Relationships between cardiorespiratory fitness, physical activity, and psychosocial variables in overweight and obese breast cancer survivors.

BACKGROUND: Breast cancer survivors not only experience distressing physical symptoms associated with treatments, but also are faced with psychosocial challenges. Despite growing scientific evidence that physical activity (PA) may mitigate psychosocial distress experienced by women treated for breast cancer, the literature is equivocal. PURPOSE: This study investigated the relationships between cardiorespiratory fitness (CRF), PA, and psychosocial factors in breast cancer survivors. METHOD: Data involving overweight or obese breast cancer survivors (N = 260) were examined. CRF was determined by a submaximal graded exercise test. PA, depressive symptoms, total fatigue, and global self-esteem were assessed with self-report measures. Pearson's correlations were conducted to determine associations among CRF, PA, depressive symptoms, total fatigue, and global self-esteem. Multiple regression models, with age and body mass index as covariates, were performed using continuous levels for CRF and PA. RESULTS: Bivariate correlations suggested that CRF and PA were unrelated to the psychosocial variables. One of the regression models identified a marginally significant (P = 0.06) inverse association between depressive symptoms and PA. CONCLUSION: CRF and PA were not associated with psychosocial factors in this sample of breast cancer survivors. However, minimal PA was reported by the majority of participants, so low PA variability likely influenced these findings.

Growth pattern and partial proteome of Mycobacterium avium subsp. paratuberculosis during the stress response to hypoxia and nutrient starvation.

Mycobacterium avium subsp. paratuberculosis is an important pathogen that causes Johne's disease in animals and has been implicated in Crohn's disease in man yet few data exist on its physiological adaptation in either the host or the environment. In this study, the proteomic responses of the two distinct strains of M. a. paratuberculosis, cattle (C) and sheep (S), to hypoxia and starvation were studied in vitro. Nutrient starvation inhibited growth of both strains and was lethal for S strain after 12 weeks. Hypoxia induced a state of very low metabolic activity but rapid resuscitation occurred upon restoration of an aerobic atmosphere, consistent with the dormancy response of other mycobacteria. A total of 55 protein spots differentially expressed in response to starvation and/or hypoxic stress in one or both strains were identified from 2D gels and classified based on biological function. Antioxidant enzymes, oxidoreducatse enzymes and proteins involved in amino acid metabolism, fatty acid metabolism, ATP and purine biosynthesis, proteolysis, cell wall synthesis, protein synthesis, signal recognition and hypothetical proteins with putative functions including dormancy response regulators and universal stress proteins were identified. These proteins are potential screening targets for future diagnosis, prevention and control of M. a. paratuberculosis infection and their identification will assist understanding the pathogenesis of diseases caused by this organism.

Enzyme-linked immunospot: an alternative method for the detection of interferon gamma in Johne's disease.

To date, the sensitivity of the interferon gamma (IFN-gamma) enzyme-linked immunosorbent assay (ELISA) to detect Johne's disease (JD) has been poor, especially in the early stages of disease. To improve the sensitivity of IFN-gamma detection in the early stages of infection, an alternate assay needs to be developed. The enzyme-linked immunospot (ELISPOT) assay is a highly sensitive technique for the detection of cytokines and has the potential to improve the diagnosis of JD. Of the variables examined, choice of capture antibody and the method by which the peripheral blood mononuclear cells were isolated significantly affected the ability to enumerate IFN-gamma-secreting cells. The ELISPOT assay was as sensitive as or better than the IFN-gamma ELISA at detecting ovine JD and could also detect disease at early time points postinoculation. The IFN-gamma ELISPOT could distinguish infected from unexposed animals; however, neither the IFN-gamma ELISA nor the ELISPOT assay could distinguish between sheep experimentally infected with Mycobacterium avium subspecies paratuberculosis and those exposed to the bacterium but diagnosed as uninfected at necropsy.

Toll-like receptor genes are differentially expressed at the sites of infection during the progression of Johne's disease in outbred sheep.

