Cytidylic Acid Research Articles

Voltammetric Study of dsDNA Modified by Multi-redox Label Based on N-methyl-4-hydrazino-7-nitrobenzofurazan

Voltammetric behaviour of novel synthetic multi-redox DNA label N-methyl-4-hydrazino-7-nitrobenzofurazan (NBF), a deoxycytidine monophosphate conjugate with NBF via formylthiophene linker (dCNBFMP), and NBF labeled model short double stranded DNA (dsDNA-NBF) were systematically studied at mercury meniscus modified silver solid amalgam electrode (m-AgSAE) for the first time in this work. Successful enzymatic incorporation of the NBF labeled deoxycytidine triphosphate into the DNA was confirmed and the dsDNA-NBF offered new more positive cathodic signals at −520mV and −1160mV, compared to the nucleic acid itself. Used modification resulted in increase of selectivity and sensitivity of the DNA voltammetric determination. Next to the pioneering voltammetric behaviour of all studied compounds at m-AgSAE, electroanalytical methods based on cyclic voltammetry, adsorptive stripping cyclic voltammetry and open circuit transfer stripping cyclic voltammetry were developed for sensitive detection of these (bio)molecules. Furthermore, utilization of variable negative vertex potential and scan rate was also observed and their appropriate selection considerably influenced registered peaks on cyclic voltammograms. These redox processes increased signal response diversity and offered utilization of signal switch on/off technique, as well. Tentative electrochemical reduction mechanisms of the NBF, dCNBFMP and dsDNA-NBF were proposed and discussed, together with possibilities and limitations of the NBF+m-AgSAE system.

Neuroprotective properties of citicoline: facts, doubts and unresolved issues.

Citicoline is the generic name of the pharmaceutical substance that chemically is cytidine-5′-diphosphocholine (CDP-choline), which is identical to the natural intracellular precursor of phospholipid phosphatidylcholine. Following injection or ingestion, citicoline is believed to undergo quick hydrolysis and dephosphorylation to yield cytidine and choline, which then enter the brain separately and are used to resynthesize CDP-choline inside brain cells. Neuroprotective activity of citicoline has been repeatedly shown in preclinical models of brain ischaemia and trauma, but two recent, large, pivotal clinical trials have revealed no benefits in ischaemic stroke and traumatic brain injury. However, the substance seems to be beneficial in some slowly advancing neurodegenerative disorders such as glaucoma and mild vascular cognitive impairment. This paper critically discusses issues related to the clinical pharmacology of citicoline, including its pharmacokinetics/biotransformation and pharmacodynamics/mode of action. It is concluded that at present, there is no adequate description of the mechanism(s) of the pharmacological actions of this substance. The possibility should be considered and tested that, in spite of apparently fast catabolism, the intact citicoline molecule or the phosphorylated intermediate products of its hydrolysis, cytidine monophosphate and phosphocholine, are pharmacologically active.

Crystal structures of IspF from Plasmodium falciparum and Burkholderia cenocepacia: comparisons inform antimicrobial drug target assessment

Background2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF) catalyzes the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate to 2C-methyl-D-erythritol-2,4-cyclodiphosphate and cytidine monophosphate in production of isoprenoid-precursors via the methylerythritol phosphate biosynthetic pathway. IspF is found in the protozoan Plasmodium falciparum, a parasite that causes cerebral malaria, as well as in many Gram-negative bacteria such as Burkholderia cenocepacia. IspF represents a potential target for development of broad-spectrum antimicrobial drugs since it is proven or inferred as essential in these pathogens and absent from mammals. Structural studies of IspF from these two important yet distinct pathogens, and comparisons with orthologues have been carried out to generate reagents, to support and inform a structure-based approach to early stage drug discovery.ResultsEfficient recombinant protein production and crystallization protocols were developed, and high-resolution crystal structures of IspF from P. falciparum (Emphasis/Emphasis>IspF) and B. cenocepacia (BcIspF) in complex with cytidine nucleotides determined. Comparisons with orthologues, indicate a high degree of order and conservation in parts of the active site where Zn2+ is bound and where recognition of the cytidine moiety of substrate occurs. However, conformational flexibility is noted in that area of the active site responsible for binding the methylerythritol component of substrate. Unexpectedly, one structure of BcIspF revealed two molecules of cytidine monophosphate in the active site, and another identified citrate coordinating to the catalytic Zn2+. In both cases interactions with ligands appear to help order a flexible loop at one side of the active site. Difficulties were encountered when attempting to derive complex structures with other ligands.ConclusionsHigh-resolution crystal structures of IspF from two important human pathogens have been obtained and compared to orthologues. The studies reveal new data on ligand binding, with citrate coordinating to the active site Zn2+ and when present in high concentrations cytidine monophosphate displays two binding modes in the active site. Ligand binding appears to order a part of the active site involved in substrate recognition. The high degree of structural conservation in and around the IspF active site suggests that any structural model might be suitable to support a program of structure-based drug discovery.

