Nucleotide increased solubility

The technique can be used to separate a wide range of biologically important molecules such as amino acids, peptides, nudeobases, nudeosides and nucleotides, water-soluble vitamins and steroid hormones etc. It is being increasingly used in the analysis of pharmaceuticals and their metabolic products. 

Since the end products of pyrimidine catabolism are highly water-soluble, pyrimidine overproduction results in few clinical signs or symptoms. In hypemricemia associated with severe overproduction of PRPP, there is overproduction of pyrimidine nucleotides and increased excretion of p-alanine. Since A, A -methyl-ene-tetrahydrofolate is required for thymidylate synthesis, disorders of folate and vitamin Bjj metabofism result in deficiencies of TMP. 

These three compounds exert many similar effects in nucleotide metabolism of chicks and rats [167]. They cause an increase of the liver RNA content and of the nucleotide content of the acid-soluble fraction in chicks [168], as well as an increase in rate of turnover of these polynucleotide structures [169,170]. Further experiments in chicks indicate that orotic acid, vitamin B12 and methionine exert a certain action on the activity of liver deoxyribonuclease, but have no effect on ribonuclease. Their effect is believed to be on the biosynthetic process rather than on catabolism [171]. Both orotic acid and vitamin Bu increase the levels of dihydrofolate reductase (EC 1.5.1.4), formyltetrahydrofolate synthetase and serine hydroxymethyl transferase in the chicken liver when added in diet. It is believed that orotic acid may act directly on the enzymes involved in the synthesis and interconversion of one-carbon folic acid derivatives [172]. The protein incorporation of serine, but not of leucine or methionine, is increased in the presence of either orotic acid or vitamin B12 [173]. In addition, these two compounds also exert a similar effect on the increased formate incorporation into the RNA of liver cell fractions in chicks [174—176]. It is therefore postulated that there may be a common role of orotic acid and vitamin Bj2 at the level of the transcription process in m-RNA biosynthesis [174—176]. 

Due to their polyanionic nature, ASOs are extremely water-soluble under neutral and basic conditions. Consequently, dmg-product concentrations are often only limited by an increase in solution viscosity at very high concentrations (e.g. >300 mg/mL) [3]. Not surprisingly, extremes of pH and ionic strength influence the apparent solubility. In acidic environments such as the stomach, inter-nucleotide linkages are partially neutralized by protonation. With this consequent decrease in their polyanionic status there is a marked decrease in oligonucleotide solubility, an event that can be easily reversed by raising the pH. 

Thrombomodulin mutations are more important in arterial diseases than in venous diseases. The thrombomodulin polymorphism, G—>A substitution at nucleotide position 127 in the gene, has been studied regarding its relation with the arterial disease. The 25 Thr allele has been reported to be more prevalent in male patients with myocardial infarction than the control population (25). Polymorphism in the thrombomodulin gene promoter (-33 G/A) influences the plasma soluble thrombomodulin levels and causes increased risk of coronary heart disease (26). Carriership of the -33A allele was also reported to cause increased occurrence of carotid atherosclerosis in patients less than 60 years (27). 

DNA is precipitated from aqueous solutions by ethanol or isopropanol in the presence of salt. The amount of alcohol and salt depends on the type of salt that one wishes to use (Table 6.2). The type of salt used depends largely on downstream applications for which the DNA is to be used. For example, precipitation in the presence of ammonium acetate removes small molecules such as nucleotides, and the DNA can be used for many enzymatic reactions. On the other hand, the phosphorylating enzyme, T4 kinase, is inhibited by ammonium ions, and unless DNA is reprecipitated in the presence of salts other than ammonium acetate, the phosphorylation reaction may be inhibited. For most routine purposes, alcohol precipitation of DNA with sodium acetate is preferred over sodium chloride because of the higher solubility of the acetate salt in ethanol. Selection of isopropanol or ethanol is more of a convenience than a rule. Although isopropanol precipitation requires an equal volume of isopropanol for the precipitation of DNA, that with ethanol requires 2 volumes and hence can increase the total volume... 

The addition of modifiers into the aqueous phase also affects selectivity. Cyclodextrins can be used in conjunction with the MECC buffer to provide selectivity for very hydrophobic analytes that would otherwise be almost totally incorporated into the micellar phase. In addition, they can be used for enantiomeric separations because of the chirality of the cyclodextrins themselves. The resolution between very hydrophobic compounds can also be improved by the addition of high concentrations of urea to the MECC buffer, which increases the solubility of hydrophobic compounds in water and breaks down hydrogen bonds in the aqueous phase. The addition of low concentrations (<20% v/v) of organic solvents, such as acetonitrile or 2-propanol, reduces the EOF and thereby expands the migration time window. Higher concentrations, however, can break down the micellar structure, so care should be taken. Finally, salts of certain metals, such as magnesium, zinc, or copper(II), can be added to enhance resolution of nucleotides. An optimization scheme for MECC separations is shown in Figure 5.9. 

The increase with time in the incorporation of radioactivity into DNA is linear between 30 and 90 min of the concomitant addition of drug and tritiated thymidine and during this time the specific activity of the acid soluble nucleotide pool remains constant and identical to that of the supplied [3H]thymidine. 

The method of circular dichroism has been applied in investigations that have determined the reactions between Pt(II) compound and nucleosides, nucleotides, and DNA. Many antimicrobial preparations are excellent ligands. The activity of some of the antimicrobial preparations has been based on their complexing with metal ions. For example, increased effectiveness of tetracycline has been observed after its coordination with Ca. The complex is more lipophilic and very oil-soluble, which explains its transportation through the cell membrane. The opposite situation occurs when the ion-metal is toxic and the coordinated antibiotic serves as carrier through the membrane. 

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