INTRODUCTION: To date, there is not generally accepted and universal indicator of activity, and functional integrity of the small intestine in patients with coeliac disease. The aim of our study was to investigate whether serum concentrations of the non-essential amino acids citrulline and ornithine might have this function. METHODS: We examined serum citrulline and ornithine concentrations in a subgroup of patients with proven coeliac disease and healthy controls (blood donors). RESULTS: A total of 94 patients with coeliac disease (29 men, mean age 53 ± 18 years; 65 women, mean age 44 ± 14 years) and 35 healthy controls (blood donors) in whom coeliac disease was serologically excluded (10 men, mean age 51 ± 14 years; 25 women, mean age 46 ± 12 years) were included in the study. Significantly lower concentrations of serum ornithine were found in patients with coeliac disease (mean 65 ± 3 μmol/L; median 63 μmol/L, IQR 34 μmol/L, p < 0.001). No statistically nor clinically significant differences were found in the citrulline concentrations between the study and control group. CONCLUSIONS: Serum ornithine (but not citrulline) may be useful for assessing the functional status of the small intestine in uncomplicated coeliac disease. Further studies involving more detailed analysis of dietary and metabolic changes in patients will be needed to reach definitive conclusions.
- MeSH
- celiakie * MeSH
- citrulin * metabolismus MeSH
- dieta MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- ornithin metabolismus MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
We adapted a radioligand receptor binding assay for measuring insulin levels in unknown samples. The assay enables rapid and accurate determination of insulin concentrations in experimental samples, such as from insulin-secreting cells. The principle of the method is based on the binding competition of insulin in a measured sample with a radiolabeled insulin for insulin receptor (IR) in IM-9 cells. Both key components, radiolabeled insulin and IM-9 cells, are commercially available. The IR binding assay was used to determine unknown amounts of insulin secreted by MIN6 β cell line after stimulation with glucose, arginine, ornithine, dopamine, and serotonin. The experimental data obtained by the IR binding assay were compared to the results determined by RIA kits and both methods showed a very good agreement of results. We observed the stimulation of glucose-induced insulin secretion from MIN6 cells by arginine, weaker stimulation by ornithine, but inhibitory effects of dopamine. Serotonin effects were either stimulatory or inhibitory, depending on the concentration of serotonin used. The results will require further investigation. The study also clearly revealed advantages of the IR binding assay that allows the measuring of a higher throughput of measured samples, with a broader range of concentrations than in the case of RIA kits. The IR binding assay can provide an alternative to standard RIA and ELISA assays for the determination of insulin levels in experimental samples and can be especially useful in scientific laboratories studying insulin production and secretion by β cells and searching for new modulators of insulin secretion.
- MeSH
- arginin metabolismus MeSH
- beta-buňky metabolismus MeSH
- buněčné linie MeSH
- dopamin metabolismus MeSH
- glukosa metabolismus MeSH
- inzulin analýza metabolismus MeSH
- krysa rodu rattus MeSH
- Langerhansovy ostrůvky metabolismus MeSH
- lidé MeSH
- myši MeSH
- ornithin metabolismus MeSH
- potkani Wistar MeSH
- radioimunoanalýza metody MeSH
- radioligandová zkouška metody MeSH
- sekrece inzulinu * MeSH
- serotonin metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Circulating extracellular DNA (ecDNA) is known to worsen the outcome of many diseases. ecDNA released from neutrophils during infection or inflammation is present in the form of neutrophil extracellular traps (NETs). It has been shown that higher ecDNA concentration occurs in a number of inflammatory diseases including inflammatory bowel disease (IBD). Enzymes such as peptidyl arginine deiminases (PADs) are crucial for NET formation. We sought to describe the dynamics of ecDNA concentrations and fragmentation, along with NETosis during a mouse model of chemically induced colitis. Plasma ecDNA concentration was highest on day seven of dextran sulfate sodium (DSS) intake and the increase was time-dependent. This increase correlated with the percentage of cells undergoing NETosis and other markers of disease activity. Relative proportion of nuclear ecDNA increased towards more severe colitis; however, absolute amount decreased. In colon explant medium, the highest concentration of ecDNA was on day three of DSS consumption. Early administration of PAD4 inhibitors did not alleviate disease activity, but lowered the ecDNA concentration. These results uncover the biological characteristics of ecDNA in IBD and support the role of ecDNA in intestinal inflammation. The therapeutic intervention aimed at NETs and/or nuclear ecDNA has yet to be fully investigated.
