Techniques for quantifying effects of dietary antioxidants on transcription factor translocation and nitric oxide production in cultured cells

Dietary antioxidants can affect cellular processes relevant to chronic inflammatory diseases such as atherosclerosis. We have used non- standard techniques to quantify effects of the antioxidant soy isoflavones genistein and daidzein on translocation of Nuclear Factor-KB (NF-KB) and nitric oxide (NO) production, which are important in these diseases. Translocation was quantified using confocal immunofluoresecence microscopy and ratiometric image analysis. NO was quantified by an electrochemical method after reduction of its oxidation products in cell culture supernatants. Activation of the RAW 264.7 murine monocyte/macrophage cell line increased the ratio of nuclear to cytoplasmic immunostaining for NF-kB. The increase was exacerbated by pre-treatment with genistein or daidzein. To show that decreases could also be detected, pre-treatment with the pine bark extract Pycnogenol (R) r was examined, and found to reduce translocation. NO production was also increased by activation, but was reduced by pre-treatment with genistein or daidzein. In the EA. hy926 human endothelial cell line, constitutive production was detectable and was increased by thrombin. The confocal and electrochemical methods gave data that agreed with results obtained using the established electromobility shift and Griess assays, but were more sensitive, more convenient, gave more detailed information and avoided the use of radioisotopes.

Rat brain serotonin neurones that express neuronal nitric oxide synthase have increased sensitivity to the substituted amphetamine serotonin toxins 3,4-methylenedioxymethamphetamine and p-chloroamphetamine

Substituted amphetamines such as p-chloroamphetamine and the abused drug methylenedioxymethamphetamine cause selective destruction of serotonin axons in rats, by unknown mechanisms. Since some serotonin neurones also express neuronal nitric oxide synthase, which has been implicated in neurotoxicity, the present study was undertaken to determine whether nitric oxide synthase expressing serotonin neurones are selectively vulnerable to methylenedioxymethamphetamine or p-chloroamphetamine. Using double-labeling immunocytochemistry and double in situ hybridization for nitric oxide synthase and the serotonin transporter, it was confirmed that about two thirds of serotonergic cell bodies in the dorsal raphe nucleus expressed nitric oxide synthase, however few if any serotonin transporter immunoreactive axons in striatum expressed nitric oxide synthase at detectable levels. Methylenedioxymethamphetamine (30 mg/kg) or p-chloroamphetamine (2 x 10 mg/kg) was administered to Sprague-Dawley rats, and 7 days after drug administration there were modest decreases in the levels of serotonin transporter protein in frontal cortex, and striatum using Western blotting, even though axonal loss could be clearly seen by immunostaining. p-Chloroamphetamine or methylenedioxymethamphetamine administration did not alter the level of nitric oxide synthase in striatum or frontal cortex, determined by Western blotting. Analysis of serotonin neuronal cell bodies 7 days after p-chloroamphetamine treatment, revealed a net down-regulation of serotonin transporter mRNA levels, and a profound change in expression of nitric oxide synthase, with 33% of serotonin transporter mRNA positive cells containing nitric oxide synthase mRNA, compared with 65% in control animals. Altogether these results support the hypothesis that serotonin neurones which express nitric oxide synthase are most vulnerable to substituted amphetamine toxicity, supporting the concept that the selective vulnerability of serotonin neurones has a molecular basis.

Eight-plex iTRAQ analysis of variant metastatic human prostate cancer cells identifies candidate biomarkers of progression: an exploratory study.

BACKGROUND: Due to the heterogeneity in the biological behavior of prostate cancer, biomarkers that can reliably distinguish indolent from aggressive disease are urgently needed to inform treatment choices. METHODS: We employed 8-plex isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to profile the proteomes of two distinct panels of isogenic prostate cancer cells with varying growth and metastatic potentials, in order to identify novel biomarkers associated with progression. The LNCaP, LNCaP-Pro5, and LNCaP-LN3 panel of cells represent a model of androgen-responsive prostate cancer, while the PC-3, PC-3M, and PC-3M-LN4 panel represent a model of androgen-insensitive disease. RESULTS: Of the 245 unique proteins identified and quantified (>or=95% confidence; >or=2 peptides/protein), 17 showed significant differential expression (>or=+/-1.5), in at least one of the variant LNCaP cells relative to parental cells. Similarly, comparisons within the PC-3 panel identified 45 proteins to show significant differential expression in at least one of the variant PC-3 cells compared with parental cells. Differential expression of selected candidates was verified by Western blotting or immunocytochemistry, and corresponding mRNA expression was determined by quantitative real-time PCR (qRT-PCR). Immunostaining of prostate tissue microarrays for ERp5, one of the candidates identified, showed a significant higher immunoexpression in pre-malignant lesions compared with non-malignant epithelium (P < 0.0001, Mann-Whitney U-test), and in high Gleason grade (4-5) versus low grade (2-3) cancers (P < 0.05). CONCLUSIONS: Our study provides proof of principle for the application of an 8-plex iTRAQ approach to uncover clinically relevant candidate biomarkers for prostate cancer progression.

