Nucleoside Phosphorylases make N7-xanthosine.

Modern, highly evolved nucleoside-processing enzymes are known to exhibit perfect regioselectivity over the glycosylation of purine nucleobases at N9. We herein report an exception to this paradigm. Wild-type nucleoside phosphorylases also furnish N7-xanthosine, a "non-native" ribosylation regioisomer of xanthosine. This unusual nucleoside possesses several atypical physicochemical properties such as redshifted absorption spectra, a high equilibrium constant of phosphorolysis and low acidity. Ultimately, the biosynthesis of this previously unknown natural product illustrates how even highly evolved, essential enzymes from primary metabolism are imperfect catalysts.

Protective Effects of Xanthine Derivatives Against Arsenic Trioxide-Induced Oxidative Stress in Mouse Hepatic and Renal Tissues.

In this study, the protective efficacy of pentoxifylline (PTX) as a xanthine derivative against arsenic trioxide (ATO)-induced kidney and liver damage in mice was investigated. Thirty-six mice were divided into six groups, receiving intraperitoneal injections of saline, ATO, PTX, or a combination for four weeks. Blood samples were analyzed for serum biochemistry, while hepatic tissue underwent examination for histopathological changes and assessment of oxidative stress markers and antioxidant gene expression through Real-Time PCR. ATO exposure significantly increased serum markers (creatinine, ALT, BUN, ALP, AST) and induced histopathological changes in the liver. Moreover, it elevated renal and hepatic nitric oxide (NO) and lipid peroxidation (LPO) levels, and reduced antioxidant enzyme expression (CAT, GSR, GPx, MPO, SOD), total thiol groups (TTGs), and total antioxidant capacity (TAC). Conversely, PTX treatment effectively lowered serum hepatic and renal markers, improved antioxidant markers, and induced histopathological alterations. Notably, PTX did not significantly affect renal and hepatic NO levels. These findings suggest that PTX offers therapeutic potential in mitigating liver and acute kidney injuries induced by various insults, including exposure to ATO.

Identification and quantification of (poly)phenol and methylxanthine metabolites in human cerebrospinal fluid: evidence of their ability to cross the BBB.

Increasing evidence suggests that dietary (poly)phenols and methylxanthines have neuroprotective effects; however, little is known about whether they can cross the blood-brain barrier (BBB) and exert direct effects on the brain. We investigated the presence of (poly)phenol and methylxanthine metabolites in plasma and cerebrospinal fluid (CSF) from 90 individuals at risk of dementia using liquid chromatography-mass spectrometry and predicted their mechanism of transport across the BBB using in silico modelling techniques. A total of 123 and 127 metabolites were detected in CSF and plasma, respectively. In silico analysis suggests that 5 of the 20 metabolites quantified in CSF can cross the BBB by passive diffusion, while at least 9 metabolites require the aid of cell transporters to cross the BBB. Our results showed that (poly)phenols and methylxanthines are bioavailable, can cross the BBB via passive diffusion or transport carriers, and can reach brain tissues to exert neuroprotective effects.

Increased genital mucosal cytokines in Canadian women associate with higher antigen-presenting cells, inflammatory metabolites, epithelial barrier disruption, and the depletion of L. crispatus.

BACKGROUND: Cervicovaginal inflammation has been linked to negative reproductive health outcomes including the acquisition of HIV, other sexually transmitted infections, and cervical carcinogenesis. While changes to the vaginal microbiome have been linked to genital inflammation, the molecular relationships between the functional components of the microbiome with cervical immunology in the reproductive tract are understudied, limiting our understanding of mucosal biology that may be important for reproductive health. RESULTS: In this study, we used a multi'-omics approach to profile cervicovaginal samples collected from 43 Canadian women to characterize host, immune, functional microbiome, and metabolome features of cervicovaginal inflammation. We demonstrate that inflammation is associated with lower amounts of L. crispatus and higher levels of cervical antigen-presenting cells (APCs). Proteomic analysis showed an upregulation of pathways related to neutrophil degranulation, complement, and leukocyte migration, with lower levels of cornified envelope and cell-cell adherens junctions. Functional microbiome analysis showed reductions in carbohydrate metabolism and lactic acid, with increases in xanthine and other metabolites. Bayesian network analysis linked L. crispatus with glycolytic and nucleotide metabolism, succinate and xanthine, and epithelial proteins SCEL and IVL as major molecular features associated with pro-inflammatory cytokines and increased APCs. CONCLUSIONS: This study identified key molecular and immunological relationships with cervicovaginal inflammation, including higher APCs, bacterial metabolism, and proteome alterations that underlie inflammation. As APCs are involved in HIV transmission, parturition, and cervical cancer progression, further studies are needed to explore the interactions between these cells, bacterial metabolism, mucosal immunity, and their relationship to reproductive health. Video Abstract.

