Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 36
Filtrar
Mais filtros

Base de dados
País/Região como assunto
Tipo de documento
Intervalo de ano de publicação
1.
Environ Microbiol ; 26(1): e16546, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38086774

RESUMO

Human activities have affected the surrounding natural ecosystems, including belowground microorganisms, for millennia. Their short- and medium-term effects on the diversity and the composition of soil microbial communities are well-documented, but their lasting effects remain unknown. When unoccupied for centuries, archaeological sites are appropriate for studying the long-term effects of past human occupancy on natural ecosystems, including the soil compartment. In this work, the soil chemical and bacterial compositions were compared between the Roman fort of Hegra (Saudi Arabia) abandoned for 1500 years, and a preserved area located at 120 m of the southern wall of the Roman fort where no human occupancy was detected. We show that the four centuries of human occupancy have deeply and lastingly modified both the soil chemical and bacterial compositions inside the Roman fort. We also highlight different bacterial putative functions between the two areas, notably associated with human occupancy. Finally, this work shows that the use of soils from archaeological sites causes little disruption and can bring relevant information, at a large scale, during the initial surveys of archaeological sites.


Assuntos
Ecossistema , Solo , Humanos , Solo/química , DNA Bacteriano/genética , Efeitos Antropogênicos , Bactérias/genética , Microbiologia do Solo
2.
Planta ; 259(6): 132, 2024 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-38662123

RESUMO

MAIN CONCLUSION: Emblematic Vachellia spp. naturally exposed to hyper-arid conditions, intensive grazing, and parasitism maintain a high nitrogen content and functional mutualistic nitrogen-fixing symbioses. AlUla region in Saudi Arabia has a rich history regarding mankind, local wildlife, and fertility islands suitable for leguminous species, such as the emblematic Vachellia spp. desert trees. In this region, we investigated the characteristics of desert legumes in two nature reserves (Sharaan and Madakhil), at one archaeological site (Hegra), and in open public domains et al. Ward and Jabal Abu Oud. Biological nitrogen fixation (BNF), isotopes, and N and C contents were investigated through multiple lenses, including parasitism, plant tissues, species identification, plant maturity, health status, and plant growth. The average BNF rates of 19 Vachellia gerrardii and 21 Vachellia tortilis trees were respectively 39 and 67%, with low signs of inner N content fluctuations (2.10-2.63% N) compared to other co-occurring plants. The BNF of 23 R. raetam was just as high, with an average of 65% and steady inner N contents of 2.25 ± 0.30%. Regarding parasitism, infected Vachellia trees were unfazed compared to uninfected trees, thereby challenging the commonly accepted detrimental role of parasites. Overall, these results suggest that Vachellia trees and R. raetam shrubs exploit BNF in hyper-arid environments to maintain a high N content when exposed to parasitism and grazing. These findings underline the pivotal role of plant-bacteria mutualistic symbioses in desert environments. All ecological traits and relationships mentioned are further arguments in favor of these legumes serving as keystone species for ecological restoration and agro-silvo-pastoralism in the AlUla region.


Assuntos
Fabaceae , Fixação de Nitrogênio , Clima Desértico , Ecossistema , Etnobotânica , Fabaceae/parasitologia , Fabaceae/fisiologia , Arábia Saudita , Simbiose
3.
Mycorrhiza ; 34(4): 251-270, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-39023766

RESUMO

Hot deserts impose extreme conditions on plants growing in arid soils. Deserts are expanding due to climate change, thereby increasing the vulnerability of ecosystems and the need to preserve them. Arbuscular mycorrhizal fungi (AMF) improve plant fitness by enhancing plant water/nutrient uptake and stress tolerance. However, few studies have focused on AMF diversity and community composition in deserts, and the soil and land use parameters affecting them. This study aimed to comprehensively describe AMF ecological features in a 5,000 km2 arid hyperalkaline region in AlUla, Saudi Arabia. We used a multimethod approach to analyse over 1,000 soil and 300 plant root samples of various species encompassing agricultural, old agricultural, urban and natural ecosystems. Our method involved metabarcoding using 18S and ITS2 markers, histological techniques for direct AMF colonization observation and soil spore extraction and observation. Our findings revealed a predominance of AMF taxa assigned to Glomeraceae, regardless of the local conditions, and an almost complete absence of Gigasporales taxa. Land use had little effect on the AMF richness, diversity and community composition, while soil texture, pH and substantial unexplained stochastic variance drove these compositions in AlUla soils. Mycorrhization was frequently observed in the studied plant species, even in usually non-mycorrhizal plant taxa (e.g. Amaranthaceae, Urticaceae). Date palms and Citrus trees, representing two major crops in the region, however, displayed a very low mycorrhizal frequency and intensity. AlUla soils had a very low concentration of spores, which were mostly small. This study generated new insight on AMF and specific behavioral features of these fungi in arid environments.


