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1.
Dokl Biochem Biophys ; 486(1): 168-170, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31367813

RESUMO

In the present study, we analyzed the uptake of radiolabeled dopamine by intact synaptosomes and purified synaptic vesicles isolated from the dorsal striatum of mice with constitutive inactivation of all three synuclein-coding genes and wild-type mice. Synuclein deficiency substantially compromised the uptake of this neurotransmitter by synaptic vesicles but had no effect on synaptosomal dopamine uptake.


Assuntos
Dopamina/metabolismo , Vesículas Sinápticas/metabolismo , Sinucleínas/deficiência , Animais , Transporte Biológico/genética , Inativação Gênica , Camundongos , Camundongos Endogâmicos C57BL , Sinucleínas/genética
2.
J Cancer Res Clin Oncol ; 145(8): 1949-1976, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31292714

RESUMO

PURPOSE: Efflux transporters of the adenosine triphosphate-binding cassette (ABC)-superfamily play an important role in the development of multidrug resistance (multidrug resistant; MDR) in cancer. The overexpression of these transporters can directly contribute to the failure of chemotherapeutic drugs. Several in vitro and in vivo models exist to screen for the efficacy of chemotherapeutic drugs against MDR cancer, specifically facilitated by efflux transporters. RESULTS: This article reviews a range of efflux transporter-based MDR models used to test the efficacy of compounds to overcome MDR in cancer. These models are classified as either in vitro or in vivo and are further categorised as the most basic, conventional models or more complex and advanced systems. Each model's origin, advantages and limitations, as well as specific efflux transporter-based MDR applications are discussed. Accordingly, future modifications to existing models or new research approaches are suggested to develop prototypes that closely resemble the true nature of multidrug resistant cancer in the human body. CONCLUSIONS: It is evident from this review that a combination of both in vitro and in vivo preclinical models can provide a better understanding of cancer itself, than using a single model only. However, there is still a clear lack of progression of these models from basic research to high-throughput clinical practice.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Antineoplásicos/isolamento & purificação , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Modelos Biológicos , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Antineoplásicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Técnicas de Cultura/métodos , Técnicas de Apoio para a Decisão , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Especificidade de Órgãos , Seleção de Pacientes
3.
BMC Plant Biol ; 19(1): 187, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064322

RESUMO

BACKGROUND: It is important to explore renewable alternatives (e.g. biofuels) that can produce energy sources to help reduce reliance on fossil oils, and reduce greenhouse gases and waste solids resulted from fossil oils consumption. Camelina sativa is an oilseed crop which has received increasing attention due to its short life cycle, broader adaptation regions, high oil content, high level of omega-3 unsaturated fatty acids, and low-input requirements in agriculture practices. To expand its Camelina production areas into arid regions, there is a need to breed for new drought-tolerant cultivars. Leaf cuticular wax is known to facilitate plant development and growth under water-limited conditions. Dissecting the genetic loci underlying leaf cuticular waxes is important to breed for cultivars with improved drought tolerance. RESULTS: Here we combined phenotypic data and single nucleotide polymorphism (SNP) data from a spring C. sativa diversity panel using genotyping-by-sequencing (GBS) technology, to perform a large-scale genome-wide association study (GWAS) on leaf wax compositions. A total of 42 SNP markers were significantly associated with 15 leaf wax traits including major wax components such as total primary alcohols, total alkanes, and total wax esters as well as their constituents. The vast majority of significant SNPs were associated with long-chain carbon monomers (carbon chain length longer than C28), indicating the important effects of long-chain carbon monomers on leaf total wax biosynthesis. These SNP markers are located on genes directly or indirectly related to wax biosynthesis such as maintaining endoplasmic reticulum (ER) morphology and enabling normal wax secretion from ER to plasma membrane or Golgi network-mediated transport. CONCLUSIONS: These loci could potentially serve as candidates for the genetic control involved in intracellular wax transport that might directly or indirectly facilitate leaf wax accumulation in C. sativa and can be used in future marker-assisted selection (MAS) to breed for the cultivars with high wax content to improve drought tolerance.


