Subtypes of non-transformed human mammary epithelial cells cultured in vitro: histo-blood group antigen H type 2 defines basal cell-derived cells

Abstract. Normal (non-transformed) human mammary epithelial cell lines derived from reduction mammoplasties were analyzed by immunocytochemistry with more than 80 monoclonal antibodies (mAbs) and other specific reagents to tissue-specific and developmentally regulated antigens at different passage levels. A subpopulation of poorly differentiated, proliferating epithelial cells, corresponding to the 'selected' cell type of late passages, is shown to be characterized by a new marker, the histo-blood group antigen H type 2, probably carried on a membrane-bound glycolipid. These cells also express a number of other onco-developmental carbohydrate antigens [Le^y, Le^x, sialosyl-Le^a, precursor of Thomsen Friedenreich antigen (T_n), but not Thomsen-Friedenreich antigen and sialosyl-T_n). Their cytokeratin (CK) phenotype, as assessed by reactivity with monospecific mAbs and two-dimensional gel electrophoresis, is CK 5, 6, 14 and 17, with CK 19 being consistently absent, and varying minor amounts of CK 7, 8 and 18, as well as 15 and 16. The reactivity of these cells with a panel of 11 mAbs specific for CK 18 varies considerably even after cloning, indicating heterogeneity of epitope expression or accessibility. Our data strongly suggest that the H type 2⁺ cells develop from the basal cell layer of the mammary gland.     

European Union’s Public Fishing Access Agreements in Developing Countries

The imperative to increase seafood supply while dealing with its overfished local stocks has pushed the European Union (EU) and its Member States to fish in the Exclusive Economic Zones of other countries through various types of fishing agreements for decades. Although European public fishing agreements are commented on regularly and considered to be transparent, this is the first global and historical study on the fee regime that governs them. We find that the EU has subsidized these agreements at an average of 75% of their cost (financial contribution agreed upon in the agreements), while private European business interests paid the equivalent of 1.5% of the value of the fish that was eventually landed. This raises questions of fisheries benefit-sharing and resource-use equity that the EU has the potential to address during the nearly completed reform of its Common Fisheries Policy.     

Arctic fisheries catches in Russia, USA, and Canada: baselines for neglected ecosystems

The Amerasian Arctic, covering northern Siberia (Russia), Arctic Alaska (USA), and the Canadian Arctic, extends over seven coastal Large Marine Ecosystems and makes up the seasonally ice-free part of FAO Statistical Area 18 (Arctic Sea). Historically, the harsh climate has limited marine fisheries (here excluding marine mammal hunting) to small-scale operations conducted mainly in estuaries and river deltas. Most of the catches have traditionally not been reported to FAO, with the result that total catch estimated here from 1950 to 2006 is 75 times higher than the sum of the catches reported for FAO 18 (Arctic). Catches were reconstructed from data on fishing communities in Canada and Alaska, and from various government and non-government sources for Siberia. Based on national data supplied to FAO since 1950, catches have been reported as 12,700 t in toto (reported on behalf of the former Soviet Union). This compares with our reconstructed catches of over 950,000 t, i.e., 770,000, 89,000, and 94,000 t by Russia, USA, and Canada, respectively for the same time period. The reconstructed catch (mainly whitefishes in Siberia, various salmonids in Alaska, and Arctic char in Canada) was 24,100 t year⁻¹ in 1950, but declined to 10,200 t year⁻¹ by the mid-2000s. Reasons for these trends are discussed by country, along with sources of uncertainty (particularly large for Siberia). Catches were allocated to Large Marine Ecosystems to present ecosystem-relevant baselines for the impact of fisheries on the Arctic, which can be expected to increase, as industrial fisheries move into a warming Arctic following the invasion of boreal species, unless countries apply precautionary ecosystem-based management approaches.     

MtNOA1/RIF1 modulates Medicago truncatula-Sinorhizobium meliloti nodule development without affecting its nitric oxide content

AtNoa1/Rif1 (formerly referred to as AtNos1) has been shown to modulate nitric oxide (NO) content in Arabidopsis. As NO generation in the legume-rhizobium symbiosis has been shown, the involvement of an AtNoa1/Rif1 orthologue from Medicago truncatula (MtNoa1/Rif1) during its symbiotic interaction with Sinorhizobium meliloti has been studied. The expression of MtNoa1/Rif1 appeared to occur mainly in nodule vascular bundles and the meristematic zone. Using an RNA interference strategy, transgenic roots exhibiting a significantly decreased level of MtNoa1/Rif1 expression were analysed. NO production was assessed using a fluorescent probe, and the symbiotic capacities of the composite plants upon infection with Sinorhizobium meliloti were determined. The decrease in MtNoa1/Rif1 expression level resulted in a decrease in NO production in roots, but not in symbiotic nodules, indicating a different regulation of NO synthesis in these organs. However, it significantly lowered the nodule number and the nitrogen fixation capacity of the functional nodules. Although having no influence on NO production in nodules, MtNOA1/RIF1 significantly affected the establishment and the functioning of the symbiotic interaction. The impairment of plastid functioning may explain this phenotype.     

