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1.
Adv Exp Med Biol ; 1211: 89-95, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31471820

RESUMO

This study investigated the effects of protein malnutrition and progesterone supplementation on the activities of a spectrum of lysosomal enzymes in tissue fragments of mouse liver and kidney. The working hypothesis was that the known anti-stress action of progesterone could have to do with the inhibition of lysosomes which are engaged in apoptotic and oxidative stress-induced responses. The study investigated the effects of exogenous progesterone in chronically (3 weeks) protein-malnourished (10% protein) mice on the activities of lysosomal hydrolases in liver and kidney tissues. Progesterone was injected intraperitoneally in a dose of 2 µg/g body mass dissolved in a vehicle volume of 10 µL/g body mass during the final 3 days of exposure to either low 10% or standard 16% protein content in the chow. After euthanizing the animals, tissue fragments of liver and kidney assayed for the content of lysosomal enzymes. The results demonstrated the stimulating effect of protein malnutrition on lysosomal activities. We further found, contrary to our hypothesis, that progesterone supplementation during both standard and low-protein conditions enhanced lysosomal activities, particularly acting in concert with protein malnutrition in kidney tissue. The effects were selective concerning both lysosomal enzymes and tissues and of highly variable magnitude. Nonetheless, we believe we have shown that progesterone assists protein malnutrition in stimulation of lysosomal enzymes, which suggests the possibility of the hormone's engagement in cleansing the cellular milieu in disorders consisting of accumulation of toxic molecules.


Assuntos
Hidrolases/metabolismo , Lisossomos/enzimologia , Progesterona/administração & dosagem , Deficiência de Proteína/enzimologia , Animais , Suplementos Nutricionais , Rim/enzimologia , Fígado/enzimologia , Camundongos
2.
Chemistry ; 25(50): 11635-11640, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31368214

RESUMO

Disulfide-containing detergents (DCDs) are introduced, which contain a disulfide bond in the hydrophobic tail. DCDs form smaller micelles than corresponding detergents with linear hydrocarbon chains, while providing good solubilization and reconstitution of membrane proteins. The use of this new class of detergents in structural biology is illustrated with solution NMR spectra of the human G protein-coupled receptor A2A AR, which is an α-helical protein, and the ß-barrel protein OmpX from E. coli.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Detergentes/química , Proteínas de Escherichia coli/química , Hidrolases/química , Receptor A2A de Adenosina/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Dissulfetos/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Humanos , Hidrolases/metabolismo , Micelas , Ressonância Magnética Nuclear Biomolecular , Estabilidade Proteica , Receptor A2A de Adenosina/metabolismo
3.
Enzyme Microb Technol ; 129: 109356, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31307580

RESUMO

Kumamolisin from Alicyclobacillus sendaiensis strain NTAP-1 is a serine protease with collagenase activity. After molecular engineering, a kumamolisin mutant, named Kuma030, was obtained with high proteolytic activity against gluten, which might cause celiac disease. Kuma030 exhibited its potential application in industrial and medicine, while challenges remained of its large-scale purification and production. In the studies here, we successfully overexpressed the Kuma030 in E. coli BL21 (DE3) by anchoring a SUMO (Small Ubiquitin-like Modifier) fusion protein at its N-terminal end. In addition, a fast protein purification procedure was developed according to the acidophilic and thermophilic properties of Alicyclobacillus sendaiensis. After a simple acid treatment followed by a heat treatment, a total of 9.9 mg functional Kuma030 was quickly obtained form 1 L LB media culture. This purified Kuma030 was confirmed to be functional to cleave the PQ sequences in a designed protein substrate, and the gluten in actual food samples, such as whole wheat bread and beer, in a fast manner. Our studies provided an efficient strategy for the overexpression and purification of functional Kuma030 in E. coli, which might expand its broad practical applications.


