Identification of novel cell-adhesion molecules in peripheral nerves using a signal-sequence trap

The development and maintenance of myelinated nerves in the PNS requires constant and reciprocal communication between Schwann cells and their associated axons. However, little is known about the nature of the cell-surface molecules that mediate axon-glial interactions at the onset of myelination and during maintenance of the myelin sheath in the adult. Based on the rationale that such molecules contain a signal sequence in order to be presented on the cell surface, we have employed a eukaryotic-based, signal-sequence-trap approach to identify novel secreted and membrane-bound molecules that are expressed in myelinating and non-myelinating Schwann cells. Using cDNA libraries derived from dbcAMP-stimulated primary Schwann cells and 3-day-old rat sciatic nerve mRNAs, we generated an extensive list of novel molecules expressed in myelinating nerves in the PNS. Many of the identified proteins are cell-adhesion molecules (CAMs) and extracellular matrix (ECM) components, most of which have not been described previously in Schwann cells. In addition, we have identified several signaling receptors, growth and differentiation factors, ecto-enzymes and proteins that are associated with the endoplasmic reticulum and the Golgi network. We further examined the expression of several of the novel molecules in Schwann cells in culture and in rat sciatic nerve by primer-specific, real-time PCR and in situ hybridization. Our results indicate that myelinating Schwann cells express a battery of novel CAMs that might mediate their interactions with the underlying axons.     

No increase in radiation-induced chromosome aberration complexity detected by m-FISH after culture in the presence of 5'-bromodeoxyuridine

The thymidine analogue, 5'-bromodeoxyuridine (BrdU), is a known mutagen that is routinely introduced into culture media for subsequent Harlequin stain analysis and determination of cell cycle status. Previously, we examined the induction of chromosome aberrations in human peripheral blood lymphocytes (PBL) known to be in their 1st cell division following exposure to a low dose (0.5 Gy, average one alpha-particle per cell) of high-LET alpha-particles. We found complex chromosome aberrations to be characteristic of exposure to high-LET radiation and suggested the features of complex exchange to reflect qualitatively the spatial deposition of this densely ionising radiation. To exclude the possibility that BrdU addition post-irradiation influenced the complexity of chromosomal damage observed by m-FISH, the effect of increasing BrdU concentration on aberration complexity was investigated. Comparisons between BrdU concentration (0, 10 and 40 microM) and between sham- and alpha-particle-irradiated PBL, were made both independently and in combination to enable discrimination between BrdU and high-LET radiation effects. Aberration type, size, complexity and completeness were assessed by m-FISH, and the relative progression through cell division was evaluated. We found no evidence of any qualitative difference in the complexity of damage as visualised by m-FISH but did observe an increase in the frequency of complex exchanges with increasing BrdU concentration indicative of altered cell cycle kinetics. The parameters measured here are consistent with findings from previous in vitro and in vivo work, indicating that each complex aberration visualised by m-FISH is characteristic of the structure of the high-LET alpha-particle track and the geometry of cell irradiated.     

Meiotic recombination: too much of a good thing?

Proper chromosome segregation in meiosis requires the right number and distribution of crossovers. Recent work in budding yeast has revealed a meiosis-specific role for RecQ helicase in limiting crossovers, distinct from its known somatic role in maintaining genome stability.     

Peroxisome proliferator-activated receptors modulate proliferation and angiogenesis in human endometrial carcinoma

Peroxisome proliferator-activated receptors (PPAR) and retinoid X receptors (RXR) are implicated in the development of several obesity-related cancers. Little is known of either the expression or function of PPARs and RXRs in endometrial cancer although this increasingly common disease is highly associated with both obesity and insulin resistance. We investigated the expression of PPAR and RXR subtypes in human endometrial cancers and normal endometrium with immunoblotting and immunohistochemistry and subsequently showed PPAR/RXR binding preferences by coimmunoprecipitation. To determine the functions of PPARs within the endometrium, we investigated proliferation, apoptosis, PTEN expression, and secretion of vascular endothelial growth factor (VEGF) in endometrial cell lines after reducing the expression of PPARα and PPARγ with antisense RNA. The functional effects of PPAR ligands were also investigated in vitro. We identified differential expression of PPAR and RXR subtypes in endometrial cancers and discovered that PPARγ expression correlated with expression of PTEN. PPARα activation influences endometrial cell growth and VEGF secretion. PPARγ activation reduces proliferation of endometrial cells via regulation of PTEN and appears to reduce VEGF secretion. We conclude that the PPAR/RXR pathway contribute to endometrial carcinogenesis by control of PTEN expression and modulation of VEGF secretion. We propose that PPAR ligands should be considered for clinical investigation in early phase studies of women with endometrial cancer.     

Alpha-Particle-Induced Complex Chromosome Exchanges Transmitted through Extra-Thymic Lymphopoiesis In Vitro Show Evidence of Emerging Genomic Instability

Human exposure to high-linear energy transfer α-particles includes environmental (e.g. radon gas and its decay progeny), medical (e.g. radiopharmaceuticals) and occupational (nuclear industry) sources. The associated health risks of α-particle exposure for lung cancer are well documented however the risk estimates for leukaemia remain uncertain. To further our understanding of α-particle effects in target cells for leukaemogenesis and also to seek general markers of individual exposure to α-particles, this study assessed the transmission of chromosomal damage initially-induced in human haemopoietic stem and progenitor cells after exposure to high-LET α-particles. Cells surviving exposure were differentiated into mature T-cells by extra-thymic T-cell differentiation in vitro. Multiplex fluorescence in situ hybridisation (M-FISH) analysis of naïve T-cell populations showed the occurrence of stable (clonal) complex chromosome aberrations consistent with those that are characteristically induced in spherical cells by the traversal of a single α-particle track. Additionally, complex chromosome exchanges were observed in the progeny of irradiated mature T-cell populations. In addition to this, newly arising de novo chromosome aberrations were detected in cells which possessed clonal markers of α-particle exposure and also in cells which did not show any evidence of previous exposure, suggesting ongoing genomic instability in these populations. Our findings support the usefulness and reliability of employing complex chromosome exchanges as indicators of past or ongoing exposure to high-LET radiation and demonstrate the potential applicability to evaluate health risks associated with α-particle exposure.     

New Medium for the Isolation and Enumeration of Pseudomonads

A new medium containing 200 ppb of 2-hydroxy-2',4,4'-trichlorodiphenyloxide (CH3565) and 10 ppm of cetyl-trimethyl-ammonium bromide (Cetrimide) in tryptic soy agar was developed and tested with 19 pure cultures of Pseudomonas, 20 microorganisms of other genera, commercially prepared ground beef, and laboratory-prepared inoculated ground beef. The new medium, CETCH agar, was compared with an antibiotic-containing medium. CETCH agar provided greater pseudomonad recoveries, a shorter incubation period prior to plate counting, and greater ease of preparation.     

Correlation of Bacterial Lipid Composition with Antibiotic Resistance

The correlation of bacterial lipid composition with antibiotic resistance was investigated with particular emphasis on those organisms in which resistance may be related to membrane or envelope structure or function, as in resistance to tetracyclines and polymyxin. Chloroform-methanol-extractable lipids, phosphatidyl ethanolamine fractions, and both fatty acids of these lipid fractions and total fatty acids were studied by using thin-layer chromatography, gas chromatography, and infrared spectroscopy. Consistent quantitative differences were found between the fatty acid compositions of sensitive and resistant strains. Most notable was the fact that, in gram-negative organisms, resistant strains showed decreases in cyclopropane acids as compared with sensitive strains. These changes were found to be inherent in the strains and not due to growth stage or culture age. No significant qualitative differences were noted. In contrast, no such variation in fatty acid content was observed in penicillin-sensitive and resistant strains of gram-positive cocci. As significant alterations of fatty acid composition were noted in gram-negative strains resistant to antibiotics, we suggest that resistance is correlated to membrane or envelope lipid composition.     

Genome-wide mapping of allele-specific protein-DNA interactions in human cells

We describe a high-throughput method, named ChIP-SNP, for the identification of allele-specific protein-DNA interactions throughout the human genome. ChIP-SNP combines chromatin immunoprecipitation (ChIP) with whole-genome single nucleotide polymorphism (SNP) genotyping microarray analysis. We demonstrated that it can be used to accurately identify allele-specific binding of RNA polymerase II (RNAP) in the human fibroblast genome, uncovering imprinted genes and other allele-specific binding events. ChIP-SNP will facilitate the study of mechanisms of allele-specific gene expression.