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At least 19 recordsLinked to original sources
PubMed1990

Leukotriene D4 and E4 induce transmembrane signaling in human epithelial cells. Single cell analysis reveals diverse pathways at the G-protein level for the influx and the intracellular mobilization of Ca2+.

Leukotrienes are recognized as important mediators of the inflammatory process. Recently, increasing attention has been paid to the role of noninflammatory cells in the regulation of the inflammatory process. To further increase our knowledge of this matter we have, in the present study, investigated leukotriene-induced Ca2+ signaling, using a single cell technique in a human epithelial cell line, Intestine 407. It was evident that both LTD4 and LTE4, at physiological concentrations (10 nM), triggered rapid and pronounced cytosolic free Ca2+ transients, due to both influx across the plasma membrane and intracellular mobilization. Preincubation with pertussis toxin (1200 ng/ml) decreased the level of agonist-induced Ca2+ transients to an extent similar to that caused by depletion of extracellular Ca2+, suggesting that the toxin affected the influx but not the intracellular mobilization of Ca2+. Indeed, by using the Mn2+ quenching technique, it could be shown that pertussis toxin totally inhibited the influx of Ca2+. The fact that, even after pertussis toxin treatment, direct G-protein activation by AIF4- was still able to trigger a cytosolic free Ca2+ transient, indicates that, in these cells, G-proteins (GTP-binding proteins) that are insensitive to pertussis toxin are capable of mediating a Ca2+ signal. In order to test the idea that such G-proteins regulate mobilization of intracellular Ca2+ induced by LTD4 and LTE4, we electropermeabilized and preincubated the Intestine 407 cells with guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), let them reseal, and, after loading with fura2, investigated the effects on agonist-stimulated Ca2+ signaling. Electropermeabiization and resealing alone did not significantly affect the Ca2+ responses triggered by LTD4 or LTE4. Addition of GDP beta S, in the presence of extracellular Ca2+, reduced the Ca2+ responses by approximately 60-70%. In Ca2(+)-depleted medium, GDP beta S also impaired the LTD4-induced response by 65%, however, it had no effect on the Ca2+ response induced by LTE4. In conclusion, LTD4 and LTE4 trigger cytosolic free Ca2+ signaling in a human epithelial cell line by causing both an influx of Ca2+ and mobilization of intracellular Ca2+. The Ca2(+)-signaling mechanism appears to consist of dual pathways, since the influx is regulated by a pertussis toxin-sensitive G-protein, but, the mobilization of Ca2+ is not. Furthermore, our data suggest that the LTD4-induced mobilization is regulated by a pertussis toxin-insensitive G-protein whereas the LTE4-induced mobilization is relatively insensitive to both pertussis toxin and GDP beta S.

Aluminum↗
PubMed1967

Autoradiographic plaques for the detection of antibody formation to soluble proteins by single cells.

Single cells that synthesize antibody to soluble proteins can be detected and counted by an autoradiographic procedure. The method is based on the specific binding of I(131)-labeled antigen to the antidogy which has been produced by and has diffused around cells in an agarose medium and which has been precipitated by an antibody to gamma globulin. The antigen-binding zones appear as dark spots on high-speed x-ray film exposed to the dried agarose preparations.

Animals↗
PubMed1986

Autonomous early differentiation of neurons and muscle cells in single cell cultures.

The extent to which early differentiation of neurons and muscle cells is autonomous or governed by soluble factors released from other cells has been examined by following development of single cells plated alone in a simple, defined culture medium. The differentiation of electrical excitability and sensitivity to neurotransmitters of amphibian spinal neurons and trunk muscle in Xenopus embryos has already been described. For both cell types, differentiation in cultures containing relatively large numbers of dispersed cells parallels development in vivo, with respect to qualitative changes in membrane properties and the time course of development. Cell contacts are not required for this process. Here we show that the differentiation of membrane properties of single, isolated cells exhibits a similar set of changes, although muscle cells develop more slowly in some respects and all cells survive for a shorter period of time. The results suggest that the continued presence of specific extracellular differentiation-promoting factors is not required for these early steps of neuronal development, although a role for such factors in development of myocytes cannot be excluded. In contrast, survival factors secreted by other cells may be necessary to prolong the lifetimes of dissociated cells.

Acetylcholine↗
PubMed1991

Characterization of the m4 muscarinic receptor Ca2+ response in a subclone of PC-12 cells by single cell flow cytometry. Inhibition of the response by bradykinin.

