiniana/genética , Segmento Externo das Células Fotorreceptoras da Retina/patologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Eletrorretinografia , Etilnitrosoureia/toxicidade , Feminino , Humanos , Masculino , Camundongos , Microscopia Eletrônica , Mutagênese , Mutação/efeitos dos fármacos , Degeneração Retiniana/diagnóstico , Degeneração Retiniana/patologia , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Epitélio Pigmentado da Retina/diagnóstico por imagem , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/ultraestrutura , Tomografia de Coerência Óptica
9.
PLoS Genet ; 16(4): e1008728, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32352966

RESUMO

Genetic studies of both the human host and Mycobacterium tuberculosis (MTB) demonstrate independent association with tuberculosis (TB) risk. However, neither explains a large portion of disease risk or severity. Based on studies in other infectious diseases and animal models of TB, we hypothesized that the genomes of the two interact to modulate risk of developing active TB or increasing the severity of disease, when present. We examined this hypothesis in our TB household contact study in Kampala, Uganda, in which there were 3 MTB lineages of which L4-Ugandan (L4.6) is the most recent. TB severity, measured using the Bandim TBscore, was modeled as a function of host SNP genotype, MTB lineage, and their interaction, within two independent cohorts of TB cases, N = 113 and 121. No association was found between lineage and severity, but association between multiple polymorphisms in IL12B and TBscore was replicated in two independent cohorts (most significant rs3212227, combined p = 0.0006), supporting previous associations of IL12B with TB susceptibility. We also observed significant interaction between a single nucleotide polymorphism (SNP) in SLC11A1 and the L4-Ugandan lineage in both cohorts (rs17235409, meta p = 0.0002). Interestingly, the presence of the L4-Uganda lineage in the presence of the ancestral human allele associated with more severe disease. These findings demonstrate that IL12B is associated with severity of TB in addition to susceptibility, and that the association between TB severity and human genetics can be due to an interaction between genes in the two species, consistent with host-pathogen coevolution in TB.


Assuntos
Coevolução Biológica , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleotídeo Único , Tuberculose/genética , Adolescente , Adulto , Idoso , Proteínas de Transporte de Cátions/genética , Evolução Molecular , Feminino , Genoma Bacteriano , Interações Hospedeiro-Patógeno , Humanos , Subunidade p40 da Interleucina-12/genética , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/patogenicidade , Tuberculose/microbiologia , Tuberculose/patologia
10.
Plant Mol Biol ; 103(4-5): 561-580, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32405802

RESUMO

KEY MESSAGE: CmHKT1;1 selectively exports Na+ from plant cells. Upon NaCl stress, its expression increased in a salt-tolerant melon cultivar. Overexpression of CmHKT1;1 increased transgenic Arabidopsis salt tolerance through improved K+/Na+ balance. High-affinity K+ transporters (HKTs) are thought to be involved in reducing Na+ in plant shoots under salt stress and modulating salt tolerance, but their function in a moderately salt-tolerant species of melon (Cucumis melo L.) remains unclear. In this study, a Na+ transporter gene, CmHKT1;1 (GenBank accession number: MK986658), was isolated from melons based on genome data. The transcript of CmHKT1;1 was relatively more abundant in roots than in stems or leaves from melon seedlings. The tobacco transient expression system showed that CmHKT1;1 was plasma-membrane localized. Upon salt stress, CmHKT1;1 expression was more strongly upregulated in a salt-tolerant melon cultivar, 'Bingxuecui' (BXC) compared with a salt-sensitive cultivar, 'Yulu' (YL). Electrophysiological evidence demonstrated that CmHKT1;1 only transported Na+, rather than K+, when expressed in Xenopus laevis oocytes. Overexpression of CmHKT1;1 increased salt sensitivity in Saccharomyces cerevisiae and salt tolerance in Arabidopsis thaliana. Under NaCl treatments, transgenic Arabidopsis plants accumulated significantly lower concentrations of Na+ in shoots than wild type plants and showed a better K+/Na+ balance, leading to better Fv/Fm, root length, biomass, and enhanced plant growth. The CmHKT1;1 gene may serve as a useful candidate for improving crop salt tolerance.


