Ctrogenetic effects of mutagens/carcinogens after activation in a microsomal system in vitro I. Induction of chromosome aberrations and sister chromatid exchanges by diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) in CHO cells in the presence of rat-liver microsomes

A rat-liver microsomal system in vitro has been used to activate two indirectly acting carcinogens, DMN and DEN. On activation, both compounds were extremely potent in inducing chromosomal aberrations as well as sister chromatid exchanges in Chinese hamster cells. The implications of these findings and the potential utility of this technique to detect mutagens/carcinogens are discussed.     

Photoreactivation of UV induced cell killing, chromosome aberrations, sister chromatid exchanges, mutations and pyrimidine dimers in Xenopus laevis fibroblasts

Summary Fibroblasts from Xenopus laevis, which possess photoreactivating enzyme were used to study the influence of photoreactivating light on the frequency of pyrimidine dimers in DNA, chromosomal aberrations, sister chromatid exchanges, cell killing and the induction of gene mutations (ouabain-resistance) induced by 254 nm ultraviolet irradiation. The frequency of all biological endpoints studied were reduced following exposure to photoreactivating light parallel to the reduction in the frequencies of pyrimidine dimers (determined as endonuclease sensitive sites). However there was not always an absolute quantitative relationship between the reduction in the frequency of pyrimidine dimers and the reduction in the biological effects. This probably reflects a fast fixation process for the biological effects prior to removal of the dimers by photoreactivation.Abbreviations UV ultraviolet - PR photoreactivating - ESS endonuclease sensitive site - SCE sister chromatid exchanges - BrdUrd 5-brothodeoxyuridine     

The prebiological paleoatmosphere: Stability and composition

In the past, it was generally assumed that the early atmosphere of the Earth contained appreciable quantities of methane (CH₄) and ammonia (NH₃). This was the type of atmosphere believed to be the most suitable environment for chemical evolution, the nonbiological formation of complex organic molecules, the precursors of living systems. Photochemical considerations suggest that a CH₄–NH₃ dominated early atmosphere was probably very short-lived, if it ever existed at all. Instead, an early atmosphere of carbon dioxide (CO₂) and nitrogen (N₂) is favored by photochemical as well as geological and geochemical considerations. Photochemical calculations also indicate that the total oxygen column density of the prebiological paleoatmosphere did not exceed 10^–7 of the present atmospheric level.Paper presented at the 6th College Park Colloquium, October 1981     

A gene that regulates DNA replication in response to DNA damage is located on human chromosome 4q.

Inhibition of replicative DNA synthesis following gamma-irradiation is observed in eukaryotic cells but is defective in cells derived from patients with the cancer-prone inherited disorder ataxia-telangiectasia (A-T) and in A-T-like Chinese hamster cell mutants. Chinese hamster cells show a less pronounced inhibition of DNA synthesis after gamma-irradiation when compared to irradiated human HeLa or mouse A9 cells. Therefore, to identify new human genes involved in the regulation of DNA replication in response to ionizing radiation in mammalian cells, single human chromosomes were introduced into Chinese hamster cells by microcell-mediated chromosome transfer. It is found that a new gene on human chromosome 4q inhibits DNA synthesis following gamma- and UV irradiation in hamster cells. However, this delay of DNA replication did not improve cell survival or the level of chromosomal aberrations induced by X-rays, indicating that the lack of the inhibition of DNA synthesis after X-irradiation is not a prerequisite for the X-ray sensitivity and chromosomal instability, which is observed in A-T and A-T-like hamster cells.     

Mechanism of cigarette smoke condensate induced adhesion of human monocytes to cultured endothelial cells

Cigarette smoking is ranked among the leading risk factors in the etiology of atherosclerotic vascular disease. The mechanisms, however, that link cigarette smoking to increased incidence of atherosclerosis are not understood. The adherence of circulating monocytes to the endothelium, migration into the subendothelium, and subsequent formation of foam cells are principal initial events in the development of atherosclerosis. We therefore determined whether cigarette smoke caused increased adherence of monocytes to endothelial cells and the cellular mechanism of this increased adherence. Cigrette smoke condensate (CSC), the particulate fraction of cigarette smoke derived from 2R1 standard research cigarettes, at a concentration of 25–30 μg/ml (average yield of CSC is 26.1 mg/cigarette), augmented (70–90%) basal adherence of human peripheral blood monocytes to a cultured monolayer of endothelial cells derived from bovine aorta (BAEC) and human umbilical vein (HUVEC). There was a concomitant increase in the expression of CD11b ligand on the surface of monocytes as determined by flow cytometry, utilizing FITC conjugated Mab MO-1 (CD11b). However, nicotine (1–15 μg/ml) and cadmium sulfate (10 μg/ml), constituents of CSC, individually or in combination had no effect either on CD11b expression or adherence of monocytes to endothelial cells. Treatment of HUVEC with CSC for 60 min also resulted in an increased expression of ICAM-1 and ELAM-1 as determined by mean fluorescence intensity of ICAM-1 and ELAM-1 labeled cells in flow cytometric analysis. The CSC induced expression of CD11b in monocytes was optimal at 25–30 min and was inhibited by protein kinase C inhibitors, staurosporine and H-7, and also by baicalein, a lipoxygenase inhibitor. Similarly, CSC induced ICAM-1 and ELAM-1 expression in HUVEC was inhibited by protein kinase C inhibitors. CSC stimulated the adherence of human monocytes but not the monocytic cell lines HL-60, U937, and THP-1 to endothelial cells. The CSC stimulated adherence of human monocytes was inhibited (80%) by MAb to CD11b and 50% by Mab to ICAM-1 and ELAM-1. These results suggest that cigarettee smoke particulate constituents activate protein kinase C, leading to increased surface expression of adhesive ligand CD11b on peripheral blood monocytes and counter receptor(s) ICAM-1 and ELAM-1 in endothelial cells. The expression of ligand and counter receptor leads to potentiated adherence of monocytes to endothelial cells, an initial event in the pathogenesis of cigarette smoke induced inflammatory response in the vessel wall. © 1994 Wiley-Liss, Inc.     

Reductive dechlorination of PCB-contaminated raisin river sediments by anaerobic microbial granules

A polychlorinated biphenyl (PCB)-dechlorinating anaerobic microbial consortium, developed in a granular form, demonstrated extensive dechlorination of PCBs present in Raisin River sediments at room (20 degrees to 22 degrees C) and at a relatively low (12 degrees C) temperature. Highly chlorinated PCB congeners were dechlorinated and less chlorinated compounds were produced. The homolog comparison showed that tri-, tetra-, penta-, hexa-, and heptachlorobiphenyl compounds decreased significantly, and mono- and dichlorobiphenyl compounds increased. After 32 weeks of incubation at 12 degrees C, the predominant less chlorinated products included 2-, 4-, 2-2/26-, 24-, 2-4-, 24-2-, 26-2-, and 26-4-CB. Among these, 24- and 24-2-CB did not accumulate at room temperature, suggesting a further dechlorination of these congeners. Predominantly meta dechlorination (i.e., pattern M) was catalyzed by the microbial consortium in the granules. Dechlorination in the control studies without granules was not extensive. This study is the first demonstration of enhanced reductive dechlorination of sediment PCBs by an exogenous anaerobic microbial consortium. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 182-190, 1997.     

