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Abstract Toxicogenomics can measure the expression of thousands of genes to identify changes associated with drug induced toxicities. It is expected that toxicogenomics can be an alternative or complementary approach in preclinical drug safety evaluation to identify or predict drug induced toxicities. One of the major concerns in applying toxicogenomics to diagnose or predict drug induced organ toxicity, is how generalizable the statistical classification model is when derived from small datasets? Here we presented that a diagnosis of kidney proximal tubule toxicity, measured by pathology, can successfully be achieved even with a study design of limited number of training studies or samples. We selected a total of ten kidney toxicants, designed the in life study with multiple dose and multiple time points to cover samples at doses and time points with or without concurrent toxicity. We employed SVM (Support Vector Machine) as the classification algorithm for the toxicogenomic diagnosis of kidney proximal tubule toxicity. Instead of applying cross validation methods, we used an independent testing set by dividing the studies or samples into independent training and testing sets to evaluate the diagnostic performance. We achieved a Sn (sensitivity) = 88% and a Sp (specificity) = 91%. The diagnosis performance underscores the potential application of toxicogenomics in a preclinical lead optimization process of drugs entering into development.

Abstract Background Neurons in the dorsal spinal cord play important roles in nociception and pain. These neurons receive input from peripheral sensory neurons and then transmit the signals to the brain, as well as receive and integrate descending control signals from the brain. Many molecules important for pain transmission have been demonstrated to be localized to the dorsal horn of the spinal cord. Further understanding of the molecular interactions and signaling pathways in the dorsal horn neurons will require a better knowledge of the molecular neuroanatomy in the dorsal spinal cord. Results A large scale screening was conducted for genes with enriched expression in the dorsal spinal cord using DNA microarray and quantitative real-time PCR. In addition to genes known to be specifically expressed in the dorsal spinal cord, other neuropeptides, receptors, ion channels, and signaling molecules were also found enriched in the dorsal spinal cord. In situ hybridization and immunohistochemistry revealed the cellular expression of a subset of these genes. The regulation of a subset of the genes was also studied in the spinal nerve ligation (SNL) neuropathic pain model. In general, we found that the genes that are enriched in the dorsal spinal cord were not among those found to be up-regulated in the spinal nerve ligation model of neuropathic pain. This study also provides a level of validation of the use of DNA microarrays in conjunction with our novel analysis algorithm (SAFER) for the identification of differences in gene expression. Conclusion This study identified molecules that are enriched in the dorsal horn of the spinal cord and provided a molecular neuroanatomy in the spinal cord, which will aid in the understanding of the molecular mechanisms important in nociception and pain.

Traditionally, chemical toxicity is determined by in vivo animal studies, which are low throughput, expensive, and sometimes fail to predict compound toxicity in humans. Due to the increasing number of chemicals in use and the high rate of drug candidate failure due to toxicity, it is imperative to develop in vitro, high-throughput screening methods to determine toxicity. The Tox21 program, a unique research consortium of federal public health agencies, was established to address and identify toxicity concerns in a high-throughput, concentration-responsive manner using a battery of in vitro assays. In this article, we review the advancements in high-throughput robotic screening methodology and informatics processes to enable the generation of toxicological data, and their impact on the field; further, we discuss the future of assessing environmental toxicity utilizing efficient and scalable methods that better represent the corresponding biological and toxicodynamic processes in humans.

Canine osteosarcoma is increasingly recognized as an informative model for human osteosarcoma. Here we show in one of the largest clinically annotated canine osteosarcoma transcriptional datasets that two previously reported, as well as de novo gene signatures devised through single sample Gene Set Enrichment Analysis (ssGSEA), have prognostic utility in both human and canine patients. Shared molecular pathway alterations are seen in immune cell signaling and activation including TH1 and TH2 signaling, interferon signaling, and inflammatory responses. Virtual cell sorting to estimate immune cell populations within canine and human tumors showed similar trends, predominantly for macrophages and CD8+ T cells. Immunohistochemical staining verified the increased presence of immune cells in tumors exhibiting immune gene enrichment. Collectively these findings further validate naturally occurring osteosarcoma of the pet dog as a translationally relevant patient model for humans and improve our understanding of the immunologic and genomic landscape of the disease in both species., (© 2023. Springer Nature Limited.)

