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1.
Int J Syst Evol Microbiol ; 70(2): 732-737, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31702538

RESUMO

An obligately anaerobic, Gram-stain-positive and coccus-shaped bacterium, designated strain YH-panp20T, was isolated from pig faeces. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the isolate belongs to the family Erysipelotrichaceae, and is most closely related to Catenisphaera adipataccumulans KCTC 15517T (93.5 % sequence similarity), followed by Faecalitalea cylindroides KCTC 5815T (92.2 %), Faecalicoccus acidiformans KCTC 15521T (90.2 %) and Holdemanella biformis KCTC 5969T (89.6 %). Average nucleotide identity values between YH-panp20T and its closest relatives were lower than 71 %. The G+C content of the isolate was 38.4 mol%, and its cell-wall peptidoglycan was found to be of A1γ type and contained meso-diaminopimelic acid. The predominant fatty acids were C18 : 1 cis 9, C18 : 0 DMA and C16 : 0. The major end-products of glucose fermentation were lactate, acetate and formate. Therefore, based on the phenotypic, phylogenetic and chemotaxonomic properties, a novel genus and species, Absicoccus porci gen. nov., sp. nov., is proposed for isolate YH-panp20T (=KCTC 15747T=JCM 32769T).


Assuntos
Firmicutes/classificação , Filogenia , Suínos/microbiologia , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , Parede Celular/química , DNA Bacteriano/genética , Ácido Diaminopimélico/química , Ácidos Graxos/química , Fezes/microbiologia , Fermentação , Firmicutes/isolamento & purificação , Bacilos Gram-Positivos/genética , Peptidoglicano/química , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA
2.
Int J Syst Evol Microbiol ; 67(11): 4352-4357, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28945536

RESUMO

Three bacterial isolates from human faeces, YS-aM39T, R2F3-3-3T and R2F3-5-1, were characterized as Gram-negative, strictly anaerobic, non-spore-forming, non-motile, and rod-shaped. Isolate YS-aM39T formed a distinct line of descent, showing greatest 16S rRNA gene sequence relatedness with R2F3-3-3T (97.5 %), R2F3-5-1 (97.5 %), Bacteroides ovatus (98.8 %) and Bacteroides xylanisolvens (97.2 %). Isolates R2F3-3-3T and R2F3-5-1 also formed a distinct line of descent, sharing greatest 16S rRNA gene sequence relatedness with B. ovatus (98.2 %) and B. xylanisolvens (97.2 %). The DNA G+C content of YS-aM39T was 44.8 mol%, that of R2F3-3-3T was 42.4 mol% and that of R2F3-5-1 was 42.6 mol%. The respiratory quinone of all three isolates was menaquinone MK-10. Polar lipid analysis identified phosphatidylethanolamine as the major lipid. The predominant fatty acids in all three isolates were anteiso-C15 : 0, iso-C15 : 0, C16 : 0 3-OH and iso-C17 : 0 3-OH. The major end products of glucose fermentation were acetic acid, lactic acid and formic acid. DNA-DNA hybridization data indicated that two isolates, YS-aM39T and R2F3-3-3T, represent a species distinct from B. ovatus and B. xylanisolvens. Finally, in this study, the two isolates represented two new species in the genus Bacteroides, for which we propose the names Bacteroides koreensis sp. nov. (type strain, YS-aM39T=KCTC 15520T=JCM 31393T) and Bacteroides kribbi sp. nov. (type strain, R2F3-3-3T=KCTC 15460T=JCM 31391T).


Assuntos
Bacteroides/classificação , Fezes/microbiologia , Filogenia , Técnicas de Tipagem Bacteriana , Bacteroides/genética , Bacteroides/isolamento & purificação , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Humanos , Hibridização de Ácido Nucleico , Fosfatidiletanolaminas/química , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Ubiquinona/química
3.
Anaerobe ; 48: 70-75, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28754474

