Nucleotide Content Research Articles

Revisiting the role of dihydroorotate dehydrogenase as a therapeutic target for cancer.

Identified as a hallmark of cancer, metabolic reprogramming allows cancer cells to rapidly proliferate, resist chemotherapies, invade, metastasize, and survive a nutrient-deprived microenvironment. Rapidly growing cells depend on sufficient concentrations of nucleotides to sustain proliferation. One enzyme essential for the de novo biosynthesis of pyrimidine-based nucleotides is dihydroorotate dehydrogenase (DHODH), a known therapeutic target for multiple diseases. Brequinar, leflunomide, and teriflunomide, all of which are potent DHODH inhibitors, have been clinically evaluated but failed to receive FDA approval for the treatment of cancer. Inhibition of DHODH depletes intracellular pyrimidine nucleotide pools and results in cell cycle arrest in S-phase, sensitization to current chemotherapies, and differentiation in neural crest cells and acute myeloid leukemia (AML). Furthermore, DHODH is a synthetic lethal susceptibility in several oncogenic backgrounds. Therefore, DHODH-targeted therapy has potential value as part of a combination therapy for the treatment of cancer. In this review, we focus on the de novo pyrimidine biosynthesis pathway as a target for cancer therapy, and in particular, DHODH. In the first part, we provide a comprehensive overview of this pathway and its regulation in cancer. We further describe the relevance of DHODH as a target for cancer therapy using bioinformatic analyses. We then explore the preclinical and clinical results of pharmacological strategies to target the de novo pyrimidine biosynthesis pathway, with an emphasis on DHODH. Finally, we discuss potential strategies to harness DHODH as a target for the treatment of cancer.

Development, validation, and clinical application of a high-performance liquid chromatography-tandem mass spectrometry assay for the quantification of total intracellular β-decitabine nucleotides and genomic DNA incorporated β-decitabine and 5-methyl-2′-deoxycytidine

DNA hypermethylation is an epigenetic event that is commonly found in malignant cells and is used as a therapeutic target for β-decitabine (β-DEC) containing hypomethylating agents (eg Dacogen® and guadecitabine). β-DEC requires cellular uptake and intracellular metabolic activation to β-DEC triphosphate before it can get incorporated into the DNA. Once incorporated in the DNA, β-DEC can exert its hypomethylating effect by trapping DNA methyltransferases (DNMTs), resulting in reduced 5-methyl-2′-deoxycytidine (5mdC) DNA content. β-DEC DNA incorporation and its effect on DNA methylation, however, have not yet been investigated in patients treated with β-DEC containing therapies. For this reason, we developed and validated a sensitive and selective LC–MS/MS method to determine total intracellular β-DEC nucleotide (β-DEC-XP) concentrations, as well as to quantify β-DEC and 5mdC DNA incorporation relative to 2′-deoxycytidine (2dC) DNA content. The assay was successfully validated according to FDA and EMA guidelines in a linear range from 0.5 to 100 ng/mL (β-DEC), 50 to 10,000 ng/mL (2dC), and 5 to 1,000 ng/mL (5mdC) in peripheral blood mononuclear cell (PBMC) lysate. An additional calibrator at a concentration of 0.1 ng/mL was added for β-DEC to serve as a limit of detection (LOD). Clinical applicability of the method was demonstrated in patients treated with guadecitabine. Our data support the use of the validated LC–MS/MS method to further explore the intracellular pharmacokinetics in patients treated with β-DEC containing hypomethylating agents.

Calcium-regulated mitochondrial ATP-Mg/Pi carriers evolved from a fusion of an EF-hand regulatory domain with a mitochondrial ADP/ATP carrier-like domain.

