Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 9.471
Filtrar
1.
Food Chem ; 328: 127112, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32470778

RESUMO

A ratiometric fluorescent probe (Probe 1) was developed for the sensitive detection of ß-galactosidase (ß-gal) activity. Probe 1 detected ß-gal activity in the range 0-1.0 U/mL, with a limit of detection of 0.025 U/mL. In addition, as different activities of ß-gal added, the luminescent intensity of Probe 1 gradually increased, as observed under a 365 nm ultraviolet lamp. Moreover, this method is low-volume, 20 µL, and time-efficient, 45 min per measurement. Probe 1 was successfully used to measure the ß-gal activity in real fruit samples in a qualitative manner, by the naked eye, fast semi-quantitative manner, by smartphone, or quantitative manner, by fluorescence spectrometer.


Assuntos
Corantes Fluorescentes/química , Análise de Alimentos/métodos , Frutas/enzimologia , Proteínas de Plantas/análise , beta-Galactosidase/análise , Limite de Detecção , Proteínas de Plantas/metabolismo , Smartphone , Espectrometria de Fluorescência/instrumentação , beta-Galactosidase/metabolismo
2.
Toxicology ; 441: 152502, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32473187

RESUMO

Cigarette smoking is a well-recognized risk factor for type 2 diabetes (T2DM), and may result in islet ß cell damage and impaired insulin secretion. However, the underlying mechanisms remain largely elusive. In the present study, we demonstrated that nicotine induced premature senescence of pancreatic ß cells in vitro and in vivo. The senescence-associated ß-galactosidase (SA-ß-Gal) assay showed that nicotine exposure induced apparent senescence phenotype of ß-TC-6 cells at an initiating dose of 100 µM and starting from 12 h. In addition, 100 and 500 µM of nicotine exposure altered the expression of senescence marker proteins, such as p16, p19 and p21. Furthermore, we uncovered that the levels of intracellular Ca2+ and reactive oxygen species (ROS) were significantly elevated in ß-TC-6 cells following exposure to 100 and 500 µM nicotine, while calcium channel blocker can reverse this effect. Furthermore, the senescence-inducing phenotype was confirmed in rat insulinoma INS-1 cells at a similar dose range, whereas blockade of nAChRs, calcium and ROS led to apparent impairment of senescence. Finally, we found that administration with 100 and 200 µg/mL nicotine in drinking water for 28 days significantly exacerbated aberrant glucose homeostasis in a mouse model of fat-induced T2DM. Of great intrigue, pancreatic ß cells exhibited significantly enhanced senescence following nicotine administration. Taken together, this study suggests that premature senescence plays a pivotal role in nicotine-triggered ß cell destruction and glucose intolerance, providing a theoretical basis for targeted prevention and treatment of smoking-induced T2DM.


Assuntos
Senescência Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 2/induzido quimicamente , Células Secretoras de Insulina/efeitos dos fármacos , Nicotina/toxicidade , Animais , Western Blotting , Cálcio/metabolismo , Progressão da Doença , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , beta-Galactosidase/metabolismo
3.
Hum Genet ; 139(5): 657-673, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32219518

RESUMO

GM1-gangliosidosis, a lysosomal storage disorder, is associated with ~ 161 missense variants in the GLB1 gene. Affected patients present with ß-galactosidase (ß-Gal) deficiency in lysosomes. Loss of function in ER-retained misfolded enzymes with missense variants is often due to subcellular mislocalization. Deoxygalactonojirimycin (DGJ) and its derivatives are pharmaceutical chaperones that directly bind to mutated ß-Gal in the ER promoting its folding and trafficking to lysosomes and thus enhancing its activity. An Emirati child has been diagnosed with infantile GM1-gangliosidosis carrying the reported p.D151Y variant. We show that p.D151Y ß-Gal in patient's fibroblasts retained < 1% residual activity due to impaired processing and trafficking. The amino acid substitution significantly affected the enzyme conformation; however, p.D151Y ß-Gal was amenable for partial rescue in the presence of glycerol or at reduced temperature where activity was enhanced with ~ 2.3 and 7 folds, respectively. The butyl (NB-DGJ) and nonyl (NN-DGJ) derivatives of DGJ chaperoning function were evaluated by measuring their IC50s and ability to stabilize the wild-type ß-Gal against thermal degradation. Although NN-DGJ showed higher affinity to ß-Gal, it did not show a significant enhancement in p.D151Y ß-Gal activity. However, NB-DGJ promoted p.D151Y ß-Gal maturation and enhanced its activity up to ~ 4.5% of control activity within 24 h which was significantly increased to ~ 10% within 6 days. NB-DGJ enhancement effect was sustained over 3 days after washing it out from culture media. We therefore conclude that NB-DGJ might be a promising therapeutic chemical chaperone in infantile GM1 amenable variants and therefore warrants further analysis for its clinical applications.


