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1.
J Chem Theory Comput ; 15(8): 4535-4546, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31264415

RESUMO

Anabaena Sensory Rhodopsin (ASR), a microbial photoactive protein featuring the retinal chromophore in two different conformations, exhibits a pH-dependent electronic absorption spectrum. Using the recently developed CpHMD-then-QM/MM multiscale protocol applied to ASR embedded in a membrane model, the pH-induced changes in its maximum absorption wavelength have been reproduced and analyzed. While the acidic tiny red-shift is essentially correlated with the deprotonation of an aspartic acid located on the ASR extracellular side, the larger blue-shift experimentally reported at pH values larger than 5 involves a cluster of titrating residues sitting on the cytoplasmic side. The ASR pH-dependent spectrum is the consequence of the competitive stabilization of retinal ground and excited states by the protein electrostatic potential.


Assuntos
Aminoácidos/química , Anabaena/química , Proteínas de Bactérias/química , Nostoc/química , Rodopsinas Sensoriais/química , Aminoácidos/análise , Ácido Aspártico/análise , Ácido Aspártico/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Prótons , Espectrofotometria , Eletricidade Estática
2.
Int J Mol Sci ; 20(15)2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31357417

RESUMO

Unlike canonical phytochromes, the GAF domain of cyanobacteriochromes (CBCRs) can bind bilins autonomously and is sufficient for functional photocycles. Despite the astonishing spectral diversity of CBCRs, the GAF1 domain of the three-GAF-domain photoreceptor all2699 from the cyanobacterium Nostoc 7120 is the only CBCR-GAF known that converts from a red-absorbing (Pr) dark state to a far-red-absorbing (Pfr) photoproduct, analogous to the more conservative phytochromes. Here we report a solid-state NMR spectroscopic study of all2699g1 in its Pr state. Conclusive NMR evidence unveils a particular stereochemical heterogeneity at the tetrahedral C31 atom, whereas the crystal structure shows exclusively the R-stereochemistry at this chiral center. Additional NMR experiments were performed on a construct comprising the GAF1 and GAF2 domains of all2699, showing a greater precision in the chromophore-protein interactions in the GAF1-2 construct. A 3D Pr structural model of the all2699g1-2 construct predicts a tongue-like region extending from the GAF2 domain (akin to canonical phytochromes) in the direction of the chromophore, shielding it from the solvent. In addition, this stabilizing element allows exclusively the R-stereochemistry for the chromophore-protein linkage. Site-directed mutagenesis performed on three conserved motifs in the hairpin-like tip confirms the interaction of the tongue region with the GAF1-bound chromophore.


Assuntos
Espectroscopia de Ressonância Magnética , Nostoc/química , Fitocromo/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomarcadores , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Molecular , Nostoc/genética , Fitocromo/metabolismo , Relação Estrutura-Atividade
3.
Mar Drugs ; 17(5)2019 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-31109094

RESUMO

The mass spectrometry-based metabolomics approach has become a powerful tool for the quantitative analysis of small-molecule metabolites in biological samples. Nostoc flagelliforme, an edible cyanobacterium with herbal value, serves as an unexploited bioresource for small molecules. In natural environments, N. flagelliforme undergoes repeated cycles of rehydration and dehydration, which are interrupted by either long- or short-term dormancy. In this study, we performed an untargeted metabolite profiling of N. flagelliforme samples at three physiological states: Dormant (S1), physiologically fully recovered after rehydration (S2), and physiologically partially inhibited following dehydration (S3). Significant metabolome differences were identified based on the OPLS-DA (orthogonal projections to latent structures discriminant analysis) model. In total, 183 differential metabolites (95 up-regulated; 88 down-regulated) were found during the rehydration process (S2 vs. S1), and 130 (seven up-regulated; 123 down-regulated) during the dehydration process (S3 vs. S2). Thus, it seemed that the metabolites' biosynthesis mainly took place in the rehydration process while the degradation or possible conversion occurred in the dehydration process. In addition, lipid profile differences were particularly prominent, implying profound membrane phase changes during the rehydration-dehydration cycle. In general, this study expands our understanding of the metabolite dynamics in N. flagelliforme and provides biotechnological clues for achieving the efficient production of those metabolites with medical potential.


