High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall

The heptapeptide Tyr-Gly- Arg-Gly-Asp- Ser-Pro containing the sequence Arg-Gly-Asp (RGD – the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (K_d1 1 nM, and K_d2 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.     

ERp57 is essential for efficient folding of glycoproteins sharing common structural domains

ERp57 is a member of the protein disulphide isomerase family of oxidoreductases, which are involved in native disulphide bond formation in the endoplasmic reticulum of mammalian cells. This enzyme has been shown to be associated with both calnexin and calreticulin and, therefore, has been proposed to be a glycoprotein-specific oxidoreductase. Here, we identify endogenous substrates for ERp57 by trapping mixed disulphide intermediates between enzyme and substrate. Our results demonstrate that the substrates for this enzyme are mostly heavily glycosylated, disulphide bonded proteins. In addition, we show that the substrate proteins share common structural domains, indicating that substrate specificity may involve specific structural features as well as the presence of an oligosaccharide side chain. We also show that the folding of two of the endogenous substrates for ERp57 is impaired in ERp57 knockout cells and that prevention of an interaction with calnexin or calreticulin perturbs the folding of some, but not all, substrates with multiple disulphide bonds. These results suggest a specific role for ERp57 in the isomerisation of non-native disulphide bonds in specific glycoprotein substrates.     

Organ-specific cellular requirements for in vivo dendritic cell generation

Bone marrow-derived dendritic cell (DC) precursors seed peripheral organs, where they encounter diverse cellular environments during their final differentiation into DCs. Flt3 ligand (Flt3-L) is critical for instructing DC generation throughout different organs. However, it remains unknown which cells produce Flt3-L and, importantly, which cellular source drives DC development in such a variety of organs. Using a novel BAC transgenic Flt3-L reporter mouse strain coexpressing enhanced GFP and luciferase, we show ubiquitous Flt3-L expression in organs and cell types. These results were further confirmed at the protein level. Although Flt3-L was produced by immune and nonimmune cells, the source required for development of the DC compartment clearly differed among organs. In lymphoid organs such as the spleen and bone marrow, Flt3-L production by hemopoietic cells was critical for generation of normal DC numbers. This was unexpected for the spleen because both immune and nonimmune cells equally contributed to the Flt3-L content in that organ. Thus, localized production rather than the total tissue content of Flt3-L in spleen dictated normal splenic DC development. No differences were observed in the number of DC precursors, suggesting that the immune source of Flt3-L promoted pre-cDC differentiation in spleen. In contrast, DC generation in the lung, kidney, and pancreas was mostly driven by nonhematopoietic cells producing Flt3-L, with little contribution by immune cells. These findings demonstrate a high degree of flexibility in Flt3-L-dependent DC generation to adapt this process to organ-specific cellular environments encountered by DC precursors during their final differentiation.     

Consequences of ERp57 deletion on oxidative folding of obligate and facultative clients of the calnexin cycle

Members of the protein-disulfide isomerase superfamily catalyze the formation of intra- and intermolecular disulfide bonds, a rate-limiting step of protein folding in the endoplasmic reticulum (ER). Here we compared maturation of one obligate and two facultative calnexin substrates in cells with and without ERp57, the calnexin-associated, glycoprotein-specific oxidoreductase. ERp57 deletion did not prevent the formation of disulfide bonds during co-translational translocation of nascent glycopolypeptides in the ER. It affected, however, the post-translational phases of oxidative influenza virus hemagglutinin (HA) folding, resulting in significant loss of folding efficiency for this obligate calnexin substrate. Without ERp57, HA also showed reduced capacity to recover from an artificially induced aberrant conformation, thus revealing a crucial role of ERp57 during post-translational reshuffling to the native set of HA disulfides. ERp57 deletion did not affect maturation of the model facultative calnexin substrates E1 and p62 (and of most cellular proteins, as shown by lack of induction of ER stress). ERp72 was identified as one of the ER-resident oxidoreductases associating with the orphan ERp57 substrates to maintain their folding competence.     

Evolving cell models for systems and synthetic biology

This paper proposes a new methodology for the automated design of cell models for systems and synthetic biology. Our modelling framework is based on P systems, a discrete, stochastic and modular formal modelling language. The automated design of biological models comprising the optimization of the model structure and its stochastic kinetic constants is performed using an evolutionary algorithm. The evolutionary algorithm evolves model structures by combining different modules taken from a predefined module library and then it fine-tunes the associated stochastic kinetic constants. We investigate four alternative objective functions for the fitness calculation within the evolutionary algorithm: (1) equally weighted sum method, (2) normalization method, (3) randomly weighted sum method, and (4) equally weighted product method. The effectiveness of the methodology is tested on four case studies of increasing complexity including negative and positive autoregulation as well as two gene networks implementing a pulse generator and a bandwidth detector. We provide a systematic analysis of the evolutionary algorithm’s results as well as of the resulting evolved cell models.     

Is There an Optimal Level of Open-Endedness in Prebiotic Evolution?

In this paper we explore the question of whether there is an optimal set up for a putative prebiotic system leading to open-ended evolution (OEE) of the events unfolding within this system. We do so by proposing two key innovations. First, we introduce a new index that measures OEE as a function of the likelihood of events unfolding within a universe given its initial conditions. Next, we apply this index to a variant of the graded autocatalysis replication domain (GARD) model, Segre et al. (P Natl Acad Sci USA 97(8):4112-4117, 2000; Markovitch and Lancet Artif Life 18(3), 2012), and use it to study - under a unified and concise prebiotic evolutionary framework - both a variety of initial conditions of the universe and the OEE of species that evolve from them.     

New approach to improving endothelial preservation in cryopreserved arterial substitutes

The endothelial loss provoked by the methods of vascular cryopreservation used at most human vessel banks is one of the main factors leading to the failure of grafting procedures performed using cryopreserved vessel substitutes. This study evaluates the effects of the storage temperature and thawing protocol on the endothelial cell loss suffered by cryopreserved vessels, and optimises the thawing temperature and protocol for cryopreserving arterial grafts in terms of that producing least endothelial loss. Segments of the common iliac artery of the minipig (n = 20) were frozen at a temperature reduction rate of 1 degrees C/min in a biological freezer. After storing the arterial fragments for 30 days, study groups were established according to the storage temperature (-80, -145 or -196 degrees C) and subsequent thawing procedure (slow or rapid thawing). Fresh vessel segments served as the control group. Once thawed, the specimens were examined by light, transmission, and scanning electron microscopy. The covered endothelial surface was determined by image analysis. Data for the different groups were compared by one way ANOVA. When cryopreservation at each of the storage temperatures was followed by slow thawing, the endothelial cells showed improved morphological features and viability over those of specimens subjected to rapid thawing. Rapidly thawed endothelial cells showed irreversible ultrastructural damage such as mitochondrial dilation and rupture, reticular fragmentation, and peripheral nuclear condensation. In contrast, slow thawing gave rise to changes compatible with reversible damage in a large proportion of the endothelial cells: general swelling, reticular dilation, mitochondrial swelling, and nuclear chromatin condensation. Gradually thawed cryopreserved arteries showed a lower proportion of damaged cells identified by the TUNEL method compared to the corresponding rapidly thawed specimens (p < 0.05, for all temperatures). In all the groups in which vessels underwent rapid thawing (except at -145 degrees C), significant differences (p < 0.05) in endothelial cover values were recorded with respect to control groups. Storage of cryopreserved vessels at -80 degrees C followed by rapid thawing led to greatest endothelial cell loss (61.36+/-9.06% covered endothelial surface), while a temperature of -145 degrees C followed by slow thawing was best at preserving the endothelium of the vessel wall (89.38+/-16.67% surface cover). In conclusion, storage at a temperature of -145 degrees C in nitrogen vapour followed by gradual automated thawing seems to be the best way of preserving the endothelial surface of the arterial cryograft. This method gives rise to best endothelial cell viability and cover values, with obvious benefits for subsequent grafting.