Adhesively-tensed cell membranes: lysis kinetics and atomic force microscopy probing

Membrane tension underlies a range of cell physiological processes. Strong adhesion of the simple red cell is used as a simple model of a spread cell with a finite membrane tension-a state which proves useful for studies of both membrane rupture kinetics and atomic force microscopy (AFM) probing of native structure. In agreement with theories of strong adhesion, the cell takes the form of a spherical cap on a substrate densely coated with poly-L-lysine. The spreading-induced tension, sigma, in the membrane is approximately 1 mN/m, which leads to rupture over many minutes; and sigma is estimated from comparable rupture times in separate micropipette aspiration experiments. Under the sharpened tip of an AFM probe, nano-Newton impingement forces (10-30 nN) are needed to penetrate the tensed erythrocyte membrane, and these forces increase exponentially with tip velocity ( approximately nm/ms). We use the results to clarify how tapping-mode AFM imaging works at high enough tip velocities to avoid rupturing the membrane while progressively compressing it to a approximately 20-nm steric core of lipid and protein. We also demonstrate novel, reproducible AFM imaging of tension-supported membranes in physiological buffer, and we describe a stable, distended network consistent with the spectrin cytoskeleton. Additionally, slow retraction of the AFM tip from the tensed membrane yields tether-extended, multipeak sawtooth patterns of average force approximately 200 pN. In sum we show how adhesive tensioning of the red cell can be used to gain novel insights into native membrane dynamics and structure.     

Über die Wirkung von ADP und einigen Kationen auf die Rotationsströmung in den Wurzelhaaren der Gerste (Hordeum vulgare L.)

Zusammenfassung In getrennten Versuchen wurde die Wirkung von ADP, Ca⁺⁺, Mg⁺⁺, K⁺ und Cu⁺⁺ auf die Rotationsströmung in den Wurzelhaaren der Gerste (Hordetim vulgare L.) untersucht. Das in verschiedenen Konzentrationen fortdauernd verabreichte ADP bedingte eine Stimulation der Plasmaströmung. Die Beschleunigung der Rotationsströmung war der ADP-Konzentration gegenüber umgekehrt proportional (Abb. 3).Von den untersuchten Kationen hatte nur Ca⁺⁺ (1·10^–3 Mol) eine Stimulationswirkung. Diese Stimulationswirkung wird der Aktivierung eines Enzyms bzw. eines kontraktilen Proteins mit ATPase-Eigenschaften zugeschrieben.Die Rolle von ADP und einigen Kationen bei der Stimulation der Rotation wurde dann mit Hilfe einer gemischten Behandlung untersucht. Diese bestand in der gleichzeitigen Verabreichung von ADP (1·10^–6 Mol) und CaCl₂, MgCl₂, KCl (1 · 10^–3 Mol) oder CuCl₂ (1·10^–6 Mol). Es wurde festgestellt, daß Mg⁺⁺ und Ca⁺⁺ eine antagonistische Wirkung ausüben. Ca⁺⁺ hebt die durch ADP induzierte Stimulation auf und reduziert die Rotationsgeschwindigkeit plötzlich bis auf den Kontrollwert. Die Mg⁺⁺-Wirkung bewirkt, nach einer zeitweiligen Beibehaltung der Stimulation, ebenfalls eine Abnahme der Geschwindigkeit. K⁺ hat eine ähnliche Wirkung wie Ca⁺⁺. Cu⁺⁺ beeinträchtigt die ADP-induzierte Stimulation in geringem Maße.Die gleichzeitige Einwirkung von ADP und einigen Kationen erlaubt die Aufstellung folgender Hypothese. Die Rotationsstimulation erfolgt dank dem ATP, das auf Kosten des von außen absorbierten ADP in den Mitochondrien synthetisiert wird. Die zusätzliche ATP-Synthese kann durch gleichzeitige Ca⁺⁺-Behandlung unterbunden werden. NachHanson und Mitarb, sollen Ca⁺⁺ und ADP um ein phosphoryliertes Zwischenprodukt in Kompetition treten, so daß es zu einer Ansammlung von Ca⁺⁺ und P_a in der Zelle kommt. Andererseits könnte teilweise auch die aktive, energieverbrauchende Salzabsorption die Geschwindigkeitsabnahme der Rotation bei gemischter Behandlung erklären.

