Development of a DNA transfer technique for human lung cells with synthetic peptide Tat-RGD and its application for regulatory and functional analysis of RELMB

The presented study is organized in two sections. In the first part a DNA-transfection procedure for human lung cells employing a peptide conjugate has been established and the corresponding transfection process characterized. In the second part this new transfection strategy has been applied for studying functional aspects of the human RELMBbeta gene. Cell-lines and primary cells exhibit a varying degree of resistance to DNA transfection strategies. In this study, we employed the synthetic peptide TatRGD (TR), composed of the HIV-1 derived translocation peptide Tat fused to the integrin binding RGD motif, as a tool for improving DNA transfer into human pulmonary cells. Binding experiments between DNA and TR and cytotoxicity measurements of TR treated cells were undertaken to optimize DNA and TR concentrations for transfection. Addition of a complex of TR and DNA (TRD) to A549 cells yielded in significant transgene expression. When combining TRD with Lipofectamine (TRDL), the expression was increased by 5-fold over Lipofectamine (DL) and by ~30-fold over TRD mediated transfections. Also, in primary smooth muscle cells (SMC) and fibroblasts (FB) derived from pulmonary arteries, an increase in TRDL mediated transfection efficiency was observed by a factor of ~ 2 and ~ 3 over that of DL. Laser scanning confocal microscopy for visualizing TR dependent DNA uptake demonstrated that the internalization of TRDL complexes is linked to caveoli in the plasma membrane. Interfering caveoli formation by methyl-b-cyclo-dextrin drastically decreased the transfection efficiency by TR. In conclusion, the TatRGD peptide mediates efficient gene delivery in human pulmonary cells, in particular when combined with standard cationic lipid based transfection reagent. The enhancement of DNA uptake by TatRGD is suggested to be mediated by caveoli dependent endocytosis. RELMBbeta(resistin-like molecule) represents the most related human homologue of mouse RELMBbeta also known as hypoxic-induced-mitogenic-factor (HIMF) for which no human orthologue gene exist. In this part of the study, we isolated RELMBbeta cDNA from human lung tissue and performed regulatory and functional expression studies with the TatRGD procedure. RELMBbeta mRNA was upregulated in hypoxia in human lung A549 cell line as well as primary cultured adventitial fibroblasts (FB) and smooth muscle cells (SMC) of pulmonary artery. Upon transfection of a RELMBbeta encoding expression plasmid into these cells, we observed significant induction of proliferation particularly in SMC and A549 cells. The results suggest that human RELMBbeta may contribute to hypoxic induced pulmonary vascular remodeling processes or hypoxia related fibrotic lung disease.Die vorgelegte Studie ist in zwei Abschnitte gegliedert. Im ersten Teil wird eine DNATransfektions Prozedur mit Hilfe eines Peptid-Konjugats etabliert und der zugehörige Transfektions Prozess charakterisiert. Im zweiten Teil wird diese Transfektions Strategie angewendet, um funktionelle Aspekte des humanen RELMBbeta Gens zu untersuchen. Zelllinien und primäre Zellen zeigen eine unterschiedliche Resistenz gegenüber DNATransfektions Methoden. In dieser Studie wurde das synthetische Peptid TatRGD (TR), das aus dem von HIV-1 abgeleitetem Translokations Peptid Tat und dem Integrin bindenden RGD Motiv zusammengesetzt ist, genutzt, um den DNA Transfer in humane pulmonale Zellen zu begünstigen. Bindungsexperimente zwischen DNA und TR, sowie Zytotoxizitäts Messungen von TR behandelten Zellen wurden durchgeführt, um die zu verwendenden DNA und TR Konzentrationen zu optimieren. Zugabe eines Komplexes von TR und DNA (TRD) zu A549 Zellen resultierte in signifikanter transgener Expression. Die Kombination von TRD und Lipofectamin (TRDL) verstärkte die Expression 5-fach gegebenüber Lipofectamin (DL) und 30-fach gegenüber TRD vermittelter Transfektion. Ebenso in primär kultivierten glatten Muskelzellen (SMC) und adventitialen Fibroblasten (FB), die aus Pulmonalarterien gewonnen wurden, wurde eine Steigerung der Transfektionseffizienz um den Faktor 2 bzw. 3 gegenüber DL beobachtet. Laser-Scan basierte konfokale Mikroskopie zeigte, dass die TR abhängige DNA Aufnahme abhängig von Caveolae in der Plasmamembran ist. Inhibition der Caveolae Bildung durch Methyl-b-Cyclo-Dextrin verminderte drastisch die Transfektions Effizienz durch TR. Zusammengefasst, vermittelt TatRGD einen effizienten DNA Transfer in humane pulmonale Zellen, insbesondere wenn es mit Transfektions Reagenzien, die auf kationischen Lipiden basieren, kombiniert wird. Die Steigerung der DNA Aufnahme wird vermutlich durch Caveolae-abhängige Endozytose vermittelt. RELMBbeta(resistin-like molecule) repräsentiert das am nächsten homologe Gen von RELMBbeta der Maus, das auch unter dem Namen HIMF (hypoxic-induced-mitogenicfactor) bekannt ist, für das aber kein orthologes humanes Gen existiert. In diesem Teil der Studie isolierten wir RELMBbeta cDNA von humanem Lungen Gewebe und führten funktionelle Expressions Studien mittels der TatRGD Methode sowie Untersuchungen zu seiner Regulation durch. Wir beobachteten eine Hochregulation von RELMBbeta mRNA in Hypoxie in der epithelialen humanen Lungenzellinie A549 sowie in primär kultivierten FB und SMC der Pulmonalarterie. Nach Transfektion der Zellen mit einem Expressions plasmid kodierend für RELMBbeta beobachteten wir eine signifikante Induktion der Proliferation insbesondere in SMC und A549 Zellen. Die Ergebnisse weisen darauf hin, dass humanes RELMBbeta am pulmonal-arteriellen Remodeling Prozess in Hypoxie oder an Hypoxie-assozierten fibrotischen Lungenerkrankungen beteiligt sein könnte

