Applications in brain proteomics: 8(th) HUPO Brain Proteome Project Workshop 7 October 2007, Seoul, Korea

What are the current approaches in brain proteomics? Can we combine different, but complementary study designs to obtain better results concerning brain diseases? What are the neuro‐hotspots, especially in Korea? These were some of the questions the participants of the 8^th HUPO Brain Proteome Project Workshop tried to answer prior to the 6^th HUPO World Congress in Seoul, Korea. Around 100 scientists came together during the afternoon of 7 October, 2007, to discuss and to catch up on the latest results and strategies concerning Huntington's disease, glioblastoma and standardization.     

Mucin secretion by the human colon cell line LS174T is regulated by bile salts

We recently reported that bile salts play a role in the regulation of mucin secretion by cultured dog gallbladder epithelial cells. In this study we have examined whether bile salts also influence mucin secretion by the human epithelial colon cell line LS174T. Solutions of bile salts were applied to monolayers of LS174T cells. Mucin secretion was quantified by measuring the secretion of [3H]GlcNAc labeled glycoproteins. Both unconjugated bile salts as well as taurine conjugated bile salts stimulated mucin secretion by the colon cells in a dose-dependent fashion. Hydrophobic bile salts were more potent stimulators than hydrophilic bile salts. Free (unconjugated) bile salts were more stimulatory compared with their taurine conjugated counterparts. Stimulation of mucin secretion by LS174T cells was found to occur at much lower bile salt concentrations than in the experiments with the dog gallbladder epithelial cells. The protein kinase C activators PMA and PDB had no stimulatory effect on mucin secretion. We conclude that mucin secretion by the human colon epithelial cell line LS174T is regulated by bile salts. We suggest that regulation of mucin secretion by bile salts might be a common mechanism, by which different epithelia protect themselves against the detergent action of bile salts, to which they are exposed throughout the gastrointestinal tract.      

Lipid-based systems for the intracellular delivery of genetic drugs.

Currently available delivery systems for genetic drugs have limited utility for systemic applications. Cationic liposome/plasmid DNA or oligonucleotide complexes are rapidly cleared from circulation, and the highest levels of activity are observed in 'first pass' organs, such as the lungs, spleen and liver. Engineered viruses can generate an immune response, which compromises transfection resulting from subsequent injections and lack target specificity. A carrier, which can accumulate at sites of diseases such as infections, inflammations and tumours, has to be a small, neutral and highly serum-stable particle, which is not readily recognized by the fixed and free macrophages of the reticuloendothelial system (RES). This review summarizes lipid-based technologies for the delivery of nucleic acid-based drugs and introduces a new class of carrier systems, which solve, at least in part, the conflicting demands of circulation longevity and intracellular delivery. Plasmid DNA and oligonucleotides are entrapped into lipid particles that contain small amounts of a positively charged lipid and are stabilized by the presence of a polythylene glycol (PEG) coating. These carriers protect nucleic acid-based drugs from degradation by nucleases, are on average 70 nm in diameter, achieve long circulation lifetimes and are capable of transfecting cells.     

Exercise by lifelong voluntary wheel running reduces subsarcolemmal and interfibrillar mitochondrial hydrogen peroxide production in the heart

Evidence suggests that mitochondrial dysfunction and oxidant production, in association with an accumulation of oxidative damage, contribute to the aging process. Regular physical activity can delay the onset of morbidity, increase mean lifespan, and reduce the risk of developing several pathological states. No studies have examined age-related changes in oxidant production and oxidative stress in both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria in combination with lifelong exercise. Therefore, we investigated whether long-term voluntary wheel running in Fischer 344 rats altered hydrogen peroxide (H2O2) production, antioxidant defenses, and oxidative damage in cardiac SSM and IFM. At 10-11 wk of age, rats were randomly assigned to one of two groups: sedentary and 8% food restriction (sedentary; n = 20) or wheel running and 8% food restriction (runners; n = 20); rats were killed at 24 mo of age. After the age of 6 mo, running activity was maintained at an average of 1,145 +/- 248 m/day. Daily energy expenditure determined by doubly labeled water technique showed that runners expended on average approximately 70% more energy per day than the sedentary rats. Long-term voluntary wheel running significantly reduced H2O2 production from both SSM (-10.0%) and IFM (-9.6%) and increased daily energy expenditure (kJ/day) significantly in runners compared with sedentary controls. Additionally, MnSOD activity was significantly lowered in SSM and IFM from wheel runners, which may reflect a reduction in mitochondrial superoxide production. Activities of the other major antioxidant enzymes (glutathione peroxidase and catalase) and glutathione levels were not altered by wheel running. Despite the reduction in mitochondrial oxidant production, no significant differences in oxidative stress levels (4-hydroxy-2-nonenal-modified proteins, protein carbonyls, and malondialdehyde) were detected between the two groups. The health benefits of chronic exercise may be, at least partially, due to a reduction in mitochondrial oxidant production; however, we could not detect a significant reduction in several selected parameters of oxidative stress.     

