Reduction Potentials of Imine-Substituted, Biologically Active Pyridines: Possible Relation To Activity

Cyclic voltammetry data were obtained for a number of biologically active compounds which incorporate imine substitution on the pyridine nucleus. The reductions in acid (iminium ion formation) were for the most part reversible, and in the range of—0.5 to—0.7 V. The toxic effect of these drugs is thought to be caused by the generation of reactive oxygen radicals that arise viacharge transfer, or by disruption of electron transport chains.     

Messina: A Novel Analysis Tool to Identify Biologically Relevant Molecules in Disease

Background

Morphologically similar cancers display heterogeneous patterns of molecular aberrations and follow substantially different clinical courses. This diversity has become the basis for the definition of molecular phenotypes, with significant implications for therapy. Microarray or proteomic expression profiling is conventionally employed to identify disease-associated genes, however, traditional approaches for the analysis of profiling experiments may miss molecular aberrations which define biologically relevant subtypes.

Methodology/Principal Findings

Here we present Messina, a method that can identify those genes that only sometimes show aberrant expression in cancer. We demonstrate with simulated data that Messina is highly sensitive and specific when used to identify genes which are aberrantly expressed in only a proportion of cancers, and compare Messina to contemporary analysis techniques. We illustrate Messina by using it to detect the aberrant expression of a gene that may play an important role in pancreatic cancer.

Conclusions/Significance

Messina allows the detection of genes with profiles typical of markers of molecular subtype, and complements existing methods to assist the identification of such markers. Messina is applicable to any global expression profiling data, and to allow its easy application has been packaged into a freely-available stand-alone software package.     

The Importance of Biologically Relevant Microclimates in Habitat Suitability Assessments

Predicting habitat suitability under climate change is vital to conserving biodiversity. However, current species distribution models rely on coarse scale climate data, whereas fine scale microclimate data may be necessary to assess habitat suitability and generate predictive models. Here, we evaluate disparities between temperature data at the coarse scale from weather stations versus fine-scale data measured in microhabitats required for a climate-sensitive mammal, the American pika (Ochotona princeps). We collected two years of temperature data in occupied talus habitats predicted to be suitable (high elevation) and unsuitable (low elevation) by the bioclimatic envelope approach. At low elevations, talus surface and interstitial microclimates drastically differed from ambient temperatures measured on-site and at a nearby weather station. Interstitial talus temperatures were frequently decoupled from high ambient temperatures, resulting in instantaneous disparities of over 30°C between these two measurements. Microhabitat temperatures were also highly heterogeneous, such that temperature measurements within the same patch of talus were not more correlated than measurements at distant patches. An experimental manipulation revealed that vegetation cover may cool the talus surface by up to 10°C during the summer, which may contribute to this spatial heterogeneity. Finally, low elevation microclimates were milder and less variable than typical alpine habitat, suggesting that, counter to species distribution model predictions, these seemingly unsuitable habitats may actually be better refugia for this species under climate change. These results highlight the importance of fine-scale microhabitat data in habitat assessments and underscore the notion that some critical refugia may be counterintuitive.     

Single-Electron-Transfer in the Reduction of 1, 2-Dioxetanes by Biologically Active Substrates

Substances of low oxidation potential, which can also make available protons and hydrogen atoms, e.g. phenothiazines. NADH, and ascorbic acid efficiently reduce 1, 2-dioxetanes to their vic-diols by single-electron-transfer; a significant side reaction is catalytic decomposition of dioxetanes into the corresponding ketone fragments     

Single-Electron-Transfer in the Reduction of 1, 2-Dioxetanes by Biologically Active Substrates

Substances of low oxidation potential, which can also make available protons and hydrogen atoms, e.g. phenothiazines. NADH, and ascorbic acid efficiently reduce 1, 2-dioxetanes to their vic-diols by single-electron-transfer; a significant side reaction is catalytic decomposition of dioxetanes into the corresponding ketone fragments     

Resveratrol is a peroxidase-mediated inactivator of COX-1 but not COX-2: a mechanistic approach to the design of COX-1 selective agents