Biochemical and biophysical analyses of tight junction permeability made of claudin-16 and claudin-19 dimerization

The molecular nature of tight junction architecture and permeability is a long-standing mystery. Here, by comprehensive biochemical, biophysical, genetic, and electron microscopic analyses of claudin-16 and -19 interactions—two claudins that play key polygenic roles in fatal human renal disease, FHHNC—we found that 1) claudin-16 and -19 form a stable dimer through cis association of transmembrane domains 3 and 4; 2) mutations disrupting the claudin-16 and -19 cis interaction increase tight junction ultrastructural complexity but reduce tight junction permeability; and 3) no claudin hemichannel or heterotypic channel made of claudin-16 and -19 trans interaction can exist. These principles can be used to artificially alter tight junction permeabilities in various epithelia by manipulating selective claudin interactions. Our study also emphasizes the use of a novel recording approach based on scanning ion conductance microscopy to resolve tight junction permeabilities with submicrometer precision

Influence of Vermiwash, Panchagavya and Weed Extract on Growth, Yield and Seed Quality Parameters of Cluster Bean (Cyamopsistetragonoloba (L).)

Cluster bean (Cyamopsistetragonoloba L.) popularly known as guar is a drought tolerant, deep-rooted, annual legume is grown for Vegetable, Food, Fodder, Green manure, Gum and as a seed. The fortification of seeds for better Growth and Yield has become important and emphasized. The study was conducted to determine the ― Effect of Vermiwash, Panchagavya and Weed Extract on Growth, Yield and Seed Quality Parameters of Cluster bean (Cyamopsistetragonoloba (L).).The experiment was carried out at Field Experimentation Centre of the Department of Genetics and Plant Breeding, Sam Higginbottom University of Agriculture, Technology & Sciences. Prayagraj (UP) during Kharif-2019. The experiment was laid out in Randomised Blocked Design and comprised of 13 treatments and 3 replications. The treatments were T0 (Control),T1 – vermiwash 5% @ 12hrs, T2 - vermin wash 10% @ 12hrs, T3 - vermiwash 15% @ 12hrs, T4 -  vermiwash 20% @ 12hrs, T5 – panchagavya 5% @ 12hrs, T6 - panchagavya 10% @ 12hrs, T7 - panchagavya 15% @ 12hrs, T8 -  panchagavya 20% @ 12hrs, T9 – weed seed extract 5% @ 12 hrs, T10 - weed seed extract 10% @ 12 hrs, T11 – weed seed extract 15% @ 12 hrs, T12 - weed seed extract 20% @ 12 hrs. View Article DOI: 10.47856/ijaast.2021.v08i10.00

Human RELMβ is a mitogenic factor in lung cells and induced in hypoxia

AbstractRELMβ (resistin-like molecule) represents the most related human homologue of mouse RELMα, also known as hypoxic-induced mitogenic factor (HIMF). In this study, we isolated RELMβ cDNA from human lung tissue and performed regulatory and functional expression studies. RELMβ mRNA was upregulated in hypoxia in human lung A549 cell line as well as primary cultured adventitial fibroblasts and smooth muscle cells (SMC) of pulmonary arteries. Upon transfection of a RELMβ encoding expression plasmid into these cells, we observed significant induction of proliferation particularly in SMC and A549 cells, which could be blocked by phosphatidyl-inositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin. The results suggest that human RELMβ may contribute to hypoxic-induced pulmonary vascular remodeling processes or hypoxia related fibrotic lung disease

Mouse model for inherited renal fibrosis associated with endoplasmic reticulum stress

Renal fibrosis is a common feature of renal failure resulting from multiple etiologies, including diabetic nephropathy, hypertension and inherited renal disorders. However, the mechanisms of renal fibrosis are incompletely understood and we therefore explored these by establishing a mouse model for a renal tubular disorder, referred to as autosomal dominant tubulointerstitial kidney disease (ADTKD) due to missense uromodulin (UMOD) mutations (ADTKD-UMOD). ADTKD-UMOD, which is associated with retention of mutant uromodulin in the endoplasmic reticulum (ER) of renal thick ascending limb cells, is characterized by hyperuricemia, interstitial fibrosis, inflammation and renal failure, and we used targeted homologous recombination to generate a knock-in mouse model with an ADTKD-causing missense cysteine to arginine uromodulin mutation (C125R). Heterozygous and homozygous mutant mice developed reduced uric acid excretion, renal fibrosis, immune cell infiltration and progressive renal failure, with decreased maturation and excretion of uromodulin, due to its retention in the ER. The ER stress marker 78 kDa glucose-regulated protein (GRP78) was elevated in cells expressing mutant uromodulin in heterozygous and homozygous mutant mice, and this was accompanied, both in vivo and ex vivo, by upregulation of two unfolded protein response pathways in primary thick ascending limb cells from homozygous mutant mice. However, this did not lead to an increase in apoptosis in vivo Thus, we have developed a novel mouse model for renal fibrosis, which will be a valuable resource to decipher the mechanisms linking uromodulin mutations with ER stress and renal fibrosis

Tubulin Binds to the Cytoplasmic Loop of TRESK Background K+ Channel In Vitro.

The cytoplasmic loop between the second and third transmembrane segments is pivotal in the regulation of TRESK (TWIK-related spinal cord K+ channel, K2P18.1, KCNK18). Calcineurin binds to this region and activates the channel by dephosphorylation in response to the calcium signal. Phosphorylation-dependent anchorage of 14-3-3 adaptor protein also modulates TRESK at this location. In the present study, we identified molecular interacting partners of the intracellular loop. By an affinity chromatography approach using the cytoplasmic loop as bait, we have verified the specific association of calcineurin and 14-3-3 to the channel. In addition to these known interacting proteins, we observed substantial binding of tubulin to the intracellular loop. Successive truncation of the polypeptide and pull-down experiments from mouse brain cytosol narrowed down the region sufficient for the binding of tubulin to a 16 amino acid sequence: LVLGRLSYSIISNLDE. The first six residues of this sequence are similar to the previously reported tubulin-binding region of P2X2 purinergic receptor. The tubulin-binding site of TRESK is located close to the protein kinase A (PKA)-dependent 14-3-3-docking motif of the channel. We provide experimental evidence suggesting that 14-3-3 competes with tubulin for the binding to the cytoplasmic loop of TRESK. It is intriguing that the 16 amino acid tubulin-binding sequence includes the serines, which were previously shown to be phosphorylated by microtubule-affinity regulating kinases (MARK kinases) and contribute to channel inhibition. Although tubulin binds to TRESK in vitro, it remains to be established whether the two proteins also interact in the living cell