Assuntos
Alicyclobacillus/enzimologia , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Glutens/metabolismo , Hidrolases/metabolismo , Alicyclobacillus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Estabilidade Enzimática , Escherichia coli/genética , Temperatura Alta , Hidrolases/química , Hidrolases/genética
4.
Curr Microbiol ; 76(10): 1161-1167, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31278426

RESUMO

The haloalkane dehalogenase DhaA can degrade sulfur mustard (2,2'-dichlorethyl sulfide; also known by its military designation HD) in a rapid and environmentally safe manner. However, DhaA is sensitive to temperature and pH, which limits its applications in natural or harsh environments. Spore surface display technology using resistant spores as a carrier to ensure enzymatic activity can reduce production costs and extend the range of applications of DhaA. To this end, we cloned recombinant Bacillus subtilis spores pHY300PLK-cotg-dhaa-6his/DB104(FH01) for the delivery of DhaA from Rhodococcus rhodochrous NCIMB 13064. A dot blotting showed that the fusion protein CotG-linker-DhaA accounted for 0.41% ± 0.03% (P < 0.01) of total spore coat proteins. Immunofluorescence analyses confirmed that DhaA was displayed on the spore surface. The hydrolyzing activity of DhaA displayed on spores towards the HD analog 2-chloroethyl ethylsulfide was 1.74 ± 0.06 U/mL (P < 0.01), with a specific activity was 0.34 ± 0.04 U/mg (P < 0.01). This is the first demonstration that DhaA displayed on the surface of B. subtilis spores retains enzymatic activity, which suggests that it can be used effectively in real-world applications including bioremediation of contaminated environments.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Hidrolases/metabolismo , Esporos Bacterianos/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Estabilidade Enzimática , Expressão Gênica , Hidrolases/genética , Gás de Mostarda/análogos & derivados , Gás de Mostarda/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rhodococcus/enzimologia , Rhodococcus/genética , Esporos Bacterianos/genética , Especificidade por Substrato
5.
Cell Mol Life Sci ; 76(20): 3987-4008, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31227845

RESUMO

Polyamines (PAs) are essential organic polycations for cell viability along the whole phylogenetic scale. In mammals, they are involved in the most important physiological processes: cell proliferation and viability, nutrition, fertility, as well as nervous and immune systems. Consequently, altered polyamine metabolism is involved in a series of pathologies. Due to their pathophysiological importance, PA metabolism has evolved to be a very robust metabolic module, interconnected with the other essential metabolic modules for gene expression and cell proliferation/differentiation. Two different PA sources exist for animals: PA coming from diet and endogenous synthesis. In the first section of this work, the molecular characteristics of PAs are presented as determinant of their roles in living organisms. In a second section, the metabolic specificities of mammalian PA metabolism are reviewed, as well as some obscure aspects on it. This second section includes information on mammalian cell/tissue-dependent PA-related gene expression and information on crosstalk with the other mammalian metabolic modules. The third section presents a synthesis of the physiological processes described as modulated by PAs in humans and/or experimental animal models, the molecular bases of these regulatory mechanisms known so far, as well as the most important gaps of information, which explain why knowledge around the specific roles of PAs in human physiology is still considered a "mysterious" subject. In spite of its robustness, PA metabolism can be altered under different exogenous and/or endogenous circumstances so leading to the loss of homeostasis and, therefore, to the promotion of a pathology. The available information will be summarized in the fourth section of this review. The different sections of this review also point out the lesser-known aspects of the topic. Finally, future prospects to advance on these still obscure gaps of knowledge on the roles on PAs on human physiopathology are discussed.


Assuntos
Fertilidade/fisiologia , Gastroenteropatias/metabolismo , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Poliaminas/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Animais , Carboxiliases/genética , Carboxiliases/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Gastroenteropatias/genética , Gastroenteropatias/fisiopatologia , Regulação da Expressão Gênica , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Mamíferos , Neoplasias/genética , Neoplasias/fisiopatologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/fisiopatologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Poliaminas/administração & dosagem , Poliaminas/farmacologia
6.
Chem Commun (Camb) ; 55(50): 7175-7178, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31162503

RESUMO

The identification and removal of senescent cells is very important to improve human health and prolong life. In this study, we introduced a novel strategy of ß-galactosidase (ß-Gal) instructed peptide self-assembly to selectively form nanofibers and hydrogels in senescent cells. We demonstrated that the in situ formed nanofibers could alleviate endothelial cell senescence by reducing p53, p21, and p16INK4a expression levels. We also demonstrated that our strategy could selectively remove senescent endothelial cells by inducing cell apoptosis, with an increase in the BAX/BCL-2 ratio and caspase-3 expression. Our study reports the first example of enzyme-instructed self-assembly (EISA) by a sugar hydrolase, which may lead to the development of supramolecular nanomaterials for the diagnosis and treatment of many diseases, such as cancer, and for other applications, such as wound healing and senescence.


