Use of a molecular beacon to track the activity of base excision repair protein OGG1 in live cells

An abundant form of DNA damage caused by reactive oxygen species is 8-oxo-7,8-dihydroguanine for which the base excision repair protein 8-oxoguanine-DNA glycosylase 1 (OGG1) is a major repair enzyme. To assess the location and intracellular activity of the OGG1 protein in response to oxidative stress, we have utilised a fluorescence–quench molecular beacon switch containing a 8-oxo-dG:C base pair and a fluorescent and quencher molecule at opposite ends of a hairpin oligonucleotide. Oxidative stress was induced by treatment with potassium bromate. Flow cytometry demonstrated a concentration-dependent increase in the activity of OGG1 that was detected by the fluorescence produced when the oligonucleotide was cleaved in the cells treated with potassium bromate. This signal is highly specific and not detectable in OGG1 knock out cells. Induction of OGG1 activity is not a result of induction of OGG1 gene expression as assessed by qPCR suggesting a role for protein stabilisation or increased OGG1 catalytic activity. High resolution confocal microscopy pinpointed the location of the fluorescent molecular beacon in live cells to perinuclear regions that were identified as mitochondria by co-staining with mitotracker dye. There is no evidence of cut beacon within the nuclear compartment of the cell. Control experiments with a positive control beacon (G:C base pair and lacking the DAB quencher) did not result in mitochondrial localisation of fluorescence signal indicating that the dye does not accumulate in mitochondria independent of OGG1 activity. Furthermore, faint nuclear staining was apparent confirming that the beacon structure is able to enter the nucleus. In conclusion, these data indicate that the mitochondria are the major site for OGG1 repair activity under conditions of oxidative stress.     

The Rat Corpulent (cp) Mutation Maps to the Same Interval on (Pgm1-Glut1) Rat Chromosome 5 as the Fatty (fa) Mutation

The autosomal recessive obesity mutations fatty (fa) and corpulent (cp) arose in separate rat strains, 13M and Koletsky, respectively. By complementation analysis, the two mutations appear to be in the same gene. The somewhat different phenotypes of fa/fa and cp/cp animals probably reflect the fact that the mutations are segregating on different rat strains. The fa mutation has been mapped to the interval between Pgm1 and Glut1 on rat Chr 5, but cp has not been mapped genetically. We mapped cp in 30 obese progeny of a LA/N-BN cp/+ intercross using microsatellite markers for these flanking genes. Cp maps to the same genetic interval as rat fa and mouse db. Cp is flanked by Glut1 and Pgm1: Pgm1——–cp——–Glut1 map distance (cM) 1.67 6.67 Thus, cp and fa map to the same ～8 cM interval of the rat genome. In conjunction with the complementation studies alluded to above, these findings indicate that cp and fa are mutations in the same gene (Lepr).     

?Microbialites in the Permian-Triassic boundary interval in central China: Structure, age and distribution

Summary A carbonate crust, with a principally digitate structure, caps latest Permian reef complexes in east Sichuan, China. The crust contains the conodontHindeodus parvus, confirming its age as earliest Triassic; it therefore closely postdates the end-Permian mass extinction, and is related to associated palaeoenvironmental change. Although its branches have lobate margins, and internal structures of radial fabrics and lobate fabrics in different specimens, an organic origin cannot be confirmed, because crust fabric is largely recrystallised. Therefore we apply the term ?microbialite to reflect uncertainty of its nature. The crust is co-eval (within theparvus Zone) with confirmed microbial biostromes and mounds in Guizhou Province, south of Sichuan. The sum of evidence, assembled by workers in several sites worldwide, indicates a sea-level rise occurred in the boundary interval, and this is corroborated by facies of the Sichuan crust. Abrupt appearance and disappearance of the crust, formed by precipitated carbonates, in east Sichuan, represents short-lived unusual post-extinction marine conditions which were switched abruptly on, then off. Microbial deposits overlying the P/T boundary in other locations in the Tethys Ocean (Iran and Japan) support the view that the unusual oceanic conditions had at least a regional distribution. Because the crust abruptly terminates, and is not succeeded by fossil-rich deposits, application of the disaster biota concept is inappropriate; an environmentally-driven control on carbonate precipitation is better supported by the evidence, whether or not it was biotically-mediated.     

