Premitotic chromosome individualization in mammalian cells depends on topoisomerase II activity

When DNA topoisomerase II (topo II) activity is inhibited with a non-DNA-damaging topo II inhibitor (ICRF-193), mammalian cells become checkpoint arrested in G2-phase. In this study, we analyzed chromosome structure in cells that bypassed this checkpoint. We observed a novel type of chromosome aberration, which we call Ω-figures. These are entangled chromosome regions that indicate the persistence of catenations between nonhomologous sequences. The number of Ω- figures per cell increased sharply as cells evaded the transient block imposed by the topo II-dependent checkpoint, and the presence of caffeine (a checkpoint-evading agent) potentiated this increase. Thus, the removal of nonreplicative catenations, a process that promotes chromosome individualization in G2, may be monitored by the topo II-dependent checkpoint in mammals. Received: 19 July 1999; in revised form: 20 October 1999 / Accepted: 7 January 2000     

Cell-specific differences in the susceptibility of potential cellular targets of human origin derived from blood and lung following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The induction of cytochrome P4501A (CYP1A1) enzyme activity is one of the best-studied direct effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds and has been shown to be a sensitive biomarker of exposure to polycyclic aromatic hydrocarbons (PAH) in different experimental animal species as well as in humans. TCDD has also been shown to modulate cytokine gene expression in human keratinocytes, including IL-1, TGF- and TFG-₂. In the present studies, the aim was to determine whether different cellular targets of human origin differed in susceptibility to TCDD as measured by CYP1A1 activity and mRNA expression, and whether cytokine gene induction/suppression correlated with TCDD susceptibility. Human airway epithelial cells, alveolar macrophages (AM), peripheral blood monocytes and lymphocytes (PBL) were exposed to 10^-10–10^-7 mol/L TCDD. CYP1A1 enzyme activity was determined by ethoxyresorufin-O-deethylase (EROD) activity, mRNA expression of CYP1A1 was measured by semiquantitative PCR assay. The secretion and/or gene expression of specific cytokines, including IL-6, IL-8, and IL-1 were also examined. Overall, there was a clear correlation between TCDD-induced enzyme activity and CYP1A1 mRNA levels, which were dose-dependently increased in the bronchoepithelial cells and PBL. The human airway epithelial cells (BEAS-S6 cell line and primary cells) appeared to be the most inducible cellular target, with up to 50-fold increases at 10^-8 mol/L TCDD with an EC₅₀ of 3×10^-11 mol/L TCDD. The pokeweed mitogen-activated peripheral blood lymphocytes revealed approximately 5-fold less capacity in CYP1A1 activity, with high interindividual variabilities (EC₅₀ 3×10^-9 mol/L TCDD). In contrast, CYP1A1 enzyme activity in both AM and purified peripheral blood monocytes, which were costimulated with LPS and/or GM-CSF, could not be detected. CYP1A1 mRNA levels, however, were detectable and only marginally enhanced in response to TCDD. The ability of all these cells to express and produce the proinflammatory cytokines IL-6 and IL-8 was neither enhanced nor impaired by TCDD. These results indicate that cell types found in human lung and peripheral blood vary in susceptibility to TCDD, with the lung epithelium being highly susceptible and the alveolar macrophage being nonsusceptible. However, expression and production of specific cytokines such as IL-6 and IL-8, which may potentiate inflammatory processes and/or work as mitogens, does not appear to be influenced by TCDD.     

Redescription of Favella ehrenbergii (Claparède and Lachmann, 1858) Jörgensen, 1924 (Ciliophora: Choreotrichia), with Phylogenetic Analyses Based on Small Subunit rRNA Gene Sequences

ABSTRACT. The identification of Favella ehrenbergii, a marine planktonic ciliate, has largely been based on its lorica features. This approach is potentially problematic given the polymorphic lorica during this organism's life cycle. We isolated a population of F. ehrenbergii from the coastal waters of Incheon, Korea, and revealed its infraciliature using the protargol staining method. Phylogenetic analysis based on small subunit rRNA gene sequences was also performed. Results showed that this population possessed 16 collar membranelles (CM) and about 100 somatic kineties. These features are highly conserved, even in later dividers. As such, the number of CM and somatic kineties can be used as key characteristics for identification of Favella species.     