Assuntos
Dessecação , Regulação Bacteriana da Expressão Gênica/fisiologia , Metaboloma/fisiologia , Nostoc/química , Membrana Celular/genética , Membrana Celular/metabolismo , Meio Ambiente , Nostoc/metabolismo , Água
4.
J Sci Food Agric ; 99(2): 639-646, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-29951991

RESUMO

BACKGROUND: Gel-like constructs can be produced using an extrusion-based 3D food printing (3D-FP) technique. Nostoc sphaeroides biomass is a natural gel material. Considering its good nutrition and rheological properties, these algae were chosen in this study as supply material (ink) for 3D-FP. With this gel material, the extrusion-based 3D printing system was set as a model, and the printing behavior was investigated. Furthermore, the 3D-FP mechanisms were explained through low-field nuclear magnetic resonance and rheological measurements. RESULTS: Results indicated that although fresh biomass gel was printable, non-uniformity and instability occurred during printing. Blanched inks showed non-smooth printing behavior, which was associated with a decrease in elasticity and viscosity. Printability was improved by increasing the rehydration time to 24 h when rehydrated powder was used. Increasing the rehydration time increased the water-binding degree. Pre-gelatinized potato starch was added to the mixture at ratios ranging from 1 to 100 g kg-1 . The best printing outcome was observed at 40 g kg-1 potato starch. CONCLUSION: We emphasize that elasticity and viscosity balance is an essential parameter to achieve printability. The strategies adopted in this work provide new insights into the development of personalized food regarding texture and nutritional additive content. © 2018 Society of Chemical Industry.


Assuntos
Nostoc/química , Polissacarídeos/química , Impressão Tridimensional/instrumentação , Biomassa , Elasticidade , Tinta , Nostoc/crescimento & desenvolvimento , Reologia , Viscosidade
5.
Proteins ; 86(10): 1047-1054, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30035823

RESUMO

Lectins are proteins of nonimmune origin, which are capable of recognizing and binding to glycoconjugate moieties. Some of them can block the interaction of viral glycoproteins to the host cell receptors acting as antiviral agents. Although cyanobacterial lectins have presented broad biotechnological potential, little research has been directed to Amazonian Cyanobacterial diversity. In order to identify new antiviral lectins, we performed genomic analysis in seven cyanobacterial strains from Coleção Amazônica de Cianobactérias e Microalgas (CACIAM). We found 75 unique CDS presenting one or more lectin domains. Since almost all were annotated as hypothetical proteins, we used homology modeling and molecular dynamics simulations to evaluate the structural and functional properties of three CDS that were more similar to known antiviral lectins. Nostoc sp. CACIAM 19 as well as Tolypothrix sp. CACIAM 22 strains presented cyanovirin-N homologues whose function was confirmed by binding free energy calculations. Asn, Glu, Thr, Lys, Leu, and Gly, which were described as binding residues for cyanovirin, were also observed on those structures. As for other known cyanovirins, those residues in both our models also made favorable interactions with dimannose. Finally, Alkalinema sp. CACIAM 70d presented one CDS, which was identified as a seven-bladed beta-propeller structure with binding sites predicted for sialic acid and N-acetylglucosamine. Despite its singular structure, our analysis suggested this molecule as a new putative antiviral lectin. Overall, the identification and the characterization of new lectins and their homologues are a promising area in antiviral research, and Amazonian cyanobacteria present biotechnological potential to be explored in this regard.


Assuntos
Antivirais/química , Proteínas de Bactérias/química , Proteínas de Transporte/química , Cianobactérias/química , Lectinas/química , Genômica , Modelos Moleculares , Simulação de Dinâmica Molecular , Nostoc/química , Termodinâmica
6.
Chembiochem ; 19(18): 1927-1933, 2018 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-29947468

