Asymmetric distribution of exogenous thymidine among pyrimidine isostichs suggests compartmentalization of replicative DNA synthesis during regeneration in rat liver

The distribution of radioactivity among pyrimidine isostichs (or isoplyths) of DNA from 24-h regenerating rat liver was studied with [³H]Thd, [¹⁴C]orotate or with inorganic ³²P_i. Expression of incorporated radioactivity as log₁₀% of total radioactivity recovered for each of the 11 pyrimidine isostichs detected showed that radioactivity from [³H]Thd was asymmetrically distributed among the isostichs, i.e., ³H radioactivity failed to access regions of DNA yielding lower molecular weight pyrimidine isostichs as efficiently as it accessed regions yielding higher molecular weight pyrimidine isostichs. The thymine (T) content of isostichs exceeded that of cytosine (C), i.e., ratios for the first 10 isostichs averaged 1.43 ± 0.08 and 1.28 ± 0.05, depending on the method of analysis; furthermore, the ratio for isostich 1 was significantly higher than ratios for isostichs 2 through 10. Asymmetric distributions of [³H]Thd radioactivity also were seen at 18 or 30 h post-partial hepatectomy. Thus, radioactivity from [³H]Thd, a DNA precursor from the salvage pathway, failed to efficiently access lower molecuar weight isostichs despite thymine enrichment, suggesting that thymine moieties were supplied from additional sources. Radioactivity from [¹⁴C]orotate accessed lower molecular weight pyrimidine tracts more efficiently than [³H]Thd, but less efficiently than it accessed higher molecular weight isostichs, resulting in an asymmetric distribution of ¹⁴C radioactivity. This result suggested that appreciable quantities of thymine and cytosine moieties utilized for DNA synthesis were supplied de novo, but other sources also were utilized. Radioactivity from ³²P_i, a de novo precursor, was distributed symmetrically, i.e., the slope among lower molecular weight isostichs increased enough that it was indistinguishable from slopes for intermediate and higher molecular weight isostichs. Since ³²P radioactivity among lower molecular weight isostichs reflects appreciable contributions of de novo phosphate moieties from both pyrimidine- and purine-containing deoxynucleoside triphosphates, opportunities for observing contributions of ³²P radioactivity from pathways other than the de novo pathways appeared to lie beyond limits of detectability. The distribution of radioactivity from labeled DNA precursors among lower molecular weight pyrimidine tracts (a) indicate that thymine moieties are contributed by both salvage and de novo pathways; (b) support the possibility that cytosine moieties also are contributed by both pathways; and (c) support the ‘replitase’ concept for channeling dNTPs to replicating forks.     

Withdrawal rates of DDT from chickens treated with diphenylhydantoin.

Male and female chickens of a broiler-type strain were fed, from 1 day old to 5 weeks of age, diets containing 0, 2.5, or 15.0 p.p.m. (mg/kg) 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (p,p'-DDT). Then the diets with pesticide were withdrawn and the chickens were fed dietary levels of diphenylhydantoin (DPH) at 0, 100, or 250 p.p.m. Adipose-tissue and liver samples were obtained on days 0, 10, 20, and 30 following withdrawal of diets with pesticides to determine DPH effect on DDT, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD) levels. DPH had no effect on the concentration of DDT and DDE in adipose tissue; their levels declined at a rate having a half-life value of 16 days. DDD was not detected in adipose tissue. DDT accounted for 87% of the adipose residues on day 0, but 66% of the residues at day 30. DPH had no effect on the concentrations of DDT and DDE in livers of chickens fed 15.0 p.p.m. DDT, but did significantly reduce the levels of DDD by 28 and 54% for levels of 100 and 250 p.p.m. DPH, respectively. The similarity of these data to studies on dairy cows and humans, and the dissimilarity to data from rat studies were discussed.     

The influence of DNA sequence on terbium (III) fluorescence enhancement by DNA

The synthetic DNA duplexes, poly(dA-dC):poly(dG-dT), poly(dG):poly(dC), poly(dG-dC):poly(dG-dC), and poly(dG-m5dC):poly(dG-m5dC), were analyzed as double- and single-strand polymers for the ability to enhance terbium fluorescence. Using conditions which limited the enhancement of Tb3+ fluorescence to that from DNA-guanosines, our results showed that (a) guanosines in single-strand DNA enhanced terbium fluorescence equally well irrespective of the primary sequence surrounding them, and (b) guanosines in either left- (Z-form) or right- (B-form) handed double helixes failed to enhance terbium fluorescence.     

Inhibition of chloramphenicol binding to Escherichia coli 70S ribosomes by 2'(3')-O-aminoacyl-dinucleoside phosphates derived from the aminoacyl-tRNA acceptor terminus.

