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At least 505 records · Page 28Linked to original sources
PubMed2006

Ion channel characterization using single cell impedance spectroscopy.

A micro electrical impedance spectroscopy system (microEIS) for single cell analysis has been developed and used to differentiate ion channel activities of bovine chromaffin cells. K+ and Ca2+ channels were blocked and their electrical impedances were measured over a frequency range of 100 Hz to 5.0 MHz and compared to that of unblocked chromaffin cells. When ion channels were blocked, an increase in magnitude and decrease in phase of the measured impedances were observed. This result demonstrates that ion channel activities can be distinguished using the developed microsystem and it is expected that this system can be used to provide positive/negative information of ion channel blockage in a high throughput screening setup.

Animals↗
PubMed1989

Bombesin stimulation of inositol 1,4,5-trisphosphate generation and intracellular calcium release is amplified in a cell line overexpressing the N-ras proto-oncogene.

Bombesin stimulation of T15 cells in which the inducible N-ras oncogene is overexpressed caused elevated production of inositol phosphates compared to uninduced cells [Wakelam, Davies, Houslay, McKay, Marshall & Hall (1986) Nature (London) 323, 173-176]. This elevated response is shown here to result from increased generation of inositol 1,4,5-trisphosphate leading to an elevated release of intracellular stored Ca2+. Single-cell analysis of Ca2+ release showed that the elevated response is not a consequence of an increased fraction of responding cells. These amplifications are consistent with p21N-ras acting like a guanine nucleotide coupling protein in this cell line.

Bombesin↗
PubMed2001

Recovery of platelet function after discontinuation of clopidogrel treatment in healthy volunteers.

AIMS: To study the recovery of platelet function after discontinuation of clopidogrel treatment in healthy volunteers. METHODS: Ten healthy volunteers were treated with clopidogrel (75 mg day(-1)) for 7 days. CD62P expression and PAC-1 binding were measured by flow cytometry. RESULTS: Adenosine diphosphate (ADP, 30 microM)-induced platelet responses were almost completely inhibited by clopidogrel. After discontinuation of the drug, platelet function gradually increased and complete recovery was seen 7 days after the last clopidogrel dose. The mean difference (95% CI) for ADP-induced PAC-1 binding (fluorescence intensity) between baseline and 7 days after the last dose was 0.01 (0.61, -0.59). Single cell analysis provides direct evidence for an irreversible mode of action of clopidogrel. CONCLUSIONS: This is the first report to directly demonstrate irreversibility of clopidogrel action in humans.

Adenosine Diphosphate↗
PubMed1997

Relevance of local Th2-type cytokine mRNA expression in immunocompetent infiltrates in inflamed gingival tissue to periodontal diseases.

It has been suggested that the types of inflammatory round cell infiltrates and the divergence in the cytokine production profile by macrophages and helper T cells regulate the course of infectious or inflammatory diseases, including periodontitis and gingivitis. We examined the expression of IL-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6 and tumour necrosis factor-alpha (TNF-alpha) mRNA in the inflamed gingiva by in situ hybridization. The results of single-cell analysis were used as data sets for statistical analyses. The density of cells expressing IL-1alpha, IL-4 and IL-5 mRNA was higher in periodontitis than in gingivitis. IL-2 mRNA-expressing cells were almost absent in gingivitis specimens. Principal component analysis disclosed three factors explaining 84.8% of the variance: one accounting for 40.5% of the variance and mainly regulated by IL-1alpha, IL-1beta, IL-6 and TNF-alpha, and two others, explaining 29.9% and 14.4% of the variance, describing the relationship between the types of cytokines derived from macrophages or Th2 type. These results suggest that the cytokines produced by inflammatory cells infiltrating in the gingival tissue are influential on the progression of gingivitis, an acute and reversible inflammatory condition, to chronic and destructive periodontitis. Thus, periodontal disease progression may be regulated by the local cytokine network, and the bias in this network towards a Th2-type cytokine dominance could be an exacerbating factor.