Assuntos
1-Desoxinojirimicina/farmacologia , Fibroblastos/metabolismo , Gangliosidose GM1/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , beta-Galactosidase/metabolismo , 1-Desoxinojirimicina/química , Pré-Escolar , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Gangliosidose GM1/tratamento farmacológico , Gangliosidose GM1/patologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Masculino , Chaperonas Moleculares/farmacologia , Proteínas Mutantes/química , Proteínas Mutantes/genética , Conformação Proteica , Transporte Proteico , beta-Galactosidase/química , beta-Galactosidase/genética
4.
Chem Commun (Camb) ; 56(18): 2731-2734, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32022000

RESUMO

We herein develop two ß-galactosidase (ß-Gal) activatable NIR fluorescent probes for visualizing ovarian cancers. Particularly, probe BOD-M-ßGal produced NIR-II emission light at 900-1300 nm upon ß-Gal activation. By using our activatable and target specific NIR-II probe for deep-tissue imaging of ß-Gal overexpressed ovarian cancer cells, rapid and accurate imaging of ovarian tumors in nude mice was achieved.


Assuntos
Corantes Fluorescentes/química , Imagem Óptica , Neoplasias Ovarianas/diagnóstico por imagem , beta-Galactosidase/química , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Corantes Fluorescentes/metabolismo , Células Endoteliais da Veia Umbilical Humana/química , Humanos , Raios Infravermelhos , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/metabolismo , Neoplasias Ovarianas/metabolismo , beta-Galactosidase/metabolismo
5.
Nucleic Acids Res ; 48(5): e28, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31980824

RESUMO

We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive ß-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased ß-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.


Assuntos
Bioensaio , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Transcrição Genética , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Sistemas CRISPR-Cas , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Repressores Lac/deficiência , Repressores Lac/genética , Plasmídeos/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
6.
Biotechnol Adv ; 39: 107465, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31689470

RESUMO

ß-Galactosidases, an important class of glycosidases, naturally catalyze the hydrolysis of ß-galactosidic bonds in oligosaccharides and polysaccharides. Traditionally, these enzymes have been used to degrade lactose in dairy products, which are beneficial for lactose-intolerant people. Attractively, ß-galactosidases exhibit glycosyl transfer activity under certain conditions in vitro. They are capable of synthesizing carbohydrates from cheap starting substrates in a facile, efficient, and environment-friendly way. The condensation of lactose into the well-known prebiotic galacto-oligosaccharides by ß-galactosidases has become a key aspect of the industrial interest in the synthetic activity in recent years. At present, the transglycosylation activity of these enzymes has been greatly extended. It can be used not only in building glycan blocks of crucial glycoconjugates to elucidate their biological functions, but also in glycosylation of vital molecules, which have been applied in food, medicine and cosmetic industries to improve solubility, stability and bioactivity. Further molecular engineering of ß-galactosidases has significantly improved their synthetic activity, expanded the substrate spectrum and made them more powerful in carbohydrate synthesis. This review covers the classification, structure and mechanism of ß-galactosidases, galactosylation reactions catalyzed by these enzymes, and various strategies of enzyme engineering, with an emphasis on recent advances.


Assuntos
beta-Galactosidase/metabolismo , Galactose , Lactose , Oligossacarídeos , Prebióticos
7.
J Enzyme Inhib Med Chem ; 35(1): 42-49, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31656110

RESUMO

Matricaria chamomilla L. contains antioxidant flavonoids that can have their bioactivity enhanced by enzymatic hydrolysis of specific glycosyl groups. This study implements an untargeted metabolomics approach based on ultra-performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight mass spectrometry technique operating in MSE mode (UPLC-QTOF-MSE) and spectrophotometric analysis of chamomile aqueous infusions, before and after hydrolysis by hesperidinase and ß-galactosidase. Several phenolic compounds were altered in the enzymatically treated infusion, with the majority being flavonoid derivatives of apigenin, esculetin, and quercetin. Although enzymatically modifying the infusion only led to a small increase in antioxidant activity (DPPH• method), its inhibitory effect on pancreatic lipase was of particular interest. The enzymatically treated infusion exhibited a greater inhibitory effect (EC50 of 35.6 µM) than unmodified infusion and kinetic analysis suggested mixed inhibition of pancreatic lipase. These results are of great relevance due to the potential of enzymatically treated functional foods in human health.