Assuntos
Arabidopsis/metabolismo , Cucumis melo/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Clorofila/análise , Clonagem Molecular , Cucumis melo/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/genética , Saccharomyces cerevisiae/genética , Tolerância ao Sal , Plântula/genética , Plântula/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Cloreto de Sódio/metabolismo , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Simportadores/genética , Simportadores/metabolismo , Tabaco/genética , Tabaco/metabolismo
11.
BMC Med Genet ; 21(1): 77, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32293312

RESUMO

BACKGROUND: Myopathy with extrapyramidal signs (MPXPS) is an autosomal recessive mitochondrial disorder which is caused by mutation in mitochondrial calcium uptake 1 (MICU1) gene located on chromosome 10q22.1. Next Generation Sequencing (NGS) technology is the most effective method for identification of pathogenic variants with the ability to overcome some limitations which Sanger sequencing may encountered. There are few reports on this rare disease around the world and here in this study we first revealed genetic identification of two affected individuals in an Iranian family with a novel mutation. CASE PRESENTATION: The proband was a 5-year-old girl from consanguenous parents. She was first clinically suspicious of affected with limb-girdle muscular dystrophy (LGMD). Muscle biopsy studies and autozygosity mapping, using four short tandem repeat (STR) markers linked to 6 genes of the most prevalent forms of LGMD, ruled out calpainopathy, dysferlinopathy, and sarcoglycanopathis. DNA sample of the proband was sent for NGS. Whole exome sequencing (WES) revealed a novel mutation c.1295delA in exon 13 of MICU1 gene. This homozygous deletion creates a frameshift and a premature stop codon downstream of canonical EF4 calcium binding motif of MICU1. According to the American College of Medical Genetics and Genomics (ACMG) guidline for sequence interpretation, this variant was a pathogenic one. Sanger sequencing in all family members confirmed the results of the WES. CONCLUSIONS: This study was the first report of MPXPS in Iranian population which also revealed a novel mutation in the MICU1 gene.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions/genética , Repetições de Microssatélites/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Doenças Musculares/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Pré-Escolar , Exoma/genética , Éxons/genética , Tratos Extrapiramidais/metabolismo , Tratos Extrapiramidais/patologia , Feminino , Mutação da Fase de Leitura/genética , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Irã (Geográfico)/epidemiologia , Doenças Musculares/patologia , Distrofia Muscular do Cíngulo dos Membros/patologia , Linhagem , Deleção de Sequência/genética , Sequenciamento Completo do Exoma
12.
Artigo em Inglês | MEDLINE | ID: mdl-32320289

RESUMO

Suboptimal lactation is a common, yet underappreciated cause for early cessation of breastfeeding. Molecular regulation of mammary gland function is critical to the process lactation; however, physiological factors underlying insufficient milk production are poorly understood. The zinc (Zn) transporter ZnT2 is critical for regulation of mammary gland development and maturation during puberty, lactation, and postlactation gland remodeling. Numerous genetic variants in the gene encoding ZnT2 (SLC30A2) are associated with low milk Zn concentration and result in severe Zn deficiency in exclusively breastfed infants. However, the functional impacts of genetic variation in ZnT2 on key mammary epithelial cell functions have not yet been systematically explored at the cellular level. Here we determined a common mutation in SLC30A2/ZnT2 substituting serine for threonine at amino acid 288 (Thr288Ser) was found in 20% of women producing low milk volume (n = 2/10) but was not identified in women producing normal volume. Exploration of cellular consequences in vitro using phosphomimetics showed the serine substitution promoted preferential phosphorylation of ZnT2, driving localization to the lysosome and increasing lysosome biogenesis and acidification. While the substitution did not initiate lysosome-mediated cell death, cellular ATP levels were significantly reduced. Our findings demonstrate the Thr288Ser mutation in SLC30A2/ZnT2 impairs critical functions of mammary epithelial cells and suggest a role for genetic variation in the regulation of milk production and lactation performance.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Metabolismo Energético , Células Epiteliais/metabolismo , Lactação/metabolismo , Lisossomos/metabolismo , Glândulas Mamárias Humanas/metabolismo , Leite Humano/metabolismo , Mutação , Trifosfato de Adenosina/metabolismo , Adulto , Estudos de Casos e Controles , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Metabolismo Energético/genética , Feminino , Humanos , Concentração de Íons de Hidrogênio , Lactação/genética , Lisossomos/genética , Biogênese de Organelas , Fosforilação , Adulto Jovem
13.
Toxicology ; 436: 152428, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32151602

RESUMO

The increase in human infertility prevalence due to male reproductive disorders has been associated with extensive endocrine-disrupting chemical (EDC) exposure. Acrylamide (AA) is a compound formed spontaneously during heat processing of some foods that are mainly consumed by children and adolescents. In this study, we evaluated the prepubertal AA exposure effects on male adult reproductive physiology using a prepubertal experimental model to analyze the pubertal development, spermatogenesis hormones levels and genes expression involved in male reproductive function. This study is the first one to use the validated protocol to correlate the AA exposure with puberty development, as well as the AA-induced endocrine disrupting effects on reproductive axis. AA did not affect the age at puberty, the reproductive organ's weight and serum hormonal levels. AA reduces spermatogenesis, induces morphological and functional defects on sperm and alters transcript expression of sexual hormone receptors (Ar and Esr2), the transcript expression of Tnf, Egr2, Rhcg and Lrrc34. These findings suggest that excessive AA consumption may impair their reproductive capacity at adulthood, despite no changes in hormonal profile being observed.


