Immunochemical properties of elongation factors 1 of plant origin

Elongation factors 1 (EF-1) have been isolated from different plants: wheat, yellow lupine, blue lupine, Chinese cabbage and Norway maple. Antibodies for EF-1 from yellow lupine have been obtained in rabbits; antibodies for wheat EF-1 were elicited in mice. The immunological properties of EF-1 were assayed by the following methods: western blotting, double immunodiffusion and rocket immunoelectrophoresis. Our results suggest that one antigenic site is similar for all plant elongation binding factors tested. This epitope probably overlaps the centre of biological activity of EF-1, as was shown for wheat EF-1. The hypothesis concerning the potential presence of plant EF-1 as a subunit of turnip yellow mosaic virus RNA replicase (similar to prokaryotic EF-Tu in the Q beta RNA replicase system) has also been tested using immunotechniques as well as tests of biological activity, but has not been confirmed.     

Isotopic evidence for the occurrence of biological nitrification and nitrogen deposition processing in forest canopies

This study examines the role of tree canopies in processing atmospheric nitrogen (N_dep) for four forests in the United Kingdom subjected to different N_dep: Scots pine and beech stands under high N_dep (HN, 13–19 kg N ha^?1 yr^?1), compared to Scots pine and beech stands under low N_dep (LN, 9 kg N ha^?1 yr^?1). Changes of NO₃‐N and NH₄‐N concentrations in rainfall (RF) and throughfall (TF) together with a quadruple isotope approach, which combines δ¹⁸O, Δ¹⁷O and δ¹⁵N in NO₃^? and δ¹⁵N in NH₄⁺, were used to assess N transformations by the canopies. Generally, HN sites showed higher NH₄‐N and NO₃‐N concentrations in RF compared to the LN sites. Similar values of δ¹⁵N‐NO₃^? and δ¹⁸O in RF suggested similar source of atmospheric NO₃^? (i.e. local traffic), while more positive values for δ¹⁵N‐NH₄⁺ at HN compared to LN likely reflected the contribution of dry NH_x deposition from intensive local farming. The isotopic signatures of the N‐forms changed after interacting with tree canopies. Indeed, ¹⁵N‐enriched NH₄⁺ in TF compared to RF at all sites suggested that canopies played an important role in buffering dry N_dep also at the low N_dep site. Using two independent methods, based on δ¹⁸O and Δ¹⁷O, we quantified for the first time the proportion of NO₃^? in TF, which derived from nitrification occurring in tree canopies at the HN site. Specifically, for Scots pine, all the considered isotope approaches detected biological nitrification. By contrast for the beech, only using the mixing model with Δ¹⁷O, we were able to depict the occurrence of nitrification within canopies. Our study suggests that tree canopies play an active role in the N cycling within forest ecosystems. Processing of N_dep within canopies should not be neglected and needs further exploration, with the combination of multiple isotope tracers, with particular reference to Δ¹⁷O.     

Site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of Hendra virus

Hendra virus (HeV) continues to cause morbidity and mortality in both humans and horses with a number of sporadic outbreaks. HeV has two structural membrane glycoproteins that mediate the infection of host cells: the attachment (G) and the fusion (F) glycoproteins that are essential for receptor binding and virion-host cell membrane fusion, respectively. N-linked glycosylation of viral envelope proteins are critical post-translation modifications that have been implicated in roles of structural integrity, virus replication and evasion of the host immune response. Deciphering the glycan composition and structure on these glycoproteins may assist in the development of glycan-targeted therapeutic intervention strategies. We examined the site occupancy and glycan composition of recombinant soluble G (sG) glycoproteins expressed in two different mammalian cell systems, transient human embryonic kidney 293 (HEK293) cells and vaccinia virus (VV)-HeLa cells, using a suite of biochemical and biophysical tools: electrophoresis, lectin binding and tandem mass spectrometry. The N-linked glycans of both VV and HEK293-derived sG glycoproteins carried predominantly mono- and disialylated complex-type N-glycans and a smaller population of high mannose-type glycans. All seven consensus sequences for N-linked glycosylation were definitively found to be occupied in the VV-derived protein, whereas only four sites were found and characterized in the HEK293-derived protein. We also report, for the first time, the existence of O-linked glycosylation sites in both proteins. The striking characteristic of both proteins was glycan heterogeneity in both N- and O-linked sites. The structural features of G protein glycosylation were also determined by X-ray crystallography and interactions with the ephrin-B2 receptor are discussed.     

Mass spectrometric approach for screening modifications of total serum N-glycome in human diseases: application to cirrhosis

Congenital and acquired modifications of glycosylation in diseases are a rapidly growing field that demonstrates the importance of glycosylation in human biology. Unfortunately, in clinical biochemistry, very few tests are available to explore oligosaccharide metabolism on a large scale. Such an assay needs to be of high throughput, rapid, and preferentially noninvasive. In the present study, we describe a method to analyze qualitative variations of N-glycosylation of human serum proteins. The method is based on direct release of N-linked oligosaccharides from patient serum samples, a single-step purification, and a matrix-assisted laser desorption ionization time of flight mass spectrometric analysis. A complementary structural study of the released oligosaccharides was achieved by enzymatic digestions, linkage analysis, and electrospray ionization ion trap mass spectrometry (ESI-IT-MS) of the permethylated N-glycome. A total of 26 oligosaccharide structures were individualized, their presence in human serum being the result of the combination of the biosynthesis and catabolic pathways. Application of the protocol to the serum of patients with cirrhosis demonstrates the ability of this assay to identify acquired modifications of glycosylation. Furthermore, we have analyzed the N-glycans and showed the increase in bisecting N-acetylglucosamine residue, core fucosylation, and the presence of an important population of neutral oligosaccharides. The study of total serum N-glycome modifications is a preliminary for the discovery of new noninvasive diagnostic or prognostic biomarkers resulting from the variations of the N-glycan metabolism during diseases.     

Reduced expression of the membrane skeleton protein beta1-spectrin (SPTBN1) is associated with worsened prognosis in pancreatic cancer

Spectrins are members of the superfamily of F-actin cross linking proteins that are important as scaffolding proteins for protein sorting, cell adhesion, and migration. In addition, spectrins have been implicated in TGF-beta signaling. The aim of the present study was to analyze the expression and localization of beta1-spectrin (SPTBN1) in pancreatic tissues. mRNA levels of SPTBN1 in cultured pancreatic cancer cell lines, as well as in normal pancreatic tissues (n=18), chronic pancreatitis (n=48) and pancreatic cancer tissues (n=66) were analyzed by real time quantitative RT-PCR. Localization of SPTBN1 in pancreatic tissues was determined by immunohistochemistry. SPTBN1 staining was assessed semi-quantitatively in 55 cancer tissues and survival analysis was carried out using the Kaplan-Meier method. Median SPTBN1 mRNA levels were 6.0-fold higher in pancreatic cancer tissues compared to the normal pancreas (p<0.0001) and 2.2-fold higher compared to chronic pancreatitis tissues (p=0.0002). In the normal pancreas, SPTBN1 was present in the cytoplasm of normal ductal cells and occasionally in pancreatic acinar and centroacinar cells. In pancreatic cancer tissues, SPTBN1 was present in the cytoplasm of pancreatic cancer cells. Low SPTBN1 protein expression indicated a tendency for worsened prognosis with a median survival of 14.0 months, versus 23.8 months for patients whose tumors expressed moderate/high levels of SPTBN1. In conclusion, reduced SPTBN1 expression correlated with shorter survival of pancreatic cancer patients, suggesting a tumor suppressor function of this gene, as has already been shown for other malignancies of the gastrointestinal tract.     

70-kDa-heat shock protein presents an adjustable lectinic activity towards O-linked N-acetylglucosamine

Numerous works demonstrated that the dynamic O-GlcNAc glycosylation could protect against the proteasomal degradation by modifying the target proteins and the proteasome itself. Considering that Hsp70 is a crucial component in the quality control of protein conformation in the proteasomal pathway, we investigated the possibility that Hsp70 physically interacts with O-GlcNAc proteins through a lectinic activity. First, we demonstrate that in HepG2 cells, Hsp70 can specifically bind to O-GlcNAc residues but also is itself modified by O-GlcNAc. Second, when cells were deprived of glucose (nutrient stress), Hsp70 lectinic activity markedly increased whereas its glycosylation dramatically decreased. On the other hand, a 42 degrees C thermic stress did not affect any of these features. Lastly, the nature of O-GlcNAc modified proteins co-immunoprecipitating with Hsp70 was similar for cells submitted to the thermic and to nutrient stress. These results strongly suggest that O-GlcNAc influences protein stability through specific interaction with 70-kDa-heat shock protein members.     

Haplotypes in the dystrophin DNA segment point to a mosaic origin of modern human diversity

Although Africa has played a central role in human evolutionary history, certain studies have suggested that not all contemporary human genetic diversity is of recent African origin. We investigated 35 simple polymorphic sites and one T(n) microsatellite in an 8-kb segment of the dystrophin gene. We found 86 haplotypes in 1,343 chromosomes from around the world. Although a classical out-of-Africa topology was observed in trees based on the variant frequencies, the tree of haplotype sequences reveals three lineages accounting for present-day diversity. The proportion of new recombinants and the diversity of the T(n) microsatellite were used to estimate the age of haplotype lineages and the time of colonization events. The lineage that underwent the great expansion originated in Africa prior to the Upper Paleolithic (27,000-56,000 years ago). A second group, of structurally distinct haplotypes that occupy a central position on the tree, has never left Africa. The third lineage is represented by the haplotype that lies closest to the root, is virtually absent in Africa, and appears older than the recent out-of-Africa expansion. We propose that this lineage could have left Africa before the expansion (as early as 160,000 years ago) and admixed, outside of Africa, with the expanding lineage. Contemporary human diversity, although dominated by the recently expanded African lineage, thus represents a mosaic of different contributions.