Neutral Red Dye Research Articles

Ablation of the secondary heart field leads to tetralogy of Fallot and pulmonary atresia

Recent studies in chick and mouse embryos have identified a previously unrecognized secondary heart field (SHF), located in the ventral midline splanchnic mesenchyme, which provides additional myocardial cells to the outflow tract as the heart tube lengthens during cardiac looping. In order to further delineate the contribution of this secondary myocardium to outflow development, we labeled the right SHF of Hamburger–Hamilton (HH) stage 14 chick embryos via microinjection of DiI/rhodamine and followed the fluorescently labeled cells over a 96-h time period. These experiments confirmed the movement of the SHF into the outflow and its spiraling migration distally, with the right side of the SHF contributing to the left side of the outflow. In contrast, when the right SHF was labeled at HH18, the fluorescence was limited to the caudal wall of the lengthening aortic sac. We then injected a combination of DiI and neutral red dye, and ablated the SHF in HH14 or 18 chick embryos. Embryos were allowed to develop until day 9, and harvested for assessment of outflow alignment. Of the embryos ablated at HH14, 76% demonstrated cardiac defects including overriding aorta and pulmonary atresia, while none of the sham-operated controls were affected. In addition, the more severely affected embryos demonstrated coronary artery anomalies. The embryos ablated at HH18 also manifested coronary artery anomalies but maintained normal outflow alignment. Therefore, the myocardium added to the outflow by the SHF at earlier stages is required for the elongation and appropriate alignment of the outflow tract. However, at later stages, the SHF contributes to the smooth muscle component of the outflow vessels above the pulmonary and aortic valves which is important for the development of the coronary artery stems. This work suggests a role for the SHF in a subset of congenital heart defects that have overriding aorta and coronary artery anomalies, such as tetralogy of Fallot and double outlet right ventricle.

Structure activity relationships of aristolochic acid analogues: Toxicity in cultured renal epithelial cells

Aristolochia species are nephrotoxic and carcinogenic. Recent studies showed that aristolochic acid (AA) could induce acute renal failure and tubular lesions in several species and available evidences demonstrate the unequivocal role of AA in so called Chinese herbs nephropathy. A series of AA derivatives isolated from Aristolochia spp. were analyzed for their nephrotoxic potential using the neutral red dye exclusion assay in cultures of LLC-PK(1) cells. The structural relationships between AA I and its analogues were compared with their cytotoxic effects to predict structural determinants for AA toxicity. Further, caspase-3 assay was performed on toxic compounds to determine if caspases, the enzymes that play a critical role in apoptosis are involved in AA-induced cytotoxicity. AA I was found to be most toxic followed by AA II, AA VIIIa, and AA Ia in decreasing levels of toxicity. The other compounds, nitrophenanthrene carboxylic acid analogues of AA I, aristolactams, and other derivatives did not exhibit considerable toxicity. The results showed significant relationships between cytotoxicity of AA compounds and the localization of functional groups in their structure. Analogues containing hydroxyl groups diminished cytotoxicity. The demethylated analogues of AA I are markedly less active. The negative impact on cytotoxicity was found on nitroreduction of AA I. AA induced caspase activation was also observed. These cytotoxic data suggest that the nitro and methoxy groups are critical determinants of nephrotoxicologic potency of AA.

Assays for the assessment of neutralizing antibody activities against Severe Acute Respiratory Syndrome (SARS) associated coronavirus (SCV)

Accurate assessment of neutralizing antibody activities is important either for patients infected with Severe Acute Respiratory Syndrome (SARS) or for animals and volunteers immunized with the experimental vaccines against the SARS associated coronavirus (SCV). However, the current assay based on the cytopathic effect (CPE) which has been frequently cited in literature has several limitations. The CPE assay relies on the visual observation on the damage of SCV infected target cells under a microscope. It is subjected to observer variations and it is difficult to generate a quantitative determination of neutralizing activities based on the level of CPE. In the current study, we established the utility of two additional assays to measure the neutralizing activities against SCV: the plaque reduction (PR) and the neutral red staining (NRS) assays. The PR assay described in this study was modified from the traditional viral plaque reduction assay by using an improved crystal staining method to achieve better plague formation in SCV infected Vero E6 cells. The NRS neutralization assay was adopted from a similar system used for detecting neutralizing antibody responses against human immunodeficiency virus type 1 (HIV-1). In this assay, the protective effect of neutralizing antibodies was determined by the cell viability which is measured by the uptake of neutral red dye at A 540. The neutralizing antibody titers can be easily determined with either of the two new assays. In this report, we described the utility of these two new neutralization assays in measuring the neutralizing activities against SCV infection from rabbit sera immunized with various forms of spike protein of SCV.

