The interaction of Bex and OMP reveals a dimer of OMP with a short half-life

Olfactory marker protein (OMP) participates in the olfactory signal transduction pathway. This is evident from the behavioral and electrophysiological deficits of OMP-null mice, which can be reversed by intranasal infection of olfactory sensory neurons with an OMP-expressing adenovirus. Bex, brain expressed X-linked protein, has been identified as a protein that interacts with OMP. We have now further characterized the interaction of OMP and Bex1/2 by in vitro binding assays and by immuno-coprecipitation experiments. OMP is a 19 kDa protein but these immunoprecipitation studies have revealed the unexpected presence of a 38 kDa band in addition to the expected 19 kDa band. Furthermore, the 38 kDa form was preferentially co-immunoprecipitated with Bex from cell extracts. In-gel tryptic digestion, mass spectrometry, and two-dimensional gel electrophoresis indicate that the 38 kDa protein behaves as a covalently cross-linked OMP-homodimer. The 38 kDa band was also identified in western blots of olfactory epithelium demonstrating its presence in vivo. The stabilities and subcellular localizations of the OMP-monomer and -dimer were studied in transfected cells. These results demonstrated that the OMP-dimer is much less stable than the monomer, and that while the monomer is present both in the nuclear and cytosolic compartments, the dimer is preferentially located in a Triton X-100 insoluble cytoskeletal fraction. These novel observations led us to hypothesize that regulation of the level of the rapidly turning-over OMP-dimer and its interaction with Bex1/2 is critical for OMP function in sensory transduction.     

Enrichment and characterization of mouse putative epidermal stem cells

Epidermis, a continuously renewing tissue, is maintained by stem cells that proliferate and replenish worn out or damaged cells in the tissue during life. Cultured epidermal stem cells have great potential in scientific research and clinical application. However, isolating a pure and viable population of epidermal stem cells and culturing them has been challenging. In this study, putative epidermal stem cells of mouse were isolated by combining Hoechst 33342 and propidium iodide staining with fluorescence-activated cell sorting. Molecular markers expression pattern analysis showed that cytokeratin 14, integrin beta1 and p63 are expressed in the sorted putative stem cells, but not active beta-catenin, nestin and involucrin. Our results provide further supporting data that mouse putative epidermal stem cells could be successfully isolated by combining Hoechst dye staining with fluorescence-activated cell sorting and cultured in vitro. The cultured mouse putative epidermal stem cells could be used as a potent tool for studying stem cell biology and testing stem cell therapy.     

Counterfactual links to the proportion of treatment effect explained by a surrogate marker

In a randomized clinical trial, a statistic that measures the proportion of treatment effect on the primary clinical outcome that is explained by the treatment effect on a surrogate outcome is a useful concept. We investigate whether a statistic proposed to estimate this proportion can be given a causal interpretation as defined by models of counterfactual variables. For the situation of binary surrogate and outcome variables, two counterfactual models are considered, both of which include the concept of the proportion of the treatment effect, which acts through the surrogate. In general, the statistic does not equal either of the two proportions from the counterfactual models, and can be substantially different. Conditions are given for which the statistic does equal the counterfactual model proportions. A randomized clinical trial with potential surrogate endpoints is undertaken in a scientific context; this context will naturally place constraints on the parameters of the counterfactual model. We conducted a simulation experiment to investigate what impact these constraints had on the relationship between the proportion explained (PE) statistic and the counterfactual model proportions. We found that observable constraints had very little impact on the agreement between the statistic and the counterfactual model proportions, whereas unobservable constraints could lead to more agreement.     

Mutation spectra in supF: approaches to elucidating sequence context effects

Shuttle vectors carrying the supF suppressor tRNA gene were originally developed for mutagenesis experiments in primate and human cells. Since then, the supF gene has been used as a mutation reporter in other mammalian cells, yeast, Escherichia coli, and transgenic mice. The widespread use of the vector for studies of many DNA reactive agents has produced a large database of mutation spectra. These provide primary information on the kinds and distribution of mutations provoked by many agents and, in many instances, allow comparisons between related agents or the same agent in different cell backgrounds. In this review we will discuss some of these data with a primary focus on the interpretation of UV mutation spectra. We will also describe our development and application of custom supF marker genes as an approach to studying the effect of sequence context on mutation hotspots and cold spots. Our studies suggest that C-C photoproducts are not mutagenic in certain sequence contexts in which T-C photoproducts are mutation hotspots. In addition, we have found several examples of sequence context effects acting as much as 80 bases away from the site of mutation. We will consider some of the problems raised by these studies and the possible resolution of some of them offered by the newly discovered family of damage bypass DNA polymerases.     

Recent advances in exploring physiology and biodiversity of ectomycorrhizas highlight the functioning of these symbioses in ecosystems

Ectomycorrhizas, the dominating mycorrhizal symbiosis in boreal, temperate and some tropical forests, are formed by 5000-6000 species of the asco- and basidiomycetes. This high diversity of fungal partners allows optimal foraging and mobilisation of various nitrogen and phosphorus forms from organic soil layers. In this review, two approaches to study the functioning of this multitude of symbiotic associations are presented. On selected culture models, physiological and molecular investigations have shown that the supply of hexoses has a key function in controlling the plant-fungus interaction via partner-specific regulation of gene expression. Environmental factors which affect fungal carbon supply, such as increased nitrogen availability, also affect mycorrhiza formation. Based on such laboratory results, the adaptative capability of ectomycorrhizas to changing field conditions is discussed. The second approach consists of analysing the distribution of mycorrhizas in ecosystem compartments and to relate distribution patterns to variations of ecological factors. Recent advances in identification of fungal partners in ectomycorrhizas by analysing the internal transcribed spacer of ribosomal DNA are presented, which can help to resolve sampling problems in field studies. The limits of the laboratory and the field approaches are discussed. Despite some problems, this combined approach is the most promising. Direct investigation of gene expression, which has been introduced for soil bacteria, will be difficult in the case of mycorrhizal fungi which constitute organisms with functionally varying structures.     