We have studied Ca2+ mobilization mediated by the constitutively expressed muscarinic receptor on a subclone of PC-12 cells. The subclone, ACH2, was isolated with a flow cytometer by selection of single cells that exhibited a strong intracellular Ca2+ response to acetylcholine (ACh). Cell to cell heterogeneity of resting Ca2+ levels was markedly reduced in the subclone and homogeneity of the population response was also dramatically improved. ACH2 cells were highly sensitive to ACh and the Ca2+ response in all cells was blocked by muscarinic antagonists. Membranes from ACH2 exhibited muscarinic binding affinities which were not typical of M1, M2, or M3 receptors but were consistent with the profile of the putative m4 receptor. The same percentage of cells responded to ACh whether or not extracellular Ca2+ was reduced with EGTA, but the response was eliminated in all cells by preincubation with pertussis toxin. Thus, the constitutive m4 receptor on ACH2 cells is efficiently coupled to intracellular Ca2+ release by a pertussis toxin-sensitive mechanism. Stimulation of the ACH2 cells by bradykinin (BK) evoked a Ca2+ response in 90% of the cells. Prestimulation with BK diminished the magnitude of the muscarinic Ca2+ response but did not reduce the number of cells which responded to ACh. Inhibition was partially attributed to inhibition of a Ca2+ influx pathway in resting cells. Thus, the signaling mechanism coupled to the m4 muscarinic receptor can be inhibited by signals initiated by the BK receptor.

Acetylcholinesterase↗
PubMed1985

Cardiac Ca channel currents at the level of single cells and single channels.

Ca channels in excitable membranes are of great importance for many cellular functions. Modulation of these channels by voltage, neurotransmitters and drugs regulates Ca influx into the cell and thereby alters the functional state of the cell (7). For many years, quantitative analysis has been seriously complicated by experimental problems (see 2, 8, 10). There has been no preparation as suitable and no drug as specific for Ca channels, as the squid axon and TTX are for Na channels (10). The advent of the isolated adult heart cell and the development of powerful new electrophysiological methods, however, have opened up the possibility to record Ca channel activity at the level of single cells and single channels and to obtain evidence on molecular mechanisms of Ca channel gating and modulation.

Animals↗
PubMed2026

Cell type resolved MR based on brain single cell eQTLs corroborated by single cell RNA sequencing uncovers neuroimmune and vascular programs in intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) lacks effective neuroprotective therapies. We integrated cell type–resolved genetic inference with single-cell profiling to map putative causal programs and multicellular circuitry relevant to ICH. METHODS: Cis-eQTLs from eight human brain cell types were used as instruments for two-sample Mendelian randomization (MR), with an ICH meta-analysis from large biobanks and a stroke consortium as the outcome. Instruments were LD-pruned and restricted to strong variants (F > 10). Inverse-variance weighting (IVW) was the primary estimator, supported by robustness methods, heterogeneity/pleiotropy diagnostics, and false discovery rate control. Experimental validation used mouse collagenase ICH single-cell RNA-seq at 24 h (n = 3 sham; n = 3 ICH) with Seurat integration, composition testing, Slingshot pseudotime, and CellChat. An independent mouse cohort underwent qRT–PCR for selected genes. RESULTS: The ICH meta-analysis showed acceptable genomic control, supporting downstream MR. We identified 524 nominal gene–cell type associations, with a glia-weighted signal landscape. Enrichment implicated autophagy/mitophagy, antigen processing, cytoskeletal and vesicular trafficking, endothelial matrix–adhesion programs, ferroptosis, and myelin stress pathways. In mouse scRNA-seq, disease-associated microglia expanded with reciprocal loss of homeostatic microglia and increased neutrophils and T cells. Prioritized genes showed directional concordance; qRT–PCR confirmed ARPC3 and EIF2AK2 upregulation and TBCK and SPECC1 downregulation in ICH versus sham. Pseudotime supported a shift toward disease-associated microglial states, and CellChat indicated increased network interaction strength with microglia and endothelium as hubs. CONCLUSIONS: Cell type–specific MR combined with single-cell validation highlights neuroimmune and neurovascular programs in ICH and links genetic signals to state transitions and inferred intercellular communication.

Animals↗
PubMed2025

Differentiation latency and dormancy signatures define fetal liver hematopoietic stem cells at single-cell resolution.