Involvement of Kv1 Potassium Channels in Spreading Acidification and Depression in the Cerebellar Cortex

Spreading acidification and depression (SAD) is a form of propagated activity in the cerebellar cortex characterized by acidification and a transient depression in excitability. This study investigated the role of Kv1 potassium channels in SAD using neutral red, flavoprotein autofluorescence, and voltage-sensitive dye optical imaging in the mouse cerebellar cortex, in vivo. The probability of evoking SAD was greatly increased by blocking Kv1.1 as well as Kv1.2 potassium channels by their specific blockers dendrotoxin K (DTX-K) and tityustoxin (TsTX), respectively. DTX-K not only greatly lowered the threshold for evoking SAD but also resulted in multiple cycles of spread and spontaneous SAD. The occurrence of spontaneous SAD originating from spontaneous parallel fiber-like beams of activity suggests that blocking Kv1 channels increased parallel fiber excitability. This was confirmed by the generation of parallel fiber-like beams with the microinjection of glutamate into the upper molecular layer in the presence of DTX-K. The dramatic effects of DTX-K suggest a possible connection between SAD and episodic ataxia type 1 (EA1), a Kv1.1 potassium channelopathy. The threshold for evoking SAD was significantly lowered in the Kv1.1 heterozygous knockout mouse compared with wild-type littermates. Carbamazepine and acetazolamide, both effective in the treatment of EA1, significantly decreased the likelihood of evoking SAD. Blocking GABAergic neurotransmission did not alter the effectiveness of DTX-K. The cyclin D2 null mouse, which lacks cerebellar stellate cells, also exhibited SAD. Therefore blocking Kv1 potassium channels establishes the conditions needed to generate SAD. Furthermore, the results are consistent with the hypothesis that SAD may underlie the transient attacks of ataxia characterizing EA1.

Antimicrobial Treatment of Dental Osseous Defects with Silver Doped Bioglass: Osteoblast Cell Response

In dentistry, chronic periodontitis often leads to bone resorption together with an increasing risk of bacteremia. Bioactive glass has found extensive application as dental graft material. A successful antimicrobial bactericidal effect has been shown from the introduction of Ag2O into the glass composition. In this study, the cytotoxicity of soluble silver, calcium and silica ions on primary human osteoblasts was investigated by measurements of mitochondrial activity and neutral red dye uptake. Silver concentrations of 4 - 6 ppm (1 mg/ml conc.) and 6 - 9 ppm (2 mg/ml conc.) have been measured in complete culture medium. It was found that the bioactive gel-glass extract with an initial concentration of 1 mg/ml (1mg glass per ml of culture medium) has no negative effect, whereas increased gel-glass concentration of 2 mg/ml seemed to have a toxic effect on the cell viability of human osteoblasts. It might be concluded that a reduction of the rate of silver dissolution from the bioactive gel-glass might preserve a maximum cell viability.

Indirect Cytotoxicity Evaluation of Silver Doped Bioglass Ag-S70C30 on Human Primary Keratinocytes

Bioactive gel-glasses, such as the silver-doped Ag-S70C30 glass, can be used to modify the inflammatory response in a local body compartment such as in acne lesions and in nonhealing dermal wounds. In this study, the cytotoxicity of soluble silver, calcium and silica ions on human epidermal keratinocytes was investigated by measurements of mitochondrial activity (MTT assay) and neutral red dye uptake (NR assay). Ag-S70C30 extracts were prepared by soaking glass powder in complete culture medium at concentrations of 1 mg/ml and 2 mg/ml (mg of glass powder per ml of culture medium). Silver concentrations for both concentrations of approximately 1 ppm were detected by inductive coupled plasma analysis (ICP). No negative effect on the cell viability was measured for an initial gel-glass concentration of 1 mg/ml and for the two shortest extraction times at a concentration of 2 mg/ml. Based on the results from MTT/ NR assays, a pH rise of approximately one unit had no negative effect on the NHEK-A cell viability. This preliminary study on keratinocyte viability merits future investigations on silver bioglass as a novel antimicrobial wound healing agent.

