pyrophosphate and triphosphoric-acid

In this review, our recent advances in the development of nucleoside di- and nucleoside triphosphate prodrugs is summarized. Previously, we had developed a successful membrane-permeable pronucleotide system for the intracellular delivery of nucleoside monophosphates as well, the so-called cycloSal-approach. In contrast to that work in which the delivery is initiated by a chemically driven hydrolysis reaction, for the di- and triphosphate delivery, an enzymatic trigger mechanism involving (carboxy)esterases had to be used. The other features of the new pronucleotide approaches are: (i) lipophilic modification was restricted to the terminal phosphate group leaving charges at the internal phosphate moieties and (ii) appropriate lipophilicity is introduced by long aliphatic residues within the bipartite prodrug moiety. The conceptional design of the di- and triphosphate prodrug systems will be described and the chemical synthesis, the hydrolysis properties, a structure-activity relationship and antiviral activity data will be discussed as well. The advantage of these new approaches is that all phosphorylation steps from the nucleoside analogue into the bioactive nucleoside triphosphate form can be bypassed in the case of the triphosphate prodrugs. Moreover, enzymatic processes like the deamination of nucleosides or nucleoside monophosphates which lead to catabolic clearance of the potential antivirally active compound can be avoided by the delivery of the higher phosphorylated nucleotides.

Topics: Antiviral Agents; Diphosphates; Humans; Microbial Sensitivity Tests; Nucleosides; Polyphosphates; Prodrugs; Viruses

To evaluate the erosive potential of orange juice modified by food additives in enamel and dentine.. Calcium lactate pentahydrate (CLP), xanthan gum (XG), sodium linear polyphosphate (LPP), sodium pyrophosphate tetrabasic (PP), sodium tripolyphosphate (STP) and some of their combinations were added to an orange juice. Pure orange juice and a calcium-modified juice were used as negative (C-) and positive (C+) controls, respectively. In phase 1, 15 modified orange juices were tested for erosive potential using pH-stat analysis. In phase 2, the additives alone and the combination with good results in phase 1 and in previous studies (CLP+LPP) were tested in an erosion-remineralization cycling model. In phase 3, the erosion and remineralization episodes were studied independently. Enamel was analysed by surface microhardness (SMH) and profilometry, whilst dentine by profilometry.. In phase 1, reduction of the erosive potential was observed for all additives and their combinations, except XG alone. In phase 2, no detectable enamel loss was observed when CLP, LPP and CLP+LPP were added to the juice. XG, STP and PP had enamel loss similar to C- (p>0.05). Amongst additives, the combination CLP+LPP showed the highest SMH values followed by CLP (p<0.05). All the other groups presented SMH values similar to C- (p>0.05). For dentine, only CLP+LPP lead to surface loss values lower than C- (p<0.05). In phase 3, CLP, LPP and CLP+LPP seemed to protect against erosion; whilst none of the tested compounds seemed to interfere with the remineralization process.. CLP and LPP reduced erosion on enamel and this effect was enhanced by their combination. For dentine, only the combination CLP+LPP reduced erosion.

Topics: Animals; Beverages; Calcium; Calcium Compounds; Cattle; Citrus sinensis; Dental Enamel; Dentin; Diphosphates; Food Additives; Fruit; Hardness; Humans; Hydrogen-Ion Concentration; Lactates; Polyphosphates; Polysaccharides, Bacterial; Protective Agents; Saliva, Artificial; Temperature; Time Factors; Tooth Erosion; Tooth Remineralization

A six-week, double-blind, cross-over clinical study was conducted to compare the effect of a new chewing gum containing pyrophosphate (1%) and tripolyphosphate (1%), versus a control chewing gum on supragingival calculus deposits.. Twenty-eight adult subjects (mean age 34 +/- 8 years) who entered the study were given a full oral prophylaxis and were assigned to chew two pieces of gum, four times a day for five minutes. The gum, either a test chewing gum or placebo chewing gum, was randomly assigned and the subjects were directed to chew their assigned gum for six weeks. All participants also received a 12-week supply of a sodium fluoride (0.32%) dentifrice (Colgate). They were then scored for calculus deposits using the modified Volpe-Manhold Calculus Index (VMI) by the same two examiners. At the end of the first six weeks, the subjects received a second oral prophylaxis and used the alternate chewing gum for a second six-week period of time. The subjects were again scored for calculus deposits and the study was completed.. The results demonstrated a mean VMI of 3.65 +/- 2.82 for the test group, and a mean of 4.24 +/- 3.25 for the placebo group. This difference was significant with paired sample t-test (p < 0.001).. These results indicated that chewing gum containing pyrophosphate and tripolyphosphate reduced supragingival calculus formation by 13.9%, compared to the placebo chewing gum.

Topics: Adult; Chewing Gum; Cross-Over Studies; Dental Calculus; Diphosphates; Double-Blind Method; Female; Humans; Male; Matched-Pair Analysis; Middle Aged; Placebos; Polyphosphates; Time Factors

Tenofovir (TFV; prescribed as TFV disoproxil fumarate and TFV alafenamide prodrugs) is currently used for HIV prevention and treatment. TFV must be phosphorylated twice into TFV-diphosphate (TFV-DP) to become pharmacologically active. Previously, we reported heterogeneity in TFV-DP distribution in colorectal tissue (a putative site of HIV infection) sections collected from research participants receiving a TFV-containing enema. This observed heterogeneity is likely multifactorial. Of note, TFV-DP is structurally similar to ATP. It is known that nucleotidases such as nucleoside triphosphate diphosphohydrolases (NTPDases) dephosphorylate ATP. Thus, it was hypothesized that NTPDase-mediated dephosphorylation plays a role in regulating TFV-DP levels in colorectal tissue. To test this hypothesis, recombinant NTPDase proteins (NTPDase 1, 3, 4, 5, 6, and 8) were incubated, individually, with TFV-DP to determine their abilities to dephosphorylate TFV-DP in vitro. Following incubations, TFV-DP dephosphorylation was determined using both malachite green phosphate assays and ultrahigh-performance liquid chromatography tandem mass spectrometry. From these, NTPDase 1 exhibited the highest activity toward TFV-DP. Further, enzyme kinetic analysis revealed Michaelis-Menten kinetics for NTPDase 1-mediated TFV-DP dephosphorylation. Next, immunoblot analyses were conducted to confirm the expression of NTPDase 1 protein in human colorectal tissue. Liquid chromatography coupled to mass spectrometry proteomics analysis was used to measure the relative abundance of NTPDases in human colorectal tissue among healthy adult individuals (

Topics: Adenosine Triphosphate; Adult; Anti-HIV Agents; Colorectal Neoplasms; Diphosphates; HIV Infections; Humans; Kinetics; Nucleosides; Nucleotides; Tenofovir

Templated assembly of small molecules into nano-structural architectures has been used extensively by nature throughout its evolution. These systems have also been studied in artificial systems to design a phosphate templated assembly. However, it is yet to be investigated how the molecules interact among themselves at the molecular level and whether the phosphate templated assembly has any role in the formation of prebiotic protocellular membranes. Here, we report the prebiotic synthesis of choline-based cationic amphiphiles (-N

Topics: Artificial Cells; Diphosphates; Polyphosphates

Pulmonary vascular tone is modulated by nucleotides, but which P2 receptors mediate these actions is largely unclear. The aim of this study, therefore, was to use subtype-selective antagonists to determine the roles of individual P2Y receptor subtypes in nucleotide-evoked pulmonary vasodilation and vasoconstriction. Isometric tension was recorded from rat intrapulmonary artery rings (i.d. 200-500 µm) mounted on a wire myograph. Nucleotides evoked concentration- and endothelium-dependent vasodilation of precontracted tissues, but the concentration-response curves were shallow and did not reach a plateau. The selective P2Y

Topics: Adenosine Triphosphate; Animals; Diphosphates; Pulmonary Artery; Rats; Receptors, Purinergic P2Y1; Receptors, Purinergic P2Y2; Uridine; Uridine Diphosphate; Uridine Triphosphate; Vasodilation

Myofibrillar protein isolated from beef muscles were treated with 3 phosphates (Sodium Hexametaphosphate, sodium tripolyphosphate, sodium pyrophosphate) with different concentrations of 0.3%, 0.6%, 0.9%, 1.2% respectively. Protein solubility, surface hydrophobicity and reactive sulfhydryl group was determined. Atomic force microscopy was used to observe the microscopic protein surface. SDS-PAGE was carried out to determine the proteolysis of myofibrillar protein. The solubility and surface hydrophobic bond of myofibrillar protein was highly increased and the diameter decreased by SHMP, TSPP, STPP. Reactive sulfhydryl groups increased after SHMP addition, but slightly decreased in STPP and TSPP treated MP. TSPP and STPP had the same effect on myofibrillar microstructure and was different from SHMP. Three phosphates all caused MP unfolding. The MP gel complexity was increased, and roughness was decreased after phosphates addition, indicating phosphates helped to construct a more ordered and smoother gel microcosmic surface.

