The biosynthesis of the lincosamide antibiotics lincomycin A and celesticetin involves the pyridoxal-5'-phosphate (PLP)-dependent enzymes LmbF and CcbF, which are responsible for bifurcation of the biosynthetic pathways. Despite recognizing the same S-glycosyl-L-cysteine structure of the substrates, LmbF catalyses thiol formation through β-elimination, whereas CcbF produces S-acetaldehyde through decarboxylation-coupled oxidative deamination. The structural basis for the diversification mechanism remains largely unexplored. Here we conduct structure-function analyses of LmbF and CcbF. X-ray crystal structures, docking and molecular dynamics simulations reveal that active-site aromatic residues play important roles in controlling the substrate binding mode and the reaction outcome. Furthermore, the reaction selectivity and oxygen-utilization of LmbF and CcbF were rationally engineered through structure- and calculation-based mutagenesis. Thus, the catalytic function of CcbF was switched to that of LmbF, and, remarkably, both LmbF and CcbF variants gained the oxidative-amidation activity to produce an unnatural S-acetamide derivative of lincosamide.
BACKGROUND & AIMS: Vitamin B6 status and mortality risk are inversely associated in different patient groups, while prospective studies in the general population are lacking. Here, for the first time, we evaluated the association between biomarkers of vitamin B6 status and mortality risk in a large population-based study. METHODS: The vitamin B6 vitamers pyridoxal-5'-phosphat (PLP) and 4-pyridoxic acid (4-PA) were measured by high-performance liquid chromatography in the National Health and Nutrition Examination Survey (NHANES) between 2005 and 2010. Participants' vital status and causes of death were recorded until December 2015. Multivariable Cox regression analyses were carried out to estimate Hazard Ratios (HRs) and 95% confidence intervals (CIs) of mortality across quintiles of PLP, 4-PA, and the ratio of 4-PA and PLP. RESULTS: Out of 15,304 study participants aged between 20 and 85 years at baseline, 1666 (7.7%) died during a median follow-up time of 7.8 years. An inverse association between PLP and mortality was found in a multivariable model adjusted for socioeconomic and lifestyle factors but became statistically non-significant upon adjustment for routine biomarkers (C-reactive protein, creatinine, albumin, and alkaline phosphatase). There was a significant linear trend for a positive association between 4-PA levels and mortality risk in the fully adjusted regression model, although a comparison of extreme quintiles (quintile 5 vs. quintile 1) did not show a significant difference (HRQ5vs.Q1 (95% CI): 1.19 (0.93, 1.51), plinear trend = 0.02). A positive association between the 4-PA/PLP ratio and all-cause mortality was observed in the multivariable model, with an HRsQ5vs.Q1 of 1.45 (95% CI: 1.14, 1.85; plinear trend<0.0001). There were no significant associations between the biomarkers and cardiovascular or cancer mortality. The association between 4-PA/PLP and mortality risk was heterogeneous across age groups, and only statistically significant among participants older than 65 years at baseline (HRQ5vs.Q1 (95% CI): 1.72 (1.29, 2.29), plinear trend<0.0001). In this group, 4-PA/PLP was also associated with cancer mortality, with an HR Q5vs.Q1 of 2.16 (1.20, 3.90), plinear trend = 0.02). CONCLUSION: Increased vitamin B6 turnover, as indicated by a higher 4-PA/PLP ratio, was associated with all-cause and cancer mortality among the older U.S. general population. Intervention trials are needed to assess whether older individuals with a high 4-PA/PLP ratio would benefit from increased vitamin B6 intake.
- MeSH
- biologické markery MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- nádory * epidemiologie MeSH
- prospektivní studie MeSH
- pyridoxalfosfát MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- vitamin B6 * MeSH
- výživa - přehledy MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- aplikace orální MeSH
- biochemická analýza krve metody MeSH
- dospělí MeSH
- kalibrace MeSH
- lidé MeSH
- pyridoxalfosfát krev MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- thiamin aplikace a dávkování krev metabolismus MeSH
- thiaminmonofosfát krev MeSH
- thiaminpyrofosfát krev MeSH
- vitamin B6 krev metabolismus MeSH
- vysokoúčinná kapalinová chromatografie přístrojové vybavení metody MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Pyridoxin dependentní epilepsie je autozomálně recesivně dědičné onemocnění, které se prenatálně, neonatálně a v časném dětství do 3 let projevuje farmakorezistentními epileptickými záchvaty. Jde o dědičné poruchy metabolizmu pyridoxinu asociované s mutacemi v genech ALDH7A1 nebo ALDH4A1. Pyridoxin dependentní epilepsie jsou úspěšně léčitelné vysokými dávkami pyridoxinu. Diagnostika je založena na genetickém a biochemickém vyšetření. Prezentovány jsou kazuistiky tři pacientů s typickým průběhem pyridoxin dependentní epilepsie a geneticky potvrzenou mutací v ALDH7A1 genu.
Pyridoxine-dependent epilepsy is a rare autosomal recessive hereditary disorder causing severe intractable epileptic seizures presenting typically in prenatal and neonatal period, rarely in early infancy (age up to 3 years). Pyridoxine-dependent epilepsy, caused by metabolic disturbance of pyridoxine, is associated with mutations in the ALDH7A1 or ALDH4A1 gene. Pyridoxine-dependent epilepsy is successfully treatable using high doses of pyridoxine. The diagnosis is based on biochemical and genetic examinations. Three case reports of patients with a typical clinical course of pyridoxine-dependent epilepsy and genetically detected mutation in the ALDH7A1 gene are presented.
- Klíčová slova
- pyridoxin dependentní epilepsie, pyridoxin dependentní záchvaty, gen ALDH7A1,
- MeSH
- aldehyddehydrogenasa * genetika MeSH
- epilepsie * etiologie farmakoterapie genetika MeSH
- genetické nemoci vrozené MeSH
- genetické testování MeSH
- lidé MeSH
- mutace MeSH
- nedostatek vitaminu B6 MeSH
- novorozenec MeSH
- pyridoxalfosfát nedostatek MeSH
- pyridoxin * metabolismus nedostatek terapeutické užití MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- Publikační typ
- kazuistiky MeSH
- práce podpořená grantem MeSH
Silver doped diamond-like carbon layers were deposited by dual pulsed laser deposition using two KrF excimer lasers. The concentration of Ag, determined by XPS and WDS, moved from zero to ~10at%. We found that the sp2/sp3ratio, film roughness and the number of droplets (SEM and AFM) increased with increasing silver concentration. The sp3content measurement (XPS) was influenced by ion cluster surface sputtering and varied from 71.0% (undoped DLC) to 36.2% (for 9.3at% Ag). Transmission was measured on the scale from 200nm to 1100nm, and decreased with increasing silver content. An increase of Ag content has an effect on the decrease of the storage modulus (E') and the indentation hardness (HIT). The highest values HIT=51.9GPa and E'=270.6GPa were measured on a sample with 0at% Ag. The lowest values HIT=26.0GPa and E'=180.2GPa were measured on a sample of 9.3at% Ag. Film adhesion was studied using the scratch test and was up to 20.8N for the highest Ag concentration. The contact angle (CA) measurements for water showed that the CA of Ag-DLC films was higher (78°-98°) that of DLC film (77°). The surface free energy of DLC and of Ag-DLC was about 40mJ·m-2. Antibacterial properties were studied using gram positive and gram negative bacteria. The antibacterial effects increased with the Ag concentration and were ~99.9% after 24h for the layers with the highest silver content (9.3at%). Our results demonstrate that the Ag-doped DLC films are potentially useful biomaterials having both good mechanical properties and antimicrobial characteristics. PRIME NOVELTY STATEMENT: Unique manufacturing technique dual pulsed laser deposition was applied on hydrogen-free diamond-like carbon doped by Ag including topological, physical and antibacterial characterization.
- MeSH
- antibakteriální látky chemie MeSH
- diamant MeSH
- povrchové vlastnosti MeSH
- pyridoxal analogy a deriváty MeSH
- stříbro MeSH
- uhlík MeSH
- Publikační typ
- časopisecké články MeSH
A large subgroup of the repeat in toxin (RTX) family of leukotoxins of Gram-negative pathogens consists of pore-forming hemolysins. These can permeabilize mammalian erythrocytes (RBCs) and provoke their colloid osmotic lysis (hemolytic activity). Recently, ATP leakage through pannexin channels and P2X receptor-mediated opening of cellular calcium and potassium channels were implicated in cell permeabilization by pore-forming toxins. In the study described here, we examined the role played by purinergic signaling in the cytolytic action of two RTX toxins that form pores of different sizes. The cytolytic potency of ApxIA hemolysin of Actinobacillus pleuropneumoniae, which forms pores about 2.4 nm wide, was clearly reduced in the presence of P2X7 receptor antagonists or an ATP scavenger, such as pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), Brilliant Blue G, ATP oxidized sodium salt, or hexokinase. In contrast, antagonists of purinergic signaling had no impact on the hemolytic potency of the adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis, which forms pores of 0.6 to 0.8 nm in diameter. Moreover, the conductance of pores formed by ApxIA increased with the toxin concentration, while the conductance of the CyaA single pore units was constant at various toxin concentrations. However, the P2X7 receptor antagonist PPADS inhibited in a concentration-dependent manner the exacerbated hemolytic activity of a CyaA-ΔN489 construct (lacking 489 N-terminal residues of CyaA), which exhibited a strongly enhanced pore-forming propensity (>20-fold) and also formed severalfold larger conductance units in planar lipid bilayers than intact CyaA. These results point to a pore size threshold of purinergic amplification involvement in cell permeabilization by pore-forming RTX toxins.
- MeSH
- Actinobacillus pleuropneumoniae metabolismus MeSH
- adenylátcyklasový toxin antagonisté a inhibitory chemie metabolismus MeSH
- bakteriální proteiny antagonisté a inhibitory chemie metabolismus MeSH
- Bordetella pertussis metabolismus MeSH
- buněčná membrána metabolismus MeSH
- erytrocyty metabolismus MeSH
- hemolýza * MeSH
- hemolyziny antagonisté a inhibitory chemie metabolismus MeSH
- hexokinasa MeSH
- kultivované buňky MeSH
- lipidové dvojvrstvy metabolismus MeSH
- makrofágy MeSH
- myši MeSH
- osmotický tlak MeSH
- permeabilita buněčné membrány MeSH
- pyridoxalfosfát analogy a deriváty MeSH
- rosanilinová barviva MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS/MS. The median enzyme activity in control plasma samples was 404 nmol/h/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol/ho/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol/hour/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.
- MeSH
- biochemická analýza krve metody normy MeSH
- chromatografie kapalinová MeSH
- cystathionin-beta-synthasa nedostatek metabolismus MeSH
- homocystinurie krev diagnóza enzymologie MeSH
- imunoenzymatické techniky metody normy MeSH
- kalibrace MeSH
- krevní plazma chemie enzymologie metabolismus MeSH
- lidé MeSH
- pyridoxalfosfát farmakologie MeSH
- S-adenosylmethionin farmakologie MeSH
- stabilita enzymů MeSH
- studie případů a kontrol MeSH
- tandemová hmotnostní spektrometrie metody normy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
Human serine racemase (hSR) is a cytosolic pyridoxal-5'-phosphate dependent enzyme responsible for production of D-serine in the central nervous system. D-Serine acts as an endogenous coagonist of N-methyl-D-aspartate receptor ion channels. Increased levels of D-serine have been linked to amyotrophic lateral sclerosis and Alzheimer's disease, indicating that SR inhibitors might be useful tools for investigation or treatment of neurodegenerative diseases. However, despite hSR's promise as a therapeutic target, relatively few specific inhibitors have been identified, which is due in part to the lack of a three-dimensional structure of the enzyme. Here, we present a strategy for the generation and screening of random hSR mutants. From a library of randomly mutated hSR variants, twenty-seven soluble mutants were selected, expressed, and evaluated for enzymatic activity. Taking three carefully characterized mutants as an example, we show how this strategy can be used to pinpoint structurally and functionally important residues. In particular, we identify S84 and P111 as residues crucial for hSR activity and C217 and K221 as residues important for binding of the Mg2+ cofactor as well as for overall stability of the enzyme.
Pyridoxin-dependentní epilepsie je autozomálně recesivně dědičné onemocnění, které se prenatálně, neonatálně a v časném dětství do 3 let projevuje farmakorezistentními epileptickými záchvaty. Jde o dědičné poruchy metabolizmu pyridoxinu asociované s mutacemi v genech ALDH7A1 nebo ALDH4A1. Podobným onemocněním je pyridoxal-fosfát dependentní epilepsie (neonatální epileptická encefalopatie) podmíněná mutacemi v PNPO genu. Pyridoxin-dependentní epilepsie jsou úspěšně léčitelné vysokými dávkami pyridoxinu. Pyridoxal-fosfát dependentní epilepsie jsou na terapii pyridoxinem refrakterní, ale reagují na léčbu pyridoxal-fosfátem. Diagnostika obou jednotek je založena na genetickém a biochemickém vyšetření.
Pyridoxine dependent epilepsy is a rare autosomal recessive hereditary disorder causing a severe intractable epileptic seizures presenting typically in prenatal and neonatal period, rarely in early infancy (age up to 3 years). Pyridoxine dependent epilepsy, caused by metabolic disturbance of pyridoxine, is associated with mutations in ALDH7A1 or ALDH4A1 gene. Similar condition, pyridoxal-phosphate dependent epilepsy (also called neonatal epileptic encephalopathy), is caused by mutations in PNPO gene. Pyridoxine dependent epilepsy is successfully treatable using high doses of pyridoxine. Neonatal epileptic encephalopathy is refractory to pyridoxine administration, however responses to treatment with pyridoxal-phosphate. The diagnosis of both pyridoxine dependent epilepsy and neonatal epileptic encephalopathy is based on biochemical and genetic examinations.
- Klíčová slova
- pyridoxal-fosfát, neonatální epileptická encefalopatie,
- MeSH
- aldehyddehydrogenasa genetika MeSH
- diagnostické techniky molekulární metody využití MeSH
- dítě MeSH
- elektroencefalografie využití MeSH
- lidé MeSH
- nemoci novorozenců genetika metabolismus MeSH
- novorozenec MeSH
- pyridoxalfosfát aplikace a dávkování metabolismus MeSH
- pyridoxin aplikace a dávkování metabolismus MeSH
- vitamin B6 aplikace a dávkování škodlivé účinky MeSH
- záchvaty diagnóza farmakoterapie genetika MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- novorozenec MeSH
- Publikační typ
- přehledy MeSH
The aim of this study was to develop and validate HPLC methods for the determination in plasma of two novel thiosemicarbazone anti-tumour drugs developed in our laboratories (Dp44mT and N4mT). The appropriate separations were achieved using a HS F5 HPLC column with the mobile phase composed of a mixture of either acetate buffer/EDTA or EDTA and acetonitrile (62:38 and 50:50, v/v, respectively). The plasma samples were pretreated with SPE (phenyl and C18, respectively). Furthermore, these methods were successfully applied to in vitro plasma stability experiments. The investigation has clearly shown that both thiosemicarbazones are markedly more stable in plasma than their aroylhydrazone forerunners.
- MeSH
- analýza rozptylu MeSH
- financování organizované MeSH
- interpretace statistických dat MeSH
- isoniazid analogy a deriváty analýza metabolismus MeSH
- králíci MeSH
- lidé MeSH
- naftaleny krev MeSH
- prasata MeSH
- protinádorové látky krev MeSH
- pyridoxal analogy a deriváty analýza metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- stabilita léku MeSH
- thiosemikarbazony krev MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé MeSH
- zvířata MeSH
