Faculty Bibliography

It remains unclear whether neo-oogenesis occurs in postnatal ovaries of mammals, based on studies in mice. We thought to test whether adult human ovaries contain germline stem cells (GSCs) and undergo neo-oogenesis. Rather than using genetic manipulation which is unethical in humans, we took the approach of analyzing the expression of meiotic marker genes and genes for germ cell proliferation, which are required for neo-oogenesis, in adult human ovaries covering an age range from 28 to 53 years old, compared to testis and fetal ovaries served as positive controls. We show that active meiosis, neo-oogenesis and GSCs are unlikely to exist in normal, adult, human ovaries. No early meiotic-specific or oogenesis-associated mRNAs for SPO11, PRDM9, SCP1, TERT and NOBOX were detectable in adult human ovaries using RT-PCR, compared to fetal ovary and adult testis controls. These findings are further corroborated by the absence of early meiocytes and proliferating germ cells in adult human ovarian cortex probed with markers for meiosis (SCP3), oogonium (OCT3/4, c-KIT), and cell cycle progression (Ki-67, PCNA), in contrast to fetal ovary controls. If postnatal oogenesis is confirmed in mice, then this species would represent an exception to the rule that neo-oogenesis does not occur in adults

The catecholamines play key roles in orchestrating the response to stress. While this is crucial to handle emergency situations, stress becomes maladaptive when prolonged or repeated, increasing allosteric load and susceptibility to a wide range of serious diseases. The time frame of the regulation of gene expression, especially as it relates to catecholamine (CA) biosynthetic enzymes are compared in three crucial catecholaminergic locations, the adrenal medulla, sympathetic ganglia and locus coeruleus in male animals. The adrenal medulla displays very rapid response to stress and gene profiling reveals a wide repertoire of target genes, many of them activated by single and not by repeated stress. In contrast to the adrenal medulla, the sympathetic ganglia are especially responsive to activation of the HPA axis, and ACTH may have a direct effect. The locus coeruleus, origin of most of the noradrenergic neurons innervating much of the brain, displays activation of additional signalling pathways and transcription factor with repeated compared to single exposure to stress. Most of the studies have been performed in males. However, there is considerable evidence that females respond differently to stress. Estradiol can regulate TH, DBH and GTPCH gene expression, as well as to modulate its response to other second messenger such as cAMP. Prior treatment with estradiol was found to alter the response of CA biosynthetic enzymes to stress. This emphasizes the tissue and sex specific features of the mechanistic underpinning of the adaptation or maladaptation of the catecholaminergic systems to stress and provides the basis for specific interventions. Key words: Adrenal medulla - Catecholamine biosynthesis - Estrogen - Sympathetic ganglia - Locus coeruleus - Stress - Transcription factors.

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19-21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work

3D cell cultures are rapidly becoming the method of choice for the physiologically relevant modeling of many aspects of non-malignant and malignant cell behavior ex vivo. Nevertheless, only a limited number of distinct cell types have been evaluated in this assay to date. Here we report the first large scale comparison of the transcriptional profiles and 3D cell culture phenotypes of a substantial panel of human breast cancer cell lines. Each cell line adopts a colony morphology of one of four main classes in 3D culture. These morphologies reflect, at least in part, the underlying gene expression profile and protein expression patterns of the cell lines, and distinct morphologies were also associated with tumor cell invasiveness and with cell lines originating from metastases. We further demonstrate that consistent differences in genes encoding signal transduction proteins emerge when even tumor cells are cultured in 3D microenvironments

The respiratory pathogen Chlamydia (Chlamydophila) pneumoniae is associated with chronic diseases, including atherosclerosis and giant-cell arteritis, which are accompanied by the occurrence of these obligate intracellular bacteria in blood vessels. There, C. pneumoniae seems to be present in a persistent state. Persistence is characterized by modified bacterial metabolism and morphology, as well as a reversible arrest of chlamydial development. In cell culture, this persistent state can be induced by gamma interferon (IFN-gamma). To elucidate this long-term interaction between chlamydiae and their host cells, microarray screening on epithelial HeLa cells was performed. Transcription of persistently (and productively) infected cells was compared with that of mock-infected cells. Sixty-six host cell genes were regulated at 24 h and/or 96 h of IFN-gamma-induced persistence. Subsequently, a set of 17 human host cell genes related to apoptosis, cell cycle, or metabolism was identified as permanently up- or down-regulated by real-time PCR. Some of these chlamydia-dependent host cell responses were diminished or even absent in the presence of rifampin. However, other expression patterns were not altered by the inhibition of bacterial RNA polymerase, suggesting two different modes of host cell activation. Thus, in the IFN-gamma model, the persisting bacteria cause long-lasting changes in the expression of genes coding for functionally important proteins. They might be potential drug targets for the treatment of persistent C. pneumoniae infections.

Embryonic heart formation requires the union of bilateral populations of cardiomyocytes and their reorganization into a simple tube. Little is known about the morphogenetic mechanisms that coordinate assembly of the heart tube and determine its dimensions. Using time-lapse confocal microscopy to track individual cardiomyocyte movements in the zebrafish embryo, we identify two morphologically and genetically separable phases of cell movement that coordinate heart tube assembly. First, all cardiomyocytes undergo coherent medial movement. Next, peripherally located cardiomyocytes change their direction of movement, angling toward the endocardial precursors and thereby establishing the initial circumference of the nascent heart tube. These two phases of cardiomyocyte behavior are independently regulated. Furthermore, we find that myocardial-endocardial interactions influence the second phase by regulating the induction, direction and duration of cardiomyocyte movement. Thus, the endocardium plays a crucial early role in cardiac morphogenesis, organizing cardiomyocytes into a configuration appropriate for heart tube assembly. Together, our data reveal a dynamic cellular mechanism by which tissue interactions establish organ architecture

Increased fat deposition in skeletal muscle is associated with insulin resistance. However, exercise increases both intramyocellular fat stores and insulin sensitivity, a phenomenon referred to as "the athlete's paradox". In this study, we provide evidence that augmenting triglyceride synthesis in skeletal muscle is intrinsically connected with increased insulin sensitivity. Exercise increased diacylglycerol (DAG) acyltransferase (DGAT) activity in skeletal muscle. Channeling fatty acid substrates into TG resulted in decreased DAG and ceramide levels. Transgenic overexpression of DGAT1 in mouse skeletal muscle replicated these findings and protected mice against high-fat diet-induced insulin resistance. Moreover, in isolated muscle, DGAT1 deficiency exacerbated insulin resistance caused by fatty acids, whereas DGAT1 overexpression mitigated the detrimental effect of fatty acids. The heightened insulin sensitivity in the transgenic mice was associated with attenuated fat-induced activation of DAG-responsive PKCs and the stress mediator JNK1. Consistent with these changes, serine phosphorylation of insulin receptor substrate 1 was reduced, and Akt activation and glucose 4 membrane translocation were increased. In conclusion, upregulation of DGAT1 in skeletal muscle is sufficient to recreate the athlete's paradox and illustrates a mechanism of exercise-induced enhancement of muscle insulin sensitivity. Thus, increasing muscle DGAT activity may offer a new approach to prevent and treat insulin resistance and type 2 diabetes mellitus.

Recent advances in cancer cell biology have focused on histone deacetylase inhibitors (HDACi's) because they target pathways critical to the development and progression of disease. In particular, HDACi's can induce expression of epigenetically silenced genes that promote growth arrest, differentiation and cell death. In glioma cells, one such repressed gene is the tetraspanin CD81, which regulates cytostasis in various cell lines and in astrocytes, the major cellular component of gliomas. Our studies show that HDACi's, trichostatin and sodium butyrate, promote growth arrest and differentiation with negligible cell death in glioma cells and induce expression of CD81 and cyclin-dependent kinase inhibitor 1A (p21(CIP/WAF-1)), another regulator of cytostasis in astrocytes. Interference RNA knock-down of CD81 abrogates cytostasis promoted by HDAC inhibition indicating that HDACi-induced CD81 is responsible for growth arrest. Induction of CD81 expression through HDAC inhibition is a novel strategy to promote growth arrest in glioma cells.

The genetic pathways that partition the developing nervous system into functional systems are largely unknown. The engrailed (En) homeobox transcription factors are candidate regulators of this process in the dorsal midbrain (tectum) and anterior hindbrain (cerebellum). En1 mutants lack most of the tectum and cerebellum and die at birth, whereas En2 mutants are viable with a smaller cerebellum and foliation defects. Our previous studies indicated that the difference in phenotypes is due to the earlier expression of En1 as compared with En2, rather than differences in protein function, since knock-in mice expressing En2 in place of En1 have a normal brain. Here, we uncovered a wider spectrum of functions for the En genes by generating a series of En mutant mice. First, using a conditional allele we demonstrate that En1 is required for cerebellum development only before embryonic day 9, but plays a sustained role in forming the tectum. Second, by removing the endogenous En2 gene in the background of En1 knock-in alleles, we show that Drosophila en is not sufficient to sustain midbrain and cerebellum development in the absence of En2, whereas En2 is more potent than En1 in cerebellum development. Third, based on a differential sensitivity to the dose of En1/2, our studies reveal a genetic subdivision of the tectum into its two functional systems and the medial cerebellum into four regions that have distinct circuitry and molecular coding. Our study suggests that an ;engrailed code' is integral to partitioning the tectum and cerebellum into functional domains