Ferroptosis is a necrotic cell death caused by lipid oxidation that may be responsible for neural degeneration in Parkinson's disease. We assessed whether three neuronal cell lines are sensitive to killing by ferroptosis. Ferroptosis inducer erastin killed LUHMES neurons at sub-micromolar concentrations, whereas neuronal cells derived from SH-SY5Y cells or neural stem cells were at least 50-fold less sensitive. LUHMES differentiated neurons were likewise sensitive to killing by RSL3 or ML210, inhibitors of the glutathione peroxidase 4 enzyme (GPX4) that consumes GSH to detoxify lipid peroxides. Additional assays showed that erastin, RSL3, and ML210 increased lipid peroxide levels, and that LUHMES neurons were protected from both peroxide accumulation and cell death by ferrostatin-1. A possible role of iron was assessed by evaluating the effects of five metal chelators on cytotoxicity of erastin and RSL3. LUHMES neurons were protected from RSL3 by three of the chelators, 2,3-dimercapto-1-propanesulfonic acid (DMPS), deferoxiprone (DFX), and deferiprone (DFP). Collectively, these results demonstrate the vulnerability of LUHMES neurons to ferroptosis by chemical treatments that disrupt glutathione synthesis, lipid peroxide detoxification, or iron metabolism. The same vulnerabilities may occur in CNS neurons, which reportedly generate low levels of GSH and metallothioneins, limiting their ability to neutralize oxidative stresses and toxic metals. These results suggest a rationale and methods to search for environmental toxicants that may exploit these vulnerabilities and promote neurodegenerative diseases., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Understanding relationships between spontaneous cancer in companion (pet) canines and humans can facilitate biomarker and drug development in both species. Towards this end we developed an experimental-bioinformatic protocol that analyzes canine transcriptomics data in the context of existing human data to evaluate comparative relevance of canine to human cancer. We used this protocol to characterize five canine cancers: melanoma, osteosarcoma, pulmonary carcinoma, B- and T-cell lymphoma, in 60 dogs. We applied an unsupervised, iterative clustering method that yielded five co-expression modules and found that each cancer exhibited a unique module expression profile. We constructed cancer models based on the co-expression modules and used the models to successfully classify the canine data. These canine-derived models also successfully classified human tumors representing the same cancers, indicating shared cancer biology between canines and humans. Annotation of the module genes identified cancer specific pathways relevant to cells-of-origin and tumor biology. For example, annotations associated with melanin production (PMEL, GPNMB, and BACE2), synthesis of bone material (COL5A2, COL6A3, and COL12A1), synthesis of pulmonary surfactant (CTSH, LPCAT1, and NAPSA), ribosomal proteins (RPL8, RPS7, and RPLP0), and epigenetic regulation (EDEM1, PTK2B, and JAK1) were unique to melanoma, osteosarcoma, pulmonary carcinoma, B- and T-cell lymphoma, respectively. In total, 152 biomarker candidates were selected from highly expressing modules for each cancer type. Many of these biomarker candidates are under-explored as drug discovery targets and warrant further study. The demonstrated transferability of classification models from canines to humans enforces the idea that tumor biology, biomarker targets, and associated therapeutics, discovered in canines, may translate to human medicine., Competing Interests: The authors have declared that no competing interests exist.