Serum Deprivation Induces Apoptotic Cell Death of Transformed Rat Retinal Ganglion Cells via Mitochondrial Signaling Pathways

Apoptosis-related signaling pathways were investigated in a cultured rat retinal ganglion cell (RGC-5) line deprived of growth factors after serum withdrawal from the culture medium. RGC-5 cells were subjected to serum deprivation for 2 to 6 days and compared with RGC-5 cells cultured in growth medium containing 10% fetal calf serum. Cell viability was determined by a neutral red dye uptake assay. Apoptosis of RGC-5 cells was established by DNA laddering. The expression of various apoptosis-related genes was investigated by immunoblot analysis, and or reverse transcription polymerase chain reaction (RT-PCR) analysis. The redox state of the cell was determined by biochemical methods, including NF-kappaB binding activity by electrophoretic mobility gel shift assays (EMSA) and mitochondrial damage by JC-1 (5,5', 6,6'-tetrachloro 1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) staining, using live cell confocal microscopy and cytosolic release of cytochrome c. Fifty percent cell loss was evident after 2 days of serum deprivation, as demonstrated by neutral red dye uptake assay. This cell loss was due to apoptotic cell death, as established by DNA laddering. The oxidative state of serum-deprived RGC-5 cells was perturbed as suggested by the increase in malonyldialdehyde (MDA) and a decrease in reduced glutathione (GSH) levels in cell lysates. The apoptosis of the RGC-5 cells was associated with the activation of caspase-3, -8, and -9, and increased levels of Bax with corresponding decreases in Bcl-2 levels and NF-kappaB (NF-kappaB) binding activity. Serum deprivation was also associated with a loss of mitochondrial function, as revealed by cytosolic release of cytochrome c and JC-1 staining of mitochondria of dying RGC-5 cells. Taken together, these results indicate that serum withdrawal induces apoptotic cell death in RGC-5 cells via mitochondrial pathways. These studies lead to the speculation that growth factor deprivation arising from blockade of retrograde transport of neurotrophins may involve similar mechanism(s) of retinal ganglion cell death in glaucoma.

Lysosomal responses as a diagnostic tool for the detection of chronic petroleum pollution at Todos os Santos Bay, Brazil

Coastal marine environments, especially semienclosed systems such as bays, are under unrelenting stress caused by urban and industrial development. Biomonitoring plays a vital role in strategies to identify, assess, and control stressors. However, due to the magnitude of the challenge there is a demand for new and innovative approaches to provide timely and accessible information to environmental managers and policy makers. The present work aimed to assess hydrocarbon levels in sediments from petroleum-related industrial areas at Todos os Santos Bay (Brazil) and associate them to toxicity-induced responses (neutral red retention (NRR) assay) by the burrowing clam Anomalocardia brasiliana. Surface sediments collected during the dry and rainy seasons were analyzed for aliphatic and aromatic hydrocarbons. At the control site, hydrocarbon levels were low and mainly biogenic. The aliphatic hydrocarbon (“total unresolved complex mixture,” alkanes, and isoprenoids) concentrations indicated a chronic situation with very little “fresh” oil contamination at the oil-related sites. The polycyclic aromatic hydrocarbons indicated sites moderately contaminated by chronic oil and some pyrolytic input. The effects of those contaminants were assessed by the lysosomal NRR assay applied to A. brasiliana hemocytes. Sediment toxicity at the oil-related sites was evidenced by the lowered capacity of the lysosomes to retain the neutral red dye compared to results from the control site. This research indicates that the NRR assay is a useful and efficient screening technique able to discriminate polluted from clean sites.