RESUMO

Gram-positive, straight or slightly curved rod-shaped bacteria, designated as strains N6H1-5T and N6H1-3, were isolated from fecal samples of old dog. The analysis of 16S rRNA gene sequences indicated that the isolates belonged to the Clostridium cluster XVI and were closely related to Eubacterium dolichum KCTC 5832T, Eubacterium tortuosum DSM 3987T, Clostridium innocuum KCTC 5183T, Allobaculum stecoricanis DSM 13633T, Eubacterium limosum KCTC 3266T, and Clostridium butyricum KCTC 1871T, with 94.0%, 93.8%, 92.0%, 84.9%, 80.7%, and 80.0% sequence similarity, respectively. Chemotaxonomic data supported placement of the strains N6H1-5T and N6H1-3 in the new taxon. The strains contained m-diaminopimelic acid cell wall peptidoglycan; the major polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and glycolipids (GL); and the major fatty acids were C18:1cis 9 (30.7%) and C16:0 (17.1%). The predominant metabolic end product was lactic acid. The G + C content was 35.8 mol%. The most closely related species, E. dolichum and E. tortuosum, were also assigned to the new taxon, based on the phylogenetic analysis and phenotypic data. Thus, the type strain N6H1-5T (=KCTC 15422 = JCM 30884) represents a novel genus and species, for which the name Absiella argi gen. nov., sp. nov is proposed. It is also proposed that E. dolichum KCTC 5832T and E. tortuosum DSM 3987T be transferred to this new genus, and named Absiella dolichum comb. nov. and Absiella tortuosum comb. nov., respectively.


Assuntos
Técnicas de Tipagem Bacteriana/veterinária , Clostridium/classificação , Eubacterium/classificação , Firmicutes/classificação , Intestinos/microbiologia , Animais , Composição de Bases/genética , Clostridium/genética , Clostridium/isolamento & purificação , DNA Bacteriano/genética , Cães , Eubacterium/genética , Eubacterium/isolamento & purificação , Ácidos Graxos/análise , Firmicutes/genética , Firmicutes/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
4.
Appl Environ Microbiol ; 80(23): 7235-43, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25239890

RESUMO

A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d- and l-sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α-d-glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3-O-ß-d-glucoside, resveratrol 4'-O-ß-d-glucoside, resveratrol 3,5-O-ß-d-diglucoside, and resveratrol 3,5,4'-O-ß-d-triglucoside. The conversion rates and numbers of products formed were found to vary with the other NDP sugar donors. Resveratrol 3-O-ß-d-2-deoxyglucoside and resveratrol 3,5-O-ß-d-di-2-deoxyglucoside were found to be produced using TDP-2-deoxyglucose as a donor; however, the monoglycosides resveratrol 4'-O-ß-d-galactoside, resveratrol 4'-O-ß-d-viosaminoside, resveratrol 3-O-ß-l-rhamnoside, and resveratrol 3-O-ß-l-fucoside were produced from the respective sugar donors. Altogether, 10 diverse glycoside derivatives of the medically important resveratrol were generated, demonstrating the capacity of YjiC to produce structurally diverse resveratrol glycosides.


Assuntos
Bacillus/enzimologia , Glucuronosiltransferase/metabolismo , Glicosídeos/metabolismo , Estilbenos/metabolismo , Bacillus/genética , Carboidratos/análise , Clonagem Molecular , Citosol/química , Expressão Gênica , Glucuronosiltransferase/genética , Glucuronosiltransferase/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Resveratrol
5.
Anaerobe ; 30: 30-4, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25094054

RESUMO

Chronic rhinosinusitis (CRS) is an inflammatory disorder of the nasal cavity and paranasal sinus related to bacterial infection. A previous study suggested that a specific bacterial group may have an important role in the course of CRS. In this study, bacteria isolated from CRS patients were characterized. A total of 15 strains were identified as Gram-positive anaerobic cocci (GPAC), which were able to utilize peptone as a sole carbon source. Sequencing of the 16S ribosomal RNA gene revealed that the isolates were closely related to members of the genus Peptoniphilus (>97% similarity) within the Clostridiales Family XI. Incertae Sedis. Genotypic and phenotypic characterization suggests that these isolates represent a novel species of the genus Peptoniphilus associated with CRS. The type strain of Peptoniphilus rhinitidis is 1-13T (=KCTC 5985T=JCM 17448T).