Assuntos
Clima Desértico , Micorrizas , Microbiologia do Solo , Micorrizas/fisiologia , Arábia Saudita , Esporos Fúngicos/fisiologia , Solo/química , Glomeromycota/fisiologia , Raízes de Plantas/microbiologia
4.
Environ Microbiol ; 24(11): 5509-5523, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35920038

RESUMO

Although rhizobia that establish a nitrogen-fixing symbiosis with legumes are also known to promote growth in non-legumes, studies on rhizobial associations with wheat roots are scarce. We searched for Rhizobium leguminosarum symbiovar viciae (Rlv) strains naturally competent to endophytically colonize wheat roots. We isolated 20 strains from surface-sterilized wheat roots and found a low diversity of Rlv compared to that observed in the Rlv species complex. We tested the ability of a subset of these Rlv for wheat root colonization when co-inoculated with other Rlv. Only a few strains, including those isolated from wheat roots, and one strain isolated from pea nodules, were efficient in colonizing roots in co-inoculation conditions, while all the strains tested in single strain inoculation conditions were found to colonize the surface and interior of roots. Furthermore, Rlv strains isolated from wheat roots were able to stimulate root development and early arbuscular mycorrhizal fungi colonization. These responses were strain and host genotype dependent. Our results suggest that wheat can be an alternative host for Rlv; nevertheless, there is a strong competition between Rlv strains for wheat root colonization. In addition, we showed that Rlv are endophytic wheat root bacteria with potential ability to modify wheat development.


Assuntos
Rhizobium leguminosarum , Rhizobium , Rhizobium leguminosarum/genética , Endófitos/genética , Triticum , Filogenia , Simbiose/genética , Bactérias/genética , Nódulos Radiculares de Plantas/microbiologia
5.
Plant Physiol ; 183(3): 1319-1330, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32376762

RESUMO

Nitrogen-fixing root nodulation in legumes challenged with nitrogen-limiting conditions requires infection of the root hairs by soil symbiotic bacteria, collectively referred to as rhizobia, and the initiation of cell divisions in the root cortex. Cytokinin hormones are critical for early nodulation to coordinate root nodule organogenesis and the progression of bacterial infections. Cytokinin signaling involves regulation of the expression of cytokinin primary response genes by type-B response regulator (RRB) transcription factors. RNA interference or mutation of MtRRB3, the RRB-encoding gene most strongly expressed in Medicago truncatula roots and nodules, significantly decreased the number of nodules formed, indicating a function of this RRB in nodulation initiation. Fewer infection events were also observed in rrb3 mutant roots associated with a reduced Nod factor induction of the Early Nodulin 11 (MtENOD11) infection marker, and of the cytokinin-regulated Nodulation Signaling Pathway 2 (Mt NSP2) gene. Rhizobial infections correlate with an expansion of the nuclear area, suggesting the activation of endoreduplication cycles linked to the cytokinin-regulated Cell Cycle Switch 52A (Mt CCS52A) gene. Although no significant difference in nucleus size and endoreduplication were detected in rhizobia-infected rrb3 mutant roots, expression of the MtCCS52A endoreduplication marker was reduced. As the MtRRB3 expression pattern overlaps with those of MtNSP2 and MtCCS52A in roots and nodule primordia, chromatin immunoprecipitation-quantitative PCR and protoplast trans-activation assays were used to show that MtRRB3 can interact with and trans-activate MtNSP2 and MtCCS52A promoters. Overall, we highlight that the MtRRB3 cytokinin signaling transcription factor coordinates the expression of key early nodulation genes.


