Mathematical models of neuronal growth

Biomechanics and Modeling in Mechanobiology - The establishment of a functioning neuronal network is a crucial step in neural development. During this process, neurons extend neurites—axons...     

Born among the ice: first morphological observations on two developmental stages of the Antarctic silverfish Pleuragramma antarcticum, a key species of the Southern Ocean

The Antarctic silverfish Pleuragramma antarcticum is a keystone species in the Southern Ocean ecosystem, providing one of the major links between lower and higher trophic levels. Despite the importance of this species, surprisingly little is known of its early development. The first spawning area for the silverfish has been recently identified in the near-shore of Terra Nova Bay (Ross Sea). Evidence indicates that spawning and embryo development occurs in the cryopelagic environment, below the seasonal pack-ice. In order to contribute to the knowledge of the life cycle of this very important Antarctic species, we carried out the first histological characterization on pre-hatching embryos and newly hatched larvae. Embryonated eggs and larvae of P. antarcticum were collected between late October and November 2005 at TNB through holes drilled into the sea ice. Embryonic stage just before hatching and the first post-hatching stage were the most abundant within our samples and thus were analysed using both macroscopic and histological approaches. Early life stages of the Antarctic silverfish revealed interesting features: the sensory system, foraging apparatus and heart appeared well developed, whereas the liver and gills were underdeveloped. Morphological details of the organogenesis were performed, providing the first substantial information on the development of P. antarcticum and representing a further steps towards the knowledge of the life cycle of this important Antarctic key species. An erratum to this article can be found at     

Immunocytochemical localization of serotonin in embryos, larvae and adults of the lancelet, Branchiostoma floridae

Serotonin (5-hydroxytryptamine) is a biogenic amine distributed throughout the metazoans and has an old evolutionary history. It is involved as a developmental signal in the early morphogenesis of both invertebrates and vertebrates, whereas in adults it acts mainly as a neurotransmitter and gastrointestinal hormone. In vertebrates, serotonin regulates the morphogenesis of the central nervous system and the specification of serotonergic as well as dopaminergic neurons. The present study uses, as an experimental model, an invertebrate chordate, the lancelet Branchiostoma floridae, characterized by its remarkable homologies with vertebrates that allows the 'bauplan' of the probable ancestor of vertebrates to be outlined. In particular, the involvement of serotonin as a developmental signal in embryos and larvae, as well as a neurotransmitter and gastrointestinal hormone in adult specimens of Branchiostoma floridae, gives further support to a common origin of cephalocordates and vertebrates.     

Granzyme B encoded by the commonly occurring human RAH allele retains pro-apoptotic activity

A key function of human granzyme B (GrB) is to induce apoptosis of target cells in conjunction with perforin. The RAH allele is the first documented variant of the human GrB gene, occurs at a frequency of 25-30%, and encodes three amino acid substitutions (Q48R, P88A, and Y245H). It was initially reported that RAH GrB is incapable of inducing apoptosis, but here we show that it has essentially identical proteolytic and cytotoxic properties to wild type GrB. Recombinant RAH and wild type GrB cleave peptide substrates with similar kinetics, are both capable of cleaving Bid and procaspase 3, and are equally inhibited by proteinase inhibitor 9, an endogenous regulator of GrB. Furthermore, cytotoxic lymphocytes from RAH heterozygotes and homozygotes have no defect in target cell killing, and in vitro RAH GrB and wild type GrB kill cells equally well in the presence of perforin. We conclude that the RAH allele represents a neutral polymorphism in the GrB gene.     

Fundamental aspects of arm repair phase in two echinoderm models

Regeneration is a post-embryonic developmental process that ensures complete morphological and functional restoration of lost body parts. The repair phase is a key step for the effectiveness of the subsequent regenerative process: in vertebrates, efficient re-epithelialisation, rapid inflammatory/immune response and post-injury tissue remodelling are fundamental aspects for the success of this phase, their impairment leading to an inhibition or total prevention of regeneration. Among deuterostomes, echinoderms display a unique combination of striking regenerative abilities and diversity of useful experimental models, although still largely unexplored.Therefore, the brittle star Amphiura filiformis and the starfish Echinaster sepositus were here used to comparatively investigate the main repair phase events after injury as well as the presence and expression of immune system and extracellular matrix (i.e. collagen) molecules using both microscopy and molecular tools.Our results showed that emergency reaction and re-epithelialisation are similar in both echinoderm models, being faster and more effective than in mammals. Moreover, in comparison to the latter, both echinoderms showed delayed and less abundant collagen deposition at the wound site (absence of fibrosis). The gene expression patterns of molecules related to the immune response, such as Ese-fib-like (starfishes) and Afi-ficolin (brittle stars), were described for the first time during echinoderm regeneration providing promising starting points to investigate the immune system role in these regeneration models.Overall, the similarities in repair events and timing within the echinoderms and the differences with what has been reported in mammals suggest that effective repair processes in echinoderms play an important role for their subsequent ability to regenerate. Targeted molecular and functional analyses will shed light on the evolution of these abilities in the deuterostomian lineage.     

Cationic sites on granzyme B contribute to cytotoxicity by promoting its uptake into target cells

Granzyme B (GrB) is a key effector of cytotoxic lymphocyte-mediated cell death. It is delivered to target cells bound to the proteoglycan serglycin, but how it crosses the plasma membrane and accesses substrates in the cytoplasm is poorly understood. Here we identify two cationic sequences on GrB that facilitate its binding and uptake. Mutation of cationic sequence 1 (cs1) prevents accumulation of GrB in a distinctive intracellular compartment and reduces cytotoxicity 20-fold. Mutation of cs2 reduces accumulation in this intracellular compartment and cytotoxicity two- to threefold. We also show that GrB-mediated cytotoxicity is abrogated by heparin and that target cells deficient in cell surface sulfate or glycosaminoglycans resist GrB. However, heparin does not completely prevent GrB internalization and chondroitin 4-sulfate does not inhibit cytotoxicity, suggesting that glycosaminoglycans are not essential GrB receptors. We propose that GrB enters cells by nonselective adsorptive pinocytosis, exchanging from chondroitin sulfate on serglycin to anionic components of the cell surface. In this electrostatic "exchange-adsorption" model, cs1 and cs2 participate in binding of GrB to the cell surface, thereby promoting its uptake and eventual release into the cytoplasm.     

Analysis of Lymphocytic DNA Damage in Early Multiple Sclerosis by Automated Gamma-H2AX and 53BP1 Foci Detection: A Case Control Study

Background

In response to DNA double-strand breaks, the histone protein H2AX becomes phosphorylated at its C-terminal serine 139 residue, referred to as γ-H2AX. Formation of γ-H2AX foci is associated with recruitment of p53-binding protein 1 (53BP1), a regulator of the cellular response to DNA double-strand breaks. γ-H2AX expression in peripheral blood mononuclear cells (PBMCs) was recently proposed as a diagnostic and disease activity marker for multiple sclerosis (MS).

Objective

To evaluate the significance of γ-H2AX and 53BP1 foci in PBMCs as diagnostic and disease activity markers in patients with clinically isolated syndrome (CIS) and early relapsing-remitting MS (RRMS) using automated γ-H2AX and 53BP1 foci detection.

Methods

Immunocytochemistry was performed on freshly isolated PBMCs of patients with CIS/early RRMS (n = 25) and healthy controls (n = 27) with γ-H2AX and 53BP1 specific antibodies. Nuclear γ-H2AX and 53BP1 foci were determined using a fully automated reading system, assessing the numbers of γ-H2AX and 53BP1 foci per total number of cells and the percentage of cells with foci. Patients underwent contrast enhanced 3 Tesla magnetic resonance imaging (MRI) and clinical examination including expanded disability status scale (EDSS) score. γ-H2AX and 53BP1 were also compared in previously frozen PBMCs of each 10 CIS/early RRMS patients with and without contrast enhancing lesions (CEL) and 10 healthy controls.

Results

The median (range) number of γ-H2AX (0.04 [0–0.5]) and 53BP1 (0.005 [0–0.2]) foci per cell in freshly isolated PBMCs across all study participants was low and similar to previously reported values of healthy individuals. For both, γ-H2AX and 53BP1, the cellular focus number as well as the percentage of positive cells did not differ between patients with CIS/RRMS and healthy controls. γ-H2AX and 53BP1 levels neither correlated with number nor volume of T2-weighted lesions on MRI, nor with the EDSS. Although γ-H2AX, but not 53BP1, levels were higher in previously frozen PBMCs of patients with than without CEL, γ-H2AX values of both groups overlapped and γ-H2AX did not correlate with the number or volume of CEL.

Conclusion

γ-H2AX and 53BP1 foci do not seem to be promising diagnostic or disease activity biomarkers in patients with early MS. Lymphocytic DNA double-strand breaks are unlikely to play a major role in the pathophysiology of MS.     

Prostaglandin E2 EP3 receptor regulates cyclooxygenase-2 expression in the kidney

Cyclooxygenase-2 (COX-2) is constitutively expressed and highly regulated in the thick ascending limb (TAL). As COX-2 inhibitors (Coxibs) increase COX-2 expression, we tested the hypothesis that a negative feedback mechanism involving PGE(2) EP3 receptors regulates COX-2 expression in the TAL. Sprague-Dawley rats were treated with a Coxib [celecoxib (20 mg·kg(-1)·day(-1)) or rofecoxib (10 mg·kg(-1)·day(-1))], with or without sulprostone (20 μg·kg(-1)·day(-1)). Sulprostone was given using two protocols, namely, previous to Coxib treatment (prevention effect; Sulp7-Coxib5 group) and 5 days after initiation of Coxib treatment (regression effect; Coxib10-Sulp5 group). Immunohistochemical and morphometric analysis revealed that the stained area for COX-2-positive TAL cells (μm(2)/field) increased in Coxib-treated rats (Sham: 412 ± 56.3, Coxib: 794 ± 153.3). The Coxib effect was inhibited when sulprostone was used in either the prevention (285 ± 56.9) or regression (345 ± 51.1) protocols. Western blot analysis revealed a 2.1 ± 0.3-fold increase in COX-2 protein expression in the Coxib-treated group, an effect abolished by sulprostone using either the prevention (1.2 ± 0.3-fold) or regression (0.6 ± 0.4-fold vs. control, P < 0.05) protocols. Similarly, the 6.4 ± 0.6-fold increase in COX-2 mRNA abundance induced by Coxibs (P < 0.05) was inhibited by sulprostone; prevention: 0.9 ± 0.3-fold (P < 0.05) and regression: 0.6 ± 0.1 (P < 0.05). Administration of a selective EP3 receptor antagonist, L-798106, also increased the area for COX-2-stained cells, COX-2 mRNA accumulation, and protein expression in the TAL. Collectively, the data suggest that COX-2 levels are regulated by a novel negative feedback loop mediated by PGE(2) acting on its EP3 receptor in the TAL.