The effect of 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate on CO2 permeability of the red blood cell membrane

It has long been assumed that the red cell membrane is highly permeable to gases because the molecules of gases are small, uncharged, and soluble in lipids, such as those of a bilayer. The disappearance of 12C18O16O from a red cell suspension as the 18O exchanges between labeled CO2 + HCO3− and unlabeled HOH provides a measure of the carbonic anhydrase (CA) activity (acceleration, or A) inside the cell and of the membrane self-exchange permeability to HCO3− (Pm,HCO−3). To test this technique, we added sufficient 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate (DIDS) to inhibit all the HCO3−/Cl− transport protein (Band III or capnophorin) in a red cell suspension. We found that DIDS reduced Pm,HCO−3 as expected, but also appeared to reduce intracellular A, although separate experiments showed it has no effect on CA activity in homogenous solution. A decrease in Pm,CO2 would explain this finding. With a more advanced computational model, which solves for CA activity and membrane permeabilities to both CO2 and HCO3−, we found that DIDS inhibited both Pm,HCO−3 and Pm,CO2, whereas intracellular CA activity remained unchanged. The mechanism by which DIDS reduces CO2 permeability may not be through an action on the lipid bilayer itself, but rather on a membrane transport protein, implying that this is a normal route for at least part of red cell CO2 exchange.

The Cratylia Mollis Seed Lectin Induces Membrane Permeability Transition In Isolated Rat Liver Mitochondria And A Cyclosporine A-insensitive Permeability Transition In Trypanosoma Cruzi Mitochondria.

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca(2+) and mitochondrial dysfunction due to matrix Ca(2+) overload. In order to investigate the mechanism of Ca(2+) -induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (∆Ψm ) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 μM Ca(2+) was significantly decreased by 50 μg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 μM Ca(2+) this lectin, at 50 μg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ∆Ψm disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca(2+) dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA-insensitive MPT in T. cruzi mitochondria.

Conditioned polarized Caco-2 cell monolayers allow to discriminate for the ability of gut-derived microorganisms to modulate permeability and antigen-induced basophil degranulation.

BACKGROUND: Food allergy is a common allergic disorder--especially in early childhood. The avoidance of the allergenic food is the only available method to prevent further reactions in sensitized patients. A better understanding of the immunologic mechanisms involved in this reaction would help to develop therapeutic approaches applicable to the prevention of food allergy. OBJECTIVE: To establish a multi-cell in vitro model of sensitized intestinal epithelium that mimics the intestinal epithelial barrier to study the capacity of probiotic microorganisms to modulate permeability, translocation and immunoreactivity of ovalbumin (OVA) used as a model antigen. METHODS: Polarized Caco-2 cell monolayers were conditioned by basolateral basophils and used to examine apical to basolateral transport of OVA by ELISA. Activation of basophils with translocated OVA was measured by beta-hexosaminidase release assay. This experimental setting was used to assess how microorganisms added apically affected these parameters. Basolateral secretion of cytokine/chemokines by polarized Caco-2 cell monolayers was analysed by ELISA. RESULTS: Basophils loaded with OVA-specific IgE responded to OVA in a dose-dependent manner. OVA transported across polarized Caco-2 cell monolayers was found to trigger basolateral basophil activation. Microorganisms including lactobacilli and Escherichia coli increased transepithelial electrical resistance while promoting OVA passage capable to trigger basophil activation. Non-inflammatory levels of IL-8 and thymic stromal lymphopoietin were produced basolaterally by Caco-2 cells exposed to microorganisms. CONCLUSION: The complex model designed in here is adequate to learn about the consequence of the interaction between microorganisms and epithelial cells vis-a-vis the barrier function and antigen translocation, two parameters essential to mucosal homeostasis. It can further serve as a direct tool to search for microorganisms with anti-allergic and anti-inflammatory properties.