Assuntos
Citocininas/metabolismo , Nodulação , Transdução de Sinais , Fatores de Transcrição/metabolismo , Tamanho do Núcleo Celular , Endorreduplicação , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Medicago truncatula/genética , Medicago truncatula/microbiologia , Fenótipo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nodulação/genética , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , Sinorhizobium meliloti/fisiologia , Ativação Transcricional/genética
6.
New Phytol ; 226(2): 555-568, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31873949

RESUMO

Fabeae legumes such as pea and faba bean form symbiotic nodules with a large diversity of soil Rhizobium leguminosarum symbiovar viciae (Rlv) bacteria. However, bacteria competitive to form root nodules (CFN) are generally not the most efficient to fix dinitrogen, resulting in a decrease in legume crop yields. Here, we investigate differential selection by host plants on the diversity of Rlv. A large collection of Rlv was collected by nodule trapping with pea and faba bean from soils at five European sites. Representative genomes were sequenced. In parallel, diversity and abundance of Rlv were estimated directly in these soils using metabarcoding. The CFN of isolates was measured with both legume hosts. Pea/faba bean CFN were associated to Rlv genomic regions. Variations of bacterial pea and/or faba bean CFN explained the differential abundance of Rlv genotypes in pea and faba bean nodules. No evidence was found for genetic association between CFN and variations in the core genome, but variations in specific regions of the nod locus, as well as in other plasmid loci, were associated with differences in CFN. These findings shed light on the genetic control of CFN in Rlv and emphasise the importance of host plants in controlling Rhizobium diversity.


Assuntos
Rhizobium leguminosarum , Rhizobium , Vicia faba , Filogenia , Rhizobium leguminosarum/genética , Simbiose
8.
Biochem J ; 475(4): 759-773, 2018 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-29358189

RESUMO

While mitochondrial mutants of the respiratory machinery are rare and often lethal, cytoplasmic male sterility (CMS), a mitochondrially inherited trait that results in pollen abortion, is frequently encountered in wild populations. It generates a breeding system called gynodioecy. In Beta vulgaris ssp. maritima, a gynodioecious species, we found CMS-G to be widespread across the distribution range of the species. Despite the sequencing of the mitochondrial genome of CMS-G, the mitochondrial sterilizing factor causing CMS-G is still unknown. By characterizing biochemically CMS-G, we found that the expression of several mitochondrial proteins is altered in CMS-G plants. In particular, Cox1, a core subunit of the cytochrome c oxidase (complex IV), is larger but can still assemble into complex IV. However, the CMS-G-specific complex IV was only detected as a stabilized dimer. We did not observe any alteration of the affinity of complex IV for cytochrome c; however, in CMS-G, complex IV capacity is reduced. Our results show that CMS-G is maintained in many natural populations despite being associated with an atypical complex IV. We suggest that the modified complex IV could incur the associated cost predicted by theoretical models to maintain gynodioecy in wild populations.


Assuntos
Beta vulgaris/genética , Citoplasma/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Infertilidade das Plantas/genética , Beta vulgaris/crescimento & desenvolvimento , Genoma Mitocondrial/genética , Mitocôndrias/enzimologia , Mitocôndrias/genética , Mutação , Pólen/genética
9.
Plant Physiol ; 171(3): 2256-76, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27217496

RESUMO

Nod factors (NFs) are lipochitooligosaccharidic signal molecules produced by rhizobia, which play a key role in the rhizobium-legume symbiotic interaction. In this study, we analyzed the gene expression reprogramming induced by purified NF (4 and 24 h of treatment) in the root epidermis of the model legume Medicago truncatula Tissue-specific transcriptome analysis was achieved by laser-capture microdissection coupled to high-depth RNA sequencing. The expression of 17,191 genes was detected in the epidermis, among which 1,070 were found to be regulated by NF addition, including previously characterized NF-induced marker genes. Many genes exhibited strong levels of transcriptional activation, sometimes only transiently at 4 h, indicating highly dynamic regulation. Expression reprogramming affected a variety of cellular processes, including perception, signaling, regulation of gene expression, as well as cell wall, cytoskeleton, transport, metabolism, and defense, with numerous NF-induced genes never identified before. Strikingly, early epidermal activation of cytokinin (CK) pathways was indicated, based on the induction of CK metabolic and signaling genes, including the CRE1 receptor essential to promote nodulation. These transcriptional activations were independently validated using promoter:ß-glucuronidase fusions with the MtCRE1 CK receptor gene and a CK response reporter (TWO COMPONENT SIGNALING SENSOR NEW). A CK pretreatment reduced the NF induction of the EARLY NODULIN11 (ENOD11) symbiotic marker, while a CK-degrading enzyme (CYTOKININ OXIDASE/DEHYDROGENASE3) ectopically expressed in the root epidermis led to increased NF induction of ENOD11 and nodulation. Therefore, CK may play both positive and negative roles in M. truncatula nodulation.


