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At least 973 records · Page 54Linked to original sources
PubMed2002

Multiparametric flow cytometric analysis in a breast cancer cell line (MCF-7).

OBJECTIVE: To investigate whether multiparameter flow cytometric analysis of solid tumor specimens, including gynecologic tumors, which were stained triply with phycoerythrin (PE), fluorescein isothiocyanate (FITC) and propidium iodide (PI), can be performed simultaneously without interference from normal diploid cell populations and spectral overlap on a standard flow cytometer. METHODS: MCF-7 breast cancer cell lines and heterogeneous cell populations mixed with MCF-7 cells and human peripheral blood lymphocytes (PBL) were fixed with 1% paraformaldehyde and permeabilized with 100% methanol. Cytokeratin and several proliferation-associated cellular antigens (proliferating cell nuclear antigen (PCNA), p53, c-erbB/2 and c-myc) were labeled with PE and FITC, which was followed by DNA staining using PI. These labeled cells were measured on a standard FACScan flow cytometer equipped with a 488 nm single laser. RESULTS: The coefficient of variation (CV) of the G0G1 peak of MCF-7 cells was 4.3 and the cell cycle phase fractions of G0G1, S and G2M were 44.9, 45.9 and 9.2%, respectively. Fluorescein isothiocyanate, PE and PI fluorescences were detected without interference. The MCF-7 cells expressed cytokeratin, PCNA, p53, c-erbB/2 and c-myc antigen. In the heterogeneous population of MCF-7 cells mixed with PBL, two cellular populations were clearly separated into diploid PBL and aneuploid MCF-7 cells without interference. The CV of G0G1 peak of PBL was 2.3 and the G0G1, S and G2M phase fractions were 85.5, 2.7 and 11.8%, respectively. The DNA index of MCF-7 cells was 1.7, which indicated that the MCF-7 cell line was composed of tumor cells with aneuploid DNA. The CV of the G0G1 peak of the MCF-7 cells was 4.2, and the cell cycle phase fractions were 47.5% for G0G1, 42.3% for S, and 10.2% for G2M. The MCF-7 cells expressed cytokeratin, but the PBL did not. CONCLUSIONS: Multiparameter flow cytometer analysis was useful to determine DNA ploidy status, phase fraction of the cell cycle and expression of cellular antigens and selective cytokeratin expression allowed epithelial originated tumor cells to be differentiated from normal stromal cells. This analysis could be performed without interference of spectral overlaps of fluorochromes using software-based algorithmic compensation of spectral overlaps. Thus, this method offers new possibilities for multiparameter flow cytometric analysis and its use should be extended to future studies of the diagnosis, treatment and prediction of prognosis of the neoplasm.

Breast Neoplasms↗
PubMed1999

Optimized fluorescent probe combinations for evaluation of proliferation and necrosis in anthracycline-treated leukaemic cell lines.

Proliferation and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukaemia (AML). Anthracyclines such as daunorubicin (DNR) are typically used to treat AML and can induce drug resistance. The goal of the studies described here was to select a combination of fluorescent probes that could be used in combination with flow cytometry to monitor cell proliferation vs. cell death/necrosis as a function of anthracycline uptake. Propidium iodide (PI), the most commonly used marker of membrane integrity, cannot be used to evaluate necrosis in DNR-containing cells because of spectral overlap. A membrane integrity probe compatible with the use of a dye dilution method using PKH67 to study cell proliferation was also selected. The results show that DAPI and Cascade Blue (CB), like PI, were able to detect necrotic cells when no DNR was present, although CB gave less resolution between viable and necrotic cells than PI or DAPI. In the presence of DNR, DAPI cannot be used owing to the fluorescence quenching by DNR. However, it was found that a combination of DNR, CB, and PKH67 allows simultaneous identification of chemoresistant cells, based on reduced DNR accumulation, necrotic cells based on CB incorporation, and proliferating cells based on partitioning of PKH67 fluorescence between daughter cells. It was also found that unless a marker of necrosis is used in combination with the dye dilution assay, a moderate decrease of fluorescence as a result of necrosis may be incorrectly interpreted as proliferation.