Identification of toxicants that underlie neurological diseases is a neglected area awaiting a valid strategy to identify such toxicants. We sought biomarkers that respond to known neurotoxicants in LUHMES immortalized neurons and evaluated these biomarkers for use in screening libraries of environmental toxicants. LUHMES immortalized human dopaminergic neurons were surveyed by RNA sequencing following challenge with parkinsonian toxicants rotenone, 6-hydroxydopamine, MPP+, and ziram (zinc dimethyldithiocarbamate; Zn 2+ DDC 2 ), as well as additional toxicants paraquat, MS275, and methylmercury. The metallothionein gene MT1G was the most dynamic gene expression response to all seven toxicants. Multiple toxicants also increased transcripts for SLC30A1 and SLC30A2 zinc secretion transporters, the SLC7A11 xCT cystine/glutamate antiporter important for glutathione synthesis, DNA damage inducible transcript 3 (DDIT3), and secreted growth factors FIBIN and CXCL12, whereas several toxicants decreased expression of the apelin growth factor (APLN). These biomarker genes revealed stress responses to many toxicants at sub-cytotoxic concentrations. Since several of these biomarker genes and prior neurological disease studies implicated disruption of metal distribution, we tested metal chelator thiram (dimethyldithiocarbamate, DDC), ziram, and several other metals and metal chelates for cytotoxicity and induction of MT1G expression. Metals and chelators that caused dynamic increases in MT1G expression also caused cytotoxicity, except Ni 2+ DDC 2 induced MT1G at 5 μM, but lacked cytotoxicity up to 100 μM. These results bolster prior work suggesting that neurons are characteristically sensitive to depletion of glutathione or to disruption of cellular metal distribution and provide biomarkers to search for such neurotoxicants in chemical libraries.

Background: A central challenge in toxicity testing is the large number of chemicals in commerce that lack toxicological assessment. In response, the Tox21 program is re-focusing toxicity testing from animal studies to less expensive and higher throughput in vitro methods using target/pathway-specific, mechanism-driven assays., Objectives: Our objective was to use an in-depth mechanistic study approach to prioritize and characterize the chemicals affecting mitochondrial function., Methods: We used a tiered testing approach to prioritize for more extensive testing 622 compounds identified from a primary, quantitative high-throughput screen of 8,300 unique small molecules, including drugs and industrial chemicals, as potential mitochondrial toxicants by their ability to significantly decrease the mitochondrial membrane potential (MMP). Based on results from secondary MMP assays in HepG2 cells and rat hepatocytes, 34 compounds were selected for testing in tertiary assays that included formation of reactive oxygen species (ROS), upregulation of p53 and nuclear erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE), mitochondrial oxygen consumption, cellular Parkin translocation, and larval development and ATP status in the nematode Caenorhabditis elegans ., Results: A group of known mitochondrial complex inhibitors (e.g., rotenone) and uncouplers (e.g., chlorfenapyr), as well as potential novel complex inhibitors and uncouplers, were detected. From this study, we identified four not well-characterized potential mitochondrial toxicants (lasalocid, picoxystrobin, pinacyanol, and triclocarban) that merit additional in vivo characterization., Conclusions: The tier-based approach for identifying and mechanistically characterizing mitochondrial toxicants can potentially reduce animal use in toxicological testing. https://doi.org/10.1289/EHP2589.

More than 75 000 man-made chemicals contaminate the environment; many of these have not been tested for toxicities. These chemicals demand quantitative high-throughput screening assays to assess them for causative roles in neurotoxicities, including Parkinson's disease and other neurodegenerative disorders. To facilitate high throughput screening for cytotoxicity to neurons, three human neuronal cellular models were compared: SH-SY5Y neuroblastoma cells, LUHMES conditionally-immortalized dopaminergic neurons, and Neural Stem Cells (NSC) derived from human fetal brain. These three cell lines were evaluated for rapidity and degree of differentiation, and sensitivity to 32 known or candidate neurotoxicants. First, expression of neural differentiation genes was assayed during a 7-day differentiation period. Of the three cell lines, LUHMES showed the highest gene expression of neuronal markers after differentiation. Both in the undifferentiated state and after 7 days of neuronal differentiation, LUHMES cells exhibited greater cytotoxic sensitivity to most of 32 suspected or known neurotoxicants than SH-SY5Y or NSCs. LUHMES cells were also unique in being more susceptible to several compounds in the differentiating state than in the undifferentiated state; including known neurotoxicants colchicine, methyl-mercury (II), and vincristine. Gene expression results suggest that differentiating LUHMES cells may be susceptible to apoptosis because they express low levels of anti-apoptotic genes BCL2 and BIRC5/survivin, whereas SH-SY5Y cells may be resistant to apoptosis because they express high levels of BCL2, BIRC5/survivin, and BIRC3 genes. Thus, LUHMES cells exhibited favorable characteristics for neuro-cytotoxicity screening: rapid differentiation into neurons that exhibit high level expression neuronal marker genes, and marked sensitivity of LUHMES cells to known neurotoxicants. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