Toll-like receptors (TLR) are engaged by ligands on microbial pathogens to initiate innate and adaptive immune responses. Little is known about TLR involvement during infection with Mycobacterium avium subsp. paratuberculosis (M. ptb), the cause of Johne's disease in ruminants, although there is a profound immunopathological response in affected animals. We have analyzed the expression of 10 TLR genes relative to validated reference genes at predilection sites in ileum, jejunum and associated lymph nodes as well as in peripheral blood, to determine if TLR expression is altered in response to infection with M. ptb in outbred sheep. Previously unexposed animals from two flocks and animals from three naturally infected flocks were used with restricted maximum likelihood linear mixed modeling applied to determine significant differences. These were related to the pathologies observed at different stages of infection in exposed sheep, after allowing for other sources of variation. In most cases there were differences in TLR expression between early paucibacillary and multibacillary groups when compared to uninfected sheep, with most TLRs for the paucibacillary group having lower expression levels than the multibacillary group. Increased expression of TLR1-5, and 8 was observed in ileum or jejunum, and TLR1-4, 6, and 8 in mesenteric lymph nodes. There was a trend for increased expression of TLR1, 2, and 6-8 in PBMCs of exposed compared to non-exposed animals. Further study of TLR expression in Johne's disease in ruminants is warranted as these observed differences may help explain pathogenesis and may be useful in the future diagnosis of M. ptb infection.

Protein extraction from Mycobacterium avium subsp. paratuberculosis: Comparison of methods for analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis, native PAGE and surface enhanced laser desorption/ionization time of flight mass spectrometry.

Mycobacterium paratuberculosis causes Johne's disease, a chronic bowel disease in ruminants worldwide and is currently incurable. This study was conducted to compare methods for examining the proteome of M. paratuberculosis. SDS-PAGE, native PAGE and SELDI-TOF-MS were compared and the efficacy of various lysis buffers was assessed. Chaotropic agents (Urea CHAPS and potassium thiocyanate) and non-ionic detergent (Tween20 and Triton X-100) extracts were compared on three different ProteinChip surfaces along with two energy absorbing molecules (EAM): EAM-1 proprietary formulation and sinapinic acid (Ciphergen). Urea CHAPS was efficient for extraction of proteins and their detection on all the ProteinChip surfaces. However, potassium thiocyanate was the most effective buffer, leading to detection of the greatest number of protein peaks on the immobilized metal affinity chromatography (IMAC) surface. Sinapinic acid was more efficient than the EAM-1 proprietary formulation and resulted in additional peaks with higher intensity for both the low and the medium molecular weight range proteins. Intra-chip and inter-chip coefficient of variation for mass/charge varied from 0.01% to 0.07% and 0.00% to 0.08%, respectively. SELDI-TOF-MS was an efficient tool for the protein profiling of M. paratuberculosis and will be useful for investigation of novel proteins, although SDS-PAGE/2D gel electrophoresis is recommended for study of high molecular weight species. All buffers were suitable for protein extraction for SDS-PAGE, while Tween20 was best for native PAGE.

Detection of Mycobacterium avium subsp. paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture.

The aims of this study were to develop a new real-time quantitative PCR (QPCR) assay based on IS900 for detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP) DNA in faeces, and to use this to detect infected sheep. Both the C and S strains of MAP were detected by the QPCR assay, and no cross reactions were detected with 51 other species of mycobacteria including 10 which contained IS900-like sequences. One copy of IS900 fragment cloned into plasmid pCR2.1 and 1 fg of MAP genomic DNA were consistently detected, while in spiked faecal samples the detection limit was 10 viable MAP per gram of ovine faeces. A total of 506 individual ovine faecal samples and 27 pooled ovine faecal samples with known culture results were tested. The QPCR assay detected 68 of 69 BACTEC culture positive individual faeces and there was a strong relation between time to detection in culture and DNA quantity measured by QPCR (r= -0.70). In pooled faecal samples, QPCR also agreed with culture (kappa=0.59). MAP DNA was detected from some culture negative faecal samples from sheep exposed to MAP, suggesting that the QPCR has very high analytical sensitivity for MAP in faecal samples and detects non-viable MAP in ovine faeces. None of the faecal samples from 176 sheep that were not exposed to MAP were positive in QPCR. This is the first report of a direct faecal QPCR assay that has similar sensitivity to a gold standard radiometric culture assay.

Validation of endogenous reference genes for expression profiling of RAW264.7 cells infected with Mycobacterium avium subsp. paratuberculosis by quantitative PCR.

