Muscle transfection and permeabilization induced by electrotransfer or pluronic® L64: Paired study by optical imaging and MRI

Background

Muscle transfection by electrotranfer is an efficient currently used procedure. Recently, the block copolymer pluronic L64 has been reported to improve muscle transfection. Both procedures are known to permeabilize muscle fibres. Relation between muscle transfection and permeabilization by electrotransfer and L64 was investigated herein.

Methods

Muscle transfection was evaluated by optical detection of the luciferase reporter gene activity. Muscle permeabilization was evaluated by the uptake of the T1 contrast agent gadolinium-Dotarem (Gd-DOTA) using Magnetic resonance imaging (MRI). Histological examination of muscle sections was also performed.

Results

Electrotransfer and L64 (at a 0.25% concentration) similarly improved muscle transfection, although the interindividual variability was higher for pluronic. On the same animals, the permeabilized volume to the Gd-DOTA was significantly increased after electrotransfer, and L64 from 0.1% to 1%. The concentration of the Gd-DOTA in the permeabilized volume was significantly increased after electrotransfer and L64 at 0.5% and 1%. By histological observation, the inflammation was maximum at day 3 after electrotransfer or L64 injection, and mostly reversed after 7 days. The permeabilized volume and the transfection level correlated for the set of all the conditions tested. However, no significant correlation was observed between Gd-DOTA concentration and transfection.

General significance

It is possible to use successively on the same animals MRI and optical imaging for paired studies of muscle transfection and permeabilization. Permeabilization is possibly not related to gene transfer but it indicates membrane modification related to transfection by the electrotransfer or co-injection of DNA with the L64.     

Anergy or cell death induced by low physiological temperature in mitogen-stimulated human T lymphocytes

1. 1. Human T cell proliferation is suppressed at 27°C, and is both diminished and delayed at 32°C.

2. 2. Temperature shift-up and viability assays indicated that concanavalin A stimulation at 27°C induced cell death in contrast to a transient unresponsiveness (anergy) induced by monoclonal anti-CD3 antibody (CD3) and the superantigen, staphylococcal exterotoxin B.

3. 3. Phytohemagglutinin also induced cell death at 27°C; however, some cells remained viable and proliferation occurred when such cultures were subsequently moved to 37°C.

4. 4. Low temperature suppression of T cell activation was not overcome by a mixture of phorbol ester and calcium ionophore indicating a probable block post-protein kinase C activation. This was confirmed in temperature shift-down assays where incubation for 18–24 h at 37°C was required to bypass the block at 27°C.

5. 5. With the exception of CD3, stimulation at 27°C with the mitogens resulted in interleukin-2 secretion, indicating that the low temperature block(s) is a relatively late event in cell activation.

     

Role of oxidative stress in lysosomal membrane permeabilization and apoptosis induced by gentamicin, an aminoglycoside antibiotic

Gentamicin, an aminoglycoside antibiotic used to treat severe bacterial infections, may cause acute renal failure. At therapeutic concentrations, gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal tubular cells. In gentamicin-treated renal LLC-PK1 cells, acridine orange release from lysosomes, previously interpreted as lysosomal membrane permeabilization, precedes the apoptotic cascade that develops during incubation with gentamicin. However, the link between gentamicin lysosomal accumulation and apoptosis remains unclear. We here examined if reactive oxygen species (ROS) production could account for gentamicin-induced acridine orange release and apoptosis, and the implication of iron in these events. We found that gentamicin induced ROS production prior to, and at lower drug concentrations than required for, acridine orange release and apoptosis. ROS antioxidant or scavenger, catalase, and N-acetylcysteine largely prevented these events. Vital confocal imaging revealed that gentamicin-induced ROS production occurs in lysosomes. Deferoxamine, an iron chelator, which is endocytosed and accumulates in lysosomes, largely prevented gentamicin-induced ROS production as well as apoptosis. Direct evidence for gentamicin-induced permeabilization of lysosomal membrane was provided by showing the release into the cytosol of Lucifer yellow, a membrane-impermeant endocytic tracer with a comparable molecular weight as gentamicin. Altogether, our data demonstrate a key role of lysosomal iron and early ROS production in gentamicin-induced lysosomal membrane permeabilization and apoptosis.     