RESUMO

Phospholipid nanodiscs are a native-like membrane mimetic that is suitable for structural studies of membrane proteins. Although nanodiscs of different sizes exist for various structural applications, their thermal and long-term stability can vary considerably. Covalently circularized nanodiscs are a perfect tool to overcome these limitations. Existing methods for the production of circularized nanodiscs can be time-consuming and technically demanding. Therefore, an easy in vivo approach, in which circularized membrane scaffold proteins (MSPs) can be directly obtained from Escherichia coli culture, is reported herein. Nostoc punctiforme DnaE split-intein fusions with MSPs of various lengths are used and consistently provide circularized nanodiscs in high yields. With this approach, a large variety of circularized nanodiscs, ranging from 7 to 26 nm in diameter, that are suitable for NMR spectroscopy and electron microscopy (EM) applications can be prepared. These nanodiscs are superior to those of the corresponding linear versions in terms of stability and size homogeneity, which affects the quality of NMR spectroscopy data and EM experiments. Due to their long-term stability and homogeneity, the presented small circular nanodiscs are suited for high-resolution NMR spectroscopy studies, as demonstrated with two membrane proteins of 17 or 32 kDa in size. The presented method will provide easy access to circularized nanodiscs for structural studies of membrane proteins and for applications in which a defined and stable nanodisc size is required.


Assuntos
Proteínas de Bactérias/química , DNA Polimerase III/química , Escherichia coli/química , Inteínas , Proteínas de Membrana/química , Nanoestruturas/química , Nostoc/química , Proteínas de Bactérias/genética , DNA Polimerase III/genética , Escherichia coli/genética , Bicamadas Lipídicas/química , Proteínas de Membrana/genética , Modelos Moleculares , Nostoc/genética , Fosfolipídeos/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
7.
J Gen Appl Microbiol ; 64(5): 203-211, 2018 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-29709901

RESUMO

The aquatic cyanobacterium Nostoc verrucosum forms macroscopic colonies in streams, and its appearance is superficially similar to that of the terrestrial cyanobacterium Nostoc commune. N. verrucosum is sensitive to desiccation, unlike N. commune, although these Nostoc cyanobacterial species share physiological features, including massive extracellular polysaccharide production and trehalose accumulation capability. In this study, water-soluble sunscreen pigments of mycosporine-like amino acids (MAAs) were characterized in N. verrucosum, and the mysABCD genes responsible for MAA biosynthesis in N. verrucosum and N. commune were compared. N. verrucosum produced porphyra-334 and shinorine, with porphyra-334 accounting for >90% of the total MAAs. Interestingly, porphyra-334 is an atypical cyanobacteial MAA, whereas shinorine is known as a common and dominant MAA in cyanobacteria. Porphyra-334 from N. verrucosum showed little or no radical scavenging activity in vitro, although the glycosylated derivatives of porphyra-334 from N. commune are potent radical scavengers. The presence of the mysABCD gene cluster in N. commune strain KU002 (genotype A) supported its porphyra-334 producing capability via the Nostoc-type mechanism, although the genotype A of N. commune mainly produces the arabinose-bound porphyra-334. The mysABC gene cluster was conserved in N. verrucosum, but the mysD gene was not included in the cluster. These results suggest that the mysABCD gene products are involved in the biosynthesis of porphyra-334 commonly in these Nostoc species, and that the genotype A of N. commune additionally acquired the glycosylation of porphyra-334.


Assuntos
Cicloexanonas , Cicloexilaminas , Glicina/análogos & derivados , Nostoc/química , Cicloexanonas/metabolismo , Cicloexilaminas/metabolismo , Glicina/biossíntese , Glicina/genética , Glicina/metabolismo , Glicosilação , Família Multigênica/genética , Nostoc/genética , Protetores Solares/química
8.
J Biol Chem ; 293(24): 9148-9161, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29632075

RESUMO

Aldehyde-deformylating oxygenase (ADO) is an essential enzyme for production of long-chain alkanes as drop-in biofuels, which are compatible with existing fuel systems. The most active ADOs are present in mesophilic cyanobacteria, especially Nostoc punctiforme Given the potential applications of thermostable enzymes in biorefineries, here we generated a thermostable (Cts)-ADO based on a consensus of ADO sequences from several thermophilic cyanobacterial strains. Using an in silico design pipeline and a metagenome library containing 41 hot-spring microbial communities, we created Cts-ADO. Cts-ADO displayed a 3.8-fold increase in pentadecane production on raising the temperature from 30 to 42 °C, whereas ADO from N. punctiforme (Np-ADO) exhibited a 1.7-fold decline. 3D structure modeling and molecular dynamics simulations of Cts- and Np-ADO at different temperatures revealed differences between the two enzymes in residues clustered on exposed loops of these variants, which affected the conformation of helices involved in forming the ADO catalytic core. In Cts-ADO, this conformational change promoted ligand binding to its preferred iron, Fe2, in the di-iron cluster at higher temperature, but the reverse was observed in Np-ADO. Detailed mapping of residues conferring Cts-ADO thermostability identified four amino acids, which we substituted individually and together in Np-ADO. Among these substitution variants, A161E was remarkably similar to Cts-ADO in terms of activity optima, kinetic parameters, and structure at higher temperature. A161E was located in loop L6, which connects helices H5 and H6, and supported ligand binding to Fe2 at higher temperatures, thereby promoting optimal activity at these temperatures and explaining the increased thermostability of Cts-ADO.


