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A novel mammalian protein that may promote apoptosis by binding to and antagonizing IAP proteins has been identified. It is called DIABLO for 'direct IAP-binding protein with low pl.' Coimmunoprecipitation and 2D immobilized pH gradient/SDS PAGE was used, followed by electrospray ionization tandem mass spectrometry.

Liver diseases are considered to predominantly possess an inherited or xenobiotic etiology. However, inheritance drives the ability to appropriately adapt to environmental stressors, and disease is the culmination of a maladaptive response. Thus "pure" genetic and "pure" xenobiotic liver diseases are modified by each other and other factors, identified or unknown. The purpose of this review is to highlight the knowledgebase of environmental exposure as a potential risk modifying agent for the development of liver disease by other causes. This exercise is not to argue that all liver diseases have an environmental component, but to challenge the assumption that the current state of our knowledge is sufficient in all cases to conclusively dismiss this as a possibility. This review also discusses key new tools and approaches that will likely be critical to address this question in the future. Taken together, identifying the key gaps in our understanding is critical for the field to move forward, or at the very least to "know what we don't know." Liver injury and disease have primary underlying causes. Liver disease is also impacted by comorbities that influence the severity and outcome. Moreover, risk modifiers (e.g., "exposome") influence interindividual susceptibility. [Display omitted] [ABSTRACT FROM AUTHOR]

Occupational and environmental exposures to industrial chemicals are well known to cause hepatotoxicity and liver injury. However, despite extensive evidence showing that exposure can lead to disease, current research approaches and regulatory policies fail to address the possibility that subtle changes caused by low level exposure to chemicals may also enhance preexisting conditions. In recent years, the conceptual understanding of the contribution of environmental chemicals to liver disease has progressed significantly. Mitochondria are often target of toxicity of environmental toxicants resulting in multisystem disorders involving different cells, tissues, and organs. Here, we review persistent maladaptive changes to mitochondria in response to environmental toxicant exposure as a mechanism of hepatotoxicity. With better understanding of the mechanism(s) and risk factors that mediate the initiation and progression of toxicant-induced liver disease, rational targeted therapy can be developed to better predict risk, as well as to treat or prevent this disease. Potential mitochondrial targets include the electron transport chain, mitochondrial membrane potential (Δ Ψ m), and organ-to-organ communication, via mitochondrial-associated membranes (MAMs). Dysfunction of any or several of these targets may perpetuate injury. [Display omitted] [ABSTRACT FROM AUTHOR]

Sterols are a class of lipid molecules that include cholesterol, oxysterols, and sterol esters. Sterol lipids play critical functional roles in mammalian biology, including the dynamic regulation of cell membrane fluidity, as precursors for the synthesis of bile acids, steroid hormones and vitamin D, as regulators of gene expression in lipid metabolism, and for cholesterol transport and storage. The most common method employed for sterol analysis is high performance liquid chromatography coupled with tandem mass spectrometry (MS/MS). However, conventional collision induced dissociation (CID) methods used for ion activation during MS/MS typically fail to provide sufficient structural information for unambiguous assignment of sterol species based on their fragmentation behaviour alone. This places a significant burden on the efficiency of the chromatographic separation methods for the effective separation of isomeric sterols. Here, toward developing an improved analysis strategy for sterol lipids, we have explored the novel use of 213 nm photodissociation MS/MS and hybrid multistage-MS/MS (i.e., MSn) data acquisition approaches for the improved structural characterization of cholesterol, representative isomeric oxysterols, and cholesteryl esters. Most notably, UVPD-MS/MS of ammoniated, lithiated and sodiated adducts of cholesterol, several representative oxysterol species, and an oxosterol lipid, are shown to give rise to abundant [M]+. radical cation products, that subsequently fragment during collision induced MS3 to yield extensive structurally informative product ions, similar to those observed by Electron Ionization, and that enable their unambiguously assignment, including isomeric differentiation of oxysterols. For cholesterol esters, a reversed hybrid collision induced-MS/MS and UVPD-MS3 approach is shown to enable assignment of the sterol backbone, and localization of the site(s) of unsaturation within esterified fatty acyl chains. Image 1 • Hybrid UV photodissociation and multistage tandem mass spectrometry is a novel analysis strategy for sterol lipids. • 213 nm UVPD-MS/MS of cholesterol and oxysterols produces abundant [M]+. radical cation product ions. • HCD-MS3 of these products enables unambiguously structural assignment, including for differentiation of isomeric oxysterols. • HCD-MS/MS and UVPD-MS3 enables detailed structural assignment of cholesterol esters, including site(s) of unsaturation. [ABSTRACT FROM AUTHOR]

Highlights: [•] A method to identify binding sites of ion channel-blocking drugs. [•] The Golden Ratio in pharmacology. [Copyright &y& Elsevier]

Abstract: Extracts from the Ginkgo biloba tree are widely used as herbal medicines, and include bilobalide (BB) and ginkgolides A and B (GA and GB). Here we examine their effects on human 5-HT3A and 5-HT3AB receptors, and compare these to the effects of the structurally related compounds picrotin (PTN) and picrotoxinin (PXN), the two components of picrotoxin (PTX), a known channel blocker of 5-HT3, nACh and GABAA receptors. The compounds inhibited 5-HT-induced responses of 5-HT3 receptors expressed in Xenopus oocytes, with IC 50 values of 470μM (BB), 730μM (GB), 470μM (PTN), 11μM (PXN) and >1mM (GA) in 5-HT3A receptors, and 3.1mM (BB), 3.9mM (GB), 2.7mM (PTN), 62 μM (PXN) and >1mM (GA) in 5-HT3AB receptors. Radioligand binding on receptors expressed in HEK 293 cells showed none of the compounds displaced the specific 5-HT3 receptor antagonist [3H]granisetron, confirming that they do not act at the agonist binding site. Inhibition by GB at 5-HT3A receptors is weakly use-dependent, and recovery is activity dependent, indicating channel block. To further probe their site of action at 5-HT3A receptors, BB and GB were applied alone or in combination with PXN, and the results fitted to a mathematical model; the data revealed partially overlapping sites of action. We conclude that BB and GB block the channel of the 5-HT3A receptor. Thus these compounds have comparable, although less potent, behaviour than at some other Cys-loop receptors, demonstrating their actions are conserved across the family. [ABSTRACT FROM AUTHOR]

An automated tandem mass spectrometry–based analysis using precursor ion and neutral loss scans in a triple quadrupole (QqQ) mass spectrometer has been used to identify and quantify changes in the abundances of glycerophospholipids extracted from retina and erythrocytes in a rat streptozotocin model of type 1 diabetes, 6 and 36 weeks after the induction of diabetes, compared with age-matched nondiabetic controls. The utility of an “internal standard” method compared with an “internal standard free” method for quantification of differences in the abundances of specific lipid ions was evaluated in both retina and erythrocyte lipid extracts. In retina, equivalent results were obtained by using the internal standard and internal standard free methods for quantification. In erythrocytes, the two methods of analysis yielded significantly different results, suggesting that factors intrinsic to particular sample types may influence the outcome of label-free lipidome quantification approaches. Overall increases (∼25–35%) in the abundances of major retina glycerophospholipid classes were demonstrated in rats at 6 weeks of diabetes, relative to control animals. However, at 36 weeks of diabetes, subsequent overall decreases in retina glycerophosphatidylcholine (GPCho) and glycerophosphatidylethanolamine (GPEtn) abundances of 16% and 33%, respectively, were observed. Additionally, retina and erythrocyte GPCho lipids at both 6 and 36 weeks of diabetes exhibited increased incorporation of linoleic acid(18:2n6) and a decrease in docosahexaenoic acid (DHA(22:6n3)) content. Finally, an approximately fivefold increase in the abundances of specific glycated GPEtn (Amadori-GPEtn) lipids were observed in the retina of 36-week diabetic rats, with a corresponding 1.6-fold increase of Amadori-GPEtn lipids in diabetic erythrocytes. [Copyright &y& Elsevier]

