Effect of diphosphonic acids on alkaline phosphatase activity

Kinetics was studied for the alkaline phosphatase activity inhibition by diphosphonic acids. When the ratio of Mg2+ and substrate (S) concentrations [( Mg2+]/[S]) is equal to 10, the process constants for methylene diphosphonic, amino methylene diphosphonic and hydroxyethylidene diphosphonic acids are 0.14, 0.12 and 0.35 mM, respectively. The inhibition is of competitive character. An increase in the Mg2+ concentration to the [Mg2+]/[S] = 40 ratio lowers the inhibition degree for all three diphosphonates; it follows a mixed mechanism. Thus, the inhibition of the alkaline phosphatase activity by diphosphonic acids is due to both competition of the inhibitor for the enzyme active centre and a decrease in the Mg2+ concentration, the phosphatase activator, because of Mg2+ complexing with diphosphonates.     

The influence of diphosphonic analogues of inorganic pyrophosphate on activity of RNA-polymerases from the calf thymus

Diphosphonic analogues of inorganic pyrophosphate (PPi): methylene-, oxyethylidene-, aminomethylenediphosphonic acids as well as phosphonacetic, imidodiphosphoric bis- (phosphonomethyl)-phosphonic acids and methylenediphosphonic and phosphonic acid monoanhydrides were studied for their effect on the RNA-synthesizing activity of thymocytes. DNA-dependent RNA-polymerases I and II from the calf thymus nuclei were used for these studies. The analogues and PPi under study are shown to be inhibitors of both RNA-polymerases in nuclei from calf thymus and of purified RNA-polymerase II, which is more sensitive to the effect of diphosphonates. Methylenediphosphonic acid is the strongest inhibitor among the studied analogues, and imidodiphosphoric and phosphonacetic acids are the weakest inhibitors. Inhibition of purified RNA-polymerase II by diphosphonates has a complex character and includes both interaction of the PPi analogues with enzymes and chelating by them of Mn ions which are cofactors for RNA polymerase.     

Diphosphonates are potent inhibitors of mammalian inorganic pyrophosphatase

Methanediphosphonate and 12 analogs thereof with different substituents at the carbon atom are potent competitive inhibitors of highly purified rat liver and bovine heart inorganic pyrophosphatases. The inhibition constants for the most effective diphosphonates, which contain an NH2 or OH group at the bridge carbon atom, are in the micromolar range. Yeast and Escherichia coli pyrophosphatases are markedly less sensitive to the diphosphonates. Pyrophosphatase inhibition may be related to the numerous biological effects exerted by diphosphonates.     

Capacity of diphosphonates to bind calcium and their effect on the osmotic permeability of the frog bladder wall

Like EDTA, diphosphonates increase permeability of the frog urinary bladder wall for water osmotic gradient. Their effect is proportional to the stability of their binding with calcium ions. The efficacy of diphosphonates falls upon pH decline. The results indicate the significance of Ca binding in cells and membrane action of diphosphonates.     

Effect of inorganic pyrophosphate and its diphosphonate analogs on glucose-6-phosphatase dehydrogenase activity of mouse organs

The glucose-6-phosphatase dehydrogenase (EC 1.1.1.49) reaction of mouse organs was studied as affected by PPi and its diphosphonate analogs. It is shown that in vitro and hydroxy-1-ethane-1,1-diphosphonic acid) inhibit the mentioned enzyme of the mouse spleen and liver. The effect of hydroxyl-1-ethane-1,1-diphosphonic acid was used as an example to show that inhibition of glucose-6-phosphate dehydrogeanse by diphosphonates belongs to the mixed type characterized by changes in the Km and Vmax values. For the spleen enzyme Km equals 0.064 mM, Vmax - 4.7 Mg of NADPH per 1 mg of protein-1. h-1. Administration of methylene diphosphonic acid causes an inhibition in vivo of the glucose-6-phosphatase dehydrogenate activity of the liver but not of the spleen and thymus. Basing on the isoenzymic composition of the enzyme for the mentioned organs, it is possible to suppose that the difference in the methylene diphosphonic acid effect in the liver and lymphoid organs may depend on the differences in its isoenzymic spectrum. The fact that in vivo methylene diphosphonic acid in a dose having an immuno-depressive action has no influence on the activity of glucose-6-phosphatase dehydrogenase in the lymphoid organs, may evidence for the absence of the indirect immunodepressive effect of diphosphonate by affecting this enzyme.     

The effects of diphosphonates on the growth and glycolysis of connective-tissue cells in culture.

