guanosine-monophosphate and guanosine-5--monophosphorothioate

The aim was to isolate a neotype bifidobacteria strain and evaluate its in vitro probiotic potential.. Bifidobacterium pseudolongum YY-26 (CGMCC 24310) was isolated from faeces of mice treated with low-molecular-weight hydrolyzed guar gum (GMPS) and identified based on 16S rRNA sequence and genome sequence. Whole-genome sequencing obtained using PacBio's single-molecular and Illumina's paired-end sequencing technology. A genome of 2.1 Mb in length, with 1877 predicted protein-coding sequences was obtained. Carbohydrate-Activity enZyme analysis revealed that YY-26 encodes 66 enzymes related to carbohydrate metabolism. Whole genome sequence analysis revealed the typical probiotic characteristics of YY-26, including safety in genetic level and ability to produce beneficial metabolites and extracellular polysaccharides. Ability of extensive carbon source utilization and short-chain fatty acid production was observed with single YY-26 cultivation. Considerable acetic acids and lactic acids were determined in GMPS utilization. YY-26 showed tolerance to simulated gastrointestinal tract and displayed appreciable antioxidant activity of free radical scavenging.. B. pseudolongum YY-26 was identified with numerous probiotic-associated genes and its probiotic characteristics were verified in vitro.. This study supplemented with limited publicly information regarding the genomes of B. pseudolongum strains and revealed the probiotic potential of YY-26.

Topics: Animals; Antioxidants; Bifidobacterium; Carbohydrates; Carbon; Fatty Acids, Volatile; Free Radicals; Guanosine Monophosphate; Mice; Probiotics; RNA, Ribosomal, 16S; Thionucleotides

It had not been reported that myocardial scar shown on gated myocardial perfusion SPECT (GMPS) might reduce after cardiac resynchronization therapy (CRT). In this study, we aim to investigate the clinical impact and characteristic of scar reduction (SR) after CRT.. Sixty-one heart failure patients following standard indication for CRT received twice GMPS as pre- and post-CRT evaluations. The patients with an absolute reduction of scar ≥ 10% after CRT were classified as the SR group while the rest were classified as the non-SR group. The SR group (N = 22, 36%) showed more improvement on LV function (∆LVEF: 18.1 ± 12.4 vs 9.4 ± 9.9 %, P = 0.007, ∆ESV: - 91.6 ± 52.6 vs - 38.1 ± 46.5 mL, P < 0.001) and dyssynchrony (ΔPSD: - 26.19 ± 18.42 vs - 5.8 ± 23.0°, P < 0.001, Δ BW: - 128.7 ± 82.8 vs - 25.2 ± 109.0°, P < 0.001) than non-SR group (N = 39, 64%). Multivariate logistic regression analysis showed baseline QRSd (95% CI 1.019-1.100, P = 0.006) and pre-CRT Reduced Wall Thickening (RWT) (95% CI 1.016-1.173, P = 0.028) were independent predictors for the development of SR.. More than one third of patients showed SR after CRT who had more post-CRT improvement on LV function and dyssynchrony than those without SR. Wider QRSd and higher RWT before CRT were related to the development of SR after CRT.

Topics: Cardiac Resynchronization Therapy; Cicatrix; Guanosine Monophosphate; Heart Failure; Humans; Myocardial Perfusion Imaging; Perfusion; Thionucleotides; Tomography, Emission-Computed, Single-Photon; Treatment Outcome

Esophageal squamous cell carcinoma (ESCC) is a malignant tumor that commonly occurs worldwide. Usually, Asia, especially China, has a high incidence of esophageal cancer. ESCC often has a poor outcome because of a late diagnosis and lack of effective treatments. To build foundations for the early diagnosis and treatment of ESCC, we used the gene expression datasets GSE20347 and GSE17351 from the GEO database and a private dataset to uncover differentially expressed genes (DEGs) and key genes in ESCC. Notably, we found that replication factor C subunit 4 (RFC4) and guanine monophosphate synthase (GMPS) were upregulated but have been rarely studied in ESCC. In particular, to the best of our knowledge, our study is the first to explore GMPS and ESCC. Furthermore, we found that high levels of RFC4 and GMPS expression may result from an increase in DNA copy number alterations. Furthermore, RFC4 and GMPS were both upregulated in the early stage and early nodal metastases of esophageal carcinoma. The expression of RFC4 was strongly correlated with GMPS. In addition, we explored the relationship between RFC4 and GMPS expression and tumor-infiltrating immune cells (TILs) in esophageal carcinoma. The results showed that the levels of RFC4 and GMPS increased with a decrease in some tumor-infiltrating cells. Upregulated RFC4 and GMPS with high TILs indicate a worse prognosis. In summary, our study shows that RFC4 and GMPS have potential as biomarkers for the early diagnosis of ESCC and may played a crucial role in the process of tumor immunity in ESCC.

