Regulation of peptidergic vesicle mobility by secretagogues

Neuropeptides are released into the extracellular space from large secretory granules. In order to reach their release sites, these granules are translocated on microtubules and thought to interact with filamentous actin as they approach the cell membrane. We have used a green fluorescent protein-tagged neuropeptide prohormone (prepro-orphanin FQ) to visualize vesicle trafficking dynamics in NS20Y cells and cultures of primary hippocampal neurons. We found that the majority of secretory granules were mobile and accumulated at both the tips of neurites as well as other apparently specialized cellular sites. We also used live-cell imaging to test the notion that peptidergic vesicle mobility was regulated by secretagogues. We show that treatment with forskolin appeared to increase vesicle rates of speed, while depolarization with high K⁺ had no effect, even though both treatments stimulated neuropeptide secretion. In cultured hippocampal neurons the green fluorescent protein-tagged secretory vesicles were routed to both dendrites and axons, indicating that peptidergic vesicle transport was not polarized. Basal peptidergic vesicle mobility rates in hippocampal neurons were the same as those in NS20Y cells. Taken together, these studies suggest that secretory vesicle mobility is regulated by specific classes of secretagogues and that neuropeptide containing secretory vesicles may be released from dendritic structures.     

Increase in ceramide level alters the lysosomal targeting of cathepsin D prior to onset of apoptosis in HT-29 colon cancer cells

Ceramide has been suggested as an important mediator of apoptosis. In HT-29 colorectal cancer cells increased ceramide levels, induced by exogenous N-acetylsphingosine (NAS, also known as C2-ceramide) or by 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol (PDMP), inhibited the transport and processing of cathepsin D (CD), a lysosomal protease implicated in apoptosis of tumour cells. C2-dihydroceramide (DH-C2), an inactive analogue of NAS, had no effect on CD transport and maturation. The treatment with either NAS or PDMP was revealed to be cytotoxic for HT-29 cells and led to cell death with classical features of apoptosis. Morphological signs of apoptosis and DNA fragmentation became apparent only between 24 and 48 h of incubation and poly(ADP ribose)-polymerase cleavage, a hallmark of caspase 3 activity, occurred no earlier than 8 h from incubation. Secretion of proCD was almost abolished and the formation of double-chain mature CD was reduced and delayed by NAS, whereas PDMP largely inhibited the lysosomal targeting and maturation of proCD. NAS- and PDMP-induced alteration of proCD transport and maturation were apparent already 2 h after incubation with the drugs, which is much earlier than when classical biochemical and morphological evidence of apoptosis could be detected. These data indicate that alteration of CD (and possibly of other glycoproteins) transport along the secretory pathway due to increased levels of cell-associated ceramide is an early event in cells undergoing apoptosis.     

Pressure drops in a distensible model of end-to-side anastomosis in systemic-to-pulmonary shunts

The modified Blalock-Taussig shunt is a surgical procedure used as a palliation to treat complex congenital heart defects. It consists of an interposing prosthetic tube between the innominate/subclavian artery and the right pulmonary artery. Previous experience indicates that the pressure drop across the shunt is affected by the pulmonary pressure at the distal anastomosis combined with the distensibility of the anastomosis. In this study, a computational fluid-structure interaction approach is presented to investigate the haemodynamic behaviour. Steady-state fluid dynamics and structural analyses were carried out using commercial codes based on the finite element method (FIDAP and ABAQUS) coupled by means of a purposely-developed procedure to transfer boundary conditions. Both prosthetic tube and artery walls were characterised by non-linear material properties. Three different pulmonary pressures (2, 5 and 15 mmHg) and two volume flow rates (0.4 and 0.8 l/min) were investigated. Results indicate that the effects of distensibility at the distal anastomosis on the shunt pressure drop are relevant only when the distal anastomosis on the shunt pressure drop are relevant only when the distal anastomosis is not fully distended, which occurs when the pulmonary pressure is lower than 5 mmHg.     

Natural and artificial chitosan-inorganic composites

Taking inspiration from many chitin-inorganic composites in nature, a number of recent articles throw light on the manufacture of such composites based on calcium carbonate, calcium phosphate and silica. These novel materials are important in the field of blood-compatible materials, bone substitutes, and cements for bone repair and reconstruction. This approach provides an attractive alternative to the processing of inorganic thin films, especially in applications where substrates cannot be exposed to high temperatures.     

Carbocysteine lysine salt monohydrate (SCMC-LYS) is a selective scavenger of reactive oxygen intermediates (ROIs)

Carbocysteine lysine salt monohydrate (SCMC-Lys) is a well-known mucoactive drug whose therapeutic efficacy is commonly related to the ability of SCMC-Lys to replace fucomucins by sialomucins. The aim of this study was to determine if SCMC-Lys could exert an anti-oxidant action by scavenging reactive oxygen intermediates (ROIs). Our results show that SCMC-Lys proved effective as a selective scavenger of hypochlorous acid (HOCl) and hydroxyl radical (OH.), this effect being related to the reactivity of the SCMC tioether group. The scavenger activity of SCMC-Lys was observed in free cellular system as well as in activated human polymorphonuclear neutrophils (PMNs). SCMC-Lys scavenger activity on HOCl was paralleled by a powerful protection from HOCl-mediated inactivation of alpha1-antitripsin (alpha1-AT) inhibitor, the main serum protease inhibitor. Production of interleukin-(IL-)8, a major mediator of PMN recruitment in inflammatory diseases, is known to be mediated by intracellular OH. SCMC-Lys significantly reduced IL-8 production on stimulated human peripheral blood mononuclear cells (PBMCs) in the same range of concentrations affecting OH. activity. It is concluded that SCMC-Lys could exert, in addition to its mucoactive capacity, an anti-oxidant action, thus contributing to the therapeutic efficacy of SCMC-Lys.     

