Gating of aquaporins by heavy metals in Allium cepa L. epidermal cells

Changes in the water permeability, aquaporin (AQP) activity, of leaf cells were investigated in response to different heavy metals (Zn2+, Pb2+, Cd2+, Hg2+). The cell pressure probe experiments were performed on onion epidermal cells as a model system. Heavy metal solutions at different concentrations (0.05 μM–2 mM) were used in our experiments. We showed that the investigated metal ions can be arranged in order of decreasing toxicity (expressed as a decrease in water permeability) as follows: Hg>Cd>Pb>Zn. Our results showed that β-mercaptoethanol treatment (10 mM solution) partially reverses the effect of AQP gating. The magnitude of this reverse differed depending on the metal and its concentration. The time course studies of the process showed that the gating of AQPs occurred within the first 10 min after the application of a metal. We also showed that after 20–40 min from the onset of metal treatment, the water flow through AQPs stabilized and remained constant. We observed that irrespective of the metal applied, the effect of AQP gating can be recorded within the first 10 min after the administration of metal ions. More generally, our results indicate that the toxic effects of investigated metal ions on the cellular level may involve AQP gating

Structure and Stability of the Spinach Aquaporin SoPIP2;1 in Detergent Micelles and Lipid Membranes

Background: SoPIP2;1 constitutes one of the major integral proteins in spinach leaf plasma membranes and belongs to the aquaporin family. SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields. In order to optimize reconstitution of the purified protein into biomimetic systems, we have here for the first time characterized the structural stability of SoPIP2;1. Methodology/Principal Finding: We have characterized the protein structural stability after purification and after reconstitution into detergent micelles and proteoliposomes using circular dichroism and fluorescence spectroscopy techniques. The structure of SoPIP2;1 was analyzed either with the protein solubilized with octyl-beta-D-glucopyranoside (OG) or reconstituted into lipid membranes formed by E. coli lipids, diphytanoylphosphatidylcholine (DPhPC), or reconstituted into lipid membranes formed from mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly a-helical in accordance with crystallographic data. The protein has a high thermal structural stability in detergent solutions, with an irreversible thermal unfolding occurring at a melting temperature of 58 degrees C. Incorporation of the protein into lipid membranes increases the structural stability as evidenced by an increased melting temperature of up to 70 degrees C. Conclusion/Significance: The results of this study provide insights into SoPIP2;1 stability in various host membranes and suggest suitable choices of detergent and lipid composition for reconstitution of SoPIP2;1 into biomimetic membranes for biotechnological applications

The Relative Importance of Topography and RGD Ligand Density for Endothelial Cell Adhesion

The morphology and function of endothelial cells depends on the physical and chemical characteristics of the extracellular environment. Here, we designed silicon surfaces on which topographical features and surface densities of the integrin binding peptide arginine-glycine-aspartic acid (RGD) could be independently controlled. We used these surfaces to investigate the relative importance of the surface chemistry of ligand presentation versus surface topography in endothelial cell adhesion. We compared cell adhesion, spreading and migration on surfaces with nano- to micro-scaled pyramids and average densities of 6×102–6×1011 RGD/mm2. We found that fewer cells adhered onto rough than flat surfaces and that the optimal average RGD density for cell adhesion was 6×105 RGD/mm2 on flat surfaces and substrata with nano-scaled roughness. Only on surfaces with micro-scaled pyramids did the topography hinder cell migration and a lower average RGD density was optimal for adhesion. In contrast, cell spreading was greatest on surfaces with 6×108 RGD/mm2 irrespectively of presence of feature and their size. In summary, our data suggest that the size of pyramids predominately control the number of endothelial cells that adhere to the substratum but the average RGD density governs the degree of cell spreading and length of focal adhesion within adherent cells. The data points towards a two-step model of cell adhesion: the initial contact of cells with a substratum may be guided by the topography while the engagement of cell surface receptors is predominately controlled by the surface chemistry

FIB patterning of stainless steel for the development of nano-structured stent surfaces for cardiovascular applications

Stent implantation is a percutaneous interventional procedure that mitigates vessel stenosis, providing mechanical support within the artery and as such a very valuable tool in the fight against coronary artery disease. However, stenting causes physical damage to the arterial wall. It is well accepted that a valuable route to reduce in-stent re-stenosis can be based on promoting cell response to nano-structured stainless steel (SS) surfaces such as by patterning nano-pits in SS. In this regard patterning by focused ion beam (FIB) milling offers several advantages for flexible prototyping. On the other hand FIB patterning of polycrystalline metals is greatly influenced by channelling effects and redeposition. Correlative microscopy methods present an opportunity to study such effects comprehensively and derive structure–property understanding that is important for developing improved patterning. In this chapter we present a FIB patterning protocol for nano-structuring features (concaves) ordered in rectangular arrays on pre-polished 316L stainless steel surfaces. An investigation based on correlative microscopy approach of the size, shape and depth of the developed arrays in relation to the crystal orientation of the underlying SS domains is presented. The correlative microscopy protocol is based on cross-correlation of top-view scanning electron microscopy, electron backscattering diffraction, atomic force microscopy and cross-sectional (serial) sectioning. Various FIB tests were performed, aiming at improved productivity by preserving nano-size accuracy of the patterned process. The optimal FIB patterning conditions for achieving reasonably high throughput (patterned rate of about 0.03 mm2/h) and nano-size accuracy in dimensions and shapes of the features are discussed as well