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a phytoalexin found in grapes that has anti-inflammatory, cardiovascular protective, and cancer chemopreventive properties. It has been shown to target prostaglandin H(2) synthase (COX)-1 and COX-2, which catalyze the first committed step in the synthesis of prostaglandins via sequential cyclooxygenase and peroxidase reactions. Resveratrol discriminates between both COX isoforms. It is a potent inhibitor of both catalytic activities of COX-1, the desired drug target for the prevention of cardiovascular disease, but only a weak inhibitor of the peroxidase activity of COX-2, the isoform target for nonsteroidal anti-inflammatory drugs. We have investigated the unique inhibitory properties of resveratrol. We find that it is a potent peroxidase-mediated mechanism-based inactivator of COX-1 only (k(inact) = 0.069 +/- 0.004 s(-1), K(i(inact)) = 1.52 +/- 0.15 microm), with a calculated partition ratio of 22. Inactivation of COX-1 was time- and concentration-dependent, it had an absolute requirement for a peroxide substrate, and it was accompanied by a concomitant oxidation of resveratrol. Resveratrol-inactivated COX-1 was devoid of both the cyclooxygenase and peroxidase activities, neither of which could be restored upon gel-filtration chromatography. Inactivation of COX-1 by [(3)H]resveratrol was not accompanied by stable covalent modification as evident by both SDS-PAGE and reverse phase-high performance liquid chromatography analysis. Structure activity relationships on methoxy-resveratrol analogs showed that the m-hydroquinone moiety was essential for irreversible inactivation of COX-1. We propose that resveratrol inactivates COX-1 by a "hit-and-run" mechanism, and offers a basis for the design of selective COX-1 inactivators that work through a mechanism-based event at the peroxidase active site.     

Non-Invasive Providencia alcalifaciens Strains Fail to Attach to HEp-2 Cells

We investigated the adherence properties of six P. alcalifaciens strains with previously characterized differential invasive capabilities in HEp-2 cells. Highly invasive strains were found to attach to HEp-2 cell monolayers within 2 h post-infection and in large numbers on the eukaryotic cell surfaces within 3 h post-infection. In contrast, weakly or non-invasive P. alcalifaciens strains were non-adherent to HEp-2 cells even at 3 h post-infection. Highly invasive isolates were found to weakly bind F-actin using the fluorescent actin staining assay although these strains were negative for Escherichia coli attachment and effacing gene (eaeA) of enteropathogenic E. coli (EPEC). These results suggest that the strain variation in the ability of P. alcalifaciens to invade HEp-2 cells previously noted by several investigators may be linked to expression of key adhesin(s) on the cell surface of invasive isolates. Received: 10 April 2001 / Accepted: 17 April 2001     

Inducible COX-2 dominates over COX-1 in prostacyclin biosynthesis: mechanisms of COX-2 inhibitor risk to heart disease

AimOur aim is to understand the molecular mechanisms of the selective nonsteroidal anti-inflammatory drugs (NSAID), cyclooxygenase-2 (COX-2) inhibitors', higher “priority” to reduce synthesis of the vascular protector, prostacyclin (PGI2), compared to that of nonselective NSAIDs.Main methodsCOX-1 or COX-2 was co-expressed with PGI2 synthase (PGIS) in COS-7 cells. The Km and initial velocity (½t Vmax) of the coupling reaction between COX-1 and COX-2 to PGIS were established. The experiment was further confirmed by a kinetics study using hybrid enzymes linking COX-1 or COX-2 to PGIS. Finally, COX-1 or COX-2 and PGIS were respectively fused to red (RFP) and cyanic (CFP) fluorescence proteins, and co-expressed in cells. The distances between COXs and PGIS were compared by FRET.Key findingsThe Km for converting arachidonic acid (AA) to PGI2 by COX-2 coupled to PGIS is ~ 2.0 μM; however, it was 3-fold more (~ 6.0 μM) for COX-1 coupled to PGIS. The Km and ½t Vmax for COX-2 linked to PGIS were ~ 2.0 μM and 20 s, respectively, which were 2–5 folds faster than that of COX-1 linked to PGIS. The FRET study found that the distance between COX-2-RFP and PGIS–CFP is shorter than that between COX-1-RFP and PGIS–CFP.SignificanceThe study provided strong evidence suggesting that the low Km, faster ½t Vmax, and closer distance are the basis for COX-2 dominance over COX-1 (coupled to PGIS) in PGI2 synthesis, and further demonstrated the mechanisms of selective COX-2 inhibitors with higher potential to reduce synthesis of the vascular protector, PGI2.     

