The p85 regulatory subunit controls sequential activation of phosphoinositide 3-kinase by Tyr kinases and Ras

Class IA phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit that regulates a variety of cell responses, including cell division and survival. PI3K is activated following Tyr kinase stimulation and by Ras. We found that the C-terminal region of p85, including the C-Src homology 2 (C-SH2) domain and part of the inter-SH2 region, protects the p110 catalytic subunit from Ras-induced activation. Although the p110 activity associated with a C-terminal p85 deletion mutant increased significantly in the presence of an active form of Ras, purified wild type p85-p110 was only slightly stimulated by active Ras. Nonetheless, incubation of purified p85-p110 with Tyr-phosphorylated peptides, which mimic the activated platelet-derived growth factor receptor, restored Ras-induced p85-p110 activation. In conclusion, p85 inhibits p110 activation by Ras; this blockage is released by Tyr kinase stimulation, showing that the classical mechanism of class IA PI3K stimulation mediated by Tyr kinases also regulates Ras-induced PI3K activation.     

Combining classical and molecular approaches elaborates on the complexity of mechanisms underpinning anterior regeneration

The current model of planarian anterior regeneration evokes the establishment of low levels of Wnt signalling at anterior wounds, promoting anterior polarity and subsequent elaboration of anterior fate through the action of the TALE class homeodomain PREP. The classical observation that decapitations positioned anteriorly will regenerate heads more rapidly than posteriorly positioned decapitations was among the first to lead to the proposal of gradients along an anteroposterior (AP) axis in a developmental context. An explicit understanding of this phenomenon is not included in the current model of anterior regeneration. This raises the question what the underlying molecular and cellular basis of this temporal gradient is, whether it can be explained by current models and whether understanding the gradient will shed light on regenerative events. Differences in anterior regeneration rate are established very early after amputation and this gradient is dependent on the activity of Hedgehog (Hh) signalling. Animals induced to produce two tails by either Smed-APC-1(RNAi) or Smed-ptc(RNAi) lose anterior fate but form previously described ectopic anterior brain structures. Later these animals form peri-pharyngeal brain structures, which in Smed-ptc(RNAi) grow out of the body establishing a new A/P axis. Combining double amputation and hydroxyurea treatment with RNAi experiments indicates that early ectopic brain structures are formed by uncommitted stem cells that have progressed through S-phase of the cell cycle at the time of amputation. Our results elaborate on the current simplistic model of both AP axis and brain regeneration. We find evidence of a gradient of hedgehog signalling that promotes posterior fate and temporarily inhibits anterior regeneration. Our data supports a model for anterior brain regeneration with distinct early and later phases of regeneration. Together these insights start to delineate the interplay between discrete existing, new, and then later homeostatic signals in AP axis regeneration.     

Amplified and Homozygously Deleted Genes in Glioblastoma: Impact on Gene Expression Levels

Background

Glioblastoma multiforme (GBM) displays multiple amplicons and homozygous deletions that involve relevant pathogenic genes and other genes whose role remains unknown.

Methodology

Single-nucleotide polymorphism (SNP)-arrays were used to determine the frequency of recurrent amplicons and homozygous deletions in GBM (n = 46), and to evaluate the impact of copy number alterations (CNA) on mRNA levels of the genes involved.

Principal Findings

Recurrent amplicons were detected for chromosomes 7 (50%), 12 (22%), 1 (11%), 4 (9%), 11 (4%), and 17 (4%), whereas homozygous deletions involved chromosomes 9p21 (52%) and 10q (22%). Most genes that displayed a high correlation between DNA CNA and mRNA levels were coded in the amplified chromosomes. For some amplicons the impact of DNA CNA on mRNA expression was restricted to a single gene (e.g., EGFR at 7p11.2), while for others it involved multiple genes (e.g., 11 and 5 genes at 12q14.1–q15 and 4q12, respectively). Despite homozygous del(9p21) and del(10q23.31) included multiple genes, association between these DNA CNA and RNA expression was restricted to the MTAP gene.

Conclusions

Overall, our results showed a high frequency of amplicons and homozygous deletions in GBM with variable impact on the expression of the genes involved, and they contributed to the identification of other potentially relevant genes.     

Structure-function relationships among human cathelicidin peptides: dissociation of antimicrobial properties from host immunostimulatory activities

Cathelicidins and other antimicrobial peptides are deployed at epithelial surfaces to defend against infection. These molecules have broad-spectrum killing activity against microbes and can have effects on specific mammalian cell types, potentially stimulating additional immune defense through direct chemotactic activity or induction of cytokine release. In humans, the cathelicidin hCAP18/LL-37 is processed to LL-37 in neutrophils, but on skin it can be further proteolytically processed to shorter forms. The influence of these cathelicidin peptides on keratinocyte function is not known. In the current study, DNA microarray analysis and confirmatory protein analysis showed that LL-37 affects the expression of several chemokines and cytokines by keratinocytes. Analysis of a synthetic peptide library derived from LL-37 showed that antimicrobial activity against bacterial, fungal, and viral skin pathogens resides within specific domains of the parent peptide, but antimicrobial activity does not directly correlate with the ability to stimulate IL-8 production in keratinocytes. IL-8 release was induced by d- and l-amino acid forms of cathelicidin and correlated with membrane permeability, suggesting that highly structure-specific binding to a cell surface receptor is not likely. However, this effect was inhibited by either pertussis toxin or AG1478, an epidermal growth factor receptor tyrosine kinase inhibitor, suggesting that cathelicidin may indirectly stimulate multiple signaling pathways associated with cell surface receptors. Taken together, these observations suggest that proteolytic processing may alter the balance between cathelicidin antimicrobial and host immunostimulatory functions.     

Characterization of monoclonal antibodies against ovine prolactin: suitability for use in immunocytochemical analysis of rat prolactin

The aim of this study was to identify a monoclonal antibody (MAb) suitable for use in the immunocytochemical localization of prolactin in rat tissues. We took advantage of the conservation of certain amino acid sequences in prolactin among species by examining the crossreactivity patterns of five MAb, originally generated to ovine prolactin, with rat prolactin by enzyme-linked immunoassay (ELISA), Western blot analysis, and immunocytochemistry. Two of five antibodies (17D9 and 6F11) showed reactivity with 100 ng of immobilized rat prolactin (NIH RP-3) by ELISA, 6F11 reacting more strongly than 17D9. Only 6F11 reacted with prolactin in lysates of GH4C1 rat pituitary tumor cells by Western blot analysis. When we examined the crossreactivity of the MAb with rat prolactin in monolayer cultures of GH4C1 cells by indirect immunofluorescence, we found that both 17D9 and 6F11 reacted strongly with the cultures. The distribution of staining with 17D9 or 6F11 was coincident with staining with a polyclonal antiserum to rat prolactin. Preabsorption of the antibodies with a 20-fold excess of purified rat prolactin abolished the staining of GH4C1 cell cultures with either antibody. Therefore, we have selected from a series of MAb raised to ovine prolactin two antibodies (17D9 and 6F11) that react specifically with rat prolactin in immunocytochemical studies, whereas 6F11 also reacts strongly with rat prolactin by ELISA and Western blot analysis.     

Leukemia inhibitory factor induces DNA synthesis in Swiss mouse 3T3 cells independently of cyclin D1 expression through a mechanism involving MEK/ERK1/2 activation

Leukemia inhibitory factor (LIF) and oncostatin M (OSM) induce DNA synthesis in Swiss 3T3 cells through common signaling mechanism(s), whereas other related cytokines such as interleukin-6 and ciliary neurotrophic factor do not cause this response. Induction of DNA replication by LIF or prostaglandin F2alpha (PGF2alpha) occurs, in part, through different signaling events. LIF and OSM specifically trigger STAT1 cytoplasmic to nuclear translocation, whereas PGF2alpha fails to do so. However, LIF and PGF2alpha can trigger increases in ERK1/2 activity, which are required for their mitogenic responses because U0126, a MEK1/2 inhibitor, prevents both ERK1/2 activation and induction of DNA synthesis by LIF or PGF2alpha treatment. PGF2alpha induces cyclin D expression and full phosphorylation of retinoblastoma protein. In contrast, LIF fails to promote increases in cyclin D mRNA/protein levels; consequently, LIF induces DNA synthesis without promoting full phosphorylation of retinoblastoma protein (Rb). However, both LIF and PGF2alpha increase cyclin E expression. Furthermore, LIF mitogenic action does not involve protein kinase C (PKC) activation, because a PKC inhibitor does not block this effect. In contrast, PKC activity is required for PGF2alpha mitogenic action. More importantly, the synergistic effect between LIF and PGF2alpha to promote S phase entry is independent of PKC activation. These results show fundamental differences between LIF- and PGF2alpha-dependent mechanism(s) that induce cellular entry into S phase. These findings are critical in understanding how LIF and other related cytokine-regulated events participate in normal cell cycle control and may also provide clues to unravel crucial processes underlying cancerous cell division.     

Benzyladenine-controlled protein synthesis and growth in apple cell suspensions

Prolific growth in cytokinin-requiring apple cell suspensions was achieved during 10 days of culture with appropriate concentrations of benzyladenine (BA). Unlike the controls, BA-treated cells showed a well developed sub-cellular organization and protein synthesis. Upon transfer to fresh cytokinin-free medium, however, these cells exhibited a rapid decrease in polyribosome formation, but with unchanged nuclear and nucleolar activities. Cells lacking added cytokinin showed a reduced metabolic activity and failed to divide. Deficiency in the translation process of cytokinin-deprived cells was overcome by exogenous BA-treatment. In cells maintained on a cytokinin-free medium for 5 days, BA-treatment enhanced protein synthesis from pre-existing mRNA in a specific way.     

Cranial Anatomy and Palaeoneurology of the Archosaur Riojasuchus tenuisceps from the Los Colorados Formation,La Rioja,Argentina

Riojasuchus tenuisceps Bonaparte 1967 is currently known from four specimens, including two complete skulls, collected in the late 1960s from the upper levels of the Los Colorados Formation (Late Triassic), La Rioja, Argentina. Computed tomography (CT) scans of the skulls of the holotype and a referred specimen of Riojasuchus tenuisceps and the repreparation of the latter allows recognition of new features for a detailed analysis of its cranial anatomy and its comparison with a wide variety of other archosauriform taxa. The diagnosis of Riojasuchus tenuisceps is emended and two autapomorphies are identified on the skull: (1) a deep antorbital fossa with its anterior and ventral edges almost coinciding with the same edges of the maxilla itself and (2) a suborbital fenestra equal in size to the palatine-pterygoid fenestra. Also, the first digital 3D reconstruction of the encephalon of Riojasuchus tenuisceps was carried out to study its neuroanatomy, showing a shape and cranial nerve disposition consistent to that of other pseudosuchians.