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1.
Materials (Basel) ; 16(5)2023 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-36902966

RESUMO

In this study, hydroxypropyl methylcellulose (HPMC) was mixed with particle-type xenografts, derived from two different species (bovine and porcine), to increase the manipulability of bone grafts and compare the bone regeneration ability. Four circular defects with a diameter of 6 mm were formed on each rabbit calvaria, and the defects were randomly divided into three groups: no treatment (control group), HPMC-mixed bovine xenograft (Bo-Hy group), and HPMC-mixed porcine xenograft (Po-Hy group). At eight weeks, micro-computed tomography (µCT) scanning and histomorphometric analyses were performed to evaluate new bone formation within the defects. The results revealed that the defects treated with the Bo-Hy and the Po-Hy showed higher bone regeneration than the control group (p < 0.05), while there was no significant difference between the two xenograft groups (p > 0.05). Within the limitations of the present study, there was no difference in new bone formation between porcine and bovine xenografts with HPMC, and bone graft material was easily moldable with the desired shape during surgery. Therefore, the moldable porcine-derived xenograft with HPMC used in this study could be a promising substitute for the currently used bone grafts as it exhibits good bone regeneration ability for bony defects.

2.
Front Microbiol ; 13: 945023, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35958135

RESUMO

Effective antibacterial substances of Aralia continentalis have anti-biofilm and bactericidal activity to the oral pathogen Streptococcus mutans. In this study, three compounds extracted from A. continentalis were identified as acanthoic acid, continentalic acid, and kaurenoic acid by NMR and were further investigated how these diterpenoids affect the physiology of the S. mutans. When S. mutans was exposed to individual or mixed fraction of diterpenoids, severe growth defects and unique morphology were observed. The proportion of unsaturated fatty acids in the cell membrane was increased compared to that of saturated fatty acids in the presence of diterpenoids. Genome-wide gene expression profiles with RNA-seq were compared to reveal the mode of action of diterpenoids. Streptococcus mutans commonly enhanced the expression of 176 genes in the presence of the individual diterpenoids, whereas the expression of 232 genes was considerably reduced. The diterpenoid treatment modulated the expression of genes or operon(s) involved in cell membrane synthesis, cell division, and carbohydrate metabolism of S. mutans. Collectively, these findings provide novel insights into the antibacterial effect of diterpenoids to control S. mutans infection, which causes human dental caries.

3.
Molecules ; 26(22)2021 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-34833935

RESUMO

Aralia continentalis has been used in Korea as a folk remedy for arthralgia, rheumatism, and inflammation. However, its anti-lymphoma effect remains uncharacterized. Here, we demonstrate that A. continentalis extract and its three diterpenes efficiently kill B-lymphoma cells. Our in vitro and in vivo results suggest that the cytotoxic activities of continentalic acid, a major diterpene from A. continentalis extract, are specific towards cancer cells while leaving normal murine cells and tissues unharmed. Mechanistically, continentalic acid represses the expression of pro-survival Bcl-2 family members, such as Mcl-1 and Bcl-xL. It dissociates the mitochondrial membrane potential, leading to the stimulation of effector caspase 3/7 activities and, ultimately, cell death. Intriguingly, this agent therapeutically synergizes with roflumilast, a pan-PDE4 inhibitor that has been successfully repurposed for the treatment of aggressive B-cell malignancies in recent clinical tests. Our findings unveiled that A. continentalis extract and three of the plant's diterpenes exhibit anti-cancer activities. We also demonstrate the synergistic inhibitory effect of continentalic acid on the survival of B-lymphoma cells when combined with roflumilast. Taken in conjunction, continentalic acid may hold significant potential for the treatment of B-cell lymphoma.


Assuntos
Antineoplásicos/farmacologia , Diterpenos/farmacologia , Linfoma de Células B/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Aralia/química , Linhagem Celular Tumoral , Humanos , Linfoma de Células B/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Extratos Vegetais/farmacologia , Raízes de Plantas/química , República da Coreia
4.
J Microbiol Biotechnol ; 31(12): 1692-1700, 2021 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-34584041

RESUMO

Glycosylation of resveratrol was carried out by using the amylosucrase of Deinococcus geothermalis, and the glycosylated products were tested for their solubility, chemical stability, and biological activities. We synthesized and identified these two major glycosylated products as resveratrol-4'-O-α-glucoside and resveratrol-3-O-α-glucoside by nuclear magnetic resonance analysis with a ratio of 5:1. The water solubilities of the two resveratrol-α-glucoside isomers (α-piceid isomers) were approximately 3.6 and 13.5 times higher than that of ß-piceid and resveratrol, respectively, and they were also highly stable in buffered solutions. The antioxidant activity of the α-piceid isomers, examined by radical scavenging capability, showed it to be initially lower than that of resveratrol, but as time passed, the α-piceid isomers' activity reached a level similar to that of resveratrol. The α-piceid isomers also showed better inhibitory activity against tyrosinase and melanin synthesis in B16F10 melanoma cells than ß-piceid. The cellular uptake of the α-piceid isomers, which was assessed by ultra-performance liquid chromatography (UPLC) analysis of the cell-free extracts of B16F10 melanoma cells, demonstrated that the glycosylated form of resveratrol was gradually converted to resveratrol inside the cells. These results indicate that the enzymatic glycosylation of resveratrol could be a useful method for enhancing the bioavailability of resveratrol.


Assuntos
Antioxidantes/metabolismo , Deinococcus/enzimologia , Glucosiltransferases/metabolismo , Resveratrol/metabolismo , Animais , Antioxidantes/química , Disponibilidade Biológica , Linhagem Celular Tumoral , Glucosídeos/química , Glucosídeos/metabolismo , Glicosilação , Melaninas/metabolismo , Camundongos , Monofenol Mono-Oxigenase/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Resveratrol/química , Solubilidade
5.
J Microbiol Biotechnol ; 31(3): 483-491, 2021 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-33622993

RESUMO

Two putative genes, lip29 and est29, encoding lipolytic enzymes from the thermophilic bacterium Geobacillus thermocatenulatus KCTC 3921 were cloned and overexpressed in Escherichia coli. The recombinant Lip29 and Est29 were purified 67.3-fold to homogeneity with specific activity of 2.27 U/mg and recovery of 5.8% and 14.4-fold with specific activity of 0.92 U/mg and recovery of 1.3%, respectively. The molecular mass of each purified enzyme was estimated to be 29 kDa by SDSPAGE. The alignment analysis of amino acid sequences revealed that both enzymes belonged to GDSL lipase/esterase family including conserved blocks with SGNH catalytic residues which was mainly identified in plants before. While Est29 showed high specificity toward short-chain fatty acids (C4-C8), Lip29 showed strong lipolytic activity to long-chain fatty acids (C12-C16). The optimal activity of Lip29 toward p-nitrophenyl palmitate as a substrate was observed at 50°C and pH 9.5, respectively, and its activity was maintained more than 24 h at optimal temperatures, indicating that Lip29 was thermostable. Lip29 exhibited high tolerance against detergents and metal ions. The homology modeling and substrate docking revealed that the long-chain substrates showed the greatest binding affinity toward enzyme. Based on the biochemical and in silico analyses, we present for the first time a GDSL-type lipase in the thermophilic bacteria group.


Assuntos
Proteínas de Bactérias/metabolismo , Geobacillus/enzimologia , Lipase/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Catálise , Clonagem Molecular , DNA Bacteriano , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Geobacillus/genética , Concentração de Íons de Hidrogênio , Lipase/genética , Simulação de Acoplamento Molecular , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Temperatura
6.
Foods ; 9(5)2020 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-32456062

RESUMO

Malva verticillata, also known as Chinese mallow, is an herbaceous plant with colorful flowers and has been used as a medicine for thousands of years. This study investigated this herb for potential antioxidant activity or an association with osteoblast differentiation. M. verticillate leaves were fermented with B. subtilis MV1 at 30 °C for 7 days to enhance their biological activities. The resultant aqueous extract (MVW) and the fermented leaves (MVB) were measured for antioxidant and osteoblast differentiation. The results showed that the total phenolic, flavonoid, and antioxidant activity, as well as the osteoblast differentiation of the MVB increased (2 to 6 times) compared with those of the MVW. MVB induced phosphorylation of p38, extracellular signal-regulated kinase in C3H10T1/2 cells, and the phosphorylation was attenuated via transforming growth factor-ß (TGF-ß) inhibitors. Moreover, runt-related transcription factor 2 and osterix in the nucleus increased in a time-dependent manner. The messenger RNA expression of alkaline phosphatase and bone sialoprotein increased about 9.4- and 65-fold, respectively, compared to the non-treated cells. MVB stimulated C3H10T1/2 cells in the osteoblasts via TGF-ß signaling. Thus, fermented M. verticillata extract exhibited enhanced antioxidant activity and osteoblast differentiation.

7.
Foods ; 9(1)2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31906298

RESUMO

The roots of Salvia miltiorrhiza are known to exhibit antioxidant and antibacterial activities. To improve the antioxidant and antibacterial activities of S. miltiorrhiza roots, the roots were fermented with Aspergillus oryzae at 25 °C for 3 weeks. The non-fermented (SME) and fermented (SMBE) roots of S. miltiorrhiza were extracted with 70% ethanol, respectively, and then fractionated with organic solvents. By fermentation, total phenolic and flavonoid contents, as well as antioxidant activity of SMBE, were increased by about 1.2 to 1.3 times compared with those of SME. The antibacterial activity of SMBE was also twice as high as that of SME. The antibacterial activity of SMBE against Bacillus cereus was lower in the n-hexane and chloroform fractions, but higher in the ethyl acetate and n-butanol fractions, compared with those of SME. These results indicate that the bioactive components of S. miltiorrhiza roots exhibiting antibacterial activity were converted to more polar compounds by fermentation of A. oryzae. Gas chromatography and mass spectrometry (GC-MS) and LC-MS analyses of SME and SMBE demonstrate that these changes are due to the acylation of dihydrofuran-2(3H)-one, dealkylation of 4-methylbenzene-1,2-diol and 4-ethylbenzene-1,2-diol, and esterification of hexadecanoic acid and (9Z, 12Z)-octadec-9,12-dienoic acid during fermentation.

8.
Foods ; 9(2)2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31979021

RESUMO

Soybean processing, e.g., by soaking, heating, and fermentation, typically results in diverse metabolic changes. Herein, multivariate analysis-based metabolic profiling was employed to investigate the effects of fermentation by Aspergillus oryzae or Bacillus subtilis on soybean substrates extracted at 4, 25, or 55 °C. As metabolic changes for both A. oryzae and B. subtilis were most pronounced for substrates extracted at 55 °C, this temperature was selected to compare the two microbial fermentation strategies, which were shown to be markedly different. Specifically, fermentation by A. oryzae increased the levels of most organic acids, γ-aminobutyric acid, and glutamine, which were ascribed to carbohydrate metabolism and conversion of glutamic acid into GABA and glutamine. In contrast, fermentation by B. subtilis increased the levels of most amino acids and isoflavones, which indicated the high activity of proteases and ß-glucosidase. Overall, the obtained results were concluded to be useful for the optimization of processing steps in terms of nutritional preferences.

9.
J Microbiol Biotechnol ; 28(4): 566-570, 2018 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-29724082

RESUMO

Because glycosylation of aesculetin and its 6-glucoside, aesculin, enhances their biological activities and physicochemical properties, whole-cell biotransformation and enzymatic synthesis methodologies using Neisseria polysaccharea amylosucrase were compared to determine the optimal production method for glycoside derivatives. High-performance liquid chromatography analysis of reaction products revealed two glycosylated products (AGG1 and AGG2) when aesculin was used as an acceptor, and three products (AG1, AG2, and AG3) when using aesculetin. The whole-cell biotransformation production yields of the major transfer products for each acceptor (AGG1 and AG1) were 85% and 25%, respectively, compared with 68% and 14% for enzymatic synthesis. These results indicate that whole-cell biotransformation is more efficient than enzymatic synthesis for the production of glycoside derivatives.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Esculina/metabolismo , Glucosiltransferases/metabolismo , Glicosídeos/biossíntese , Neisseria/enzimologia , Proteínas Recombinantes , Umbeliferonas/biossíntese , Biotransformação , Cromatografia Líquida de Alta Pressão , Esculina/química , Regulação Bacteriana da Expressão Gênica , Glucosídeos/metabolismo , Glicosídeos/química , Glicosilação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Umbeliferonas/química
10.
J Microbiol Biotechnol ; 28(6): 909-916, 2018 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-29642287

RESUMO

Previously, a cytosolic trehalase (TreH) from the hyperthermophilic archaeon Sulfolobus acidocaldarius was reported; however, the gene responsible for the trehalase activity was not identified. Two genes, saci_1816 and saci_1250, that encode the glycoside hydrolase family 15 type glucoamylase-like proteins in S. acidocaldarius were targeted and expressed in Escherichia coli, and their abilities to hydrolyze trehalose were examined. Recombinant Saci_1816 hydrolyzed trehalose exclusively without any help from a cofactor. The mass spectrometric analysis of partially purified native TreH also confirmed that Saci_1816 was involved in proteins exhibiting trehalase activity. Optimal trehalose hydrolysis activity of the recombinant Saci_1816 was observed at pH 4.0 and 60°C. The pH dependence of the recombinant enzyme was similar to that of the native enzyme, but its optimal temperature was 20-25°C lower, and its thermostability was also slightly reduced. From the biochemical and structural results, Saci_1816 was identified as a trehalase responsible for trehalose degradation in S. acidocaldarius. Identification of the treH gene confirms that the degradation of trehalose in Sulfolobus species occurs via the TreH pathway.


Assuntos
Sulfolobus acidocaldarius/enzimologia , Trealase/metabolismo , Trealose/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Hidrólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura , Trealase/química , Trealase/genética
11.
Artigo em Inglês | MEDLINE | ID: mdl-29385663

RESUMO

Because glycosylation of aesculetin and its 6-glucoside, aesculin, enhances their biological activities and physicochemical properties, whole-cell biotransformation and enzymatic synthesis methodologies using Neisseria polysaccharea amylosucrase were compared to determine the optimal production method for glycoside derivatives. High performance liquid chromatography analysis of reaction products revealed two glycosylated products (AGG1 and AGG2) when aesculin was used as an acceptor and three products (AG1, AG2, and AG3) when using aesculetin. The whole-cell biotransformation production yields of the major transfer products for each acceptor (AGG1 and AG1) were 85% and 25%, respectively, compared to 68% and 14% for enzymatic synthesis. These results indicate that whole-cell biotransformation is more efficient than enzymatic synthesis for the production of glycoside derivatives.

12.
J Microbiol Biotechnol ; 27(11): 1916-1924, 2017 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-28870007

RESUMO

In this study, we synthesized a glycosylated derivative of caffeic acid phenethyl ester (CAPE) using the amylosucrase from Deinococcus geothermalis with sucrose as a substrate and examined its solubility, chemical stability, and anti-inflammatory activity. Nuclear magnetic resonance spectroscopy showed that the resulting glycosylated CAPE (G-CAPE) was the new compound caffeic acid phenethyl ester-4-O-α-D-glucopyranoside. G-CAPE was 770 times more soluble than CAPE and highly stable in Dulbecco's modified Eagle's medium and buffered solutions, as estimated by its half-life. The glycosylation of CAPE did not significantly affect its anti-inflammatory activity, which was assessed by examining lipopolysaccharide-induced nitric oxide production and using a nuclear factor erythroid 2-related factor 2 reporter assay. Furthermore, a cellular uptake experiment using high-performance liquid chromatography analysis of the cell-free extracts of RAW 264.7 cells demonstrated that G-CAPE was gradually converted to CAPE within the cells. These results demonstrate that the glycosylation of CAPE increases its bioavailability by helping to protect this vital molecule from chemical or enzymatic oxidation, indicating that G-CAPE is a promising candidate for prodrug therapy.


Assuntos
Anti-Inflamatórios/farmacologia , Ácidos Cafeicos/química , Ácidos Cafeicos/metabolismo , Ácidos Cafeicos/farmacologia , Estabilidade Enzimática , Álcool Feniletílico/análogos & derivados , Animais , Disponibilidade Biológica , Sobrevivência Celular , Deinococcus/enzimologia , Regulação da Expressão Gênica , Glucosiltransferases/metabolismo , Glicosídeos/metabolismo , Glicosilação , Concentração de Íons de Hidrogênio , Lipopolissacarídeos/farmacologia , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Óxido Nítrico/metabolismo , Álcool Feniletílico/química , Álcool Feniletílico/metabolismo , Álcool Feniletílico/farmacologia , Células RAW 264.7/efeitos dos fármacos , Solubilidade , Temperatura
13.
Materials (Basel) ; 10(1)2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-28772381

RESUMO

In this study, bisphasic calcium phosphate (BCP) and two types of polysaccharide, carboxymethyl cellulose (CMC) and hyaluronic acid (HyA), were used to fabricate composite block bone grafts, and their physical and biological features and performances were compared and evaluated in vitro and in vivo. Specimens of the following were prepared as 6 mm diameter, 2 mm thick discs; BPC mixed with CMC (the BCP/CMC group), BCP mixed with crosslinked CMC (the BCP/c-CMC group) and BCP mixed with HyA (the BCP/HyA group) and a control group (specimens were prepared using particle type BCP). A scanning electron microscope study, a compressive strength analysis, and a cytotoxicity assessment were conducted. Graft materials were implanted in each of four circular defects of 6 mm diameter in calvarial bone in seven rabbits. Animals were sacrificed after four weeks for micro-CT and histomorphometric analyses, and the findings obtained were used to calculate new bone volumes (mm³) and area percentages (%). It was found that these two values were significantly higher in the BCP/c-CMC group than in the other three groups (p < 0.05). Within the limitations of this study, BCP composite block bone graft material incorporating crosslinked CMC has potential utility when bone augmentation is needed.

14.
Eur J Pharmacol ; 744: 147-56, 2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25446915

RESUMO

Baicalein-6-α-glucoside (BG), a glycosylated derivative of baicalein, was synthesized by using sucrose and the amylosucrase of Deinococcus geothermalis and tested for its solubility, chemical stability, and anti-inflammatory activity. BG was 26.3 times more soluble than baicalein and highly stable in buffered solutions and Dulbecco׳s modified Eagle medium containing 10% fetal bovine serum. BG treatment decreased the production of nitric oxide in RAW 264.7 cells treated with lipopolysaccharide (LPS). Luciferase reporter assays, western blots, reverse transcription-polymerase chain reaction, and flow cytometric analyses indicated that BG activated nuclear factor erythroid 2-related factor 2 (Nrf2), an antioxidant transcription factor that confers protection from various inflammatory diseases, induced Nrf2-dependent gene expression, and suppressed the production of reactive oxygen species elicited by LPS more effectively than baicalein. Cellular uptake of BG assessed by confocal microscopy and HPLC analysis of the cell-free extracts of RAW 264.7 cells demonstrated that BG was gradually converted to baicalein inside the cells. These results explain that glycosylation increased the bioavailability of baicalein by helping to protect this vital molecule from chemical or enzymatic oxidation. Therefore, BG, a glycosylated derivative of baicalein, can be an alternative to baicalein as a therapeutic drug.


Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Flavanonas/química , Flavanonas/farmacologia , Glicosídeos/química , Glicosídeos/farmacologia , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo
15.
J Microbiol Biotechnol ; 18(6): 1064-9, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18600048

RESUMO

Levan fructotransferase (LFTase) preferentially catalyzes the transfructosylation reaction in addition to levan hydrolysis, whereas other levan-degrading enzymes hydrolyze levan into a levan-oligosaccharide and fructose. Based on sequence comparisons and enzymatic properties, the fructosyl transfer activity of LFTase is proposed to have evolved from levanase. In order to probe the residues that are critical to the intramolecular fructosyl transfer reaction of the Microbacterium sp. AL-210 LFTase, an error-prone PCR mutagenesis process was carried out, and the mutants that led to a shift in activity from transfructosylation towards hydrolysis of levan were screened by the DNS method. After two rounds of mutagenesis, TLC and HPLC analyses of the reaction products by the selected mutants revealed two major products; one is a di-D-fructose- 2,6':6,2'-dianhydride (DFAIV) and the other is a levanbiose. The newly detected levanbiose corresponds to the reaction product from LFTase lacking transferring activity. Two mutants (2-F8 and 2-G9) showed a high yield of levanbiose (38-40%) compared with the wild-type enzyme, and thus behaved as levanases. Sequence analysis of the individual mutants responsible for the enhanced hydrolytic activity indicated that Asn-85 was highly involved in the transfructosylation activity of LFTase.


Assuntos
Actinomycetales/enzimologia , Actinomycetales/genética , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Dissacarídeos/biossíntese , Escherichia coli/enzimologia , Escherichia coli/genética , Frutanos/biossíntese , Frutanos/metabolismo , Hidrólise , Mutagênese , Reação em Cadeia da Polimerase
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