Nano-C60 cytotoxicity is due to lipid peroxidation

This study examines the biological effects of water-soluble fullerene aggregates in an effort to evaluate the fundamental mechanisms that contribute to the cytotoxicity of a classic engineered nanomaterial. For this work we used a water-soluble fullerene species, nano-C₆₀, a fullerene aggregate that readily forms when pristine C₆₀ is added to water. Nano-C₆₀ was cytotoxic to human dermal fibroblasts, human liver carcinoma cells (HepG2), and neuronal human astrocytes at doses 50 ppb (LC₅₀=2–50 ppb, depending on cell type) after 48 h exposure. This water-soluble nano-C₆₀ colloidal suspension disrupts normal cellular function through lipid peroxidation; reactive oxygen species are responsible for the membrane damage. Cellular viability was determined through live/dead staining and LDH release. DNA concentration and mitochondrial activity were not affected by the nano-C₆₀ inoculations to cells in culture. The integrity of cellular membrane was examined by monitoring the peroxy-radicals on the lipid bilayer. Subsequently, glutathione production was measured to assess the cell's reaction to membrane oxidation. The damage to cell membranes was observed both with chemical assays, and confirmed physically by visualizing membrane permeability with high molecular weight dyes. With the addition of an antioxidant, l-ascorbic acid, the oxidative damage and resultant toxicity of nano-C₆₀ was completely prevented.     

Organic anion permeation at the proximal tubule of Necturus

This study deals with the electrical responses of the peritubular membrane of the Necturus proximal tubule to 8 organic anions, in NaHCO₃-free (trismaleate-buffered) and NaHCO₃-containing solutions. The anions glutamate and gluconate brought about a small depolarization, but only in NaHCO₃-free media. Benzene sulfonate did not alter significantly membrane p.d. The 5 other test-anions produced hyperpolarization. The magnitude of membrane depolarization elicited by high-K media was proportionally larger in the presence of the test-anions propionate, lactate, pyruvate, acetate and formate than with chloride: it is inferred that these anions increasedT _K. The same 5 anions shifted in the negative direction the p.d. achieved at peak K-depolarization; according to a previous analysis (Anagnostopoulos, 1977), this observation suggests that their permeabilities (P _A) are greater thanP _Cl, at least during the substitution. The association ofP _A>P _Cl with an increase ofT _K, upon exposure of the kidney to test-anions, is at best accounted for by a decrease ofP _Cl. The pattern of voltage attenuation along the epithelial cable during anionic substitutions is also consistent with an increase ofT _K via a decrease ofP _Cl. In conclusion, the apparent sequence of relative anionic permeabilities, as obtained from the responses of the tissue to a single anion, irrespective of buffering procedures, is:P _acet,P _lact,P _pyruv,P _prop,P _form>P _ClP _gluc,P _glut. The test-anions propionate, lactate, pyruvate, acetate and formate tend to increaseT _K, mainly by reducingP _Cl. The effect of glutamate and gluconate on physiologic ion permeabilities is too small to be specified with accuracy: it depends to some extent on the buffer used in the solutions.     

Effect of stretch on calcium channel currents recorded from the antral circular myocytes of guinea-pig stomach

The effect of membrane stretch on voltage-activated Ba²⁺ current (I _Ba) was studied in antral circular myocytes of guinea-pig using the whole- cell patch-clamp technique. The changes in cell volume were elicited by superfusing the myocytes with anisosmotic solutions. Hyposmotic superfusate (202 mosmol/l) induced cell swelling and increased peak values of I _Ba at 0 mV (from −406.6 ± 45.5 pA to −547.5 ± 65.6 pA, mean ± SEM, n = 8) and hyperosmotic superfusate (350 mosmol/l) induced cell shrinkage and decreased peak values of I _Ba at 0 mV (to −269.5 ± 39.1 pA, n = 8). Such changes were reversible and the extent of change was dependent on the osmolarity of superfusate. The values of normalized I _Ba at 0 mV were 1.43 ± 0.04, 1.30 ± 0.06, 1.23 ± 0.04, 1.19 ± 0.04, 1 and 0.68 ± 0.06 at 202, 220, 245, 267, 290 and 350 mosmol/l, respectively (n = 8). I _Ba was almost completely blocked by nicardipine (5 μM) under hyposmotic conditions. The values of steady-state half-inactivation voltage (−37.7 ± 3.3 and −36.5 ± 2.6 mV, under control and hyposmotic conditions, respectively) or the half-activation voltage (−13.6 ± 2.3 and −13.9 ± 1.9 mV) of I _Ba were not significantly changed (P > 0.05, n = 6). Cell membrane capacitance was slightly increased from 50.00 ± 2.86 pF to 50.22 ± 2.82 pF by a hyposmotic superfusate (P < 0.05, n = 6). It is suggested that cell swelling increases voltage-operated L-type calcium channel current and that such a property is related to the response of gastric smooth muscle to mechanical stimuli. Received: 14 November 1995/Received after revision and accepted: 8 January 1996     