Assuntos
Citocininas/metabolismo , Lipopolissacarídeos/metabolismo , Medicago truncatula/metabolismo , Epiderme Vegetal/metabolismo , Raízes de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Lasers , Lipopolissacarídeos/farmacologia , Medicago truncatula/genética , Epiderme Vegetal/efeitos dos fármacos , Epiderme Vegetal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Análise de Sequência de RNA/métodos , Transdução de Sinais
10.
Methods ; 95: 70-7, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26255961

RESUMO

The characterization of macromolecular samples at synchrotrons has traditionally been restricted to direct exposure to X-rays, but beamline automation and diversification of the user community has led to the establishment of complementary characterization facilities off-line. The Sample Preparation and Characterization (SPC) facility at the EMBL@PETRA3 synchrotron provides synchrotron users access to a range of biophysical techniques for preliminary or parallel sample characterization, to optimize sample usage at the beamlines. Here we describe a sample pipeline from bench to beamline, to assist successful structural characterization using small angle X-ray scattering (SAXS) or macromolecular X-ray crystallography (MX). The SPC has developed a range of quality control protocols to assess incoming samples and to suggest optimization protocols. A high-throughput crystallization platform has been adapted to reach a broader user community, to include chemists and biologists that are not experts in structural biology. The SPC in combination with the beamline and computational facilities at EMBL Hamburg provide a full package of integrated facilities for structural biology and can serve as model for implementation of such resources for other infrastructures.


Assuntos
Cristalografia por Raios X/normas , Substâncias Macromoleculares/ultraestrutura , Síncrotrons/instrumentação , Difração de Raios X/normas , Humanos , Substâncias Macromoleculares/química , Controle de Qualidade , Espalhamento a Baixo Ângulo , Software , Manejo de Espécimes/normas
11.
J Biomol NMR ; 64(4): 281-9, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-26984476

RESUMO

Maintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor(®) provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Fluorometria/métodos , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Estabilidade Proteica , Temperatura
12.
Plant Cell Environ ; 39(10): 2198-209, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27341695

RESUMO

Legume plants adapt to low nitrogen by developing an endosymbiosis with nitrogen-fixing soil bacteria to form a new specific organ: the nitrogen-fixing nodule. In the Medicago truncatula model legume, the MtCRE1 cytokinin receptor is essential for this symbiotic interaction. As three other putative CHASE-domain containing histidine kinase (CHK) cytokinin receptors exist in M. truncatula, we determined their potential contribution to this symbiotic interaction. The four CHKs have extensive redundant expression patterns at early nodulation stages but diverge in differentiated nodules, even though MtCHK1/MtCRE1 has the strongest expression at all stages. Mutant and knock-down analyses revealed that other CHKs than MtCHK1/CRE1 are positively involved in nodule initiation, which explains the delayed nodulation phenotype of the chk1/cre1 mutant. In addition, cre1 nodules exhibit an increased growth, whereas other chk mutants have no detectable phenotype, and the maintained nitrogen fixation capacity in cre1 requires other CHK genes. Interestingly, an AHK4/CRE1 genomic locus from the aposymbiotic Arabidopsis plant rescues nodule initiation but not the nitrogen fixation capacity. This indicates that different CHK cytokinin signalling pathways regulate not only nodule initiation but also later developmental stages, and that legume-specific determinants encoded by the MtCRE1 gene are required for later nodulation stages than initiation.