Assuntos
Aldeídos/metabolismo , Alcanos/metabolismo , Cianobactérias/enzimologia , Oxigenases/metabolismo , Biocombustíveis/microbiologia , Cianobactérias/química , Cianobactérias/genética , Cianobactérias/metabolismo , Estabilidade Enzimática , Escherichia coli/química , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Fontes Termais/microbiologia , Temperatura Alta , Metagenoma , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Nostoc/química , Nostoc/enzimologia , Nostoc/genética , Nostoc/metabolismo , Oxigenases/química , Oxigenases/genética , Conformação Proteica
9.
Proc Natl Acad Sci U S A ; 115(17): 4387-4392, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29632180

RESUMO

Phytochrome photoreceptors control plant growth, development, and the shade avoidance response that limits crop yield in high-density agricultural plantings. Cyanobacteriochromes (CBCRs) are distantly related photosensory proteins that control cyanobacterial metabolism and behavior in response to light. Photoreceptors in both families reversibly photoconvert between two photostates via photoisomerization of linear tetrapyrrole (bilin) chromophores. Spectroscopic and biochemical studies have demonstrated heterogeneity in both photostates, but the structural basis for such heterogeneity remains unclear. We report solution NMR structures for both photostates of the red/green CBCR NpR6012g4 from Nostoc punctiforme In addition to identifying structural changes accompanying photoconversion, these structures reveal structural heterogeneity for residues Trp655 and Asp657 in the red-absorbing NpR6012g4 dark state, yielding two distinct environments for the phycocyanobilin chromophore. We use site-directed mutagenesis and fluorescence and absorbance spectroscopy to assign an orange-absorbing population in the NpR6012g4 dark state to the minority configuration for Asp657. This population does not undergo full, productive photoconversion, as shown by time-resolved spectroscopy and absorption spectroscopy at cryogenic temperature. Our studies thus elucidate the spectral and photochemical consequences of structural heterogeneity in a member of the phytochrome superfamily, insights that should inform efforts to improve photochemical or fluorescence quantum yields in the phytochrome superfamily.


Assuntos
Proteínas de Bactérias/química , Nostoc/química , Fitocromo/química , Proteínas de Bactérias/genética , Mutagênese Sítio-Dirigida , Nostoc/genética , Fitocromo/genética , Domínios Proteicos
10.
Biochemistry ; 57(18): 2636-2648, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29633829

RESUMO

Forward and reverse primary (<10 ns) and secondary (>10 ns) photodynamics of cyanobacteriochrome (CBCR) NpF2164g7 were characterized by global analysis of ultrafast broadband transient absorption measurements. NpF2164g7 is the most C-terminal bilin-binding GAF domain in the Nostoc punctiforme phototaxis sensor PtxD (locus Npun_F2164). Although a member of the canonical red/green CBCR subfamily phylogenetically, NpF2164g7 exhibits an orange-absorbing 15ZPo dark-adapted state instead of the typical red-absorbing 15ZPr dark-adapted state characteristic of this subfamily. The green-absorbing 15EPg photoproduct of NpF2164g7 is unstable, allowing this CBCR domain to function as a power sensor. Photoexcitation of the 15ZPo state triggers inhomogeneous excited-state dynamics with three spectrally and temporally distinguishable pathways to generate the light-adapted 15EPg state in high yield (estimated at 25-30%). Although observed in other CBCR domains, the inhomogeneity in NpF2164g7 extends far into secondary relaxation dynamics (10 ns -1 ms) through to formation of 15EPg. In the reverse direction, the primary dynamics after photoexcitation of 15EPg are qualitatively similar to those of other red/green CBCRs, but secondary dynamics involve a "pre-equilibrium" step before regenerating 15ZPo. The anomalous photodynamics of NpF2164g7 may reflect an evolutionary adaptation of CBCR sensors that function as broadband light intensity sensors.