Candida albicans inactivation and cell membrane integrity damage by microwave irradiation

In indicating the microwave irradiation for disinfecting dentures it is necessary to see how this procedure influences Candida albicans integrity and viability. The aim of this study was to evaluate the ability of microwaves to inactivate C. albicans and damage cell membrane integrity. Two 200-ml C. albicans (ATCC 10231) suspensions were obtained. A sterile denture was placed in a beaker containing the Experimental (ES) or the Control suspension (CS). ES was microwaved at 650 W for 6 min. Suspensions were optically counted using methylene blue dye uptake as indicative of membrane-damaged cells; spread on Agar Sabouraud dextrose (ASD) for viability assay; or spectrophotometrically measured at 550 nm. Cell-free solutions were submitted to content analyses of protein (Bradford and Pyrogallol red methods); Ca++ (Cresolftaleine complexone method); DNA (spectrophotometer measurements at 260 nm) and K + (selective electrode technique). Data were analysed by Student's t- or Wilcoxon z-tests (α = 0.05). All ES cells demonstrated cell membrane damage. Viable cells were non-existent in the ES ASD plates. No significant difference in optical density between ES and CS was observed (P = 0.272). ES cells released significantly high protein (P < 0.001, Bradford; P = 0.005, Pyrogallol red), K+ (P < 0.001), Ca++ (P = 0.012) and DNA (P = 0.046) contents. Microwaves inactivated C. albicans and damaged cell membrane integrity. © 2007 The Authors.

Reconstruction of mandibular segmental defects using the guided-bone regeneration technique with polylactide membranes and/or autogenous bone graft: A preliminary study on the influence of membrane permeability

Purpose: Bone maintenance after mandibular reconstruction with autogenous iliac crest may be disappointing due to extensive resorption in the long term. The potential of the guided-bone regeneration (GBR) technique to enhance the healing process in segmental defects lacks comprehensive scientific documentation. This study aimed to investigate the influence of polylactide membrane permeability on the fate of iliac bone graft (BG) used to treat mandibular segmental defects. Materials and Methods: Unilateral 10-mm-wide segmental defects were created through the mandibles of 34 mongrel dogs. All defects were mechanically stabilized, and the animals were divided into 6 treatment groups: control, BG alone, microporous membrane (poly L/DL-lactide 80/20%) (Mi); Mi plus BG; microporous laser-perforated (15 cm(2) ratio) membrane (Mip), and Mip plus BG. Calcein fluorochrome was injected intravenously at 3 months, and animal euthanasia was carried out at 6 months postoperatively. Results: Histomorphometry showed that BG protected by Mip was consistently related to larger amounts of bone compared with other groups (P <= .0001). No difference was found between defects treated with Mip alone and BG alone. Mi alone rendered the least bone area and reduced the amount of grafted bone to control levels. Data from bone labeling indicated that the bone formation process was incipient in the BG group at 3 months postoperatively regardless of whether or not it was covered by membrane. In contrast, GBR with Mip tended to enhance bone formation activity at 3 months. Conclusions: The use of Mip alone could be a useful alternative to BG. The combination of Mip membrane and BG efficiently delivered increased bone amounts in segmental defects compared with other treatment modalities. (C) 2008 American Association of Oral and Maxillofacial Surgeons.

Force-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusion.

The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVβ3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process. Copyright © 2014 John Wiley & Sons, Ltd.

Membrane fluidization triggers membrane remodeling which affects the thermotolerance in Escherichia coli.

Treatment of Escherichia coli with non-lethal doses of heat or benzyl alcohol (BA) causes transient membrane fluidization and permeabilization, and induces the rapid transcription of heat-shock genes in a sigma32-dependent manner. This early response is followed by a rapid adaptation (priming) of the cells to otherwise lethal elevated temperature, in strong correlation with an observed remodeling of the composition and alkyl chain unsaturation of membrane lipids. The acquisition of cellular thermotolerance in BA-primed cells is unrelated to protein denaturation and is not accompanied by the formation of major heat-shock proteins, such as GroEL and DnaK. This suggests that the rapid remodeling of membrane composition is sufficient for the short-term bacterial thermotolerance.

Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity.

As an approved vaccine adjuvant for use in humans, alum has vast health implications, but, as it is a crystal, questions remain regarding its mechanism. Furthermore, little is known about the target cells, receptors, and signaling pathways engaged by alum. Here we report that, independent of inflammasome and membrane proteins, alum binds dendritic cell (DC) plasma membrane lipids with substantial force. Subsequent lipid sorting activates an abortive phagocytic response that leads to antigen uptake. Such activated DCs, without further association with alum, show high affinity and stable binding with CD4(+) T cells via the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1). We propose that alum triggers DC responses by altering membrane lipid structures. This study therefore suggests an unexpected mechanism for how this crystalline structure interacts with the immune system and how the DC plasma membrane may behave as a general sensor for solid structures.

Seed priming and sulfur effects on soybean cell membrane stability and yield in saline soil

The objective of this work was to determine the effects of seed priming and sulfur application on cell membrane characteristics, seedling emergence, chlorophyll content and grain yield of soybean (Glycine max) in saline soil. A complete-block design in 4x3 factorial arrangement with three replicates was used to test four types of seed priming (water, auxin, gibberellin and non-priming) and three levels of sulfate availability (0, 70 and 140 kg ha-1 K2SO4). The soil had a silty loam texture with an electrical conductivity of 3.61 ds m-1, a pH of 8.2 and a saturation percentage of about 46%. Seed priming had significant effects on mean emergence rate (MER), emergence percentage, relative water content (RWC) of leaves, relative chlorophyll content, time of maturity, shoot length and grain yield. The highest values for these variables were observed in the priming treatments, except for the time of maturity. Sulfur application had significant effects on MER, shoot length, RWC, membrane injury index and grain yield. Priming treatments provide greater emergence rates and grain yields and interact sinergicaly with sulfur rates.

Cell-permeable peptides induce dose- and length-dependent cytotoxic effects.

We have explored the threshold of tolerance of three unrelated cell types to treatments with potential cytoprotective peptides bound to Tat(48-57) and Antp(43-58) cell-permeable peptide carriers. Both Tat(48-57) and Antp(43-58) are well known for their good efficacy at crossing membranes of different cell types, their overall low toxicity, and their absence of leakage once internalised. Here, we show that concentrations of up to 100 microM of Tat(48-57) were essentially harmless in all cells tested, whereas Antp(43-58) was significantly more toxic. Moreover, all peptides bound to Tat(48-57) and Antp(43-58) triggered significant and length-dependent cytotoxicity when used at concentrations above 10 microM in all but one cell types (208F rat fibroblasts), irrespective of the sequence of the cargo. Absence of cytotoxicity in 208F fibroblasts correlated with poor intracellular peptide uptake, as monitored by confocal laser scanning fluorescence microscopy. Our data further suggest that the onset of cytotoxicity correlates with the activation of two intracellular stress signalling pathways, namely those involving JNK, and to a lesser extent p38 mitogen-activated protein kinases. These responses are of particular concern for cells that are especially sensitive to the activation of stress kinases. Collectively, these results indicate that in order to avoid unwanted and unspecific cytotoxicity, effector molecules bound to Tat(48-57) should be designed with the shortest possible sequence and the highest possible affinity for their binding partners or targets, so that concentrations below 10 microM can be successfully applied to cells without harm. Considering that cytotoxicity associated to Tat(48-57)- and Antp(43-58) bound peptide conjugates was not restricted to a particular type of cells, our data provide a general framework for the design of cell-penetrating peptides that may apply to broader uses of intracellular peptide and drug delivery.

Clinical implications in the shift of syndecan-1 expression from the cell membrane to the cytoplasm in bladder cancer

Background To determine the diagnostic and prognostic capability of urinary and tumoral syndecan-1 (SDC-1) levels in patients with cancer of the urinary bladder. Methods SDC-1 levels were quantitated by enzyme-linked immunosorbent assay (ELISA) in 308 subjects (102 cancer subjects and 206 non-cancer subjects) to assess its diagnostic capabilities in voided urine. The performance of SDC-1 was evaluated using the area under the curve of a receiver operating characteristic curve. In addition, immunohistochemical (IHC) staining assessed SDC-1 protein expression in 193 bladder specimens (185 cancer subjects and 8 non-cancer subjects). Outcomes were correlated to SDC-1 levels. Results Mean urinary levels of SDC-1 did not differ between the cancer subjects and the non-cancer subjects, however, the mean urinary levels of SDC-1 were reduced in high-grade compared to low-grade disease (p < 0.0001), and in muscle invasive bladder cancer (MIBC) compared to non-muscle invasive bladder cancer (NMIBC) (p = 0.005). Correspondingly, preliminary data note a shift from a membranous cellular localization of SDC-1 in normal tissue, low-grade tumors and NMIBC, to a distinctly cytoplasmic localization in high-grade tumors and MIBC was observed in tissue specimens. Conclusion Alone urinary SDC-1 may not be a diagnostic biomarker for bladder cancer, but its urinary levels and cellular localization were associated with the differentiation status of patients with bladder tumors. Further studies are warranted to define the potential role for SDC-1 in bladder cancer progression.

Clinical implications in the shift of syndecan-1 expression from the cell membrane to the cytoplasm in bladder cancer

Background To determine the diagnostic and prognostic capability of urinary and tumoral syndecan-1 (SDC-1) levels in patients with cancer of the urinary bladder. Methods SDC-1 levels were quantitated by enzyme-linked immunosorbent assay (ELISA) in 308 subjects (102 cancer subjects and 206 non-cancer subjects) to assess its diagnostic capabilities in voided urine. The performance of SDC-1 was evaluated using the area under the curve of a receiver operating characteristic curve. In addition, immunohistochemical (IHC) staining assessed SDC-1 protein expression in 193 bladder specimens (185 cancer subjects and 8 non-cancer subjects). Outcomes were correlated to SDC-1 levels. Results Mean urinary levels of SDC-1 did not differ between the cancer subjects and the non-cancer subjects, however, the mean urinary levels of SDC-1 were reduced in high-grade compared to low-grade disease (p < 0.0001), and in muscle invasive bladder cancer (MIBC) compared to non-muscle invasive bladder cancer (NMIBC) (p = 0.005). Correspondingly, preliminary data note a shift from a membranous cellular localization of SDC-1 in normal tissue, low-grade tumors and NMIBC, to a distinctly cytoplasmic localization in high-grade tumors and MIBC was observed in tissue specimens. Conclusion Alone urinary SDC-1 may not be a diagnostic biomarker for bladder cancer, but its urinary levels and cellular localization were associated with the differentiation status of patients with bladder tumors. Further studies are warranted to define the potential role for SDC-1 in bladder cancer progression.

Melatonin and N-acetyl-serotonin cross the red blood cell membrane and evoke calcium mobilization in malarial parasites

The duration of the intraerythrocytic cycle of Plasmodium is a key factor in the pathogenicity of this parasite. The simultaneous attack of the host red blood cells by the parasites depends on the synchronicity of their development. Unraveling the signals at the basis of this synchronicity represents a challenging biological question and may be very important to develop alternative strategies for therapeutic approaches. Recently, we reported that the synchrony of Plasmodium is modulated by melatonin, a host hormone that is synthesized only during the dark phases. Here we report that N-acetyl-serotonin, a melatonin precursor, also releases Ca2+ from isolated P. chabaudi parasites at micro- and nanomolar concentrations and that the release is blocked by 250 mM luzindole, an antagonist of melatonin receptors, and 20 mM U73122, a phospholipase C inhibitor. On the basis of confocal microscopy, we also report the ability of 0.1 µM melatonin and 0.1 µM N-acetyl-serotonin to cross the red blood cell membrane and to mobilize intracellular calcium in parasites previously loaded with the fluorescent calcium indicator Fluo-3 AM. The present data represent a step forward into the understanding of the signal transduction process in the host-parasite relationship by supporting the idea that the host hormone melatonin and N-acetyl-serotonin generate IP3 and therefore mobilize intracellular Ca2+ in Plasmodium inside red blood cells.

TRP channels, omega-3 fatty acids, and oxidative stress in neurodegeneration: from the cell membrane to intracellular cross-links

The transient receptor potential channels family (TRP channels) is a relatively new group of cation channels that modulate a large range of physiological mechanisms. In the nervous system, the functions of TRP channels have been associated with thermosensation, pain transduction, neurotransmitter release, and redox signaling, among others. However, they have also been extensively correlated with the pathogenesis of several innate and acquired diseases. On the other hand, the omega-3 polyunsaturated fatty acids (n-3 fatty acids) have also been associated with several processes that seem to counterbalance or to contribute to the function of several TRPs. In this short review, we discuss some of the remarkable new findings in this field. We also review the possible roles played by n-3 fatty acids in cell signaling that can both control or be controlled by TRP channels in neurodegenerative processes, as well as both the direct and indirect actions of n-3 fatty acids on TRP channels.