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At least 307 records · Page 17Linked to original sources
PubMed1995

Analysis of single cells by capillary electrophoresis with on-column derivatization and laser-induced fluorescence detection.

On-column derivatization of single mammalian cells with capillary electrophoretic separation and laser-induced fluorescence detection is described. Individual cells are electrophorectically injected into the front end of the separation capillary, which is used as a chamber to lyse the cell and derivatize its contents for subsequent separation and detection. Reagents to lyse the cell and derivatize its contents are electrophoretically injected into the front end of the capillary (2.7 mm, 600-pL volume for a 17-microns-i.d. capillary) after the cell has been injected. Dopamine and five amino acids have been quantitatively determined in individual rat pheochromocytoma cells after on-column derivatization with naphthalene-2,3-dicarboxaldehyde and CN-. Average values of compounds determined in these cells range from 180 +/- 110 amol/cell for aspartic acid to 5.1 +/- 1.5 fmol/cell for taurine.

Amines↗
PubMed1994

Repair of O6-alkylguanines in the nuclear DNA of human lymphocytes and leukaemic cells: analysis at the single-cell level.

Inter-individual and cell-cell variability of repair of O6-alkylguanines (O6-AlkGua) in nuclear DNA was studied at the single-cell level in peripheral lymphocytes from healthy donors and in leukaemic cells isolated from patients with chronic lymphatic leukaemia (CLL) or acute myeloid leukaemia (AML). Cells were pulse exposed to N-ethyl- or N-(n-)butyl-N-nitrosourea in vitro, and O6-AlkGua residues in DNA were quantified using an anti-(O6-AlkGua) monoclonal antibody and electronically intensified fluorescence. The kinetics of O6-AlkGua elimination revealed considerable inter-individual differences in O6-ethylguanine (O6-EtGua) half-life (t1/2) values in DNA, ranging from 1.5 to 4.5 h (five AML patients), from 0.8 to 2.8 h (five CLL patients) and from 1.2 to 7.3 h (five healthy donors). The elimination from DNA of equimolar amounts of O6-butylguanine was generally 3-5 times slower in comparison with O6-EtGua. The t1/2 values of individual samples varied in parallel for both DNA alkylation products. Upon preincubation with O6-benzylguanine, the activity of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AT) in both lymphocytes and leukaemic blasts was reduced to < or = 1%. However, while the rate of O6-EtGua elimination from DNA was decelerated it was not abolished, suggesting the possible involvement of additional repair systems that might be co-regulated with AT. Within individual samples, no major cell subpopulations were observed whose repair kinetics would differ significantly from the remaining cells.

Alkylating Agents↗
PubMed2002

Binding and transport in norepinephrine transporters. Real-time, spatially resolved analysis in single cells using a fluorescent substrate.

Monoamine transporters, the molecular targets for drugs of abuse and antidepressants, clear norepinephrine, dopamine, or serotonin from the synaptic cleft. Neurotransmitters, amphetamines, and neurotoxins bind before being transported, whereas cocaine and antidepressants bind to block transport. Although binding is crucial to transport, few assays separate binding from transport, nor do they provide adequate temporal or spatial resolution to describe real-time kinetics or localize sites of active uptake. Here, we report a new method that distinguishes substrate binding from substrate transport using single-cell, space-resolved, real-time fluorescence microscopy. For these studies we use a fluorescent analogue of 1-methyl-4-phenylpyridinium, a neurotoxic metabolite and known substrate of monoamine transporters, to assess binding and transport with 50-ms, sub-micron resolution. We show that ASP(+) (4-(4-(dimethylamino)styrl)-N-methylpyridinium) has micromolar potency for the human norepinephrine transporter, that ASP(+) accumulation is Na(+)-, Cl(-)-, cocaine-, and desipramine-sensitive and temperature-dependent, and that ASP(+) competes with norepinephrine uptake. Using this method we demonstrate that norepinephrine transporters are efficient buffers for substrate, with binding rates exceeding transport rates by 100-fold. Furthermore, substrates bind deep within the transporter, isolated from both the bath and the lipid bilayer. Although transport per se depends on Na(+) and Cl(-), binding is independent of Na(+) and actually increases in low Cl(-). We further demonstrate that ASP(+) interacts with transporters not only in transfected cells but in cultured neurons. ASP(+) is also a substrate for dopamine and serotonin transporters and therefore represents a powerful new technique for studying the biophysical properties of monoamine transporters, an approach also amenable to high throughput assays for drug discovery.

Adrenergic Uptake Inhibitors↗
PubMed1995

Whole genome amplification of single cells: mathematical analysis of PEP and tagged PCR.

We construct a mathematical model for two whole genome amplification strategies, primer extension preamplification (PEP) and tagged polymerase chain reaction (tagged PCR). An explicit formula for the expected target yield of PEP is obtained. The distribution of the target yield and the coverage properties of these two strategies are studied by simulations. From our studies we find that polymerase with high processivity may increase the efficiency of PEP and tagged PCR.

Computer Simulation↗
PubMed2006

Cardiomyocyte function after burn injury and lipopolysaccharide exposure: single-cell contraction analysis and cytokine secretion profile.

A component of multiorgan dysfunction in burned patients is heart failure. Burn trauma induces cytokine synthesis of interleukin (IL) 1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) which can negatively impact cardiac function. Infectious complications are common following severe burn injury. We hypothesized that burn injury and lipopolysaccharide (LPS) exposure independently influence peak cardiomyocyte contraction and cytokine secretion. Rats underwent a full-thickness 30% total body surface area scald or sham burn. At 1, 6, 12, and 24 h after burn, cardiomyocytes were isolated and incubated with increasing LPS doses. Peak sarcomere shortening and contractile velocity parameters were recorded using a variable-rate video camera with sarcomere length detection software. Supernatants were assayed for IL-1beta, IL-6, and TNF-alpha by ELISA. Peak sarcomere shortening was decreased in the burn group at 1, 6, 12, and 24 h after burn. IL-1beta, IL-6, and TNF-alpha levels were increased in cardiomyocytes isolated 1 h after burn compared with sham controls, but returned to sham levels at 6, 12, and 24 h after burn. LPS exposure caused dose-dependent decreases in sarcomere shortening in sham and burn animals. LPS exposure did not produce increased cardiomyocyte cytokine expression. Burn injury diminished peak sarcomere shortening. Whereas exposure to LPS did not have an effect on cardiomyocyte cytokine expression, LPS significantly inhibited sarcomere shortening in a dose-dependent fashion. Combined burn and LPS exposure inhibited sarcomere shortening more than each alone. These results demonstrate that LPS exposure and burn injury independently decrease peak cardiac shortening. These decreases did not directly correlate with the levels of cytokines released in response to each stressor.

Animals↗
PubMed1996

Behavioral analysis of single cells of Myxococcus xanthus in response to prey cells of Escherichia coli.

Myxococcus xanthus cells move over surfaces by gliding motility. The frz signal transduction system is used to control the reversal frequency, and thus the overall direction of movement of M. xanthus cells. We analyzed the behavior of wild-type and frz mutant cells in response to prey bacteria (Escherichia coli). Wild-type cells of M. xanthus did not respond to microcolonies of E. coli until they made physical contact. Cells which penetrated a colony remained in the colony until all of the prey cells were digested. Cells of frz mutants also penetrated E. coli microcolonies and digested some of the E. coli cells, but they invariably abandoned the microcolony leaving their food source behind. These observations illustrate the importance of the frz system of signal transduction for the feeding behavior of M. xanthus cells.

Bacterial Adhesion↗
PubMed2000

Superantigen-induced T cell death by apoptosis: analysis on a single cell level and effect of IFN-gamma and IL-4 treatment.

BACKGROUND: A role of bacterial superantigens in several chronic inflammatory diseases has previously been proposed. Many of these diseases are associated with an imbalance of the T helper cell subsets and their cytokine production. METHODS: Peripheral blood mononuclear cells from healthy donors were incubated with various concentrations of staphylococcal enterotoxin B (SEB) with or without IL-4 or IFN-gamma. After different time points cell activation, proliferation, Fas expression, cytokine release and cell death via apoptosis were detected. RESULTS: SEB treatment resulted in sequential T cell activation, proliferation, Fas expression, cytokine release, subsequently followed by cell death via apoptosis in a dose- and time-dependent manner. This biphasic effect occurred preferentially in SEB-responsive cells represented by the expression of Vbeta3 and Vbeta12 on T cells. A strong relationship between T cell activation and apoptosis was observed. The amplitude between these events increased with the dose of SEB. The highest rate of apoptotic T cells was observed at a dose of 1,000 ng/ml SEB. Addition of IFN-gamma to SEB-treated cells significantly reduced the rate of apoptotic cells, whereas IL-4 prevented apoptosis only in SEB-untreated cells. CONCLUSION: These results support the concept that the dose of superantigen exposure determines the rate of T cell proliferation and subsequent cell death. This T cell immune response is modulated by the presence and the type of cytokines.