Topics: Animals; Cattle; Diphosphates; Hydrophobic and Hydrophilic Interactions; Microscopy, Atomic Force; Muscle Proteins; Phosphates; Polyphosphates; Red Meat; Solubility

Topics: Adenosine Triphosphate; Binding Sites; Diphosphates; Enzyme Inhibitors; Humans; Hydrolysis; Kinetics; Methionine Adenosyltransferase; Polyphosphates; Protein Conformation; S-Adenosylmethionine

Staphylococcus aureus is a well-known clinical pathogenic bacterium. In recent years, due to the emergence of multiple drug-resistant strains of S. aureus in clinical practice, S. aureus infections have become an increasingly severe clinical problem. Ntdp (nucleoside tri- and diphosphatase, also known as Sa1684) is a nucleotide phosphatase that has a significant effect on the proliferation of S. aureus colonies and the killing ability of the host. Here, we identified the nucleoside tri- and diphosphate hydrolysis activity of Ntdp and obtained the three-dimensional structures of apo-Ntdp and three substrate analog (ATP

Topics: Bacterial Proteins; Diphosphates; Gene Expression Regulation, Bacterial; Hydrolysis; Nucleosides; Polyphosphates; Staphylococcus aureus; Trans-Activators; Virulence Factors

The objective of this research was to investigate the influence of various levels (0.0, 0.1, 0.2, 0.3, 0.4, 0.5%) of added encapsulated polyphosphates (sodium tripolyphosphate; sodium pyrophosphate) combined with unencapsulated polyphosphate to total 0.5% on the inhibition of lipid oxidation in cooked ground meat (beef, chicken) during refrigerated storage (0, 1, 7 d). The use of sodium tripolyphosphate (encapsulated sodium tripolyphosphate, unencapsulated sodium tripolyphosphate) led to lower cooking loss compared to sodium pyrophosphate in both meat species (p < 0.05). Increasing encapsulated sodium tripolyphosphate up to 0.3% decreased cooking loss in ground beef (p < 0.05). Added encapsulated polyphosphate at 0.5% had the same effect on pH as 0.5% unencapsulated polyphosphate in the cooked ground beef and chicken. A higher accumulation of orthophosphate was determined in the samples with sodium tripolyphosphate compared to those with sodium pyrophosphate (p < 0.05). Inclusion of a minimum of 0.1% encapsulated polyphosphate decreased thiobarbituric acid reactive substances and lipid hydroperoxides on 7 d. Increasing encapsulated sodium tripolyphosphate and encapsulated sodium pyrophosphate up to 0.2% in beef decreased thiobarbituric acid reactive substances at 7 d. Addition of 0.4% encapsulated sodium tripolyphosphate and 0.3% encapsulated sodium pyrophosphate in chicken prevented any increase in TBARS during storage. Incorporating encapsulated sodium pyrophosphate at 0.3% inhibited lipid hydroperoxide formation in beef and chicken. The meat industry could achieve enhanced lipid oxidation inhibition by replacing some of the unencapsulated polyphosphate with encapsulated polyphosphate in their product formulations.

Topics: Animals; Antioxidants; Capsules; Cattle; Chickens; Cooking; Diphosphates; Food Handling; Food Preservation; Food Preservatives; Food Storage; Humans; Hydrogen-Ion Concentration; Lipid Peroxidation; Meat; Phosphates; Polyphosphates; Red Meat; Refrigeration; Thiobarbituric Acid Reactive Substances

The ability of polyvalent anions to influence protein-protein interactions and protein net charge was investigated through solubility and turbidity experiments, determination of osmotic second virial coefficients ( B

Topics: Chemical Precipitation; Chlorides; Citric Acid; Diphosphates; Muramidase; Polyphosphates; Protein Binding; Protein Multimerization; Scattering, Radiation; Sulfates

The effect of levels (0.1%, 0.2%, 0.3%, 0.4%, 0.5%) of added encapsulated (e) phosphate (sodium tripolyphosphate, STP; sodium hexametaphosphate, HMP; sodium pyrophosphate, SPP) on lipid oxidation inhibition during storage (0, 1, and 7 d) of ground meat (chicken, beef) was evaluated. The use of eSTP and eSPP resulted in lower and higher cooking loss (CL) compared to eHMP, respectively (P < 0.05). Increasing encapsulated phosphate level (PL) enhanced the impact of phosphates on CL in both chicken and beef samples (P < 0.05). Encapsulated STP increased pH, whereas eSPP decreased pH (P < 0.05). pH was not affected by PL. The highest orthophosphate (OP) was obtained with eSTP, followed by eSPP and eHMP (P < 0.05). The level of OP determined in both chicken and beef samples increased (P < 0.05) during storage. Increasing PL caused an increase in OP (P < 0.05). The highest reduction rate in the formation of thiobarbituric acid reactive substances (TBARS) and LPO for both meat species were obtained with eSPP, followed by eSTP and eHMP (P < 0.05). Increasing PL resulted in lower TBARS and LPO (P < 0.05). Findings suggest that encapsulated phosphates can be a strategy to inhibit lipid oxidation for the meat industry and the efficiency of encapsulated phosphates on lipid oxidation inhibition can be enhanced by increasing PL.

Topics: Animals; Capsules; Cattle; Chickens; Cooking; Diphosphates; Food Handling; Food Preservation; Food Storage; Humans; Lipid Metabolism; Lipid Peroxidation; Lipids; Meat; Oxidation-Reduction; Phosphates; Polyphosphates; Thiobarbituric Acid Reactive Substances

Topics: Chitosan; Circular Dichroism; Cross-Linking Reagents; Diffusion; Diphosphates; Gels; Hydrogels; Polyphosphates; Scattering, Radiation; Solutions

The coformulation of the nucleos(t)ide analogs (NA) tenofovir (TFV) disoproxil fumarate (TDF) and emtricitabine (FTC) is approved for HIV-infection treatment and prevention. Plasma TFV and FTC undergo complicated hybrid processes to form, accumulate, and retain as their active intracellular anabolites: TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP). Such complexities manifest in nonlinear intracellular pharmacokinetics (PK). In target cells, TFV-DP/FTC-TP compete with endogenous deoxynucleoside triphosphates (dNTP) at the active site of HIV reverse transcriptase, underscoring the importance of analog:dNTP ratios for antiviral efficacy. However, NA such as TFV and FTC have the potential to disturb the dNTP pool, which could augment or reduce their efficacies. We conducted a pharmacokinetics-pharmacodynamics (PKPD) study among forty subjects receiving daily TDF/FTC (300 mg/200 mg) from the first-dose to pharmacological intracellular steady-state (30 days). TFV/FTC in plasma, TFV-DP/FTC-TP and dNTPs in peripheral blood mononuclear cells (PBMC) were quantified using validated LC/MS/MS methodologies. Concentration-time data were analyzed using nonlinear mixed effects modeling (NONMEM). Formations and the accumulation of intracellular TFV-DP/FTC-TP was driven by plasma TFV/FTC, which was described by a hybrid of first-order formation and saturation. An indirect response link model described the interplay between TFV-DP/FTC-TP and the dNTP pool change. The EC50 (interindividual variability, (%CV)) of TFV-DP and FTC-TP on the inhibition of deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate (dCTP) production were 1020 fmol/106 cells (130%) and 44.4 pmol/106 cells (82.5%), resulting in (90% prediction interval) 11% (0.45%, 53%) and 14% (2.6%, 35%) reductions. Model simulations of analog:dNTP molar ratios using IPERGAY dosing suggested that FTC significantly contributes to the protective effect of preexposure prophylaxis (PrEP). Simulation-based intracellular operational multiple dosing half-lives of TFV-DP and FTC-TP were 6.7 days and 33 hours. This model described the formation of intracellular TFV-DP/FTC-TP and the interaction with dNTPs, and can be used to simulate analog:dNTP time course for various dosing strategies.