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The effect of ADP and some cations on rotational streaming in barley (Hordeum vulgare L.) root hairs

Summary The effect of ADP, Ca⁺⁺, Mg⁺⁺, K⁺, and Cu⁺⁺ upon rotational streaming within barley (Hordeum vulgäre L.) root hairs was separately studied. It was shown that various solutions of ADP may stimulate the streaming after continuous treatment. The rate increase of the rotational streaming was inverse proportional to ADP concentration (Fig. 3).From the investigated cations only Ca⁺⁺ (1·10^–3M) caused a stimulation of streaming after continuous treatment. This effect is probably due to enzymic activation of a contractile proteine which has ATPase feature.The role of ADP and of the investigated cations in the stimulation of the rotational streaming was studied by means of mixed treatment. This kind of treatment consists in a simultaneous administration of ADP (1 · 10^–6M) and CaCl₂, MgCl₂, KCl (1 · 10^–3M), or CuCl₂ (1 · 10^–6M) solutions. Ca⁺⁺ and Mg⁺⁺ showed an antagonistic action. Ca⁺⁺ brings about an immediately suppress of ADP induced stimulation. Suddenly the rate of streaming comes back to control. Mg⁺⁺ after a temporary maintaining of stimulation, also causes the lowering of the streaming. The action of K⁺ was very similar to those of Ca⁺⁺. Cu⁺⁺ changes to a little extent the stimulation caused by ADP.The simultaneous action of ADP and of the investigated cations allow us to express the following hypothesis. The stimulation of the rotational streaming after ADP treatment probably is due to ATP synthetized in mitochondria on the account of ADP. The additional synthesis of ATP can be prevented by simultaneous administration of Ca⁺⁺. According toHanson and his coworkers Ca⁺⁺ would compete with ADP for a phosphorylated intermediate product. From a such competition would result the Ca⁺⁺ and P_i accumulation. The active uptake of salts which require energy would also explain the lowering of the rotational streaming rate after the mixed treatment.

     

Imaging proteins inside cells with fluorescent tags

Watching biological molecules provides clues to their function and regulation. Some of the most powerful methods of labeling proteins for imaging use genetically encoded fluorescent fusion tags. There are four standard genetic methods of covalently tagging a protein with a fluorescent probe for cellular imaging. These use (i) autofluorescent proteins, (ii) self-labeling enzymes, (iii) enzymes that catalyze the attachment of a probe to a target sequence, and (iv) biarsenical dyes that target tetracysteine motifs. Each of these techniques has advantages and disadvantages. In this review, we cover new developments in these methods and discuss practical considerations for their use in imaging proteins inside living cells.     

Numerical simulation of the insertion process of an uncemented hip prosthesis in order to evaluate the influence of residual stress and contact distribution on the stem initial stability

The long-term success of a cementless total hip arthroplasty depends on the implant geometry and interface bonding characteristics (fit, coating and ingrowth) and on stem stiffness. This study evaluates the influence of stem geometry and fitting conditions on the evolution and distribution of the bone–stem contact, stress and strain during and after the hip stem insertion, by means of dynamic finite element techniques. Next, the influence of the mechanical state (bone–stem contact, stress and strain) resulted from the insertion process on the stem initial resistance to subsidence is investigated. In addition, a study on the influence of bone–stem interface conditions (friction) on the insertion process and on the initial stem stability under physiological loading is performed. The results indicate that for a stem with tapered shape the contact in the proximal part of the stem was improved, but contact in the calcar region was achieved only when extra press-fit conditions were considered. Changes in stem geometry towards a more tapered shape and extra press fit and variation in the bone–stem interface conditions (contact amount and high friction) led to a raise in the total insertion force. A direct positive relationship was found between the stem resistance to subsidence and stem geometry (tapering and press fit), bone–stem interface conditions (bone–stem contact and friction interface) and the mechanical status at the end of the insertion (residual stress and strain). Therefore, further studies on evaluating the initial performance of different stem types should consider the parameters describing the bone–stem interface conditions and the mechanical state resulted from the insertion process.     

