Early Postmortem Detection of Exudative Pork Meat Based on Nucleotide Content

The content of nucleotide metabolites in the muscle L. dorsi was analyzed for the detection of pale, soft and exudative (PSE) pork meats at just 2 h postmortem. PSE meat was characterized by significantly lower (p < 0.05) amounts of adenosine 5'-triphosphate (ATP) and higher adenosine 5'-monophosphate (AMP), inosine 5'-monophosphate (IMP), inosine (ino) and hypoxanthine (hyp) than normal meat. IMP and ino classified all the samples from both groups with statistical significance (p < 0.05). The K₀ value, R' value and IMP/ATP ratios were also useful indicators for full distinction of PSE meats. Thus, the assay of any of these nucleotide metabolites may allow a good detection of PSE meats at very short, only 2 h, postmortem time.     

Polymerase Synthesis of DNA Containing Iodinated Pyrimidine or 7-Deazapurine Nucleobases and Their Post-synthetic Modifications through the Suzuki-Miyaura Cross-Coupling Reactions

All four iodinated 2′-deoxyribonucleoside triphosphates (dNTPs) derived from 5-iodouracil, 5-iodocytosine, 7-iodo-7-deazaadenine and 7-iodo-7-deazaguanine were prepared and studied as substrates for KOD XL DNA polymerase. All of the nucleotides were readily incorporated by primer extension and by PCR amplification to form DNA containing iodinated nucleobases. Systematic study of the Suzuki-Miyaura cross-coupling reactions with two bulkier arylboronic acids revealed that the 5-iodopyrimidines were more reactive and gave cross-coupling products both in the terminal or internal position in single-stranded oligonucleotides (ssONs) and in the terminal position of double-stranded DNA (dsDNA), whereas the 7-iodo-7-deazapurines were less reactive and gave cross-coupling products only in the terminal position. None of the four iodinated bases reacted in an internal position of dsDNA. These findings are useful for the use of the iodinated nucleobases for post-synthetic modification of DNA with functional groups for various applications.     

Reaction of cytidine nucleotides with cyanoacetylene: support for the intermediacy of nucleoside-2',3'-cyclic phosphates in the prebiotic synthesis of RNA

A robust and prebiotically plausible synthesis of RNA is a key requirement of the "RNA World" hypothesis, but, to date, no such synthesis has been demonstrated. Monomer synthesis strategies involving attachment of preformed nucleobases to sugars have failed, and, even if activated 5'-nucleotides could be made, the hydrolysis of these intermediates in water makes their efficient oligomerisation appear unlikely. We recently reported a synthesis of cytidine-2',3'-cyclic phosphate 1 (C>p) in which the nucleobase was assembled in stages on a sugar-phosphate template. However, 2',3'-cyclic nucleotides (N>p's) also undergo hydrolysis, in this case giving a mixture of the 2'- and 3'-monophosphates. This hydrolysis has previously been seen as making the, otherwise promising, oligomerisation of N>p's seem as unlikely as that of the 5'-activated nucleotides. We now find that cyanoacetylene, the reagent used for the second stage of nucleobase assembly in the synthesis of C>p, also reverses the effect of the hydrolysis by driving efficient cyclisation of C2'p and C3'p back to C>p. Excess cyanoacetylene also derivatises the nucleobase, but this modification is reversible at neutral pH. These findings significantly strengthen the case for N>p's in a prebiotic synthesis of RNA.     

A new photoactive building block for investigation of DNA backbone interactions: photoaffinity labeling of human DNA polymerase beta

The cross-linking of target proteins or nucleic acids to light-activatable ligands is an important tool for elucidating molecular interactions. Through the use of photoaffinity-labeling reagents, several new insights into nucleic acid interactions have been obtained, for example in DNA replication and repair. In most known photoprobes, the applied light-sensitive functionalities are placed directly at the nucleobase or are attached via linkers to either the nucleobase or the phosphate backbone. Here we describe the first photoprobe that bears a light-sensitive aryl(trifluoromethyl)diazirine at the sugar moiety of a DNA oligonucleotide. We devised a route for the synthesis of the modified nucleoside and its incorporation into an oligonucleotide. The photoactive species was proven to be stable under the conditions employed in routine automated DNA synthesis. The modified oligonucleotide was shown by subsequent photolabeling studies of human DNA polymerase beta to form a covalent complex to the enzyme upon irradiation with near-UV light.     

Synthesis of C8‐N‐Arylamine‐Modified 2′‐Deoxyguanosine‐5′‐Triphosphates and Their Effects on Primer Extension by DNA Polymerases

C8‐N‐arylamine adducts of 2′‐deoxyguanosine (2′‐dG) play an important role in the induction of the chemical carcinogenesis caused by aromatic amines. C8‐N‐acetyl‐N‐arylamine dG adducts that differ in their substitution pattern in the aniline moiety were converted by cycloSal technology into the corresponding C8‐N‐acetyl‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates and C8‐NH‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates. Their conformation preference has been investigated by NOE spectroscopy and DFT calculations. The substrate properties of the C8‐dG adducts were studied in primer‐extension assays by using Klenow fragment exo^? of Escherichia coli DNA polymerase I and human DNA polymerase β. It was shown that the incorporation was independent of the substitution pattern in the aryl moiety and the N‐acetyl group. Although the triphosphates were poor substrates for the human polymerases, they were incorporated twice before the termination of the elongation process occurred; this might demonstrate the importance of C8‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates in chemical carcinogenesis.     

A Quantitative Comparison of Wild‐Type and Gatekeeper Mutant Cdk2 for Chemical Genetic Studies with ATP Analogues

Mutant kinase kinetics : Protein kinases with enlarged ATP binding sites are increasingly being used as tools to probe the functioning signal transduction cascades. Using human cyclin‐dependent kinase 2 as a model system, we demonstrate that enlargement of the ATP binding site does not substantially alter either the catalysis kinetics nor substrate or phosphorylation site selection.

     

Effect of gamma irradiation on the keeping quality and nutrients of tilapia (Oreochromis mossambicus) and silver carp (Hypophthalmichthys molitrix) stored at 1.C

Fillets (skin on) obtained from freshly killed tilapia (Oreochromis mossambicus Peters) and silver carp (Hypophthalmichythys molitrix Curier & Valenciennes) were gamma irradiated at 1.0 kGy at 2–4.C. The chemical con position and thiamin and riboflavin contents of irradiated fish fillets were analysed and compared with those of the non-irradiated fish. Loss of thiamin content of silver carp fillets was significant whereas the thiamin content of tilapia and other constituents of tilapia and silver carp were unaffected. Irradiation at 1.0 kGy markedly reduced the bacterial level of tilapia and silver carp. No significant increase in total counts in both irradiated and non-irradiated samples was found during 5 days' storage at 1.C. The pattern of nucleotide changes in tilapia and silver carp was different. Irradiation had little effect on the concentrations of nucleotide catabolites in both species. Sensory evaluation of flesh colour and flavour was conducted after 5 days' refrigerated storage. Except that the colour of irradiated carp was more reddish, there were no significant differences in other qualities.