The mitochondrial ATP‐Mg/Pi carrier is responsible for the calcium‐dependent regulation of adenosine nucleotide concentrations in the mitochondrial matrix, which allows mitochondria to respond to changing energy requirements of the cell. The carrier is expressed in mitochondria of fungi, plants and animals and belongs to the family of mitochondrial carriers. The carrier is unusual as it consists of three separate domains: (i) an N‐terminal regulatory domain with four calcium‐binding EF‐hands similar to calmodulin, (ii) a loop domain containing an amphipathic α‐helix and (iii) a mitochondrial carrier domain related to the mitochondrial ADP/ATP carrier. This striking example of three domains coming together from different origins to provide new functions represents an interesting quirk of evolution. In this review, we outline how the carrier was identified and how its physiological role was established with a focus on human isoforms. We exploit the sequence and structural information of the domains to explore the similarities and differences to their closest counterparts; mitochondrial ADP/ATP carriers and proteins with four EF‐hands. We discuss how their combined function has led to a mechanism for calcium‐regulated transport of adenosine nucleotides. Finally, we compare the ATP‐Mg/Pi carrier with the mitochondrial aspartate/glutamate carrier, the only other mitochondrial carrier regulated by calcium, and we will argue that they have arisen by convergent rather than divergent evolution. © 2018 The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(12):1222–1232, 2018

Validation of a LC/MSMS method for simultaneous quantification of 9 nucleotides in biological matrices

Nucleotides play a role in inflammation processes: cAMP and cGMP in the endothelial barrier function, ADP in platelet aggregation, ATP and UTP in vasodilatation and/or vasoconstriction of blood vessels, UDP in macrophages activation. The aim of this study is to develop and validate a LC/MS-MS method able to quantify simultaneously nine nucleotides (AMP, cAMP, ADP, ATP, GMP, cGMP, UMP, UDP and UTP) in biological matrixes (cells and plasma). The method we developed, has lower LOQ's than others and has the main advantage to quantify all nucleotides within one single injection in less than 10 min. The measured nucleotides concentrations obtained with this method are similar to those obtained with assay kits commercially available. Analysis of plasma and red blood cells from healthy donors permits to estimate the physiological concentration of those nucleotides in human plasma and red blood cells, such information being poorly available in the literature. Furthermore, the protocol presented in this paper allowed us to observe that AMP, ADP, ATP concentrations are modified in human red blood cells and plasma after a venous stasis of 4 min compared to physiological blood circulation. Therefore, this specific method enables future studies on nucleotides implications in chronic inflammatory diseases but also in other pathologies where nucleotides are implicated in.

Long-read sequencing across the C9orf72 \u2018GGGGCC\u2019 repeat expansion: implications for clinical use and genetic discovery efforts in human disease

BackgroundMany neurodegenerative diseases are caused by nucleotide repeat expansions, but most expansions, like the C9orf72 ‘GGGGCC’ (G4C2) repeat that causes approximately 5–7% of all amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, are too long to sequence using short-read sequencing technologies. It is unclear whether long-read sequencing technologies can traverse these long, challenging repeat expansions. Here, we demonstrate that two long-read sequencing technologies, Pacific Biosciences’ (PacBio) and Oxford Nanopore Technologies’ (ONT), can sequence through disease-causing repeats cloned into plasmids, including the FTD/ALS-causing G4C2 repeat expansion. We also report the first long-read sequencing data characterizing the C9orf72 G4C2 repeat expansion at the nucleotide level in two symptomatic expansion carriers using PacBio whole-genome sequencing and a no-amplification (No-Amp) targeted approach based on CRISPR/Cas9.ResultsBoth the PacBio and ONT platforms successfully sequenced through the repeat expansions in plasmids. Throughput on the MinION was a challenge for whole-genome sequencing; we were unable to attain reads covering the human C9orf72 repeat expansion using 15 flow cells. We obtained 8× coverage across the C9orf72 locus using the PacBio Sequel, accurately reporting the unexpanded allele at eight repeats, and reading through the entire expansion with 1324 repeats (7941 nucleotides). Using the No-Amp targeted approach, we attained > 800× coverage and were able to identify the unexpanded allele, closely estimate expansion size, and assess nucleotide content in a single experiment. We estimate the individual’s repeat region was > 99% G4C2 content, though we cannot rule out small interruptions.ConclusionsOur findings indicate that long-read sequencing is well suited to characterizing known repeat expansions, and for discovering new disease-causing, disease-modifying, or risk-modifying repeat expansions that have gone undetected with conventional short-read sequencing. The PacBio No-Amp targeted approach may have future potential in clinical and genetic counseling environments. Larger and deeper long-read sequencing studies in C9orf72 expansion carriers will be important to determine heterogeneity and whether the repeats are interrupted by non-G4C2 content, potentially mitigating or modifying disease course or age of onset, as interruptions are known to do in other repeat-expansion disorders. These results have broad implications across all diseases where the genetic etiology remains unclear.

