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1.
Chemosphere ; 240: 124902, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31563721

RESUMO

Eisenia fetida earthworm is an ecotoxicologically important test species to monitor various pollutants. However, there is a little knowledge about the effects of cadmium (Cd) on earthworms at the transcriptional level. Firstly, we exposed E. fetida to soils supplemented with different concentrations (10, 30, 60 mg/kg soil) of Cd. Moreover, we depicted the characterization of gene expressions with E. fetida using high-throughput profiling of gene expression. In addition, a comparison of the gene expression profiles between each Cd treatment group and the control group suggested that differential expressional genes (DEGs) mainly enriched in enzyme activity, metabolism, oxidative stress, regeneration and apoptosis pathways. 8 DEGs from these pathways had been selected randomly to confirm the data of RNA-seq. Among these DEGs, six genes (metallothionein-2, phytochelatin synthase 1a, CuZn superoxide dismutase, sex determining region Y-box 2, sex determining region Y-box 4b, TP53-regulated inhibitor of apoptosis 1-like) up-regulated and 2 genes (beta-1,4-endoglucanase, apoptosis-stimulating of p53 protein 2-like) down-regulated in response to Cd exposure. The alteration of them indicated that earthworms could reduce the toxicity and bioavailability of Cd in polluted soil ecosystems through different pathways. This work lays an important foundation for linking earthworm transcriptional level with the ecological risk of Cd in soil ecosystem.


Assuntos
Cádmio/toxicidade , Oligoquetos/fisiologia , Poluentes do Solo/toxicidade , Aminoaciltransferases , Animais , Disponibilidade Biológica , Cádmio/análise , Ecossistema , Perfilação da Expressão Gênica , Metalotioneína/genética , Metalotioneína/metabolismo , Oligoquetos/efeitos dos fármacos , Oligoquetos/genética , Estresse Oxidativo/efeitos dos fármacos , Solo , Poluentes do Solo/análise , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
2.
Arq Gastroenterol ; 56(4): 367-371, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31721970

RESUMO

BACKGROUND: Gastric cancer is the fourth most common cause of worldwide cancer. Also in contrast to the huge advances in curing, the chance of living is very low even in surgery cases. Having a genetic predisposition plays an important role in cancer development. The association between Metallothionein-2A gene polymorphisms and the risk of adenocarcinoma has been widely studied, yet there is only one study on stomach diseases. OBJECTIVE: In this study, we aimed to investigate the association between 2 (MT-2A) polymorphisms and adenocarcinoma. METHODS: This cross-sectional case control study was performed between Mach 2014 and January 2015 at the Tuba Hospital of Sari, Iran. Peripheral blood samples were collected in EDTA tube. DNA extraction was performed using the spin column procedure. The MT-2A polymorphisms MT-2A (rs1610216), (rs28366003) were determined by polymerase chain reaction-restriction fragment length polymorphism analysis in 95 a topic adenocarcinoma patients and 90 healthy individuals from Iranian population. RESULTS: The MT-2A rs1610216 polymorphism increased the risk of adeno carcinoma in our Iranian population [OR: 3.8533; 95%CI, 1.3155-11.2869; P=0.0139] and rs28366003 [OR: 4.0978; 95%CI, 1.2521-13.4108; P=0.0197]. CONCLUSION: The MT-2A gene polymorphism was associated with the risk of adenocarcinoma in the Iranian population.


Assuntos
Adenocarcinoma/genética , Predisposição Genética para Doença/genética , Metalotioneína/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Estudos Transversais , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único/genética , Neoplasias Gástricas/genética , Adulto Jovem
3.
Protein Pept Lett ; 26(10): 768-775, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618171

RESUMO

INTRODUCTION: Metallothioneins (MTs) are members of a family of low molecular weight and cysteine-rich proteins that are involved in heavy metal homeostasis and detoxification in living organisms. Plants have multiple MT types that are generally divided into four subgroups according to the arrangement of Cys residues. METHODS: In the present study the E. coli cells which heterologously express four different rice MT (OsMT) isoforms were analyzed for the accumulation of two forms of chromium, Cr3+ and Cr6+. RESULTS: The results show that the transgenic bacteria were more tolerant than control cells when they were grown up in the medium comprising Cr(NO3)3.9H2O or Na2CrO4. The cells expressing OsMT1, OsMT2, OsMT3 and OsMT4 give rise to 6.5-, 2.7-, 5.5- and 2.1-fold improvements on the accumulation capacity for Cr3+ and 9-, 3-, 5- and 3- fold Cr6+ respectively compared with comparison to the control strain. Furthermore, the purified recombinant GST-OsMTs were tested for their binding ability to Cr+3 and Cr+6 in vitro. DISCUSSION: The data show that the recombinant GST-OsMT1 and GST-OsMT2 were able to bind both Cr3+ and Cr6+, in vitro. However, their binding strength was low with respect to previous tested divalent ions like Cd2+.