Changes on proteomic and metabolomic profiling of cryopreserved sperm effected by melatonin.

Cryopreservation may reduce sperm fertility due to cryodamage including physical-chemical and oxidative stress damages. As a powerful antioxidant, melatonin has been reported to improve cryoprotective effect of sperm. However, the molecular mechanism of melatonin on cryopreserved ram sperm hasn't been fully understand. Give this, this study aimed to investigate the postthaw motility parameters, antioxidative enzyme activities and lipid peroxidation, as well as proteomic, metabolomic changes of Huang-huai ram spermatozoa with freezing medium supplemented with melatonin. Melatonin was firstly replenished to the medium to yield five different final concentrations: 0.1, 0.5, 1.0, 1.5, and 2.0 mM. A control (NC) group without melatonin replenishment was included. Protective effects of melatonin as evidenced by postthaw motility, activities of T-AOC, T-SOD, GSH-Px, CAT, contents of MDA, 4-HNE, as well as acrosome integrity, plasma membrane integrity, with 0.5 mM being the most effective concentration (MC group). Furthermore, 29 differentially abundant proteins involving in sperm functions were screened among Fresh, NC and MC groups of samples (n = 5) based on the 4D-LFQ, with 7 of them upregulated in Fresh and MC groups. 26 differentially abundant metabolites were obtained involving in sperm metabolism among the three groups of samples (n = 8) based on the UHPLC-QE-MS, with 18 of them upregulated in Fresh and MC groups. According to the bioinformatic analysis, melatonin may have positive effects on frozen ram spermatozoa by regulating the abundance changes of vital proteins and metabolites related to sperm function. Particularly, several proteins such as PRCP, NDUFB8, NDUFB9, SDHC, DCTN1, TUBB6, TUBA3E, SSNA1, as well as metabolites like L-histidine, L-targinine, ursolic acid, xanthine may be potential novel biomarkers for evaluating the postthaw quality of ram spermatozoa. In conclusion, a dose-dependent replenishment of melatonin to freezing medium protected ram spermatozoa during cryopreservation, which can improve motility, antioxidant enzyme activities, reduce levels of lipid peroxidation products, modify the proteomic and metabolomic profiling of cryopreserved ram spermatozoa through reduction of oxidative stress, maintenance of OXPHOS and microtubule structure. SIGNIFICANCE: Melatonin, a powerful antioxidant protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner, with 0.5 mM being the most effective concentration. Furthermore, sequencing results based on the 4D-LFQ combined with the UHPLC-QE-MS indicated that melatonin modifies proteomic and metabolomic profiling of ram sperm during cryopreservation. According to the bioinformatic analysis, melatonin may have positive effects on frozen ram spermatozoa by regulating the expression changes of vital proteins and metabolites related to sperm metabolism and function. Particularly, several potential novel biomarkers for evaluating the postthaw quality of ram spermatozoa were acquired, proteins such as PRCP, NDUFB8, NDUFB9, SDHC, DCTN1, TUBB6, TUBA3E, SSNA1, as well as metabolites like L-histidine, L-targinine, ursolic acid, xanthine.

Effects of polystyrene microplastics acute exposure in the liver of swordtail fish (Xiphophorus helleri) revealed by LC-MS metabolomics.

