Direct observation of trapping and release of nitric oxide by glutathione and cysteine with electron paramagnetic resonance spectroscopy

While the biosynthesis of nitric oxide (NO) is well established, one of the key issues that remains to be solved is whether NO participates in the biological responses right after generation through biosynthesis or there is a "secret passage" via which NO itself is trapped, transported, and released to exert its functions. It has been shown that NO reacts with thiol-containing biomolecules (RSH), like cysteine (Cys), glutathione (GSH), etc., to form S-nitrosothiols (RSNOs), which then release nitrogen compounds, including NO. The direct observation of trapping of NO and its release by RSNO has not been well documented, as most of the detection techniques measure the content of NO as well as nitrite and nitrate. Here we use spin-trapping electron paramagnetic resonance (EPR) technique to measure NO content directly in the reaction time course of samples of GSH and Cys ( approximately mM) mixed with NO ( approximately microM) in the presence of metal ion chelator, which pertains to physiological conditions. We demonstrate that NO is readily trapped by these thiols in less than 10 min and approximately 70-90% is released afterward. These data imply that approximately 10-30% of the reaction product of NO does not exist in the free radical form. The NO release versus time curves are slightly pH dependent in the presence of metal ion chelator. Because GSH and Cys exist in high molar concentrations in blood and in mammalian cells, the trapping and release passage of NO by these thiols may provide a mechanism for temporal and spatial sequestration of NO to overcome its concentration gradient-dependent diffusion, so as to exert its multiple biological effects by reacting with various targets through regeneration.     

Spin label EPR structural studies of the N-terminus of alpha-spectrin

Spectrin, a vital component in human erythrocyte, is composed of alpha- and beta-subunits, which associate to form (alphabeta)2 tetramers. The tetramerization site is believed to involve the alpha-spectrin N-terminus and the beta-spectrin C-terminus. Abnormal interactions in this region may lead to blood disorders. It has been proposed that both termini consist of partial structural domains and that tetramerization involves the association of these partial domains. We have studied the N-terminal region of a model peptide for alpha-spectrin by making a series of double spin-labeled peptides and studying their dipolar interaction by electron paramagnetic resonance methods. Our results indicate that residues 21-42 of the N-terminus region exhibit an alpha-helical conformation, even in the absence of B-spectrin.     

Nitrite inhalation in rats elevates tissue NOS III expression and alters tyrosine nitration and phosphorylation

Organic nitrites are nitric oxide (NO) donors that are used predominantly as inhalant drugs of abuse and have been shown to have immunomodulating effects. NO donors can modulate NOS activity and expression, thus altering the level of endogenous NO production. NO can react with superoxide (O(*)(2)(-)) to form peroxynitrite (ONOO(-)), which can nitrate tyrosine residues in proteins and alter tyrosine phosphorylation. We investigated the effects of inhaled isobutyl nitrite (ISBN) on NOS expression, tyrosine nitration, and tyrosine phosphorylation in selected organs of rats. Following exposures of 109 and 1517 ppm ISBN for 4 h, the lung, spleen, liver, and kidney were removed and assayed by SDS-PAGE for NOS III (eNOS), NOS II (iNOS), nitrotyrosine (NT)- and phosphotyrosine (PT)-immunoreactive proteins using specific antibodies. ISBN at 1517 ppm, but not 109 ppm, caused an increase in NOS III expression in the liver and kidney, but not in the lung and spleen. No apparent effect on NOS II expression was observed in these organs. The expressions of NT and PT protein bands (30-200 kDa) were increased in the liver and kidney, but not in the lung and spleen. This increase in NT persisted for 24 h post-exposure. Increased NOS III expression in the liver and kidney may promote peroxynitrite formation and contribute to the increase in NT and PT immunoreactivity. ISBN inhalation may thus cause changes in cellular signaling involving tyrosine phosphorylation. These findings may suggest a mechanistic basis for the apparent immunotoxicity associated with nitrite abuse.     

Morin hydrate: a potential antioxidant in minimizing the free-radicals-mediated damage to cardiovascular cells by anti-tumor drugs

The co-incubation of morin hydrate with either doxorubicin or mitomycin C could minimize the toxicity of these anti-tumor drugs on cardiovascular cells, such as red blood cells, human umbilical vein endothelial cells (ECV304) and primary mouse cardiomyocytes, whereas morin hydrate did not lower the cytotoxicity of the drugs on human hepatocellular carcinoma cells (HepG2). Morin hydrate may not exert its antioxidant effect by enhancing the antioxidant enzymatic activity because it did not cause any induction on the mRNA levels of manganese superoxide dismutase expression in ECV304 cells and HepG2 cells.     

