Morphine and its pharmacological derivatives are the most prescribed analgesics for moderate to severe pain management. However, chronic use of morphine reduces pathogen clearance and induces ...bacterial translocation across the gut barrier. The enteric microbiome has been shown to have a critical role in the preservation of the mucosal barrier function and metabolic homeostasis. Here, we show for the first time, using bacterial 16s rDNA sequencing, that chronic morphine treatment significantly alters the gut microbial composition and induces preferential expansion of Gram-positive pathogenic and reduction in bile-deconjugating bacterial strains. A significant reduction in both primary and secondary bile acid levels was seen in the gut, but not in the liver with morphine treatment. Morphine-induced microbial dysbiosis and gut barrier disruption was rescued by transplanting placebo-treated microbiota into morphine-treated animals, indicating that microbiome modulation could be exploited as a therapeutic strategy for patients using morphine for pain management.
Carbon dots (CDs) are emerging as the material of choice in a range of applications due to their excellent photoluminescence properties, ease of preparation from inexpensive precursors, and low ...toxicity. However, the precise nature of the mechanism for the fluorescence is still under debate, and several molecular fluorophores have been reported. In this work, a new blue fluorophore, 5-oxopyrrolidine-3-carboxylic acid, was discovered in carbon dots synthesized from the most commonly used precursors: citric acid and urea. The molecular product alone has demonstrated interesting aggregation-enhanced emission (AEE), making it unique compared to other fluorophores known to be generated in CDs. We propose that this molecular fluorophore is associated with a polymer backbone within the CDs, and its fluorescence behavior is largely dependent on intermolecular interactions with the polymers or other fluorophores. Thus, a new class of non-traditional fluorophores is now relevant to the consideration of the CD fluorescence mechanism, providing both an additional challenge to the community in resolving the mechanism and an opportunity for a greater range of CD design schemes and applications.
An unconventional fluorophore, 5-oxopyrrolidine-3-carboxylic acid, was discovered as the fluorescence origin of polymeric carbon dots. This unique fluorophore has aggregation-enhanced emission which contributes to carbon dot fluorescence.
Substrates homoprotocatechuate (HPCA) and O2 bind to the FeII of homoprotocatechuate 2,3-dioxygenase (FeHPCD) in adjacent coordination sites. Transfer of an electron(s) from HPCA to O2 via the iron ...is proposed to activate the substrates for reaction with each other to initiate aromatic ring cleavage. Here, rapid-freeze-quench methods are used to trap and spectroscopically characterize intermediates in the reactions of the HPCA complexes of FeHPCD and the variant His200Asn (FeHPCD–HPCA and H200N–HPCA, respectively) with O2. A blue intermediate forms within 20 ms of mixing of O2 with H200N–HPCA (H200NInt1 HPCA). Parallel mode electron paramagnetic resonance and Mössbauer spectroscopies show that this intermediate contains high-spin FeIII (S = 5/2) antiferromagnetically coupled to a radical (S R = 1/2) to yield an S = 2 state. Together, optical and Mössbauer spectra of the intermediate support assignment of the radical as an HPCA semiquinone, implying that oxygen is bound as a (hydro)peroxo ligand. H200NInt1 HPCA decays over the next 2 s, possibly through an FeII intermediate (H200NInt2 HPCA), to yield the product and the resting FeII enzyme. Reaction of FeHPCD–HPCA with O2 results in rapid formation of a colorless FeII intermediate (FeHPCDInt1 HPCA). This species decays within 1 s to yield the product and the resting enzyme. The absence of a chromophore from a semiquinone or evidence of a spin-coupled species in FeHPCDInt1 HPCA suggests it is an intermediate occurring after O2 activation and attack. The similar Mössbauer parameters for FeHPCDInt1 HPCA and H200NInt2 HPCA suggest these are similar intermediates. The results show that transfer of an electron from the substrate to the O2 via the iron does occur, leading to aromatic ring cleavage.
The arginine decarboxylase pathway, which converts arginine to agmatine, is present in both humans and most bacterial pathogens. In humans agmatine is a neurotransmitter with affinities towards ...α2-adrenoreceptors, serotonin receptors, and may inhibit nitric oxide synthase. In bacteria agmatine serves as a precursor to polyamine synthesis and was recently shown to enhance biofilm development in some strains of the respiratory pathogen Pseudomonas aeruginosa. We determined agmatine is at the center of a competing metabolism in the human lung during airways infections and is influenced by the metabolic phenotypes of the infecting pathogens. Ultra performance liquid chromatography with mass spectrometry detection was used to measure agmatine in human sputum samples from patients with cystic fibrosis, spent supernatant from clinical sputum isolates, and from bronchoalvelolar lavage fluid from mice infected with P. aeruginosa agmatine mutants. Agmatine in human sputum peaks during illness, decreased with treatment and is positively correlated with inflammatory cytokines. Analysis of the agmatine metabolic phenotype in clinical sputum isolates revealed most deplete agmatine when grown in its presence; however a minority appeared to generate large amounts of agmatine presumably driving sputum agmatine to high levels. Agmatine exposure to inflammatory cells and in mice demonstrated its role as a direct immune activator with effects on TNF-α production, likely through NF-κB activation. P. aeruginosa mutants for agmatine detection and metabolism were constructed and show the real-time evolution of host-derived agmatine in the airways during acute lung infection. These experiments also demonstrated pathogen agmatine production can upregulate the inflammatory response. As some clinical isolates have adapted to hypersecrete agmatine, these combined data would suggest agmatine is a novel target for immune modulation in the host-pathogen dynamic.
Determination of tea catechins Dalluge, Joseph J; Nelson, Bryant C
Journal of Chromatography A,
06/2000, Letnik:
881, Številka:
1
Book Review, Journal Article
Recenzirano
An overview of analytical methods for the measurement of biologically important tea catechins is presented. Liquid chromatography and capillary electrophoresis are the most cited techniques for ...catechin separation, identification and quantitation. Liquid chromatography with ultraviolet detection is frequently used; however, mass spectrometry, electrochemical, fluorescence and chemiluminescence detection are also utilized in cases where more sensitive or selective detection is needed. Two modes of capillary electrophoresis, capillary zone electrophoresis and micellar electrokinetic capillary chromatography, have been employed for the determination of catechins. Both modes of capillary electrophoresis are based on ultraviolet detection. Additional analytical techniques, such as gas chromatography, thin-layer chromatography, paper chromatography, spectrophotometry, biosensing, chemiluminescence and nuclear magnetic resonance spectroscopy have also been utilized for the determination of catechins and are reviewed herein.
A study of a variety of stationary phases and elution conditions for the liquid chromatographic (LC) determination of six biologically active green tea catechins has resulted in the development of ...two well-defined, reproducible systems for such analyses which overcome limitations of previously described methods. Comparison of six reversed-phase columns indicates that deactivated stationary phases, which utilize ultrapure silica and maximize coverage of the silica support, provide significantly improved separation and chromatographic efficiencies for catechin analyses using LC, compared to conventional monomeric or polymeric C
18 columns. Evaluation of elution conditions used for the separations reveals that the presence of acid in the mobile phase (0.05% trifluoroacetic acid) is essential for both the complete resolution of the catechins present in tea and the efficient chromatography of these compounds. The efficacy of one of the developed systems was demonstrated by the quantitative measurement of the six biologically active catechins in aqueous infusions of green tea (
Camellia sinensis). Overall precision values for the analyses were within the range 0.3–1% (relative standard deviation).
A method has been developed for the direct microscale determination of 12 catechins in green and black tea infusions. The method is based on liquid chromatography/atmospheric pressure chemical ...ionization-mass spectrometry (LC/APCI-MS). Standard catechin mixtures and tea infusions were analyzed by LC/APCI-MS with detection of protonated molecular ions and characteristic fragment ions for each compound. The identities of eight major catechins and caffeine in tea were established based on LC retention times and simultaneously recorded mass spectra. In addition, monitoring of the catechin-specific retro Diels−Alder fragment ion at m/z 139 throughout the chromatogram provided a unique fingerprint for catechin content in the samples that led to the identification of four minor chemically modified catechin derivatives in the infusions. This report is the first to describe the comprehensive determination of all 12 reported catechins in a single analysis. The utility of LC/APCI-MS for providing routine separation and identification of catechins at femtomole to low-picomole levels without extraction or sample pretreatment, and its potential as a standard analytical tool for the determination of polyphenols in natural products and biological fluids, are discussed.
To investigate the potential of matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS) as a platform to support biodiversity and phylogenetic studies of psychrophilic yeasts in ...cold environments, the technique was employed to rapidly characterize and distinguish three psychrophilic yeasts (
Rhodotorula mucilaginosa
,
Naganishia vishniacii
, and
Dioszegia cryoxerica
) from three mesophilic counterparts (
Saccharomyces cerevisiae
Cry Havoc,
S. cerevisiae
California V Ale, and
S. pastorianus
). A detailed workflow for providing reproducible mass spectral fingerprints of low molecular weight protein/peptide features specific to the organisms studied is presented. The potential of this approach as a tool in the study of biodiversity, systematics, and phylogeny of psychrophilic microorganisms is highlighted.
The influence of posttranscriptional modification on structural stabilization of tRNA from hyperthermophilic archaea was studied, using Pyrococcus furiosus (growth optimum 100 degrees C) as a primary ...model. Optical melting temperatures (Tm) of unfractionated tRNA in 20 mM Mg2+ are 97 degrees C for P. furiosus and 101.5 degrees C for Pyrodictium occultum (growth optimum, 105 degrees C). These values are approximately 20 degrees C higher than predicted solely from G-C content and are attributed primarily to posttranscriptional modification. Twenty-three modified nucleosides were determined in total digests of P. furiosus tRNA by combined HPLC-mass spectrometry. From cells cultured at 70, 85, and 100 degrees C, progressively increased levels of modification were observed within three families of nucleosides, the most highly modified forms of which were N4-acetyl-2'-O-methylcytidine (ac4Cm), N2,N2,2'-O-trimethylguanosine (m2(2)Gm), and 5-methyl-2-thiouridine (m5s2U). Nucleosides ac4Cm and m2(2)Gm, which are unique to the archaeal hyperthermophiles, were shown in earlier NMR studies to exhibit unusually high conformational stabilities that favor the C3'-endo form Kawai, G., et al. (1991) Nucleic Acids Symp. Ser. 21, 49-50; (1992) Nucleosides Nucleotides 11, 759-771. The sequence location of m5s2U was determined by mass spectrometry to be primarily at tRNA position 54, a site of known thermal stabilization in the bacterial thermophile Thermus thermophilus Horie, N., et al. (1985) Biochemistry 24, 5711-5715. It is concluded that selected posttranscriptional modifications in archaeal thermophiles play major stabilizing roles beyond the effects of Mg2+ binding and G-C content, and are proportionally more important and have evolved with greater structural diversity at the nucleoside level in the bacterial thermophiles.
The title peptide, oxytocin trisulfide (
3
), was required on a multi-100 mg scale, as part of an analytical method validation process for the clinically and commercially important product “Oxytocin ...Injection,” which is a sterile isotonic injectable formulation of oxytocin (
2
). We report here that previous chemistry from our academic laboratory can be successfully scaled up in the biotechnology sector to indeed provide pure
3
(overall yield 6
–
7%, > 95% purity). The scaled-up synthesis also gave rise, somewhat unexpectedly, to modest levels of oxytocin tetrasulfide (
4
), and even higher homologues. Protocols for synthesis, purification, and characterization by HPLC, mass spectrometry and amino acid analysis are described and discussed, along with possible mechanistic explanations for our findings.
PDF