Assuntos
Cromo/química , Metalotioneína/química , Oryza/química , Proteínas de Plantas/química , Proteínas Recombinantes/química , Sequência de Aminoácidos , Cátions/química , Cisteína/química , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Metalotioneína/genética , Proteínas de Plantas/genética , Ligação Proteica , Isoformas de Proteínas , Proteínas Recombinantes/genética
4.
J Agric Food Chem ; 67(41): 11373-11379, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31539240

RESUMO

Cadmium bioremediation with metal-binding proteins is primarily conducted using metallothioneins (MTs). However, in the present study, we investigated a non-MT cadmium-binding protein from Lentinula edodes (LECBP) as a remediation tool for cadmium biosorption in Escherichia coli. The results indicated that the expression of LECBP significantly enhanced the cadmium biosorption capacity of transgenic E. coli. The secondary structure and conformation of LECBP were changed after binding with cadmium as evidenced by circular dichroism and fluorescence spectroscopy. The results of Fourier transform infrared spectroscopy indicated that carboxyl oxygen and amino nitrogen atoms were involved in the interaction between LECBP and cadmium. The results further demonstrated that glutamic acid and histidine residues are the potential binding sites. Our results have thus provided new insights into cadmium bioremediation in an aquatic environment.


Assuntos
Cádmio/metabolismo , Metalotioneína/metabolismo , Proteínas de Plantas/metabolismo , Cogumelos Shiitake/genética , Sítios de Ligação , Biodegradação Ambiental , Cádmio/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Metalotioneína/química , Metalotioneína/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Cogumelos Shiitake/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier
5.
Genes Dev ; 33(21-22): 1525-1538, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31530651

RESUMO

Cellular homeostasis requires transcriptional outputs to be coordinated, and many events post-transcription initiation can dictate the levels and functions of mature transcripts. To systematically identify regulators of inducible gene expression, we performed high-throughput RNAi screening of the Drosophila Metallothionein A (MtnA) promoter. This revealed that the Integrator complex, which has a well-established role in 3' end processing of small nuclear RNAs (snRNAs), attenuates MtnA transcription during copper stress. Integrator complex subunit 11 (IntS11) endonucleolytically cleaves MtnA transcripts, resulting in premature transcription termination and degradation of the nascent RNAs by the RNA exosome, a complex also identified in the screen. Using RNA-seq, we then identified >400 additional Drosophila protein-coding genes whose expression increases upon Integrator depletion. We focused on a subset of these genes and confirmed that Integrator is bound to their 5' ends and negatively regulates their transcription via IntS11 endonuclease activity. Many noncatalytic Integrator subunits, which are largely dispensable for snRNA processing, also have regulatory roles at these protein-coding genes, possibly by controlling Integrator recruitment or RNA polymerase II dynamics. Altogether, our results suggest that attenuation via Integrator cleavage limits production of many full-length mRNAs, allowing precise control of transcription outputs.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Regulação da Expressão Gênica , Metalotioneína/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Animais , Linhagem Celular , Cobre/toxicidade , Endorribonucleases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ligação Proteica , Clivagem do RNA , Estresse Fisiológico/efeitos dos fármacos
6.
Life Sci ; 234: 116735, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31394124

RESUMO

AIMS: The present study was to investigate the protective effects of Zn supplementation in OTA-induced apoptosis of Madin-Darby canine kidney (MDCK) epithelial cells and explore the potential mechanisms. Aiming to provides a new insight into the treatment strategy of OTA-induced nephrotoxicity by nutritional regulation. MAIN METHODS: Initially, through MTT and LDH assay revealed that Zn supplementation significantly suppressed OTA-induced cytotoxicity in MDCK cells. Then, the production of reactive oxygen species (ROS) was detected by using a DCFH-DA assay. Annexin V-FITC/PI, Hoechst 33258 staining and Flow cytometry were used to detect the apoptosis. The expressions of apoptosis-related molecules were determined by RT-PCR, Western blotting. Interestingly, OTA treatment slightly increased the levels of Metallothionein-1 (MT-1) and Metallothionein-2 (MT-2) by using RT-PCR, Western blotting assay; while Zn supplementation further improved the increase of MT-1 and MT-2 induced by OTA. However, the inhibitive effects of Zn supplementation were significantly blocked after double knockdown of MT-1 and MT-2 by using Small Interfering RNA (siRNA) Transfection method. KEY FINDINGS: Our study provides supportive data for the potential roles of Zn in reducing OTA-induced oxidative stress and apoptosis in MDCK cells. SIGNIFICANCE: Zn is one of the key structural components of many proteins, which plays an important role in several physiological processes such as cell survival and apoptosis. This metal is expected to contribute to the conservative and adjuvant treatment of kidney disease and should therefore be investigated further.


