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1.
Sci Rep ; 11(1): 16430, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385527

RESUMO

Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.


Assuntos
Teste para COVID-19/métodos , COVID-19/virologia , Colorimetria/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/isolamento & purificação , COVID-19/diagnóstico , Humanos , Pandemias , Portugal/epidemiologia , RNA Viral/genética , SARS-CoV-2/genética , Saliva/química , Saliva/virologia , Sensibilidade e Especificidade
2.
Molecules ; 26(2)2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33445716

RESUMO

A new synthetic route for the quorum sensing signal Autoinducer-2 (AI-2) is described and used for the preparation of [4-13C]-AI-2 starting from [1-13C]-bromoacetic acid. The key step in this process was the enantioselective reduction of an intermediate ketone. This synthesis provides, selectively, both enantiomers of the labelled or unlabelled parent compound, (R) or (S)-4,5-dihydroxypentane-2,3-dione (DPD) and was used for an improved synthesis of [1-13C]-AI-2.


Assuntos
Homosserina/análogos & derivados , Lactonas/síntese química , Lactonas/farmacologia , Fenômenos Ópticos , Percepção de Quorum , Ciclização , Homosserina/síntese química , Homosserina/farmacologia , Percepção de Quorum/efeitos dos fármacos
3.
Plants (Basel) ; 9(8)2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32824217

RESUMO

Carotenoids are a class of pigments with a biological role in light capture and antioxidant activities. High value ketocarotenoids, such as astaxanthin and canthaxanthin, are highly appealing for applications in human nutraceutical, cosmetic, and animal feed industries due to their color- and health-related properties. In this review, recent advances in metabolic engineering and synthetic biology towards the production of ketocarotenoids, in particular the red-orange canthaxanthin, are highlighted. Also reviewed and discussed are the properties of canthaxanthin, its natural producers, and various strategies for its chemical synthesis. We review the de novo synthesis of canthaxanthin and the functional ß-carotene ketolase enzyme across organisms, supported by a protein-sequence-based phylogenetic analysis. Various possible modifications of the carotenoid biosynthesis pathway and the present sustainable cost-effective alternative platforms for ketocarotenoids biosynthesis are also discussed.

4.
Bioorg Chem ; 94: 103452, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31810755

RESUMO

Histone deacetylases are involved in chromatin remodelling and thus play a vital role in the epigenetic regulation of gene expression. HDAC inhibitors alter the acetylation status of histone and non-histone proteins to regulate various cellular events such as transcription. Novel HDAC inhibitors were designed and synthesised to promote higher levels of recombinant protein production in tobacco cell cultures. The effect of these chemical enhancers on the epigenetic profiles in plant cells has been evaluated by molecular docking, in vitro and in vivo studies. The addition of these novel enhancers led to an increase in histone H3 acetylation levels that promoted an increase in the accumulation levels of the recombinant protein in cell culture. These results can pave the way for the application of these enhancers to improve the production of high value products in plant cell based systems.


Assuntos
Butiratos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Tabaco/efeitos dos fármacos , Butiratos/síntese química , Butiratos/química , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Proteínas Recombinantes/biossíntese , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Tabaco/metabolismo
5.
Bioorg Chem ; 92: 103200, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31470199

RESUMO

In processes regulated by quorum sensing (QS) bacteria respond to the concentration of autoinducers in the environment to engage in group behaviours. Autoinducer-2 (AI-2) is unique as it can foster interspecies communication. Currently, two AI-2 receptors are known, LuxP and LsrB, but bacteria lacking these receptors can also respond to AI-2. In this work, we present an efficient and reproducible synthesis of a novel chemical probe, d-desthiobiotin-AI-2. This probe binds both LuxP and LsrB receptors from different species of bacteria. Thus, this probe is able to bind receptors that recognise the two known biologically active forms of AI-2, presenting the plasticity essential for the identification of novel unknown AI-2 receptors. Moreover, a protocol to pull down receptors bound to d-desthiobiotin-AI-2 with anti-biotin antibodies has also been established. Altogether, this work highlights the potential of conjugating chemical signals to biotinylated derivatives to identify and tag signal receptors involved in quorum sensing or other chemical signalling processes.