- MeSH
- biologické markery metabolismus MeSH
- deoxyribonukleasy metabolismus MeSH
- DNA krev metabolismus MeSH
- endoskopie MeSH
- extracelulární pasti účinky léků metabolismus MeSH
- extracelulární prostor metabolismus MeSH
- kolitida krev chemicky indukované patologie MeSH
- mitochondriální DNA krev MeSH
- myši inbrední C57BL MeSH
- ornithin analogy a deriváty farmakologie MeSH
- peptidylarginindeiminasa typu 4 metabolismus MeSH
- síran dextranu MeSH
- streptonigrin farmakologie MeSH
- střeva účinky léků patologie MeSH
- střevní sliznice účinky léků patologie MeSH
- stupeň závažnosti nemoci MeSH
- zánět krev patologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We present the solid-phase synthesis of 1,2-dihydroquinazoline-2-carboxylate derivatives with a quaternary carbon in position 2 and their subsequent cyclization in solution into compounds with unique 3D architectures and pharmacological relevance-spiroquinazolines, namely, 1' H-spiro[pyrrolidine-3,2'-quinazolin]-2-ones and 1' H-spiro[piperidine-3,2'-quinazolin]-2-ones. Acyclic precursors were prepared from commercially available building blocks: protected amino acids (2,4-diaminobutyric acid and ornithine), 2-nitrobenzensulfonyl chlorides and α-bromoacetophenones. The crucial step of the synthesis was a base-mediated tandem reaction including C-arylation followed by cyclization into indazole oxides, and the formation of a 5-membered heterocycle was accomplished by ring expansion into quinazolines. These derivatives were cyclized into spiro compounds in solution after cleavage from the resin.
- MeSH
- acetofenony chemie MeSH
- aminobutyráty chemie MeSH
- chinazoliny chemická syntéza MeSH
- cyklizace MeSH
- nitrobenzeny chemie MeSH
- ornithin chemie MeSH
- piperidiny chemická syntéza MeSH
- pyrrolidiny chemická syntéza MeSH
- spirosloučeniny chemická syntéza MeSH
- techniky syntézy na pevné fázi metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Sucrose and molasses are attractive raw materials for industrial fermentation. Although Corynebacterium glutamicum shows sucrose-utilizing activity, sucrose or molasses is only a fraction of carbon source used in the fermentation medium in most works. An engineered C. glutamicum strain was constructed for producing L-ornithine with sucrose or molasses as a sole carbon source by transferring Mannheimia succiniciproducens β-fructofuranosidase gene (sacC). The engineered strain, C. glutamicum ΔAPE6937R42 (pEC-sacC), produced 22.0 g/L of L-ornithine with sucrose as the sole carbon source, which is on par with that obtained by the parent strain C. glutamicum ΔAPE6937R42 with glucose as the sole carbon. The resulting strain C. glutamicum ΔAPE6937R42 (pEC-sacC) produced 27.0 g/L of L-ornithine with molasses as the sole carbon source, which is higher than that obtained by the parent strain C. glutamicum ΔAPE6937R42 with glucose as the sole carbon. This strategy can be applied for developing sucrose- or molasses-utilizing industrial strains.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Corynebacterium glutamicum genetika metabolismus MeSH
- fermentace MeSH
- melasa analýza mikrobiologie MeSH
- ornithin biosyntéza MeSH
- průmyslová mikrobiologie MeSH
- sacharosa metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Citrulline ureidase (CTU, EC3.5.1.20) degrades citrulline into ornithine, carbon dioxide, and ammonia. Here, we present the report on expression of recombinant CTU in Escherichia coli. The soluble and active recombinant CTU was expressed in the periplasmic space with the vector pET-22b and the His-tagged CTU was purified with Ni-Affinity Chromatography. The yield of soluble recombinant protein was significantly increased when 1% sorbitol was supplemented in medium. By using phenylisothiocyanate (PITC) pre-column derivatization HPLC, the enzyme activity of recombinant CTU was determined via measuring of the substrate citrulline and the corresponding products. Our results could be useful in the study of CTU biochemical characteristics, enzymatic preparation of ornithine and development of an enzymatic detection method of citrulline.