Electronic circular dichroism of monomethyl [O, O, O]-phosphate and [O, O, O]-thiophosphate revisited

Phosphoryl-transfer reactions have long been of interest due to their importance in maintaining numerous cellular functions. A phosphoryl-transfer reaction results in two possible stereochemical outcomes: either retention or inversion of configuration at the transferred phosphorus atom. When the product is phosphate, isotopically-labeled [¹⁶O, ¹⁷O, ¹⁸O]-phosphate derivatives can be used to distinguish these outcomes; one oxygen must be replaced by sulfur or esterified to achieve isotopic chirality. Conventionally, stereochemical analysis of isotopically chiral phosphate has been based on ³¹P NMR spectroscopy and involves complex chemical or enzymatic transformations. An attractive alternative would be direct determination of the enantiomeric excess using chiroptical spectroscopy. (S)-Methyl-[¹⁶O, ¹⁷O, ¹⁸O]-phosphate (MePi^∗), 7 and enantiomeric [¹⁶O, ¹⁷O, ¹⁸O]-thiophosphate (TPi^∗), 10, were previously reported to exhibit weak electronic circular dichroism (ECD), although with 10 the result was considered to be uncertain. We have now re-examined the possibility that excesses of 7 and 10 enantiomers can be detected by ECD spectrometry, using both experimental and theoretical approaches. 7 and both the (R) and (S) enantiomers of 10 (10a, 10b) were synthesized by the ‘Oxford route’ and characterized by ¹H, ³¹P and ¹⁷O NMR, and by MS analysis. Weak ECD could be found for 7, with suboptimal S/N. No significant ECD could be detected for the 10 enantiomers.Time-dependent DFT (TDDFT) calculations of the electronic excitation energies and rotational strengths of the same three enantiomers were carried out using the functional B3LYP and the basis set 6-311G^∗∗. The isotopically-perturbed geometries were predicted using the anharmonic vibrational frequency calculational code in GAUSSIAN 03. In the case of 10, calculations were also carried out for the hexahydrated complex to investigate the influence of the aqueous solvent. The predicted excitation wavelengths are greater than the observed wavelengths, a not unusual result of TDDFT calculations. The predicted anisotropy ratios are 2.9 × 10⁻⁵ for 7, −5.3 × 10⁻⁶ for 10a/b, and 1.7 × 10⁻⁶ for 10a/b⋅(H₂O)₆. For 7 the predicted anisotropy ratio approximates that observed in this work, 4.5 × 10⁻⁵ at 208 nm. For 10a/b, the upper limits of the experimental anisotropy ratios (<5 × 10⁻⁶ at 225 nm, pH 9; <5 × 10⁻⁶ at 236 nm, pH 12) are comparable to the predicted magnitude of the value for 10a/b. The lower predicted value for 10a/b · (H₂O)₆ suggests that the aqueous environment affects the ECD significantly. Altogether, the TDDFT calculations together with a stereochemical analysis based on NMR and the MS data support the conclusion that the experimental ECD results for MePi^∗ and TPi^∗ may be reliable in order of magnitude.     

Hypermethylation of Fads2 and altered hepatic fatty acid and phospholipid metabolism in mice with hyperhomocysteinemia

Alterations in lipid metabolism may play a role in the vascular pathology associated with hyperhomocysteinemia (HHcy). Homocysteine is linked to lipid metabolism through the methionine cycle and the synthesis of phosphatidylcholine (PC) by phosphatidylethanolamine (PE) methyltransferase, which is responsible for the synthesis of 20-40% of liver PC. The goal of the present study was to determine if the reduced methylation capacity in HHcy is associated with alterations in liver phospholipid and fatty acid metabolism. Mice heterozygous for disruption of cystathionine beta-synthase (Cbs+/-) fed a diet to induce HHcy (HH diet) had higher (p<0.001) plasma total homocysteine (30.8+/-4.4 microM, mean+/-S.E.) than C57BL/6 mice (Cbs+/+) fed the HH diet (7.0+/-1.1 microM) or Cbs+/+ mice fed a control diet (2.3+/-0.3 microM). Mild and moderate HHcy was accompanied by lower adenosylmethionine/adenosylhomocysteine ratios (p<0.05), higher PE (p<0.05) and PE/PC ratios (p<0.01), lower PE methyltransferase activity (p<0.001), and higher linoleic acid (p<0.05) and lower arachidonic acid (p<0.05) in PE. Mice with moderate HHcy also had higher linoleic acid and alpha-linolenic acid (p<0.05) and lower arachidonic acid and docosahexaenoic acid (p<0.05) in liver PC. The first step in the desaturation and elongation of linoleic acid and linolenic acid to arachidonic acid and docosahexaenoic acid, respectively, is catalyzed by Delta6-desaturase (encoded by Fads2). We found hypermethylation of the Fads2 promoter (p<0.01), lower Fads2 mRNA (p<0.05), and lower Delta6-desaturase activity (p<0.001) in liver from mice with HHcy. These findings suggest that methylation silencing of liver Fads2 expression and changes in liver fatty acids may contribute to the pathology of HHcy.     

