Tannase-producing filamentous fungi residing alongside tannin-rich ambient in the Northwest Himalayas were isolated at laboratory conditions and further identified by 18S ribosomal RNA gene sequencing. Five most potent tannase producing strains (EI ≥ 2.0), designated Aspergillus fumigatus AN1, Fusarium redolens AN2, Penicillium crustosum AN3, Penicillium restrictum AN4, and Penicillium commune AN5, were characterized. The strain Penicillium crustosum AN3 exhibited a maximum zone dia (25.66 mm ± 0.38). During solid-state fermentation, a maximal amount of tannase was attained with Penicillium crustosum AN3 using pine needles (substrate) by adopting response surface methodology for culture parameter optimization. Gel filtration chromatography yielded 46.48% of the partially purified enzyme with 3.94-fold of tannase purification. We found two subunits in enzyme-117.76 KDa and 88.51 KDa, respectively, in the SDS-PAGE. Furthermore, the characterization of partially purified tannase revealed a maximum enzyme activity of 8.36 U/mL at 30 °C using a substrate concentration (methyl gallate) of 10 mM. To broaden the knowledge of crude enzyme application, dye degradation studies were subjected to extracellular crude tannase from Penicillium crustosum AN3 where the maximum degradation achieved at a low enzyme concentration (5 ppm).
- MeSH
- barvicí látky metabolismus chemie MeSH
- fermentace MeSH
- fungální proteiny genetika metabolismus izolace a purifikace chemie MeSH
- Fusarium enzymologie genetika MeSH
- fylogeneze MeSH
- houby enzymologie genetika MeSH
- karboxylesterhydrolasy * metabolismus genetika izolace a purifikace chemie MeSH
- kultivační média chemie MeSH
- molekulová hmotnost MeSH
- Penicillium * enzymologie genetika MeSH
- RNA ribozomální 18S genetika MeSH
- stabilita enzymů MeSH
- substrátová specifita MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Dabigatran directly inhibits thrombin and is used in primary and secondary stroke prevention in individuals with nonvalvular atrial fibrillation. The prodrug dabigatran etexilate is absorbed by enteral P-glycoprotein (ABCB1) and then activated by hepatic and intestinal carboxylesterases (CES1) to produce active metabolites. Variations in dabigatran metabolism because of genetics may affect concentration levels and clinical outcomes. STUDY QUESTION: We conducted a study to assess how polymorphisms in the CES1 (rs2244613) and ABCB1 (rs4148738) genes affect the through plasma level (c min ) of dabigatran and its correlation to clinical outcomes. STUDY DESIGN: Retrospective multicentric study of consecutive patients on dabigatran therapy. Examination of CES1 rs2244613 and ABCB1 rs4148738 polymorphisms, c min 12 hours after administration, clinical follow-up (ischemic stroke, major or clinically relevant hemorrhage, myocardial infarction, other thromboembolism, and death). MEASURES AND OUTCOMES: A total of 432 patients received treatment for an average of 19.78 months (SD of 20.165). The sex distribution of the patients was 56.5% male, and the average age was 67.56 years (SD of 14.7). The ABCB1 variant genotype was present in 67.8% of patients, whereas 37.5% carried the CES1 polymorphism. RESULTS: Compared with wild-type patients, patients with the CES1 variant had significantly lower dabigatran plasma levels (with a mean difference of 16.986; 95% confidence interval, 5.794-28.178 ng/mL, P = 0.003). We also found a significant risk of major bleeding in patients carrying the ABCB1 rs4148738 allele (hazard ratio = 1.99, confidence interval 95% 1.10 to 3.59, P = 0.024). CONCLUSIONS: The CES1 variant genotype rs2244613 is closely linked with reduced c min of dabigatran. Carriers of the ABCB1 rs4148738 polymorphism exhibit a tendency toward higher plasma levels of dabigatran, which leads to a significantly increased risk of bleeding.
