New observations concerning the chloroacetaldehyde reaction with some tRNA constituents. Stable intermediates, kinetics and selectivity of the reaction.

The stable intermediates formed in the reaction of cytosine, cytidine and adenosine with chloracetaldehyde were isolated. The -CH2CH/OH/- bridge between the exo and endo nitrogen atoms of the parent base was found in these compounds by means of PMR spectroscopy. Their acid-induced dehydration resulted in formation of appropriate ethenoderivatives. The rate constants of the intermediate formation and its dehydration were found to be 38x10(-4) and 47x10(-4) /min-1/ for adenosine, and 33x10(-4) and 10x10(-4) /min-1/ for cytidine. The PH range of 4.5--5.0 was found to be optimum for both adenosine and cytidine reactions. The quantitative modification of these two nucleosides in the presence of guanosine may be achieved with high selectivity only at a low pH of 3.0--4.0 N6-methyladenosine and N4-methylcytidine react quantitatively with chloroacetaldehyde and the reaction rate is higher than in the case of the parent nucleosides. The structure of the reaction products was assigned on the basis of PMR spectroscopy.     

Characterization of cytoplasmic arginine-rich basic protein of Guerin epithelioma

Summary Arginine-rich basic protein from cytoplasma of Guerin epitheliomas has been isolated and characterized. It contains five amino acids: arginine, lysine, glycine, alanine and glutamic acid which make together 74 per cent of all amino acid residues. The protein has a cationic character with an isoelectric point of 8.2. No carbohydrate component was found in this protein. The significance of arginine-rich basic protein in the cytoplasma of Guerin epithelioma is discussed briefly.     

Uptake of immune RNA by normal mouse spleen cells

Summary Normal mouse spleen cells take up in vitro radioactively labeled immune RNA. RNA taken up is present in nuclei, polysomes, membranes and cytoplasm. About 20–40% of immune RNA is nonspecifically associated with cell surface. 45% of RNA taken up is degraded and reutilized inside the cells within 2 hours.This work was supported by the Polish Academy of Sciences within the project 09.7.4.1.1.     

Genome-scale patterns in the loss of heterozygosity incidence in Saccharomyces cerevisiae

Former studies have established that loss of heterozygosity can be a key driver of sequence evolution in unicellular eukaryotes and tissues of metazoans. However, little is known about whether the distribution of loss of heterozygosity events is largely random or forms discernible patterns across genomes. To initiate our experiments, we introduced selectable markers to both arms of all chromosomes of the budding yeast. Subsequent extensive assays, repeated over several genetic backgrounds and environments, provided a wealth of information on the genetic and environmental determinants of loss of heterozygosity. Three findings stand out. First, the number of loss of heterozygosity events per unit time was more than 25 times higher for growing than starving cells. Second, loss of heterozygosity was most frequent when regions of homology around a recombination site were identical, about a half-% sequence divergence was sufficient to reduce its incidence. Finally, the density of loss of heterozygosity events was highly dependent on the genome’s physical architecture. It was several-fold higher on short chromosomal arms than on long ones. Comparably large differences were seen within a single arm where regions close to a centromere were visibly less affected than regions close, though usually not strictly adjacent, to a telomere. We suggest that the observed uneven distribution of loss of heterozygosity events could have been caused not only by an uneven density of initial DNA damages. Location-depended differences in the mode of DNA repair, or its effect on fitness, were likely to operate as well.     

The X-ray structure and absolute configuration of the anticancer drug (+)-4-ketocyclophosphamide

4-Ketocyclophosphamide (4-keto CP), C7H12Cl2N2O3P, monoclinic, P2(1), a = 11.909 (2), b = 10.254 (1), c = 9.873 (1) A, beta = 91.08 (1) degrees, V = 1205.45 (3) A3 Z = 4, Dc = 1.51 Mg-m-3, Cu K alpha, lambda = 1.54178 A, alpha 25 = +53.8 degrees (c = 3.0, MeOH), m.p. 107 degrees C, mu = 61.8 cm-1, F (000) = 564, R = 0.064 for 2961 observed reflexions with I greater than 1.96 sigma(I). Dextrarotatory enantiomer of 4-keto CP has S configuration at the stereogenic center. One of the two crystallographically independent molecules is disordered both in a six-membered ring and in --N(CH2CH2Cl)2 moiety. With the exception of a less populated conformer of a disordered molecule, 4-keto CP molecules adopt a conformation in which 1,3,2-oxazophosphorinane ring is in the sofa form with C(6) deviating from the plane through the remaining five ring atoms while an exocyclic N atom with its three substituents is nearly coplanar with the phosphoryl oxygen atom O(8). In a less populated conformer, the six membered ring takes the form of sofa with C(5) as a flap while an exocyclic N atom and its substituents are oriented toward the P--N(3) bond.     

