Dye Entry Research Articles

Adapting techniques for calcium imaging in muscles of adult Brugia malayi.

Brugia malayi is a human filarial nematode parasite that causes lymphatic filariasis or 'elephantiasis' a disfiguring neglected tropical disease. This parasite is a more tractable nematode parasite for the experimental study of anthelmintic drugs and has been studied with patch-clamp and RNAi techniques. Unlike in C. elegans however, calcium signaling in B. malayi or other nematode parasites has not been achieved, limiting the studies of the mode of action of anthelmintic drugs. We describe here the development of calcium imaging methods that allow us to characterize changes in cellular calcium in the muscles of B. malayi. This is a powerful technique that can help in elucidating the mode of action of selected anthelmintics. We developed two approaches that allow the recording of calcium signals in the muscles of adult B. malayi: (a) soaking the muscles with Fluo-3AM, promoting large-scale imaging of multiple cells simultaneously and, (b) direct insertion of Fluo-3 using microinjection, providing the possibility of performing dual calcium and electrophysiological recordings. Here, we describe the techniques used to optimize dye entry into the muscle cells and demonstrate that detectable increases in Fluo-3 fluorescence to elevated calcium concentrations can be achieved in B. malayi using both techniques.

The role of membrane tension in the action of antimicrobial peptides and cell-penetrating peptides in biomembranes.

For antimicrobial peptides (AMPs) with antimicrobial and bactericidal activities and cell-penetrating peptides (CPPs) with activity to permeate through plasma membrane, their interactions with lipid bilayer region in plasma membrane play important roles in these functions. However, the elementary processes and mechanisms of their functions have not been clear. The single giant unilamellar vesicle (GUV) method has revealed the details of elementary processes of interaction of some AMPs and CPPs with lipid vesicles. In this review, we summarize the mode of action of AMPs such as magainin 2 (Mag) and CPPs such as transportan 10 (TP10), revealed by the single GUV methods, and especially we focus on the role of membrane tension in actions of Mag and TP10 and the mechanisms of their actions. First, we explain the characteristics of the single GUV method briefly. Next, we summarize the recent view on the effect of tension on physical properties of lipid bilayers and describe the role of tension in actions of Mag and TP10. Some experimental results indicate that Mag-induced pore is a stretch-activated pore. The effect of packing of transbilayer asymmetric lipid on Mag-induced pore formation is described. On the other hand, entry of fluorescent dye, carboxyfluorescein (CF)-labeled TP10 (i.e., CF-TP10), into single GUVs without pore formation is affected by tension and high concentration of cholesterol. Pre-pore model for translocation of CF-TP10 across lipid bilayer is described. The experimental methods and their analysis described here are useful for investigation of functions of the other types of AMPs, CPPs, and proteins.

Trafficking pathway between plasma membrane and mitochondria via clathrin-mediated endocytosis

Endocytosis is a basic cellular process that describes a form of active transport across the plasma membrane into the cell. The endocytic pathway consists of distinct membrane compartments; internalized molecules are delivered to early endosomes, and some of them are recycled back to the surface, whereas other molecules are sent to late endosomes and lysosomes for degradation. However, little is known about how mitochondria are involved in the endocytic pathway. Here, we report that FM dyes, membrane-impermeant fluorescent lipid probes, can traffic to mitochondria directly from the plasma membrane by clathrin-mediated endocytosis. FM dye entry into mitochondria uses microtubule-dependent active transport, but the mechanism is different from the classical endocytic pathway. Hence, this study reveals a previously unrealized lipid trafficking pathway from the plasma membrane to mitochondria.

Dynasore protects the ocular surface against damaging oxidative stress.