Supramolecular polylactides – methods of preparation and selected properties

Artykuł stanowi podsumowanie prac naszego zespołu dotyczących supramolekularnych polilaktydów. Przedstawiono nowe metody syntezy i zjawiska towarzyszące tworzeniu supramolekularnych agregatów enancjomerów polilaktydów oraz ich stereokompleksów. Połączenie dwóch supramolekularnych oddziaływań: oddziaływania enancjomerycznych łańcuchów polilaktydowych o przeciwnej konfiguracji i oddziaływania wzajemnie się uzupełniających grup przyłączonych do końców makrocząsteczek polilaktydów prowadziło do powstania materiałów o zróżnicowanej morfologii i właściwościach termicznych. Opisano najważniejsze metody wykorzystywane do badania procesu samoorganizacji enancjomerycznych polilaktydów i ich stereokompleksów.This paper presents an overview of our recent work concerning supramolecular polylactides. It is based on the lecture given at 58th Annual Scientific Meeting of the Polish Chemical Society in September 2015. New synthetic methods and the phenomena related to the formation of supramolecular aggregates of enantiomeric polylactides and their stereocomplexes are presented. A combination of two kinds of supramolecular interactions: the ones between enantiomeric polylactide chains with opposite configuration and those between the complementary end groups of polylactide macromolecules, leads to the formation of materials with different morphology and thermal properties. The main methods used to investigate the process of self-assembly of enantiomeric polylactides and their stereocomplexes are also presented

Stereocomplexation of polylactides containing ionic liquid end-groups

Linear and cyclic oligomers of epichlorohydrin were synthesized and converted by reaction with N-methylimidazole into linear or cyclic derivatives fitted with imidazolium ionic liquid groups. These oligomers containing hydroxyl groups were used as macroinitiators of cationic polymerization of L- and D-lactide. Two types of products were obtained: linear polylactides (PLA's) containing a few ionic liquid groups at the chain end and star PLA's containing a few ionic liquid groups at the core. The enantiomeric forms of PLA, poly(L-lactide) (PLLA) and poly(D-lactide) (PDLA) were used for stereocomplexation studies following earlier observation that if PLLA and PDLA containing one imidazolium ionic liquid group at the chain end are mixed in 1,4-dioxane solution, stereocomplex precipitates spontaneously in form of uniform microspheres. In a present contribution it is shown that if more than one ionic liquid group is introduced at the chain ends of PLLA and PDLA, morphology of precipitated stereocomplex is similar to that observed for PLA's fitted with one ionic liquid group but microspheres are slightly less regular. When few ionic liquid groups are present at the core of star PLLA no specific morphology is observed for precipitating stereocomplex with linear PDLA fitted with C4H9O- group. Only when end-groups able to interacting with each other are introduced, specific morphology appears. With ionic liquid end-groups in linear component, stereocomplex precipitates in form of irregular granules while with strongly interacting ureidopirymidine end-groups, microspheres, although not very regular, are formed. These results indicate that well defined morphology (regular spherical particles) is observed when interacting groups are present at the freely moving chain ends but not, when the same groups are embedded and immobilized at the core of star polymers and through it probably are hidden inside polymer random coil.Otrzymano liniowe i cykliczne oligomery epichlorohydryny, następnie grupy chlorometylowe przekształcono – w reakcji z N-metyloimidazolem – w imidazoliowe, analogiczne do grup występujących w cieczach jonowych. Oligomery zawierające grupy hydroksylowe zastosowano jako makroinicjatory kationowej polimeryzacji L- i D-laktydu, w wyniku której uzyskano dwa rodzaje produktów: liniowe polimery laktydu (PLA) z udziałem kilku grup cieczy jonowej na jednym zkońców łańcucha oraz gwiaździste polimery laktydu zawierające kilka grup cieczy jonowej w centrum gwiaździstej makrocząsteczki. Enancjomeryczne formy PLA: poli(L-laktyd) (PLLA) i poli(D-laktyd) (PDLA) zastosowano do wytworzenia stereokompleksów. Wykazano, że jeśli na końcu łańcucha znajduje się więcej niż jedna grupa cieczy jonowej, morfologia wytrącającego się stereokompleksu jest zbliżona do morfologii obserwowanej w wypadku obecności tylko jednej grupy cieczy jonowej, ale mikrosfery są mniej jednorodne. Wyniki wskazują, że regularne mikrosfery powstają jeśli grupy zdolne do wzajemnych oddziaływań znajdują się na końcach liniowych makrocząsteczek, nie powstają natomiast wtedy, gdy grupy te znajdują się w centrum gwiaździstej makrocząsteczki i są otoczone jej ramionami

Stereokompleksowanie polilaktydów zawierających ciecze jonowe jako grupy końcowe

Linear and cyclic oligomers of epichlorohydrin were synthesized and converted by reaction with N-methylimidazole into linear or cyclic derivatives fitted with imidazolium ionic liquid groups. These oligomers containing hydroxyl groups were used as macroinitiators of cationic polymerization of L- and D-lactide. Two types of products were obtained: linear polylactides (PLA's) containing a few ionic liquid groups at the chain end and star PLA's containing a few ionic liquid groups at the core. The enantiomeric forms of PLA, poly(L-lactide) (PLLA) and poly(D-lactide) (PDLA) were used for stereocomplexation studies following earlier observation that if PLLA and PDLA containing one imidazolium ionic liquid group at the chain end are mixed in 1,4-dioxane solution, stereocomplex precipitates spontaneously in form of uniform microspheres. In a present contribution it is shown that if more than one ionic liquid group is introduced at the chain ends of PLLA and PDLA, morphology of precipitated stereocomplex is similar to that observed for PLA's fitted with one ionic liquid group but microspheres are slightly less regular. When few ionic liquid groups are present at the core of star PLLA no specific morphology is observed for precipitating stereocomplex with linear PDLA fitted with C4H9O- group. Only when end-groups able to interacting with each other are introduced, specific morphology appears. With ionic liquid end-groups in linear component, stereocomplex precipitates in form of irregular granules while with strongly interacting ureidopirymidine end-groups, microspheres, although not very regular, are formed. These results indicate that well defined morphology (regular spherical particles) is observed when interacting groups are present at the freely moving chain ends but not, when the same groups are embedded and immobilized at the core of star polymers and through it probably are hidden inside polymer random coil.Otrzymano liniowe i cykliczne oligomery epichlorohydryny, następnie grupy chlorometylowe przekształcono – w reakcji z N-metyloimidazolem – w imidazoliowe, analogiczne do grup występujących w cieczach jonowych. Oligomery zawierające grupy hydroksylowe zastosowano jako makroinicjatory kationowej polimeryzacji L- i D-laktydu, w wyniku której uzyskano dwa rodzaje produktów: liniowe polimery laktydu (PLA) z udziałem kilku grup cieczy jonowej na jednym zkońców łańcucha oraz gwiaździste polimery laktydu zawierające kilka grup cieczy jonowej w centrum gwiaździstej makrocząsteczki. Enancjomeryczne formy PLA: poli(L-laktyd) (PLLA) i poli(D-laktyd) (PDLA) zastosowano do wytworzenia stereokompleksów. Wykazano, że jeśli na końcu łańcucha znajduje się więcej niż jedna grupa cieczy jonowej, morfologia wytrącającego się stereokompleksu jest zbliżona do morfologii obserwowanej w wypadku obecności tylko jednej grupy cieczy jonowej, ale mikrosfery są mniej jednorodne. Wyniki wskazują, że regularne mikrosfery powstają jeśli grupy zdolne do wzajemnych oddziaływań znajdują się na końcach liniowych makrocząsteczek, nie powstają natomiast wtedy, gdy grupy te znajdują się w centrum gwiaździstej makrocząsteczki i są otoczone jej ramionami

Supramolecular Polylactides by the Cooperative Interaction of the End Groups and Stereocomplexation

The controlled ring-opening polymerization of l-lactide and d-lactide using 2-aminopyridine (AP), isocytosine (IC), uridine (U), and 2-ureido-4­[<i>1H</i>]-pyrimidinone (UPy-OH) initiators with stannous­(II) octanoate as catalyst leads to polylactides (AP-PLA-OH, IC-PLA-OH, U-PLA-OH, and UPy-PLA-OH), which are capable of forming strong hydrogen bonds and consequently self-assemble. By means of “¹H NMR titration”, the association constant (<i>K</i>_a) was determined for chosen model system composed of polylactides with uridine and aminopyridine end groups. The coupling reaction of UPy-PLA-OH with diisocyanates was used to obtain PLLA and PDLA with UPy end groups on both sides of the chain: UPy-PLLA-UPy and UPy-PDLA-UPy. The SEC analysis of the telechelic UPy-PLA-UPy revealed the presence of a supramolecular polymer with a high-molecular-weight fraction (<i>M</i>_n ≈ 70 000 g/mol) compared with the molar mass of the starting dimer (UPy-PLA-OH, <i>M</i>_n ≈ 7000 g/mol), which confirms the strong complementary interactions of the end groups. Moreover, the influence of the end groups of enantiomeric PLAs on their thermal properties and morphology was investigated. Additionally, the influence of the modified PLA enantiomers on the stereocomplexation phenomenon was analyzed. It was observed that stereocomplexes prepared from PLAs with UPy or U end groups on one chain end lead to the formation of microspheres during precipitation from <i>N</i>-methylpyrrolidone into methanol. The microscopic analysis of the telechelic UPy-PLLA-UPy/UPy-PDLA-UPy stereocomplex resulting from the precipitation from chloroform into methanol revealed a fibrous morphology. The probable mechanism of formation of the hierarchical structure of this stereocomplex material, which can be related to the specific geometry of the enantiomeric chains (mainly in a parallel arrangement), was proposed