Changes in tumor-specific nuclear antigen activity in carcinogen-treated colon by tumor promoter and carcinogen inhibitors.

Changes in the immunospecificity of the nuclear antigens were demonstrated in the colon chromatin of rats treated with 1,2-dimethylhydrazine. Tumor-specific nuclear antigen appeared in the early stages of chemically induced colon carcinogenesis. Sodium barbiturate, in conjunction with the carcinogen, induced a higher level of nuclear antigen activity than that obtained with carcinogen alone. The rise of immunoactivity in carcinogen-treated colon chromatin can be abolished by simultaneous treatment with disulfiram or butylated hydroxytoluene.

Co-evolution of hydrological components under climate change scenarios in the Mediterranean area.

The Mediterranean area is historically characterized by high human pressure on water resources. Today, while climate is projected to be modified in the future, through precipitation decrease and temperature increase, that jointly and non-linearly may affect runoff, concerns about water availability are increasing. For these reasons, quantitative assessment of future modifications in the mean annual water availability are important; likewise, the description of the future interannual variability of some hydrological components such as runoff and evapotranspiration are highly wished for water management and ecosystems dynamics analyses. This study investigates at basin spatial scale future runoff and evapotranspiration, exploring their probability density functions and their interdependence as functions of climatic changes. In order to do that, a parsimonious conceptual lumped model is here used. The model is forced by different future climate scenarios, generated through a weather generator based on a stochastic downscaling of an ensemble of General Circulation Models (GCMs) realizations. The use of the adopted hydrological model, under reliable stochastic future climate scenarios, allows to project future values of evapotranspiration and runoff in a probabilistic framework and, at the same time, the evaluation of their bivariate frequency distributions for changes through the Multivariate Kernel Density Estimation method. As a case study, a benchmark Mediterranean watershed has been proposed (Imera Meridionale, Italy). Results suggest a radical shift and shape modification of the annual runoff and evapotranspiration probability density functions. Possible implications and impacts on water resources management are here addressed and discussed.

Stimulated phosphorylation of non-histone phosphoproteins in SV-40 transformed WI-38 human diploid fibroblasts.

Because the phosphorylation of non-histone proteins has been suggested to play a role in the regulation of eukaryotic gene transcription, we have compared the phosphorylation of these proteins in normal and SV-40 transformed WI-38 human diploid fibroblasts. The rate of phosphorylation was found to be roughly ten-fold higher in the transformed cells, and this striking difference could not be accounted for by either an increased rate of phosphate transport or by the synthesis of new species of non-histone proteins which subsequently become phosphorylated. To our knowledge this is the most dramatic alteration in non-histone protein phosphorylation ever described, and therefore may have important implications for our understanding of malignant transformation.

Reproducible amplification of RAPD markers from vertebrate DNA.

Randomly amplified polymorphic DNA (RAPD) is a modification of PCR that uses short, randomly generated primers to amplify genomic DNA. Generally, many bands of mixed intensity (i.e., strong, faint, fuzzy or sharp) are generated with each primer. Mixed-intensity bands are inherent with the RAPD technique because (i) the target DNAs are undefined, (ii) one or more copies of the target DNA may exist per genome and (iii) the percentage of hybridization of primer to target may vary. The problem of mixed-intensity band exacerbates the well-known sensitivity of PCRs to reagent and template concentrations, pH and other reaction parameters. These complications have discouraged many investigators from using RAPD. Our goal was to optimize the RAPD amplification conditions for vertebrate DNA. We present the optimized protocol along with an experimental strategy for obtaining reproducible, interpretable RAPD banding patterns in vertebrates.

Colon tumor specific nuclear antigen with potential as a pre-tumor diagnostic probe.

Tumor specific nuclear antigen was demonstrated in early stages of chemically-induced colon carcinogenesis. At these early stages, there is no observable nuclear or cytoplasmic alteration in the colon mucosae. The rise in tumor specific nuclear antigen in carcinogen-treated animals can be abolished by simultaneous treatment with carcinogen inhibitor. The potential for tumor specific nuclear antigen to be used as a pretumor diagnostic probe is discussed.

Randomly amplified polymorphic DNA (RAPD) analysis of Atlantic Coast striped bass.

Atlantic Coast striped bass exhibit exceptionally low levels of genetic variation. The ability of the randomly amplified polymorphic DNA (RAPD) method to uncover genetic variation in this highly conserved species was investigated. Sufficient levels of variation were detected to allow a population genetic analysis of the four migratory populations of Atlantic Coast striped bass. Lynch's analogue of Wright's FST (F'ST) suggests that Atlantic Coast striped bass are genetically subdivided (F'ST for pooled Atlantic samples = 0.44). Significant heterogeneity was detected in the frequencies of 32 per cent of surveyed RAPD markers. A modification of Slatkin's conditional average frequency method suggests that gene flow is present among the sampled Atlantic Coast striped bass. Results of the RAPD analysis suggest that gene flow is sufficient to prevent fixation of alternate genetic markers, but not sufficient to prevent the development of significant divergence in frequencies of these markers.

Enzyme-linked immunosorbent assay for use with nuclear protein-DNA complex antigens.

An enzyme-linked immunosorbent assay (ELISA) was developed for identification and quantification of nuclear antigens (nonhistone protein-DNA complexes from chromatin). Until now, the complement fixation assay has been the only immunoassay routinely applied to nonhistone protein-DNA complexes. The ELISA is considerably more sensitive than the micro-complement fixation test for assaying the immunospecificity of nuclear protein-DNA complexes. Dilutions of rabbit antisera as great as 1:6400 could be used to detect nanogram quantities of antigen, chicken reticulocyte chromatin or dehistonized rat liver chromatin.

Chicken reticulocyte nuclear antigen: its identification and relation to transcriptive activity in erythropoietic cells.

Antibodies to chicken reticulocyte dehistonized chromatin were produced. These antibodies can distinguish between chromatin from reticulocytes and erythrocytes and have no activity with chicken liver chromatin. The antibodies will also differentiate between reticulocytes and erythrocytes by the the horseradish peroxidase bridge localization technique. The nonhistone protein components of the antigenic complex are very tightly bound to the DNA, removable by extraction with sodium dodecyl sulfate but not 2.5 M NaCl-5 M urea. Our data indicate that the antigen complexes could actually be present in both reticulocytes and erythrocytes but that chromatin condensation causes a structural masking of the complexes which coincides with the known decline in transcriptive activity of the erythrocyte.

Antigenic changes in nuclear chromatin in 1,2-dimethylhydrazine-induced colon carcinogenesis.

Chromosomal nonhistone protein-DNA complexes prepared from rat colon adenocarcinoma were used to produce tumor specific antisera in rabbits. The antisera reacted specifically in complement fixation tests with chromatins isolated from rat colon tumor and not with those from a number of other rodent tumors and normal tissues, including normal rat colon epithelial cells. Tumor specificity of this antisera was also demonstrated by their cross reactivity with human and mouse colon adenocarcinomas. Immunohistochemical localization of the antigens by the horseradish peroxidase bridge technique demonstrated their presence in the nuclei. The changes in the immunospecificity of the nuclear antigens in chromatin were studied in colon of rats treated with 1,2-dimethylhydrazine. A significant change in the immunospecificity of the chromatin occurred as early as the fourth week after 1,2-dimethylhydrazine treatment was begun.

Phosphorylation of nonhistone chromosomal proteins early during the prereplicative phase of the cell cycle of WI-38 human diploid fibroblasts.

Stimulation of confluent monolayers of nondividing WI-38 cells to proliferate is accompanied by a doubling in the rate of phosphorylation of nonhistone chromosomal proteins during the first hour. The use of cycloheximide to inhibit protein synthesis does not appear to affect phosphorylation of the nonhistones. Nondividing and serum-stimulated cells exhibit qualitative differences in the phosphorylation and dephosphorylation of individual polypeptide peaks resolved by polyacrylamide gel electrophoresis. These results are consistent with other evidence implicating nonhistone protein phosphorylation as an early event involved in the activation of transcription occurring during cell proliferation.

Three patterns of mitochondrial DNA nucleotide divergence in the meadow vole, Microtus pennsylvanicus.

The DNA sequence was determined for the cytochrome c oxidase II (COII), tRNALys, and ATPase 8 genes from the mitochondrial genome of the meadow vole, Microtus pennsylvanicus. When compared to other rodents, three different patterns of evolutionary divergence were found. Nucleotide variation in tRNALys is concentrated in the T psi C loop. Nucleotide variation in the COII gene in three genera of rodents (Microtus, Mus, Rattus) consists predominantly of transitions in the third base positions of codons. The predicted amino acid sequence in highly conserved (greater than 92% similarity). Analysis of the ATPase 8 gene among four genera (Microtus, Cricetulus, Mus, Rattus) revealed more detectable transversions than transitions, many fixed first and second position mutations, and considerable amino acid divergence. The rate of nucleotide substitution at nonsynonymous sites in the ATPase 8 gene is 10 times the rate in the COII gene. In contrast, the estimated absolute mutation rate as determined by analysis of nucleotide substitutions at fourfold degenerate sites probably is the same for the two genes. The primary sequences of the ATPase 8 and COII peptides are constrained differently, but each peptide is conserved in terms of predicted secondary-level configuration.

Nutritional requirements of papillomavirus-transformed mouse cells and an uninfected parent line in serum-free culture.

A serum-free culture system was used to compare the nutritional requirements of mouse mammary cells transformed by bovine papillomavirus type 1 (ID13 cells) and the uninfected parent line (C127 cells). The serum-free, chemically defined medium used for this study was an MCDB 151-based medium (MCDB 151+S+I), supplemented with epidermal growth factor, transferrin, hydrocortisone, ethanolamine, phosphoethanolamine, retinoic acid, trace metals, and insulin. Proliferation of either cell type in serum-free culture required the addition of 250 micrograms/ml of insulin. ID13 cells have a doubling time of greater than 96 h in MCDB 151+S+I, whereas C127 cells have a doubling time of 60 h. This is in sharp contrast to the growth characteristics of the two cell types in 10% fetal bovine serum, where doubling times for the ID13 and C127 cells are 24 and 30 h, respectively. Culture of the cells in a serum-free medium has therefore revealed that the papillomavirus-transformed cells have more stringent growth requirement than the uninfected parent line.

Mitochondrial DNA polymorphism in three Antillean island populations of the fruit bat, Artibeus jamaicensis.

The Neotropical fruit bat, Artibeus jamaicensis, occurs throughout Latin America and on many islands in the Caribbean. Populations from Jamaica (in the Greater Antilles) to Barbados (in the Lesser Antilles) have been classified as a subspecies (A.j. jamaicensis) separate from that on the Lesser Antillean island of St. Vincent (A.j. schwartzi). Mitochondrial DNA (mtDNA) was isolated from 54 individuals collected on these islands, analyzed by digestion with restriction endonucleases, and the restriction sites were mapped. Three different mtDNA genotypes (16,000 +/- 200 bp) were identified: J-1 (16 animals from Jamaica, one from St. Vincent, 15 from Barbados), J-2 (two animals from Jamaica), and SV-1 (18 animals from St. Vincent, two from Barbados). The J-1 and J-2 genotypes were estimated to differ from each other by only 0.4%, but the SV-1 genotype differed from J-1 and J-2 by 8.1%-10.5%. The estimated sequence divergence between SV-1 and J-1 is unusually large for mammals that are regarded as conspecific. Restriction mapping showed that the differences among the genotypes (presence or absence of particular restriction sites) were located throughout the genome. The presence of the J-1 mtDNA genotype on Jamaica and on St. Vincent and Barbados (1,400 km away) demonstrates that maternal lineages in these bats are not necessarily confined to single islands or limited geographic regions. The presence of the J-1 mtDNA genotype within the A. j. schwartzi population on St. Vincent and the presence of the SV-1 genotype in two specimens of A. j. jamaicensis from Barbados document genetic exchange between subspecific populations on these islands, which are separated by 180 km of open water.

Immunospecificity of nuclear antigens in chicken erythroid cells.

Specific antisera were produced against chicken reticulocyte dehistonized chromatin. The antisera reacts strongly with chicken reticulocyte chromatin, but only marginally with chicken erythrocyte chromatin. There is no reticulocyte antigen detected in chicken liver. Reticulocyte maturation is accompanied by a gradual decrease in the chromatin immunological activity and template capacity. The reduction of immunological activity is due to the change of chromatin conformation during erythrocyte maturation. Dehistonization and sonication of erythrocyte chromatin raises the erythrocyte chromatin immunological activity to levels similar to those of reticulocyte chromatin. The erythrocyte nuclear antigens are class specific, not being found in frog erythroid cell or murine Friend leukemia cell chromatins.

Complete mitochondrial genome of a neotropical fruit bat, Artibeus jamaicensis, and a new hypothesis of the relationships of bats to other eutherian mammals.

The complete mitochondrial genome was obtained from a microchiropteran bat, Artibeus jamaicensis. The presumptive amino acid sequence for the protein-coding genes was compared with predicted amino acid sequences from several representatives of other mammalian orders. Data were analyzed using maximum parsimony, maximum likelihood, and neighbor joining. All analyses placed bats as the sister group of carnivores, perissodactyls, artiodactyls, and cetaceans (e.g., 100% bootstrap value with both maximum parsimony and neighbor joining). The data strongly support a new hypothesis about the origin of bats, specifically a bat/ferungulate grouping. None of the analyses supported the superorder Archonta (bats, flying lemurs, primates, and tree shrews). Our hypothesis regarding the relationship of bats to other eutherian mammals is concordant with previous molecular studies and contrasts with hypotheses based solely on morphological criteria and an incomplete fossil record. The A. jamaicensis mitochondrial DNA control region has a complex pattern of tandem repeats that differs from previously reported chiropteran control regions.

Age and skill differences in adaptive competence.

Previous research has documented qualitative changes in certain cognitive abilities during the older adult years, such as in short-term memory, perceptual and motor skills, and attentional capacities. Other work has suggested that a number of significant age-related changes, across a variety of cognitive abilities, are based on social experiences, such as occupational or recreational activities. The current study is based on earlier research by Perlmutter and her colleagues (1990) and examines age and skill-related differences among adults engaged in a social-recreational activity. BINGO players, ranging in age from nineteen to seventy-four, and having from less than two months to over twenty years of playing experience, were given a variety of psychometric, cognitive, and experimental measures. The participants were also observed as they played real BINGO games. No age-related differences were found on the psychometric or memory measures, suggesting that BINGO playing experience may have positive benefits for many older adults. Skilled players at all age levels were found to be more efficient in their game-playing actions. The oldest and most experienced players did not differ from the younger, equally experienced, players on the cognitive and skill-based tasks. These findings demonstrate the need to investigate adaptive competence in those situations in which social-environmental factors play a role in enhancing older adults' cognitive skills.

Habitat islands, genetic diversity, and gene flow in a Patagonian rodent.

The effects of terrestrial habitat islands on gene flow and genetic diversity in animal populations have been predicted and discussed in theoretical terms, but empirical data are needed to test these predictions and provide an understanding of the relationships of life-history characteristics to genetics of insular species. We studied saxicolous mice (Phyllotis xanthopygus) in Patagonia to explore genetic structure, phylogeography, and gene flow in a species inhabiting natural habitat islands. Phylogeographic analyses based on mtDNA sequences revealed two haplotype clades, which presumably reflect early Pleistocene factors that temporarily separated the mice into two geographically isolated groups. The Río Chubut, which lies within a glacial drainage basin bisecting northern Patagonia, might have affected gene flow in the species. Although we anticipated isolation by distance and founder phenomena associated with habitat islands, in some habitat patches we found evidence of high local genetic diversity. The amount of divergence in the mitochondrial cytochrome b gene (approximately 3.4%) in animals at a single locality could best be explained through a combination of historical factors and metapopulation source-sink theory. Demographic shifts, dispersal, and episodic recolonization are important in the life history and genetic population structure of P. xanthopygus.