Assuntos
Acrilamida/toxicidade , Disruptores Endócrinos/toxicidade , Contaminação de Alimentos , Infertilidade Masculina/induzido quimicamente , Desenvolvimento Sexual/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Fatores Etários , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Relação Dose-Resposta a Droga , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Infertilidade Masculina/fisiopatologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratos Wistar , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Medição de Risco , Espermatozoides/metabolismo , Espermatozoides/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
14.
Nat Chem Biol ; 16(4): 469-478, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32152546

RESUMO

Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR-Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.


Assuntos
Resistência a Medicamentos/genética , Proteínas Carreadoras de Solutos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Antineoplásicos , Fenômenos Bioquímicos , Transporte Biológico/genética , Transporte Biológico/fisiologia , Sistemas CRISPR-Cas , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Resistência a Medicamentos/fisiologia , Testes Genéticos , Humanos , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Transporte Proteico/fisiologia , Proteínas Carreadoras de Solutos/fisiologia , Simportadores/genética , Simportadores/metabolismo
15.
Arch Biochem Biophys ; 685: 108350, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32220566

RESUMO

Iron is an essential requirement for the survival and virulence of most bacteria. The bacterial ferrous iron transporter protein FeoB functions as a major reduced iron transporter in prokaryotes, but its biochemical mechanism has not been fully elucidated. In the present study, we compared enzymatic properties of the cytosolic portions of pathogenic bacterial FeoBs to elucidate each bacterial strain-specific characteristic of the Feo system. We show that bacterial FeoBs are classified into two distinct groups that possess either a sole GTPase or an NTPase with a substrate promiscuity. This difference in nucleotide preference alters cellular requirements for monovalent and divalent cations. While the hydrolytic activity of the GTP-dependent FeoBs was stimulated by potassium, the action of the NTP-dependent FeoBs was not significantly affected by the presence of monovalent cations. Mutation of Asn11, having a role in potassium-dependent GTP hydrolysis, changed nucleotide specificity of the NTP-dependent FeoB, resulting in loss of ATPase activity. Sequence analysis suggested a possible association of alanine in the G5 motif for the NTP-dependent activity in FeoBs. This demonstration of the distinct enzymatic properties of bacterial FeoBs provides important insights into mechanistic details of Feo iron transport processes, as well as offers a promising species-specific anti-virulence target.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte de Cátions/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/química , Hidrólise , Mutagênese Sítio-Dirigida , Mutação , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Potássio/metabolismo , Ligação Proteica , Alinhamento de Sequência , Especificidade por Substrato
16.
Insect Biochem Mol Biol ; 120: 103360, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32126276

RESUMO

Ammonia is one of the principal kairomones originating from human and other animal emanations and in that context, plays an essential role in the host-seeking behaviors of the malaria vector mosquito Anopheles gambiae. Nevertheless, despite its importance in directing host-seeking, the mechanisms underlying ammonia detection in the mosquito olfactory system remains largely unknown. In addition to ongoing efforts to identify and characterize the molecular receptors that underlie ammonia sensitivity, previous studies have revealed a prominent role for ammonium transporters (Amt) in modulating antennal and behavioral responses in Drosophila melanogaster and An. gambiae. In the former, localization of DmAmt in antennal sensilla to auxiliary cells surrounding the ammonia sensory neurons led to the hypothesis that its role was to clear excess ammonium ions in the sensillar lymph. In the latter, RT-PCR and heterologous expression have been used to examine the expression and functional characteristics of the An. gambiae ammonium transporter, AgAmt. We now employ advanced transgenic tools to comprehensively examine AgAmt spatial localization across the peripheral chemosensory appendages in larvae and adult female An. gambiae. In the larval antennae, AgAmt appears localized in both neuronal and auxiliary cells. In contrast to D. melanogaster, in the adult antennae, AgAmt-derived signals are observed in both non-neuronal auxiliary cells and in sensory neurons in ammonia-responsive basiconic and coeloconic sensilla. In the maxillary palps, labella, and tarsi, AgAmt appears restricted to sensory neurons. We have also characterized the responses to ammonia of adult antennal coeloconic sensilla and maxillary palp capitate pegs revealing a correlation between sensillar AgAmt expression and ammonia sensitivity. Taken together, these data suggest that AgAmt may play heterogeneous roles in the adult and larval chemosensory apparatus and potentially broad utility as a supra-receptor target in mosquito control.