Decoding the mechanisms governing the self-renewal of hematopoietic stem cells (HSCs) during their expansion in the fetal liver (FL) could unlock novel therapeutic strategies to expand transplantable HSCs, a long-standing challenge. To explore intrinsic and extrinsic regulation of FL-HSC self-renewal at single-cell resolution, we engineered a culture platform replicating the FL endothelial niche that supports the amplification of serially engraftable HSCs. Leveraging this platform together with single-cell index flow cytometry, live imaging, transplantation assays, and single-cell RNA sequencing, we demonstrate that differentiation latency, cell-division symmetry, and transcriptional signatures of biosynthetic dormancy are distinguishing properties of rare FL-HSCs capable of serial multilineage hematopoietic reconstitution. Our findings support a paradigm in which intrinsic programs and niche-derived signals together facilitate the symmetric self-renewal of FL-HSCs while delaying their active participation in hematopoiesis. Our study also provides a resource for future investigations into intrinsic and extrinsic signaling pathways governing FL-HSC self-renewal.

Hematopoietic Stem Cells↗
PubMed1992

Electrophysiology of nitrous oxide on cerebellar granule cells: a single-cell study.

We recorded 18 single cells in the granule cell layer of the cat. Each single cell was screened and identified as a granule cell based on a set of criteria derived from known electrophysiological properties of granule cells. We then monitored the effects of nitrous oxide on the spontaneous activities and the auditory responses of these cells. Nitrous oxide consistently caused a severe inhibition of spontaneous activities as well as responses to sound in these cells. Furthermore, the amplitudes of their action potentials decreased during the inhibition. Three of the 18 cells were subsequently injected intracellularly with HRP. All three were verified to be granule cells. In those granule cells we recorded intracellularly, nitrous oxide did not change the resting membrane potentials. The gradual decrease in the amplitudes of action potentials suggested that some of the mechanisms leading to the genesis of action potentials were being altered by nitrous oxide. It is also possible that nitrous oxide may act on synaptic transmission at a site located postsynaptically on the granule cells.

Acoustic Stimulation↗
PubMed1992

High frequency, heat treatment-induced inactivation of the phosphinothricin resistance gene in transgenic single cell suspension cultures of Medicago sativa.

One descendant of the Medicago sativa Ra-3 transformant T304 was analysed with respect to the somatic stability of the synthetic phosphinothricin-N-acetyltransferase (pat) gene which was used as a selective marker and was under the control of the 5'/3' expression signals of the cauliflower mosaic virus (CaMV) gene VI. In order to quantify gene instability, we developed a system for culturing and regenerating individual cells. Single cell suspension cultures derived from T304 and the ancestral non-transgenic M. sativa cultivar Ra-3, were established. The cells were regenerated into monoclonal calli. In transgenic calli, the phosphinothricin (Pt)-resistance phenotype was retained after more than 2 months of non-selective growth. In contrast, up to 12% of the suspension culture cells grown under nonselective conditions and at constant temperature (25 degrees C) lost the herbicide-resistance phenotype within 150 days. Surprisingly, a heat treatment (37 degrees C), lasting for 10 days, during the culture period resulted in an almost complete (95%) loss of the Pt resistance of the suspension culture cells. However, the frequency of cell division was identical in cultures grown under normal and heat treatment conditions. A biochemical test revealed that no phosphinothricin-N-acetyltransferase activity was present in heat treated, Pt-sensitive cells. The resistance level of the Pt-sensitive transgenic cells was equivalent to that of the wild-type cells. A PCR analysis confirmed the presence of the pat gene in heat treated, Pt-sensitive cells. From these results it is concluded that the Pt resistance gene was heat-inactivated at a high frequency in the M. sativa suspension cultures.

Acetyltransferases↗
PubMed1984

Removal of rat dendritic cells from single cell suspensions by passage through columns of Sephadex G-10.

Rat T lymphocytes require dendritic cells as accessory cells in order to respond to the mitogen sodium periodate. Passage of a lymph node cell suspension through a column of Sephadex G-10 reduced the mitogenic response by greater than 90%, despite a cell recovery of 75%. An even greater reduction in the response occurred after a second passage over Sephadex G-10; addition of purified dendritic cells restored the response. Lymph node cell suspensions and preparations enriched in dendritic cells were nearly depleted of these cells by passage over Sephadex G-10. After passage of lymph node cells over Sephadex G-10, a limited number of retained cells could be recovered; these included both dendritic cells and macrophages. Enumeration of macrophages in the various passed and retained fractions by non-specific esterase staining of smears confirmed that macrophages were effectively removed from lymph node cell suspensions by repeated passage over Sephadex G-10. Cell preparations that pass through Sephadex G-10 are therefore depleted of both dendritic cells and macrophages.

