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1.
Gene ; 712: 143962, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31288057

RESUMO

Veratrum nigrum is protected plant of Melanthiaceae family, able to synthetize unique steroidal alkaloids important for pharmacy. Transcriptomes from leaves, stems and rhizomes of in vitro maintained V. nigrum plants were sequenced and annotated for genes and markers discovery. Sequencing of samples derived from the different organs resulted in a total of 108,511 contigs with a mean length of 596 bp. Transcripts derived from leaf and stalk were annotated at 28%, and 38% in Nr nucleotide database, respectively. The sequencing revealed 949 unigenes related with lipid metabolism, including 73 transcripts involved in steroids and genus-specific steroid alkaloids biosynthesis. Additionally, 3203 candidate SSRs markers we identified in unigenes with average density of one SSR locus every 6.2 kb sequence. Unraveling of biochemical machinery of the pathway responsible for steroidal alkaloids will open possibility to design and optimize biotechnological process. The transcriptomic data provide valuable resources for biochemical, molecular genetics, comparative transcriptomics, functional genomics, ecological and evolutionary studies of V. nigrum.


Assuntos
Alcaloides/biossíntese , Regulação da Expressão Gênica de Plantas , Esteroides/biossíntese , Transcriptoma , Veratrum/metabolismo , Mapeamento de Sequências Contíguas , DNA Complementar/metabolismo , Biblioteca Gênica , Ontologia Genética , Marcadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Repetições de Microssatélites , Anotação de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Análise de Sequência de RNA
2.
Plant Mol Biol ; 101(3): 257-268, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31302867

RESUMO

KEY MESSAGE: The C-terminal cysteine-rich motif of NYE1/SGR1 affects chlorophyll degradation likely by mediating its self-interaction and conformational change, and somehow altering its Mg-dechelating activity in response to the changing redox potential. During green organ senescence in plants, the most prominent phenomenon is the degreening caused by net chlorophyll (Chl) loss. NON-YELLOWING1/STAY-GREEN1 (NYE1/SGR1) was recently reported to be able to dechelates magnesium (Mg) from Chl a to initiate its degradation, but little is known about the domain/motif basis of its functionality. In this study, we carried out a protein truncation assay and identified a conserved cysteine-rich motif (CRM, P-X3-C-X3-C-X-C2-F-P-X5-P) at its C terminus, which is essential for its function. Genetic analysis showed that all four cysteines in the CRM were irreplaceable, and enzymatic assays demonstrated that the mutation of each of the four cysteines affected its Mg-dechelating activity. The CRM plays a critical role in the conformational change and self-interaction of NYE1 via the formation of inter- and intra-molecular disulfide bonds. Our results may provide insight into how NYE1 responds to rapid redox changes during leaf senescence and in response to various environmental stresses.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Clorofila/química , Proteínas de Cloroplastos/metabolismo , Motivos de Aminoácidos , Quelantes/química , DNA Complementar/metabolismo , Dissulfetos , Regulação da Expressão Gênica de Plantas , Magnésio/química , Oxirredução , Fenótipo , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Conformação Proteica , Domínios Proteicos , Estresse Fisiológico
3.
Plant Mol Biol ; 100(6): 647-658, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31154655