Assuntos
Compostos de Amônio/metabolismo , Anopheles/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Insetos/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Anopheles/crescimento & desenvolvimento , Anopheles/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Feminino , Perfilação da Expressão Gênica , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Malária , Mosquitos Vetores/genética , Mosquitos Vetores/crescimento & desenvolvimento , Mosquitos Vetores/metabolismo , Sensilas/metabolismo
17.
Cell Physiol Biochem ; 54(2): 180-194, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32068980

RESUMO

BACKGROUND/AIMS: Still in 1999 the first hints were published for the pharmacoresistant Cav2.3 calcium channel to be involved in the generation of epileptic seizures, as transcripts of alpha1E (Cav2.3) and alpha1G (Cav3.1) are changed in the brain of genetic absence epilepsy rats from Strasbourg (GAERS). Consecutively, the seizure susceptibility of mice lacking Cav2.3 was analyzed in great detail by using 4-aminopyridine, pentylene-tetrazol, N-methyl-D-aspartate and kainic acid to induce experimentally convulsive seizures. Further, γ-hydroxybutyrolactone was used for the induction of non-convulsive absence seizures. For all substances tested, Cav2.3-competent mice differed from their knockout counterparts in the sense that for convulsive seizures the deletion of the pharmacoresistant channel was beneficial for the outcome during experimentally induced seizures [1]. The antiepileptic drug lamotrigine reduces seizure activity in Cav2.3-competent but increases it in Cav2.3-deficient mice. In vivo, Cav2.3 must be under tight control by endogenous trace metal cations (Zn2+ and Cu2+). The dyshomeostasis of either of them, especially of Cu2+, may alter the regulation of Cav2.3 severely and its activity for Ca2+ conductance, and thus may change hippocampal and neocortical signaling to hypo- or hyperexcitation. METHODS: To investigate by telemetric EEG recordings the mechanism of generating hyperexcitation by kainate, mice were tested for their sensitivity of changes in neuronal (intracerebroventricular) concentrations of the trace metal cation Zn2+. As the blood-brain barrier limits the distribution of bioavailable Zn2+ or Cu2+ into the brain, we administered micromolar Zn2+ ions intracerebroventricularly in the presence of 1 mM histidine as carrier and compared the effects on behavior and EEG activity in both genotypes. RESULTS: Kainate seizures are more severe in Cav2.3-competent mice than in KO mice and histidine lessens seizure severity in competent but not in Cav2.3-deficient mice. Surprisingly, Zn2+ plus histidine resembles the kainate only control with more seizure severity in Cav2.3-competent than in deficient mice. CONCLUSION: Cav2.3 represents one important Zn2+-sensitive target, which is useful for modulating convulsive seizures.


Assuntos
Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Convulsões/tratamento farmacológico , Zinco/uso terapêutico , Animais , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Eletroencefalografia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Histidina/farmacologia , Íons/química , Ácido Caínico/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Convulsões/induzido quimicamente , Convulsões/patologia , Índice de Gravidade de Doença , Zinco/farmacologia , Ácido gama-Aminobutírico/metabolismo
18.
Plant Mol Biol ; 102(6): 603-614, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32052233

RESUMO

The WRKY transcription factor family is involved in responding to biotic and abiotic stresses. Its members contain a typical WRKY domain and can regulate plant physiological responses by binding to W-boxes in the promoter regions of downstream target genes. We identified the sweet sorghum SbWRKY50 (Sb09g005700) gene, which encodes a typical class II of the WRKY family protein that localizes to the nucleus and has transcriptional activation activity. The expression of SbWRKY50 in sweet sorghum was reduced by salt stress, and its ectopic expression reduced the salt tolerance of Arabidopsis thaliana plants. Compared with the wild type, the germination rate, root length, biomass and potassium ion content of SbWRKY50 over-expression plants decreased significantly under salt-stress conditions, while the hydrogen peroxide, superoxide anion and sodium ion contents increased. Real-time PCR results showed that the expression levels of AtSOS1, AtHKT1 and genes related to osmotic and oxidative stresses in over-expression strains decreased under salt-stress conditions. Luciferase complementation imaging and yeast one-hybrid assays confirmed that SbWRKY50 could directly bind to the upstream promoter of the SOS1 gene in A. thaliana. However, in sweet sorghum, SbWRKY50 could directly bind to the upstream promoters of SOS1 and HKT1. These results suggest that the new WRKY transcription factor SbWRKY50 participates in plant salt response by controlling ion homeostasis. However, the regulatory mechanisms are different in sweet sorghum and Arabidopsis, which may explain their different salt tolerance levels. The data provide information that can be applied to genetically modifying salt tolerance in different crop varieties.