Association of anti-oxidized LDL and candidate genes with severity of coronary stenosis in the Women's Ischemia Syndrome Evaluation study

Atherosclerosis is the major cause of coronary artery disease (CAD), and oxidized LDL (oxLDL) is believed to play a key role in the initiation of the atherosclerotic process. Recent studies show that inflammation and autoimmune reactions are also relevant in atherosclerosis. In this study, we examined the association of antibodies against oxLDL (anti-oxLDL) with the severity of CAD in 558 Women's Ischemia Syndrome Evaluation (WISE) study samples (465 whites; 93 blacks) determined by coronary stenosis (< 20%, 20%-49%, > 50% stenosis). We also examined the relationship of anti-oxLDL with serum lipid levels and nine candidate genes including APOE, APOH, APOA5, LPL, LRP1, HL, CETP, PON1, and OLR1. IgM anti-oxLDL levels were significantly higher in the >20% stenosis group than in the ≥ 20% stenosis group in whites (0.69 ± 0.02 vs. 0.64 ± 0.01, respectively; P = 0.02). IgM anti-oxLDL levels correlated significantly with total cholesterol (r2 = 0.01; P = 0.03) and LDL cholesterol (r2 = 0.017; P = 0.004) in whites. Multiple regression analysis revealed a suggestive association of LPL/S447X single-nucleotide polymorphism (SNP) with both IgG anti-oxLDL (P = 0.02) and IgM anti-oxLDL (P = 0.07), as well as between IgM anti-oxLDL and the OLR1/3'UTR SNP (P = 0.020). Our data suggest that higher IgM anti-oxLDL levels may provide protection against coronary stenosis and that genetic variation in some candidate genes are determinants of anti-oxLDL levels.     

Loss-of-function mutation of REDUCED WALL ACETYLATION2 in Arabidopsis leads to reduced cell wall acetylation and increased resistance to Botrytis cinerea

Nearly all polysaccharides in plant cell walls are O-acetylated, including the various pectic polysaccharides and the hemicelluloses xylan, mannan, and xyloglucan. However, the enzymes involved in the polysaccharide acetylation have not been identified. While the role of polysaccharide acetylation in vivo is unclear, it is known to reduce biofuel yield from lignocellulosic biomass by the inhibition of microorganisms used for fermentation. We have analyzed four Arabidopsis (Arabidopsis thaliana) homologs of the protein Cas1p known to be involved in polysaccharide O-acetylation in Cryptococcus neoformans. Loss-of-function mutants in one of the genes, designated REDUCED WALL ACETYLATION2 (RWA2), had decreased levels of acetylated cell wall polymers. Cell wall material isolated from mutant leaves and treated with alkali released about 20% lower amounts of acetic acid when compared with the wild type. The same level of acetate deficiency was found in several pectic polymers and in xyloglucan. Thus, the rwa2 mutations affect different polymers to the same extent. There were no obvious morphological or growth differences observed between the wild type and rwa2 mutants. However, both alleles of rwa2 displayed increased tolerance toward the necrotrophic fungal pathogen Botrytis cinerea.     

The Role of the Plant-Specific ALTERED XYLOGLUCAN9 Protein in Arabidopsis Cell Wall Polysaccharide O-Acetylation