“Vital” dyes such as fluorescein and rose bengal are used clinically to evaluate ocular surface health; however, staining mechanisms remain poorly understood. Recent evidence suggests that sublethal cell damage stimulates fluorescein dye uptake. Since damage can also stimulate reparative plasma membrane remodeling, we hypothesized that dye uptake occurs via endocytic vesicles. Using an oxidative stress model, we show that damage to relatively undifferentiated monolayer cultures of human corneal epithelial cells stimulates uptake of fluorescein and rose bengal dyes and also stimulates endocytosis. Importantly, dye uptake was blocked by co-treatment with three different endocytosis inhibitors. Damage to stratified and differentiated corneal epithelial cell cultures, which are a better model of the ocular surface, also stimulated dye uptake; however, endocytosis was not stimulated in this case, and two of the inhibitors did not block dye uptake. The exception was the inhibitor Dynasore and its more potent analogue Dyngo-4a, small molecules that target dynamin family GTPases, but also have off-target effects on the plasma membrane. Significantly, while Dynasore blocked stress-stimulated dye uptake at the ocular surface of ex vivo mouse eyes when treatment was performed at the same time as eyes were stressed, it had no effect when used after stress was applied and the ocular surface was already damaged. Thus, Dynasore could not be working by inhibiting endocytosis. Employing cytotoxicity and western blotting assays, we demonstrate an alternative mechanism, showing that Dynasore is remarkably protective of cells and their surface glycocalyx, preventing damage due to oxidative stress, and thus precluding dye entry. These unexpected and novel findings provide greater insight into mechanisms of vital dye uptake and emphasize the importance of using a differentiated cell culture model for such studies. They also suggest that Dynasore and analogues might be used therapeutically to protect the ocular surface and to treat ocular surface disease.

Fragile X Mental Retardation Protein Restricts Small Dye Iontophoresis Entry into Central Neurons.

Fragile X mental retardation protein (FMRP) loss causes Fragile X syndrome (FXS), a major disorder characterized by autism, intellectual disability, hyperactivity, and seizures. FMRP is both an RNA- and channel-binding regulator, with critical roles in neural circuit formation and function. However, it remains unclear how these FMRP activities relate to each other and how dysfunction in their absence underlies FXS neurological symptoms. In testing circuit level defects in the Drosophila FXS model, we discovered a completely unexpected and highly robust neuronal dye iontophoresis phenotype in the well mapped giant fiber (GF) circuit. Controlled dye injection into the GF interneuron results in a dramatic increase in dye uptake in neurons lacking FMRP. Transgenic wild-type FMRP reintroduction rescues the mutant defect, demonstrating a specific FMRP requirement. This phenotype affects only small dyes, but is independent of dye charge polarity. Surprisingly, the elevated dye iontophoresis persists in shaking B mutants that eliminate gap junctions and dye coupling among GF circuit neurons. We therefore used a wide range of manipulations to investigate the dye uptake defect, including timed injection series, pharmacology and ion replacement, and optogenetic activity studies. The results show that FMRP strongly limits the rate of dye entry via a cytosolic mechanism. This study reveals an unexpected new phenotype in a physical property of central neurons lacking FMRP that could underlie aspects of FXS disruption of neural function.SIGNIFICANCE STATEMENT FXS is a leading heritable cause of intellectual disability and autism spectrum disorders. Although researchers established the causal link with FMRP loss >;25 years ago, studies continue to reveal diverse FMRP functions. The Drosophila FXS model is key to discovering new FMRP roles, because of its genetic malleability and individually identified neuron maps. Taking advantage of a well characterized Drosophila neural circuit, we discovered that neurons lacking FMRP take up dramatically more current-injected small dye. After examining many neuronal properties, we determined that this dye defect is cytoplasmic and occurs due to a highly elevated dye iontophoresis rate. We also report several new factors affecting neuron dye uptake. Understanding how FMRP regulates iontophoresis should reveal new molecular factors underpinning FXS dysfunction.

A.O.7 - Research for the pathogenesis and therapy of dysferlinopathy using proteomics approach

