XIPHOPHORUS ENZYME GENE EXPRESSION: TISSUE SPECIFICITY, GENETIC CONTROL, AND STABILITY IN CULTURED CELLS

Five permanent cell lines were developed from Xiphophorus maculatus, X. helleri, and their hybrids using three tissue sources, including adults and embryos of different stages. To evaluate cell line gene expression for retention of either tissue-of-origin-specific or ontogenetic stage-specific characters, the activity distribution of 44 enzyme loci was determined in 11 X. maculatus tissues, and the developmental genetics of 17 enzyme loci was charted in X. helleri and in helleri x maculatus hybrids using starch gel electrophoresis. In the process, eight new loci were discovered and characterized for Xiphophorus.^ No Xiphophorus cell line showed retention of tissue-of-origin-specific or ontogenetic stage-specific enzyme gene expressional traits. Instead, gene expression was similar among the cell lines. One enzyme, adenosine deaminase (ADA) was an exception. Two adult-origin cell lines expressed ADA, whereas, three cell lines derived independently from embryos did not. ADA('-) expression of Xiphophorus embryo-derived cell lines may represent retention of an embryonic gene expressional trait. In one cell line (T(,3)) derived from 13 pooled interspecific hybrid (F(,2)) embryos, shifts with time were observed at enzyme loci polymorphic between the two species. This suggested shifts in ratios of cells of different genotypes in the population rather than unstable gene expression in one dominant cell type.^ Verification of this hypothesis was attempted by cloning the culture--seeking clones having different genetic signatures. The large number of loci electrophoretically polymorphic between the two species and whose alleles segregated independently into the 13 progeny from which this culture originated almost guaranteed the presence of different genetic signatures (lineages) in T(,3).^ Seven lineages of cells were found within T(,3), each expressing genotypes at some loci not characteristic of the expression of the culture-as-a-whole, supporting the hypothesis tested. Quantitative studies of ADA expression in the whole culture (ADA('-)) and in clones of these seven lineages suggested the predominance in T(,3) of ADA deficient cell lineages, although moderate to high ADA output clones also occurred. Thus, T(,3) has the potential to shift phenotypes from ADA('-) to ADA('+) by simply changing proportions of its constituent cell types, demonstrating that such shifts can occur in any cell culture containing cells of mixed expressional characteristics.^

HEMATOCRIT AND HEMOGLOBIN, ATP AND DPG CONCENTRATIONS IN ANDEAN MAN: VARIABILITY BY SEX, AGE, VILLAGE, ALTITUDE, WEIGHT, SMOKING HABITS AND GENETIC CONSTITUTION

The Departmento de Arica in northern Chile was chosen as the investigation site for a study of the role of certain hematologic and glycolytic variables in the physiological and genetic adaptation to hypoxia.^ The population studied comprised 876 individuals, residents of seven villages at three altitudes: coast (0-500m), sierra (2,500-3,500m) and altiplano (> 4,000m). There was an equal number of males and females ranging in ages from six to 90 years. Although predominantly Aymara, those of mixed or Spanish origin were also examined. The specimens were collected in heparinized vacutainers precipitated with cold trichloroacetic acid (TCA) and immediately frozen to -196(DEGREES)C. Six variables were measured. Three were hematologic: hemoglobin, hematocrit and mean cell hemoglobin concentration. The three others were glycolytic: erythrocyte 2,3-diphosphoglycerate (DPG), adenosine triphosphate (ATP) and the percentage of phosphates (DPG + ATP) in the form of DPG.^ Hemoglobin and hematocrit were measured on site. The DPG and ATP content was assayed in specimens which had been frozen at -196(DEGREES)C and transported to Houston. Structured interviews on site provided information as to lifestyle and family pedigrees.^ The following results were obtained: (1) The actual village, rather than the altitude, of examination accounted for the greatest proportion of the variance in all variables. In the coast, a large difference in levels of ionic lithium in the drinking water exists. The chemical environment of food and drink is postulated to account, in part, for the importance of geographic location in explaining the observed variance. (2) Measurements of individuals from the two extreme altitudes, coast and altiplano, did not exhibit the same relationship with age and body mass. The hematologic variables were significantly related to both age and body build in the coast. The glycolytic variables were significantly related to age and body mass in the altiplano. (3) The environment modified male values more than female values in all variables. The two sexes responded quite differently to age and changes in body mass as well. The question of differing adaptability of the two sexes is discussed. (4) Environmental factors explained a significantly higher proportion of total variability in the altiplano than in the coast for hemoglobin, hematocrit and DPG. Most of the ATP variability at both altitudes is explained by genetic factors. ^

DEVELOPMENT AND APPLICATION OF SHORT-TERM MUTAGENICITY AND CYTOTOXICITY BIOASSAYS FOR INTEGRATED HEALTH ASSESSMENT STUDIES OF COAL FLY ASH EMISSIONS