Development and Evaluation of New Microemulsion-Based Hydrogel Formulations for Topical Delivery of Fluconazole

The aim of the present investigation was to develop and evaluate microemulsion-loaded hydrogels (MEHs) for the topical delivery of fluconazole (FZ). The solubility of FZ in oils, surfactants and cosurfactants was evaluated to identify the components of the microemulsion. The pseudo-ternary phase diagrams were constructed using the novel phase diagram by micro-plate dilution method. Carbopol EDT 2020 was used to convert FZ-loaded microemulsions into gel form without affecting their structure. The selected microemulsions were assessed for globule size, zeta potential and polidispersity index. Besides this, the microemulsion-loaded hydrogel (MEH) formulations were evaluated for drug content, pH, rheological properties and in vitro drug release through synthetic membrane and excised pig ear skin in comparison with a conventional hydrogel. The optimised MEH FZ formulations consisting of FZ 2%, Transcutol P 11.5% and 11%, respectively, as oil phase, Lansurf SML 20-propyleneglycol 52% and 50%, respectively, as surfactant–cosurfactant (2:1), Carbopol EDT 2020 1.5% as gelling agent and water 34.5% and 37%, respectively, showed highest flux values and high release rate values, and furthermore, they had low surfactant content. The in vitro FZ permeation through synthetic membrane and excised pig ear skin from the studied MEHs was best described by the zero-order and first-order models. Finally, the optimised MEH FZ formulations showed similar or slightly higher antifungal activity as compared to that of conventional hydrogel and Nizoral® cream, respectively. The results suggest the potential use of developed MEHs as vehicles for topical delivery of FZ, encouraging further in vitro and in vivo evaluation.KEY WORDS: fluconazole, in vitro skin permeation, microemulsion, microemulsion-loaded hydrogel, topical     

Initiation of ribosome degradation during starvation in Escherichia coli

Ribosomes are known to be degraded under conditions of nutrient limitation. However, the mechanism by which a normally stable ribosome becomes a substrate for the degradation machinery has remained elusive. Here, we present in vitro and in vivo data demonstrating that free ribosome subunits are the actual targets of the degradative enzymes, whereas 70S particles are protected from such degradation. Conditions that increase the formation of subunits both in vitro and in vivo lead to enhanced degradation, while conditions favoring the presence of intact 70S ribosomes prevent or reduce breakdown. Thus, the simple formation of free 50S and 30S subunits is sufficient to serve as the initiation mechanism that allows endoribonuclease cleavage and subsequent ribosome breakdown.     

Amphiphilic and thermosensitive copolymers based on pullulan and Jeffamine: Synthesis, characterization and physicochemical properties

The synthesis of thermosensitive copolymers based on pullulan and polyether amine was performed in water using a water-soluble carbodiimide and N-hydroxysuccinimide as activators. Jeffamine^® M2005 was chosen as a polyether to impart thermosensitive character to the copolymer. Pullulan was modified into carboxymethylpullulan, to bring carboxylate groups to the polysaccharide so as to further the grafting reaction. The copolymers were characterized by FT-IR, ¹H NMR spectroscopy and molecular weights measurements (by SEC coupled with MALS/DRI/Viscometer lines). The thermosensitive behaviour of CMP-g-M2005 copolymers was studied by fluorescence spectroscopy of pyrene, by rheometry and microDSC measurements. The sol-gel transition temperature was found dependent on the solvent, the grafting degree of M2005 and the concentration of the copolymer. For example it was 35 °C in water, 28 °C in acid buffer (0.1 M, pH 5.4) and 26 °C in saline phosphate buffer (0.15 M, pH 7.4) for a grafting degree of 0.20 at a concentration of 5 wt%.     

Invasion of tumorigenic HT1080 cells is impeded by blocking or downregulating the 37-kDa/67-kDa laminin receptor

The 37-kDa/67-kDa laminin receptor precursor/laminin receptor (LRP/LR) acting as a receptor for prions and viruses is overexpressed in various cancer cell lines, and their metastatic potential correlates with LRP/LR levels. We analyzed the tumorigenic fibrosarcoma cell line HT1080 regarding 37-kDa/67-kDa LRP/LR levels and its invasive potential. Compared to the less invasive embryonic fibroblast cell line NIH3T3, the tumorigenic HT1080 cells display approximately 1.6-fold higher cell-surface levels of LRP/LR. We show that anti-LRP/LR tools interfere with the invasive potential of HT1080 cells. Anti-LRP/LR single-chain variable fragment antibody (scFv) iS18 generated by chain shuffling from parental scFv S18 and its full-length version immunoglobulin G1-iS18 reduced the invasive potential of HT1080 cells significantly by 37% and 38%, respectively. HT1080 cells transfected with lentiviral plasmids expressing small interfering RNAs directed against LRP mRNA showed reduced LRP levels by approximately 44%, concomitant with a significant decrease in the invasive potential by approximately 37%. The polysulfated glycans HM2602 and pentosan polysulfate (SP-54), both capable of blocking LRP/LR, reduced the invasive potential by 20% and 35%, respectively. Adhesion of HT1080 cells to laminin-1 was significantly impeded by scFv iS18 and immunoglobulin G1-iS18 by 60% and 68%, respectively, and by SP-54 and HM2602 by 80%, suggesting that the reduced invasive capacity achieved by these tools is due to the perturbation of the LRP/LR-laminin interaction on the cell surface. Our in vitro data suggest that reagents directed against LRP/LR or LRP mRNA such as antibodies, polysulfated glycans, or small interfering RNAs, previously shown to encompass an anti-prion activity by blocking or downregulating the prion receptor LRP/LR, might also be potential cancer therapeutics blocking metastasis by interfering with the LRP/LR-laminin interaction in neoplastic tissues.     

Substrate recognition by AAA+ ATPases: distinct substrate binding modes in ATP-dependent protease Yme1 of the mitochondrial intermembrane space

The energy-dependent proteolysis of cellular proteins is mediated by conserved proteolytic AAA(+) complexes. Two such machines, the m- and i-AAA proteases, are present in the mitochondrial inner membrane. They exert chaperone-like properties and specifically degrade nonnative membrane proteins. However, molecular mechanisms of substrate engagement by AAA proteases remained elusive. Here, we define initial steps of substrate recognition and identify two distinct substrate binding sites in the i-AAA protease subunit Yme1. Misfolded polypeptides are recognized by conserved helices in proteolytic and AAA domains. Structural modeling reveals a lattice-like arrangement of these helices at the surface of hexameric AAA protease ring complexes. While helices within the AAA domain apparently play a general role for substrate binding, the requirement for binding to surface-exposed helices within the proteolytic domain is determined by the folding and membrane association of substrates. Moreover, an assembly factor of cytochrome c oxidase, Cox20, serves as a substrate-specific cofactor during proteolysis and modulates the initial interaction of nonassembled Cox2 with the protease. Our findings therefore reveal the existence of alternative substrate recognition pathways within AAA proteases and shed new light on molecular mechanisms ensuring the specificity of proteolysis by energy-dependent proteases.