The Effect of Chitosan as Internal or External Coating on the 5-ASA Release from Calcium Alginate Microparticles

The effect of chitosan as internal or external coating on the mesalamine (5-ASA) release from calcium alginate microparticles (CaAl) was studied, and a delayed release of 5-ASA system intended for colonic drug delivery was developed. The external chitosan coating was developed by immersion of wetted CaAl in chitosan solution and the internal coating by mixing 5-ASA with chitosan solution and drying before the preparation of CaAl. Both systems were coated with Acryl-EZE^® using combined fluid bed coating and immersion procedure. The results showed that in phosphate medium (pH 7.5), chitosan as 5-ASA coating promotes a quick erosion process accelerating drug release, but chitosan as external coating (CaAlCS) does not increase the T ₅₀ value compared with the microparticles without chitosan (CaAl). Chitosan as internal or external coating was not effective to avoid the quick 5-ASA release in acidic medium (pH 1.2). The presence of β-glucosidase enzymes increases significantly the 5-ASA release for CaAl, while no effect was observed with chitosan as internal or external coating. Fourier transform infrared spectroscopy, thermogravimetric analysis, and X-ray data revealed that 5-ASA did not form a solid solution but was dispersed in the microparticles. The Acryl-EZE^® coating of microparticles was effective because all the formulations showed a low release, less than 15%, of 5-ASA in acid medium at pH 1.2. Significant differences in the percentage of 5-ASA released between formulations were observed in phosphate buffer at pH 6.0. In phosphate buffer at pH 7.2, all the formulations released 100% of 5-ASA.     

Metabolic processes sustaining the reviviscence of lichen <Emphasis Type="Italic">Xanthoria elegans</Emphasis> (Link) in high mountain environments

To survive in high mountain environments lichens must adapt themselves to alternating periods of desiccation and hydration. Respiration and photosynthesis of the foliaceous lichen, Xanthoria elegans, in the dehydrated state were below the threshold of CO₂-detection by infrared gas analysis. Following hydration, respiration totally recovered within seconds and photosynthesis within minutes. In order to identify metabolic processes that may contribute to the quick and efficient reactivation of lichen physiological processes, we analysed the metabolite profile of lichen thalli step by step during hydration/dehydration cycles, using ³¹P- and ¹³C-NMR. It appeared that the recovery of respiration was prepared during dehydration by the accumulation of a reserve of gluconate 6-P (glcn-6-P) and by the preservation of nucleotide pools, whereas glycolytic and photosynthetic intermediates like glucose 6-P and ribulose 1,5-diphosphate were absent. The large pools of polyols present in both X. elegans photo- and mycobiont are likely to contribute to the protection of cell constituents like nucleotides, proteins, and membrane lipids, and to preserve the integrity of intracellular structures during desiccation. Our data indicate that glcn-6-P accumulated due to activation of the oxidative pentose phosphate pathway, in response to a need for reducing power (NADPH) during the dehydration-triggered down-regulation of cell metabolism. On the contrary, glcn-6-P was metabolised immediately after hydration, supplying respiration with substrates during the replenishment of pools of glycolytic and photosynthetic intermediates. Finally, the high net photosynthetic activity of wet X. elegans thalli at low temperature may help this alpine lichen to take advantage of brief hydration opportunities such as ice melting, thus favouring its growth in harsh high mountain climates.     

Both the suppressor of cytokine signaling 1 (SOCS-1) kinase inhibitory region and SOCS-1 mimetic bind to JAK2 autophosphorylation site: implications for the development of a SOCS-1 antagonist