Antibiotics, Antineoplastic↗
PubMed2004

Annexin V binding to plasma membrane predicts the quality of human cryopreserved spermatozoa.

Cryopreservation-induced stress may result in membrane injury with consequent decrease of sperm motility and fertilizing capacity. This study has investigated the relationship between human spermatozoa tolerance to cryopreservation and the loss of plasma membrane asymmetry, especially translocation of phosphatidylserine (PS) from the inner to the outer leaflet. The prospective study was performed on semen samples from 31 men. Conventional characteristics of 20 semen were analysed, before and after cryopreservation as well as externalization of PS assessed by annexin V-staining in combination with the propidium iodide which stains dead cells. The fertilizing capacity was evaluated by a zona free hamster egg penetration test in 11 freeze/thaw spermatozoa samples. The percentages of vital annexin V-negative and annexin V-positive spermatozoa in post-thaw samples were significantly lower than in pre-freeze ones (10 +/- 3 vs. 25 +/- 5% and 22 +/- 3 vs. 34 +/- 4% respectively), while the percentages of dead spermatozoa annexin V-negative and annexin V-positive had increased (42 +/- 4 vs. 23 +/- 4% and 23 +/- 4 vs. 16 +/- 2% respectively). The percentages of progressive and total motile spermatozoa were significantly correlated with the percentage of vital annexin V-negative spermatozoa before as well as after cryopreservation. Furthermore, recovery of motile spermatozoa after freeze/thaw was strongly correlated (p < 0.002) with the proportion of vital annexin V-negative spermatozoa in fresh semen. The percentage of penetration of zona-free hamster eggs was correlated (p < 0.02) with the percentage of live annexin V-negative cryopreserved spermatozoa capacitated for 3 h. These findings provide evidence that annexinV-binding staining in combination with PI brings additional information to predict the outcome of cryopreserved ejaculated sperm and may be used as a novel and reliable marker to study the freeze/thaw process.

Animals↗
PubMed1998

Assessment of bacterial viability status by flow cytometry and single cell sorting.

Rapid bacterial detection and viability measurements have been greatly enhanced by recent advances in the use of fluorescent stains in cytometry. It has previously been shown that four physiological states can be distinguished: reproductively viable, metabolically active, intact and permeabilized. Previous sorting experiments have shown that not all intact cells readily grow, but some intact cells can grow even when they fail to show metabolic activity, as determined by esterase turnover. To circumvent the limitations imposed by active dye extrusion or cell dormancy on viability measurements used to date (e.g., enzyme activity or cell polarization), a fast triple fluorochrome staining procedure has been developed that takes account of these problems. This allows further cellular characterization of intact cells by: active exclusion of ethidium bromide (EB) (metabolically active cells), uptake of EB but exclusion of bis-oxonol (BOX) (de-energized but with a polarized cell membrane) and uptake of both dyes (depolarized). Permeabilized cells were identified by propidium iodide (PI) uptake. The method was validated using an electronically programmable single cell sorter (EPICS Elite) and aged Salmonella typhimurium cells. Reproductive viability was determined by sorting single cells to their staining pattern directly onto agar plates. Most polarized cells could be recovered as well as a significant fraction of the depolarized cells, demonstrating that depolarization is a sensitive measure of cell damage but a poor indicator of cell death.

Bacteriological Techniques↗
PubMed2000

The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil).