Assuntos
Antioxidantes/farmacologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Lipase/antagonistas & inibidores , Matricaria/química , Antioxidantes/química , Antioxidantes/metabolismo , Compostos de Bifenilo/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Glicosídeo Hidrolases/metabolismo , Humanos , Hidrólise , Lipase/metabolismo , Matricaria/metabolismo , Metabolômica , Estrutura Molecular , Picratos/antagonistas & inibidores , Relação Estrutura-Atividade , beta-Galactosidase/metabolismo
8.
Food Chem ; 303: 125388, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31454757

RESUMO

Saponins are known for their bioactive and surfactant properties, showing applicability to the food, cosmetic and pharmaceutical industries. This work evaluated the saponins effects on Kluyveromyces lactis ß-galactosidase activity and correlated these changes to the protein structure. Enzyme kinetic was evaluated by catalytic assay, protein structure was studied by circular dichroism and fluorescence, and isothermal titration calorimetry was used to evaluate the interactions forces. In vitro enzymatic activity assays indicated an increase in the protein activity due to the saponin-protein interaction. Circular dichroism shows that saponin changes the ß-galactosidase secondary structure, favoring its protein-substrate interaction. Besides, changes in protein microenvironment due to the presence of saponin was observed by fluorescence spectroscopy. Isothermal titration calorimetry analyses suggested that saponins increased the affinity of ß-galactosidase with the artificial substrate o-nitrophenyl-ß-galactoside. The increase in the enzyme activity by saponins, demonstrated here, is important to new products development in food, cosmetic, and pharmaceutical industries.


Assuntos
Kluyveromyces/enzimologia , Saponinas/farmacologia , beta-Galactosidase/efeitos dos fármacos , Calorimetria , Dicroísmo Circular , Cinética , Nitrofenilgalactosídeos/metabolismo , Casca de Planta/química , Estrutura Secundária de Proteína , Quillaja/química , Espectrometria de Fluorescência , beta-Galactosidase/metabolismo
9.
Food Chem ; 305: 125481, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31525592

RESUMO

Prebiotics are rising in interest in commercial scale productions due to increasing health awareness of consumers. Under bio-economic aspects, sweet and acid whey provide a suitable feed medium for the enzymatic generation of prebiotic lactulose. Since whey has a broad variation in composition, the influence of the feed composition on the concentration of generated lactulose was investigated. The influence of lactose and fructose concentration as well as enzymatic activity of two commercially available ß-galactosidases were investigated. The results were evaluated via response surface analysis with a quadratic model containing pairwise interaction terms. The optimal feed composition yielding a theoretical maximal amount of lactulose was determined as 1.28 or 0.74 mol/kg fructose and 0.17 or 0.19 mol/kg lactose with an enzymatic activity of 2.0 or 2.8 µkat/kg for acid (pH 4.4) or sweet (pH 6.6) whey. Furthermore, the major reaction product was isolated and subsequently, the structural identity was elucidated and verified via extensive NMR analysis.


Assuntos
Lactulose/metabolismo , Soro do Leite/metabolismo , beta-Galactosidase/metabolismo , Frutose/metabolismo , Concentração de Íons de Hidrogênio , Isomerismo , Lactose/metabolismo , Lactulose/isolamento & purificação , Espectroscopia de Ressonância Magnética , Soro do Leite/química
10.
J Dairy Sci ; 103(1): 166-171, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31704010

