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PubMed2000

Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentrations.

We studied fluorescence resonance energy transfer between donors and acceptors bound to double-helical DNA. The donor Hoechst 33258 binds to the minor groove of DNA and the acceptor propidium iodide (PI) is an intercalator. The time-resolved donor decays were measured in the frequency domain. The donor decays were consistent with a random 1-dimensional distribution of acceptors. The decays were analyzed in terms of three 1-dimensional models: a random continuous acceptor distribution; acceptors placed on discrete lattice sites; and a cylindrical model with the acceptor in the center, and the donors on a cylinder surface. The data were well described by all three models. Interpretation in terms of continuous distribution of acceptors revealed a minimum donor to acceptor distance of 13 A, which is 3 bp from the center of Hoechst 33252. These results suggest that PI is excluded from the 4 bp covered by Hoechst 33252 when it is bound to the minor groove of DNA.

Animals↗
PubMed2000

Multiparameter analysis of progeny of individual cells by laser scanning cytometry.

BACKGROUND: Effectiveness of antitumor drugs to suppress unrestricted proliferation of cancer cells is commonly measured by cell clonogenicity assays. Assays of clonogenicity are also used in studies of stem/progenitor cells and in analysis of carcinogenic transformation. The conventional assays are limited to providing information about frequency of colonies (cloning efficiency) and do not reveal the qualitative (phenotype) attributes of individual colonies that may yield clues on mechanisms by which cell proliferation was affected by the studied agent. METHODS: Laser scanning cytometry (LSC) was adapted to identify and characterize size and phenotype of colonies of MCF-7 cells growing in microscope slide chambers, untreated and treated with the cytotoxic ribonuclease, onconase (Onc). Individual colonies were located and data representing each colony were segmented based on >650-nm fluorescence excited by a He-Ne laser of the cells whose protein was stained with BODIPY 630/650-X. The DNA of the cells was stained with propidium iodide (red fluorescence) whereas specific proteins (estrogen receptor [ER] or tumor suppressor p53) were detected immunocytochemically (green fluorescence), each excited by an Ar ion laser. RESULTS: A plethora of attributes of individual colonies were measured, such as (a) morphometric features (area, circumference, area/circumference ratio, DNA or protein content per area ratio), (b) number of cells (nuclei), (c) DNA content, (d) protein content and protein/DNA ratio, and (e) expression of ER or p53 per colony, per total protein, per nucleus or per DNA, within a colony. Also cell cycle distribution within individual colonies and heterogeneity of colonies with respect to all the measured features could be assessed. The colonies growing in the presence of Onc had many of the above attributes different than the colonies from the untreated cultures. CONCLUSIONS: Analysis of the features of cell colonies by LSC provides a wealth of information about the progeny of individual cells. Changes in colony size and phenotype, reflecting altered cell shape, cell size, colony protein/DNA ratio, and expression of individual proteins, may reveal mechanisms by which drugs suppress the proliferative capacity of the cells. This may include inducing growth imbalance and differentiation and modulating expression of the genes that may be associated with cell cycle, apoptosis, or differentiation in a progeny of individual cells. Extensions of LSC may make it applicable for automatic analysis of cloning efficiency and multiparameter analysis of cell colonies in soft agar. Such analyses may be useful in studies of the mechanisms and effectiveness of antitumor drugs, in the field of carcinogenesis, and for analyzing primary cultures and assessing tumor prognosis and drug sensitivity. The assay can also be adapted to analysis of microbial colonies.

Boron Compounds↗
PubMed2000

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells.

BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.

3T3 Cells↗
PubMed2001

Quantitative assessment of phagocytic activity of hemocytes in the prawn, Penaeus merguiensis, by flow cytometric analysis.

