Fluorescein Diacetate-propidium Iodide Research Articles

Methanolic extract of Entada phaseoloides inhibits Colorectal carcinoma cells proliferation of HT-29 cell by Modulating mitochondria-mediated apoptotic pathway

Colorectal cancer (CRC) is among the leading three diseases with higher death rates worldwide, with an expected 2.2 million new cases continuously in 2030. The expanding utilization of common plant-inferred parts, investigating the counter proliferative impacts of phytochemicals is progressively picking up significance in planning anticancer medications. This study aimed to examine the effect of methanolic extracts of Entada phaseoloides (MEEP) on the apoptotic pathway in human colon carcinoma cells (HT-29 cells). MTT assay and live/dead staining with fluorescein diacetate/propidium iodide (FDA/PI) were utilized to quantify cell viability in cancer cells. This research facility's exploratory investigation showed the effects of colon malignant growth cells (HT-29) exposed to various portions (1.23, 3.70, 11.11, 33.33, and 100µg/mL) of MEEP. The pure compounds isolated from the extracts includes Oleic acid, Entadamide A, Entadamide A-beta-D-glucopyranoside, Phaseoloidin. The result showed that MEEP actuated the concealment of cell viability and apoptosis in HT-29 cells in a dose-dependent manner. This included characteristic changes in nuclear morphology, the breakdown of mitochondrial membrane potential (Δψm), up-regulation of pro-apoptotic BAX, and down-regulation of anti-apoptotic Bcl-2, which initiates the transformation of caspase-3 to cleaved caspase-3, thus actuating PARP promoting apoptosis. Furthermore, it was found that MEEP does not affect ROS production. Thus overall findings applies against proliferative impact through various signalling pathways, is an outstanding possibility for hostile to colon cancer therapy with the help of natural sources.

The effect of collagen hydrogels on chondrocyte behaviors through restricting the contraction of cell/hydrogel constructs.

Collagen is a promising material for tissue engineering, but the poor mechanical properties of collagen hydrogels, which tend to cause contraction under the action of cellular activity, make its application challengeable. In this study, the amino group of type I collagen (Col I) was modified with methacrylic anhydride (MA) and the photo-crosslinkable methacrylate anhydride modified type I collagen (CM) with three different degrees of substitution (DS) was prepared. The physical properties of CM and Col I hydrogels were tested, including micromorphology, mechanical properties and degradation properties. The results showed that the storage modulus and degradation rate of hydrogels could be adjusted by changing the DS of CM. In vitro, chondrocytes were seeded into these four groups of hydrogels and subjected to fluorescein diacetate/propidium iodide (FDA/PI) staining, cell counting kit-8 (CCK-8) test, histological staining and cartilage-related gene expression analysis. In vivo, these hydrogels encapsulating chondrocytes were implanted subcutaneously into nude mice, then histological staining and sulfated glycosaminoglycan (sGAG)/DNA assays were performed. The results demonstrated that contraction of hydrogels affected behaviors of chondrocytes, and CM hydrogels with suitable DS could resist contraction of hydrogels and promote the secretion of cartilage-specific matrix in vitro and in vivo.

4-Methylumbelliferone as a potent and selective antitumor drug on a glioblastoma model.

Glioblastoma (GBM), the most frequent primary tumor of the central nervous system, has a median survival of 14.6months. 4-Methylumbelliferone (4MU) is a coumarin derivative widely used as a hyaluronan synthesis inhibitor with proven antitumor activity and without toxic effects reported. We aim to evaluate the antitumor effect of 4MU alone or combined with temozolomide (TMZ) on a GBM cell line, its absence of toxicity on brain cells and its selectivity for tumor cells. The antitumor effect of 4MU alone or combined with TMZ was evaluated on GL26 cells by assessing the metabolic activity through the XTT assay, cell proliferation by BrdU incorporation assay, migration by the wound healing assay, cell death by fluorescein diacetate/propidium iodide (FDA/PI) staining, apoptosis by membrane asymmetry and DNA fragmentation and metalloproteinase activity by zymography. The levels of hyaluronan and its capacity to counteract the effects of 4MU and the expression of RHAMM and CD44 were also determined. The toxicity and selectivity of 4MU were determined by XTT assay and PI staining on normal brain primary cell culture (NBPC-GFP) and GL26/NBPC-GFP cocultures. The GL26 cells expressed RHAMM but not CD44 while synthetized hyaluronan. 4MU decreased hyaluronan synthesis, diminished proliferation and induced apoptosis while reducing cell migration and the activity of metalloproteinases, which was restored by addition of hyaluronic acid. Furthermore, 4MU sensitized GL26 cells to the TMZ effect and showed selective toxicity on tumor cells without exhibiting neurotoxic effects. We demonstrated for the first time the cytotoxic effect of 4MU on GBM cells, highlighting its potential usefulness to improve GBM treatment.