Assuntos
Apoptose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Metalotioneína/genética , Ocratoxinas/toxicidade , Substâncias Protetoras/farmacologia , Zinco/farmacologia , Animais , Citoproteção/efeitos dos fármacos , Cães , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Madin Darby de Rim Canino , Estresse Oxidativo/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
7.
Int J Mol Sci ; 20(16)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394742

RESUMO

Colorectal cancer (CRC) is a heterogeneous disease resulting from the combined influence of many genetic factors. This complexity has caused the molecular characterization of CRC to remain uncharacterized, with a lack of clear gene markers associated with CRC and the prognosis of this disease. Thus, highly sensitive tumor markers for the detection of CRC are the most essential determinants of survival. In this study, we examined the simultaneous downregulation of the mRNA levels of six metallothionein (MT) genes in CRC cell lines and public CRC datasets for the first time. In addition, we detected downregulation of these six MT mRNAs' levels in 30 pairs of tumor (T) and adjacent non-tumor (N) CRC specimens. In order to understand the potential prognostic relevance of these six MT genes and CRC, we presented a four-gene signature to evaluate the prognosis of CRC patients. Further discovery suggested that the four-gene signature (MT1F, MT1G, MT1L, and MT1X) predicted survival better than any combination of two-, three-, four-, five-, or six-gene models. In conclusion, this study is the first to report that simultaneous downregulation of six MT mRNAs' levels in CRC patients, and their aberrant expression together, accurately predicted CRC patients' outcomes.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Perfilação da Expressão Gênica , Metalotioneína/genética , Transcriptoma , Biomarcadores Tumorais , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Metalotioneína/metabolismo , Prognóstico , RNA Mensageiro/genética
8.
J Basic Microbiol ; 59(10): 1016-1030, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31430397

RESUMO

Heavy metals such as lead, chromium, and metalloid like arsenic dominate the pinnacle in posing a threat to life. Being environment-friendly, elucidating the mechanism by which microorganisms detoxify such elements has always been an active field of research hitherto. In the present study, we have investigated the capability of nitrogen-deprived Papiliotrema laurentii strain RY1 toward enhanced tolerance and neutralizing toxic elements. There were biosorption and bioprecipitation of lead and chromium at the cell surfaces. Bioprecipitation mechanisms included the formation of lead phosphates and pyromorphites from lead, grimaldite from chromium. Transcripts such as metallothionein, aquaporins, and arsenical pump-driving ATPase have been surmised to be involved in the detoxification of elements. Furthermore, activation of antioxidant defense mechanisms for the cells for each of the elements should contribute towards yeast's propagation. The efficiency of removal of elements for live cells and immobilized cells were high for lead and chromium. To the best of our knowledge, this is the first report of such high tolerance of lead, arsenic, and chromium for any yeast. The yeast showed such varied response under dual stress due to nitrogen starvation and in the presence of respective elements. The yeast possesses promising potentials in nitrogen deprived and enriched environments to aid in bioremediation sectors.


Assuntos
Arsênico/metabolismo , Basidiomycota/metabolismo , Poluentes Ambientais/metabolismo , Metais Pesados/metabolismo , Nitrogênio/metabolismo , Antioxidantes/metabolismo , Arsênico/toxicidade , Basidiomycota/efeitos dos fármacos , Basidiomycota/crescimento & desenvolvimento , Biodegradação Ambiental , Transporte Biológico/genética , Cádmio/metabolismo , Cádmio/toxicidade , Poluentes Ambientais/toxicidade , Expressão Gênica , Inativação Metabólica , Chumbo/metabolismo , Chumbo/toxicidade , Metalotioneína/genética , Metais Pesados/toxicidade , Testes de Sensibilidade Microbiana
9.
Biomed Res Int ; 2019: 2957821, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31380415