Isolation from a sheep of an attaching and effacing Escherichia coli O115:H- with a novel combination of virulence factors

Attaching and effacing (AE) lesions were observed in the caecum, proximal colon and rectum of one of four lambs experimentally inoculated at 6 weeks. of age with Escherichia coli O157:H7. However, the attached bacteria did not immunostain with O157-specific antiserum. Subsequent bacteriological analysis of samples from this animal yielded two E. coli O115:H- strains, one from the colon (CO) and one from the rectum (RC), and those bacteria forming the AE lesions were shown to be of the O115 serogroup by immunostaining. The O115:H(-)isolates formed microcolonies and attaching and effacing lesions, as demonstrated by the fluorescence actin staining test, on HEp-2 tissue culture cells. Both isolates were confirmed by PCR to encode the epsilon (epsilon) subtype of intimin. Supernates of both O115:H- isolates induced cytopathic effects on Vero cell monolayers, and PCR analysis verified that both isolates encoded EAST1, CNF1 and CNF2 toxins but not Shiga-like toxins. Both isolates harboured similar sized plasmids but-PCR analysis indicated that only one of the O115:H- isolates (CO) possessed the plasmid-associated virulence determinants ehxA and etpD. Neither strain possessed the espP, katP or bfpA plasmid-associated virulence determinants. These E. coli O115:H- strains exhibited a novel combination of virulence determinants and are the first isolates found to possess both CNF1 and CNF2.

Morphological characterization of periodontium-derived human stem cells

The aim of this study has been to characterize adult human somatic periodontium-derived stem cells (PDSCS) isolated from human periodontium and to follow their differentiation after cell culture. PDSCS were isolated from human periodontal tissue and cultured as spheres in serum-free medium. After 10 days the primary spheres were dissociated and the secondary spheres sub-cultured for another 1-2 weeks. Cells from different time points were analyzed, and immunohistochemical and electron microscopic investigations carried out. Histological analysis showed differentiation of spheres deriving from the PDSCS with central production of extracellular matrix beginning 3 days after sub-culturing. Isolated PDSCS developed pseudopodia which contained actin. Tubulin was found in the central portion of the cells. Pseudopodia between different cells anastomosed, indicating intercellular transport. Immunostaining for osteopontin demonstrated a positive reaction in primary spheres and within extracellular matrix vesicles after sub-culturing. In cell culture under serum-free conditions human PDSCS form spheres which are capable of producing extracellular matrix. Further investigations have do be carried out to investigate the capability of these cells to differentiate into osteogenic progenitor cells.

Dystrophin abnormalities in polymyositis and dermatomyositis.

The expression of dystrophin in muscle biopsies from nine cases of polymyositis, ten cases of juvenile dermatomyositis and three adults with dermatomyositis was studied by Western blot analysis and immunocytochemistry. Five antibodies corresponding to different N- and C-terminal regions of the dystrophin gene were used. Sixteen of the 22 cases (73%) showed an abnormality in the expression of dystrophin on Western blot analysis, either with a reduced molecular weight protein or a reduced amount. Immunostaining was abnormal in 11 out of 19 cases (58%) and showed varying degrees of discontinuity or loss of sarcolemmal staining. Immunolabelling of these areas with antibodies to beta-spectrin was normal implying that the changes were not caused by a loss of the sarcolemma. These results show that secondary changes in the expression of dystrophin can occur in the absence of an abnormality in the corresponding gene and that dystrophin cannot be used in isolation as a diagnostic marker for muscular dystrophy.