RESUMO

The ability to use lactose is critical for the application of Streptococcus thermophilus in fermented dairy products. Most studies have evaluated the use of lactose of S. thermophilus by measuring lactose utilization, but its correlation with ß-galactosidase and urease has rarely been investigated. In this study, 10 strains of S. thermophilus isolated from fermented yak milk exhibited a diversity of ß-galactosidase and urease activities, growth, and acid production in de Man, Rogosa, and Sharpe-lactose. Among the strains, 15G5 possessed the highest ß-galactosidase activity and showed the highest cell growth, lactic acid production, and titratable acidity during fermentation. In contrast, 7G10, with the weakest ß-galactosidase activity, produced the lowest lactic acid content and change in titratable acidity. Further investigation indicated that ß-galactosidase activity of S. thermophilus showed significant positive correlations with the growth of cell densities, the production of lactic acid, and titratable acidity, and urease activity of S. thermophilus showed a significant correlation with the use of lactose and the production of lactic acid and acetaldehyde. These findings suggest that the differences of ß-galactosidase and urease activities are essential for the performance in the lactose metabolism, growth, and acid production of S. thermophilus, providing new insights into strain selection and application.


Assuntos
Ácido Láctico/metabolismo , Lactose/metabolismo , Leite/enzimologia , Streptococcus thermophilus/enzimologia , Urease/metabolismo , beta-Galactosidase/metabolismo , Animais , Metabolismo dos Carboidratos , Fermentação
11.
Appl Microbiol Biotechnol ; 104(3): 1135-1148, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31853563

RESUMO

Three recombinant ß-galactosidases (BGALs; PcBGAL35A, PcBGAL35B, and PcGALX35C) belonging to the glycoside hydrolase (GH) family 35 derived from Penicillium chrysogenum 31B were expressed using Pichia pastoris and characterized. PcBGAL35A showed a unique substrate specificity that has not been reported so far. Based on the results of enzymological tests and 1H-nuclear magnetic resonance, PcBGAL35A was found to hydrolyze ß-1,4-galactosyl residues linked to L-rhamnose in rhamnogalacturonan-I (RG-I) of pectin, as well as p-nitrophenyl-ß-D-galactopyranoside and ß-D-galactosyl oligosaccharides. PcBGAL35B was determined to be a common BGAL through molecular phylogenetic tree and substrate specificity analysis. PcGALX35C was found to have similar catalytic capacities for the ß-1,4-galactosyl oligomer and polymer. Furthermore, PcGALX35C hydrolyzed RG-I-linked ß-1,4-galactosyl oligosaccharide side chains with a degree of polymerization of 2 or higher in pectin. The amino acid sequence similarity of PcBGAL35A was approximately 30% with most GH35 BGALs, whose enzymatic properties have been characterized. The amino acid sequence of PcBGAL35B was approximately 80% identical to those of BGALs from Penicillium sp. The amino acid sequence of PcGALX35C was classified into the same phylogenetic group as PcBGAL35A. Pfam analysis revealed that the three BGALs had five domains including a catalytic domain. Our findings suggest that PcBGAL35A and PcGALX35C are enzymes involved in the degradation of galactosylated RG-I in pectin. The enzymes characterized in this study may be applied for products that require pectin processing and for the structural analysis of pectin.


Assuntos
Pectinas/metabolismo , Penicillium chrysogenum/enzimologia , beta-Galactosidase/metabolismo , Sequência de Aminoácidos , Hidrólise , Penicillium chrysogenum/genética , Filogenia , Pichia/genética , Especificidade por Substrato , beta-Galactosidase/genética
12.
Carbohydr Polym ; 229: 115549, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31826450

RESUMO

An acid-extracted polysaccharide from alchohol-insoluble solids of leek was obtained. The sugar composition indicated that galactose and galacturonic acid were the major sugars, followed by small amounts of rhamnose and arabinose. The fraction contained a relatively high methyl-esterified homogalacturonan next to rhamnogalacturonan type I decorated with galactose-rich side chains. The fraction consisted of three high Mw populations, covering the range of 10-100 kDa. Enzymatic fingerprinting was performed with HG/RG-I degrading enzymes to elucidate the structure. The oligomers were analysed using LC-HILIC-MS, HPAEC, and MALDI-TOF MS. The data revealed the presence of GalA sequences, having different patterns of methyl-esterification, RG-I composed of unbranched segments and segments heavily substituted with ß-(1→4)-linked galactan chains of varying length. The rheological study showed the shear-thinning, weak thixotropic, anti-thixotropic, and non-Newtonian behavior of the polysaccharide. The pectin exhibited higher water holding capacity than oil-holding capacity and the fraction did form stable foams at high concentration.