Two respirable coal fly ash samples ((LESSTHEQ) 3(mu)m), one from a pressurized fluidized-bed combustion miniplant and one from a conventional combustion power plant, were investigated for physical properties, chemical composition and biological activity. Electron microscopy illustrated irregularity in fluidized-bed combustion fly ash and sphericity in conventional combustion fly ash. Elemental analysis of these samples showed differences in trace elements. Both fly ash samples were toxic in rabbit alveolar macrophage and Chinese hamster ovary cell systems in vitro. The macrophages were more sensitive to toxicity of fly ash than the ovary cells. For measuring the cytotoxicity of fly ash, the most sensitive parameters were adenosine triphosphate in the alveolar macrophage system and viability index in the hamster ovary system. Intact fluidized-bed combustion fly-ash particles showed mutagenicity only in strains TA98 and TA1538 without metabolic activation in the Ames Salmonella assay. No mutagenicity was detected in bioassay of conventional combustion fly ash particles. Solvent extraction yielded more mass from fluidized-bed combustion fly ash than from conventional combustion fly ash. The extracts of fluidized-bed combustion fly ash showed higher mutagenic activity than conventional combustion fly ash. These samples contained direct-acting, frameshift mutagens.^ Fly ash samples collected from the same fluidized-bed source by cyclones, a fabric filter, and a electrostatic precipitator at various temperatures were compared for particle size, toxicity, and mutagenicity. Results demonstrated that the biological activity of coal fly ash were affected by the collection site, device, and temperature.^ Coal fly ash vapor-coated with 1-nitropyrene was developed as a model system to study the bioavailability and recovery of nitroaromatic compounds in fly ash. The effects of vapor deposition on toxicity and mutagenicity of fly ash were examined. The nitropyrene coating did not significantly alter the ash's cytotoxicity. Nitropyrene was bioavailable in the biological media, and a significant percentage was not recovered after the coated fly ash was cultured with alveolar macrophages. 1-Nitropyrene loss increased as the number of macrophages was increased, suggesting that the macrophages are capable of metabolizing or binding 1-nitropyrene present in coal fly ash. ^

Advanced Protein Modeling Method: Benchmarking and Applications in Computer-Aided Drug Discovery

Development of homology modeling methods will remain an area of active research. These methods aim to develop and model increasingly accurate three-dimensional structures of yet uncrystallized therapeutically relevant proteins e.g. Class A G-Protein Coupled Receptors. Incorporating protein flexibility is one way to achieve this goal. Here, I will discuss the enhancement and validation of the ligand-steered modeling, originally developed by Dr. Claudio Cavasotto, via cross modeling of the newly crystallized GPCR structures. This method uses known ligands and known experimental information to optimize relevant protein binding sites by incorporating protein flexibility. The ligand-steered models were able to model, reasonably reproduce binding sites and the co-crystallized native ligand poses of the β2 adrenergic and Adenosine 2A receptors using a single template structure. They also performed better than the choice of template, and crude models in a small scale high-throughput docking experiments and compound selectivity studies. Next, the application of this method to develop high-quality homology models of Cannabinoid Receptor 2, an emerging non-psychotic pain management target, is discussed. These models were validated by their ability to rationalize structure activity relationship data of two, inverse agonist and agonist, series of compounds. The method was also applied to improve the virtual screening performance of the β2 adrenergic crystal structure by optimizing the binding site using β2 specific compounds. These results show the feasibility of optimizing only the pharmacologically relevant protein binding sites and applicability to structure-based drug design projects.