Assuntos
Senescência Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Nanofibras , beta-Galactosidase/metabolismo , Regulação da Expressão Gênica , Humanos , Hidrolases/metabolismo , Lipopolissacarídeos/toxicidade
7.
BMC Plant Biol ; 19(1): 233, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159738

RESUMO

BACKGROUND: Auxin conjugates are hydrolyzed to release free auxin to ensure defined cellular auxin levels or gradients within tissues for proper development or response to environmental signals. The auxin concentration in the abscission zone (AZ) is thought to play an important role in mediating the abscission lag phase. RESULTS: In this study, the full cDNA sequences of seven tomato ILR1-like SlILL genes were identified and characterized, All SlILLs were found to have auxin conjugate hydrolysis activity. The effects of different auxin conjugates on abscission identified IAA-Ile as a candidate to determine the auxin conjugate and auxin conjugate hydrolysis functions in abscission. Treatment of pedicel explants with IAA-Ile for different times showed that application before 6 h could effectively delay abscission. IAA-Ile pre-incubation for 2 h was sufficient to inhibit abscission. These results showed that there is not sufficient auxin conjugates in the AZ to inhibit abscission, and the optimal time to inhibit abscission by the application of exogenous auxin conjugates is before 6 h. Treatment with cycloheximide (CHX, a protein biosynthesis inhibitor) indicated that de novo synthesis of auxin conjugate hydrolases is also required to delay abscission. During abscission, SlILL1, 5, and 6 showed abscission-related gene expression patterns, and SlILL1, 3, 5, 6, and 7 showed increasing expression trends, which collectively might contribute to delay abscission. Silencing the expression of SlILL1, 3, 5, 6, and 7 using virus-induced gene silencing showed that SlILL1, 5, and 6 are major mediators of abscission in tomato. CONCLUSIONS: In the process of abscission, auxin inhibition is concentration dependent, and the concentration of auxin in the AZ was regulated by hydrolyzed auxin conjugates. SlILR1, 5, and 6 play a key role in flower pedicel abscission.


Assuntos
Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Hidrolases/metabolismo , Ácidos Indolacéticos/metabolismo , Lycopersicon esculentum/genética , Inibidores da Síntese de Proteínas/farmacologia , Cicloeximida/farmacologia , Flores/genética , Lycopersicon esculentum/enzimologia
8.
Appl Microbiol Biotechnol ; 103(13): 5401-5410, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31065754

RESUMO

Cyanide is toxic to most living organisms. The toxicity of cyanide derives from its ability to inhibit the enzyme cytochrome C oxidase of the electronic transport chain. Despite its high toxicity, several industrial processes rely on the use of cyanide, and considerable amounts of industrial waste must be adequately treated before discharge. Biological treatments for the decontamination of cyanide waste include the use of microorganisms and enzymes. Regarding the use of enzymes, cyanide dihydratase (CynD), which catalyzes the conversion of cyanide into ammonia and formate, is an attractive candidate. Nevertheless, the main impediment to the effective use of this enzyme for the biodegradation of cyanide is the marked intolerance to the alkaline pH at which cyanide waste is kept. In this work, we explore the operational capabilities of whole E. coli cells overexpressing Bacillus pumilus CynD immobilized in three organic polymer matrices: chitosan, polyacrylamide, and agar. Remarkably, the immobilized cells on agar and polyacrylamide retained more than 80% activity even at pH 10 and displayed high reusability. Conversely, the cells immobilized on chitosan were not active. Finally, the suitability of the active complexes for the degradation of free cyanide from a solution derived from the gold processing industry was demonstrated.


Assuntos
Bacillus pumilus/enzimologia , Biodegradação Ambiental , Células Imobilizadas , Hidrolases/genética , Polímeros , Resinas Acrílicas , Ágar , Bacillus pumilus/genética , Proteínas de Bactérias/metabolismo , Quitosana , Cianetos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ouro , Concentração de Íons de Hidrogênio , Hidrolases/metabolismo , Mineração
9.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 324-331, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045561

RESUMO

Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.