- MeSH
- antithrombiny * škodlivé účinky krev farmakokinetika aplikace a dávkování MeSH
- dabigatran * škodlivé účinky farmakokinetika krev aplikace a dávkování MeSH
- fibrilace síní farmakoterapie genetika komplikace krev MeSH
- ischemická cévní mozková příhoda * prevence a kontrola genetika krev MeSH
- jednonukleotidový polymorfismus MeSH
- karboxylesterhydrolasy * genetika krev MeSH
- krvácení * chemicky indukované krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- P-glykoproteiny * genetika MeSH
- retrospektivní studie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
Numerous functions in pathogenic Pectobacterium are regulated by quorum sensing (QS). Two different aiiA genes isolated from Bacillus sp. A24(aiiAA24) and Bacillus sp. DMS133(aiiADMS133) were used. Both genes encode acyl-homoserine lactonase (AiiA), which disrupts QS in Pectobacterium. To investigate the effect of different AiiAs on the inhibition of Pectobacterium carotovorum pathogenicity, two aiiA genes from different Bacillus strains were cloned and the resulting plasmids pME6863 (aiiAA24) and pME7080 (aiiADMS133) were transformed into P. carotovorum EMPCC cells. The effects of different lactonases on virulence features such as enzymatic activity, twitching and swimming motilities, and production of pellicle and biofilm formation were investigated. In EMPCC/pME6863, twitching and swimming motilities, and pellicle production were significantly reduced compared with EMPCC/pME7080. Quantitative real-time PCR (qRT-PCR) was used to measure virulence gene expression in transformed cells compared with expression levels in wild-type EMPCC. The expression of peh and hrpL genes was greatly reduced in EMPCC/pME6863 compared with EMPCC/pME7080. The sequence alignment and molecular dynamic modeling of two different AiiAA24 and AiiADMS133 proteins suggested that the replacement of proline 210 from AiiAA24 to serine in AiiADMS133 caused the reduction of enzyme activity in AiiADMS133.
- MeSH
- Bacillus * genetika enzymologie MeSH
- bakteriální proteiny * genetika metabolismus MeSH
- biofilmy růst a vývoj MeSH
- karboxylesterhydrolasy * genetika metabolismus MeSH
- klonování DNA MeSH
- metaloendopeptidasy MeSH
- Pectobacterium carotovorum genetika enzymologie patogenita MeSH
- quorum sensing * MeSH
- regulace genové exprese u bakterií MeSH
- virulence MeSH
- Publikační typ
- časopisecké články MeSH
6-Diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist with robust anticancer efficacy; however, its therapeutic potential was hampered by its biodistribution and toxicity to normal tissues, specifically gastrointestinal (GI) tissues. To circumvent DON's toxicity, we synthesized a series of tumor-targeted DON prodrugs designed to circulate inert in plasma and preferentially activate over DON in tumor. Our best prodrug 6 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate) showed stability in plasma, liver, and intestinal homogenates yet was readily cleaved to DON in P493B lymphoma cells, exhibiting a 55-fold enhanced tumor cell-to-plasma ratio versus that of DON and resulting in a dose-dependent inhibition of cell proliferation. Using carboxylesterase 1 knockout mice that were shown to mimic human prodrug metabolism, systemic administration of 6 delivered 11-fold higher DON exposure to tumor (target tissue; AUC0- t = 5.1 nmol h/g) versus GI tissues (toxicity tissue; AUC0- t = 0.45 nmol h/g). In summary, these studies describe the discovery of a glutamine antagonist prodrug that provides selective tumor exposure.
- MeSH
- acetylace MeSH
- antitumorózní látky aplikace a dávkování MeSH
- diazooxonorleucin aplikace a dávkování farmakokinetika MeSH
- karboxylesterhydrolasy genetika MeSH
- lidé MeSH
- lysin chemie MeSH
- myši knockoutované MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- plocha pod křivkou MeSH
- prasata MeSH
- prekurzory léčiv chemie MeSH
- systémy cílené aplikace léků * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Pectin methylesterase (PME) controls the methylesterification status of pectins and thereby determines the biophysical properties of plant cell walls, which are important for tissue growth and weakening processes. We demonstrate here that tissue-specific and spatiotemporal alterations in cell wall pectin methylesterification occur during the germination of garden cress (Lepidium sativum). These cell wall changes are associated with characteristic expression patterns of PME genes and resultant enzyme activities in the key seed compartments CAP (micropylar endosperm) and RAD (radicle plus lower hypocotyl). Transcriptome and quantitative real-time reverse transcription-polymerase chain reaction analysis as well as PME enzyme activity measurements of separated seed compartments, including CAP and RAD, revealed distinct phases during germination. These were associated with hormonal and compartment-specific regulation of PME group 1, PME group 2, and PME inhibitor transcript expression and total PME activity. The regulatory patterns indicated a role for PME activity in testa rupture (TR). Consistent with a role for cell wall pectin methylesterification in TR, treatment of seeds with PME resulted in enhanced testa permeability and promoted TR. Mathematical modeling of transcript expression changes in germinating garden cress and Arabidopsis (Arabidopsis thaliana) seeds suggested that group 2 PMEs make a major contribution to the overall PME activity rather than acting as PME inhibitors. It is concluded that regulated changes in the degree of pectin methylesterification through CAP- and RAD-specific PME and PME inhibitor expression play a crucial role during Brassicaceae seed germination.