The essential oil of Melaleuca alternifolia (tea tree) exhibits broad-spectrum antimicrobial activity. Its mode of action against the Gram-negative bacterium Escherichia coli AG100, the Gram-positive bacterium Staphylococcus aureus NCTC 8325, and the yeast Candida albicans has been investigated using a range of methods. We report that exposing these organisms to minimum inhibitory and minimum bactericidal/fungicidal concentrations of tea tree oil inhibited respiration and increased the permeability of bacterial cytoplasmic and yeast plasma membranes as indicated by uptake of propidium iodide. In the case of E. coli and Staph. aureus, tea tree oil also caused potassium ion leakage. Differences in the susceptibility of the test organisms to tea tree oil were also observed and these are interpreted in terms of variations in the rate of monoterpene penetration through cell wall and cell membrane structures. The ability of tea tree oil to disrupt the permeability barrier of cell membrane structures and the accompanying loss of chemiosmotic control is the most likely source of its lethal action at minimum inhibitory levels.

Anti-Bacterial Agents↗
PubMed2000

Viability of Escherichia coli O157:H7 in natural river water determined by the use of flow cytometry.

The enzymatic activity and viability of Escherichia coli O157:H7 in natural river water was determined by flow cytometry. River water was collected at two sites (an agricultural area and an industrial area) on the Aigawa River (Osaka, Japan). To facilitate estimation of the physiology of E. coli O157 in natural river water, bacterial cells in the water were stained with 6-carboxyfluorescein diacetate (6CFDA) and propidium iodide (PI). The cells were sorted into two populations, using a flow cytometer, based on their esterase activity. Each population was stained with E. coli O157:H7 fluorescent antibody (FA), and E. coli O157:H7 cells were observed in the esterase-active population. River water samples collected at the same points were incubated with yeast extract containing antibiotics to prevent cell division, and bacterial cells in the incubated samples were stained with PI and FA. Escherichia coli O157:H7 existed in both the viable (elongated and/or fattened) and inactive bacterial population determined by flow cytometry. These results indicate that E. coli O157:H7 may retain metabolic activity and growth potential in the natural aquatic environment.

Colony Count, Microbial↗
PubMed2001

Heterogeneity of stress gene expression and stress resistance among individual cells of Saccharomyces cerevisiae.

Knowledge of gene expression and cellular responses in microorganisms is derived from analyses of populations consisting of millions of cells. Analytical techniques that provide data as population averages fail to inform of culture heterogeneity. Flow cytometry and fluorescence techniques were used to provide information on the heterogeneity of stress-responsive gene expression and stress tolerance in individual cells within populations. A sequence of DNA encoding the heat shock and stress response elements of the Saccharomyces cerevisiae HSP104 gene was used to express enhanced green fluorescent protein (EGFP). When integrated into the genome of yeast strain W303-1A, intrinsic expression of EGFP increased about twofold as cells progressed from growth on glucose to ethanol utilization in aerobic batch cultures. Staining of cells with orange/red fluorescent propidium iodide (PI), which only enters cells that have compromised membrane integrity, revealed that the population became more tolerant to 52 degrees C heat stress as it progressed from growth on glucose and through the ethanol utilization phase of aerobic batch culture. Exposure of cultures growing on glucose to a mild heat shock (shift from 25 degrees C to 37 degrees C) resulted in significantly increased expression of EGFP in the population. However, there was heterogeneity in the intensity of fluorescence of individual cells from heat-shocked cultures, indicating variability in the strength of stress response in the clonal population. Detailed analysis of the heterogeneity showed a clear positive trend between intensity of stress response and individual cell resistance, measured in terms of PI exclusion, to heat stress at 52 degrees C. Further experiments indicated that, although the mean gene expression by a population is influenced by the genetic background, the heterogeneity among individual cells in clonal populations is largely physiologically based.

Base Sequence↗
PubMed1999

Technical advance: near-infrared femtosecond laser pulses as a novel non-invasive means for dye-permeation and 3D imaging of localised dye-coupling in the Arabidopsis root meristem.