Glucosamine hydrochloride specifically inhibits COX-2 by preventing COX-2 N-glycosylation and by increasing COX-2 protein turnover in a proteasome-dependent manner

COX-2 and its products, including prostaglandin E(2), are involved in many inflammatory processes. Glucosamine (GS) is an amino monosaccharide and has been widely used for alternative regimen of (osteo) arthritis. However, the mechanism of action of GS on COX-2 expression remains unclear. Here we describe a new action mechanism of glucosamine hydrochloride (GS-HCl) to tackle endogenous and agonist-driven COX-2 at protein level. GS-HCl (but not GS sulfate, N-acetyl GS, or galactosamine HCl) resulted in a shift in the molecular mass of COX-2 from 72-74 to 66-70 kDa and concomitant inhibition of prostaglandin E(2) production in a concentration-dependent manner in interleukin (IL)-1beta-treated A549 human lung epithelial cells. Remarkably, GS-HCl-mediated decrease in COX-2 molecular mass was associated with inhibition of COX-2 N-glycosylation during translation, as assessed by the effect of tunicamycin, the protein N-glycosylation inhibitor, or of cycloheximide, the translation inhibitor, on COX-2 modification. Specifically, the effect of low concentration of GS-HCl (1 mM) or of tunicamycin (0.1 microg/ml) to produce the aglycosylated COX-2 was rescued by the proteasomal inhibitor MG132 but not by the lysosomal or caspase inhibitors. However, the proteasomal inhibitors did not show an effect at 5 mM GS-HCl, which produced the aglycosylated or completely deglycosylated form of COX-2. Notably, GS-HCl (5 mM) also facilitated degradation of the higher molecular species of COX-2 in IL-1beta-treated A549 cells that was retarded by MG132. GS-HCl (5 mM) was also able to decrease the molecular mass of endogenous and IL-1beta- or tumor necrosis factor-alpha-driven COX-2 in different human cell lines, including Hep2 (bronchial) and H292 (laryngeal). However, GS-HCl did not affect COX-1 protein expression. These results demonstrate for the first time that GS-HCl inhibits COX-2 activity by preventing COX-2 co-translational N-glycosylation and by facilitating COX-2 protein turnover during translation in a proteasome-dependent manner.     

Inactivation of the Antibacterial and Cytotoxic Properties of Silver Ions by Biologically Relevant Compounds

There has been a recent surge in the use of silver as an antimicrobial agent in a wide range of domestic and clinical products, intended to prevent or treat bacterial infections and reduce bacterial colonization of surfaces. It has been reported that the antibacterial and cytotoxic properties of silver are affected by the assay conditions, particularly the type of growth media used in vitro. The toxicity of Ag⁺ to bacterial cells is comparable to that of human cells. We demonstrate that biologically relevant compounds such as glutathione, cysteine and human blood components significantly reduce the toxicity of silver ions to clinically relevant pathogenic bacteria and primary human dermal fibroblasts (skin cells). Bacteria are able to grow normally in the presence of silver nitrate at >20-fold the minimum inhibitory concentration (MIC) if Ag⁺ and thiols are added in a 1∶1 ratio because the reaction of Ag⁺ with extracellular thiols prevents silver ions from interacting with cells. Extracellular thiols and human serum also significantly reduce the antimicrobial activity of silver wound dressings Aquacel-Ag (Convatec) and Acticoat (Smith & Nephew) to Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli in vitro. These results have important implications for the deployment of silver as an antimicrobial agent in environments exposed to biological tissue or secretions. Significant amounts of money and effort have been directed at the development of silver-coated medical devices (e.g. dressings, catheters, implants). We believe our findings are essential for the effective design and testing of antimicrobial silver coatings.     

Sox9 Duplications Are a Relevant Cause of Sry-Negative XX Sex Reversal Dogs

Sexual development in mammals is based on a complicated and delicate network of genes and hormones that have to collaborate in a precise manner. The dark side of this pathway is represented by pathological conditions, wherein sexual development does not occur properly either in the XX and the XY background. Among them a conundrum is represented by the XX individuals with at least a partial testis differentiation even in absence of SRY. This particular condition is present in various mammals including the dog. Seven dogs characterized by XX karyotype, absence of SRY gene, and testicular tissue development were analysed by Array-CGH. In two cases the array-CGH analysis detected an interstitial heterozygous duplication of chromosome 9. The duplication contained the SOX9 coding region. In this work we provide for the first time a causative mutation for the XXSR condition in the dog. Moreover this report supports the idea that the dog represents a good animal model for the study of XXSR condition caused by abnormalities in the SOX9 locus.     

Getting to the heart of COX-2 inhibition

Aspirin protects against heart disease, while COX-2 Inhibitors appear to injure the heart. The studies by [this Issue of Cell Metabolism]) shed light on two distinct prostaglandin receptors contributing to these conflicting cardiovascular effects.     

Potentially Pathogenic Escherichia coli Can Form a Biofilm under Conditions Relevant to the Food Production Chain

The biofilm-producing abilities of potentially human-pathogenic serotypes of Escherichia coli from the ovine reservoir were studied at different temperatures and on different surfaces. A possible influence of the hydrophobicity of the bacterial cells, as well as the presence of two virulence factors, the Shiga toxin-encoding (Stx) bacteriophage and the eae gene, was also studied. A total of 99 E. coli isolates of serotypes O26:H11, O103:H2, and O103:H25 isolated from sheep feces were included. The results show that isolates of all three E. coli serotypes investigated can produce biofilm on stainless steel, glass, and polystyrene at 12, 20, and 37°C. There was a good general correlation between the results obtained on the different surfaces. E. coli O103:H2 isolates produced much more biofilm than those of the other two serotypes at all three temperatures. In addition, isolates of serotype O26:H11 produced more biofilm than those of O103:H25 at 37°C. The hydrophobicity of the isolates varied between serotypes and was also influenced by temperature. The results strongly indicated that hydrophobicity influenced the attachment of the bacteria rather than their ability to form biofilm once attached. Isolates with the eae gene produced less biofilm at 37°C than isolates without this gene. The presence of a Stx bacteriophage did not influence biofilm production. In conclusion, our results show that potentially human-pathogenic E. coli from the ovine reservoir can form biofilm on various surfaces and at several temperatures relevant for food production and handling.     

植物多酚的防癌抗癌作用

植物多酚是植物中广泛存在的一大类多酚化合物的总称,包括多酚（如单宁）、黄酮、酚酸等。体外实验研究表明植物多酚对多种人癌细胞具有增殖抑制作用,抑制动物体内肿瘤生长。其作用机理与抗氧化、调控细胞周期、诱导肿瘤细胞凋亡和分化、影响血管生成和肿瘤细胞信号传导等有关。对植物多酚的防癌抗癌作用,值得进一步深入研究。     

4-hydroxy-2-nonenal as a COX-2 inducer

4-hydroxy-2-nonenal (HNE) activates a variety of signaling pathways. We have recently evaluated the effect of oxidized fatty acid metabolites on cyclooxygenase-2 (COX-2) induction in rat liver epithelial RL34 cells and found that, among the compounds tested, HNE most dramatically induced COX-2. A p38 mitogen-activated protein kinase (p38 MAPK) pathway has been shown to play a key role in the mechanism of the HNE-induced COX-2 expression. It appears that the HNE-induced activation of p38 MAPK leads to the stabilization of COX-2 mRNA.     

Hybrid fluorescent conjugates of COX-2 inhibitors: Search for a COX-2 isozyme imaging cancer biomarker

The observation that the cyclooxygenase-2 (COX-2) isozyme is over-expressed in multiple types of cancer, relative to that in adjacent non-cancerous tissue, prompted this investigation to prepare a group of hybrid fluorescent conjugates wherein the COX inhibitors ibuprofen, (S)-naproxen, acetyl salicylic acid, a chlororofecoxib analog and celecoxib were coupled via a linker group to an acridone, dansyl or rhodamine B fluorophore. Within this group of compounds, the ibuprofen-acridone conjugate (10) showed potent and selective COX-2 inhibition (COX-2 IC₅₀ = 0.67 μM; SI = 110.6), but its fluorescence emission (λ_em = 417, 440 nm) was not suitable for fluorescent imaging of cancer cells that over-express the COX-2 isozyme. In comparison, the celecoxib-dansyl conjugate (25) showed a slightly lower COX-2 potency and selectivity (COX-2 IC₅₀ = 1.1 μM; SI > 90) than the conjugate 10, and it possesses a better fluorescence emission (λ_em = 500 nm). Ultimately, a celecoxib-rhodamine B conjugate (28) that exhibited moderate COX-2 potency and selectivity (COX-2 IC₅₀ = 3.9 μM; SI > 25) having the best fluorescence emission (λ_em = 580 nm) emerged as the most promising biomarker for fluorescence imaging using a colon cancer cell line that over-expresses the COX-2 isozyme.