Assuntos
Galactose/metabolismo , Cebolas/metabolismo , Polissacarídeos/química , Proteínas de Bactérias/metabolismo , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Esterificação , Ácidos Hexurônicos/metabolismo , Peso Molecular , Polissacarídeos/metabolismo , Reologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , beta-Galactosidase/metabolismo
13.
Int J Mol Sci ; 21(1)2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31878234

RESUMO

Calorie restriction can extend lifespan by increasing intracellular nicotinamide adenine dinucleotide (NAD+), thereby upregulating the activity of sirtuins (Caenorhabditis elegans Sir-2.1; human SIRT1). Nicotinic acid (NA) can be metabolized to NAD+; however, the calorie restriction mimetic (CRM) potential of NA is unclear. This study explored the ability and mechanism of NA to extend the lifespan of human Hs68 cells and C. elegans. We found that NA can efficiently increase the intracellular NAD+ levels in Hs68 cells and C. elegans; however, NA was only able to extend the lifespan of C. elegans. The steady-state NAD+ level in C. elegans was approximately 55 µM. When intracellular NAD+ was increased by a mutation of pme-1 (poly (ADP-ribose) metabolism enzyme 1) or by pretreatment with NAD+ in the medium, the lifespan extension ability of NA disappeared. Additionally, the saturating concentration of NAD+ required by SIRT1 was approximately 200 µM; however, the steady-state concentration of NAD+ in Hs68 cells reached up to 460 µM. These results demonstrate that the lifespan extension ability of NA depends on whether the intracellular level of NAD+ is lower than the sirtuin-saturating concentration in Hs68 cells and in C. elegans. Thus, the CRM potential of NA should be limited to individuals with lower intracellular NAD+.


Assuntos
NAD/metabolismo , Niacina/metabolismo , Sirtuínas/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Restrição Calórica/métodos , Linhagem Celular , Humanos , beta-Galactosidase/metabolismo
14.
J Pediatr ; 215: 152-157.e3, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31761138

RESUMO

OBJECTIVE: To evaluate the clinical presentation of patients with GM1 gangliosidosis and to determine whether specific clinical or biochemical signs could lead to a prompt diagnosis. STUDY DESIGN: We retrospectively analyzed clinical, biochemical, and genetic data of 22 patients with GM1 gangliosidosis from 5 metabolic centers in Germany and Austria. RESULTS: Eight patients were classified as infantile, 11 as late-infantile, and 3 as juvenile form. Delay of diagnosis was 6 ± 2.6 months in the infantile, 2.6 ± 3.79 years in the late-infantile, and 14 ± 3.48 years in the juvenile form. Coarse facial features, cherry red spots, and visceromegaly occurred only in patients with the infantile form. Patients with the late-infantile and juvenile forms presented with variable neurologic symptoms. Seventeen patients presented with dystonia and 14 with dysphagia. Laboratory analysis revealed an increased ASAT concentration (13/20), chitotriosidase activity (12/15), and pathologic urinary oligosaccharides (10/19). Genotype analyses revealed 23 causative or likely causative mutations in 19 patients, 7 of them being novel variants. In the majority, a clear genotype-phenotype correlation was found. CONCLUSIONS: Diagnosis of GM1 gangliosidosis often is delayed, especially in patients with milder forms of the disease. GM1 gangliosidosis should be considered in patients with progressive neurodegeneration and spastic-dystonic movement disorders, even in the absence of visceral symptoms or cherry red spots. ASAT serum concentrations and chitotriosidase activity may be of value in screening for GM1 gangliosidosis.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , DNA/genética , Gangliosidose GM1/genética , Mutação , beta-Galactosidase/genética , Adolescente , Áustria/epidemiologia , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Seguimentos , Gangliosidose GM1/diagnóstico , Gangliosidose GM1/epidemiologia , Genótipo , Alemanha/epidemiologia , Humanos , Incidência , Lactente , Masculino , Fenótipo , Estudos Retrospectivos , Adulto Jovem , beta-Galactosidase/metabolismo
15.
Chem Commun (Camb) ; 55(99): 15000-15003, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31777880

RESUMO

Reported herein is a novel p-quinone methide-based self-immobilizing fluorogenic probe for the visualization of ß-galactosidase activities in live cells. This easily prepared imaging reagent massively increases the fluorescence intensity and covalently links to the activation site with high efficiency upon enzymatic trigger.


Assuntos
Corantes Fluorescentes/metabolismo , Indolquinonas/metabolismo , beta-Galactosidase/metabolismo , Células HEK293 , Células HeLa , Humanos , Metilação , Espectrometria de Fluorescência
16.
Anal Bioanal Chem ; 411(30): 7957-7966, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31732786

RESUMO

ß-Galactosidase (ß-gal) has captured the attention of biologists, chemists, and medical researchers as an important biomarker for cell senescence and primary ovarian cancer. Therefore, many fluorescent probes with visible light emission have been developed for the detection and imaging of ß-gal in living cells. However, near-infrared (NIR) ratiometric probes are more suitable for bioimaging because near-infrared light can effectively avoid the interference of autofluorescence and the ratiometric approach can improve sensitivity and accuracy of the detection. In this work, we designed an NIR ratiometric probe (TMG) for the highly sensitive detection of ß-gal. Using a spontaneous degradation mechanism based on the ICT effect, the change in ratio (F650/F580) exhibited a prominent ß-gal-dependent performance and proved a strong linear response to the activity of ß-gal at an enzyme concentration between 0 and 200 U L-1, with a limit of detection as low as 0.86 U L-1, and the response speed is much faster than the same type of probes previously reported. The probe also revealed an excellent biocompatibility and a large Stokes shift. Moreover, fluorescence microscopy imaging experiments confirmed that this probe could be successfully used for the detection of endogenous ß-gal in living cells. Graphical abstract.


Assuntos
Corantes Fluorescentes/química , Análise Espectral/métodos , beta-Galactosidase/metabolismo , Animais , Linhagem Celular , Humanos , Limite de Detecção , Microscopia de Fluorescência
17.
Nat Commun ; 10(1): 5002, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676820

RESUMO

Metal-organic frameworks (MOFs) have recently garnered consideration as an attractive solid substrate because the highly tunable MOF framework can not only serve as an inert host but also enhance the selectivity, stability, and/or activity of the enzymes. Herein, we demonstrate the advantages of using a mechanochemical strategy to encapsulate enzymes into robust MOFs. A range of enzymes, namely ß-glucosidase, invertase, ß-galactosidase, and catalase, are encapsulated in ZIF-8, UiO-66-NH2, or Zn-MOF-74 via a ball milling process. The solid-state mechanochemical strategy is rapid and minimizes the use of organic solvents and strong acids during synthesis, allowing the encapsulation of enzymes into three prototypical robust MOFs while maintaining enzymatic biological activity. The activity of encapsulated enzyme is demonstrated and shows increased resistance to proteases, even under acidic conditions. This work represents a step toward the creation of a suite of biomolecule-in-MOF composites for application in a variety of industrial processes.


Assuntos
Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Estruturas Metalorgânicas/química , Metais/química , Biocatálise , Catalase/química , Catalase/metabolismo , Catalase/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Enzimas Imobilizadas/ultraestrutura , Estruturas Metalorgânicas/síntese química , Microscopia Eletrônica de Varredura , Difração de Pó , beta-Frutofuranosidase/química , beta-Frutofuranosidase/metabolismo , beta-Frutofuranosidase/ultraestrutura , beta-Galactosidase/química , beta-Galactosidase/metabolismo , beta-Galactosidase/ultraestrutura , beta-Glucosidase/química , beta-Glucosidase/metabolismo , beta-Glucosidase/ultraestrutura
18.
Invest Ophthalmol Vis Sci ; 60(14): 4643-4651, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31682715

RESUMO

Purpose: Conjunctivochalasis (CCH) is a common ocular disease and has received extensive attention recently. However, its exact pathogenesis remains largely unknown. Owing to the high morbidity of CCH in older people, this study aimed to investigate whether cellular senescence contributes to CCH progression and the underlying mechanism. Methods: Loose conjunctival tissues from CCH patients (n = 13) and normal conjunctival tissues from age-matched persons (n = 12) were obtained and the fibroblasts were separately induced and obtained. Cellular senescence, and the expression of senescence-associated genes (p53 and p21) and p38 in CCH conjunctival tissues and normal controls, were determined by senescence-associated ß-galactosidase (SA-ß-Gal) staining and quantitative (q)RT-PCR, respectively. To explore the effects of p38 on cellular senescence in CCH fibroblasts, small interfering RNA (siRNA) targeting p38 (siP38) and p38-specific inhibitor SB203580 was performed in CCH fibroblasts. Then, cellular senescence, cell viability, reactive oxygen species (ROS) production, and gene expression were detected according to the corresponding methods. Results: CCH conjunctival tissues had significantly more senescent cells, evidenced by more SA-ß-Gal-positive cells, and higher expression of senescence-associated genes (p53 and p21) and p38. CCH fibroblasts transfected with siP38 or treated with SB203580 had obviously reduced numbers of senescent cells, decreased ROS production, and increased cell viability, as well as reduced expression of senescence-associated genes. Meanwhile, blocking p38 signaling decreased the expression of p53 and p21. Conclusions: Therefore, these findings indicate that cellular senescence might be a causative factor for CCH. P38 signaling might play an important role in the progress of cellular senescence in CCH fibroblasts via manipulation of p53/p21 signaling.


Assuntos
Senescência Celular/fisiologia , Doenças da Túnica Conjuntiva/enzimologia , Fibroblastos/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Western Blotting , Contagem de Células , Proliferação de Células , Células Cultivadas , Doenças da Túnica Conjuntiva/patologia , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Superóxido Dismutase/metabolismo , beta-Galactosidase/metabolismo
19.
J Microbiol Biotechnol ; 29(11): 1717-1728, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31581381

RESUMO

The gene encoding ß-galactosidase was cloned from Bifidobacterium longum RD47 with combinations of several bifidobacterial promoters and expressed in B. bifidum BGN4. Among the recombinant bifidobacteria, BGN4+G1 showed the highest ß-galactosidase level, for which the hydrolytic activity was continuously 2.5 to 4.2 times higher than that of BGN4 and 4.3 to 9.6 times higher than that of RD47. The ß-galactosidase activity of BGN4+G1 was exceedingly superior to that of any of the other 35 lactic acid bacteria. When commercial whole milk and BGN4+G1 were reacted, BGN4+G1 removed nearly 50% of the lactose in the milk by the 63-h time point, and a final 61% at 93 h. These figures are about twice the lactose removal rate of conventional fermented milk. As for the reaction of commercial whole milk and crude enzyme extract from BGN4+G1, the ß-galactosidase of BGN4+G1 eliminated 51% of the lactose in milk in 2 h. As shown below, we also compared the strengths and characteristics of the strong bifidobacterial promoters reported by previous studies.


Assuntos
Proteínas de Bactérias/genética , Bifidobacterium/genética , Bifidobacterium/metabolismo , beta-Galactosidase/genética , Animais , Proteínas de Bactérias/metabolismo , Bifidobacterium/enzimologia , Clonagem Molecular , Fermentação , Expressão Gênica , Hidrólise , Lactose/metabolismo , Leite/química , Leite/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Galactosidase/metabolismo
20.
Int J Mol Sci ; 20(17)2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31484304

RESUMO

ArthßDG is a dimeric, cold-adapted ß-d-galactosidase that exhibits high hydrolytic and transglycosylation activity. A series of crystal structures of its wild form, as well as its ArthßDG_E441Q mutein complexes with ligands were obtained in order to describe the mode of its action. The ArthßDG_E441Q mutein is an inactive form of the enzyme designed to enable observation of enzyme interaction with its substrate. The resulting three-dimensional structures of complexes: ArthßDG_E441Q/LACs and ArthßDG/IPTG (ligand bound in shallow mode) and structures of complexes ArthßDG_E441Q/LACd, ArthßDG/ONPG (ligands bound in deep mode), and galactose ArthßDG/GAL and their analysis enabled structural characterization of the hydrolysis reaction mechanism. Furthermore, comparative analysis with mesophilic analogs revealed the most striking differences in catalysis mechanisms. The key role in substrate transfer from shallow to deep binding mode involves rotation of the F581 side chain. It is worth noting that the 10-aa loop restricting access to the active site in mesophilic GH2 ßDGs, in ArthßDG is moved outward. This facilitates access of substrate to active site. Such a permanent exposure of the entrance to the active site may be a key factor for improved turnover rate of the cold adapted enzyme and thus a structural feature related to its cold adaptation.


Assuntos
Arthrobacter/enzimologia , Arthrobacter/metabolismo , beta-Galactosidase/metabolismo , Sequência de Aminoácidos , Arthrobacter/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Temperatura Baixa , Hidrólise , beta-Galactosidase/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA