The Big Blue(R) transgenic mouse mutation detection assay: the mutation pattern of sectored mutant plaques

There are mutational artifacts in the Big Blue(R) assay and it is important to characterize the source and nature of these mutations. Differences were reported in the mutation patterns of a small sample of 23 sectored and 91 circular mutant plaques derived from skin using the Big Blue(R) transgenic mouse mutation detection system [G. R. Stuart, N.J. Gorelick, J.L. Andrews, J.G. de Boer, B.W. Glickman, The genetic analysis of lacI mutations in sectored plaques from Big Blue transgenic mice, Environ. Mol. Mutagen 28 (1996) 385-392.]. We have extended these observations by analyzing 46 sectored and 224 circular mutant plaques derived from seven tissues. The frequency of sectored mutant plaques is estimated to be 16% with no significant variation with tissue type. However, the patterns of mutation for sectored mutants and mouse-derived mutations differed significantly (p=0.04). Base substitutions in sectored mutant plaques do not show the asymmetries found in circular mutants consistent with integration of a GC rich transgene into the AT rich mammalian genome. Sectored mutants have mutation patterns consistent with a mixture of mouse, in vitro and Escherichia coli-derived mutations. Data on the relative frequencies of different mutant plaque morphologies suggests that overlapped plaques are substantially contaminated by sectored plaques at recommended plating densities.     

Placental failure and impaired vasculogenesis result in embryonic lethality for neuropathy target esterase-deficient mice

Age-dependent neurodegeneration resulting from widespread apoptosis of neurons and glia characterize the Drosophila Swiss Cheese (SWS) mutant. Neuropathy target esterase (NTE), the vertebrate homologue of SWS, reacts with organophosphates which initiate a syndrome of axonal degeneration. NTE is expressed in neurons and a variety of nonneuronal cell types in adults and fetal mice. To investigate the physiological functions of NTE, we inactivated its gene by targeted mutagenesis in embryonic stem cells. Heterozygous NTE(+/-) mice displayed a 50% reduction in NTE activity but underwent normal organ development. Complete inactivation of the NTE gene resulted in embryonic lethality, which became evident after gastrulation at embryonic day 9 postcoitum (E9). As early as E7.5, mutant embryos revealed growth retardation which did not reflect impaired cell proliferation but rather resulted from failed placental development; as a consequence, massive apoptosis within the developing embryo preceded its resorption. Histological analysis indicated that NTE is essential for the formation of the labyrinth layer and survival and differentiation of secondary giant cells. Additionally, impairment of vasculogenesis in the yolk sacs and embryos of null mutant conceptuses suggested that NTE is also required for normal blood vessel development.     

Analysis of imprinting in mice with uniparental duplication of proximal chromosomes 7 and 15 by use of a custom oligonucleotide microarray

We have developed an imprinting assay combining the use of mice carrying maternal or paternal duplication of chromosomal regions of interest with custom oligonucleotide microarrays. As a model system, we analyzed RNA from CNS tissue of neonatal mice carrying the reciprocal translocation T(7;15)9H and uniparental duplication of proximal Chr 7 and 15. The duplicated region includes the locus on proximal Chr 7 corresponding to the human Prader-Willi/Angelman Syndrome. The microarray contained 322 oligonucleotides, including probes to detect major genes involved in neural excitability and synaptic transmission, as well as known imprinted genes mapping to proximal Chr 7: Ndn, Snrpn, Mkrn3, Magel2, Peg3, and Ube3a. Imprinting of these genes in neonatal cortex and cerebellum was first confirmed by quantitative RT-PCR. Their inclusion on the microarray thus provided positive controls for evaluating the effect of background on the sensitivity of the assay, and for establishing the minimum level of expression required to detect imprinting. Our analysis extended previous work by revealing bi-allelic expression in CNS tissue of those queried genes mapping to proximal Chr 7 or 15, including the Gabrb3 gene, for which there have been conflicting reports. Microarray analysis also revealed no effect of the maternal or paternal disomy on expression levels of the unlinked genes detected, including those potentially implicated in the Prader-Willi or Angelman Syndrome. In addition, quantitative RT-PCR revealed a gene dosage effect in both cerebellum and cortex for all of the known imprinted genes assayed, except for Ube3a in cerebellum.     