Rheological and structural properties of Hemiramphus far skin gelatin: Potential use as an active fish coating agent

The present study aims to characterize black-barred (Hemiramphus far) skin gelatin (BG) gel enriched or not with its hydrolysate (BGH) in terms of their rheological and micro-structural properties, as well as to evaluate their effect on smooth hound fillet quality during chilled storage. The results of the rheological properties showed that peptides addition influenced elastic and loss modulus as observed from temperature sweep. Based on the microstructure, BG gel had a fine network with small voids that were filled by the presence of BGH peptides. On the other hand, when used as coating agents for smooth hound fillets, BG and BG-BGH showed that, in comparison to control group, fish spoilage was significantly delayed in coated samples. In fact, after 8 days of storage, samples coated with BG and BG+BGH showed the lowest weight loss levels, and preserved their initial water activity (aw) values. In addition, fillet coating was able to significantly reduce TVB-N content and oxidation process, as reflected by the low malondialdehyde and carbonyl contents. Furthermore, compared to the control group, all the treatments were found to reduce the degree of microbial deterioration of the fillets, leading to low the free amino acids and nucleotides contents in wrapped fish samples. Therefore, gelatin coating helps to prevent fish fillets from deterioration and oxidation processes.

How to influence the mesenchymal stem cells fate? Emerging role of ectoenzymes metabolizing nucleotides.

Extracellular purines, principally adenosine triphosphate and adenosine, are among the oldest evolutionary and widespread chemical messengers. The integrative view of purinergic signaling as a multistage coordinated cascade involves the participation of nucleotides/nucleosides, their receptors, enzymes metabolizing extracellular nucleosides and nucleotides as well as several membrane transporters taking part in the release and/or uptake of these molecules. In view of the emerging data, it is evident and widely accepted that an extensive network of diverse enzymatic activities exists in the extracellular space. The enzymes regulate the availability of nucleotide and adenosine receptor agonists, and consequently, the course of signaling events. The current data indicate that mesenchymal stem cells (MSCs) and cells induced to differentiate exhibit different sensitivity to purinergic ligands as well as a distinct activity and expression profiles of ectonucleotidases than mature cells. In the proposed review, we postulate for a critical role of these enzymatic players which, by orchestrating a fine-tune regulation of nucleotides concentrations, are integrally involved in modulation and diversification of purinergic signals. This specific hallmark of the MSC purinome should be linked with cell-specific biological potential and capacity for tissue regeneration. We anticipate this publication to be a starting point for scientific discussion and novel approach to the in vitro and in vivo regulation of the MSC properties.

CRX directs photoreceptor differentiation by accelerating chromatin remodeling at specific target sites

BackgroundRecent technological advances have delivered the genome-wide targets of many important transcription factors (TFs). However, increasing evidence suggests that not all target sites mediate regulatory function, raising the questions of how to determine which sites are active, what are the epigenetic consequences of TF binding at these sites, and how the specificity is coded. To address these questions, we focused on CRX, a disease-associated homeodomain TF required for photoreceptor gene expression and development. Since CRX binds more than 6000 sites across the genome in the retina, we profiled chromatin landscape changes at each binding site during normal development and in the absence of CRX and interpreted the results by thorough investigation of other epigenomic datasets and sequence features.ResultsCRX is required for chromatin remodeling at only a subset of its binding sites, which undergo retina or neuronal specific activation during photoreceptor differentiation. Genes near these “CRX Dependent” sites code for proteins important for photoreceptor physiology and function, and their transcription is significantly reduced in Crx deficient retinas. In addition, the nucleotide and motif content distinguish these CRX Dependent sites from other CRX-bound sites.ConclusionsTogether, our results suggest that CRX acts only at select, uniquely-coded binding sites to accelerate chromatin remodeling during photoreceptor differentiation. This study emphasizes the importance of connecting TF binding with its functional consequences and provides a framework for making such a connection using comparative analyses of available genomic datasets. Finally, this study prioritizes sets of non-coding DNA sites for future functional interrogation and identification of mutations associated with retinal disease.

Metformin inhibits gluconeogenesis via a redox-dependent mechanism in vivo.

Metformin, the universal first-line treatment for type 2 diabetes, exerts its therapeutic glucose-lowering effects by inhibiting hepatic gluconeogenesis. However, the primary molecular mechanism of this biguanide remains unresolved, though it has been suggested to act, at least partially, by mitochondrial complex I inhibition. Here, we show that clinically-relevant plasma metformin concentrations achieved by acute intravenous, acute intraportal or chronic oral administration in awake normal and diabetic rats inhibit gluconeogenesis from lactate and glycerol but not from pyruvate and alanine, implicating an increased cytosolic redox state in mediating metformin’s antihyperglycemic effect. All of these effects occurred independent of complex I inhibition, evidenced by unaltered hepatic energy charge and citrate synthase flux. Normalizing the cytosolic redox state by infusion of methylene blue or substrates that contribute to gluconeogenesis independently of the cytosolic redox state abrogated metformin-mediated inhibition of gluconeogenesis in vivo. Additionally, in mice expressing constitutively active acetyl-CoA carboxylase, metformin acutely decreased hepatic glucose production and increased the hepatic cytosolic redox state without altering hepatic triglyceride content or gluconeogenic enzyme expression. These studies demonstrate that metformin, at clinically-relevant plasma concentrations, inhibits hepatic gluconeogenesis in a redox-dependent manner independent of reductions in citrate synthase flux, hepatic nucleotide concentrations, acetyl-CoA carboxylase activity, or gluconeogenic enzyme protein expression.

Cytosolic 5'-Nucleotidase II Silencing in a Human Lung Carcinoma Cell Line Opposes Cancer Phenotype with a Concomitant Increase in p53 Phosphorylation.

Purine homeostasis is maintained by a purine cycle in which the regulated member is a cytosolic 5′-nucleotidase II (cN-II) hydrolyzing IMP and GMP. Its expression is particularly high in proliferating cells, indeed high cN-II activity or expression in hematological malignancy has been associated to poor prognosis and chemoresistance. Therefore, a strong interest has grown in developing cN-II inhibitors, as potential drugs alone or in combination with other compounds. As a model to study the effect of cN-II inhibition we utilized a lung carcinoma cell line (A549) in which the enzyme was partially silenced and its low activity conformation was stabilized through incubation with 2-deoxyglucose. We measured nucleotide content, reduced glutathione, activities of enzymes involved in glycolysis and Krebs cycle, protein synthesis, mitochondrial function, cellular proliferation, migration and viability. Our results demonstrate that high cN-II expression is associated with a glycolytic, highly proliferating phenotype, while silencing causes a reduction of proliferation, protein synthesis and migration ability, and an increase of oxidative performances. Similar results were obtained in a human astrocytoma cell line. Moreover, we demonstrate that cN-II silencing is concomitant with p53 phosphorylation, suggesting a possible involvement of this pathway in mediating some of cN-II roles in cancer cell biology.

Sequence-dependent DNA condensation as a driving force of DNA phase separation.

The physical properties of DNA have been suggested to play a central role in spatio-temporal organization of eukaryotic chromosomes. Experimental correlations have been established between the local nucleotide content of DNA and the frequency of inter- and intra-chromosomal contacts but the underlying physical mechanism remains unknown. Here, we combine fluorescence resonance energy transfer (FRET) measurements, precipitation assays, and molecular dynamics simulations to characterize the effect of DNA nucleotide content, sequence, and methylation on inter-DNA association and its correlation with DNA looping. First, we show that the strength of DNA condensation mediated by poly-lysine peptides as a reduced model of histone tails depends on the DNA’s global nucleotide content but also on the local nucleotide sequence, which turns out to be qualitatively same as the condensation by spermine. Next, we show that the presence and spatial arrangement of C5 methyl groups determines the strength of inter-DNA attraction, partially explaining why RNA resists condensation. Interestingly, multi-color single molecule FRET measurements reveal strong anti-correlation between DNA looping and DNA–DNA association, suggesting that a common biophysical mechanism underlies them. We propose that the differential affinity between DNA regions of varying sequence pattern may drive the phase separation of chromatin into chromosomal subdomains.

Karakteristik Gen Sitokrom C Oksidase Sub Unit I (CO1) Lebah Liar Apis cerena (Hymenoptera: apidae) Asal Pulau Hoga Sulawesi Tenggara

The study was conducted to assess the caracteristic of cytochrome C oxidase subunit I (COI) gene on wild honey bee Apis cerena, and detection of barcode sites from these gene. A total fifteen individual A. cerena were collected from Hoga Island, Southeast Sulawesi. Genomic DNA was extracted from torax, then amplified by PCR method and than sequenced. Sequencing result characterized their nucleotide and amino acid content. The results showed that 595 nucleotides at the 5' end of COI gene of A. cerena very conserved at the most of the sites. Nucleotide dominated by thymine and adenine bases (± 70%). There are 25 barcoding sites for A. cerena. There are two of these sites are diagnostics for A. cerena from of the Hoga Island. From of 595 basepairs nucleotide were prediction encode 198 amino acid, and only eleven percent of the these are varied between species. Amino acid 91th (treonine) and 103th (asparagin) were diagnostics amino acid for A. cerena from Hoga Island, Southeast Sulawesi. Molecular phylogeny reconstructed based on both nucleotide and amino acid sequence placing apis cerena from Hoga Island, Souteast Sulawesi is closely related to Apis cerena from Genbank source.

Understanding the mechanism of DNA threshold elongation

The mechanism of threshold elongation of DNA macromolecule (overstretching) is studied within the framework of the phenomenological approach, accounting both external (stretching) and internal (conformational) displacement components. As shown, the overstretching of DNA under the action of an external force can occur in two stages. Firstly, due to the coupling between the components, at a some critical value of external force a conformational bistability is formed in the macromolecule structure. In turn, the appearance of bistability stimulates the formation of domains in the DNA chain with two different conformations (B and S). Secondly, under favorable boundary conditions, the conformationally induced deformation acquires the possibility to propagate along the macromolecule as domain walls. In this way the bistability occurrence in the macromolecule conformation provides a threshold effect of elongation. The calculated contributions in DNA overstretching show agreement with the observed data, and allow to explain the dependence of macromolecule threshold elongation on nucleotide content.

Abstract 1541: Directed, unbiased mapping of lentiviral integrations with next generation sequencing in CAR-T cells

Abstract During manufacturing of an exemplary CD19-directed CAR-T cell product, patient T cells were transduced with a viral vector encoding the CD19-directed, 41BBζ endodomain-containing chimeric antigen receptor (CAR) construct, enabling permanent genetic modification of the target T cells. Lentiviral transduction with a self-inactivating (SIN) vector can offer certain advantages in safety and consistency over other DNA delivery methods. Although the integration patterns of wildtype HIV-1 have been well-characterized and shown to have a low risk of insertional transformation, the addition of an artificial construct may have the potential to affect integration patterns. Existing techniques for viral integration mapping, such as ligation-mediated polymerase chain reaction (LM-PCR), have limitations precluding comprehensive, unbiased, high-throughput insertion site mapping. To characterize insertional events in engineered CAR-T cells, we developed an unbiased NGS assay and computational pipeline capable of characterizing viral integration events genome-wide. The method leverages Tn5 transposase, coupled with a long terminal repeat-specific PCR, to map integration events down to nulceotide resolution. This assay identified over 150,000 high-confidence integration events across 23 patient-derived CAR-T cell products. Analysis of these sites revealed a similar integration preference to lentiviruses described in the literature: driven by GC content, local nucleotide content, chromatin status, and transcriptional activity, with no indication of preferential integration at oncogenic loci. In conclusion, in this assay a CD19-directed, CAR-containing lentivirus exhibited a low risk integration profile with no significant evidence of construct-specific risk factors. Furthermore, the findings suggest that the NGS assay may be useful for more efficient, cost-effective, and sensitive measurement of CAR-T integration events for a range of constructs. Citation Format: Timothy G. Johnstone, Rajagopal Chari, David Koppstein, Ronald J. Hause, Rafael Ponce, Adrian W. Briggs, Francois Vigneault. Directed, unbiased mapping of lentiviral integrations with next generation sequencing in CAR-T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1541.

Physiological responses of Chinese hamster ovary cells to a productivity-enhancing yeast extract

Hydrolysates play important roles in enhancing the productivity of recombinant proteins in mammalian cell cultures. Lacking of detailed understanding of the mechanisms, hydrolysate is commonly regarded as an unstable factor which should be used with cautions. A yeast extract (YE) was approved to improve the Fc-fusion protein productivity in a recombinant Chinese hamster ovary (CHO) cell line. To elucidate the responses of cells to hydrolysates, we further elaborate their physiological changes during the processes in the presence and absence of YE. Firstly, cell sizes and the cellular components including dry cell weight, cellular fatty acid, and total cellular protein were increased in the presence of YE. Then, by comparing the extracellular and intracellular concentrations of the main metabolites and their consumption rates, we excluded the possibility of nutrient depletion in the absence of YE and observed a distinct improvement on the net consumption rates of metabolites in the presence of YE. Furthermore, the increase on the contents of intracellular nucleotides illustrated an abundance of the nucleic acid precursors and energy charge for recombinant protein synthesis in the presence of YE. In conclusion, this study systematically elucidated YE enhanced cell mass and capacity, activated substrate and energy metabolism of cells in addition to a boost in product synthesis process. The findings provide valuable information for process optimization and cell engineering.

Effect of flow on ATP/ADP concentration at the endothelial cell surface: interplay between shear stress and mass transport

The nucleotides ATP and ADP regulate many aspects of endothelial cell (EC) biology, including intracellular calcium concentrations, focal adhesion activation, cytoskeletal organization, and cellular motility. In vivo, ECs are constantly under flow, and the concentration of ATP/ADP on the EC surface is determined by the combined effects of nucleotide convective and diffusive transport as well as hydrolysis by ectonucleotidases on the EC surface. In addition, experiments have demonstrated that flow induces ATP release from the cells. Previously computational models have incorporated the above effects and thus described concentration at the EC surface. However, it remains unclear what physical processes are responsible for nucleotide regulation. While some EC responses to flow have been shown to be directly driven by shear stress, others appear to also involve a non-negligible contribution of transport. In the present work, we develop a mathematical model and perform numerical simulations to investigate the relative contributions of shear stress and transport to nucleotide concentration at the EC surface, with the effect of cell density. Because in vitro experiments are performed by using confluent cells in some cases and subconfluent cells in other cases, we also investigate the effect of cell density on the results. The outcomes of the simulations demonstrate a complex interplay between shear stress and transport such that transport has a significant contribution at certain shear stress values but not at others. The effect of transport on nucleotide concentration increases with cell density. The present findings enhance our understanding of the mechanisms that govern the regulation of such molecules at the EC surface under flow. The implications of these findings for downstream responses such as cellular motility merit future investigation.

Different dietary protein levels affect flesh quality, fatty acids and alter gene expression of Nrf2-mediated antioxidant enzymes in the muscle of grass carp (Ctenopharyngodon idella)

This study investigated the effects of dietary protein levels on the flesh quality, fatty acids, antioxidant capacity and antioxidant-related signalling molecule gene expression in the muscle of grass carp (Ctenopharyngodon idella). A total of 540 grass carp (264.11 ± 0.76 g) were fed six diets containing graded levels of protein (143.1, 176.7, 217.2, 257.5, 292.2 and 322.8 g digestible protein kg−1 diet) for 8 weeks. The results indicated that optimal dietary protein levels: (1) improved physical (or chemical) and flavor quality of grass carp fillets by increasing shear force, pH, hydroxyproline, protein, lipid, free amino acids (aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, lysine, histidine, arginine and proline) and nucleotide (5′-inosinic acid) contents, and decreasing cooking loss, cathepsin B and L activities and lactate content. (2) enhanced the potential health benefits to humans of grass carp fillets by decreasing the saturated fatty acid (C14:0, C15:0, C16:0, C18:0 and C20:0) concentrations, and increasing unsaturated fatty acids [monounsaturated fatty acids (C14:1 and C16:1) and polyunsaturated fatty acids (C18:2c+t, C18:3n-3, C20:4 and C22:6)] concentrations. (3) enhanced antioxidant capacity of grass carp fillets by decreasing reactive oxygen species, malondialdehyde and protein carbonyl contents, and increasing the activities and gene expression of copper/zinc superoxide dismutase, manganese superoxide dismutase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase and the content of glutathione. (4) regulated antioxidant-related signalling molecules gene expression by up-regulating the mRNA levels of NF-E2-related factor 2, casein kinase 2α and 2β, and decreasing Kelch-like-ECH-associated protein 1a and 1b in the muscle of grass carp. In conclusion, optimal dietary protein level improved the physical and flavor characteristics, fatty acid profile, antioxidant capacity and regulated the gene expression of antioxidant-related signalling molecules in fish fillets.

RNase I regulates Escherichia coli 2',3'-cyclic nucleotide monophosphate levels and biofilm formation.

Regulation of nucleotide and nucleoside concentrations is critical for faithful DNA replication, transcription, and translation in all organisms, and has been linked to bacterial biofilm formation. Unusual 2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs) recently were quantified in mammalian systems, and previous reports have linked these nucleotides to cellular stress and damage in eukaryotes, suggesting an intriguing connection with nucleotide/nucleoside pools and/or cyclic nucleotide signaling. This work reports the first quantification of 2',3'-cNMPs in Escherichia coli and demonstrates that 2',3'-cNMP levels in E. coli are generated specifically from RNase I-catalyzed RNA degradation, presumably as part of a previously unidentified nucleotide salvage pathway. Furthermore, RNase I and 2',3'-cNMP levels are demonstrated to play an important role in controlling biofilm formation. This work identifies a physiological role for cytoplasmic RNase I and constitutes the first progress toward elucidating the biological functions of bacterial 2',3'-cNMPs.

Cryo-electron microscopy structures and progress toward a dynamic understanding of KATP channels.

Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels are molecular sensors of cell metabolism. These hetero-octameric channels, comprising four inward rectifier K+ channel subunits (Kir6.1 or Kir6.2) and four sulfonylurea receptor (SUR1 or SUR2A/B) subunits, detect metabolic changes via three classes of intracellular adenine nucleotide (ATP/ADP) binding site. One site, located on the Kir subunit, causes inhibition of the channel when ATP or ADP is bound. The other two sites, located on the SUR subunit, excite the channel when bound to Mg nucleotides. In pancreatic β cells, an increase in extracellular glucose causes a change in oxidative metabolism and thus turnover of adenine nucleotides in the cytoplasm. This leads to the closure of KATP channels, which depolarizes the plasma membrane and permits Ca2+ influx and insulin secretion. Many of the molecular details regarding the assembly of the KATP complex, and how changes in nucleotide concentrations affect gating, have recently been uncovered by several single-particle cryo-electron microscopy structures of the pancreatic KATP channel (Kir6.2/SUR1) at near-atomic resolution. Here, the author discusses the detailed picture of excitatory and inhibitory ligand binding to KATP that these structures present and suggests a possible mechanism by which channel activation may proceed from the ligand-binding domains of SUR to the channel pore.

The effects of adenine nucleotide perfusion on interstitial adenosine production in rat skeletal muscle.

The purpose of the present study was to utilize the microdialysis technique in rat skeletal muscle to perfuse varying concentrations of AMP, ADP, and ATP into the interstitium to examine the effects that these adenine nucleotides have on the production of adenosine in the interstitial space. Interstitial adenosine production appears to be related to the type (ATP, ADP, or AMP) and concentration (2-60 μmol/L) of the adenine nucleotide perfused. Interstitial adenosine levels increased (P < 0.05) from baseline (0.18 ± 0.02 and 0.22 ± 0.02 μmol/L) to 0.23 ± 0.02 and 0.41 ± 0.05 μmol/L following 5 and 30 μmol/L AMP perfusion, respectively. Similarly, perfusion with 30 μmol/L ADP and 30, 40, and 60 μmol/L ATP resulted in an increase (P < 0.05) in interstitial adenosine concentration from baseline (0.25 ± 0.02, 0.26 ± 0.02, 0.19 ± 0.03, and 0.14 ± 0.02 μmol/L) to 0.30 ± 0.02, 0.32 ± 0.02, 0.36 ± 0.04, and 0.33 ± 0.04 μmol/L, respectively. Interestingly, the most prominent increase in interstitial adenosine production occurred during the perfusion of 60 μmol/L ATP (126% increase from baseline). These data strongly suggest that interstitial ATP may play a more potent role in stimulating interstitial adenosine production as compared with ADP or AMP. In addition, interstitial adenosine production can occur independent of muscle contraction (voluntary or involuntary) or hypoxia when adequate concentrations of adenine nucleotides are available.