Adult↗
PubMed1999

Progressive polarization towards a T helper/cytotoxic type-1 cytokine pattern during age-dependent maturation of the immune response inversely correlates with CD30 cell expression and serum concentration.

In order to investigate the T cell cytokine profile during age-dependent maturation of the immune response, we evaluated the cytokine expression of CD4+ and CD8+ circulating cells by flow cytometric single-cell analysis after non-specific stimulation in vitro in different age groups of normal individuals, from cord blood to adulthood. Moreover, we correlated these lymphocyte cytokine patterns with the expression/release of CD30, a member of the tumour necrosis factor (TNF) receptor superfamily, which has been suggested to be related to the T helper/cytotoxic (Th(c))2-type immune responses, in order to verify this association in vivo, in non-pathological conditions. The results showed a progressive increase of circulating Th(c)1-type, interferon-gamma (IFN-gamma)- and/or IL-2-producing T cells along with ageing and, conversely, a stable number, although higher than in cord blood samples, of CD4+/IL-4+ T cells in the post-natal groups. In addition, serum levels of soluble CD30 (sCD30) and numbers of circulating CD4+/CD30+ and CD8+/CD30+ T cells were significantly higher in children aged < 5 years in comparison with those found either in cord blood or in blood from both older children and adults. These data support the concept of a progressive polarization of the Th(c) cell cytokine profile towards the Th(c)1 pattern during age-dependent maturation of the immune response. Moreover, the peak of CD30 expression/release in early infancy before the Th(c)1 shifting occurs, although not associated with a significant increase of circulating IL-4+ T cells, raises the question of the possible relationship in vivo between CD30 and Th(c)2-type immune responses.

Adolescent↗
PubMed2001

T cell responses to highly active antiretroviral therapy defined by chemokine receptors expression, cytokine production, T cell receptor repertoire and anti-HIV T-lymphocyte activity.

The immunological correlates of highly active antiretroviral therapy (HAART)-induced suppression of human immunodeficiency virus type 1 (HIV-1) replication have been investigated. 20 HIV-1-infected patients with mean CD4+ T cell count of 298/microl, plasma viral load of 4.7 log10 copies/ml and naive for protease inhibitors (PI) were studied during12 months of HAART. An increased number of both CD4+ and CD8+ naive T cells and a normalization of the frequency of CCR5- and CXCR4-expressing CD4+ T cells were readily observed after starting therapy. Single cell analysis of cytokine production after 12 months of HAART showed an increased number of interleukin (IL)-2-, but not IL-4- and (IFN)-gamma-, producing T cells and a decreased percentage of CD8+ IFN-gamma + cells. A correlation between the frequency of IFN-gamma-producing T cells and that of memory, CCR5+ and CD95+ T cells was demonstrated in both CD4+ and CD8+ subsets. The diversity of T cell receptor (TCR) variable beta (BV) chain repertoire significantly increased after 12 months of HAART within the CD4+ but not the CD8+ T cell subset. However, the level of perturbation of the third complementarity-determining region (CDR3), was not significantly modified by effective therapy. The number of anti-HIV Gag and Pol cytotoxic T lymphocytes precursors (CTLp) decreased during HAART and highly correlated with the CD8 IFN-gamma response. Ameliorated clinical conditions were observed in all patients in absence of any opportunistic infections during all the study period. These observations indicate that a better restoration of immunity may be obtained in patients starting HAART at less advanced stages of the disease.

Adult↗
PubMed1998

Transgenic plants changed in carbon allocation pattern display a shift in diurnal growth pattern.

Photosynthesis, partitioning of carbohydrates and growth have to be highly orchestrated to enable an efficient performance of plants. To study the diurnal relationships between carbon distribution and growth, we analysed transgenic potato plants with altered carbon allocation patterns. To modify carbohydrate supply of growing sinks, we used plants that accumulated starch as a consequence of inhibition in triose-phosphate export from chloroplasts and plants that were genetically inhibited in starch production. Carbon assimilation was analysed by gas exchange and single cell analysis of source leaves. Export was determined by microanalysis of phloem exudates and internodal growth rates were measured by displacement transducers. Gas exchange measurements showed similar assimilation rates in the wild-type and transgenic plants during the light period. Sugar analysis of phloem exudates and epidermal cells revealed a severe shift of sucrose concentrations in the individual plant lines. Moreover, epidermal cells turned out to be a potential storage site for carbohydrates in potato. Finally, we could demonstrate that changing the diurnal rhythm of carbon allocation results in a change in the diurnal growth pattern.