Animals↗
PubMed1983

Characteristics and requirements of the interaction between human monocytes and tumor cells on the single cell level.

Highly purified (97%-99%) and viable (99%) peripheral blood monocytes obtained by EDTA-reversible adherence to autologous-serum-precoated plastic surfaces could rapidly lyse a variety of tumor cells in a 3-4 hr 51Cr release assay. Using these monocytes as effectors, a short-term agarose/conjugate assay was utilized, permitting us to examine the interaction between fresh human monocytes and neoplastic target cells on a single cell level. That the tumor-bound effector cells were indeed monocytes was confirmed by employing the monocyte-specific monoclonal antibody 61D3, which stained 95%-99% of the mononuclear cells bound to conjugated and killed K562 tumor targets. The binding of monocytes to target cells appeared to be temperature dependent and was extremely rapid, reaching a plateau after 5 min at 30 degrees C. Our findings demonstrated for the first time that only a proportion of human blood monocytes can bind to a particular target cell and that only a fraction of the binding cells have the intrinsic potential to kill those neoplastic targets. The proportion of monocytes capable of binding and killing varies between individuals and also depends on the tumor cell used, indicating heterogeneity in the monocyte and tumor cell populations. The highest proportion of monocytes bind to the human erythromyeloid leukemia K562 cell line (13%-50%). The frequency of monocytes capable of killing K562 tumor cells is relatively low (7%-13%). The system described here should be useful to study the heterogeneity of mononuclear phagocytes and to analyze the molecular basis of the interaction between those effector cells and neoplastic target cells.

Antibodies, Monoclonal↗
PubMed1988

A complex mechanism of facilitation in pituitary ACTH cells: recent single-cell studies.

The transfer of information by chemical signals during complex biological processes can, with increasing frequency, be described in terms of interacting signal pairs. External signalling is rarely monolithic; rather, signal pairs are utilized in processes such as hormone secretion, neurotransmission, cell growth and differentiation. The dualism of external signalling often results in the occurrence of synergy. One signal appears to turn the cell on or off, and its synergistic partner increases cell responsiveness, providing gain control of the cellular response. ACTH release provoked by certain stressors arises from a synergistic interaction between two hypothalamic hormones: corticotropin releasing hormone (CRH) and a modulator such as vasopressin (AVP). The pituitary ACTH cell has been used to unravel the intracellular messenger equivalents of an external signal interaction that generates synergy. Research emphasizes the single cell approach. Direct measurements of intracellular free Ca2+ were performed using the Ca2+-sensitive fluorescent probe fura-2/AM and instrumentation for digital image processing. A reverse haemolytic plaque assay was used to measure cumulative ACTH release from single pituitary cells in culture. What is the physiological role of intracellular Ca2+ as a messenger? What are the feedforward and feedback relationships between major second messengers [cyclic AMP, diacylglycerol (DAG), inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)] and intracellular Ca2+? That is, how are individual messenger circuits "wired' within ACTH cells. Intracellular Ca2+ may act as a common signal into which interacting second messenger signals [cyclic AMP, Ins(1,4,5)P3, DAG] are transduced and integrated to govern ACTH release. A novel circuit of messenger pathways linked by Ca2+ is proposed as the intracellular basis for the synergistic interaction of CRH- and AVP-regulated ACTH release.

Adrenocorticotropic Hormone↗
PubMed2026

ELISA (Embedding-Linked Interactive Single-cell Agent): an interpretable hybrid generative Artificial Intelligence agent for expression-grounded discovery in single-cell genomics.