Autoantibodies specific for apoptotic U1-70K are superior serological markers for mixed connective tissue disease

Modifications occurring on autoantigens during cell death have been proposed to have a role in the initiation of autoimmune diseases. Patients suffering from mixed connective tissue disease (MCTD) produce autoantibodies directed to U1 small nuclear ribonucleoprotein (snRNP), and antibodies against a 70 kDa protein component, the U1-70K (70K) protein, are the most prominent. During apoptosis, 70K is cleaved by caspase-3 to a 40 kDa product, which remains associated with the complex. Autoantibodies preferentially recognizing the apoptotic form of 70K have been described previously, and an apoptosis-specific epitope on 70K has been identified. This study shows that 29 of 53 (54%) MCTD sera preferentially recognize the apoptotic form of 70K over intact 70K. Moreover, we show that antibodies directed to an apoptosis-specific epitope on 70K are more specifically associated with MCTD than other anti-70K antibodies, suggesting that apoptotic 70K is a better antigen for the detection of these antibodies in MCTD patients. Longitudinal analysis of 12 MCTD patients showed in several patients that early sera are relatively enriched with antibodies recognizing an apoptosis-specific epitope, and that the levels of these apoptosis-specific antibodies decrease in time. These findings indicate that the early detection of apoptotic 70K is of considerable interest for anti-U1 snRNP-positive patients.     

Adipose Triglyceride Lipase Deficiency Causes Tissue-specific Changes in Insulin Signaling

Triacylglycerol accumulation in insulin target tissues is associated with insulin resistance. Paradoxically, mice with global targeted deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in triacylglycerol hydrolysis, display improved glucose tolerance and insulin sensitivity despite triacylglycerol accumulation in multiple tissues. To determine the molecular mechanisms for this phenotype, ATGL-deficient (ATGL^−/−) and wild-type mice were injected with saline or insulin (10 units/kg, intraperitoneally), and then phosphorylation and activities of key insulin-signaling proteins were determined in insulin target tissues (liver, adipose tissue, and muscle). Insulin signaling and/or glucose transport was also evaluated in isolated adipocytes and skeletal muscle ex vivo. In ATGL^−/− mice, insulin-stimulated phosphatidylinositol 3-kinase and Akt activities as well as phosphorylation of critical residues of IRS1 (Tyr(P)-612) and Akt (Ser(P)-473) were increased in skeletal muscle in vivo. Insulin-stimulated phosphatidylinositol 3-kinase activity and total insulin receptor and insulin receptor substrate 1, but not other parameters, were also increased in white adipose tissue in vivo. In contrast, in vivo measures of insulin signaling were decreased in brown adipose tissue and liver. Interestingly, the enhanced components of insulin signaling identified in skeletal muscle and white adipose tissue in vivo and their expected downstream effects on glucose transport were not present ex vivo. ATGL deficiency altered intramyocellular lipids as well as serum factors known to influence insulin sensitivity. Thus, skeletal muscle, rather than other tissues, primarily contributes to enhanced insulin sensitivity in ATGL^−/− mice in vivo despite triacylglycerol accumulation, and both local and systemic factors contribute to tissue-specific effects of global ATGL deficiency on insulin action.Triacylglycerols (TAGs)⁴ are the predominant form of energy storage in animals. The ability to store and release this energy in response to variable energy availability requires a carefully regulated balance between TAG synthesis and hydrolysis. In the setting of chronic energy excess, however, TAGs and other lipid metabolites accumulate in adipose tissue as well as in metabolically relevant non-adipose tissues where they have been proposed to contribute to cellular dysfunction via a process known as lipotoxicity (1–3). Indeed, intracellular TAG accumulation has been repeatedly associated with metabolic dysfunction, a relationship that is particularly strong for insulin resistance (1–3). Despite this strong association, however, intracellular TAG accumulation is not always associated with insulin resistance (4) and may even be associated with insulin sensitivity, as is the case with highly trained endurance athletes (the so-called “athlete paradox”) (5). Thus, the contribution of intracellular TAGs and TAG metabolism per se to lipotoxicity remains controversial. What is clear is that lipid-induced insulin resistance is a major risk factor for morbidity and mortality from a variety of causes, including overt diabetes mellitus, nonalcoholic fatty liver disease, and cardiovascular disease. Hence, understanding the mechanisms by which dysregulated TAG metabolism contributes to steatosis, lipotoxicity, and insulin resistance is essential to understanding and treating these increasingly prevalent disorders.Although no mechanistic data have been identified directly linking intracellular TAGs per se to insulin resistance, lipotoxicity may occur when the capacity of the lipid droplets to effectively store TAGs is exceeded. Several other lipid metabolites that are products of TAG hydrolysis (i.e. diacylglyerols (DAGs), fatty acids (FAs), fatty acyl-CoAs (FA-CoAs), and ceramides) have been shown to directly or indirectly interfere with insulin signaling and glucose transport via a variety of mechanisms (6–9). Under normal physiological circumstances, insulin binds to the insulin receptor (IR), thereby triggering its intrinsic protein-tyrosine kinase activity. The subsequent autophosphorylation of several IR tyrosine residues promotes the recruitment and tyrosine phosphorylation of IR substrates (IRSs) followed by activation of phosphatidylinositol 3-kinase (PI3K) and Akt, which in turn promote the pleiotrophic downstream effects of insulin. The above lipid metabolites have been shown to increase serine/threonine phosphorylation and decrease tyrosine phosphorylation of IRS1, decrease serine/threonine phosphorylation of Akt, decrease IRS1-associated PI3K activity and Akt activity, and decrease Glut4 translocation (6–9). Possible mechanisms by which these lipid metabolites may influence glucose homeostasis and insulin action include competition for substrate oxidation, interference with cellular energy sensing, regulation of gene expression, promotion of oxidative stress and mitochondrial dysfunction, and activation of inflammatory and apoptotic pathways (6–9). However, most studies evaluating the role of lipotoxicity in insulin resistance have focused on cellular lipid uptake or oxidation, both of which produce unidirectional changes in intracellular TAGs and other intracellular lipid metabolites and hence do not adequately address the role of intracellular TAGs and TAG metabolism per se to this process.Understanding the role of TAG metabolism in lipotoxicity and insulin resistance has been further complicated by the fact that the rate-limiting enzyme for TAG hydrolysis, adipose triglyceride lipase (ATGL), has only recently been identified (10–12). ATGL has been most extensively studied in adipose tissue where it mediates the hydrolysis of long chain fatty acyl TAGs (10). ATGL is also expressed in other tissues, including liver, muscle, and pancreas (13), where its contribution to tissue-specific and systemic metabolism is less well understood. Mice with global targeted deletion of ATGL (ATGL^−/− mice) have severe defects in TAG hydrolysis, leading to TAG accumulation in virtually all tissues (14). Surprisingly, despite increased adiposity and “ectopic” TAG accumulation, which are characteristically associated with insulin resistance, ATGL^−/− mice paradoxically exhibit enhanced glucose tolerance and insulin sensitivity (14). This finding has largely been attributed to the effect of reduced systemic FA delivery on energy substrate availability (14). However, the contribution of altered tissue-specific insulin action to this phenotype has not been evaluated.ATGL^−/− mice represent a unique model for examining the contribution of intracellular TAG accumulation to glucose homeostasis and insulin action because intracellular TAG accumulation is dissociated from systemic FA delivery, and presumably also from the production/accumulation of other intracellular lipid metabolites. In addition, ATGL^−/− mice differ from the other models in which increased adiposity is paradoxically associated with insulin sensitivity in that enhanced expansion of adipose tissue mass and reduced systemic FA delivery do not protect against ectopic lipid deposition in ATGL^−/− mice (15, 16). The aims of this study were to evaluate the mechanisms by which impaired TAG hydrolysis and intracellular TAG accumulation because of global ATGL deficiency promote whole-body glucose tolerance and insulin sensitivity and to define the contribution of tissue-specific changes in insulin action to this phenotype. Here we demonstrate that global ATGL deficiency in mice not only reduces energy substrate availability but also produces tissue-specific changes in insulin action.     