The hyperpolarisation-activated current,I f,is not required for pacemaking in single cells from the rabbit atrioventricular node

Using electrophysiological and radiotracer studies in parallel, we have investigated the characteristics of the endogenous Na⁺-dependent amino acid transporter (system B^0,+) in Xenopus oocytes with regard to ion dependence, voltage dependence and transport stoichiometry. In voltage-clamped oocytes (–60 mV) superfusion with saturating concentrations of amino acids (1 mM) in 100 mM NaCl resulted in reversible, inward currents (mean±SEM): alanine, 1.83±0.09 nA (n=21); arginine, 2.54±0.18 nA (n=17); glutamine, 1.73±0.10 nA (n=19). Only arginine evoked a current in choline medium (0.50±0.13 nA, n=10), whereas Cl^– replacement had no effect on evoked currents. The glutamine-evoked current was saturable (I _max=1.73 nA, glutamine K _m=0.12 mM) and linearly dependent upon voltage between –90 and –30 mV. Using direct and indirect (activation) methods, we found that transport can proceed with Na⁺/amino acid coupling stoichiometry of either 11 or 21, but coupling was the same for each amino acid tested (alanine, arginine and glutamine) within a batch of oocytes (i.e. from a single toad). Despite the net single positive charge on arginine, the magnitude of the net transmembrane charge movement during Na⁺-coupled arginine transport was identical to that for the zwitterionic neutral amino acids glutamine and alanine; this may be explained by a concomitant stimulation of K⁺ efflux during arginine transport with a putative coupling of 1 K⁺1 arginine.     

Lipid components in the detergent-resistant membrane microdomain (DRM) obtained from the synaptic plasma membrane of rat brain

Lateral association of sphingolipids and cholesterol is considered to form membrane microdomains such as “lipid rafts” obtainable as a detergent-resistant membrane microdomain (DRM) fraction after solubilization with a non-ionic detergent and density gradient centrifugation. Since not only sphinogolipids and cholesterol, but also functional lipids such as phosphatidylinositol 4,5-bisphosphate (PIP₂) are reported to be localized in DRM prepared from several cultured cells, this domain is considered to be a platform mediating lipid-signaling. Although PIP₂ is considered to have pivotal roles in the nervous system, little information is available on the localization of PIP₂ in the DRM within the synaptic plasma membrane (SPM) obtained from matured rat brains. In this study, in order to know the localization of PIP₂ in SPM-derived DRM, we measured the amount of PIP₂ in SPM and SPM-derived DRM, by the thin-layer chromatography blotting method, using a GST-fusion protein of the pleckstrin-homology domain of phospholipase Cδ1 as a PIP₂ binding probe. About 10% of the PIP₂ in SPM was recovered in DRM. In contrast, over 40% recovery was observed for the membrane cholesterol and sphingomyelin, and about 30% recovery was observed for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in the DRM were detected using the thin-layer chromatography method. Since the recovery of proteins in DRM was about 10%, the result indicates that there occurs no enrichment of PIP₂ in DRM prepared from SPM.     

The theoretical small signal impedance of the frog node,Rana pipiens

Summary The small signal impedance of the frog node is calculated for frequencies from 1 Hz to 10,000 Hz and transmembrane potentials from –80 mV to –30 mV by linearizing the voltage clamp equations of Dodge [7] and Hille [8]. The modulus of the impedance is presented for the total system, and separately for the potassium and sodium systems as a function of frequency and voltage. There is a broad resonance in the total impedance with a voltage-dependent peak frequency. At 22°C, in the range –75 mV to –45 mV, the peak frequencies occur between 50 and 500 Hz. Removing the potassium system leaves a relatively shapr resonance centered around 200 Hz at –45 mV.     

