Dioecious <Emphasis Type="Italic">Silene latifolia</Emphasis> plants show sexual dimorphism in the vegetative stage

Background  

Prior to this study, no differences in gene expression between male and female dioecious plants in the vegetative state had been detected. Among dioecious plants displaying sexual dimorphism, Silene latifolia is one of the most studied species. Although many sexually dimorphic traits have been described in S. latifolia, all of them are quantitative, and they usually become apparent only after the initiation of flowering.     

Clustered O-Glycans of IgA1: DEFINING MACRO- AND MICROHETEROGENEITY BY USE OF ELECTRON CAPTURE/TRANSFER DISSOCIATION*

IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) O-glycans, plays a pivotal role in the pathogenesis of the disease. It is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. We have described the utility of activated ion-electron capture dissociation (AI-ECD) mass spectrometric analysis of IgA1 O-glycosylation. However, locating and characterizing the entire range of O-glycan attachment sites are analytically challenging due to the clustered serine and threonine residues in the HR of IgA1 heavy chain. To address this problem, we analyzed all glycoforms of the HR glycopeptides of a Gal-deficient IgA1 myeloma protein, mimicking the aberrant IgA1 in patients with IgAN, by use of a combination of IgA-specific proteases + trypsin and AI-ECD Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS). The IgA-specific proteases provided a variety of IgA1 HR fragments that allowed unambiguous localization of all O-glycosylation sites in the six most abundant glycoforms, including the sites deficient in Gal. Additionally, this protocol was adapted for on-line liquid chromatography (LC)-AI-ECD MS/MS and LC-electron transfer dissociation MS/MS analysis. Our results thus represent a new clinically relevant approach that requires ECD/electron transfer dissociation-type fragmentation to define the molecular events leading to pathogenesis of a chronic kidney disease. Furthermore, this work offers generally applicable principles for the analysis of clustered sites of O-glycosylation.Glycosylation is one of the most common post-translational modifications of proteins. It is estimated that over half of mammalian proteins are glycosylated. Patients with several autoimmune disorders, chronic inflammatory diseases, and some infectious diseases exhibit abnormal glycosylation of serum immunoglobulins and other glycoproteins (1–5). The biological functions of these modifications in health and disease have become a significant area of interest in biomedical research (6). A subset of these glycoproteins has clustered sites of O-glycosylation with serine- and threonine-rich stretches within the amino acid sequence. Mucins, such as membrane-associated MUC1, are perhaps the best known family of proteins that are heavily O-glycosylated. Their altered expression and aberrant glycosylation have made them potential targets as biomarkers for early detection of cancer (7). Immunoglobulin A1 (IgA1)1 contains both O- and N-glycans (Fig. 1). Aberrant O-glycosylation of IgA1 is involved in the pathogenesis of IgA nephropathy (IgAN) and the closely related Henoch-Schönlein purpura nephritis (1, 8). Interestingly, the aberrantly glycosylated molecules, IgA1 in IgAN and MUC1 in cancer, are recognized by the immune system as neoepitopes as evidenced by formation of specific antibodies (9–11). Mucin-like bacterial surface proteins exhibit similar properties: the molecules have clustered bacterial O-glycans that mediate cellular adhesion, and blocking antibodies target these glycan-containing epitopes (12).Open in a separate windowFig. 