RESUMO

Background: Metallothioneins (MTs) family comprises many isoforms, most of which are frequently dysregulated in a wide range of cancers. However, the expression pattern and exact role of each distinct MT family isoform which contributes to tumorigenesis, progression, and drug resistance of gastric cancer (GC) are still unclear. Methods: Publicly available databases including Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), Kaplan-Meier plotter, SurvExpress, MethHC, cBioportal, and GeneMANIA were accessed to perform an integrated bioinformatic analysis and try to detect fundamental relationships between each MT family member and GC. Results: Bioinformatic data indicated that the mRNA expression of all MT family members was almost lowly expressed in GC compared with normal gastric tissue (P<0.05), and patients with reduced mRNA expression of each individual MT member had inconsistent prognostic value (OS, FP, PPS), which depended on the individual isoform of MT. A negative correlation between the methylation in promoter region of majority of MT members and their mRNA expression was detected from MethHC database (p<0.001). Data downloaded from TCGA revealed that MTs were rarely mutated in GC patients and MT2A was frequently regulated by other three genes (FOS, JUN, SP1) in GC patients. Conclusion: MTs were nearly downregulated, and distinct type of MT harbored different prognostic role in GC patients. Methylation in gene promoter region of MTs partially contributed to their reduced expression in GC. Our comprehensive analyses from multiple independent databases may further lead researches to explore MT-targeting reagents or potential diagnostic and prognostic markers for GC patients.


Assuntos
Biomarcadores Tumorais/genética , Carcinogênese/genética , Metalotioneína/genética , Neoplasias Gástricas/genética , Metilação de DNA/genética , Progressão da Doença , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Família Multigênica/genética , Prognóstico , RNA Mensageiro/genética , Neoplasias Gástricas/epidemiologia , Neoplasias Gástricas/patologia
10.
J Basic Microbiol ; 59(9): 879-889, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31339587

RESUMO

Metallothionein (MT) is a low-molecular-weight protein with a high metal binding capacity and plays a key role in organism adaptation to heavy metals. In this study, a metallothionein gene was successfully cloned and sequenced from Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5. Nucleotide sequencing and analysis revealed that the gene had four exons interrupted by three introns. MTs complementary DNA (named as RmMT) had an open reading frame of 321 bp encoding a 106 amino acid protein with a predicted molecular weight of 10.3 kDa and pI of 8.49. The number of amino acids and distribution of cysteine residues indicated that RmMT was a novel family of fungal MTs. Quantitative real-time polymerase chain reaction analysis showed that RmMT expression was elevated under copper-induced stress. The RmMT gene was transferred into E. coli and the RmMT expressing bacteria showed improved tolerance to copper ion and increased accumulation of heavy metals, such as Cu2+ , Pb2+ , Zn2+ , Cd2+ , and Ag+ . Moreover, in vitro studies, purified recombinant RmMT demonstrated that it could be used as a good scavenger of superoxide anion, hydroxyl, and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radicals. In summary, these results demonstrate that RmMT plays a key role in the tolerance and bioaccumulation of heavy metals.


Assuntos
Camada de Gelo/microbiologia , Metalotioneína/genética , Metalotioneína/metabolismo , Metais Pesados/metabolismo , Rhodotorula/genética , Adaptação Fisiológica/genética , Regiões Antárticas , Antioxidantes/isolamento & purificação , Antioxidantes/metabolismo , Sequência de Bases , Clonagem Molecular , Cobre/metabolismo , Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Expressão Gênica , Metalotioneína/isolamento & purificação , Fases de Leitura Aberta , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rhodotorula/fisiologia
11.
EMBO J ; 38(16): e102003, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31313851

RESUMO

Many eukaryotic proteins are regulated by modification with the ubiquitin-like protein small ubiquitin-like modifier (SUMO). This linkage is reversed by SUMO proteases, of which there are two in Saccharomyces cerevisiae, Ulp1 and Ulp2. SUMO-protein conjugation regulates transcription, but the roles of SUMO proteases in transcription remain unclear. We report that Ulp2 is recruited to transcriptionally active genes to control local polysumoylation. Mutant ulp2 cells show impaired association of RNA polymerase II (RNAPII) with, and diminished expression of, constitutively active genes and the inducible CUP1 gene. Ulp2 loss sensitizes cells to 6-azauracil, a hallmark of transcriptional elongation defects. We also describe a novel chromatin regulatory mechanism whereby histone-H2B ubiquitylation stimulates histone sumoylation, which in turn appears to inhibit nucleosome association of the Ctk1 kinase. Ctk1 phosphorylates serine-2 (S2) in the RNAPII C-terminal domain (CTD) and promotes transcript elongation. Removal of both ubiquitin and SUMO from histones is needed to overcome the impediment to S2 phosphorylation. These results suggest sequential ubiquitin-histone and SUMO-histone modifications recruit Ulp2, which removes polySUMO chains and promotes RNAPII transcription elongation.


