Characteristic glycopeptides associated with extreme human longevity identified through plasma glycoproteomics

Background

Glycosylation is highly susceptible to changes of the physiological conditions, and accordingly, is a potential biomarker associated with several diseases and/or longevity. Semi-supercentenarians (SSCs; older than 105?years) are thought to be a model of human longevity. Thus, we performed glycoproteomics using plasma samples of SSCs, and identified proteins and conjugated N-glycans that are characteristic of extreme human longevity.

Diverse Legionella‐Like Bacteria Associated with Testate Amoebae of the Genus Arcella (Arcellinida: Amoebozoa)

Diverse species of Legionella and Legionella‐like amoebal pathogens (LLAPs) have been identified as intracellular bacteria in many amoeboid protists. There are, however, other amoeboid groups such as testate amoeba for which we know little about their potential to host such bacteria. In this study, we assessed the occurrence and diversity of Legionella spp. in cultures and environmental isolates of freshwater arcellinid testate amoebae species, Arcella hemispherica, Arcella intermedia, and Arcella vulgaris, via 16S rRNA gene sequence analyses and fluorescent in situ hybridization (FISH). Analysis of the 16S rRNA gene sequences indicated that A. hemispherica, A. intermedia, and A. vulgaris host Legionella‐like bacteria with 94–98% identity to other Legionella spp. based on NCBI BLAST search. Phylogenetic analysis placed Legionella‐like Arcella‐associated bacteria (LLAB) in three different clusters within a tree containing all other members of Legionella and LLAPs. The intracellular localization of the Legionella within Arcella hosts was confirmed using FISH with a Legionella‐specific probe. This study demonstrates that the host range of Legionella and Legionella‐like bacteria in the Amoebozoa extends beyond members of “naked” amoebae species, with members of the testate amoebae potentially serving an ecological role in the dispersal, protection, and replication of Legionella spp. in natural environments.     

As thick as three in a bed

During the evolution of the lichen symbiosis, shifts from one main type of photobiont to another were infrequent (Miadlikowska et al. 2006 ) but some remarkable transitions from green algal to diazotrophic cyanobacterial photobionts are known from unrelated fungal clades within the ascomycetes. Cyanobacterial, including tripartite, associations (green algal and cyanobacterial photobionts in one lichen individual) facilitate these holobionts to live as C‐ and N‐autotrophs. Tripartite lichens are among the most productive lichens, which provide N‐fertilization to forest ecosystems under oceanic climates (Peltigerales) or deliver low, but ecologically significant N‐input into subarctic and alpine soil communities (Lecanorales, Agyriales). In this issue of Molecular Ecology, Schneider et al. (2016) mapped morphometric data against an eight‐locus fungal phylogeny across a transition of photobiont interactions from green algal to a tripartite association and used a phylogenetic comparative framework to explore the role of nitrogen‐fixing cyanobacteria in size differences in the Trapelia–Placopsis clade (Agyriales). Within the group of tripartite species, the volume of cyanobacteria‐containing structures (cephalodia) correlates with thallus thickness in both phylogenetic generalized least squares and phylogenetic generalized linear mixed‐effects analyses, and the fruiting body core volume increased ninefold. The authors conclude that cyanobacterial symbiosis appears to have enabled lichens to overcome size constraints in oligotrophic environments such as rock surfaces. The Trapelia–Placopsis clade analyzed by Schneider et al. (2016) is an exciting example of interactions between ecology, phylogeny and lichen biology including development – from thin crustose green algal microlichens to thick placodioid, tripartite macrolichens: as thick as three in a bed (Scott 1820 ).     