Sp1-regulated expression of p11 contributes to motor neuron degeneration by membrane insertion of TASK1

Disruption in membrane excitability contributes to malfunction and differential vulnerability of specific neuronal subpopulations in a number of neurological diseases. The adaptor protein p11, and background potassium channel TASK1, have overlapping distributions in the CNS. Here, we report that the transcription factor Sp1 controls p11 expression, which impacts on excitability by hampering functional expression of TASK1. In the SOD1-G93A mouse model of ALS, Sp1-p11-TASK1 dysregulation contributes to increased excitability and vulnerability of motor neurons. Interference with either Sp1 or p11 is neuroprotective, delaying neuron loss and prolonging lifespan in this model. Nitrosative stress, a potential factor in human neurodegeneration, stimulated Sp1 expression and human p11 promoter activity, at least in part, through a Sp1-binding site. Disruption of Sp1 or p11 also has neuroprotective effects in a traumatic model of motor neuron degeneration. Together our work suggests the Sp1-p11- TASK1 pathway is a potential target for treatment of degeneration of motor neurons

Pranlukast is a novel small molecule activator of the two-pore domain potassium channel TREK2

TREK2 (KCNK10, K2P10.1) is a two-pore domain potassium (K2P) channel and a potential target for the treatment of pain. Like the majority of the K2P superfamily, there is currently a lack of useful pharmacological tools to study TREK2. Here we present a strategy for identifying novel TREK2 activators. A cell-based thallium flux assay was developed and used to screen a library of drug-like molecules, from which we identified the CysLT1 antagonist Pranlukast as a novel activator of TREK2. This compound was selective for TREK2 versus TREK1 and showed no activity at TRAAK. Pranlukast was also screened against other members of the K2P superfamily. Several close analogues of Pranlukast and other CysLT1 antagonists were also tested for their ability to activate K2P channels. Consistent with previous work, structure activity relationships showed that subtle structural changes to these analogues completely attenuated the activation of TREK2, whereas for TREK1, analogues moved from activators to inhibitors. Pranlukast's activity was also confirmed using whole-cell patch clamp electrophysiology. Studies using mutant forms of TREK2 suggest Pranlukast does not bind in the K2P modulator pocket or the BL-1249 binding site. Pranlukast therefore represents a novel tool by which to study the mechanism of TREK2 activation

GI-530159, a novel, selective, mechanosensitive two‐pore‐domain potassium (K2P_{2P}) channel opener, reduces rat dorsal root ganglion neuron excitability

Background and Purpose: TREK two pore domain potassium channels play a critical role in regulating the excitability of somatosensory nociceptive neurons and are important mediators of pain perception. An understanding of the roles of TREK channels in pain perception and, indeed, in other pathophysiological conditions, has been severely hampered by the lack of potent and/or selective activators and inhibitors. In this study we describe a new, selective opener of TREK channels, GI-530159 (4,4?-(Hexafluoroisopropylidene)bis(p-phenyleneoxy)dianiline). Experimental Approach: The effect of GI-530159 on TREK channels was demonstrated using 86Rb efflux assays, whole-cell and single channel patch clamp recordings from recombinant TREK channels. TREK1, TREK2 and TRAAK expression was determined using transcriptome analysis from single dorsal root ganglion (DRG) cells. Current-clamp recordings from cultured rat DRG neurons were used to measure the effect of GI-530159 on neuronal excitability. Key Results: For recombinant human TREK1 channels GI-530159 had an EC50 of 0.8?M from Rb efflux experiments and 0.9?M from electrophysiological recordings. The compound activated TREK2 channels but it had no detectable action on TRAAK channels nor any significant effect on other K channels tested. Current clamp recordings from cultured rat DRG neurones showed that application of GI-530159 at 1?M resulted in a significant reduction in firing frequency and a small hyperpolarisation of resting membrane potential. Conclusions and Implications: This study provides pharmacological evidence for the presence of mechano-sensitive TREK K2P channels in sensory neurones and suggests that development of selective K2P channel openers like GI-530159 could aid to the development of novel analgesic agents