gamma-radiolysis of N2O-saturated aqueous solutions of trehalose and trehalose/amino acid mixtures.

gamma-radiolysis of aqueous N2O-saturated solutions of alpha, alpha-trehalose (10(-2) M) with doses of 500 krad yielded glucose (G = 2.3), gluconic acid lactone (G = 0.25) and--after reduction--iditol (G = 0.15), mannitol (G = 0.05), 5-deoxy-glucitol (G = 0.34) and 2-deoxy-glucitol (G = 0.14). The decomposition of trehalose (G = -5.9) was reduced, if equimolar amounts of amino acids (alanine, leucine, phenylalanine, methionine or cysteine) were present during irradiation. The extent of this reduction has been correlated with the .OH radical-scavenging properties of the added amino acids. Cysteine also protected trehalose by the repair of initially formed trehalose radicals and almost completely suppressed the formation of products. The addition of the remaining amino acids led to an increase of molar product yields (glucose, 5-deoxy-glucitol and 2-deoxy-glucitol), which was related to the decomposition of trehalose. This finding was explained by hydrogen transfer from the amino acids to precursor radicals of the products.     

Radiation-induced conversion of 5,6-dihydropyrimidines to the corresponding pyrimidines in N2O-saturated aqueous solutions

The effect of transition metal salts on the radiation-induced conversion of 5,6-dihydropyrimidines to the corresponding parent pyrimidines was studied in N2O-saturated aqueous solution at pH 7.0. The yield of the pyrimidines increased in sigmoidal forms with the increased one-electron reduction potential of the transition metal salts. The radiolysis of 5,6-dihydroorotic acid suggested that the 6-yl radical of the acid undergoes oxidation by transition metal salts to give orotic acid, whereas the corresponding 5-yl radical readily liberates CO2 to give uracil radical anion.     

Radiolytic hydroxylation of 1-substituted thymines promoted by transition metal salts in N2- and N2O-saturated aqueous solutions

The effect of high-valent transition metal salts on the radiation-induced hydroxylation of thymine (1a), 1-methylthymine (1b), and thymidine (1c) to the corresponding thymine glycol derivatives (2a-c) was studied at pH 7.0 in N2- and N2O-saturated aqueous solutions. The selectivities of (2a-c) based on converted (1a-c) increased to attain the maxima of 35-69% and then decreased, with increasing the one-electron reduction potential [E(M (n+1)+/Mn+)] of metal salts in the range of 0-1.0 V vs. NHE less than. Metal salts with E(M (n+1)+/Mn+) 0 or greater 1.0 V vs. NHE caused little change in the yields of (2a-c).     

Radiation-induced generation of chlorine derivatives in N2O-saturated phosphate buffered saline: toxic effects on Escherichia coli cells.

The radiolysis of aqueous chloride solutions has been investigated using pulse and steady-state methods. We have found a correlation between the yields of Cl2- and HOCl formed in pulse-irradiated N2O-saturated solutions. The yields increased with the increasing concentrations of Cl- and phosphate. Phosphate enhanced the yield of Cl2- in neutral solutions because of a proton transfer from H2PO4- to HOCl- with a rate constant of (2.6 +/- 0.5) x 10(8) M-1s-1. HOCl could not be detected in pulse-irradiated He or air-saturated, phosphate-buffered saline (PBS) solutions or in gamma-irradiated N2O, He, or air-saturated PBS solutions. The results are discussed in light of previously suggested mechanisms for the formation and decay of Cl2-. Pulse-irradiated N2O-saturated PBS solutions have a lethal effect on Escherichia coli cells, which is proportional to the amount of HOCl in the solutions. Gamma-irradiation of cells in N2O-saturated PBS solution also raises the radiosensitivity of the cells, although HOCl does not accumulate in this system. The effects of the radiation-induced toxic products on E. coli cells are similar to the effects of NaOCl. The cell membrane is probably the site of physiological injury induced by the radiation products.     