1.IgA1 structural elements. IgA1 has N-linked glycans (filled circles) and O-linked glycans (open circles). The O-glycosylated sites are in the HR between the first and second constant region domains of the heavy chains. The HR is a Pro-rich segment with nine possible sites of O-glycan attachment. Underlined serine and threonine residues are usually glycosylated (31). Arrows show cleavage sites of trypsin and IgA-specific proteases.An O-glycosylated protein from a single source contains a population of variably O-glycosylated isoforms that show a distinct distribution of microheterogeneity of the O-glycan chains in terms of number, sites of attachment, and composition. Characterizing these clustered sites and understanding how the distributions change under different biological conditions or disease states are an analytical challenge. Enzymatic or chemical release of O-glycans is not selective. The heterogeneity, composition, and quantitative aspects of different O-glycan chains can be assessed and quantified by gas chromatographic and/or mass spectrometric techniques. However, the site-specific information and context of location and composition of adjacent chains are lost. Carbohydrate-specific lectin analysis of O-glycoproteins can provide information on glycan composition and comparative differences between samples, such as those from healthy controls and patients with various disease states. We have successfully demonstrated this in the analysis of IgA1 O-glycans from patients with IgAN versus healthy controls and disease controls (13–15). This included proximal assessment of sites with galactose (Gal)-deficient O-glycans after digests with IgA-specific proteases (8). Several studies have demonstrated the value of mass spectrometry (MS) in identifying Gal-deficient IgA1 in patients with IgAN (16–21), including our work that demonstrated the first direct localization of native sites of O-glycan chains in the hinge region (HR) of IgA1 by use of electron capture dissociation (ECD) (20, 22). ECD and the more recently developed electron transfer dissociation (ETD) have been used to identify sites of O-glycosylation on a variety of proteins (23–26). This includes the analysis of sites of O-glycosylation by on-line LC-ECD/ETD MS/MS methods (23, 26, 27).IgAN is the most common primary glomerulonephritis worldwide (28) with about 20–40% of patients developing end stage renal failure. It is characterized by mesangial deposits of IgA1-containing immune complexes (28). The distinctive O-glycan chains of IgA1 molecules play a pivotal role in the pathogenesis of IgAN (1, 10, 14–16, 29, 30). IgA1 contains an HR between the first and second heavy chain constant region domains with a high content of Ser, Thr, and Pro. This segment usually has three to five O-glycan chains per HR (31) (see Fig. 1). Aberrantly glycosylated IgA1, deficient in Gal in some of the O-glycans in the HR, in serum is rare in healthy individuals but is present at elevated levels in IgAN patients (13, 15). This distinctive IgA1 is in circulating immune complexes (8, 10, 15) and in the glomerular deposits of IgAN patients (16, 29). The absence of Gal apparently leads to the exposure of neoepitopes, including terminal and sialylated N-acetylgalactosamine (GalNAc) residues (9, 10). These epitopes are recognized by naturally occurring anti-glycan IgG or IgA1 antibodies and, consequently, circulating immune complexes are formed (9, 10, 15) that can deposit in the glomerular mesangia. To identify the pathogenic forms of IgA1, a thorough analysis of O-glycan microheterogeneity, including identification of the attachment sites, will be required.In this work, we demonstrate the complete analysis of O-glycoform microheterogeneity and site localization of the glycoforms in a naturally Gal-deficient IgA1 (Ale) myeloma protein that mimics the nephritogenic IgA1 in patients with IgAN (8, 9). Reversed phase (RP) LC FT-ICR MS successfully identified 10 distinct IgA1 HR fragments representing >99% of total IgA1. AI-ECD of the six most abundant IgA1 HR glycoforms (>95% of total IgA1) was accomplished with three distinct IgA-specific protease + trypsin digestions, identifying sites of Gal deficiency across four distinct IgA1 O-glycoforms. Based on the success of the ECD fragmentation of these IgA1 HR fragments, we adapted the analysis for on-line LC-MS/MS methods for both ECD and ETD. The variety of IgA1 HR proteolytic fragments provides a practical set of guidelines for the ECD/ETD analysis of clustered sites of O-glycosylation on this and other proteins. These results also provide insight into the order of attachment of the O-glycans in the IgA1 HR.     

RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins

Accumulation of repair proteins on damaged chromosomes is required to restore genomic integrity. However, the mechanisms of protein retention at the most destructive chromosomal lesions, the DNA double-strand breaks (DSBs), are poorly understood. We show that RNF8, a RING-finger ubiquitin ligase, rapidly assembles at DSBs via interaction of its FHA domain with the phosphorylated adaptor protein MDC1. This is accompanied by an increase in DSB-associated ubiquitylations and followed by accumulation of 53BP1 and BRCA1 repair proteins. Knockdown of RNF8 or disruption of its FHA or RING domains impaired DSB-associated ubiquitylation and inhibited retention of 53BP1 and BRCA1 at the DSB sites. In addition, we show that RNF8 can ubiquitylate histone H2A and H2AX, and that its depletion sensitizes cells to ionizing radiation. These data suggest that MDC1-mediated and RNF8-executed histone ubiquitylation protects genome integrity by licensing the DSB-flanking chromatin to concentrate repair factors near the DNA lesions.     

Spatial distribution of the Daphnia longispina species complex and other planktonic crustaceans in the heterogeneous environment of canyon-shaped reservoirs

Canyon-shaped reservoirs are often characterised by longitudinalgradients of environmental factors (including trophic level,phytoplankton and zooplankton biomass and abundance of planktivorousfish) affecting the taxonomic composition of the pelagic community.We tested the hypothesis that the spatial distribution of differentspecies and interspecific hybrids of the Daphnia longispinaspecies complex is non-random under such conditions. Duringthe summer stratification, we sampled crustacean zooplanktonfrom 11 reservoirs, covering both longitudinal (upstream, middle,dam) and vertical (epi-, meta- and hypolimnion) environmentalgradients. Allozyme electrophoresis was used to discriminateamong different Daphnia taxa. All three frequently hybridizingEuropean species of the complex (galeata, cucullata, longispina= hyalina) and hybrids with Daphnia galeata were commonly recorded.Smaller-bodied Daphnia cucullata and its hybrids, when present,preferred mostly the nutrient- and food-rich upstream regions;D. longispina and its hybrids were more commonly found in thedownstream part, and often dominated in the meta- or hypolimnion.Redundancy analyses confirmed significant differences in theDaphnia taxon composition, as well as in spatial distributionof other crustacean species, along both gradients. For the firsttime, we demonstrate regular patterns in the horizontal distributionof Daphnia species and hybrids within a water body, thus acceptingour hypothesis. Such spatial distributional patterns may stronglyimpact local hybridization processes.     

Proteomic analysis of hepatic iron overload in mice suggests dysregulation of urea cycle, impairment of fatty acid oxidation, and changes in the methylation cycle

Liver iron overload can be found in hereditary hemochromatosis, chronic liver diseases such as alcoholic liver disease, and chronic viral hepatitis or secondary to repeated blood transfusions. The excess iron promotes liver damage, including fibrosis, cirrhosis, and hepatocellular carcinoma. Despite significant research effort, we remain largely ignorant of the cellular consequences of liver iron overload and the cellular processes that result in the observed pathological changes. In addition, the variability in outcome and the compensatory response that likely modulates the effect of increased iron levels are not understood. To provide insight into these critical questions, we undertook a study to determine the consequences of iron overload on protein levels in liver using a proteomic approach. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we studied hepatic iron overload induced by carbonyl iron-rich diet in mice and identified 30 liver proteins whose quantity changes in condition of excess liver iron. Among the identified proteins were enzymes involved in several important metabolic pathways, namely the urea cycle, fatty acid oxidation, and the methylation cycle. This pattern of changes likely reflects compensatory and pathological changes associated with liver iron overload and provides a window into these processes.     

Heterosubtypic immunity to influenza A virus infection requires a properly diversified antibody repertoire

Heterosubtypic immunity (HSI) is defined as cross-protection to infection with an influenza A virus serotype other than the one used for primary infection. Although HSI has been thought to be mediated by serotype cross-reactive cytotoxic T lymphocytes (CTL) that recognize conserved epitopes of structural proteins, recent studies suggest that antibodies (Abs) may make a significant contribution. In this study, we provide further evidence for the role of Abs in HSI using transgenic mice lacking terminal deoxyribonucleotidyltransferase (TdT), which adds N nucleotides to V-D and D-J junctions of the complementary determining region 3 (CDR3) (TdT(-/-)) and mice with altered Ab repertoires due to replacement of the complete locus of heavy chain diversity segments (D(H)) with an altered D(H) segment (namely, Delta D-iD). Both types of mice failed to generate complete HSI, although they were able to mount protective immunity to a homologous challenge. Lower levels of virus-specific antibodies along with more severely impaired HSI were observed in TdT(-/-) mice compared to those in Delta D-iD mice, while CTL activity remained unchanged in both types of mice. These findings indicate that a properly diversified antibody repertoire is required for HSI and that N addition by TdT is a more effective mechanism in the induction of a properly diversified antibody repertoire and, therefore, complete HSI. The results suggest that the diversity of the antibody repertoire as determined by the composition of the D region of HCDR3 and by N addition are among the mechanisms selected for in evolution to create a favorable environment to resolve infections with mutated viruses.     

Irradiated cationic mesoporphyrin induces larger damage to isolated rat liver mitochondria than the anionic form

The action of irradiated cationic Fe(III)TMPyP and anionic Fe(III)TPPS4 forms of mesoporphyrins on mitochondrial functions was investigated using experimental conditions that caused minimal effects on mitochondria in the dark. Treatment of mitochondria with 1 microM Fe(III)TMPyP for 2 min decreased the respiratory control by 3% in the dark and 28% after irradiation. Fe(III)TPPS4 (1 microM) had no significant effect on respiratory control under any of the above conditions. Both porphyrins increased the mitochondrial production of reactive oxygen species in the presence of Ca2+; however, the effect of Fe(III)TMPyP was significantly stronger. In both cases, this overproduction was associated with membrane lipid peroxidation. It was also observed that the association constant of Fe(III)TMPyP with mitochondria was 11 times higher than that of Fe(III)TPPS4. In conclusion, the damage to isolated mitochondria induced by Fe(III)TMPyP under illumination was larger than by Fe(III)TPPS4, probably because its cationic charge favors association with the mitochondrial membrane. This is supported by the decrease in the association constant of Fe(III)TMPyP with mitochondria in higher salt medium.