Topics: Biomarkers, Tumor; Computational Biology; Datasets as Topic; DNA Copy Number Variations; DNA, Neoplasm; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Guanosine Monophosphate; Humans; Male; Middle Aged; Prognosis; Replication Protein C; ROC Curve; Thionucleotides; Up-Regulation

Hydrogen sulfide (H2S) is synthesized in perivascular adipose tissue (PVAT) and induces vasorelaxation. We examined whether the sulfur-containing AMP and GMP analogs AMPS and GMPS can serve as the H2S donors in PVAT. H2S production by isolated rat periaortic adipose tissue (PAT) was measured with a polarographic sensor. In addition, phenylephrine-induced contractility of aortic rings with (+) or without (-) PAT was examined. Isolated PAT produced H2S from AMPS or GMPS in the presence of the P2X7 receptor agonist BzATP. Phenylephrine-induced contractility of PAT(+) rings was lower than of PAT(-) rings. AMPS or GMPS had no effect on the contractility of PAT(-) rings, but used together with BzATP reduced the contractility of PAT(+) rings when endogenous H2S production was inhibited with propargylglycine. A high-fat diet reduced endogenous H2S production by PAT. Interestingly, AMPS and GMPS were converted to H2S by PAT of obese rats, and reduced contractility of PAT(+) aortic rings isolated from these animals even in the absence of BzATP. We conclude that (i) AMPS and GMPS can be hydrolyzed to H2S by PAT when P2X7 receptors are activated, (ii) a high-fat diet impairs endogenous H2S production by PAT, (iii) AMPS and GMPS restore the anticontractile effects of PAT in obese animals without P2X7 stimulation.

Topics: Adenosine Monophosphate; Adipose Tissue; Animals; Aorta; Aorta, Abdominal; Aorta, Thoracic; Guanosine Monophosphate; Hydrogen Sulfide; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth, Vascular; Obesity; Rats, Wistar; Thionucleotides; Vasodilation

Hydrogen sulfide (H2S) is the gasotransmitter enzymatically synthesized in mammalian tissues from l-cysteine. H2S donors are considered as the potential drugs for the treatment of cardiovascular, neurological and inflammatory diseases. Recently, it has been demonstrated that synthetic nucleotide analogs, adenosine- and guanosine 5'-monophosphorothioates (AMPS and GMPS) can be converted to H2S and AMP or GMP, respectively, by purified histidine triad nucleotide-binding (Hint) proteins. We examined if AMPS and GMPS can be used as the H2S donors in intact biological systems. H2S production by isolated rat kidney glomeruli was measured by the specific polarographic sensor. H2S production was detected when glomeruli were incubated with AMPS or GMPS and ionotropic purinergic P2X7 receptor/channel agonist, BzATP. More H2S was generated from GMPS than from equimolar amount of AMPS. Nucleoside phosphorothioates together with BzATP relaxed angiotensin II-preconstricted glomeruli. In addition, infusion of AMPS or GMPS together with BzATP into the renal artery increased filtration fraction and glomerular filtration rate but had no effect on renal vascular resistance or renal blood flow. AMPS but not GMPS was converted to adenosine by isolated glomeruli, however, adenosine was not involved in AMPS-induced H2S synthesis because neither adenosine nor specific adenosine receptor agonists had any effect on H2S production. AMPS, but not GMPS, increased phosphorylation level of AMP-stimulated protein kinase (AMPK), but AMPK inhibitor, compound C, had no effect on AMPS-induced H2S production. In conclusion, nucleoside phosphorothioates are converted to H2S which relaxes isolated kidney glomeruli in vitro and increases glomerular filtration rate in vivo. AMPS and GMPS can be used as the H2S donors in experimental studies and possibly also as the H2S-releasing drugs.