1. The effects of two diphosphonates (compounds containing a P-C-P bond), disodium dichloromethanediphosphonate and disodium 1-hydroxyethane-1,1-diphosphonate, on the metabolism of cultured rat calvaria cells, rabbit ear cartilage cells and rat skin fibroblasts were investigated. 2. The diphosphonates had no effect on the growth of cartilage cells and on the exponential growth of the calvaria cells and the fibroblasts. However, dichloromethanediphosphonate stopped the growth of the calvaria cells and the fibroblasts after the beginning of confluence, whereas the untreated cells were still growing to a certain extent. This inhibition was dose-dependent. After the drug was withdrawn, the cells recovered slowly. 1-Hydroxyethane-1,1-diphosphonate had no detectable effect on the growth of any of the cell types studied. Both diphosphonates decreased the cloning efficiency of calvaria cells and fibroblasts. 3. The K+ content of cartilage, calvaria and skin cells was diminished only by the highest (0.25 mM) concentration of dichloromethanediphosphonate. 4. Radioactive dichloromethanediphosphonate and 1-hydroxyethane-1,1-diphosphonate were taken up linearly with time for at least 48 h by calvaria cells and fibroblasts. The diphosphonate concentration in the cells depended on its concentration in the medium. 5. Both diphosphonates, in a dose-dependent fashion, markedly inhibited glycolysis, dichloromethanediphosphonate being more effective than 1-hydroxyethane-1,1-diphosphonate, at drug doses that had no effect on cell growth or cellular K+ content. Calvaria cells were much more sensitive than cartilage cells. When cartilage cells were cultured in an N2 atmosphere, these effects on glucose and lactate metabolism disappeared. 6. As increased acid production appears to be associated with resorption of bone, this decrease in lactate may explain why diphosphonates are effective inhibitors of bone resorption in vivo.     

Effect of chelators of bivalent metal ions on the reaction of myometrium actomoysin superprecipitation]

Kinetic regularities of the reaction of superprecipitation of myometrium actomyosin, as well as the effect of different concentrations of EGTA, EDTA and diphosphonic acids on this process have been studied. Results obtained are of interest from the viewpoint of possible practical use of diphosphonates as factors modifying interaction of the contractile proteins of the uterus smooth muscles under the pathology of contractile response.     

The effect of nitrofurantoin on deoxyribonucleic acid synthesis in plant mitochondria.

Nitrofurantoin (1-([(5-nitrofuran-2-yl)methylene]amino)imidazolidine-2,4-dione), a widely used drug and also a well-known bacterial mutagen, inhibits DNA synthesis in mitochondria from 48 h etiolated seedlings of Vigna sinensis (Linn.) Savi (snake bean). The effect appears at the level of the uptake of radioactive deoxynucleoside triphosphates by the plant mitochondria. Nitrofurantoin does not inhibit DNA synthesis in vitro by homogeneous Escherichia coli DNA polymerase I and DNA polymerase from avian-myeloblastosis virus. No specific nitroreductase activity could be detected in mitochondria.     

Aspects des complications ostéoarticulaires de la drépanocytose en scintigraphie osseuse planaire (infection exclue). À propos de trois observations

Skeletal complications of sickle-cell anemia are multiple and can appear on the acute (osseous infarction, acute osteomyelitis) or chronic mode (osteonecrosis, chronic osteomyelitis). The radio-labelled diphosphonates bone scintigraphy remains an important tool in the early diagnosis and in the follow-up of these complications and must form part of the initial assessment of the disease. Through clinical observations, the authors undertake to sum up the bone scintigraphy aspects of these complications.     

Repair of damaged DNA by Arabidopsis cell extract

All living organisms have to protect the integrity of their genomes from a wide range of genotoxic stresses to which they are inevitably exposed. However, understanding of DNA repair in plants lags far behind such knowledge in bacteria, yeast, and mammals, partially as a result of the absence of efficient in vitro systems. Here, we report the experimental setup for an Arabidopsis in vitro repair synthesis assay. The repair of plasmid DNA treated with three different DNA-damaging agents, UV light, cisplatin, and methylene blue, after incubation with whole-cell extract was monitored. To validate the reliability of our assay, we analyzed the repair proficiency of plants depleted in AtRAD1 activity. The reduced repair of UV light- and cisplatin-damaged DNA confirmed the deficiency of these plants in nucleotide excision repair. Decreased repair of methylene blue-induced oxidative lesions, which are believed to be processed by the base excision repair machinery in mammalian cells, may indicate a possible involvement of AtRAD1 in the repair of oxidative damage. Differences in sensitivity to DNA polymerase inhibitors (aphidicolin and dideoxy TTP) between plant and human cell extracts were observed with this assay.     

Thermodynamic studies on anion binding to apotransferrin and to recombinant transferrin N-lobe half molecules

Equilibrium constants for the binding of anions to apotransferrin, to the recombinant N-lobe half transferrin molecule (Tf/2N), and to a series of mutants of Tf/2N have been determined by difference UV titrations of samples in 0.1 M Hepes buffer at pH 7.4 and 25 degrees C. The anions included in this study are phosphate, sulfate, bicarbonate, pyrophosphate, methylenediphosphonic acid, and ethylenediphosphonic acid. There are no significant differences between anion binding to Tf/2N and anion binding to the N-lobe of apotransferrin. The binding of simple anions like phosphate appears to be essentially equivalent for the two apotransferrin binding sites. The binding of pyrophosphate and the diphosphonates is inequivalent, and the studies on the recombinant Tf/2N show that the stronger binding is associated with the N-terminal site. Anion binding constants for phosphate, pyrophosphate, and the diphosphonates with the N-lobe mutants K206A, K296A, and R124A have been determined. Anion binding tends to be weakest for the K296A mutant, but the variation in log K values among the three mutants is surprisingly small. It appears that the side chains of K206, K296, and R124 all make comparable contributions to anion binding. There are significant variations in the intensities of the peaks in the difference UV spectra that are generated by the titrations of the mutant apoproteins with these anions. These differences appear to be related more to variations in the molar extinction coefficients of the anion-protein complexes rather than to differences in binding constants.     

Boradiazaindacene dyes for technology of biological microchips

A number of boradiazaindacene dyes containing a carboxyl group separated from a fluorophore by two methylene units were synthesized. The compounds have narrow spectral bands with absorption maxima at 480–530 nm and fluorescence maxima at 500–550 nm. Succinimide esters of these compounds and the corresponding fluorescent-labeled oligonucleotides were also prepared. Boradiazaindacene dyes can be used as fluorescent labels for oligonucleotides for analysis of melting curves of duplexes on microchips either by themselves or in combination with Texas Red. They can also be applied for labeling primers for polymerase chain reaction.     

Boradiazaindacene dyes for technology of biological microchips

A number of boradiazaindacene dyes containing a carboxyl group separated from a fluorophore by two methylene units were synthesized. The compounds have narrow spectral bands with absorption maxima at 480-530 nm and fluorescence maxima at 500-550 nm. Succinimide esters of these compounds and the corresponding fluorescent-labeled olgionucleotides were also prepared. Boradiazaindacene dyes can be used as fluorescent labels for oligonucleotides for analysis of melting curves of duplexes on microchips either by themselves or in combination with Texas Red. They can also be applied for labeling primers for polymerase chain reaction.     

Inhibitor-induced structural change of the active site of human poly(ADP-ribose) polymerase

The crystal structure of human recombinant poly(ADP-ribose) polymerase (PARP) complexed with a potent inhibitor, FR257517, was solved at 3.0 A resolution. The fluorophenyl part of the inhibitor induces an amazing conformational change in the active site of PARP by motion of the side chain of the amino acid, Arg878, which forms the bottom of the active site. Consequently, a corn-shaped hydrophobic subsite, which consists of the side chains of Leu769, Ile879, Pro881, and the methylene chain of Arg878, newly emerges from the well-known active site.     

Increase in fatty acid oxidation in calvaria cells cultured with diphosphonates.

1. Cultured calvaria cells oxidized palmitate and octanoate to CO2 and water-soluble products. 2. When these cells were treated for 6 days with 0.025 and 0.25 mM-dichloromethanediphosphonate, oxidation of palmitate was increased, whereas that of octanoate was influenced less. 3. When the rate of oxidation was raised by increasing the palmitate concentration in the medium, the effect of the diphosphonate was decreased and finally disappeared. 4. 1-Hydroxyethane-1,1-diphosphonate had only minor effects. 5. The increase in palmitate oxidation appeared 2 days after the addition of dichloromethanediphosphonate, simultaneously with a fall in lactate production. (Inhibition of glycolysis by diphosphonates has already been shown.) 6. Cycloheximide, an inhibitor of protein synthesis, did not influence the effect of dichloromethanediphosphonate on the oxidation of palmitate and the production of lactate. 7. Cells cultured with dichloromethanediphosphonate showed a faster uptake of palmitic acid than did control cells. However, this observation did not explain the increased palmitate oxidation, since uptake was much faster than oxidation, and was therefore not the rate-limiting step. 8. 2-Bromopalmitate, an inhibitor of fatty acid oxidation, did not influence the inhibition of glycolysis by the diphosphonates. This inhibition, therefore, did not result from the increased oxidation of palmitate. It is also unlikely that the increased oxidation of palmitate is connected with the inhibition of glycolysis.