We have used near-infrared femtosecond Titanium: Sapphire laser pulses as novel non-invasive means for dye loading into various cell types of the Arabidopsis root meristem, and by 3D imaging have assessed the extent of dye coupling between the meristematic cells. The post-embryonic primary root of Arabidopsis thaliana has an invariant ontogeny and fixed cellular organisation which makes it an attractive model system to study developmental events involving cell fate determination, cellular differentiation and pattern formation. Local intercellular communication and local transmission of positional signals are likely to play a pivotal role in cell proliferation and regulation of differentiation. We have therefore examined the extent to which the constituent cells in the root meristem are symplastically coupled. Following laser-assisted loading of membrane impermeate fluorescent dye propidium iodide (PI) in single cells, we show by time-lapse and 3D imaging that in the root tip all undifferentiated cells are dye-coupled. When PI is permeated into the central cells, it rapidly moved into the adjacent initials of the columella, cortex, pericycle and stele. Interestingly, when only either of the initials were loaded with the dye, it never moved into any of the central cells. Amongst the epidermal cells, the differentiated hair cells are symplastically isolated. Our data provide evidence (1) for differential dye-coupling behaviour between quiescent centre cells and the neighbouring initials; (2) that cells in the root are coupled during stages at which the cell-lineage pattern is formed and that it becomes progressively secluded as they differentiate and the pattern is fixed. Taken together, our NIR-laser mediated approach is highly efficient and has numerous potential applications for non-invasive permeation of dyes in different cell types.

Arabidopsis↗
PubMed2003

The origin and identification of unknown events associated with low-level leucocyte counting by flow cytometry.

BACKGROUND AND OBJECTIVES: Flow cytometric enumeration of residual leucocytes (WBC) in leucocyte-depleted components occasionally reveals a population of events that stain with propidium iodide, but which are outside the main counting region. These extraregional events may result in discrepancies in residual WBC counts. MATERIALS AND METHODS: To identify the origin of these unknown events, separate populations of mononuclear cells and polymorphonuclear neutrophils (PMNs) (prepared by density-gradient centrifugation) were spiked into leucocyte-depleted red cell concentrates (RCC) to a concentration of 150 cells/ microl and assessed, using LeucoCount and DNA Prep reagents, by flow cytometry. In addition, isolated WBC nuclei, DNA in saline, or enzymatically digested free DNA, was spiked into diluted whole blood (final concentration 5 microg/ml). RESULTS: Isolated WBC nuclei fell in the main counting region of the dot plot. Extraregional events were generated in leucocyte-depleted RCC spiked with PMN (17/ microl by day 4), but not with mononuclear cells, and increased with sample storage (4 days). A significantly (P < 0.01) greater number of extraregional events was observed using the LeucoCount reagent, compared with the DNA Prep reagent. Enzymatically digested WBC nuclei or free DNA, added to diluted whole blood, generated extraregional events using LeucoCount, but not the DNA Prep reagent. When free DNA was enzymatically digested, no extraregional events were observed. CONCLUSIONS: These previously unidentified events are probably fragmented nuclei or free DNA originating from PMN owing to a combination of ageing and reagent addition. Currently, in our protocols, the region used for WBC enumeration counts intact WBC nuclei. To achieve WBC counting consistency, flow cytometric gating protocols must be standardized, and a decision taken as to whether to include extraregional events in the count.

Cell Nucleus↗
PubMed2000

Determinants of substrate specificity of a second non-neuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis.