Animals↗
PubMed2004

Activation of caspase-3 by the Dot/Icm virulence system is essential for arrested biogenesis of the Legionella-containing phagosome.

The Dot/Icm type IV secretion system of Legionella pneumophila is essential for evasion of endocytic fusion and for activation of caspase-3 during early stages of infection of macrophages, but the mechanisms of manipulating these host cell processes are not known. Here, we show that caspase-3 activation by L. pneumophila is independent of all the known apoptotic pathways that converge on the activation of caspase-3. The cytoplasmic proteins IcmS, IcmR and IcmQ, which are involved in secretion of Dot/Icm effectors, are required for caspase-3 activation. Pretreatment of U937 macrophages and human peripheral blood monocytes (hPBM) with the capase-3 inhibitor (DEVD-fmk) or the paninhibitor of caspases (Z-VAD-fmk) before infection blocks intracellular replication of L. pneumophila in a dose-dependent manner. Inhibition of caspase-3 results in co-localization of the L. pneumophila-containing phagosome (LCP) with the late endosomal/lysosomal marker Lamp-2, and the LCP contains lysosomal enzymes, similar to the dotA mutant, which is defective in caspase-3 activation. However, activation of caspase-3 before infection does not rescue the replication defect of the dotA mutant. Interestingly, inhibition of caspase-3 after a 15 or 30 min infection period by the parental strain has no detectable effect on the formation of a replicative niche. The Dot/Icm-mediated activation of caspase-3 by L. pneumophila specifically cleaves, in a dose- and time-dependent manner, the Rab5 effector Rabaptin-5, which maintains Rab5-GTP on the endosomal membrane. In addition, PI3 kinase, which is a crucial effector of Rab5 downstream of Rababptin-5, is not required for intracellular replication. Using single-cell analysis, we show that apoptosis is not evident in the infected cell until bacterial replication results in > 20 bacteria per cell. We conclude that activation of caspase-3 by the Dot/Icm virulence system of L. pneumophila is essential for halting biogenesis of the LCP through the endosomal/lysosomal pathway, and that this is associated with the cleavage of Rabpatin-5.

Antigens, CD↗
PubMed2006

Early establishment of diverse T cell receptor profiles for influenza-specific CD8(+)CD62L(hi) memory T cells.

Single-cell analysis of endogenous, primary CD8(+) T cell responses to the influenza D(b)NP(366) and D(b)PA(224) epitopes indicates that prominent clonotypes bearing "public" or "shared" T cell receptors (TCRs) subset early into CD62L(hi) and CD62L(lo) populations. The CD62L(lo) effectors divide more and are rapidly eliminated during the contraction phase, whereas stable CD62L(hi) memory populations persist in the long-term. Reflecting the high frequency of small CD62L(hi) clones expressing "private" TCRs, the TCR diversity range per mouse is generally two times higher within the CD62L(hi)CD8(+)D(b)NP(366)(+) set (1.6 times higher for CD62L(hi)CD8(+)D(b)PA(224)(+)) from 8 to >180 days after antigen challenge. Memory CD8(+)CD62L(hi) T cell precursors thus segregate from the outset into populations expressing "best-fit" and "suboptimal" TCR characteristics, with this pattern being maintained stably thereafter. Hence, our analysis suggests that early establishment of influenza-specific memory within the CD8(+)CD62L(hi) subset preserves clonal diversity and prevents "overdominance" by a few public, or shared, clones.

Amino Acid Sequence↗
PubMed2002

Chromosome translocations and covert leukemic clones are generated during normal fetal development.