The impact of β‐azido(or 1‐piperidinyl)methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues

The synthesis of new dermorphin analogues is described. The (R)‐alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α‐methyl‐β‐azidoalanine or α‐benzyl‐β‐azido(1‐piperidinyl)alanine residues. The potency and selectivity of the new analogues were evaluated by a competitive receptor binding assay in rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). The most active analogue in this series, Tyr‐(R)‐Ala‐(R)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ and its epimer were analysed by ¹H and ¹³C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around C^α in the α‐benzyl‐β‐azidoAla residue in position 3. The (R) configuration led to the formation of a type I β‐turn, whilst switching to the (S) configuration gave rise to an inverse β‐turn of type I′, followed by the formation of a very short β‐sheet. The selectivity of Tyr‐(R)‐Ala‐(R) and (S)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

蛇毒中γ-谷氨酰转肽酶

γ-谷氨酰转肽酶 (简称γ-GT)广泛存在各种哺乳动物组织中,在氨基酸转运中起重要作用。我们对分属于眼镜蛇科、蝰科和海蛇科的九种毒蛇的蛇毒进行研究,发现蛇毒中存在γ-GT,但各种蛇毒的总酶活力差异很大,以眼镜王蛇和眼镜蛇毒含量较丰富。聚丙烯酰胺凝胶电泳和凝胶等电聚焦电泳分析指出,大多数蛇毒含多种形式的γ-GT,存在分子量与等电点不同的酶区带。     

A characteristic electrophoretic pattern of cytosolic polypeptides from hepatocyte nodules generated during liver carcinogenesis in several models

The cytosolic polypeptides of hepatocyte nodules in six models of liver carcinogenesis were analysed by SDS-polyacrylamide gel electrophoresis and their patterns compared with these of control and variously treated livers. The amount of a polypeptide of Mr 21,000 was about tenfold elevated in the cytosol of five of the six types of nodules and moderately elevated in the sixth. Certain other polypeptides, particularly one of Mr 26,000, also varied in amount, so that all of the nodules analysed could be distinguished from liver by their electrophoretic patterns. Some possible identities of the two polypeptides are discussed. Their study may have mechanistic as well as diagnostic importance.     

Glutathione and glutathione conjugate efflux from cultured liver cells

Efflux of glutathione (GSH) and GSH-conjugates from cultured rat liver epithelial cell lines; the non-tumorigenic ARL-15C₁ and the -glutamyl transpeptidase containing, tumorigenic ARL-16T₂, has been assessed under basal condition and during chronic treatment with 75 and 150 M ethacrynic acid (EA). The intracellular level of GSH increased in proportion to EA concentration during chronic exposure. The rates of GSH and GSH-EA conjugate efflux increased with intracellular GSH in both ARL cell lines.Glutathione-S-transferase activity measured with EA as substrate increased over the experimental time course after treatment with 150, but not 75 M EA. When intracellular GSH content was increased by treatment with the cysteine pro-drug, 2-L-oxothiazolidine 4-carboxylic acid, the rate of GSH efflux was increased, but not the rate of GS-EA conjugate export. Inhibition of -glutamyl transpeptidase by acivicin (AT-125) increased the GSH and GS-EA conjugate efflux rate in ARL-16T₂ cells by factors of approximately 2 and 15, respectively. Acivicin treatment of ARL-16T₂ cells chronically treated with EA elevated GSH efflux rate by 10-fold and GS-EA efflux by 40-fold versus control samples. These studies show that GSH and GSH conjugate efflux are accomplished as independently regulated processes. Efflux of GSH is enhanced by increased in racellular GSH, but increase in the conjugate transport rate requires the presence of the GSH conjugate. The response of the efflux process to treatment with a chronic GSH depleting agent was identical in two cell lines in which the metabolic fate of glutathione is known to differ fundamentally.Abbreviations GSH reduced glutathione - GSSG oxidized glutathione - GS-EA the glutathione conjugate of ethacrynic acid - EA ethacrynic acid - CDNB 1-chloro 2,4-dinitrobenzene - HBS HEPES buffered saline - OTC 2-L-oxothiazolidine 4-carboxylic acid - CYSSG cysteinyl-glutathione mixed disulfide - FDNB 1-fluoro-2,4-dinitrobenzene - GCS -glutamyl cysteine synthetase - GST glutathione-S-transferase - BCA bicinchoninic acid - SDS sodium dodecyl sulfate - PCA perchloric acid     

Induction by butylated hydroxytoluene of rat liver gamma-glutamyl transpeptidase activity in comparison to expression in carcinogen-induced altered lesions