Effects of PRP and LyPRP on osteogenic differentiation of MSCs.

Platelet-rich plasma (PRP) is rich in a variety of growth factors and plays an important role in the proliferation and differentiation of mesenchymal stem cells (MSCs). It has been reported that the preparation of freeze-dried platelets (lyophilized platelets [LyPRP]) from platelets could be an effective strategy to preserve the bioactivity of platelets for a long time. In this study, the osteogenic induction effects of PRP and LyPRP on MSCs were evaluated. The rabbit arterial blood was drawing to preparation of PRP by secondary centrifugation. Whole blood was prepared by lyophilization buffer to prepare LyPRP, which were activated by chloride and their surface morphology was observed. It was observed using a scanning electron microscope that platelets were evenly distributed on the surface of PRP and LyPRP. Growth factors were slowly released from PRP and LyPRP during the first 7 days and detected by the enzyme-linked immunosorbent assay kit. Cell proliferation assays and fluoresceindiacetate/propidium iodide (FDA/PI) staining demonstrated that PRP and LyPRP could promote cell proliferation. PRP and LyPRP were also shown to promote osteogenic differentiation of MSCs in vitro by osteogenesis characteristic staining and qPCR quantitative detection of osteogenic related gene expression. Both PRP and LyPRP could promote the proliferation and osteogenic differentiation of MSCs effectively. Moreover, PRP exhibited a better osteogenic induction effect on MSC than LyPRP.

Cell-laden interpenetrating network hydrogels formed from methacrylated gelatin and silk fibroin via a combination of sonication and photocrosslinking approaches

The regeneration of load-bearing soft tissues has long driven the research and development of bioactive hydrogels. A major challenge facing the application of hydrogels to load-bearing tissues is the development of hydrogels with appropriate biological functionality and biomechanical stability that closely mimic the host tissue. In this paper, we describe a newly synthesized cell-laden interpenetrating polymer network (IPN) hydrogel based on gelatin methacrylate (GelMA) and silk fibroin (SF) that was formed via sequential sonication and photocrosslinking. The experimental results revealed that SF-GelMA IPN hydrogels exhibited high swelling ratios, excellent mechanical properties, resistance to enzymatic degradation by collagenase, and porous internal microstructures. Moreover, these properties could be tailored by changing the prepolymer components. MC3T3-E1 pre-osteoblasts attached to and subsequently proliferated on the IPN hydrogels, as demonstrated by fluorescein diacetate/propidium iodide (FDA/PI) staining and Cell Counting Kit-8 (CCK-8) analysis. In addition, the encapsulation of MC3T3-E1 pre-osteoblasts and a subsequent cell viability assay demonstrated that the entire IPN formation process was compatible with cells and that the growth of encapsulated cells could be tuned by adjusting the GelMA concentration, underlining their versatility for various load-bearing soft tissue engineering. Overall, this study introduces a class of mechanically robust and tunable cell-laden IPN hydrogels which have great potential as load-bearing soft tissue engineering scaffold.

Modification of Polyurethane Scaffolds for Localised Immunosuppression of Subcutaneous Islet Transplantation.