Assuntos
Proteínas de Bactérias/química , Hidrocarbonetos Halogenados/química , Hidrolases/química , Psychrobacter/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Temperatura Baixa , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hidrocarbonetos Halogenados/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Psychrobacter/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica
11.
Yakugaku Zasshi ; 139(5): 837-844, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31061351

RESUMO

The hydrolysis activity and expression level of carboxylesterase (CES) in skin were compared with liver and intestine in the same individual of beagle dog and cynomolgus monkey, and their aging effects were studied. CES1 isozymes were mainly present in skin of both animals. The dermal hydrolysis activity was about 10 and 40% of hepatic activity in beagle dog and cynomolgus monkey, respectively. In beagle dog, the hydrolysis activity and the expression level of CES isozyme in liver and skin were nearly the same between 2- and 11-year-old individuals. On the other hand, the dermal hydrolase activity was lower in young individual than in old, in contrast to slight increase of hepatic and intestinal activity in old cynomolgus monkey. These differences by aging in cynomolgus monkey were related to the expression of CES1 proteins and their mRNA. Furthermore, mRNA level of human CES was investigated using total RNA of two individuals (63 and 85 years old). The two individuals showed approximately 2-fold higher expression of hCE2 than hCE1 in human skin.


Assuntos
Envelhecimento/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Intestinos/enzimologia , Fígado/enzimologia , Pele/enzimologia , Idoso de 80 Anos ou mais , Animais , Cães , Feminino , Expressão Gênica , Humanos , Hidrólise , Isoenzimas/genética , Isoenzimas/metabolismo , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
Molecules ; 24(8)2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-31014025

RESUMO

Snakebite envenoming is a serious medical problem in different areas of the world. In Latin America, the major prevalence is due to snakes of the family Viperidae, where rattlesnakes (Crotalus) are included. They produce hemotoxic venom which causes bleeding, tissue degradation and necrosis. Each venom has several enzymatic activities, producing different effects in the envenoming, doing its clinical effects difficult to study. Comparison between venom molecules is also difficult when different techniques are used, and therefore, their identification/characterization using the same methodology is necessary. In this work, a general biochemical characterization in snake venom of serine proteases (SVSP), phospholipases A2 (PLA2), metalloproteases (SVMP) and hyaluronidases (SVH) of Crotalus aquilus (Ca), Crotalus polystictus (Cp) and Crotalus molossus nigrescens (Cmn) was done. Differences in protein pattern, enzyme content and enzymatic activities were observed. All the venoms showed high PLA2 activity, high molecular weight SVSP, and a wide variety of SVMP and SVH forms. Ca and Cp showed the highest enzymatic activities of SVMP and SVSP trypsin-like and chymotrypsin-like, whereas Cmn showed the highest SVH and similar PLA2 activity with Ca. All the venoms showed peptides with similar molecular weight to crotamine-like myotoxins. No previous biochemical characterization of C. aquilus has been reported and there are no previous analyses that include these four protein families in these Crotalus venoms.


Assuntos
Hidrolases/metabolismo , Hidrolases/toxicidade , Venenos de Serpentes/enzimologia , Animais , Crotalus , Metaloproteases/análise , México , Serina Proteases/análise , Especificidade da Espécie
13.
Prep Biochem Biotechnol ; 49(7): 649-658, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31012794

RESUMO

In this study, various levels of ultra-high pressure (UHP) were combined with the enzymatic synthesis of the fructooligosaccharide (FOS) using Pectinex Ultra SP-L and inulinase. The combination enhanced the FOS yields up to 2.5- and 1.5-fold, respectively, compared to atmospheric condition (0.1 MPa). However, the enzymatic reaction was dependent on the levels of pressure, the reaction times, and the initial sucrose concentrations. The combined UHP and inulinase showed that the maximum FOS yield (71.81%) was obtained under UHP at 200 MPa for 20 min with 300 g/L of initial sucrose as a substrate, while the FOS yield (57.13%) using Pectinex Ultra SP-L was obtained under UHP at 300 MPa for 15 min with 600 g/L of initial sucrose as a substrate. The FOS composition produced by Pectinex Ultra SP-L under the UHP was 1-kestose (GF2), nystose (GF3), and 1F-fructofuranosylnystose (GF4), whereas the FOS produced by inulinase composed of only GF2 and GF3. The combined UHP is a useful tool in the industrial application for FOS production. Highlights UHP activated the activity of Pectinex Ultra SP-L yet inactivated inulinase Pressure level, time, and sucrose concentration significantly affect FOS yields under UHP UHP enhanced FOS production with time-saving benefits within 15-20 min.


