Priprava i ex vivo evaluacija TEC kao promotora apsorpcije tvari u kolonu

In previous studies, it was established that chitosan and its quaternized derivatives are potent enhancers of hydrophilic compounds absorption across intestinal epithelia. The aim of this study was to evaluate the application of a new quaternized chitosan, triethyl chitosan (TEC), in pharmaceutical approaches. TEC was synthesized by a one step process via a 22 factorial design to optimize the preparation conditions. In ex vivo experiments, everted rat colon sac was used to determine the effect of TEC on the penetration of hydrophilic compounds of different molecular masses (e.g., sodium fluorescein and brilliant blue) through colonic epithelia in comparison with chitosan at pH 7.4. These studies indicated a significant increase in absorption of sodium fluorescein and brilliant blue in the presence of TEC compared to chitosan. TEC bearing positive charge is able to interact with the tight junctions of colon epithelia and hence increase the permeation of sodium fluorescein and brilliant blue through the tight junctions. This investigation has shown that triethyl chitosan could be used as a penetration enhancer for poorly absorbable compounds in the colon drug delivery system.U ranijim istraživanjima utrđeno je da su kitozan i njegovi kvartenizirani derivati snažni promotori apsorpcije hidrofilnih spojeva kroz intestinalnu sluznicu. Cilj rada bio je evaluirati novi kvartenizirani kitozan, trietil kitozan (TEC ).TEC je sintetiziran u jednom stupnju. U ex vivo eksperimentima na kolonu štakora praćen je učinak tog polimera na penetraciju hidrofilnih spojeva različitih molekulskih masa (fluorescein natrija i briljant plavila). Rezultati su uspoređivani s učinkom kitozana na pH 7,4. Primjećeno je da TEC značajno povećava apsorpciju ispitivanih tvari u odnosu na nemodificirani kitozan. TEC svojim pozitivnim nabojem dolazi u interakciju s epitelom kolona i tako povećava njegovu permeabilnost. Ispitivanja ukazuju da se trietil kitozan može upotrijebiti kao promotor penetracije za spojeve koji se slabo apsorbiraju u kolonu

Priprava i ex vivo evaluacija TEC kao promotora apsorpcije tvari u kolonu

In previous studies, it was established that chitosan and its quaternized derivatives are potent enhancers of hydrophilic compounds absorption across intestinal epithelia. The aim of this study was to evaluate the application of a new quaternized chitosan, triethyl chitosan (TEC), in pharmaceutical approaches. TEC was synthesized by a one step process via a 22 factorial design to optimize the preparation conditions. In ex vivo experiments, everted rat colon sac was used to determine the effect of TEC on the penetration of hydrophilic compounds of different molecular masses (e.g., sodium fluorescein and brilliant blue) through colonic epithelia in comparison with chitosan at pH 7.4. These studies indicated a significant increase in absorption of sodium fluorescein and brilliant blue in the presence of TEC compared to chitosan. TEC bearing positive charge is able to interact with the tight junctions of colon epithelia and hence increase the permeation of sodium fluorescein and brilliant blue through the tight junctions. This investigation has shown that triethyl chitosan could be used as a penetration enhancer for poorly absorbable compounds in the colon drug delivery system.U ranijim istraživanjima utrđeno je da su kitozan i njegovi kvartenizirani derivati snažni promotori apsorpcije hidrofilnih spojeva kroz intestinalnu sluznicu. Cilj rada bio je evaluirati novi kvartenizirani kitozan, trietil kitozan (TEC ).TEC je sintetiziran u jednom stupnju. U ex vivo eksperimentima na kolonu štakora praćen je učinak tog polimera na penetraciju hidrofilnih spojeva različitih molekulskih masa (fluorescein natrija i briljant plavila). Rezultati su uspoređivani s učinkom kitozana na pH 7,4. Primjećeno je da TEC značajno povećava apsorpciju ispitivanih tvari u odnosu na nemodificirani kitozan. TEC svojim pozitivnim nabojem dolazi u interakciju s epitelom kolona i tako povećava njegovu permeabilnost. Ispitivanja ukazuju da se trietil kitozan može upotrijebiti kao promotor penetracije za spojeve koji se slabo apsorbiraju u kolonu