Assuntos
Brassicaceae/genética , Folhas de Planta/química , Polimorfismo de Nucleotídeo Único , Ceras/química , Ceras/metabolismo , Álcoois/metabolismo , Aldeídos/metabolismo , Algoritmos , Alcanos/metabolismo , Transporte Biológico/genética , Genética Populacional , Estudo de Associação Genômica Ampla , Desequilíbrio de Ligação , Fenótipo , Folhas de Planta/genética
4.
Plant Cell Rep ; 38(8): 951-963, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31062133

RESUMO

KEY MESSAGE: Overexpression of SlMBP9 reduced auxin biosynthesis and transport, and negatively regulated lateral root formation and apical dominance. MADS-box transcription factors play a critical role in plant development. In this study, we describe SlMBP9, a novel MADS-box gene that is expressed in the roots of tomato plants. Tomato lines that over- or under-expressed SlMBP9 were generated using a transgenic approach. The number of lateral roots (LRs) were reduced in SlMBP9-overexpressing lines but slightly increased in SlMBP9-silenced lines. A physiological index revealed that the auxin content significantly decreased in the root maturation zone of the overexpression lines. In addition, gene expression analysis revealed that the expression of the polar auxin transporter genes PIN1 and ABCB19/MDR1 and genes involved in auxin biosynthesis was downregulated in the stems of overexpression lines, which is consistent with the reduced accumulation of auxin in the root maturation zone. Exogenous indole-3-acetic acid (auximone) rescued the lateral root phenotypes of the SlMBP9-overexpressing lines. Overexpression of SlMBP9 resulted in dwarf plants, enhanced lateral buds and reduced the gibberellin content in the stems. Together, these results suggest that SlMBP9 plays a negative role in the process of auxin biosynthesis and transport.


Assuntos
Ácidos Indolacéticos/metabolismo , Lycopersicon esculentum/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Transporte Biológico/genética , Transporte Biológico/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Lycopersicon esculentum/genética , Proteínas de Plantas/genética , Raízes de Plantas/genética
5.
Genes Cells ; 24(6): 408-421, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30963674

RESUMO

The flagellar protein export apparatus switches its substrate specificity when hook length has reached approximately 55 nm in Salmonella. The C-terminal cytoplasmic domain of FlhB (FlhBC ) is involved in this switching process. FlhBC consists of FlhBCN and FlhBCC polypeptides. FlhBCC has a flexible C-terminal tail (FlhBCCT ). FlhBCC is involved in substrate recognition, and conformational rearrangements of FlhBCN -FlhBCC boundary are postulated to be required for the export switching. However, it remains unknown how it occurs. To clarify this question, we carried out mutational analysis of highly conserved residues in FlhBC . The flhB(E230A) mutation reduced the FlhB function. The flhB(E11S) mutation restored the protein transport activity of the flhB(E230A) mutant to the wild-type level, suggesting that the interaction of FlhBCN with the extreme N-terminal region of FlhB is required for flagellar protein export. The flhB(R320A) mutation affected hydrophobic interaction networks in FlhBCC , thereby increasing insolubility of FlhBC . The R320A mutation also affected the export switching, thereby producing longer hooks with the filament attached. C-terminal truncations of FlhBCCT induced a conformational change of FlhBCN -FlhBCC boundary, resulting in a loose hook length control. We propose that FlhBCCT may control conformational arrangements of FlhBCN -FlhBCC boundary through the hydrophobic interaction networks of FlhBCC .


Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Salmonella typhi/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Transporte Biológico/genética , Análise Mutacional de DNA/métodos , Flagelos/genética , Flagelos/fisiologia , Proteínas de Membrana/metabolismo , Mutação , Domínios Proteicos , Transporte Proteico/genética , Salmonella/genética , Salmonella/metabolismo , Salmonella typhi/metabolismo , Especificidade por Substrato
6.
Biochim Biophys Acta Bioenerg ; 1860(4): 317-324, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30707884