Characterisation of dystrophin in carriers of Duchenne muscular dystrophy.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, was studied in needle biopsy samples taken from the quadriceps muscle of 15 asymptomatic carriers of DMD (13 adults and 2 young girls) and one symptomatic adult carrier. Antibodies to N- and C-terminal regions of dystrophin were used for both Western blot analysis and immunocytochemistry and a monoclonal antibody to beta-spectrin used to assess membrane integrity. All asymptomatic adult carriers showed some abnormality in dystrophin immunostaining but very few negative fibres were present. A clear mosaic of dystrophin positive and negative fibres was seen only in the adult symptomatic carrier and the two young girls. On a Western blot, all carriers studied had dystrophin of normal molecular weight, but most had reduced abundance. In adult carriers, the amount of dystrophin relative to normal controls varied, but it was unrelated to age, serum creatine kinase (CK) levels or to the degree of pathology. Carriers with normal CK showed abnormalities in dystrophin expression. The dystrophin immunoblotting profile of the 2 young girls was very similar to that of their mothers, but the mosaic pattern of immunostaining was not apparent in the older carriers. In conclusion, dystrophin immunostaining and Western blot analysis of biopsy samples from asymptomatic carriers is often abnormal and they may be useful additional aids for establishing carrier status, particularly in younger girls.

Dystrophin immunoreactivity in normal and Duchenne human fetal neurons in culture.

Dystrophin, the protein product defective in Duchenne muscular dystrophy (DMD), is present in all types of muscle and in the brain. The function of the protein is unknown and its role in the brain is unclear, although 30% of DMD patients show nonprogressive mental retardation. We have therefore studied the localisation of dystrophin in cultures of normal and DMD human fetal neurons using antibodies raised to different regions of the protein. Dystrophin immunoreactivity was demonstrated in the soma and axon hillock of normal neurons and appeared to be associated with the inner part of the cell membrane, although some intracellular staining was also observed. Positive dystrophin staining was present only in cells with fully developed neuronal features, although not all the neurons were positive. Glial cells were always negative for the antigen. Immunostaining with antibodies to the brain spectrins indicate that the dystrophin antibodies did not crossreact with these proteins. The possibility of cross-reactivity with other proteins is discussed. Studies of cells cultured from a DMD fetus also showed specific dystrophin immunostaining in neurons, although the muscle was generally negative for dystrophin. However, the localisation of dystrophin immunostaining and that of the brain spectrins and neurofilaments appeared abnormal, as did the overall morphology of the cells. This suggests that dystrophin may play a role during brain development and dystrophin deficiency results in abnormal neuronal features. This would be consistent with the nonprogressive nature of the mental retardation observed in DMD patients.

Characterisation of dystrophin during development of human skeletal muscle.

Dystrophin, the 427 x 10(3) Mr product of the Duchenne muscular dystrophy (DMD) gene, was studied in human foetal skeletal muscle from 9 to 26 weeks of gestation. Dystrophin could be detected from at least 9 weeks of gestation at the sarcolemmal membrane of most myotubes, though there was differential staining with antibodies raised to various regions of the protein. Dystrophin immunostaining increased and became more uniform with age and by 26 weeks of gestation there was intense sarcolemmal staining of all myotubes. On a Western blot, a doublet of smaller relative molecular mass than that seen in adult tissue was detected in all foetuses studied. There was a gradual increase in abundance of the upper band from 9 to 26 weeks, and the lower band, although present in low amounts in young foetuses, increased significantly between 20 and 26 weeks of gestation. These data indicate that there are several specific isoforms of dystrophin present in developing skeletal muscle, though the role of these is unknown.

Cell stress-induced phosphorylation of ATF2 and c-Jun transcription factors in rat ventricular myocytes.

Ventricular myocytes are exposed to various pathologically important cell stresses in vivo. In vitro, extreme stresses (sorbitol-induced hyperosmotic shock in the presence or absence of okadaic acid, and anisomycin) were applied to ventricular myocytes cultured from neonatal rat hearts to induce a robust activation of the 46 and 54 kDa stress-activated protein kinases (SAPKs). These activities were increased in nuclear extracts of cells in the absence of any net import of SAPK protein. Phosphorylation of ATF2 and c-Jun was increased as shown by the appearance of reduced-mobility species on SDS/PAGE, which were sensitive to treatment with protein phosphatase 2A. Hyperosmotic shock and anisomycin had no effect on the abundance of ATF2. In contrast, cell stresses induced a greater than 10-fold increase in total c-Jun immunoreactivity detected on Western blots with antibody to c-Jun (KM-1). Cycloheximide did not inhibit this increase, which we conclude represents phosphorylation of c-Jun. This conclusion was supported by use of a c-Jun(phospho-Ser-73) antibody. Immunostaining of cells also showed increases in nuclear phospho-c-Jun in response to hyperosmotic stress. Severe stress (hyperosmotic shock+okadaic acid for 2 h) induced proteins (migrating at approx. 51 and 57 kDa) that cross-reacted strongly with KM-1 antibodies in both the nucleus and the cytosol. These may represent forms of c-Jun that had undergone further modification. These studies show that stresses induce phosphorylation of transcription factors in ventricular myocytes and we suggest that this response may be pathologically relevant.