Comparison of the differential context-dependence of DNA deamination by APOBEC enzymes: correlation with mutation spectra in vivo

To investigate the extent to which in vivo mutation spectra might reflect the intrinsic specificities of active mutators, genetic and biochemical assays were used to analyse the DNA target specificities of cytidine deaminases of the APOBEC family. The results reveal the critical importance of nucleotides immediately 5' of the targeted C for the specificity of all three enzymes studied (AID, APOBEC1 and APOBEC3G). At position -1, APOBEC1 showed a marked preference for dT, AID for dA/dG and APOBEC3G a strong preference for dC. Furthermore, AID and APOBEC3G showed distinct dependence on the nucleotide at position -2 with dA/dT being favoured by AID and dC by APOBEC3G. Most if not all activity of the recombinant deaminases on free dC could be attributed to low-level contamination by host enzymes. The target preference of APOBEC3G supports it being a major but possibly not sole contributor to HIV hypermutation without making it a dominant contribution to general HIV sequence variation. The specificity of AID as deduced from the genetic assay (which relies on inactivation of sacB of Bacillus subtilis) agrees well with that deduced by Pham et al. using an in vitro assay although we postulate that major intrinsic mutational hotspots in immunoglobulin V genes in vivo might reflect favoured sites of AID action being generated by proximal DNA targets located on opposite DNA strands. The target specificity of AID also accords with the spectrum of mutations observed in B lymphoma-associated oncogenes. The possibility of deaminase involvement in non-lymphoid human tumours is hinted at by tissue-specific differences in the spectra of dC transitions in tumour-suppressor genes. Thus, the patterns of hypermutation in antibodies and retroviruses owe much to the intrinsic sequence preferences of the AID/APOBEC family of DNA deaminases: analogous biases might also contribute to the spectra of cancer-associated mutation.     

Thermal dissociation and unfolding of insulin

The thermal stability of human insulin was studied by differential scanning microcalorimetry and near-UV circular dichroism as a function of zinc/protein ratio, to elucidate the dissociation and unfolding processes of insulin in different association states. Zinc-free insulin, which is primarily dimeric at room temperature, unfolded at approximately 70 degrees C. The two monomeric insulin mutants Asp(B28) and Asp(B9),Glu(B27) unfolded at higher temperatures, but with enthalpies of unfolding that were approximately 30% smaller. Small amounts of zinc caused a biphasic thermal denaturation pattern of insulin. The biphasic denaturation is caused by a redistribution of zinc ions during the heating process and results in two distinct transitions with T(m)'s of approximately 70 and approximately 87 degrees C corresponding to monomer/dimer and hexamer, respectively. At high zinc concentrations (>or=5 Zn(2+) ions/hexamer), only the hexamer transition is observed. The results of this study show that the thermal stability of insulin is closely linked to the association state and that the zinc hexamer remains stable at much higher temperatures than the monomer. This is in contrast to studies with chemical denaturants where it has been shown that monomer unfolding takes place at much higher denaturant concentrations than the dissociation of higher oligomers [Ahmad, A., et al. (2004) J. Biol. Chem. 279, 14999-15013].     

Development of real-time PCR for detection of <Emphasis Type="Italic">Mycoplasma hominis</Emphasis>

Background  

Mycoplasma hominis is associated with pelvic inflammatory disease, bacterial vaginosis, post partum fever, sepsis and infections of the central nervous system often leading to serious conditions. Association with development of female infertility has also been suggested, but different publications present different results. We developed a sensitive and fast diagnostic real-time PCR to test clinical samples from women undergoing laparoscopic examination before fertility treatment. To develop a test for the detection and quantification of M. hominis we selected a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gap), as a target.     