RESUMO

KEY MESSAGE: Found a trans-splicing of PHYTOENE SYNTHASE 1 alters tomato fruit color by map-based cloning, functional complementation and RACE providing an insight into fruit color development. Color is an important fruit quality trait and a major determinant of the economic value of tomato (Solanum lycopersicum). Fruit color inheritance in a yellow-fruited cherry tomato (cv. No. 22), named yellow-fruited tomato 2 (yft2), was shown to be controlled by a single recessive gene, YFT2. The YFT2 gene was mapped in a 95.7 kb region on chromosome 3, and the candidate gene, PHYTOENE SYNTHASE 1 (PSY1), was confirmed by functional complementation analysis. Constitutive over expression of PSY1 in yft2 increased the accumulation of carotenoids and resulted in a red fruit color, while no causal mutation was detected in the YFT2 allele of yft2, compared with red-fruited SL1995 cherry tomato or cultivated variety (cv. M82). Expression of YFT2 3' region in yft2 was significantly lower than in SL1995, and further studies revealed a difference in YFT2 post-transcriptional processing in yft2 compared with SL1995 and cv. M82, resulting in a longer YFT2 transcript. The alternatively trans-spliced allele of YFT2 in yft2 is predicted to encode a novel LT-YFT2 protein of 432 amino acid (AA) residues, compared to the 412 AA YFT2 protein of SL1995. The trans-spliced event also resulted in significantly down regulated expression of YFT2 in yft2 tomato, and the YFT2 allele suppressed expression of the downstream genes involved in the carotenoid biosynthesis pathway and carotenoids synthesis by a mechanism of the feed-forward regulation. In conclusion, we found that trans-splicing of YFT2 alters tomato fruit color, providing new insights into fruit color development.


Assuntos
Lycopersicon esculentum/metabolismo , Pigmentação/genética , Proteínas de Plantas/metabolismo , Processamento Alternativo , Carotenoides/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Cor , DNA Complementar/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genes Recessivos , Teste de Complementação Genética , Genótipo , Lycopersicon esculentum/genética , Mutação , Proteínas de Plantas/genética , Trans-Splicing
4.
Plant Physiol Biochem ; 141: 206-214, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31176880

RESUMO

SUMOylation is an important protein modification that regulates the properties of substrate proteins in a variety of cellular processes. SUMOylation is catalyzed via a cascade of enzymes and is usually stimulated by SUMO E3 ligases. However, the molecular functions and regulatory mechanisms of SUMOylation in forage crops are unknown. Here, we isolated and functionally characterized DiMMS21, a homolog of the Arabidopsis thaliana SUMO ligase AtMMS21, from the forage legume Desmodium intortum. DiMMS21 is expressed ubiquitously in various D. intortum organs and its encoded protein is found in the cytoplasm and nucleus. Bioinformatics analysis indicated that DiMMS21 contains a conserved SP-RING domain that is required for its activity. Biochemical evidence supports the notion that this protein is a functional SUMO ligase. When expressed in an Arabidopsis mms21 mutant, DiMMS21 completely rescued the defects in root, leaf, and silique development. The results from cotyledon greening and marker gene expression suggested that DiMMS21 can only partially complements the role of AtMMS21 in abscisic acid (ABA) responses. In summary, we characterized the molecular features of DiMMS21 and uncovered potential roles of this SUMO ligase in development and ABA responses, increasing our understanding on the function of SUMOylation in forage crops.


Assuntos
Fabaceae/enzimologia , Regulação da Expressão Gênica de Plantas , Ligases/metabolismo , Proteínas de Plantas/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Biologia Computacional , Citoplasma/metabolismo , DNA Complementar/metabolismo , Perfilação da Expressão Gênica , Mutação , Filogenia , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Sementes/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo
5.
Plant Physiol Biochem ; 141: 193-201, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31174036

RESUMO

Centipedegrass (Eremochloa ophiuroides [Munro] Hack.) is a warm-season turfgrass, widely planted in residential lawns and recreational fields. Here, we uncovered three major terpenes released from the shoots of Eo: (E)-ß-ocimene (6%), α-muurolene (87.8%), and eremophilene (6.2%). Methyl jasmonate (MeJA) treatment increased the emission of monoterpenes, including (E)- and (Z)-ß-ocimene, limonene, and myrcene, as well as sesquiterpene blends of (E)-caryophyllene, α-copaene, (+)-cyclosativene, and α-farnesene. RNA sequencing analysis predicted 14 putative Eo terpene synthase (EoTPS) genes, and two full-length EoTPS were successfully amplified: Eo7816 (1722 bp) and Eo6039 (1701 bp). Phylogenetic analysis revealed that Eo7816 and Eo6039 belonged to the clades of TPS-b and TPS-a, respectively. The Arabidopsis transgenic plants overexpressing Eo7816 exclusively released (E)-ß-ocimene (96%) with (Z)-ß-ocimene and myrcene. In contrast, Eo6039-overexpressing Arabidopsis plants emitted significant amounts of α-muurolene (69.4%) and eremophilene (21.8%). Together, we demonstrated that the two TPSs play roles in producing major volatile terpenes in Eo.