Introduction Hepatic islet transplantation, as a curative therapy for Type 1 Diabetes, is suboptimal for islet function and viability. Subcutaneous engraftment presents an attractive alternative, yet, is currently incapable of supporting islet survival without modification. The use of a polyurethane (PU) scaffold has previously been shown to generate a hyper-vascularised dermal layer, and co-opting this may overcome inherent dermal hypoxia and hypovascularity. However, mitigating the cutaneous immune response is crucial. The aim of this study was to evaluate scaffold loading of the immunosuppressant rapamycin (Rapa), generating a localised immunosuppressive microenvironment supporting islet engraftment. Methods PU disks (8mmx2mm) were loaded topically with 2nM of Rapa. Structural changes to the PU were evaluated using SEM. UV-VIS spectroscopy was utilised to detail Rapa absorption (278nm) over 7 days evaluating drug release kinetics. In vitro biocompatibility of the Rapa-PU was investigated through co-culture with BTC-6 murine β-cell line, murine and human islets. Islet viability was evaluated using fluorescein diacetate/propidium iodide (FDA/PI) staining. T-cell inhibitory properties were evaluated in murine and human cells using a MLR and anti-CD3/28 antibody stimulation assay. Murine in vivo biocompatibility of subcutaneously implanted Rapa-PU was assessed with immunohistochemistry. Results/Discussion SEM of Rapa-PU showed increased pore density with surface integrity disruption, due to methanol treatment, and crystal deposition. Rapa-PU release kinetics demonstrated steady release of up to 70% (60nM) in 7 days following the Higuchi and Ritger-Peppas model, indicating a time dependent release caused by scaffold degradation. In vitro no phenotypically, acute, negative effects were observed upon BTC-6 cell, murine and human islet viability with exposure to PU alone and Rapa-PU. T cell proliferation was potently suppressed by the PU alone, however, immunosuppression was also observed from Rapa released from the scaffold through conditioned media. In vivo scaffolds were well tolerated, and blood vessel formation was detected in unloaded and Rapa-PU scaffolds evaluated by CD31 immunofluorescence staining, indicating that Rapa loading does not abrogate angiogenesis and is comparable to control scaffolds. Conclusion PU scaffolds provide a stable platform for the slow elution of rapamycin, neovascularisation and graft survival. Both the scaffold and rapamycin provides local immunosuppression. In doing so, this model could allow for initial local immunosuppression to prevent early immune infiltration of the graft. Additionally, this model may reduce the requirement for initial systemic immunosuppression, and deliver a higher, less toxic, immunosuppression to the site of transplantation.

Neuro-differentiated Ntera2 cancer stem cells encapsulated in alginate beads: First evidence of biological functionality

The present communication investigates an application of alginate encapsulation technology to the differentiation of the embryonic cancer stem NTera2 cells (NT2) into dopamine-producing cells. The encapsulation of cells in polymeric beads allows their immune isolation and makes them eligible for transplantation, thus representing a promising biotech tool for the delivery of biologically active compounds to the brain. The polysaccharide alginate is one of the most commonly used material for this procedure since it is well tolerated by various tissues, including the brain. Two different initial cell concentrations (i.e. 0.5∗106/ml and 1.0∗106/ml) were tested, in order to identify which one could better reflect the homogeneous cell distribution into the alginate beads and guarantee a good cell viability at different times of culture. As evidenced, the higher number of cells promoted the formation of clusters resulting in a better interaction among encapsulated cells and the subsequent promotion of mitotic activity. The distribution of alive/dead cells into the alginate beads was verified and followed at different time points through the fluorescein diacetate/propidium iodide (FDA/PI) staining, confirming the presence of living neuronal positive cells, as determined from fluorescence microscopy imaging. The functionality of the encapsulated NT2 cells was confirmed by their dopamine production capability as assessed by UV–Vis spectrophotometric analysis and by liquid chromatography–mass spectrometry (LC-MS). The NT2/microspheres system can be considered a groundbreaking experimental procedure, a functionally active platform, able to produce and release dopamine, and thus potentially exploitable for therapy in Parkinson's disease.

High Recovery of Functional Islets Stored at Low and Ultralow Temperatures.

