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1.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206508

RESUMO

Plants of the genus Allium developed a diversity of defense mechanisms against pathogenic fungi of the genus Fusarium, including transcriptional activation of pathogenesis-related (PR) genes. However, the information on the regulation of PR factors in garlic (Allium sativum L.) is limited. In the present study, we identified AsPR genes putatively encoding PR1, PR2, PR4, and PR5 proteins in A. sativum cv. Ershuizao, which may be involved in the defense against Fusarium infection. The promoters of the AsPR1-5 genes contained jasmonic acid-, salicylic acid-, gibberellin-, abscisic acid-, auxin-, ethylene-, and stress-responsive elements associated with the response to plant parasites. The expression of AsPR1c, d, g, k, AsPR2b, AsPR5a, c (in roots), and AsPR4a(c), b, and AsPR2c (in stems and cloves) significantly differed between garlic cultivars resistant and susceptible to Fusarium rot, suggesting that it could define the PR protein-mediated protection against Fusarium infection in garlic. Our results provide insights into the role of PR factors in A. sativum and may be useful for breeding programs to increase the resistance of Allium crops to Fusarium infections.


Assuntos
Fusarium , Alho/genética , Alho/microbiologia , Genes de Plantas , Interações Hospedeiro-Patógeno/genética , Família Multigênica , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Clonagem Molecular , Fusarium/fisiologia , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Genômica/métodos , Regiões Promotoras Genéticas
2.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209905

RESUMO

Both non-immune "natural" and antigen-induced "immune" IgM are important for protection against pathogens and for regulation of immune responses to self-antigens. Since the bona fide IgM Fc receptor (FcµR) was identified in humans by a functional cloning strategy in 2009, the roles of FcµR in these IgM effector functions have begun to be explored. In this short essay, we describe the differences between human and mouse FcµRs in terms of their identification processes, cellular distributions and ligand binding activities with emphasis on our recent findings from the mutational analysis of human FcµR. We have identified at least three sites of human FcµR, i.e., Asn66 in the CDR2, Lys79 to Arg83 in the DE loop and Asn109 in the CDR3, responsible for its constitutive IgM-ligand binding. Results of computational structural modeling analysis are consistent with these mutational data and a model of the ligand binding, Ig-like domain of human FcµR is proposed. Serendipitously, substitution of Glu41 and Met42 in the CDR1 of human FcµR with mouse equivalents Gln and Leu, either single or more prominently in combination, enhances both the receptor expression and IgM binding. These findings would help in the future development of preventive and therapeutic interventions targeting FcµR.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Animais , Proteínas Reguladoras de Apoptose/química , Sítios de Ligação , Clonagem Molecular , Humanos , Imunoglobulina M/metabolismo , Ligantes , Proteínas de Membrana/química , Camundongos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica
3.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209706

RESUMO

The plant transcription factor WRINKLED1 (WRI1), a member of AP2/EREBP, is involved in the regulation of glycolysis and the expression of genes related to the de novo synthesis of fatty acids in plastids. In this study, the key regulator of seed oil synthesis and accumulation transcription factor gene PoWRI1 was identified and cloned, having a complete open reading frame of 1269 bp and encoding 422 amino acids. Subcellular localization analysis showed that PoWRI1 is located at the nucleus. After the expression vector of PoWRI1 was constructed and transformed into wild-type Arabidopsis thaliana, it was found that the overexpression of PoWRI1 increased the expression level of downstream target genes such as BCCP2, KAS1, and PKP-ß1. As a result, the seeds of transgenic plants became larger, the oil content increased significantly, and the unsaturated fatty acid content increased, which provide a scientific theoretical basis for the subsequent use of genetic engineering methods to improve the fatty acid composition and content of plant seeds.


Assuntos
Regulação da Expressão Gênica de Plantas , Paeonia/genética , Paeonia/metabolismo , Óleos Vegetais/metabolismo , Proteínas de Plantas/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Vias Biossintéticas/genética , Clonagem Molecular , Ácidos Graxos/metabolismo , Fenótipo , Filogenia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Transporte Proteico , Sementes/genética , Sementes/metabolismo , Análise de Sequência de DNA
4.
Int J Mol Sci ; 22(12)2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207582

RESUMO

A forward genetic approach is a powerful tool for identifying the genes underlying the phenotypes of interest. However, the conventional map-based cloning method is lengthy, requires a large mapping population and confirmation of many candidate genes in a broad genetic region to clone the causal variant. The whole-genome sequencing method clones the variants with a certain failure probability for multiple reasons, especially for heterozygotes, and could not be used to clone the mutation of epigenetic modifications. Here, we applied the highly complementary characteristics of these two methods and developed a sequencing-based mapping method (SBM) for identifying the location of plant variants effectively with a small population and low cost, which is very user-friendly for most popular laboratories. This method used the whole-genome sequencing data of two pooled populations to screen out enough markers. These markers were used to identify and narrow the candidate region by analyzing the marker-indexes and recombinants. Finally, the possible mutational sites were identified using the whole-genome sequencing data and verified in individual mutants. To elaborate the new method, we displayed the cloned processes in one Arabidopsis heterozygous mutant and two rice homozygous mutants. Thus, the sequencing-based mapping method could clone effectively different types of plant mutations and was a powerful tool for studying the functions of plant genes in the species with known genomic sequences.


Assuntos
Arabidopsis/genética , Mapeamento Cromossômico , Clonagem Molecular , Genes de Plantas , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Oryza/genética
5.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208617

RESUMO

Retinitis pigmentosa (RP) is an inherited form of retinal degeneration characterized by primary rod photoreceptor cell death followed by cone loss. Mutations in several genes linked to the disease cause increased levels of cyclic guanosine monophosphate (cGMP) and calcium ion influxes. The purpose of this project was to develop a new in vitro photoreceptor degeneration model for molecular studies of RP. 661W cells were genetically modified to stably express the neural retina leucine zipper (NRL) transcription factor. One clone (661W-A11) was selected based on the expression of Nrl target genes. 661W-A11 showed a significant increase in expression of rod-specific genes but not of cone-specific genes, compared with 661W cells. Zaprinast was used to inhibit phosphodiesterase 6 (PDE6) activity to mimic photoreceptor degeneration in vitro. The activation of cell death pathways resulting from PDE6 inhibition was confirmed by detection of decreased viability and increased intracellular cGMP and calcium, as well as activation of protein kinase G (PKG) and calpains. In this new in vitro system, we validated the effects of previously published neuroprotective drugs. The 661W-A11 cells may serve as a new model for molecular studies of RP and for high-throughput drug screening.


Assuntos
Retinite Pigmentosa/etiologia , Retinite Pigmentosa/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Biomarcadores , Linhagem Celular , Células Cultivadas , Clonagem Molecular , Suscetibilidade a Doenças , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Citometria de Fluxo , Imunofluorescência , Expressão Gênica , Humanos , Camundongos , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/etiologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinite Pigmentosa/tratamento farmacológico , Retinite Pigmentosa/patologia
6.
Int J Mol Sci ; 22(13)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34281277

RESUMO

The aim of this research was to analyze the heterologous expression, purification, and immunoregulatory activity of recombinant YGP40 (rYGP40), the potential precursor of the yolkin peptide complex. The ygp40 coding sequence was codon optimized, successfully expressed in the E. coli system, and purified from inclusion bodies with a yield of about 1.1 mg/L of culture. This study showed that the protein exhibits immunomodulatory activity, expressed by the stimulation of TNF-α and IL-10 production and nitric oxide induction at a level comparable to that of the natural yolkin peptide complex obtained by other authors from hen egg yolk. At the highest dose of 100 µg/mL, rYGP40 also caused the up-regulation of iNOS expression in murine bone marrow-derived macrophages (BMDM). Moreover, no cytotoxic effects of rYGP40 on the BMDM cell line were observed.


Assuntos
Vitelogeninas/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Clonagem Molecular , Gema de Ovo/química , Feminino , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/genética , Fatores Imunológicos/farmacologia , Técnicas In Vitro , Interleucina-10/biossíntese , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Peso Molecular , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Vitelogeninas/genética , Vitelogeninas/farmacologia
7.
Gene ; 799: 145852, 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34274480

RESUMO

Cerebellins (CBLN1-4), together with C1qTNF proteins, belong to the CBLN subfamily of C1q proteins. Cerebellin-1 (CBLN1) is active in synapse formation and functions at the parallel fiber-Purkinje cell synapses. Cerebellins form tripartite complexes with neurexins and the glutamate-receptor-related proteins GluD1 and GluD2, playing a role as trans-synaptic cell-adhesion molecules that critically contribute to both synapse formation and functioning and brain development. In this study, I present a molecular characterization of the four porcine CBLN genes. Experimental data and in silico analyses collectively describes the gene structure, chromosomal localization, and expression of CBLN1-4. Two cDNAs encoding the cerebellins CBLN1 and CBLN3 were RT-PCR cloned and sequenced. The nucleotide sequence of the CBLN1 clone contains an open reading frame of 582 nucleotides and encodes a protein of 193 amino acids. The deduced amino acid of the porcine CBLN1 protein was 99% identical to both mouse CBLN1 and to human CBLN1. The deduced CBLN1 protein contains a putative signal sequence of 21 residues, two conserved cysteine residues, and C1q domain. The nucleotide sequence of the CBLN3 cDNA clone comprises an open reading frame of 618 nucleotides and encodes a protein of 205 amino acids. The deduced amino acid sequence of the porcine CBLN3 protein was 88% identical to mouse CBLN3 and 94% identical to human CBLN3. The amino terminal ends of both the CBLN1 and CBLN3 proteins contain three possible N-linked glycosylation sites. The genomic organization of both porcine CBLN1 and CBLN3 is very similar to those of their human counterparts. The expression analyses demonstrated that CBLN1 and CBLN3 transcripts are predominantly expressed in the cerebellum. The sequences of the porcine precerebellin genes and cDNAs were submitted to DDBJ/EMBL/GenBank under the following accession numbers: CBLN1 gene (GenBank ID: FJ621565), CBLN1 cDNA (GenBank ID: EF577504), CBLN3 gene (GenBank ID: FJ621566), CBLN3 cDNA (GenBank ID: EF577505) and CBLN4 cDNA (GenBank ID: FJ196070).


Assuntos
Encéfalo/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Animais , Encéfalo/fisiologia , Clonagem Molecular , Feminino , Glicosilação , Família Multigênica , Proteínas do Tecido Nervoso/metabolismo , Fases de Leitura Aberta , Precursores de Proteínas/genética , Suínos
8.
Structure ; 29(7): 655-663.e4, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34111408

RESUMO

Emerging SARS-CoV-2 strains, B.1.1.7 and B.1.351, from the UK and South Africa, respectively, show decreased neutralization by monoclonal antibodies and convalescent or vaccinee sera raised against the original wild-type virus, and are thus of clinical concern. However, the neutralization potency of two antibodies, 1-57 and 2-7, which target the receptor-binding domain (RBD) of the spike, was unaffected by these emerging strains. Here, we report cryo-EM structures of 1-57 and 2-7 in complex with spike, revealing each of these antibodies to utilize a distinct mechanism to bypass or accommodate RBD mutations. Notably, each antibody represented an immune response with recognition distinct from those of frequent antibody classes. Moreover, many epitope residues recognized by 1-57 and 2-7 were outside hotspots of evolutionary pressure for ACE2 binding and neutralizing antibody escape. We suggest the therapeutic use of antibodies, such as 1-57 and 2-7, which target less prevalent epitopes, could ameliorate issues of monoclonal antibody escape.


Assuntos
Enzima de Conversão de Angiotensina 2/química , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Receptores Virais/química , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Epitopos/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/genética , Receptores Virais/imunologia , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
9.
BMC Plant Biol ; 21(1): 284, 2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34157974

RESUMO

BACKGROUND: Identifying genes involved in salt tolerance in the recretohalophyte Limonium bicolor could facilitate the breeding of crops with enhanced salt tolerance. Here we cloned the previously uncharacterized gene LbHLH and explored its role in salt tolerance. RESULTS: The 2,067-bp open reading frame of LbHLH encodes a 688-amino-acid protein with a typical helix-loop-helix (HLH) domain. In situ hybridization showed that LbHLH is expressed in salt glands of L. bicolor. LbHLH localizes to the nucleus, and LbHLH is highly expressed during salt gland development and in response to NaCl treatment. To further explore its function, we heterologously expressed LbHLH in Arabidopsis thaliana under the 35S promoter. The overexpression lines showed significantly increased trichome number and reduced root hair number. LbHLH might interact with GLABRA1 to influence trichome and root hair development, as revealed by yeast two-hybrid analysis. The transgenic lines showed higher germination percentages and longer roots than the wild type under NaCl treatment. Analysis of seedlings grown on medium containing sorbitol with the same osmotic pressure as 100 mM NaCl demonstrated that overexpressing LbHLH enhanced osmotic resistance. CONCLUSION: These results indicate that LbHLH enhances salt tolerance by reducing root hair development and enhancing osmotic resistance under NaCl stress.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Genes de Plantas/genética , Proteínas de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plumbaginaceae/genética , Plantas Tolerantes a Sal/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Clonagem Molecular , Genes de Plantas/fisiologia , Hibridização In Situ , Pressão Osmótica , Proteínas de Plantas/fisiologia , Plumbaginaceae/metabolismo , Plumbaginaceae/fisiologia , Reação em Cadeia da Polimerase , Estresse Salino , Tolerância ao Sal/genética , Plantas Tolerantes a Sal/metabolismo , Plantas Tolerantes a Sal/fisiologia , Técnicas do Sistema de Duplo-Híbrido
10.
BMC Plant Biol ; 21(1): 301, 2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34187365

RESUMO

BACKGROUND: Mustard (Brassica juncea) is an important economic vegetable, and some cultivars have purple leaves and accumulate more anthocyanins than the green. The genetic and evolution of purple trait in mustard has not been well studied. RESULT: In this study, free-hand sections and metabolomics showed that the purple leaves of mustard accumulated more anthocyanins than green ones. The gene controlling purple leaves in mustard, Mustard Purple Leaves (MPL), was genetically mapped and a MYB113-like homolog was identified as the candidate gene. We identified three alleles of the MYB113-like gene, BjMYB113a from a purple cultivar, BjMYB113b and BjMYB113c from green cultivars. A total of 45 single nucleotide polymorphisms (SNPs) and 8 InDels were found between the promoter sequences of the purple allele BjMYB113a and the green allele BjMYB113b. On the other hand, the only sequence variation between the purple allele BjMYB113a and the green allele BjMYB113c is an insertion of 1,033-bp fragment in the 3'region of BjMYB113c. Transgenic assay and promoter activity studies showed that the polymorphism in the promoter region was responsible for the up-regulation of the purple allele BjMYB113a and high accumulation of anthocyanin in the purple cultivar. The up-regulation of BjMYB113a increased the expression of genes in the anthocyanin biosynthesis pathway including BjCHS, BjF3H, BjF3'H, BjDFR, BjANS and BjUGFT, and consequently led to high accumulation of anthocyanin. However, the up-regulation of BjMYB113 was compromised by the insertion of 1,033-bp in 3'region of the allele BjMYB113c. CONCLUSIONS: Our results contribute to a better understanding of the genetics and evolution of the BjMYB113 gene controlling purple leaves and provide useful information for further breeding programs of mustard.


Assuntos
Genes de Plantas/genética , Mutação com Perda de Função/genética , Mostardeira/genética , Folhas de Planta/anatomia & histologia , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Alelos , Antocianinas/metabolismo , Arabidopsis , Clonagem Molecular , Cor , Genes de Plantas/fisiologia , Mostardeira/anatomia & histologia , Mostardeira/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Fatores de Transcrição/fisiologia
11.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070875

RESUMO

TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.


Assuntos
Subunidades Proteicas/química , Fator 2 Associado a Receptor de TNF/química , Tirosina/química , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Pró-Proteína Convertases/química , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteína de Domínio de Morte Associada a Receptor de TNF/química , Proteína de Domínio de Morte Associada a Receptor de TNF/genética , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Termodinâmica , Tirosina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
12.
In Vivo ; 35(4): 2025-2033, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34182477

RESUMO

BACKGROUND/AIM: The reproducibility of athero - sclerotic lesions was evaluated after the production of cloned-microminipigs and their offspring. MATERIALS AND METHODS: Cloned-microminipig-parents were produced by microminipigsomatic cell nuclei. These parents were crossbred and delivered males (F1-offspring) were divided into two groups: normal chow diet (NcD)-fed and high-fat/high-cholesterol diet (HcD)-fed groups. One of the F1-offsprings was subjected to cloning, and delivered males (F1-clones) were fed with HcD. After 8 weeks, all animals were necropsied for patho - physiological studies compared to non-cloned-microminipigs. RESULTS: HcD-fed F1-offspring and F1-clones, but not NcD-fed F1-offspring, exhibited increased serum lipid levels and systemic atherosclerosis, which were comparable to those of HcD-fed non-cloned-microminipigs. Homogeneity of variance analysis demonstrated that standard deviation values of serum lipoprotein and aortic atherosclerosis area from HcD-fed animals decreased in F1-offspring and F1-clones. CONCLUSION: HcD-induced atherogenesis was highly reproducible in F1-offsprings and F1-clones, indicating that the atherosclerosis-prone genomic background was preserved in the cloned-microminipigs, which can be used for studies on human atherosclerosis and related diseases.


Assuntos
Aterosclerose , Animais , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Colesterol , Clonagem Molecular , Humanos , Masculino , Reprodutibilidade dos Testes , Tecnologia
13.
BMC Plant Biol ; 21(1): 275, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34134615

RESUMO

BACKGROUND: Flavonoid biosynthesis in plants is primarily regulated at the transcriptional level by transcription factors modulating the expression of genes encoding enzymes in the flavonoid pathway. One of the most studied transcription factor complexes involved in this regulation consists of a MYB, bHLH and WD40. However, in Chinese Narcissus (Narcissus tazetta L. var. chinensis), a popular monocot bulb flower, the regulatory mechanism of flavonoid biosynthesis remains unclear. RESULTS: In this work, genes related to the regulatory complex, NtbHLH1 and a R2R3-MYB NtMYB6, were cloned from Chinese Narcissus. Phylogenetic analysis indicated that NtbHLH1 belongs to the JAF13 clade of bHLH IIIf subgroup, while NtMYB6 was highly homologous to positive regulators of proanthocyanidin biosynthesis. Both NtbHLH1 and NtMYB6 have highest expression levels in basal plates of Narcissus, where there is an accumulation of proanthocyanidin. Ectopic over expression of NtbHLH1 in tobacco resulted in an increase in anthocyanin accumulation in flowers, and an up-regulation of expression of the endogenous tobacco bHLH AN1 and flavonoid biosynthesis genes. In contrast, the expression level of LAR gene was significantly increased in NtMYB6-transgenic tobacco. Dual luciferase assays showed that co-infiltration of NtbHLH1 and NtMYB6 significantly activated the promoter of Chinese Narcissus DFR gene. Furthermore, a yeast two-hybrid assay confirmed that NtbHLH1 interacts with NtMYB6. CONCLUSIONS: Our results suggest that NtbHLH1 may function as a regulatory partner by interacting directly with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Narcissus/metabolismo , Proteínas de Plantas/metabolismo , Proantocianidinas/biossíntese , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Narcissus/genética , Ligação Proteica , RNA de Plantas , RNA-Seq , Tabaco/genética
14.
Parasitol Res ; 120(7): 2617-2629, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34142223

RESUMO

Proteins containing WD40 domains play important roles in the formation of multiprotein complexes. Little is known about WD40 proteins in the malaria parasite. This report contains the initial description of a WD40 protein that is unique to the genus Plasmodium and possibly closely related genera. The N-terminal portion of this protein consists of seven WD40 repeats that are highly conserved in all Plasmodium species. Following the N-terminal region is a central region that is conserved within the major Plasmodium clades, such as parasites of great apes, monkeys, rodents, and birds, but partially conserved across all Plasmodium species. This central region contains extensive low-complexity sequence and is predicted to have a disordered structure. Proteins with disordered structure generally function in molecular interactions. The C-terminal region is semi-conserved across all Plasmodium species and has no notable features. This WD40 repeat protein likely functions in some aspect of parasite biology that is unique to Plasmodium and this uniqueness makes the protein a possible target for therapeutic intervention.


Assuntos
Plasmodium/genética , Proteínas de Protozoários/isolamento & purificação , Repetições WD40 , Sequência de Aminoácidos , Animais , Aves , Clonagem Molecular , Epitopos/química , Regulação da Expressão Gênica , Modelos Químicos , Parasitos/metabolismo , Peptídeo Hidrolases/química , Plasmodium/classificação , Proteínas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Técnicas do Sistema de Duplo-Híbrido
15.
Plant Mol Biol ; 106(4-5): 433-448, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34142302

RESUMO

KEY MESSAGE: Endogenous and exogenous GA3 responses to DoEXP and DoXTH depend on the DoGA20ox1, DoGA3ox1, DoGA2ox3, DoGA2ox4, DoGID1a, and DoDELLA1 to regulate yam tuber growth. Yam tuber undergoes significant alteration in morphogenesis and functions during growth, and gibberellins (GA) are considered potentially important regulators of tuber growth. However, it is little known about the regulation of GA metabolism and GA signaling components genes in tuber growth of yam. In this study, the cloning and expressions of GA3 level, GA metabolism and signaling genes, and cell wall genes in tuber growth in response to GA3 and GA biosynthesis inhibitor paclobutrazol (PP333) treatments were studied. The contents of GA3 accumulated at the tuber growth, with the highest levels in the early expansion stage. DoGA20ox1, DoGA3ox1, and four DoGA2ox genes were significantly abundant in the early expansion stage of tuber and gradually declined along with tuber growth. Three DoGID1 and three DoDELLA genes were showed different expression patterns in the early expansion stage of tuber and gradually declined along with tuber growth. Five DoEXP and three DoXTH genes expression levels were higher in the early expansion stage than in other stages. Exogenous GA3 increased endogenous GA3 levels, whereas the expression levels of DoGA20ox1, DoGA3ox1, DoGID1a, and DoDELLA1 were down-regulated in the early expansion stage of tuber by GA3 treatment, DoGA2ox3 and DoGA2ox4 were up-regulated. PP333 application exhibited opposite consequences. Thus, a mechanism of GA3 regulating yam tuber growth by DELLA-dependent pathway is established.