Assuntos
Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/isolamento & purificação , Infecções por Bactérias Gram-Positivas/microbiologia , Rinite/microbiologia , Sinusite/microbiologia , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Doença Crônica , Análise por Conglomerados , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Humanos , Masculino , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
6.
Metab Eng ; 18: 44-52, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23563322

RESUMO

Most current metabolic engineering applications rely on the inactivation of unwanted reactions and the amplification of product-oriented reactions. All of the biochemical reactions involved with cellular metabolism are tightly coordinated with the electron flow, which depends on the cellular energy status. Thus, the cellular metabolic flux can be controlled either by modulation of the electron flow or the regulation of redox reactions. This study analyzed the genome-wide anaerobic fermentation products of 472 Escherichia coli single gene knockouts, which comprised mainly of dehydrogenases, oxidoreductases, and redox-related proteins. Many metabolic pathways that were located far from anaerobic mixed-acid fermentation significantly affected the profiles of lactic acid, succinic acid, acetic acid, formic acid, and ethanol. Unexpectedly, D-lactate overproduction was determined by a single gene deletion in dehydrogenases (e.g., guaB, pyrD, and serA) involved with nucleotide and amino acid metabolism. Furthermore, the combined knockouts of guaB, pyrD, serA, fnr, arcA, or arcB genes, which are involved with anaerobic transcription regulation, enhanced D-lactate overproduction. These results suggest that the anaerobic fermentation profiles of E. coli can be tuned via the disruption of peripheral dehydrogenases in anaerobic conditions.


Assuntos
Escherichia coli K12/metabolismo , Ácido Láctico/biossíntese , Engenharia Metabólica , Anaerobiose/fisiologia , Escherichia coli K12/genética , Escherichia coli K12/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Estudo de Associação Genômica Ampla , Oxirredutases/genética , Oxirredutases/metabolismo
7.
Appl Environ Microbiol ; 79(11): 3516-21, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23542617

RESUMO

A UDP-glycosyltransferase from Bacillus licheniformis was exploited for the glycosylation of phloretin. The in vitro glycosylation reaction confirmed the production of five phloretin glucosides, including three novel glucosides. Consequently, we demonstrated the application of the same glycosyltransferase for the efficient whole-cell biocatalysis of phloretin in engineered Escherichia coli.


Assuntos
Bacillus/enzimologia , Proteínas de Escherichia coli/metabolismo , Glicosiltransferases/metabolismo , Floretina/metabolismo , Cromatografia Líquida de Alta Pressão , Escherichia coli , Engenharia Genética , Glicosilação , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Espectrometria de Massas em Tandem , Açúcares de Uridina Difosfato/metabolismo
8.
Appl Environ Microbiol ; 78(21): 7680-6, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22923401

RESUMO

Geldanamycin (GM) is a naturally occurring anticancer agent isolated from several strains of Streptomyces hygroscopicus. However, its potential clinical utility is compromised by its severe toxicity and poor water solubility. For this reason, considerable efforts are under way to make new derivatives that have both good clinical efficacy and high water solubility. On the other hand, glycosylation is often a step that improves the water solubility and/or biological activity in many natural products of biosynthesis. Here, we report the facile production of glucose-conjugated nonbenzoquinone GM analogs using the Bacillus UDP-glycosyltransferase BL-C. Five aglycon substrates containing nonbenzoquinone aromatic rings were chosen to validate the in vitro glycosylation reaction. Putative glucoside compounds were determined through the presence of a product peak(s) and were also verified using LC/MS analyses. Further, the chemical structures of new glucoside compounds 6 and 7 were elucidated using spectroscopy data. These glucoside compounds showed a dramatic improvement in water solubility compared with that of the original aglycon, nonbenzoquinone GM.


Assuntos
Antibióticos Antineoplásicos/metabolismo , Bacillus/enzimologia , Benzoquinonas/metabolismo , Glicosiltransferases/metabolismo , Lactamas Macrocíclicas/metabolismo , Bacillus/genética , Desenho de Fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose/metabolismo , Glicosilação , Glicosiltransferases/genética , Dados de Sequência Molecular , Solubilidade
9.
Int J Syst Evol Microbiol ; 62(Pt 10): 2347-2355, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22140163

RESUMO

A Gram-positive, rod-shaped, endospore-forming bacterium, designated strain BLB-1(T), was isolated from samples of tidal flat sediment from the Yellow Sea. 16S rRNA gene sequence analysis demonstrated that the isolate belonged to the Bacillus rRNA group 2 and was closely related to Bacillus massiliensis CIP 108446(T) (97.4%), Bacillus odysseyi ATCC PTA-4993(T) (96.7%), Lysinibacillus fusiformis DSM 2898(T) (96.2%) and Lysinibacillus boronitolerans DSM 17140(T) (95.9%). Sequence similarities with related species in other genera, including Caryophanon, Sporosarcina and Solibacillus, were <96.1%. Chemotaxonomic data supported the affiliation of strain BLB-1(T) with the genus Lysinibacillus. The major menaquinone was MK-7, the cell-wall sugars were glucose and xylose, the cell-wall peptidoglycan type was A4α (L-Lys-D-Asp), the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and several unknown phospholipids, and the major fatty acids were anteiso-C(15:0) (35.6%), iso-C(15:0) (25.6%) and anteiso-C(17:0) (16.5%). The most closely related species, Bacillus massiliensis and Bacillus odysseyi, were also assigned to this genus based on phylogenetic analysis and phenotypic data. The results of DNA-DNA hybridizations and phenotypic tests supported the differentiation of all three taxa from species of the genus Lysinibacillus with validly published names. Thus, strain BLB-1(T) ( = KCTC 13296(T)  = JCM 15800(T)) represents a novel species, for which the name Lysinibacillus sinduriensis sp. nov. is proposed. It is also proposed that Bacillus massiliensis CIP 108446(T) ( =4400831(T) = CCUG49529(T)  =KCTC 13178(T)) and Bacillus odysseyi NBRC 100172(T) ( =34hs-1(T)  =ATCC PTA-4993(T)  =NRRL B-30641(T)  =DSM 18869(T)  =CIP 108263(T)  =KCTC 3961(T)) be transferred to the genus Lysinibacillus as Lysinibacillus massiliensis comb. nov. and Lysinibacillus odysseyi comb. nov., respectively.


Assuntos
Bacillaceae/classificação , Peptidoglicano/análise , Filogenia , Microbiologia do Solo , Bacillaceae/genética , Bacillaceae/isolamento & purificação , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/análise , Sedimentos Geológicos/microbiologia , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fosfolipídeos/análise , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA
10.
J Immunol ; 182(9): 5823-35, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19380831

RESUMO

Surface molecules of pathogens play an important role in stimulating host immune responses. Elucidation of the signaling pathways activated by critical surface molecules in host cells provides insight into the molecular pathogenesis resulting from bacteria-host interactions. MspTL is the most abundant outer membrane protein of Treponema lecithinolyticum, which is associated with periodontitis, and induces expression of a variety of proinflammatory factors. Although bacteria and bacterial components like LPS and flagellin are known to induce IFN-beta, induction by bacterial surface proteins has not been reported. In the present study, we investigated MspTL-mediated activation of signaling pathways stimulating up-regulation of IFN-beta and IFN-stimulated genes in a human monocytic cell line, THP-1 cells, and primary cultured human gingival fibroblasts. MspTL treatment of the cells induced IFN-beta and the IFN-stimulated genes IFN-gamma-inducible protein-10 (IP-10) and RANTES. A neutralizing anti-IFN-beta Ab significantly reduced the expression of IP-10 and RANTES, as well as STAT-1 activation, which was also induced by MspTL. Experiments using specific small interfering RNA showed that MspTL activated TANK-binding kinase 1 (TBK1), but not inducible IkappaB kinase (IKKi). MspTL also induced dimerization of IFN regulatory factor-3 (IRF-3) and translocation into the nucleus. The lipid rapid-disrupting agents methyl-beta-cyclodextrin, nystatin, and filipin inhibited the MspTL internalization and cellular responses, demonstrating that lipid raft activation was a prerequisite for MspTL cellular signaling. Our results demonstrate that MspTL, the major outer protein of T. lecithinolyticum, induced IFN-beta expression and subsequent up-regulation of IP-10 and RANTES via TBK1/IRF-3/STAT-1 signaling secondary to lipid raft activation.


Assuntos
Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica/imunologia , Fator Regulador 3 de Interferon/fisiologia , Interferon beta/genética , Microdomínios da Membrana/imunologia , Monócitos/imunologia , Porinas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Treponema/imunologia , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Linhagem Celular Tumoral , Quimiocina CCL5/biossíntese , Quimiocina CCL5/genética , Quimiocina CXCL10/biossíntese , Quimiocina CXCL10/fisiologia , Dimerização , Fibroblastos/enzimologia , Fibroblastos/imunologia , Fibroblastos/microbiologia , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/biossíntese , Interferon beta/fisiologia , Microdomínios da Membrana/enzimologia , Microdomínios da Membrana/microbiologia , Monócitos/enzimologia , Monócitos/microbiologia , Periodontite/enzimologia , Periodontite/imunologia , Periodontite/microbiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Treponema/patogenicidade , Infecções por Treponema/enzimologia , Infecções por Treponema/imunologia , Infecções por Treponema/microbiologia , Regulação para Cima/genética , Regulação para Cima/imunologia
11.
Mol Cancer ; 9: 97, 2010 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-20433747

