Direct Visualization Of The Action Of Triton X-100 On Giant Vesicles Of Erythrocyte Membrane Lipids.

The raft hypothesis proposes that microdomains enriched in sphingolipids, cholesterol, and specific proteins are transiently formed to accomplish important cellular tasks. Equivocally, detergent-resistant membranes were initially assumed to be identical to membrane rafts, because of similarities between their compositions. In fact, the impact of detergents in membrane organization is still controversial. Here, we use phase contrast and fluorescence microscopy to observe giant unilamellar vesicles (GUVs) made of erythrocyte membrane lipids (erythro-GUVs) when exposed to the detergent Triton X-100 (TX-100). We clearly show that TX-100 has a restructuring action on biomembranes. Contact with TX-100 readily induces domain formation on the previously homogeneous membrane of erythro-GUVs at physiological and room temperatures. The shape and dynamics of the formed domains point to liquid-ordered/liquid-disordered (Lo/Ld) phase separation, typically found in raft-like ternary lipid mixtures. The Ld domains are then separated from the original vesicle and completely solubilized by TX-100. The insoluble vesicle left, in the Lo phase, represents around 2/3 of the original vesicle surface at room temperature and decreases to almost 1/2 at physiological temperature. This chain of events could be entirely reproduced with biomimetic GUVs of a simple ternary lipid mixture, 2:1:2 POPC/SM/chol (phosphatidylcholine/sphyngomyelin/cholesterol), showing that this behavior will arise because of fundamental physicochemical properties of simple lipid mixtures. This work provides direct visualization of TX-100-induced domain formation followed by selective (Ld phase) solubilization in a model system with a complex biological lipid composition.

Oligomerization, Membrane Association, And In Vivo Phosphorylation Of Sugarcane Udp-glucose Pyrophosphorylase.

Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.

Enhanced Glucose-induced Intracellular Signaling Promotes Insulin Hypersecretion: Pancreatic Beta-cell Functional Adaptations In A Model Of Genetic Obesity And Prediabetes.

Obesity is associated with insulin resistance and is known to be a risk factor for type-2 diabetes. In obese individuals, pancreatic beta-cells try to compensate for the increased insulin demand in order to maintain euglycemia. Most studies have reported that this adaptation is due to morphological changes. However, the involvement of beta-cell functional adaptations in this process needs to be clarified. For this purpose, we evaluated different key steps in the glucose-stimulated insulin secretion (GSIS) in intact islets from female ob/ob obese mice and lean controls. Obese mice showed increased body weight, insulin resistance, hyperinsulinemia, glucose intolerance and fed hyperglycemia. Islets from ob/ob mice exhibited increased glucose-induced mitochondrial activity, reflected by enhanced NAD(P)H production and mitochondrial membrane potential hyperpolarization. Perforated patch-clamp examination of beta-cells within intact islets revealed several alterations in the electrical activity such as increased firing frequency and higher sensitivity to low glucose concentrations. A higher intracellular Ca(2+) mobilization in response to glucose was also found in ob/ob islets. Additionally, they displayed a change in the oscillatory pattern and Ca(2+) signals at low glucose levels. Capacitance experiments in intact islets revealed increased exocytosis in individual ob/ob beta-cells. All these up-regulated processes led to increased GSIS. In contrast, we found a lack of beta-cell Ca(2+) signal coupling, which could be a manifestation of early defects that lead to beta-cell malfunction in the progression to diabetes. These findings indicate that beta-cell functional adaptations are an important process in the compensatory response to obesity.

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.

Integrated Proteomics Identified Up-regulated Focal Adhesion-mediated Proteins In Human Squamous Cell Carcinoma In An Orthotopic Murine Model.

Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.

Electronic And Structural Study Of Pt-modified Au Vicinal Surfaces: A Model System For Pt-au Catalysts.

