Cell wall acidification in growing barley (Hordeum vulgare L.) leaves

Apoplast acidification associated with growth is well-documented in roots and coleoptiles but not in leaves. In the present study, advantage was taken of high cuticle permeability in the elongation zone of barley leaves to measure apoplast pH and acidification and the role which the plasma membrane H+ ATPase (PM-ATPase) plays in this process. An in-vitro gel system and pH-microelectrodes were used to monitor pH, and growth was measured with a linear variable differential transformer (LVDT). Test reagents which blocked (vanadate) or stimulated (fusicoccin) PM-ATPase were applied to the leaf elongation zone. In addition, the expression level (qPCR) and activity of PM-ATPase was determined. Apoplast pH was lower in growing compared with non-growing tissue. Growth and apoplast acidification were stimulated by fusicoccin and reduced by vanadate. Expression of PM-ATPase, as judged from Ct-values, was almost identical in the two leaf regions. In contrast, activity of PM-ATPase, expressed per unit plasma membrane protein, was about twice as high in growing tissue. It is concluded that PM-ATPase is required in growing leaf cells to achieve maximum rates of elongation and apoplast acidification and that this is due in part to a higher plasma membrane surface density of PM-ATPase activity

Recent advances in understanding of the mechanism of paraquat resistance

Cationic amino acid/polyamine transporter CAT4 is supposed to play essential part in paraquat resistance of horseweed. In order to get better understanding on its role in resistance mechanism here we examined and compared the expression level of this transporter in the susceptible rape Brassica napus and in the different (susceptible and resistant) biotypes of horseweed. We found that paraquat induced an increase in expression level of CAT4 in rape, similarly to its upregulation in horseweed

Apoplast acidification in growing barley (Hordeum vulgare L.) leaves

Apoplast acidification associated with growth is well-documented in roots, coleoptiles and internodes but not in leaves. In the present project on barley (Hordeum vulgare L.) advantage was taken of the high cuticle permeability in the elongation zone of leaves to measure apoplast pH and growth in response to application of test reagents. The role of the plasma membrane H+-ATPase (PM-H+-ATPase) and K+ in this process was of particular interest. An in vitro gel system with bromocresol purple as pH indicator, pH microelectrodes and pH-sensitive fluorescence dye combined with confocal microscopy were used to monitor apoplast pH. Growth was measured in parallel or in separate experiments using a linear variable differential transformer (LVDT). Test reagents which blocked (vanadate) or stimulated (fusicoccin) PM-H+-ATPase, or which reduced (NH4 +, Cs+, tetraethylammonium) K+ uptake were applied. Plasma membranes were isolated from growing and mature leaf tissue and used to determine the activity (ATPase assay) and abundance (Western blotting) of PM-H+-ATPase protein. Protein localisation was studied by immunohistochemistry and expression of mRNA quantified using real time PCR (qPCR). Apoplast pH was by up to 1.0 pH unit lower in growing compared to nongrowing leaf tissue. Depending on the K+ concentration in the bathing medium used during electrophysiological analyses, apoplast pH in the elongation zone ranged from pH 4.8 (0.1 mM K+) to pH 5.8 (10 mM K+). In the emerged blade, apoplast pH remained at about pH 5.8 irrespective of the K+ concentration in the bathing medium Growth was more responsive to test reagents than to changes in apoplast pH. Expression of PM-H+- ATPase was comparable between growing and non-growing leaf regions when expression was related to per unit extracted RNA or cell number. However, when expression was related to per unit surface area of plasma membrane, expression of PM-H+-ATPase was about twice as high in growing compared to non-growing leaf tissue. The same applied to the protein level and activity of PM-H+-ATPase. Immunohistochemical analyses showed that PM-H+-ATPase was present in all living leaf tissues, particular in those (guard cells, phloem, and xylem parenchyma) associated with high rates of trans-membrane solute transport. It is concluded that leaf cell expansion in barley depends on the activity of the PM-H+-ATPase and K+ transport processes. The higher surface density of PM-H+-ATPase activity in growing barley leaf tissue aids apoplast acidification and growth. A H+ / K+ co-transport system may play a key role in linking growth with apoplast pH, H+ pump activity and K+-uptake

Mechanoreceptor sejtek a Mimosa pudica tercier pulvinusán

Annak ellenére, hogy feltűnően gyors mozgása miatt a Mimosa pudica kedvelt kísérleti objektum, mégis sok vele kapcsolatban a megválaszolatlan kérdés. Máig sem sikerült megtalálni azokat a mechanoreceptor sejteket, melyek érzékelik a külvilágból érkező mechanikai jeleket és továbbítják a növény felé. Munkánk során a növény tercier pulvinusán ilyen mechanoreceptív sejteket fedeztünk fel. Megfigyeltük ezeknek a sejteknek a fény- és elektronmikroszkópos szerkezetét, igyekeztünk felderíteni a receptor sejtek és az azok közelében lévő szomszédos sejtek között meglévő sejt-sejt kapcsolatokat is. Elektrofiziológiai vizsgálataink igazolták, hogy az általunk felfedezett sejtek mechanikai ingerek segítségével ingerületbe hozhatók, és a keletkező akciós potenciálok tovaterjedve alkalmasak a levelek mozgatását végző motorsejtek irányítására

An improved 96 well plate format lipid quantification assay for standardization of experiments with extracellular vesicles

The field of extracellular vesicles is an exponentially growing segment of biomedical sciences. However, the problems of normalisation and quantification of extracellular vesicle samples have not been completely solved yet. Currently, extracellular vesicle samples are standardized on the basis of their protein content sometimes combined with determination of the particle number. However, even this combined approach may result in inaccuracy and overestimation of the extracellular vesicle concentration. Lipid bilayers are indispensable components of extracellular vesicles. Therefore, a lipidbased quantification, in combination with the determination of particle count and/or protein content, appears to be a straightforward and logical approach for the extracellular vesicle field. In this study we set the goal to improve the previously reported sulfo-phospho-vanillin total lipid assay. We introduced an aqueous phase liposome standard (DOPC) to replace the purified lipid standards in organic solvents (used commonly in previous studies). Furthermore, we optimized the concentration of the vanillin reagent in the assay. We found that elimination of organic solvents from the reaction mixture could abolish the background colour that interfered with the assay. Comparison of the optimised assay with a commercial lipid kit (based on the original sulfo-phospho-vanillin lipid assay) showed an increase of sensitivity by approximately one order of magnitude. Thus, here we report a quick, reliable and sensitive test that may fill an existing gap in extracellular vesicle standardisation. When using the optimised total lipid assay reported here, extracellular vesicle lipid measurements can be as easy as measuring proteins with a simple BCA test

Extracellular Vesicle Release and Uptake by the Liver Under Normo‐ and Hyperlipidemia

Liver plays a central role in elimination of circulating extracellular vesicles (EVs), and it also significantly contributes to EV release. However, the involvement of the different liver cell populations remains unknown. Here, we investigated EV uptake and release both in normolipemia and hyperlipidemia. C57BL/6 mice were kept on high fat diet for 20–30 weeks before circulating EV profiles were determined. In addition, control mice were intravenously injected with (99m)Tc-HYNIC-Duramycin labeled EVs, and an hour later, biodistribution was analyzed by SPECT/CT. In vitro, isolated liver cell types were tested for EV release and uptake with/without prior fatty acid treatment. We detected an elevated circulating EV number after the high fat diet. To clarify the differential involvement of liver cell types, we carried out in vitro experiments. We found an increased release of EVs by primary hepatocytes at concentrations of fatty acids comparable to what is characteristic for hyperlipidemia. When investigating EV biodistribution with (99m)Tc-labeled EVs, we detected EV accumulation primarily in the liver upon intravenous injection of mice with medium (326.3 ± 19.8 nm) and small EVs (130.5 ± 5.8 nm). In vitro, we found that medium and small EVs were preferentially taken up by Kupffer cells, and liver sinusoidal endothelial cells, respectively. Finally, we demonstrated that in hyperlipidemia, there was a decreased EV uptake both by Kupffer cells and liver sinusoidal endothelial cells. Our data suggest that hyperlipidema increases the release and reduces the uptake of EVs by liver cells. We also provide evidence for a size-dependent differential EV uptake by the different cell types of the liver. The EV radiolabeling protocol using (99m)Tc-Duramycin may provide a fast and simple labeling approach for SPECT/CT imaging of EVs biodistribution. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00018-021-03969-6

Systematic transcriptomic and phenotypic characterization of human and murine cardiac myocyte cell lines and primary cardiomyocytes reveals serious limitations and low resemblances to adult cardiac phenotype

Background Cardiac cell lines and primary cells are widely used in cardiovascular research. Despite increasing number of publications using these models, comparative characterization of these cell lines has not been performed, therefore, their limitations are undetermined. We aimed to compare cardiac cell lines to primary cardiomyocytes and to mature cardiac tissues in a systematic manner. Methods and results Cardiac cell lines (H9C2, AC16, HL-1) were differentiated with widely used protocols. Left ventricular tissue, neonatal primary cardiomyocytes, and human induced pluripotent stem cell-derived cardiomyocytes served as reference tissue or cells. RNA expression of cardiac markers (e.g. Tnnt2, Ryr2) was markedly lower in cell lines compared to references. Differentiation induced increase in cardiac- and decrease in embryonic markers however, the overall transcriptomic profile and annotation to relevant biological processes showed consistently less pronounced cardiac phenotype in all cell lines in comparison to the corresponding references. Immunocytochemistry confirmed low expressions of structural protein sarcomeric alpha-actinin, troponin I and caveolin-3 in cell lines. Susceptibility of cell lines to sI/R injury in terms of viability as well as mitochondrial polarization differed from the primary cells irrespective of their degree of differentiation. Conclusion Expression patterns of cardiomyocyte markers and whole transcriptomic profile, as well as response to sI/R, and to hypertrophic stimuli indicate low-to-moderate similarity of cell lines to primary cells/cardiac tissues regardless their differentiation. Low resemblance of cell lines to mature adult cardiac tissue limits their potential use. Low translational value should be taken into account while choosing a particular cell line to model cardiomyocytes