Assuntos
Acetatos/química , Alquil e Aril Transferases/metabolismo , Ciclopentanos/química , Oxilipinas/química , Poaceae/enzimologia , Terpenos/química , Transcriptoma , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Análise por Conglomerados , DNA Complementar/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Fenótipo , Filogenia , Proteínas de Plantas/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Sesquiterpenos/química
6.
Parasitol Res ; 118(6): 1849-1863, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31055672

RESUMO

In the search for immunoprophylactics for the control of human lymphatic filariasis, we recently identified troponin 1 (Tn1) in Brugia malayi adult worms. The present study reports the cloning and expression of the B. malayi Tn1 (Tn1bm), its immunoprophylactic efficacy against B. malayi infection, and the immunological responses of the host. The Tn1bm gene was cloned (Acc no. JF912447) and expressed, and the purified recombinant Tn1bm (rTn1bm) presented a single ~ 27 kDa band. Parasite load in rTn1bm-immunized BALB/c mice challenged with B. malayi infective larvae (L3) was assessed. In rTn1bm-immunized animals, IgE, IgG, and IgG subclasses in the serum, cell proliferative response, Th1 and Th2 cytokine secretion (from splenocytes), and NO release (from peritoneal macrophages) were determined. Antibody-dependent cell-mediated cytotoxicity (ADCC) to L3 was assayed using rTn1bm-immune serum. The innate immune response markers MHC class-I, MHC class-II, TLR2, TLR4, and TLR6 expression in peritoneal macrophages and CD3+, CD4+, CD8+, and CD19+ in the splenocyte population were determined in Tn1bm-exposed cells from naïve mice. rTn1bm-immunized L3-challenged animals showed a 60% reduction in parasite burden. Immunization upregulated cellular proliferation, cytokine (IFN-γ, TNF-α, IL-1ß, IL-4, IL-6, and IL-10) secretion, NO release, and antigen-specific IgG, IgG1, and IgG2b antibody levels. rTn1bm-immune serum killed > 65% of L3 in the ADCC assay. Increased MHC class-II, TLR2, and TLR6 expression and the relative CD4+ and CD19+ cell populations of naïve animal cells indicated the ability of rTn1bm to mobilize innate immune responses. This is the first report of the immunoprophylactic potential of rTn1bm against B. malayi.


Assuntos
Anticorpos Antiprotozoários/sangue , Brugia Malayi/imunologia , Filariose Linfática/imunologia , Filariose Linfática/prevenção & controle , Troponina I/genética , Troponina I/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Brugia Malayi/genética , Clonagem Molecular , Citocinas/sangue , Citocinas/imunologia , DNA Complementar/metabolismo , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Células Th1/imunologia , Células Th2/imunologia , Vacinação
7.
J Zhejiang Univ Sci B ; 20(4): 322-331, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30932377

RESUMO

We investigated the microRNA172 (miR172)-mediated regulatory network for the perception of changes in external and endogenous signals to identify a universally applicable floral regulation system in ornamental plants, manipulation of which could be economically beneficial. Transgenic gloxinia plants, in which miR172 was either overexpressed or suppressed, were generated using Agrobacterium-mediated transformation. They were used to study the effect of altering the expression of this miRNA on time of flowering and to identify its mRNA target. Early or late flowering was observed in transgenic plants in which miR172 was overexpressed or suppressed, respectively. A full-length complementary DNA (cDNA) of gloxinia (Sinningia speciosa) APETALA2-like (SsAP2-like) was identified as a target of miR172. The altered expression levels of miR172 caused up- or down-regulation of SsAP2-like during flower development, which affected the time of flowering. Quantitative real-time reverse transcription PCR analysis of different gloxinia tissues revealed that the accumulation of SsAP2-like was negatively correlated with the expression of miR172a, whereas the expression pattern of miR172a was negatively correlated with that of miR156a. Our results suggest that transgenic manipulation of miR172 could be used as a universal strategy for regulating time of flowering in ornamental plants.