Topics: Adult; Anti-HIV Agents; Computer Simulation; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Diphosphates; Emtricitabine; Female; HIV; HIV Infections; Humans; Male; Middle Aged; Models, Biological; Polyphosphates; Prospective Studies; Tenofovir; Young Adult

Effects of 0.5% encapsulated (e) phosphates (sodium tripolyphosphate, STP; sodium hexametaphosphate, HMP; sodium pyrophosphate, SPP) on lipid oxidation during storage (0, 1, and 7 d) of ground meat (chicken, beef) after being cooked to 3 end-point cooking temperatures (EPCT; 71, 74, and 77 °C) were evaluated. The use of STP or eSTP resulted in lower (P < 0.05) cooking loss (CL) compared to encapsulated or unencapsulated forms of HMP and SPP. Increasing EPCT led to a significant increase in CL (P < 0.05). Both STP and eSTP increased pH, whereas SPP and eSPP decreased pH (P < 0.05). The higher orthophosphate (OP) was obtained with STP or SPP compared to their encapsulated counterparts (P < 0.05). The lowest OP was determined in samples with HMP or eHMP (P < 0.05). A 77 °C EPCT resulted in lower OP in chicken compared to 74 and 71 °C (P < 0.05), dissimilar to beef, where EPCT did not affect OP. In encapsulated or unencapsulated form, using STP and SPP enhanced reduction in TBARS and lipid hydroperoxides (LPO) compared with HMP (P < 0.05). Regardless of the phosphate type, more effective lipid oxidation inhibition was achieved by the use of encapsulated forms (P < 0.05). Increasing EPCT resulted in lower TBARS in beef and higher LPO values in both beef and chicken samples (P < 0.05). Findings suggest that encapsulated phosphates can be a strategy to inhibit lipid oxidation for meat industry and the efficiency of encapsulated phosphates on lipid oxidation inhibition can be enhanced by lowering EPCT.

Topics: Animals; Capsules; Cattle; Chickens; Cooking; Diphosphates; Food Handling; Food Preservation; Food Preservatives; Hot Temperature; Lipid Metabolism; Lipid Peroxidation; Lipid Peroxides; Meat; Oxidation-Reduction; Phosphates; Polyphosphates; Temperature; Thiobarbituric Acid Reactive Substances

The dynamic hydrolysis of tetrasodium pyrophosphate (TSPP), sodium tripolyphosphate (STPP) and polyphosphate compound, which was catalyzed by purified pyrophosphatase (PPase) and myosin- tripolyphosphatase (TPPase) from the silver carp dorsal muscle, was studied using (31) P NMR spectroscopy. In the PPase + TSPP system, the pyrophosphate (PP) was hydrolyzed quickly and completely within 8 h and the hydrolysis rate of PP was 12.51%/h. In the TPPase + STPP system, the first-order hydrolysis of tripolyphosphate (TPP) was not yet complete after 48 h, and the derived PP accumulated progressively. Given the coexistence of PPase and TPPase, only 1.20% of TPP in STPP alone remained after 48 h. However, the generation rate of Pi in the polyphosphate compound (TSPP: STPP: sodium hexametaphosphate = 1: 8: 1) was 0.76%/h, which was less than 0.88%/h in STPP alone. In the presence of polyphosphatases, the decrease of PP or TPP content in the polyphosphate compound was not as rapid as that in TSPP or STPP alone due to the inhibitory effect of PP on TPPase and the effect of low system pH on PPase. The understanding of polyphosphates hydrolysis mechanism was capable of developing the advanced polyphosphate mixture in order to reduce the phosphate residue in fish products.. Processors appreciate the proven value of phosphates to increase the yield and functionality of the fish meat products. Our studies showed that the hydrolysis rate of PP or TPP in the blend was slower than that of polyphosphate alone. Thus, it is likely that the addition of PP and TPP in a polyphosphate blend had a prolonged interaction with proteins in fish meat processing and the effectiveness of polyphosphates was enhanced.

Topics: Acid Anhydride Hydrolases; Animals; Carps; Diphosphates; Fish Products; Food Additives; Hydrogen-Ion Concentration; Hydrolysis; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Meat; Muscle, Skeletal; Myosins; Phosphates; Polyphosphates

The objective of this study was to describe the dependence of textural properties (hardness, cohesiveness, and relative adhesiveness) of processed cheese spreads on the proportion of disodium phosphate (DSP), tetrasodium diphosphate (TSPP), and sodium salts of polyphosphate in ternary mixtures of emulsifying salts. Sodium salts of polyphosphate with different mean lengths (n ≈ 5, 9, 13, 20, and 28) were used. Pentasodium triphosphate (PSTP) was used instead of TSPP in the second part of the study. Products with and without pH adjustment were tested (the target pH value was 5.60-5.80). Textural properties of the processed cheese were observed after 2, 9, and 30 d of storage at 6°C. Hardness of the processed cheese with a low content of polyphosphate increased at a specific DSP:TSPP ratio (~1:1 to 3:4). This trend was the same for all the polyphosphates used; only the absolute values of texture parameters were different. The same trends were observed in the ternary mixtures with PSTP, showing lower final values of hardness compared with samples containing TSPP. Hardness and cohesiveness decreased and relative adhesiveness increased in the samples with increased pH values and vice versa; the main trend remained unchanged.

Topics: Animals; Cheese; Diphosphates; Emulsions; Food Handling; Food Storage; Hardness; Hydrogen-Ion Concentration; Phosphates; Polyphosphates; Salts

Ionically crosslinked chitosan particles with submicron dimensions attract widespread interest as materials for controlled release. To this end, we have examined the formation and dissolution of nanoparticles prepared by crosslinking chitosan with pyrophosphate (PPi). The formation of these particles required a critical PPi concentration (which increased with the chitosan concentration), and their z-average hydrodynamic diameters could be predictably tuned from roughly 60 to 220 nm by varying the concentration of the parent chitosan solutions. Unlike the nanoparticles crosslinked with the commonly used tripolyphosphate (TPP), which coagulated and precipitated when TPP was in excess, the chitosan/PPi nanoparticles remained colloidally stable even at high PPi concentrations. Moreover, the analysis of their dissolution revealed hysteresis in the particle formation/dissolution cycle, where portions of the crosslinked chitosan/PPi complexes remained stably intact at PPi concentrations below those required for their formation. This irreversible behavior was surmised to reflect the cooperativity of chitosan/PPi binding and was qualitatively modeled using the Bragg-Williams theory.

Topics: Chitosan; Colloids; Cross-Linking Reagents; Diphosphates; Hydrodynamics; Ions; Kinetics; Light; Models, Theoretical; Nanoparticles; Particle Size; Polyphosphates; Scattering, Radiation; Solubility; Solutions

Effects of encapsulated sodium tripolyphosphate (STP), sodium hexametaphosphate (HMP) and sodium pyrophosphate (SPP) on lipid oxidation in uncooked (0, 2, 24h) and cooked (0, 1, 7 d) ground chicken and beef during storage were determined. Ten phosphate treatments included a control (no phosphate), three unencapsulated (u) at 0.5% and three encapsulated (e) phosphates (0.5%) each at a low (e-low) and high (e-high) coating level. Two heating rates (slow, fast) were investigated. Cooking loss (CL), pH, color, orthophosphate (OP), TBARS and lipid hydroperoxides (LPO) were determined. A fast heating and uSTP resulted in lower CL (p<0.05). Orthophosphate increased with phosphate incorporation, slow heating and storage (p<0.05). Encapsulated phosphates and increased coating level reduced OP (p<0.05). Unencapsulated STP increased CIE a* and pH, whereas uSPP decreased CIE a* and pH (p<0.05). Encapsulated phosphates and the greater coating level had no effect on the pH in cooked samples. Not increased coating level but encapsulated phosphates decreased lipid oxidation in cooked samples (p<0.05).

Topics: Animals; Cattle; Chickens; Color; Cooking; Diphosphates; Hydrogen-Ion Concentration; Lipid Metabolism; Meat; Oxidation-Reduction; Phosphates; Polyphosphates; Thiobarbituric Acid Reactive Substances

Ground beef with 1% NaCl was incorporated with 0.5% unencapsulated sodium tripolyphosphate (uSTP), 0.5% encapsulated sodium tripolyphosphate (eSTP), 0.5% unencapsulated sodium acid pyrophosphate (uSAPP), or 0.5% encapsulated sodium acid pyrophosphate (eSAPP) prior to being cooked and stored (0 or 6 d, 3 °C). The pH was higher (P<0.05) for sodium tripolyphosphate samples (6 d: uSTP 5.98; eSTP 5.89) and lower (P<0.05) for sodium acid pyrophosphate (6 d: uSAPP 5.31, eSAPP 5.33) samples than control sample (6 d, 5.50). Overall, samples with uSTP had the least cooking loss and lowest TBARS values. TBARS (mg/kg) for the phosphate treatments were lower (P<0.05; ave. 1.78, 0 d; 3.49, 6 d) than for the control samples (3.07, 0 d; 22.85, 6 d). Therefore, phosphate incorporation into ground beef prior to cooking aids in the reduction of oxidation in the cooked, stored product, although a longer period of time before thermal processing may be necessary for the encapsulated phosphate to have significant benefits.