Topics: Adenosine; Adenosine Monophosphate; Adenosine Triphosphate; AMP-Activated Protein Kinases; Animals; Glomerular Filtration Rate; Guanosine Monophosphate; Hydrogen Sulfide; In Vitro Techniques; Kidney Glomerulus; Male; Purinergic P2X Receptor Agonists; Rats, Wistar; Thionucleotides

Here we present the in vitro selection of a novel ribozyme specific for Zn2+-dependent catalysis on hydrolysis of a phosphorothiolate thiolester bond. The ribozyme, called the TW17 ribozyme, was evolved and selected from an artificial RNA pool covalently linked to a biotin-containing substrate through the phosphorothiolate thiolester bond. The secondary structure for the evolved ribozyme consisted of three major helices and three loops. Biochemical and chemical studies of ribozyme-catalyzed reaction products provided evidence that the ribozyme specifically catalyzes hydrolysis of the phosphorothiolate thiolester linkage. A successful ribozyme construct with active catalysis in trans further supported the determined ribozyme structure and indicated the potential of the ribozyme for multiple-substrate turnover. The ribozyme also requires Zn2+ and Mg2+ for maximal catalysis. The TW17 ribozyme, in the presence of Zn2+ and Mg2+, conferred a rate enhancement of at least 5 orders of magnitude when compared to the estimated rate of the uncatalyzed reaction. The ribozyme completely lost catalytic activity in the absence of Zn2+, like Zn2+-dependent protein hydrolases. The discovery and characterization of the TW17 ribozyme suggest additional roles for Zn2+ in ribozyme catalysts.

Topics: Aptamers, Nucleotide; Base Sequence; Biotin; Carbon-Nitrogen Ligases; Catalysis; Guanosine Monophosphate; Mutagenesis, Site-Directed; NADH, NADPH Oxidoreductases; RNA, Catalytic; Substrate Specificity; Thiolester Hydrolases; Thionucleotides; Trans-Activators; Zinc

Ubiquitin-specific proteases (USPs) are papain-like isopeptidases with variable inter- and intramolecular regulatory domains. To understand the effect of these domains on USP activity, we have analyzed the enzyme kinetics of 12 USPs in the presence and absence of modulators using synthetic reagents. This revealed variations of several orders of magnitude in both the catalytic turnover (k(cat)) and ubiquitin (Ub) binding (K(M)) between USPs. Further activity modulation by intramolecular domains affects both the k(cat) and K(M), whereas the intermolecular activators UAF1 and GMPS mainly increase the k(cat). Also, we provide the first comprehensive analysis comparing Ub chain preference. USPs can hydrolyze all linkages and show modest Ub-chain preferences, although some show a lack of activity toward linear di-Ub. This comprehensive kinetic analysis highlights the variability within the USP family.

Topics: Amino Acid Sequence; Catalytic Domain; Endopeptidases; Guanosine Monophosphate; Humans; Kinetics; Nuclear Proteins; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins; Thionucleotides; Ubiquitin; Ubiquitin-Specific Proteases

The present study used a mechanistic approach to control the phase transformation of guanosine 5'-monophosphate (GMP) via the operating conditions of agitation and feed concentration during drowning-out crystallization. First, Fourier transform infrared and UV/vis spectrophotometry were successfully applied to monitor the mass fraction of GMP polymorphs (amorphous and hydrate crystalline GMPs) and GMP supersaturation, respectively, during the crystallization. The phase transformation of amorphous GMP into hydrate crystals was significantly influenced by the agitation, which promoted the mass transfer of GMP dissolution and growth. Therefore, the phase transformation was quickly finished when increasing the agitation speed. However, a high agitation caused breakage of the hydrate crystals, resulting in a reduced crystal size with a bimodal distribution. The phase transformation was also influenced by the GMP feed concentration, as the crystal growth was promoted and the crystal size increased when increasing the feed concentration up to 61 g/l. However, a further increase in the feed concentration caused secondary nucleation due to the induction of a high supersaturation level during the phase transformation, leading to a small crystal size with a bimodal distribution. In addition, the rectangular-shaped hydrate GMP crystals exhibited a higher growth rate in the b direction rather than the a direction. Therefore, the crystal morphology shifted from a long rectangle to a square when increasing the feed concentration.