Dysferlinopathies are a group of autosomal recessive muscular dystrophies caused by mutations in dysferlin gene (DYSF). There are two main phenotypes including Miyoshi muscular dystrophy and limb-girdle muscular dystrophy type 2B. DYSF is composed of 55 exons coding for 2,080 amino acid residues. DYSF is expressed in the plasma membrane of skeletal muscles and is involved in calcium-mediated membrane fusion events and membrane repair. We previously identified over 50 different mutations across the entire DYSF in approximately 60% of the patients. Our research theme is focusing on these DYSF and DYSF-related proteins. Recent advances have identified some components of the cell membrane repair process, particularly those involved in a putative pathway specific to striated muscles. Two important members are DYSF and Mitsugumin 53 (MG53). Compared to DYSF, molecular size of MG53 is rather small and can be expressed and work as membrane repair molecules. To find the binding partner of DYSF, which can be working as the similar functional role, would be a good clue to find the therapeutic strategy of dysferlinopathy. Using recombinant proteins and affinity column binding methods combined with LC/MS/MS, we identified novel DYSF-binding proteins. Protein X, one of the novel proteins, is considered to be an essential component of the membrane repair process. To directly evaluate the membrane repair capacity of the X knockdown C2C12 cells, we measured FM fluorescent dye entry after laser-induced damage to cells. The wild-type cells could effectively reseal sarcolemmal membranes, as they showed only minimal FM dye entry after laser damage. In contrast, significant entry of FM dye into the X knockdown cells was observed after laser-induced damage, suggesting insufficient membrane repair in X-deficient cells. To elucidate the molecular function of protein X in dysferlinopathy condition, we will also use several kinds of myoblasts originated from dysferlinopathy patients.

Ion channel-mediated uptake of cationic vital dyes into live cells: a potential source of error when assessing cell viability.

Ionic "vital dyes" are commonly used to assess cell viability based on the idea that their permeation is contingent on a loss of membrane integrity. However, the possibility that dye entry is conducted into live cells by endogenous membrane transporters must be recognized and controlled for. Several cation-selective plasma membrane-localized ion channels, including the adenosine 5'-triphosphate (ATP)-gated P2X receptors, have been reported to conduct entry of the DNA-binding fluorescence dye, YO-PRO-1, into live cells. Extracellular ATP often becomes elevated as a result of release from dying cells, and so it is possible that activation of P2X channels on neighboring live cells could lead to exaggerated estimation of cytotoxicity. Here, we screened a number of fluorescent vital dyes for ion channel-mediated uptake in HEK293 cells expressing recombinant P2X2, P2X7, or TRPV1 channels. Our data shows that activation of all three channels caused substantial uptake and nuclear accumulation of YO-PRO-1, 4',6-diamidino-2-phenylindole (DAPI), and Hoechst 33258 into transfected cells and did so well within the time period usually used for incubation of cells with vital dyes. In contrast, channel activation in the presence of propidium iodide and SYTOX Green caused no measurable uptake and accumulation during a 20-min exposure, suggesting that these dyes are not likely to exhibit measurable uptake through these particular ion channels during a conventional cell viability assay. Caution is encouraged when choosing and employing cationic dyes for the purpose of cell viability assessment, particularly when there is a likelihood of cells expressing ion channels permeable to large ions.

Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p < 0.05) as well as the immortalized cell line (p < 0.001). This difference in sensitivity was also observed when a viability assay was performed one day after plasma membrane permeabilization by electroporation. Viability in the primary normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81–88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment.

Abstract 626: Oxidized Low-density Lipoprotein-induced Cell Membrane Damage in Bone Marrow Stem Cells is Independent of ROS Formation

Objectives: Membrane repair mechanism is important to the cell survival and function. MG53, also known as TRIM72, is a critical component in the membrane repair process. We have demonstrated that cell membrane damage was involved in the impaired survival of bone marrow stem cells (BMSCs) by oxidized low-density lipoprotein (ox-LDL) in vitro, and recombinant human MG53 protein (rhMG53) significantly protected the cell membrane of BMSCs and enhanced their survival. However, the mechanism(s) for ox-LDL-induced membrane damage in BMSCs is unclear. It is well known that ox-LDL is a potent oxidative agent with formation of abundant reactive oxygen species ( ROS ). The present study was designed to determine the role of ROS in ox-LDL-induced membrane damage in BMSCs. Methods: Rat bone marrow multipotent adult progenitor cells ( MAPCs ) were cultured with and without ox-LDL (0-20 ug/ml) for up to 48 hrs. Exposure of MAPCs to ox-LDL led to increased LDH release to the media and entry of fluorescent dye FM1-43 measured under confocal microscope, suggesting damage to the plasma membrane. Ox-LDL also generated excessive ROS as measured with electron paramagnetic resonance spectroscopy. While antioxidant N-acetylcysteine (NAC) completely blocked ROS production from ox-LDL, it failed to prevent ox-LDL-induced cell death or membrane damage. When MAPCs were treated with rhMG53, ox-LDL induced LDH release and FM1-43 dye entry were significantly reduced. Interestingly, NAC treatment significantly potentiated the protective effect of rhMG53 on ox-LDL-induced membrane damage with enhanced cell survival. Western blot analysis showed that ox-LDL significantly reduced the level of rhMG53 both in vitro and in vivo that was substantially prevented with NAC treatment. Conclusion: Our data suggest that membrane damage induced by ox-LDL contributed to the impaired survival of MAPCs independent of ROS production. NAC potentiated the protective effect of rhMG53 on ox-LDL-induced membrane damage of BMSCs via preventing ox-LDL-induced reduction of rhMG53 level.

Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells.

Tunneling nanotubes (TNTs) are F-actin-based membrane tubes that form between cells in culture and in tissues. They mediate intercellular communication ranging from electrical signalling to the transfer of organelles. Here, we studied the role of TNTs in the interaction between apoptotic and healthy cells. We found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when cocultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterized by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. In addition, these TNTs show different biophysical properties, for example, increased diameter allowing dye entry, prolonged lifetime and decreased membrane fluidity. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c but did not enter into the execution phase of apoptosis characterized by caspase-3 activation. Moreover, mitochondria colocalized with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the coculture. We conclude that TNT-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context.

Clinical staining of the ocular surface: mechanisms and interpretations.

In this article we review the mechanism of ocular surface staining. Water-soluble dyes are excluded from the normal epithelium by tight junctions, the plasma membranes and the surface glycocalyx. Shed cells can take up dye. A proportion of normal corneas show sparse, scattered time-dependent, punctate fluorescein uptake, which, we hypothesise, is due to a graded loss of the glycocalyx barrier, permitting transcellular entry into pre-shed cells. In pathological staining, there is little evidence of 'micropooling' at sites of shedding and the term 'punctate erosion' may be a misnomer. It is more likely that the initial event involves transcellular dye entry and, in addition, diffusion across defective tight junctions. Different dye-staining characteristics probably reflect differences in molecular size and other physical properties of each dye, coupled with differences in visibility under the conditions of illumination used. This is most relevant to the rapid epithelial spread of fluorescein from sites of punctate staining, compared to the apparent confinement of dyes to staining cells with dyes such as lissamine green and rose bengal. We assume that fluorescein, with its lower molecular weight, spreads initially by a paracellular route and then by transcellular diffusion. Solution-Induced Corneal Staining (SICS), related to the use of certain contact lens care solutions, may have a different basis, involving the non-pathological uptake of cationic preservatives, such as biguanides, into epithelial membranes and secondary binding of the fluorescein anion. It is transient and may not imply corneal toxicity. Understanding the mechanism of staining is relevant to the standardisation of grading, to monitoring disease and to the conduct of clinical trials.

Cell membrane damage is involved in the impaired survival of bone marrow stem cells by oxidized low-density lipoprotein.

Cell therapy with bone marrow stem cells (BMSCs) remains a viable option for tissue repair and regeneration. A major challenge for cell therapy is the limited cell survival after implantation. This study was to investigate the effect of oxidized low-density lipoprotein (ox-LDL, naturally present in human blood) on BMSC injury and the effect of MG53, a tissue repair protein, for the improvement of stem cell survival. Rat bone marrow multipotent adult progenitor cells (MAPCs) were treated with ox-LDL, which caused significant cell death as reflected by the increased LDH release to the media. Exposure of MAPCs to ox-LDL led to entry of fluorescent dye FM1-43 measured under confocal microscope, suggesting damage to the plasma membrane. Ox-LDL also generated reactive oxygen species (ROS) as measured with electron paramagnetic resonance spectroscopy. While antioxidant N-acetylcysteine completely blocked ROS production from ox-LDL, it failed to prevent ox-LDL-induced cell death. When MAPCs were treated with the recombinant human MG53 protein (rhMG53) ox-LDL induced LDH release and FM1-43 dye entry were significantly reduced. In the presence of rhMG53, the MAPCs showed enhanced cell survival and proliferation. Our data suggest that membrane damage induced by ox-LDL contributed to the impaired survival of MAPCs. rhMG53 treatment protected MAPCs against membrane damage and enhanced their survival which might represent a novel means for improving efficacy for stem cell-based therapy for treatment of diseases, especially in setting of hyperlipidemia.