Assuntos
Biotina/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Homosserina/análogos & derivados , Lactonas/síntese química , Percepção de Quorum/efeitos dos fármacos , Alcinos/química , Biotina/síntese química , Biotina/química , Biotina/metabolismo , Proteínas de Transporte/metabolismo , Escherichia coli/genética , Homosserina/síntese química , Homosserina/metabolismo , Lactonas/metabolismo , Ligantes , Estrutura Molecular , Transdução de Sinais
6.
IUCrJ ; 6(Pt 4): 572-585, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31316802

RESUMO

Bacteria are challenged to adapt to environmental variations in order to survive. Under nutritional stress, several bacteria are able to slow down their metabolism into a nonreplicating state and wait for favourable conditions. It is almost universal that bacteria accumulate carbon stores to survive during this nonreplicating state and to fuel rapid proliferation when the growth-limiting stress disappears. Mycobacteria are exceedingly successful in their ability to become dormant under harsh circumstances and to be able to resume growth when conditions are favourable. Rapidly growing mycobacteria accumulate glucosylglycerate under nitrogen-limiting conditions and quickly mobilize it when nitrogen availability is restored. The depletion of intracellular glucosyl-glycerate levels in Mycolicibacterium hassiacum (basonym Mycobacterium hassiacum) was associated with the up-regulation of the gene coding for glucosylglycerate hydrolase (GgH), an enzyme that is able to hydrolyse glucosylglycerate to glycerate and glucose, a source of readily available energy. Highly conserved among unrelated phyla, GgH is likely to be involved in bacterial reactivation following nitrogen starvation, which in addition to other factors driving mycobacterial recovery may also provide an opportunity for therapeutic intervention, especially in the serious infections caused by some emerging opportunistic pathogens of this group, such as Mycobacteroides abscessus (basonym Mycobacterium abscessus). Using a combination of biochemical methods and hybrid structural approaches, the oligomeric organization of M. hassiacum GgH was determined and molecular determinants of its substrate binding and specificity were unveiled.

7.
Int J Pharm ; 565: 162-173, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31054877

RESUMO

Determining the stability of downstream process (DSP) intermediates is an extremely important parameter used to maintain product quality attributes within their acceptance ranges. The IgG4 monoclonal antibody studied (mAb1) showed aggregation under acidic conditions, inhibiting the use of low pH treatment to inactivate endogenous retroviruses, and poor virus filtration performance. Both manufacturing steps are included in mAb DSP for viral clearance. The impact of several new compounds on the aggregation and stabilization of mAb1 in process intermediate pools encountered during these critical DSP steps was investigated. Results showed that, in the presence of a protein stabilizer at pH 3.2, 27% less aggregation was observed compared to controls, during the low pH treatment for viral inactivation. The impact of a novel protein stabilizer on virus filter throughput during mAb1 filtration was compared to L-arginine using an innovative high-throughput automation technique. Compared to control experiments without additives, conditions were found where a 70% increase in filter volumetric throughput was achieved in the presence of the novel stabilizer, and a 56% decrease in volumetric throughput observed with L-arginine. These findings present the possibility of using these novel compounds to stabilize proteins during DSP and permitting the use of platform DSP elements such as low pH treatment and high-throughput virus filtration to challenging and unstable proteins.


Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Química Farmacêutica , Estabilidade de Medicamentos , Filtração , Concentração de Íons de Hidrogênio , Vírus
8.
Proc Natl Acad Sci U S A ; 116(3): 835-844, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30606802

RESUMO

Mycobacteria are a wide group of organisms that includes strict pathogens, such as Mycobacterium tuberculosis, as well as environmental species known as nontuberculous mycobacteria (NTM), some of which-namely Mycobacterium avium-are important opportunistic pathogens. In addition to a distinctive cell envelope mediating critical interactions with the host immune system and largely responsible for their formidable resistance to antimicrobials, mycobacteria synthesize rare intracellular polymethylated polysaccharides implicated in the modulation of fatty acid metabolism, thus critical players in cell envelope assembly. These are the 6-O-methylglucose lipopolysaccharides (MGLP) ubiquitously detected across the Mycobacterium genus, and the 3-O-methylmannose polysaccharides (MMP) identified only in NTM. The polymethylated nature of these polysaccharides renders the intervening methyltransferases essential for their optimal function. Although the knowledge of MGLP biogenesis is greater than that of MMP biosynthesis, the methyltransferases of both pathways remain uncharacterized. Here, we report the identification and characterization of a unique S-adenosyl-l-methionine-dependent sugar 1-O-methyltransferase (MeT1) from Mycobacterium hassiacum that specifically blocks the 1-OH position of 3,3'-di-O-methyl-4α-mannobiose, a probable early precursor of MMP, which we chemically synthesized. The high-resolution 3D structure of MeT1 in complex with its exhausted cofactor, S-adenosyl-l-homocysteine, together with mutagenesis studies and molecular docking simulations, unveiled the enzyme's reaction mechanism. The functional and structural properties of this unique sugar methyltransferase further our knowledge of MMP biosynthesis and provide important tools to dissect the role of MMP in NTM physiology and resilience.


Assuntos
Metilmanosídeos/metabolismo , Metiltransferases/metabolismo , Mycobacterium/metabolismo , Polissacarídeos Bacterianos/biossíntese , Domínio Catalítico , Metiltransferases/genética , Família Multigênica , Mycobacterium/genética
9.
Bioorg Chem ; 85: 75-81, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30602129

RESUMO

Quorum sensing (QS) regulates population-dependent bacterial behaviours, such as toxin production, biofilm formation and virulence. Autoinducer-2 (AI-2) is to date the only signalling molecule known to foster inter-species bacterial communication across distantly related bacterial species. In this work, the synthesis of pure enantiomers of C4-propoxy-HPD and C4-ethoxy-HPD, known AI-2 analogues, has been developed. The optimised synthesis is efficient, reproducible and short. The (4S) enantiomer of C4-propoxy-HPD was the most active compound being approximately twice as efficient as (4S)-DPD and ten-times more potent than the (4R) enantiomer. Additionally, the specificity of this analogue to bacteria with LuxP receptors makes it a good candidate for clinical applications, because it is not susceptible to scavenging by LsrB-containing bacteria that degrade the natural AI-2. All in all, this study provides a new brief and effective synthesis of isomerically pure analogues for QS modulation that include the most active AI-2 agonist described so far.


Assuntos
Antibacterianos/farmacologia , Pentanonas/farmacologia , Percepção de Quorum/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Escherichia coli/fisiologia , Proteínas de Escherichia coli/metabolismo , Pentanonas/síntese química , Pentanonas/metabolismo , Estereoisomerismo , Vibrio/fisiologia
10.
Org Biomol Chem ; 16(38): 6860-6864, 2018 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-30226251

RESUMO

The plant hormone conjugate 2-O-(indole-3-acetyl)-myo-inositol (IAInos) has been selectively prepared for the first time by two routes from myo-inositol. One of the syntheses depended upon the construction of the 3-indoleacetyl group by a Fischer indole synthesis on an unreactive axial hydroxyl group, while the other via a direct acylation of the equatorially orientated hydroxy group created by conformational constraint of the cyclohexane ring. The latter synthesis produced IAInos in 5 steps and 29% overall yield.


