Aminopeptidase N during the ontogeny of the chick

Little is known about the production and function of metallopeptidases in embryonic development. One such enzyme, aminopeptidase N (APN), is present in several epithelia, the brain and angiogenic vessels in adults. APN promotes vascular growth and endothelial cell proliferation in physiological and pathological models of angiogenesis. However, its possible role in embryonic angiogenesis or other developmental processes is unknown. Its expression profile in the early phase of embryonic development has not been reported. We report here the expression of this enzyme during the early development of the chick embryo, using complementary techniques for monitoring APN mRNA, protein, and enzymatic activity. We detected APN in the embryo as early as gastrulation. In addition to the known sites of APN production identified in both adults and rat fetuses toward the end of gestation, APN was found in unexpected sites, such as the primitive streak, the dorsal folds of the neural tube, the somites, and the primordia of several organs. APN was present mostly in the cardiovascular compartment during the first 13 days of incubation, and in the hematopoietic compartment (yolk sac and aorta-gonad-mesonephros region) early in development. This study provides clues as to the possible role of APN in embryonic development.     

Preparative laser capture microdissection and single-pot cell wall material preparation: a novel method for tissue-specific analysis

In adaptation to their function the walls of plant cell display tissue-specific variations of composition according to their developmental stage, cell type and stress of various origin. It is therefore important to obtain a precise analytical data describing the cell wall composition with respect to these different factors. In the present work, laser capture microdissection (LCM) was used for isolating different tissues from the stem of Urtica dioica L. at a semi-preparative scale. The technique was associated for the first time to a one-pot sequential cell wall preparation and hydrolysis for the carbohydrate analysis of each cell type. The results demonstrate that the combination of LCM and micro-analytical methods can provide individual cell type composition and should improve our knowledge of the biochemical diversity of cell walls in plants. This approach will be of potential interest for the understanding of the effects of stress or genetic engineering on the composition of the cell walls.     

The effect of hydrodynamic shear on 3D engineered chondrocyte systems subject to direct perfusion

Bioreactors allowing direct-perfusion of culture medium through tissue-engineered constructs may overcome diffusion limitations associated with static culturing, and may provide flow-mediated mechanical stimuli. The hydrodynamic stress imposed on cells within scaffolds is directly dependent on scaffold microstructure and on bioreactor configuration. Aim of this study is to investigate optimal shear stress ranges and to quantitatively predict the levels of hydrodynamic shear imposed to cells during the experiments. Bovine articular chondrocytes were seeded on polyestherurethane foams and cultured for 2 weeks in a direct perfusion bioreactor designed to impose 4 different values of shear level at a single flow rate (0.5 ml/min). Computational fluid dynamics (CFD) simulations were carried out on reconstructions of the scaffold obtained from micro-computed tomography images. Biochemistry analyses for DNA and sGAG were performed, along with electron microscopy. The hydrodynamic shear induced on cells within constructs, as estimated by CFD simulations, ranged from 4.6 to 56 mPa. This 12-fold increase in the level of applied shear stress determined a 1.7-fold increase in the mean content in DNA and a 2.9-fold increase in the mean content in sGAG. In contrast, the mean sGAG/DNA ratio showed a tendency to decrease for increasing shear levels. Our results suggest that the optimal condition to favour sGAG synthesis in engineered constructs, at least at the beginning of culture, is direct perfusion at the lowest level of hydrodynamic shear. In conclusion, the presented results represent a first attempt to quantitatively correlate the imposed hydrodynamic shear level and the invoked biosynthetic response in 3D engineered chondrocyte systems.     

Homeostasis of natural killer cells

Natural killer (NK) cells are important players of innate immunity, dedicated to the host defense against viruses and also involved in the immune surveillance of tumors. NK cells are widely distributed in the body and their number may increase locally during infection. They develop mainly in the bone marrow and perhaps in other lymphoid organs. They are constantly renewed, with a half-life of about 17 days at the periphery. In this article, we review the factors that regulate the homeostasis of NK cells including their development, differentiation, export to the periphery, their turnover, their homeostatic or antigen-induced proliferation and their survival before or after activation. In addition, we discuss the homeostasis of recently described so-called "memory" NK cells.     

Specificity and Structure of a High Affinity Activin Receptor-like Kinase 1 (ALK1) Signaling Complex

Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-β superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-β family ligands BMP9 and BMP10 as well as the three type II TGF-β family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies.     

Aqueous dispersions of DMPG in low salt contain leaky vesicles

Aqueous dispersions of dimyristoyl phosphatidylglycerol (DMPG), at low ionic strength, display uncommon thermal behavior. Models for such behavior need to assign a form to the lipid aggregate. Although most studies accept the presence of lipid vesicles in the lipid gel and fluid phases, this is still controversial. With electron spin resonance (ESR) spectra of spin labels incorporated into DMPG aggregates, quantification of [(14)C]sucrose entrapped by the aggregates, and viscosity measurements, we demonstrate the existence of leaky vesicles in dispersions of DMPG at low ionic strength, in both gel and fluid phases of the lipid. As a control system, the ubiquitous lipid dimyristoyl phosphatidylcholine (DMPC) was used. For DMPG in the gel phase, spin labeling only indicated the presence of lipid bilayers, strongly suggesting that DMPG molecules are organized as vesicles and not micelles or bilayer fragments (bicelles), as the latter has a non-bilayer structure at the edges. Quantification of [(14)C]sucrose entrapping by DMPG aggregates revealed the presence of highly leaky vesicles. Due to the short hydrocarbon chains ((14)C atoms), DMPC vesicles were also found to be partially permeable to sucrose, but not as much as DMPG vesicles. Viscosity measurements, with the calculation of the intrinsic viscosity of the lipid aggregate, showed that DMPG vesicles are rather similar in the gel and fluid phases, and quite different from aggregates observed along the gel-fluid transition. Taken together, our data strongly supports that DMPG forms leaky vesicles at both gel and fluid phases.