Assuntos
Medicago truncatula/microbiologia , Receptores de Superfície Celular/fisiologia , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Arabidopsis/genética , Citocininas/metabolismo , Genoma de Planta , Medicago truncatula/metabolismo , Fixação de Nitrogênio , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/fisiologia , Plantas Geneticamente Modificadas/microbiologia , Receptores de Superfície Celular/metabolismo , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Transdução de Sinais , Sinorhizobium/fisiologia , Simbiose
13.
J Struct Biol ; 191(3): 290-8, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26208466

RESUMO

Levansucrases are members of the glycoside hydrolase family and catalyse both the hydrolysis of the substrate sucrose and the transfer of fructosyl units to acceptor molecules. In the presence of sufficient sucrose, this may either lead to the production of fructooligosaccharides or fructose polymers. Aim of this study is to rationalise the differences in the polymerisation properties of bacterial levansucrases and in particular to identify structural features that determine different product spectrum in the levansucrase of the Gram-negative bacterium Erwinia amylovora (Ea Lsc, EC 2.4.1.10) as compared to Gram-positive bacteria such as Bacillus subtilis levansucrase. Ea is an enterobacterial pathogen responsible for the Fire Blight disease in rosaceous plants (e.g., apple and pear) with considerable interest for the agricultural industry. The crystal structure of Ea Lsc was solved at 2.77 Å resolution and compared to those of other fructosyltransferases from Gram-positive and Gram-negative bacteria. We propose the structural features, determining the different reaction products, to reside in just a few loops at the rim of the active site funnel. Moreover we propose that loop 8 may have a role in product length determination in Gluconacetobacter diazotrophicus LsdA and Microbacterium saccharophilum FFase. The Ea Lsc structure shows for the first time the products of sucrose hydrolysis still bound in the active site.


Assuntos
Erwinia amylovora/metabolismo , Hexosiltransferases/química , Hexosiltransferases/metabolismo , Sacarose/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/metabolismo , Domínio Catalítico , Gluconacetobacter/metabolismo , Hidrolases/metabolismo , Hidrólise , Dados de Sequência Molecular , Alinhamento de Sequência
14.
J Am Chem Soc ; 137(48): 15122-34, 2015 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-26424125

RESUMO

Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Förster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin α and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin α. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.


Assuntos
Virus da Influenza A Subtipo H5N1/enzimologia , Carioferinas/metabolismo , Proteínas Virais/metabolismo , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Soluções , Proteínas Virais/química
15.
Biopolymers ; 104(5): 552-9, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26095000

RESUMO

The IgY antibodies offer an attractive alternative to mammalian IgGs in research, diagnosis and medicine. The isolation of immunoglobulin Y from the egg yolks is efficient and economical, causing minimal suffering to animals. Here we present the methodology for the production of IgY antibodies specific to Staphylococcus aureus fibrinogen binding protein (Efb) and its peptidyl epitope (spanning residues 127-140). The Efb is an extracellular, adhesion protein which binds both human fibrinogen and complement C3 protein thus contributing to the high infectious potential of this pathogen. The selected epitope of Efb protein is responsible for the interaction with C3. The immunochemical characterization of both anti-Efb and epitope-specific IgY antibodies revealed their similar avidity, titer, and reactivity profile, although some differences in the hen's immune response to administered antigens is discussed.


Assuntos
Formação de Anticorpos , Fibrinogênio/imunologia , Imunoglobulinas/biossíntese , Staphylococcus aureus/imunologia , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/imunologia , Galinhas , Epitopos/imunologia , Feminino , Humanos , Peptídeos/imunologia , Ligação Proteica
16.
J Immunol ; 189(11): 5367-81, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23089396