Assuntos
Ácidos Indolacéticos/síntese química , Indóis/síntese química , Inositol/síntese química , Reguladores de Crescimento de Plantas/síntese química , Acilação , Técnicas de Química Sintética , Ácidos Indolacéticos/química , Indóis/química , Inositol/análogos & derivados , Reguladores de Crescimento de Plantas/química
11.
J Agric Food Chem ; 65(31): 6460-6466, 2017 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-28198187

RESUMO

(Poly)phenols are a large group of dietary compounds present in fruits and vegetables; their consumption is associated with health beneficial effects. After ingestion, (poly)phenols suffer extensive metabolization, and the identification of their metabolites is an emerging area, because these metabolites are considered the effective bioactive molecules in the human organism. However, a lack of commercially available standards has hampered the study of metabolite bioactivity and the exact structural confirmation in biological samples. New (poly)phenol metabolites previously identified in human samples after the intake of berry juice were chemically synthesized. Efficient chemical reactions were performed with moderate to excellent yields and selectivities. These new compounds could be used as standard chemicals for confirmation of the structure of metabolites in biological samples and will also allow mechanistic studies in cellular models.


Assuntos
Glucuronatos/metabolismo , Polifenóis/metabolismo , Sulfatos/metabolismo , Ingestão de Alimentos , Glucuronatos/química , Humanos , Estrutura Molecular , Polifenóis/síntese química , Polifenóis/química , Sulfatos/química
12.
Mol Nutr Food Res ; 60(10): 2130-2140, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27242317

RESUMO

SCOPE: Cranberries are rich in potentially bioactive (poly)phenols. The aim of this paper was to investigate whether cranberry juice intake can improve vascular function in healthy men in a dose- and time-dependent manner, and to understand which of the circulating (poly)phenol metabolites correlate with vascular effects. METHODS AND RESULTS: A double-blind randomized controlled crossover trial was conducted in ten healthy males. Flow-mediated dilation (FMD), blood pressure, pulse wave velocity and augmentation index were investigated at baseline, 1, 2, 4, 6, and 8 h post-consumption of cranberry juices containing 409, 787, 1238, 1534, and 1910 mg of total cranberry (poly)phenols (TP), and a control drink. Plasma (poly)phenol metabolites were analyzed by UPLC-Q-TOF MS using authentic standards. We observed dose-dependent increases in FMD at 1, 2, 4, 6, and 8 h with a peak at 4 h and maximal effects with juice containing 1238 mg TP. A total of 60 metabolites were quantified in plasma after cranberry consumption. Twelve (poly)phenol metabolites significantly correlated with the increases in FMD, including ferulic and caffeic acid sulfates, quercetin-3-O-ß-D-glucuronide and a γ-valerolactone sulfate. CONCLUSION: (Poly)phenols in cranberry juice can improve vascular function in healthy males and this is linked to the presence of specific newly identified plasma metabolites.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Sucos de Frutas e Vegetais , Polifenóis/farmacologia , Vaccinium macrocarpon/química , Adulto , Pressão Sanguínea/efeitos dos fármacos , Estudos Cross-Over , Relação Dose-Resposta a Droga , Método Duplo-Cego , Endotélio Vascular/fisiologia , Sucos de Frutas e Vegetais/análise , Humanos , Masculino , Polifenóis/administração & dosagem , Polifenóis/metabolismo , Análise de Onda de Pulso , Vasodilatação/efeitos dos fármacos
13.
Arch Biochem Biophys ; 599: 31-41, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-26836705

RESUMO

Cranberries are a rich source of (poly)phenols, in particular proanthocyanidins, anthocyanins, flavonols, and phenolic acids. However, little is known about their bioavailability in humans. We investigated the absorption, metabolism, and excretion of cranberry (poly)phenols in plasma and urine of healthy young men after consumption of a cranberry juice (787 mg (poly)phenols). A total of 60 cranberry-derived phenolic metabolites were identified using UPLC-Q-TOF-MS analysis with authentic standards. These included sulfates of pyrogallol, valerolactone, benzoic acids, phenylacetic acids, glucuronides of flavonols, as well as sulfates and glucuronides of cinnamic acids. The most abundant plasma metabolites were small phenolic compounds, in particular hippuric acid, catechol-O-sulfate, 2,3-dihydroxybenzoic acid, phenylacetic acid, isoferulic acid, 4-methylcatechol-O-sulfate, α-hydroxyhippuric acid, ferulic acid 4-O-sulfate, benzoic acid, 4-hydroxyphenyl acetic acid, dihydrocaffeic acid 3-O-sulfate, and vanillic acid-4-O-sulfate. Some benzoic acids, cinnamic acids, and flavonol metabolites appeared in plasma early, at 1-2 h post-consumption. Others such as phenylacetic acids, benzaldehydes, pyrogallols, catechols, hippuric and dihydrocinnamic acid derivatives appear in plasma later (Tmax 4-22 h). The 24 h urinary recovery with respect to the amount of (poly)phenols consumed was 6.2%. Our extensive description of the bioavailability of cranberry (poly)phenols lays important groundwork necessary to start understanding the fate of these compounds in humans.