A mutation in the ALTERED XYLOGLUCAN9 (AXY9) gene was found to be causative for the decreased xyloglucan acetylation phenotype of the axy9.1 mutant, which was identified in a forward genetic screen for Arabidopsis (Arabidopsis thaliana) mutants. The axy9.1 mutant also exhibits decreased O-acetylation of xylan, implying that the AXY9 protein has a broad role in polysaccharide acetylation. An axy9 insertional mutant exhibits severe growth defects and collapsed xylem, demonstrating the importance of wall polysaccharide O-acetylation for normal plant growth and development. Localization and topological experiments indicate that the active site of the AXY9 protein resides within the Golgi lumen. The AXY9 protein appears to be a component of the plant cell wall polysaccharide acetylation pathway, which also includes the REDUCED WALL ACETYLATION and TRICHOME BIREFRINGENCE-LIKE proteins. The AXY9 protein is distinct from the TRICHOME BIREFRINGENCE-LIKE proteins, reported to be polysaccharide acetyltransferases, but does share homology with them and other acetyltransferases, suggesting that the AXY9 protein may act to produce an acetylated intermediate that is part of the O-acetylation pathway.The plant cell wall is a complex composite of polysaccharides, glycoproteins, and polyphenols, with the fine structure and quantity of each varying by species, tissue, and developmental time point (Knox, 2008; Burton et al., 2010). Cellulose, hemicelluloses, and pectic polysaccharides are the three major classes of polysaccharides observed in the wall. Current models of the wall have cellulose microfibrils as the major structural component, with hemicelluloses binding to the microfibrils and pectins as an amorphous matrix in which the cellulose/hemicellulose network is embedded (Pauly et al., 1999a; Somerville et al., 2004; Cosgrove, 2005). Unlike the linear β-1,4-glucan chains making up cellulose microfibrils, hemicelluloses and pectins consist of a diverse set of glycosyl units and linkages as well as other modifications such as methylation and acetylation (Caffall and Mohnen, 2009; Scheller and Ulvskov, 2010; Pauly et al., 2013).The O-acetyl substitutions on hemicelluloses and pectins occur on a variety of specific glycosyl residues. The hemicellulose xyloglucan (XyG) consists of a β-1,4-glucan backbone with a regular pattern of xylosyl branches, with additional galactosyl, fucosyl, arabinosyl, and/or galacturonosyl substitution depending on the tissue and plant species (Obel et al., 2009; Pauly et al., 2013; Schultink et al., 2014). XyG O-acetylation has been reported on the β-1,4-glucan backbone (Sims et al., 1996; York et al., 1996) as well as on specific galactosyl or arabinosyl side chains (Kiefer et al., 1989; Vierhuis et al., 2001). The hemicellulose xylan is heavily acetylated at positions O2 and O3 of the backbone β-1,4-xylosyl residues, with the degree of acetylation (O-acetyl groups per backbone of xylosyl residue) ranging from approximately 0.4 to 0.6 depending on the species (Teleman et al., 2002; Evtuguin et al., 2003; Prozil et al., 2012; Chong et al., 2014; Lee et al., 2014). The glycosyl substituents of xylan, including glucuronosyl, arabinosyl, and xylosyl groups, have not been reported to be acetylated. The backbone β-1,4-mannosyl residues of the hemicellulosic polysaccharide mannan also can be acetylated (Manna and McAnalley, 1993). The predominant location of O-acetyl groups in pectin has been reported to be on galacturonic acid residues at positions O2 and O3 (Ralet et al., 2005). O-Acetylation of pectin also has been observed on rhamnosyl (Sengkhamparn et al., 2009), fucosyl, and aceric acid residues (Glushka et al., 2003).The functional significance and biosynthetic pathway of wall polysaccharide O-acetylation are not fully understood. O-Acetylation has been shown to influence the solubility, gelation, and enzymatic accessibility of polysaccharides in vitro (Biely et al., 1986; Huang et al., 2002). These properties are likely to be important for appropriate function in planta. Recently identified Arabidopsis (Arabidopsis thaliana) mutants with polysaccharide O-acetylation deficiencies (reduced wall acetylation [rwa] and trichome birefringence-like [tbl]; Gille and Pauly, 2012) have allowed for testing of the in vivo role of this substituent. The ALTERED XYLOGLUCAN4 (AXY4 [TBL27]) gene from the TBL family was identified in a forward genetic screen of Arabidopsis and is believed to code for a XyG acetyltransferase (Gille et al., 2011). The growth morphology of this mutant, which lacks XyG O-acetylation in leaves, etiolated seedlings, and roots, was not affected under laboratory growth conditions. Arabidopsis mutants deficient for a putative xylan acetyltransferase (TBL29/ESKIMO1 [ESK1]) were reported to have reduced growth and irregular xylem and to be freezing tolerant (Xin et al., 2007; Xiong et al., 2013; Yuan et al., 2013). Arabidopsis mutants deficient for other TBL genes have been reported to exhibit phenotypes such as aberrant trichomes (Bischoff et al., 2010a) and resistance to powdery mildew (Vogel et al., 2004), but polysaccharide acetylation defects have not been demonstrated in these cases. The variation in the morphological phenotypes of different tbl mutants suggests that the function of polysaccharide acetylation is specific to the particular polysaccharide and tissue.While the TBL gene products seem to affect single wall polysaccharides, Arabidopsis mutants defective for one or more of the four RWA genes have decreased acetylation of multiple polysaccharides and growth phenotypes ranging from mild to severe (Lee et al., 2011; Manabe et al., 2011, 2013). For this reason, and because the RWA proteins are integral membrane proteins with 10 predicted transmembrane domains, it has been hypothesized that they may act as transporters for an activated form of acetate into the Golgi apparatus (Manabe et al., 2011). It has been demonstrated that acetyl-CoA is involved in the pathway of pectin acetylation (Pauly and Scheller, 2000); however, it is not clear if acetyl-CoA is transported into the Golgi or there is an alternative donor substrate that acts as a carrier.In this study, we report the identification and characterization of AXY9, an additional component of the plant cell wall polysaccharide acetylation pathway.