Assuntos
Proteínas de Bactérias/química , Cianobactérias/química , Nostoc/química , Fotorreceptores Microbianos/química , Cinética , Luz , Processos Fotoquímicos , Fototaxia/efeitos dos fármacos , Fototaxia/efeitos da radiação
11.
Molecules ; 23(2)2018 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-29495302

RESUMO

Nostoc colonies have been used as food and medicine for centuries, and their main supporting matrix is polysaccharides, which help Nostoc cells resist various environmental stresses including oxidative stress. Here we isolated a polysaccharide, nostoglycan, from cultured Nostocsphaeroides colonies and determined its physicochemical properties, which revealed a characteristic infrared absorption spectrum typical of polysaccharides and an amorphous morphology with rough surfaces. We also show that nostoglycan has strong moisture absorption and retention capacities and a high relative viscosity. Using Caenorhabditis elegans models, we then demonstrate that nostoglycan is capable of improving overall survival rate of the animals under increased oxidative stress caused by paraquat. Nostoglycan also reduces reactive oxygen species level, inhibits protein carbonyl formation and lipid peroxidation, and increases activities of superoxide dismutase and catalase in paraquat-exposed nematodes. As oxidative stress may drive tumor progression, we further demonstrate that nostoglycan can suppress the proliferation of several types of tumor cells and induce apoptosis of human lung adenocarcinoma A549 cells via caspase-3 activation. Together, our results yield important information on the physicochemical characteristics and demonstrate the antioxidant and anti-proliferative functions of nostoglycan, and thus provide an insight into its potential in food and health industries.


Assuntos
Microalgas/química , Nostoc/química , Polissacarídeos/química , Polissacarídeos/farmacologia , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fenômenos Químicos , Humanos , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Superóxido Dismutase/metabolismo
12.
J Nat Prod ; 81(2): 410-413, 2018 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-29432010

RESUMO

Cybastacines A (1) and B (2) were discovered as a novel pentacyclic sesterterpenoid-alkaloid skeleton structure, with a guanidinium group. These molecules were isolated from a Nostoc sp. cyanobacterium collected in the Canary Islands. Their structures were elucidated primarily by a combination of spectroscopic analyses and X-ray diffraction. These compounds showed antibiotic activities against several clinically relevant bacterial strains.


Assuntos
Antibacterianos/química , Nostoc/química , Sesterterpenos/química , Guanidina/química , Espanha
13.
J Nat Prod ; 81(3): 572-578, 2018 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-29381355

RESUMO

The cell extracts of two cultured freshwater Nostoc spp., UIC 10279 and UIC 10366, both from the suburbs of Chicago, showed antiproliferative activity against MDA-MB-231 and MDA-MB-435 cancer cell lines. Bioassay-guided fractionation led to the isolation of five glycosylated cylindrocyclophanes, named ribocyclophanes A-E (1-5) and cylindrocyclophane D (6). The structure determination was carried out by HRESIMS and 1D and 2D NMR analyses and confirmed by single-crystal X-ray crystallography. The structures of ribocyclophanes A-E (1-5) contain a ß-d-ribopyranose glycone in the rare 1 C4 conformation. Among isolated compounds, ribocyclophane D (4) showed antiproliferative activity against MDA-MB-435 and MDA-MB-231 cancer cells with an IC50 value of less than 1 µM.


Assuntos
Éteres Cíclicos/química , Éteres Cíclicos/farmacologia , Nostoc/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X/métodos , Ensaios de Seleção de Medicamentos Antitumorais , Água Doce/microbiologia , Glicosilação , Humanos , Ressonância Magnética Nuclear Biomolecular
14.
Toxicon ; 143: 81-89, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29366868