The effect of 2' and 3'-O-aminoacyl-dinucleoside phosphates cytidylyl(3'-5')-2'(3')-O-L-phenyl-alanyladenosine (I), cytidylyl(3'-5')-3'-deoxy-2'-O-L-phenylalanyladenosine (IIa), cytidylyl(3'-5')-2'-deoxy-3'-O-L-phenylalanyladenosine (IIIa), cytidylyl(3'-5')-3'-deoxy-2'-O-glycyladenosine (IIb), cytidylyl(3'-5')-2'-deoxy-3'-O-glycyladenosine (IIIb), cytidylyl(3'-5')-3'-deoxy-2'-O-L-leucyladenosine (IIc), cytidylyl(3'-5')-2'-deoxy-3'-O-L-leucyladenosine (IIIc), cytidylyl(3'-5')-3'-O-L-phenylalanyladenosine (IIId) as analogs of the 2'(3')-aminoacyl-tRNA termini, on chloramphenicol binding to 70S Excherichia coli ribosomes was investigated. The association constants (Kb) of the investigated compounds were determined by the equilibrium dialysis method. Based on the constancy of Kb over the range of inhibitor concentration, it was determined that the binding site of the 2' isomers IIa-IIc overlaps with the chloramphenicol site, whereas the variability of Kb for the 3' isomers IIIb, IIIc and especially IIIa seems to indicate that they do not achieve a complete fit. The consistently higher values of the Kb values for the 3' isomers IIIa-IIIc relative to that of the 2' isomers IIa-IIc also indicate a stabilization of the binding of the former due to a specific interaction between its amino acid portion and a ribosomal site.     

HPV testing in liquid cytology specimens: comparison of analytic sensitivty and specificity for in situ hybridization and chemiluminescent nucleic acid testing

OBJECTIVE: To compare the analytic sensitivity and specificity of Hybrid Capture 2 (HC2) (Digene Corporation, Gaithersburg, Maryland, U.S.A.) with in situ hybridization (ISH) in detecting high-risk types of HPV (HR-HPV). STUDY DESIGN: Performance characteristics of ISH and HC2 were compared in 99 consecutive cervical cytology samples diagnosed as low grade squamous intraepithelial lesion and processed by either the ThinPrep (Cytyc Corporation, Boxborough, Massachusetts, U.S.A.) or SurePath (TriPath Imaging Systems, Research Triangle Park, North Carolina, U.S.A.) method at 2 geographically different centers. Polymerase chain reaction (PCR) was used as the gold standard. RESULTS: Of the samples, 67% were positive for HR-HPV viral types by PCR. ISH had a sensitivity of 0.87 as compared to 0.95 (p = 0.11) for HC2. The specificity of ISH was significantly different from that of HC2 (0.57 vs. 0.13, respectively; p = 0.0004). The performance characteristics of ISH were not affected by the processing method or population tested. CONCLUSION: The sensitivity of ISH is comparable to that of HC2, with significantly superior specificity, and is therefore an efficacious alternative to HC2 for triaging patients with abnormal cervical cytology results.     

Monoterpene double-bond reductases of the (-)-menthol biosynthetic pathway: isolation and characterization of cDNAs encoding (-)-isopiperitenone reductase and (+)-pulegone reductase of peppermint

Random sequencing of a peppermint essential oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes. Full-length acquisitions of each type were screened by functional expression in Escherichia coli using a newly developed in situ assay. cDNA clones encoding the monoterpene double-bond reductases (-)-isopiperitenone reductase and (+)-pulegone reductase were isolated, representing two central steps in the biosynthesis of (-)-menthol, the principal component of peppermint essential oil, and the first reductase genes of terpenoid metabolism to be described. The (-)-isopiperitenone reductase cDNA has an open reading frame of 942 nucleotides that encodes a 314 residue protein with a calculated molecular weight of 34,409. The recombinant reductase has an optimum pH of 5.5, and K(m) values of 1.0 and 2.2 microM for (-)-isopiperitenone and NADPH, respectively, with k(cat) of 1.3s(-1) for the formation of the product (+)-cis-isopulegone. The (+)-pulegone reductase cDNA has an open reading frame of 1026 nucleotides and encodes a 342 residue protein with a calculated molecular weight of 37,914. This recombinant reductase catalyzes the reduction of the 4(8)-double bond of (+)-pulegone to produce both (-)-menthone and (+)-isomenthone in a 55:45 ratio, has an optimum pH of 5.0, and K(m) values of 2.3 and 6.9 microM for (+)-pulegone and NADPH, respectively, with k(cat) of 1.8s(-1). Deduced sequence comparison revealed that these two highly substrate specific double-bond reductases show less than 12% identity. (-)-Isopiperitenone reductase is a member of the short-chain dehydrogenase/reductase superfamily and (+)-pulegone reductase is a member of the medium-chain dehydrogenase/reductase superfamily, implying very different evolutionary origins in spite of the similarity in substrates utilized and reactions catalyzed.