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1.
Nat Commun ; 11(1): 285, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941905

RESUMO

Predator-prey interactions play important roles in the cycling of marine organic matter. Here we show that a Gram-negative bacterium isolated from marine sediments (Pseudoalteromonas sp. strain CF6-2) can kill Gram-positive bacteria of diverse peptidoglycan (PG) chemotypes by secreting the metalloprotease pseudoalterin. Secretion of the enzyme requires a Type II secretion system. Pseudoalterin binds to the glycan strands of Gram positive bacterial PG and degrades the PG peptide chains, leading to cell death. The released nutrients, including PG-derived D-amino acids, can then be utilized by strain CF6-2 for growth. Pseudoalterin synthesis is induced by PG degradation products such as glycine and glycine-rich oligopeptides. Genes encoding putative pseudoalterin-like proteins are found in many other marine bacteria. This study reveals a new microbial interaction in the ocean.

2.
J Infect ; 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31812703

RESUMO

OBJECTIVE: Viral fitness plays an important role in HIV-1 evolution, transmission and pathogenesis. However, how mutations accumulated during early infection affect viral fitness has not been well studied. METHODS: Paired infectious molecular clones (IMCs) for transmitted/founder (T/F) and 6-month (6-mo) viruses post infection were generated from 10 infected individuals to investigate the impact of accumulated mutations on viral fitness by comparing 6-mo viruses to their cognate T/F viruses. RESULTS: All ten 6-mo viruses were less fit than their cognate T/F viruses. Moreover, the fitness losses of the 6-mo viruses correlated with the decrease in viral loads from the peak of viremia. CONCLUSION: These results show that the mutations accumulated during half a year post infection collectively reduce viral fitness and thereby contribute to lowering viral loads.

3.
Int J Mol Sci ; 20(23)2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31817029

RESUMO

A triphenylmethane reductase derived from Citrobacter sp. KCTC 18061P was coupled with a glucose 1-dehydrogenase from Bacillus sp. ZJ to construct a cofactor self-sufficient bienzyme biocatalytic system for dye decolorization. Fed-batch experiments showed that the system is robust to maintain its activity after 15 cycles without the addition of any expensive exogenous NADH. Subsequently, three different machine learning approaches, including multiple linear regression (MLR), random forest (RF), and artificial neural network (ANN), were employed to explore the response of decolorization efficiency to the variables of the bienzyme system. Statistical parameters of these models suggested that a three-layered ANN model with six hidden neurons was capable of predicting the dye decolorization efficiency with the best accuracy, compared with the models constructed by MLR and RF. Weights analysis of the ANN model showed that the ratio between two enzymes appeared to be the most influential factor, with a relative importance of 54.99% during the decolorization process. The modeling results confirmed that the neural networks could effectively reproduce experimental data and predict the behavior of the decolorization process, especially for complex systems containing multienzymes.

4.
Mar Drugs ; 17(12)2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31835449

RESUMO

To obtain chitinase-producing microorganisms with high chitinolytic activity at low temperature, samples collected from Fildes Peninsula in Antarctica were used as sources for bioprospecting of chitinolytic microorganisms. A cold-adapted strain, designated as GWSMS-1, was isolated from marine sediment and further characterized as Pseudomonas. To improve the chitinase production, one-factor-at-a-time and orthogonal test approaches were adopted to optimize the medium components and culture conditions. The results showed that the highest chitinolytic activity (6.36 times higher than that before optimization) was obtained with 95.41 U L-1 with 15 g L-1 of glucose, 1 g L-1 of peptone, 15 g L-1 of colloid chitin and 0.25 g L-1 of magnesium ions contained in the medium, cultivated under pH 7.0 and a temperature of 20 °C. To better understand the application potential of this strain, the enzymatic properties and the antifungal activity of the crude chitinase secreted by the strain were further investigated. The crude enzyme showed the maximum catalytic activity at 35 °C and pH 4.5, and it also exhibited excellent low-temperature activity, which still displayed more than 50% of its maximal activity at 0 °C. Furthermore, the crude chitinase showed significant inhibition of fungi Verticillium dahlia CICC 2534 and Fusarium oxysporum f. sp. cucumerinum CICC 2532, which can cause cotton wilt and cucumber blight, respectively, suggesting that strain GWSMS-1 could be a competitive candidate for biological control in agriculture, especially at low temperature.