Membrane orientation of the precursor 74-kDa form of L-histidine decarboxylase

Objective We previously demonstrated that, when expressed in COS-7 cells, L-histidine decarboxylase (HDC), which has neither an amino terminal signal sequence nor a hydrophobic membrane anchor, was localized in the endoplasmic reticulum (ER), although its orientation in the membrane remains to be clarified. Methods & Results Protease digestion and immunofluorescence analyses of the cells, of which plasma membrane was selectively permeabilized, revealed that the amino terminal 50-kDa portion of HDC is hardly accessible to proteases and antibodies added exogenously from the cytosolic side. Green fluorescent protein fused with the carboxyl terminal 20-kDa region of HDC at its carboxyl terminus exhibited the same characteristics as native HDC. Conclusion These results indicate that HDC is tightly associated with the ER membrane with its carboxyl terminal region exposed on the cytosolic side. Received 22 November 2005; returned for revision 28 December 2005; accepted by A. Falus 22 January 2006     

Membrane attack complex of complement and 20 kDa homologous restriction factor (CD59) in myocardial infarction

In order to investigate the mechanism of deposition of the complement membrane attack complex (MAC) in cardiomyocytes in areas of human myocardial infarction, the 20 kDA homologous restriction factor of complement (HRF20; CD59) and complement components (C1q, C3d and MAC) were analysed immunohistochemically using specific antibodies. Myocardial tissues obtained at autopsy from nine patients who died of acute myocardial infarction were fixed in acetone and embedded in paraffin. The ages of the infarcts ranged from about 3.5 h to 12 days. In cases of myocardial infarction of 20 h or less, MAC deposition was shown in the infarcted cardiomyocytes without loss of HRF20. Where the duration was 4 days or more, the cardiomyocytes with MAC deposition in the infarcted areas also showed complete loss of HRF20. Outside the infarcts, HRF20 in the cardiomyocytes was well preserved without MAC deposition. The present study suggests that the initial MAC deposition in dead cardiomyocytes can occur as a result of degradation of plasma-membrane by a mechanism independent of complement-mediated injury to the membrane. Loss of HRF20 from dead cardiomyocytes may not be the initial cause of MAC deposition, but may accelerate the deposition process of MAC in later stages of infarction.     

A model of electrical activity and cytosolic calcium dynamics in vascular endothelial cells in response to fluid shear stress

A mathematical model is proposed to describe the intracellularCa ²⁺ (Ca _i) transient and electrical activity of vascular endothelial cells (VEC) elicited by fluid shear stress (τ). The intracellularCa ²⁺ store of the model VEC is comprised of aCa _i-sensitive (sc) and an inositol (1,4,5)-trisphosphate (IP ₃)-sensitive compartment (dc). The dc [Ca ²⁺] is refilled by the sc whose [Ca ²⁺] is the same as extracellular [Ca ²⁺].IP ₃ produced by the τ-deformed mechanoreceptors discharges the dcCa ²⁺ into the cytosol. The increase of cytosolic[Ca ²⁺] inducesCa ²⁺ release (CICR) from the sc. The raisedCa _i activates aCa _i-activatedK ⁺ current (I _{K, Ca}) and inhibitsIP ₃ production. The cell membrane potential is determined byI _{K, Ca}, voltage-dependentNa ⁺ andK ⁺ currents. Steady τ>0.1 dyne/cm² elicits aCa _i varies sigmoidally withLog ₁₀(τ) with a maximal peakCa _i of 150 nM at τ=4 dynes/cm². Step increases of τ fail to elicit aCa ²⁺ response in cells previously stimulated by a lower shear. TheCa ²⁺ response gradually decreases with repetitive τ stimuli. Pulsatile shear elicits two to three times higherCa _i and hyperpolarizes the cell more than steady shear of the same magnitude. The simulatedCa ²⁺ responses to τ are quantitatively and qualitatively similar to those observed in cultured VEC. The model provides a possible explanation of why the vasodilating stimulus is greater for pulsatile flow than for nonpulsatile flow.     

温敏型智能化靶向式药物载体研究（I）——具有温敏开关的多孔膜

研究了膜孔内接枝聚异丙基丙烯酰胺 (PNIPAM)“开关”的温敏型智能膜的制备 ,并对其进行了温度感应开关性能实验。实验中采用等离子体接枝填孔聚合法将 PNIPAM接枝在多孔平板膜的膜孔中 ,结果表明 ,这种接枝了 PNIPAM“开关”的多孔膜具有温度感应特性 ,其利用膜孔内 PNIPAM接枝链的膨胀 -收缩特性实现了感温性开关性能。当环境温度低于 PNIPAM的低临界溶解温度 (L CST)时 ,膜孔内 PNIPAM分子链膨胀而使膜孔呈“关闭”状态 ;而当环境温度高于 L CST时 ,PNIPAM分子链变为收缩状态而使膜孔“开启”。温敏开关的 L CST可通过添加丙烯酰胺 (AAM)与异丙基丙烯酰胺 (NIPAM)共聚来调节 ,AAM与 NIPAM共聚开关的 L CST随 AAM添加量的增加而单调上升。