Web-accessible proteome databases for microbial research

The analysis of proteomes of biological organisms represents a major challenge of the post-genome era. Classical proteomics combines two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) for the identification of proteins. Novel technologies such as isotope coded affinity tag (ICAT)-liquid chromatography/mass spectrometry (LC/MS) open new insights into protein alterations. The vast amount and diverse types of proteomic data require adequate web-accessible computational and database technologies for storage, integration, dissemination, analysis and visualization. A proteome database system (http://www.mpiib-berlin.mpg.de/2D-PAGE) for microbial research has been constructed which integrates 2-DE/MS, ICAT-LC/MS and functional classification data of proteins with genomic, metabolic and other biological knowledge sources. The two-dimensional polyacrylamide gel electrophoresis database delivers experimental data on microbial proteins including mass spectra for the validation of protein identification. The ICAT-LC/MS database comprises experimental data for protein alterations of mycobacterial strains BCG vs. H37Rv. By formulating complex queries within a functional protein classification database "FUNC_CLASS" for Mycobacterium tuberculosis and Helicobacter pylori the researcher can gather precise information on genes, proteins, protein classes and metabolic pathways. The use of the R language in the database architecture allows high-level data analysis and visualization to be performed "on-the-fly". The database system is centrally administrated, and investigators without specific bioinformatic competence in database construction can submit their data. The database system also serves as a template for a prototype of a European Proteome Database of Pathogenic Bacteria. Currently, the database system includes proteome information for six strains of microorganisms.     

Overexpression of manganese superoxide dismutase promotes the survival of prostate cancer cells exposed to hyperthermia

It has been hypothesized that exposure of cells to hyperthermia results in an increased flux of reactive oxygen species (ROS), primarily superoxide anion radicals, and that increasing antioxidant enzyme levels will result in protection of cells from the toxicity of these ROS. In this study, the prostate cancer cell line, PC-3, and its manganese superoxide dismutase (MnSOD)-overexpressing clones were subjected to hyperthermia (43°C, 1 h). Increased expression of MnSOD increased the mitochondrial membrane potential (MMP). Hyperthermic exposure of PC-3 cells resulted in increased ROS production, as determined by aconitase inactivation, lipid peroxidation, and H₂O₂ formation with a reduction in cell survival. In contrast, PC-3 cells overexpressing MnSOD had less ROS production, less lipid peroxidation, and greater cell survival compared to PC-3 Wt cells. Since MnSOD removes superoxide, these results suggest that superoxide free radical or its reaction products are responsible for part of the cytotoxicity associated with hyperthermia and that MnSOD can reduce cellular injury and thereby enhance heat tolerance.     

Backbone dynamics of the human MIA protein studied by (15)N NMR relaxation: implications for extended interactions of SH3 domains

The melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma as enhanced values diagnose metastatic melanoma stages III and IV. Here, we report the backbone dynamics of human MIA studied by (15)N NMR relaxation experiments. The folded core of human MIA is found to be rigid, but several loops connecting beta-sheets, such as the RT-loop for example, display increased mobility on picosecond to nanosecond time scales. One of the most important dynamic features is the pronounced flexibility of the distal loop, comprising residues Asp 68 to Ala 75, where motions on time scales up to milliseconds occur. Further, significant exchange contributions are observed for residues of the canonical binding site of SH3 domains including the RT-loop, the n-Src loop, for the loop comprising residues 13 to 19, which we refer to as the"disulfide loop", in part for the distal loop, and the carboxyl terminus of human MIA. The functional importance of this dynamic behavior is discussed with respect to the biological activity of several point mutations of human MIA. The results of this study suggest that the MIA protein and the recently identified highly homologous fibrocyte-derived protein (FDP)/MIA-like (MIAL) constitute a new family of secreted proteins that adopt an SH3 domain-like fold in solution with expanded ligand interactions.     

The iodothyronine selenodeiodinases are thioredoxin-fold family proteins containing a glycoside hydrolase clan GH-A-like structure

The three iodothyronine selenodeiodinases catalyze the initiation and termination of thyroid hormone effects in vertebrates. Structural analyses of these proteins have been hindered by their integral membrane nature and the inefficient eukaryotic-specific pathway for selenoprotein synthesis. Hydrophobic cluster analysis used in combination with Position-specific Iterated BLAST reveals that their extramembrane portion belongs to the thioredoxin-fold superfamily for which experimental structure information exists. Moreover, a large deiodinase region imbedded in the thioredoxin fold shares strong similarities with the active site of iduronidase, a member of the clan GH-A-fold of glycoside hydrolases. This model can explain a number of results from previous mutagenesis analyses and permits new verifiable insights into the structural and functional properties of these enzymes.     

Comparing beta-carotene,vitamin E and nitric oxide as membrane antioxidants

Singlet oxygen initiates lipid peroxidation via a nonfree radical mechanism by reacting directly with unsaturated lipids to form lipid hydroperoxides (LOOHs). These LOOHs can initiate free radical chain reactions leading to membrane leakage and cell death. Here we compare the ability and mechanism by which three small-molecule membrane antioxidants (beta-carotene, alpha-tocopherol and nitric oxide) inhibit lipid peroxidation in membranes. We demonstrate that beta-carotene provides protection against singlet oxygen-mediated lipid peroxidation, but does not slow free radical-mediated lipid peroxidation. Alpha-Tocopherol does not protect cells from singlet oxygen, but does inhibit free radical formation in cell membranes. Nitric oxide provides no direct protection against singlet oxygen exposure, but is an exceptional chain-breaking antioxidant as evident from its ability to blunt oxygen consumption during free radical-mediated lipid peroxidation. These three small-molecule antioxidants appear to have complementary mechanisms for the protection of cell membranes from detrimental oxidations.     

Correction of diet-induced hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance by moderate hyperleptinemia

Human obesity and high fat feeding in rats are associated with the development of insulin resistance and perturbed carbohydrate and lipid metabolism. It has been proposed that these metabolic abnormalities may be reversible by interventions that increase plasma leptin. Up to now, studies in nongenetic animal models of obesity and in human obesity have concentrated on multiple injection therapy with mixed results. Our study sought to determine whether a sustained, moderate increase in plasma leptin, achieved by administration of a recombinant adenovirus containing the leptin cDNA (AdCMV-leptin) would be effective in reversing the metabolic abnormalities of the obese phenotype. Wistar rats fed a high-fat diet (HF) were heavier (P < 0.05), had increased fat mass and intramuscular triglycerides (mTG), and had elevated plasma glucose, insulin, triglyceride, and free fatty acids compared with standard chow-fed (SC) control animals (all P < 0.01). HF rats also had impaired glucose tolerance and were markedly insulin resistant, as demonstrated by a 40% reduction in insulin-stimulated muscle glucose uptake (P < 0.001). Increasing plasma leptin levels to 29.0 +/- 1.5 ng/ml (from 7.0 +/- 1.4 ng/ml, P < 0.001) for a period of 6 days decreased adipose mass by 40% and normalized plasma glucose and insulin levels. In addition, insulin-stimulated skeletal muscle glucose uptake was normalized in hyperleptinemic rats, an effect that correlated closely with a 60% (P < 0.001) decrease in mTG. Importantly, HF rats that received a control adenovirus containing the beta-galactosidase cDNA and were calorically matched to AdCMV-leptin-treated animals remained hyperglycemic, hyperinsulinemic, insulin resistant, and maintained elevated mTG. We conclude that a gene-therapeutic intervention that elevates plasma leptin moderately for a sustained period reverses diet-induced hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance, and that these improvements are tightly linked to leptin-induced reductions in mTG.     

EPR detection of lipid-derived free radicals from PUFA, LDL, and cell oxidations

We have used the spin trap 5,5-dimethyl-pyrroline-1-oxide (DMPO) and EPR to detect lipid-derived radicals (Ld*) during peroxidation of polyunsaturated fatty acids (PUFA), low-density lipoprotein (LDL), and cells (K-562 and MCF-7). All oxygen-centered radical adducts of DMPO from our oxidizable targets have short lifetimes (<20 min). We hypothesized that the short lifetimes of these spin adducts are due in part to their reaction with radicals formed during lipid peroxidation. We proposed that stopping the lipid peroxidation processes by separating oxidation-mediator from oxidation-substrate with an appropriate extraction would stabilize the spin adducts. To test this hypothesis we used ethyl acetate to extract the lipid-derived radical adducts of DMPO (DMPO/Ld*) from an oxidizing docosahexaenioc acid (DHA) solution; Folch extraction was used for LDL and cell experiments. The lifetimes of DMPO spin adducts post-extraction are much longer (>10 h) than the spin adducts detected without extraction. In iron-mediated DHA oxidation we observed three DMPO adducts in the aqueous phase and two in the organic phase. The aqueous phase contains DMPO/HO* aN approximately aH approximately 14.8 G) and two carbon-centered radical adducts (aN1 approximately 15.8 G, aH1 approximately 22.6 G; aN2 approximately 15.2 G, aH2 approximately 18.9 G). The organic phase contains two long-chain lipid radical adducts (aN approximately 13.5 G, aH approximately 10.2 G; and aN approximately 12.8 G; aH approximately 6.85 G, 1.9 G). We conclude that extraction significantly increases the lifetimes of the spin adducts, allowing detection of a variety of lipid-derived radicals by EPR.