We recently reported on a non-neuronal secreted acetylcholinesterase (AChE B) from the nematode parasite Nippostrongylus brasiliensis. Here we describe the primary structure and enzymatic properties of a second secreted variant, termed AChE C after the designation of native AChE isoforms from this parasite. As for the former enzyme, AChE C is truncated at the carboxyl terminus in comparison with the Torpedo AChE, and three of the 14 aromatic residues that line the active site gorge are substituted by nonaromatic residues, corresponding to Tyr70 (Ser), Trp279 (Asn) and Phe288 (Met). A recombinant form of AChE C was highly expressed by Pichia pastoris. The enzyme was monomeric and hydrophilic, and displayed a marked preference for acetylthiocholine as substrate. A double mutation (W302F/W345F, corresponding to positions 290 and 331 in Torpedo) rendered the enzyme 10-fold less sensitive to excess substrate inhibition and two times less susceptible to the bis quaternary inhibitor BW284C51, but did not radically affect substrate specificity or sensitivity to the 'peripheral site' inhibitor propidium iodide. In contrast, a triple mutant (M300G/W302F/W345F) efficiently hydrolysed propionylthiocholine and butyrylthiocholine in addition to acetylthiocholine, while remaining insensitive to the butyrylcholinesterase-specific inhibitor iso-OMPA and displaying a similar profile of excess substrate inhibition as the double mutant. These data highlight a conserved pattern of active site architecture for nematode secreted AChEs characterized to date, and provide an explanation for the substrate specificity that might otherwise appear inconsistent with the primary structure in comparison to other invertebrate AChEs.

Acetylcholinesterase↗
PubMedDate not supplied

Induction of apoptosis in human corneal epithelial cells in vitro.

PURPOSE: The purpose of this study was to evaluate exhausted-medium-induced apoptosis in human corneal epithelial (HCE) cells in vitro. METHODS: Confluent HCE cells were maintained in tissue-culture medium for 5-7days. The supernatant was harvested and fresh HCE cells were exposed to the exhausted supernatant for 24-48 h over a range of pH conditions. The exhausted medium was assayed for cytokines interleukin 6 (IL-6), IL-8, interferon-gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) by ELISA. Apoptotic cells were assessed using Hoechst 33342 Propidium iodide and AnnexinV-FITC staining and by the TUNEL assay. RESULTS: Human corneal epithelial (HCE) cells exposed to exhausted medium demonstrated a high incidence of apoptosis, which increased over time to 87+/-5% after 48 h. Apoptosis was independent of pH. Cytokine levels were not elevated in the exhausted medium. CONCLUSIONS: This study suggests that exhausted medium can be used as a simple, rapid and potent positive control for apoptosis of HCE cells.

Annexin A5↗
PubMed1999

Calpain activation and inhibition in organotypic rat hippocampal slice cultures deprived of oxygen and glucose.

It has been suggested that, after ischaemia, activation of proteases such as calpains could be involved in cytoskeletal degradation leading to neuronal cell death. In vivo, calpain inhibitors at high doses have been shown to reduce ischaemic damage and traumatic brain injury, however, the relationship between calpain activation and cell death remains unclear. We have investigated the role of calpain activation in a model of ischaemia based on organotypic hippocampal slice cultures using the appearance of spectrin breakdown products (BDPs) as a measure of calpain I activation. Calpain I activity was detected on Western blot immediately after a 1-h exposure to ischaemia. Up to 4 h post ischaemia, BDPs were found mainly in the CA1 region and appeared before uptake of the vital dye propidium iodide (PI). 24 h after the insult, BDPs were detected extensively in CA1 and CA3 pyramidal cells, all of which was PI-positive. However, there were many more PI-positive cells that did not have BDPs, indicating that the appearance of BDPs does not necessarily accompany ischaemic cell death. Inhibition of BDP formation by the broad-spectrum protease inhibitor leupeptin was not accompanied by any neuroprotective effects. The more specific and more cell-permeant calpain inhibitor MDL 28170 had a clear neuroprotective effect when added after the ischaemic insult. In contrast, when MDL 28170 was present throughout the entire pre- and post-incubation phases, PI labelling actually increased, indicating a toxic effect. These results suggest that calpain activation is not always associated with cell death and that, while inhibition of calpains can be neuroprotective under some conditions, it may not always lead to beneficial outcomes in ischaemia.

Animals↗
PubMed2003

The immunosuppressant mycophenolate mofetil attenuates neuronal damage after excitotoxic injury in hippocampal slice cultures.