The role of hydroxo-bridged dinuclear species and the influence of "innocent" buffers in the reactivity of cis-[Co(III)(cyclen)(H₂O)₂]³⁺ and [Co(III)(tren)(H₂O)₂]³⁺ complexes with biologically relevant ligands at physiological pH.

In view of the relevance of the reactivity of inert tetraamine Co(III) complexes having two substitutionally active cis positions capable of interact with biologically relevant ligands, the study of the reaction of cis-[Co(cyclen)(H2O)2](3+) and [Co(tren)(H2O)2](3+) with chlorides, inorganic phosphate and 5'-CMP (5'-cytidinemonophosphate) has been pursued at physiological pH. The results indicate that, in addition to the actuation of the expected labilising conjugate-base mechanism, the formation of mono and inert bis hydroxo-bridged species is relevant for understanding their speciation and reactivity. The reactivity pattern observed also indicates the key role played by the "innocent" buffers frequently used in most in vitro studies, which can make the results unreliable in many cases. The differences between the reactivity of inorganic and biologically relevant phosphates has also been found to be remarkable, with outer-sphere hydrogen bonding interactions being a dominant factor for the process. While for the inorganic phosphate substitution process the formation of μ-η(2)-OPO2O represents the termination of the reactivity monitored, for 5'-CMP only the formation of η(1)-OPO3 species is observed, which evolve with time to the final dead-end bis hydroxo-bridged complexes. The promoted hydrolysis of the 5'-CMP phosphate has not been observed in any of the processes studied.

Ca2+metal ion adducts with cytosine, cytidine and cytidine 5′-monophosphate: a comprehensive study of calcium reactivity towards building units of nucleic acids

Six new Ca(ii) adducts with cytosine (cyt), cytidine (H2cyd) and cytidine 5′-monophosphate (CMP) are presented. H2cyd and CMP show unprecedented binding sites for the calcium ion.

Symptomatic improvement in an acute, non-traumatic spine pain model with a combination of uridine triphosphate, cytidine monophosphate, and hydroxocobalamin

Rationale: In a previously published trial on spinal acute non-traumatic pain, peripheral neuro- regenerative combination of UTP, CMP and hydroxocobalamin presented unexpected analgesicproperties. Objective: To corroborate analgesiceffects of UTP, CMP and hydroxocobalamin combination in a self-paired evolutionary model. Methods: Mean VAS scores from pretreatment, V2 (5th treatment day) and V3 (10th treatment day) were plotted and statistically analyzed (ANOVA) for differences. PFQ scores from pretreatment, V2, and V3 were analyzed using the chisquare test. Results: The difference between V3 and pretreatment mean VAS scores was statistically significant (p < 0.0001). The improvement in PFQ scores throughout the study was found to be statistically significant (p < 0.0001). Conclusion: The combination of UTP, CMP and hydroxocobalamin seems to have analgesic properties in mediumterm use. The complex peripheral neu-roregenerative pharmacodynamics of this combination provides a plausible basis for this finding. Further randomized studies are needed to explore this combination for the indication of neuropathic pain due to spinal structure involvement.

Human Rev1 polymerase disrupts G-quadruplex DNA

The Y-family DNA polymerase Rev1 is required for successful replication of G-quadruplex DNA (G4 DNA) in higher eukaryotes. Here we show that human Rev1 (hRev1) disrupts G4 DNA structures and prevents refolding in vitro. Nucleotidyl transfer by hRev1 is not necessary for mechanical unfolding to occur. hRev1 binds G4 DNA substrates with Kd,DNA values that are 4–15-fold lower than those of non-G4 DNA substrates. The pre-steady-state rate constant of deoxycytidine monophosphate (dCMP) insertion opposite the first tetrad-guanine by hRev1 is ∼56% as fast as that observed for non-G4 DNA substrates. Thus, hRev1 can promote fork progression by either dislodging tetrad guanines to unfold the G4 DNA, which could assist in extension by other DNA polymerases, or hRev1 can prevent refolding of G4 DNA structures. The hRev1 mechanism of action against G-quadruplexes helps explain why replication progress is impeded at G4 DNA sites in Rev1-deficient cells and illustrates another unique feature of this enzyme with important implications for genome maintenance.

Effect of phosphate group in switching off electron transfer in different media

We have studied the interactions of individual nucleoside 5′-monophosphates of all the five nucleic acid bases, adenosine 5′-monophosphate (AMP), guanosine 5′-monophosphate (GMP), thymidine 5′-monophosphate (TMP), cytidine 5′-monophosphate (CMP) and uridine 5′-monophosphate (UMP) with two quinones, 9,10-anthraquinone (AQ) and 2-methyl 1,4-naphthoquinone (MQ) commonly known as menadione using laser flash photolysis in two different types of media. Although electron transfer (ET) in excited state is a well-known phenomenon in DNA with quinones, this work reveals a total failure of ET with these isolated 5′-monophosphate molecules in a homogeneous medium (acetonitrile/water), except GMP. Similar experiments in SDS micelles have been repeated but here even GMP failed to transfer electrons. We have attributed the failure of ET in homogeneous medium to a semi-circular conformation adopted by the 5′-monophosphates, which is assumed to pull back the electrons from the electron donor center, the nitrogenous base. In SDS micelles a rapid spin exchange within an abnormally short inter-radical distance has been considered to prevent ET.

Effect of 5- AZn-2 ′-deoxycytidine on proliferation of human lung adenocarcinoma cell line A549 in vitro

ObjectiveTo explore effect of 5-AZn-2 ′-deoxycytidine on proliferation of human lung adenocarcinoma cell line A549 in vitro. MethodsSuperoxide dismutase (SOD) activity was measured by hydroxylamine colorimetric method. Inhibition effect of 5-AZn-2′ deoxycytidylic acid at different concentration and different time on growth of A549 cell was determined by MTT assay. Methylene dioxyamphetamine (MDA) was measured by thiobarbituric acid colorimetric method. Effect of 5-AZn-2′ deoxycytidylic acid on apoptosis of A549 cell was determined by Hoechst 33258 dyeing detection. Results5-AZn-2′ deoxycytidylic acid had significant inhibition effect on proliferation of A549 cells in vitro, and the inhibition was notably dependent on time and dosage during 48-72 h; SOD level was significantly lower than those of control group (P<0.05, P<0.01), MDA level was significantly higher than those in the control group (P<0.05, P<0.01). A549 cells began to be in apoptosis after using 5-AZn-2′deoxycytidylic acid. Conclusions5- AZn-2′ deoxycytidylic acid has significant inhibition effect on growth of A549 cell, and can lead the change of lipid peroxidation. It indicates that the mechanism has relationship with A549 cell cycle tissue and induction factor of apoptosis.

IFIT5 potentiates anti-viral response through enhancing innate immune signaling pathways

Humans have a distinct combination of IFIT (IFN-induced protein with tetratricopeptide repeats) family orthologs, including IFIT1 (ISG56), IFIT2 (ISG54), IFIT3 (ISG60), and IFIT5 (ISG58). The function of IFIT1/IFIT2/IFIT3 has been intensively investigated. However, little is known about the role of IFIT5 in any cellular processes. In this study, we reported that both the mRNA and protein levels of IFIT5 are up-regulated in response to RNA virus infection or polyinosinic-cytidylic acid stimulation. Ectopic expression of IFIT5 could synergize IRF3- and NF-κB-mediated gene expression, whereas knockdown of IFIT5 impairs the transcription of these genes. Consistently, anti-viral responses of host cells are significantly increased or decreased in the presence or absence of IFIT5. Mechanistically, IFIT5 co-localizes partly with mitochondria and interacts with RIG-I and MAVS. Our study identified that IFIT5 is an important enhancer in innate immune response.

Engineering of a Cytidine 5′‐Monophosphate‐Sialic Acid Synthetase for Improved Tolerance to Functional Sialic Acids

AbstractSialic acid‐containing glycoconjugates at the cell surface are of high importance in carbohydrate‐mediated recognition phenomena in physiological and pathological events, as well as in bacterial or viral infection. A key step in the enzymatic synthesis of natural sialoconjugates and functional synthetic analogues is the activation of sialic acids to cytidine 5′‐monophosphate (CMP)‐sialic acid intermediates catalyzed by CMP‐sialic acid synthetase (CSS). Based on our recently developed aligned protein model of substrate binding and a simple colorimetric screening assay, we have engineered the CSS from Neisseria meningitidis by structure‐guided site‐specific saturation mutagenesis at positions 192/193 to generate enzymes with broadened substrate scope. Top hits, including the F192S/F193Y variant, display an improvement of up to 70‐fold catalytic efficiency relative to wild‐type CSS for the conversion of sterically demanding N‐acyl modified sialic acid analogues, without compromising protein stability. Such significantly enhanced substrate capacity is a major step forward to realizing a generalized chemo‐enzymatic strategy for the efficient preparation of neo‐sialoconjugate libraries, demonstrated by the highly efficient, regio‐ and stereospecific synthesis of 2,6‐sialyllactose analogues by enzymatic coupling to the highly substrate tolerant α2,6‐sialyltransferase from Photobacterium leiognathi JT‐SHIZ‐145. Our results further document the unusual versatility of the N. meningitidis CSS and engineered variants for a common synthetic approach to sialoconjugates comprising a large diversity of natural and non‐natural sialic acid forms without the need for post‐synthetic enzymatic modification.magnified image

Effects of dietary nucleotides on growth performance, intestinal mor-phology and anti-oxidative activities of juvenile Litopenaeus vannamei

There has been extensive research into the role of nucleotides and their related metabolic products in aquatic feeds.Nucleotides and metabolites play key roles in many biological processes and are considered condi-tionally essential nutrients.During periods of rapid growth or certain disease states,dietary nucleotides may spare the cost of de novo nucleotides synthesis and optimize the function of rapidly dividing tissues,such as those in the gastrointestinal and immune systems.Research on dietary nucleotides in aquatic animals has illustrated that they may improve diet palatability,enhance growth in early stages of development,and maintain intestinal and liver health,as well as increase immunity and disease resistance.Despite their apparent importance,little is known about the benefits of supplementary nucleotides in Litopenaeus vannamei.We evaluated the effects of dietary nu-cleotides on growth,body composition,midgut morphology,and anti-oxidant activity in the hepatopancreas and serum in juvenile L.vannamei.We randomly assigned 960 shrimp(mean body weight: 1.01 g±0.02 g) into 8 trip-licate groups.Group G0(control) was fed a base diet and the remaining seven groups(G0.1,G0.2,G0.4,G0.6,G0.8,G1.0,and G1.2) were fed the base diet supplemented with 0.1,0.2,0.4,0.6,0.8,1.0,or 1.2 g/kg,respec-tively,of a nucleotide mixture containing adenosine-5′-monophosphate(AMP),cytidine-5′-monophosphate(CMP),uridine-5′-monophosphate disodium salt(UMP),inosine-5′-monophosphate disodium salt(IMP),and guanosine-5′-monophosphate disodium salt(GMP)(1 : 1 : 1 : 1 : 1 w/w,mix-NT).All groups were fed their re-spective diets three times a day(8:00,15:00,and 20:00) at the same fixed rate,which ranged from 4% to 6% of body weight,for 7 weeks.Specific growth rate(SGR) and survival(SR) tended to increase as the concentration of the dietary mix-NT increased,peaking in the group supplemented with 0.6 g/kg,though the differences among the groups were not significant(P0.05).The feed conversion rate(FCR) was lower in the treatment groups than in the control group,but not significantly(P0.05).The whole body moisture content was significantly affected by the level of dietary mix-NT supplementation(P0.05),but there were no significant differences in the content of crude protein,ether extract,or ash(P0.05).The dietary mix-NT had a significant effect on the hepatosomatic index(HSI) in the group supplemented with 0.6 g/kg(P0.05).Glutamic-oxaloacetic transaminease(GOT),glu-tamic-pyruvic transaminase(GPT),and uric acid(UA) content were significantly lower in shrimp fed 0.4 g/kg mix-NT(P0.05).There was a significant increase in the midgut jejunum wall thickness in shrimp fed 0.1–0.8 g/kg mix-NT,but there no significant difference in villus height of all groups.Alkaline phosphatase(AKP) activity and total antioxidant capacity(T-AOC) in the hepatopancreas tended to increase as the level of supplementation with dietary mix-NT increased,and were significantly higher in groups G0.2 and G0.4,respectively,than in the control(P0.05).Adding mix-NT had no significant effect on serum alkaline phosphatase(AKP) and superoxide dismutase(SOD) activity(P0.05).In conclusion,supplementing the diet with a mixture of 5 nucleotides im-proves hepatopancreas function,maintains intestinal morphology,and enhances anti-oxidant activity in the hepa-topancreas of juvenile L.vannamei.It would be interesting to determine the effects of nucleotide supplementation on immune function,particularly with respect to white spot syndrome virus(WSSV).

Pharmacological Inhibition of polysialyltransferase ST8SiaII Modulates Tumour Cell Migration

Polysialic acid (polySia), an α-2,8-glycosidically linked polymer of sialic acid, is a developmentally regulated post-translational modification predominantly found on NCAM (neuronal cell adhesion molecule). Whilst high levels are expressed during development, peripheral adult organs do not express polySia-NCAM. However, tumours of neural crest-origin re-express polySia-NCAM: its occurrence correlates with aggressive and invasive disease and poor clinical prognosis in different cancer types, notably including small cell lung cancer (SCLC), pancreatic cancer and neuroblastoma. In neuronal development, polySia-NCAM biosynthesis is catalysed by two polysialyltransferases, ST8SiaII and ST8SiaIV, but it is ST8SiaII that is the prominent enzyme in tumours. The aim of this study was to determine the effect of ST8SiaII inhibition by a small molecule on tumour cell migration, utilising cytidine monophosphate (CMP) as a tool compound. Using immunoblotting we showed that CMP reduced ST8iaII-mediated polysialylation of NCAM. Utilizing a novel HPLC-based assay to quantify polysialylation of a fluorescent acceptor (DMB-DP3), we demonstrated that CMP is a competitive inhibitor of ST8SiaII (K i = 10 µM). Importantly, we have shown that CMP causes a concentration-dependent reduction in tumour cell-surface polySia expression, with an absence of toxicity. When ST8SiaII-expressing tumour cells (SH-SY5Y and C6-STX) were evaluated in 2D cell migration assays, ST8SiaII inhibition led to significant reductions in migration, while CMP had no effect on cells not expressing ST8SiaII (DLD-1 and C6-WT). The study demonstrates for the first time that a polysialyltransferase inhibitor can modulate migration in ST8SiaII-expressing tumour cells. We conclude that ST8SiaII can be considered a druggable target with the potential for interfering with a critical mechanism in tumour cell dissemination in metastatic cancers.

Toll-Like Receptor-3 Activation Increases the Vulnerability of the Neonatal Brain to Hypoxia-Ischemia

Susceptibility and progression of brain injury in the newborn is closely associated with an exacerbated innate immune response, but the underlying mechanisms are often unclear. Toll-like receptors (TLRs) are important innate immune sensors that may influence the vulnerability of the developing brain. In the current study, we provide novel data to show that activation of the viral innate immune receptor TLR-3 sensitizes the neonatal brain to subsequent hypoxic-ischemic (HI) damage. Poly inosinic:poly cytidylic acid (Poly I:C), a synthetic ligand for TLR-3, was administered to neonatal mice 14 h before cerebral HI. Activation of TLR-3 before HI increased infarct volume from 3.0 ± 0.5 to 15.4 ± 2.1 mm³ and augmented loss of myelin basic protein from 13.4 ± 6.0 to 70.6 ± 5.3%. The sensitizing effect of Poly I:C was specific for the TLR-3 pathway because mice deficient in the TLR-3 adaptor protein Toll/IL-1R domain-containing adaptor molecule-1 (TRIF) did not develop larger brain damage. The increased vulnerability was associated with a TRIF-dependent heightened inflammatory response, including proinflammatory cytokines, chemokines, and the apoptosis-associated mediator Fas, whereas there was a decrease in reparative M2-like CD11b⁺ microglia and phosphorylation of Akt. Because TLR-3 is activated via double-stranded RNA during most viral infections, the present study provides evidence that viral infections during pregnancy or in the neonate could have great impact on subsequent HI brain injury.

Kinetic mechanism and energetics of binding of phosphoryl group acceptors to Mycobacterium tuberculosis cytidine monophosphate kinase

Cytidine monophosphate kinase from Mycobacterium tuberculosis (MtCMK) likely plays a role in supplying precursors for nucleic acid synthesis. MtCMK catalyzes the ATP-dependent phosphoryl group transfer preferentially to CMP and dCMP. Initial velocity studies and Isothermal titration calorimetry (ITC) measurements showed that MtCMK follows a random-order mechanism of substrate (CMP and ATP) binding, and an ordered mechanism for product release, in which ADP is released first followed by CDP. The thermodynamic signatures of CMP and CDP binding to MtCMK showed favorable enthalpy and unfavorable entropy, and ATP binding was characterized by favorable changes in enthalpy and entropy. The contribution of linked protonation events to the energetics of MtCMK:phosphoryl group acceptor binary complex formation suggested a net gain of protons. Values for the pKa of a likely chemical group involved in proton exchange and for the intrinsic binding enthalpy were calculated. The Asp187 side chain of MtCMK is suggested as the likely candidate for the protonation event. Data on thermodynamics of binary complex formation were collected to evaluate the contribution of 2′-OH group to intermolecular interactions. The data are discussed in light of functional and structural comparisons between CMP/dCMP kinases and UMP/CMP ones.

OP0083 RO52/TRIM21 Expression in Salivary Gland Epithelial Cells (SGECS) is Upregulated by TLR3-Signaling

BackgroundRo52/TRIM21 is a major target of autoantigenic responses in Sjögren’s syndrome (SS). Up-regulated epithelial Ro52 expression has been reported at the salivary gland inflammatory lesions of SS patients; however, the...

Surfactant Protein (SP)-A Suppresses Preterm Delivery and Inflammation via TLR2

Toll like receptors (TLRs) are pattern-recognition molecules that initiate the innate immune response to pathogens. Pulmonary surfactant protein (SP)-A is an endogenously produced ligand for TLR2 and TLR4. SP-A has been proposed as a fetally produced signal for the onset of parturition in the mouse. We examined the effect of interactions between SP-A and the pathogenic TLR agonists lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic:cytidylic acid (poly(I:C)) (ligands for TLR4, TLR2 and TLR3, respectively) on the expression of inflammatory mediators and preterm delivery. Three types of mouse macrophages (the cell line RAW 264.7, and fresh amniotic fluid and peritoneal macrophages, including macrophages from TLR4 and TLR2 knockout mice) were treated for up to 7 hours with pathogenic TLR agonists with or without SP-A. SP-A alone had no effect upon inflammatory mediators in mouse macrophages and did not independently induce preterm labor. SP-A significantly suppressed TLR ligand-induced expression of inflammatory mediators (interleukin (IL)-1β, tumor necrosis factor (TNF)-α and the chemokine CCL5) via a TLR2 dependent mechanism. In a mouse inflammation-induced preterm delivery model, intrauterine administration of SP-A significantly inhibited preterm delivery, suppressed the expression of proinflammatory mediators and enhanced the expression of the CXCL1 and anti-inflammatory mediator IL-10. We conclude that SP-A acts via TLR2 to suppress TLR ligand-induced preterm delivery and inflammatory responses.

Study on Electron Transfer Across Surface of Nucleobases Terminated Self-assembled Film

Abstract 3-Mercaptopropionic acid, Mg 2+ and nucleotide monophosphate (NTMP) including guanosine monophosphate (GMP), adenosine monophosphate (AMP), cytidine monophosphate (CMP) and uridine-5′-monophosphate (UMP) were adsorbed onto Au substrates stepwise to form nucleobases terminated surface. Electron transfer (ET) process across the NTMP/Mg 2+ /MPA/Au multilayer self-assembled film was studied and the ET kinetics were determined using electrochemical methods including cyclic voltammetry (CV), chronoamperometry (CA) and electrochemical impedance spectroscopy (EIS). The experimental results showed that the rate of ET kinetics decreased in the following order: GMP/Mg 2+ /MPA/Au > AMP/Mg 2+ /MPA/Au > UMP/Mg 2+ /MPA/Au > CMP/Mg 2+ /MPA/Au. The calculated energy gaps (Δ E gap = E LUMO - E HOMO ) of their corresponding Mg-NTMP complexes motif increased in the order: Δ E Mg-GMP (0.89 eV) E Mg-AMP (1.40 eV) E Mg-UMP (1.57 eV) E Mg-CMP (1.64 eV). The results suggest that the complexation of Mg with the NTMP, and their corresponding Δ E gap may play important role in their ET process across the nucleobases terminated multilayer self-assembled film.

Equilibrium studies of thallium(I) complexes with cytidine 5′-monophosphate in different aqueous solutions of methanol

The protonation equilibria of cytidine 5′-monophosphate (CMP) and its complex formation with Tl(I) ion were studied over a wide pH range (1.5 to 10.5), using a combination of spectrophotometric and potentiometric methods at constant temperature (25°C), different methanol–water mixtures (0 to 50% v/v), and constant ionic strength (0.1moldm−3 sodium perchlorate). Least squares regression calculations are consistent with the formation of TlHL and TlL− species, where L2− represents the fully dissociated ligand. The protonation of CMP and the formation constants of the formed complexes in different media were analyzed in terms of Kamlet, Abboud, and Taft (KAT) parameters. A single-parameter correlation of the formation constants versus α (hydrogen-bond donor acidity), β (hydrogen-bond acceptor basicity) and π* (dipolarity/polarizability) are relatively poor in all solutions, but dual and multi-parameter correlations represent significant improvements with regard to the single-parameter model. Linear correlation is observed when the experimental formation constant values (in logarithm scale) are plotted versus the calculated ones, while the KAT parameters are considered. Finally, the results are discussed in terms of the effect of solvent on protonation and complexation.

Mucosal Expression of the Cytochromes P450 (CYP) and Nucleotide Kinases Involved in the Biotransformation of Drugs Used in Human Immunodeficiency Virus (HIV) Pre‐exposure Prophylaxis (PrEP)

Attempts to prevent HIV infection through PrEP have included topical application of anti‐HIV drugs to the mucosal sites of infection. However, clinical studies have revealed marked anatomic variability in drug exposure in vaginal tissue versus colorectal tissue. Thus, studies were performed to determine whether the CYPs and nucleotide kinases responsible for the metabolism and activation of drugs used in HIV PrEP were differentially expressed in human vaginal versus colonic tissue. Quantitative reverse transcriptase‐PCR revealed that colonic mRNA expression of CYPs 1A1, 2B6, and 3A4/5 were 14‐, 37‐ and 4‐fold greater, respectively, than vaginal mRNA. Immunoblotting confirmed the presence of CYPs 3A4 and 3A5 protein in vagina. The vaginal mRNA abundance of adenylate kinase 2, the primary enzyme responsible for the activation of the HIV PrEP drug tenofovir, was 1.5‐fold greater than in colonic tissue. Cytidine monophosphate kinase mRNAs were 8‐fold greater in colonic tissue and phosphoglycerate kinase mRNAs were 4‐fold greater in vaginal tissue. These kinases have been proposed to activate emtricitabine, which is also under development as a topical microbicide for HIV PrEP. These data indicate that enzymes that modulate exposure to anti‐HIV drugs may be differentially expressed in mucosal tissues that are sites of topical drug application for PrEP. Funding: NIH U01 AI068613 (HIV Prevention Trials Network).