Eps8 controls actin-based motility by capping the barbed ends of actin filaments

Actin filament barbed-end capping proteins are essential for cell motility, as they regulate the growth of actin filaments to generate propulsive force. One family of capping proteins, whose prototype is gelsolin, shares modular architecture, mechanism of action, and regulation through signalling-dependent mechanisms, such as Ca(2+) or phosphatidylinositol-4,5-phosphate binding. Here we show that proteins of another family, the Eps8 family, also show barbed-end capping activity, which resides in their conserved carboxy-terminal effector domain. The isolated effector domain of Eps8 caps barbed ends with an affinity in the nanomolar range. Conversely, full-length Eps8 is auto-inhibited in vitro, and interaction with the Abi1 protein relieves this inhibition. In vivo, Eps8 is recruited to actin dynamic sites, and its removal impairs actin-based propulsion. Eps8-family proteins do not show any similarity to gelsolin-like proteins. Thus, our results identify a new family of actin cappers, and unveil novel modalities of regulation of capping through protein-protein interactions. One established function of the Eps8-Abi1 complex is to participate in the activation of the small GTPase Rac, suggesting a multifaceted role for this complex in actin dynamics, possibly through the participation in alternative larger complexes.     

Purification and characterization of a beta-D-mannosidase from the marine anaspidean Aplysia fasciata

A beta-D-mannosidase was purified to homogeneity from visceral mass extract of Aplysia fasciata a mollusc belonging to the order Anaspidea. The purified enzyme is a homodimer with a subunit mass of 130 kDa. Temperature and pH optima of this enzyme were 45 degrees C and 4.5, respectively. Substrate specificity tests revealed that the enzyme exerts only beta-D-mannosidase activity. The K(M) and V(max) values for p-nitrophenyl beta-D-mannopyranoside were determined to be 2.4 mM and 50.3 micromol min(-1)mg(-1), respectively. The catalytic efficiency of this beta-mannosidase (11,519 min(-1)) was significantly higher than those reported for beta-mannosidases from other sources. It was verified that this is an exo-acting glycosyl hydrolase with transglycosidase activity. When the enzyme was incubated in the presence of p-nitrophenyl beta-D-mannopyranoside, self-transfer of the mannosyl group was observed, and a 10-15% yield of a beta-1-4 disaccharide was obtained. When the reaction was performed in the presence of o-nitrophenyl alpha-D-2-deoxy-N-acetyl glucopyranoside in 3:1 molar ratio with respect to the p-nitrophenyl beta-D-mannopyranoside, two regioisomers (85:15, 12% yield) due to the beta-mannosylation of the heteroacceptor in 4 and in 6 positions were formed.     

Salinivibrio costicola subsp. alcaliphilus subsp. nov., a haloalkaliphilic aerobe from Campania Region (Italy)

Phenotypic and phylogenetic studies were performed on unidentified Gram-negative staining, haloalkaliphilic aerobe and protease producer Salinivibrio-like organism recovered from a saltish spring with algal mat in the “Pozzo del Sale” site (Salt's Well) in the Campania Region (South Italy). Phylogenetic analysis based on comparison of 16S rRNA gene sequences demonstrated that the isolate was related to species of Salinivibrio genus. The DNA–DNA hybridization of the type strain 18AG^T with the most related Salinivibrio costicola subsp. costicola showed a re-association value of 72%. Based on the phenotypic distinctiveness of 18AG^T strain and molecular, chemical and genetic evidence, it is proposed that strain 18AG^T can be classified as S. costicola subsp. alcaliphilus, subsp. nov. The type strain of S. costicola subsp. alcaliphilus, is ATCC BAA-952^T; DSM 16359^T.     

Halomonas campaniensis sp. nov., a haloalkaliphilic bacterium isolated from a mineral pool of Campania Region, Italy

A Gram-negative, aerobic, motile and rod-shaped haloalkaliphilic bacterial strain 5AGT (DSM 15293 and ATCC BAA-966) was isolated from water with algal mat of a mineral pool in Malvizza site (Campania-Italy) and was subjected to a polyphasic study. The isolate grew at temperature of 10.0-43.0 degrees C with an optimum at 37.0 degrees C. Strain 5AGT grew optimally in the presence of 10% NaCl and grew also in the absence of salt. The isolate grew in the pH range 7.0-10.0 with an optimum at pH 9.0. It accumulated glycine-betaine, ectoine, and glutamate, as osmoprotectants. Strain 5AGT was also characterized chemotaxonomically by having ubiquinone-8 (Q8) as the predominant isoprenoid quinone, phosphoethanolamine (PEA), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG), as major polar lipids and aiC16:0 and C18:1cis as the major fatty acids. The DNA G+C content was 63.7mol%. Phylogenetic analyses based on 16S rRNA gene sequence showed that the isolate belonged to the genus Halomonas. The DNA-DNA hybridization of the type strain 5AGT with the most related Halomonas campisalis showed a re-association value of 35.0%.On the basis of phenotypic properties and phylogeny, strain 5AGT should be placed in the genus Halomonas as a member of a novel species for which we propose the name Halomonas campaniensis sp. nov.     

Effects of Microenvironment on Morphology and Function of the Microglial Cell Line BV-2

Effects of microenvironmental changes were examined in the microglial cell line BV-2. In serum supplemented medium cells were ameboid shaped and exhibited thin cytoplasmatic processes at lower concentration or in absence of serum. High levels of acetylated low-density lipoprotein (LDL) receptor and of phagocytic and proliferative activity were detected. Lipopolysaccharide (LPS) and the neuropeptide substance P (SP) induced secretion of interleukin-6. Low interleukin-3 secretion was detected only occasionally and was not influenced by LPS and SP. In defined medium, process-bearing cells were evident. Compared to cultures in serum supplemented medium, the cells expressed lower acetylated LDL-binding and phagocytic activity while actively proliferated, the response to LPS was reduced and to SP absent. Granulocyte/macrophage colony-stimulating factor increased the number of process-bearing cells, of acetylated LDL-binding and of IL-6 secretion induced by LPS. Cell morphology was not influenced by neurotrophins like nerve growth factor and brain-derived neurotrophic factor. The described phenotypical and functional plasticity makes the BV-2 cell line a useful model to investigate mechanisms of microglial activation.     

Rasche Bildung von Entmischungskörpern in Zellsäften vonBuxus sempervirens

Zusammenfassung Die Zellen der unteren Blattepidermis vonBuxus sempervirens zeigen bei Behandlung mit starken (2—4 mol) KCNS-Lösungen nicht bloß die bekannten Erscheinungen der Kappen- und Tonoplastenplasmolyse, sondern es treten verschiedene Entmischungen in der Vakuole auf, und schließlich bleibt nach dem Platzen des Tonoplasten ein kugeliger, fester Körper in der Zelle als Rückstand liegen. Besser lassen sich diese Vorgänge an vitalgefärbten Zellen beobachten. Der Entmischungskörper behält in diesem Falle den größten Teil des vorher von der Vakuole gespeicherten Farbstoffes in sich eingeschlossen.Bei Färbung der Zellen mit Akridinorange pH 8,0, 110.000, kommt es zunächst zur gewohnten Diffusfärbung, nach etwa 15 Minuten Färbezeit tritt jedoch in manchen Zellen ein rotorange gefärbtes netzartiges oder rundliches Gebilde auf, das im Fluoreszenzmikroskop grellgelb fluoresziert.     

Phenotype modulation in cultures of vascular smooth muscle cells from diabetic rats: association with increased nitric oxide synthase expression and superoxide anion generation

Proliferative modification of vascular smooth muscle cell (vSMC) and impaired bioavailability of nitric oxide (NO) have both been proposed among the mechanisms linking diabetes and atherosclerosis. However, diabetes induced modifications in phenotype and nitric oxide synthase(s) (NOS) expression and activity in vSMC have not been fully characterized. In this study, cell morphology, proliferative response to serum, alpha-SMactin levels, eNOS expression and activity, cGMP intracellular content, and superoxide anion release were measured in cultures of vSMC obtained from aorta medial layer of ten diabetic (90% pancreatectomy, DR) and ten control (sham surgery, CR) rats. Vascular SMC from DR showed a less evident "hill and valley" culture morphology, increased growth response to serum, greater saturation density, and lower levels of alpha-SMactin. In the same cells, as compared to CR cells, eNOS mRNA levels and NOS activity were increased, while intracellular cGMP level was lower and superoxide anion production was significantly greater. These data indicate that chronic hyperglycemia might induce, in the vascular wall, an increased number of vSMC proliferative clones which persist in culture and are associated with increased eNOS expression and activity. However, upregulation of eNOS and increased NO synthesis occur in the presence of a marked concomitant increase of O(2-) production. Since NO bioavailability, as reflected by cGMP levels, was not increased in DR cells, it is tempting to hypothesize that the proliferative phenotype observed in DR cells is associated with a redox imbalance responsible quenching and/or trapping of NO, with the consequent loss of its biological activity.     

Synthesis,characterization and cytotoxic activity on breast cancer cells of new half-titanocene derivatives

A series of novel titanocene-complexes has been prepared and evaluated for their growth regulatory effects in MCF7 and SkBr3 breast cancer cells. The capability of some of these compound to elicit relevant repressive effects on cancer cell growth could be taken into account towards novel pharmacological approaches in cancer therapy.     

High prevalence of Cag-A positive H. pylori strains in ischemic stroke: a primary care multicenter study

Background: Previous studies suggested an association between CagA-positive H. pylori strains and ischemic stroke. The aim of the present study was to assess the prevalence of Helicobacter pylori infection and CagA status in patients with atherosclerotic stroke in the primary care setting.
Materials and methods: A total of 106 consecutive patients (age 76.6 ± 8 years; males 52%) with well-documented history of atherosclerotic stroke and 106 sex–age- (age 76.5 ± 9 years; males 52%) and social background-matched controls without relevant vascular diseases. Risk factors for ischemic stroke were recorded in all subjects. H. pylori infection was assessed by[13]C-urea breath test. A serologic assay for specific IgG against CagA was performed in infected subjects.
Results: A trend toward a higher prevalence of H. pylori was observed in cases (63%) with respect to controls (54%) without reaching a statistical significance. CagA positivity was associated to a higher risk of atherosclerotic stroke (adjusted odds ratio 2.69, 95% confidence interval 1.37–5.30).
Conclusions: Our findings suggest that CagA-positive strains of H. pylori are significantly associated to atherosclerotic stroke. This is not a merely confirmative study since it has been performed for the first time in the primary care setting and included only subjects with an active infection.