Assuntos
Aspergillus niger/enzimologia , Glicosídeo Hidrolases/metabolismo , Microbiologia Industrial , Oligossacarídeos/metabolismo , Hidrolases/metabolismo , Hidrólise , Microbiologia Industrial/métodos , Pressão , Sacarose/metabolismo , Trissacarídeos/metabolismo
14.
FEMS Microbiol Rev ; 43(4): 389-400, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980074

RESUMO

Bacteria use dedicated mechanisms to respond adequately to fluctuating environments and to optimize their chances of survival in harsh conditions. One of the major stress responses used by virtually all bacteria relies on the sharp accumulation of an alarmone, the guanosine penta- or tetra-phosphate commonly referred to as (p)ppGpp. Under stressful conditions, essentially nutrient starvation, these second messengers completely reshape the metabolism and physiology by coordinately modulating growth, transcription, translation and cell cycle. As a central regulator of bacterial stress response, the alarmone is also involved in biofilm formation, virulence, antibiotics tolerance and resistance in many pathogenic bacteria. Intracellular concentrations of (p)ppGpp are determined by a highly conserved and widely distributed family of proteins called RelA-SpoT Homologs (RSH). Recently, several studies uncovering mechanisms that regulate RSH activities have renewed a strong interest in this field. In this review, we outline the diversity of the RSH protein family as well as the molecular devices used by bacteria to integrate and transform environmental cues into intracellular (p)ppGpp levels.


Assuntos
Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Hidrolases/genética , Ligases/genética , Proteínas de Bactérias/metabolismo , Hidrolases/metabolismo , Ligases/metabolismo , Estresse Fisiológico/genética , Virulência/genética
15.
Nat Commun ; 10(1): 1717, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30979881

RESUMO

The extreme durability of polyethylene terephthalate (PET) debris has rendered it a long-term environmental burden. At the same time, current recycling efforts still lack sustainability. Two recently discovered bacterial enzymes that specifically degrade PET represent a promising solution. First, Ideonella sakaiensis PETase, a structurally well-characterized consensus α/ß-hydrolase fold enzyme, converts PET to mono-(2-hydroxyethyl) terephthalate (MHET). MHETase, the second key enzyme, hydrolyzes MHET to the PET educts terephthalate and ethylene glycol. Here, we report the crystal structures of active ligand-free MHETase and MHETase bound to a nonhydrolyzable MHET analog. MHETase, which is reminiscent of feruloyl esterases, possesses a classic α/ß-hydrolase domain and a lid domain conferring substrate specificity. In the light of structure-based mapping of the active site, activity assays, mutagenesis studies and a first structure-guided alteration of substrate specificity towards bis-(2-hydroxyethyl) terephthalate (BHET) reported here, we anticipate MHETase to be a valuable resource to further advance enzymatic plastic degradation.


Assuntos
Burkholderiales/enzimologia , Hidrolases/metabolismo , Plásticos/química , Polietilenotereftalatos/química , Biodegradação Ambiental , Domínio Catalítico , Enzimas , Etilenoglicol/química , Fluorometria , Hidrólise , Ligantes , Mutagênese , Mutagênese Sítio-Dirigida , Ácidos Ftálicos/química , Filogenia , Domínios Proteicos , Dobramento de Proteína , Estrutura Secundária de Proteína , Especificidade por Substrato
16.
Food Chem Toxicol ; 129: 229-238, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31034933