Radiolytic reduction of protein and nonprotein disulfides in the presence of formate: a chain reaction

We have reported recently that the disulfide groups in bovine serum albumin can be reduced by a radiolytic chain reaction which occurs in deoxygenated solutions containing formate ions. This reaction, which involves the reduction of disulfide groups by hydrated electrons and carbon dioxide radical anions, has now been studied in greater detail and compared with an analogous reaction in small, disulfide containing molecules over a range of pH values and substrate concentrations. A two-step reaction is proposed to account for the reduction of disulfides in reactions which can have chain lengths of 20 or more. Thiols produced by the disulfide reduction are stable to the conditions of the reaction. For example, a biological assay showed that the integrity of glutathione was maintained even at radiation doses much larger than those required to achieve complete reduction of glutathione disulfide. It was found that the extent of disulfide reduction could easily be controlled by varying the radiation dose delivered to the solutions. Radiolytic reduction is a very useful way of reducing protein and low molecular weight disulfides without the use of excess quantities of reagents such as dithiothreitol. In many cases, the reaction solutions could be used directly for subsequent reactions and this may be of considerable value in modifying the structure of hormones, enzymes, membrane receptors, and other disulfide containing proteins. If ammonium formate is used, freeze drying is an effective way to remove the formate salt, should this be required.     

A coalescent approach to the polymerase chain reaction.

A versatile algorithm is developed to model PCR on a computer. The method is based on a modification of the coalescent process and provides a general framework to analyse data from PCR. It allows for incorporation of the dynamics of the replication process as described in terms of the number of starting template molecules and cycle-dependent PCR efficiency. The simulation method generates, as a first step, the genealogy of a set of sequences sampled from a final PCR product. In a second step a mutation process is superimposed and the resulting data set is analysed. The efficiency of our algorithm enables us to get reliable approximations of various sample distributions. We demonstrate the relevance of our method with two applications: maximum likelihood estimation of the error rate in PCR and a test of homogeneity of the template.     

Gamma-radiolysis of D-glucose in aerated, aqueous solution.

Gamma-Radiolysis of D-glucose in aerated, aqueous solution gives mainly D-glucono-1,5-lactone, D-arabino-hexosulose, and D-ribo-hexos-3-ulose, together with D-xylo-hexos-4-ulose, D-xylo-hexos-5-ulose, and other pentose, tetrose, and triose derivatives as minor products, which were estimated by mass spectrometry of their alditol-d acetates. These hexose derivatives appear to be produced by the decomposition of D-glucose peroxy-radicals which are formed by the reaction of the primary radicals of D-glucose with oxygen. Bond scission of the peroxy-radicals yields triose, tetrose, and pentose. A radiolysis mechanism for the degradation of D-glucose in aerated, aqueous solution is proposed, based on the reaction of several kinds of D-glucose radical with oxygen.     

Quantitative analysis of formate in solutions containing phosphate

Standard colorimetric methods based on the initial reduction of formate to formaldehyde were found to yield erratic results when applied to the analysis of millimolar concentrations of formate in a microbial culture medium. The source of interference was identified as inorganic orthophosphate inhibition of the magnesium/hydrochloric acid reduction stage. Passivation of magnesium by millimolar concentrations of phosphate is known to occur at low pH and it is proposed that this phenomenon is responsible for the inhibition of the reduction process. The presence of orthophosphate in biological extracts is almost universal and would lead to acceptance of spuriously low values for formate concentration if the previously unreported inhibitory effect is not recognized. The colorimetric method of Barker and Somers in which formate is reacted directly with 2-thiobarbituric acid to form the chromophore was evaluated and proved to be entirely free from interference by orthophosphate and other medium components. This method although less sensitive than the formate reduction methods is therefore suggested as the method of choice for the determination of formate in biological or other solutions containing phosphate.     

Targeted gene walking polymerase chain reaction.

We describe a modification of a polymerase chain reaction method called 'targeted gene walking' that can be used for the amplification of unknown DNA sequences adjacent to a short stretch of known sequence by using the combination of a single, targeted sequence specific PCR primer with a second, nonspecific 'walking' primer. This technique can replace conventional cloning and screening methods with a single step PCR protocol to greatly expedite the isolation of sequences either upstream or downstream from a known sequence. A number of potential applications are discussed, including its utility as an alternative to cloning and screening for new genes or cDNAs, as a method for searching for polymorphic sites, restriction endonuclease or regulatory regions, and its adaptation to rapidly sequence DNA of lengthy unknown regions that are contiguous to known genes.