In this study we investigated whether treatment with the immunosuppressant mycophenolate mofetil (MMF) has beneficial effects on neuronal damage after excitotoxic injury. Organotypic hippocampal slice culture (OHSC), lesioned by the application of N-methyl-d-aspartate (NMDA) after 6 days in vitro, showed an improved preservation of the hippocampal cytoarchitecture after continuous treatment with MMF for 3 further days (10 or 100 micro g/mL). Treatment with NMDA and MMF (100 microg/mL) reduced the number of damaged propidium iodide (PI)+ neurons by 50.7% and the number of microglial cells by 52%. Continuous treatment of lesioned OHSCs with MMF for 3 days almost abrogated the glial proliferative response, reflected by the 91.5% reduction in the number of bromo-desoxy-uridine (BrdU)-labelled microglial cells and astrocytes. Microglial cells in MMF-treated OHSCs contained fragmented nuclei, indicating apoptotic cell death, an effect which was also found in isolated microglial cells treated with MMF. The beneficial effect of MMF on neuronal survival apparently does not reflect a direct antiexcitotoxic effect, as short-term treatment of OHSCs with NMDA and MMF for 4 h did not reduce the number of PI+ neurons. In conclusion, MMF inhibits proliferation and activation of microglia and astrocytes and protects neurons after excitotoxic injury.

Animals↗
PubMed2003

Clostridium perfringens epsilon toxin rapidly decreases membrane barrier permeability of polarized MDCK cells.

Epsilon toxin is produced by Clostridium perfringens types B and D which are responsible for fatal intestinal diseases in animals. The main biological activity of epsilon toxin is the production of oedema in various organs. We have previously found that epsilon toxin forms a large membrane complex in MDCK cells which is not internalized into cell, and induces cell volume enlargement and loss of cell viability (Petit, L., Gibert, M., Gillet, D., Laurent-Winter, C., Boquet, P., Popoff, M. R. (1997) J Bacteriol 179, 6480-6487). Here, we show that epsilon toxin is very potent to decrease the trans-epithelial electrical resistance of polarized MDCK cells grown on filters without altering the organization of the junctional complexes. The dose-dependent decrease in trans-epithelial electrical resistance, more marked when the toxin was applied to the apical side than to the basal side of MDCK cells, was associated with a moderate increase of the paracellular permeability to low-molecular-weight compounds but not to macromolecules. Epsilon toxin probably acts by forming large membrane pores which permit the flux of ions and other molecules such as the entry of propidium iodide and finally to the loss of cell viability.

Actins↗
PubMed1994

Transcription and motoneuron size.

Nuclear size and total RNA synthesis were compared in single lumbar motoneurons isolated from the grass frog. Transcription was found to correlate significantly, but not exclusively, with nuclear area or volume over a wide range of nuclear size, the largest nuclei having the highest mean transcriptional activity. Flow cytometric analysis of propidium iodide-stained nuclei excluded polyploidy or polyteny as an explanation for the increased transcription, but left open the possibility of a small increase in DNA with increasing nuclear size. Alternatively, motoneurons may increase transcription and nuclear size without increasing their DNA content, possibly by increasing the proportion of dispersed chromatin (euchromatin). These two mechanisms for size-related changes in RNA synthesis in motoneurons present an interesting contrast to mechanisms used by many other large animal cells.

Animals↗
PubMed1998

Optical imaging of hippocampal neurons with a chloride-sensitive dye: early effects of in vitro ischemia.