BACKGROUND: The blood cells of crustaceans are involved in phagocytosis of invading microorganisms, contributing to their defense mechanisms. In this study, phagocytic activity of hemocytes of the prawn, Penaeus merguiensis, was quantitated by means of flow cytometric analysis. METHOD: This study was done in vitro. Hemolymph, which was extracted from prawns, was mixed with an equal volume of anticoagulant. Heat-killed Escherichia coli prestained with propidium iodide (PI) was then added. Hemocytes were fixed at various time intervals for flow cytometric analysis. This study was supplemented with electron micrographs using transmission electron microscopy (TEM), which showed three populations of hemocytes. RESULTS: It was observed that those hemocytes that were more active engulfed and digested bacteria readily, thus having higher red fluorescence intensity. The phagocytic activity was expressed as fluorescence unit or engulfed E. coli number per hemocyte. CONCLUSIONS: With this approach, the phagocytic and cellular activity of individual hemocyte populations could be studied quantitatively.

Animals↗
PubMed2001

Oncosis is associated with exposure of phosphatidylserine residues on the outside layer of the plasma membrane: a reconsideration of the specificity of the annexin V/propidium iodide assay.

BACKGROUND: Following a lethal injury, two modes of cell death can be distinguished, apoptosis and primary necrosis. Cells pass through a prelethal stage characterized by a preservation of membrane integrity, in which they shrink (apoptosis) or swell (oncosis, the early phase of primary necrosis). During apoptosis, a loss of phospholipid asymmetry leads to exposure of phosphatidylserine (PS) residues on the outer leaflet of the plasma membrane. We examined whether the external PS exposure, initially supposed to be specific for apoptosis, was also observed in oncotic cells. METHODS: Human peripheral lymphocytes, Jurkat T cells, U937 cells, or HeLa cells were submitted to either apoptotic or oncotic stimuli. PS external exposure was assessed after binding of FITC-conjugated annexin V as was the loss of membrane integrity after propidium iodide (PI) uptake. Morphological examination was performed by optical or electron microscopy. RESULTS: Similarly to apoptotic cells, oncotic cells expose external PS residues while preserving membrane integrity. Consequently, oncotic cells exhibit the annexin V+ PI- phenotype, previously considered to be specific for apoptotic cells. CONCLUSIONS: This study concludes that the annexin V/PI assay does not discriminate between apoptosis and oncosis and that it can be a useful tool to study oncosis by flow cytometry.

Annexin A5↗
PubMed2001

Effects of paclitaxel on cultured synovial cells from patients with rheumatoid arthritis.

BACKGROUND: Proliferation of synovial cells is considered to play a key role in rheumatoid arthritis (RA). Using paclitaxel, a unique antineoplastic agent known to suppress collagen-induced arthritis, we conducted an in vitro study of cell kinetics on cultured synovial cells from patients with RA. METHODS: Alterations of the cell cycle of cultured fibroblast-like synovial cells (FLSs) from patients with RA were studied using flow cytometry and laser scanning cytometry. Apoptosis and accumulation of cyclin concerning effects of paclitaxel were detected. RESULTS: Paclitaxel induced arrest of the cell cycle at G2/M phase and apoptosis in FLSs. The late stage of apoptosis was determined by the positivity of terminal deoxynucleotidyl transferase assay. Morphological observation by combined usage of both annexin V and propidium iodide on FLSs on a slide glass showed early apoptotic changes in detail. FLSs arrested at G2/M phase showed marked accumulation of cyclin B1. The effects of paclitaxel decreased on FLSs, which diminished proliferative activity. CONCLUSIONS: These data indicate that paclitaxel induces cell arrest at G2/M phase followed by apoptosis in human FLSs, which have high proliferative activity, and possible therapeutic effects of paclitaxel on RA.

Adult↗
PubMed2001

"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: potential application at microgravity conditions in space.

BACKGROUND: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable-e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat. METHODS: The "liquidless" staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm2) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by laser scanning cytometry (LSC). RESULTS: Satisfactory cell and tissue staining, with minimal background, was achieved after 10-20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was found to be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G1, S, and G2/M cell populations and 2:1 ratio of G2/M to G1 peak fluorescence. Individual gels can be saturated with more than a single dye-e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in "aqueous" medium was satisfactory. CONCLUSIONS: Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions.