Molecular analysis of skewed Tcra-V gene use in T-cell receptor β-chain transgenic mice

 The influence of β-chain diversity on the expressed T-cell receptor (TCR) α-chain repertoire was investigated using transgenic mice which exclusively express a single rearranged TCR β-chain gene. Analysis of these mice using α-chain-specific recombinant cDNA libraries showed that expression of the transgene-encoded β chain results in significant skewing in Tcra-V gene segment usage vs nontransgenic mice. Skewing was most pronounced towards α chains using TCRA-V segments. Sequence analysis of Tcra-V8-containing genes from transgenic T cells revealed predominant use of a single Tcra-J segment (Tcra-J24), which was not detected in Tcra-V8 containing genes isolated from nontransgenic T cells. Further analysis revealed that co-expression of Tcra-V8 with Tcra-J24 in β-transgenic mice is exhibited almost exclusively by CD4⁺ T cells, and is associated with a limited number of closely related N-regions. Analysis of transgenic CD8⁺ T cells demonstrated predominant co-expression of Tcra-V8 with another Tcra-J (Tcra-J30), together with a different, limited N-region sequence. We conclude that the composition of expressed β chains can profoundly influence the selection of companion α chains expressed in the periphery, and that α-chain N and J regions play a crucial role in discriminating between class I vs class II major histocompatibility complex (MHC)-restricted recognition. Further, these results are in agreement with recent data concerning the crystal structure of the TCR, and most consistent with a model for TCR structure in which the complementarity determining region (CDR)3α domain participates in direct contact with the MHC. Received: 28 January 1997 / Revised: 22 July 1997     

Fluorescencein SituHybridization with Comets

We have adapted the fluorescencein situhybridization technique to single-cell gel electrophoresis (comet assayed) preparations. Since cells were embedded in agarose, probed regions could be visualized in three dimensions. This system makes it possible to determine the spatial distribution of chromosome-specific DNA sequences at the level of the individual nucleus (nonelectrophoresed) as well as in chromatin fibers of comets (electrostretched chromosomal DNA). This methodology is likely to bring new insights into the field of interphase nuclear ultrastructure. Here, we present the preliminary data obtained with human blood lymphocytes in G₀after they have been electrophoresed for different times. Chromosome-specific areas (all centromeres, all telomeres, chromosome 7-specific centromere, and long arm of chromosome 3-specific telomere, as well as three segments of theO⁶-methylguanine–DNA methyltransferase gene) were investigated. Our results are in agreement with the concept that telomeres are in close association with the nuclear membrane and suggest that centromeres are relatively less condensed structures located in the center of the interphase nucleus.     

Regulation of phospholipase D by tyrosine kinases

Activation of phospholipase D (PLD) represents part of an important signalling pathway in mammalian cells, Phospholipase D catalyzed hydrolysis of phospholipids generates phosphatidic acid (PA) which is subsequently metabolized to lyso-PA (LPA) or diacylglycerol (DAG). While DAG is an endogenous activator of protein kinase C (PKC), PA and LPA have been recognized as second messengers as well, Activation of PLD in response to an external stimulus may involve PKC, Ca²⁺, G-proteins and/or tyrosine kinases. In this review, we will address the role of protein tyrosine phosphorylation in growth factor-, agonist- and oxidant-mediated activation of PLD. Furthermore, a possible link between PKC, Ca²⁺, G-proteins and tyrosine kinases is discussed to indicate the complexity involved in the regulation of PLD in mammalian cells.     

Mutations affecting RNA splicing in man are detected more frequently in somatic than in germ cells

The spectrum of DNA sequence alterations in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of HPRTase-deficient T-lymphocytes isolated from the blood of healthy male donors was determined and compared with the spectrum found in patients suffering from genetic diseases (Lesch-Nyhan syndrome or gouty arthritis) associated with a mutation in the same gene. Most of the T-cell mutants still produced hprt mRNA which was converted into cDNA and used for DNA sequence analysis after amplification using the polymerase chain reaction (PCR). In 39% of the 31 analyzed T-cell mutants of normal donors 1 or 2 exons were completely or partially deleted from hprt mRNA, probably because of a mutation in a splice acceptor site. Among patients suffering from the Lesch-Nyhan syndrome or gouty arthritis, the class of splice mutations amounts only to 7%. These data suggest that carriers of splice mutations often do not show the characteristics of HPRTase deficiency associated with these genetic diseases, because correctly spliced hprt mRNA is still produced at a low level.