Catalase and NO CATALASE ACTIVITY1 Promote Autophagy-Dependent Cell Death in Arabidopsis

Programmed cell death often depends on generation of reactive oxygen species, which can be detoxified by antioxidative enzymes, including catalases. We previously isolated catalase-deficient mutants (cat2) in a screen for resistance to hydroxyurea-induced cell death. Here, we identify an Arabidopsis thaliana hydroxyurea-resistant autophagy mutant, atg2, which also shows reduced sensitivity to cell death triggered by the bacterial effector avrRpm1. To test if catalase deficiency likewise affected both hydroxyurea and avrRpm1 sensitivity, we selected mutants with extremely low catalase activities and showed that they carried mutations in a gene that we named NO CATALASE ACTIVITY1 (NCA1). nca1 mutants showed severely reduced activities of all three catalase isoforms in Arabidopsis, and loss of NCA1 function led to strong suppression of RPM1-triggered cell death. Basal and starvation-induced autophagy appeared normal in the nca1 and cat2 mutants. By contrast, autophagic degradation induced by avrRpm1 challenge was compromised, indicating that catalase acted upstream of immunity-triggered autophagy. The direct interaction of catalase with reactive oxygen species could allow catalase to act as a molecular link between reactive oxygen species and the promotion of autophagy-dependent cell death.     

Influence of the Chlamydia pneumoniae AR39 bacteriophage ϕCPAR39 on chlamydial inclusion morphology

The human respiratory tract pathogen Chlamydia pneumoniae AR39 is naturally infected by the bacteriophage ?CPAR39. The phage genome encodes six ORFs, [ORF8, ORF4, ORF5, and viral protein (VP) 1, VP2 and VP3]. To study the growth of the phage, antibodies were generated to VP1 and used to investigate the ?CPAR39 infection. Using immunofluorescence laser confocal microscopy and two-dimensional gel electrophoresis, we investigated the ?CPAR39 infection of C. pneumoniae AR39. It was observed that ?CPAR39 infection differentially suppressed the C. pneumoniae protein synthesis as the polymorphic membrane protein 10 and the secreted chlamydial protein Cpn0796 was hardly expressed while the secreted chlamydial protein Cpaf was expressed, but not secreted. The inclusion membrane protein, IncA, was demonstrated to surround the phage-infected abnormal reticulate bodies (RB) as well as being located in the inclusion membrane. As IncA is secreted by the type 3 secretion (T3S) system, it is likely that the T3S is disrupted in the phage-infected chlamydiae such that it accumulates around the infected RB.     

Epithelial cell kinetics of the gastric mucosa during Helicobacter pylori infection

Helicobacter pylori is an important pathogen in major gastroduodenal diseases, including inflammation with ulceration and gastric malignancies. Alterations in H. pylori associated cell turnover in gastric epithelial cells are examined in relation to inflammatory activity, bacteria load and cytokines which may improve knowledge concerning the outcome of gastric diseases caused by H. pylori. Antral biopsies from 42 dyspeptic patients including 27 H. pylori-positive and 15 H. pylori-negative patients were tested for apoptotic activity by the TUNEL assay, and immuno-histochemically for p53 and the proliferative marker Ki-67. H. pylori infection, bacteria load and inflammatory activity were associated with increased cell turnover as judged by enhanced activities of TUNEL, p53 and Ki-67. Only p53 was significantly correlated to IFN-gamma, IL-8 and IL-10. The H. pylori-positive state was furthermore accompanied by varying degrees of altered distribution pattern of the markers studied, with occasional presence of apoptosis in the deeper pit zones, upward extension of Ki-67 and to a lesser degree of p53. Given a similar pattern of change in proliferation and apoptosis in some neoplastic lesions in other parts of the gastrointestinal tract, such studies in cell turnover may provide insights valuable in the investigations of potential precursors of gastric malignancies.     

