Investigations on mRNA maturation disturbancies in the pathogenesis of psoriasis

Despite the advances in the field of psoriasis research, several questions remain that need to be answered considering the exact molecular pathogenesis of the disorder, including the description of molecular triggers and the mechanism of disorder initiation. However, the altered response of keratinocytes to the T-cell signals is undoubtful an essential factor of psoriasis development. To date, relatively few studies have been conducted on mRNA maturation disturbances in psoriasis. In the study of Ting et al. was shown that the EDA+ domain containing fibronectin isoform (EDA+ fibronectin) is overexpressed in psoriatic non-involved epidermis. We found that proliferating keratinocytes (both normal cultured keratinocytes and HaCaT cells) are able to produce this isoform. EDA+ fibronectin expression was modest in healthy keratinocytes whereas keratinocytes from psoriatic non-involved epidermis proved to be effective producers of this splice variant. In our cDNA microarray study we examinated the T-lymphokine induced gene expression changes. In the applied experimental setup, organotypic cultures were generated from both healthy and psoriatic epidermis samples, and half of them were treated with a lymphokine mixture containing GM-CSF, IFN-gamma and IL-3. It was previously proven that IFN-gamma in the presence of GM-CSF and IL-3 is able to promote proliferation of the keratinocyte precursors originating from the non-involved epidermis, therefore, these mediators could play a crucial role in the early steps of disorder development. In the following analysis, a comparison has been made between the autologuous pairs of untreated and treated samples, and the differentially regulated genes among healthy and psoriatic non-involved epidermis samples were selected for further analyses. An important outcome of the study was that due to T-lymphokine treatment, several identified genes showed upregulation in healthy epidermis, while in the non-involved epidermis, downregulation or unchanged gene expression was experienced. Moreover, we have also proved that certain SR-rich splicing regulators showed altered responsiveness to T-lymphokine stimuli. In this experiment, we identified splicing factor, luc-7 like protein 3 (LUC7L3), peptidyl-prolyl cis-trans isomerase G (PPIG) and arginine/serine-rich 18 (SFRS18), differentially regulated among healthy and psoriatic non-involved epidermis, therefore they could contribute to the responsiveness changes of keratinocytes

PPIG, SFRS-18 és LUC7L3 Splicing regulárok vizsgálata szinkronizált, immortalizált sejtvonalakban és pikkelysömörben

Az mRNS érés (splicing) folyamatának vizsgálata egészen újfajta megközelítésnek számít a pikkelysömör kutatásban: a splicing mechanizmus e betegségben tapasztalt eltéréseiről mindössze néhány közlemény jelent meg napjainkig. Egy nemrég elvégzett microarray vizsgálatban több olyan gént is azonosítottunk, amelyek T-limfokin kezelés hatására eltérő kifejeződés változással reagálnak, majd bioinformatikai módszerekkel tovább elemeztük azokat. A gének között három olyat is találtunk (serine/ arginine-rich splicing factor 18 (SFRS18), peptydilpropyl isomerase G (PPIG), luc-7 like 3 (LUC7L3)) amelyek funkciója az mRNS érés szabályozásához kötődik. A PPIG, SFRS18 és LUC7L3 gének kifejeződését szinkronizált, immortalizált sejtvonalak segítségével követtük. Két független sejtvonal (HaCaT és HPV-KER) esetében is azt tapasztaltuk, hogy a splicing regulátorok kifejeződési mintázata jelentős hasonlóságot mutat. Ezekből az eredményekből arra következtettünk, hogy a gének upstream regulációja közös transzkripciós faktorok útján valósulhat meg. A splicing gének fehérjeszintű expresszióját is meghatároztuk Western blot technika segítségével. Előkísérleteinkben azt találtuk, hogy az SFRS18 és a PPIG expressziója fehérje szinten is hasonlóságot mutat a szinkronizált, immortalizált HPV-KER sejtekben. Az in vitro analízisek mellett egészséges és pikkelysömörös betegekből származó tünetes és tünetmentes mintákon is megvizsgáltuk, miként alakul a három fehérje expressziója. Kísérleteink során megmutattuk, hogy a PPIG fehérje expressziós mintázata és kifejeződésének mennyisége is jelentősen megváltozik: a tünetmentes bőrben a PPIG festődése fokozottnak mutatkozott és eloszlása is eltér az egészséges bőrétől. Eredményeink alapján úgy gondoljuk, hogy a splicing szabályozás eltéréseinek fontos szerepe lehet a pikkelysömör tüneteinek kifejlődésében. A splicing folyamatának további tanulmányozása nagyban segítheti a betegség komplex molekuláris hátterének megértését

What have we learned about non-involved psoriatic skin from large-scale gene expression studies?