Assuntos
Proteínas de Arabidopsis/genética , Flores/fisiologia , Proteínas de Homeodomínio/genética , Lamiales/fisiologia , MicroRNAs/genética , Proteínas Nucleares/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular , DNA Complementar/metabolismo , Flores/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/metabolismo , Lamiales/genética , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Transgenes
8.
Gene ; 706: 19-31, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31028869

RESUMO

The fem-1 gene in Caenorhabditis elegans is involved in sex differentiation; it is specifically required for all aspects of male development. In this study, the full-length cDNA of the fem-1 (Pvfem-1) gene was isolated from the Pacific whiteleg shrimp Penaeus vannamei. The Pvfem-1 transcript is 3778 nt long and encodes a putative protein (PvFEM-1) of 638 amino acids that presented eight ankyrin repeats. The translated protein showed a significant (P < 0.05) structural similitude by superposition with C. elegans FEM-1 protein. Pvfem-1 expression was evaluated by qPCR and in situ hybridization (ISH) during embryogenesis, larval development, and gonads of both genders in subadult and adult life stages. Pvfem-1 was found expressed in brain, intestine, hepatopancreas, and in the gonads of both genders in subadults and adults when quantified by RT-qPCR. A significant finding was the discovery of a natural antisense transcript (NAT) of Pvfem-1 by ISH. It was present in the oocyte nucleus of subadult female shrimp gonads but was not seen within oocytes from adult females, although it was detected in follicular cells, suggesting a possible post-transcriptional regulation of Pvfem-1 in female gonad. Conversely, in males, no NAT was observed, and Pvfem-1 was found expressed in spermatogonia of both, subadult and adult shrimps indicating a function in male sexual differentiation and gametes generation. This study represents the first step for future functional analysis that is expected to contribute to clarifying the role of Pvfem-1 in sex differentiation and determination.


Assuntos
Elementos Antissenso (Genética)/fisiologia , Penaeidae/genética , Processos de Determinação Sexual/genética , Sequência de Aminoácidos , Animais , Elementos Antissenso (Genética)/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , DNA Complementar/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Gônadas/metabolismo , Hibridização In Situ , Masculino , Ovário/metabolismo , Filogenia , RNA Mensageiro/genética , Alinhamento de Sequência , Análise de Sequência de DNA/métodos , Fatores Sexuais
9.
Methods Mol Biol ; 1979: 25-44, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028630

RESUMO

In the last few years single-cell RNA sequencing (scRNA-seq) has enabled the investigation of cellular heterogeneity at the transcriptional level, the characterization of rare cell types as well as the detailed analysis of the stochastic nature of gene expression. A large number of methods have been developed, varying in their throughput, sensitivity, and scalability. A major distinction is whether they profile only 5'- or 3'-terminal part of the transcripts or allow for the characterization of the entire length of the transcripts. Among the latter, Smart-seq2 is still considered the "gold standard" due to its sensitivity, precision, lower cost, scalability and for being easy to set up on automated platforms. In this chapter I describe how to efficiently generate sequencing-ready libraries, highlight common issues and pitfalls, and offer solutions for generating high-quality data.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , Análise de Célula Única/métodos , Animais , DNA Complementar/genética , DNA Complementar/metabolismo , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Humanos , Transcrição Reversa , Transcriptoma , Transposases/metabolismo
10.
FEBS Open Bio ; 9(4): 618-628, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30984537

RESUMO

The enzyme 5,10-methylenetetrahydrofolate dehydrogenase (MTHFD) is essential for the production of certain amino acids (glycine, serine, and methionine) and nucleic acids (thymidylate and purine). Here, we identified a cDNA encoding this enzyme from the silkworm Bombyx mori. The recombinant B. mori MTHFD (bmMTHFD) expressed in Escherichia coli recognized 5,10-methylenetetrahydrofolate and 5,10-methenyltetrahydrofolate as substrate in the presence of NADP + as well as NAD +. The bmMTHFD structure was determined at a resolution of 1.75 Å by X-ray crystallography. Site-directed mutagenesis indicated that the amino acid residue Tyr49 contributed to its catalytic activity. Our findings provide insight into the mechanism underlying the activity of MTHFD from B. mori and potentially other insects and may therefore facilitate the development of inhibitors specific to MTHFD as insecticides.


Assuntos
Bombyx/genética , Proteínas de Insetos/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Sequência de Aminoácidos , Animais , Bombyx/enzimologia , Bombyx/metabolismo , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/metabolismo , Escherichia coli/genética , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/química , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Estrutura Molecular , Mutagênese Sítio-Dirigida , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
11.
Pestic Biochem Physiol ; 156: 56-62, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31027581

RESUMO

Glutamate-gated chloride channels (GluCls) mediate inhibitory synaptic transmission in invertebrate nervous systems, and only one GluCl gene has been found in insects. Therefore, insect GluCls are one of the major targets of insecticides including avermectins. In the present study, a 1347 bp full-length cDNA encoding a 449-amino acid protein (named MsGluCl, GenBank ID: MK336885) was cloned from the oriental armyworm, Mythimna separata, and characterized two alternative splicing variants of MsGluCl. The protein shares 76.9-98.6% identity with other insect GluCl isoforms. Spatial and temporal expression analysis revealed that MsGluCl was highly expressed in the 3rd instar and adult head. Dietary ingestion of dsMsGluCl significantly reduced the mRNA level of MsGluCl and decreased abamectin mortality. Thus, our results reveal that MsGluCl could be the molecular target of abamectin and provide the basis for further understanding the resistance mechanism to abamectin in arthropods.


Assuntos
Processamento Alternativo/genética , Canais de Cloreto/metabolismo , Clonagem Molecular/métodos , Mariposas/genética , Animais , Canais de Cloreto/genética , DNA Complementar/genética , DNA Complementar/metabolismo , Inseticidas/farmacologia , Ivermectina/análogos & derivados , Ivermectina/farmacologia , Mariposas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
Int J Mol Sci ; 20(7)2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30965614

RESUMO

Luteinizing hormone (LH), a pituitary gonadotropin, coupled with LH receptor (LHR) is essential for the regulation of the gonadal maturation in vertebrates. Although LH homolog has been detected by immunocytochemical analysis, and its possible role in ovarian maturation was revealed in decapod crustacean, so far there is no molecular evidence for the existence of LHR. In this study, we cloned a novel LHR homolog (named EsLHR) from the Chinese mitten crab Eriocheir sinensis. The complete sequence of the EsLHR cDNA was 2775bp, encoding a protein of 924 amino acids, sharing 71% amino acids identity with the ant Zootermopsis nevadensis LHR. EsLHR expression was found to be high in the ovary, while low in testis, gill, brain, and heart, and no expression in the thoracic ganglion, eye stalk, muscle, and hepatopancreas. Quantitative PCR revealed that the expression level of EsLHR mRNA was significantly higher in the ovaries in previtellogenic (Pvt), late vitellogenic (Lvt), and germinal vesicle breakdown (GVBD) stages than that in the vitellogenic (Mvt) and early vitellogenic (Evt) stages (P < 0.05), and, the highest and the lowest expression were in Lvt, and Evt, respectively. The strong signal was mainly localized in the ooplasm of Pvt oocyte as detected by in situ hybridization. The crab GnRH homolog can significantly induce the expression of EsLHR mRNA at 36 hours post injection in vivo (P < 0.01), suggesting that EsLHR may be involved in regulating ovarian development through GnRH signaling pathway in the mitten crab.


Assuntos
Braquiúros/metabolismo , Receptores do LH/metabolismo , Animais , Braquiúros/embriologia , DNA Complementar/metabolismo , Feminino , Masculino , Ovário/embriologia , Ovário/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Receptores do LH/genética , Testículo/embriologia , Testículo/metabolismo
13.
Pestic Biochem Physiol ; 155: 36-44, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30857625

RESUMO

Ionotropic γ-aminobutyric acid (GABA) receptors (GABARs) mediate rapid inhibitory neurotransmission in both vertebrates and invertebrates, and are important molecular targets of insecticides. However, components of insect GABARs remain elusive. In addition to CsRDL1 and CsRDL2, the complementary DNAs (cDNAs) of another two GABA receptor-like subunits, CsLCCH3 and Cs8916, were identified from the rice striped stem borer, Chilo suppressalis Walker in the present study. Both CsLCCH3 and Cs8916 subunits shared common structural features, such as a highly-conserved Cys-loop structure, six distinct regions involved in ligand binding (loops A-F), and four transmembrane domains (TM 1-4). Transcript analysis demonstrated that the relative mRNA expression levels of both CsLCCH3 and Cs8916 subunits were the highest in the ventral nerve cord. Regarding developmental stage, transcript levels of both subunits were highest in eggs. Injections of double-stranded RNAs (dsRNAs), including dsRDL1, dsRDL2, dsLCCH3, or ds8916, significantly reduced mRNA abundance after 24 and 48 h. However, no observable effects on the development of C. suppressalis were observed. Injection of dsRDL1 or dsRDL2 did significantly reduce the mortality of C. suppressalis treated with fluralaner. Our results indicated that CsRDLs mediated the susceptibility of C. suppressalis to fluralaner, whereas CsLCCH3 and CsL8916 did not. The current investigation enhances our knowledge of Lepidopteran GABARs and offers a molecular basis for the development of novel insecticides to control C. suppressalis.


Assuntos
Lepidópteros/metabolismo , Receptores de GABA/metabolismo , Animais , DNA Complementar/metabolismo , Mariposas , RNA Mensageiro/metabolismo
14.
Methods Mol Biol ; 1957: 159-168, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30919353

RESUMO

ADP-ribosylation factors (ARF) are GTPases that act to control the activation of numerous signaling events and cellular responses. The ARF6 isoform, present at the plasma membrane, can be activated by the angiotensin II type 1 receptor (AT1R), a process dependent upon ß-arrestin recruitment to the activated receptor. Here, we describe classical methods used to assess ß-arrestin-dependent activation of ARF6 following agonist stimulation of cells. In addition, because ARF6 and ß-arrestin can form a complex, we describe the procedures used to detect the interaction of ß-arrestin with this GTPase.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Citoesqueleto de Actina/metabolismo , Biologia Molecular/métodos , beta-Arrestinas/metabolismo , DNA Complementar/metabolismo , Ativação Enzimática , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/metabolismo
15.
Gen Comp Endocrinol ; 276: 52-59, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30849410

RESUMO

Neurotransmitters such as serotonin (5-hydroxytryptamine; 5-HT) in the central nervous system regulate diverse physiological functions, including reproduction, feeding, learning, and memory, in diverse animal phyla. 5-HT and the 5-HT1 subtype receptor play important roles in sexual maturation and in the initiation of gamete release in mollusks. However, little is known about the involvement of other 5-HT receptor subfamilies in the reproduction process. In the present study, we identified the cDNAs encoding eight subtypes of 5-HT receptors from the ganglia tissues of the Pacific abalone Haliotis discus hannai (Mollusca; Gastropoda; Haliotidae), and examined the gonadal expression of the transcripts of 5-HT receptors. A phylogenetic analysis indicated that the molluskan 5-HT receptors are largely classified into four major clades: 5-HT1/5/7, 5-HT2, 5-HT4, and 5-HT6. Among the H. discus hannai (Hdh) 5-HT1-7 transcripts, Hdh5-HT1B, 4A, 4B, and 6 were the major subtypes detected in the mature ovary. Estradiol-17ß injection into the pedal sinus induced the downregulation of 5-HT4B and upregulation of 5-HT6 transcripts in the ovary of mature abalone within 72 h. In HEK293 cells overexpressing Hdh5-HT1B, forskolin-stimulated cAMP response element luciferase (CRE-Luc) reporter activity was inhibited by 5-HT in a dose-dependent manner, whereas serum response element luciferase (SRE-Luc) activity was not affected. In Hdh5-HT4A-expressing HEK293 cells, forskolin-stimulated CRE-Luc and SRE-Luc reporter activities were both marginally increased by treatment with a high dose of 5-HT. Our results provide new insights into the roles of 5-HT through diverse G protein-coupled 5-HT receptors in the reproductive process of mollusks.


Assuntos
Gastrópodes/genética , Regulação da Expressão Gênica , Receptores de Serotonina/genética , Animais , DNA Complementar/metabolismo , Estradiol/farmacologia , Feminino , Gastrópodes/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Serotonina/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
Toxicon ; 163: 59-69, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30902682

RESUMO

Phoneutria nigriventer spider venom has been studied for more than 40 years and several components with pharmacological potential have been described in it. However, studies on venoms from other species of the Phoneutria genus are scarce. In this work, a conventional cDNA library from the species Phoneutria pertyi venom glands was constructed, aiming to identify novel putative cysteine-rich peptide toxins for the genus Phoneutria. 296 unique sequences were identified and 51 sequences corresponded to putative cysteine-rich peptide toxins. Besides cysteine-rich peptide toxins, other putative venom components such as protease inhibitors, defensins and serine proteinases were identified. Furthermore, by manual curation of the sequences with no match at UniProt, we were able to identify glycine-rich proteins (GRP), a class of venom component never described in Phoneutria genus. This work describes the first complete sequences of toxins from the venom of P. pertyi and reveals that, despite most of the retrieved toxins show a high identity to toxins identified in Phoneutria genus, novel putative toxins remains to be described.


Assuntos
Venenos de Aranha/química , Transcriptoma , Animais , Proteínas de Artrópodes/análise , DNA Complementar/genética , DNA Complementar/metabolismo , Defensinas/análise , Perfilação da Expressão Gênica , Peptídeos/análise , Inibidores de Proteases/análise , Serina Proteases/análise , Aranhas/genética , Aranhas/metabolismo
17.
Anal Cell Pathol (Amst) ; 2019: 9365654, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30775237

RESUMO

After breast and colon cancer, cervical cancer is the third most common cancer of women worldwide. Since human papillomavirus (HPV) infection is known to be the predominant cause of cervical cancer, molecular HPV screening is currently used along with cytological and histological examination methods for precancer diagnosis. Nevertheless, the sensitivity of the current HPV test is less than 80%; thus, many cervical cancer cases are not able to be diagnosed by HPV screening alone, and likewise, patients with cervical cancer are often determined to be HPV-negative by the current screening methods. Therefore, human telomerase reverse transcriptase (hTERT) and Ki67 previously identified as cancer markers were attempted. And cervical exfoliated cells of high-grade squamous intraepithelial lesion (HSIL), the most severe precancerous lesion of cancer, were used in the study. However, it takes a long time to collect enough specimens to conduct statistical analysis. Therefore, in the present study, microscope slides, cervical exfoliated cells on glass slides, were attempted. The results of the analysis demonstrated that hTERT and Ki67 expression levels were useful in distinguishing between cancerous and normal specimens, exhibiting a higher sensitivity and specificity than conventional HPV E6/E7 testing. And the study suggests clinical slide cell samples could be effectively used in the context of retrospective studies to identify novel biomarkers.