Poor recovery of islets upon cryopreservation is the main hurdle in islet banking. Pancreatic islets have a poor antioxidative defense mechanism, and exposure of islets to low temperature leads to oxidative stress. We aimed to investigate whether known compounds such as metformin, γ aminobutyric acid (GABA), docosahexanoic acid (DHA), or eicosapentaenoic acid (EPA) alone or in combination are capable of reducing oxidative stress for better islet recovery upon storage at suboptimal temperatures. Islets isolated from mouse pancreas were stored at low temperature (4°C) for 15 days and at ultralow temperature (-196°C) for 30 days with or without additives. After revival from cold storage, islets were assessed by using three methods: (1) specificity by dithizone (DTZ), (2) viability by fluorescein diacetate/propidium iodide (FDA/PI) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay, and (3) functionality by glucose-stimulated insulin secretion (GSIS). The oxidative status of the islets stored at suboptimal temperatures was determined by both intracellular free radical release (fluorometric analysis) and lipid peroxidation (enzymatic determination). Supplementation with additives led to an improvement in islet survival upon storage at suboptimal temperatures, without depletion of insulin secretory activity, which was comparable to that of controls. The additives acted as cryoprotectants and antioxidants as revealed by high recovery of viable islets and reduction in total reactive oxygen species (ROS) and malonidealdehyde (MDA), respectively. Our results demonstrate for the first time that supplementation with EPA, DHA, and metformin may lead to higher islet recovery from -196°C storage, enabling proper islet banking.

Evaluation of Berberine’s Algicidal Effects on Toxic Microcystis aeruginosa Growth using a Double Fluorescein Staining Method

An accurate and convenient method for determining Microcystis cell viability is necessary to control blooms in small aquaculture water bodies. The fluorescein diacetate-propidium iodide (FDA-PI) staining method was developed to determine the viability of toxic Microcystis aeruginosa (FACHB905). Live M. aeruginosa 905 cells, stained with FDA, emitted bright green fluorescence after excitation at 470 nm. Dead cells, stained with PI, produced bright red fluorescence after excitation at 540 nm. Berberine inhibited M. aeruginosas 905 growth and the inhibition rate determined by FDA-PI fluorescein staining and counting on a dark field was much higher than when determined by counting on a bright field because some berberine-killed cells were misidentified as live cells. Thus, less berberine is needed to control Microcystis bloom when the accurate and reliable FDA-PI fluorescein staining method is used to judge the viability of the algae cells.

Evaluation of Cell‐Material Interactions on Newly Designed, Printable Polymers for Tissue Engineering Applications

AbstractThe generation of advanced scaffolds for tissue engineering is increasingly implemented by rapid prototyping techniques and therefore designed materials. Two novel polymeric networks based on newly synthesized carboxyfunctionalized polyacrylates were created to fit the requirements of 3D printing processes regarding viscosity and their surface tension. The polyacrylates presented carboxylic groups either without any spacer, or, in the case of mono‐(2‐acryloxyethyl)‐succinate, with a substantial spacing from the polymer backbone. The material properties were characterized with rheometry, FTIR, XPS, and contact angle measurements. Polymer surfaces were prepared and seeded with primary human dermal microvascular endothelial cells (HDMEC). Cell–material interaction with the different polymer surfaces were investigated by light microscopy, water soluble tetrazolium‐1 (WST‐1) viability assay, fluorescein diacetate/propidium iodide (FDA/PI) staining, and acetylated low‐density lipoprotein (acLDL) assay. Thus, the cell confluence, viability, and functionality of the HDMEC were evaluated. The non‐spaced methacrylic acid derivative was proved in cell culture regarding its biocompatibility. Along with its appropriate physical properties, the novel network‐generating polymeric blends constitute promising candidates for future scaffold design in tissue engineering.

