Cytosine Monophosphate Research Articles

P0006 Developmentally endothelial locus-1 promotes intestinal inflammation resolution through inhibition of the Cmpk2-cGAS-STING pathway and regulates reparatory macrophage transition

Abstract Background Abnormalities in inflammation resolution function are closely associated with chronic inflammation, and proresolution therapies may provide new opportunities for the treatment of inflammatory bowel disease (IBD). Developmental endothelial locus 1 (DEL-1) is an important endogenous molecule that regulates tissue immune homeostasis and promotes resolution of inflammation, but its effect on IBD remains unknown. Here, we aimed to explore the expression and roles of DEL-1 in IBD. Methods DEL-1 levels were examined in patients with IBD, mice with colitis, and LPS-induced RAW264.7 cells. DEL-1 overexpression plasmids and/or DEL-1-Fc intervened in cells and mice to explore it effects in the acute and recovery phases of inflammation. Moreover, flow cytometry, RNA-Seq, chromatin immunoprecipitation (ChIP), dual-luciferase reporter assays and 16S rRNA were used to illustrate the potential mechanism of DEL-1 in colitis. Results DEL-1 levels were remarkably lower in IBD patients, colitis mice, and LPS-induced RAW264.7 cells, while the levels increased with inflammation to resolve. Transfection with DEL-1 overexpression plasmid and/or DEL-1-Fc intervention reduces levels of inflammatory cytokines in both phases and upregulates reparative gene levels in the recovery phase. Mechanistically, we demonstrated that DEL-1 inhibits Cytosine monophosphate kinase 2 (Cmpk2)-dependent mtDNA synthesis, thereby inhibiting the cGAS-STING pathway to ameliorate intestinal inflammation. Moreover, DEL-1 promoted reparative macrophage transition in the repair model of colitis. Spi1 was identified as a transcription factor that regulates Cmpk2 and the reparative gene Il10. Intervention with overexpression plasmid of Spi1 or Cmpk2 or the STING agonist DMXAA reverses the effects of DEL-1. DEL-1 also inhibits neutrophil recruitment, repairs the intestinal barrier, and improves intestinal microbiota dysbiosis. Conclusion This is the first study to show that DEL-1 effectively attenuates colonic inflammation in mice with DSS-induced colitis by inhibits the Cmpk2-cGAS-STING pathway and regulates reparatory macrophage transition, thus providing a novel therapeutic target for IBD.

Adenovirus infection in allogeneic hematopoietic cell transplantation.

Adenovirus (AdV) infection occurs in 0-20% of patients in the first 3-4 months after allogeneic hematopoietic cell transplantation (HCT), being higher in pediatric than in adult patients. About 50% of AdV infections involve the blood, which in turn, correlates with an increased risk developing AdV diseases, end-organ damage, and 6-month overall mortality. The main risk factors for AdV infection are T-cell depletion of the graft by ex vivo selection procedures or in vivo use of alemtuzumab or antithymocyte serum, development of graft versus host disease (GVHD) grade III-IV, donor type (haploidentical or human leucocyte antigen mismatched related donor > cord blood> unrelated matched donor) and severe lymphopenia (<0.2×109 /L). The prevention of AdV disease relies on early diagnosis of increasing viral replication in blood or stool and the pre-emptive start of cidofovir as viral load exceeds the threshold of ≥102-3 copies/mL in blood and/or 106 copies/g stool in the stool. Cidofovir (CDV), a cytosine monophosphate nucleotide analog, is currently the only antiviral recommended for AdV infection despite limited efficacy and moderate risk of nephrotoxicity. Brincidofovir, a lipid derivative of CDV with more favorable pharmacokinetics properties and superior efficacy, is not available and currently is being investigated for other viral infections. The enhancement of virus-specific T-cell immunity in the first few months post-HCT by the administration of donor-derived or third-party-donor-derived virus-specific T-cells represents an innovative and promising modality of intervention and data of efficacy and safety of the ongoing prospective randomized studies are eagerly awaited.