The first guidance on Good Cell Culture Practice (GCCP) dates back to 2005. This document expands this to include aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice that can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance to facilitate the generation of reliable data from cell culture systems, and is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when it is necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and the safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered a first step toward a revised GCCP 2.0.

Drug-induced phospholipidosis is characterized by the accumulation of intracellular phospholipids in cells exposed to cationic amphiphilic drugs. The appearance of unicentric or multicentric multilamellar bodies viewed under an electron microscope (EM) is the morphological hallmark of phospholipidosis. Although the EM method is the gold standard for detecting cellular phospholipidosis, this method has its drawbacks, including low throughput, high cost, and unsuitability for screening a large chemical library. In this study, a cell-based phospholipidosis assay has been developed using the LipidTOX Red reagent in HepG2 cells and miniaturized into a 1536-well plate format. To validate this assay for high-throughput screening (HTS), the LOPAC library of 1280 compounds was screened using a quantitative HTS platform. A group of known phospholipidosis inducers, such as amiodarone, propranolol, chlorpromazine, desipramine, promazine, clomipramine, and amitriptyline, was identified by the screen, consistent with previous reports. Several novel phospholipidosis inducers, including NAN-190, ebastine, GR127935, and cis-(Z)-flupentixol, were identified in this study and confirmed using the EM method. These results demonstrate that this assay can be used to evaluate and profile large numbers of chemicals for drug-induced phospholipidosis.

Despite wide-spread consensus on the need to transform toxicology and risk assessment in order to keep pace with technological and computational changes that have revolutionized the life sciences, there remains much work to be done to achieve the vision of toxicology based on a mechanistic foundation. To this end, a workshop was organized to explore one key aspect of this transformation - the development of Pathways of Toxicity as a key tool for hazard identification based on systems biology. Several issues were discussed in depth in the workshop: The first was the challenge of formally defining the concept of a Pathway of Toxicity (PoT), as distinct from, but complementary to, other toxicological pathway concepts such as mode of action (MoA). The workshop came up with a preliminary definition of PoT as "A molecular definition of cellular processes shown to mediate adverse outcomes of toxicants". It is further recognized that normal physiological pathways exist that maintain homeostasis and these, sufficiently perturbed, can become PoT. Second, the workshop sought to define the adequate public and commercial resources for PoT information, including data, visualization, analyses, tools, and use-cases, as well as the kinds of efforts that will be necessary to enable the creation of such a resource. Third, the workshop explored ways in which systems biology approaches could inform pathway annotation, and which resources are needed and available that can provide relevant PoT information to the diverse user communities.

Application of any new biomarker to support safety-related decisions during regulated phases of drug development requires provision of a substantial data set that critically assesses analytical and biological performance of that biomarker. Such an approach enables stakeholders from industry and regulatory bodies to objectively evaluate whether superior standards of performance have been met and whether specific claims of fit-for-purpose use are supported. It is therefore important during the biomarker evaluation process that stakeholders seek agreement on which critical experiments are needed to test that a biomarker meets specific performance claims, how new biomarker and traditional comparators will be measured and how the resulting data will be merged, analyzed and interpreted.

