Uridine Monophosphate Research Articles

Frequency of genotypic markers for genetic disorders, colour, polledness, and major genes in Blanco Orejinegro cattle.

The purpose of this work was to establish DNA marker frequencies for genetic disorders, colour, horned/polled trait, and major genes of importance for productive and reproductive traits in Blanco Orejinegro (BON) cattle. The Blanco Orejinegro breed is a Colombian creole breed characterized by a white hair coat on black skin with black ears, black hair on the middle part of the legs, and absence of horns. We genotyped 420 animals of Colombia, 70 with the GGP-Bovine 150K chip and 350 with the GGP-Bovine 50K chip. The markers were associated with 50 genetic diseases, 52 major gene variants related to productive traits, and 12 variants related to coat coloration, presence of horns, and adaptation, selected from the information contained in the chips. Genotype frequencies were estimated using the R statistical program. Genetic disorder annotations were derived using the Online Mendelian Inheritance in Animals tool (OMIA) and the average inbreeding coefficient (F) (n = 7799) using the MTDFNRM program. Carriers were found for 16 of the genetic disorders evaluated but with low frequencies (0.24 to 2.46%); no homozygous animals were found for the disorders. Carriers were associated with disorders such as bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), syndactyly, and epidermolysis bullosa (EB). The F was 4.41%. Concerning the genes associated with colour (TYR, MC1R, and PMEL), alleles related to black pigmentation, the absence of horns (polledness), and slick coat (an adaptive trait) were highly frequent (> 81.90%). Markers associated with milk production and quality, yellow fat, and fertility showed variable frequencies, indicating selection potential. Allele frequency of genetic disorders in BON cattle was low, suggesting few genetic disorder problems, with syndactyly being the most frequent condition. The markers associated with colour and polledness were almost fixed, with a frequency at or near 100%. Production and reproduction markers showed variability for selection.

Identification and characterization of novel infection associated transcripts in macrophages

ABSTRACT By analysis of lncRNA expression profiles of macrophages in response to Mycobacterium tuberculosis (Mtb) infection, we identified novel highly expressed transcripts, unique in encompassing a protein coding transcript- Cytidine Monophosphate Kinase 2 (CMPK2) and a previously identified lncRNA- Negative Regulator of Interferon Response (NRIR). While these transcripts (TILT1, 2,3 – TLR4 and Infection induced Long Transcript) are induced by virulent Mtb as well as lipopolysaccharide (LPS) early, lack of/delayed expression in non-viable Mtb/BCG infected cells, respectively, suggest an important role in macrophage responses. The elevated expression by 3 hr in response to fast growing bacteria further emphasizes the importance of these RNAs in the macrophage infection response. Overall, we provide evidence for the presence of multiple transcripts that form a part of the early infection response programme of macrophages. Abbreviations: IFN: Interferon; NRIR: negative regulator of interferon response; CMPK2: cytidine/ uridine monophosphate kinase; LPS: lipopolysaccharide; LAM: Lipoarabinomannan; PIMs: Phosphatidylinositol Mannosides; TILT1, 2,3: TLR4 and Infection induced Long Transcript; TLR4: Toll-like receptor 4; Mtb: Mycobacterium tuberculosis; BCG: Mycobacterium bovis BCG; MDMs: human monocyte derived macrophages

Treatment of neurological complications in patients with type 2 diabetes mellitus at the stage ofrehabilitation after COVID-19

Although the predominant clinical manifestation of COVID-19 is a respiratory disease, various neurological symptoms are increasingly being diagnosed, in particular, diabetic polyneuropathy is diagnosed in most patients with diabetes, affecting large and small nerve fibers. Drugs that are traditionally used for neuropathic pain (tricyclic antidepressants, gabapentinoids, etc.), despite their positive effect in eliminating the symptoms of polyneuropathy, often cause side effects and do not impact nerve regeneration. Over the last decade, a group of nucleotides has been used quite actively. Additional information on the effects of this group of drugs was accumulated and there is a gradual transformation, including their compositions. Thus, recently the attention of researchers has been devoted to the study of the effectiveness of the combination of uridine, choline, vitamins B1, B6, B12, and folic acid, which is characterized by a fairly high safety profile and regenerative potential. The review highlights the mechanisms of action and results of clinical use of this combination. Uridine monophosphate, B vitamins, folic acid are involved in metabolic processes, enhancing nerve regeneration. This contributes to the development of indirect (secondary) analgesic effect. In addition, the data of new studies indicate the ability of uridine monophosphate derivatives to impact purinergic P2Y receptors, which causes a direct analgesic and direct regenerative effect. Studies have demonstrated the clinical efficacy of this combination in the main types of peripheral neuropathic pain. The combination did not cause side effects and was well tolerated. There was a reduction or complete withdrawal of concomitant analgesics against the background of improving the quality of life of patients. The combination of uridine, choline, vitamin B1, vitamin B6, vitamin B12, and folic acid is a very effective addition to the standard therapy of peripheral neuropathic pain of various genesis and rehabilitation after COVID-19.