Genome-wide linkage analyses and candidate gene fine mapping for HDL3 cholesterol: the Framingham Study

High density lipoprotein cholesterol (HDL-C) is inversely associated with coronary heart disease and has a genetic component; however, linkage to HDL-C is not conclusive. Subfractions of HDL, such as HDL(3)-C, may be better phenotypes for linkage studies. Using HDL(3)-C levels measured on 907 Framingham Heart Study subjects from 330 families around 1987, we conducted a genome-wide variance components linkage analysis with 401 microsatellite markers spaced approximately 10 centimorgan (cM) apart. Nine candidate genes were identified and annotated using a bioinformatics approach in the region of the highest linkage peak. Twenty-eight single nucleotide polymorphisms (SNPs) were selected from these candidate genes, and linkage and family-based association fine mapping were conducted using these SNPs. The highest multipoint log-of-the-odds (LOD) score from the initial linkage analysis was 3.7 at 133 cM on chromosome 6. Linkage analyses with additional SNPs yielded the highest LOD score of 4.0 at 129 cM on chromosome 6. Family-based association analysis revealed that SNP rs2257104 in PLAGL1 at approximately 143 cM was associated with multivariable adjusted HDL(3) (P = 0.03). Further study of the linkage region and exploration of other variants in PLAGL1 are warranted to define the potential functional variants of HDL-C metabolism.     

Construction and characterization of a live, attenuated apxIICA inactivation mutant of Actinobacillus pleuropneumoniae lacking a drug resistance marker

The apxIIC gene of Actinobacillus pleuropneumoniae serotype 7 was inactivated by homologous recombination using a sucrose counter-selectable marker system, resulting in a mutant strain that had no antibiotic resistance marker and expressed an inactivated ApxII toxin. The safety and immunogenicity of the mutant were evaluated in mice. The mutant strain caused no adverse effects in mice at doses up to 2 x 10(9) CFU via the intraperitoneal route while the parental strain induced total mortality at a dose of 2 x 10(7) CFU. Mice vaccinated intraperitoneally with the mutant strain had 100% and 70% protection against homologous (serotype 7) or heterologous (serotype 1, 3) challenge with A. pleuropneumoniae, respectively. The A. pleuropneumoniae mutant strain HB04C- and the counterselection method used in the study show promise in developing effective live vaccines for porcine pleuropneumonia and for other infections diseases of the respiratory system.     

Rapid and quantitative detection of blood Serratia marcescens by a real-time PCR assay: its clinical application and evaluation in a mouse infection model

Large-scale nosocomial outbreaks of Serratia marcescens septicaemia in Japan have had a fatality rate of 20-60% within 48 h. As a countermeasure, a real-time PCR assay was constructed for the rapid diagnosis of S. marcescens septicaemia. This assay indeed detected S. marcescens in clinical blood specimens (at ca. 10(2)CFU ml(-1)), at a frequency of 0.5% in suspected cases of septicaemia. In mice, the assay provided estimates of blood S. marcescens levels at various infectious stages: namely, 10(7) to 10(8)CFU ml(-1) at a fatal stage (resulting in 100% death), 10(4)-10(5)CFU ml(-1) at a moderately fatal stage (resulting in 50% or more death), and <10(3)CFU ml(-1) at a mild stage (resulting in 100% survival), consistent with actual CFU measurements. Blood bacterial levels could be an important clinical marker that reflects the severity of septicaemia. The simultaneous detection of S. marcescens and the carbapenem resistance gene was also demonstrated.     

Challenges in the Testing of Non-Heart-Beating Cadavers for Viral Markers: Implications for the Safety of Tissue Donors

Natural changes that occur in blood and tissue after death may result in false positive results in antigen and antibody detection tests performed to identify markers of viral infection in potential tissue donors. Such tissue, which might otherwise be acceptable for therapeutic purposes, would not meet current standards for safe tissue banking. This is especially important in the context of insufficiency in the tissue supply. In this study, a series of blood samples collected during routine post-mortem examination was assayed using a range of commercially available kits for the detection of HBsAg, anti-HCV and anti-HIV 1 + 2 antibody/antigen. Results of tests on 104 samples collected from 97 individuals indicate that some kits result in a higher number of initial reactive samples than others. Approximately 40% of samples were reactive in one or more HBsAg assay, less than 10% in at least one anti-HIV kit and only 1 sample at low level on an anti-HCV kit. Liver or lymph node samples from individuals whose serum sample gave reactive results in antigen/antibody assays were tested for viral nucleic acid in the corresponding nucleic acid amplification test. Only one individual’s sample was confirmed to test positive for HBsAg in a confirmatory neutralisation test and by nucleic acid amplification technology, and a second individual whose serum was scored reactive for anti-HCV, but negative for HBsAg, had a liver sample which was HBV DNA positive and HCV RNA negative. The results of the study indicate that antibody/antigen assays are not as specific as NAT using state of the art DNA extraction techniques. Both types of assay complement each other and used together will help assure the safety of tissues for transplantation.