Background: Gene expression profiling distinguishes the absence or presence of moderate to severe grades of acute cellular rejection in cardiac allograft recipients using a 20-gene classifier. We explored the hypothesis that the rejection classifier also differentiates various forms of mild rejection and we performed sub-analyses based on time post-transplant and confirmatory pathology interpretations. Methods: A post hoc analysis of 265 CARGO study patients and 714 clinical encounters focused on the correlation of rejection classifier–derived gene expression (GE) scores for blood samples accompanying endomyocardial biopsies. Biopsy grades assigned by a study center pathologist (center) were re-interpreted by three pathologists (panel) in a blinded manner. Results: Mean GE scores not only differentiated Grades ≥3A from Grade 0 (p < 0.00001, center or panel), but also from Grades 1A or 2 (p < 0.05, center or panel), based on mild rejection sub-groups defined by the ISHLT 1990 grading system. In contrast, mean GE scores for Grades 1B and ≥3A were indistinguishable, using either center or panel interpretation. Sub-group analyses of encounters from 2 to 6 months or >6 months post-transplant showed similar results for the classifier’s ability to discriminate moderate to severe rejection from Grades 1A and 2 mild rejection, but indistinguishable mean GE scores for Grades ≥3A and the Grade 1B sub-group. Of the classifier’s 11 informative genes, expression of MIR and WDR40 showed statistically significant increases for both Grade 1B and Grade ≥3A rejection, while expression of PDCD1 or SEMA7A showed similar directional patterns without achieving statistical significance. Conclusions: These data demonstrate that GE scores discriminate moderate to severe rejection from Grades 1A and 2 mild rejection. However, a sub-group of mild rejection cases, defined as Grade 1B according to the 1990 grading system, share a molecular signature more consistent with moderate to severe rejection. The clinical relevance of these data remains to be defined. [Copyright &y& Elsevier]

Abstract: An efficient modular strategy has been developed for the synthesis of β-functionalised terthiophene monomers using Suzuki–Miyaura and Wittig/Horner–Emmons chemistries. This paper discusses the problems encountered with converting the β-terthiophene aldehyde building block to the β-terthiophene phosphonium salt and the use of this material in a Wittig condensation. An improved strategy using the β-terthiophene phosphonate building block constructed via Suzuki–Miyaura coupling protocols was developed. We have synthesised and characterised a broad range of functionalised terthiophene materials that have been designed for specific end-use applications. The availability of these building blocks has dramatically increased access to a range of key monomers. [Copyright &y& Elsevier]

The gas-phase fragmentation reactions of various anionic adducts of phosphatidylcholine lipids have been examined by electrospray ionization and multistage tandem mass spectrometry (MSn) in a linear quadrupole ion trap mass spectrometer. The MS/MS and MSn fragmentation reactions of the anionic adducts were found to be highly dependent on the identity of the anions (A). Consistent with previous reports, MS/MS of phosphatidylcholine lipid ions formed by anionic adduction with formate, acetate and chloride resulted in a dominant product ion corresponding to the loss of (A + CH3) from the [M + A]− precursor ions. In contrast, MS/MS of other anion adducted precursor ions gave rise to multiple products, including [M + A—(A + CH3)]−, [M + A—(AH)]−, [M + A—(AH + CH3)]−, [M + A—(AH + (CH3)3N)]−, [M + A—(A + (CH3)3NCHCH2)]− and [M + A(CH3)3N)]−. In order to gain evidence for the structures of these product ions, and the mechanisms by which they were formed, each of the products formed by MS/MS were subjected to further analysis by MS3 and MS4. The MS/MS and MSn fragmentation reactions of several deuterium isotope labeled phosphatidylcholine lipid standards, as well as authentic or structurally analogous lipid standards corresponding to the proposed product ion structures were also examined to gain evidence to support the proposed mechanisms. The results from this study indicate that great care must be taken to control the identity of the anions employed during lipid isolation, purification and separation, in order to allow the development and application of sensitive and robust neutral loss scan methods for mass spectrometry based lipid identification and quantitative analysis. [Copyright &y& Elsevier]