Metabolism and actions of 2 -chloro-2$\sp\prime$-fluoro-arabinosyladenine

2-Chloro-9-(2-deoxy-2-fluoro-$\beta $-D-arabinofuranosyl)adenine(Cl-F-ara-A) is a new deoxyadenosine analogue which is resistant to phosphorolytic cleavage and deamination, and exhibits therapeutic activity for both leukemia and solid tumors in experimental systems. To characterize its mechanism of cytotoxicity, the present study investigated the cellular pharmacology and the biochemical and molecular mechanisms of action of Cl-F-ara-A, from entrance of the drug into the cell, chemical changes to active metabolites, targeting on different cellular enzymes, to final programmed cell death response to the drug treatment.^ Cl-F-ara-A exhibited potent inhibitory action on DNA synthesis in a concentration-dependent and irreversible manner. The mono-, di-, and triphosphates of Cl-F-ara-A accumulated in cells, and their elimination was non-linear with a prolonged terminal phase, which resulted in prolonged dNTP depression. Ribonucleotide reductase activity was inversely correlated with the cellular Cl-F-ara-ATP level, and the inhibition of the reductase was saturated at higher cellular Cl-F-ara-ATP concentrations. The sustained inhibition of ribonucleotide reductase and the consequent depletion of deoxynucleotide triphosphate pools result in a cellular Cl-F-ara-ATP to dATP ratio which favors analogue incorporation into DNA.^ Incubation of CCRF-CEM cells with Cl-F-ara-A resulted in the incorporation of Cl-F-ara-AMP into DNA. A much lesser amount was associated with RNA, suggesting that Cl-F-ara-A is a more DNA-directed compound. The site of Cl-F-ara-AMP in DNA was related to the ratio of the cellular concentrations of the analogue triphosphate and the natural substrate dATP. Clonogenicity assays showed a strong inverse correlation between cell survival and Cl-F-ara-AMP incorporation into DNA, suggesting that the incorporation of Cl-F-ara-A monophosphate into DNA is critical for the cytotoxicity of Cl-F-ara-A.^ Cl-F-ara-ATP competed with dATP for incorporation into the A-site of the extending DNA strand catalyzed by both DNA polymerase $\alpha$ and $\varepsilon$. The incorporation of Cl-F-ara-AMP into DNA resulted in termination of DNA strand elongation, with the most pronounced effect being observed at Cl-F-ara-ATP:dATP ratio $>$1. The presence of Cl-F-ara-AMP at the 3$\sp\prime$-terminus of DNA also resulted in an increased incidence of nucleotide misincorporation in the following nucleotide position. The DNA termination and the nucleotide misincorporation induced by the incorporation of Cl-F-ara-AMP into DNA may contribute to the cytotoxicity of Cl-F-ara-A. ^

A role for p53 in sensing DNA damage and triggering apoptotic responses to anticancer agents

The p53 tumor suppressor protein plays a major role in cellular responses to anticancer agents that target DNA. DNA damage triggers the accumulation of p53, resulting in the transactivation of genes, which induce cell cycle arrest to allow for repair of the damaged DNA, or signal apoptosis. The exact role that p53 plays in sensing DNA damage and the functional consequences remain to be investigated. The main goal of this project was to determine if p53 is directly involved in sensing DNA damage induced by anticancer agents and in mediating down-stream cellular responses. This was tested in two experimental models of DNA damage: (1) DNA strand termination caused by anticancer nucleoside analogs and (2) oxidative DNA damage induced by reactive oxygen species (ROS). Mobility shift assays demonstrated that p53 and DNA-PK/Ku form a complex that binds DNA containing the anticancer nucleoside analog gemcitabine monophosphate in vitro. Binding of the p53-DNA-PK/Ku complex to the analog-containing DNA inhibited DNA strand elongation. Furthermore, treatment of cells with gemcitabine resulted in the induction of apoptosis, which was associated with the accumulation of p53 protein, its phosphorylation, and nuclear localization, suggesting the activation of p53 to trigger apoptosis following gemcitabine induced DNA strand termination. The role of p53 as a DNA damage sensor was further demonstrated in response to oxidative DNA damage. Protein pull-down assays demonstrated that p53 complexes with OGG1 and APE, and binds DNA containing the oxidized DNA base 8-oxoG. Importantly, p53 enhances the activities of APE and OGG1 in excising the 8-oxoG residue as shown by functional assays in vitro. This correlated with the more rapid removal of 8-oxoG from DNA in intact cells with wild-type p53 exposed to exogenous ROS stress. Interestingly, persistent exposure to ROS resulted in the accelerated onset of apoptosis in cells with wild-type p53 when compared to isogenic cells lacking p53. Apoptosis in p53+/+ cells was associated with accumulation and phosphorylation of p53 and its nuclear localization. Taken together, these results indicate that p53 plays a key role in sensing DNA damage induced by anticancer nucleoside analogs and ROS, and in triggering down-stream apoptotic responses. This study provides new mechanistic insights into the functions of p53 in cellular responses to anticancer agents. ^

ATP, chlorophyll "a", and size structure of phytolpankton in seawater at stations in the Atlantic sector of the Southern Ocean

Chlorophyll "a" and adenozine triphosphate (ATP) concentrations together with size structure of microplankton were investigated in January-April 1989 in the Indian Ocean and in the Weddell Sea. ATP values varied from 11 to 92 ng/l, and chlorophyll "a" concentrations varied from 0.04 to 0.27 µg/l in the Indian Ocean, with prevailing nanoplankton and picoplankton fractions. Both ATP and chlorophyll "a" concentrations increased 2 times to the south of 40°S; in the Weddell Sea they exceeded 400 ng/l and 0.6 µg/l, respectively. Cells of nanophytoplankton and microphytoplankton (mainly diatoms) prevailed in size spectra. Spatial variabilities of the parameters were within one order of magnitude; their values decreased 3-4 times during 1 month. Size structure changed due to increased portion of nanoplankton and picolankton. ATP concentrations in the photic layer (0-200 m) varied from 31.96 mg/m**2 in February to 8.02 mg/m**2 in March to April. ATP concentrations were 61.5 and 98.8 mg/m**2 at depths of 4200 and 4700 m, respectively.