RESUMO

In the native and most mutant reaction centers of bacterial photosynthesis, the electron transfer is coupled to proton transfer and is rate limiting for the second reduction of QB- → QBH2. In the presence of divalent metal ions (e.g. Cd2+) or in some ("proton transfer") mutants (L210DN/M17DN or L213DN), the proton delivery to QB- is made rate limiting and the properties of the proton pathway can be directly examined. We found that small weak acids and buffers in large concentrations (up to 1 M) were able to rescue the severely impaired proton transfer capability differently depending on the location of the defects: lesions at the protein surface (proton gate H126H/H128H + Cd2+), beneath the surface (M17DN + Cd2+, L210DN/M17DN) or deep inside the protein (L213DN) could be completely, partially or to very small extent recovered, respectively. Small zwitterionic acids (azide/hydrazoic acid) and buffers (tricine) proved to be highly effective rescuers consistent with their enhanced binding affinity and access to any of the proton acceptors (including QB- itself) in the pathway. As a consequence, back titration of the protons at L212Glu could be observed as a pH-dependence of the rate constant of the charge recombination in the presence of azide or formate. Model calculations support the collective influence of the acid cluster on the change of the protonation states upon extension of the cluster with the bound small acid. In proton transfer mutants, the rescuing agents decreased the free energy of activation together with their enthalpic and entropic components. This is in agreement with the hypothesis that they function as protein-penetrating protonophores delivering protons into the chain and select dominating paths out of many alternate routes. We estimate that the proton delivery will be accelerated in one pathway out of 100-200 alternate pathways. The implications for design of the chemical recovery of impaired intra-protein proton transfer pathways in proton transfer mutants are discussed.


Assuntos
Proteínas de Bactérias/metabolismo , Mutação de Sentido Incorreto , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Prótons , Rhodobacter sphaeroides/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Transporte Biológico/genética , Concentração de Íons de Hidrogênio , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Rhodobacter sphaeroides/genética
7.
J Biosci Bioeng ; 128(1): 13-21, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30704918

RESUMO

We cloned a set of genes encoding alcohol oxidase from Ochrobactrum sp. AIU 033 (OcAOD), which exhibits the appropriate substrate specificity for glyoxylic acid production from glycolic acid. The set of genes for OcAOD contained two open reading frames consisting of 555-bp (aodB) and 1572-bp (aodA) nucleotides, which encode the precursor for the ß-subunit and α-subunit of OcAOD, respectively. We expressed the cloned genes as an active product in Escherichia coli BL21(DE3). The recombinant OcAOD oxidized glycolic acid and primary alcohols with C2-C8 but not glyoxylic acid (as is the case for native OcAOD), whereas the Km and Vmax values for glycolic acid and the pH stability were higher than those of native OcAOD. A consensus sequence for the twin-arginine translocation (Tat) pathway was identified in the N-terminal region of the precursor for the ß-subunit, and the active form of OcAOD was localized in the periplasm of recombinant E. coli, which indicated that OcAOD would be transported from the cytoplasm to the periplasm by the hitchhiker mechanism through the Tat pathway. The OcAOD productivity of the recombinant E. coli was 24-fold higher than that of Ochrobactrum sp. AIU 033, and it was further enhanced by 1.2 times by the co-expression of additional tatABC from E. coli BL21(DE3). Our findings thus suggest a function of the ß-subunit of OcAOD in membrane translocation, and that the recombinant OcAOD has characteristics that are suitable for the enzymatic synthesis of glyoxylic acid as well as native OcAOD.


Assuntos
Oxirredutases do Álcool/genética , Arginina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ochrobactrum/genética , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/fisiologia , Transporte Biológico/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Glioxilatos/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Ochrobactrum/enzimologia , Organismos Geneticamente Modificados , Periplasma/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA
8.
Nano Lett ; 19(3): 1467-1478, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30730145

RESUMO

Sustaining blood retention for theranostic nanoparticles is a big challenge. Various approaches have been attempted and have demonstrated some success but limitations remain. We hypothesized that peptides capable of increasing blood residence time for M13 bacteriophage, a rod-shaped nanoparticle self-assembled from proteins and nucleic acids, should also prolong blood circulation for engineered nanoparticles. Here we demonstrate the feasibility of this approach by identifying a series of blood circulation-prolonging (BCP) peptides through in vivo screening of an M13 peptide phage display library. Intriguingly, the majority of the identified BCP peptides contained an arginine-glycine-aspartic acid (RGD) motif, which was necessary but insufficient for the circulation-prolonging activity. We further demonstrated that the RGD-mediated specific binding to platelets was primarily responsible for the enhanced blood retention of BCP1. The utility of the BCP1 peptide was demonstrated by fusion of the peptide to human heavy-chain ferritin (HFn), leading to significantly improved pharmacokinetic profile, enhanced tumor cell uptake and optimum anticancer efficacy for doxorubicin encapsulated in the HFn nanocage. Our results provided a proof-of-concept for an innovative yet simple strategy, which utilizes phage display to discover novel peptides with the capability of substantially prolonging blood circulation for engineered theranostic nanoparticles.