As global pollution, microplastics pollution has aroused growing concerns. In our experiment, the effect of microplastics acute exposure on the liver of swordtail fish was investigated by using LC-MS metabolomics. Fishes treated with high concentration polystyrene microspheres (1 µm) for 72 h were divided into three concentration groups: (A) no microplastics, (B): 1 × 106 microspheres L-1, (C): 1 × 107 microspheres L-1. Metabolomic analysis indicated that exposure to microplastics caused alterations of metabolic profiles in swordtail fish, including 37 differential metabolites were identified in B vs. A, screened out ten significant metabolites, which involved 14 metabolic pathways. One hundred three differential metabolites were identified in C vs. A, screened out 16 significant metabolites, which involved 30 metabolic pathways. Six significant metabolites were overlapping in group B vs. A and C vs. A; they are 3-hydroxyanthranilic acid, l-histidine, citrulline, linoleic acid, pantothenate, and xanthine. In addition, four metabolic pathways are overlapping in group B vs. A and C vs. A; they are beta-alanine metabolism, biosynthesis of amino acids, linoleic acid metabolism, and aminoacyl-tRNA biosynthesis. These differential metabolites were involved in oxidative stress, immune function, energy metabolism, sugar metabolism, lipid metabolism, molecule transport, and weakened feed utilization, growth performance, nutrient metabolism, and animal growth. Furthermore, we found that the number of interfered amino acids and microplastics showed a dose-effect. In summary, great attention should be paid to the potential impact of microplastics on aquatic organisms.

Discovery and Biochemical Characterization of N-methyltransferase Genes Involved in Purine Alkaloid Biosynthetic Pathway of Camellia gymnogyna Hung T.Chang (Theaceae) from Dayao Mountain.

In the present study, purine alkaloid analysis and transcriptome of Camellia gymnogyna Hung T. Chang (Theaceae) from Dayao Mountain were performed by high-performance liquid chromatography (HPLC) and RNA-Seq, respectively. The results showed that the major purine alkaloids accumulated in Camellia gymnogyna Hung T. Chang (Theaceae) were theobromine together with a small amount of theacrine and caffeine. Through polymerase chain reaction (PCR), three types of cDNA encoding N-methyltransferases were isolated from the leaves of Camellia gymnogyna Hung T. Chang (Theaceae) and designated GCS1, GCS2, and GCS3. We subsequently expressed GCS1, GCS2, and GCS3 in Escherichia coli and incubated lysates of the bacterial cells with a variety of xanthine substrates in the presence of S-adenosyl-L-methionine as the methyl donor. We found that the recombinant GCS1 proteins catalyzed 1,3,7-trimethyluric acid to produce theacrine, the recombinant GCS3 proteins catalyzed 7-methylxanthine to produce theobromine, while the recombinant GCS2 proteins did not catalyze any xanthine derivatives. Simultaneous analysis of the expressions of GCS1, GCS2, GCS3, and a caffeine synthase gene (TCS1) in Camellia gymnogyna Hung T. Chang (Theaceae) and other tea plants provided a reference for further research on the functions of these genes.

SLC23A3 is a renal hypoxanthine transporter.

LLC-PK1 renal cells show Na+-dependent and Na+-independent hypoxanthine uptake. While the latter is inhibited by adenine, neither are inhibited by xanthine. In rats, intestinal Na+-dependent hypoxanthine transporter Slc23a4 is not expressed in the kidney, and its action is inhibited by xanthine. This study aimed to clone Slc23a4-paralog SLC23A3 from the human kidney and investigate its hypoxanthine transport activity. We observed Na+-dependent 10 nM [3H]-hypoxanthine uptake in SLC23A3 RNA-injected Xenopus oocytes. Moreover, 100 µM xanthine did not inhibit Na+-independent 300 nM [3H]-hypoxanthine uptake, whereas 100 µM adenine did. These results confirm that SLC23A3 is a hypoxanthine transporter in the human kidney.

Identification of Co-Expressed Genes Related to Theacrine Synthesis in Tea Flowers at Different Developmental Stages.