The Effect of Human Umbilical Cord Blood Cells on Survival and Cytokine Production by Post-Ischemic Astrocytes in Vitro

Cerebral ischemia induces death of all neural cell types within the region affected by the loss of blood flow. We have shown that administering human umbilical cord blood cells after a middle cerebral artery occlusion in rats significantly reduces infarct size, presumably by rescuing cells within the penumbra. In this study we examined whether the cord blood cells enhanced astrocyte survival in an in vitro model of hypoxia with reduced glucose availability. Primary astrocyte cultures were incubated for 2 h in no oxygen (95% N, 5% CO(2)) and low glucose (1% compared to 4.5%) media. Cord blood mononuclear cells were added to half the cultures at the beginning of hypoxia. Astrocyte viability was determined using fluorescein diacetate/propidium iodide (FDA/PI) labeling and cytokine production by the astrocytes measured using ELISA. In some studies, T cells, B cells or monocytes/macrophages isolated from the cord blood mononuclear fraction with magnetic antibody cell sorting (MACS) were used instead to determine which cellular component of the cord blood mononuclear fraction was responsible for the observed effects. Co-culturing mononuclear cord blood cells with astrocytes during hypoxia stimulated production of IL-6 and IL-10 during hypoxia. The cord blood T cells decreased survival of the astrocytes after hypoxia but had no effect on the examined cytokines. Our data demonstrate that the tested cord blood fractions do not enhance astrocyte survival when delivered individually, suggesting there is either another cellular component that is neuroprotective or an interaction of all the cells is essential for protection.

Optimization of FDA–PI method using flow cytometry to measure metabolic activity of the cyanobacteria, Microcystis aeruginosa

A rapid toxicity test based on inhibition of esterase activity in the harmful freshwater microalgae – Microcystis aeruginosa was developed using flow cytometry. The hydrolysis rate of fluorescein diacetate (FDA) by intracellular esterase to fluorescein was used to indicate the metabolic activity of algae. Uptake of FDA was optimized at different concentrations and incubation times. Propidium iodide (PI) was utilized to assess cell membrane integrity. The optimized FDA/PI staining dosages were 10 mg/L and 10 μM, respectively, lower than the reported concentrations. Correspondingly, the proper incubation time was 14–21 min at the optimal FDA dosage determined in this study. A new procedure based on optimized FDA/PI condition, called “whole algal culture flow cytometry with fluorescence triggering”, was developed for short-term bioassays. This new procedure, taking account of working conditions such as pH and impure cultures, is able to avoid algal cell damages in sample preparation and separate algal cells from non-algal particles by fluorescence triggering. This newly-developed procedure was then used to assess the toxicity of copper on M. aeruginosa in a short-term exposure (36 h). As copper concentrations increased, it was found that the esterase activity decreased in a concentration-dependent manner with increased membrane fragments. Moreover, esterase activity was a good indicator of copper toxicity in M. aeruginosa. The EC 50 value based on mean fluorescence intensity (MFI) was 123.3 μg/L (95% confidence limits 101.5–146.2 μg/L). Therefore, the new-developed procedure could be used for sublethal endpoints detection, and has the potential to be a rapid and cost-effective bioassay for selecting M. aeruginosa control methods or exploring the M. aeruginosa activity inhibition mechanism.

Human Umbilical Cord Blood Cells Decrease Microglial Survival In Vitro

When human umbilical cord blood (HUCB) cells are systemically administered following middle cerebral artery occlusion (MCAO) in rats, they produce a reduction in infarct size resulting in recovery of motor function. Rats receiving HUCB cells have a less severe inflammatory response compared to MCAO stroke rats. The purpose of this study was to determine the interaction between HUCB cells and the main resident immune cells of the brain (microglia) under normoxic and hypoxic conditions in vitro. Primary microglial cultures were incubated for 2 h in no oxygen (95% N, 5% CO(2)) and low glucose (1%) media. Mononuclear HUCB cells were added to half the cultures at the beginning of the hypoxia conditions. Microglial viability was determined using fluorescein diacetate/propidium iodide (FDA/PI) labeling and cytokine expression using ELISA. In some studies, CD11b+ or CD19+ cells isolated from the HUCB mononuclear fraction with magnetic antibody cell sorting (MACS) were used instead of the mononuclear fraction. Co-culturing mononuclear HUCB cells with microglia decreased viability of the microglia during hypoxia. In the microglial monocultures, hypoxia significantly increased release of IL-1beta compared to normoxia, while adding HUCB cells in the hypoxia condition decreased IL-1beta concentrations to the same level as in the normoxia monocultures. Both CD11b+ and CD19+ HUCB cells decreased microglial viability during normoxia and hypoxia. Our data suggest that HUCB cells may produce a soluble factor that decreases viability of microglia.

Mammalian Tissue-Free Liberase: A New GMP-Graded Enzyme Blend for Human Islet Isolation

Islet transplantation has found its niche in diabetes treatment. It has contributed to a better quality of life and better glycemic control of patients with diabetes suffering from severe hypoglycemia that are not eligible for vascularized pancreas transplantation. Islet isolation is a technically challenging procedure. The different studies within this doctoral thesis aim to improve and standardize different steps in the isolation procedure. They are in particular looking to improve human pancreas preservation during cold storage, to optimize islet release from the exocrine tissue and to assess whether the isolated islet yield can be predicted from a biopsy. We found that pancreas preservation with pre-oxygenated perfluorodecalin (two-layer method) did not improve the ischemic tolerance of the human pancreas as compared to cold storage with the University of Wisconsin (UW) solution. Furthermore, in pancreas with long cold ischemia time (CIT) (>10 hours), Histidine-Tryptophan-Ketoglutarate (HTK) had a limited preservation capacity as compared with the UW solution with respect to isolation outcome. We also found that during enzymatic pancreas digestion, Vitacyte HA was able to provide a similar islet yield and quality as Serva NB1 with less collagenase activity and shorter digestion time. We further describe the first experience with a new GMP manufactured enzyme called Liberase MTF-S for successful human islet isolation. Finally, we found that the isolated islet yield could not be predicted from a biopsy taken from the head of the pancreas concerning solely morphological parameters of the islets tissue. The improvement of pancreas preservation will allow for marginal organs with prolonged cold ischemia time to expand the donor pool. Better knowledge of how the pancreatic extracellular matrix is digested by collagenase will lead to a fast and predictable islet release from the exocrine tissue. By standardizing the isolation procedure and improving organ selection we will increase the success rate in human islet isolation, thereby making islet transplantation available for more patients.

Effect of methanol and Me 2SO exposure on mitochondrial activity and distribution in stage III ovarian follicles of zebrafish ( Danio rerio)

In this study the effect of cryoprotectants that have been shown to be the least toxic to zebrafish ovarian follicles (methanol and Me 2SO), on mitochondria of stage III ovarian follicles was evaluated. The mitochondrial distributional arrangement, mitochondrial membrane potential, mtDNA copy number, ATP levels and ADP/ATP ratios were assessed following exposure to cryoprotectants for 30 min at room temperature. Results obtained by confocal microscopy showed that 30 min exposure to 2 M methanol induced a loss of membrane potential, although viability tests showed no decrease in survival even after 5 h post-exposure incubation. Higher concentrations of methanol (3 and 4 M) induced not only a decrease in mitochondrial membrane potential but also the loss of mitochondrial distributional arrangement, which suggested a compromised mitochondrial function. Furthermore 3 and 4 M treatments resulted in a decrease in viability assessed by Fluorescein diacetate–Propidium iodide (FDA–PI) and in a decrease in mtDNA copy number and ADP/ATP ratio after 5 h incubation following methanol exposure, indicating a delayed effect. The use of Me 2SO, which is considered to be a more toxic CPA to zebrafish ovarian follicles than methanol, caused a decrease in viability and a sustained decrease in ATP levels accompanied by failure to maintain mtDNA copy number within 1 h post-exposure incubation. These results indicated that even CPAs that are considered to have no toxicity as determined by Trypan blue (TB) and FDA–PI tests can have a deleterious effect on mitochondrial activity, potentially compromising oocyte growth and embryo development.

Estradiol and lithium chloride specifically alter NMDA receptor subunit NR1 mRNA and excitotoxicity in primary cultures

Glutamate facilitates calcium influx via NMDAR, and excess calcium influx increases excitotoxicity — a pathological characteristic of neurological diseases. Both 17β-estradiol (E2) and lithium influence NMDAR expression/signaling and excitotoxicity. This led us to hypothesize that combined E2 and lithium will alter NMDAR expression and excitotoxicity. We tested this hypothesis using primary cell cultures from the cortex and hippocampus of C57BL/6J fetal mice pretreated with E2, lithium chloride (LiCl) and combined E2/LiCl for 12, 24 or 48 h. We examined cultures for brain cell type and changes in cell type caused by experimental procedures using glia and neuron gene specific primers. These cultures expressed increased glial fibrillary acidic protein (GFAP) mRNA with low neurofilament-heavy chain (NF-H) mRNA expression. Subsequent analysis of cortical cell cultures indicated that combined E2/LiCl decreased NR1 mRNA expression after a 12 and 48 h treatment period. Combined E2/LiCl also reduced NR1 mRNA expression in hippocampal cultures but only after a 48 h treatment period. LiCl-treated hippocampal cultures also reduced NR1 mRNA expression after a 24 and 48 h treatment. We next examined the response of 48 h pretreated cultures to a toxic level of glutamate. Excitotoxicity was measured using fluorescein diacetate/propidium iodide (FDA/PI) cell viability assay. Results from FDA/PI assay revealed that LiCl pretreatment increased viability for cortical cultures while E2 and combined E2/LiCl reduced viability. All pretreatments for hippocampal cultures failed to increase viability. Our results showed combined E2/LiCl reduced NR1 mRNA and prevented protection against glutamate excitotoxicity in glial primary cultures.

Quinclorac-induced cell death is accompanied by generation of reactive oxygen species in maize root tissue

The importance of reactive oxygen species for herbicide quinclorac (3,7-dichloro-8-quinolinecarboxylic acid)-induced cell death in roots was investigated. This was in order to understand its mode of action in grass species grown in the dark. Under these dark conditions, quinclorac suppressed the shoot and root growth of maize ( Zea mays L. cv. Honey Bantam) in a concentration-dependent manner (⩽50 μM), although the inhibition level was less than that observed under growth conditions in the light. Analysis of cell viability using Evans blue or fluorescein diacetate–propidium iodide (FDA–PI) staining showed that the maize root cells significantly lost their viability after 14 h root treatment with 10 μM quinclorac, but not 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Determination of reactive oxygen species (ROS) in maize roots using a superoxide anion ( O 2 - )-specific indicator, dihydroethidium (DHE), indicated that 50 μM quinclorac induced a high level of O 2 - production in maize roots after 14 h root treatment than that of either the control (non-treated) or with 50 μM 2,4-D. Moreover, either cell death or ethane evolution, an indicator of lipid peroxide formation, in maize root segments was significantly enhanced by 50 μM quinclorac, but not by 50 μM 2,4-D. On the other hand, the 50 μM 2,4-D treatment induced much higher ethylene and cyanide production in the root segments than with the 50 μM quinclorac. These results suggest that quinclorac-induced cell death in maize roots may be caused by ROS and lipid peroxidation, but not by ethylene and its biosynthetic pathway-related substances including cyanide, which have been thought to be the causative factor of quinclorac-induced phytotoxicity in susceptible grass weeds such as Echinochloa, Digitaria, and Setaria.

Mist culture for mass harvesting of root border cells: aluminum effects

AbstractRoot border cells, which form a cell layer around the root tip, seem to play multiple roles in the rhizosphere of the apical root. As these cells (species‐dependent dozens to several thousand per root tip) are rapidly sloughed off in water, studies in hydroponic culture fail to elucidate their role in most conventional physiological studies. The common method for harvesting these cells consists in germination of seeds in a humid atmosphere (usually a Petri dish), but labor and time constraints allow to yield only very limited amounts of uniform cells. We thus developed a low‐cost mist‐culture method, where intact border cells can be collected in the range of several grams. We applied this technique in a preliminary experiment where the influence of aluminum (Al) supply on calcium (Ca) release and viability of this cell type was studied. Purified detached border cells of pea were incubated with 0, 50, and 500 mmol m–3 AlCl3 solution (pH 4.5) for 90 min at a ratio of 3 × 105 cells (4 mL)–1. After incubation, cells contained 4.27 and 13.28 mg Al g–1 C at 50 mmol m–3 and 500 mmol m–3 AlCl3, respectively, while their total Ca content decreased correspondingly. Cell viability of border cells as tested by fluorescein diacetate‐propidium iodide (FDA‐PI) fluorescence yielded unexpected results: the test exhibited significantly lower vitality at 50, but not at 500 mmol m–3 AlCl3. Assessing mitochondrial activity by 3‐(4,5)‐dimethylthiazol‐2‐yl‐2,5 diphenyl‐tetrazolium‐bromide (MTT) reduction showed that viability decreased in a dose‐dependent manner with increasing Al concentrations. This apparent contradiction is attributed to the formation of dense mucilage around border cells at high Al concentrations, which likely inhibits the access of the dye PI or may chemically inactivate this compound and thus wrongly suggest higher viability. Mist culture allows harvesting selectively large amounts of homogeneous border cells quickly and to study their physiological reactions separated from the root tip.

Role of Protein Phosphatase 2A in mGluR5-regulated MEK/ERK Phosphorylation in Neurons

The regulation of protein phosphorylation requires coordinated interaction between protein kinases and protein phosphatases (PPs). Recent evidence has shown that the Galphaq-protein-coupled metabotropic glutamate receptor (mGluR) 5 up-regulates phosphorylation of MAPK/ERK1/2. However, signaling mechanisms linking mGluR5 to ERK are poorly understood. In this study, roles of a major serine/threonine PP, PP2A, in this event were evaluated in cultured neurons. We found that the PP1/2A inhibitors okadaic acid and calyculin A mimicked the effect of the mGluR5 agonists (RS)-3,5-dihydroxyphenylglycine and (RS)-2-chloro-5-hydroxyphenylglycine in facilitating phosphorylation of ERK1/2 and its upstream kinase, MEK1/2, in a PP2A-dependent but not PP1-dependent manner. Co-administration of either inhibitor with an mGluR5 agonist produced additive phosphorylation of ERK1/2. Enzymatic assays showed a basal level of phosphatase activity of PP2A under normal conditions, and activation of mGluR5 selectively inhibited PP2A, but not PP1, activity. In addition, a physical association of the cytoplasmic C terminus of mGluR5 with PP2A was observed, and ligand activation of mGluR5 reduced mGluR5-PP2A binding. Additional mechanistic studies revealed that mGluR5 activation increased tyrosine (Tyr307) phosphorylation of PP2A, which was dependent on activation of a p60c-Src family tyrosine kinase, but not the epidermal growth factor receptor tyrosine kinase and resulted in dissociation of PP2A from mGluR5 and reduced PP2A activity. Together, we have identified a novel, mGluR5-triggered signaling mechanism involving use- and Src-dependent inactivation of PP2A, which contributes to mGluR5 activation of MEK1/2 and ERK1/2.

Variation in Human Islet Viability Based on Different Membrane Integrity Stains

Membrane integrity fluorescent staining is used routinely to evaluate islet viability. Results are used as one of the determining factors in islet product release criteria, and are used to assess the efficacy of different culture conditions. Recently, it has been observed that there is variation in the viability staining of freshly isolated islets based on which viability assay is used. This investigation compares three membrane integrity stains for the viability assessment of isolated human islets. Fluorescein diacetate/propidium iodide (FDA/ PI), the current standard method for assessing islet viability, demonstrates intense extracellular fluorescence, reducing the differential staining of intact islets. We further evaluated SYTO-13/ethidium bromide (SYTO/ EB) and calcein AM/ethidium homodimer (C/EthD) as alternative viability assays, and found considerable variation between FDA/PI and either SYTO/EB or C/EthD staining. Preparations of human islets were obtained from cadaveric pancreata after collagenase digestion, mechanical separation, and purification by continuous Ficoll gradient centrifugation. For each preparation, two replicate samples of 50 islets were counted for each stain, and the percent viability calculated. The results for SYTO/EB and C/EthD were nearly identical [57.6 +/- 7.3% and 57.9 +/- 7.2%, respectively (mean +/- SEM), N = 11]. FDA/PI-stained islets, however, showed consistently elevated values when compared to SYTO/EB. Accurate assessment of islet viability remains a critical determinant of islet product release. The discrepancies found between FDA/PI scoring and visual quality, compared with alternative stains, suggests that the FDA/PI stain may not be the optimal approach to assess islet viability.