Molecular cloning and characterization of a hypoxia-responsive CITED3 cDNA from grass carp

We have isolated a 1586-bp full-length CITED3 cDNA from grass carp which specifies for a cAMP-responsive element-binding protein/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain protein. The cDNA, designated as gcCITED3, has an open reading frame of 762 bp and encodes a protein of 253 amino acids with a predicted molecular mass of 28.3 kDa and pI of 6.4. Pairwise comparison showed that gcCITED3 shares high sequence identity with the CITED3 of zebrafish (94%), chicken (72%) and Xenopus (59%). Northern blot analysis indicated that gcCITED3 is most highly expressed and responsive to hypoxia in the carp kidney. Hypoxic induction was also observed in heart, albeit at a lower level. This is the first report on the isolation of a hypoxia-responsive CITED3 gene from fish.     

Ozone-induced inactivation of antioxidant enzymes

Ozone is an air pollutant that damages a variety of biomolecules. We investigated ozone-induced inactivation of three major antioxidant enzymes. Cu/Zn superoxide dismutase was inactivated by ozone in a concentration-dependent manner. The concentration of ozone for 50% inactivation was approximately 45 microM when 10 microM Cu/Zn superoxide dismutase was incubated for 30 min in the presence of ozone. SDS-polyacrylamide gel electrophoresis (PAGE) showed that the enzyme was randomly fragmented. Both ascorbate and glutathione were very effective in protecting Cu/Zn superoxide dismutase from ozone-induced inactivation. The other two enzymes, catalase and glutathione peroxidase, were much more resistant to ozone than Cu/Zn superoxide dismutase. The ozone concentrations for 50% inactivation of 10 microM catalase and glutathione peroxidase were 500 and 240 microM, respectively. SDS-PAGE demonstrated that ozone caused formation of high molecular weight aggregates in catalase and dimerization in glutathione peroxidase. Glutathione protected catalase and glutathione peroxidase from ozone but the effective concentrations were much higher than that for Cu/Zn superoxide dismutase. Ascorbate was almost ineffective. The result suggests that, among the three antioxidant enzymes, Cu/Zn superoxide dismutase is a major target for ozone-induced inactivation and both glutathione and ascorbate are very effective in protecting the enzyme from ozone.     

Doxorubicin treatment in vivo activates caspase-12 mediated cardiac apoptosis in both male and female rats

We investigated in vivo the chemotherapeutic anthracycline agents doxorubicin and its ability to activate mitochondrial-mediated, receptor-mediated and endoplasmic/sarcoplasmic reticulum-mediated apoptosis transduction pathways in cardiac tissue from male and female rats. We administered a single low dose of doxorubicin (10 mg/kg of body weight, i.p.) and then isolated mitochondrial and cytosolic proteins one and four days later from the heart. Caspase-3 protein content and caspase-3 activity were significantly increased after day four of doxorubicin treatment in both male and female rats. However, while males had DNA fragmentation at day one but not day four following doxorubicin administration, females showed no significant increase in DNA fragmentation at either time. Caspase-12, localized in the SR, is considered a central caspase, and its activation by cleavage via calpain indicates activation of the SR-mediated pathway of apoptosis. Cleaved caspase-12 content and calpain activity significantly increased after day four of doxorubicin treatment in both sexes. In the mitochondrial-mediated pathway, there were no significant treatment effects observed in cytosolic cytochrome c and cleaved (active) caspase-9 in either sex. In control rats (saline injection), glutathione peroxidase (GPX) activity and hydrogen peroxide (H2O2) production were lower in females compared to males. Doxorubicin treatment did not significantly affect H2O2, GPX activity or ATP production in isolated mitochondria in either sex. Female rats produced significantly lower levels of H2O2 production one day after doxorubicin treatment, whereas male rats produced significantly less mitochondrial H2O2 four days after doxorubicin treatment. The receptor-mediated pathway (caspase-8 and c-FLIP) showed no evidence of being significantly activated by doxorubicin treatment. Hence, doxorubicin-induced apoptosis in vivo is mediated by the SR to a greater extent than other apoptotic pathways and should therefore be considered for targeted therapeutic interventions. Moreover, no major sex differences exist in apoptosis signaling transduction cascade due to doxorubicin treatment.