Assuntos
Homeostase , Tolerância ao Sal/fisiologia , Sorghum/genética , Sorghum/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biomassa , Proteínas de Transporte , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação , Peróxido de Hidrogênio/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Potássio/metabolismo , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio/metabolismo , Sementes , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Estresse Fisiológico , Superóxidos/metabolismo , Simportadores/genética , Simportadores/metabolismo
19.
PLoS Negl Trop Dis ; 14(2): e0007991, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32023254

RESUMO

BACKGROUND: During infections involving intracellular pathogens, iron performs a double-edged function by providing the pathogen with nutrients, but also boosts the host's antimicrobial arsenal. Although the role of iron has been described in visceral leishmaniasis, information regarding its status in the dermal sequel, Post Kala-azar Dermal Leishmaniasis (PKDL) remains limited. Accordingly, this study aimed to establish the status of iron within monocytes/macrophages of PKDL cases. METHODOLOGY/PRINCIPAL FINDINGS: The intramonocytic labile iron pool (LIP), status of CD163 (hemoglobin-haptoglobin scavenging receptor) and CD71 (transferrin receptor, Tfr) were evaluated within CD14+ monocytes by flow cytometry, and soluble CD163 by ELISA. At the lesional sites, Fe3+ status was evaluated by Prussian blue staining, parasite load by qPCR, while the mRNA expression of Tfr (TfR1/CD71), CD163, divalent metal transporter-1 (DMT-1), Lipocalin-2 (Lcn-2), Heme-oxygenase-1 (HO-1), Ferritin, Natural resistance-associated macrophage protein (NRAMP-1) and Ferroportin (Fpn-1) was evaluated by droplet digital PCR. Circulating monocytes demonstrated elevated levels of CD71, CD163 and soluble CD163, which corroborated with an enhanced lesional mRNA expression of TfR, CD163, DMT1 and Lcn-2. Additionally, the LIP was raised along with an elevated mRNA expression of ferritin and HO-1, as also iron exporters NRAMP-1 and Fpn-1. CONCLUSIONS/SIGNIFICANCE: In monocytes/macrophages of PKDL cases, enhancement of the iron influx gateways (TfR, CD163, DMT-1 and Lcn-2) possibly accounted for the enhanced LIP. However, enhancement of the iron exporters (NRAMP-1 and Fpn-1) defied the classical Ferritinlow/Ferroportinhigh phenotype of alternatively activated macrophages. The creation of such a pro-parasitic environment suggests incorporation of chemotherapeutic strategies wherein the availability of iron to the parasite can be restricted.


Assuntos
Ferro/metabolismo , Leishmaniose Cutânea/metabolismo , Adolescente , Adulto , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Feminino , Humanos , Índia , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/fisiologia , Leishmaniose Cutânea/parasitologia , Lipocalina-2/genética , Lipocalina-2/metabolismo , Macrófagos/metabolismo , Masculino , Monócitos/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Adulto Jovem
20.
Nat Commun ; 11(1): 626, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005818

RESUMO

Potassium homeostasis is vital for all organisms, but is challenging in single-celled organisms like bacteria and yeast and immobile organisms like plants that constantly need to adapt to changing external conditions. KUP transporters facilitate potassium uptake by the co-transport of protons. Here, we uncover the molecular basis for transport in this widely distributed family. We identify the potassium importer KimA from Bacillus subtilis as a member of the KUP family, demonstrate that it functions as a K+/H+ symporter and report a 3.7 Å cryo-EM structure of the KimA homodimer in an inward-occluded, trans-inhibited conformation. By introducing point mutations, we identify key residues for potassium and proton binding, which are conserved among other KUP proteins.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Transporte de Cátions/química , Potássio/metabolismo , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Transporte Biológico , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Dimerização , Transporte de Íons , Modelos Moleculares , Família Multigênica , Domínios Proteicos
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