Elucidating the Role of Transport Processes in Leaf Glucosinolate Distribution

In Arabidopsis (Arabidopsis thaliana), a strategy to defend its leaves against herbivores is to accumulate glucosinolates along the midrib and at the margin. Although it is generally assumed that glucosinolates are synthesized along the vasculature in an Arabidopsis leaf, thereby suggesting that the margin accumulation is established through transport, little is known about these transport processes. Here, we show through leaf apoplastic fluid analysis and glucosinolate feeding experiments that two glucosinolate transporters, GTR1 and GTR2, essential for long-distance transport of glucosinolates in Arabidopsis, also play key roles in glucosinolate allocation within a mature leaf by effectively importing apoplastically localized glucosinolates into appropriate cells. Detection of glucosinolates in root xylem sap unambiguously shows that this transport route is involved in root-to-shoot glucosinolate allocation. Detailed leaf dissections show that in the absence of GTR1 and GTR2 transport activity, glucosinolates accumulate predominantly in leaf margins and leaf tips. Furthermore, we show that glucosinolates accumulate in the leaf abaxial epidermis in a GTR-independent manner. Based on our results, we propose a model for how glucosinolates accumulate in the leaf margin and epidermis, which includes symplasmic movement through plasmodesmata, coupled with the activity of putative vacuolar glucosinolate importers in these peripheral cell layers.Feeding behavior of herbivorous insects and distribution of defense compounds in plants have been suggested to be a result of an arms race between plants and insects that has spanned millions of years (Ehrlich and Raven, 1964). Whether insects adapted first to plants or the other way around is an ongoing debate in this research field (Schoonhoven et al., 2005; Ali and Agrawal, 2012). Leaf margin accumulation of defense compounds has been demonstrated in various plant species (Gutterman and Chauser-Volfson, 2000; Chauser-Volfson et al., 2002; Kester et al., 2002; Cooney et al., 2012). In the model plant Arabidopsis (Arabidopsis thaliana), higher concentration of glucosinolates, which constitute a major part of the chemical defense system in this plant (Kliebenstein et al., 2001a; Halkier and Gershenzon, 2006), was found at the leaf midrib and margins compared with the leaf lamina (Shroff et al., 2008; Sønderby et al., 2010). This nonuniform leaf distribution of glucosinolates appeared to explain the feeding pattern of a generalist herbivore (Helicoverpa armigera), as it avoided feeding at the leaf margin and midrib (Shroff et al., 2008). A similar feeding pattern on Arabidopsis was observed for a different generalist herbivore, Spodoptera littoralis (Schweizer et al., 2013). Interestingly, S. littoralis was shown to favor feeding from Arabidopsis leaf margins in glucosinolate-deficient mutants (Schweizer et al., 2013), which could indicate an inherent preference for margin feeding and that Arabidopsis adapted to such behavior by accumulating defense compounds here. A damaged leaf margin may be more critical for leaf stability than damage to inner leaf parts (Shroff et al., 2008), further motivating protection of this tissue. The margin-feeding preference of S. littoralis might be explained by better nutritional value of the leaf margin cells (Schweizer et al., 2013), which has been shown to consist of specialized elongated cell files (Koroleva et al., 2010; Nakata and Okada, 2013).Other distribution patterns have been reported for glucosinolates in an Arabidopsis leaf. A study investigating spatiotemporal metabolic shifts during senescence in Arabidopsis reported that fully expanded mature leaves exhibited a glucosinolate gradient from base to tip, with highest level of glucosinolates at the leaf base (Watanabe et al., 2013). In contrast to the horizontal plane, less has been reported on distribution of glucosinolates in the vertical plane of a leaf. A localization study of cyanogenic glucosides, defense molecules related to glucosinolates (Halkier and Gershenzon, 2006), determined that these compounds primarily were located in the epidermis of sorghum (Sorghum bicolor; Kojima et al., 1979). Whereas epidermis-derived trichomes in Arabidopsis were recently demonstrated to contain glucosinolates and to express glucosinolate biosynthetic genes (Frerigmann et al., 2012), no studies have investigated glucosinolates in the epidermal cell layer.Based on promoter-GUS studies, biosynthesis of glucosinolates in leaves of Arabidopsis has been