RESUMO

Failure to induce synthesis of neutralizing Abs to the CD4 binding determinant (CD4BD) of gp120, a central objective in HIV vaccine research, has been alternately ascribed to insufficient immunogen binding to Abs in their germline V region configuration expressed as BCRs, insufficient adaptive mutations in Ab V regions, and conformational instability of gp120. We employed peptide analogs of gp120 residues 421-433 within the CD4BD (CD4BD(core)) to identify Abs produced without prior exposure to HIV (constitutive Abs). The CD4BD(core) peptide was recognized by single-chain Fv fragments from noninfected humans with lupus that neutralized genetically diverse strains belonging to various HIV subtypes. Replacing the framework region (FR) of a V(H)4-family single-chain Fv with the corresponding V(H)3-family FRs from single-chain Fv JL427 improved the CD4BD(core) peptide-binding activity, suggesting a CD4BD(core) binding site outside the pocket formed by the CDRs. Replacement mutations in the FR site vicinity suggested the potential for adaptive improvement. A very small subset of serum CD4BD(core)-specific serum IgAs from noninfected humans without autoimmune disease isolated by epitope-specific chromatography neutralized the virus potently. A CD4BD(core)-specific, HIV neutralizing murine IgM with H and L chain V regions (V(H) and V(L) regions) free of immunogen-driven somatic mutations was induced by immunization with a CD4BD(core) peptide analog containing an electrophilic group that binds B cells covalently. The studies indicate broad and potent HIV neutralization by constitutive Abs as an innate, germline-encoded activity directed to the superantigenic CD4BD(core) epitope that is available for amplification for vaccination against HIV.


Assuntos
Vacinas contra a AIDS/biossíntese , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Superantígenos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/biossíntese , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Epitopos/imunologia , Epitopos/metabolismo , Anticorpos Anti-HIV/biossíntese , Proteína gp120 do Envelope de HIV/química , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/química , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/imunologia , Superantígenos/química
17.
Environ Microbiome ; 19(1): 65, 2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-39223675

RESUMO

In nature, microbes do not thrive in seclusion but are involved in complex interactions within- and between-microbial kingdoms. Among these, symbiotic associations with mycorrhizal fungi and nitrogen-fixing bacteria are namely known to improve plant health, while providing resources to benefit other microbial members. Yet, it is not clear how these microbial symbionts interact with each other or how they impact the microbiota network architecture. We used an extensive co-occurrence network analysis, including rhizosphere and roots samples from six plant species in a natural desert in AlUla region (Kingdom of Saudi Arabia) and described how these symbionts were structured within the plant microbiota network. We found that the plant species was a significant driver of its microbiota composition and also of the specificity of its interactions in networks at the microbial taxa level. Despite this specificity, a motif was conserved across all networks, i.e., mycorrhizal fungi highly covaried with other mycorrhizal fungi, especially in plant roots-this pattern is known as assortativity. This structural property might reflect their ecological niche preference or their ability to opportunistically colonize roots of plant species considered non symbiotic e.g., H. salicornicum, an Amaranthaceae. Furthermore, these results are consistent with previous findings regarding the architecture of the gut microbiome network, where a high level of assortativity at the level of bacterial and fungal orders was also identified, suggesting the existence of general rules of microbiome assembly. Otherwise, the bacterial symbionts Rhizobiales and Frankiales covaried with other bacterial and fungal members, and were highly structural to the intra- and inter-kingdom networks. Our extensive co-occurrence network analysis of plant microbiota and study of symbiont assortativity, provided further evidence on the importance of bacterial and fungal symbionts in structuring the global plant microbiota network.

18.
Protein Expr Purif ; 91(2): 192-206, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23948764

RESUMO

The efficient large scale production of recombinant proteins depends on the careful conditioning of the protein as it is isolated and purified to homogeneity. Low protein stability leads to low purification yields as a result of protein degradation, precipitation and folding instability. It is often necessary to go through several iterations of trial-and-error to optimize the homogeneity, stability and solubility of the protein sample. We have set up Thermofluor assays to identify customized protocols for the preparation and characterization of individual protein constructs. We apply a two-step approach: we first screen for global parameters, followed by a search for protein-specific additives. The first screen has been designed in such a way, that it is possible to discern global stability trends according to pH, salt concentration, buffer type and concentration. The second screen contains small molecules that can affect the folding, aggregation state and solubility of the protein construct and also includes small molecules that specifically bind and stabilize proteins. The screens are designed to evaluate purification and storage protocols, and aim to provide hints to optimize these protocols. The home-made screens have been tested on more than 200 different protein constructs at the Sample Preparation and Characterization (SPC) facility at EMBL Hamburg. We describe which RT-PCR machines can be adapted to perform Thermofluor assays, what are the necessary experimental conditions to set up a screen, some leads on how to interpret the data and we give several examples of Thermofluor applications beyond stability screens.