5.
Microb Ecol ; 2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31863131

RESUMO

Recently, an increasing number of studies have focused on the biogeographic distribution of marine microorganisms. However, the extent to which geographic distance can affect marine microbial communities is still unclear, especially for the microbial communities in well-connected surface seawaters. In this study, the bacterial community compositions of 21 surface seawater samples, that were distributed over a distance of 7800 km, were surveyed to investigate how bacterial community similarity changes with increasing geographical distance. Proteobacteria and Bacteroidetes were the dominant bacterial phyla, with Proteobacteria accounting for 52.6-92.5% and Bacteroidetes comprising 3.5-46.9% of the bacterial communities. A significant bacterial distance-decay relationship was observed in the well-connected Southern Ocean surface seawater. The number of pairwise shared operational taxonomic units (OTUs), and community similarities tended to decrease with increasing geographic distance. Calculation of the similarity indices with all, abundant or rare OTUs did not affect the observed distance-decay relationship. Spatial distance can largely explain the observed bacterial community variation. This study shows that even in well-connected surface waters, bacterial distance-decay patterns can be found as long as the geographical distance is great enough. The biogeographic patterns should then be present for marine microorganisms considering the large size and complexity of the marine ecosystem.

7.
Biochem Biophys Res Commun ; 509(3): 641-646, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30616888

RESUMO

Oxidative damage plays a critical role in cochlear cell apoptosis, which is central to the physiopathology of noise-induced hearing loss (NIHL). Sirtuin 2 (SIRT2) is an NAD-dependent deacetylase that regulates cellular response to oxidative stress, however, its role in NIHL remains poorly understood. Here, we report that SIRT2 is upregulated in the cochlea after noise exposure. Functionally, the treatment of AK-7, one specific SIRT2 inhibitor, attenuates the progression of NIHL. In addition, AK-7 treatment reduces oxidative nuclear DNA damage and apoptosis in the cochlea after noise exposure. Moreover, AK-7 treatment reduces apoptosis of mouse inner ear HEI-OC1 cells exposed to oxidative stress in vitro. Taken together, these results suggest that SIRT2 inhibition with AK-7 reduces cochlear cell apoptosis through attenuating oxidative stress-induced damage, which may underlie its protective role against NIHL. This study also implies that AK-7 may have potential therapeutic significance in the intervention of NIHL.


Assuntos
Apoptose/efeitos dos fármacos , Cóclea/efeitos dos fármacos , Perda Auditiva Provocada por Ruído/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Sirtuína 2/antagonistas & inibidores , Animais , Linhagem Celular , Cóclea/citologia , Cóclea/metabolismo , Cóclea/patologia , Perda Auditiva Provocada por Ruído/metabolismo , Perda Auditiva Provocada por Ruído/patologia , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Sirtuína 2/análise , Sirtuína 2/metabolismo
8.
Mar Drugs ; 16(11)2018 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-30380738