Assuntos
Sucos de Frutas e Vegetais , Polifenóis/sangue , Polifenóis/urina , Vaccinium macrocarpon/química , Adolescente , Adulto , Humanos , Masculino
14.
Sci Rep ; 5: 13610, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26324178

RESUMO

Mycobacteria synthesize unique intracellular methylglucose lipopolysaccharides (MGLP) proposed to modulate fatty acid metabolism. In addition to the partial esterification of glucose or methylglucose units with short-chain fatty acids, octanoate was invariably detected on the MGLP reducing end. We have identified a novel sugar octanoyltransferase (OctT) that efficiently transfers octanoate to glucosylglycerate (GG) and diglucosylglycerate (DGG), the earliest intermediates in MGLP biosynthesis. Enzymatic studies, synthetic chemistry, NMR spectroscopy and mass spectrometry approaches suggest that, in contrast to the prevailing consensus, octanoate is not esterified to the primary hydroxyl group of glycerate but instead to the C6 OH of the second glucose in DGG. These observations raise important new questions about the MGLP reducing end architecture and about subsequent biosynthetic steps. Functional characterization of this unique octanoyltransferase, whose gene has been proposed to be essential for M. tuberculosis growth, adds new insights into a vital mycobacterial pathway, which may inspire new drug discovery strategies.


Assuntos
Lipopolissacarídeos/biossíntese , Mycobacterium/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácidos Graxos/metabolismo , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Mycobacterium/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato
15.
J Med Chem ; 58(14): 5408-18, 2015 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-26061085

RESUMO

Alzheimer's disease is a grave social problem in an aging population. A major problem is the passage of drugs through the blood-brain barrier. This work tests the hypothesis that the conjugation of peptidomimetic ß-secretase inhibitors with a fragment of amyloid-ß peptide facilitates entrance into the central nervous system. HVR-3 (compound 4), one of the conjugation products, was found to be as potent as OM00-3, a known peptidomimetic inhibitor, 4-fold more selective toward ß-secretase 1 in relation to ß-secretase 2 and 3-fold more resistant to in vitro metabolization in human serum. Its intravenous administration to mice and Wistar rats generated an active metabolite recovered from the rodent's brains.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Acilação , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Feminino , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Peptidomiméticos/farmacocinética , Peptidomiméticos/uso terapêutico , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/uso terapêutico , Ratos , Relação Estrutura-Atividade
16.
PLoS One ; 10(3): e0121042, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25826206

RESUMO

The human pathogen Streptococcus pneumoniae is a strictly fermentative organism that relies on glycolytic metabolism to obtain energy. In the human nasopharynx S. pneumoniae encounters glycoconjugates composed of a variety of monosaccharides, which can potentially be used as nutrients once depolymerized by glycosidases. Therefore, it is reasonable to hypothesise that the pneumococcus would rely on these glycan-derived sugars to grow. Here, we identified the sugar-specific catabolic pathways used by S. pneumoniae during growth on mucin. Transcriptome analysis of cells grown on mucin showed specific upregulation of genes likely to be involved in deglycosylation, transport and catabolism of galactose, mannose and N acetylglucosamine. In contrast to growth on mannose and N-acetylglucosamine, S. pneumoniae grown on galactose re-route their metabolic pathway from homolactic fermentation to a truly mixed acid fermentation regime. By measuring intracellular metabolites, enzymatic activities and mutant analysis, we provide an accurate map of the biochemical pathways for galactose, mannose and N-acetylglucosamine catabolism in S. pneumoniae. Intranasal mouse infection models of pneumococcal colonisation and disease showed that only mutants in galactose catabolic genes were attenuated. Our data pinpoint galactose as a key nutrient for growth in the respiratory tract and highlights the importance of central carbon metabolism for pneumococcal pathogenesis.