RESUMO

BACKGROUND: Myc is a well known driver of lymphomagenesis, and Myc-activating chromosomal translocation is the recognized hallmark of Burkitt lymphoma, an aggressive form of non-Hodgkin's lymphoma. We developed a model that mimics this translocation event by inserting a mouse Myc cDNA gene into the immunoglobulin heavy chain locus, just upstream of the intronic Emu enhancer. These mice, designated iMyc E mu, readily develop B-cell lymphoma. To study the mechanism of Myc-induced lymphoma, we analyzed signaling pathways in lymphoblastic B-cell lymphomas (LBLs) from iMyc E mu mice, and an LBL-derived cell line, iMyc E mu-1. RESULTS: Nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription 3 (STAT3) were constitutively activated in iMyc E mu mice, not only in LBLs but also in the splenic B-lymphocytes of young animals months before tumors developed. Moreover, inhibition of either transcription factor in iMyc E mu-1 cells suppressed growth and caused apoptosis, and the abrogation of NF-kappaB activity reduced DNA binding by both STAT3 and Myc, as well as Myc expression. Inhibition of STAT3 signaling eliminated the activity of both NF-kappaB and Myc, and resulted in a corresponding decrease in the level of Myc. Thus, in iMyc E mu-1 cells NF-kappaB and STAT3 are co-dependent and can both regulate Myc. Consistent with this, NF-kappaB and phosphorylated STAT3 were physically associated with one another. In addition, LBLs and iMyc E mu-1 cells also showed constitutive AKT phosphorylation. Blocking AKT activation by inhibiting PI3K reduced iMyc E mu-1 cell proliferation and caused apoptosis, via downregulation of NF-kappaB and STAT3 activity and a reduction of Myc levels. Co-treatment with NF-kappaB, STAT3 or/and PI3K inhibitors led to additive inhibition of iMyc E mu-1 cell proliferation, suggesting that these signaling pathways converge. CONCLUSIONS: Our findings support the notion that constitutive activation of NF-kappaB and STAT3 depends on upstream signaling through PI3K, and that this activation is important for cell survival and proliferation, as well as for maintaining the level of Myc. Together, these data implicate crosstalk among NF-kappaB, STAT3 and PI3K in the development of iMyc E mu B-cell lymphomas.


Assuntos
Linfoma de Células B/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptor Cross-Talk/fisiologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética , Animais , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Fragmentação do DNA , Modelos Animais de Doenças , Ensaio de Desvio de Mobilidade Eletroforética , Elementos Facilitadores Genéticos , Ativação Enzimática/fisiologia , Ensaio de Imunoadsorção Enzimática , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes de Cadeia Pesada de Imunoglobulina , Genes myc , Imunoprecipitação , Linfoma de Células B/genética , Camundongos , NF-kappa B/genética , Fosfatidilinositol 3-Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética
12.
Anal Biochem ; 397(2): 259-61, 2010 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-19799850

RESUMO

A new zymography for detecting nisin-like acidic bacteriocins was developed using a tricine-sodium dodecyl sulfate (SDS) gel and an acidic gel matrix (pH 4.0). After electrophoresis, proteins in the tricine gel were electrotransferred to an optimal pH-conditioned gel matrix (OP-CGM). The OP-CGM was overlaid with indicator cells (Bacillus cereus) embedded in nutrient broth soft agar (0.8%, w/v). Antibacterial activity shown as a growth inhibition using B. cereus was detected at approximately 3.8kDa. Because nisin is unstable in buffers at pH values over 6.0, the common electrophoretic systems, SDS-polyacrylamide gel electrophoresis and tricine gel, are not suitable for detection of nisin-like acidic bacteriocins.


Assuntos
Bacteriocinas/análise , Ácidos , Bacillus cereus/efeitos dos fármacos , Bacteriocinas/farmacologia , Eletroforese em Gel de Poliacrilamida/métodos , Concentração de Íons de Hidrogênio , Nisina/análise
13.
J Microbiol Biotechnol ; 20(6): 995-1000, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20622498

RESUMO

A novel deoC gene was identified from Paenibacillus sp. EA001 isolated from soil. The gene had an open reading frame (ORF) of 663 base pairs encoding 220 amino acids with a molecular mass of 24.5 kDa. The amino acid sequence was 79 % identical to that of deoxyribose 5-phosphate aldolase (DERA) from Geobacillus sp. Y412MC10. The deoC gene encoding DERA was cloned into expression vector and the protein was expressed in Escherichia coli. The recombinant DERA was purified by using Ni-NTA affinity chromatography and characterized. The optimum temperature and pH for DERA were 50 degrees C and 6.0, respectively. The specific activity for deoxyribose 5-phosphate (DR5P), substrate, was 62 micronmol/min/mg. The Km value for DR5P was determined to be 145 mM with the Kcat value of 3.2 times 10(2 /sec) from Lineweaver-Burk plots. The EA001 DERA showed stability toward a high concentration of acetaldehyde (100 mM).


Assuntos
Aldeído Liases/química , Proteínas de Bactérias/química , Expressão Gênica , Paenibacillus/enzimologia , Aldeído Liases/genética , Aldeído Liases/isolamento & purificação , Aldeído Liases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Cinética , Dados de Sequência Molecular , Paenibacillus/classificação , Paenibacillus/genética , Paenibacillus/isolamento & purificação , Filogenia , Homologia de Sequência de Aminoácidos , Microbiologia do Solo
14.
Electrophoresis ; 30(12): 2234-7, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19544489

RESUMO

A new zymography method for simultaneous detection of two different enzymatic activities (lipolytic and proteolytic) using a single SDS-containing or native-conformation gel and a mixed-substrate (glycerol tributyrate and fibrin) (MS)(1) gel was developed. After routine electrophoresis, SDS in the gel was removed by treatment with Triton X-100. Gel proteins were electrotransferred to the MS gel. To visualize lipolytic activity, the MS gel was incubated at 37 degrees C (for 6 or 24 h) until clear bands against an opaque background were observed. To detect proteolytic activity, the same MS gel was stained with Coomassie brilliant blue. Using this method, we show that six lipolytic enzymes from Staphylococcus pasteuri NJ-1 and four proteolytic enzymes from two Bacillus strains, B. licheniformis DJ-2 and B. licheniformis NJ-5, isolated from soil, can be simultaneously detected.


Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Fibrina/química , Lipase/análise , Peptídeo Hidrolases/análise , Triglicerídeos/química , Bacillus/enzimologia , Fibrina/metabolismo , Lipase/química , Lipase/metabolismo , Nefelometria e Turbidimetria , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Corantes de Rosanilina , Staphylococcus/enzimologia , Triglicerídeos/metabolismo
15.
Protein Expr Purif ; 68(2): 196-200, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19505577

RESUMO

A new deoC gene encoding deoxyribose 5-phosphate aldolase (DERA) was identified in Yersinia sp. EA015 isolated from soil. The DERA gene had an open reading frame (ORF) of 672 base pairs encoding 223 amino acids to yield a protein of molecular mass 24.8 kDa. The amino acid sequence was 94% identical to that of DERA from Yersinia intermedia ATCC 29909. DERA was over-expressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific activity was 137 micromol/min/mg. The Michaelis constant (k(m) value) of DERA was 9.1 mM. DERA was optimally active at pH 6.0 and 50 degrees C. DERA was tolerant to a high concentration (300 mM) of acetaldehyde.


Assuntos
Aldeído Liases/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Yersinia/enzimologia , Acetaldeído/metabolismo , Aldeído Liases/química , Aldeído Liases/genética , Aldeído Liases/metabolismo , Sequência de Aminoácidos , Cromatografia de Afinidade , Escherichia coli/enzimologia , Escherichia coli/genética , Gliceraldeído 3-Fosfato/metabolismo , Histidina/genética , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Microbiologia do Solo , Temperatura , Yersinia/genética , Yersinia/isolamento & purificação
16.
Extremophiles ; 13(4): 657-67, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19412677

RESUMO

The family B DNA polymerase gene from the archaeon Thermococcus marinus (Tma) contains a long open reading frame of 3,939 bp that encodes 1,312 amino acid residues. The gene is split by one intervening sequence that forms a continuous open reading frame with the two polymerase exteins. In this study, the Tma DNA polymerase gene both with (precursor form) and without (mature form) its intein was expressed in Escherichia coli, purified by heat treatment and HiTrap Heparin HP column chromatography and characterized. Primary sequence analysis of the mature Tma polymerase showed high sequence identity with DNA polymerases in the genus Thermococcus. The expressed precursor form was easily spliced during purification steps. The molecular mass of the purified Tma DNA polymerases is about 90 kDa, as estimated by SDS-PAGE. Both Tma DNA polymerases showed the same properties. PCR performed with this enzyme was found to be optimal in the presence of 50 mM Tris-HCl (pH 8.4), 40 mM KCl, 12.5 mM (NH(4))(2)SO(4,) 2 mM MgCl(2,) 0.05% Triton X-100 and 0.0075% BSA. Furthermore, long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tma DNA polymerases (Tma plus DNA polymerase).


Assuntos
DNA Polimerase Dirigida por DNA/química , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Thermococcus/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Primers do DNA/química , Exonucleases/metabolismo , Perfilação da Expressão Gênica , Genes Arqueais , Concentração de Íons de Hidrogênio , Modelos Genéticos , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Análise de Sequência de DNA
17.
J Food Sci ; 84(11): 3186-3193, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31589348

RESUMO

Rebaudioside A was modified via glucosylation by recombinant dextransucrase of Leuconostoc lactis EG001 in Escherichia coli BL21 (DE3), forming single O-α-D-glucosyl-(1″→6') rebaudioside A with yield of 86%. O-α-D-glucosyl-(1″→6') rebaudioside A was purified using HPLC and Diaion HP-20 and its properties were characterized for possible use as a food ingredient. Almost 98% of O-α-D-glucosyl-(1″→6') rebaudioside A was dissolved after 15 days of storage at room temperature, compared to only 11% for rebaudioside A. Compared to rebaudioside A, O-α-D-glucosyl-(1″→6') rebaudioside A showed similar or improved acidic or thermal stability in commercial drinks. Thus, O-α-D-glucosyl-(1″→6') rebaudioside A could be used as a highly pure and improved sweetener with high stability in commercial drinks. PRACTICAL APPLICATION: The proposed method can be used to generate glucosyl rebaudioside A by enzymatic glucosylation. Simple glucosyl rebaudioside A exhibited high acid/thermal stability and improved sweetener in commercialized drinks. This method can be applied to obtain high value-added bioactive compounds by enzymatic modification.


Assuntos
Proteínas de Bactérias/química , Diterpenos do Tipo Caurano/química , Glucosiltransferases/química , Leuconostoc/enzimologia , Edulcorantes/química , Biocatálise , Cromatografia Líquida de Alta Pressão
18.
J Clin Invest ; 113(12): 1763-73, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15199411

RESUMO

Deregulated expression of both Myc and Bcl-X(L) are consistent features of human plasma cell neoplasms (PCNs). To investigate whether targeted expression of Myc and Bcl-X(L) in mouse plasma cells might lead to an improved model of human PCN, we generated Myc transgenics by inserting a single-copy histidine-tagged mouse Myc gene, Myc(His), into the mouse Ig heavy-chain Calpha locus. We also generated Bcl-X(L) transgenic mice that contain a multicopy Flag-tagged mouse Bcl-x(Flag) transgene driven by the mouse Ig kappa light-chain 3' enhancer. Single-transgenic Bcl-X(L) mice remained tumor free by 380 days of age, whereas single-transgenic Myc mice developed B cell tumors infrequently (4 of 43, 9.3%). In contrast, double-transgenic Myc/Bcl-X(L) mice developed plasma cell tumors with short onset (135 days on average) and full penetrance (100% tumor incidence). These tumors produced monoclonal Ig, infiltrated the bone marrow, and contained elevated amounts of Myc(His) and Bcl-X(L)(Flag) proteins compared with the plasma cells that accumulated in large numbers in young tumor-free Myc/Bcl-X(L) mice. Our findings demonstrate that the enforced expression of Myc and Bcl-X(L) by Ig enhancers with peak activity in plasma cells generates a mouse model of human PCN that recapitulates some features of human multiple myeloma.