RESUMO

A thermostable ß-1,3-galactosidase from Marinomonas sp. BSi20414 was successfully heterologously expressed in Escherichia coli BL21 (DE3), with optimum over-expression conditions as follows: the recombinant cells were induced by adding 0.1 mM of IPTG to the medium when the OD600 of the culture reached between 0.6 and 0.9, followed by 22 h incubation at 20 °C. The recombinant enzyme ß-1,3-galactosidase (rMaBGA) was further purified to electrophoretic purity by immobilized metal affinity chromatography and size exclusion chromatography. The specific activity of the purified enzyme was 126.4 U mg-1 at 37 °C using ONPG (o-nitrophenyl-ß-galactoside) as a substrate. The optimum temperature and pH of rMaBGA were determined as 60 °C and 6.0, respectively, resembling with its wild-type counterpart, wild type (wt)MaBGA. However, rMaBGA and wtMaBGA displayed different thermal stability and steady-state kinetics, although they share identical primary structures. It is postulated that the stability of the enzyme was altered by heterologous expression with the absence of post-translational modifications such as glycosylation, as well as the steady-state kinetics. To evaluate the potential of the enzyme in synthesis of galactooligosaccharides (GOS), the purified recombinant enzyme was employed to catalyze the transgalactosylation reaction at the lab scale. One of the transgalactosylation products was resolved as 3'-galactosyl-lactose, which had been proven to be a better bifidogenic effector than GOS with ß-1,4 linkage and ß-1,6 linkages. The results indicated that the recombinant enzyme would be a promising alternative for biosynthesis of GOS mainly with ß-1,3 linkage.


Assuntos
Proteínas de Bactérias/metabolismo , Galactose/biossíntese , Marinomonas/química , Oligossacarídeos/biossíntese , Proteínas Recombinantes/metabolismo , beta-Galactosidase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Ensaios Enzimáticos , Estabilidade Enzimática , Galactose/química , Glicosilação , Cinética , Marinomonas/genética , Oligossacarídeos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Temperatura Ambiente , beta-Galactosidase/química , beta-Galactosidase/genética , beta-Galactosidase/isolamento & purificação
9.
Zhongguo Gu Shang ; 31(10): 933-936, 2018 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-30373347

RESUMO

OBJECTIVE: To investigate expression features and correlation of genes expression on MyD88-dependent signaling pathway in synovial membrane (SM) of progression of knee osteoarthritis (OA). METHODS: Sixty Wistar rats were randomly divided into 6 groups, including blank group (N), false surgical group, model groups[2 weeks (2W), 4 weeks (4W), 8 weeks (8W) and 12 weeks (12W)], with 10 rats in each group. The models were established by using Hulth method. Control group was experienced no surgery, while false surgical group was only opened joint cavity and sutured. The SM samples was collected according to the time designed above. The relative expression quantity of MyD88, TLR4 and NF-κB was detected by Real-time PCR after the extraction of the total RNA and reverse transcription. The correlation analysis was obtained by SPSS. RESULTS: There was no significant difference in each gene mRNA expression between false surgical and blank group(P> 0.05), while enhanced expression was found in the model groups(P<0.05). The correlation index among MyD88, TLR4 and NF-κB was 0.91 and 0.86 respectively, and had significant difference among them. CONCLUSIONS: Positively relative among MyD88, TLR4 and NF-κB played main role in TLR4/NF-κB signal passway, and could predicate the expression of other genes in the passway. It also could further provide the basis for clarify the pathologic mechanism of knee OA.


Assuntos
Osteoartrite do Joelho , Animais , Fator 88 de Diferenciação Mieloide , NF-kappa B , Ratos , Ratos Wistar , Transdução de Sinais , Receptor 4 Toll-Like
10.
Biochim Biophys Acta Biomembr ; 1860(5): 1193-1204, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29425673

RESUMO

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is an ABC transporter containing two transmembrane domains forming a chloride ion channel, and two nucleotide binding domains (NBD1 and NBD2). CFTR has presented a formidable challenge to obtain monodisperse, biophysically stable protein. Here we report a comprehensive study comparing effects of single and multiple NBD1 mutations on stability of both the NBD1 domain alone and on purified full length human CFTR. Single mutations S492P, A534P, I539T acted additively, and when combined with M470V, S495P, and R555K cumulatively yielded an NBD1 with highly improved structural stability. Strategic combinations of these mutations strongly stabilized the domain to attain a calorimetric Tm > 70 °C. Replica exchange molecular dynamics simulations on the most stable 6SS-NBD1 variant implicated fluctuations, electrostatic interactions and side chain packing as potential contributors to improved stability. Progressive stabilization of NBD1 directly correlated with enhanced structural stability of full-length CFTR protein. Thermal unfolding of the stabilized CFTR mutants, monitored by changes in intrinsic fluorescence, demonstrated that Tm could be shifted as high as 67.4 °C in 6SS-CFTR, more than 20 °C higher than wild-type. H1402S, an NBD2 mutation, conferred CFTR with additional thermal stability, possibly by stabilizing an NBD-dimerized conformation. CFTR variants with NBD1-stabilizing mutations were expressed at the cell surface in mammalian cells, exhibited ATPase and channel activity, and retained these functions to higher temperatures. The capability to produce enzymatically active CFTR with improved structural stability amenable to biophysical and structural studies will advance mechanistic investigations and future cystic fibrosis drug development.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Mutação , Nucleotídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Sítios de Ligação/genética , Células CHO , Cricetinae , Cricetulus , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/isolamento & purificação , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Estabilidade Enzimática/genética , Células HEK293 , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas/genética , Estabilidade Proteica , Temperatura Ambiente
11.
J Cell Physiol ; 233(2): 1342-1358, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28513840

RESUMO

Osteoarthritis (OA) is a common chronic degenerative disease that affects all joints. At present, the pathological processes and mechanisms of OA are still unclear. Innate immunity, a key player in damage to the structure of the joint and the mechanism by which the host attempts to repair OA, affects all pathological stages of the disease. In the present study, our aim was to assess changes in innate immunity during the pathological processes of OA in articular cartilage (AC) and the synovial membrane (SM), which are the major structures in joints, and to systematically examine the histological changes in AC and SM in mild, moderate and severe cases of OA, in order to further speculate about the manner in which the interactions of AC and SM are facilitated by innate immunity. Histological methods (including HE and Safranin O-fast green staining), immunofluorescent double staining, TUNEL stain, and Western blots were used to assess the morphological changes within AC and SM tissues in healthy and mild, moderate, or severe OA rats. Our results showed that the damage to AC and SM within the joints progressively worsened in different degrees during the course of the disease, and that the innate immune system was closely involved in the AC and SM during each stage of OA. These findings also confirmed that SM may affect the pathological changes in AC through the innate immune system, and therefore affect the progress of OA.


Assuntos
Cartilagem Articular/imunologia , Cartilagem Articular/patologia , Imunidade Inata , Osteoartrite/imunologia , Osteoartrite/patologia , Sinovite/imunologia , Sinovite/patologia , Animais , Comportamento Animal , Cartilagem Articular/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Masculino , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Osteoartrite/metabolismo , Ratos , Ratos Wistar , Índice de Gravidade de Doença , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Sinovite/metabolismo , Receptor 4 Toll-Like/metabolismo
12.
Int J Mol Sci ; 18(6)2017 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-28587256

RESUMO

Due to the dual cofactor specificity, glucose 1-dehydrogenase (GDH) has been considered as a promising alternative for coenzyme regeneration in biocatalysis. To mine for potential GDHs for practical applications, several genes encoding for GDH had been heterogeneously expressed in Escherichia coli BL21 (DE3) for primary screening. Of all the candidates, GDH from Bacillus sp. ZJ (BzGDH) was one of the most robust enzymes. BzGDH was then purified to homogeneity by immobilized metal affinity chromatography and characterized biochemically. It displayed maximum activity at 45 °C and pH 9.0, and was stable at temperatures below 50 °C. BzGDH also exhibited a broad pH stability, especially in the acidic region, which could maintain around 80% of its initial activity at the pH range of 4.0-8.5 after incubating for 1 hour. Molecular dynamics simulation was conducted for better understanding the stability feature of BzGDH against the structural context. The in-silico simulation shows that BzGDH is stable and can maintain its overall structure against heat during the simulation at 323 K, which is consistent with the biochemical studies. In brief, the robust stability of BzGDH made it an attractive participant for cofactor regeneration on practical applications, especially for the catalysis implemented in acidic pH and high temperature.