Coloring Agents↗
PubMed2001

A flow cytometry assay simultaneously detects independent apoptotic parameters.

BACKGROUND: Apoptosis regulation is of fundamental importance in tissue homeostasis and in the pathogenesis of a variety of diseases. Different cytofluorometric methods are used to investigate apoptotic events. We set up a method to simultaneously evaluate mitochondria depolarization, cell morphology changes, and loss of plasma membrane asymmetry and integrity, thus increasing the information and minimizing errors in the analysis of the apoptotic process. METHODS: Jurkat T cells were induced to undergo apoptosis with different agents. They were labeled with (1) the mitochondrion-selective probes tetramethylrhodamine methyl ester (TMRM) or chloromethyl X-rosamine (CMXRos), which do not accumulate in depolarized mitochondria; (2) Annexin V-fluorescein isothyocianate (FITC) to detect phosphatidylserine (PS) exposure on the cell surface; and (3) propidium iodide (PI) to assess loss of plasma membrane integrity. Cell morphology changes were studied following variations in light scatter parameters. RESULTS: This is a fast, reliable, and reproducible technique to detect simultaneously independent apoptotic changes by cytofluorometric inspection. TMRM is more effective than CMXRos in responding to variations in the electrochemical gradient of mitochondria. CONCLUSIONS: This technique allows us to integrate the analysis and to follow the kinetics of different apoptotic cell changes.

Annexin A5↗
PubMed2001

Measurement of DNA damage associated with apoptosis by laser scanning cytometry.

BACKGROUND: Phosphatidylserine (PS) binding by annexin V (AV) is an early membrane marker of apoptosis. Using laser scanning cytometry (LSC) and the comet assay, we showed that the DNA of AV(+) cells is so highly fragmented that it cannot be quantified by the comet assay (Bacso et al.: Cancer Res 60:4623-8, 2000). METHODS: The "halo" assay was used instead of the comet assay to quantify DNA damage associated with apoptosis. The LSC was used to measure both AV fluorescence and DNA damage on the same Jurkat cells following treatment with anti-Fas. The data from both sets of measurements were merged, allowing direct correlation of membrane and nuclear markers of cell death. RESULTS: AV(+) cells had significant DNA damage determined by the ratio between nuclear DNA and peripheral (migrated) DNA. Cells in the early and late stages of apoptosis could be discriminated on the basis of DNA content. In addition, it was possible to distinguish between apoptotic and necrotic cells in the AV(+) propidium iodide-positive population based on DNA content and DNA damage. The addition of specific inhibitors for caspases-8, 9, and 3 blocked both PS externalization and DNA fragmentation, indicating these events are downstream from caspase activation. CONCLUSIONS: This technique allows accurate distinction between apoptotic and necrotic cells and cytometric grading of apoptosis.

Annexin A5↗
PubMed2001

Flow cytometry-based assay for determination of teleost cytotoxic cell lysis of target cells.

BACKGROUND: The nonradiometric assays previously developed to detect cellular cytotoxic activity have been hindered by many difficulties. Among the problems are the requirement for expensive commercial kits and the use of techniques that produce high background noise and decreased sensitivity. In addition, these assays did not account for bidirectional apoptosis (activation-induced cell death [AICD]). Most attempts to derive cytometry-based cytotoxicity assays have been unsuccessful because individual effectors and targets could not be identified (i.e., "separated") using gating techniques. METHODS: In the present study, teleost nonspecific cytotoxic (NCC) and mammalian target cells were each sufficiently different in size to identify them by flow cytometry (FCM). Using appropriate gating and discriminator techniques, these two cell populations were differentiated based on scatter properties and propidium iodide (PI) binding. Total capacity for PI binding was obtained by permeabilization of the targets with ice-cold acetone. Spontaneous PI binding was relatively low. This technique detected cytotoxicity at effector-to-target ratios (E:T) of 1:1 and after only 30 min cocultivation. RESULTS: Tilapia NCC from peripheral blood kill human transformed target cells by necrosis and apoptosis as identified by PI binding. Maximum killing of HL-60 targets (approximately 100%) occurred by 180 min cocultivation. For the same time, the killing of IM-9 did not exceed 60%. Almost 90% of IM-9 targets are lysed following 14 h of cocultivation. The maximum killing of both HL-60 and IM-9 targets was observed at a 25:1 E:T ratio after 14 h. Comparisons of the chromium(>51) release assay with flow detection of cytotoxicity revealed that FCM detected 55% lysis of the target cells compared with 2% cytotoxicity by chromium release, after a cocultivation time of 240 min. DISCUSSION: FCM detection of (teleost) NCC lysis of target cells using PI uptake is more sensitive than standard chromium release assays. This level of sensitivity was observed because NCC and targets were sufficiently different in size such that they could be resolved by scatter plots. Using FCM, cytotoxicity was detected earlier and at lower E:T ratios than previously reported for chromium release assays. Although tilapia were reported previously to be not capable of lysing IM-9 targets by chromium release detection, the more sensitive method of FCM detected cytotoxicity using PI uptake. HL-60 lysis by tilapia NCC exhibited saturable kinetics but occurred at different times post-cocultivation.

Animals↗
PubMed2000

Chlorpromazine induces apoptosis in activated human lymphoblasts: a mechanism supporting the induction of drug-induced lupus erythematosus?

OBJECTIVE: Drug-induced lupus erythematosus is a serious side effect of certain medications, such as procainamide, quinidine, hydralazine, chlorpromazine, and isoniazid, the underlying pathogenesis of which is unresolved. In this study, we examined the influence of these drugs on the regulation of apoptosis, or programmed cell death, in quiescent and activated human lymphocytes. We also discuss the dysregulation of apoptosis as a pathogenetic factor in systemic lupus erythematosus. METHODS: Peripheral blood mononuclear cells or activated lymphoblasts from normal donors were incubated with different concentrations of each of the above-mentioned drugs. RESULTS: We did not find induction of apoptosis in quiescent cells over a broad concentration range. In contrast, lymphoblasts readily underwent apoptosis when cultured with chlorpromazine, but not any of the other drugs, after stimulation with interleukin-2 (IL-2) in a dose-, time- and cell cycle-dependent manner. By several lines of evidence, toxicity was ruled out. Characteristic features of apoptosis-like incorporation of propidium iodide (PI), such as increased annexin V binding, changes in mitochondrial membrane potential, and induction of DNA breaks (as evidenced by TUNEL techniques), could be induced in lymphoblasts after chlorpromazine treatment. Chlorpromazine did not cause apoptosis by inhibition of cytokine binding or blockade of early intracellular signaling. The protease inhibitor Z-VAD and the ceramide inhibitor sphingosine 1-phosphate effectively blocked chlorpromazine-induced apoptosis (by PI staining and by externalization of phosphatidylserine), in contrast to the caspase 3/CPP32 inhibitor DEVD, which had only minor effects. Western blot analysis revealed IL-2-mediated phosphorylation of extracellular signal-regulated kinase, which was sensitive to chlorpromazine. Using lymphoblasts from a patient with Canale-Smith syndrome, we found that chlorpromazine-mediated apoptosis is Fas/ APO-1 independent. CONCLUSION: These data suggest that chlorpromazine mediates apoptosis in human lymphoblasts through specific activation of intracellular proapoptotic signaling cascades. This mechanism might lead to an unsynchronized inflow of apoptotic break-down products and thereby to the induction of (auto)immunity against nuclear components.

Apoptosis↗
PubMed1990

Changes in numbers of pancreatic acinar cell nuclei and in DNA content during raw soya flour feeding in mice.

Nuclei of pancreatic cells were isolated by trypsin-detergent digestion of fresh tissue and stained with propidium iodide, and nuclear DNA was measured by flow cytometry. Samples were isolated from mice fed either chow or raw soya flour (RSF) for periods ranging from 1 day to 48 weeks, beginning at 4 weeks of age. In chow-fed mice, the pancreas contained about 80% diploid (2N) and 20% tetraploid (4N) cells at the start of the study, but tetraploidy gradually increased to about 40% 2 weeks later (6 weeks of age) and remained at this level from that time onwards. Low levels of octaploid nuclei (8N) were also present in some animals after 2 weeks. In RSF-fed mice, about 20% tetraploid nuclei were also present for 1 and 2 days after starting RSF, but by 4 days tetraploidy had increased significantly to 40% and by 14 days had further increased to 50%. This level was significantly higher than that seen in chow-fed animals and was maintained for up to 48 weeks. Significantly higher numbers of octaploid nuclei were also present in the RSF-fed animals. In both chow- and RSF-fed mice, most cells were mononuclear, averaging 70% in chow-fed and 64% in RSF-fed animals. This difference was significant. This study shows that the mouse pancreas differs from the rat pancreas in the absence of a large population of binucleate acinar cells and the presence of considerable nuclear tetraploidy. Raw soya flour feeding leads to significant changes in these features, but in this species these changes do not appear to predispose to neoplasia.

Animal Feed↗
PubMed1993

Flow cytometric analysis of the ploidy of normoblasts in the peripheral blood of patients with beta-thalassemia.

The chronic severe anemia of patients with beta-thalassemia major stimulates extensive erythropoiesis, which results in circulating nucleated normoblasts. We devised a dual staining flow cytometric procedure in order to analyse the cell cycle and ploidy of these normoblasts. Peripheral blood cells of O blood-group type were first stained with Fluorescein Isothiocyanate (FITC)-conjugated anti-H lectin which labels erythroid cells (RBC and normoblasts) by green fluorescence, and then with propidium iodide (PI) which binds to DNA and thereby labels nucleated cells (leukocytes and normoblasts) by red fluorescence. The leukocytes and normoblasts present in the blood sample of thalassemic patients could be distinguished and "gated" based on their green fluorescence. The PI (red) fluorescence, i.e., the DNA histogram of each population, was thus obtained. The results indicated no statistically significant difference in the PI fluorescence of these two populations. Thus, in spite of the abnormal erythropoiesis in beta-thalassemia, the resultant orthochromatic normoblasts are normal with respect to their DNA content.

Erythroblasts↗
PubMed2005

Synthesis and biological evaluation of tacrine-thiadiazolidinone hybrids as dual acetylcholinesterase inhibitors.

The synthesis of tacrine-thiadiazolidinone hybrids is described. These compounds are designed as dual acetylcholinesterase inhibitors binding simultaneously to the peripheral and catalytic sites of the enzyme. All tested compounds exhibit significant AChE inhibitory activity. Competition assays using propidium as reference of selective ligand for the peripheral anionic site on acetylcholinesterase indicates the influence of the designed compounds over the peripheral site. They can be considered as new leads in the optimization of Alzheimer's disease modifying agents.

Acetylcholinesterase↗
PubMed2003

Chemical heterogeneity in cell death: combined synchrotron IR and fluorescence microscopy studies of single apoptotic and necrotic cells.

The combination of synchrotron IR microspectroscopy and fluorescence microscopy has led to the identification of specific IR signatures of apoptosis and necrosis at a single cell level. Apoptosis was induced by treatment of Fas+ tumor cell lines with anti-Fas monoclonal antibodies. Detection of the early and late stages of apoptosis was performed using conjugated annexin V-fluorescein isothiocyanate (AV-FITC) and propidium iodide. Very early cellular changes were detected by IR before externalization of phosphatidylserine and AV-FITC labeling, and they were probably linked to DNA unwinding. The IR signals at 1044, 1177, and 1222 cm(-1), as well as an intensity variation in the CHx stretching region, are the main signature changes of early and late apoptosis, in line with the hypothesis of DNA fragmentation. The increased intensity of the CHx stretching bands of the lipids was observed only at an early stage of apoptosis. Changes in the relative intensity of CH3 and CH2 stretching accompany this increased intensity, suggesting changes in the relative amount and/or type of lipids concomitant with an increased lipid content. Finally, necrotic cells were characterized by marked changes in their chemical composition because several new vibrational features were observed.

Animals↗
PubMed1994

Effects of DNA charge and length on the electrophoretic mobility of intercalated DNA.

DNA molecules ranging in size from 1 to 630 kilobase pair and intercalated with either ethidium bromide (EtBr) or propidium iodide (PI) were electrophoresed in 1% agarose at four different electric field strengths. The extent of intercalation of EtBr under the conditions of our electrophoresis experiments was determined by a spectroscopic technique, whereas the extent of intercalation of PI was inferred from previous studies. The effects of the increase in DNA contour length and the concomitant decrease of linear charge density were separated based on our analysis of the mobility data. We conclude that the main factor responsible for the reduced electrophoretic mobility of intercalated DNA is the diminished linear charge density and not the increased contour length.

DNA↗
PubMed2005

Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation.

The key to optimizing productivity during industrial fermentations is the ability to rapidly monitor and interpret the physiological state of single microbial cells in a population and to recognize and characterize different sub-populations. Here, a flow cytometry-based method for the reproducible detection of changes in membrane function and/or structure of recombinant E. coli JM101 (pSPZ3) expressing xylene monooxygenase (XMO), was developed. XMO expression led to compromised but not permeabilized cell membranes. This was deduced from the fact that recombinant cells only stained with ethidium bromide (EB) and not with propidium iodide (PI). During the glucose-limited fedbatch cultivation, an increase from 25% to 95% of EB-stained cells was observed, occurring between 2 and 5 h after induction. Control experiments confirmed that this increase was due to the recombinant protein production and not caused by any possible effects of varying substrate availability, high cell density, plasmid replication or the presence of the inducing agent. We hypothesize that the integration of the recombinant protein into the cell membrane physically disrupted the functionality of the efflux pumps, thus resulting in EB-staining of the recombinant cells. This method enabled us to detect changes in the physiological state of single cells 2-4 h before other indications of partial cell damage, such as unbalanced growth, acetate accumulation and an increased CO(2) production rate, were observed. This method therefore shows promise with respect to the further development of an early-warning system to prevent sudden productivity decreases in processes with recombinant E. coli expressing heterologous membrane proteins.

Acetates↗
PubMed1986

Choline acetyltransferase immunoreactivity suggests that ganglion cells in the goldfish retina are not cholinergic.

Published evidence that ganglion cells in the retinae of nonmammalian species are cholinergic is strong but indirect. In this paper we report results of attempts to demonstrate choline acetyltransferase immunoreactivity in ganglion cells of goldfish retina using two different antisera against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme. We obtained ChAT-immunoreactive staining of amacrine and displaced amacrine cells in the retina and type XIV cells in the tectum, but we obtained no direct immunocytochemical evidence that ganglion cells in the goldfish retina are cholinergic. Thus, ganglion cells identified by retrograde transport of propidium iodide were never ChAT-immunoreactive. Intraocular injections of colchicine did not result in the appearance of a population of ChAT-immunoreactive neurons in the ganglion cell layer. ChAT-immunoreactive axons were not observed in intact, ligated, or transected optic nerves. And finally, the ChAT immunoreactivity of cells and fibers in the optic tectum was unaffected by deafferentation. These experiments provide no positive evidence that any ganglion cells in goldfish retina contain the acetylcholine-synthesizing enzyme, ChAT. While it is possible that our method is too insensitive to detect the enzyme in ganglion cell somata or too specific to recognize the form of ChAT present in these cells, the fact that we can stain putatively cholinergic retinal amacrine cells and tectal neurons makes these alternative explanations improbable. We conclude that it is unlikely that any of the ganglion cells in the retina are cholinergic and that alternative explanations should be sought for previously published results that suggest that they are.

Acetylcholine↗