Topics: Animals; Antioxidants; Cattle; Cooking; Diphosphates; Hot Temperature; Hydrogen-Ion Concentration; Lipid Metabolism; Meat; Oxidation-Reduction; Phosphoric Monoester Hydrolases; Polyphosphates; Thiobarbituric Acid Reactive Substances

The active metabolites of tenofovir (TFV) and emtricitabine (FTC) in peripheral blood mononuclear cells (PBMCs) have been used as markers of long-term antiretroviral (ARV) adherence. However, the process of isolating PBMCs is expensive, complex, and not feasible in many settings. We compared concentrations of TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP) in the upper layer packed cells (ULPCs) obtained after whole blood centrifugation to isolated PBMCs as a possible alternative marker of adherence.. Ten HIV+ adults with HIV RNA <50 copies/mL on a TDF/FTC-containing regimen provided 5 paired PBMC and ULPC samples over 6 hours. TFV-DP and FTC-TP concentrations were analyzed by liquid chromatography/mass spectrometry. Partial areas under the curve were calculated using noncompartmental methods and Spearman Rank Correlations (rho) between PBMC and ULPC were determined.. The median (25th-75th percentile) concentration of TFV-DP in PBMCs was 143 (103-248) fmol/10(6) cells and in ULPC was 227 (160-394) fmol/10(6) cells (rho = 0.65; P < 0.0001). The concentration of FTC-TP in PBMCs was 6660 (5650-10,000) fmol/10(6) cells and in ULPC was 19.0 (12.0-27.8) fmol/10(6) cells (rho = 0.55; P < 0.0001). Compared to PBMCs, ULPC TFV-DP was 64% higher and FTC-TP was 99.7% lower. ULPC concentrations of TFV-DP and FTC-TP in one additional subject receiving a single dose of TDF/FTC were only 0.05% and 25%, of the other 10 subjects, respectively.. ULPC concentrations significantly correlated with PBMC concentrations. Preliminary single-dose data suggest some discrimination between intermittent versus consistent dosing. ULPC concentrations of TFV-DP and FTC-TP should be further investigated as a simply collected surrogate measure of ARV adherence.

Topics: Adenine; Adult; Anti-HIV Agents; Blood Cells; Deoxycytidine; Diphosphates; Emtricitabine; Female; HIV Infections; Humans; Leukocytes, Mononuclear; Male; Medication Adherence; Middle Aged; Organophosphonates; Polyphosphates; Tenofovir

Calcium is a mineral naturally present in water and may be included into meat products during processing thereby influencing meat quality. Phosphates improve myofibril swelling and meat water-holding capacity (WHC) but can be sensitive to calcium precipitation. In this study, pork shoulder meat was used to investigate the impact of calcium at 0, 250, and 500 ppm and phosphate type [sodium pyrophosphate (PP), tripolyphosphate (TPP), and hexametaphopshate (HMP)] at 10 mM on nitrite-cured protein extract color at various pH levels (5.5, 6.0, and 6.5) and crude myofibril WHC at pH 6.0. Neither calcium nor phosphates present in the curing brines significantly affected the cured color. Increasing the pH tended to promote the formation of metmyoglobin instead of nitrosylmyoglobin. The ability of PP to enhance myofibril WHC was hampered (P < 0.05) by increasing the calcium concentration due to PP precipitation. Calcium also decreased the solubility of TPP but did not influence its enhancement of WHC. On the other hand, HMP was more tolerant of calcium but the soluble Ca-HMP complex was less effective than free HMP to promote water binding by myofibrils. The depressed muscle fiber swelling responding to added calcium as evidenced by phase contrast microscopy substantiated, to a certain extent, the deleterious effect of calcium, suggesting that hardness of curing water can significantly affect the quality of cured meat products.. Although not affecting nitrite-cured color, calcium hampers the efficacy of phosphates to promote water binding by muscle proteins, underscoring the importance of water quality for brine-enhanced meat products.

Topics: Animals; Calcium; Color; Diphosphates; Food Handling; Hydrogen-Ion Concentration; Meat Products; Muscle Proteins; Myofibrils; Myoglobin; Nitrites; Phosphates; Polyphosphates; Salts; Solubility; Solutions; Swine; Water; Water Quality

The role of different types of emulsifying salts-sodium citrate (TSC), sodium hexametaphosphate (SHMP), sodium tripolyphosphate (STPP) and tetrasodium pyrophosphate (TSPP)-on microstructure and rheology of "requeijão cremoso" processed cheese was determined. The cheeses manufactured with TSC, TSPP, and STPP behaved like concentrated solutions, while the cheese manufactured with SHMP exhibited weak gel behavior and the lowest values for the phase angle (G"/G'). This means that SHMP cheese had the protein network with the largest amount of molecular interactions, which can be explained by its highest degree of fat emulsification. Rotational viscometry indicated that all the spreadable cheeses behaved like pseudoplastic fluids. The cheeses made with SHMP and TSPP presented low values for the flow behavior index, meaning that viscosity was more dependent on shear rate. Regarding the consistency index, TSPP cheese showed the highest value, which could be attributed to the combined effect of its high pH and homogeneous fat particle size distribution.

Topics: Cheese; Citrates; Diphosphates; Emulsions; Food Handling; Hydrogen-Ion Concentration; Phosphates; Polyphosphates; Rheology; Salts; Viscosity

The aim of this study was to investigate the effect of different types and concentrations of emulsifying salts (trisodium citrate, tetrasodium pyrophosphate, sodium tripolyphosphate, sodium hexametaphosphate, and disodium orthophosphate) on the physicochemical properties of processed cheese. The physicochemical composition, texture profile, degree of casein dissociation, fat particle size, color, and nuclear magnetic resonance profile (NMR) of processed cheese were determined. Hardness, degree of casein dissociation, and pH increased as the concentration of emulsifying salts increased. The fat particle size of processed cheese was significantly influenced by the type of emulsifying salts, with processed cheese made with sodium hexametaphosphate having larger particles (4.68 µm) than cheeses made with the other salts (from 2.71 to 3.30 µm). The processed cheese prepared with trisodium citrate was whiter than those prepared with the other emulsifying salts. The NMR analysis showed that the relaxation time of processed cheese of 10 to 100 ms accounted for a major proportion, indicating that the moisture in processed cheese was mainly bound water combined with the fat globule and hydrated casein.

Topics: Cheese; Citrates; Diphosphates; Emulsifying Agents; Food Handling; Phosphates; Polyphosphates

It has been shown previously that [M-H](-) anions of small peptides containing two phosphate residues undergo cyclisation of the phosphate groups, following collision-induced dissociation (CID), to form a characteristic singly charged anion A (H3P2O7(-), m/z 177). In the present study it is shown that the precursor anions derived from the diphosphopeptides of caerin 1.1 [GLLSVLGSVAKHVLPHVVPVIAEHL(NH2)] and frenatin 3 [GLMSVLGHAVGNVLGGLFKPKS(OH)] also form the characteristic product anion A (m/z 177). Both of the precursor peptides show random structures in water, but partial helices in membrane-mimicking solvents [e.g. in d3-trifluoroethanol/water (1:1)]. In both cases the diphosphopeptide precursor anions must have flexible conformations in order to allow approach of the phosphate groups with consequent formation of A: for example, the two pSer groups of 4,22-diphosphofrenatin 3 are seventeen residues apart. Finally, CID tandem mass spectrometric (MS/MS) data from the [M-H](-) anion of the model triphosphoSer-containing peptide GpSGLGpSGLGpSGL(OH) show the presence of both product anions A (m/z 177) and D (m/z 257, H4P3O10(-)). Ab initio calculations at the HF/6-31+G(d)//AM1 level of theory suggest that cyclisation of the three phosphate groups occurs by a stepwise cascade mechanism in an energetically favourable reaction (ΔG = -245 kJ mol(-1)) with a maximum barrier of +123 kJ mol(-1).

Topics: Diphosphates; Molecular Conformation; Phosphopeptides; Polyphosphates; Spectrometry, Mass, Electrospray Ionization

Among the numerous chemosensors available for diphosphate (P(2)O(7)(4-), PPi) and nucleoside triphosphates (NTPs), only a few can distinguish between PPi and NTPs. Hence, very few bioanalytical applications based on such selective chemosensors have been realized. We have developed a new fluorescence sensing system for distinction between PPi and NTPs based on the combination of two sensors, a binuclear Zn(II) complex (1·2Zn) and boronic acid (BA), in which one chemosensor (1·2Zn) shows signal changes depending on the PPi (or NTP) concentration, and the other (BA) blocks the signal change caused by NTPs; this system enables the distinction of PPi from NTPs and is sensitive to nanomolar concentrations of PPi. The new sensing system has been successfully used for the direct quantification of RNA polymerase activity.