RESUMO

Nostoc sp. is one of the most widely distributed cyanobacterial genera that produce potentially protein phosphatase (PP) inhibitor; microcystins (MCs). MCs have posed a worldwide concern due to predominant hepatotoxicity to human health. We have previously isolated a Nostoc strain (NR1) from the Nile River (the main water supply in Egypt) and this strain exerted production of rare and highly toxic MC; demethylated microcystin-LR. There is no data concerning risk factors of liver diseases for human and animal exposure to NR1-contaminated drinking water yet. It is thus important to evaluate acute (LD50 dose), subacute (0.01% and 10% of LD50 dose) and subchronic (0.01% and 10% of LD50 dose) hepatotoxicity's NR1 extract using experimental mice. Mice groups, who orally received 0.01% LD50, represented a permissible concentration of the World Health Organization (WHO) for MC in drinking water. Several parameters were detected, including hepatotoxicity (i.e. PP activity, liver function, oxidative stress markers and DNA fragmentation), pro-inflammatory cytokine (TNF-α) and liver histopathology. Our results demonstrated LD50 of NR1 extract was at 15,350 mg/kg body weight and caused hepatotoxicity that attributed to PP inhibition and a significant increase of hepatic damage biomarkers with lipid accumulation. Moreover, NR1 extract induced hepatic oxidative damage that may have led to DNA fragmentation and production of TNF-α. As demonstrated from the histopathological study, NR1 extract caused a severe collapse of cytoskeleton with subsequent focal degeneration of hepatocytes, necroinflammation and steatosis. The grade of hepatotoxicity in subacute (10% of LD50) group was higher than that in the subchronic (10% of LD50 and 0.01% of LD50, WHOch, respectively) groups. No significant hepatotoxicity was detectable for subacute (0.01% of LD50, WHOac) group. NR1 is therefore considered as one of the harmful and life-threatening cyanobacteria for Egyptian people being exposed to dose above WHO guideline. Thus, biological indicators and thresholds for water treatment are extremely needed.


Assuntos
Fígado/efeitos dos fármacos , Microcistinas/toxicidade , Nostoc/química , Animais , Citoesqueleto/patologia , Fragmentação do DNA/efeitos dos fármacos , Água Potável , Egito , Dose Letal Mediana , Fígado/patologia , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fator de Necrose Tumoral alfa/análise
15.
J Sci Food Agric ; 98(5): 1719-1724, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28858376

RESUMO

BACKGROUND: Nostoc sphaeroides has been used as a highly effective herbal medicine and dietary supplement for thousands of years. The desired dark green colour of fresh N. sphaeroides is converted into an undesirable dark brown during conventional high pressure (HP) steam sterilisation. Radio frequency (RF) sterilisation technology was used in this study to determine its effectiveness in sterilising N. sphaeroides and to achieve better preservation of natural colour and desirable flavour. Sterilisation was carried out using a 6 kW, 27 MHz RF instrument for 10, 20 and 30 min. The degree of microbial kill and the effects of RF sterilisation on colour and flavour were determined and compared with those obtained from HP steam (121 °C, 30 min) sterilisation. RESULTS: The effects of RF sterilisation on colour and flavour (measured using electronic nose) parameters were significantly lower than that in HP steam sterilisation. The RF sterilisation carried out for 20 min achieved logarithmic reduction of bacterial population and met China's national standard while preserving the colour and flavour better. CONCLUSION: Results of the present study indicated that application of RF sterilisation would improve the quality of sterilised N. sphaeroides and broaden its application in the food and health food industries. © 2017 Society of Chemical Industry.


Assuntos
Fatores Biológicos/isolamento & purificação , Aromatizantes/isolamento & purificação , Manipulação de Alimentos/métodos , Nostoc/química , Fatores Biológicos/química , Cor , Suplementos Nutricionais/análise , Aromatizantes/química , Humanos , Nostoc/efeitos da radiação , Pressão , Controle de Qualidade , Ondas de Rádio , Vapor/análise , Esterilização , Paladar
16.
Cell Mol Biol (Noisy-le-grand) ; 63(9): 96-105, 2017 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-28980928