Assuntos
Endopeptidases/metabolismo , Histonas/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Elongação da Transcrição Genética , Endopeptidases/genética , Regulação Fúngica da Expressão Gênica , Metalotioneína/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação , Elongação da Transcrição Genética/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Uracila/análogos & derivados , Uracila/farmacologia
12.
Ecotoxicol Environ Saf ; 181: 301-307, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31202929

RESUMO

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins, which play important roles in metal homeostasis and heavy metal detoxification. In our previous study, a novel full length MT cDNA was successfully cloned from the freshwater crab (Sinopotamon henanense). In the present study, tandem repeats of two and three copies of the crab MT gene were integrated by overlap extension PCR (SOE-PCR) and expressed in Escherichia coli. The SUMO fusion expression system was adopted to increase the stability and solubility of the recombinant MT proteins. The recombinant proteins were purified and their metal-binding abilities were further analyzed by the ultraviolet absorption spectral scan. Furthermore, the metal tolerance and bioaccumulation of E. coli cells expressing oligomeric MTs were determined. Results showed that the recombinant plasmids pET28a-SUMO-2MT and pET28a-SUMO-3MT were successfully constructed. SDS-PAGE analysis showed that the SUMO-2MT and SUMO-3MT were expressed mainly in the soluble forms. Oligomeric MTs expression significantly enhanced Cu, Cd or Zn tolerance and accumulation in E. coli in the order: SUMO-3MT˃SUMO-2MT˃SUMO-MT˃control. Cells harboring pET28a-SUMO -3MT exhibited the highest Cu, Cd or Zn bioaccumulation at 5.8-fold, 3.1-fold or 6.7-fold higher than that of the control cells. Our research could lay a foundation for large-scale preparation of MTs and provide a scientific basis for bioremediation of heavy metal pollution by oligomeric MTs.


Assuntos
Escherichia coli/genética , Metalotioneína/genética , Metais Pesados/metabolismo , Animais , Braquiúros/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Metalotioneína/metabolismo , Metais Pesados/toxicidade , Proteínas Recombinantes de Fusão/metabolismo
13.
Biochemistry (Mosc) ; 84(4): 441-451, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228936

RESUMO

Prions are proteins that can exist under the same conditions in two or more conformations, at least one of them is infectious. Usually, acquisition of infectious prion conformation is associated with the formation of amyloids - protein aggregates with a characteristic spatial structure. About 10 prions have been identified in the yeast Saccharomyces cerevisiae. The Gln3 protein, which is one of the key regulators of nitrogen metabolism in S. cerevisiae, contains an amyloidogenic region manifesting prion-like properties. The prion properties of the full-length Gln3 have not been studied. We have found that the amyloidogenic region of Gln3 acts as a template and initiates aggregation of the full-length Gln3 in the presence of the [PIN+] prion when Gln3 is overexpressed. Full-length Gln3 in its aggregated form manifests prion-like properties, including infectivity and dependence on the anti-prion agents; however, unlike other known yeast prions, prion-like state of Gln3 is observed only upon the protein overproduction. Here, we suggest the term "conditional prions" for proteins, whose prion state is maintained exclusively under non-physiological conditions.


Assuntos
Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Metalotioneína/genética , Microscopia Confocal , Agregados Proteicos/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Fatores de Transcrição/genética
14.
Int J Mol Sci ; 20(12)2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31212812

RESUMO

Although the date palm tree is an extremophile with tolerance to drought and certain levels of salinity, the damage caused by extreme salt concentrations in the soil, has created a need to explore stress-responsive traits and decode their mechanisms. Metallothioneins (MTs) are low-molecular-weight cysteine-rich proteins that are known to play a role in decreasing oxidative damage during abiotic stress conditions. Our previous study identified date palm metallothionein 2A (PdMT2A) as a salt-responsive gene, which has been functionally characterized in yeast and Arabidopsis in this study. The recombinant PdMT2A protein produced in Escherichia coli showed high reactivity against the substrate 5'-dithiobis-2-nitrobenzoic acid (DTNB), implying that the protein has the property of scavenging reactive oxygen species (ROS). Heterologous overexpression of PdMT2A in yeast (Saccharomyces cerevisiae) conferred tolerance to drought, salinity and oxidative stresses. The PdMT2A gene was also overexpressed in Arabidopsis, to assess its stress protective function in planta. Compared to the wild-type control, the transgenic plants accumulated less Na+ and maintained a high K+/Na+ ratio, which could be attributed to the regulatory role of the transgene on transporters such as HKT, as demonstrated by qPCR assay. In addition, transgenic lines exhibited higher chlorophyll content, higher superoxide dismutase (SOD) activity and improved scavenging ability for reactive oxygen species (ROS), coupled with a better survival rate during salt stress conditions. Similarly, the transgenic plants also displayed better drought and oxidative stress tolerance. Collectively, both in vitro and in planta studies revealed a role for PdMT2A in salt, drought, and oxidative stress tolerance.