Assuntos
Galactose/metabolismo , Polissacarídeos/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus pneumoniae/metabolismo , Regulação Bacteriana da Expressão Gênica , Mucinas/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crescimento & desenvolvimento , Virulência
17.
Extremophiles ; 19(2): 373-82, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25555708

RESUMO

The solute pool of the actinobacterium Rubrobacter xylanophilus has been investigated as a function of the growth temperature and concentration of NaCl in the medium (Empadinhas et al. Extremophiles 11: 667-673, 2007). Changing the carbon source from glucose to maltose in a minimal growth medium led to the accumulation of an unknown organic compound whose structure was investigated by NMR and confirmed by chemical synthesis in the present study as: (2R)-2-(1-O-α-D-mannopyranosyl)-3-(1-O-α-D-glucopyranosyl)-D-glycerate (MGlyG). In addition to this newly identified diglycoside, the solute pool of R. xylanophilus included trehalose, mannosylglycerate, di-myo-inositol phosphate and di-N-acetyl-glucosamine phosphate. The structure of MGlyG was established by NMR and confirmed by chemical synthesis. The availability of g-amounts of the synthetic material allowed us to perform stabilization tests on three model enzymes (malate dehydrogenase, staphylococcal nuclease, and lysozyme), and compare the efficacy of MGlyG with other natural glyceryl glycosides, such as α-D-mannosyl-D-glycerate, α-D-glucosyl-D-glycerate and α-D-glucosyl-(1 → 6)-α-D-glucosyl-(1 → 2)-D-glycerate.


Assuntos
Actinobacteria/metabolismo , Ácidos Glicéricos/química , Glicolipídeos/química , Glicosídeos/química , Actinobacteria/química , Sequência de Carboidratos , Ácidos Glicéricos/metabolismo , Glicolipídeos/síntese química , Glicolipídeos/metabolismo , Glicosídeos/metabolismo , Dados de Sequência Molecular
18.
Br J Nutr ; 113(3): 454-63, 2015 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-25571923

RESUMO

Bioavailability studies are vital to assess the potential impact of bioactive compounds on human health. Although conjugated phenolic metabolites derived from colonic metabolism have been identified in the urine, the quantification and appearance of these compounds in plasma is less well studied. In this regard, it is important to further assess their potential biological activity in vivo. To address this gap, a cross-over intervention study with a mixed fruit purée (blueberry, blackberry, raspberry, strawberry tree fruit and Portuguese crowberry) and a standard polyphenol-free meal was conducted in thirteen volunteers (ten females and three males), who received each test meal once, and plasma metabolites were identified by HPLC-MS/MS. Sulfated compounds were chemically synthesised and used as standards to facilitate quantification. Gallic and caffeic acid conjugates were absorbed rapidly, reaching a maximum concentration between 1 and 2 h. The concentrations of sulfated metabolites resulting from the colonic degradation of more complex polyphenols increased in plasma from 4 h, and pyrogallol sulfate and catechol sulfate reached concentrations ranging from 5 to 20 µm at 6 h. In conclusion, phenolic sulfates reached high concentrations in plasma, as opposed to their undetected parent compounds. These compounds have potential use as biomarkers of polyphenol intake, and their biological activities need to be considered.