Crystal Structures of an Oligopeptide-Binding Protein from the Biosynthetic Pathway of the β-Lactamase Inhibitor Clavulanic Acid

Clavulanic acid (CA) is a clinically important β-lactamase inhibitor that is produced by fermentation of Streptomyces clavuligerus. The CA biosynthesis pathway starts from arginine and glyceraldehyde-3-phosphate and proceeds via (3S,5S)-clavaminic acid, which is converted to (3R,5R)-clavaldehyde, the immediate precursor of (3R,5R)-CA. Open reading frames 7 (orf7) and 15 (orf15) of the CA biosynthesis cluster encode oligopeptide-binding proteins (OppA1 and OppA2), which are essential for CA biosynthesis. OppA1/2 are proposed to be involved in the binding and/or transport of peptides across the S. clavuligerus cell membrane. Peptide binding assays reveal that recombinant OppA1 and OppA2 bind di-/tripeptides containing arginine and certain nonapeptides including bradykinin. Crystal structures of OppA2 in its apo form and in complex with arginine or bradykinin were solved to 1.45, 1.7, and 1.7 Å resolution, respectively. The overall fold of OppA2 consists of two lobes with a deep cavity in the center, as observed for other oligopeptide-binding proteins. The large cavity creates a peptide/arginine binding cleft. The crystal structures of OppA2 in complex with arginine or bradykinin reveal that the C-terminal arginine of bradykinin binds similarly to arginine. The results are discussed in terms of the possible roles of OppA1/2 in CA biosynthesis.