Butylated hydroxytoluene (BHT) at concentrations of 300-6000 ppm in the diet caused a dose-dependent increase in gamma-glutamyl transpeptidase (GGT) activity in normal F344 male rat liver at 18 weeks. However, the activities of glutathione S-transferases (GSTs) of rat liver cytosol were enhanced only at concentrations of 3000 or 6000 ppm BHT. Histochemically, the enhanced GGT activity was localized to hepatocytes surrounding the portal areas. Autoradiographic measurements of DNA synthesis showed that dietary BHT did not increase the level of cell proliferation and the GGT-positive hepatocytes did not exhibit different rates of DNA synthesis from those of GGT-negative cells. Feeding of the liver carcinogen N-2-fluorenylacetamide (FAA) induced foci and nodules of GGT-positive altered cells which exhibited higher rates of DNA synthesis than those of surrounding GGT-negative hepatocytes. Following iron loading, the periportal GGT-positive hepatocytes produced by BHT accumulated cellular iron, whereas the cells in FAA-induced lesions excluded iron. These results suggest that dietary BHT induces GGT activity in periportal hepatocytes without proliferation of the cells and that induction does not represent fetal expression or a preneoplastic alteration.     

The symmetry of competitive interactions in mixed Norway spruce,silver fir and European beech forests

Questions

We aim for a better understanding of the different modes of intra‐ and inter‐specific competition in two‐ and three‐species mixed‐forests. How can the effect of different modes of competitive interactions be detected and integrated into individual tree growth models? Are species interactions in spruce–fir–beech forests more associated with size‐symmetric or size‐asymmetric competition? Do competitive interactions between two of these species change from two‐ to three‐species mixtures?

Location

Temperate mixed‐species forests in Central Europe (Switzerland).

Methods

We used data from the Swiss National Forest Inventory to fit basal area increment models at the individual tree level, including the effect of ecological site conditions and indices of size‐symmetric and size‐asymmetric competition. Interaction terms between species‐specific competition indices were used to disentangle significant differences in species interactions from two‐ to three‐species mixtures.

Results

The growth of spruce and fir was positively affected by increasing proportions of the other species in spruce–fir mixtures, but negative effects were detected with increasing presence of beech. We found that competitive interactions for spruce and fir were more related to size‐symmetric competition, indicating that species interactions might be more associated with competition for below‐ground resources. Under constant amounts of stand basal area, the growth of beech clearly benefited from the increasing admixture of spruce and fir. For this species, patterns of size‐symmetric and size‐asymmetric competitive interactions were similar, indicating that beech is a strong self‐competitor for both above‐ground and below‐ground resources. Only for silver fir and beech, we found significant changes in species interactions from two‐ to three‐species mixtures, but these were not as prominent as the effects due to differences between intra‐ and inter‐specific competition.

Conclusions

Species interactions in spruce–fir–beech, or other mixed forests, can be characterized depending on the mode of competition, allowing interpretations of whether they occur mainly above or below ground level. Our outcomes illustrate that species‐specific competition indices can be integrated in individual tree growth functions to express the different modes of competition between species, and highlight the importance of considering the symmetry of competition alongside competitive interactions in models aimed at depicting growth in mixed‐species forests.

     

Characterization of γ-glutamyl transpeptidase activity of cultured endothelial cells from porcine brain capillaries

Summary Endothelial cells were isolated with high viability (>93%) from porcine brain capillaries by Percoll gradient centrifugation after purely enzymatic digestion. Primary cultures were grown to confluent cell monolayers and quantitated for the activity of -glutamyl transpeptidase. The -glutamyl transpeptidase activity starts from a high enzymatic level, decreases with time in culture to about 15% of the initial value, and remains constant at this level after day 10 in culture. The activity progression depends on surface conditions. In the presence of collagen, an exponential decrease starts immediately after seeding, with a time constant of 70±10h. In the absence of collagen, -glutamyl transpeptidase activity first decreases on day 1 after plating, recovers to the initial value on day 2 and 3 and afterwards declines exponentially to a low and constant activity level. Ethanol added to the cell culture at a time when low constant activity is reached, reactivates the -glutamyl transpeptidase to 30% of the initial value.