Assuntos
Dieta , Frutas , Fenóis/sangue , Sulfatos/sangue , Adulto , Mirtilos Azuis (Planta)/química , Catecóis/sangue , Cromatografia Líquida de Alta Pressão , Estudos Cross-Over , Feminino , Fragaria/química , Frutas/química , Humanos , Masculino , Pessoa de Meia-Idade , Polifenóis/sangue , Pirogalol/sangue , Rubus/química , Espectrometria de Massas em Tandem
19.
Sci Rep ; 4: 6766, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25341489

RESUMO

Some microorganisms accumulate glucosylglycerate (GG) during growth under nitrogen deprivation. However, the molecular mechanisms underlying the role of GG and the regulation of its levels in the nitrogen stress response are elusive. Since GG is required for biosynthesis of mycobacterial methylglucose lipopolysaccharides (MGLP) we examined the molecular mechanisms linking replenishment of assimilable nitrogen to nitrogen-starved M. hassiacum with depletion of GG accumulated during nitrogen deficiency. To probe the involvement of a newly identified glycoside hydrolase in GG depletion, we produced the mycobacterial enzyme recombinantly and confirmed the specific hydrolysis of GG (GG hydrolase, GgH) in vitro. We have also observed a pronounced up-regulation of GgH mRNA in response to the nitrogen shock, which positively correlates with GG depletion in vivo and growth stimulation, implicating GgH in the recovery process. Since GgH orthologs seem to be absent from most slowly-growing mycobacteria including M. tuberculosis, the disclosure of the GgH function allows reconfiguration of the MGLP pathway in rapidly-growing species and accommodation of this possible regulatory step. This new link between GG metabolism, MGLP biosynthesis and recovery from nitrogen stress furthers our knowledge on the mycobacterial strategies to endure a frequent stress faced in some environments and during long-term infection.


Assuntos
Ácidos Glicéricos/metabolismo , Hidrolases/metabolismo , Mycobacterium/metabolismo , Nitrogênio/metabolismo , Meios de Cultura , Ordem dos Genes , Genes Bacterianos , Genoma Bacteriano , Hidrolases/química , Hidrolases/genética , Hidrólise , Espaço Intracelular/metabolismo , Mycobacterium/genética , Mycobacterium/crescimento & desenvolvimento , Filogenia , Biossíntese de Proteínas , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Especificidade por Substrato , Termodinâmica , Transcrição Genética
20.
Proc Natl Acad Sci U S A ; 111(39): 14235-40, 2014 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-25225400

RESUMO

The quorum sensing signal autoinducer-2 (AI-2) regulates important bacterial behaviors, including biofilm formation and the production of virulence factors. Some bacteria, such as Escherichia coli, can quench the AI-2 signal produced by a variety of species present in the environment, and thus can influence AI-2-dependent bacterial behaviors. This process involves uptake of AI-2 via the Lsr transporter, followed by phosphorylation and consequent intracellular sequestration. Here we determine the metabolic fate of intracellular AI-2 by characterizing LsrF, the terminal protein in the Lsr AI-2 processing pathway. We identify the substrates of LsrF as 3-hydroxy-2,4-pentadione-5-phosphate (P-HPD, an isomer of AI-2-phosphate) and coenzyme A, determine the crystal structure of an LsrF catalytic mutant bound to P-HPD, and identify the reaction products. We show that LsrF catalyzes the transfer of an acetyl group from P-HPD to coenzyme A yielding dihydroxyacetone phosphate and acetyl-CoA, two key central metabolites. We further propose that LsrF, despite strong structural homology to aldolases, acts as a thiolase, an activity previously undescribed for this family of enzymes. With this work, we have fully characterized the biological pathway for AI-2 processing in E. coli, a pathway that can be used to quench AI-2 and control quorum-sensing-regulated bacterial behaviors.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Homosserina/análogos & derivados , Lactonas/metabolismo , Acetiltransferases/química , Acetiltransferases/genética , Acetiltransferases/metabolismo , Substituição de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Coenzima A/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Homosserina/metabolismo , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Processamento de Proteína Pós-Traducional , Percepção de Quorum
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