We determined if changes in intraneuronal Cl- occur early after ischemia in the hippocampal slice. Slices from juvenile rats (14-19 days old) were loaded with the cell-permeant form of 6-methoxy-N-ethylquinolinium chloride (MEQ), a Cl(-)-sensitive fluorescent dye. Real-time changes in intracellular chloride concentration ([Cl-]i) were measured with UV laser scanning confocal microscopy in multiple neurons within each slice. In vitro ischemia (26-28 degrees C, 10 min) was confirmed by the loss of synaptic transmission (evoked field excitatory postsynaptic potentials) from pyramidal cells in area CA1. After ischemia and reoxygenation (10 min), MEQ fluorescence decreased significantly in CA1 pyramidal cells and interneurons. The decreased fluorescence corresponded to an ischemia-induced increase in [Cl-]i of approximately 10 mM. Pretreatment with the GABA(A)-gated Cl- channel antagonist picrotoxin (100 microM) blocked the ischemia-induced change in [Cl-]i. Analysis of the superfusates indicated that ischemia also caused a transient amino acid (GABA, glutamate, and aspartate) release that was maximal at approximately 10 min, returning to baseline shortly thereafter. Recovery from ischemia was confirmed by the return of synaptic transmission in area CA1, the return toward baseline of the ischemia-induced decrease in MEQ fluorescence, and exclusion of propidium iodide from MEQ fluorescent cells. Furthermore, pyramidal cells did not undergo cell swelling during this early phase of reoxygenation, as indicated by the volume-sensitive dye calcein. Thus, mild ischemia induces the accumulation of [Cl-]i secondary to GABA(A) receptor activation, in the absence of cellular swelling or death. In contrast, depolarization of the slice with K+ (50 mM) decreased MEQ fluorescence significantly but caused cell swelling. Picrotoxin did not prevent the K+-induced increase in [Cl-]i. It is possible that an increased [Cl-]i, following either an ischemic event or an episode of depolarization, would reduce the Cl- driving force and thereby limit synaptic transmission by GABA. To support this hypothesis, ischemia caused a reduction in the ability of the GABA agonist muscimol to increase [Cl-]i after 20-min reoxygenation.

Animals↗
PubMed2003

Tetrahydrobiopterin precursor sepiapterin provides protection against neurotoxicity of 1-methyl-4-phenylpyridinium in nigral slice cultures.

Complex-I inhibition and oxidative processes have been implicated in the loss of nigral dopamine neurones in Parkinson's disease and the toxicity of MPTP and its metabolite MPP+. Tetrahydrobiopterin, an essential cofactor for tyrosine hydroxylase, may act as an antioxidant in dopaminergic neurones and protects against the toxic consequences of glutathione depletion. Here we studied the effects of manipulating tetrahydrobiopterin levels on MPP+ toxicity in organotypic, rat ventral mesencephalic slice cultures. In cultures exposed to 30 micro m MPP+ for 2 days, followed by 8 days 'recovery' in control medium, we measured dopamine and its metabolites in the tissue and culture medium by HPLC, lactate dehydrogenase release to the culture medium, cellular uptake of propidium iodide and counted the tyrosine hydroxylase-immunoreactive neurones. Inhibition of tetrahydrobiopterin synthesis by 2,4-diamino-6-hydroxypyrimidine had no significant synergistic effect on MPP+ toxicity. In contrast, the tetrahydrobiopterin precursor l-sepiapterin attenuated the MPP+-induced dopamine depletion and loss of tyrosine hydroxylase-positive cells in a dose-dependent manner with 40 micro m l-sepiapterin providing maximal protection. Accordingly, increasing intracellular tetrahydrobiopterin levels may protect against oxidative stress by complex-I inhibition.

1-Methyl-4-phenylpyridinium↗
PubMed1997

Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell division and injury.

Flow cytometry in combination with fluorescent molecular markers 5- (and 6-) carboxyfluorescein succinimidylester (CFSE) and propidium iodide (PI) have been applied to determine lag times, numbers of cell divisions and injury after mild heat (50 degrees C, 5 min) and nisin treatments (0.1 and 1.0 microgram ml-1) of Lactobacillus plantarum. Initial labelling with covalently bound dye CFSE (20 and 100 micrograms ml-1) allowed determination of lag times and cell proliferation for up to eight generations. Double-labelling with CFSE and PI (5 micrograms ml-1) provided additional information about damage levels and distributions within populations. Subpopulations surviving treatment could be identified easily and selectively sorted.

Cell Division↗