Orotic acid production by Yarrowia lipolytica under conditions of limited pyrimidine.

Orotic acid (OA) is an intermediate of the pyrimidine biosynthesis with high industrial relevance due to its use as precursor for production of biochemical pyrimidines or its use as carrier molecule in drug formulations. It can be produced by fermentation of microorganisms with engineered pyrimidine metabolism. In this study, we surprisingly discovered the yeast Yarrowia lipolytica as a powerful producer of OA. The overproduction of OA in the Y.lipolytica strain PO1f was found to be caused by the deletion of the URA3 gene which prevents the irreversible decarboxylation of OA to uridine monophosphate. It was shown that the lack of orotidine-5'-phosphate decarboxylase was the reason for the accumulation of OA inside the cell since a rescue mutant of the URA3 deletion in Y.lipolytica PO1f completely prevented the OA secretion into the medium. In addition, pyrimidine limitation in the cell massively enhanced the OA accumulation followed by secretion due to intense overflow metabolism during bioreactor cultivations. Accordingly, supplementation of the medium with 200 mg/L uracil drastically decreased the OA overproduction by 91%. OA productivity was further enhanced in fed-batch cultivation with glucose and ammonium sulfate feed to a maximal yield of 9.62 ± 0.21 g/L. Y.lipolytica is one of three OA overproducing yeasts described in the literature so far, and in this study, the highest productivity was shown. This work demonstrates the potential of Y.lipolytica as a possible production organism for OA and provides a basis for further metabolic pathway engineering to optimize OA productivity.

The risk assessment of the combined medicine in the capsule dosage form at the pharmaceutical development stage

Aim. To determine the potential risk factors associated with the critical quality indicators of the combined medicine “Neuronucleos” in the capsule dosage form for the treatment of polyneuropathies using the general risk assessment methodology while planning the drug quality at the stage of pharmaceutical development (PD). Materials and methods. The series of the combined medicine “Neuronucleos” in the capsule dosage form, critical indicators of the drug quality, the flowchart of the drug production, critical control points of the drug manufacturing process were developed. The method of causality was used. The quantitative assessment of risk factors was carriedout using the FMECA methodology. Results and discussion. The main objective of this study was to apply the Quality-by-Design (QbD) approach to PD of the combined medicine in the capsule dosage form based on uridine-5 monophosphate disodium salt (UMP), cytidine-5-monophosphate disodium salt (CMF), vitamin B6, thioctic acid and magnesium lactate dihydrate. For better patient compliance and the product quality, a target quality profile as a base for PD planning, as well as critical quality attributes (CQA) related to the product safety and efficacy were determined. The criticality of each CQA was assessed using a special scale. It was shown that “Quantitative content”, “Uniformity of dosage units”, “Dissolution”, “Impurities” were defined as the most CQA due to the minimum amount of UMF and CMF in the capsule and the possibility of their decomposition and increase in the quantity of impurities. The critical attributes of materials (CMA) were identified, and the characteristics required to control them were determined in order to ensure the expected product quality. The primary assessment of the quality indicator risks of the active ingredients was performed. It was found that the particle size affected the homogeneity, the quantitative content of API and dissolution in FPP, and it was shown that the solubility of active substances had a high risk when performing the “Dissolution” test. To determine the potential factors with a significant impact on the drug quality the maximum number of factors was found, and the Ishikawa diagram was constructed. The risk factors associated with the quality and compatibility of active substances and excipients, the quality of primary packaging, production conditions, the drug quality control and the technological process were identified. These factors are the causes of risk and can lead to a situation with negative consequences for the quality of a medicinal product. The FMECA process assessment allowed us to determine the impact of the manufacturing process on the CQA. Conclusions. At the stage of PD for the combined medicine in the capsule dosage form the potential critical indicators of the drug quality have been determined. The critical parameters of the quality of the initial components and the properties of the product have been assessed; the most probable risks for the drug quality have been identified, analyzed and assessed. Further research is advisable to focus on studying the effect of process parameters on critical parameters of the product quality and assessing risks for quality and creating a validation plan and its implementation.