Assuntos
Dioscorea/crescimento & desenvolvimento , Giberelinas/metabolismo , Proteínas de Plantas/fisiologia , Tubérculos/crescimento & desenvolvimento , Clonagem Molecular , Dioscorea/genética , Dioscorea/metabolismo , Proteínas de Plantas/genética , Tubérculos/genética , Tubérculos/metabolismo
16.
Nat Commun ; 12(1): 3287, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078893

RESUMO

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.


Assuntos
Magnésio/química , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Magnésio/metabolismo , Metilação , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética
17.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072501

RESUMO

d-type cyclins (CYCDs) are a special class of cyclins and play extremely important roles in plant growth and development. In the plant kingdom, most of the existing studies on CYCDs have been done on herbaceous plants, with few on perennial woody plants. Here, we identified a Populus d-type cyclin gene, PsnCYCD1;1, which is mainly transcribed in leaf buds and stems. The promoter of PsnCYCD1;1 activated GUS gene expression and transgenic Arabidopsis lines were strongly GUS stained in whole seedlings and mature anthers. Moreover, subcellular localization analysis showed the fluorescence signal of PsnCYCD1;1-GFP fusion protein is present in the nucleus. Furthermore, overexpression of the PsnCYCD1;1 gene in Arabidopsis can promote cell division and lead to small cell generation and cytokinin response, resulting in curved leaves and twisted inflorescence stems. Moreover, the transcriptional levels of endogenous genes, such as ASs, KNATs, EXP10, and PHB, were upregulated by PsnCYCD1;1. Together, our results indicated that PsnCYCD1;1 participates in cell division by cytokinin response, providing new information on controlling plant architecture in woody plants.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Divisão Celular/genética , Ciclina D3/genética , Expressão Gênica , Folhas de Planta/genética , Populus/genética , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular , Ciclina D3/metabolismo , Regulação da Expressão Gênica de Plantas , Morfogênese/genética , Especificidade de Órgãos , Fenótipo , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Transporte Proteico
18.
Dis Aquat Organ ; 145: 89-100, 2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34137379

RESUMO

Protease inhibitors are proteins or small polypeptides functioning in numerous biological processes in all organisms. The I84 family of protease inhibitors in the MEROPS database represents a novel protease inhibitor family that has been reported in 2 bivalves, Crassostrea virginica and Sinonovacula constricta, and is believed to play a role in host defense. In the present study, 7 new members of Family I84 were identified in 2 bivalves, Meretrix meretrix and Mytilus galloprovincialis, and 1 gastropod, Haliotis discus hannai, at the mRNA level via cDNA cloning. The expression patterns of the newly identified genes varied in response to salinity stresses and pathogen-associated molecular pattern stimulations, suggesting their involvement in the host defense of related species. Additionally, analyses of sequence data in public databases did not reveal any Family I84 protease inhibitor molecules in non-molluscan animals. The results indicated that Family I84 protease inhibitors are likely mollusk specific, constituting a unique host defense mechanism in molluscan species.


Assuntos
Fenômenos Biológicos , Gastrópodes , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar , Filogenia , Inibidores de Proteases
19.
Braz J Biol ; 82: e239449, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34105678

RESUMO

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% ß-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.


Assuntos
Escherichia coli , alfa-Amilases , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Geobacillus , Concentração de Íons de Hidrogênio , Temperatura , alfa-Amilases/genética , alfa-Amilases/metabolismo
20.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065885

RESUMO

Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of Nicotiana tabacum (tobacco) transformed by Agrobacterium tumefaciens with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells.


Assuntos
Agrobacterium tumefaciens/fisiologia , Proteínas de Fluorescência Verde/genética , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina/metabolismo , Tabaco/microbiologia , Agrobacterium tumefaciens/genética , Clonagem Molecular , Proteínas de Fluorescência Verde/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Plasmídeos/genética , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tabaco/genética , Tabaco/metabolismo , Transformação Bacteriana
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