Endothelial Gata6 deletion reduces monocyte recruitment and proinflammatory macrophage formation and attenuates atherosclerosis through Cmpk2-Nlrp3 pathways

Endothelial dysfunction results in chronic vascular inflammation, which is critical for the development of atherosclerotic diseases. Transcription factor Gata6 has been reported to regulate vascular endothelial cell activation and inflammation in vitro. Here, we aimed to explore the roles and mechanisms of endothelial Gata6 in atherogenesis.Endothelial cell (EC) specific Gata6 deletion was generated in the ApoeKO hyperlipidemic atherosclerosis mouse model. Atherosclerotic lesion formation, endothelial inflammatory signaling, and endothelial-macrophage interaction were examined in vivo and in vitro by using cellular and molecular biological approaches. EC-GATA6 deletion mice exhibited a significant decrease in monocyte infiltration and atherosclerotic lesion compared to littermate control mice. Cytosine monophosphate kinase 2 (Cmpk2) was identified as a direct target gene of GATA6 and EC-GATA6 deletion decreased monocyte adherence, migration and pro-inflammatory macrophage foam cell formation through regulation of the CMPK2-Nlrp3 pathway. Endothelial target delivery of Cmpk2-shRNA by intercellular adhesion molecule 2 (Icam-2) promoter-driven AAV9 carrying the shRNA reversed the Gata6 upregulation mediated elevated Cmpk2 expression and further Nlrp3 activation and thus attenuated atherosclerosis. In addition, C–C motif chemokine ligand 5 (Ccl5) was also identified as a direct target gene of Gata6 to regulate monocyte adherence and migration influencing atherogenesis.This study provides direct in vivo evidence of EC-GATA6 involvement in the regulation of Cmpk2-Nlrp3, as well as Ccl5, on monocyte adherence and migration in atherosclerosis development and advances our understanding of the in vivo mechanisms of atherosclerotic lesion development, and meanwhile provides opportunities for future therapeutic interventions.

Electroacupuncture Inhibits NLRP3 Activation by Regulating CMPK2 After Spinal Cord Injury.

ObjectivesThis study aimed to evaluate the expression of cytosine monophosphate kinase 2 (CMPK2) and activation of the NLRP3 inflammasome in rats with spinal cord injury (SCI) and to characterize the effects of electroacupuncture on CMPK2-associated regulation of the NLRP3 inflammasome.MethodsAn SCI model was established in Sprague–Dawley (SD) rats. The expression levels of NLRP3 and CMPK2 were measured at different time points following induction of SCI. The rats were randomly divided into a sham group (Sham), a model group (Model), an electroacupuncture group (EA), an adeno-associated virus (AAV) CMPK2 group, and an AAV NC group. Electroacupuncture was performed at jiaji points on both sides of T9 and T11 for 20 min each day for 3 consecutive days. In the AAV CMPK2 and AAV NC groups, the viruses were injected into the T9 spinal cord via a microneedle using a microscope and a stereotactic syringe. The Basso–Beattie–Bresnahan (BBB) score was used to evaluate the motor function of rats in each group. Histopathological changes in spinal cord tissue were detected using H&E staining, and the expression levels of NLRP3, CMPK2, ASC, caspase-1, IL-18, and IL-1β were quantified using Western blotting (WB), immunofluorescence (IF), and RT-PCR.ResultsThe expression levels of NLRP3 and CMPK2 in the spinal cords of the model group were significantly increased at day 1 compared with those in the sham group (p < 0.05). The expression levels of NLRP3 and CMPK2 decreased gradually over time and remained low at 14 days post-SCI. We successfully constructed AAV CMPK2 and showed that CMPK2 was significantly knocked down following 2 dilutions. Finally, treatment with EA or AAV CMPK2 resulted in significantly increased BBB scores compared to those in the model group and the AAV NC group (p < 0.05). The histomorphology of the spinal cord in the EA and AAV CMPK2 groups was significantly different than that in the model and AAV NC groups. WB, IF, and PCR analyses showed that the expression levels of CMPK2, NLRP3, ASC, caspase-1, IL-18, and IL-1β were significantly lower in the EA and AAV CMPK2 groups compared with those in the model and AAV NC groups (p < 0.05).ConclusionOur study showed that CMPK2 regulated NLRP3 expression in rats with SCI. Activation of NLRP3 is a critical mechanism of inflammasome activation and the inflammatory response following SCI. Electroacupuncture downregulated the expression of CMPK2 and inhibited activation of NLRP3, which could improve motor function in rats with SCI.