Assuntos
Adaptação Biológica , Resistência à Doença/genética , Expressão Gênica , Metalotioneína/genética , Phoeniceae/fisiologia , Doenças das Plantas/genética , Estresse Fisiológico/genética , Sequência de Aminoácidos , Arabidopsis/microbiologia , Arabidopsis/parasitologia , Arabidopsis/fisiologia , Secas , Metalotioneína/química , Estresse Oxidativo , Fenótipo , Phoeniceae/classificação , Phoeniceae/microbiologia , Phoeniceae/parasitologia , Filogenia , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Plantas Geneticamente Modificadas , Salinidade , Plantas Tolerantes a Sal , Plântula , Solo
15.
Mycorrhiza ; 29(4): 389-395, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31218402

RESUMO

Arbuscular mycorrhizal fungi (AMF) are considered a potential biotechnological tool for mitigating heavy metal (HM) toxicity. A greenhouse experiment was conducted to evaluate the impacts of the AM fungus Rhizophagus irregularis on cadmium (Cd) uptake, mycorrhizal colonization, and some plant growth parameters of Medicago sativa (alfalfa) in Cd-polluted soils. In addition, expression of two metal chelators (MsPCS1 (phytochelatin synthase) and MsMT2 (metallothionein)) and two metal transporter genes (MsIRT1 and MsNramp1) was analyzed using quantitative real-time PCR (qRT-PCR). Cd addition had a significant negative effect on mycorrhizal colonization. However, AMF symbiosis promoted the accumulation of biomass under both stressed and unstressed conditions compared with non-mycorrhizal (NM) plants. Results also showed that inoculation with R. irregularis significantly reduced shoot Cd concentration in polluted soils. Transcripts abundance of MsPCS1, MsMT2, MsIRT1, and MsNRAMP1 genes were downregulated compared with NM plants indicating that metal sequestration within hyphal fungi probably made Cd concentration insufficient in root cells for induction of these genes. These results suggest that reduction of shoot Cd concentration in M. sativa colonized by R. irregularis could be a promising strategy for safe production of this plant in Cd-polluted soils.


Assuntos
Aminoaciltransferases/genética , Cádmio/metabolismo , Glomeromycota/fisiologia , Medicago sativa/metabolismo , Metalotioneína/genética , Micorrizas/fisiologia , Proteínas de Plantas/genética , Poluentes do Solo/metabolismo , Aminoaciltransferases/metabolismo , Transporte Biológico , Medicago sativa/genética , Medicago sativa/microbiologia , Metalotioneína/metabolismo , Proteínas de Plantas/metabolismo , Simbiose
16.
Chemosphere ; 230: 144-156, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31103860

RESUMO

Ability of hexavalent chromium to accumulate and induce oxidative stress has been studied in the gills of Ctenopharyngodon idellus, with the resulting damage in the form of altered endogenous antioxidant enzyme activity and, histopathology in the tissue. The fish were exposed to 5.3 (C1) and 10.63 mg/L (C2) of hexavalent chromium and were scrutinised on 15th, 30th and 45th day of toxicant exposure. Oxidative stress studied in terms of lipid peroxidation and glutathione levels and the antioxidant enzymes activity also exhibited alterations. The histopathological modifications in gills announced lesions in the form of hyperplasia, aneurysm, lamellar fusion, focal proliferation, epithelial degeneration and necrosis with loss of lamellae, bringing irreversible damage on 45th day with mean degree of tissue change value of 100.35 ±â€¯10.69. Bioaccumulation of chromium, and increased anomalies in branchial tissue exhibited damage in concentration and time-dependent manner. The ultrastructural anomalies in the cellular morphology in the epithelial cells of filaments and lamellae, exhibited pleomorphic nuclei, swollen mitochondria, extensive vacuolation and loss of microridges in pavement cells. The tissue also displayed altered regulation of Nrf2 and Mt2 following Cr(VI) exposure with maximum downregulation on 45th day by 61 and 53%, respectively. PCA generated two principal components, PC1 (GSH, GST, CAT and SOD) and PC2 (DTC, MDA and Cr(VI) concentration). Thus, it can be concluded that accumulation of Cr(VI) induces alteration in the gene expression of Nrf2 and Mt2 leading to the development of oxidative stress, ensuing various pathological changes creating hindrance in fish survival.