Assuntos
Glucose 1-Desidrogenase/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Motivos de Aminoácidos , Sequência de Aminoácidos , Bacillus/enzimologia , Biocatálise , Sequência Conservada , Estabilidade Enzimática , Glucose 1-Desidrogenase/genética , Glucose 1-Desidrogenase/metabolismo , Cinética , Filogenia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Temperatura Ambiente
13.
PLoS One ; 12(2): e0170672, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28151945

RESUMO

The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the viral envelope glycoproteins (Envs), which are derived by the proteolytic cleavage of a trimeric gp160 Env precursor. The mature Env trimer is a major target for entry inhibitors and vaccine-induced neutralizing antibodies. Env interstrain variability, conformational flexibility and heavy glycosylation contribute to evasion of the host immune response, and create challenges for structural characterization and vaccine development. Here we investigate variables associated with reconstitution of the HIV-1 Env precursor into nanodiscs, nanoscale lipid bilayer discs enclosed by membrane scaffolding proteins. We identified detergents, as well as lipids similar in composition to the viral lipidome, that allowed efficient formation of Env-nanodiscs (Env-NDs). Env-NDs were created with the full-length Env precursor and with an Env precursor with the majority of the cytoplasmic tail intact. The self-association of Env-NDs was decreased by glutaraldehyde crosslinking. The Env-NDs exhibited an antigenic profile expected for the HIV-1 Env precursor. Env-NDs were recognized by broadly neutralizing antibodies. Of note, neutralizing antibody epitopes in the gp41 membrane-proximal external region and in the gp120:gp41 interface were well exposed on Env-NDs compared with Env expressed on cell surfaces. Most Env epitopes recognized by non-neutralizing antibodies were masked on the Env-NDs. This antigenic profile was stable for several days, exhibiting a considerably longer half-life than that of Env solubilized in detergents. Negative selection with weak neutralizing antibodies could be used to improve the antigenic profile of the Env-NDs. Finally, we show that lipid adjuvants can be incorporated into Env-NDs. These results indicate that Env-NDs represent a potentially useful platform for investigating the structural, functional and antigenic properties of the HIV-1 Env trimer in a membrane context.


Assuntos
Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/imunologia , Bicamadas Lipídicas/imunologia , Animais , Células CHO , Linhagem Celular , Cricetulus , Epitopos/imunologia , Humanos , Bicamadas Lipídicas/metabolismo , Nanoestruturas , Internalização do Vírus
14.
Virol J ; 14(1): 33, 2017 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-28209172

RESUMO

BACKGROUND: The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env), a Type 1 transmembrane protein, assembles into a trimeric spike complex that mediates virus entry into host cells. The high potential energy of the metastable, unliganded Env trimer is maintained by multiple non-covalent contacts among the gp120 exterior and gp41 transmembrane Env subunits. Structural studies suggest that the gp41 transmembrane region forms a left-handed coiled coil that contributes to the Env trimer interprotomer contacts. Here we evaluate the contribution of the gp41 transmembrane region to the folding and stability of Env trimers. METHODS: Multiple polar/charged amino acid residues, which hypothetically disrupt the stop-transfer signal, were introduced in the proposed lipid-interactive face of the transmembrane coiled coil, allowing release of soluble cleavage-negative Envs containing the modified transmembrane region (TMmod). We also examined effects of cleavage, the cytoplasmic tail and a C-terminal fibritin trimerization (FT) motif on oligomerization, antigenicity and functionality of soluble and membrane-bound Envs. RESULTS: The introduction of polar/charged amino acids into the transmembrane region resulted in the secretion of soluble Envs from the cell. However, these TMmod Envs primarily formed dimers. By contrast, control cleavage-negative sgp140 Envs lacking the transmembrane region formed soluble trimers, dimers and monomers. TMmod and sgp140 trimers were stabilized by the addition of a C-terminal FT sequence, but still exhibited carbohydrate and antigenic signatures of a flexible ectodomain structure. On the other hand, detergent-solubilized cleaved and uncleaved Envs isolated from the membranes of expressing cells exhibited "tighter" ectodomain structures, based on carbohydrate modifications. These trimers were found to be unstable in detergent solutions, but could be stabilized by the addition of a C-terminal FT moiety. The C-terminal FT domain decreased Env cleavage and syncytium-forming ability by approximately three-fold; alteration of the FT trimerization interface restored Env cleavage and syncytium formation to near-wild-type levels. CONCLUSION: The modified transmembrane region was not conducive to trimerization of soluble Envs. However, for HIV-1 Env ectodomains that are minimally modified, membrane-anchored Envs exhibit the most native structures and can be stabilized by appropriately positioned FT domains.


Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Dobramento de Proteína , Multimerização Proteica , Substituição de Aminoácidos , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica
15.
J Virol ; 91(9)2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28202756

RESUMO

HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry (MS)-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals. (i) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs. (ii) We identified the variables that impact Env's glycosylation profile at sites with divergent glycosylation. Over half of the gp120 glycosylation sites on 11 different trimeric Envs have a conserved glycan profile, indicating that a native consensus glycosylation profile does indeed exist among trimers. We showed that some soluble gp120s and gp140s exhibit highly divergent glycosylation profiles compared to trimeric Env. We also assessed the impact of several variables on Env glycosylation: truncating the full-length Env; producing Env, instead of the more virologically relevant T lymphocytes, in CHO cells; and purifying Env with different chromatographic platforms, including nickel-nitrilotriacetic acid (Ni-NTA), 2G12, and PGT151 affinity. This report provides the first consensus glycosylation profile of Env trimers, which should serve as a useful benchmark for HIV-1 vaccine developers. This report also defines the sites where glycosylation may be impacted when Env trimers are truncated or produced in CHO cells.IMPORTANCE A protective HIV-1 vaccine will likely include a recombinant version of the viral envelope glycoprotein (Env). Env is highly glycosylated, and yet vaccine developers have lacked guidance on how to assess whether their immunogens have optimal glycosylation. The following important questions are still unanswered. (i) What is the "target" glycosylation profile, when the goal is to generate a natively glycosylated protein? (ii) What variables exert the greatest influence on Env glycosylation? We identified numerous sites on Env where the glycosylation profile does not deviate in 11 different Env trimers, and we investigated the impact on the divergent glycosylation profiles of changing the genotype of the Env sequence, the construct design, the purification method, and the producer cell type. The data presented here give vaccine developers a "glycosylation target" for their immunogens, and they show how protein production variables can impact Env glycosylation.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/imunologia , Sequência de Aminoácidos , Animais , Benchmarking , Células CHO , Cricetinae , Cricetulus , Epitopos/imunologia , Glicosilação , HIV-1/química , Evasão da Resposta Imune/imunologia
16.
Mar Drugs ; 15(1)2017 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-28075353

RESUMO

A novel ß-1,3-galactosidase, designated as MaBGA (ß-galactosidase from Marinomonas sp. BSi20414), was successfully purified to homogeneity from Marinomonas sp. BSi20414 isolated from Arctic sea ice by ammonium sulfate precipitation and anion exchange chromatography, resulting in an 8.12-fold increase in specific activity and 9.9% recovery in total activity. MaBGA displayed its maximum activity at pH 6.0 and 60 °C, and maintained at least 90% of its initial activity over the pH range of 5.0-8.0 after incubating for 1 h. It also exhibited considerable thermal stability, which retained 76% of its initial activity after incubating at 50 °C for 6 h. In contrast to other ß-galactosidases, MaBGA displayed strict substrate specificity, not only for the glycosyl group, but also for the linkage type. To better understand the structure-function relationship, the encoding gene of MaBGA was obtained and subject to bioinformatics analysis. Multiple alignments and phylogenetic analysis revealed that MaBGA belonged to the glycoside hydrolase family 42 and had closer genetic relationships with thermophilic ß-galactosidases of extremophiles. With the aid of homology modeling and molecular docking, we proposed a reasonable explanation for the linkage selectivity of MaBGA from a structural perspective. On account of the robust stability and 1,3-linkage selectivity, MaBGA would be a promising candidate in the biosynthesis of galacto-oligosaccharide with ß1-3 linkage.