Topics: Crystallization; Guanosine Monophosphate; Phase Transition; Thionucleotides

Thiopurines (such as azathioprine and 6-mercaptopurine) are widely used for the treatment of patients suffering from malignancies, rheumatic disease, inflammatory bowel disease and solid organ transplant rejection. These drugs are activated and eliminated by a number of enzymes in the human body. This analyzes all the exons and exon-intron junctions of 5 enzyme genes (hypoxanthine-guanine phosphoribosyltransferase, HGPRT; inosine triphosphate pyrophosphatase, ITPA; inosine monophosphate dehydrogenases 1 and 2, IMPDH1 and IMPDH2 and guanosine monophosphate synthetase, GMPS) involved in the metabolism of thiopurine drugs. Twelve novel single nucleotide polymorphisms (SNPs) (HGPRT: IVS6-12C>A (frequency:0.003); ITPA: 569T>C (Phe189Phe, 0.003); IMPDH1: IVS8-15C>A (0.003), IVS9+227A>G (0.003), IVS17+115C>T (0.003), and 930C>T (Thr310Thr, 0.005); IMPDH2: IVS1+50G>T (0.003), IVS2+15G>A (0.010), IVS3-20G>A (0.003), 609C>T (Arg203Arg, 0.003), and 1534C>T (Arg512Trp, 0.003); and GMPS: 1563T>C (Gly521Gly, 0.003)) and 7 known SNPs (ITPA: 94C>A (Pro32Thr, 0.005), 138G>A (Gln46Gln, 0.586), and 563G>A (Glu187Glu, 0.433); IMPDH1: 987G>C (Leu329Leu, 0.113) and 1575A>G (Ala525Ala, 0.620) and GMPS: IVS5-7T>C (0.153), 993A>G (Thr331Thr, 0.153)) were identified in 200 Japanese subjects. These data should provide useful information for thiopurine therapy in the Japanese and as well as other Asian populations.

Topics: Asian People; Azathioprine; Carbon-Nitrogen Ligases; Exons; Gene Frequency; Genes, Recessive; Genetic Variation; Genotype; Guanosine Monophosphate; Humans; Hypoxanthine Phosphoribosyltransferase; Inflammatory Bowel Diseases; Inosine Triphosphatase; Introns; Mercaptopurine; Methyltransferases; Molecular Sequence Data; Mutation; Myeloid-Lymphoid Leukemia Protein; Oncogene Proteins, Fusion; Pharmacogenetics; Phenotype; Polymorphism, Single Nucleotide; Pyrophosphatases; Thionucleotides

Topics: Alkylation; Bradykinin; Catalysis; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Genetic Techniques; Guanosine Monophosphate; Kinetics; Peptides; Phenylmercury Compounds; RNA; Sepharose; Sulfur; Thionucleotides; Transcription, Genetic

Photocrosslinking allows first-order structural analysis with relatively small amounts of biological material and can be applied in complex in vitro systems. In this article we describe methods for positioning both arylazide and thionucleotide photoagents within an RNA of interest by end modification of circularly permuted RNAs. Application of this technique provided a library of constraints that, together with biochemical and phylogenetic comparative data, were used to develop a structure model of the bacterial ribonuclease P ribozyme-substrate complex. Circularly permuted genes for in vitro transcription are generated by PCR from tandem genes. Circularly permuted RNA transcripts can be modified with high efficiency at both the 5' and 3' termini with arylazide crosslinking reagents, or transcription can be primed with photoactive nucleotide analog monophosphates such as 6-thioguanosine. These crosslinking agents can be used over a wide range of experimental conditions but remain inert until they are activated by UV light. Crosslinked sites are subsequently mapped by reverse transcriptase primer extension of gel-purified crosslinked species. In addition to providing basic protocols for these methods, we discuss approaches for establishing the relevance of crosslinking data to native RNA structure.

Topics: Aminophylline; Cross-Linking Reagents; Diphenhydramine; Drug Combinations; Endoribonucleases; Guanosine Monophosphate; Methamphetamine; Models, Chemical; Models, Genetic; Models, Molecular; Molecular Biology; Nucleic Acid Conformation; Photoaffinity Labels; Reproducibility of Results; Ribonuclease P; RNA; RNA, Catalytic; Thionucleotides

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well-established, and a low-resolution model of the three-dimensional structure of the ribozyme-substrate complex has been proposed based on site-specific crosslinking and phylogenetic comparative data [Harris ME et al., 1994 EMBO J 13:3953-3963]. However, several substructures of that model were poorly constrained by the available data. In the present analysis, additional constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. Circularly permuted RNase P RNAs were used to position an azidophenacyl photoactive crosslinking agent specifically at strategic sites within the ribozyme-substrate complex. Crosslink sites were mapped by primer extension and confirmed by analysis of the mobility of the crosslinked RNA lariats on denaturing acrylamide gels relative to circular and linear RNA standards. Crosslinked species generally retained significant catalytic activity, indicating that the results reflect the native ribozyme structure. The crosslinking results support the general configuration of the structure model and predicate new positions and orientations for helices that were previously poorly constrained by the data set. The expanded library of crosslinking constraints was used, together with secondary and tertiary structure identified by phylogenetic sequence comparisons, to refine significantly the model of RNase P RNA with bound substrate pre-tRNA. The crosslinking results and data from chemical-modification and mutational studies are discussed in the context of the current structural perspective on this ribozyme.