Assuntos
Regulação Neoplásica da Expressão Gênica , Plasmocitoma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Medula Óssea/patologia , Humanos , Linfonodos/citologia , Linfonodos/metabolismo , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasmocitoma/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/genética , Baço/citologia , Baço/metabolismo , Baço/patologia , Proteína bcl-X
19.
Cancer Res ; 65(17): 7644-52, 2005 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16140930

RESUMO

Gene-targeted mice that contain a His6-tagged mouse c-Myc cDNA, Myc(His), inserted head to head into different sites of the mouse immunoglobulin heavy-chain locus, Igh, mimic the chromosomal T(12;15)(Igh-Myc) translocation that results in the activation of Myc in the great majority of mouse plasmacytomas. Mice carrying Myc(His) just 5' of the intronic heavy-chain enhancer Emu (strain iMyc(Emu)) provide a specific model of the type of T(12;15) found in a subset (approximately 20%) of plasmacytomas that develop "spontaneously" in the gut-associated lymphoid tissue (GALT) of interleukin-6 transgenic BALB/c (C) mice. Here we show that the transfer of the iMyc(Emu) transgene from a mixed genetic background of segregating C57BL/6 x 129/SvJ alleles to the background of C increased the incidence of GALT plasmacytomas by a factor of 2.5 in first-generation backcross mice (C.iMyc(Emu) N1). Third-generation backcross mice (C.iMyc(Emu) N3, approximately 94% C alleles) were hypersusceptible to inflammation-induced peritoneal plasmacytomas (tumor incidence, 100%; mean tumor onset, 86 +/- 28 days) compared with inbred C mice (tumor incidence, 5% on day 150 after tumor induction). Peritoneal plasmacytomas of C.iMyc(Emu) N3 mice overexpressed Myc(His), produced monoclonal immunoglobulin, and exhibited a unique plasma cell signature upon gene expression profiling on mouse Lymphochip cDNA microarrays. These findings indicated that the iMyc(Emu) transgene accelerates plasmacytoma development by collaborating with tumor susceptibility alleles of strain C and circumventing the requirement for tumor precursors to acquire deregulated Myc by chromosomal translocation.


Assuntos
Genes de Imunoglobulinas/genética , Genes myc/genética , Neoplasias Peritoneais/genética , Plasmocitoma/genética , Alelos , Animais , Expressão Gênica , Tecido Linfoide/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Neoplasias Peritoneais/patologia , Plasmocitoma/patologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Transgenes/genética
20.
Cancer Res ; 65(4): 1306-15, 2005 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-15735016

RESUMO

We used gene targeting in mice to insert a His(6)-tagged mouse c-Myc cDNA, Myc(His), head to head into the mouse immunoglobulin heavy-chain locus, Igh, just 5' of the intronic enhancer, Emu. The insertion of Myc(His) mimicked both the human t(8;14)(q24;q32) translocation that results in the activation of MYC in human endemic Burkitt lymphomas and the homologous mouse T(12;15) translocation that deregulates Myc in certain mouse plasmacytomas. Beginning at the age of 6 months, Myc(His) transgenic mice developed B-cell and plasma neoplasms, such as IgM(+) lymphoblastic B-cell lymphomas, Bcl-6(+) diffuse large B-cell lymphomas, and CD138(+) plasmacytomas, with an overall incidence of 68% by 21 months. Molecular studies of lymphoblastic B-cell lymphoma, the most prevalent neoplasm (50% of all tumors), showed that the lymphomas were clonal, overexpressed Myc(His), and exhibited the P2 to P1 promoter shift in Myc expression, a hallmark of MYC/Myc deregulation in human endemic Burkitt lymphoma and mouse plasmacytoma. Only 1 (6.3%) of 16 lymphoblastic B-cell lymphomas contained a BL-typical point mutation in the amino-terminal transactivation domain of Myc(His), suggesting that most of these tumors are derived from naive, pregerminal center B cells. Twelve (46%) of 26 lymphoblastic B-cell lymphomas exhibited changes in the p19(Arf)-Mdm2-p53 tumor suppressor axis, an important pathway for Myc-dependent apoptosis. We conclude that Myc(His) insertion into Igh predictably induces B-cell and plasma-cell tumors in mice, providing a valuable mouse model for understanding the transformation-inducing consequences of the MYC/Myc-activating endemic Burkitt lymphoma t(8;14)/plasmacytoma T(12;15) translocation.


Assuntos
Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 8/genética , Genes myc/genética , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma de Células B/genética , Plasmocitoma/genética , Translocação Genética/genética , Sequência de Aminoácidos , Animais , Linfócitos B/fisiologia , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Modelos Animais de Doenças , Genes Supressores de Tumor/fisiologia , Humanos , Linfoma de Células B/patologia , Camundongos , Dados de Sequência Molecular , Plasmocitoma/patologia , Mutação Puntual , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Regiões Promotoras Genéticas , Transgenes
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