ALFRED: an allele frequency database for diverse populations and DNA polymorphisms--an update.

ALFRED (the ALelle FREquency Database) is designed to store and disseminate frequencies of alleles at human polymorphic sites for multiple populations, primarily for the population genetics and molecular anthropology communities. Currently ALFRED has information on over 180 polymorphic sites for more than 70 populations. Since our initial release of the database we have focussed on increasing the quantity and quality of data, making reciprocal links between ALFRED and other related databases, and providing useful tools to make the data more comprehensible to the end user. ALFRED is accessible from the Kidd Lab home page (http://info.med.yale. edu/genetics/kkidd/) or from ALFRED directly (http://alfred.med.yale. edu/alfred/index.asp).

ALFRED: the ALelle FREquency Database. Update.

Elaboration of ALFRED (http://alfred.med.yale.edu) is being continued in two directions. One of which is developing tools for efficiently annotating the entries and checking the integrity of the data already in the database while the other is to increase the quantity and accessibility of data. Information contained in ALFRED such as, polymorphic sites, number of populations and frequency tables (one sample typed for one site) has significantly increased.

The specificity determinant of the Y mating-type proteins of Schizophyllum commune is also essential for Y-Z protein binding.

This paper concerns the manner in which combinatorial mating proteins of the fungus, Schizophyllum commune, recognize one another to form complexes that regulate target gene expression. In Schizophyllum, tightly linked Y and Z mating-type genes do not promote development in the combinations present in haploid strains (i.e., self combinations). When the Y and Z genes from two different mating types are brought together by the fusion of two haploid cells, the Y and Z proteins from different mating types recognize one another as nonself, form a complex and activate development. Several Y and Z alleles are present in the population and all nonself combinations of Y and Z alleles are equally functional. We have made chimeric genes among Y1, Y3, Y4 and Y5 and examined their mating-type specificities by transformation and mating tests. These studies show that the specificity of Y protein recognized by Z protein is encoded within a short region of N-terminal amino acids. The critical region is not precisely the same in each Y protein and in each Y-Z protein interaction. For Y3 protein compared with Y4 protein, the critical residues are in an N-terminal region of 56 amino acids (residues 17-72), with 40% identity and 65% similarity. Two-hybrid studies show that: the first 144 amino acids of Y4 protein are sufficient to bind Z3 and Z5 proteins, but not Z4 protein, and proteins deleted of the Y4 specificity region do not bind Z3, Z4 or Z5 protein. Thus the specificity determinant of the Y protein is essential for protein-protein recognition, Y-Z protein binding and mating activity.

Intratracheal instillation versus intratracheal inhalation: influence of cytokines on inflammatory response.

Our laboratory has developed a method of particle exposure whereby anesthetized rats intratracheally inhale, at a regulated breathing rate and pressure, an aerosolized test material. This method is capable of delivering considerable doses in a short time period and, unlike the commonly used method of intratracheal instillation, does so with an even particle distribution throughout the lung. Early studies comparing the response of male Fischer 344 rats exposed to TiO2 particles of two differing primary particle sizes showed that at similar particle doses animals exposed by the two methods showed differences in response, as measured by bronchoalveolar lavage (BAL) parameters. Building on this, we sought to study the roles that macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor alpha (TNF-alpha), two cytokines thought to have proinflammatory roles in the lung, may play in the differences observed. Increases in MIP-2 protein levels in the lavaged cells, but not the supernatant, were observed in those groups where increased polymorphonuclear cells (PMN) in the lung lavage were found, but not in those where no increase in PMN levels was observed. BAL TNF-alpha levels, measured by enzyme-linked immunosorbent assay, showed no apparent correlation with cellular or biochemical BAL parameters for either particle size or dosing method. Increases in immunocytochemical staining for TNF-alpha, compared to unexposed controls, were observed in several particle-exposed groups. Thus, it appears that increased BAL MIP-2 protein levels, but not TNF-alpha, correlate well with the inflammatory response, as measured by PMN numbers in lavaged cells, for both exposure systems.

Suppressed oxidant-induced apoptosis in cadmium adapted alveolar epithelial cells and its potential involvement in cadmium carcinogenesis.

Apoptosis involves a series of genetically programmed events associated with endonucleolytic cleavage of DNA. This process is triggered by a variety of agents, including oxidants such as hydrogen peroxide (H(2)O(2)) and it plays a key role in eliminating pre-neoplastic cells from the lung. Failure to do so could favor tumor promotion. The current study demonstrated that alveolar epithelial cells, adapted to cadmium (CdCl(2)) by repeated in vitro exposure, exhibit lower levels of H(2)O(2)-induced apoptosis than similarly challenged non-adapted cells. An immunologic assay, measuring cytoplasmic histone-associated DNA fragments, indicated maximal apoptosis 24 h after exposure to 400 microM H(2)O(2). Non-adapted cells showed a 13-fold increase in oxidant-induced apoptosis while Cd-adapted cells had only a 4-fold elevation. A terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method was used to assess the percentage of cells with DNA breaks consistent with apoptosis. Cd-adapted and non-adapted cells that were not exposed to H(2)O(2) did not differ in TUNEL positivity. However, after H(2)O(2) treatment, the percentage of TUNEL positive cells was 4-fold higher in non-adapted cultures than in adapted ones. Suppression of oxidant-induced apoptosis is due, in part, to up-regulation in the gene expression of several resistance factors including metallothioneins (MT-1 and MT-2), glutathione S-transferases (GST-alpha and GST-pi), and gamma-glutamylcysteine synthetase catalytic subunit (gamma-GCS). These steady-state mRNA changes, determined by Northern blotting, were accompanied by increased levels of MT and gamma-GCS protein, GST activity, and glutathione (GSH). Suppressed oxidant-induced apoptosis, resulting at least in part from these response modifications, could leave pre-neoplastic or neoplastic cells alive, favor clonal expansion, and ultimately lead to cancer development.

Characterization of cadmium-induced apoptosis in rat lung epithelial cells: evidence for the participation of oxidant stress.

The mode of cadmium-induced cell death was investigated in a rat lung epithelial cell line. Cells, grown to near confluence, were exposed to 0-30 microM CdCl2 for 0-72 h. Phase contrast microscopy and fluorescent nuclear staining showed that Cd caused morphological alterations in lung epithelial cells that are characteristic of apoptosis. These changes included cell shrinkage, detachment of the cell from its neighbors, cytoplasmic and chromatin condensation, and fragmentation of the nucleus into multiple chromatin bodies surrounded by remnants of the nuclear envelope. Apoptotic DNA degradation was validated and quantitated using a sensitive enzyme-linked immunosorbent assay (ELISA) which measures the amount of histone-bound DNA fragments in the cytosol. Using this technique, a maximum level of apoptosis (5-fold higher than control) was observed in cultures exposed for 48 h to 20 microM CdCl2. The terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling method (TUNEL) was subsequently used to determine the percentage of cells that contained Cd-induced DNA strand breaks. After 48 h, approximately 54% of the cells exposed to 20 microM Cd were TUNEL positive compared to less than 2% for control cells. Although the mechanisms by which Cd initiates apoptosis in these cells are presently not known, reactive oxygen species are likely to play a role. This possibility is supported by the finding that the first morphological features indicative of apoptosis were preceded by the up-regulation of oxidant stress genes (glutathione S-transferase-alpha, gamma-glutamylcysteine synthetase, and metallothionein-1), activation of redox sensitive transcription factors (AP-1 and NF-kappaB), and changes in various forms of glutathione (reduced, oxidized, and protein-bound).

Intratracheal inhalation vs intratracheal instillation: differences in particle effects.

Our laboratory has developed a method of intratracheal inhalation whereby rats can be exposed to high aerosol concentrations, resulting in high lung particle burdens in a short time period with deposition occurring directly in the lower respiratory tract, thus avoiding many drawbacks of larger nose-only or whole body inhalation systems. In this report, we compare the response of rats exposed by intratracheal inhalation to "fine" (approximately 250 nm) and "ultrafine" (approximately 21 nm) titanium dioxide particles with rats exposed to similar doses by intratracheal instillation. Animals receiving particles through inhalation showed a decreased pulmonary response, measured by bronchoalveolar lavage parameters, in both severity and persistence, when compared with those receiving particles through instillation. These results demonstrate a difference in pulmonary response to an inhaled vs an instilled dose, which may be due to differences in dose rate, particle distribution, or altered clearance between the two methods.

Possible epistatic role of ADH7 in the protection against alcoholism.

In recent studies of the role of the alcohol dehydrogenase genes (ADH) in alcoholism the ADH1B Arg47His polymorphism appears to affect risk via a protective effect associated with the ADH1B*47His. Here we present evidence for an additional effect from outside the Class I ADH genes, presumably from functional variation at the ADH7 gene. The protective effect is restricted to one of two haplotypes identical at ADH1B but differing at an intronic SNP at ADH7 suggesting epistasis or strong linkage disequilibrium (LD).

Cadmium-mediated oxidative stress in alveolar epithelial cells induces the expression of gamma-glutamylcysteine synthetase catalytic subunit and glutathione S-transferase alpha and pi isoforms: potential role of activator protein-1.

Exposure of rat alveolar epithelial cells to 10 micromol/L CdCl2 causes time-dependent increases in steady-state mRNA levels of the gamma-glutamylcysteine synthetase catalytic (heavy) subunit (gamma-GCS) and of glutathione S-transferase isoforms (GST-alpha and GST-pi). The expression of gamma-GCS was significantly increased as early as 2 h after addition of cadmium. Maximal induction of gamma-GCS mRNA (approximately 4-fold), at 8 h, was subsequently followed by increases in gamma-GCS activity/protein and glutathione (GSH) levels. Maximal elevations in GST-pi (approximately 2-fold) and GST-alpha (approximately 10-fold) transcripts, at 8 and 24 h, respectively, were also accompanied by enhanced GST activity. Cadmium-induced oxidative stress, assessed by alterations in GSH homeostasis and an accelerated rate of intracellular oxidant production, could constitute early events in the signal transduction pathway mediating these responses. The dimeric transcription factor, activator protein-1 (AP-1), may also play a regulatory role in this process. This association is suggested by transcriptional activation of the immediate-early response genes, c-fos and c-jun, within 15 min after exposure to cadmium and by the enhancement of AP-1 DNA binding activity, involving a c-Jun protein complex, which is maximally induced (approximately 4-fold) by 2 h. These molecular changes likely function together to protect alveolar epithelial cells against cadmium toxicity.

ALFRED: a Web-accessible allele frequency database.

We present a Web-accessible database (ALFRED) that allows public access to gene frequency data for a diverse set of population samples and genetic systems. The data in ALFRED are modeled based on the experience and needs of a single laboratory, but with the expectation that the database will meet the needs of a much broader scientific community that needs population-specific gene frequency estimates. Our database currently contains data on more than 40 populations representing most major regions of the world and data on more than 150 genetic systems including SNPs, STRPs, and insertion-deletion polymorphisms. While data are not available for all population-genetic system combinations, over 2000 allele frequency tables already exist. In this paper, we enumerate the broad needs in the scientific domain, describe their significance, and describe how we have designed the database to meet those needs. We compare our database with dbSNP, the NCBI database that has a broader but overlapping purpose.

ALFRED: an allele frequency database for diverse populations and DNA polymorphisms.

We have developed a publicly accessible database (ALFRED, the ALlele FREquency Database) that catalogues allele frequency data for a wide range of population samples and DNA polymorphisms. This database is web-accessible through our laboratory (Kidd Lab) Web site: http://info.med.yale.edu/genetics/kkidd. ALFRED currently contains data on 60 populations and 156 genetic systems including single nucleotide polymorphisms (SNPs), short tandem repeat polymorphisms (STRPs), variable number of tandem repeats (VNTRs) and insertion-deletion polymorphisms. While data are not available for all population-DNA polymorphism combinations, over 2000 allele frequency tables have been entered. Our database is designed (i) to address our specific research requirements as well as broader scientific objectives; (ii) to allow researchers and interested educators to easily navigate and retrieve data of interest to them; and (iii) to integrate links to other related public databases such as dbSNP, GenBank and PubMed.

Linkage disequilibrium at the ADH2 and ADH3 loci and risk of alcoholism.

Two of the three class I alcohol dehydrogenase (ADH) genes (ADH2 and ADH3) encode known functional variants that act on alcohol with different efficiencies. Variants at both these genes have been implicated in alcoholism in some populations because allele frequencies differ between alcoholics and controls. Specifically, controls have higher frequencies of the variants with higher Vmax (ADH2*2 and ADH3*1). In samples both of alcoholics and of controls from three Taiwanese populations (Chinese, Ami, and Atayal) we found significant pairwise disequilibrium for all comparisons of the two functional polymorphisms and a third, presumably neutral, intronic polymorphism in ADH2. The class I ADH genes all lie within 80 kb on chromosome 4; thus, variants are not inherited independently, and haplotypes must be analyzed when evaluating the risk of alcoholism. In the Taiwanese Chinese we found that, only among those chromosomes containing the ADH3*1 variant (high Vmax), the proportions of chromosomes with ADH2*1 (low Vmax) and those with ADH2*2 (high Vmax) are significantly different between alcoholics and controls (P<10-5). The proportions of chromosomes with ADH3*1 and those with ADH3*2 are not significantly different between alcoholics and controls, on a constant ADH2 background (with ADH2*1, P=.83; with ADH2*2, P=.53). Thus, the observed differences in the frequency of the functional polymorphism at ADH3, between alcoholics and controls, can be accounted for by the disequilibrium with ADH2 in this population.