Topics: Affinity Labels; Base Sequence; Binding Sites; Computer Simulation; Cross-Linking Reagents; Endoribonucleases; Escherichia coli; Escherichia coli Proteins; Guanosine Monophosphate; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Ribonuclease P; Ribonucleoproteins; RNA; RNA Precursors; RNA, Bacterial; RNA, Catalytic; RNA, Circular; RNA, Transfer; Thionucleotides

An in vitro RNA selection for catalytic activity was used to co-select for binding activity to a small peptide. 5'-phosphorothioate-modified RNA (GMPS-RNA) sequences were selected from a randomized pool of oligoribonucleotides for their ability to accelerate a halide substitution reaction with N-bromoacetyl-bradykinin (BrBK). One RNA selected shows a 2,420-fold increase in rate of reaction with BrBK relative to the starting pool. This reaction is specifically inhibited by free bradykinin (Ki 230 microM), indicating that interactions with bradykinin contribute to the rate enhancement. Inhibition of the reaction by the peptide requires both the amino- and carboxy-terminal arginine residues of the peptide for optimal inhibition activity. Reaction with N-bromoacetamide is not enhanced, indicating that the intrinsic reactivity of the 5' phosphorothioate is not increased in the selected RNA. Through 3'-end boundary analysis, much of the catalytic activity of the selected GMPS-RNA is shown to reside in a hairpin structure in the selected region of the molecule. This hairpin structure is also implicated in the recognition of the peptide substrate.

Topics: Aldehydes; Alkylation; Arginine; Base Sequence; Bradykinin; Butanones; Directed Molecular Evolution; Guanosine Monophosphate; Kinetics; Ligands; Molecular Sequence Data; Nucleic Acid Conformation; Oligoribonucleotides; Protein Binding; RNA; RNA-Binding Proteins; RNA, Catalytic; Substrate Specificity; Sulfur; Thionucleotides

Ribonuclease P RNA is the catalytic moiety of the ribonucleoprotein enzyme that removes precursor sequences from 5'-ends of pre-tRNAs. A photoaffinity cross-linking agent was coupled to the substrate phosphate on which RNase P acts and used to map nucleotides in the vicinity of the catalytic site of this ribozyme. Mature tRNA(Phe) containing a 5'-thiophosphate was synthesized by transcription in vitro using phage T7 RNA polymerase in the presence of guanosine 5'-phosphorothioate. The photoagent (azidophenacyl) was coupled uniquely to the 5'-thiophosphate of the tRNA, the site of action by RNase P. The photoagent-containing tRNA binds to RNase P RNA and is cross-linked by UV irradiation to it at high efficiency (10-30%). Cross-linked conjugates are enzymatically inactive, consistent with the occupancy of the active site of the RNase P RNA by the tRNA. Reversal of the cross-link by phenylmercuric acetate restores activity. The sites of cross-linking in RNase P RNA were determined by primer extension. In order to identify generalities and detect idiosyncrasies, analyses were carried out using RNase P RNAs from three phylogenetically diverse organisms: Bacillus subtilis, Chromatium vinosum and Escherichia coli. In the context of a phylogenetic structure model, two regions of cross-linking are observed in all three RNAs. Two of the RNAs cross-link to a lesser extent at a third structural region and one of the RNAs is cross-linked to a small extent to a fourth region. All the sites of cross-linking between the substrate phosphate in tRNA and the RNase P RNAs are in the conserved core of the structure model, consistent with the importance of the cross-linked residues to the action of this RNA enzyme.

Topics: Affinity Labels; Bacillus subtilis; Base Sequence; Binding Sites; Chromatium; Cross-Linking Reagents; Endoribonucleases; Escherichia coli Proteins; Guanosine Monophosphate; Molecular Sequence Data; Nucleic Acid Conformation; Oligonucleotide Probes; Ribonuclease P; RNA, Bacterial; RNA, Transfer, Phe; Thionucleotides; Transcription, Genetic