- MeSH
- Bacteria MeSH
- bakteriologické techniky metody využití MeSH
- chromatografie afinitní metody využití MeSH
- citrulin * izolace a purifikace metabolismus MeSH
- enzymatické testy metody využití MeSH
- Escherichia coli enzymologie imunologie izolace a purifikace MeSH
- Francisella tularensis * enzymologie izolace a purifikace metabolismus MeSH
- hydrolasy izolace a purifikace MeSH
- ornithin * izolace a purifikace metabolismus MeSH
- rekombinantní proteiny genetika izolace a purifikace metabolismus MeSH
- sorbitol diagnostické užití MeSH
- statistika jako téma MeSH
- tularemie diagnóza etiologie mikrobiologie MeSH
- ureasa izolace a purifikace metabolismus MeSH
- vysokoúčinná kapalinová chromatografie metody využití MeSH
- Publikační typ
- práce podpořená grantem MeSH
BACKGROUND: Escherichia coli is a frequent causative agent of urinary tract infections, and increasing resistance of E. coli to antimicrobials presents a growing challenge. METHODS: Here we compare phenotypes of extended-spectrum β-lactamase (ESBL) producers (n = 220) with a control group of sensitive strains (non-ESBL producers; n = 150). For each strain, we assessed the presence of O25 antigen, hemolysis, biofilm production, sensitivity to antibiotics, and biochemical profile. RESULTS: Compared to the control group, ESBL producers were more frequently O25 positive (6.0% vs. 42.3%) and less frequently hemolytic (34.7% vs. 6.4%). Comparison of biofilm production in brain-heart infusion (BHI) and in BHI with 4% glucose supplementation showed that ESBL-positive strains produced biofilm in BHI with glucose less intensely than the control group (p < 0.05). Most ESBL producers were ciprofloxacin-resistant (91.8%). Biochemical analyses revealed that ESBL producers more frequently utilized inositol, ornithine, sorbitol, melibiose, and saccharose, whereas the control group more frequently used esculin, lysine, arginine, and dulcitol. The control group strains with O25 antigen were more commonly resistant to ciprofloxacin (p < 0.05). Pulsed-field gel electrophoresis results showed higher variability among the control group of sensitive strains. CONCLUSION: These findings suggest a potential to detect ESBL strains based on virulence factors and biochemical properties, which could be useful in shaping proper empiric antimicrobial therapy, and for initiating such therapy as soon as possible.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence MeSH
- beta-laktamasy sekrece MeSH
- biofilmy růst a vývoj MeSH
- buněčná stěna chemie MeSH
- ciprofloxacin farmakologie MeSH
- dospělí MeSH
- Escherichia coli klasifikace genetika izolace a purifikace fyziologie MeSH
- fenotyp MeSH
- genetická variace MeSH
- genotyp MeSH
- hemolýza MeSH
- infekce močového ústrojí mikrobiologie MeSH
- infekce vyvolané Escherichia coli mikrobiologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- mladiství MeSH
- mladý dospělý MeSH
- moč mikrobiologie MeSH
- O-antigeny analýza MeSH
- ornithin analýza MeSH
- pulzní gelová elektroforéza MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- techniky typizace bakterií MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- arginin biosyntéza fyziologie MeSH
- biochemické jevy MeSH
- lidé MeSH
- močovina * dějiny metabolismus MeSH
- ornithin biosyntéza fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- historické články MeSH
- O autorovi
- Krebs, Hans Adolf, 1900-1981 Autorita