RESUMO

Cyanovirin-N (CVN) is well known as an anti-HIV protein. The efficient production of low cost microbicides for preventing the HIV-infection  has lately become a requirement worldwide. The aim of the present study was to optimize the expression of antiviral Cyanovirin-N homology gene found in the indigenous strain of Nostoc ellipsospourum LZN using Response Surface Methodology (RSM) and Protein Structure Analysis. Optimization of three induction factors (IPTG concentration (0.1, 0.55 and 1mM), temperature for bacterial growth (20, 28.5 and 37°C) and induction time (4, 10 and 16h) was done using RSM and Box-Behnken Design. Total RNA extraction was performed and mRNA levels were quantified in each experimental design by one-step SYBR qPCR. Protein structure was predicted using I-TASSER server. The full-length sequence of LZN-CVN gene is 306 bp in length, due mostly to five mutations. RSM analysis showed that the optimum condition to obtain maximum fold change was a concentration of 0.6mM IPTG, temperature set to 29°C and a 12h long induction time. The extracted protein from periplasmic fraction (8 kDa) was verified via SDS-PAGE. The high percentage of LZN-CVN similarity was demonstrated with PDB (Protein Data Bank) accession code of 2rp3A (CVN domain B mutant) and the ligand binding sites were related to N42, V43, D44, G45, S52, N53 and E56 residues. Different expression systems could assist in the development of anti-HIV proteins in a large scale. The LZN-CVN protein was successfully expressed in the E.coli system. RSM could be applied to a series of mathematical and statistical methods for modeling and analysis of responses which are influenced by various variables of interest.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Clonagem Molecular/métodos , Nostoc/genética , Sequência de Aminoácidos , Antivirais/química , Antivirais/metabolismo , Proteínas de Bactérias/química , Sequência de Bases , Proteínas de Transporte/química , Genes Bacterianos , Modelos Moleculares , Mutação , Nostoc/química , Conformação Proteica , Alinhamento de Sequência
17.
Chembiochem ; 18(22): 2242-2246, 2017 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-28914478

RESUMO

Inteins carry out protein-splicing reactions, which are used in protein chemistry, protein engineering and biotechnological applications. Rearrangement of the order of the domains in split-inteins results in head-to-tail cyclisation of the target sequence, which can be used for genetic encoding and expression of libraries of cyclic peptides (CPs). The efficiency of the splicing reaction depends on the target sequence. Here we used mass spectrometry to assess in vivo cyclic peptide formation from different hexameric target sequences by the DnaE split-inteins from Synechocystis sp. and Nostoc punctiforme, revealing a strong impact of the target sequence and of the intein on the intracellular peptide concentration. Furthermore, we determined the crystal structures of their pre-splicing complexes, which allowed us to identify F-block Asp17 as crucial for the DnaE-mediated splicing reaction.


Assuntos
DNA Polimerase III/química , DNA Polimerase III/metabolismo , Inteínas , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/química , Espectrometria de Massas , Modelos Moleculares , Nostoc/química , Synechocystis/química
18.
J Photochem Photobiol B ; 172: 102-108, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28544967

RESUMO

A UV-absorbing compound was purified and identified as a novel glycosylated mycosporine-like amino acid (MAA), 13-O-ß-galactosyl-porphyra-334 (ß-Gal-P334) from the edible cyanobacterium Nostoc sphaericum, known as "ge xian mi" in China and "cushuro" in Peru. Occurrence of the hexosylated derivative of shinorine (hexosyl-shinorine) was also supported by LC-MS/MS analysis. ß-Gal-P334 accounted for about 86.5% of total MAA in N. sphaericum, followed by hexosyl-shinorine (13.2%) and porphyra-334 (0.2%). ß-Gal-P334 had an absorption maximum at 334nm and molecular absorption coefficient was 46,700 at 334nm. Protection activity of ß-Gal-P334 from UVB and UVA+8-methoxypsoralen induced cell damage on human keratinocytes (HaCaT) was assayed in comparison with other MAA (porphyra-334, shinorine, palythine and mycosporine-glycine). The UVB protection activity was highest in mycosporine-glycine, followed by palythine, ß-Gal-P334, porphyra-334 and shinorine in order. ß-Gal-P334 had highest protection activity from UVA+8-methoxypsoralen induced cell damage followed by porphyra-334, shinorine, mycosporine-glycine and palythine. We also found an antioxidant (radical-scavenging) activity of ß-Gal-P334 by colorimetric and ESR methods. From these findings, ß-Gal-P334 was suggested to play important roles in stress tolerant mechanisms such as UV and oxidative stress in N. sphaericum as a major MAA. We also consider that the newly identified MAA, ß-Gal-P334 has a potential for use as an ingredient of cosmetics and toiletries.


Assuntos
Cicloexanonas/química , Glicina/análogos & derivados , Nostoc/química , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Raios Ultravioleta , Aminoácidos/química , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cromatografia Líquida de Alta Pressão , Cicloexanonas/isolamento & purificação , Cicloexanonas/farmacologia , Cicloexilaminas/química , Glicina/química , Glicina/isolamento & purificação , Glicina/farmacologia , Glicosilação , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Conformação Molecular , Nostoc/metabolismo , Estresse Oxidativo/efeitos da radiação , Substâncias Protetoras/química , Substâncias Protetoras/isolamento & purificação , Espectrometria de Massas em Tandem
19.
Appl Environ Microbiol ; 83(13)2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28455341

RESUMO

To investigate the function of 2-methylhopanoids in modern cyanobacteria, the hpnP gene coding for the radical S-adenosyl methionine (SAM) methylase protein that acts on the C-2 position of hopanoids was deleted from the filamentous cyanobacterium Nostoc punctiforme ATCC 29133S. The resulting ΔhpnP mutant lacked all 2-methylhopanoids but was found to produce much higher levels of two bacteriohopanepentol isomers than the wild type. Growth rates of the ΔhpnP mutant cultures were not significantly different from those of the wild type under standard growth conditions. Akinete formation was also not impeded by the absence of 2-methylhopanoids. The relative abundances of the different hopanoid structures in akinete-dominated cultures of the wild-type and ΔhpnP mutant strains were similar to those of vegetative cell-dominated cultures. However, the ΔhpnP mutant was found to have decreased growth rates under both pH and osmotic stress, confirming a role for 2-methylhopanoids in stress tolerance. Evidence of elevated photosystem II yield and NAD(P)H-dependent oxidoreductase activity in the ΔhpnP mutant under stress conditions, compared to the wild type, suggested that the absence of 2-methylhopanoids increases cellular metabolic rates under stress conditions.IMPORTANCE As the first group of organisms to develop oxygenic photosynthesis, Cyanobacteria are central to the evolutionary history of life on Earth and the subsequent oxygenation of the atmosphere. To investigate the origin of cyanobacteria and the emergence of oxygenic photosynthesis, geobiologists use biomarkers, the remnants of lipids produced by different organisms that are found in geologic sediments. 2-Methylhopanes have been considered indicative of cyanobacteria in some environmental settings, with the parent lipids 2-methylhopanoids being present in many contemporary cyanobacteria. We have created a Nostoc punctiforme ΔhpnP mutant strain that does not produce 2-methylhopanoids to assess the influence of 2-methylhopanoids on stress tolerance. Increased metabolic activity in the mutant under stress indicates compensatory alterations in metabolism in the absence of 2-methylhopanoids.


Assuntos
Nostoc/metabolismo , Triterpenos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Concentração de Íons de Hidrogênio , Isomerismo , Metilação , Nostoc/química , Nostoc/genética , Nostoc/crescimento & desenvolvimento , Osmose , Triterpenos/química
20.
J Nat Prod ; 80(4): 1073-1080, 2017 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-28252962

RESUMO

Merocyclophanes C and D (1 and 2) were isolated from the cell extract of the cultured cyanobacterium UIC 10110. The structures were determined by one-dimensional nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry and confirmed by 2D NMR techniques. The absolute configurations were determined using electronic circular dichroism spectroscopy. Merocyclophanes C and D represent the first known analogues of the merocyclophane core structure, a recently discovered scaffold of [7,7] paracyclophanes characterized by an α-branched methyl at C-1/C-14; 1 and 2 showed antiproliferative activity against the MDA-MB-435 cell line with IC50 values of 1.6 and 0.9 µM, respectively. Partial 16S analysis determined UIC 10110 to be a Nostoc sp., and it was found to clade with UIC 10062 Nostoc sp., the only other strain known to produce merocyclophanes. The genome of UIC 10110 was sequenced, and a biosynthetic gene cluster was identified that is proposed to encode type I and type III polyketide synthases that are potentially responsible for production of the merocyclophanes; however, further experiments will be required to verify the true function of the gene cluster. The gene cluster provides a genetic basis for the observed structural differences of the [7,7] paracyclophane core structures.


Assuntos
Compostos Macrocíclicos/isolamento & purificação , Nostoc/química , Animais , Antibacterianos/química , Colorado , Água Doce/microbiologia , Concentração Inibidora 50 , Compostos Macrocíclicos/química , Camundongos , Estrutura Molecular , Nostoc/genética , Ressonância Magnética Nuclear Biomolecular , Espectrometria de Massas por Ionização por Electrospray
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