Assuntos
Análise Diferencial Térmica , Fluorometria , Proteínas Recombinantes/química , Corantes Fluorescentes , Reação em Cadeia da Polimerase , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/normas
19.
J Biol Chem ; 286(12): 10439-48, 2011 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-21216958

RESUMO

In the adaptation of avian viruses to mammalian hosts, mutations in the viral polymerase, notably in the PB2 subunit, play an important role. A PB2 C-terminal domain rich in putative host adaptation residues has been shown to bind importin α nuclear import receptors. Adaptation has been proposed to involve binding of PB2 to importins of the new host. To date PB2-importin complexes have been characterized semiquantitatively with no precise measurement of binding parameters. To investigate the effects of adaptive mutations on importin interaction and selectivity, surface plasmon resonance was used to compare the binding rate constants and affinities of avian H5N1 and human H3N2 PB2 C-terminal variants with importin isoforms human α 1, 3, 5 and 7, and avian α 1. Using purified proteins eliminates host environment effects and permits measurement of intrinsic affinities and rates of complex formation and dissociation. Two effects were observed: first, adaptive mutations D701N, R702K, and S714R in the nuclear localization signal domain increased 2-4-fold the association rates with avian and human importins; second, measurement of different structural forms of the PB2 C terminus demonstrated that the upstream 627 domain reduced binding affinity, consistent with a steric clash predicted from crystal structures. From these kinetic data, structural analyses, and the data of others, a model is proposed in which an increase in charged surface residues during host adaptation increases the association rate of PB2 to cytoplasmic importins and where the C-terminal 627-nuclear localization signal domain may reorganize upon importin binding, consistent with a role in active polymerase assembly.


Assuntos
Adaptação Fisiológica/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Vírus da Influenza A Subtipo H3N2/enzimologia , Virus da Influenza A Subtipo H5N1/enzimologia , RNA Polimerase Dependente de RNA/química , Proteínas Virais/química , alfa Carioferinas/química , Substituição de Aminoácidos , Contagem de Células , Humanos , Vírus da Influenza A Subtipo H3N2/genética , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Modelos Moleculares , Mutação de Sentido Incorreto , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , alfa Carioferinas/genética , alfa Carioferinas/metabolismo
20.
J Biol Chem ; 285(37): 28411-7, 2010 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-20538599

RESUMO

The heterotrimeric RNA-dependent RNA polymerase of influenza viruses catalyzes RNA replication and transcription activities in infected cell nuclei. The nucleotide polymerization activity is common to both replication and transcription processes, with an additional cap-snatching function being employed during transcription to steal short 5'-capped RNA primers from host mRNAs. Cap-binding, endonuclease, and polymerase activities have long been studied biochemically, but structural studies on the polymerase and its subunits have been hindered by difficulties in producing sufficient quantities of material. Recently, because of heightened effort and advances in expression and crystallization technologies, a series of high resolution structures of individual domains have been determined. These shed light on intrinsic activities of the polymerase, including cap snatching, subunit association, and nucleocytoplasmic transport, and open up the possibility of structure-guided development of new polymerase inhibitors. Furthermore, the activity of influenza polymerase is highly host- and cell type-specific, being dependent on the identity of a few key amino acid positions in the different subunits, especially in the C-terminal region of PB2. New structures demonstrate the surface exposure of these residues, consistent with ideas that they might modulate interactions with host-specific factors that enhance or restrict activity. Recent proteomic and genome-wide interactome and RNA interference screens have suggested the identities of some of these potential regulators of polymerase function.


Assuntos
Vírus da Influenza A/enzimologia , DNA Polimerase Dirigida por RNA/química , Transporte Ativo do Núcleo Celular , Animais , Domínio Catalítico , Núcleo Celular/enzimologia , Núcleo Celular/virologia , Cristalografia por Raios X , Humanos , Estrutura Quaternária de Proteína , Capuzes de RNA/biossíntese , RNA Viral/biossíntese , DNA Polimerase Dirigida por RNA/metabolismo , Relação Estrutura-Atividade , Transcrição Gênica , Replicação Viral/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA