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This study aimed to assess the influence of rice grain in the total mixed ration (TMR) on the growth performance, blood metabolites, rumen fermentation, and rumen microbial community of fattening Hanwoo steers. Two experimental diets were prepared: (i) a TMR containing 33% dry matter (DM) corn grains (Corn TMR) and (ii) a TMR containing 33% DM rice grains (Rice TMR). Twenty-two Hanwoo steers (body weight [BW], 498 ± 32 kg; months, 17 ± 0.5) were distributed into two treatment groups in a completely randomized block design according to BW. The Rice TMR group had a higher final BW and DM intake (DMI) compared to those in the Corn TMR group (p < 0.01). However, no difference was observed in the average daily gain (ADG) and feed conversion ratio (FCR) between the two treatments. For the rumen fermentation parameters, the molar portion of butyrate in the Rice TMR was higher than in the Corn TMR (p < 0.01). Streptococcus bovis tended to be higher in the Rice TMR (p = 0.09). The results of this study suggest that using rice grain as the primary starch source in TMRs may be an alternative option for fattening Hanwoo steers.
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OBJECTIVE: This study was realized to evaluate the nutritional value of rice grains as a replacement for corn grains in the diet of growing Hanwoo steers. METHODS: Two experimental diets were prepared: i) Corn total mixed ration (TMR) consisting of 20% corn grains and ii) Rice TMR consisting of 20% rice grains, in a dry matter (DM) basis. These treatments were used for in vitro rumen fermentation and in vivo growth trials. In the rumen fermentation experiment, the in vitro DM digestibility (IVDMD), in vitro crude protein digestibility (IVCPD), in vitro neutral detergent fiber digestibility, pH, ammonia nitrogen, and volatile fatty acids (VFA) were estimated at 48 h, and the gas production was measured at 3, 6, 12, 24, and 48 h. Twenty four growing Hanwoo steers (9 months old; body weight [BW]: 259±13 kg) were randomly divided into two treatment groups and the BW, dry matter intake (DMI), average daily gain (ADG), and feed conversion ratio (FCR) were measured. RESULTS: The in vitro experiment showed that the IVDMD, IVCPD, and VFA production of the Rice TMR were higher than those of the Corn TMR (p<0.05). The growth trial showed no differences (p>0.05) in the final BW, ADG, DMI, and FCR between the two TMRs. CONCLUSION: The use of rice grains instead of corn grains did not exhibit any negative effects on the rumen fermentation or growth performance, thereby rice grains with a DM of less than 20% could be used as a starch source in the diet of growing steers.
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PURPOSE: To report two cases of retinal vasculitis associated with CREST syndrome, a novel ocular finding. OBSERVATIONS: We report two cases of patients with CREST syndrome with ocular inflammatory disease. Patient 1 presented with a right unilateral panuveitis with extensive retinal vasculitis and evidence of prior uveitis in the contralateral eye. Patient 2 presented with a left branch retinal artery occlusion and bilateral retinal vasculitis. Both patients underwent treatment with prednisone and mycophenolate motefil. CONCLUSIONS AND IMPORTANCE: Retinal vasculitis has not been previously reported in CREST syndrome. Prompt therapy with immunomodulatory therapy can potentially minimize ocular morbidity.
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Among the enzymes involved in the post-translational modification of Ras, isoprenyl carboxyl methyltransferase (ICMT) has been explored by a number of researchers as a significant enzyme controlling the activation of Ras. Indeed, inhibition of ICMT exhibited promising anti-cancer activity against various cancer cell lines. This paper reviews patents and research articles published between 2009 and 2016 that reported inhibitors of ICMT as potential chemotherapeutic agents targeting Ras-induced growth factor signaling. Since ICMT inhibitors can modulate Ras signaling pathway, it might be possible to develop a new class of anti-cancer drugs targeting Ras-related cancers. Researchers have discovered indole-based small-molecular ICMT inhibitors through high-throughput screening. Researchers at Duke University identified a prototypical inhibitor, cysmethynil. At Singapore University, Ramanujulu and his colleagues patented more potent compounds by optimizing cysmethynil. In addition, Rodriguez and Stevenson at Universidad Complutense De Madrid and Cancer Therapeutics CRC PTY Ltd., respectively, have developed inhibitors based on formulas other than the indole base. However, further optimization of chemicals targeted to functional groups is needed to improve the characteristics of ICMT inhibitors related to their application as drugs, such as solubility, effectiveness, and safety, to facilitate clinical use.
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Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica , Indóis/farmacologia , Neoplasias/tratamento farmacológico , Proteínas Metiltransferases/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Animais , Antineoplásicos/síntese química , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Humanos , Indóis/síntese química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Patentes como Assunto , Proteínas Metiltransferases/genética , Proteínas Metiltransferases/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Proteínas ras/antagonistas & inibidores , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
(E)-3-Phenyl-1-(2-pyrrolyl)-2-propenone (PPP) is a pyrrole derivative of chalcone, in which the B-ring of chalcone linked to ß-carbon is replaced by pyrrole group. While pyrrole has been studied for possible Src inhibition activity, chalcone, especially the substituents on the B-ring, has shown pharmaceutical, anti-inflammatory, and anti-oxidant properties via inhibition of NF-κB activity. Our study is aimed to investigate whether this novel synthetic compound retains or enhances the pharmaceutically beneficial activities from the both structures. For this purpose, inflammatory responses of lipopolysaccharide (LPS)-treated RAW264.7 cells were analyzed. Nitric oxide (NO) production, inducible NO synthase (iNOS) and tumor necrosis factor-α (TNF-α) mRNA expression, and the intracellular inflammatory signaling cascade were measured. Interestingly, PPP strongly inhibited NO release in a dose-dependent manner. To further investigate this anti-inflammatory activity, we identified molecular pathways by immunoblot analyses of nuclear fractions and whole cell lysates prepared from LPS-stimulated RAW264.7 cells with or without PPP pretreatment. The nuclear levels of p50, c-Jun, and c-Fos were significantly inhibited when cells were exposed to PPP. Moreover, according to the luciferase reporter gene assay after cotransfection with either TRIF or MyD88 in HEK293 cells, NF-κB-mediated luciferase activity dose-dependently diminished. Additionally, it was confirmed that PPP dampens the upstream signaling cascade of NF-κB and AP-1 activation. Thus, PPP inhibited Syk, Src, and TAK1 activities induced by LPS or induced by overexpression of these genes. Therefore, our results suggest that PPP displays anti-inflammatory activity via inhibition of Syk, Src, and TAK1 activity, which may be developed as a novel anti-inflammatory drug.
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Aripiprazole (ARI) is a commonly prescribed medication used to treat schizophrenia and bipolar disorder. To date, there have been no studies regarding the molecular pathological and immunotoxicological profiling of aripiprazole. Thus, in the present study, we prepared two different formulas of aripiprazole [Free base crystal of aripiprazole (ARPGCB) and cocrystal of aripiprazole (GCB3004)], and explored their effects on the patterns of survival and apoptosis-regulatory proteins under acute toxicity and cytotoxicity test conditions. Furthermore, we also evaluated the modulatory activity of the different formulations on the immunological responses in macrophages primed by various stimulators such as lipopolysaccharide (LPS), pam3CSK, and poly(I:C) via toll-like receptor 4 (TLR4), TLR2, and TLR3 pathways, respectively. In liver, both ARPGCB and GCB3004 produced similar toxicity profiles. In particular, these two formulas exhibited similar phospho-protein profiling of p65/nuclear factor (NF)-κB, c-Jun/activator protein (AP)-1, ERK, JNK, p38, caspase 3, and bcl-2 in brain. In contrast, the patterns of these phospho-proteins were variable in other tissues. Moreover, these two formulas did not exhibit any cytotoxicity in C6 glioma cells. Finally, the two formulations at available in vivo concentrations did not block nitric oxide (NO) production from activated macrophage-like RAW264.7 cells stimulated with LPS, pam3CSK, or poly(I:C), nor did they alter the morphological changes of the activated macrophages. Taken together, our present work, as a comparative study of two different formulas of aripiprazole, suggests that these two formulas can be used to achieve similar functional activation of brain proteins related to cell survival and apoptosis and immunotoxicological activities of macrophages.
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BACKGROUND: Korean ginseng is an ethnopharmacologically valuable herbal plant with various biological properties including anticancer, antiatherosclerosis, antidiabetic, and anti-inflammatory activities. Since there is currently no drug or therapeutic remedy derived from Korean ginseng, we developed a ginsenoside-enriched fraction (AP-SF) for prevention of various inflammatory symptoms. METHODS: The anti-inflammatory efficacy of AP-SF was tested under in vitro inflammatory conditions including nitric oxide (NO) production and inflammatory gene expression. The molecular events of inflammatory responses were explored by immunoblot analysis. RESULTS: AP-SF led to a significant suppression of NO production compared with a conventional Korean ginseng saponin fraction, induced by both lipopolysaccharide and zymosan A. Interestingly, AP-SF strongly downregulated the mRNA levels of genes for inducible NO synthase, tumor necrosis factor-α, and cyclooxygenase) without affecting cell viability. In agreement with these observations, AP-SF blocked the nuclear translocation of c-Jun at 2 h and also reduced phosphorylation of p38, c-Jun N-terminal kinase, and TAK-1, all of which are important for c-Jun translocation. CONCLUSION: Our results suggest that AP-SF inhibits activation of c-Jun-dependent inflammatory events. Thus, AP-SF may be useful as a novel anti-inflammatory remedy.
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ETHNOPHARMACOLOGICAL RELEVANCE: Phyllanthus acidus (L.) Skeels (Phyllanthaceae) has traditionally been used to treat gastric trouble, rheumatism, bronchitis, asthma, respiratory disorders, and hepatitis. Despite this widespread use, the pharmacological activities of this plant and their molecular mechanisms are poorly understood. Therefore, we evaluated the immunopharmacological activities of the methanolic extract of the aerial parts of this plant (Pa-ME) and validated its pharmacological targets. MATERIALS AND METHODS: Lipopolysaccharide (LPS)-treated macrophages, an HCl/EtOH-induced gastritis model, and an acetic acid-injected capillary permeability mouse model were employed to evaluate the anti-inflammatory activity of Pa-ME. Potentially active anti-inflammatory components of this extract were identified by HPLC. The molecular mechanisms of the anti-inflammatory activity were studied by kinase assays, reporter gene assays, immunoprecipitation analysis, and overexpression of target enzymes. RESULTS: Pa-ME suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2) and prevented morphological changes in LPS-treated RAW264.7 cells. Moreover, both HCl/EtOH-induced gastric damage and acetic acid-triggered vascular permeability were restored by orally administered Pa-ME. Furthermore, this extract downregulated the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 and reduced the nuclear levels of NF-κB. Signalling events upstream of NF-κB translocation, such as phosphorylation of Src and Syk and formation of Src/Syk signalling complexes, were also inhibited by Pa-ME. The enzymatic activities of Src and Syk were also suppressed by Pa-ME. Moreover, Src-induced and Syk-induced luciferase activity and p85/Akt phosphorylation were also inhibited by Pa-ME. Of the identified flavonoids, kaempferol and quercetin were revealed as partially active anti-inflammatory components in Pa-ME. CONCLUSION: Pa-ME exerts anti-inflammatory activity in vitro and in vivo by suppressing Src, Syk, and their downstream transcription factor, NF-κB.
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Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Phyllanthus , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Ácido Acético , Animais , Permeabilidade Capilar/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Dinoprostona/metabolismo , Etanol , Gastrite/induzido quimicamente , Células HEK293 , Humanos , Ácido Clorídrico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Lipopolissacarídeos , Metanol/química , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Solventes/química , Quinase Syk , Células U937 , Quinases da Família src/antagonistas & inibidoresRESUMO
Kaempferol (KF) is the most abundant polyphenol in tea, fruits, vegetables, and beans. However, little is known about its in vivo anti-inflammatory efficacy and mechanisms of action. To study these, several acute mouse inflammatory and nociceptive models, including gastritis, pancreatitis, and abdominal pain were employed. Kaempferol was shown to attenuate the expansion of inflammatory lesions seen in ethanol (EtOH)/HCl- and aspirin-induced gastritis, LPS/caerulein (CA) triggered pancreatitis, and acetic acid-induced writhing.
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Dor Abdominal/tratamento farmacológico , Gastrite/tratamento farmacológico , Quempferóis/farmacologia , Nociceptividade/efeitos dos fármacos , Pancreatite/tratamento farmacológico , Ácido Acético/toxicidade , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/efeitos adversos , Ceruletídeo/efeitos adversos , Modelos Animais de Doenças , Gastrite/induzido quimicamente , Gastrite/patologia , Camundongos , Pancreatite/induzido quimicamenteRESUMO
The Cordyceps species have been widely used for treating various cancer diseases. Although the Cordyceps species have been widely known as an alternative anticancer remedy, which compounds are responsible for their anticancer activity is not fully understood. In this study, therefore, we examined the anticancer activity of 5 isolated compounds derived from the butanol fraction (Cb-BF) of Cordyceps bassiana. For this purpose, several cancer cell lines such as C6 glioma, MDA-MB-231, and A549 cells were employed and details of anticancer mechanism were further investigated. Of 5 compounds isolated by activity-guided fractionation from BF of Cb-EE, KTH-13, and 4-isopropyl-2,6-bis(1-phenylethyl)phenol, Cb-BF was found to be the most potent antiproliferative inhibitor of C6 glioma and MDA-MB-231 cell growth. KTH-13 treatment increased DNA laddering, upregulated the level of Annexin V positive cells, and altered morphological changes of C6 glioma and MDA-MB-231 cells. In addition, KTH-13 increased the levels of caspase 3, caspase 7, and caspase 9 cleaved forms as well as the protein level of Bax but not Bcl-2. It was also found that the phosphorylation of AKT and p85/PI3K was also clearly reduced by KTH-13 exposure. Therefore, our results suggest KTH-13 can act as a potent antiproliferative and apoptosis-inducing component from Cordyceps bassiana, contributing to the anticancer activity of this mushroom.
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INTRODUCTION: 3-Phosphoinositide-dependent kinase 1 (PDK1) is a master regulator of the AGC protein kinase family and is a critical activator of multiple pro-survival and oncogenic protein kinases, for which it has garnered considerable interest as an oncology drug target. AREAS COVERED: This manuscript reviews small molecule patent literature disclosures between October 2011 and September 2014 for both PDK1 activators and inhibitors and restates the selective patents published before September 2011. PDK1 modulators are organized according to pharmaceutical company and chemical structural class. EXPERT OPINION: Many academic institutions and pharmaceutical companies continue to research into the development of small molecules that can function as PDK1 inhibitors or modulators. To date, >50 patent publications on PDK1 disruptors and modulators have been published since the protein was first discovered in 1998. Most of these molecules act as ATP mimetics, forming similar hydrogen bonding patterns to PDK1 as ATP and functioning as hydrophobic pharmacophores. To achieve selectivity in PDK1 inhibition, the discovery of binding pockets structurally distinctive from the ATP site is a challenging but promising strategy.
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Proteínas Quinases Dependentes de 3-Fosfoinositídeo/efeitos dos fármacos , Desenho de Fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Ligação de Hidrogênio , Patentes como AssuntoRESUMO
INTRODUCTION: Nitric oxide synthases (NOSs) are a family of enzymes that play an essential role in synthesizing nitric oxide (NO) by oxidizing l-arginine. As previously reported, NO is a significant mediator in cellular signaling pathways. It serves as a crucial regulator in insulin secretion, vascular tone, peristalsis, angiogenesis, neural development and inflammation. Due to its important role, the inhibition of these vital enzymes provides, as tools, the opportunity to gain an insight into potential therapeutic applications targeting NOSs. AREAS COVERED: This paper reviews the patent literature between 2011 and mid-2014 that specified inhibitors of NOS family members as the significant targets. Google and Baidu search engines were used to find relevant patents and clinical information using NOSs or NOS inhibitor as search terms. EXPERT OPINION: Considerable recent progress has been made in the development of NOS inhibitors with pharmacodynamic and pharmacokinetic properties, and such development is likely to continue. The patented compounds attenuated mostly embodying evidence from in vitro and in vivo trials that demonstrate good potential for future clinical human trials and industrial applications. Furthermore, new techniques such as X-ray ligand crystallographic study and structure-activity relationship were popularly utilized, which give new insights for developing novel, safe, efficient and selective NOS inhibitors.
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Indústria Farmacêutica/legislação & jurisprudência , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Patentes como Assunto , Animais , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Humanos , Estrutura Molecular , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/metabolismo , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
21-O-Angeloyltheasapogenol E3 (ATS-E3) is a triterpenoid saponin recently isolated from the seeds of the tea tree Camellia sinensis (L.) O. Kuntze. ATS-E3 has several beneficial properties including anti-inflammatory, antidiabetic, antiatherosclerotic, and anticancer effects. Unlike other phenolic compounds isolated from tea plants, there are no studies reporting the pharmacological action of ATS-E3. In this study, we therefore aimed to explore the cellular and molecular inhibitory activities of ATS-E3 in macrophage-mediated inflammatory responses. ATS-E3 remarkably diminished cellular responses of macrophages such as FITC-dextran-induced phagocytic uptake, sodium nitroprusside- (SNP-) induced radical generation, and LPS-induced nitric oxide (NO) production. Analysis of its molecular activity showed that this compound significantly suppressed the expression of inducible NO synthase (iNOS), nuclear translocation of nuclear factor- (NF-) κB subunits (p50 and p65), phosphorylation of inhibitor of κB kinase (IKK), and the enzyme activity of AKT1. Taken together, the novel triterpenoid saponin compound ATS-E3 contributes to the beneficial effects of tea plants by exerting anti-inflammatory and antioxidative activities in an AKT/IKK/NF-κB-dependent manner.
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Anti-Inflamatórios/farmacologia , Macrófagos/efeitos dos fármacos , Saponinas/farmacologia , Triterpenos/farmacologia , Animais , Anti-Inflamatórios/isolamento & purificação , Camellia sinensis , Linhagem Celular , Células HEK293 , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Saponinas/isolamento & purificação , Sementes , Transdução de Sinais/efeitos dos fármacos , Triterpenos/isolamento & purificaçãoRESUMO
AP736 was identified as an antimelanogenic drug that can be used for the prevention of melasma, freckles, and dark spots in skin by acting as a suppressor of melanin synthesis and tyrosinase expression. Since macrophage-mediated inflammatory responses are critical for skin health, here we investigated the potential anti-inflammatory activity of AP736. The effects of AP736 on various inflammatory events such as nitric oxide (NO)/prostaglandin (PG) E2 production, inflammatory gene expression, phagocytic uptake, and morphological changes were examined in RAW264.7 cells. AP736 was found to strongly inhibit the production of both NO and PGE2 in lipopolysaccharide- (LPS-) treated RAW264.7 cells. In addition, AP736 strongly inhibited both LPS-induced morphological changes and FITC-dextran-induced phagocytic uptake. Furthermore, AP736 also downregulated the expression of multiple inflammatory genes, such as inducible NO synthase (iNOS), cyclooxygenase- (COX-) 2, and interleukin- (IL-) 1ß in LPS-treated RAW264.7 cells. Transcription factor analysis, including upstream signalling events, revealed that both NF-κB and AP-1 were targeted by AP736 via inhibition of the IKK/IκBα and IRAK1/TAK1 pathways. Therefore, our results strongly suggest that AP736 is a potential anti-inflammatory drug due to its suppression of NF-κB-IKK/IκBα and AP-1-IRAK1/TAK1 signalling, which may make AP736 useful for the treatment of macrophage-mediated skin inflammation.
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Adamantano/análogos & derivados , Anti-Inflamatórios não Esteroides/farmacologia , Benzamidas/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , NF-kappa B/antagonistas & inibidores , Fator de Transcrição AP-1/antagonistas & inibidores , Adamantano/química , Adamantano/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Benzamidas/química , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/imunologia , Interleucina-1beta/genética , Lipopolissacarídeos/toxicidade , Macrófagos/imunologia , Melaninas/biossíntese , Camundongos , Estrutura Molecular , Óxido Nítrico Sintase Tipo II/genética , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/imunologia , Pele/metabolismo , Pigmentação da Pele/efeitos dos fármacosRESUMO
The inhibitory activities of the Cordyceps pruinosa butanol fraction (Cp-BF) were investigated by determining inflammatory responses of lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells and by evaluating HCl/ethanol (EtOH)-triggered gastric ulcers in mice. The molecular mechanisms of the inhibitory effects of Cp-BF were investigated by identifying target enzymes using biochemical and molecular biological approaches. Cp-BF strongly inhibited the production of NO and TNF-α, release of reactive oxygen species (ROS), phagocytic uptake of FITC-dextran, and mRNA expression levels of interleukin (IL)-6, inducible NO synthase (iNOS), and tumour necrosis factor-alpha (TNF)-α in activated RAW264.7 cells. Cp-BF also strongly downregulated the NF-κB pathway by suppressing IKKß according to luciferase reporter assays and immunoblot analysis. Furthermore, Cp-BF blocked both increased levels of NF-κB-mediated luciferase activities and phosphorylation of p65/p50 observed by IKKß overexpression. Finally, orally administered Cp-BF was found to attenuate gastric ulcer and block the phosphorylation of IκBα induced by HCl/EtOH. Therefore, these results suggest that the anti-inflammatory activity of Cp-BF may be mediated by suppression of IKKα and its downstream NF-κB activation. Since our group has established the mass cultivation conditions by developing culture conditions for Cordyceps pruinosa, the information presented in this study may be useful for developing new anti-inflammatory agents.
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Gusakov critiques our methodology for comparing the cellulolytic activity of the bacterial cellulase CelA with the fungal cellulase Cel7A. We address his concerns by clarifying some misconceptions, carefully referencing the literature, and justifying our approach to point out that the results from our study still stand.
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Bactérias/enzimologia , Proteínas de Bactérias/química , Celulase/química , Celulose/químicaRESUMO
Most fungi and bacteria degrade plant cell walls by secreting free, complementary enzymes that hydrolyze cellulose; however, some bacteria use large enzymatic assemblies called cellulosomes, which recruit complementary enzymes to protein scaffolds. The thermophilic bacterium Caldicellulosiruptor bescii uses an intermediate strategy, secreting many free cellulases that contain multiple catalytic domains. One of these, CelA, comprises a glycoside hydrolase family 9 and a family 48 catalytic domain, as well as three type III cellulose-binding modules. In the saccharification of a common cellulose standard, Avicel, CelA outperforms mixtures of commercially relevant exo- and endoglucanases. From transmission electron microscopy studies of cellulose after incubation with CelA, we report morphological features that suggest that CelA not only exploits the common surface ablation mechanism driven by general cellulase processivity, but also excavates extensive cavities into the surface of the substrate. These results suggest that nature's repertoire of cellulose digestion paradigms remain only partially discovered and understood.
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Bactérias/enzimologia , Proteínas de Bactérias/química , Celulase/química , Celulose/química , Proteínas de Bactérias/isolamento & purificação , Catálise , Domínio Catalítico , Celulase/isolamento & purificação , Temperatura Alta , Hidrólise , Especificidade por SubstratoRESUMO
Piceid is widely used in food, cosmetics, and pharmaceuticals because of its therapeutic benefits. However, the use of piceid as a drug is limited because of its low solubility. To increase solubility, we synthesized piceid glucosides using maltosyltransferase from Caldicellulosiruptor bescii. The MTase gene was cloned and expressed in Escherichia coli. The enzyme had a unique transfer specificity to the transfer of maltosyl units. Four piceid transglycosylation products were present and identified by thin-layer chromatography and recycling preparative high-performance liquid chromatography. The major product was purified by C(18) and gel filtration chromatography, and its molecular structure was determined using nuclear magnetic resonance spectroscopy to be α-D-maltosyl-(1â4)-piceid. The solubility of maltosyl piceid was 8.54 × 10(3) and 1.86 × 10(3) times those of natural resveratrol and piceid, respectively, suggesting that the transglycosylation greatly increased the water solubility. This suggests that dietary intake of this compound can enhance the bioavailability of resveratrol in the human body.
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Bactérias/enzimologia , Glucosídeos/biossíntese , Glucosiltransferases/metabolismo , Disponibilidade Biológica , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Escherichia coli/genética , Escherichia coli/metabolismo , Glucosídeos/química , Glucosiltransferases/genética , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resveratrol , Solubilidade , Estilbenos/química , Estilbenos/farmacocinética , alfa-Glucosidases/metabolismoRESUMO
Caldicellulosiruptor bescii DSM 6725 utilizes various polysaccharides and grows efficiently on untreated high-lignin grasses and hardwood at an optimum temperature of â¼ 80 °C. It is a promising anaerobic bacterium for studying high-temperature biomass conversion. Its genome contains 2666 protein-coding sequences organized into 1209 operons. Expression of 2196 genes (83%) was confirmed experimentally. At least 322 genes appear to have been obtained by lateral gene transfer (LGT). Putative functions were assigned to 364 conserved/hypothetical protein (C/HP) genes. The genome contains 171 and 88 genes related to carbohydrate transport and utilization, respectively. Growth on cellulose led to the up-regulation of 32 carbohydrate-active (CAZy), 61 sugar transport, 25 transcription factor and 234 C/HP genes. Some C/HPs were overproduced on cellulose or xylan, suggesting their involvement in polysaccharide conversion. A unique feature of the genome is enrichment with genes encoding multi-modular, multi-functional CAZy proteins organized into one large cluster, the products of which are proposed to act synergistically on different components of plant cell walls and to aid the ability of C. bescii to convert plant biomass. The high duplication of CAZy domains coupled with the ability to acquire foreign genes by LGT may have allowed the bacterium to rapidly adapt to changing plant biomass-rich environments.
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Metabolismo dos Carboidratos/genética , Genoma Bacteriano , Bactérias Gram-Positivas/genética , Aderência Bacteriana , Proteínas de Bactérias/genética , Biomassa , Perfilação da Expressão Gênica , Genes Bacterianos , Genômica , Bactérias Gram-Positivas/metabolismo , Bactérias Gram-Positivas/ultraestrutura , Plantas/metabolismo , ProteômicaRESUMO
The thermophilic, cellulolytic, anaerobic bacterium 'Anaerocellum thermophilum' strain Z-1320 was isolated from a hot spring almost two decades ago and deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) as DSM 6725. The organism was classified as representing a new genus, 'Anaerocellum', primarily on its growth physiology, cell-wall type and morphology. The results of recent physiological studies and of phylogenetic and genome sequence analyses of strain DSM 6725 of 'A. thermophilum' obtained from the DSMZ showed that its properties differed from those originally described for strain Z-1320. In particular, when compared with strain Z-1320, strain DSM 6725 grew at higher temperatures and had an expanded range of growth substrates. Moreover, the 16S rRNA gene sequence of strain DSM 6725 fell within the Caldicellulosiruptor clade. It is therefore suggested that 'Anaerocellum thermophilum' should be classified as a member of the genus Caldicellulosiruptor, for which the name Caldicellulosiruptor bescii sp. nov. is proposed (type strain DSM 6725(T)=ATCC BAA-1888(T)). C. bescii sp. nov. DSM 6725(T) is the most thermophilic cellulose-degrading organism known. The strain was able to grow up to 90 degrees C (pH 7.2) and degraded crystalline cellulose and xylan as well as untreated plant biomass, including potential bioenergy plants such as poplar and switchgrass.