Two GCC boxes and AP2/ERF-domain transcription factor ORA59 in jasmonate/ethylene-mediated activation of the PDF1.2 promoter in Arabidopsis

Plant defense against microbial pathogens depends on the action of several endogenously produced hormones, including jasmonic acid (JA) and ethylene (ET). In defense against necrotrophic pathogens, the JA and ET signaling pathways synergize to activate a specific set of defense genes including PLANT DEFENSIN1.2 (PDF1.2). The APETALA2/Ethylene Response Factor (AP2/ERF)-domain transcription factor ORA59 acts as the integrator of the JA and ET signaling pathways and is the key regulator of JA- and ET-responsive PDF1.2 expression. The present study was aimed at the identification of elements in the PDF1.2 promoter conferring the synergistic response to JA/ET and interacting with ORA59. We show that the PDF1.2 promoter was activated synergistically by JA and the ET-releasing agent ethephon due to the activity of two GCC boxes. ORA59 bound in vitro to these GCC boxes and trans-activated the PDF1.2 promoter in transient assays via these two boxes. Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo. Finally, we show that a tetramer of a single GCC box conferred JA/ethephon-responsive expression, demonstrating that the JA and ET signaling pathways converge to a single type of GCC box. Therefore ORA59 and two functionally equivalent GCC box binding sites form the module that enables the PDF1.2 gene to respond synergistically to simultaneous activation of the JA and ET signaling pathways

Effect of glycated proteins and inhibitory compounds intervening with the AGE-RAGE pathway on macrophages

Glycation is a specific type of protein modification in ageing. The reaction of reducing sugar with a primary amino group is the most common nonenzymatic modification of proteins. Subsequent rearrangement, oxidation and dehydration yield a heterogeneous group of mostly coloured and fluorescent compounds, termed “Maillard products” or “advanced glycation end products (AGEs)”. AGEs are thought to play a role in the aetiology of various age-related diseases such as diabetes mellitus (DM) and Alzheimer's disease (AD). AD is a multifactorial disease, in which the rate of synapse loss and neuronal cell death determines the onset and/or progression of dementia. Activation of microglia and astrocytes with subsequent oxidative stress and cytokine release may be an important progression factor for AD. It is also suggested that the receptor for advanced glycation end products (RAGE) ligands-AGEs interaction might be another cause of glial activation, cytokine and reactive oxygen species (ROS) release. Different antioxidants, receptor mediated compounds and ROS scavenging enzymes might be able to intervene with the AGE-RAGE signalling pathway and slow down the progression of AD. The aim of this study is to investigate the effect of different compounds of antioxidants, receptor mediated compounds and ROS scavenging enzymes on inflammation induced by glycated proteins and to explore the suggested AGE-RAGE signalling pathway by examining inhibition of different parts of the signalling network

Rhinovirus 3C protease facilitates specific nucleoporin cleavage and mislocalisation of nuclear proteins in infected host cells

<div><p>Human Rhinovirus (HRV) infection results in shut down of essential cellular processes, in part through disruption of nucleocytoplasmic transport by cleavage of the nucleoporin proteins (Nups) that make up the host cell nuclear pore. Although the HRV genome encodes two proteases (2A and 3C) able to cleave host proteins such as Nup62, little is known regarding the specific contribution of each. Here we use transfected as well as HRV-infected cells to establish for the first time that 3C protease is most likely the mediator of cleavage of Nup153 during HRV infection, while Nup62 and Nup98 are likely to be targets of HRV2A protease. HRV16 3C protease was also able to elicit changes in the appearance and distribution of the nuclear speckle protein SC35 in transfected cells, implicating it as a key mediator of the mislocalisation of SC35 in HRV16-infected cells. In addition, 3C protease activity led to the redistribution of the nucleolin protein out of the nucleolus, but did not affect nuclear localisation of hnRNP proteins, implying that complete disruption of nucleocytoplasmic transport leading to relocalisation of hnRNP proteins from the nucleus to the cytoplasm in HRV-infected cells almost certainly requires 2A in addition to 3C protease. Thus, a specific role for HRV 3C protease in cleavage and mislocalisation of host cell nuclear proteins, in concert with 2A, is implicated for the first time in HRV pathogenesis.</p></div

Active 3C protease is sufficient to degrade nuclear proteins/nucleoporins in transfected cells.

<p>(<b>Ai</b>) COS-7 cells transfected to express either GFP-3C or GFP-3Cinac were trypsinized 18 h after transfection, harvested in ice-cold PBS and FACS sorted to collect GFP-expressing cells. Cells were then lysed using RIPA buffer plus protease and phosphatase inhibitors and lysates were subjected to Western analysis as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g001" target="_blank">Figure 1A</a>; untransfected cells were lysed similarly and used as control. The antibodies used are shown on the left of the figure. The arrow in the nucleolin blot denotes a clear cleavage product, with the approximate molecular weight (kDa) indicated on the right. (<b>Aii</b>) Results for densitometric analysis of nucleolin and Nup153 protein bands such as those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g003" target="_blank">Figure 3A</a>i, where data were normalised to the corresponding value of tubulin, relative to the corresponding value for the control sample. Densitometric analyses were performed using ImageJ; values represent the mean (+ SD) from two independent experiments (<b>Bi</b>) Ohio-Hela whole cell lysates were incubated with 4 units of HRV14 3C protease at 37°C for the indicated incubation times, subsequent to SDS-PAGE on 10% gels and Western analysis as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g001" target="_blank">Figure 1A</a>. (<b>Bii</b>) Plot of densitometric analysis of protein bands in (Bi), where data were normalised to the corresponding values for tubulin, relative to 0 h samples. Densitometric analyses were performed using Image J and values were the mean of two different experiments (± SD). (<b>C</b>) COS-7 cells transfected to express either GFP-3C or GFP-3Cinac were fixed and permeabilized 18 h post-transfection, and immunostained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g002" target="_blank">Figure 2</a> with the indicated primary and Alexa-568 conjugated secondary antibodies. Fluorescence was imaged by CLSM (see Materials and Methods). In each panel, images on the left depict localisation of HRV16 proteins (green channel) and the images in the middle depict localisation of cellular proteins (red channel), with the merged image on the right.</p

Specific nucleoporins are degraded in HRV16-infected cells.

<p>(<b>A</b>) Ohio-HeLa cells were infected without (mock) or with HRV16 (MOI of 1) and cells lysed using RIPA buffer containing protease and phosphatase inhibitors at the time points shown. Cell lysates were subjected to SDS-PAGE on 4–20% gradient gels and Western analysis using the indicated primary antibodies/horseradish peroxidise-conjugated secondary antibodies and enhanced chemiluminescence (Perkin Elmer). The specificity of the antibodies is indicated on the left. Bands corresponding to 3C, 3CD’ and 3CD are indicated on the right. p.i. - post-infection. (<b>B</b>) Results for densitometric analysis of FG-Nup protein bands (left) and non-FG-Nups (right) such as those shown in (A), where data were normalised to the corresponding values for tubulin, relative to the corresponding values for the mock sample. Densitometric analyses were performed using Image J; values represent the mean (± SD) from two independent experiments.</p

HRV16 infection leads to mislocalisation of nuclear proteins.

<p>Ohio-HeLa cells grown on coverslips were infected without (mock) or with HRV16 as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g001" target="_blank">Figure 1</a>; cells were fixed at the indicated times and permeabilized, and then probed with the indicated pairs of primary antibodies, followed by Alexa 488 and Alexa-568 conjugated secondary antibodies. Fluorescence was imaged by CLSM (see Materials and Methods). In each panel, images on the left depict localisation of HRV16 proteins (green channel) and the images in the middle depict localisation of cellular proteins (red channel), with the merged image on the right.</p