Evaluation of in vitro endocytosis and antibody synthesis by rainbow trout head kidney cells treated with bovine lactoferrin

Bovine lactoferrin (LF) was evaluated for its capacity to modulate the in vitro endocytosis (phagocytosis and pinocytosis) and antibody synthesis by head kidney cells of rainbow trout Oncorhynchus mykiss. Phagocytic activity and phagocytic index of head kidney macrophages, determined by measurement of ingested yeast, were influenced by bovine LF starting from the LF concentration of 1 and 0·1 μg ml−1, respectively. Endocytosis, determined by the evaluation of droplet uptake of neutral red dye solution, was significantly enhanced by 10 μg ml−1 of LF. In contrast, antibody synthesis by head kidney cells, evaluated by immunoenzymatic assay, from fish immunized against human‐γ‐globulins (HGG)in vivo was not affected by bovine LF. Although these results showed that bovine LF had no effect on specific immunoglobulin production in vitro, an enhancement of the acquired immune response may be assumed in LF‐treated fish in vivo, as observed in higher vertebrates.

Adaptations to Foliar Absorption of Faeces: a Pathway in Plant Carnivory

Roridula plants capture insects but have no digestive enzymes. It has been hypothesized that Roridula leaves absorb nitrogen from the faeces of obligately associated, carnivorous hemipterans. But rapid movement across the leaf surfaces of most plant leaves is prevented by the presence of an impermeable cuticle. However, in carnivorous plants, cuticular gaps or pores in digestive/absorptive cells allow rapid movement across the leaf surface. Recently, it was suggested that the hemipteran-plant interaction constituted a new pathway for plant carnivory. Here, a further adaptation to this pathway is described by demonstrating how Roridula plants probably absorb hemipteran faeces rapidly through their leaf cuticles. The dye neutral red was used to document the rapidity of foliar absorption and TEM to determine the nature of cuticular discontinuities in the leaf of Roridula. Aqueous compounds diffuse rapidly across the cuticle of Roridula's leaves but not across the cuticles of co-occurring, non-carnivorous plant leaves. Furthermore, immature Roridula leaves were unable to absorb neutral red whereas mature leaves could. Using TEM, cuticular gaps and pores similar to those in other carnivorous plants were found in the epidermal cells of mature Roridula leaves. The leaf cuticle of Roridula is very thin (0-120 nm) and cell wall elements project close to the leaf surface, possibly enhancing foliar absorption. In addition to these, cuticular gaps were frequently seen and probably perform a function similar to those found in other carnivorous plants: namely the absorption of aqueous compounds. The cuticular gaps of Roridula are probably an adaptation to plant carnivory, supporting the newly described pathway.

COMPARATIVE CYTOTOXICITY POTENTIAL OF SOFT CONTACT LENS CARE PRODUCTS

ABSTRACTPurpose. To determine the cytotoxicity potential of soft contact lens care products and benzalkonium chloride (BAK) by two colorimetric in vitro assays on an immortalized human corneal epithelial cell line (HCE-T). Methods. Four commercial soft contact lens care solutions were tested at 1:3 dilution in growth medium. The positive controls for cytotoxicity were BAK in Dulbecco's phosphate buffered saline (DPBS) at 10, 5, 2.5, and 1.25 ppm and 1:3 dilution of DPBS as negative control. Cell viability was assayed using a novel tetrazolium compound and an electron coupling reagent (MTS/PES), and by cell membrane integrity using neutral red dye uptake and release (NRUR). Mean spectrophotometric optical density of the test samples was compared to mean optical density of the controls at 24 and 48 hours of exposure to test product. Significance was measured by ANOVA/Tukey HSD test. Results. Cell viability and cell membrane integrity tests were not significantly different between lens care solutions and the negative controls for CIBA Vision SOLOcare™ PLUS, Advanced Medical Optics COMPLETE® Comfort PLUS™, or BAK at 1.25 ppm. Exposure to Bausch and Lomb ReNu MultiPlus®, Alcon OPTI-FREE® Express® with Aldox™, BAK 10, 5, and 2.5 ppm yielded significant reduction in cell viability and membrane integrity compared to negative controls. Discussion. Assays of HCE-T cell viability by MTS/PES, and cell membrane integrity by NRUR cells yielded data that were similar to that previously reported with mouse L929 cells in tests based on the USP Elution Test. For the MTS/PES and NRUR assay methods, the solutions in order of increasing cytotoxicity potential were: SOLOCare = COMPLETE Comfort Plus < ReNu < < OPTI-FREE® Express® with Aldox.

4-Nonylphenol induces immunomodulation and apoptotic events in the clam Tapes philippinarum

The effects of 4-nonylphenol (NP) on functional responses of haemocytes from the clam Tapes philippinarum were investigated after 7 d exposure to sublethal NP concentrations (0, 0 + ace- tone, 0.025, 0.05, 0.1 and 0.2 mg l -1 NP). Haemocytes from both controls and exposed clams were col- lected, and the effects of NP on uptake of the vital dye Neutral Red (NR), superoxide dismutase (SOD) and lysozyme activities, total haemocyte count (THC) and volume of circulating cells were evaluated. The capability of NP to induce apoptosis was also investigated. Exposure of clams to 0.2 mg l -1 NP significantly increased (p < 0.05) NR uptake when compared with controls, suggesting that NP caused alterations in cell membrane stability. Significant decreases in both SOD and lysozyme activ- ity were observed from 0.05 mg l -1 NP (p < 0.01 and p < 0.05, respectively) with respect to controls, indicating that NP causes oxidative stress and reduces the immunocompetence of the exposed clams. From 0.05 mg l -1 NP, the apoptotic index (percentage of haemocytes showing positivity to the TUNEL reaction) significantly increased (p < 0.001). Apoptotic haemocytes showed shrinkage and lost their amoeboid shape. Moreover, NP exposure significantly increased THC (p < 0.05 at 0.2 mg l -1 NP) and caused a different distribution in size frequency. On the basis of the threshold concentration values, the most sensitive endpoints for NP were SOD, lysozyme and apoptosis assays. Our results highlight a relationship between NP exposure and changes in the functional responses of haemocytes, sug- gesting that the contaminant induces immunomodulation in T. philippinarum, mainly by altering cell morphology, cell membrane stability and enzymatic activities, and by promoting oxidative stress and apoptotic events.

The Use of Heterogeneous Advanced Oxidation Processes to Degrade Neutral Red Dye in Aqueous Solution

The photodecolorization of neutral red dye has been carried out using a combination between ZnO and UV radiation (λ = 254 nm) at a pH of 6. The dye decolorization was also assessed by adding low concentrations of inorganic oxidants such as Na2S2O8 and NaIO4 to ZnO in the presence of UV radiation. It was observed that the dye decolorization was enhanced using UV/ZnO/Na2S2O8 and UV/ZnO/NaIO4 catalytic systems over UV/ZnO alone. First-order kinetics adequately described the disappearance of the dye. Reaction rate orders with respect to the oxidant species were 0.6, 0.5, and 1.2 for, respectively, ZnO, Na2S2O8, and NaIO4. The most effective catalytic system for dye decolorization was found to be UV/ZnO/NaIO4.

Laser-induced micro-bubbles in cells

We report experimental study and detection of laser-induced micro-bubbles in individual live cells and in confined micro-volumes of solutions. Using pulsed laser radiation (532 nm, 10 ns) as the source of local heating we detected micro-bubbles in light-absorbing media that were caused at least by two mechanisms—heating of the media above (more than 300 °C) critical temperature (referred as ‘hot’ bubbles) and action of rarefaction pressure waves (referred as ‘cold’ bubbles) at much lower temperatures (30–150 °C). Bubble generation thresholds, probabilities of bubble forming and bubble lifetimes were experimentally studied with two photothermal (PT) methods—thermal lensing and PT-time-resolved imaging for human red blood cells, homogeneous solutions of haemoglobin, neutral red dye and for suspension of polystyrene nano-particles.

Antiviral activity of Undaria pinnatifida against herpes simplex virus.