Kidney toxicity accounts both for the failure of many drug candidates as well as considerable patient morbidity. Whereas histopathology remains the gold standard for nephrotoxicity in animal systems, serum creatinine (SCr) and blood urea nitrogen (BUN) are the primary options for monitoring kidney dysfunction in humans. The transmembrane tubular protein kidney injury molecule-1 (Kim-1) was previously reported to be markedly induced in response to renal injury. Owing to the poor sensitivity and specificity of SCr and BUN, we used rat toxicology studies to compare the diagnostic performance of urinary Kim-1 to BUN, SCr and urinary N-acetyl-beta-D-glucosaminidase (NAG) as predictors of kidney tubular damage scored by histopathology. Kim-1 outperforms SCr, BUN and urinary NAG in multiple rat models of kidney injury. Urinary Kim-1 measurements may facilitate sensitive, specific and accurate prediction of human nephrotoxicity in preclinical drug screens. This should enable early identification and elimination of compounds that are potentially nephrotoxic.

Dahl-Iwai (DI) salt-sensitive rats were studied using microarrays to identify sex-specific differences in the kidney, both basal differences and differences in responses to a high-salt diet. In DI rat kidneys, gene expression profiles demonstrated inflammatory and fibrotic responses selectively in females. Gonadectomy of DI rats abrogated sex differences in gene expression. Gonadectomized female and gonadectomized male DI rats both responded to high salt with the same spectrum of gene expression changes as intact female DI rats. Androgens dominated the sex-selective responses to salt. Several androgen-responsive genes with roles potentiating the differential responses to salt were identified, including increased male expression of angiotensin-vasopressin receptor and prolactin receptor, decreased 5 alpha-reductase, and mixed increases and decreases in expression of Cyp4a genes that can produce eicosanoid hormones. These sex differences potentiate sodium retention by males and increase kidney function during gestation in females.

The androgen receptor (AR) is expressed in the uterus; however, the role of AR in female reproductive physiology is poorly understood. Here we examined the effects of androgens on uterine growth and gene expression in adult ovariectomized rats. Nonaromatizable AR-selective agonists potently stimulate hypertrophy and induce significant myometrial expansion distinct from that induced by 17beta-estradiol (E2). In the endometrium, androgens only modestly increase epithelial cell height and antagonize the trophic effects of E2. To identify underlying mechanisms, global changes in RNA levels 24 h after stimulation with E2 and 5alpha-dihydrotestosterone (DHT) were compared. A total of 491 genes were differentially expressed after E2 treatment, including key regulators of tissue remodeling, cell signaling, metabolism, and gene expression. Of the 164 transcripts regulated by DHT, 86% were also affected by E2, including trophic genes like IGF-I and epithelial secretory genes such as uterocalin. In estrogen receptor (ER)alpha knockout mice, DHT cannot induce uterine growth, suggesting a key role for ERalpha. However, DHT appears not to activate ERalpha directly because DHT induction of IGF-I is blocked by the AR antagonist bicalutamide, and multiple genes regulated directly by ERalpha were not induced by DHT. The similarity between estrogens and androgens instead could reflect general trophic signaling in reproductive tissues because 93 of the 503 genes regulated in the uterus are similarly affected during prostate growth. Thus androgens regulate the trophic environment and architecture of the rodent uterus via a gene expression program that is overlapping but distinct from the estrogen response.

We have combined protein motif search and gene finding methods to identify genes encoding proteins containing specific domains. Particularly, we have focused on finding new human genes of the cadherin superfamily proteins, which represent a major group of cell-cell adhesion receptors contributing to embryonic neuronal morphogenesis. Models for three cadherin protein motifs were generated from over 100 already annotated cadherin domains and used to search the complete translated human genome. The genomic sequence regions containing motif "hits" were analyzed by eukaryotic GeneMark.hmm to identify the exon-intron structure of new genes. Three new genes CDH-J, PCDH-J and FAT-J were found. The predicted proteins PCDH-J and FAT-J were classified into protocadherin and FAT-like subfamilies, respectively, based on the number and organization of cadherin domains and presence of subfamily-specific conserved amino acid residues. Expression of FAT-J was shown in almost all tested tissues. The exon-intron organization of CDH-J was experimentally verified by PCR with specifically designed primers and its tissue-specific expression was demonstrated. The described methodology can be applied to discover new genes encoding proteins from families with well-characterized structural and functional domains.

The androgen receptor (AR), when complexed with 5alpha-dihydrotestosterone (DHT), supports the survival and proliferation of prostate cells, a process critical for normal development, benign prostatic hypertrophy, and tumorigenesis. However, the androgen-responsive genetic pathways that control prostate cell division and differentiation are largely unknown. To identify such pathways, we examined gene expression in the ventral prostate 6 and 24 h after DHT administration to androgen-depleted rats. 234 transcripts were expressed significantly differently from controls (p < 0.05) at both time points and were subjected to extensive data mining. Functional clustering of the data reveals that the majority of these genes can be classified as participating in induction of secretory activity, metabolic activation, and intracellular signaling/signal transduction, indicating that AR rapidly modulates the expression of genes involved in proliferation and differentiation in the prostate. Notably AR represses the expression of several key cell cycle inhibitors, while modulating members of the wnt and notch signaling pathways, multiple growth factors, and peptide hormone signaling systems, and genes involved in MAP kinase and calcium signaling. Analysis of these data also suggested that p53 activity is negatively regulated by AR activation even though p53 RNA was unchanged. Experiments in LNCaP prostate cancer cells reveal that AR inhibits p53 protein accumulation in the nucleus, providing a post-transcriptional mechanism by which androgens control prostate cell growth and survival. In summary these data provide a comprehensive view of the earliest events in AR-mediated prostate cell proliferation in vivo, and suggest that nuclear exclusion of p53 is a critical step in prostate growth.

Neuropathic pain is induced by injury or disease of the nervous system. Studies aimed at understanding the molecular pathophysiology of neuropathic pain have so far focused on a few known molecules and signaling pathways in neurons. However, the pathophysiology of neuropathic pain appears to be very complex and remains poorly understood. A global understanding of the molecular mechanisms involved in neuropathic pain is needed for a better understanding of the pathophysiology and treatment of neuropathic pain. Towards this end, we examined global gene expression changes as well as the pathobiology at the cellular level in a spinal nerve ligation neuropathic pain model using DNA microarray, quantitative real-time PCR and immunohistochemistry. We found that the behavioral hypersensitivity that is manifested in the persistent pain state is accompanied by previously undescribed changes in gene expression. In the DRG, we found regulation of: (1) immediate early genes; (2) genes such as ion channels and signaling molecules that contribute to the excitability of neurons; and (3) genes that are indicative of secondary events such as neuroinflammation. In addition, we studied gene regulation in both injured and uninjured DRG by quantitative PCR, and observed differential gene regulation in these two populations of DRGs. Furthermore, we demonstrated unexpected co-regulation of many genes, especially the activation of neuroinflammation markers in both the PNS and CNS. The results of our study provide a new picture of the molecular mechanisms that underlie the complexity of neuropathic pain and suggest that chronic pain shares common pathobiology with progressive neurodegenerative disease., (Copyright 2002 IBRO)

Oligonucleotide DNA microarrays were investigated for utility in measuring global expression profiles of drug metabolism genes. This study was performed to investigate the feasibility of using microarray technology to minimize the long, expensive process of testing drug candidates for safety in animals. In an evaluation of hybridization specificity, microarray technology from Affymetrix distinguished genes up to a threshold of approximately 90% DNA identity. Oligonucleotides representing human cytochrome P-450 gene CYP3A5 showed heterologous hybridization to CYP3A4 and CYP3A7 RNAs. These genes could be clearly distinguished by selecting a subset of oligonucleotides that hybridized selectively to CYP3A5. Further validation of the technology was performed by measuring gene expression profiles in livers of rats treated with vehicle, 3-methylcholanthrene (3MC), phenobarbital, dexamethasone, or clofibrate and by confirming data for six genes using quantitative RT-PCR. Responses of drug metabolism genes, including CYPs, epoxide hydrolases (EHs), UDP-glucuronosyl transferases (UGTs), glutathione sulfotransferases (GSTs), sulfotransferases (STs), drug transporter genes, and peroxisomal genes, to these well-studied compounds agreed well with, and extended, published observations. Additional gene regulatory responses were noted that characterize metabolic effects or stress responses to these compounds. Thus microarray technology can provide a facile overview of gene expression responses relevant to drug metabolism and toxicology.

11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) is an enzyme that converts cortisone to the active glucocorticoid, cortisol. Cortisol-cortisone interconversion plays a key role in the regulation of glucose metabolism, since mice deficient in 11betaHSD1 are resistant to diet-induced hyperglycemia. Peroxisome proliferator activator receptors (PPAR) are key regulators of glucose and lipid homeostasis. We observed a striking downregulation of murine hepatic 11betaHSD1 expression and activity after chronic treatment of wild-type mice with PPARalpha agonists, while 11betaHSD1 in the livers of PPARalpha knockout mice, or in mice treated for only 7 h with PPARalpha agonists, was unaltered. Our results are the first to show PPARalpha agonists can affect glucocorticoid metabolism in the liver by altering 11betaHSD1 expression after chronic treatment. Regulation of active glucocorticoid levels in the liver by PPARalpha agonists may in turn affect glucose metabolism, consistent with reports of their antidiabetic effects., (Copyright 2000 Academic Press.)

DNA chips are glass surfaces that represent thousands of DNA fragments arrayed at discrete sites. Hybridization of RNA or DNA-derived samples to DNA chips allows us to monitor expression of mRNAs or the occurrence of polymorphisms in genomic DNA. The technology holds great promise for identifying gene polymorphisms that predispose man to disease, gene regulation events involved in disease progression, and more-effective disease treatments.

The structural gene HMP1 encoding a cruciform DNA binding protein from Ustilago maydis has been cloned. Gene isolation was enabled by a polymerase chain reaction procedure using primers designed from amino acid sequence obtained from the purified protein. DNA sequence determination has revealed that the gene encodes a protein containing 98 amino acids with a calculated molecular weight of 10151. Comparison of the cDNA and genomic sequences indicated the presence of a single intron in the 5' coding region of the gene. The gene was over-expressed as a translational fusion with a hexahistidine leader sequence enabling affinity purification of the protein on an immobilized metal matrix. Protein isolated after over-expression exhibited cruciform binding activity, conforming earlier purified native protein results. Sequence analysis indicated that no HMG box was present and very little homology to other known cruciform binding proteins was found. It is plausible that HMP1 represents a novel class of proteins that recognize such secondary structures.

We report here the cloning of the Candida albicans genomic topoisomerase I gene (TOP1) by use of PCR and subsequent hybridization. The predicted protein sequence shared 58.8% identity with the Saccharomyces cerevisiae topoisomerase I and 30-50% identity with other eukaryotic topoisomerase I proteins. A conditional gene disruption strain (CWJ477) was constructed so that one copy of TOP1 was deleted and the other copy of TOP1 was placed under a regulatable promoter. Under repressed conditions, cells grew slowly and cell morphology was abnormal. The virulence of CWJ477 was markedly reduced in a mouse model system, and that of the single gene knockout-strain was slightly attenuated, indicating that TOP1 might play a role in the infection of C. albicans in mice in a dose-dependent manner. Despite the reduced virulence of both the single and double knockout strains, viable cells of the pathogen were recovered from the kidneys as late as 22 d post-infection.

ESTs or 'expressed sequence tags' are DNA sequences read from both ends of expressed gene fragments. The Merck-WashU EST Project and several other public EST projects are being performed to rapidly discover the complement of human genes, and make them easily accessible. These ESTs are widely used to discover novel members of gene families, to map genes to chromosomes as 'sequence-tagged sites' (STSs), and to identify mutations leading to heritable diseases. Informatic strategies for querying the EST databases are discussed, as well as the strengths and weaknesses of the EST data. There is a compelling need to build on the informatic synthesis of human gene data, and to devise facile methods for determining gene functions.

An in vitro nucleosome assembly system has been established from cell-free extracts of the fungus Ustilago maydis. The extract catalyzed DNA supercoiling in the absence of exogenously added co-factors such as ATP and MgCl2 and was inhibited by moderate concentrations (200 mM) of KCl or NaCl. DNA supercoiling occurs via the formation of nucleosomes. Similar extracts, displaying the same activity, were prepared from Saccharomyces cerevisiae and Candida albicans, suggesting that the extract preparation protocol may be useful for many lower eukaryotic systems. An extract prepared from a strain of U. maydis lacking topoisomerase I failed to catalyze nucleosome assembly, clearly implicating this enzyme in this process. Addition of purified topoisomerase I, and, to a lesser extent, topoisomerase II, to the top1- extract regenerated the supercoiling activity. Our results provide a method for preparing assembly extracts from organisms, that are particularly amenable to genetic manipulation.

The Ustilago maydis genomic TOP1 gene encoding DNA topoisomerase I was cloned by amplifying a gene fragment using the polymerase chain reaction, and using this fragment to search a genomic DNA library by hybridization. The predicted peptide sequence exhibited 30-40% identity to other eukaryotic TOP1 genes, yet differed in several features. First, an unusually long acidic region was identified near the amino terminus (28/29 residues are acidic), which resembles other nucleolar peptide motifs. Second, an atypical carboxy-terminal 'tail', absent in other TOP1 genes, followed the active site tyrosine residue. A top1 gene disruption mutant was constructed by replacing the genomic TOP1 gene, with a top1::HygR null allele. This mutant lost the abundant topoisomerase I activity evident in wild-type U.maydis, and displayed a subtle coloration phenotype evident during cell senescence.

Avirulence gene D (avrD) in strain PT23 of Pseudomonas syringae pv. tomato (Pst) specifies the production of syringolides, which are elicitors of plant defense reactions. An 83-kb indigenous plasmid (pPT23B) that carries avrD has been mapped and characterized and a putative par region was identified. pPT23B contains a large amount of DNA that is repeated in other native plasmids in PT23. A putative mobile insertion element that occurs on plasmid pPT23A as well as on the chromosome was also identified in strain PT23. New broad-host-range expression vectors that functioned in Pst were constructed for overexpression of the cloned avrD gene and high-level production of the syringolides. Introduction of an avrD overexpression plasmid into PT23 or plasmid-cured strains led to identical syringolide peaks on HPLC with no new peaks observed. These results suggested that neither pPT23B nor other indigenous plasmids in Pst carry additional genes required for syringolide production or metabolism. Pst strains lacking pPT23B were not impaired in virulence on tomato plants.

The nodulation regulon of Rhizobium meliloti AK631 includes several operons (nodABC, hsnABC, hsnD, efn locus) which have in common a consensus promoter sequence called the nod box. A synthetic nod box probe was used to identify two additional nod boxes, n4 and n5, which were subcloned for study. By constructing lac fusions, we show that n4 and n5 sponsor induction of downstream regions as previously shown for n1-nodABC and n2-hsnABC. Using site-directed Tn5 mutagenesis, we find that the n5 locus plays a significant role in nodulation of alfalfa and sweetclover, whereas the n4 locus is important for alfalfa, but not for sweetclover. Hybridization data suggest that the n5 locus is conserved among Rhizobium species. In contrast, the n4 locus seems to be unique to Rhizobium meliloti strains, in agreement with the host-specific phenotype of n4 locus mutants. Thus, the use of a promoter probe allows us to identify nodulation genes which may be overlooked by standard methods such as random Tn5 mutagenesis.

Trichome exudate from mite-resistant geraniums (Pelargonium horlorum) was analyzed, principally by mass spectrometry and NMR spectroscopy. The exudate was found to consist of two anacardic acid derivatives,o-pentadecenylsalicylic acid ando-heptadecenylsalicylic acid. Bioassays established a moderate toxicity of these compounds to the two-spotted spider mite,Tetranychus urticae. The production of these compounds in geraniums was correlated with the two complementary dominant genes previously reported for host resistance to spider mites.