- MeSH
- endosperm enzymologie fyziologie MeSH
- hypokotyl enzymologie fyziologie MeSH
- karboxylesterhydrolasy biosyntéza genetika fyziologie MeSH
- klíčení genetika fyziologie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- Lepidium sativum enzymologie genetika fyziologie MeSH
- regulace genové exprese u rostlin genetika fyziologie MeSH
- rostlinné proteiny genetika fyziologie MeSH
- semena rostlinná enzymologie fyziologie MeSH
- stanovení celkové genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
It is hypothesized that oligosaccharides are another potential source of immunological cross-reaction between different plant allergens. Patatin is the most abundant glycoprotein in potato and has been described to have an oligosaccharide of composition Man3(Xyl)GlcNAc2(Fuc). In this work, N-glycosylation profiles of patatin proteins isolated from tubers of different potato species were investigated and compared. Oligosaccharides were released by enzymatic digestion with PNAGase A and analyzed primarily by matrix-assisted laser desorption ionization mass spectrometry. For glycan labeling, a modified version of on-target derivatization with phenylhydrazine was applied. This study found the presence of glycan structures not described previously in patatins of potato tubers, and their glycan profiles significantly differed. This knowledge about the glycosylation of potato patatins may be helpful for correct choice of potato species to decrease the presence of specific glycan epitopes causing food allergy as well as for utilization of potatoes for the manufacture of therapeutic proteins.
- MeSH
- glukany chemie metabolismus MeSH
- glykosylace MeSH
- hlízy rostlin chemie klasifikace genetika metabolismus MeSH
- karboxylesterhydrolasy chemie genetika metabolismus MeSH
- rostlinné proteiny chemie genetika metabolismus MeSH
- Solanum chemie klasifikace genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Pseudomonas chlororaphis strain 449 isolated from the rhizosphere of maize suppresses numerous plant pathogens in vitro. The strain produces phenazine antibiotics and synthesizes at least three types of quorum sensing signaling molecules, N-acylhomoserine lactones. Here we have shown that the rhizospheric P. chlororaphis strains 449, well known strain 30-84 as well as two other P. chlororaphis strains exhibit polygalacturonase activity. Using mini-Tn5 transposon mutagenesis, four independent mutants of strain P. chlororaphis 449 with insertion of mini-Tn5 Km2 in gene gacS of two-component GacA-GacS system of global regulation were selected. All these mutant strains were deficient in production of extracellular proteinase(s), phenazines, N-acylhomoserine lactones synthesis, and did not inhibit the growth of G(+) bacteria in comparison with the wild type strain. The P. chlororaphis 449-06 gacS (-) mutant studied in greater detail was deficient in polygalacturonase, pectin methylesterase activities, swarming motility and antifungal activity. It is the first time the involvement of GacA-GacS system in the regulation of enzymes of pectin metabolism, polygalacturonase and pectin methylesterase, was demonstrated in fluorescent pseudomonads.
- MeSH
- antibióza MeSH
- bakteriální proteiny genetika metabolismus MeSH
- houby fyziologie MeSH
- karboxylesterhydrolasy genetika metabolismus MeSH
- laktony metabolismus MeSH
- mutace MeSH
- polygalakturonasa genetika metabolismus MeSH
- Pseudomonas enzymologie fyziologie genetika MeSH
- půdní mikrobiologie MeSH
- regulace genové exprese u bakterií MeSH
- transkripční faktory genetika metabolismus MeSH
- vývojová regulace genové exprese MeSH
- MeSH
- DNA primery MeSH
- genetická vazba MeSH
- inbrední kmeny potkanů * genetika MeSH
- karboxylesterasa MeSH
- karboxylesterhydrolasy * genetika MeSH
- krysa rodu rattus MeSH
- mapování chromozomů * MeSH
- molekulární sekvence - údaje MeSH
- polymerázová řetězová reakce MeSH
- satelitní DNA genetika MeSH
- sekvence nukleotidů MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