Translating single-cell RNA sequencing (scRNA-seq) data into mechanistic biological hypotheses remains a critical bottleneck, as agentic AI systems lack direct access to transcriptomic representations while expression foundation models remain opaque to natural language. Here, we introduce ELISA (Embedding-Linked Interactive Single-cell Agent), an interpretable framework that unifies single-cell generative pretrained transformer expression embeddings with biomedical bidirectional encoder representations from transformers-based semantic retrieval and large-language model (LLM)-mediated interpretation for interactive single-cell discovery. An automatic query classifier routes inputs to gene marker scoring, semantic matching, or reciprocal rank fusion pipelines depending on whether the query is a gene signature, natural language concept, or mixture of both. Integrated analytical modules perform pathway activity scoring across 60+ gene sets, ligand-receptor interaction prediction using 280+ curated pairs, condition-aware comparative analysis, and cell-type proportion estimation, all operating directly on embedded data without access to the original count matrix. Benchmarked across six diverse scRNA-seq datasets spanning inflammatory lung disease, pediatric and adult cancers, organoid models, healthy tissue, and neurodevelopment, ELISA significantly outperforms CellWhisperer, a classical lexical retriever (BM25), and a random baseline in cell type retrieval (combined permutation test, $p < 2\times 10^{-5}$ for each), with particularly large gains on gene-signature queries (Cohen's $d = 5.98$ for mean reciprocal rank). ELISA replicates published biological findings (mean composite score 0.88), and generates candidate hypotheses through grounded LLM reasoning, bridging the gap between transcriptomic data exploration and biological discovery.

Generative Artificial Intelligence↗
PubMedDate not supplied

Effect of antiepileptic drugs on growth of murine lymphoid tumor cells in single-cell culture.

The effect of three commonly used antiepileptic drugs (AEDs), phenytoin (PHT), carbamazepine (CBZ), and valproate (VPA), on the growth of lymphoid tumor cells was assessed in vitro. A single-cell culture method was used to determine growth rates by direct visualization. The amount of free drug was determined by ultrafiltration to ascertain its correlation to therapeutic drug levels. VPA slowed the growth of B-myeloma (FO) and T-lymphoma (AKR-1) cells significantly within the range of therapeutic drug levels. CBZ and PHT likewise inhibited cell growth in both lineages but at two to four times the therapeutic level of free drug. CBZ was shown to have long-term effects on FO and AKR-1 cells, demonstrated by the reduced growth rates of cloned lines for 2-3 months after drug removal. Cloned sublines of myeloma cells secreting lambda light chain (J558L) treated with CBZ or PHT had a higher frequency of lambda light chain secretion loss mutations than the nontreated parent line.

Animals↗
PubMed1984

Mechanism of cell-mediated cytotoxicity at the single cell level. VI. Direct assessment of the cytotoxic potential of human peripheral blood non-lytic effector-target cell conjugates.

Single cell cytotoxicity assays reveal that a large percentage of lymphocytes are unable to kill attached targets in a 4- to 18-hr assay. Additional signals (in the form of lectin or anti-target antibody) delivered to target-bound lymphocytes enable these previously non-lytic lymphocytes to kill attached target cells. This finding was obtained by using a modification of the single cell assay, in which lectin or target cell antibody is incorporated into agarose with preformed lymphocyte-target conjugates. Human peripheral blood lymphocytes (PBL) or Percoll density gradient-enriched large granular lymphocytes (LGL) were used as effector cells in natural killer (NK), antibody-dependent cellular cytotoxicity (LDCC) assay systems. The targets used were NK-sensitive K562 and Molt-4 and NK-insensitive Raji. Several findings were made in the modified single cell assay, namely a) the frequency of cytotoxic NK or ADCC effector cells was not augmented, suggesting that the initial trigger was sufficient for lytic expression in these instances. Furthermore, these results showed that the NK-sensitive targets used do not bind nonspecifically to the LDCC effector cells. K562 coated with Con A, however, serve as LDCC targets. b) The frequency of two target conjugate lysis by NK/K effectors was not augmented by Con A. These results suggest that Con A does not potentiate the killing of multiple targets bound to a single cytotoxic lymphocyte. c) Although conjugates formed between LGL or PBL and NK-insensitive Raji are non-lethal, significant lysis was observed when these conjugates were suspended in Con A or antibody agarose. These results demonstrate that Raji bind to cytotoxic NK, K, and LDCC effector cells, but are lysed only when the appropriate trigger is provided. d) The cytotoxic potential of non-lytic conjugates appears to lie within the low density Percoll fraction, although the high density lymphocytes are able to nonlethally bind to targets. Altogether the results demonstrate that target recognition and/or binding by the effector cells is a distinct event from the trigger or lytic process. The implications of these findings are discussed.

Animals↗