Under våren 2015 och hösten 2014 har en mängd trafikmätningar, fasadreduktionsmätningar och modellering gjorts av trafik och byggnader i Stockholms stad. Syftet med mätningarna har varit att undersöka trafikbullersituationen som har varit en del av många debatter senaste åren. Mätningarna och modelleringen har snabbt visat en bild av hur problem uppstår i bostäder på grund av lågfrekvent buller som skapas av långsamtgående trafik. Allteftersom arbetet fortskritt har det blivit tydligare att just lågfrekvent buller bör beaktas i större grad än vad det gör idag vid stadsplanering, projektering av, och besiktning av byggnader. Detta arbete presenterar och diskuterar problematik kring regelverk om trafikbuller i Stockholm där hastighetsbegränsning är maximalt 50 km/h. Problematiken är kopplad till det lågfrekventa buller som många utsätts för i sina hem och avsaknaden av riktlinjer som behandlar det. Genom att presentera och diskutera dagens byggnormer, riktvärden, krav och skrivelser som behandlar trafikbuller samt ställa dessa mot mätdata och modellering av typfasader som uppförts senaste århundradet ges en tydlig bild av problematiken som uppstår med lågfrekvent trafikbuller.A great number of traffic noise measurements, façade transmission loss measurements and modelling of buildings in Stockholm have been undergone in the spring of 2015 and late autumn of 2014. The main purpose of these has been to investigate the traffic noise situation which has been the subject of many [also ongoing] debates. The measurements together with the modelling have shown existing problems with low frequency noise present in dwellings due to slow moving traffic. As the investigation has proceeded it’s become evident that in particular low frequency noise should be emphasized and considered in the planning of cities and execution and surveying of buildings. This work presents and discusses issues related to governmental guidelines and rules regarding traffic noise in Stockholm where the speed limit does not exceed 50 km/h. The issue is connected to the low frequency noise that people experience in their dwelling and the lack of guidelines controlling it. By presenting and discussing current building standards, guidelines, governmental rules and literature regarding traffic noise control and by demonstrating the findings from the acquired data, the reader will understand the problems related to low frequency traffic noise

Splicing regulation disturbances in psoriasis pathogenesis

In a recently performed cDNA microarray experiment we identified three splicing regulators (serine/arginine-rich splicing factor 18 (SFRS18), peptydilpropyl isomerase G (PPIG), luc-7 like3 (LUC7L3) that where differentially expressed in response to T-lymphokines in healthy and psoriatic non-involved epidermis samples. We and others have previously shown that the oncofetal splice variant of fibronectin - the isoform containing the EDA domain (EDA+) - is overexpressed in psoriatic non-involved epidermis. In this study we investigated whether SFRS18, LUC7L3 and PPIG are able to alter the ratio of the normal (EDA-) and psoriasis-associated (EDA+) variant. To this aim the expressions of the splicing regulators were silenced in immortalized keratinocytes (HPV-KER). We found that the EDA+/EDA- ratio was altered upon silencing of LUC7L3 and PPIG: before silencing the expression level of EDA+ fibronectin isoform was higher than the EDA- fibronectin, but as a result of silencing the amount of the two splice variants became comparable. In addition, the expression patterns of the splicing regulators were compared in two different synchronized, immortalized cell lines, HaCaT and HPV-KER cells. Gene and protein expression patterns of the three regulators were very similar in both cell lines during their proliferation and differentiation suggesting that they may share common regulation

Splicing factors differentially expressed in psoriasis alter mRNA maturation of disease-associated EDA+ fibronectin.