Topics: Adenosine Triphosphate; Biosensing Techniques; Diphosphates; Fluorescence; Magnetic Resonance Spectroscopy; Molecular Structure; Nucleosides; Organometallic Compounds; Polyphosphates; Zinc

Human ribonucleotide reductases (hRNRs) catalyze the conversion of nucleotides to deoxynucleotides and are composed of α- and β-subunits that form active α(n)β(m) (n, m = 2 or 6) complexes. α binds NDP substrates (CDP, UDP, ADP, and GDP, C site) as well as ATP and dNTPs (dATP, dGTP, TTP) allosteric effectors that control enzyme activity (A site) and substrate specificity (S site). Clofarabine (ClF), an adenosine analog, is used in the treatment of refractory leukemias. Its mode of cytotoxicity is thought to be associated in part with the triphosphate functioning as an allosteric inhibitor of hRNR. Studies on the mechanism of inhibition of hRNR by ClF di- and triphosphates (ClFDP and ClFTP) are presented. ClFTP is a reversible inhibitor (K(i) = 40 nM) that rapidly inactivates hRNR. However, with time, 50% of the activity is recovered. D57N-α, a mutant with an altered A site, prevents inhibition by ClFTP, suggesting its A site binding. ClFDP is a slow-binding, reversible inhibitor ( K(i)*; t(1/2) = 23 min). CDP protects α from its inhibition. The altered off-rate of ClFDP from E•ClFDP* by ClFTP (A site) or dGTP (S site) and its inhibition of D57N-α together implicate its C site binding. Size exclusion chromatography of hRNR or α alone with ClFDP or ClFTP, ± ATP or dGTP, reveals in each case that α forms a kinetically stable hexameric state. This is the first example of hexamerization of α induced by an NDP analog that reversibly binds at the active site.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Algorithms; Allosteric Regulation; Antineoplastic Agents; Arabinonucleosides; Binding Sites; Biocatalysis; Catalytic Domain; Clofarabine; Diphosphates; Guanosine Triphosphate; Humans; Kinetics; Molecular Structure; Mutation; Polyphosphates; Protein Multimerization; Protein Structure, Quaternary; Protein Subunits; Ribonucleotide Reductases; Substrate Specificity; Time Factors

In this study, we investigated the tripolyphosphatase (TPPase) activity responsible for the hydrolysis of tripolyphosphates (TPP) in rabbit Psoas major muscle tissue. After a series of extraction and purification steps, myosin was identified to be a TPPase. Optimum pH and temperature for myosin-TPPase activity were 6.0 and 35°C, respectively. We also found that myosin-TPPase activity was significantly influenced by Mg(2+) and Ca(2+) levels, whose optimal concentrations were determined to be 3 and 6mM, respectively. Furthermore, myosin-TPPase was strongly inhibited by EDTA-4Na(+) and KIO(3), and was slightly activated by EDTA-2Na(+). These results suggest that it may be useful to regulate tripolyphosphate hydrolysis to enhance its function in meat processing.

Topics: Acid Anhydride Hydrolases; Animals; Calcium; Diphosphates; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Female; Hydrogen-Ion Concentration; Hydrolysis; Magnesium; Muscle Proteins; Myosins; Polyphosphates; Psoas Muscles; Rabbits; Temperature

Compounds of the mevalonate pathway containing a terminal di- or triphosphate (mev-PP or mev-PPP) were tested as substrates of several enzyme ligases (T4 RNA ligase, T4 DNA ligase, firefly luciferase and other ligases) for the synthesis of ATP derivatives of the mev-pppA or mev-ppppA type. T4 RNA ligase, in the presence of ATP and the substrates: geranyl, farnesyl or isopentenyl triphosphates, and geranyl, farnesyl, dimethylallyl or isopentenyl diphosphates, all at 0.3 mM concentration, catalyzed the synthesis of the corresponding ATP derivatives at a relative rate of activity of: 7.6+/-1.4 mU/mg or 100%; 39%; 42%; 24%; 18%; 12% and 6%, respectively. Inhibition (%) of the synthesis by excess of substrate (0.8 mM vs. 0.3 mM) was observed with farnesyl diphosphate (99%); farnesyl triphosphate (96%) and geranyl triphosphate (32%). V(max), K(m), K(cat) and K(cat)/K(m) values were also determined. The K(cat)/K(m) values calculated were for: farnesyl triphosphate, 166; geranyl triphosphate, 52.2; farnesyl diphosphate, 12.1; geranyl diphosphate, 8.6; isopentenyl triphosphate, 6.7; dimethylallyl diphosphate, 3.1 and isopentenyl diphosphate, 0.9. Similar results were obtained with T4 DNA ligase. The above-mentioned compounds were also substrates of firefly luciferase synthesizing the mev-pppA or mev-ppppA derivatives. In our hands, neither the acyl- or acetyl-CoA synthetases nor the ubiquiting activating enzyme (E1) catalyzed the synthesis of ATP derivatives of these compounds. The results here presented could be related with the mechanism of action of bisphosphonates on osteoclasts or tumor cells.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Chromatography, High Pressure Liquid; Diphosphates; Diphosphonates; Diterpenes; DNA Ligases; Hemiterpenes; Mevalonic Acid; Organophosphorus Compounds; Osteoclasts; Polyisoprenyl Phosphates; Polyphosphates; RNA Ligase (ATP); Sesquiterpenes; Substrate Specificity

Triphosphate or pyrophosphate can be recognised by a diZn(2+) complex of bis(BPEA)-appended intramolecular charge transfer fluorophore 4-amino-7-aminosulfonyl-2,1,3-benzoxadiazole, displaying a 5-6 fold fluorescent enhancement at 576 nm.

Topics: Benzoxazoles; Diphosphates; Fluorescent Dyes; Light; Polyphosphates; Pyridines; Spectrometry, Fluorescence; Zinc

The activating and stabilizing effects of inorganic pyrophosphate, tripolyphosphate and nucleoside triphosphates on firefly luciferase bioluminescence were studied. The results obtained show that those effects are a consequence of the luciferase-catalyzed splitting of dehydroluciferyl-adenylate, a powerful inhibitor formed as a side product in the course of the bioluminescence reaction. Inorganic pyrophosphate, tripolyphosphate, CTP and UTP antagonize the inhibitory effect of dehydroluciferyl-adenylate because they react with it giving rise to products that are, at least, less powerful inhibitors. Moreover, we demonstrate that the antagonizing effects depended on the rate of the splitting reactions being higher in the cases of inorganic pyrophosphate and tripolyphosphate and lower in the cases of CTP and UTP. In the case of inorganic pyrophosphate, the correlation between the rate of dehydroluciferyl-adenylate pyrophosphorolysis and the activating effect on bioluminescence only occurs for low concentrations because inorganic pyrophosphate is, simultaneously, an inhibitor of the bioluminescence reaction. Our results demonstrate that previous reports concerning the activating effects of several nucleotides (including some that do not react with dehydroluciferyl-adenylate) on bioluminescence were caused by the presence of inorganic pyrophosphate contamination in the preparations used.

Topics: Allosteric Regulation; Animals; Diphosphates; Fireflies; Luciferases, Firefly; Luminescence; Luminescent Agents; Polyphosphates; Pyrophosphatases; Substrate Specificity

Eukaryotic cells produce a family of diverse inositol polyphosphates (IPs) containing pyrophosphate bonds. Inositol pyrophosphates have been linked to a wide range of cellular functions, and there is growing evidence that they act as second messengers. Inositol hexakisphosphate kinase (IP6K) is able to convert the natural substrates inositol pentakisphosphate (IP 5) and inositol hexakisphosphate (IP 6) to several products with an increasing number of phospho-anhydride bonds. In this study, we structurally analyzed IPs synthesized by three mammalian isoforms of IP6K from IP 5 and IP 6. The NMR and mass analyses showed a number of products with diverse, yet specific, stereochemistry, defined by the architecture of IP6K's active site. We now report that IP6K synthesizes both pyrophosphate (diphospho) as well as triphospho groups on the inositol ring. All three IP6K isoforms share the same activities both in vitro and in vivo.

Topics: Cacodylic Acid; Chromatography, High Pressure Liquid; Diphosphates; Inositol Phosphates; Isoenzymes; Mass Spectrometry; Phosphotransferases (Phosphate Group Acceptor); Phytic Acid; Polyphosphates; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism; Substrate Specificity

In this study, chitosan capsules with asymmetric membrane to induce osmotic effects and in situ formation of the delivery orifice were optimally prepared and characterized. Chitosan capsules were formed on stainless steel mold pins by dipping the pins into a chitosan solution followed by forming asymmetric structure by dipping into a quenching solution containing tripolyphosphate (TPP) to cause an ionic cross-linking reaction between the outer layer of chitosan and TPP. Factors influencing the properties of the capsule membrane, such as the molecular weight of chitosan, the dipping solution and dipping time, and the quenching solution and time, were optimized to successfully produce osmotic chitosan capsules with asymmetric membrane using chitosans that possessed different viscosities. In situ formation of a delivery orifice on the asymmetric membrane of the chitosan capsule was proven by the observation of a jet stream of chlorophyll being released from the capsule. Drugs with different solubility were selected, and a linear correlation between drug solubility and the initial drug release rate calculated from the slope of the drug release profile was used to verify that the delivery orifices that were in situ formed on the asymmetric membrane of the chitosan capsules induced by osmotic effect was responsible for the drug release. Water permeability across the optimally produced asymmetric membrane of the capsule from chitosan of 500 cps (300-700 cps) quenched with TPP for 30 min (C500/TPP30) was determined to be 1.40 x 10(-6)cm(2)h(-1)atm(-1) at 37.0+/-0.5 degrees C. The encapsulation of poorly water-soluble drugs, felodipine (FE) and nifedipine (NF), in such an asymmetric chitosan capsule was capable of creating a sufficient osmotic effect to activate the release of the drug with the addition of SLS and HPMC. The multiple regression equations of maximal release percent at 24h for FE and NF confirmed that both sodium lauryl sulfate (SLS) and hydroxypropyl methylcellulose (HPMC) positively influenced this response factor, and the effect of SLS was greater than that of HPMC.

Topics: Capsules; Chitosan; Cross-Linking Reagents; Delayed-Action Preparations; Diphosphates; Drug Carriers; Drug Compounding; Felodipine; Hydrogen-Ion Concentration; Hypromellose Derivatives; Linear Models; Membranes, Artificial; Methylcellulose; Nifedipine; Osmosis; Permeability; Pharmaceutical Preparations; Polyphosphates; Sodium Dodecyl Sulfate; Solubility; Surface Properties; Surface-Active Agents; Time Factors; Viscosity; Water

Numerous natural and artificial ribozymes have been shown to facilitate reactions that invert stereochemistry. Here, we demonstrate that an RNA-capping ribozyme retains stereochemistry at a phosphorus reaction center. The ribozyme synthesizes a broad range of 5'-5' RNA caps by exchanging phosphate groups around the alpha-phosphate found at the 5' terminus of the ribozyme. A ribozyme prepared with an Rp adenosine(5')alpha-thiotetraphosphate cap was found to exchange this cap for an Rp 4-thiouridine(5')alpha-thiotetraphosphate cap when incubated with 4-thiouridine triphosphate. The same Rp capped construct, when incubated with [gamma-(32)P]-ATP, exchanged the unlabeled ATP for a radiolabeled one while maintaining the same stereoconfiguration. In contrast, ribozymes prepared with an Sp cap failed to react even in the presence of thiophilic metal ions such as manganese. The kinetics of capping was also unusual as compared to inverting ribozymes. When the ribozyme was prepared with a triphosphate, capping was found to follow Michaelis-Menten-type kinetics even though the rate of pyrophosphate release was completely independent of nucleotide substrate concentration. Interestingly, the rate of capping and hydrolysis, when summed, was found to be indistinguishable from the rate of pyrophosphate release, indicating that an early rate-limiting step precedes both capping and hydrolysis. Together the retention of stereochemistry and kinetics imply that capping utilizes two inverting chemical steps that are separated by the transient formation of a rate-limiting covalent intermediate. As all protein enzymes that mediate similar capping reactions utilize a covalent intermediate, chemical necessity may have strongly guided the evolution of both protein and RNA-capping catalysts.

Topics: Adenosine Triphosphate; Base Sequence; Catalysis; Diphosphates; Isotope Labeling; Kinetics; Models, Chemical; Polyphosphates; RNA Cap Analogs; RNA, Catalytic; Uridine Triphosphate

Rate constants for the removal of iron from N-terminal monoferric transferrin have been measured for a series of phosphate and phosphonocarboxylic acids in pH 7.4 0.1 M hepes buffer at 25 degrees C. The bidentate ligands pyrophosphate and phosphonoacetic acid (PAA) show a combination of saturation and first-order kinetics with respect to the ligand concentration. Similar results are observed following a single substitution at the 2-position of PAA to give 2-benzyl-PAA and phosphonosuccinic acid. In contrast, disubstitution at the 2-position to form 2,2-dibenzyl-PAA leads to a marked reduction in iron removal via the first-order pathway. Rate constants were also measured for tripolyphosphate and phosphonodiacetic acid, which are elongated versions of PP(i) and PAA. In both cases, this elongation completely eliminates the first-order component for iron release while having relatively little impact on the saturation pathway. The sensitivity of the first-order component to the structure of the ligand strongly indicates that this pathway involves the binding of the ligand to a specific site on the protein and cannot be attributed to changes in the overall ionic strength of the solution as the ligand concentration increases. It is proposed that this structural sensitivity reflects steric restrictions on the ability of the incoming ligand to substitute for the synergistic carbonate anion to form a relatively unstable Fe-ligand-Tf ternary intermediate, which then dissociates to FeL and apoTf.

Topics: Binding Sites; Diphosphates; Humans; Hydrogen-Ion Concentration; Iron; Kinetics; Ligands; Magnetic Resonance Spectroscopy; Osmolar Concentration; Oxidation-Reduction; Phosphonoacetic Acid; Polyphosphates; Transferrin

N4-Acetyl-1-(2, 3-di-O-acetyl-4-thio-beta-D-arabinofuranosyl) cytosine (2) was synthesized in three steps from 1-(4-thio-beta-D-arabinofuranosyl) cytosine (1). The reaction of this partially blocked 4'-thio-ara-C derivative 2 with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one gave the 5-phosphitylate derivative 3, which on reaction with pyrophosphate gave the 5'-nucleosidylcyclotriphosphite 4. Product 4 was then oxidized with iodine/pyridine/water and deblocked with concentrated ammonium hydroxide to provide the desired 4'-thio-ara-C-5'-triphosphate 5. This triphosphate 5 was converted to 4'-thio-ara-C -5'-monophosphate 6 by treatment with snake venom phosphodiesterase I. The details of the synthesis, purification, and characterization of both nucleotides are described.

Topics: Ammonium Hydroxide; Arabinofuranosylcytosine Triphosphate; Arabinonucleotides; Chemistry, Pharmaceutical; Chromatography, Ion Exchange; Cytidine Monophosphate; Cytidine Triphosphate; Diphosphates; Drug Design; Hydroxides; Models, Chemical; Molecular Structure; Phosphodiesterase I; Polyphosphates; Water

Cells of two heat-resistant strains of Enterococcus faecium were heated and incubated in meat suspensions containing curing ingredients. The concentrations of the curing ingredients were those frequently used for pasteurized ham-type products, except that the concentrations of the oligophosphates (triphosphate and diphosphate) varied. Heating tests at 69 degrees C were performed with inoculated meat suspensions in heat-sealed plastic pouches. Numbers of bacteria were counted immediately after heating and in parallel series of heated pouches incubated at 37 degrees C. Plating was performed in Tryptone Dextrose Yeast Meat Peptonised Milk Agar (TDYMP); in TDYMP Agar to which the curing ingredients were added; and in TDYMP Agar to which the curing ingredients except oligophosphates were added. The inclusion of oligophosphates in the heating medium increased the heat-injury sustained by the E. faecium cells, and in combination with rather severe heat treatment even completely blocked the growth of surviving organisms in the meat suspension incubated at 37 degrees C. The presence of oligophosphates in the culture medium TDYMP Agar severely reduced the counts of freshly heated cells; however, this effect disappeared after repair and growth of the surviving organisms in the meat suspension.