Prolin-rich tyrosine kinase 2 (PYK2) expression and localization in mouse testis

Prolin-rich kinase 2 (PYK2) is a nonreceptor tyrosine kinase related to the focal adhesion kinase (FAK) p125(FAK). PYK2 is rapidly phosphorylated on tyrosine residues in response to various stimuli, such as tumor necrosis factor-alpha (TNF-alpha), changes in osmolarity, elevation in intracellular calcium concentration, angiotensin, and UV irradiation. PYK2 has ligand sequences for Src homology 2 and 3 (SH-2 and SH-3), and has binding sites for paxillin and p130(cas). Activation of PYK2 leads to modulation of ion channel function, phosphorylation of tyrosine residues, and activation of the MAP kinase signaling pathways. Immunocytochemistry shows that PYK2 is present in mouse germinal and Sertoli cells (ser). Northern blot and immunoprecipitation analysis demonstrate that, among germinal cells, PYK2 is more abundant in spermatocytes (spc) and spermatids (spt); in addition, immunofluorescence analysis clearly shows that the diffuse cytoplasmic localization of PYK2 changes in a specific cellular compartment in spt and spermatozoa.     

Antioxidant activity of galloyl quinic derivatives isolated from P. lentiscus leaves

The antioxidant properties of galloyl quinic derivatives isolated from Pistacia lentiscus L. leaves have been investigated by means of Electron Paramagnetic Resonance spectroscopy (EPR) and UV-Vis spectrophotometry. Antioxidant properties have been also estimated using the biologically relevant LDL test. The scavenger activities of gallic acid, 5- O -galloyl, 3,5- O -digalloyl, 3,4,5- O -trigalloyl quinic acid derivatives, have been estimated against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide ( O 2 - ) radical, and hydroxyl (OH) radical. On the whole, the scavenger activity raised as the number of galloyl groups on the quinic acid skeleton increased. The half-inhibition concentrations (IC 50 ) of di- and tri-galloyl derivatives did not exceed 30 μM for all the tested free radicals. All the tested metabolites strongly reduced the oxidation of low-density lipoproteins (LDL), following a trend similar to that observed for the scavenger ability against OH radical.     

Peristalsis regulation by tachykinin NK1 receptors in the rabbit isolated distal colon

In the gastrointestinal tract, tachykinin NK1 receptors are widely distributed in a number of neuronal and nonneuronal cells involved in the control of gut motor activity. In particular, in the rabbit isolated distal colon, which is a suitable model system to investigate the contribution of tachykinins as noncholinergic excitatory transmitters, the influence of NK1 receptors in the regulation of peristalsis is not known. The selective NK1-receptor antagonists SR-140333 (0.3 and 1 nM) and MEN-10930 (0.3-10 nM) significantly enhanced the velocity of rabbit colonic propulsion to submaximal stimulation. The prokinetic effect of SR-140333 was prevented by N(omega)-nitro-L-arginine (L-NNA), a nitric oxide synthase inhibitor, indicating that NK1 receptors located on nitrergic innervation exert a functional inhibitory restraint on the circular muscle and probably on descending excitatory and inhibitory pathways during propulsion. Conversely, the selective NK1-receptor agonist septide (3-10 nM) significantly inhibited colonic propulsion. In the presence of L-NNA, the inhibitory effect of septide was reverted into a prokinetic effect, which is probably mediated by the activation of postjunctional excitatory NK1 receptors.     

Potential role for ADAM15 in pathological neovascularization in mice

ADAM15 (named for a disintegrin and metalloprotease 15, metargidin) is a membrane-anchored glycoprotein that has been implicated in cell-cell or cell-matrix interactions and in the proteolysis of molecules on the cell surface or extracellular matrix. To characterize the potential roles of ADAM15 during development and in adult mice, we analyzed its expression pattern by mRNA in situ hybridization and generated mice carrying a targeted deletion of ADAM15 (adam15(-/-) mice). A high level of expression of ADAM15 was found in vascular cells, the endocardium, hypertrophic cells in developing bone, and specific areas of the hippocampus and cerebellum. However, despite the pronounced expression of ADAM15 in these tissues, no major developmental defects or pathological phenotypes were evident in adam15(-/-) mice. The elevated levels of ADAM15 in endothelial cells prompted an evaluation of its role in neovascularization. In a mouse model for retinopathy of prematurity, adam15(-/-) mice had a major reduction in neovascularization compared to wild-type controls. Furthermore, the size of tumors resulting from implanted B16F0 mouse melanoma cells was significantly smaller in adam15(-/-) mice than in wild-type controls. Since ADAM15 does not appear to be required for developmental angiogenesis or for adult homeostasis, it may represent a novel target for the design of inhibitors of pathological neovascularization.