Assuntos
Antioxidantes/metabolismo , Carpas/fisiologia , Cromo/toxicidade , Metalotioneína/genética , Poluentes Químicos da Água/toxicidade , Animais , Carpas/genética , Carpas/metabolismo , Expressão Gênica , Brânquias/metabolismo , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Metalotioneína/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos
17.
Planta ; 250(2): 427-443, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31037485

RESUMO

MAIN CONCLUSION: Physcomitrella patens contains four metallothionein-like genes. Three were shown to confer metal tolerance in yeast. Transcript profiling suggests their roles in senescence and reproductive development or cadmium and oxidative stress. Metallothioneins (MTs) have been suggested to play various roles including metal detoxification, nutrient remobilization, ROS scavenging, stress tolerance, and plant development. However, little is known about the forms and functions of MTs in bryophytes. The moss Physcomitrella patens genome was found to contain four MT-like genes. Amino acid sequence composition showed that the P. patens MTs (PpMTs) were clustered with Type 1 plant MTs, and could be further classified into two sub-types, herein referred to as sub-type 1: PpMT1.1a and PpMT1.1b and sub-type 2: PpMT1.2a and PpMT1.2b. Transcript abundance of PpMT1.1b and PpMT1.2b was upregulated in the gametophore compared to protonema, and all, except PpMT1.2a, were highly induced in senescing gametophytes. PpMT1.1a and PpMT1.1b transcripts were upregulated in protonema treated with cadmium and hydrogen peroxide. Unlike many higher plant MTs, the PpMT transcript abundance was not strongly induced in response to copper and zinc. These results suggest that PpMTs may play a role in protecting P. patens from cadmium and oxidative stress and may be involved in tissues senescence and reproductive development. The PpMTs, except PpMT1.2b, were also able to confer metal tolerance and accumulation when heterologously expressed in the ∆cup1 yeast. A P. patens mutant lacking PpMT1.2a through targeted gene disruption was generated. However, it did not show any alteration in growth phenotypes under senescence-induced conditions or hypersensitivity to cadmium, copper, zinc, H2O2, and NaCl stresses. Further characterization of additional P. patens mutants lacking single or multiple PpMTs may provide insight into the physiological roles of bryophytic MTs.


Assuntos
Bryopsida/genética , Metalotioneína/metabolismo , Metais Pesados/toxicidade , Sequência de Aminoácidos , Bryopsida/fisiologia , Cádmio/toxicidade , Cobre/toxicidade , Técnicas de Inativação de Genes , Peróxido de Hidrogênio/toxicidade , Metalotioneína/genética , Estresse Oxidativo/efeitos dos fármacos , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Isoformas de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Alinhamento de Sequência , Cloreto de Sódio/toxicidade , Estresse Fisiológico , Zinco/toxicidade
18.
Oncol Rep ; 42(1): 253-262, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31059101

RESUMO

Lung cancer is the most commonly diagnosed cancer and the most frequent cause of death worldwide. Tesmin (testis­specific metallothionein­like protein; MTL­5) is a 60­kDa protein which has cysteine­rich motifs (CXC domain), characteristic of metallothioneins (MTs). Tesmin expression has been observed in germ cells during spermatogenesis, oogenesis and also in various cell nuclei after exposure to heavy metal ions. Yet, the role of tesmin in carcinogenesis is unknown. The aim of the present study was to evaluate the localization and intensity of tesmin expression in non­small cell lung cancer (NSCLC) and its association with the clinicopathological data of patients. A total of 121 cases of NSCLC and 20 cases of non­cancerous tissue samples from the surgical margin (control) were used for immunohistochemistry (IHC). In addition, 20 cases of frozen NSCLC tissues and 20 cases of control were used for the in vivo study. Normal lung fibroblasts (IMR­90) and lung cancer cell lines NCI­H1703 (lung squamous cell carcinoma), NCI­H522 and A549 (both adenocarcinomas of the lung) were used for western blot analysis (WB) and RT­PCR studies. Positive cytoplasmic tesmin expression was observed in 88.42% of the examined cases of NSCLC. Statistical analysis showed increased IHC tesmin expression in cancer cells compared to that noted in the controls. In addition, MTL5 mRNA and WB tesmin protein expression were also higher in cancer cases compared to the controls. A positive correlation between tesmin and Ki­67 IHC expression was demonstrated (r=0.32; P<0.001). Higher WB tesmin expression was also associated with shorter overall survival (P<0.05, Mantel­Cox test). The in vitro study revealed higher tesmin protein (WB) and MTL5 (qPCR) in lung cancer cell lines compared to the lung fibroblast control cell line. Higher tesmin expression in cancer cells compared to control cells may suggest a role of tesmin in NSCLC carcinogenesis. A positive correlation between tesmin and Ki­67 could indicate a possible role of tesmin in the proliferation of NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Metalotioneína/genética , Metalotioneína/metabolismo , Células A549 , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Sobrevivência Celular , Citoplasma/genética , Citoplasma/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Prognóstico
19.
J Toxicol Sci ; 44(5): 327-333, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31068538