ATP-sensitive K+ channels in an insulin-secreting cell line are inhibited byd-glyceraldehyde and activated by membrane permeabilization

Summary The control of K⁺ channels in the insulin-secreting cell line RINm5F has been investigated by patch-clamp singlechannel current recording experiments. The unitary current events recorded from cell-attached patches are due to large and small inwardly rectifying ATP-sensitive K⁺ channels with conductance properties similar to the two channels previously identified in primary cultured rat islet cells (Findlay, I., Dunne, M.J., & Petersen, O. H.J. Membrane Biol. 88:165–172, 1985). Cell permeabilization through brief exposure to 10 m digitonin or 0.05% saponin (outside the isolated membrane patch area) results in a dramatic increase in current through the cell-attached patch due to opening of many large and small K⁺-selective channels. These channels are inhibited in a dose-dependent manner by ATP applied to the bath (near-complete inhibition by 5mm ATP). During prolonged ATP exposure (1–5 min) the initial inhibition is followed by partial recovery of channel activity, although further activation does occur when ATP is subsequently removed. From the maximal number of coincident channel openings in the permeabilized cells (in the absence of ATP), it is estimated that there are on average 12 large ATP-sensitive K⁺ channels per membrane patch, but in the intact cells less than 5% of the membrane patches exhibited three or more coincident K⁺ channel openings, indicating the degree to which the channels are inhibited in the resting condition by endogenous ATP. Stimulation of RINm5F cells to secrete insulin was carried out by challenging intact cells with 10mm d-glyceraldehyde.d-glyceraldehyde induced depolarization of the membrane from about –70 to –20 mV and evoked a marked reduction in the open-state probability of both the large and small ATP-sensitive channels.d-glyceraldehyde also induced action potentials in a number of cases. All effects of stimulation were largely transient, lasting about 100 sec. The two ATP-sensitive K⁺ channels are probably responsible for the resting potential and play a crucial role in coupling metabolism to membrane depolarization.     

Effect of low temperature preservation and cell density on metabolic function in a bioartificial liver

Difficulties associated with bioartificial liver (BAL) preservation limit not only the commercialization of BAL, but also its clinical trials. In this study, the possibility of cold preservation of BAL cartridges containing porcine hepatocytes was examined at 4 °C. In anin vitro perfusion culture system, BAL cartridges maintained cytochrome P450 metabolic function for at least 50 days. However, all BAL cartridges completely lost their ammonia eliminating ability when stored at 4 °C. We also studied the effect of cell density on the maintenance of BAL liver function in a highly differentiated and healthy state. As expected, BALs containing a larger number of hepatocytes demonstrated higher metabolic functions. When metabolic functions were compared per gram of hepatocytes, no large differences were observed between devices containing different densities of hepatocytes. Decreased cell density did not successfully prolong BAL function. The viability and function of isolated hepatocytes highly depend on the culture conditions, such as cell density, substrata, culture media, and additives to the culture media. Perfusion culture of BAL cartridges at 4°C gave a promosing result with respect to the maintenance of P450 activity. However, as indicated by the rapid loss of ammonia metabolic activity, many factors still remain to be optimized for preservation of BAL keeping high metabolic functions for a longer time.     

Plasma and mitochondrial membrane perturbation induced by aluminum in human peripheral blood lymphocytes

Aluminum is a redox-inert element that could induce cell damage via activation of oxidative stress. In this work, the effect of aluminum on different cellular compartments of human peripheral blood lymphocytes was studied. The presence of aluminum induced a lipid peroxidation and physico-chemical modifications at the membrane level. A decrease in fluorescence anisotropy of TMA-DPH and in the polarity of the lipid bilayer with a concomitant shift toward a gel phase was observed, while the pyrene excimerization coefficient (Kex) increased.Flow cytometry measurements, using JC-1, Rhodamine 123 and H₂-DCFDA as fluorescent probes, indicated that aluminum induces a slight mitochondrial membrane depolarization that was associated with a moderate increase in reactive oxygen species production. A significative influence on these parameters was measured only at high aluminum concentration.     