Reference genes are frequently used to normalize between different biological samples the levels of mRNA measured using quantitative PCR (qPCR). The expression level of many commonly used reference genes has been shown to vary between tissues or cells, or following exposure to various treatments including infection with microbes. The selection of an appropriate reference gene for an individual experiment is therefore a crucial step in the process of accurately determining changes in gene expression. For this purpose, we analyzed the expression of nine commonly used reference genes in a murine macrophage cell line, RAW264.7, for their potential use in the analysis of differential gene expression by quantitative polymerase chain reaction (qPCR) following experimental infection with Mycobacterium avium subsp. paratuberculosis. Only one of nine putative reference genes tested, casc3a, was found to be suitable, and combinations of two or more reference genes were disadvantageous. Based on data from the study, we recommend an approach for selection of reference genes, conducting assays with technical replicates in duplicate rather than triplicate, determining decision-limit quality control criteria for technical replicates and assessing the significance of gene expression fold differences using DeltaDeltaC(t) based on knowledge of the variation in the reference gene.

Stressful military training: endocrine reactivity, performance, and psychological impact.

INTRODUCTION: We examined the responsiveness of both cortisol and dehydroepiandrosterone sulfate (DHEAS) to the stress of survival training in military men and evaluated relationships to performance, peritraumatic dissociation, and the subsequent impact of stressful events. METHODS: Baseline salivary cortisol samples were self-collected by 19 men at 0900 and 1930 in a free-living (FL) environment. DHEAS samples were also collected in a subset of this sample (N = 12). Samples were subsequently taken at similar time points during a stressful captivity (SC) phase of training. Repeated-measures analyses of variance with follow-up paired t-tests examined differences across time and conditions. RESULTS: Significant increases were observed at both time points (0900 and 1930) from FL to SC in both cortisol (0900: 9.2 +/- 3.4 nmol x L(-1) vs. 18.4 +/- 10.5 nmol x L(-1); 1930: 3.5 +/- 3.0 nmol x L(-1) vs. 27.7 +/- 10.9 nmol x L(-1)) and DHEAS (0900: 1.7 +/- 1.3 ng x ml(-1) vs.6.7 +/- 3.5 ngx ml(-1); 1930: 1.5 0.84 ng x ml(-1) vs. 4.5 +/- 3.0 ng x ml(-1)). Also, overall performance during a high-intensity captivity-related challenge was inversely related to the DHEAS-cortisol ratio; conversely, overall performance during a low-intensity captivity-related challenge was positively related to DHEAS at the 0900 time point during SC. Dissociation was unrelated to endocrine indices measured during SC, while total impact of events was inversely related to percent change in DHEAS from FL to SC. CONCLUSIONS: Cortisol and DHEAS increase in response to allostatic load, and may relate to human performance during SC as well as PTSD symptoms.

The S. pombe translation initiation factor eIF4G is Sumoylated and associates with the SUMO protease Ulp2.

SUMO is a small post-translational modifier, that is attached to lysine residues in target proteins. It acts by altering protein-protein interactions, protein localisation and protein activity. SUMO chains can also act as substrates for ubiquitination, resulting in proteasome-mediated degradation of the target protein. SUMO is removed from target proteins by one of a number of specific proteases. The processes of sumoylation and desumoylation have well documented roles in DNA metabolism and in the maintenance of chromatin structure. To further analyse the role of this modification, we have purified protein complexes containing the S. pombe SUMO protease, Ulp2. These complexes contain proteins required for ribosome biogenesis, RNA stability and protein synthesis. Here we have focussed on two translation initiation factors that we identified as co-purifying with Ulp2, eIF4G and eIF3h. We demonstrate that eIF4G, but not eIF3h, is sumoylated. This modification is increased under conditions that produce cytoplasmic stress granules. Consistent with this we observe partial co-localisation of eIF4G and SUMO in stressed cells. Using HeLa cells, we demonstrate that human eIF4GI is also sumoylated; in vitro studies indicate that human eIF4GI is modified on K1368 and K1588, that are located in the C-terminal eIF4A- and Mnk-binding sites respectively.

Proteomic profiling of ovine serum by SELDI-TOF MS: optimisation, reproducibility and feasibility of biomarker discovery using routinely collected samples.

The diagnosis of infectious diseases in animals may be enhanced by study of the serum proteome in which myriad components are influenced by physiological and pathological processes. Surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS) has the capacity to detect known and unknown immunologically relevant molecules in the serum proteome. Optimum combinations of ProteinChip array surfaces, energy absorbing molecules, sample dilutions and instrument settings were determined for spectral generation from whole ovine sera. The coefficient of variation for within and between chip mass/charge and peak intensity were <0.03% and <23%, respectively. There were minor alterations in spectra associated with storage of chips or machine drift. Clotting times of 30 min to 3h did not greatly alter protein spectra although storage of sera at -20 degrees C led to alterations. However, routinely collected serum samples stored at -20 degrees C were useful for identification of biomarkers associated with vaccination with a bacterial antigen. This information will inform future studies on serum proteome profiling in livestock, but independent assessments are recommended for each species.