The EDA+ fibronectin splicing variant is overexpressed in psoriatic non-lesional epidermis and sensitizes keratinocytes to mitogenic signals. However, regulation of its abundance is only partially understood. In our recent cDNA microarray experiment, we identified three SR-rich splicing factors-splicing factor, arginine/serine-rich 18 (SFRS18), peptidyl-prolyl cis-trans isomerase G (PPIG), and luc-7 like protein 3 (LUC7L3)-which might be implicated in the preactivated states of keratinocytes in psoriatic non-involved skin and could also contribute to the regulation of fibronectin mRNA maturation. In this study, we investigated the role of LUC7L3, PPIG, and SFRS18 in psoriasis and in the mRNA maturation process of fibronectin. Regarding tissue staining experiments, we were able to demonstrate a characteristic distribution of the splicing factors in healthy, psoriatic non-involved and involved epidermis. Moreover, the expression profiles of these SR-rich proteins were found to be very similar in synchronized keratinocytes. Contribution of splicing facwwtors to the EDA+ fibronectin formation was also confirmed: their siRNA silencing leads to altered fibronectin mRNA and protein expression patterns, suggesting the participation in the EDA domain inclusion. Our results indicate that LUC7L3, PPIG, and SFRS18 are not only implicated in EDA+ fibronectin formation, but also that they could possess multiple roles in psoriasis-associated molecular abnormalities

Inhibition of Opioid Receptor Mediated G-Protein Activity After Chronic Administration of Kynurenic Acid and its Derivative without Direct Binding to Opioid Receptors.

There is an increasing number of evidence showing analgesic properties of the kynurenic acid (KYNA), and also some studies demonstrate that kynurenine might interact with the opioid system. Therefore in this study, for the first time we investigated the direct binding affinity of KYNA and its structural analog KYNA-1 towards mu, kappa and delta opioid receptor in competition binding experiments applying opioid receptor specific radioligands. The binding affinity measurements were performed in Chinese hamster ovary cell lines overexpressing the corresponding opioid receptor (mu and kappa opioid receptor were rat, delta opioid receptor were mouse sequence). Additionally we also examined the chronic effect of these compounds on mu, kappa and delta opioid receptor and also nociceptin peptide receptor mediated G-protein activity in [(35)S]GTPgammaS binding assays performed in mouse cortex and striatum membranes. Our results showed that KYNA and KYNA-1 had no affinity towards any of the three classic opioid receptors. On the other hand the compounds significantly decreased opioid and nociceptin receptor mediated G-protein activity or in some cases enhanced the potency of the activating ligand. Moreover, the alterations were receptor and brain region specific. Accordingly, we conclude that KYNA and KYNA-1 do not interact directly with the opioid receptors, but more likely alter the receptor functions intracellularly

Further Characterization of Hemopressin Peptide Fragments in the Opioid and Cannabinoid Systems

BACKGROUND:: Hemopressin, so-called because of its hypotensive effect, belongs to the derivatives of the hemoglobin α-chain. It was isolated from rat brain membrane homogenate by the use of catalytically inactive forms of endopeptidase 24.15 and neurolysin. Hemopressin has antihyperalgesic features that cannot be prevented by the opioid receptor antagonist, naloxone. METHODS:: In the present study, we investigated whether hemopressin (PVNFKFLSH) and its C-terminally truncated fragment hemopressin 1–7 (PVNFKFL) have any influence on opioid-dependent signaling. Peptides have been analyzed using G-protein–stimulating functional and receptor bindings in this experimental setup. RESULTS:: These 2 compounds efficiently activated the G-proteins, and naloxone slightly blocked this stimulation. At the same time, they were able to displace radiolabeled [H]DAMGO, a selective ligand for μ-opioid system, at micromolar concentrations. Displacement caused by the heptapeptide was more modest compared with hemopressin. Experiments performed on cell lines overexpressing μ-opioid receptors verified the opioid activity of both hemopressins. Moreover, the CB1 cannabinoid receptor antagonist, AM251, significantly decreased their G-protein stimulatory effect. CONCLUSIONS:: Here, we further confirm that hemopressins can modulate CB1 receptors and can have a slight modulatory effect on the opioid system. © 2015 International Anesthesia Research Society