RESUMO

Metallothionein (MT) is a low-molecular-weight, cysteine-rich, and metal-binding protein that protects cells from the cytotoxic effects of heavy metals and reactive oxygen species. Previously, we found that transcriptional induction of endothelial MT-1A was mediated by not only the metal-regulatory transcription factor 1 (MTF-1)-metal responsive element (MRE) pathway but also the nuclear factor-erythroid 2-related factor 2 (Nrf2)-antioxidant response element/electrophile responsive element (ARE) pathway, whereas that of MT-2A was mediated only by the MTF-1-MRE pathway, using the organopnictogen compounds tris(pentafluorophenyl)stibane, tris(pentafluorophenyl)arsane, and tris(pentafluorophenyl)phosphane as molecular probes in vascular endothelial cells. In the present study, we investigated the binding sites of MTF-1 and Nrf2 in the promoter regions of MTs in cultured bovine aortic endothelial cells treated with these organopnictogen compounds. We propose potential mechanisms underlying transcriptional induction of endothelial MT isoforms. Specifically, both MRE activation by MTF-1 and that of ARE in the promoter region of the MT-2A gene by Nrf2 are involved in transcriptional induction of MT-1A, whereas only MRE activation by MTF-1 or other transcriptional factor(s) is required for transcriptional induction of MT-2A in vascular endothelial cells.


Assuntos
Células Endoteliais/efeitos dos fármacos , Metalotioneína/genética , Fosfinas/toxicidade , Animais , Aorta/citologia , Bovinos , Células Cultivadas , Proteínas de Ligação a DNA/genética , Células Endoteliais/metabolismo , Fator 2 Relacionado a NF-E2/genética , Isoformas de Proteínas/genética , Fatores de Transcrição/genética , Transcrição Genética
20.
Environ Monit Assess ; 191(6): 372, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101990

RESUMO

The expression levels of Esterase Isoenzyme and total soluble protein fractionation were studied in two coleopteran insects Blaps polycresta and Trachyderma hispida to evaluate the possible hazards from ceramic and plastic factories in the Khorshed Region, East of Alexandria, Egypt. Two insect collection sites were selected. The first site was the garden of the Faculty of Science, Moharram Bek, Alexandria University, which is considered a non-polluted site, and Khorshed district, considered as the polluted site. Percentages of heavy metals were estimated using SEM-X-ray microanalysis of soft tissues of both sexes of the two coleopteran insects. Esterase Isoenzymes were found to be overexpressed in B. Polycresta but not T. hispida. Female B. polycresta from the polluted site exhibited overexpression of the second and third loci. Furthermore, the females were found to be more affected than males, which only showed the overexpression of the second loci. T. hispida (females and males) collected from the reference site were found to have increased esterase activity compared with those sampled from the polluted site. The Snake-Skin™ Dialysis tubing technique, used for optimizing the protein extraction method, reflected the highest quantified proteins compared to other, traditional methods. SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the whole-body protein reflected definite variations between T. hispida and B. polycresta in fraction number and activity at the two sites. Varied expression levels for metallothionein (MT) heavy metal resistance proteins for B. polycresta and T. hispida were also detected in the study. Based on these results, we suggest that biochemical biomarkers could infer environmental hazards, B. polycresta and T. hispida are successful biomarkers for heavy metal pollution.


Assuntos
Cerâmica , Besouros/efeitos dos fármacos , Monitoramento Ambiental/métodos , Poluição Ambiental/análise , Proteínas de Insetos/metabolismo , Isoenzimas/metabolismo , Animais , Biomarcadores/metabolismo , Besouros/genética , Besouros/metabolismo , Egito , Poluição Ambiental/efeitos adversos , Feminino , Expressão Gênica/efeitos dos fármacos , Indústrias , Proteínas de Insetos/genética , Isoenzimas/genética , Masculino , Metalotioneína/genética , Metalotioneína/metabolismo , Metais Pesados/análise , Metais Pesados/toxicidade , Fatores Sexuais , Especificidade da Espécie
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