Experimental infection model for Johne's disease using a lyophilised, pure culture, seedstock of Mycobacterium avium subspecies paratuberculosis.

Johne's disease is a severe chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (Map). Repeatable infections of known duration are required for validation of new diagnostic tests, evaluation of pathogenesis and development of improved vaccines. In the first study of its type, a standardised experimental model for Johne's disease was developed based on a lyophilised, low passage, pure culture, seedstock of Map. Experimental inoculations of sheep with accurately enumerated doses of Map resulted in infection outcomes across multiple trials that were modulated by the interval between inoculation and examination. Compared to an inoculum consisting of an intestinal mucosal homogenate from a naturally affected sheep, clinical signs from the pure culture of Map were manifested later, but other measures of infection were similar. Immunological assays showed that most of the inoculated animals were IFN-gamma positive in the early stages of the infection. Over time, an increasing number of sheep became Map-specific antibody positive, developed typical histopathological lesions and shed Map in their faeces. The repeatability and utility of this experimental infection model will enable study of many aspects of Johne's disease. It is the first study to show that models for Johne's disease can be standardised in relevant species using traditional microbiological approaches to production and storage of seedstock. It is recommended that an international bank of master seedstock be established, containing low passage isolates that are representative of the major strains of Map, S and C.

Evaluation of the immunogenicity of recombinant stress-associated proteins during Mycobacterium avium subsp. paratuberculosis infection: Implications for pathogenesis and diagnosis.

The aim of this study was to assess the immunogenicity of recombinant stress-associated proteins of Mycobacterium avium subsp. paratuberculosis in sheep infected with the organism compared to control sheep. Five proteins - MAP2411, ClpP, Ppa, MAP0593c and GreA - which were identified previously in in vitro stress or dormancy responses of M. paratuberculosis to hypoxia, nutrient starvation and heat, were cloned, expressed and purified as His-tag recombinant proteins from the pET-15b vector in a BL21(DE3)pLysS strain of E. coli. The immunogenicity of MAP2411 did not differ between infected and control sheep. However, the serological reactivity of the other recombinant antigens, and combinations of them, varied according to the histological stage of paratuberculosis. Interestingly, the sera from some animals with paucibacillary lesions, which were not immunoreactive in a commercial paratuberculosis ELISA that was based on non-defined native antigens, recognised the recombinant antigens. We infer from their differential immunogenicity in infected and control sheep that four of the stress-associated proteins were expressed by M. paratuberculosis in vivo. These data provide fundamental information on host-mycobacterial interactions and have conceptual implications for the development of future diagnostic tests for early immune responses in animals infected with mycobacteria.

Cell-cycle-dependent localisation of Ulp1, a Schizosaccharomyces pombe Pmt3 (SUMO)-specific protease.

We report here on the characterisation of Ulp1, a component of the SUMO modification process in S. pombe. Recombinant S. pombe Ulp1 has de-sumoylating activity; it is involved in the processing of Pmt3 (S. pombe SUMO) and can, to a limited extent, remove Pmt3 from modified targets in S. pombe cell extracts. ulp1 is not essential for cell viability, but cells lacking the gene display severe cell and nuclear abnormalities. ulp1-null (ulp1.d) cells are sensitive to ultraviolet radiation in a manner similar to rad31.d and hus5.62, which have mutations in one subunit of the activator and the conjugator for the ubiquitin-like protein SUMO respectively. However ulp1.d cells are less sensitive to ionising radiation and hydroxyurea (HU) than are rad31.d and hus5.62. ulp1-null cells are defective in processing precursor Pmt3 and display reduced levels of Pmt3 conjugates compared with wild-type cells. The slow growth phenotype of ulp1 null cells is not substantially rescued by over-expression of the mature form of Pmt3 (Pmt3-GG), suggesting that the de-conjugating activity of Ulp1 is required for normal cell cycle progression. During the S and G2 phases of the cell cycle the Ulp1 protein is localised to the nuclear periphery. However, during mitosis the pattern of staining alters, and during anaphase, Ulp1 is observed within the nucleus. Ulp1 localisation at the nuclear periphery is generally re-established by the time of septation (S phase).