Oxidative DNA Base Modifications and Polycyclic Aromatic Hydrocarbon DNA Adducts in Squamous Cell Carcinoma of Larynx

Tobacco smoke, recognized as a major etiological factor for cancers of the upper aerodigestive tract, represents an abundant source of reactive oxygen species (ROS), which are believed to play a significant role in mutagenesis and carcinogenesis. An additional source of ROS in tissues exposed to tobacco smoke may be metabolic oxidation of polycyclic aromatic hydrocarbons (PAH). To investigate the relationships between oxidative DNA lesions and aromatic DNA adducts, six modified DNA bases 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine and the total level of PAH-related DNA adducts were measured in cancerous and the surrounding normal larynx tissues (68 subjects), using gas chromatography/isotope-dilution mass spectroscopy with selected ion monitoring and the 32 P-postlabeling-HPLC assay, respectively. The levels of oxidative DNA lesions in cancerous and adjacent tissue were comparable; the differences between the two types of tissue were significant only for 5-hydroxypyrimidines (slightly higher levels were observed in the adjacent tissue). Comparable levels of DNA lesions in cancerous and the surrounding normal tissues observed in the larynx tumors support a field cancerization theory. The surrounding tissues may still be recognized as normal by histological criteria. However, molecular alterations resulting from the chronic tobacco smoke exposure, which equally affects larynx epithelia, may lead to multiple premalignant lesions. Thus, a demonstration of similar levels of DNA damage in cancerous and the adjacent tissue could explain a frequent formation of secondary tumors in the larynx and the frequent recurrence in this type of cancer. A weak, but distinct effect of tumor grading and metastatic status was observed in both kinds of tissue in the case of 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine. This effect was displayed as a gradual shift in the data distribution toward high values from G1 through G2-G3 and from non-metastatic to metastatic tumors. Since the levels of oxidative DNA base modifications tended to increase with the tumor aggressiveness, we postulate that the oxidative DNA lesions increase genetic instability and thus contribute to tumor progression in laryngeal cancer. No associations between aromatic adduct levels and oxidative DNA lesions were present, suggesting that the metabolism of PAH does not contribute significantly to the oxidative stress in larynx tissues, remaining the tobacco smoke ROS as a major source of oxidative DNA damage in the exposed tissue.     

Synthesis of 5′-O-Dimeth0Xytrityl-4-N-/6-Trifluoroacetamidohexyl/-2′-Deoxycytidine and its Application in the Synthesis of Biotin-Labeled Oligonucleotides

Abstract

5′3′-O-protected 4-N-tosyl-2′-deoxycyt id ine was converted with 1,6-diaminohexane to 4-N-/6-ami nohexyl/-2′-deoxycyt id i ne and then into 5′-0-d imethoxytr i ty 1 -k-N-/(-tr if luoroacetamidohexyl 1–2 ′-deoxycyt id ine, The latter was used to prepare oligonucleotides by the phosphoramidite approach. Deprotected oligomers were labeled with biotin.     

Postsynthetic Transformations of Oligodeoxynucleotides Originated at 6-Methylthio-purine Site

Abstract

New route to oligodeoxynucleotides labeled with fluorescent luminarine was explored. Regioselective oxidation of 6-methylthio-purines to 6-methylsulphoxides reactive toward pyridine was achieved. Upon UV irradiation of 6-pyridinium-purines oligonucleotide cleavage instead of phototransformation to luminarine was observed.     

Synthesis of,and Conformational Studies on, 2-Trifluoromethyl,Substituted Benzimidazole Ribofuranosides

Abstract

The 1-β-D-ribofuranosides of several 2-trifluoromethyl benzimidazoles were prepared by the fusion method, and their conformations, particularly about the glycosidic bond, determined by ¹H NMR spectroscopy.