Assuntos
Antígeno Ki-67/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Telomerase/metabolismo , Neoplasias do Colo do Útero/metabolismo , Adolescente , Adulto , Idoso , Apoptose/genética , Apoptose/fisiologia , Criança , DNA Complementar/metabolismo , Feminino , Genótipo , Humanos , Antígeno Ki-67/genética , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/genética , RNA Mensageiro/metabolismo , Estudos Retrospectivos , Telomerase/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Adulto Jovem
19.
Exp Parasitol ; 199: 24-29, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30796912

RESUMO

The genus Acanthamoeba, which may cause different infections in humans, occurs widely in the environment. Lung inflammation caused by these parasites induces pulmonary pathological changes such as pulmonary necrosis, peribronchial plasma cell infiltration, moderate desquamation of alveolar cells and partial destruction of bronchial epithelial cells, and presence of numerous trophozoites and cysts among inflammatory cells. The aim of this study was to assess the influence of plant extracts from Artemisia annua L. on expression of the toll-like receptors TLR2 and TLR4 in lungs of mice with acanthamoebiasis. A. annua, which belongs to the family Asteraceae, is an annual plant that grows wild in Asia. In this study, statistically significant changes of expression of TLR2 and TLR4 were demonstrated. In the lungs of infected mice after application of extract from A. annua the expression of TLRs was observed mainly in bronchial epithelial cells, pneumocytes (to a lesser extent during the outbreak of infection), and in the course of high general TLR expression. TLR4 in particular was also visible in stromal cells of lung parenchyma. In conclusion, we confirmed that a plant extract of A. annua has a modulatory effect on components of the immune system such as TLR2 and TLR4.


Assuntos
Acanthamoeba/fisiologia , Amebíase/tratamento farmacológico , Artemisia annua/química , Pneumopatias Parasitárias/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Receptores Toll-Like/metabolismo , Amebíase/metabolismo , Animais , DNA Complementar/metabolismo , Imuno-Histoquímica , Pulmão/parasitologia , Pulmão/patologia , Pneumopatias Parasitárias/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Extratos Vegetais/farmacologia , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Reversa , Receptor 2 Toll-Like/efeitos dos fármacos , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Receptores Toll-Like/efeitos dos fármacos , Receptores Toll-Like/genética
20.
Anticancer Res ; 39(2): 771-780, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30711956

RESUMO

BACKGROUND/AIM: Radiotherapy (RT) can lead to cardiovascular disease (CVD). Evidence suggests that radiation modulates miRNA levels. Our purpose was to assess the acute response to radiation-induced modulation of the expression of miRNA-146a, miRNA-155, miRNA-221, and miRNA-222, inflammatory response and endothelial dysfunction on endothelial cells. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to 2 Gy RT, and intracellular levels of selected miRNAs were measured by real-time polymerase chain reaction at 2 and 24 h. Cytokine and adhesion molecule release were also assessed. RESULTS: Results showed that 2 Gy significantly increased the expression of miRNA-221 and miRNA-222, and reduced the level of miRNA-155 after 2 h; whereas miRNA-146a and miRNA-155 were significantly overexpressed and miRNA-222 was significantly down-regulated at 24 h. Interleukin-8 and soluble vascular cell adhesion molecule 1 levels were not affected by the studied RT. CONCLUSION: RT at 2 Gy modulated expression of selected miRNAs by endothelial cells after 2 and 24 h, which might be related to CVD development in patients who receive RT.


Assuntos
Doenças Cardiovasculares/metabolismo , Endotélio Vascular/patologia , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Inflamação/tratamento farmacológico , MicroRNAs/metabolismo , MicroRNAs/efeitos da radiação , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/radioterapia , Adesão Celular , Citocinas/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta à Radiação , Humanos , Molécula 1 de Adesão de Célula Vascular/metabolismo
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