Suppressor of cytokine signaling (SOCS)-1 protein modulates signaling by IFN-gamma by binding to the autophosphorylation site of JAK2 and by targeting bound JAK2 to the proteosome for degradation. We have developed a small tyrosine kinase inhibitor peptide (Tkip) that is a SOCS-1 mimetic. Tkip is compared in this study with the kinase inhibitory region (KIR) of SOCS-1 for JAK2 recognition, inhibition of kinase activity, and regulation of IFN-gamma-induced biological activity. Tkip and a peptide corresponding to the KIR of SOCS-1, ((53))DTHFRTFRSHSDYRRI((68)) (SOCS1-KIR), both bound similarly to the autophosphorylation site of JAK2, JAK2(1001-1013). The peptides also bound to JAK2 peptide phosphorylated at Tyr(1007), pJAK2(1001-1013). Dose-response competitions suggest that Tkip and SOCS1-KIR similarly recognize the autophosphorylation site of JAK2, but probably not precisely the same way. Although Tkip inhibited JAK2 autophosphorylation as well as IFN-gamma-induced STAT1-alpha phosphorylation, SOCS1-KIR, like SOCS-1, did not inhibit JAK2 autophosphorylation but inhibited STAT1-alpha activation. Both Tkip and SOCS1-KIR inhibited IFN-gamma activation of Raw 264.7 murine macrophages and inhibited Ag-specific splenocyte proliferation. The fact that SOCS1-KIR binds to pJAK2(1001-1013) suggests that the JAK2 peptide could function as an antagonist of SOCS-1. Thus, pJAK2(1001-1013) enhanced suboptimal IFN-gamma activity, blocked SOCS-1-induced inhibition of STAT3 phosphorylation in IL-6-treated cells, enhanced IFN-gamma activation site promoter activity, and enhanced Ag-specific proliferation. Furthermore, SOCS-1 competed with SOCS1-KIR for pJAK2(1001-1013). Thus, the KIR region of SOCS-1 binds directly to the autophosphorylation site of JAK2 and a peptide corresponding to this site can function as an antagonist of SOCS-1.     

Roles of alternative splicing in the functional properties of inner ear-specific KCNQ4 channels

The function of the KCNQ4 channel in the auditory setting is crucial to hearing, underpinned by the finding that mutations of the channel result in an autosomal dominant form of nonsyndromic progressive high frequency hearing loss. The precise function of KCNQ4 in the inner ear has not been established. However, recently we demonstrated that there is differential expression among four splice variants of KCNQ4 (KCNQ4_v1-v4) along the tonotopic axis of the cochlea. Alternative splicing specifies the outcome of functional channels by modifying the amino acid sequences within the C terminus at a site designated as the membrane proximal region. We show that variations within the C terminus of splice variants produce profound differences in the voltage-dependent phenotype and functional expression of the channel. KCNQ4_v4 lacks exons 9-11, resulting in deletion of 54 amino acid residues adjacent to the S6 domain compared with KCNQ4_v1. Consequently, the voltage-dependent activation of KCNQ4_v4 is shifted leftward by approximately 20 mV, and the number of functional channels is increased severalfold compared with KCNQ4_v1. The properties of KCNQ4_v2 and KCNQ4_v3 fall between KCNQ4_v1 and KCNQ4_v4. Because of variations in the calmodulin binding domains of the splice variants, the channels are differentially modulated by calmodulin. Co-expression of these splice variants yielded current magnitudes suggesting that the channels are composed of heterotetramers. Indeed, a dominant negative mutant of KCNQ4_v1 cripples the currents of the entire KCNQ4 channel family. Furthermore, the dominant negative KCNQ4 mutant stifles the activity of KCNQ2-5, raising the possibility of a global disruption of KCNQ channel activity and the ensuing auditory phenotype.     

Structural basis for innate immune sensing by M-ficolin and its control by a pH-dependent conformational switch

Ficolins are soluble oligomeric proteins with lectin-like activity, assembled from collagen fibers prolonged by fibrinogen-like recognition domains. They act as innate immune sensors by recognizing conserved molecular markers exposed on microbial surfaces and thereby triggering effector mechanisms such as enhanced phagocytosis and inflammation. In humans, L- and H-ficolins have been characterized in plasma, whereas a third species, M-ficolin, is secreted by monocytes and macrophages. To decipher the molecular mechanisms underlying their recognition properties, we previously solved the structures of the recognition domains of L- and H-ficolins, in complex with various model ligands (Garlatti, V., Belloy, N., Martin, L., Lacroix, M., Matsushita, M., Endo, Y., Fujita, T., Fontecilla-Camps, J. C., Arlaud, G. J., Thielens, N. M., and Gaboriaud, C. (2007) EMBO J. 24, 623-633). We now report the ligand-bound crystal structures of the recognition domain of M-ficolin, determined at high resolution (1.75-1.8 A), which provides the first structural insights into its binding properties. Interaction with acetylated carbohydrates differs from the one previously described for L-ficolin. This study also reveals the structural determinants for binding to sialylated compounds, a property restricted to human M-ficolin and its mouse counterpart, ficolin B. Finally, comparison between the ligand-bound structures obtained at neutral pH and nonbinding conformations observed at pH 5.6 reveals how the ligand binding site is dislocated at acidic pH. This means that the binding function of M-ficolin is subject to a pH-sensitive conformational switch. Considering that the homologous ficolin B is found in the lysosomes of activated macrophages (Runza, V. L., Hehlgans, T., Echtenacher, B., Zahringer, U., Schwaeble, W. J., and Mannel, D. N. (2006) J. Endotoxin Res. 12, 120-126), we propose that this switch could play a physiological role in such acidic compartments.     

Triggering of dendritic cell responses after exposure to activated, but not resting, apoptotic PBMCs

Dendritic cells (DCs) can be activated by signaling via pathogen receptors, by interaction with activated T cells or by exposure to inflammatory mediators. Clearance of apoptotic cells by DCs is generally considered a silent event that is not associated with an inflammatory response. Necrotic cell death, in contrast, leads to induction of inflammation. However, emerging data challenge the view of apoptotic cells as inherently nonimmunogenic. In this study, we report that the activation state of the apoptotic cell may determine whether the exposed DC becomes activated and rendered proficient in Ag presentation. We show that coculture with activated, but not resting, apoptotic PBMCs leads to up-regulation of surface expression of the costimulatory molecules CD80, CD83, and CD86 in human DCs as well as release of proinflammatory cytokines. Furthermore, we show that DCs exposed to allogeneic, activated apoptotic PBMCs induce proliferation and IFN-gamma production in autologous T cells. Together, these findings show that activated apoptotic PBMCs per se provide an activation/maturation signal to DCs, suggesting that activated apoptotic PBMCs possess endogenous adjuvant properties.     

Genome structure impacts molecular evolution at the AvrLm1 avirulence locus of the plant pathogen Leptosphaeria maculans

Leptosphaeria maculans, a dothideomycete fungus causing stem canker on oilseed rape, develops gene-for-gene interactions with its host plants. It has the ability to rapidly adapt to selection pressure exerted by cultivars harbouring novel resistance genes as exemplified recently by the 3-year evolution towards virulence at the AvrLm1 locus in French populations. The AvrLm1 avirulence gene was recently cloned and shown to be a solo gene within a 269 kb non-coding, heterochromatin-like region. Here we describe the sequencing of the AvrLm1 genomic region in one avirulent and two virulent isolates to investigate the molecular basis of evolution towards virulence at the AvrLm1 locus. For these virulent isolates, the gain of virulence was linked to a 260 kb deletion of a chromosomal segment spanning AvrLm1 and deletion breakpoints were identical or similar. Among the 460 isolates analysed from France, Australia and Mexico, a similar large deletion was apparent in > 90% of the virulent isolates. Deletion breakpoints were also strongly conserved in most of the virulent isolates, which led to the hypothesis that a unique deletion event leading to the avrLm1 virulence has diffused in pathogen populations. These data finally suggest that retrotransposons are key drivers in genome evolution and adaptation to novel selection pressure in L. maculans.     

Flaxseed alone or in combination with tamoxifen inhibits MCF-7 breast tumor growth in ovariectomized athymic mice with high circulating levels of estrogen

Flaxseed (FS) is rich in mammalian lignan precursors and alpha-linolenic acid, which have been suggested as having anticancer effects. Previous studies have shown that 10% FS inhibits the growth of human estrogen-dependent breast cancer (MCF-7) in athymic mice, and it enhances the inhibitory effect of tamoxifen (TAM). This study determined whether the effect of FS, alone or in combination with TAM, is dose dependent, and it explored the potential mechanism of action. Ovariectomized athymic mice with estradiol (E2) supplementation (1.7 mg/pellet, 60-day release) and established MCF-7 tumors were treated with basal diet control (0FS), 5% FS (5FS), 10% FS (10FS), and TAM (TAM/ 0FS; 5 mg/pellet, 60-day release), alone or in combination (TAM/ 5FS and TAM/10FS) for 8 weeks. Compared with control, 5FS and 10FS significantly inhibited tumor growth by 26% and 38%, respectively. TAM/0FS had an effect similar to the 10FS. TAM/ 5FS and TAM/10FS, respectively, induced significant 48% and 43% reductions in tumor size compared with 0FS, and 18% and 10% reductions compared with TAM/0FS. The relative uterine weight was significantly lower in all TAM groups compared with the control. The reduction of tumor growth resulted from decreased cell proliferation and increased cell apoptosis. TAM/ 5FS caused a significantly higher expression of estrogen receptor-alpha (ERalpha) compared with 5FS and TAM/0FS, whereas TAM/10FS had a higher ERalpha than 10FS and TAM/0FS. Compared with the control, progesterone receptor (PgR) expression was significantly reduced in all treatment groups, but insulin-like growth factor-1 (IGF-1) expression was reduced only by 10FS, TAM/5FS and TAM/10FS. Tumor cell proliferation was significantly positively associated with expression of PgR and IGF-1 and negatively associated with apoptosis and ERalpha. Apoptosis was only associated with ERalpha. In conclusion, FS inhibited MCF-7 tumor growth in a dose-dependent manner and enhanced the inhibitory effect of TAM due to the modulation of ER and growth factor signal transduction pathways.     

Petrosal and inner ear anatomy and allometry amongst specimens referred to Litopterna (Placentalia)

New isolated petrosals from the Itaboraí beds of Brazil (late Palaeocene or early Eocene) are here described and referred to the early diverging litoptern Miguelsoria parayirunhor, based on phylogenetic, size, and abundance arguments. Both the external and internal anatomy of these specimens were investigated, which for the first time document many details of the auditory region of a Palaeogene litoptern. Our cladistic analysis, which included our new observations, failed to recover a monophyletic Litopterna but did not exclude it. A constrained analysis for the monophyly of this order showed that several features such as a (sub)quadrangular and anteroposteriorly elongated tensor tympani fossa and a large notch in the vicinity of the external opening of the cochlear canaliculus may constitute synapomorphies for Litopterna. The evolution of several other auditory characters amongst Litopterna is discussed and the relative dimensions of the inner ear and surrounding petrosal in the group were also investigated. This allowed detection of negative allometry of the bony labyrinth within the petrosal, which was confirmed by measurements and regression analysis across a larger sample of placental mammals. This scaling effect probably has an important influence on several characters of the bony labyrinth and petrosal, amongst which are the length of the vestibular aqueduct and cochlear canaliculus. It demonstrates that many aspects of the morphological variation of the bony labyrinth need to be thoroughly investigated before being incorporated into phylogenetic analyses. © 2015 The Linnean Society of London     

Uncoupling phosphate deficiency from its major effects on growth and transcriptome via PHO1 expression in Arabidopsis

Inorganic phosphate (Pi) is one of the most limiting nutrients for plant growth in both natural and agricultural contexts. Pi‐deficiency leads to a strong decrease in shoot growth, and triggers extensive changes at the developmental, biochemical and gene expression levels that are presumably aimed at improving the acquisition of this nutrient and sustaining growth. The Arabidopsis thaliana PHO1 gene has previously been shown to participate in the transport of Pi from roots to shoots, and the null pho1 mutant has all the hallmarks associated with shoot Pi deficiency. We show here that A. thaliana plants with a reduced expression of PHO1 in roots have shoot growth similar to Pi‐sufficient plants, despite leaves being strongly Pi deficient. Furthermore, the gene expression profile normally triggered by Pi deficiency is suppressed in plants with low PHO1 expression. At comparable levels of shoot Pi supply, the wild type reduces shoot growth but maintains adequate shoot vacuolar Pi content, whereas the PHO1 underexpressor maintains maximal growth with strongly depleted Pi reserves. Expression of the Oryza sativa (rice) PHO1 ortholog in the pho1 null mutant also leads to plants that maintain normal growth and suppression of the Pi‐deficiency response, despite the low shoot Pi. These data show that it is possible to unlink low shoot Pi content with the responses normally associated with Pi deficiency through the modulation of PHO1 expression or activity. These data also show that reduced shoot growth is not a direct consequence of Pi deficiency, but is more likely to be a result of extensive gene expression reprogramming triggered by Pi deficiency.