Two single crystalline surfaces of Au vicinal to the (111) plane were modified with Pt and studied using scanning tunneling microscopy (STM) and X-ray photoemission spectroscopy (XPS) in ultra-high vacuum environment. The vicinal surfaces studied are Au(332) and Au(887) and different Pt coverage (θPt) were deposited on each surface. From STM images we determine that Pt deposits on both surfaces as nanoislands with heights ranging from 1 ML to 3 ML depending on θPt. On both surfaces the early growth of Pt ad-islands occurs at the lower part of the step edge, with Pt ad-atoms being incorporated into the steps in some cases. XPS results indicate that partial alloying of Pt occurs at the interface at room temperature and at all coverage, as suggested by the negative chemical shift of Pt 4f core line, indicating an upward shift of the d-band center of the alloyed Pt. Also, the existence of a segregated Pt phase especially at higher coverage is detected by XPS. Sample annealing indicates that the temperature rise promotes a further incorporation of Pt atoms into the Au substrate as supported by STM and XPS results. Additionally, the catalytic activity of different PtAu systems reported in the literature for some electrochemical reactions is discussed considering our findings.

The Effect Of Celastrol, A Triterpene With Antitumorigenic Activity, On Conformational And Functional Aspects Of The Human 90kda Heat Shock Protein Hsp90α, A Chaperone Implicated In The Stabilization Of The Tumor Phenotype.

Hsp90 is a molecular chaperone essential for cell viability in eukaryotes that is associated with the maturation of proteins involved in important cell functions and implicated in the stabilization of the tumor phenotype of various cancers, making this chaperone a notably interesting therapeutic target. Celastrol is a plant-derived pentacyclic triterpenoid compound with potent antioxidant, anti-inflammatory and anticancer activities; however, celastrol's action mode is still elusive. In this work, we investigated the effect of celastrol on the conformational and functional aspects of Hsp90α. Interestingly, celastrol appeared to target Hsp90α directly as the compound induced the oligomerization of the chaperone via the C-terminal domain as demonstrated by experiments using a deletion mutant. The nature of the oligomers was investigated by biophysical tools demonstrating that a two-fold excess of celastrol induced the formation of a decameric Hsp90α bound throughout the C-terminal domain. When bound, celastrol destabilized the C-terminal domain. Surprisingly, standard chaperone functional investigations demonstrated that neither the in vitro chaperone activity of protecting against aggregation nor the ability to bind a TPR co-chaperone, which binds to the C-terminus of Hsp90α, were affected by celastrol. Celastrol interferes with specific biological functions of Hsp90α. Our results suggest a model in which celastrol binds directly to the C-terminal domain of Hsp90α causing oligomerization. However, the ability to protect against protein aggregation (supported by our results) and to bind to TPR co-chaperones are not affected by celastrol. Therefore celastrol may act primarily by inducing specific oligomerization that affects some, but not all, of the functions of Hsp90α. To the best of our knowledge, this study is the first work to use multiple probes to investigate the effect that celastrol has on the stability and oligomerization of Hsp90α and on the binding of this chaperone to Tom70. This work provides a novel mechanism by which celastrol binds Hsp90α.

The Soluble Guanylyl Cyclase Activator Bay 60-2770 Potently Relaxes The Pulmonary Artery On Congenital Diaphragmatic Hernia Rabbit Model.

Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension which is often difficult to manage, and a significant cause of morbidity and mortality. In this study, we have used a rabbit model of CDH to evaluate the effects of BAY 60-2770 on the in vitro reactivity of left pulmonary artery. CDH was performed in New Zealand rabbit fetuses (n = 10 per group) and compared to controls. Measurements of body, total and left lung weights (BW, TLW, LLW) were done. Pulmonary artery rings were pre-contracted with phenylephrine (10 μM), after which cumulative concentration-response curves to glyceryl trinitrate (GTN; NO donor), tadalafil (PDE5 inhibitor) and BAY 60-2770 (sGC activator) were obtained as well as the levels of NO (NO3/NO2). LLW, TLW and LBR were decreased in CDH (p < 0.05). In left pulmonary artery, the potency (pEC50) for GTN was markedly lower in CDH (8.25 ± 0.02 versus 9.27 ± 0.03; p < 0.01). In contrast, the potency for BAY 60-2770 was markedly greater in CDH (11.7 ± 0.03 versus 10.5 ± 0.06; p < 0.01). The NO2/NO3 levels were 62 % higher in CDH (p < 0.05). BAY 60-2770 exhibits a greater potency to relax the pulmonary artery in CDH, indicating a potential use for pulmonary hypertension in this disease.

Bay 41-2272, A Soluble Guanylate Cyclase Stimulator, Relaxes Isolated Human Ureter In A Standardized In Vitro Model.

