LOCALIZATION AND ORIENTATION OF METHOXY FLAVONOIDS IN DPPC BILAYERS: EFFECT ON THEIR ANTI-PROLIFERATIVE ACTIVITY

Objective: Flavones and flavonols are an important class of naturally occurring flavonoids. They are well known for their pharmacological activity. This activity is associated with the ability of flavones and flavonols to influence membrane–dependent processes. In this paper, we have reported localization, orientation and interaction, of four synthesized flavone/flavonols with 1, 2–dipalmitoyl–sn–glycero–3–phosphocholine (DPPC) bilayers. These are compared with standard flavone; chrysin (CHY) and flavonol Quercetin (QUE).Methods: The molecules studied are 4ʹ–methoxy flavone (MF), 3ʹ,4ʹ–dimethoxyflavone (DMF), 4ʹ–methoxyflavonol (MF–ol) and 3ʹ,4ʹ–dimethoxyflavonol (DMF–ol). The techniques used are Differential Scanning Calorimetry (DSC) and multi–nuclear NMR.Results: Highest binding to lipid bilayers is shown by DMF, followed by QUE. Based on DSC studies it is seen, that maximum interaction of MF and DMF, takes place with the hydrophobic core of lipid bilayers. DMF–ol shows formation of a heterogeneous system at higher concentrations. The 1H NMR spectra of unilamellar vesicles of DPPC, incorporated with MF, DMF and MF–ol shows significant interaction of these compounds with the alkyl chain of the hydrophobic core. MF, DMF and MF–ol acquire parallel orientation in bilayers with the B–ring pointing towards hydrophobic core, while DMF–ol acquire mixed orientation. This may be ascribed to the presence of two methoxy and one hydroxyl group on the B–ring of DMF–ol which hinders its partitioning inside the hydrophobic core of lipid bilayer. Multi–lamellar vesicles (MLV) of DPPC incorporated with flavones, show maximum increase in Chemical Shift Anisotropy in 31P spectrum of DMF. This is followed by MF. DSC.Conclusion: NMR and binding studies indicate that DMF is partitioned deeply inside the hydrophobic core, while MF, MF–ol and DMF–ol are mostly located in the vicinity of sn–glycero region. Therefore, we conclude that DMF which penetrates deepest inside the hydrophobic core also shows the highest anti–proliferative activity against K562 and MCF–7 cancer cell lines. Its activity is also better than CHY

EFFECT OF METHYL SUBSTITUTION IN FLAVONES ON ITS LOCALIZATION AND INTERACTION WITH DPPC MODEL MEMBRANE: IMPLICATIONS FOR ANTI-PROLIFERATIVE ACTIVITY

Objective: Flavones are an important class of naturally occurring molecules possessing multiple pharmacological activities. The anti-proliferative activity is associated with the ability of flavones to influence membrane–dependent processes. We have investigated the localization and interaction of the synthesized flavones: 4΄–methylflavone (4MF) and 4΄–methyl–7–hydroxy flavone (4M7HF) with 1,2–dipalmitoyl–sn–glycero–3–phosphocholine (DPPC) model membrane. Methods: Diferential Scanning Calorimetry (DSC) and multi nuclear NMR were used to study the interactions with DPPC model membrane. The extent of interaction of these compounds has been compared with the parent molecules: flavone (FLV) and 7–hydroxy flavone (7HF). Results: Results of DSC and NMR indicate that FLV partitions deepest inside the hydrophobic core and 7HF is localized mostly at the lipid/water interface. 4MF and 4M7HF lying in between the hydrophilic and hydrophobic core. All four molecules assume a mixed orientation with respect to the bilayer normal as indicated by chemical shifts of the lipid protons in NMR. Interaction with the membrane follows the order FLV>4MF>4M7HF>7HF. Radical scavenging activity parallels the presence of hydroxyl groups. Although FLV interacts highest with the membrane, it does not show highest antiproliferative activity. Interaction of the compounds with protons 3, 5a and 7 of DPPC is improved by the methyl substitution on the B-ring, so is the antiproliferative activity. Conclusion: That's antiproliferative activity of the compounds is at least partially related to the interaction of these molecules with the lipid water interface region

Potential Mechanisms Underlying TGF-β-mediated Complement Activation in Lung Fibrosis

While our previous studies suggest that limiting bleomycin-induced complement activation suppresses TGF-β signaling, the specific hierarchical interactions between TGF-β and complement in lung fibrosis are unclear. Herein, we investigated the mechanisms underlying TGF-β-induced complement activation in the pathogenesis of lung fibrosis. C57-BL6 mice were given intratracheal instillations of adenoviral vectors overexpressing TGF-β (Ad-TGFβ) or the firefly gene-luciferase (Ad-Luc; control). Two weeks later, mice with fibrotic lungs were instilled RNAi specific to receptors for C3a or C5a-C3ar or C5ar, and sacrificed at day 28. Histopathological analyses revealed that genetic silencing of C3ar or C5ar arrested the progression of TGF-β-induced lung fibrosis, collagen deposition and content (hydroxyproline, col1a1/2); and significantly suppressed local complement activation. With genetic silencing of either C3ar or C5ar, in Ad-TGFβ-injured lungs: we detected the recovery of Smad7 (TGF-β inhibitor) and diminished local release of DAF (membrane-bound complement inhibitor); in vitro: TGF-β-mediated loss of DAF was prevented. Conversely, blockade of the TGF-β receptor prevented C3a-mediated loss of DAF in both normal primary human alveolar and small airway epithelial cells. Of the 52 miRNAs analyzed as part of the Affymetrix array, normal primary human SAECs exposed to C3a, C5a or TGF-β caused discrete and overlapping miRNA regulation related to epithelial proliferation or apoptosis (miR-891A, miR-4442, miR-548, miR-4633), cellular contractility (miR-1197) and lung fibrosis (miR-21, miR-200C, miR-31HG, miR-503). Our studies present potential mechanisms by which TGF-β activates complement and promotes lung fibrosis

Developmental disruption and restoration of brain synaptome architecture in the murine Pax6 neurodevelopmental disease model

Neurodevelopmental disorders of genetic origin delay the acquisition of normal abilities and cause disabling phenotypes. Nevertheless, spontaneous attenuation and even complete amelioration of symptoms in early childhood and adolescence can occur in many disorders, suggesting that brain circuits possess an intrinsic capacity to overcome the deficits arising from some germline mutations. We examined the molecular composition of almost a trillion excitatory synapses on a brain-wide scale between birth and adulthood in mice carrying a mutation in the homeobox transcription factor Pax6, a neurodevelopmental disorder model. Pax6 haploinsufficiency had no impact on total synapse number at any age. By contrast, the molecular composition of excitatory synapses, the postnatal expansion of synapse diversity and the acquisition of normal synaptome architecture were delayed in all brain regions, interfering with networks and electrophysiological simulations of cognitive functions. Specific excitatory synapse types and subtypes were affected in two key developmental age-windows. These phenotypes were reversed within 2-3 weeks of onset, restoring synapse diversity and synaptome architecture to the normal developmental trajectory. Synapse subtypes with rapid protein turnover mediated the synaptome remodeling. This brain-wide capacity for remodeling of synapse molecular composition to recover and maintain the developmental trajectory of synaptome architecture may help confer resilience to neurodevelopmental genetic disorders

Neutron Diffraction Study of Field Cooling Effects on Relaxor Ferroelectrics Pb[(Zn_{1/3} Nb_{2/3})_{0.92} Ti_{0.08}] O_{3}

High-temperature (T) and high-electric-field (E) effects on Pb[(Zn_{1/3} Nb_{2/3})_{0.92} Ti_{0.08}]O_3 (PZN-8%PT) were studied comprehensively by neutron diffraction in the ranges 300 <= T <= 550 K and 0 <= E <= 15 kV/cm. We have focused on how phase transitions depend on preceding thermal and electrical sequences. In the field cooling process (FC, E parallel [001] >= 0.5 kV/cm), a successive cubic (C) --> tetragonal (T) --> monoclinic (M_C) transition was observed. In the zero field cooling process (ZFC), however, we have found that the system does not transform to the rhombohedral (R) phase as widely believed, but to a new, unidentified phase, which we call X. X gives a Bragg peak profile similar to that expected for R, but the c-axis is always slightly shorter than the a-axis. As for field effects on the X phase, we found an irreversible X --> M_C transition via another monoclinic phase (M_A) as expected from a previous report [Noheda et al. Phys. Rev. Lett. 86, 3891 (2001)]. At a higher electric field, we confirmed a c-axis jump associated with the field-induced M_C --> T transition, which was observed by strain and x-ray diffraction measurements.Comment: 8 pages, 9 figures, revise

Evidence for MBM_B and MCM_C phases in the morphotropic phase boundary region of (1−x)[Pb(Mg1/3Nb2/3)O3]−xPbTiO3(1-x)[Pb(Mg_{1/3}Nb_{2/3})O_3]-xPbTiO_3 : A Rietveld study

We present here the results of the room temperature dielectric constant measurements and Rietveld analysis of the powder x-ray diffraction data on $(1-x)[Pb(Mg_{1/3}Nb_{2/3})O_3]-xPbTiO_3$(PMN-$x$PT) in the composition range $0.20 \leq x \leq 0.45$ to show that the morphotropic phase boundary (MPB) region contains two monoclinic phases with space groups Cm (or $M_B$ type) and Pm (or $M_C$ type) stable in the composition ranges $0.27 \leq x \leq 0.30$ and $0.31 \leq x \leq 0.34$, respectively. The structure of PMN-$x$PT in the composition ranges $0 \leq x \leq$ 0.26, and $0.35 \leq x \leq1$ is found to be rhombohedral (R3m) and tetragonal (P4mm), respectively. These results are compared with the predictions of Vanderbilt & Cohen's theory.Comment: 20 pages, 11 pdf figure

Low temperature superlattice in monoclinic PZT

TEM has shown that the strongly piezoelectric material Pb(Zr0.52Ti0.48)O3 separates into two phases at low temperatures. The majority phase is the monoclinic phase previously found by x-ray diffraction. The minority phase, with a nanoscale coherence length, is a slightly distorted variant of the first resulting from the anti-phase rotation of the oxygen octahedra about [111]. This work clears up a recent controversy about the origin of superlattice peaks in these materials, and supports recent theoretical results predicting the coexistence of ferroelectric and rotational instabilities.Comment: REVTeX4, 4 eps figures embedded. JPG version of figs. 2&4 is also include

Developmental disruption and restoration of brain synaptome architecture in the murine Pax6 neurodevelopmental disease model

Neurodevelopmental disorders of genetic origin delay the acquisition of normal abilities and cause disabling phenotypes. Nevertheless, spontaneous attenuation and even complete amelioration of symptoms in early childhood and adolescence can occur in many disorders, suggesting that brain circuits possess an intrinsic capacity to overcome the deficits arising from some germline mutations. We examined the molecular composition of almost a trillion excitatory synapses on a brain-wide scale between birth and adulthood in mice carrying a mutation in the homeobox transcription factor Pax6, a neurodevelopmental disorder model. Pax6 haploinsufficiency had no impact on total synapse number at any age. By contrast, the molecular composition of excitatory synapses, the postnatal expansion of synapse diversity and the acquisition of normal synaptome architecture were delayed in all brain regions, interfering with networks and electrophysiological simulations of cognitive functions. Specific excitatory synapse types and subtypes were affected in two key developmental age-windows. These phenotypes were reversed within 2-3 weeks of onset, restoring synapse diversity and synaptome architecture to the normal developmental trajectory. Synapse subtypes with rapid protein turnover mediated the synaptome remodeling. This brain-wide capacity for remodeling of synapse molecular composition to recover and maintain the developmental trajectory of synaptome architecture may help confer resilience to neurodevelopmental genetic disorders

Crosstalk between TGF-β1 and complement activation augments epithelial injury in pulmonary fibrosis

The epithelial complement inhibitory proteins (CIPs) cluster of differentiation 46 and 55 (CD46 and CD55) regulate circulating immune complex-mediated complement activation in idiopathic pulmonary fibrosis (IPF). Our previous studies demonstrated that IL-17A mediates epithelial injury via transforming growth factor β1 (TGF-β1) and down-regulates CIPs. In the current study, we examined the mechanistic role of TGF-β1 in complement activation-mediated airway epithelial injury in IPF pathogenesis. We observed lower epithelial CIP expression in IPF lungs compared to normal lungs, associated with elevated levels of complement component 3a and 5a (C3a and C5a), locally and systemically. In normal primary human small airway epithelial cells (SAECs) treated with TGF-β1 (10 ng/ml), C3a, or C5a (100 nM), we observed loss of CIPs and increased poly(ADP-ribose) polymerase (PARP) activation [also observed with RNA interference (RNAi) of CD46/CD55]. TGF-β1-mediated loss of CIPs and Snail induction [SNAI1; a transcriptional repressor of E-cadherin (E-CAD)] was blocked by inhibiting mitogen-activated protein kinase (p38MAPK; SB203580) and RNAi silencing of SNAI1. C3a- and C5a-mediated loss of CIPs was also blocked by p38MAPK inhibition. While C3a upregulated TGFb transcripts, both C3a and C5a down-regulated SMAD7 (negative regulator of TGF-β), and whereas TGF-β1 induced C3a/C5a receptor (C3aR/C5aR) expression, pharmacologic C3aR/C5aR inhibition protected against C3a-/C5a-mediated loss of CIPs. Taken together, our results suggest that epithelial injury in IPF can be collectively amplified as a result of TGF-β1-induced loss of CIPs leading to complement activation that down-regulates CIPs and induces TGF-β1 expressio

IL‐17A deficiency mitigates bleomycin‐induced complement activation during lung fibrosis

Interleukin 17A (IL‐17A) and complement (C′) activation have each been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). We have reported that IL‐17A induces epithelial injury via TGF‐β in murine bronchiolitis obliterans; that TGF‐β and the C′ cascade present signaling interactions in mediating epithelial injury; and that the blockade of C′ receptors mitigates lung fibrosis. In the present study, we investigated the role of IL‐17A in regulating C′ in lung fibrosis. Microarray analyses of mRNA isolated from primary normal human small airway epithelial cells indicated that IL‐17A (100 ng/ml; 24 h; n = 5 donor lungs) induces C′ components (C′ factor B, C3, and GPCR kinase isoform 5), cytokines (IL8, ‐6, and ‐1B), and cytokine ligands (CXCL1, ‐2, ‐3, ‐5, ‐6, and ‐16). IL‐17A induces protein and mRNA regulation of C′ components and the synthesis of active C′ 3a (C3a) in normal primary human alveolar type II epithelial cells (AECs). Wild‐type mice subjected to IL‐17A neutralization and IL‐17A knockout (i717a−/−) mice were protected against bleomycin (BLEO)‐induced fibrosis and collagen deposition. Further, BLEO‐injured i17a−/− mice had diminished levels of circulating Krebs Von Den Lungen 6 (alveolar epithelial injury marker), local caspase‐3/7, and local endoplasmic reticular stress‐related genes. BLEO‐induced local C′ activation [C3a, C5a, and terminal C′ complex (C5b‐9)] was attenuated in il17a−/− mice, and IL‐17A neutralization prevented the loss of epithelial C′ inhibitors (C′ receptor‐1 related isoform Y and decay accelerating factor), and an increase in local TUNEL levels. RNAi‐mediated gene silencing of il17a in fibrotic mice arrested the progression of lung fibrosis, attenuated cellular apoptosis (caspase‐3/7) and lung deposition of collagen and C′ (C5b‐9). Compared to normals, plasma from IPF patients showed significantly higher hemolytic activity. Our findings demonstrate that limiting complement activation by neutralizing IL‐17A is a potential mechanism in ameliorating lung fibrosis.—Cipolla, E., Fisher, A. J., Gu, H., Mickler, E. A., Agarwal, M., Wilke, C. A., Kim, K. K., Moore, B. B., Vittal, R. IL‐17A deficiency mitigates bleomycin‐induced complement activation during lung fibrosis. FASEB J. 31, 5543–5556 (2017). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154482/1/fsb2fj201700289r-sup-0001.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154482/2/fsb2fj201700289r.pd