Putative Nucleotide-Based Second Messengers in the Archaeal Model Organisms Haloferax volcanii and Sulfolobus acidocaldarius.

Research on nucleotide-based second messengers began in 1956 with the discovery of cyclic adenosine monophosphate (3′,5′-cAMP) by Earl Wilbur Sutherland and his co-workers. Since then, a broad variety of different signaling molecules composed of nucleotides has been discovered. These molecules fulfill crucial tasks in the context of intracellular signal transduction. The vast majority of the currently available knowledge about nucleotide-based second messengers originates from model organisms belonging either to the domain of eukaryotes or to the domain of bacteria, while the archaeal domain is significantly underrepresented in the field of nucleotide-based second messenger research. For several well-stablished eukaryotic and/or bacterial nucleotide-based second messengers, it is currently not clear whether these signaling molecules are present in archaea. In order to shed some light on this issue, this study analyzed cell extracts of two major archaeal model organisms, the euryarchaeon Haloferax volcanii and the crenarchaeon Sulfolobus acidocaldarius, using a modern mass spectrometry method to detect a broad variety of currently known nucleotide-based second messengers. The nucleotides 3′,5′-cAMP, cyclic guanosine monophosphate (3′,5′-cGMP), 5′-phosphoadenylyl-3′,5′-adenosine (5′-pApA), diadenosine tetraphosphate (Ap4A) as well as the 2′,3′-cyclic isomers of all four RNA building blocks (2′,3′-cNMPs) were present in both species. In addition, H. volcanii cell extracts also contain cyclic cytosine monophosphate (3′,5′-cCMP), cyclic uridine monophosphate (3′,5′-cUMP) and cyclic diadenosine monophosphate (3′,5′-c-di-AMP). The widely distributed bacterial second messengers cyclic diguanosine monophosphate (3′,5′-c-di-GMP) and guanosine (penta-)/tetraphosphate [(p)ppGpp] could not be detected. In summary, this study gives a comprehensive overview on the presence of a large set of currently established or putative nucleotide-based second messengers in an eury- and a crenarchaeal model organism.

Toxic effects of sublethal acetochlor with sublethal doses on earthworms in soil using multi-endpoints system

In this study, we examined the toxic effect of sublethal doses of acetochlor (1, 2, 4, 8 mg·kg-1) on earthworms by exogenous addition. The growth inhibition rate, cytochrome P450 isozymes (CYP1A2, 2C9 and 3A4) activities and the metabolomics were analyzed after seven days of exposure, to infer the toxicity threshold of acetochlor, screen the sensitive biomarkers from the levels of the individual, detoxified enzymes and small molecular metabolites, and elucidate the underlying toxicity mechanism. The results showed that CYP1A2, 2C9 and 3A4 activities were all significantly inhibited, and that the levels of ten metabolites (fructose-6-diphosphate, cytosine monophosphate, uridine monophosphate, adenosine monophosphate, adenosine, xanthine, fumaric acid, dihydroxyglutaric acid, ornithine and 16-hydroxyeicosatetraenoic acid) were significantly decreased by acetochlor exposure. The levels of six metabolites (adenosine succinic acid, succinic acid, arginine, tryptophan, asparagine and phenylalanine) were significantly increased when earthworms being exposed to 2-8 mg·kg-1 acetochlor. Acetochlor exposure caused oxidative damage to earthworms, weakened the glycolysis, disturbed the tricarboxylic acid cycle, disordered the purine and pyrimidine metabolism, and impaired the amino acids metabolism. Compared with the end point at individual level, the above 16 small molecule metabolites and CYP isozymes activities were more sensitive to acetochlor exposure. It was thus recommended that CYP isozymes (1A2, 2C9, and 3A4) activities and small molecular metabolites could be used as a set of biomarkers to diagnose the acetochlor pollution, given their high sensitivity and accuracy.

Electronic Transport through DNA Nucleotides in Atomically Thin Phosphorene Electrodes for Rapid DNA Sequencing.

