The Next Generation Scientist program: capacity-building for future scientific leaders in low- and middle-income countries.

Background: Scientific and professional development opportunities for early career scientists in low- and middle- income countries (LMICs) are limited and not consistent. There is a disproportionately low number of biomedical and clinical researchers in LMIC's relative to their high burden of disease, a disparity that is aggravated by emigration of up to 70% of scientists from their countries of birth for education and employment elsewhere. To help address this need, a novel University-accredited, immersive fellowship program was established by a large public-academic-private network.

Postnatal ontogeny of hippocampal expression of the mineralocorticoid and glucocorticoid receptors in the common marmoset monkey.

The mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) are nuclear transcription factors that mediate many of the basal and stress functions and effects of the corticosteroid hormones, including those related to brain development. Despite this, relatively little is known about the postnatal ontogeny of MR and GR gene and protein expression in the central nervous system, and this is particularly true of the primates, including humans. Here we describe the postnatal ontogeny of central MR and GR gene and protein expression in the common marmoset monkey.

Transcriptional response of yeast to aflatoxin B1: recombinational repair involving RAD51 and RAD1.

The potent carcinogen aflatoxin B(1) is a weak mutagen but a strong recombinagen in Saccharomyces cerevisiae. Aflatoxin B(1) exposure greatly increases frequencies of both heteroallelic recombination and chromosomal translocations. We analyzed the gene expression pattern of diploid cells exposed to aflatoxin B(1) using high-density oligonucleotide arrays comprising specific probes for all 6218 open reading frames.

Expression of human microsomal epoxide hydrolase in Saccharomyces cerevisiae reveals a functional role in aflatoxin B1 detoxification.

The metabolism and genotoxicity of the carcinogenic mycotoxin, aflatoxin B1 (AFB), was studied in the lower eukaryotic yeast Saccharomyces cerevisiae. Recombinant strains of yeast were engineered to express human cDNAs for CYP1A1, CYP1A2, and microsomal epoxide hydrolase (mEH). Coexpression of mEH with CYP1A1 or CYP1A2 resulted in significant decreases in measurements of AFB genotoxicity.

Liver carcinogen aflatoxin B1 as an inducer of mitotic recombination in a human cell line.

The mycotoxin aflatoxin B1 (AFB1) is one of the most potent rodent and human liver carcinogens. Upon cytochrome P450-specific metabolism, it induces mutations as well as mitotic recombination events in in vitro systems. We have found that in the lower eukaryote yeast, the recombinagenic activity of AFB1 surpasses its mutagenic activity, and we speculated on possible consequences in terms of the mechanism of liver carcinogenesis.

The potential use of mutation spectra in cancer related genes in genetic toxicology: a statement of a GUM working group.

In recent years, there has been widespread interest in the relationship between carcinogenic exposure and mutation spectra in cancer-related genes. To evaluate potential benefits and/or limitations in the use of mutation spectra in genetic toxicology, a GUM working group has been established to discuss this subject. Based on methodological possibilities and limitations, the impact of mutation spectra in the interpretation of animal experiments and in the identification of etiological agents in human cancer has been considered.

Codon 249 of the human TP53 tumor suppressor gene is no hot spot for aflatoxin B1 in a heterologous background.

Mutations in the TP53 tumor suppressor gene are the most common alteration in cancer, and human primary liver cancers related to previous dietary exposure to the mycotoxin aflatoxin B1 (AFB1) exhibit a specific hot spot mutation at TP53 codon 249. We have asked whether the 249 hot spot is related to a particular susceptibility to AFB1 of this TP53 region or whether it is related to a phenotype of the 249S p53 mutant protein. This was addressed by constructing a metabolically competent variant of Saccharomyces cerevisiae strain yIG397 expressing human cytochrome P450 1A2 and P450-reductase and isolating AFB1-induced mutants that failed to express the genomic ADE2 reporter gene.

Heterocyclic aromatic amines efficiently induce mitotic recombination in metabolically competent Saccharomyces cerevisiae strains.

Heterocyclic aromatic amines (HAs) represent a class of potent bacterial mutagens and rodent carcinogens which gain their biological activity upon metabolic conversion by phase I and phase II enzymes. Subsequent to cytochrome P450 (CYP)-dependent hydroxylation, mainly catalyzed by CYP1A2, acetylation mediated by the activity of N-acetyltransferase, NAT2, produces the ultimate electrophilic product that may react with DNA. In addition to point mutations observed in HA-exposed cells as genotoxic endpoint in vitro, loss of heterozygosity (LOH) has often been identified in HA-related rodent tumors as another endpoint in vivo.

Rearrangements in minisatellite sequences induced by aflatoxin B1 in a metabolically competent strain of Saccharomyces cerevisiae.

The role of aflatoxin B1 (AFB1) in the induction of rearrangements affecting minisatellite sequences was studied in an in vitro yeast model. The Saccharomyces cerevisiae strain used expresses human cytochrome P450 1A2 and NADPH-cytochrome P450 oxidoreductase and has previously been used to study genetic recombination events induced by AFB1. DNA multilocus fingerprinting was performed using probe M13 core hybridizing to a set of hypervariable minisatellite sequences in S.

The Sge1 protein of Saccharomyces cerevisiae is a membrane-associated multidrug transporter.

In this study, we report the further characterization of the Saccharomyces cerevisiae crystal violet-resistance protein Sge1. Sge1 is a highly hydrophobic 59 kDa protein with 14 predicted membrane-spanning domains. It shares homologies with several drug-resistance proteins and sugar transporters of the major facilitator superfamily.

A genetic system to detect mitotic recombination between repeated chromosomal sequences in Drosophila Schneider line 2 cells.

In order to study mitotic homologous recombination in somatic Drosophila melanogaster cells in vitro and to learn more on the question how recombination is influenced by mutagens, a genetic system was developed where spontaneous and drug-induced recombination could be monitored. Two recombination reporter substrates were stably introduced in multiple copies into the genome of established D. melanogaster Schneider line 2 cells: one plasmid (pSB310) contained the 5' and 3' deleted neomycin phosphoribosyltransferase alleles neoL and neoR as direct repeats; the other (pSB485) contained similar deletions (lacZL and lacZR) of the beta-galactosidase gene (lacZ).

Ectopic mitotic recombination in Drosophila probed with bacterial beta-galactosidase gene-based reporter transgenes.

Plasmids were constructed to investigate homologous mitotic recombination in Drosophila cells. Heteroalleles containing truncated but overlapping segments of the bacterial beta-galactosidase gene (lacZ) were positioned either on separate plasmids or as direct repeats on the same chromosome. Recombination reconstituted a functional lacZgene leading to expression of LacZ+activity detectable by histochemical staining.

Genotoxicity of ethyl carbamate (urethane) in Salmonella, yeast and human lymphoblastoid cells.

Ethyl carbamate is a known carcinogen occurring in fermented food and beverages and is therefore of interest for food safety assurance. We studied the genotoxicity of ethyl carbamate in Salmonella typhimurium, in Saccharomyces cerevisiae and in human lymphoblastoid TK6 cells. In absence of cytochrome P450 enzymes, no ethyl carbamate-mediated genotoxicity was observed in any of the three test systems in the non-cytotoxic range.

Genotoxicity of aflatoxin B1: evidence for a recombination-mediated mechanism in Saccharomyces cerevisiae.

The potent liver carcinogen aflatoxin B1 (AFB1) is metabolized by cytochrome P450 to the mutagenic epoxide. We have observed that activated AFB1 also strongly induced mitotic recombination in the yeast Saccharomyces cerevisiae. To compare the recombinogenicity of AFB1 to its mutagenicity, three metabolically competent S.

Metabolism of promutagens catalyzed by Drosophila melanogaster CYP6A2 enzyme in Saccharomyces cerevisiae.

The somatic mutation and recombination test (SMART) in Drosophila melanogaster allows screening of chemicals for genotoxicity in a multicellular organism. In order to correlate data obtained in the SMART with those from genotoxicity tests in rodents, it is important to learn more on the variety of drug-metabolizing enzymes present in this insect and to identify their substrate specificities. In this study we have concentrated on the phase I enzyme cytochrome P450 6A2, which is the first cytochrome P450 cloned from Drosophila.

Identification of the sequences in HMG-CoA reductase required for karmellae assembly.

In all eukaryotic cells that have been examined, specific membrane arrays are induced in response to increased levels of the ER membrane protein, HMG-CoA reductase. Analysis of these inducible membranes has the potential to reveal basic insights into general membrane assembly. Yeast express two HMG-CoA reductase isozymes, and each isozyme induces a morphologically distinct proliferation of the endoplasmic reticulum.

Targeting of heterologous membrane proteins into proliferated internal membranes in Saccharomyces cerevisiae.

Overproduction of chimeric proteins containing the HMG2/1 peptide, which comprises the seven transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl-CoA reductase isozymes 1 and 2, has previously been observed to induce the proliferation of internal endoplasmic reticulum-like membranes. In order to exploit this amplified membrane surface area for the accommodation of heterologous microsomal proteins, we fused sequences coding for human cytochrome P4501A1 (CYP1A1) to sequences encoding the HMG2/1 peptide and expressed the hybrid genes in yeast. The heterologous hybrid proteins were targeted into strongly proliferated membranes, as shown by electron microscopic and immunofluorescent analysis.

Functional expression of fused enzymes between human cytochrome P4501A1 and human NADPH-cytochrome P450 oxidoreductase in Saccharomyces cerevisiae.

The activity of human cytochrome P450 enzymes heterologously expressed in Saccaromyces cerevisiae cells is limited by the yeast endogenous cytochrome P450 oxidoreductase (yOR). To overcome these limitations, we constructed hybrids between human P4501A1 (CYP1A1) and human P450 oxidoreductase (hOR) by combining the cDNA encoding hOR with the CYP1A1 cDNA. In addition, in one construct, the amino terminus of hOR was replaced by the membrane anchor domain of a yeast protein.

DNA recombination induced by aflatoxin B1 activated by cytochrome P450 1A enzymes.

Mutations in tumor suppressor genes are intricately associated with the etiology of neoplasia. Often, such mutations are followed by the loss of the second, functional alleles of tumor suppressor genes, a phenomenon known as loss of heterozygosity. Loss of heterozygosity may occur by different molecular mechanisms, including mitotic recombination, and it is conceivable that these molecular events are influenced by endogenous as well as exogenous factors.

The Saccharomyces cerevisiae SGE1 gene product: a novel drug-resistance protein within the major facilitator superfamily.

Several pleiotropic drug sensitivities have been described in yeast. Some involve the loss of putative drug efflux pumps analogous to mammalian P-glycoproteins, others are caused by defects in sterol synthesis resulting in higher plasma membrane permeability. We have constructed a Saccharomyces cerevisiae strain that exhibits a strong crystal violet-sensitive phenotype.

Characterization of the trp5-27 allele used to monitor drug-induced mitotic gene conversion in the Saccharomyces cerevisiae tester strain D7.

Mitotic gene conversions, among other recombinagenic events, can play an important role in the multistep process of carcinogenesis. The ability of chemicals to induce such gene conversions can easily be monitored in the Saccharomyces cerevisiae tester strain YHE2, a derivative of strain D7. For the detection of drug-induced gene conversions, two mutations in the TRP5 locus are used, trp5-12 and trp5-27.

High promutagen activating capacity of yeast microsomes containing human cytochrome P-450 1A and human NADPH-cytochrome P-450 reductase.

Yeast Saccharomyces cerevisiae strains have been constructed that co-express cDNAs coding for the human cytochrome P-450 enzymes CYP1A1 or CYP1A2 in combination with human NADPH-cytochrome P-450 reductase (oxidoreductase). Microsomal fractions prepared from the strains were able to efficiently activate various drugs to Salmonella mutagens. These experiments demonstrated that a functional interaction occurred between the respective human enzymes in the yeast microsomes.

The role of mitotic recombination in carcinogenesis.

Genetic recombination systems are present in all living cells and viruses and generally contribute to their hosts' flexibility with respect to changing environmental conditions. Recombination systems not only help highly developed organisms to protect themselves from microbial attack via an elaborate immune system, but conversely, recombination systems also enable microorganisms to escape from such an immune system. Recombination enzymes act with a high specificity on DNA sequences that either exhibit extended stretches of homology or contain characteristic signal sequences.