Jiaocheng kucha is the first reported tea germplasm resource which contains theacrine founded in Fujian Province. Currently, the anabolic mechanism of theacrine within tea leaves is clear, but there are few studies focused on its flowers. In order to further explore the mechanism of theacrine synthesis and related genes in flowers, current study applied Jiaocheng kucha flowers (JC) as test materials and Fuding Dabaicha flowers (FD) as control materials to make transcriptome sequencing, and determination of purine alkaloid content in three different developmental periods (flower bud stage, whitening stage and full opening stage). The results showed that the flower in all stages of JC contained theacrine. The theacrine in the flower bud stage was significantly higher than in the other stages. The differentially expressed genes (DEGs) at three different developmental stages were screened from the transcriptome data, and were in a total of 5642, 8640 and 8465. These DEGs related to the synthesis of theacrine were primarily annotated to the pathways of purine alkaloids. Among them, the number of DEGs in xanthine synthesis pathway was the largest and upregulated in JC, while it was the smallest in caffeine synthesis pathway and downregulated in JC. Further weighted gene co-expression network (WGCNA) indicated that ADSL (CsTGY03G0002327), ADSL (CsTGY09G0001824) and UAZ (CsTGY06G0002694) may be a hub gene for the regulation of theacrine metabolism in JC. Our results will contribute to the identification of candidate genes related to the synthesis of theacrine in tea flowers, and explore the molecular mechanism of theacrine synthesis in JC at different developmental stages.

Systemic Medications in Chronic Obstructive Pulmonary Disease: Use and Outcomes.

Inhaled therapy remains the cornerstone of chronic obstructive pulmonary disease pharmacologic care, but some systemic treatments can be of help when the burden of the disease remains high. Azithromycin, phosphodiesterase-4 inhibitors, and mucoactive agents can be used in such situations. The major difficulty remains in the identification of the optimal target populations. Another difficulty is to determine how these treatments should be positioned in the global treatment algorithm. For instance, should they be prescribed in addition to other antiinflammatory agents or should they replace them in some cases? Research is ongoing to identify new therapeutic targets.

Convergent Biochemical Pathways for Xanthine Alkaloid Production in Plants Evolved from Ancestral Enzymes with Different Catalytic Properties.

Convergent evolution is widespread but the extent to which common ancestral conditions are necessary to facilitate the independent acquisition of similar traits remains unclear. In order to better understand how ancestral biosynthetic catalytic capabilities might lead to convergent evolution of similar modern-day biochemical pathways, we resurrected ancient enzymes of the caffeine synthase (CS) methyltransferases that are responsible for theobromine and caffeine production in flowering plants. Ancestral CS enzymes of Theobroma, Paullinia, and Camellia exhibited similar substrate preferences but these resulted in the formation of different sets of products. From these ancestral enzymes, descendants with similar substrate preference and product formation independently evolved after gene duplication events in Theobroma and Paullinia. Thus, it appears that the convergent modern-day pathways likely originated from ancestral pathways with different inferred flux. Subsequently, the modern-day enzymes originated independently via gene duplication and their convergent catalytic characteristics evolved to partition the multiple ancestral activities by different mutations that occurred in homologous regions of the ancestral proteins. These results show that even when modern-day pathways and recruited genes are similar, the antecedent conditions may be distinctive such that different evolutionary steps are required to generate convergence.

Association Between Kidney Clearance of Secretory Solutes and Cardiovascular Events: The Chronic Renal Insufficiency Cohort (CRIC) Study.

RATIONALE & OBJECTIVE: The clearance of protein-bound solutes by the proximal tubules is an innate kidney mechanism for removing putative uremic toxins that could exert cardiovascular toxicity in humans. However, potential associations between impaired kidney clearances of secretory solutes and cardiovascular events among patients with chronic kidney disease (CKD) remains uncertain. STUDY DESIGN: A multicenter, prospective, cohort study. SETTING & PARTICIPANTS: We evaluated 3,407 participants from the Chronic Renal Insufficiency Cohort (CRIC) study. EXPOSURES: Baseline kidney clearances of 8 secretory solutes. We measured concentrations of secretory solutes in plasma and paired 24-hour urine specimens using liquid chromatography-tandem mass spectrometry (LC-MS/MS). OUTCOMES: Incident heart failure, myocardial infarction, and stroke events. ANALYTICAL APPROACH: We used Cox regression to evaluate associations of baseline secretory solute clearances with incident study outcomes adjusting for estimated GFR (eGFR) and other confounders. RESULTS: Participants had a mean age of 56 years; 45% were women; 41% were Black; and the median estimated glomerular filtration rate (eGFR) was 43 mL/min/1.73 m2. Lower 24-hour kidney clearance of secretory solutes were associated with incident heart failure and myocardial infarction but not incident stroke over long-term follow-up after controlling for demographics and traditional risk factors. However, these associations were attenuated and not statistically significant after adjustment for eGFR. LIMITATIONS: Exclusion of patients with severely reduced eGFR at baseline; measurement variability in secretory solutes clearances. CONCLUSIONS: In a national cohort study of CKD, no clinically or statistically relevant associations were observed between the kidney clearances of endogenous secretory solutes and incident heart failure, myocardial infarction, or stroke after adjustment for eGFR. These findings suggest that tubular secretory clearance provides little additional information about the development of cardiovascular disease events beyond glomerular measures of GFR and albuminuria among patients with mild-to-moderate CKD.

Human TRPC5 structures reveal interaction of a xanthine-based TRPC1/4/5 inhibitor with a conserved lipid binding site.

TRPC1/4/5 channels are non-specific cation channels implicated in a wide variety of diseases, and TRPC1/4/5 inhibitors have recently entered clinical trials. However, fundamental and translational studies require a better understanding of TRPC1/4/5 channel regulation by endogenous and exogenous factors. Although several potent and selective TRPC1/4/5 modulators have been reported, the paucity of mechanistic insights into their modes-of-action remains a barrier to the development of new chemical probes and drug candidates. Xanthine-based modulators include the most potent and selective TRPC1/4/5 inhibitors described to date, as well as TRPC5 activators. Our previous studies suggest that xanthines interact with a, so far, elusive pocket of TRPC1/4/5 channels that is essential to channel gating. Here we report the structure of a small-molecule-bound TRPC1/4/5 channel-human TRPC5 in complex with the xanthine Pico145-to 3.0 Å. We found that Pico145 binds to a conserved lipid binding site of TRPC5, where it displaces a bound phospholipid. Our findings explain the mode-of-action of xanthine-based TRPC1/4/5 modulators, and suggest a structural basis for TRPC1/4/5 modulation by endogenous factors such as (phospho)lipids and Zn2+ ions. These studies lay the foundations for the structure-based design of new generations of TRPC1/4/5 modulators.

3-Methylxanthine production through biodegradation of theobromine by Aspergillus sydowii PT-2.

BACKGROUND: Methylxanthines, including caffeine, theobromine and theophylline, are natural and synthetic compounds in tea, which could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several microbial isolates from Pu-erh tea could degrade and convert caffeine and theophylline. We speculated that these candidate isolates also could degrade and convert theobromine through N-demethylation and oxidation. In this study, seven tea-derived fungal strains were inoculated into various theobromine agar medias and theobromine liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture to investigate their potential application in the production of 3-methylxanthine. RESULTS: Based on theobromine utilization capacity, Aspergillus niger PT-1, Aspergillus sydowii PT-2, Aspergillus ustus PT-6 and Aspergillus tamarii PT-7 have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii PT-2 and A. tamarii PT-7 could degrade theobromine significantly (p < 0.05) in all given liquid mediums. 3,7-Dimethyluric acid, 3-methylxanthine, 7-methylxanthine, 3-methyluric acid, xanthine, and uric acid were detected in A. sydowii PT-2 and A. tamarii PT-7 culture, respectively, which confirmed the existence of N-demethylation and oxidation in theobromine catabolism. 3-Methylxanthine was common and main demethylated metabolite of theobromine in the liquid culture. 3-Methylxanthine in A. sydowii PT-2 culture showed a linear relation with initial theobromine concentrations that 177.12 ± 14.06 mg/L 3-methylxanthine was accumulated in TLM-S with 300 mg/L theobromine. Additionally, pH at 5 and metal ion of Fe2+ promoted 3-methylxanthine production significantly (p < 0.05). CONCLUSIONS: This study is the first to confirm that A. sydowii PT-2 and A. tamarii PT-7 degrade theobromine through N-demethylation and oxidation, respectively. A. sydowii PT-2 showed the potential application in 3-methylxanthine production with theobromine as feedstock through the N-demethylation at N-7 position.

Detection of caffeine and its main metabolites for early diagnosis of Parkinson's disease using micellar electrokinetic capillary chromatography.

Caffeine (CA) is a common xanthine alkaloid found in tea leaves, coffee beans, and other natural plants, and is the most widely used psychotropic substance in the world. Accumulating evidence suggests that low plasma levels of CA and its metabolites may serve as reliable diagnostic markers for early Parkinson's disease (PD) patients. In this study, we demonstrated a new MEKC method for determining CA and its three main downstream metabolites, paraxanthine (PX), theobromine (TB), and theophylline (TP), in human plasma. Plasma samples were collected, and analyzed using MEKC, after SPE. The running buffer was composed of 35 mM phosphate, pH of 10.5, and 25 mM SDS. The separation voltage was 15 kV and the detection wavelength was at 210 nm. Under the optimum conditions, four distinct analytes were completely separated and detected in less than 12 min. Method limits of detection were as low as 7.5 ng/mL for CA, 5.0 ng/mL for TB, and 4.0 ng/mL for both PX and TP. The recoveries were between 88.0% and 105.9%. This method was successfully applied to 27 human plasma samples. The results indicate that the plasma concentrations of the four analytes are significantly lower in patients with early PD than in control subjects (p < 0.05). The area under curve was improved to 0.839 when CA and its three main metabolites were included, suggesting that MEKC testing of CA, TP, TB, and PX may serve as a potential method for early diagnosis of PD.

A bacterial riboswitch class senses xanthine and uric acid to regulate genes associated with purine oxidation.

Dozens of candidate orphan riboswitch classes have been discovered previously by using comparative sequence analysis algorithms to search bacterial genomic sequence databases. Each orphan is classified by the presence of distinct conserved nucleotide sequences and secondary structure features, and by its association with particular types of genes. One previously reported orphan riboswitch candidate is the "NMT1 motif," which forms a hairpin structure with an internal bulge that includes numerous highly conserved nucleotides. This motif associates with genes annotated to encode various dioxygenase enzymes, transporters, or proteins that have roles associated with thiamin or histidine metabolism. Biochemical evaluation of numerous ligand candidates revealed that NMT1 motif RNA constructs most tightly bind 8-azaxanthine, xanthine, and uric acid, whereas most other closely related compounds are strongly rejected. Genetic assays revealed that NMT1 motif RNAs function to turn off gene expression upon ligand binding, likely by regulating translation initiation. These results suggest that NMT1 motif RNAs function as aptamer domains for a riboswitch class that specifically responds to high concentrations of oxidized purines. Members of this "xanthine riboswitch" class appear to regulate genes predominantly related to purine transport and oxidation, thus avoiding the effects of overproduction of these common purine derivatives.

Isolation, characterization and application of theophylline-degrading Aspergillus fungi.

BACKGROUND: Caffeine, theobromine and theophylline are main purine alkaloid in tea. Theophylline is the downstream metabolite and it remains at a very low level in Camellia sinensis. In our previous study, Aspergillus sydowii could convert caffeine into theophylline in solid-state fermentation of pu-erh tea through N-demethylation. In this study, tea-derived fungi caused theophylline degradation in the solid-state fermentation. The purpose of this study is identify and isolate theophylline-degrading fungi and investigate their application in production of methylxanthines with theophylline as feedstock through microbial conversion. RESULTS: Seven tea-derived fungi were collected and identified by ITS, ß-tubulin and calmodulin gene sequences, Aspergillus ustus, Aspergillus tamarii, Aspergillus niger and A. sydowii associated with solid-state fermentation of pu-erh tea have shown ability to degrade theophylline in liquid culture. Particularly, A. ustus and A. tamarii could degrade theophylline highly significantly (p < 0.01). 1,3-dimethyluric acid, 3-methylxanthine, 3-methyluric acid, xanthine and uric acid were detected consecutively by HPLC in A. ustus and A. tamarii, respectively. The data from absolute quantification analysis suggested that 3-methylxanthine and xanthine were the main degraded metabolites in A. ustus and A. tamarii, respectively. 129.48 ± 5.81 mg/L of 3-methylxanthine and 159.11 ± 10.8 mg/L of xanthine were produced by A. ustus and A. tamarii in 300 mg/L of theophylline liquid medium, respectively. CONCLUSIONS: For the first time, we confirmed that isolated A. ustus, A. tamarii degrade theophylline through N-demethylation and oxidation. We were able to biologically produce 3-methylxanthine and xanthine efficiently from theophylline through a new microbial synthesis platform with A. ustus and A. tamarii as appropriate starter strains.

Novel caffeine degradation gene cluster is mega-plasmid encoded in Paraburkholderia caffeinilytica CF1.

The widespread use of caffeine in food and drug industries has caused great environmental pollution. Herein, an efficient caffeine-degrading strain Paraburkholderia caffeinilytica CF1 isolated from a tea garden in China can utilize caffeine as its sole carbon and nitrogen source. Combination of chromatographic and spectrophotometric techniques confirmed that strain CF1 adopts N-demethylation pathway for caffeine degradation. Whole genome sequencing of strain CF1 reveals that it has two chromosomes with sizes 3.62 Mb and 4.53 Mb, and a 174-kb mega-plasmid. The plasmid P1 specifically harbors the genes essential for caffeine metabolism. By analyzing the sequence alignment and quantitative real-time PCR data, the redundant gene cluster of caffeine degradation was elucidated. Genes related to catalyzing the N1-demethylation of caffeine to theobromine, the first step of caffeine degradation were heterologously expressed, and methylxanthine N1-demethylase was purified and characterized. Above all, this study systematically unravels the molecular mechanism of caffeine degradation by Paraburkholderia. KEY POINTS: • Caffeine degradation cluster in Paraburkholderia caffeinilytica CF1 was located in mega-plasmid P1. • The whole genome and the caffeine degrading pathway of P. caffeinilytica CF1 were sequenced and elucidated, respectively. • This study succeeded in heterologous expression of methylxanthine N1-demethylase (CdnA) and Rieske oxygenase reductase (CdnD) and illuminated the roles of CdnA and CdnD in caffeine degradation of P. caffeinilytica CF1.

Increased Adenine Nucleotide Degradation in Skeletal Muscle Atrophy.

Adenine nucleotides (AdNs: ATP, ADP, AMP) are essential biological compounds that facilitate many necessary cellular processes by providing chemical energy, mediating intracellular signaling, and regulating protein metabolism and solubilization. A dramatic reduction in total AdNs is observed in atrophic skeletal muscle across numerous disease states and conditions, such as cancer, diabetes, chronic kidney disease, heart failure, COPD, sepsis, muscular dystrophy, denervation, disuse, and sarcopenia. The reduced AdNs in atrophic skeletal muscle are accompanied by increased expression/activities of AdN degrading enzymes and the accumulation of degradation products (IMP, hypoxanthine, xanthine, uric acid), suggesting that the lower AdN content is largely the result of increased nucleotide degradation. Furthermore, this characteristic decrease of AdNs suggests that increased nucleotide degradation contributes to the general pathophysiology of skeletal muscle atrophy. In view of the numerous energetic, and non-energetic, roles of AdNs in skeletal muscle, investigations into the physiological consequences of AdN degradation may provide valuable insight into the mechanisms of muscle atrophy.

Synthesis of new isoquinoline-base-oxadiazole derivatives as potent inhibitors of thymidine phosphorylase and molecular docking study.

Here in this study regarding the over expression of TP, which causes some physical, mental and socio problems like psoriasis, chronic inflammatory disease, tumor angiogenesis and rheumatoid arthritis etc. By this consideration, the inhibition of this enzyme is vital to secure life from serious threats. In connection with this, we have synthesized twenty derivatives of isoquinoline bearing oxadiazole (1-20), characterized through different spectroscopic techniques such as HREI-MS, 1H- NMR and 13C-NMR and evaluated for thymidine phosphorylase inhibition. All analogues showed outstanding inhibitory potential ranging in between 1.10 ± 0.05 to 54.60 ± 1.50 µM. 7-Deazaxanthine (IC50 = 38.68 ± 1.12 µM) was used as a positive control. Through limited structure activity relationships study, it has been observed that the difference in inhibitory activities of screened analogs are mainly affected by different substitutions on phenyl ring. The effective binding interactions of the most active analogs were confirmed through docking study.