RESUMO

Natural compounds are often characterized by high biological activity and sometimes toxicity. This also applies to compounds contained in the herb mistletoe. The objective of this study was to investigate short-term effects (up to 48 h) of mistletoe toxins on mouse hepatocytes. Standardized mistletoe extract Iscador P was given to female mice as a single injection (0.1 mg/kg b.w., 1 mg/kg b.w., or 2 mg/kg b.w). Activities of lysosomal hydrolases: acid phosphatase, cathepsins D and L, N-acetyl-ß-D-hexosaminidase, ß-D-glucuronidase, ß-D-glucosidase and cytosolic proteases: arginine and leucine aminopeptidases were analyzed in the liver fractions 24 and 48 h after the injection. The morphology of hepatocytes was examined by light and transmission electron microscopy. Iscador P caused a decrease in the activity of all lysosomal hydrolases (except cathepsins) in the lysosomal pellet, and an increase in the activity of both aminopeptidases and ß-D-glucuronidase in the cytosol. However, despite membranotropic properties of the viscotoxins, we did not find a significant labilising effect on the lysosomal membranes. Only ß-D-glucuronidase activity was relocated to the supernatant of lysosomal fraction. Microscopic examinations revealed that hepatocyte mitochondria were enlarged and increased in number, whereas the surface of the rough endoplasmic reticulum was decreased significantly.


Assuntos
Fígado/efeitos dos fármacos , Erva-de-Passarinho/química , Toxinas Biológicas/toxicidade , Animais , Citosol/enzimologia , Feminino , Hepatócitos/efeitos dos fármacos , Hidrolases/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Camundongos
17.
Artif Cells Nanomed Biotechnol ; 47(1): 1149-1172, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30942100

RESUMO

In this review, the concept and various strategies in molecular imprinting is discussed briefly. How the concept of transition state analogue can be used to design a template to prepare catalytic imprinted polymers is described in detail. The use of the "bait and switch" approach and alternative covalent template strategies show how functional groups which assist in the catalytic properties can be assembled within the imprint. Thus, there are so many reports on P catalyzed reactions. Owing to their advantageous properties over natural biological recognition agents, molecularly imprinted polymers (MIPs) therefore offer great potential for various applications.


Assuntos
Materiais Biomiméticos/química , Materiais Biomiméticos/síntese química , Desenho de Drogas , Hidrolases/metabolismo , Polímeros/química , Polímeros/síntese química , Catálise , Técnicas de Química Sintética
18.
BMC Evol Biol ; 19(1): 83, 2019 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-30917781

RESUMO

BACKGROUND: The genus Streptococcus comprises pathogens that strongly influence the health of humans and animals. Genome sequencing of multiple Streptococcus strains demonstrated high variability in gene content and order even in closely related strains of the same species and created a newly emerged object for genomic analysis, the pan-genome. Here we analysed the genome evolution of 25 strains of Streptococcus suis, 50 strains of Streptococcus pyogenes and 28 strains of Streptococcus pneumoniae. RESULTS: Fractions of the pan-genome, unique, periphery, and universal genes differ in size, functional composition, the level of nucleotide substitutions, and predisposition to horizontal gene transfer and genomic rearrangements. The density of substitutions in intergenic regions appears to be correlated with selection acting on adjacent genes, implying that more conserved genes tend to have more conserved regulatory regions. The total pan-genome of the genus is open, but only due to strain-specific genes, whereas other pan-genome fractions reach saturation. We have identified the set of genes with phylogenies inconsistent with species and non-conserved location in the chromosome; these genes are rare in at least one species and have likely experienced recent horizontal transfer between species. The strain-specific fraction is enriched with mobile elements and hypothetical proteins, but also contains a number of candidate virulence-related genes, so it may have a strong impact on adaptability and pathogenicity. Mapping the rearrangements to the phylogenetic tree revealed large parallel inversions in all species. A parallel inversion of length 15 kB with breakpoints formed by genes encoding surface antigen proteins PhtD and PhtB in S. pneumoniae leads to replacement of gene fragments that likely indicates the action of an antigen variation mechanism. CONCLUSIONS: Members of genus Streptococcus have a highly dynamic, open pan-genome, that potentially confers them with the ability to adapt to changing environmental conditions, i.e. antibiotic resistance or transmission between different hosts. Hence, integrated analysis of all aspects of genome evolution is important for the identification of potential pathogens and design of drugs and vaccines.


Assuntos
Variação Antigênica/genética , Evolução Biológica , Transferência Genética Horizontal , Seleção Genética , Streptococcus/genética , Animais , Sequência Conservada/genética , DNA Intergênico , Fluxo Gênico , Ontologia Genética , Rearranjo Gênico/genética , Genes Bacterianos , Tamanho do Genoma , Humanos , Hidrolases/metabolismo , Nucleotídeos/genética , Filogenia , Deleção de Sequência , Especificidade da Espécie , Streptococcus pneumoniae/genética , Virulência/genética
19.
World J Microbiol Biotechnol ; 35(4): 60, 2019 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-30919119

RESUMO

Acidithiobacillus ferrooxidans is a gram-negative, autotrophic and rod-shaped bacterium. It can gain energy through the oxidation of Fe(II) and reduced inorganic sulfur compounds for bacterial growth when oxygen is sufficient. It can be used for bio-leaching and bio-oxidation and contributes to the geobiochemical circulation of metal elements and nutrients in acid mine drainage environments. The iron and sulfur oxidation pathways of A. ferrooxidans play key roles in bacterial growth and survival under extreme circumstances. Here, the electrons transported through the thermodynamically favourable pathway for the reduction to H2O (downhill pathway) and against the redox potential gradient reduce to NAD(P)(H) (uphill pathway) during the oxidation of Fe(II) were reviewed, mainly including the electron transport carrier, relevant operon and regulation of its expression. Similar to the electron transfer pathway, the sulfur oxidation pathway of A. ferrooxidans, related genes and operons, sulfur oxidation mechanism and sulfur oxidase system are systematically discussed.


Assuntos
Acidithiobacillus/enzimologia , Acidithiobacillus/metabolismo , Ferro/metabolismo , Enxofre/metabolismo , Acidithiobacillus/genética , Acidithiobacillus/crescimento & desenvolvimento , Azurina/metabolismo , Transporte Biológico Ativo , Citocromos c/metabolismo , Dioxigenases/metabolismo , Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Hidrolases/metabolismo , Redes e Vias Metabólicas/genética , Óperon/genética , Oxirredução , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Oxigênio/metabolismo , Compostos de Enxofre/metabolismo
20.
Mucosal Immunol ; 12(3): 761-771, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30710097

RESUMO

Peptidyl arginine deiminase-4 (PAD4) is indispensable for generation of neutrophil extracellular traps (NETs), which can provide antimicrobial effects during host innate immune response; however, the role of PAD4 against gastrointestinal infection is largely unknown. Herein, we challenged PAD4-deficient (Pad4-/-) mice and wild-type (WT) littermates with Citrobacter rodentium (CR), and investigated bacteria clearance and gut pathology. Luminal colonization of CR in Pad4-/- mice peaked between 11-14 days post-infection, whereas WT mice suppressed the infection by 14 days. We demonstrated that Pad4-/- mice were unable to form NETs, whereas WT mice showed increased NETs formation in the colon during infection. Pad4-/- mice showed aggravated CR-associated inflammation as indicated by elevated systemic and colonic pro-inflammatory markers. Histological analysis revealed that transmissible colonic hyperplasia, goblet cell depletion, and apoptotic cell death were more pronounced in the colon of CR-infected Pad4-/- mice. Treating WT mice with deoxyribonuclease I, which can disrupt NETs generation, recapitulated the exacerbated CR infection and gut pathology associated with the loss of PAD4. Administration of the PAD4 inhibitor, Cl-amidine also aggravated CR infection, but to a lesser extent. Taken together, our findings highlight the importance of PAD4 in the mucosal clearance of CR and in resolving gut-associated inflammation.


Assuntos
Citrobacter rodentium/fisiologia , Colo/patologia , Infecções por Enterobacteriaceae/imunologia , Armadilhas Extracelulares/metabolismo , Hidrolases/metabolismo , Inflamação/imunologia , Intestinos/imunologia , Neutrófilos/imunologia , Animais , Carga Bacteriana , Desoxirribonuclease I/administração & dosagem , Hidrolases/genética , Imunidade Inata , Intestinos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
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