Assuntos
Doxorrubicina/farmacologia , Ferritinas/química , Nanopartículas/química , Peptídeos/química , Sequência de Aminoácidos/genética , Arginina/química , Ácido Aspártico/química , Bacteriófago M13/química , Transporte Biológico/genética , Técnicas de Visualização da Superfície Celular , Doxorrubicina/química , Glicina/química , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Biblioteca de Peptídeos , Peptídeos/sangue
9.
Plant Cell Rep ; 38(5): 597-607, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30725161

RESUMO

KEY MESSAGE: TuCAX1a and TuCAX1b improved Ca2+ and Zn2+ translocation and TuCAX1b enhanced Ca2+, Zn2+, Mn2+ and Fe2+ content when exposed to Cd2+; Cd2+ translocation was inhibited under Ca2+ and Zn2+. Cation/H+ antiporters (CAXs) are involved in the translocation of Ca2+ and various metal ions in higher plants. In the present study, TuCAX1a and TuCAX1b, two cation/H+ antiporters, were isolated from the diploid wheat Triticum urartu, and their metal cation translocation functions investigated. TuCAX1a and TuCAX1b showed abundant tissue-specific expression in the internode and beard, respectively, and their expression levels were increased in shoots exposed to Cd2+, Zn2+ and Ca2+. Plant phenotype analysis showed that overexpression of TuCAX1a and TuCAX1b could improve the tolerance of Arabidopsis to exogenous Ca2+ and Zn2+. In the plant shoots and roots, the contents of Ca2+ and Zn2+ were higher than wild-type plants under Ca2+ and Zn2+ treatments, indicating that TuCAX1a and TuCAX1b can enhance Ca2+ and Zn2+ translocation. Ca2+, Zn2+, Mn2+ and Fe2+ contents showed higher accumulation in TuCAX1b-transgenic Arabidopsis shoots than in wild-type plants exposed to Cd2+, and the translocation of Cd2+ was inhibited under Ca2+ and Zn2+. Overall, the present study provides a novel genetic resource for improving the uptake of microelements and reducing accumulation of toxic heavy metals in wheat.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cálcio/metabolismo , Zinco/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico/genética , Transporte Biológico/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Metais Pesados/metabolismo
10.
J Reprod Dev ; 65(2): 155-162, 2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-30662012

RESUMO

The mitochondrial sheath is composed of mitochondria that coil tightly around the midpiece of sperm flagellum. These mitochondria are recruited from the cytoplasm to the flagellum late in spermatogenesis. Initially, recruited mitochondria are spherical-shaped but then elongate laterally to become crescent-like in shape. Subsequently, crescent-like mitochondria elongate continuously to coil tightly around the flagellum. Recently, disorganization of the mitochondrial sheath was reported in Glycerol kinase 2 (Gk2) disrupted mice. To analyze the disorganization of the mitochondrial sheath further, we generated Gk2-deficient mice using the CRISPR/Cas9 system and observed sperm mitochondria in testis using a freeze-fracture method with scanning electron microscopy. Gk2-disrupted spermatids show abnormal localization of crescent-like mitochondria, in spite of the initial proper alignment of spherical mitochondria around the flagellum, which causes abnormal mitochondrial sheath formation leading to exposure of the outer dense fibers. These results indicate that GK2 is essential for proper arrangement of crescent-like mitochondria to form the mitochondrial sheath during mouse spermatogenesis.


Assuntos
Glicerol Quinase/fisiologia , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Cauda do Espermatozoide/metabolismo , Espermatogênese/fisiologia , Animais , Transporte Biológico/genética , Citoplasma/metabolismo , Feminino , Fertilização In Vitro/veterinária , Glicerol Quinase/genética , Isoenzimas/genética , Isoenzimas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos ICR , Camundongos Knockout , Mitocôndrias/genética , Cauda do Espermatozoide/ultraestrutura , Espermatogênese/genética , Espermatozoides/citologia , Espermatozoides/metabolismo , Espermatozoides/ultraestrutura
11.
Plant Mol Biol ; 99(4-5): 347-362, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30644059

RESUMO

KEY MESSAGE: We compared the transcriptomes of parenchymal and vascular cells of Sedum alfredii stem under Cd stress to reveal gene regulatory networks underlying Cd hyperaccumulation. Cadmium (Cd) hyperaccumulation in plants is a complex biological process controlled by gene regulatory networks. Efficient transport through vascular systems and storage by parenchymal cells are vital for Cd hyperaccumulation in the Cd hyperaccumulator Sedum alfredii, but the genes involved are poorly understood. We investigated the spatial gene expression profiles of transport and storage sites in S. alfredii stem using laser-capture microdissection coupled with RNA sequencing. Gene expression patterns in response to Cd were distinct in vascular and parenchymal cells, indicating functional divisions that corresponded to Cd transportation and storage, respectively. In vascular cells, plasma membrane-related terms enriched a large number of differentially-expressed genes (DEGs) for foundational roles in Cd transportation. Parenchymal cells contained considerable DEGs specifically concentrated on vacuole-related terms associated with Cd sequestration and detoxification. In both cell types, DEGs were classified into different metabolic pathways in a similar way, indicating the role of Cd in activating a systemic stress signalling network where ATP-binding cassette transporters and Ca2+ signal pathways were probably involved. This study identified site-specific regulation of transcriptional responses to Cd stress in S. alfredii and analysed a collection of genes that possibly function in Cd transportation and detoxification, thus providing systemic information and direction for further investigation of Cd hyperaccumulation molecular mechanisms.


Assuntos
Cádmio/toxicidade , Sedum/efeitos dos fármacos , Sedum/genética , Sedum/metabolismo , Estresse Psicológico/genética , Estresse Psicológico/metabolismo , Transcriptoma , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/genética , Cádmio/metabolismo , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Redes e Vias Metabólicas , Floema/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Caules de Planta/citologia , Caules de Planta/efeitos dos fármacos , Análise de Sequência de RNA , Transdução de Sinais , Transcrição Genética/genética
12.
Nat Commun ; 10(1): 407, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30679446

RESUMO

Plants are dependent on controlled sugar uptake for correct organ development and sugar storage, and apoplastic sugar depletion is a defense strategy against microbial infections like rust and mildew. Uptake of glucose and other monosaccharides is mediated by Sugar Transport Proteins, proton-coupled symporters from the Monosaccharide Transporter (MST) superfamily. We present the 2.4 Å structure of Arabidopsis thaliana high affinity sugar transport protein, STP10, with glucose bound. The structure explains high affinity sugar recognition and suggests a proton donor/acceptor pair that links sugar transport to proton translocation. It contains a Lid domain, conserved in all STPs, that locks the mobile transmembrane domains through a disulfide bridge, and creates a protected environment which allows efficient coupling of the proton gradient to drive sugar uptake. The STP10 structure illuminates fundamental principles of sugar transport in the MST superfamily with implications for both plant antimicrobial defense, organ development and sugar storage.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Monossacarídeos/metabolismo , Açúcares/metabolismo , Simportadores/metabolismo , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico/genética , Transporte Biológico/fisiologia , Glucose/metabolismo , Transporte de Íons/fisiologia , Modelos Moleculares , Proteínas de Transporte de Monossacarídeos/genética , Conformação Proteica , Simportadores/genética , Xenopus
13.
Biochim Biophys Acta Biomembr ; 1861(3): 610-618, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30576623

RESUMO

BACKGROUND: VhChiP is a sugar-specific-porin present in the outer membrane of the marine bacterium Vibrio harveyi and responsible for chitin uptake, with a high selectivity for chitohexaose. METHODS: VhChiP and its mutants were expressed and purified from BL21 (DE3) Omp8 Rosetta strain. After reconstitution into planar lipid bilayers, the ion current fluctuations caused by chitohexaose entering the channel were measured in deuterium oxide and in water. RESULTS: The role of hydrogen-bonding in sugar binding was investigated by comparing channel occlusion by chitohexaose in buffers containing H2O and D2O. The BLM results revealed the significant contribution of hydrogen bonding to the binding of chitohexaose in the constriction zone of VhChiP. Replacing H2O as solvent by D2O significantly decreased the on- and off-rates of sugar penetration into the channel. The importance of hydrogen bonding inside the channel was more noticeable when the hydrophobicity of the constriction zone was diminished by replacing Trp136 with the charged residues Asp or Arg. The on- and off-rates decreased up to 2.5-fold and 4-fold when Trp136 was replaced by Arg, or 5-fold and 3-fold for Trp136 replacement by Asp, respectively. Measuring the on-rate at different temperatures and for different channel mutants revealed the activation energy for chitohexaose entrance into VhChiP channel. CONCLUSIONS: Hydrogen-bonds contribute to sugar permeation.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Hidrogênio/farmacologia , Porinas/metabolismo , Açúcares/metabolismo , Vibrio , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Fenômenos Biofísicos/efeitos dos fármacos , Fenômenos Biofísicos/genética , Metabolismo dos Carboidratos/efeitos dos fármacos , Metabolismo dos Carboidratos/genética , Hidrogênio/química , Ligações de Hidrogênio , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Organismos Geneticamente Modificados , Porinas/química , Porinas/genética , Ligação Proteica/efeitos dos fármacos , Vibrio/genética , Vibrio/metabolismo
14.
EMBO Rep ; 20(1)2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30552148

RESUMO

RNA-binding proteins (RBPs) determine spatiotemporal gene expression by mediating active transport and local translation of cargo mRNAs. Here, we cast a transcriptome-wide view on the transported mRNAs and cognate RBP binding sites during endosomal messenger ribonucleoprotein (mRNP) transport in Ustilago maydis Using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP), we compare the key transport RBP Rrm4 and the newly identified endosomal mRNP component Grp1 that is crucial to coordinate hyphal growth. Both RBPs bind predominantly in the 3' untranslated region of thousands of shared cargo mRNAs, often in close proximity. Intriguingly, Rrm4 precisely binds at stop codons, which constitute landmark sites of translation, suggesting an intimate connection of mRNA transport and translation. Towards uncovering the code of recognition, we identify UAUG as specific binding motif of Rrm4 that is bound by its third RRM domain. Altogether, we provide first insights into the positional organisation of co-localising RBPs on individual cargo mRNAs.


Assuntos
Proteínas Fúngicas/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Ustilago/genética , Sítios de Ligação , Transporte Biológico/genética , Endossomos/genética , Regulação da Expressão Gênica , Microtúbulos/genética , Transporte de RNA/genética , RNA Mensageiro/genética , Transcriptoma/genética
15.
Fungal Genet Biol ; 123: 78-86, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30550852

RESUMO

Coatomer-I (COPI) is a heteromeric protein coat that facilitates the budding of membranous carriers mediating Golgi-to-ER and intra-Golgi transport. While the structural features of COPI have been thoroughly investigated, its physiological role is insufficiently understood. Here we exploit the amenability of A. nidulans for studying intracellular traffic, taking up previous studies by Breakspear et al. (2007) with the α-COP/CopA subunit of COPI. Endogenously tagged α-COP/CopA largely localizes to SedVSed5 syntaxin-containing early Golgi cisterna, and acute inactivation of ER-to-Golgi traffic delocalizes COPI to a haze, consistent with the cisternal maturation model. In contrast, the Golgi localization of COPI is independent of the TGN regulators HypBSec7 and HypATrs120, implying that COPI budding predominates at the SedVSed5 early Golgi, with lesser contribution of the TGN. This finding agrees with the proposed role of COPI-mediated intra-Golgi retrograde traffic in driving cisternal maturation, which predicts that the capacity of the TGN to generate COPI carriers is low. The COPI early Golgi compartments intimately associates with Sec13-containing ER exit sites. Characterization of the heat-sensitive copA1ts (sodVIC1) mutation showed that it results in a single residue substitution in the ε-COP-binding Carboxyl-Terminal-Domain of α-COP that likely destabilizes its folding. However, we show that Golgi disorganization by copA1ts necessitates >150 min-long incubation at 42 °C. This weak subcellular phenotype makes it unsuitable for inactivating COPI traffic acutely for microscopy studies, and explains the aneuploidy-stabilizing role of the mutation at subrestrictive temperatures.


Assuntos
Aspergillus nidulans/ultraestrutura , Complexo I de Proteína do Envoltório/química , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Aspergillus nidulans/química , Aspergillus nidulans/genética , Transporte Biológico/genética , Complexo I de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/química , Complexo de Golgi/química , Microscopia de Fluorescência , Mutação , Fenótipo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética
16.
Appl Microbiol Biotechnol ; 103(3): 1339-1350, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30539257

RESUMO

Efflux pumps are recognized as an important mechanism for decreased susceptibility of benzalkonium chloride (BC) in Listeria monocytogenes. Previous studies showed that the efflux pump MdrL was overexpressed in L. monocytogenes exposed to BC. In the present work, we aimed to clarify the role of MdrL in tolerance to BC and environmental stresses including acid, alkali, osmotic, ethanol, and oxidative stresses, as well as resistance to other antimicrobial agents in L. monocytogenes EGD-e. In addition, regulation of the expression of mdrL by LadR was investigated. Gene deletion mutants were constructed by homologous recombination strategy. For the wild-type and mutant strains, minimum inhibitory concentrations (MICs) of antimicrobial agents were determined by the agar dilution, and the growth and survival analysis were also performed. LadR was expressed and the interaction between LadR and the mdrL promoter was investigated by electrophoretic mobility shift assay (EMSA). Compared to the wild-type strain, the growth of ΔmdrL deletion mutant strain was impaired in the presence of sublethal concentration of BC. Moreover, the mutant showed a lower level of survival than that of the wild-type strain in the presence of lethal concentration of BC. However, the deletion of mdrL had no impact on cefotaxime resistance and ethidium bromide efflux. BC could induce the expression of mdrL in L. monocytogenes and the mdrL expression was regulated by LadR instead of SigmaB. LadR was able to specifically bind to the mdrL promoter. The results showed that efflux pump MdrL was associated with BC tolerance in L. monocytogenes EGD-e. Moreover, our results also provided strong evidence that LadR negatively regulated the expression of mdrL. Since BC is commonly used in the food industry, efflux pump MdrL is beneficial for L. monocytogenes to survive this stress in food processing environments.


Assuntos
Antibacterianos/farmacologia , Compostos de Benzalcônio/farmacologia , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico/genética , Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica/genética , Listeria monocytogenes/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana
17.
Microbiol Res ; 218: 108-117, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30454652

RESUMO

All three domains of life have an ordered plasma membrane which is pivotal in the selective fitness of primitive life. Like cholesterol in eukaryotes, hopanoids are important in bacteria to modulate membrane order. Hopanoids are pentacyclic triterpenoid lipids biosynthesised in many eubacteria, few ferns and lichens. Hopanoid modulates outer membrane order and hopanoid deficiency results in the weakened structural integrity of the membrane which may in turn affect the other structures within or spanning the cell envelope and contributing to various membrane functions. Hence, to decipher the role of hopanoid, genome-wide transcriptome of wild-type and Δshc mutant of Rhodopseudomonas palustris TIE-1 was studied which indicated 299 genes were upregulated and 306 genes were downregulated in hopanoid deficient mutant, representing ∼11.5% of the genome. Thirty-eight genes involved in chemotaxis, response to stimuli and signal transduction were differentially regulated and impaired motility in hopanoid deficient mutant showed that hopanoid plays a crucial role in chemotaxis. The docking study demonstrated that diguanylate cyclase which catalyses the synthesis of secondary messenger exhibited the capability to interact with hopanoids and might be confederating in chemotaxis and signal transduction. Seventy-four genes involved in membrane transport were differentially expressed and cell assays also explicit that the multidrug transport is compromised in Δshc mutant. Membrane transport is reliant on hopanoids which may explain the basis for previous observations linking hopanoids to antibiotic resistance. Disturbing the membrane order by targeting lipid synthesis can be a possible novel approach in developing new antimicrobials and hopanoid biosynthesis could be a potential target.


Assuntos
Transporte Biológico/genética , Membrana Celular/fisiologia , Quimiotaxia/genética , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Membrana Transportadoras/genética , Rodopseudomonas/genética , Rodopseudomonas/metabolismo , Triterpenos/metabolismo , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Transdução de Sinais/genética
18.
Ecotoxicol Environ Saf ; 167: 95-106, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30312890

RESUMO

Sedum alfredii Hance is a Zn/Cd co-hyperaccumulator and its underlying molecular mechanism of Cd tolerance is worthy to be elucidated. Although numerous studies have reported the uptake, sequestration and detoxification of Cd in S. alfredii Hance, how it senses Cd-stress stimuli and transfers signals within tissues remains unclear. Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are vital for plant growth, development, immunity and signal transduction. Till now, there is lack of comprehensive studies addressing their functions in S. alfredii Hance responding to Cd stress. In the present study, we identified 60 LRR-RLK genes in S. alfredii Hance based on transcriptome analysis under Cd stress. They were categorized into 11 subfamilies and most of them had highly conserved protein structures and motif compositions. The inter-family diversity provided evidence for their functional divergence, supported by their expression level and profile in tissues under Cd stress. Co-expression network analysis revealed that the most highly connected hubs, Sa0F.522, Sa0F.1036, Sa28F.115 and Sa1F.472, were closely related with other genes involved in metal transport, stimulus response and transcription regulations. Of the ten hub genes exhibiting differential expression dynamics under the short-term Cd stress (Sa0F.522, Sa0F.1036 and Sa28F.115) were dramatically induced in the whole plant. Among them, Sa0F.522 gene was heterologously expressed in a Cd-sensitive yeast cell line and its function in Cd signal perception was confirmed. For the first time, our findings performed a comprehensive analysis of LRR-RLKs in S. alfredii Hance, mapped their expression patterns under Cd stress, and identified the key roles of Sa0F.522, Sa0F.1036 and Sa28F.115 in Cd signal transduction.


Assuntos
Cádmio/toxicidade , Proteínas de Plantas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas/genética , Sedum/genética , Transdução de Sinais/genética , Transporte Biológico/genética , Cádmio/metabolismo , Perfilação da Expressão Gênica , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Sedum/metabolismo , Estresse Fisiológico , Transcrição Genética/genética
19.
Orv Hetil ; 159(32): 1295-1302, 2018 Aug.
Artigo em Húngaro | MEDLINE | ID: mdl-30078355

RESUMO

Etoposide is a topoisomerase II inhibitor antitumor agent which is widely used in the treatment of several hematologic malignancies and solid tumors. The therapeutic index of etoposide is quite high, thus its application causes several short-term and long-term side effects which can decrease the chance to cure patients. Drug dosing is based on body surface area calculation; recommendations for individual dosing do not exist yet. The biotransformation and transportation of etoposide are carried out by enzymes and transporters as reported in pharmacogenomic studies published in this area. Nowadays pharmacoepigenetics research has come to the fore. The authors wish to give an insight into the research of the epigenetical changes of the etoposide pathways, especially focusing on published findings on enzymes and transporters with pharmacokinetic relevance. In the future, epigenetical changes of the etoposide pathway might have a great role in diagnostics, prognostics and personalized medicine. Orv Hetil. 2018; 159(32): 1295-1302.


Assuntos
Antineoplásicos Fitogênicos/metabolismo , Transporte Biológico/genética , Epigênese Genética , Etoposídeo/metabolismo , Antineoplásicos Fitogênicos/farmacocinética , Etoposídeo/farmacocinética , Corpo Humano , Humanos
20.
Dev Cell ; 46(4): 481-494.e6, 2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-30057273

RESUMO

The biogenesis of the RNA payload of mature sperm is of great interest, because RNAs delivered to the zygote at fertilization can affect early development. Here, we tested the hypothesis that small RNAs are trafficked to mammalian sperm during the process of post-testicular maturation in the epididymis. By characterizing small RNA dynamics during germ cell maturation in mice, we confirm and extend prior observations that sperm undergo a dramatic switch in the RNA payload from piRNAs to tRNA fragments (tRFs) upon exiting the testis and entering the epididymis. Small RNA delivery to sperm could be recapitulated in vitro by incubating testicular spermatozoa with caput epididymosomes. Finally, tissue-specific metabolic labeling of RNAs in intact mice definitively shows that mature sperm carry RNAs that were originally synthesized in the epididymal epithelium. These data demonstrate that soma-germline RNA transfer occurs in male mammals, most likely via vesicular transport from the epididymis to maturing sperm.


Assuntos
Movimento Celular/genética , Epididimo/crescimento & desenvolvimento , MicroRNAs/genética , Maturação do Esperma/genética , Animais , Transporte Biológico/genética , Masculino , Mamíferos/metabolismo , Camundongos Transgênicos , Transporte Proteico/genética , Espermatozoides/metabolismo , Testículo/metabolismo
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