The major component of an aqueous extract of the seaweed Undaria pinnati fi da has been identified previously as a galactofucan (GFS), a sulfated polysaccharide. The galactofucan was partially purified and the material tested in this study is 75% pure galactofucan sulfate. GFS was evaluated for antiviral activity against 32 clinical strains of herpes simplex virus (HSV): 14 strains of HSV-1 and 18 strains of HSV-2. Twelve strains (four HSV-1 and eight HSV-2) were resistant to acyclovir (ACV-R) and 20 strains (10 HSV-1 and 10 HSV-2) were susceptible to ACV (ACV-S). The median IC(50) of GFS for the 14 strains of HSV-1 was 32 micro g/mL. The median IC(50) of GFS for the 18 strains of HSV-2 was 0.5 micro g/mL. GFS is significantly more active against clinical strains of HSV-2 than HSV-1, p < 0.001. The mode of action of the GFS was shown to be the inhibition of viral binding and entry into the host cell. The cytotoxicity of GFS was >4.0 mg/mL in the neutral red dye uptake assay indicating that GFS is non-toxic in this assay.

Structure of polysaccharide from Polygonatum cyrtonema Hua and the antiherpetic activity of its hydrolyzed fragments

A neutral polysaccharide named PD was isolated from the traditional Chinese medicinal herb, Polygonatum cyrtonema Hua. Five fragments were isolated by Bio-Gel P4 chromatography from hydrolysates of PD. Using assays of cytopathic effect inhibition, neutral red dye uptake and plaque forming inhibition, it was proved that the fragments with degree of polymerization (DP) of 4 and 5 were the shortest ones which retained the activity against herpes simplex virus type 2 (HSV-2) in vero cell culture. The structures of PD and one of its activity-retaining fragments, B3, were determined by permethylation followed with reductive cleavage, mass spectrometry and nuclear magnetic resonance spectrometry. It was shown that PD was a branched fructan with average DP of 28. There was one two-residue side chain composed of (2 → 6)-linked β- d-fructofuranosyl (Fru f) residues every three (2 → 1)-linked β- d-Fru f residues in the backbone of PD, whereas B3 was a mixture containing 1-kestose and neokestose series of oligosaccharides of DP 3–5 without branches.

Evaluation of the neutral red assay as a stress response indicator in cultivated mussels ( Mytilus spp.) in relation to post-harvest processing activities and storage conditions

The neutral red assay (NRA) was evaluated and used as a biochemical indicator of stress response in cultured mussels that were subjected to various post-harvest processing and storage conditions. The NRA, which measures retention time of neutral red dye in the hemocyte organelle, the lysosome, can be correlated to the condition of mussels under various circumstances. Shelf life also provides an index of mussel condition and quality. The objectives of this study were to evaluate mussel stress response in relation to post-harvest conditions of (1) handling, (2) processing, and (3) storage practices. Neutral red retention was altered in mussels that underwent washing and declumping compared to unprocessed mussels. However, the process of debyssing substantially decreased neutral red retention in mussels. Mussels held under chilled or iced storage conditions displayed decreased neutral red retention compared to those held under wet storage. The results demonstrated that NRA was a useful index of physiological stress response in mussels subjected to conditions under various culture practices. The implications of this study for growers are that the practice of reimmersion of mussels for 24 h, especially after debyssing, and storage of mussels in water or on ice can reduce stress response and provide a better quality product with longer shelf life.

The in vitro biocompatibility performance of a 25 mmol/L bicarbonate/10 mmol/L lactate-buffered peritoneal dialysis fluid