Studies on monozygotic twins with concordant leukemia and retrospective scrutiny of neonatal blood spots of patients with leukemia indicate that chromosomal translocations characteristic of pediatric leukemia often arise prenatally, probably as initiating events. The modest concordance rate for leukemia in identical twins ( approximately 5%), protracted latency, and transgenic modeling all suggest that additional postnatal exposure and/or genetic events are required for clinically overt leukemia development. This notion leads to the prediction that chromosome translocations, functional fusion genes, and preleukemic clones should be present in the blood of healthy newborns at a rate that is significantly greater than the cumulative risk of the corresponding leukemia. Using parallel reverse transcriptase-PCR and real-time PCR (Taqman) screening, we find that the common leukemia fusion genes, TEL-AML1 or AML1-ETO, are present in cord bloods at a frequency that is 100-fold greater than the risk of the corresponding leukemia. Single-cell analysis by cell enrichment and immunophenotype/fluorescence in situ hybridization multicolor staining confirmed the presence of translocations in restricted cell types corresponding to the B lymphoid or myeloid lineage of the leukemias that normally harbor these fusion genes. The frequency of positive cells (10(-4) to 10(-3)) indicates substantial clonal expansion of a progenitor population. These data have significant implications for the pathogenesis, natural history, and etiology of childhood leukemia.

Base Sequence↗
PubMed2003

Microtubule destabilization and nuclear entry are sequential steps leading to toxicity in Huntington's disease.

There has been a longstanding debate regarding the role of proteolysis in Huntington's disease. The toxic peptide theory posits that N-terminal cleavage fragments of mutant Huntington's disease protein [mutant huntingtin (mhtt)] enter the nucleus to cause transcriptional dysfunction. However, recent data suggest a second model in which proteolysis of full-length mhtt is inhibited. Importantly, the two competing theories differ with respect to subcellular distribution of mhtt at initiation of toxicity: nuclear if cleaved and cytoplasmic in the absence of cleavage. Using quantitative single-cell analysis and time-lapse imaging, we show here that transcriptional dysfunction is "downstream" of cytoplasmic dysfunction. Primary and reversible toxic events involve destabilization of microtubules mediated by full-length mhtt before cleavage. Restoration of microtubule structure by taxol inhibits nuclear entry and increases cell survival.

Active Transport, Cell Nucleus↗
PubMed1994

Feedback regulation of cell-substratum adhesion by integrin-mediated intracellular Ca2+ signaling.

Integrin binding to extracellular matrix (ECM) regulates cell migration and gene expression in embryogenesis, metastasis, would healing, and the inflammatory response. In many cases, binding of integrins to ECM triggers intracellular signaling pathways. The regulatory roles of intracellular signaling mechanisms in these events are poorly understood. Using single-cell analysis, we demonstrate that beads coated with peptide containing Arg-Gly-Asp (RGD), an integrin recognition motif found in many ECM proteins, elicit a rapid transient increase in intracellular calcium in Madin-Darby canine kidney (MDCK) epithelial cells. Also, significantly more beads bind to responding cells than to nonresponders. Several independent methods that inhibit RGD-induced Ca2+ signaling decrease both the number of beads bound and the strength of adhesion to an RGD-coated substratum. These results indicate that intracellular Ca2+ signaling participates in a positive feedback loop that enhances integrin-mediated cell adhesion.

Amino Acid Sequence↗
PubMed1997

The yeast Cac1 protein is required for the stable inheritance of transcriptionally repressed chromatin at telomeres.