Background & Aims: The biguanide drug metformin has recently been found to improve steatosis and liver damage in animal models and in humans with nonalcoholic steatohepatitis. Methods: The aim of the present study was to determine whether metformin also prevents steatosis and liver damage in mouse models of acute and chronic alcohol exposure. Results: Acute ethanol exposure caused a >20-fold increase in hepatic lipids, peaking 12 hours after administration. Metformin treatment significantly blunted the ethanol effect by >60%. Although metformin is a known inducer of AMP kinase (AMPK) activity, the hepatoprotective property of metformin did not correlate with activation of AMPK or of AMPK-dependent pathways. Instead, the protective effects of metformin correlated with complete prevention of the upregulation of plasminogen activator inhibitor (PAI)-1 caused by ethanol. Indeed, a similar protective effect against acute alcohol-induced lipid accumulation was observed in PAI-1−/− mice. Hepatic fat accumulation caused by chronic enteral ethanol feeding was also prevented by metformin or by knocking out PAI-1. Under these conditions, necroinflammatory changes caused by ethanol were also significantly attenuated. Conclusions: Taken together, these findings suggest a novel mechanism of action for metformin and identify a new role of PAI-1 in hepatic injury caused by ethanol. [Copyright &y& Elsevier]

Summary: The E. coli pyruvate and 2-oxoglutarate dehydrogenases are two closely related, large complexes that exemplify a growing number of multiprotein “machines” whose domains have been studied extensively and modeled in atomic detail, but whose quaternary structures have remained unclear for lack of an effective imaging technology. Here, electron cryotomography was used to show that the E1 and E3 subunits of these complexes are flexibly tethered ∼11 nm away from the E2 core. This result demonstrates unambiguously that electron cryotomography can reveal the relative positions of features as small as 80 kDa in individual complexes, elucidating quaternary structure and conformational flexibility. [Copyright &y& Elsevier]

Abstract: Reaction of an appropriate ortho-halo tosylate precursor with organolithium reagents provides the first conclusive route to the intermediate, 7,8-quinolyne. The transient existence of this hetaryne was confirmed by Diels–Alder reactions with furan derivatives that provide endoxide adducts. Chemical induced rearrangement of these adducts allows entry to key compounds of 10-hydroxy[h]benzoquinoline and its 7-substituted derivatives in modest yields. [Copyright &y& Elsevier]

There is a growing recognition of choroid plexus functioning as a source of neuropeptides, cytokines and growth factors in cerebrospinal fluid (CSF) with diffusional access into brain parenchyma. In this study, choroid plexus and other components of the CSF circulatory system were investigated by Western blotting, reverse transcriptase polymerase chain reaction and immunohistochemistry for production of interleukin-6-related cytokines characterized by neuroactivity [cardiotrophin-1 (CT-1), ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M] and signaling through the gp130/leukemia inhibitory factor receptor-β receptor heterodimer. Western blot analysis showed that CT-1 was the only cytokine family member detectable in adult rat choroid plexus, as in leptomeninges. The specificity of detection was verified with blots of the same tissues from CT-1-deficient mice. Levels of both CT-1 mRNA and protein were constitutively high in rat from birth through adulthood in choroid plexus, up-regulated postnatally in leptomeninges and undetectable in brain parenchyma. Using antigen retrieval, CT-1 immunolocalized to choroid epithelial cells in all choroid plexuses in addition to leptomeninges (arachnoid and pial–glial membranes). Ependymal cells lining the ventricular neuroaxis, unlike the central canal, were also CT-1-immunoreactive. Western blots indicated rat choroid epithelial cells express and release CT-1 immunoreactivity under defined culture conditions and also revealed the presence of a CT-1-like protein in human choroid plexus and CSF. Previously, CT-1 has been conceptualized to function as a target-derived factor for PNS neurons. Our study clearly demonstrates production of CT-1 in the postnatal and adult CNS, specifically by cell types comprising the blood–CSF barrier, and its accumulation in ventricular ependyma. This finding has broad implications for CT-1 functioning apart from other leukemia inhibitory factor receptor ligands as a CSF-borne signal of brain homeostasis, one possibly involving regulation of the barrier itself, the ependyma or target cells in the surrounding parenchyma, including the subventricular zone. A rationale for studies examining CT-1-deficient mice in these respects is provided by the data. [Copyright &y& Elsevier]