Abstract 130: Ox-LDL Impairs The Survival Of Bone Marrow Stem Cells Partially Through Membrane Damage Independent Of ROS Production In Vitro

Aims: cell-based therapy with bone marrow stem cells (MSCs) remains a viable option for tissue repair and regeneration. One of the major challenges for cell-based therapy is the limited survival of the cells after in vivo administration. The exact mechanism(s) for impaired in vivo survival of the implanted MSCs remains to be defined. Oxidized low-density lipid protein (ox-LDL) is a natural product in human blood, and the major contributor to the development of atherosclerosis. The present study was to investigate the effect of ox-LDL on the survival of bone marrow stem cells and the mechanisms in vitro. Methods and Results: Rat bone marrow multipotent adult progenitor cells (MAPCs) were treated with ox-LDL (with the final concentration of 10 and 20 ug/ml) for up to 48 hours. Exposure to ox-LDL resulted in significant cell death and apoptosis of MAPCs in association with a significant increase in LDH release in the conditioned media in a dose- and time-dependent manner, indicating significant cell membrane damage. The membrane damage was further confirmed with the rapid entry of the small fluorescent dye FM1-43 as detected using confocal microscope. Ox-LDL generated a significant amount of reactive oxygen species (ROS) in the culture system as measured with electron paramagnetic resonance spectroscopy. The antioxidant N-acetylcysteine (NAC, 0.1 mM) completely inhibited the production of ROS from ox-LDL. However, it didn’t prevent ox-LDL-induced cell death or apoptosis. However, pre-treatment of the cells with the specific membrane protective recombinant human MG53 protein (rhMG53)(66 ug/ml, final concentration) significantly, reduced LDH release and the entry of FM1-43 dye into the cells exposed to ox-LDL. Conclusion: Ox-LDL enhanced cell death and apoptosis of MAPCs with a mechanism independent of ROS generation in vitro. Ox-LDL impaired the survival of MAPCs partially through cell membrane damage in vitro.

Where do dyes go inside living cells? Predicting uptake, intracellular localisation, and accumulation using QSAR models

Uptake of dyes into living cells and organisms is of concern to several diverse groups of people. These include those not wishing dyes to enter cells (e.g. manufacturers and users of textile dyes, or laboratory workers using dyes as analytical reagents) and those requiring dye entry (e.g. biologists imaging cell contents, or clinicians using photoactive dyes as antitumour drugs). This diversity results in the need to consider an extremely wide range of dyes – and indeed of cells and organisms. An overview of methods for predicting uptake and intracellular localisation is provided, followed by a more detailed account of the concepts and procedures involved in decision‐rule quantitative structure–activity relationship (QSAR) models. Some of these models permit the prediction of which dyes are likely to enter cells, and which dyes will be excluded. Other models predict where internalised dyes will localise within the live cells. Use of QSAR models to understand intracellular accumulation, redistribution, loss from the cell, and metabolic modification of dyes is also considered. In particular, the relationship of such predictions to toxicity is discussed. An extended case example is provided, describing the modelling of dye binding to nucleic acids in single‐cell systems. A further case example then illustrates dye localisation in multicellular organisms. Finally, conclusions, critiques, and probable future directions concerning the QSAR modelling approach to dye uptake and localisation are given. A summary of key QSAR decision rules in the form of decision logic tabulations is provided.

Hydraulic resistance of developing Actinidia fruit

Xylem flows into most fruits decline as the fruit develop, with important effects on mineral and carbohydrate accumulation. It has been hypothesized that an increase in xylem hydraulic resistance (RT) contributes to this process. This study examined changes in RT that occur during development of the berry of kiwifruit (Actinidia deliciosa), identified the region within the fruit where changes were occurring, and tested whether a decrease in irradiance during fruit development caused an increase in RT, potentially contributing to decreased mineral accumulation in shaded fruit. RT was measured using pressure chamber and flow meter methods, the two methods were compared, and the flow meter was also used to partition RT between the pedicel, receptacle and proximal and distal portions of the berry. Dye was used as a tracer for xylem function. Artificial shading was used to test the effect of light on RT, dye entry and mineral accumulation. RT decreased during the early phase of rapid fruit growth, but increased again as the fruit transitioned to a final period of slower growth. The most significant changes in resistance occurred in the receptacle, which initially contributed 20 % to RT, increasing to 90 % later in development. Dye also ceased moving beyond the receptacle from 70 d after anthesis. The two methods for measuring RT agreed in terms of the direction and timing of developmental changes in RT, but pressure chamber measurements were consistently higher than flow meter estimates of RT, prompting questions regarding which method is most appropriate for measuring fruit RT. Shading had no effect on berry growth but increased RT and decreased dye movement and calcium concentration. Increased RT in the receptacle zone coincides with slowing fresh weight growth, reduced transpiration and rapid starch accumulation by the fruit. Developmental changes in RT may be connected to changes in phloem functioning and the maintenance of water potential gradients between the stem and the fruit. The effect of shade on RT extends earlier reports that shading can affect fruit vascular differentiation, xylem flows and mineral accumulation independently of effects on transpiration.

P2X7 Receptor Channels Allow Direct Permeation of Nanometer-Sized Dyes

P2X receptors are widely distributed in the nervous system, and P2X7 receptors have roles in neuropathic pain and in the release of cytokines from microglia. They are trimeric membrane proteins, which open an integral ion channel when ligated by extracellular ATP. This channel is preferentially permeable to small cations (sodium, potassium, calcium) but also allows permeation of larger cations such as N-methyl-d-glucamine. ATP also leads to entry of fluorescent dyes in many cells expressing P2X7 receptors, but controversy persists as to whether such large molecules pass directly through the open ion channel or enter the cell by a different route. We measured cellular fluorescence and membrane currents in individual human embryonic kidney cells expressing rat P2X7 receptors. Introduction of positive side chains by mutagenesis into the inner half of the pore-forming second transmembrane domain of the receptor (T348K, D352N, D352K) increased relative permeability of chloride ions. It also promoted entry of the large (>1 nm) negative dye fluorescein-5-isothiocyanate while decreasing entry of the structurally similar but positive dye ethidium. Furthermore, larger cysteine-reactive methanethiosulfonates [sulforhodamine-methanethiosulfonate and 2-((biotinoyl)amino)ethyl methanethiosulfonate] reduced both ATP-evoked currents and dye entry when applied to open P2X7[G345C] receptors. The results demonstrate that the open channel of the P2X7 receptor can allow passage of molecules with sizes up to 1.4 nm.

An osmotic model of the growing pollen tube.

Pollen tube growth is central to the sexual reproduction of plants and is a longstanding model for cellular tip growth. For rapid tip growth, cell wall deposition and hardening must balance the rate of osmotic water uptake, and this involves the control of turgor pressure. Pressure contributes directly to both the driving force for water entry and tip expansion causing thinning of wall material. Understanding tip growth requires an analysis of the coordination of these processes and their regulation. Here we develop a quantitative physiological model which includes water entry by osmosis, the incorporation of cell wall material and the spreading of that material as a film at the tip. Parameters of the model have been determined from the literature and from measurements, by light, confocal and electron microscopy, together with results from experiments made on dye entry and plasmolysis in Lilium longiflorum. The model yields values of variables such as osmotic and turgor pressure, growth rates and wall thickness. The model and its predictive capacity were tested by comparing programmed simulations with experimental observations following perturbations of the growth medium. The model explains the role of turgor pressure and its observed constancy during oscillations; the stability of wall thickness under different conditions, without which the cell would burst; and some surprising properties such as the need for restricting osmotic permeability to a constant area near the tip, which was experimentally confirmed. To achieve both constancy of pressure and wall thickness under the range of conditions observed in steady-state growth the model reveals the need for a sensor that detects the driving potential for water entry and controls the deposition rate of wall material at the tip.

Additive Phenotype of MG53 and Dysferlin Deficiencies in Membrane Repair Function of Skeletal Muscle Fibers

Dysferlin and MG53 have been implicated as members of the sarcolemma repair machinery. Skeletal muscle fibers isolated from mice lacking expression of one or the other protein display defective membrane repair process following acute injuries. Our previous studies show that MG53 can nucleate the assembly of the membrane repair machinery by facilitating translocation of intracellular vesicles to membrane injury sites. Dysferlin appears to participate in Ca2+-dependent fusion of these vesicles for formation of a repair patch, but dysferlin itself cannot translocate to the injury site in the absence of MG53. To test the complementary function of MG53 and dysferlin in cell membrane repair, we generated a double knockout mouse lacking both MG53 and dysferlin. While serum creatine kinase (CK) levels of dysferlin-/-, mg53-/-, and wild-type mice were not significantly different from each other, CK levels of the mg53-/-dysferlin-/- mice were significantly elevated at resting condition. This suggests a more severe membrane repair defect. Plasma membrane targeted UV-irradiation of isolated skeletal muscle fibers in the presence of a membrane impermeant dye (FM1-43) was used to directly assay membrane repair. This experimental procedure was modified from previously established protocols for a quantitative assessment of the muscle membrane repair capacity. Compared with dysferlin-/- and mg53-/- fibers, significant elevated entry of FM1-43 dye was observed in mg53-/-dysferlin-/- muscle following UV-irradiation. These results suggest that the involvement of these two proteins in the membrane repair mechanism is more complicated than that as two points along a linear pathway. The additive effects of MG53 and dysferlin in muscle membrane repair suggest the possibility that these two proteins may either sense different membrane injury signals, or that they may act at different compartments of the sarcolemma membrane (caveolae or transverse-tubule network).

Appearance Time of Methylene Blue in the Aorta: Intra-osseous vs Peripheral Intravenous Route.

Background:The intra-osseous (IO) route serves as an appropriate venous access site if access is needed in an emergency.Objectives:In this study, we compared the appearance time of methylene blue (MB) in the aorta following IO and peripheral intravenous (IV) routes in a rabbit model to assess a novel idea and compare the speed of IV and IO route of serum delivery into the main circulation.Materials and Methods:Twenty rabbits were used in our study. They were divided into two groups (odds as G1, n=10, evens as G2, n=10). After laparotomy, the aorta was located and cannulated by a 16 gauge angiocatheter. For IV injection in GII, the marginal vein of either ear was accessed. For IO injection in G1, the medial surface of the proximal extremity of left tibia was used. Once satisfied with positioning, 10 ml of methylene blue solution at a concentration of 10 mg /ml was injected and the time was recorded. The time taken from injection to appearance of MB in the aorta was measured.Results:All rabbits survived until the end of the experiment. There was no significant difference between the groups regarding the body weight. There was no significant difference between mean time of dye entry into the aorta in either group. It was 9.66 ± 2.51 seconds in G1 and 10.24 ± 1.95 seconds in G2 (P = 0.56).Conclusions:Our study demonstrated that there was no significant difference between the time taken for MB to reach the central circulation via IO or IV routes.

Highly dynamic biodegradable micelles capable of lysing Gram-positive and Gram-negative bacterial membrane

The development of biodegradable antimicrobial polymers adds to the toolbox of attractive antimicrobial agents against antibiotic-resistant microbes. To this end, the potential of polycarbonate polymers as such materials were explored. A series of random polycarbonate polymers consisting of monomers MTC-OEt and MTC-CH2CH3Cl were designed and synthesized using metal-free organocatalytic ring-opening polymerization. Random polycarbonate polymers self-assembled in solution but appeared highly dynamic; such behaviors are desirable as ready disassembly of polymers at the microbial membrane facilitates membrane disruption. Their activities against clinically relevant Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (E.coli and Pseudomonas aeruginosa) revealed that the hydrophobic-hydrophilic composition balance in polymers are important to render antimicrobial potency. Scanning electron microscopy (SEM) studies indicated microbial cell surface damage after treatment with polymers, and confocal microscopy studies also showed entry of FITC-dextran dye in Escherichia coli as a result of membrane disruption. On the other hand, the polymers exhibited minimal toxicity against red blood cells in hemolysis tests. Therefore, these random polycarbonate polymers are promising antimicrobial agents against both Gram-positive and Gram-negative bacteria for various biomedical applications.