Cac1p is a subunit of yeast chromatin assembly factor I (yCAF-I) that is thought to assemble nucleosomes containing diacetylated histones onto newly replicated DNA [Kaufman, P. D., Kobayashi, R. & Stillman, B. (1997) Genes Dev. 11, 345-357]. Although cac1 delta cells could establish and maintain transcriptional repression at telomeres, they displayed a reduced heritability of the repressed state. Single-cell analysis revealed that individual cac1 delta cells switch from transcriptionally "off" to transcriptionally "on" more often per cell cycle than wild-type cells. In addition, cac1 delta cells were defective for transcriptional silencing near internal tracts of C(1-3)A sequence, but they showed no defect in silencing at the silent mating type loci when analyzed by a reverse transcription-PCR assay. Despite the loss of transcriptional silencing at telomeres and internal C(1-3)A tracts, subtelomeric DNA was organized into nucleosomes that had all of the features characteristic of silent chromatin, such as hypoacetylation of histone H4 and protection from methylation by the Escherichia coli dam methylase. Thus, these features of silent chromatin are not sufficient for stable maintenance of a silent chromatin state. We propose that the inheritance of the transcriptionally repressed state requires the specific pattern of histone acetylation conferred by yCAF-I-mediated nucleosome assembly.

Chromatin↗
PubMed1999

A mutation in the transmembrane/luminal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease.

Central core disease is a rare, nonprogressive myopathy that is characterized by hypotonia and proximal muscle weakness. In a large Mexican kindred with an unusually severe and highly penetrant form of the disorder, DNA sequencing identified an I4898T mutation in the C-terminal transmembrane/luminal region of the RyR1 protein that constitutes the skeletal muscle ryanodine receptor. All previously reported RYR1 mutations are located either in the cytoplasmic N terminus or in a central cytoplasmic region of the 5,038-aa protein. The I4898T mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. The response of the mutant RyR1 Ca2+ channel to the agonists halothane and caffeine in a Ca2+ photometry assay was completely abolished. Coexpression of normal and mutant RYR1 cDNAs in a 1:1 ratio, however, produced RyR1 channels with normal halothane and caffeine sensitivities, but maximal levels of Ca2+ release were reduced by 67%. [3H]Ryanodine binding indicated that the heterozygous channel is activated by Ca2+ concentrations 4-fold lower than normal. Single-cell analysis of cotransfected cells showed a significantly increased resting cytoplasmic Ca2+ level and a significantly reduced luminal Ca2+ level. These data are indicative of a leaky channel, possibly caused by a reduction in the Ca2+ concentration required for channel activation. Comparison with two other coexpressed mutant/normal channels suggests that the I4898T mutation produces one of the most abnormal RyR1 channels yet investigated, and this level of abnormality is reflected in the severe and penetrant phenotype of affected central core disease individuals.

Amino Acid Sequence↗
PubMed1996

Anti-Ig-induced calcium influx in rat B lymphocytes mediated by cGMP through a dihydropyridine-sensitive channel.

In contrast to excitable tissues where calcium channels are well characterized, the nature of the B lymphocyte calcium channel is unresolved. Here, we demonstrate by single cell analysis of freshly isolated rat B cells that the anti-immunoglobulin (Ig)-induced calcium influx takes place through a channel which shares pharmacologic and serologic properties with the L-type calcium channel found in excitable tissues. It is sensitive to the dihydropyridines nicardipine and Bay K 8644, to calciseptine, and to an anti-peptide antibody raised against the alpha1 subunit of the L-type calcium channel, but is voltage-insensitive. Anti-alpha1 and anti-alpha2 antibodies stain B but not T lymphocytes. Application of a cGMP agonist, measurement of cGMP levels in anti-Ig-stimulated B cells, and examining the effect of a guanylyl cyclase inhibitor on the anti-Ig response show that cGMP mediates the influx. This possibly involves a cGMP-dependent protein kinase. The anti-Ig-induced response is not abolished by prior treatment of B cells with a high dose of thapsigargin. These findings undermine the widely held belief of a categorical divide between excitable and non-excitable tissue calcium channels, demonstrate the limitations of the capacitative calcium influx theory, and point to a distinction between the calcium response mechanisms utilized by B and T lymphocytes.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2001

Differentiated 3T3L1 adipocytes are composed of heterogenous cell populations with distinct receptor tyrosine kinase signaling properties.