Adult↗
PubMed1976

Methods for analysis of acid alpha-1,4-glucosidase activity in single hybrid cells.

Two methods are described which allow the quantitative assay of the lysosomal enzyme alpha-1,4-glucosidase in single fibroblasts. In the first procedure the substrate was maltose, and liberated glucose was measured with an enzymatic cycling procedure for reduced nicotinamide adenine dinucleotide phosphate. Single cultured fibroblasts were found to have enzyme activities in the range of 0.5-10 X 10(-13) moles glucose/hr. In the second procedure the artificial substrate 4-methylumbelliferyl-alpha-D-glucopyransodie was used. It is hydrolyzed in a single step reaction to the fluorescent product 4-methylumbelliferone (MU). By reducing the incubation volume and by measuring the fluorescence in microdroplets with a microscope fluorometer, a sensitivity of 10(-14) moles MU could be obtained. Activities were found ranging from 0.5-10 X 10(-14) moles MU/hr/cell. Both procedures for single cell analysis proved to be reliable when compared with conventional assays on cell homogenates. Cocultivation and cell fusion studies were performed to demonstrate that these methods can be used to study the metabolic and genetic interaction between normal and enzyme-deficient fibroblasts derived from patients with glycogenosis II.

Fibroblasts↗
PubMed2003

Th1 and Th2 subsets in orthopaedic surgery by single-cell cytokine analysis.

Significant immunosuppression can occur following allogeneic blood transfusion or surgery. Cytokine stimulation controls immune responses and determines their type and intensity. Infusion of autologous or allogeneic blood provides elements, including cytokines, which may result in transfusion-associated immunomodulation. This study investigates to what extent autologous/cell salvage transfusions affect levels of intracellular cytokines interferon-gamma and interleukin-4, and if this indicates a shift in the T-helper 1/T-helper 2 cell ratio using a novel method of detecting intracellular cytokines, the Magnetic Activated Cell Sorter Cytokine Secretion Assay (MACS Assay). Comparisons were made between patients receiving autologous blood or no blood transfusion, for pre- and post-operation levels of interferon-gamma and interleukin-4. Interferon-gamma producing T-helper 1 cells decreased post-operatively. Concomitantly, interleukin-4 producing T-helper 2 cells increase. These results demonstrate a measurable shift from T-helper 1 to T-helper 2 cells post-operatively. Secondly, the study showed surgery alone instigates the same level of immunomodulation as autologous/cell salvage blood transfusion in combination with surgery.

Adult↗
PubMed1984

Studies of natural killer cells in pregnancy. I. Analysis at the single cell level.

In using both a 51Cr-release assay and a single cell technique we measured different parameters of the effector lymphocytes killing process in pregnant and control women on a cell population and single cell level. Total NK activity was lower in pregnancy, but the difference was not significant. Parity showed no cumulative effect. Pregnant women had normal percentages of potentially cytotoxic target binding cells, however, the relative number of lymphocytes that could kill bound targets (i.e. active NK cells) was significantly depressed in pregnancy when compared with normal controls (p less than 0.05). This diminished number of active NK lymphocytes was, however, normal in all phases of the lytic procedure. Interferon (IFN) treatment of effector cells in pregnant women in vitro did not alter target cell binding, but did increase the percentage of active NK cells. The level of stimulation by IFN was the same in pregnancy and control patients. The kinetics of response were the same for both cell populations and different doses of interferon caused a similar level of augmentation. In conclusion we suggest that the here demonstrated deficiency of active NK lymphocytes in pregnancy may represent a function of pregnancy associated immunoregulatory molecules which prevent a population of pre-NK cells to express their cytotoxic potential.

Cytotoxicity, Immunologic↗