The in vitro biocompatibility performance of a 25 mmol/L bicarbonate/10 mmol/L lactate-buffered peritoneal dialysis fluid.The biocompatibility profile of a new peritoneal dialysis (PD) solution (Physioneal 35) was determined using a selection of in vitro assay systems. Physioneal 35 is buffered by a combination of 25 mmol/L bicarbonate and 10 mmol/L lactate, thereby providing a solution with a total of 35 mmol/L of alkali to complement the currently available 25 mmol/L bicarbonate and 15 mmol/L lactate combination solution, Physioneal 40. In addition, the new solution contains a calcium concentration of 1.75 mmol/L rather than 1.25 mmol/L present in Physioneal 40. Physioneal 35 and 40 are manufactured in double chamber bag systems that permit separation of glucose from the buffer during sterilization. When the two chambers are mixed just before patient use, the resulting solution has a neutral pH and reduced glucose degradation content. Physioneal 35 was evaluated for its cytotoxicity potential using a murine fibroblast assay, its acute effect on human neutrophil and human peritoneal mesothelial cell function, and its in vitro potential to form advanced glycation end products (AGE). The biocompatibility characteristics of this new formulation were compared with that of a conventional, lactate-based solution and to that of its parent formulation, Physioneal 40.Proliferation of murine fibroblasts was determined after exposure to dialysis fluids for 72 hours. Cell viability was assayed by the ability to take up neutral red dye. Human neutrophils were exposed for 15 minutes to dialysis fluids, and their ATP content and phorbol 12-myristate 13-acetate (PMA) stimulated chemiluminescence response was determined as a measure of viability and respiratory burst activity, respectively. Cellular interleukin (IL)-1beta-driven IL-8 synthesis by human mesothelial cells following acute exposure to dialysis fluids was also assessed. Advanced glycation end product formation in the dialysis fluids was measured after 5 and 20 days of incubation with human serum albumin (HSA) as the model protein.In all assays employed, the biocompatibility profile of Physioneal 35 was similar to that of the Physioneal 40 parent formulation. Physioneal 35 showed a significant improvement in biocompatibility performance compared to a pH neutralized conventional lactate-buffered peritoneal dialysis solution in the murine fibroblast assay. In the acute exposure assays, human neutrophil viability and respiratory burst were significantly improved compared with the acidic, conventional solution; however, no statistically significant improvement were seen in mesothelial cells. AGE formation, which is thought to be an important mechanism by which glucose and glucose degradation products cause structural and functional changes of the peritoneal membrane, was significantly lower in Physioneal 35 compared with the conventional dialysis solution.The biocompatibility profile of Physioneal 35 was similar to that of the original Physioneal 40 bicarbonate/ lactate-buffered dialysis solution, confirming that differences in both buffer content and calcium concentration do not affect biocompatibility performance. Both bicarbonate/lactate formulations (Physioneal 35 and Physioneal 40) were more biocompatible than a conventional lactate-buffered dialysis solution in this in vitro biocompatibility assessment.

Synergism Between Magnetic Fields And Lead Overload On ThePermeability Of The Blood Brain Barrier In Mice

Extremely low frequency electromagnetic fields (EMF, 50 Hz) are emitted mainly by high tension power lines and domestic appliances. There are opposing opinions as to the possible toxicity of this radiation. Various epidemiological studies point to a possible direct involvement of EMF in certain childhood illnesses detected in the immediate vicinity of high tension pylons. However other studies deny any such link, and disaffm the epidemiological results for varying reasons. The aim of this study is to show the effect of exposure to a magnetic field of 50 Hz and 27.5 G (2.75 mT) intensity, on the permeability of the blood brain barrier in mice, as well as to consider the influence that the presence or absence of lead in the organism of the mice might have on the brain's response to this radiation. The synergic effect of lead is established as a neurotoxic agent, with the transfer of the neutral red dye to the brain. This metal has been chosen because it is one of the most frequent environmental pollutants, both in water and on land.

Involvement of Syntaxin 7 in Human Gastric Epithelial Cell Vacuolation Induced by the Helicobacter pylori-produced Cytotoxin VacA

The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The formed vacuole is assumed to be a hybrid of late endosome and lysosome. To elucidate the molecular mechanism of VacA-induced vacuolation, we examined the participation of syntaxin 7 in the human gastric epithelial cell line AGS. Immunocytochemistry revealed that endogenous syntaxin 7 was localized to vacuoles induced by VacA. Northern and Western blotting demonstrated that VacA intoxication increased syntaxin 7 mRNA and protein expression, respectively, in a time-dependent manner. Transient transfection of dominant-negative mutant syntaxin 7, which lacks a carboxyl-terminal transmembrane domain, inhibited VacA-induced vacuolation. In contrast, transient transfection of wild-type syntaxin 7, dominant-negative mutant syntaxin 1a, or dominant-negative mutant syntaxin 4 did not alter VacA-induced vacuolation. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with mutant syntaxin 7 but not in cells stably transfected with wild-type syntaxin 7, mutant syntaxin 1a, or mutant syntaxin 4. Sequential immunocytochemical observation confirmed that expression of mutant syntaxin 7 did not affect VacA attachment to or internalization into AGS cells. We suggest that syntaxin 7 is involved in the intracellular vacuolation induced by VacA.