Characterization of a Helicobacter pylori vaccine candidate by proteome techniques

In a previous two-dimensional (2D) gel electrophoretic study of protein antigens of the gastric pathogen, Helicobacter pylori recognized by human sera, one of the highly and consistently reactive antigens, a protein with M_r of approximately 30?000 (Spot 15) seemed to be of special interest because of low yields on N-terminal protein sequencing. This suggested possible N-terminal modification, as the N-terminal sequence analysis of this 30?000 protein (Spot 15) did not provide a definitive match within the H. pylori genomic database. This protein was isolated by 2D polyacrylamide gel electrophoresis, evaluated by liquid chromatography–mass spectrometry, and found to consist of two related species of approximately 28?100 and 26?500. In parallel, the proteins within this spot were digested in situ with the endoprotease Lys-C. Analysis of the Lys-C digest by matrix-assisted laser desorption time-of-flight mass spectrometry, peptide mapping, and sequence analysis was conducted. Comparison of the mass and sequence of the Lys-C peptides with those derived from a H. pylori genomic library identified an open reading frame of approximately 300 base pairs as the source of the Spot 15 protein. This corresponded to HP0175 in the recently reported H. pylori genome sequence, an open reading frame with some homology to Campylobacter jejeuni cell binding protein 2. Mass spectral and sequence analysis indicated that Spot 15 was a processed product generated by proteolytic cleavage at both the carboxy and amino termini of the 34 open reading frame precursor.     

Design,synthesis and evaluation of constrained tetrahydroimidazopyrimidine derivatives as antagonists of corticotropin-releasing factor type 1 receptor (CRF1R)

Several tetrahydroimidazopyrimidines were prepared using silver assisted cyclization as the key step. The binding affinities of compounds thus prepared were evaluated in vitro toward hCRF₁R. Initial lead compound 16 (K_i = 32 nM) demonstrated modest putative anxiolytic effects in the mouse canopy test. Further optimization using parallel synthesis provided compounds with K_i’s <50 nM.     

Blood-Based Protein Biomarker Panel for the Detection of Colorectal Cancer

Background

The majority of colorectal cancer (CRC) cases are preventable by early detection and removal of precancerous polyps. Even though CRC is the second most common internal cancer in Australia, only 30 per cent of the population considered to have risk factors participate in stool-based test screening programs. Evidence indicates a robust, blood-based, diagnostic assay would increase screening compliance. A number of potential diagnostic blood-based protein biomarkers for CRC have been reported, but all lack sensitivity or specificity for use as a stand-alone diagnostic. The aim of this study was to identify and validate a panel of protein-based biomarkers in independent cohorts that could be translated to a reliable, non-invasive blood-based screening test.

Principal Findings

In two independent cohorts (n = 145 and n = 197), we evaluated seven single biomarkers in serum of CRC patients and age/gender matched controls that showed a significant difference between controls and CRC, but individually lack the sensitivity for diagnostic application. Using logistic regression strategies, we identified a panel of three biomarkers that discriminated between controls and CRC with 73% sensitivity at 95% specificity, when applied to either of the two cohorts. This panel comprised of Insulin like growth factor binding protein 2 (IGFBP2), Dickkopf-3 (DKK3), and Pyruvate kinase M2(PKM2).

Conclusions

Due to the heterogeneous nature of CRC, a single biomarker is unlikely to have sufficient sensitivity or specificity for use as a stand-alone diagnostic screening test and a panel of markers may be more effective. We have identified a 3 biomarker panel that has higher sensitivity and specificity for early stage (Stage I and -II) disease than the faecal occult blood test, raising the possibility for its use as a non-invasive blood diagnostic or screening test.     

Chinese Social Media Reaction to Information about 42 Notifiable Infectious Diseases

This study aimed to identify what information triggered social media users’ responses regarding infectious diseases. Chinese microblogs in 2012 regarding 42 infectious diseases were obtained through a keyword search in the Weiboscope database. Qualitative content analysis was performed for the posts pertinent to each keyword of the day of the year with the highest daily count. Similar posts were grouped and coded. We identified five categories of information that increased microblog traffic pertaining to infectious diseases: news of an outbreak or a case; health education / information; alternative health information / Traditional Chinese Medicine; commercial advertisement / entertainment; and social issues. News unrelated to the specified infectious diseases also led to elevated microblog traffic. Our study showcases the diverse contexts from which increased social media traffic occur. Our results will facilitate better health communication as causes underlying increased social media traffic are revealed.     

Revealing Pathway Dynamics in Heart Diseases by Analyzing Multiple Differential Networks

Development of heart diseases is driven by dynamic changes in both the activity and connectivity of gene pathways. Understanding these dynamic events is critical for understanding pathogenic mechanisms and development of effective treatment. Currently, there is a lack of computational methods that enable analysis of multiple gene networks, each of which exhibits differential activity compared to the network of the baseline/healthy condition. We describe the iMDM algorithm to identify both unique and shared gene modules across multiple differential co-expression networks, termed M-DMs (multiple differential modules). We applied iMDM to a time-course RNA-Seq dataset generated using a murine heart failure model generated on two genotypes. We showed that iMDM achieves higher accuracy in inferring gene modules compared to using single or multiple co-expression networks. We found that condition-specific M-DMs exhibit differential activities, mediate different biological processes, and are enriched for genes with known cardiovascular phenotypes. By analyzing M-DMs that are present in multiple conditions, we revealed dynamic changes in pathway activity and connectivity across heart failure conditions. We further showed that module dynamics were correlated with the dynamics of disease phenotypes during the development of heart failure. Thus, pathway dynamics is a powerful measure for understanding pathogenesis. iMDM provides a principled way to dissect the dynamics of gene pathways and its relationship to the dynamics of disease phenotype. With the exponential growth of omics data, our method can aid in generating systems-level insights into disease progression.