To characterize the relaxation induced by BAY 41-2272 in human ureteral segments. Ureter specimens (n = 17) from multiple organ human deceased donors (mean age 40 ± 3.2 years, male/female ratio 2:1) were used to characterize the relaxing response of BAY 41-2272. Immunohistochemical analysis for endothelial and neuronal nitric oxide synthase, guanylate cyclase stimulator (sGC) and type 5 phosphodiesterase was also performed. The potency values were determined as the negative log of the molar to produce 50% of the maximal relaxation in potassium chloride-precontracted specimens. The unpaired Student t test was used for the comparisons. Immunohistochemistry revealed the presence of endothelial nitric oxide synthase in vessel endothelia and neuronal nitric oxide synthase in urothelium and nerve structures. sGC was expressed in the smooth muscle and urothelium layer, and type 5 phosphodiesterase was present in the smooth muscle only. BAY 41-2272 (0.001-100 μM) relaxed the isolated ureter in a concentration dependent manner, with a potency and maximal relaxation value of 5.82 ± 0.14 and 84% ± 5%, respectively. The addition of nitric oxide synthase and sGC inhibitors reduced the maximal relaxation values by 21% and 45%, respectively. However, the presence of sildenafil (100 nM) significantly potentiated (6.47 ± 0.10, P <.05) this response. Neither glibenclamide or tetraethylammonium nor ureteral urothelium removal influenced the relaxation response by BAY 41-2272. BAY 41-2272 relaxes the human isolated ureter in a concentration-dependent manner, mainly by activating the sGC enzyme in smooth muscle cells rather than in the urothelium, although a cyclic guanosine monophosphate-independent mechanism might have a role. The potassium channels do not seem to be involved.

Membrane Lipid Profile Monitored By Mass Spectrometry Detected Differences Between Fresh And Vitrified In Vitro-produced Bovine Embryos.

Summary This study aimed to evaluate the impact of vitrification on membrane lipid profile obtained by mass spectrometry (MS) of in vitro-produced bovine embryos. Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) has been used to obtain individual embryo membrane lipid profiles. Due to conditions of analysis, mainly membrane lipids, most favorably phosphatidylcholines (PCs) and sphingomyelins (SMs) have been detected. The following ions described by their mass-to-charge ratio (m/z) and respective attribution presented increased relative abundance (1.2-20×) in the vitrified group: 703.5 [SM (16:0) + H]+; 722.5 [PC (40:3) + Na]+; 758.5 [PC (34:2) + H]+; 762.5 [PC (34:0) + H]+; 790.5 [PC (36:0) + H]+ and 810.5 [PC (38:4) + H]+ and/or [PC (36:1) + Na]+. The ion with a m/z 744.5 [PCp (34:1) and/or PCe (34:2)] was 3.4-fold more abundant in the fresh group. Interestingly, ions with m/z 722.5 or 744.5 indicate the presence of lipid species, which are more resistant to enzymatic degradation as they contain fatty acyl residues linked through ether type bonds (alkyl ether or plasmalogens, indicated by the lowercase 'e' and 'p', respectively) to the glycerol structure. The results indicate that cryopreservation impacts the membrane lipid profile, and that these alterations can be properly monitored by MALDI-MS. Membrane lipids can therefore be evaluated by MALDI-MS to monitor the effect of cryopreservation on membrane lipids, and to investigate changes in lipid profile that may reflect the metabolic response to the cryopreservation stress or changes in the environmental conditions.

Brij Detergents Reveal New Aspects Of Membrane Microdomain In Erythrocytes.

Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4 °C and 37 °C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs - a detergent that preferentially solubilizes the membrane inner leaflet - while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.

Atomic Charge Transfer-counter Polarization Effects Determine Infrared Ch Intensities Of Hydrocarbons: A Quantum Theory Of Atoms In Molecules Model.

Atomic charge transfer-counter polarization effects determine most of the infrared fundamental CH intensities of simple hydrocarbons, methane, ethylene, ethane, propyne, cyclopropane and allene. The quantum theory of atoms in molecules/charge-charge flux-dipole flux model predicted the values of 30 CH intensities ranging from 0 to 123 km mol(-1) with a root mean square (rms) error of only 4.2 km mol(-1) without including a specific equilibrium atomic charge term. Sums of the contributions from terms involving charge flux and/or dipole flux averaged 20.3 km mol(-1), about ten times larger than the average charge contribution of 2.0 km mol(-1). The only notable exceptions are the CH stretching and bending intensities of acetylene and two of the propyne vibrations for hydrogens bound to sp hybridized carbon atoms. Calculations were carried out at four quantum levels, MP2/6-311++G(3d,3p), MP2/cc-pVTZ, QCISD/6-311++G(3d,3p) and QCISD/cc-pVTZ. The results calculated at the QCISD level are the most accurate among the four with root mean square errors of 4.7 and 5.0 km mol(-1) for the 6-311++G(3d,3p) and cc-pVTZ basis sets. These values are close to the estimated aggregate experimental error of the hydrocarbon intensities, 4.0 km mol(-1). The atomic charge transfer-counter polarization effect is much larger than the charge effect for the results of all four quantum levels. Charge transfer-counter polarization effects are expected to also be important in vibrations of more polar molecules for which equilibrium charge contributions can be large.

Mesenchymal Stromal Cells From Adipose Tissue Attached To Suture Material Enhance The Closure Of Enterocutaneous Fistulas In A Rat Model.

Surgical treatment for enterocutaneous fistulas (EF) frequently fails. Cell therapy may represent a new approach to treatment. Mesenchymal stromal cells (MSCs) have high proliferative and differentiation capacity. This study aimed to investigate whether MSCs could adhere to suture filament (SF), promoting better EF healing. MSCs, 1 × 10(6), from adipose tissue (ATMSCs) were adhered to a Polyvicryl SF by adding a specific fibrin glue formulation. Adhesion was confirmed by confocal and scanning electron microscopy (SEM). A cecal fistula was created in 22 Wistar rats by incising the cecum and suturing the opening to the surgical wound subcutaneously with four separate stitches. The animals were randomly allocated to three groups: control (CG)-five animals, EF performed; injection (IG)-eight animals 1 × 10(6) ATMSCs injected around EF borders; and suture filament (SG): nine animals, sutured with 1 × 10(6) ATMSCs attached to the filaments with fibrin glue. Fistulas were photographed on the operation day and every 3 days until the 21st day and analyzed by two observers using ImageJ Software. Confocal and SEM results demonstrated ATMSCs adhered to SF (ATMSCs-SF). The average reduction size of the fistula area at 21st day was greater for the SG group (90.34%, P < 0.05) than the IG (71.80%) and CG (46.54%) groups. ATMSCs adhered to SF maintain viability and proliferative capacity. EF submitted to ATMSCs-SF procedure showed greater recovery and healing. This approach might be a new and effective tool for EF treatment.

Changes In Motor Behavior During Pregnancy In Rats: The Basis For A Possible Animal Model Of Restless Legs Syndrome.

Pregnant women have a 2-3 fold higher probability of developing restless legs syndrome (RLS - sleep-related movement disorders) than general population. This study aims to evaluate the behavior and locomotion of rats during pregnancy in order to verify if part of these animals exhibit some RLS-like features. We used 14 female 80-day-old Wistar rats that weighed between 200 and 250 g. The rats were distributed into control (CTRL) and pregnant (PN) groups. After a baseline evaluation of their behavior and locomotor activity in an open-field environment, the PN group was inducted into pregnancy, and their behavior and locomotor activity were evaluated on days 3, 10 and 19 of pregnancy and in the post-lactation period in parallel with the CTRL group. The serum iron and transferrin levels in the CTRL and PN groups were analyzed in blood collected after euthanasia by decapitation. There were no significant differences in the total ambulation, grooming events, fecal boli or urine pools between the CTRL and PN groups. However, the PN group exhibited fewer rearing events, increased grooming time and reduced immobilization time than the CTRL group (ANOVA, p<0.05). These results suggest that pregnant rats show behavioral and locomotor alterations similar to those observed in animal models of RLS, demonstrating to be a possible animal model of this sleep disorder.

Epiretinal Membrane Formation Associated With Idiopathic Macular Telangiectasia: Case Report.

A 46-year-old woman complained of blurred and distorted vision in both eyes. Ophthalmic examination showed that visual acuity was 20/200 for the right eye and counting fingers left eye. Fundoscopy revealed perimacular hemorrhages, aneurismal dilatation of the vessels in the posterior pole, and a white and elevated lesion adjacent to vascular changes. We report a case of idiopathic macular telangiectasia and epiretinal membrane that occurs concomitantly. To our knowledge, this is the first report that describes an association between idiopathic macular telangiectasia and epiretinal membrane formation.