associated with primarily major and minor veins in leaves and silique walls (Mikkelsen et al., 2000; Reintanz et al., 2001; Tantikanjana et al., 2001; Chen et al., 2003; Grubb et al., 2004; Schuster et al., 2006; Gigolashvili et al., 2007; Li et al., 2011; Redovniković et al., 2012). The discrepancy between vasculature-associated glucosinolate biosynthesis and margin accumulation of glucosinolates suggests that transport processes must be involved in establishing the distribution pattern of glucosinolates within a leaf.Plant transport systems include the apoplastic xylem, the symplastic phloem, and plasmodesmata. Xylem transport is mainly driven by an upward pull generated by transpiration from aerial plant organs, thereby directing transport to sites of rapid evaporation (such as leaf margins; Sattelmacher, 2001). Phloem flow is facilitated by an osmosis-regulated hydrostatic pressure difference between source and sink tissue, primarily generated by Suc bulk flow (Lucas et al., 2013). Plasmodesmata are intercellular channels that establish symplasmic pathways between neighboring cells, and most cell types in a plant are symplastically connected via plasmodesmata (Roberts and Oparka, 2003). Translocation of small molecules in these channels is driven by diffusion and is regulated developmentally as well as spatially to form symplastically connected domains (Roberts and Oparka, 2003; Christensen et al., 2009). To what extent any of these transport processes are involved in establishing specific distribution patterns of glucosinolates within leaves is not known.Recently, two plasma membrane-localized, glucosinolate-specific importers, GLUCOSINOLATE TRANSPORTER1 (GTR1) and GTR2, were identified in Arabidopsis (Nour-Eldin et al., 2012). In leaf, their expression patterns were shown to be in leaf veins (GTR1 and GTR2) and surrounding mesophyll cells (GTR1; Nour-Eldin et al., 2012). Absence of aliphatic and indole glucosinolates in seeds of the gtr1gtr2 double knockout (dKO) mutant (gtr1gtr2 dKO) demonstrated that these transporters are essential for long-distance glucosinolate transport to the seeds and indicates a role in phloem loading (Nour-Eldin et al., 2012). Another study investigating long-distance transport of glucosinolates in the 3-week-old wild type and gtr1gtr2 dKO indicated that GTR1 and GTR2 were involved in bidirectional transport of aliphatic glucosinolates between root and shoot via both phloem and xylem pathways (Andersen et al., 2013). The authors suggested a role for GTR1 and GTR2 in the retention of long-chained aliphatic glucosinolates in roots by removing the compounds from the xylem (Andersen et al., 2013).Identification of the glucosinolate transporters GTR1 and GTR2 has provided a molecular tool to investigate the role of transport processes in establishing leaf glucosinolate distribution. In this study, we have performed a detailed spatial investigation of the distribution of an exogenously fed glucosinolate (sinigrin) and endogenous glucosinolates within mature wild-type and gtr1gtr2 dKO Arabidopsis leaves, achieved by collecting and analyzing leaf parts at the horizontal (y axis: petiole, base, and tip; x axis: midrib, lamina, and margin) as well as at the vertical leaf plane (z axis: abaxial epidermis). Furthermore, we analyze wild-type and gtr1gtr2 dKO root xylem sap and leaf apoplastic fluids for glucosinolates. Based on our results, we propose a model where GTR1 and GTR2 import glucosinolates from the apoplast to the symplast and where the glucosinolate distribution pattern within an Arabidopsis leaf is established via symplasmic movement of glucosinolates through plasmodesmata, coupled with the activity of putative vacuolar glucosinolate importers in peripheral cell layers.     

AID Enzymatic Activity Is Inversely Proportional to the Size of Cytosine C5 Orbital Cloud

Activation induced deaminase (AID) deaminates cytosine to uracil, which is required for a functional humoral immune system. Previous work demonstrated, that AID also deaminates 5-methylcytosine (5 mC). Recently, a novel vertebrate modification (5-hydroxymethylcytosine - 5 hmC) has been implicated in functioning in epigenetic reprogramming, yet no molecular pathway explaining the removal of 5 hmC has been identified. AID has been suggested to deaminate 5 hmC, with the 5 hmU product being repaired by base excision repair pathways back to cytosine. Here we demonstrate that AID's enzymatic activity is inversely proportional to the electron cloud size of C5-cytosine - H > F > methyl > hydroxymethyl. This makes AID an unlikely candidate to be part of 5 hmC removal.