Rapid progresses in developing the fast, low-cost, and reliable methods for DNA sequencing are envisaged for development of personalized medicine. In this respect, nanotechnology has paved the role for the development of advanced DNA sequencing techniques including sequencing with solid-state nanopores or nanogaps. Herein, we have explored the application of a black phosphorene based nanogap-device for DNA sequencing. Using density-functional-theory based non-equilibrium Green's function approach, we have computed transverse transmission and current-voltage ( I- V) characteristics of all the four DNA nucleotides (deoxy adenosine monophosphate, deoxy guanidine monophosphate, deoxy thymidine monophosphate, and deoxy cytosine monophosphate) as functions of applied bias voltages. We deduce that it is in principle; possible to differentiate between all the four nucleotides by three sequencing runs at distinct applied bias voltages, i.e., at 0.2, 1.4, and 1.6 V, where individual identification of all the four nucleotides may be possible. Hence, we believe our study might be helpful for experimentalist towards the development of a phosphorene based nanodevice for DNA sequencing to diagnose critical diseases.

Improving the Diet for the Rearing of Glossina brevipalpis Newstead and Glossina austeni Newstead: Blood Source and Collection - Processing - Feeding Procedures.

One of the challenges to maintain tsetse fly (Diptera: Glossinidae) colonies is the sustainable supply of high quality blood meals. The effect of using anticoagulants during collection of the blood, the addition of phagostimulants to the blood meals as well as using mixtures of bovine and porcine blood in different proportions for feeding on colony productivity was assessed. Defibrinated bovine blood was found to be suitable to maintain both the Glossina brevipalpis Newstead and Glossina austeni Newstead colonies. Blood collected with the anticoagulants sodium citrate, citric sodium combination, citrate phosphate dextrose adenine and citric acid did not affect colony performance of both species. Defibrinated bovine and porcine blood in a 1:1 ratio or the feeding of either bovine or porcine blood on alternating days improved pupae production of G. austeni and can be used to enhance colony growth. Bovine blood is appropriate to maintain G. brevipalpis colonies, however, feeding either bovine or porcine blood on alternating days did improve productivity. Adding the phagostimulants inosine tri-phosphate, cytosine mono-phosphate and guanosine mono-phosphate to the blood at a concentration of 10−4 M improved pupae production of the G. brevipalpis colony. The addition of adenosine tri-phosphate and inosine tri-phosphate improved the performance of the G. austeni colony. Decisions on the most suitable rearing diet and feeding protocols will not only depend on the biological requirements of the species but also on the continuous supply of a suitable blood source that can be collected and processed in a cost-effective way.

Folding Properties of Cytosine Monophosphate Kinase from E. coli Indicate Stabilization through an Additional Insert in the NMP Binding Domain

The globular 25 kDa protein cytosine monophosphate kinase (CMPK, EC ID: 2.7.4.14) from E. coli belongs to the family of nucleoside monophosphate (NMP) kinases (NMPK). Many proteins of this family share medium to high sequence and high structure similarity including the frequently found α/β topology. A unique feature of CMPK in the family of NMPKs is the positioning of a single cis-proline residue in the CORE-domain (cis-Pro124) in conjunction with a large insert in the NMP binding domain. This insert is not found in other well studied NMPKs such as AMPK or UMP/CMPK. We have analyzed the folding pathway of CMPK using time resolved tryptophan and FRET fluorescence as well as CD. Our results indicate that unfolding at high urea concentrations is governed by a single process, whereas refolding in low urea concentrations follows at least a three step process which we interpret as follows: Pro124 in the CORE-domain is in cis in the native state (Nc) and equilibrates with its trans-isomer in the unfolded state (Uc - Ut). Under refolding conditions, at least the Ut species and possibly also the Uc species undergo a fast initial collapse to form intermediates with significant amount of secondary structure, from which the trans-Pro124 fraction folds to the native state with a 100-fold lower rate constant than the cis-Pro124 species. CMPK thus differs from homologous NMP kinases like UMP/CMP kinase or AMP kinase, where folding intermediates show much lower content of secondary structure. Importantly also unfolding is up to 100-fold faster compared to CMPK. We therefore propose that the stabilizing effect of the long NMP-domain insert in conjunction with a subtle twist in the positioning of a single cis-Pro residue allows for substantial stabilization compared to other NMP kinases with α/β topology.

Structure-based Mechanism of CMP-2-keto-3-deoxymanno-octulonic Acid Synthetase

The enzyme CMP-Kdo synthetase (KdsB) catalyzes the addition of 2-keto-3-deoxymanno-octulonic acid (Kdo) to CTP to form CMP-Kdo, a key reaction in the biosynthesis of lipopolysaccharide. The reaction catalyzed by KdsB and the related CMP-acylneuraminate synthase is unique among the sugar-activating enzymes in that the respective sugars are directly coupled to a cytosine monophosphate. Using inhibition studies, in combination with isothermal calorimetry, we show the substrate analogue 2beta-deoxy-Kdo to be a potent competitive inhibitor. The ligand-free Escherichia coli KdsB and ternary complex KdsB-CTP-2beta-deoxy-Kdo crystal structures reveal that Kdo binding leads to active site closure and repositioning of the CTP phosphates and associated Mg(2+) ion (Mg-B). Both ligands occupy conformations compatible with an S(n)2-type attack on the alpha-phosphate by the Kdo 2-hydroxyl group. Based on strong similarity with DNA/RNA polymerases, both in terms of overall chemistry catalyzed as well as active site configuration, we postulate a second Mg(2+) ion (Mg-A) is bound by the catalytically competent KdsB-CTP-Kdo ternary complex. Modeling of this complex reveals the Mg-A coordinated to the conserved Asp(100) and Asp(235) in addition to the CTP alpha-phosphate and both the Kdo carboxylic and 2-hydroxyl groups. EPR measurements on the Mn(2+)-substituted ternary complex support this model. We propose the KdsB/CNS sugar-activating enzymes catalyze the formation of activated sugars, such as the abundant CMP-5-N-acetylneuraminic acid, by recruitment of two Mg(2+) to the active site. Although each metal ion assists in correct positioning of the substrates and activation of the alpha-phosphate, Mg-A is responsible for activation of the sugar-hydroxyl group.

Phosphodiester bond rupture in 5′ and 3′ cytosine monophosphate in aqueous environment and the effect of low-energy electron attachment: A Car–Parrinello QM/MM molecular dynamics study

In this study we have explored the effect of low-energy electrons (LEEs) when rupturing the C3′–O3′ and C5′–O5′ bonds in 3′ and 5′ cytosine monophosphate in an aqueous environment. This has been done using a hybrid quantum mechanics/classical mechanics (QM/MM) setup within the framework of Car–Parrinello molecular dynamics (CPMD). Our results are in agreement with experimental findings and indicate that LEEs do not drastically lower the energy barrier for breaking the 3′ or 5′ phosphodiester bonds for single cytosine nucleotides in aqueous environment.

Efecto de los nucleótidos CMP y UMP en la fatiga en ratas

The aetiology of muscle fatigue has yet not been clearly established. Administration of two nucleotides, cytosine monophosphate (CMP) and uridine monophosphate (UMP), has been prescribed for the treatment of neuromuscular affections in humans. Patients treated with CMP/UMP recover from altered neurological functions and experience pain relief, thus the interest to investigate the possible effect of the drug on exhausting exercise. With such aim, we have determined, in exercised rats treated with CMP/UMP, exercise endurance, levels of lactate, glucose and glycogen, and the activity of several metabolic enzymes such as, creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST). Our results show that rats treated with CMP/UMP are able to endure longer periods of exercise (treadmill-run). Before exercise, muscle glucose level is significantly higher in treated rats, suggesting that the administration of CMP/UMP favours the entry of glucose in the muscle. Liver glycogen levels remains unaltered during exercise, suggesting that CMP/UMP may be implicated in maintaining the level of hepatic glycogen constant during exercise. Lactate dehydrogenase and aspartate aminotransferase activity is significantly lower in the liver of treated rats. These results suggest that administration of CMP/UMP enable rats to endure exercise by altering some metabolic parameters.

Cloning and expression of the sucrose phosphorylase gene from Leuconostoc mesenteroides in Escherichia coli

The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K (m) of 3 mM with sucrose as a substrate; optimum activity was at 37 degrees C and pH 6.7. The purified 742SPase transferred the glucosyl moiety of sucrose to cytosine monophosphate (CMP).

Complete crystallographic analysis of the dynamics of CCA sequence addition

CCA-adding polymerase matures the essential 3'-CCA terminus of transfer RNA without any nucleic-acid template. However, it remains unclear how the correct nucleotide triphosphate is selected in each reaction step and how the polymerization is driven by the protein and RNA dynamics. Here we present complete sequential snapshots of six complex structures of CCA-adding enzyme and four distinct RNA substrates with and without CTP (cytosine triphosphate) or ATP (adenosine triphosphate). The CCA-lacking RNA stem extends by one base pair to force the discriminator nucleoside into the active-site pocket, and then tracks back after incorporation of the first cytosine monophosphate (CMP). Accommodation of the second CTP clamps the catalytic cleft, inducing a reorientation of the turn, which flips C74 to allow CMP to be accepted. In contrast, after the second CMP is added, the polymerase and RNA primer are locked in the closed state, which directs the subsequent A addition. Between the CTP- and ATP-binding stages, the side-chain conformation of Arg 224 changes markedly; this is controlled by the global motion of the enzyme and position of the primer terminus, and is likely to achieve the CTP/ATP discrimination, depending on the polymerization stage. Throughout the CCA-adding reaction, the enzyme tail domain firmly anchors the TPsiC-loop of the tRNA, which ensures accurate polymerization and termination.

Kinetic modeling for chromatographic separation of cytosine monophosphate and uracil monophosphate

Kinetic modeling for preparative chromatography is a topic of present interest in the fine chemicals and pharmaceutical industries. In this study, chromatographic separation of the two nucleotides CMP and UMP was simulated by the equilibrium-dispersive (ED) model, and the adsorption isotherms in the ED model were determined by the inverse method. Prediction performance of the model was validated under three different kinds of conditions and the importance of selecting isotherms was discussed in detail. Excellent agreement was achieved with the experimental band profiles and the prediction of the ED model. The ED model with bi-Langmuir isotherm was especially suitable for simulating chromatographic separation of CMP and UMP. The error of prediction by the ED model with bi-Langmuir isotherm was about 9.4 times smaller than that with Langmuir isotherm.

Sensory and Chemical Analyses of Oyster Mushrooms (Pleurotus sajor-caju) Harvested from Different Substrates

: The sensory characteristics of dehydrated oyster mushrooms harvested from 8 different substrates and chemical compounds related to sensory attributes were analyzed and compared. The sensory attributes rubbery, sweet pea, and bitter as well as the chemical content of total lipids, palmitic acid, benzaldehyde, mannitol, 14 free amino acids, and 5′-adenosine monophosphate were found to be significantly affected by the substrates used for cultivating oyster mushrooms. This study demonstrated that the substrate composition for oyster mushroom production had minor effects on sensory and quality characteristics. Chemical analysis indicated that benzaldehyde, some free amino acids, 5′-cytosine monophosphate (CMP), 5′-guanosine monophosphate (GMP), and 5′-inosine monophosphate (IMP) showed correlations with the sensory attributes perceived by the sensory panel; however, some of these compounds were not detected in all samples.

Effects of elevated ammonium on glycosylation gene expression in CHO cells

The negative effects of ammonium on recombinant protein productivity and glycosylation have been well documented, but the interaction of ammonium on glycosylation genes has not been completely elucidated. In this study, the effects of elevated ammonium on 12 glycosylation related genes in Chinese hamster ovary cells were evaluated by quantitative real time reverse transcriptase polymerase chain reaction. Numerous cytosol and endoplasmic reticulum (ER) localized genes associated with early glycosylation steps were insensitive to the ammonium condition. The initial expression of uridine diphosphate (UDP)-galactose transporter was higher for the ammonium-treated culture, while the initial expressions of cytosine monophosphate (CMP)-sialic acid transporter, β(1,4)-galactosyltransferase, and UDP-glucose pyrophosphorylase were higher for the control culture. α(2,3)-sialyltransferase was observed to have lower expression level under the elevated ammonium condition compared to the control culture. This study indicates that galactosylation and sialylation inhibition is mainly due to decreased gene expression of galactosyltransferase, sialyltransferase, and CMP-sialic acid transporter and not due to sialidase. These unbalanced initial glycosylation and branching steps can explain the higher molecular heterogeneity under ammonium stress. Moreover, this study indicates that elevated ammonium has limited effects on the glycosylation genes associated with the ER and cytosol compared to the genes associated with the Golgi.

Molecular cloning, expression, and characterization of adenylate isopentenyltransferase from hop ( Humulus lupulus L.)

A cDNA encoding adenylate isopentenyltransferase (AIPT) was cloned and sequenced from cones of hop ( Humulus lupulus L.) by RT-PCR using oligonucleotide primers based on the conserved sequences of Arabidopsis thaliana AIPT isozymes (AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7 and AtIPT8). A full-length cDNA contained a 990-bp open reading frame encoding a molecular mass of 36,603 Da protein with 329 amino acids. Further, DNA sequencing of genomic DNA revealed absence of introns in the frame. On Southern blot analysis, a single AIPT gene was detected in H. lupulus, while RT-PCR analyses demonstrated that the gene was equally expressed in almost all tissues in the plant including roots, stems, leaves and cones. The deduced amino acid sequence shares 38–51% identity to those of A. thaliana AtIPTs. A recombinant enzyme expressed in Escherichia coli catalyzed isopentenyl transfer reaction from dimethylallyldiphosphate (DMAPP) to the N 6 amino group of adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP), respectively. In contrast, other nucleotides; guanosine monophosphate (GMP), inosine monophosphate (IMP), cytosine monophosphate (CMP), uridine monophosphate (UMP), were not accepted as a substrate. Interestingly, steady-state kinetic analyses revealed that the isopentenylation of ADP and ATP were more efficient than that of AMP as previously reported for A. thaliana AtIPT4. Finally, H. lupulus AIPT contains the putative ATP/GTP binding motif at the N-terminal as in the case of other known isopentenyltransferases. Site-directed mutagenesis of a conserved Asp62, located right after the ATP/GTP binding motif, with Ala resulted in complete loss of enzyme activity.

Quantum capping potentials with point charges: a simple QM/MM approach for the calculation of large-molecule NMR shielding tensors.

A simple quantum-mechanics/molecular-mechanics (QM/MM) approach for calculating NMR shielding tensors (sigma) is presented. The method involves capping the QM region with quantum capping potentials (QCPs) and representing the MM region with point charges. Test calculations on simple systems without MM charges show that calculated sigma values improve relative to the full QM results with increasing distance between the capped bond and chromophore. Calculations on the histidine amino acid and cytosine monophosphate (CMP) nucleic acid show that the use of QCPs with point charges result in mean errors in the isotropic component of sigma that are less than 1.6 ppm. The results also reveal that, contrary to previous work, the explicit effect of point charges on sigma through coupling with gauge factors, as in the gauge including atomic orbital approach, is minimal for the CMP molecule. The present QM/MM approach for calculating sigma is easy to apply and requires no code modification.

Dietary Nucleotides Accelerate Changes in Intestinal Lymphocyte Maturation in Weanling Mice

ABSTRACTObjectiveNucleotides, the building blocks of nucleic acids, are normal components of the mammalian diet. These molecules have been implicated in biologic processes, such as the stimulation of the immunologic response. Nucleotides have also been considered as conditionally essential nutrients for infant formulas. The authors evaluated the influence of dietary nucleotides on the expression of several surface antigens by different intestinal lymphocyte populations in weanling mice.MethodsMice at weaning were fed a semipurified diet with or without 3 g/kg of each of the following nucleotides: adenosine monophosphate, cytosine monophosphate, guanosine monophosphate, and uridine monophosphate. Animals were killed at different times (0, 4, 7, 12, and 18 days) after weaning, and lymphocytes from intestinal Peyer's patches, epithelium, and lamina propria were isolated. The expression of different antigens (CD3, CD4, CD8α, CD8β, TCRαβ, TCRγδ, CD5, CD22 and CD45R) was analyzed by flow cytometry.ResultsThe expression of these antigens changed parallel to the maturation of the lymphocytes from Peyer's patches, epithelium, and lamina propria. However, developmental changes of expression for most of the antigens occurred sooner in the animals fed the diet supplemented with nucleotides. The expression of T and B antigens was different in the lymphocyte populations analyzed and also changed according to the diet within each population. In general, nucleotides promoted the expression of B‐ and T‐helper cell antigens.ConclusionsThe authors conclude that dietary nucleotides may affect the process of maturation and differentiation of intestinal lymphocytes, which usually takes place at weaning.