We have previously described a guinea-pig model where pigmented nevi similar to human nevi can be produced by application of low-dose topical 7,12-dimethylbenzanthracene (DMBA) followed by solar-simulated light. Five groups of guinea-pigs were used to test the effect of various spectral bands of solar-simulated light on low-dose DMBA-induced melanocytic nevi. Animals were irradiated with either UVB to near UVA2 (290–325 nm), UVA, visible light, full solar spectrum or no irradiation three times per wk for 12 mo to determine the broad-band effect of nevi-inducing irradiation. There was a significant increase in nevi/animal in the UVB-treated group (mean 1.53) compared with all groups ( versus UVA 0.3, p<0.001; versus visible light 0.24, p<0.001; versus full spectrum (UVB+UVA+visible) 0.68, p=0.02; versus control (nil irradiation) 0.37, p=0.01). No differences in skin thickness were found between any group (p=0.11). In conclusion, we present a report of the active waveband of melanocytic nevi induction; where UVB to near UVA2 is the likely responsible waveband. Furthermore, because there was a significant decrease in nevi/animal receiving the full solar spectrum compared with the UVB group, it is possible that broad-band UVA and or visible light may be inhibitory wavebands for nevi induction. [ABSTRACT FROM AUTHOR]

It has become increasingly evident that one of the most fruitful approaches to understanding cellular processes and their relation to disease consists of large-scale gene profiling of cells, tissues, and organs. This also constitutes a first step in exploring the molecular biologic basis of various diseases. In the current study, we used cDNA microarray technology to assess possible changes in the expression of a large number of genes in the liver of rats fed alcohol (ethanol) chronically (4 weeks) by means of intragastric infusion. This animal model resembles closely the alcoholic liver disease in human beings. Of a total of 8,740 probe sets arrayed on the microchip, 2,069 were expressed by the liver. After a correction for false discovery rate at 10%, 72 genes were found to be significantly up-regulated (40) or down-regulated (32). Forty-two genes were suppressed, and four genes were induced, by alcohol. These genes are involved in a wide spectrum of cellular functions. Also, the genes that underwent significant changes were categorized into two groups: genes that have been implicated in alcoholic liver disease and genes that have not been tested for possible changes in expression. Large-scale gene profiling of the liver reveals changes in the expression of a number of genes that have never been implicated in alcohol-induced injury. Further investigation of such genes may cast light on mechanisms underlying alcohol-induced liver injury and help in the design of new therapeutic approaches. [Copyright &y& Elsevier]

Coral reef fish communities in the Seychelles are highly diverse and remain less affected by the direct impacts of human activities than those on many other coral reefs in the Indian Ocean. These factors make them highly suitable for a detailed survey of the impacts of the 1998 mass coral mortality, which devastated the coral faunas of the region. Using underwater visual census (UVC) techniques, fish communities were sampled in three localities in the southern Seychelles and one locality in the northern (granitic) Seychelles. Initial surveys were undertaken from the latter site in 1997. Surveys were undertaken at all sites during the coral bleaching episode in 1998 prior to any major changes in the reef fish communities. Repeat surveys were undertaken in 1999 one year after the coral mortality. Over 250 fish species were sampled from 35 families. Results suggest that changes in the overall fish community structures are not great, despite massive changes in the benthic cover. Significant changes have been observed in a number of individual species. These include those most heavily dependent on live coral cover for shelter or sustenance. Future potential changes are discussed, and potential management interventions are considered. [Copyright &y& Elsevier]

Presents information on a study which examined the ability of procynidin oligomers to protect against peroxynitrite oxidation and nitration. Discussion on oxidative stress and properties of flavonoids; Characteristics of the reaction of peroxynitrite with epicatechin and the tetrametric compound; Role of dietary phenols in health and disease.