Assuntos
Marinomonas/química , beta-Galactosidase/química , Sequência de Aminoácidos , Regiões Árticas , Clonagem Molecular/métodos , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Camada de Gelo , Marinomonas/genética , Simulação de Acoplamento Molecular/métodos , Filogenia , Alinhamento de Sequência , Especificidade por Substrato
17.
Appl Microbiol Biotechnol ; 101(3): 1217-1226, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27785540

RESUMO

Cupriavidus basilensis is a species with diverse metabolic capabilities, including degradation of xenobiotics and heavy metal resistance. Although the genomes of several strains of this species have been sequenced, no plasmid has yet been constructed for genetic engineering in this species. In this study, we identified a novel plasmid, designated pWS, from C. basilensis WS with a copy number of 1-3 per cell and a length of 2150 bp. pWS contained three protein-coding genes, among which only rep was required for plasmid replication. Rep showed no homology with known plasmid replication initiators. Unlike most plasmids, pWS did not have a cis-acting replication origin outside the region of rep. The minimal replicon of pWS was stable in C. basilensis WS without selection. A conjugative C. basilensis/Escherichia coli shuttle vector, pCB5, was constructed using the minimal replicon of pWS. Interestingly, the copy number of pCB5 was flexible and could be manipulated. Enhancing the expression level of Rep in pCB5 by either doubling the promoter or coding region of rep resulted in doubling of the plasmid copy number. Moreover, replacing the native promoter of rep with the lac promoter increased the copy number by over fivefold. Finally, using two different ß-galactosidase reporting systems constructed with pCB5, we successfully demonstrated the different regulatory patterns of bph and dmp operons during diphenyl ether (DE) degradation in C. basilensis WS. Thus, this shuttle vector provided an efficient tool for DNA cloning and metabolic engineering in C. basilensis.


Assuntos
Cupriavidus/genética , Vetores Genéticos , Plasmídeos/genética , Origem de Replicação , Sequência de Bases , Clonagem Molecular , Escherichia/genética , Dosagem de Genes , Engenharia Genética , Engenharia Metabólica , Óperon , Éteres Fenílicos/metabolismo , Plasmídeos/isolamento & purificação , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de DNA
18.
Genet Mol Biol ; 39(4): 567-572, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27727359

RESUMO

The aim was to study the frequencies of common deafness-related mutations and their contribution to hearing loss in different regions of Inner Mongolia. A total of 738 deaf children were recruited from five different ethnic groups of Inner Mongolia, including Han Chinese (n=486), Mongolian (n=216), Manchurian (n=24), Hui (n=6) and Daur (n=6). Nine common mutations in four genes (GJB2, SLC26A4, GJB3 and mitochondrial MT-RNR1 gene) were detected by allele-specific PCR and universal array. At least one mutated allele was detected in 282 patients. Pathogenic mutations were detected in 168 patients: 114 were homozygotes and 54 were compound heterozygotes. The 114 patients were carriers of only one mutated allele. The frequency of GJB2 variants in Han Chinese (21.0%) was higher than that in Mongolians (16.7%), but not significantly different. On the other hand, the frequency of SLC26A4 variants in Han Chinese (14.8%) was lower than that in Mongolians (19.4%), but also not significantly different. The frequency of patients with pathogenic mutations was different in Ulanqab (21.4%), Xilingol (40.0%), Chifeng (40.0%), Hulunbeier (30.0%), Hohhot (26.3%), and in Baotou (0%). In conclusion, the frequency of mutated alleles in deafness-related genes did not differ between Han Chinese and Mongolians. However, differences in the distribution of common deafness-related mutations were found among the investigated areas of Inner Mongolia.

19.
Environ Sci Pollut Res Int ; 23(21): 21319-21326, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27502455

RESUMO

Biodegradation of triphenylmethane dyes by microorganisms is hampered by the transport barrier imposed by cell membranes. On the other hand, cell-free systems using enzyme-based biodegradation strategy are costly. Therefore, an efficient and inexpensive approach circumventing these problems is highly desirable. Here, we constructed a self-sufficient system for synthetic dye removal by coupling of spore surface-displayed triphenylmethane reductase (TMR) and glucose 1-dehydrogenase (GDH) for the first time. Display of both TMR and GDH significantly enhanced their stability under conditions of extreme pH and temperature. These engineered spores also exhibited more robust long-term stability than their purified counterparts. Furthermore, we observed that a high ratio of spore-displayed GDH is necessary for high dye degradation efficiency. These results indicate that this continuous dye removal system with cofactor regeneration offers a promising solution for dye biodegradation applications.


Assuntos
Bacillus subtilis/metabolismo , Corantes/isolamento & purificação , Corantes/metabolismo , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Esporos Bacterianos/metabolismo , Compostos de Tritil/metabolismo , Biodegradação Ambiental , Poluentes Ambientais/isolamento & purificação , Poluentes Ambientais/metabolismo , Glucose 1-Desidrogenase/metabolismo , Temperatura Ambiente
20.
Zhongguo Gu Shang ; 28(8): 722-6, 2015 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-26502523

RESUMO

OBJECTIVE: To evaluate the clinical effects of the massage method of micro-regulating with vertical cross pressing lying on one side in treating cervicogenic headache (CEH). METHODS: Total 136 patients with CEH were collected in the study from August 2012 to April 2014. They were divided randomly into two groups according to random digits table. Sixty-nine patients accepted the treatment of micro-regulating with vertical cross pressing lying on one side (pressing micro-regulating group), including 29 males and 40 females with an average age of (50.55 ± 11.38) years old; 67 patients received the treatment of traditional massage (traditional massage group), including 28 males and 39 females with an average age of (51.20 ± 11.90) years old. Clinical effect was observed according to the standard of curative effect of State Administration of Traditional Chinese Medicine; the function of cervical vertebra and all body status were evaluated according to NDI score. VAS score, frequency and time of headache were recorded and compared before and after treatment. RESULTS: No adverse reactions were found after treatment, all patients were followed up from 1 to 6 months with an average of 3.1 months. In pressing micro-regulating group, 25 cases got fully recover, 26 excellence, 14 effectiveness and 4 inefficiency; and in traditional massage group, the results were 12,21,22, 12;clinical effect of pressing micro-regulating group was better than that of traditional massage group (P<0.01). NDI score in pressing micro-regulating group decreased from preoperative 13.48 ± 4.83 to postoperative 6.23 ± 3.76; in traditional massage group also decreased from preoperative 13.82 ± 5.78 to postoperative 8.25 ± 4.75; the improvement of the pressing micro-regulating group was obviously better than that of traditional massage group (P < 0.01). VAS score in pressing micro-regulating group decreased from preoperative 4.75 ± 0.97 to postoperative 1.88 ± 1.78; and in traditional massage group decreased from pre-operative 4.78 ± 0.98 to postoperative 2.84 ± 1.94; pressing micro-regulating group was more notable than that of traditional massage group (P < 0.01). The frequency per week,the pain time in pressing micro-regulating group decreased from preoperative (5.38 ± 1.96) times and (6.87 ± 3.67) hours to postoperative (1.71 ± 2.04) times and (0.97 ± 1.74) hours,respectively,in traditional massage group the above parameters decreased from preoperative (5.22 ± 1.81) times and (6.90 ± 3.79) hours to postoperative (2.81 ± 2.42) times and (1.83 ± 2.21) hours;pressing micr-regulating group was more notable than that of traditional massage group (P < 0.01 or P < 0.05). CONCLUSION: Using the tuina method of micro-regulating with vertical cross pressing lying on one side to treat CEH can improve function of cervical vertebra and all body status, lessen the intensity, frequency, duration time of pain, and had advantage of higher security, simple operation, and evident effect.


Assuntos
Massagem/métodos , Modalidades de Fisioterapia , Cefaleia Pós-Traumática/terapia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
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