EGF regulates a complex pattern of gene expression and represses smooth muscle differentiation during the neurotypic conversion of the neural-crest-derived TC-1S cell line

EGF, known to sustain CNS neuronal progenitors, also promotes a neurotypic response in the thymic neural-crest-derived TC-1S cell line. We report here the use of TC-1S cells as a model to identify the genetic programs regulated during the neurotypic response induced by EGF and to isolate 23 EGF-responsive genes. Among them 5 represent novel cDNAs, while 18 are known genes, whose regulation by EGF is associated with the mitogenic or differentiating effects of the growth factor. The repression of smooth muscle alpha-actin and SM22alpha genes by EGF and their increase by TGFbeta suggest that the TC-1S line includes neural crest multipotent cells whose smooth muscle differentiation is repressed upon EGF treatment and stimulated by TGFbeta. Therefore, we identified a complex pattern of EGF-target genes and propose EGF as a novel signal able to recruit postmigratory neural-crest-derived cells along proliferation and cell lineage choice pathways.     

Targeting Src Family Kinases Inhibits Bevacizumab-Induced Glioma Cell Invasion

Anti-VEGF antibody therapy with bevacizumab provides significant clinical benefit in patients with recurrent glioblastoma multiforme (GBM). Unfortunately, progression on bevacizumab therapy is often associated with a diffuse disease recurrence pattern, which limits subsequent therapeutic options. Therefore, there is an urgent need to understand bevacizumab''s influence on glioma biology and block it''s actions towards cell invasion.To explore the mechanism(s) of GBM cell invasion we have examined a panel of serially transplanted human GBM lines grown either in short-term culture, as xenografts in mouse flank, or injected orthotopically in mouse brain. Using an orthotopic xenograft model that exhibits increased invasiveness upon bevacizumab treatment, we also tested the effect of dasatinib, a broad spectrum SFK inhibitor, on bevacizumab-induced invasion.We show that 1) activation of Src family kinases (SFKs) is common in GBM, 2) the relative invasiveness of 17 serially transplanted GBM xenografts correlates strongly with p120 catenin phosphorylation at Y228, a Src kinase site, and 3) SFK activation assessed immunohistochemically in orthotopic xenografts, as well as the phosphorylation of downstream substrates occurs specifically at the invasive tumor edge. Further, we show that SFK signaling is markedly elevated at the invasive tumor front upon bevacizumab administration, and that dasatinib treatment effectively blocked the increased invasion induced by bevacizumab.Our data are consistent with the hypothesis that the increased invasiveness associated with anti-VEGF therapy is due to increased SFK signaling, and support testing the combination of dasatinib with bevacizumab in the clinic.     

Sheets of Vertically Aligned BaTiO3 Nanotubes Reduce Cell Proliferation but Not Viability of NIH-3T3 Cells

All biomaterials initiate a tissue response when implanted in living tissues. Ultimately this reaction causes fibrous encapsulation and hence isolation of the material, leading to failure of the intended therapeutic effect of the implant. There has been extensive bioengineering research aimed at overcoming or delaying the onset of encapsulation. Nanotechnology has the potential to address this problem by virtue of the ability of some nanomaterials to modulate interactions with cells, thereby inducing specific biological responses to implanted foreign materials. To this effect in the present study, we have characterised the growth of fibroblasts on nano-structured sheets constituted by BaTiO₃, a material extensively used in biomedical applications. We found that sheets of vertically aligned BaTiO₃ nanotubes inhibit cell cycle progression - without impairing cell viability - of NIH-3T3 fibroblast cells. We postulate that the 3D organization of the material surface acts by increasing the availability of adhesion sites, promoting cell attachment and inhibition of cell proliferation. This finding could be of relevance for biomedical applications designed to prevent or minimize fibrous encasement by uncontrolled proliferation of fibroblastic cells with loss of material-tissue interface underpinning long-term function of implants.     

Microbial transformation of spirolaxine, a bioactive undecaketide fungal metabolite from the basidiomycete Sporotrichum laxum

Incubation of (+)-spirolaxine (= (3R)-5-hydroxy-7-methoxy-3-{5-[(2R,5R,7R)-2-methyl-1,6-dioxaspiro[4.5]dec-7-yl]pentyl}-2-benzofuran-1(3H)-one; 1a) with Bacillus megaterium afforded two new mono- and one new dihydroxylated metabolite(s), all OH groups being introduced on the non-activated six-membered ring. In contrast, exposure of 1a to Cunninghamella echinulata gave rise to hydroxylation on the five-membered ring of the parent structure. The structures and absolute configurations of the new products 1b-e were deduced on the basis of MS and NMR data. The metabolite 1b was investigated, in comparison to 1a, for its cytotoxicity (sulforhodamin-B test) and for its antiproliferative activity towards bovine microvascular endothelial cells (BMEC).     

Natural killer cell dysfunction is a distinguishing feature of systemic onset juvenile rheumatoid arthritis and macrophage activation syndrome

Macrophage activation syndrome (MAS) has been reported in association with many rheumatic diseases, most commonly in systemic juvenile rheumatoid arthritis (sJRA). Clinically, MAS is similar to hemophagocytic lymphohistiocytosis (HLH), a genetic disorder with absent or depressed natural killer (NK) function. We have previously reported that, as in HLH, patients with MAS have profoundly decreased NK activity, suggesting that this abnormality might be relevant to the pathogenesis of the syndrome. Here we examined the extent of NK dysfunction across the spectrum of diseases that comprise juvenile rheumatoid arthritis (JRA). Peripheral blood mononuclear cells (PBMC) were collected from patients with pauciarticular (n = 4), polyarticular (n = 16), and systemic (n = 20) forms of JRA. NK cytolytic activity was measured after co-incubation of PBMC with the NK-sensitive K562 cell line. NK cells (CD56+/T cell receptor [TCR]-alphabeta-), NK T cells (CD56+/TCR-alphabeta+), and CD8+ T cells were also assessed for perforin and granzyme B expression by flow cytometry. Overall, NK cytolytic activity was significantly lower in patients with sJRA than in other JRA patients and controls. In a subgroup of patients with predominantly sJRA, NK cell activity was profoundly decreased: in 10 of 20 patients with sJRA and in only 1 of 20 patients with other JRA, levels of NK activity were below two standard deviations of pediatric controls (P = 0.002). Some decrease in perforin expression in NK cells and cytotoxic T lymphocytes was seen in patients within each of the JRA groups with no statistically significant differences. There was a profound decrease in the proportion of circulating CD56bright NK cells in three sJRA patients, a pattern similar to that previously observed in MAS and HLH. In conclusion, a subgroup of patients with JRA who have not yet had an episode of MAS showed decreased NK function and an absence of circulating CD56bright population, similar to the abnormalities observed in patients with MAS and HLH. This phenomenon was particularly common in the systemic form of JRA, a clinical entity strongly associated with MAS.     

Chiniquodontid cynodonts: systematic and morphometric considerations

Systematic and morphometric reappraisal of most specimens previously assigned to the cynodont clade Chiniquodontidae reveal that the group is diagnosed by two autapomorphies: (1) the distinctive angulation between the posterior portion of the maxillary and the anterior portion of the zygomatic arch and (2) very extended pterygoid flanges, ending in a thin projection; and a combination of features including: posterior postcanines sectorial with principal cusps backwardly recurved, and a long osseous palate. The species Chiniquodon theotonicus (including Belesodon magnificus , Probelesodon kitchingi , P. lewisi and P. minor ) and C. sanjuanensis (comb. nov.) were the only recognised members of the family on the basis of qualitative characters. The South American species Cromptodon mamiferoides , Probainognathus jenseni and Thrinaxodon brasiliensis are thus excluded from the group, as are the African taxa Aleodon brachyrhamphus and Cistecynodon parvus . Allometric analysis of chiniquodontids sensu stricto reveals that: (1) all specimens can be confidently arranged in a single growth series, irrespective of their original species assignments; and (2) most of the measurements change isometrically with respect to skull length. Adults are virtually scaled–up juveniles, showing a deepened zygoma, a longer osseous palate, and a wider anterior muzzle in the canine region. Thus, allometric data reinforce the proposed synonymy of all forms, with the specific status of C. sanjuanensis supported by qualitative diagnostic traits.     

Structure–activity relationship study of nitrosopyrimidines acting as antifungal agents

The design, synthesis, in vitro evaluation, and conformational study of nitrosopyrimidine derivatives acting as antifungal agents are reported. Different compounds structurally related with 4,6-bis(alkyl or arylamino)-5-nitrosopyrimidines were evaluated. Some of these nitrosopyrimidines have displayed a significant antifungal activity against human pathogenic strains. In this paper, we report a new group of nitrosopyrimidines acting as antifungal agents. Among them, compounds 2a, 2b and 15, the latter obtained from a molecular modeling study, exhibited antifungal activity against Candida albicans, Candida tropicalis and Cryptococcus neoformans. We have performed a conformational and electronic analysis on these compounds by using quantum mechanics calculations in conjunction with Molecular Electrostatic Potentials (MEP) obtained from B3LYP/6–31G(d) calculations. Our experimental and theoretical results have led us to identify a topographical template which may provide a guide for the design of new nitrosopyrimidines with antifungal effects.     

Kallikrein 6 is a novel molecular trigger of reactive astrogliosis

Kallikrein-related peptidase 6 (KLK6) is a trypsin-like serine protease upregulated at sites of central nervous system (CNS) injury, including de novo expression by reactive astrocytes, yet its physiological actions are largely undefined. Taken with recent evidence that KLK6 activates G-protein-coupled protease-activated receptors (PARs), we hypothesized that injury-induced elevations in KLK6 contribute to the development of astrogliosis and that this occurs in a PAR-dependent fashion. Using primary murine astrocytes and the Neu7 astrocyte cell line, we show that KLK6 causes astrocytes to transform from an epitheliod to a stellate morphology and to secrete interleukin 6 (IL-6). By contrast, KLK6 reduced expression of glial fibrillary acidic protein (GFAP). The stellation-promoting activities of KLK6 were shown to be dependent on activation of the thrombin receptor, PAR1, as a PAR1-specific inhibitor, SCH79797, blocked KLK6-induced morphological changes. The ability of KLK6 to promote astrocyte stellation was also shown to be linked to activation of protein kinase C (PKC). These studies indicate that KLK6 is positioned to serve as a molecular trigger of select physiological processes involved in the development of astrogliosis and that this is likely to occur at least in part by activation of the G-protein-coupled receptor, PAR1.