Accuracy of radioanalytical procedures used to determine the biobased content of manufactured products

Radiocarbon analyses were used to determine the "biobased content" of a variety of diverse samples. The theoretical biobased contents of those samples were compared to the biobased content values obtained by radiocarbon analyses. Results of this work indicated that the radiocarbon analyses provided accurate (within +/-3%, absolute) biobased content values for the samples tested. It is not practical to examine the accuracy of the radiocarbon analyses for every possible type of sample matrix. However, based on analyses performed on various types of samples, every indication is that the analyses provide accurate and reliable results on the biobased content of liquid and solid materials.     

Upper thermal tolerance of wild‐type,domesticated and growth hormone‐transgenic coho salmon Oncorhynchus kisutch

In coho salmon Oncorhynchus kisutch, no significant differences in critical thermal maximum (c. 26·9° C, C_Tmax) were observed among size‐matched wild‐type, domesticated, growth hormone (GH)‐transgenic fish fed to satiation, and GH‐transgenic fish on a ration‐restricted diet. Instead, GH‐transgenic fish fed to satiation had significantly higher maximum heart rate and Arrhenius breakpoint temperature (mean ± s.e. = 17·3 ± 0·1° C, T_AB). These results provide insight into effects of modified growth rate on temperature tolerance in salmonids, and can be used to assess the potential ecological consequences of GH‐transgenic fishes should they enter natural environments with temperatures near their thermal tolerance limits.     

Standing genetic variation and compensatory evolution in transgenic organisms: a growth-enhanced salmon simulation

Genetically modified strains usually are generated within defined genetic backgrounds to minimize variation for the engineered characteristic in order to facilitate basic research investigations or for commercial application. However, interactions between transgenes and genetic background have been documented in both model and commercial agricultural species, indicating that allelic variation at transgene-modifying loci are not uncommon in genomes. Engineered organisms that have the potential to allow entry of transgenes into natural populations may cause changes to ecosystems via the interaction of their specific phenotypes with ecosystem components and services. A transgene introgressing through natural populations is likely to encounter a range of natural genetic variation (among individuals or sub-populations) that could result in changes in phenotype, concomitant with effects on fitness and ecosystem consequences that differ from that seen in the progenitor transgenic strain. In the present study, using a growth hormone transgenic salmon example, we have modeled selection of modifier loci (single and multiple) in the presence of a transgene and have found that accounting for genetic background can significantly affect the persistence of transgenes in populations, potentially reducing or reversing a "Trojan gene" effect. Influences from altered life history characteristics (e.g., developmental timing, age of maturation) and compensatory demographic/ecosystem controls (e.g., density dependence) also were found to have a strong influence on transgene effects. Further, with the presence of a transgene in a population, genetic backgrounds were found to shift in non-transgenic individuals as well, an effect expected to direct phenotypes away from naturally selected optima. The present model has revealed the importance of understanding effects of selection for background genetics on the evolution of phenotypes in populations harbouring transgenes.     

Testing for an unusual distribution of rare variants

Technological advances make it possible to use high-throughput sequencing as a primary discovery tool of medical genetics, specifically for assaying rare variation. Still this approach faces the analytic challenge that the influence of very rare variants can only be evaluated effectively as a group. A further complication is that any given rare variant could have no effect, could increase risk, or could be protective. We propose here the C-alpha test statistic as a novel approach for testing for the presence of this mixture of effects across a set of rare variants. Unlike existing burden tests, C-alpha, by testing the variance rather than the mean, maintains consistent power when the target set contains both risk and protective variants. Through simulations and analysis of case/control data, we demonstrate good power relative to existing methods that assess the burden of rare variants in individuals.     

Linkage analysis of a completely ascertained sample of familial schizophrenics and bipolars from Palau, Micronesia

We report on linkage analysis of a completely ascertained population of familial psychosis derived from the oceanic nation of Palau. Palau, an archipelago of islands in the Southern Pacific, currently has a population of approximately 23,000 individuals. The peoples of Palau populated these islands recently in human history, approximately 2,000 years ago. As both historical and genetic evidence suggest, the population is far more homogeneous than most other populations undergoing genetic studies, and should therefore prove quite useful for mapping genetic variants having a meaningful impact on susceptibility to psychotic disorders. Moreover, for our study, essentially all on-island schizophrenics (150) and individuals with other psychotic disorders (25) participated. By analysis of narrow (only schizophrenia) and broad (all psychosis) diagnostic schemes, two-point linkage analyses suggest that two regions of the genome harbor genetic variants affecting liability in most families, 3q28 (LOD=3.03) and 17q32.2 (LOD=2.80). Results from individual pedigrees also support 2q37.2, 2p14, and 17p13 as potentially harboring important genetic variants. Most of these regions have been implicated in other genetic studies of psychosis in populations physically quite distant from this Oceanic population, although some (e.g., 3q28) appear to be novel results for schizophrenia linkage analyses.