Topics: Animals; Colony Count, Microbial; Culture Media; Diphosphates; Enterococcus faecium; Food Contamination; Food Microbiology; Hot Temperature; Meat Products; Polyphosphates; Swine; Time Factors

1. Heteromeric P2X2/3 receptors are much more sensitive than homomeric P2X2 receptors to alphabeta-methylene-ATP, and this ATP analogue is widely used to discriminate the two receptors on sensory neurons and other cells. 2. We sought to determine the structural basis for this selectivity by synthesising ADP and ATP analogues in which the alphabeta and/or betagamma oxygen atoms were replaced by other moieties (including -CH2-, -CHF-, -CHCl-, -CHBr-, -CF2-, -CCl2-, -CBr2-, -CHSO3-, -CHPO3-, -CFPO3-, -CClPO3-, -CH2-CH2-, C triple bond C, -NH-, -CHCOOH-). 3. We tested their actions as agonists or antagonists by whole-cell recording from human embryonic kidney cells expressing P2X2 subunits alone (homomeric P2X2 receptors), or cells expressing both P2X2 and P2X3 subunits, in which the current through heteromeric P2X2/3 receptors was isolated. 4. ADP analogues had no agonist or antagonist effect at either P2X2 or P2X2/3 receptors. All the ATP analogues tested were without agonist or antagonist activity at homomeric P2X2 receptors, except betagamma-difluoromethylene-ATP, which was a weak agonist. 5. At P2X2/3 receptors, betagamma-imido-ATP, betagamma-methylene-ATP, and betagamma-acetylene-ATP were weak agonists, whereas alphabeta,betagamma- and betagamma,gammadelta-bismethylene-AP4 were potent full agonists. betagamma-Carboxymethylene-ATP and betagamma-chlorophosphonomethylene-ATP were weak antagonists at P2X2/3 receptors (IC50 about 10 microm). 6. The results indicate (a). that the homomeric P2X2 receptor presents very stringent structural requirements with respect to its activation by ATP; (b). that the heteromeric P2X2/3 receptor is much more tolerant of alphabeta and betagamma substitution; and (c). that a P2X2/3-selective antagonist can be obtained by introduction of additional negativity at the betagamma-methylene.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Cell Line; Child, Preschool; Dimerization; Diphosphates; Dose-Response Relationship, Drug; Humans; Membrane Potentials; Polyphosphates; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3

The aim of this paper was to investigate the electrostatic interactions between multivalent phosphates (phosphate (Phos), pyrophosphate (Pyro) and tripolyphosphate (TPP)) and chitosan, as well as the influence of electrostatic interactions on the properties of chitosan films ionically cross-linked by the above mentioned phosphates. The charge number of Phos was too low to interact with chitosan, while Pyro and TPP with more negative charges showed a significant ability to ionically cross-link chitosan. Solution pH played an important role on the charge numbers carried by Pyro, TPP and chitosan, especially for Pyro/chitosan. For instance, at pH less than 2.0 the interaction between Pyro and chitosan disappeared, while for TPP/chitosan even in solutions at pH less than 0.5 it still existed. Media pH and ionic strength also had a significant influence on the properties of cross-linked chitosan film with multivalent phosphates. Usually these films swelled and drug was released quickly in acidic conditions (such as in simulated gastric fluid) while under neutral conditions (such as in simulated intestinal fluid) they remained in a shrinkage state and drug was released slowly. Compared to TPP/chitosan films, Pyro/chitosan films exhibited much better pH-sensitive swelling and controlled release properties due to their relatively weak electrostatic interaction. The same reasoning was used to explain the significant acceleration of Pyro/chitosan film swelling and model drug release observed on adding sodium chloride. These films may be promising for site-specific drug delivery in the stomach.

Topics: Anions; Biocompatible Materials; Chitin; Chitosan; Cross-Linking Reagents; Delayed-Action Preparations; Diphosphates; Hydrogen-Ion Concentration; Nephelometry and Turbidimetry; Osmolar Concentration; Pharmaceutic Aids; Phosphates; Polyphosphates; Potentiometry; Riboflavin; Rosaniline Dyes; Time Factors

RNA triphosphatase catalyzes the first step in mRNA cap formation which entails the cleavage of the beta-gamma phosphoanhydride bond of triphosphate-terminated RNA to yield a diphosphate end that is then capped with GMP by RNA guanylyltransferase. Here we characterize a 303 amino acid RNA triphosphatase (Pct1p) encoded by the fission yeast SCHIZOSACCHAROMYCES: pombe. Pct1p hydrolyzes the gamma phosphate of triphosphate-terminated poly(A) in the presence of magnesium. Pct1p also hydrolyzes ATP to ADP and P(i) in the presence of manganese or cobalt (K(m) = 19 microM ATP; k(cat) = 67 s(-1)). Hydrolysis of 1 mM ATP is inhibited with increasing potency by inorganic phosphate (I(0.5) = 1 mM), pyrophosphate (I(0.5) = 0.4 mM) and tripolyphosphate (I(0.5) = 30 microM). Velocity sedimentation indicates that Pct1p is a homodimer. Pct1p is biochemically and structurally similar to the catalytic domain of Saccharomyces cerevisiae RNA triphosphatase Cet1p. Mechanistic conservation between Pct1p and Cet1p is underscored by a mutational analysis of the putative metal-binding site of Pct1p. Pct1p is functional in vivo in S.cerevisiae in lieu of Cet1p, provided that it is coexpressed with the S.pombe guanylyltransferase. Pct1p and other yeast RNA triphosphatases are completely unrelated, mechanistically and structurally, to the metazoan RNA triphosphatases, suggesting an abrupt evolutionary divergence of the capping apparatus during the transition from fungal to metazoan species.

Topics: Acid Anhydride Hydrolases; Adenosine Triphosphate; Amino Acid Motifs; Amino Acid Sequence; Animals; Artificial Gene Fusion; Diphosphates; Fungal Proteins; Genetic Complementation Test; Hydrolysis; Kinetics; Metals; Mice; Molecular Sequence Data; Nucleotidyltransferases; Phosphates; Phosphoric Monoester Hydrolases; Polyphosphates; RNA Caps; Saccharomyces cerevisiae; Schizosaccharomyces; Sequence Homology, Amino Acid

S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase) catalyzes the only known route of biosynthesis of the primary biological alkylating agent. The internal thermodynamics of the Escherichia coli S-adenosylmethionine (AdoMet) synthetase catalyzed formation of AdoMet, pyrophosphate (PP(i)), and phosphate (P(i)) from ATP, methionine, and water have been determined by a combination of pre-steady-state kinetics, solvent isotope incorporation, and equilibrium binding measurements in conjunction with computer modeling. These studies provided the rate constants for substrate binding, the two chemical interconversion steps [AdoMet formation and subsequent tripolyphosphate (PPP(i)) hydrolysis], and product release. The data demonstrate the presence of a kinetically significant isomerization of the E.AdoMet.PP(i).P(i) complex before product release. The free energy profile for the enzyme-catalyzed reaction under physiological conditions has been constructed using these experimental values and in vivo concentrations of substrates and products. The free energy profile reveals that the AdoMet formation reaction, which has an equilibrium constant of 10(4), does not have well-balanced transition state and ground state energies. In contrast, the subsequent PPP(i) hydrolytic reaction is energetically better balanced. The thermodynamic profile indicates the use of binding energies for catalysis of AdoMet formation and the necessity for subsequent PPP(i) hydrolysis to allow enzyme turnover. Crystallographic studies have shown that a mobile protein loop gates access to the active site. The present kinetic studies indicate that this loop movement is rapid with respect to k(cat) and with respect to substrate binding at physiological concentrations. The uniformly slow binding rates of 10(4)-10(5) M(-)(1) s(-)(1) for ligands with different structures suggest that loop movement may be an intrinsic property of the protein rather than being ligand induced.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Binding Sites; Catalysis; Computer Simulation; Diphosphates; Escherichia coli; Fluorescence; Hydrolysis; Isomerism; Kinetics; Ligands; Methionine; Methionine Adenosyltransferase; Oxygen; Oxygen Isotopes; Phosphates; Polyphosphates; S-Adenosylmethionine; Solvents; Thermodynamics; Titrimetry; Water

S-Adenosylmethionine (AdoMet) synthetase catalyzes the only known route of biosynthesis of the primary in vivo alkylating agent. Inhibitors of this enzyme could provide useful modifiers of biological methylation and polyamine biosynthetic processes. The AdoMet synthetase catalyzed reaction converts ATP and L-methionine to AdoMet, PP(i), and P(i), with formation of tripolyphosphate as a tightly bound intermediate. This work describes a nonhydrolyzable analogue of the tripolyphosphate (PPP(i)) reaction intermediate, diimidotriphosphate (O(3)P-NH-PO(2)-NH-PO(3)(5)(-)), as a potent inhibitor. In the presence of AdoMet, PNPNP is a slow-binding inhibitor with an overall inhibition constant (K(i)) of 2 nM and a dissociation rate of 0.6 h(-)(1). In contrast, in the absence of AdoMet PNPNP is a classical competitive inhibitor with a K(i) of 0.5 microM, a slightly higher affinity than PPP(i) itself (K(i) = 3 microM). The imido analogue of the product pyrophosphate, imidodiphosphate (O(3)P-NH-PO(3)(4)(-)) also displays slow onset inhibition only in the presence of AdoMet, with a K(i) of 0.8 microM, compared to K(i) of 250 microM for PP(i). Circular dichroism spectra of the unliganded enzyme and various complexes are indistinguishable indicating that the protein secondary structure is not greatly altered upon complex formation, suggesting local rearrangements at the active site during the slow binding process. A model based on ionization of the bridging -NH- moiety is presented which could account for the potent inhibition by PNP and PNPNP.

Topics: Acid Anhydride Hydrolases; Adenosine Triphosphate; Adenylyl Imidodiphosphate; Amino Acid Substitution; Arginine; Binding, Competitive; Diphosphates; Diphosphonates; Enzyme Inhibitors; Hydrolysis; Leucine; Methionine Adenosyltransferase; Mutagenesis, Site-Directed; Polyphosphates

In view of the importance of a prebiotic source of high energy phosphates, we have investigated a number of potentially prebiotic processes to produce condensed phosphates from orthophosphate and cyclic trimetaphosphate from tripolyphosphate. The reagents investigated include polymerizing nitriles, acid anhydrides, lactones, hexamethylene tetramine and carbon suboxide. A number of these processes give substantial yields of pyrophosphate from orthophosphate and trimetaphosphate from tripolyphosphate. Although these reactions may have been applicable in local areas, they are not sufficiently robust to have been of importance in the prebiotic open ocean.

Topics: 4-Butyrolactone; Acetic Anhydrides; Calcium; Diphosphates; Evolution, Chemical; Formaldehyde; Formates; Hydrogen Cyanide; Imidazoles; Magnesium; Maleic Anhydrides; Methenamine; Nitriles; Phosphates; Polyphosphates; Thiocyanates

The formation of carbamyl phosphate (CAP) in dilute solutions of cyanate (NCO-) and orthophosphate (Pi) was measured both in the absence and in the presence of a precipitated matrix of calcium phosphate (Pi.Ca). The second-order rate constant and the free energy of CAP synthesis were not modified by the presence of the solid matrix, indicating that synthesis occurs in the homogeneous Pi-containing solution. The elimination reaction of CAP to form NCO- and Pi followed first-order kinetics and the rate constant was the same whether or not calcium phosphate was present. Elimination was not complete, and the steady level of remaining CAP was that expected from the free energy of synthesis. The formation of pyrophosphate (PPi) was detected in CAP-containing medium only in the presence of calcium, showing a close correlation with the amount of precipitated Pi.Ca. Phosphorolysis of CAP followed a sigmoidal time course, compatible with adsorption of CAP to the solid matrix as a prelude to transphosphorylation. Addition of 5'-AMP and of short linear polyphosphates inhibited phosphorolysis of CAP. It is proposed that the presence of a solid phosphate matrix and the relative concentrations of cyano compounds, as well as those of nucleotides and inorganic polyphosphates, could have played a crucial role in the conservation of chemical energy of CAP and in its use in prebiotic phosphorylation reactions.

Topics: Adenosine Monophosphate; Biopolymers; Calcium Phosphates; Carbamyl Phosphate; Chemical Precipitation; Crystallization; Cyanates; Diphosphates; Evolution, Chemical; Kinetics; Phosphates; Phosphorylation; Polyphosphates

Difference ultraviolet spectroscopy has been used to monitor the binding of a series of phosphonate ligands to human apotransferrin. The ligands consist of pyrophosphate as well as the phosphonic acids (aminomethyl)phosphonic acid (AMPA), (hydroxymethyl)phosphonic acid (HMP), (phosphonomethyl)-iminodiacetic acid (PIDA), N,N-bis(phosphonomethyl)glycine (DPG), and nitrilotris(methylenephosphonic acid) (NTP). Equilibrium constants have been measured for the sequential binding of two ligands per molecule of apotransferrin. In addition, site-specific equilibrium constants have been measured for the binding of AMPA, HMP, and PIDA to the vacant binding site of both forms of monoferric transferrin. Since titrations of diferric transferrin produce no difference UV spectrum, it is proposed that the primary binding site for phosphonic acids includes the protein groups that bind the synergistic bicarbonate anion that is required for formation of a stable ferric transferrin complex. It is further proposed that those ligands with two phosphonate groups can simultaneously bind to cationic amino acid side chains that extend into the cleft between the two domains of each lobe of transferrin. From an inspection of the ferric transferrin crystal structure, the most likely anion binding residues in the cleft are Arg-632 and Lys-534 in the C-terminal lobe and Lys-206 and Lys-296 in the N-terminal lobe.

Topics: Animals; Apoproteins; Binding Sites; Chelating Agents; Diphosphates; Ferric Compounds; Kinetics; Ligands; Organophosphonates; Polyphosphates; Protein Conformation; Rabbits; Transferrin; X-Ray Diffraction

After the removal of the exchangeable guanine nucleotides by chromatography on phenyl-Sepharose [Hanssens, I., Baert, J., and Van Cauwelaert, F. (1990) Biochemistry 29, 5160-5165] tubulin polymerizations with GTP, GDP, tripolyphosphate, pyrophosphate or orthophosphate as possible stimulants are compared. It is demonstrated that, besides GTP and pyrophosphate, also tripolyphosphate stimulates the assembly into microtubules at high concentrations (4.65 mM) of Mg2+. The influence of Mg2+ is more pronounced in combination with pyrophosphate and tripolyphosphate than with GTP. The microtubules assembled in combination with Mg2+ and tripolyphosphate or pyrophosphate are short, suggesting that especially the nucleation step of microtubule assembly is favoured.

Topics: Animals; Brain Chemistry; Diphosphates; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Magnesium; Microscopy, Electron; Microtubules; Negative Staining; Phosphates; Polyphosphates; Swine; Tubulin

Topics: Animals; Dental Calculus; Diphosphates; Polyphosphates; Rats

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Carbon Isotopes; Chromatography; Diphosphates; Escherichia coli; Hot Temperature; Ligases; Magnesium; Metabolism; Phosphorus Isotopes; Polyphosphates; Research; RNA

Topics: Chemical Precipitation; Crystallization; Diphosphates; Kidney Calculi; Kidney Tubules; Milk; Nephrocalcinosis; Pathology; Pharmacology; Phosphates; Polyphosphates; Rats; Research; Solubility; Toxicology; Urinary Calculi; Urolithiasis

Topics: Animals; Cytosine Nucleotides; Diabetes Mellitus, Experimental; Diphosphates; Glucose; Glucose-6-Phosphatase; Inorganic Pyrophosphatase; Insulin; Kinetics; Liver; Microsomes; Microsomes, Liver; Phosphotransferases; Polyphosphates; Pyrophosphatases; Rats; Research; Surface-Active Agents; Toxicology

Topics: Adenine Nucleotides; Adenosine Triphosphate; Carbon Isotopes; Chromatography; Cytosine Nucleotides; Diphosphates; Glucosephosphate Dehydrogenase; Guanine Nucleotides; Ion Exchange Resins; Nucleoside-Diphosphate Kinase; Nucleotides; Phosphotransferases; Polyphosphates; Research; Saccharomyces; Saccharomyces cerevisiae; Spectrophotometry

Topics: Adenosine; Adenosine Triphosphate; Calcium; Cations, Divalent; Chelating Agents; Cytokinesis; Diphosphates; Ions; Magnesium; Manganese; Morpholines; Phosphates; Polyphosphates; Research

Topics: Adenine Nucleotides; Adenosine Triphosphate; Bacillus subtilis; Carbon Isotopes; Diphosphates; Enzyme Inhibitors; NAD; Niacin; Nicotinic Acids; Nucleotides; Nucleotidyltransferases; Pentosyltransferases; Pharmacology; Polyphosphates; Research

Topics: Dactinomycin; Diphosphates; Humans; Nucleosides; Nucleotides; Polyphosphates

Topics: Chemistry, Pharmaceutical; Diphosphates; Morpholines; Nucleosides; Nucleotides; Polyphosphates; Research; Thymine Nucleotides

Topics: Adenine Nucleotides; Adenosine; Diphosphates; Female; Humans; Labor, Obstetric; Polyphosphates; Pre-Eclampsia; Pregnancy; Work

Topics: Adenine Nucleotides; Adenosine; Digitalis; Digitalis Glycosides; Digitoxin; Diphosphates; Myocardium; Phosphates; Phosphocreatine; Polyphosphates

Topics: Cytokinesis; Diphosphates; Hydrolysis; Nucleosides; Nucleotides; Phosphates; Polyphosphates; Pyrophosphatases

Topics: Diphosphates; Nucleotides; Polyphosphates

Topics: Diphosphates; Enzymes; Phosphorus; Phosphorus Compounds; Phosphorus, Dietary; Polyphosphates

Topics: Acids; Diphosphates; Enzymes; Polyphosphates; Thiamine; Thiamine Pyrophosphate