Interleukin-8 secretion by fibroblasts induced by low density lipoproteins is p38 MAPK-dependent and leads to cell spreading and wound closure

We have previously reported (Dobreva, I., Waeber, G., Mooser, V., James, R. W., and Widmann, C. (2003) J. Lipid Res. 44, 2382-2390) that low density lipoproteins (LDLs) induce activation of the p38 MAPK pathway, resulting in fibroblast spreading and lamellipodia formation. Here, we show that LDL-stimulated fibroblast spreading and wound sealing are due to secretion of a soluble factor. Using an antibody-based human protein array, interleukin-8 (IL-8) was identified as the main cytokine whose concentration was increased in supernatants from LDL-stimulated cells. Incubation of supernatants from LDL-treated cells with an anti-IL-8 blocking antibody completely abolished their ability to induce cell spreading and mediate wound closure. In addition, fibroblasts treated with recombinant IL-8 spread to the same extent as cells incubated with LDL or supernatants from LDL-treated cells. The ability of LDL and IL-8 to induce fibroblast spreading was mediated by the IL-8 receptor type II (CXCR-2). Furthermore, LDL-induced IL-8 production and subsequent wound closure required the activation of the p38 MAPK pathway, because both processes were abrogated by a specific p38 inhibitor. Therefore, the capacity of LDLs to induce fibroblast spreading and accelerate wound closure relies on their ability to stimulate IL-8 secretion in a p38 MAPK-dependent manner. Regulation of fibroblast shape and migration by lipoproteins may be relevant to atherosclerosis that is characterized by increased LDL cholesterol levels, IL-8 production, and extensive remodeling of the vessel wall.     

Bovine lactoferricin causes apoptosis in Jurkat T-leukemia cells by sequential permeabilization of the cell membrane and targeting of mitochondria

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that kills Jurkat T-leukemia cells by the mitochondrial pathway of apoptosis. However, the process by which LfcinB triggers mitochondria-dependent apoptosis is not well understood. Here, we show that LfcinB-induced apoptosis in Jurkat T-leukemia cells was preceded by LfcinB binding to, and progressive permeabilization of the cell membrane. Colloidal gold electron microscopy revealed that LfcinB entered the cytoplasm of Jurkat T-leukemia cells prior to the onset of mitochondrial depolarization. LfcinB was not internalized by endocytosis because endocytosis inhibitors did not prevent LfcinB-induced cytotoxicity. Furthermore, intracellular delivery of LfcinB via fusogenic liposomes caused the death of Jurkat T-leukemia cells, as well as normal human fibroblasts. Collectively, these findings suggest that LfcinB caused damage to the cell membrane that allowed LfcinB to enter the cytoplasm of Jurkat T-leukemia cells and mediate cytotoxicity. In addition, confocal microscopy showed that intracellular LfcinB co-localized with mitochondria in Jurkat T-leukemia cells, while flow cytometry and colloidal gold electron microscopy showed that LfcinB rapidly associated with purified mitochondria. Furthermore, purified mitochondria treated with LfcinB rapidly lost transmembrane potential and released cytochrome c. We conclude that LfcinB-induced apoptosis in Jurkat T-leukemia cells resulted from cell membrane damage and the subsequent disruption of mitochondrial membranes by internalized LfcinB.     

A separation chamber to sort cells and cell organelles by weak physical forces: III. Preparative electrophoresis of cells in stationary density gradients at low electric field strength

An apparatus was designed for preparative density gradient electrophoresis of mammalian cells. In a low conductivity isotonic Ficoll density gradient of 1.5 cm length, human erythrocytes treated with neuraminidase were separated from untreated erythrocytes at an electric field strength of approximately 2.7 v/cm. Within 5 min two bands of erythrocytes were visible. Electrophoretic separation was completed within 25 min. The fractionation is performed in a design consisting of three Perspex circular plates, bottom and top plates of which can be displaced simultaneously relative to the stationary middle plate by a worm-gear mechanism. The middle plate contains a cylindrical separation chamber of 50 cm² and 1.5 cm high. Top and bottom plates contain cones and flow deflectors for the undisturbed thin layering of cell suspensions and for introduction of the density gradient. Also present in top and bottom plates are electrode compartments containing a large platinum electrode and a cellophane membrane that isolates the separation chamber hydrodynamically but not electrically from the electrode compartment. The electrode compartments were flushed with electrophoresis buffer to remove products of electrophoresis as well as the (low) generated Joule heat.     

Variations in transmembrane Ca2+ gradient and apoptosis of macrophages induced by oxidized low density lipoprotein

While Ca²⁺ has been proposed to be a messenger in OxLDL-induced cell death, few studies have addressed the possibility that it may influence the occurrence of apoptosis and necrosis of macrophages induced by OxLDL in virtue of change of transmembrane Ca²⁺ gradient including that across plasma membrane and intracellular organelle membranes. In this paper, various lipophilic Ca²⁺ fluorescent indicators and specific organelle markers were used to study the relationship between the changes of the transmembrane Ca²⁺ gradients and the OxLDL induced apoptosis of macrophages. Our results showed that following exposure of low dose OxLDL to macrophages, the transmembrane Ca²⁺ gradient across the plasma membrane, as well as the membrane-proximal Ca²⁺ gradient, the transnuclear, and the transmitochondrial membrane Ca²⁺ gradient were all changed significantly. These data suggested that changes in transmembrane Ca²⁺ gradients might be involved in the apoptosis of macrophages induced by OxLDL.     

Cheddar cheese cooking temperature induces differential lactococcal cell permeabilization and autolytic responses as detected by flow cytometry: implications for intracellular enzyme accessibility

AIMS: To determine the influence of cheese cooking temperature on autolysis and permeabilization of two lactococcal starter strains in broth and in Cheddar cheese juice during ripening. METHODS AND RESULTS: Flow cytometry (FCM) was used to identify and enumerate intact and permeabilized cells in broth and in Cheddar cheese juice. Levels of intracellular enzyme activities were quantified concurrently. Permeabilized cell numbers increased for both strains in broth following a temperature shift from 32 to 38 degrees C and was accompanied by an increase in the level of accessible intracellular enzyme activities. The relative proportions of intact and permeabilized cell populations, as detected by FCM in cheese juice, changed during 42-day ripening. Permeabilized cell populations increased during ripening for both strains; however, an increase in accessible intracellular enzyme activity was observed only for the highly autolytic strain Lactococcus lactis AM2. CONCLUSIONS: Differences in the autolytic and permeabilization response induced by cooking temperature in two lactococcal strains affects intracellular enzyme accessibility in Cheddar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the importance of the autolytic and permeabilization properties of lactic acid bacteria starter strains and their impact on cheese ripening.     

Growth regulators differentially affect photosensitivity induced by low temperature and osmotic stresses in germinating white clover seeds

Negative photoblastism induced in white clover seeds at 5°C or by lowered water potential (–0.3 MPa, polyethylene glycol) was affected by ethrel, gibberellin A₃, benzylaminopurine and kinetin treatments. The effects were different for water and temperature stressed seeds. The observed synergistic and additive effects of light and growth regulators confirm the earlier suggestion that there are two different mechanisms involved in the light inhibition of white clover seed germination induced by various adverse environmental conditions.Abbreviations ABA abscisic acid - BAP benzylaminopurine - GA₃ gibberellin A₃ - PEG polyethylene glycol     

Changes in cell membrane permeability and lipid content of wheat root cortex cells induced by NaCl

Wheat seedlings were grown hydroponically in absence and presence of 100 mM NaCl for 7 d. Cell membrane permeability to nonelectrolytes and water was determined by the plasmometric method for individual intact cells. NaCl increased membrane permeability to urea, methylurea and ethylurea and decreased permeability to water. Membrane lipid partiality was decreased by NaCl. The effects of NaCl on cell permeability parallel changes in the lipid composition of the plasma membranes induced by NaCl stress suggesting that nonelectrolyte permeability is a useful tool to probe alterations in the lipid matrix of the membrane.     

Effect of nitrogen,phosphorus and potassium on spikelet sterility induced by low temperature at the reproductive stage of rice

Spikelet sterility induced by low temperature at the reproductive stage of rice increased further with the increase of nitrogen supply. Spikelet sterility in Fujisaka-5 did not increase due to low temperature when nitrogen supply was increased from 10 to 40 ppm and at 80 ppm nitrogen supply it was less affected than IR36. Total nitrogen content in the leaves increased with the increase of nitrogen supply and was forced to be associated with the spikelet sterility induced by low temperature. Based on auricle distance between the last two leaves, the most sensitive stage to low temperature damage differed in Fujisaka-5 and IR36. Spikelet sterility induced by low temperature for 10 days was very high in both the varieties and the effect of nitrogen was not clear.The effect of phosphorus on the spikelet sterility induced by low temperature at reproductive stage was not clear except that at the highest phosphorus (P) level (10 ppm) the spikelet sterility increased both in Fujisaka-5 and IR36.Spikelet sterility induced by low temperature at the reproductive stage of rice decreased with the increase of Potassium (K) supply in both Fujisaka-5 and IR36. With an increase of potassium supply, nitrogen (N) content decreased in the leaves and panicles and spikelet sterility induced by low temperature decreased with an increase of the K to N ratio in the leaves and panicles. The results suggest that potassium might play a major role to counteract the low temperature damage at the reproductive stage of rice.     

冷锻炼对低温胁迫下夏威夷椰子膜脂过氧化及保护酶活性的影响

在－5℃低温胁迫下,夏威夷椰子（Pritchardia gaudichaudii H.Wendl.)幼苗叶片的丙二醛（MDA）含量逐渐增加,表明膜脂过氧化作用逐渐增强;含水量不断下降;细胞保护酶中的超氧化物歧化酶（SOD）、过氧化物酶（POD) 过氧化氢酶（CAT）酶活性均先升高,然后下降。冷锻炼处理可以减缓夏威夷椰子膜脂过氧化作用的增强,促进SOD酶活性的提高,同时抑制POD和CAT酶活性的变化,因而使夏威夷椰子幼苗的抗寒性得以提高,在－5℃低温胁迫下的半致死时间从1.6d延长到2.2d。     

Human basement membrane heparan sulfate proteoglycan core protein: a 467-kD protein containing multiple domains resembling elements of the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor

The primary structure of the large human basement membrane heparan sulfate proteoglycan (HSPG) core protein was determined from cDNA clones. The cDNA sequence codes for a 467-kD protein with a 21-residue signal peptide. Analysis of the amino acid sequence showed that the protein consists of five domains. The amino-terminal domain I contains three putative heparan sulfate attachment sites; domain II has four LDL receptor-like repeats; domain III contains repeats similar to those in the short arms of laminin; domain IV has lg-like repeats resembling those in neural cell adhesion molecules; and domain V contains sequences resembling repeats in the G domain of the laminin A chain and repeats in the EGF. The domain structure of the human basement membrane HSPG core protein suggests that this mosaic protein has evolved through shuffling of at least four different functional elements previously identified in other proteins and through duplication of these elements to form the functional domains. Comparison of the human amino acid sequence with a partial amino acid sequence from the corresponding mouse protein (Noonan, D. M., E. A. Horigan, S. R. Ledbetter, G. Vogeli, M. Sasaki, Y. Yamada, and J. R. Hassell. 1988. J. Biol. Chem. 263:16379-16387) shows a major difference between the species in domain IV, which contains the Ig repeats: seven additional repeats are found in the human protein inserted in the middle of the second repeat in the mouse sequence. This suggests either alternative splicing or a very recent duplication event in evolution. The multidomain structure of the basement membrane HSPG implies a versatile role for this protein. The heparan sulfate chains presumably participate in the selective permeability of basement membranes and, additionally, the core protein may be involved in a number of biological functions such as cell binding, LDL-metabolism, basement membrane assembly, calcium binding, and growth- and neurite-promoting activities.     

Reversible phosphorylation and turnover of the D1 protein under various redox states of Photosystem II induced by low temperature photoinhibition

Reversible phosphorylation and turnover of the D1 protein in vivo were studied under low-temperature photoinhibition of pumpkin leaves and under subsequent recovery at low light at 4 °C or 23 °C. The inactivation of PS II and photodamage to D1 were not enhanced during low-temperature photoinhibition when compared to that at room temperature. The PS II repair cycle, however, was completely blocked at 4 °C at the level of D1 degradation. Both the recovery of the photochemical activity of PS II and the degradation of the damaged D1 protein at low light at 23 °C were delayed about 1 hour after low-temperature photoinhibition, suggesting that in addition to the decrease in catalytic turnover of the enzyme, the protease was specifically inactivated in vivo at low temperature. The effect of low temperature on the other regulatory enzymes of PS II repair, protein kinase and phosphatase [Rintamäki et al. (1996) J Biol Chem 271: 14870-14875] was variable. The D1 protein kinase was operational at low temperature while dephosphorylation of the D1 protein seemed to be completely inhibited during low temperature treatment. Under subsequent recovery conditions at low light and 23 °C, the high phosphorylation level of D1 was sustained in leaf discs photoinhibited at low temperature, despite the recovery of the phosphatase activity. This high phosphorylation level of D1 was due to the persistently active kinase. The D1 kinase, previously shown to get activated by reduction of plastoquinone, was, however, found to be maximally active already at relatively low redox state of the plastoquinone pool. We suggest that phosphorylation of PS II centers increases the stability of PS II complexes and concomitantly improves their survival under stress conditions.     

Oxidized low density lipoprotein impairs endothelial progenitor cell function by downregulation of E-selectin and integrin alpha(v)beta5

Background

Oxidized low density lipoprotein (oxLDL) has been shown to induce apoptosis and senescence of endothelial progenitor cells (EPC). In the present study, we hypothesized that even sub-apoptotic concentrations of oxLDL impair the angiogenic potential of EPC and investigated if this effect is mediated by affecting adhesion and incorporation.

Methods

A co-culture system of human microvascular endothelial cells and EPC was used to study the effect of sub-apoptotic concentrations of native (nLDL) and oxLDL on cell-cell interaction. The expression and the functional role of angiogenic adhesion molecules and integrins was monitored by FACS and neutralizing assay, respectively.

Results

We observed an inhibition of tube formation and impairment of EPC integration into the vascular network of mature endothelial cells by oxLDL. In contrast, nLDL did not affect angiogenic properties of EPC. Incubation of EPC with sub-apoptotic oxLDL concentrations significantly decreased E-selectin and integrin α_vβ₅ expression (37.6% positive events vs. 71.5% and 24.3% vs. 49.9% compared to control culture media without oxLDL). Interestingly, expression of α_vβ₃, VE-cadherin and CD31 remained unchanged. Blocking of E-selectin and integrin α_vβ₅ by neutralizing antibody effectively inhibited adhesion of EPC to differentiated endothelial cells (56.5% and 41.9% of control; p < 0.001).

Conclusion

In conclusion, oxidative alteration of LDL impairs angiogenic properties of EPC at sub-apoptotic levels by downregulation of E-selectin and integrin α_vβ₅, both substantial mediators of EPC-endothelial cell interaction.     

Oxidative modification of low density lipoprotein (LDL) by activated human monocytes and the cell lines U937 and HL60

Summary Human peripheral blood monocytes, upon activation, have the capacity to oxidize low density lipoprotein (LDL) and render the LDL toxic to cultured cells. Previous studies by our laboratory indicate that this process is mediated by free radicals in that it can be prevented by addition of free radical scavengers and antioxidants during the incubation of monocytes with LDL. Here we report that optimal modification of LDL by monocytes was influenced by media composition. In the absence of added metal ions, oxidation was distinctly dependent on the concentration of monocytes as well as LDL concentration. Exposure of monocytes to lipopolysaccharide or stimulation of phagocytosis by opsonized zymosan resulted in marked enhancement of LDL oxidation compared to other activating agents. After exposure to activated monocytes, lipid oxidation products in the supernatant were found both in a high molecular weight fraction containing LDL (>30 000 Daltons) and in a lipoprotein-free, low molecular weight fraction (<30 000 Daltons), yet only the high molecular weight, LDL-containing fraction was toxic to target cells. In addition, human myelomonocytic cell lines U937 and HL60 were shown to mediate oxidation of LDL. As with monocytes, exposing these cells to opsonized zymosan caused the level of LDL oxidation to be significantly enhanced. These findings offer further insight into the mechanisms of monocyte-mediated oxidation of lipoproteins and will facilitate studies investigating the role of monocyte-modified LDL in tissue injury. This project was funded by grants form the American Heart Association-Northeast Ohio Affiliate and the National Institutes of Health, Bethesda, MD (HL-29582).