Various studies have demonstrated that the platelet-derived growth factor (PDGF) receptor in adipocytes can activate PI 3-kinase activity without affecting insulin-responsive glucose transporter (GLUT4) translocation. To investigate this phenomenon of receptor signaling specificity, we utilized single cell analysis to determine the cellular distribution and signaling properties of PDGF and insulin in differentiated 3T3L1 adipocytes. The insulin receptor was highly expressed in a large percentage of the cell population (>95%) that also expressed caveolin 2 and GLUT4 with very low levels of the PDGF receptor. In contrast, the PDGF receptor was only expressed in approximately 10% of the differentiated 3T3L1 cell population with relatively low levels of the insulin receptor, caveolin 2, and GLUT4. Consistent with this observation, insulin stimulated the phosphorylation of Akt in the caveolin 2- and GLUT4-positive cells, whereas PDGF primarily stimulated Akt phosphorylation in the caveolin 2- and GLUT4-negative cell population. Furthermore, transfection of the PDGF receptor in the insulin receptor-, GLUT4-, and caveolin 2-positive cells resulted in the ability of PDGF to stimulate GLUT4 translocation. These data demonstrate that differentiated 3T3L1 adipocytes are not a homogeneous population of cells, and the lack of PDGF receptor expression in the GLUT4-positive cell population accounts for the inability of the endogenous PDGF receptor to activate GLUT4 translocation.

3T3 Cells↗
PubMed2006

SLCO/OATP-like transport of glutathione in FasL-induced apoptosis: glutathione efflux is coupled to an organic anion exchange and is necessary for the progression of the execution phase of apoptosis.

Apoptosis is characterized by the activation of specific biochemical pathways that lead to the organized demise of cells. Intracellular GSH depletion has been observed during apoptosis; however, neither the mechanisms involved in the reduction of the intracellular GSH concentration, [GSH](i), nor its link to the progression of apoptosis have been elucidated. We have studied this issue using Fas ligand (FasL)-induced apoptosis in Jurkat cells where changes in [GSH](i) can be analyzed biochemically and at the single cell level by flow cytometry. A reduction in the total [GSH](i) in response to FasL occurs in two distinct stages prior to the loss of membrane integrity. Jurkat cells express several members of the multidrug resistance protein (ABCC/MRP), and the organic anion-transporting polypeptide protein (SLCO/OATP) families of GSH efflux pumps at the mRNA level. Glutathione loss and its accumulation in the extracellular medium, induced by FasL, was trans-stimulated by the organic substrates MK571, probenecid, taurocholic acid, estrone sulfate, and bromosulfophthalein and inhibited by high concentrations of extracellular GSH. Single cell analysis demonstrated that intracellular GSH loss was paralleled by the activation of an organic anion uptake process, supporting the role of an anion exchange mechanism (SLCO/OATP-like transport) in GSH efflux induced by FasL. Additionally, high extracellular GSH inhibited the activation of the execution caspases, the cleavage of their substrates poly(ADP-ribose) polymerase (PARP) and alpha-fodrin, and DNA degradation. In contrast, the trans-stimulation of GSH efflux by MK571 increased the cleavage of the execution caspases and their substrates. Together these results suggest that GSH efflux during FasL-induced apoptosis is mediated by a SLCO/OATP-like transport mechanism that modulates the progression of the execution phase of apoptosis.

Apoptosis↗
PubMed2004

Preimplantation genetic diagnosis and the welfare of the child.

Preimplantation genetic diagnosis is a form of very early prenatal diagnosis. The technique combines assisted reproductive technology with molecular genetics and cytogenetics to allow the identification of abnormalities in embryos prior to implantation. Since its introduction in 1990 this approach has been applied to an increasing number of single gene disorders, chromosomal rearrangements, and more recent indications such as aneuploidy screening and HLA matching. Since its inception the technology has attracted much attention: geneticists have expressed concerns about the robustness and validity of diagnosis based on single cell analysis, perinatologists were anxious about the effect of embryo biopsy on normal fetal development; and philosophers and ethicists have argued the cases for and against embryo selection. This article attempts to highlight the difficult choices and ethical challenges confronting patients and clinicians in an effort to balance the recognition of parental autonomy with the obligation of clinics to consider the welfare of any child born as a result of this treatment.

Child↗