Cyclic CMP and cyclic UMP mediate bacterial immunity against phages

The cyclic pyrimidines 3',5'-cyclic cytidine monophosphate (cCMP) and 3',5'-cyclic uridine monophosphate (cUMP) have been reported in multiple organisms and cell types. As opposed to the cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP), which are second messenger molecules with well-established regulatory roles across all domains of life, the biological role of cyclic pyrimidines has remained unclear. Here we report that cCMP and cUMP are second messengers functioning in bacterial immunity against viruses. We discovered a family of bacterial pyrimidine cyclase enzymes that specifically synthesize cCMP and cUMP following phage infection and demonstrate that these molecules activate immune effectors that execute an antiviral response. A crystal structure of a uridylate cyclase enzyme from this family explains the molecular mechanism of selectivity for pyrimidines as cyclization substrates. Defense systems encoding pyrimidine cyclases, denoted here Pycsar (pyrimidine cyclase system for antiphage resistance), are widespread in prokaryotes. Our results assign clear biological function to cCMP and cUMP as immunity signaling molecules in bacteria.

Untargeted LC-MS-based metabonomic analysis of the effect of photoperiod on the testes of broiler roosters.

Photoperiod is an important factor that stimulates the reproductive performance of broiler breeder roosters. However, the mechanism by which photoperiod affects the reproductive performance of broiler breeder roosters has not been fully studied. To study the effects of different photoperiods on the reproductive performance of broiler breeder roosters, 120 Arbor Acres broiler breeder roosters aged 20weeks were randomly assigned to three groups (n=40), and the three groups were treated with different photoperiod regimes: control (CTR; 12.5h of light and 11.5h of dark, 12.5 L: 11.5 D), short day (SD; 16 L: 8 D) and long day (LD; 8 L: 16 D). Serum and testes were collected after 4weeks of feeding, and testosterone-related indices were detected. We found that testosterone synthesis in the testes of broiler roosters was boosted with prolonged of photoperiod. Subsequently, metabonomics was used to identify the differential endogenous metabolites that may affect the function of the testes in breeder roosters. We found compared with other groups, the concentrations of creatine, uridine monophosphate, phosphoribosyl pyrophosphate, dCMP, α-D-glucose and citric acid in the SD group decreased significantly (p<0.05), and glyoxylic acid, D-ribose 5-phosphate, deoxyuridine and orotic acid in the SD group increased significantly (p<0.05), while the CTR group and LD group showed no significant difference (p>0.05). The concentrations of linoleic acid and α-linolenic acid in the LD group were increased significantly (p<0.05) than those in the CTR and SD groups. Compared with the CTR group, the concentrations of histamine in the SD and LD groups were significant increased (p<0.05). The 13 of the different metabolites could be used as candidate biomarkers for different photoperiods affecting testosterone synthesis, may be used to molecular breeding of high reproductive performance broiler roosters.

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.

X-ray induced luminescence spectroscopy for DNA damaging intermediates aided by a monochromatic synchrotron radiation

Purpose To identify the bonding sites of initial radiation interaction with DNA and to trace the following chemical reaction sequences on the pathway of damage induction, we carry out a spectroscopy XIL (X-ray induced luminescence) using soft X-ray synchrotron radiation. This is a nondestructive analysis of the excited intermediate species produced in a molecular mechanism on the damage induction pathway. Materials and Methods We introduce aqueous samples of UMP (uridine-5’-monophosphate) in the vacuum by the use of a liquid micro-jet technique. The luminescence in the region of UV-VIS (from visible to ultraviolet) radiation induced after the absorption of monochromatic soft X-ray by aqueous UMP is measured with sweeping the soft X-ray energy in the region of 370–560 eV. Results The enhanced XIL intensities for aqueous UMP in the region of soft X-ray of 410–530 eV (in “water window” region) are obtained. The enhancement of XIL intensities in the UV-VIS region, relative to the water control, is explained by the excitation and ionization of a K-shell electron of nitrogen atoms in the uracil moiety. The enhanced XIL intensities do not match the structure of XANES (X-ray absorption near-edge structure) of the aqueous UMP. This suggests that the XIL intensities reflect the quantum yields of luminescence, or the quantum yields for conversion by UMP of an absorbed X-ray into UV-VIS radiation. In this paper, spectra of luminescence are shown to be resolved by combining low pass filters. The filtered luminescence spectra are obtained at the center of gravity (λc) of the band pass wavelength regions at λc = 270nm, 295 nm, 340 nm, 385 nm, 450 nm, and 525 nm., which show a trend similar to the fluorescence of nucleobases induced by ultraviolet radiation. Conclusion It is concluded that the origin of the observed XIL is the hydrated uracil moiety in aqueous UMP, decomposition of which is suppressed by the migration of excess charge and internal energy after the double ionization due to Auger decay.

Computational Study on DNA Repair: The Roles of Electrostatic Interactions Between Uracil-DNA Glycosylase (UDG) and DNA.

Uracil-DNA glycosylase (UDG) is one of the most important base excision repair (BER) enzymes involved in the repair of uracil-induced DNA lesion by removing uracil from the damaged DNA. Uracil in DNA may occur due to cytosine deamination or deoxy uridine monophosphate (dUMP) residue misincorporation during DNA synthesis. Medical evidences show that an abnormal expression of UDG is related to different types of cancer, including colorectal cancer, lung cancer, and liver cancer. Therefore, the research of UDG is crucial in cancer treatment and prevention as well as other clinical activities. Here we applied multiple computational methods to study UDG in several perspectives: Understanding the stability of the UDG enzyme in different pH conditions; studying the differences in charge distribution between the pocket side and non-pocket side of UDG; analyzing the field line distribution at the interfacial area between UDG and DNA; and performing electrostatic binding force analyses of the special region of UDG (pocket area) and the target DNA base (uracil) as well as investigating the charged residues on the UDG binding pocket and binding interface. Our results show that the whole UDG binding interface, and not the UDG binding pocket area alone, provides the binding attractive force to the damaged DNA at the uracil base.

Novel mutation in UMPS gene leads to false positive result of DUMPS (genetic disorder) in buffaloes: need for gene sequencing before confirming results of RFLP in new species

Deficiency of uridine monophosphate synthase (DUMPS) is a lethal genetic disorder associated with early embryonic mortality. Murrah and Mehsana male buffaloes (n = 594) were screened for DUMPS by PCR-RFLP technique. A few Murrah buffalo male calves were found to be carriers of DUMPS in RFLP, which has not been reported earlier. On the Sanger sequencing, a novel A to G substitution mutation was identified in AvaI restriction recognition site of UMPS gene in buffaloes. This mutation hinders digestion of DNA by AvaI which leds to false positive results for DUMPS carrier in RFLP. The results indicated that genome sequencing must be performed before confirming results of RFLP in any new species. All the buffaloes that were tested had only wild-type genotype in exon 5 for DUMPS specific allele.

Interactive effect of dietary fish oil and pyrimidine nucleotide supplementation on the fatty acid composition of juvenile rainbow trout Oncorhynchus mykiss : Enhancement of ARA and DHA contents in the fillet of fish fed‐supplemented diet

The present study investigated interactive effect of different levels of dietary fish oil and pyrimidine nucleotide supplementation on growth and fatty acid composition of juvenile rainbow trout. Six experimental diets were formulated with two different levels of fish oil. High fish oil (HFO) diet contains 120 g/kg fish oil, while low fish oil (LFO) diet contains 40, 60 and 20 g/kg fish, linseed and rapeseed oils respectively. Uridine 5’-monophosphate (UMP) or cytidine 5’-monophosphate (CMP) disodium salt was supplemented onto each diet at 1.5 g/kg respectively. For control diet, UMP and CMP were not supplemented to HFO and LFO diets respectively (HFO-Cont. and LFO-Cont.). Rainbow trout with initial average body weight of 13.5 g were randomly placed into 12 tanks and offered one of the six diets for 12 weeks. Growth, feed utilization and hepatosomatic index (HSI) of fish were not influenced by either fish oil or pyrimidine nucleotide supplementation. This trend was also similar on the crude lipid content in the liver and proximate composition in the muscle and whole fish body. In addition, fatty acid composition in the liver was influenced by fish oil level, but not by pyrimidine nucleotide supplementation. Interestingly, pyrimidine nucleotide supplementation affected 20:4n-6 and 22:6n-3 contents in the muscle of fish. In summary, although dietary UMP and CMP have no positive effect on growth, it affected on the fatty acid composition of fish by increasing muscular 20:4n-6 and 22:6n-3 contents particularly when it is supplemented in low fish oil-based diet.

Pathogenic variants in uridine diphosphate nucleotidase ( CANT1 ) or glucuronyltransferase ( B3GAT3 ) causes a pseudodiastrophic dysplasia

Calcium-activated uridine diphosphate nucleotidase 1 (CANT1) hydrolyzes UDP to uridine 5’-monophosphate (UMP) and an inorganic phosphate. Glucuronyltransferase-I (GlcAT-I) transfers a glucuronic acid (GlcA) from UDP-GlcA to galactose-galactose-xylose in core proteins of proteoglycans. Biallelic mutations in CANT1 or GlcAT-I cause Desbuquois dysplasia and multiple joint dislocations, respectively [1, 2]. Here, we identified the biallelic mutations of the CANT1 (Glu215Lys) or GlcAT-I (Arg169Trp/Arg225X) in the patients with pseudodiastrophic dysplasia [3], which is a severe skeletal dysplasia associated with prenatal manifestation and early lethality [4]. The GlcAT-I activities of recombinant Arg169Trp- and Arg225X-GlcAT-I proteins were significantly decreased compared to that of wild type-GlcAT-I [3]. Furthermore, cell lysates from the patient fibroblasts showed significantly reduced GlcAT-I activity compared with those from control fibroblasts. The amount of chondroitin sulfate and dermatan sulfate in the fibroblasts from a patient was markedly lower than that in control fibroblasts. Furthermore, the UDP-nucleotidase activity of recombinant Glu215Lys-CANT1was significantly reduced compared with that of wild type enzyme. These results suggest that the variants, Arg169Trp/Arg225X and Glu215Lys, in GlcAT-I and CANT1, respectively, are severe loss-of-function mutations, and cause the disturbance of biosynthesis of proteoglycans, which results in a pseudodiastrophic dysplasia with the significant phenotypic overlap of conditions within the biosynthetic pathway for proteoglycans. Refs. 1) Huber et al., Am J Hum Genet, 85, 706, 2009; 2) Baasanjav et al., Am J Hum Genet, 89, 15, 2011; 3) Byrne, Mizumoto et al., J Med Genet, 57, 454, 2020; 4) Yap et al., Am J Med Genet A, 170, 1363, 2016.

Simultaneous Genetic Screening of Bovine Leukocyte Adhesion Deficiency, Complex Vertebral Malformation, Deficiency of Uridine Monophosphate Synthase, and Mule foot in Holstein Cows in Taiwan

Simultaneous Genetic Screening of Bovine Leukocyte Adhesion Deficiency, Complex Vertebral Malformation, Deficiency of Uridine Monophosphate Synthase, and Mule foot in Holstein Cows in Taiwan

Występowanie chorób genetycznych (BLAD, DUMPS) w polskiej populacji bydła rasy holsztyńsko-fryzyjskiej

BLAD (bovine leukocyte adhesion deficiency) and DUMPS (deficiency of uridine monophosphate synthase) are hereditary lethal autosomal recessive disorders that may affect Holstein cattle. The aim of the present study was to evaluate the frequency of BLAD and DUMPS genotypes in the Polish population of dairy cows. In the study 5 732 Polish Holstein-Friesian females were genetically tested for BLAD and DUMPS. Results of the present study indicate that the Polish population of dairy cattle is free from DUMPS as there were not carriers nor sick animals. For BLAD the study confirmed the present of carriers in the population to be at a low value (0.21%). Both BLAD and DUMPS may strongly affect the profitability of a dairy farm, therefore, in order to avoid the economic losses caused by the presence of these genetic disorders in the herd it is important to screen the population and detect carriers as soon as possible.

Prebiotic studies on the interaction of zirconia nanoparticles and ribose nucleotides and their role in chemical evolution

AbstractStudies on the interaction of biomolecules with inorganic compounds, mainly mineral surfaces, are of great concern in identifying their role in chemical evolution and origins of life. Metal oxides are the major constituents of earth and earth-like planets. Hence, studies on the interaction of biomolecules with these minerals are the point of concern for the study of the emergence of life on different planets. Zirconium oxide is one of the metal oxides present in earth's crust as it is a part of several types of rocks found in sandy areas such as beaches and riverbeds, e.g. pebbles of baddeleyite. Different metal oxides have been studied for their role in chemical evolution but no studies have been reported about the role of zirconium oxide in chemical evolution and origins of life. Therefore, studies were carried out on the interaction of ribonucleic acid constituents, 5′-CMP (cytidine monophosphate), 5′-UMP (uridine monophosphate), 5′-GMP (guanosine monophosphate) and 5′-AMP (adenosine monophosphate), with zirconium oxide. Synthesized zirconium oxide particles were characterized by using vibrating sample magnetometer, X-Ray Diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy. Zirconia particles were in the nanometre range, from 14 to 27 nm. The interaction of zirconium oxide with ribonucleic acid constituents was performed in the concentration range of 5 × 10−5–300 × 10−5 M. Interaction studies were carried out in three mediums; acidic (pH 4.0), neutral (pH 7.0) and basic (pH 9.0). At neutral pH, maximum interaction was observed. The interaction of zirconium oxide with 5′-UMP was 49.45% and with 5′-CMP 67.98%, while with others it was in between. Interaction studies were Langmurian in nature. Xm and KL values were calculated. Infrared spectral studies of ribonucleotides, metal oxide and ribonucleotide–metal oxide adducts were carried out to find out the interactive sites. It was observed that the nitrogen base and phosphate moiety of ribonucleotides interact with the positive charge surface of metal oxide. SEM was also carried out to study the adsorption. The results of the present study favour the important role of zirconium oxide in concentrating the organic molecules from their dilute aqueous solutions in primeval seas.

Mechanisms of feedback inhibition and sequential firing of active sites in plant aspartate transcarbamoylase

Aspartate transcarbamoylase (ATC), an essential enzyme for de novo pyrimidine biosynthesis, is uniquely regulated in plants by feedback inhibition of uridine 5-monophosphate (UMP). Despite its importance in plant growth, the structure of this UMP-controlled ATC and the regulatory mechanism remain unknown. Here, we report the crystal structures of Arabidopsis ATC trimer free and bound to UMP, complexed to a transition-state analog or bearing a mutation that turns the enzyme insensitive to UMP. We found that UMP binds and blocks the ATC active site, directly competing with the binding of the substrates. We also prove that UMP recognition relies on a loop exclusively conserved in plants that is also responsible for the sequential firing of the active sites. In this work, we describe unique regulatory and catalytic properties of plant ATCs that could be exploited to modulate de novo pyrimidine synthesis and plant growth.

Mechanisms of Cd (II) binding to GMP and UMP: a combined conductometry, isothermal titration calorimetry and NMR study

In this research, conductometry was applied as a rapid method to screen functional ligands of cadmium ion-imprinted polymers for the first time. To better understand the binding mechanisms of Cd(II) to nucleoside monophosphates (NMPs), isothermal titration calorimetry (ITC) and nuclear magnetic resonance spectroscopy (NMR) experiments were carried out. First, the magnitude of conductivity change, Δσ, was found to be proportional to the ligand’s binding capacity to cadmium, and the ΔσGMP ≈ ΔσdGMP = 103.5 μs $$\cdot $$ cm−1. Second, the ITC experiments indicated that the coordination binding of Cd(II) and guanosine monophosphate (GMP) in 4-(2-hydroxyerhyl)piperazine-1-erhanesulfonic acid (HEPES)/boric acid buffers were demonstrated as different thermodynamic process: an exothermic process (ΔH = -1.446 kcal $$\cdot $$ mol−1) in HEPES, and an exothermic process (ΔH = − 1.495 kcal $$\cdot $$ mol−1) followed by an endothermic one (ΔH = 1.383 kcal mol−1) in boric acid. In contrast, the formation of the Cd(II)–uridine monophosphate (UMP) system was an endothermic process in both buffers. NMR experiments indicated that the shift Δδ of the GMP’s proton signals were different, especially for H1: 0.37 ppm in HEPES, and 0.03 ppm in boric acid. Referring to the results of ITC experiments, the major Cd(II) binding sites of GMP in HEPES buffer was the guanosine group, and phosphate group was another site in the boric acid buffer. By contrast, the major Cd(II) binding site of UMP was the phosphate group in both buffers. In general, investigating the functional ligand–cadmium binding mechanism has provided important theoretical and experimental guidance for the subsequent preparation of cadmium ion-imprinted materials.

Photochemistry of potassium ferrocyanide and its reaction with uridine 5′-monophosphate in aqueous solution under ultraviolet irradiation

The photochemical reaction of potassium ferrocyanide (K4Fe(CN)6) exhibits excitation wavelength dependence and non-Kasha rule behavior. In this study, the excited-state dynamics of K4Fe(CN)6 were studied by transient absorption spectroscopy. Excited state electron detachment (ESED) and photoaquation reactions were clarified by comparing the results of 260, 320, 340, and 350 nm excitations. ESED is the path to generate a hydrated electron (eaq−). ESED energy barrier varies with the excited state, and it occurs even at the first singlet excited state (1T1g). The 1T1g state shows ∼0.2 ps lifetime and converts into triplet [Fe(CN)6]4− by intersystem crossing. Subsequently, 3[Fe(CN)5]3− appears after one CN− ligand is ejected. In sequence, H2O attacks [Fe(CN)5]3− to generate [Fe(CN)5H2O]3− with a time constant of approximately 20 ps. The 1T1g state and eaq− exhibit strong reducing power. The addition of uridine 5′-monophosphate (UMP) to the K4Fe(CN)6 solution decrease the yield of eaq− and reduce the lifetimes of the eaq− and 1T1g state. The obtained reaction rate constant of 1T1g state and UMP is 1.7×1014 (mol/L)−1·s−1, and the eaq− attachment to UMP is ∼8×109 (mol/L)−1·s−1. Our results indicate that the reductive damage of K4Fe(CN)6 solution to nucleic acids under ultraviolet irradiation cannot be neglected.

The mystery of sub-picosecond charge transfer following irradiation of hydrated uridine monophosphate.

The early mechanisms by which ionizing rays damage biological structures by so-called direct effects are largely elusive. In a recent picosecond pulse radiolysis study of concentrated uridine monophosphate solutions [J. Ma, S. A. Denisov, J.-L. Marignier, P. Pernot, A. Adhikary, S. Seki and M. Mostafavi, J. Phys. Chem. Lett., 2018, 9, 5105], unexpected results were found regarding the oxidation of the nucleobase. The signature of the oxidized nucleobase could not be detected 5 ps after the electron pulse, but only the oxidized phosphate, raising intriguing questions about the identity of charge-transfer mechanisms that could explain the absence of U+. We address here this question by means of advanced first-principles atomistic simulations of solvated uridine monophosphate, combining Density Functional Theory (DFT) with polarizable embedding schemes. We contrast three very distinct mechanisms of charge transfer covering the atto-, femto- and pico-second timescales. We first investigate the ionization mechanism and subsequent hole/charge migrations on a timescale of attoseconds to a few femtoseconds under the frozen nuclei approximation. We then consider a nuclear-driven phosphate-to-oxidized-nucleobase electron transfer, showing that it is an uncompetitive reaction channel on the sub-picosecond timescale, despite its high exothermicity and significant electronic coupling. Finally, we show that non-adiabatic charge transfer is enabled by femtosecond nuclear relaxation after ionization. We show that electronic decoherence and the electronic coupling strength are the key parameters that determine the hopping probabilities. Our results provide important insight into the interplay between electronics and nuclear motions in the early stages of the multiscale responses of biological matter subjected to ionizing radiation.