A series of N (alpha)-acyl (alkyl)- and N (alpha)-alkoxycarbonyl-derivatives of L- and D-ornithine were prepared, characterized, and analyzed for their potency toward the bacterial enzyme N (alpha)-acetyl-L-ornithine deacetylase (ArgE). ArgE catalyzes the conversion of N (alpha)-acetyl-L-ornithine to L-ornithine in the fifth step of the biosynthetic pathway for arginine, a necessary step for bacterial growth. Most of the compounds tested provided IC(50) values in the muM range toward ArgE, indicating that they are moderately strong inhibitors. N (alpha)-chloroacetyl-L-ornithine (1g) was the best inhibitor tested toward ArgE providing an IC(50) value of 85 microM while N (alpha)-trifluoroacetyl-L-ornithine (1f), N (alpha)-ethoxycarbonyl-L-ornithine (2b), and N (alpha)-acetyl-D-ornithine (1a) weakly inhibited ArgE activity providing IC(50) values between 200 and 410 microM. Weak inhibitory potency toward Bacillus subtilis-168 for N (alpha)-acetyl-D-ornithine (1a) and N (alpha)-fluoro- (1f), N (alpha)-chloro- (1g), N (alpha)-dichloro- (1h), and N (alpha)-trichloroacetyl-ornithine (1i) was also observed. These data correlate well with the IC(50) values determined for ArgE, suggesting that these compounds might be capable of getting across the cell membrane and that ArgE is likely the bacterial enzymatic target.
- MeSH
- amidohydrolasy antagonisté a inhibitory MeSH
- antibakteriální látky chemická syntéza chemie farmakologie MeSH
- Bacillus subtilis účinky léků MeSH
- fosgen analogy a deriváty chemie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- inhibitory enzymů chemická syntéza chemie farmakologie MeSH
- kinetika MeSH
- magnetická rezonanční spektroskopie MeSH
- mikrobiální testy citlivosti MeSH
- molekulární struktura MeSH
- molekulová hmotnost MeSH
- ornithin analogy a deriváty chemická syntéza chemie farmakologie MeSH
- polystyreny chemie MeSH
- proteiny z Escherichia coli antagonisté a inhibitory MeSH
- racionální návrh léčiv MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Glycation is a process closely related to the aging and pathogenesis of diabetic complications. Reactive alpha-dicarbonyl compounds (e.g., methylglyoxal) are formed during middle stage of glycation reaction. Compounds that would inhibit the glycation process have been seeked for years. The objective of this study was to investigate the inhibitory effect of hydroxycitric (0.25-2.5 mM) and uric acid (0.4-1.2 mM) on middle stage of protein glycation in vitro using the model containing aspartate aminotransferase (AST) and 0.5 mM methylglyoxal. Hydroxycitric acid, at all tested concentrations, reduced AST activity decrease and formation of fluorescent AGEs during incubation of the enzyme with methylglyoxal at 37 degrees C. This compound also prevented formation of high-molecular weight protein cross-links and changes in molecular charge of AST caused by glycation. Uric acid showed no positive anti-glycation activity. The results support the hypothesis that hydroxycitric acid has beneficial effects in controlling protein glycation.
- MeSH
- aspartátaminotransferasy chemie metabolismus MeSH
- citráty farmakologie MeSH
- fluorescence MeSH
- glykosylace účinky léků MeSH
- kvarterní struktura proteinů MeSH
- kyselina močová farmakologie MeSH
- ornithin analogy a deriváty metabolismus MeSH
- produkty pokročilé glykace metabolismus MeSH
- pyrimidiny metabolismus MeSH
- pyruvaldehyd farmakologie MeSH
- reagencia zkříženě vázaná farmakologie MeSH
- Sus scrofa MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH