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At least 415 records · Page 23Linked to original sources
PubMed2000

Expression of T-cell receptor beta-chain mRNA and protein in gamma/delta T-cells from euthymic and athymic rats: implications for T-cell lineage divergence.

The relationship between alpha/beta and gamma/delta T-cell lineages was studied in rats using RT-PCR analysis of TCRbeta transcripts in gamma/delta T-cell hybridomas and an intracellular staining technique to detect TCRbeta protein in primary gamma/delta T-cells. We report the presence of functional TCRbeta transcripts in 2/9 gamma/delta T-cell hybridomas. About 15% of peripheral gamma/delta T-cells and thymocytes also express TCRbeta protein, giving a minimum estimate for successful Tcrb rearrangement based on ex vivo single cell analysis. In athymic rats, gamma/delta T-cells expressing intracellular beta protein are present but at a lower frequency than in euthymic controls, suggesting that in the thymus, more gamma/delta T-cell precursors pass through a stage where functional beta rearrangement has occurred than in extrathymic sites. Analysis of TCR expression in purified transitory immature CD4-8+ (iCD8SP) thymocytes and their spontaneously developing CD4+8+ (DP) progeny showed that TCRy mRNA is expressed in iCD8SP cells but not in their immediate DP progeny that reinitiate RAG-I transcription and commence alpha/betaTCR expression. We conclude that rat gamma/delta T cells can separate from the alpha/beta lineage after TCRbeta expression, but not after entry into the DP compartment.

Animals↗
PubMed1998

A simple combined microdissection and aspiration device for the rapid procurement of single cells from clinical peripheral blood smears.

Molecular analysis of cells from cytology specimens can help to establish a diagnosis in ambiguous cases. However, mutations in heterogeneous samples might not be detected because of the diluting effect of DNA from normal background cells. Even if a mutation were detected, it could not be traced back to a specific cell type. Molecular analysis of single cells circumvents this problem. Both mechanical and laser assisted methods have been described for the selective procurement of cells from histology slides; however, they have the drawback of either being technically demanding or expensive. Furthermore, it is nuclear whether they can be applied to cytology specimens. Finally, few of these techniques are able to procure single cells. Therefore, we developed a simplified combined microdissection and aspiration device for the rapid procurement of single cells from clinical cytology specimens. The principle of this device, called the cytopicker, is the combination of the microdissection tool, a steel cannula, with the aspiration tool, a glass capillary connected to a vacuum, into one device. Steel cannulae are optimal for microdissection of cells from the hard matrix of cytology specimens but aspirate poorly. On the other hand, glass capillaries are suboptimal for dissecting but aspirate very well. Combining both tools into one by inserting the capillary into the cannula allows optimal dissection using the cannula (with the glass capillary with-drawn and thus protected), followed by optimal aspiration using the capillary (after being advanced through the cannula). All movements of the device are controlled by just one micromanipulator, making the cytopicker inexpensive to manufacture. The cytopicker can rapidly and simply procure single cells, such as lymphoblasts, from cytology specimens, such as peripheral blood smears. DNA from these cells can be amplified by PCR. However, precautions have to be taken to avoid contamination. Once improved further, the cytopicker might facilitate molecular analysis in the routine cytology laboratory.

Blood Specimen Collection↗
PubMedDate not supplied

Automated morphometric endothelial analysis combined with video specular microscopy.

Contact and noncontact specular microscopes were tested in combination with a highly sensitive video residual-light camera and with the Kontron IPS (Image Processing System). The video information is recorded with a video recorder or with the IPS frame grabber, a digital image storage system. Examples of experimental and clinical applications are demonstrated. Nine linear and structural parameters are analyzed. Two endothelium-typical parameters are presented, which allow topological as well as hexagonal-specific geometric endothelial single-cell analysis.

Animals↗
PubMed2003

Polyclonal proliferation and apoptosis of CCR5+ T lymphocytes during primary human immunodeficiency virus type 1 infection: regulation by interleukin (IL)-2, IL-15, and Bcl-2.

We measured apoptosis of subsets of T lymphocytes by single-cell analysis of caspase activation, to confirm high turnover of chemokine receptor CCR5(+) T cells in subjects with acute, primary human immunodeficiency virus type 1 (HIV-1) infection (PHI). High levels of spontaneous apoptosis, consisting mainly of CD8(+) T lymphocytes, were closely associated with increases in the activation markers Ki-67, CD38, and the HIV coreceptor CCR5 and with decreases in Bcl-2 and the interleukin (IL)-7 receptor at the single-cell level. Increased expression of Ki-67 and CCR5 ex vivo, as well as increased apoptosis, was seen in all T cell receptor beta-chain variable region (TCRBV) subfamilies studied. The addition of IL-2 or IL-15, but not IL-7, significantly inhibited caspase activation, increased Bcl-2 expression, and rapidly initiated proliferation in vitro of CD8(+) T cells expressing CCR5 and multiple TCRBV subfamilies. Furthermore, IL-15 receptor alpha-chain messenger RNA levels were increased in peripheral blood mononuclear cells during PHI. These results suggest that CCR5(+)Ki-67(+)Bcl-2(dim) activated T cells generated during PHI traffic via blood to tissue sites, where the cells may survive and/or further proliferate under the local influence of IL-2 or IL-15. Understanding cytokine effects on CCR5(+) T cells will be important in understanding chronic HIV-1 replication and pathogenesis.

Adult↗
PubMed2004

Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications.

Analysis of protein phosphorylation with flow cytometric techniques has emerged as a powerful tool in the field of immunological signaling, allowing cellular subsets in complex populations to be analyzed accurately and rapidly. In this review, we examine the development of phospho-epitope, or phospho-specific, flow cytometry and the premises upon which the technique is based. Phospho-specific flow cytometry is compared to traditional biochemical methods, and its advantages, such as single cell analysis, multiparameter data acquisition, rapid protocols, and the ability to analyze rare cell subsets, are detailed. We also discuss the many technical considerations that must be addressed when developing new antibodies or analyzing new epitopes including antigen accessibility, stability of the phospho-epitope, fluorophore selection, surface phenotype integrity, and antibody suitability for staining epitopes inside fixed and permeabilized cells. The methods that have been used to date are described in light of these technical considerations. The importance of developing bioinformatic platforms in parallel with these techniques is emphasized due to the large, multiparameter datasets that are rapidly accumulated and which require more efficient data viewing and complex clustering methods than currently available for flow cytometric data. Finally, we discuss the potential clinical applications of phospho-specific flow cytometry in analyzing immune cell development and antigen-specific immune responses, as well as pharmacodynamic profiling of disease states or drug efficacy and specificity against particular signaling proteins.

Animals↗
PubMed2006

Charging it up: global analysis of protein phosphorylation.

Protein phosphorylation affects most, if not all, cellular activities in eukaryotes and is essential for cell proliferation and development. An estimated 30% of cellular proteins are phosphorylated, representing the phosphoproteome, and phosphorylation can alter a protein's function, activity, localization and stability. Recent studies for large-scale identification of phosphosites using mass spectrometry are revealing the components of the phosphoproteome. The development of new tools, such as kinase assays using modified kinases or protein microarrays, enables rapid kinase substrate identification. The dynamics of specific phosphorylation events can now be monitored using mass spectrometry, single-cell analysis of flow cytometry, or fluorescent reporters. Together, these techniques are beginning to elucidate cellular processes and pathways regulated by phosphorylation, in addition to global regulatory networks.

Animals↗
PubMed2026

Construction and validation of a β-hydroxybutyrylation-related molecular model for predicting prognosis of papillary thyroid carcinoma.

BACKGROUND: Papillary thyroid carcinoma (PTC) usually has a favorable prognosis, yet a subset of patients develops persistent, recurrent, or biologically aggressive disease. The clinical relevance of lysine β-hydroxybutyrylation (Kbhb)-related transcriptional programs in PTC remains unclear. Accordingly, this study aimed to characterize Kbhb-related molecular heterogeneity in PTC, construct a prognostic signature, and explore its association with the tumor microenvironment (TME). METHODS: Transcriptomic and clinical data from PTC samples within The Cancer Genome Atlas Thyroid Carcinoma (TCGA-THCA) cohort were analyzed to identify Kbhb-related differentially expressed genes (DEGs), define molecular subtypes, construct a prognostic signature, and characterize tumor microenvironmental features. Single-cell RNA-sequencing data from PTC were further used to explore the cellular distribution of representative genes. RESULTS: We identified 51 Kbhb-related DEGs in PTC and defined two Kbhb molecular subtypes. The Kbhb_C2 subtype showed shorter progression-free interval (PFI) and a more immune- and stroma-enriched microenvironment. A six-gene prognostic signature comprising TARID, CDSN, PIMREG, KLRC1, SYT13, and NPR3 was then established. High-risk patients had significantly worse PFI in the full, training, and testing cohorts, with 1-, 3-, and 5-year areas under the curve (AUCs) of 0.715, 0.793, and 0.771, respectively, in the full cohort. High-risk tumors also exhibited higher stromal, immune, and ESTIMATE scores, altered immune infiltration, and increased expression of multiple immune checkpoint molecules. Single-cell analysis confirmed distinct cell-type-specific expression patterns of representative genes. CONCLUSIONS: Kbhb-related transcriptional programs define clinically relevant molecular heterogeneity in PTC and are closely associated with prognosis and TME remodeling. The identified six-gene signature provides a biologically interpretable framework for risk stratification in PTC.

Papillary thyroid carcinoma (PTC)↗
PubMed2006

Cultured smooth muscle cells of the human vesical sphincter are more sensitive to histamine than are detrusor smooth muscle cells.

OBJECTIVES: To compare histamine receptor expression in cultured smooth muscle cells from the human detrusor and internal sphincter using receptor-specific agonists. METHODS: Smooth muscle cells from the bladder dome and internal sphincter were cultured from 5 male patients undergoing cystectomy for bladder cancer therapy. Calcium transients in cells stimulated with carbachol, histamine, histamine receptor 1 (H1R)-specific heptanecarboxamide (HTMT), dimaprit (H2R), and R-(alpha)-methylhistamine (H3R) were measured by calcium imaging. Histamine receptor proteins were detected by Western blot analysis and immunocytochemistry. RESULTS: H1R, H2R, and H3R expression was found in tissue and cultured cells. Carbachol stimulated equal numbers of detrusor and sphincter cells (60% and 51%, respectively). Histamine stimulated significantly more cells than carbachol in detrusor (100%) and sphincter (99.34%) cells. Calcium responses to carbachol in detrusor and sphincter cells were comparable and did not differ from those to histamine in detrusor cells. However, histamine and specific agonists stimulated more sphincter cells than did carbachol (P <0.001), and the calcium increase was greater in sphincter cells than in detrusor cells. Single cell analysis revealed comparable H2R responses in detrusor and sphincter cells, but H1R and H3R-mediated calcium reactions were significantly greater in sphincter cells. CONCLUSIONS: Histamine very effectively induces calcium release in smooth muscle cells. In sphincter cells, histamine is even more effective than carbachol regarding the number of reacting cells and the intracellular calcium increase. Some of the variability in the outcome of antihistaminic interstitial cystitis therapies might be caused by the ineffectiveness of the chosen antihistaminic or unintentional weakening of sphincteric function.

Calcium↗
PubMed1997

Subcellular analysis of Ca2+ homeostasis in primary cultures of skeletal muscle myotubes.

Specifically targeted aequorin chimeras were used for studying the dynamic changes of Ca2+ concentration in different subcellular compartments of differentiated skeletal muscle myotubes. For the cytosol, mitochondria, and nucleus, the previously described chimeric aequorins were utilized; for the sarcoplasmic reticulum (SR), a new chimera (srAEQ) was developed by fusing an aequorin mutant with low Ca2+ affinity to the resident protein calsequestrin. By using an appropriate transfection procedure, the expression of the recombinant proteins was restricted, within the culture, to the differentiated myotubes, and the correct sorting of the various chimeras was verified with immunocytochemical techniques. Single-cell analysis of cytosolic Ca2+ concentration ([Ca2+]c) with fura-2 showed that the myotubes responded, as predicted, to stimuli known to be characteristic of skeletal muscle fibers, i.e., KCl-induced depolarization, caffeine, and carbamylcholine. Using these stimuli in cultures transfected with the various aequorin chimeras, we show that: 1) the nucleoplasmic Ca2+ concentration ([Ca2+]n) closely mimics the [Ca2+]c, at rest and after stimulation, indicating a rapid equilibration of the two compartments also in this cell type; 2) on the contrary, mitochondria amplify 4-6-fold the [Ca2+]c increases; and 3) the lumenal concentration of Ca2+ within the SR ([Ca2+]sr) is much higher than in the other compartments (> 100 microM), too high to be accurately measured also with the aequorin mutant with low Ca2+ affinity. An indirect estimate of the resting value (approximately 1-2 mM) was obtained using Sr2+, a surrogate of Ca2+ which, because of the lower affinity of the photoprotein for this cation, elicits a lower rate of aequorin consumption. With Sr2+, the kinetics and amplitudes of the changes in [cation2+]sr evoked by the various stimuli could also be directly analyzed.

Aequorin↗
PubMed2003

Studies on neuronal apoptosis in primary forebrain cultures: neuroprotective/anti-apoptotic action of NR2B NMDA antagonists.

While the role of apoptosis in neuronal injury is continually being re-defined, approaches to intervene in the progression of apoptotic injury have been documented to provide neuroprotection against a variety of insults. The present studies were undertaken to systematically study the effects of certain neuroprotective agents against neuronal apoptosis mediated by staurosporine (ST). ST (0.01-5 micro M) produced a dose-related apoptotic injury (as characterized by cellular morphology, 'Comet' assay analysis [single cell gel electrophoresis] and caspase-3 activation) in primary cultures of forebrain neurons. ST significantly increased caspase-3 activity. The NMDA receptor subtype non-selective antagonist dizocilpine [(+) MK-801; 0.1-50 micro M] and a novel sodium channel blocker RS100642 (1.0-250 micro M) had no significant effects against ST-induced neurotoxicity. Conversely, NR2B-selective NMDA receptor antagonists CGX-1007 (0.01-50 micro M) and ifenprodil (0.01-50 micro M) provided dose-dependent neuroprotection against ST-induced neurotoxicity (as measured by neuronal viability and comet assay analysis). CGX-1007 had no significant effect on ST-induced caspase-3 activity; however, ifenprodil did block activation of caspase-3. These studies demonstrate that NR2B NMDA receptor antagonists are anti-apoptotic and may mediate their action via mechanism(s) that are dependent or independent of caspase-3 activation.

Animals↗
PubMed2026

Hepatic metabolic adaptation to endurance exercise: temporal and sex differences by multiomics integration and validation.

BACKGROUND: Although endurance exercise benefits liver health, sex-specific adaptive trajectories remain unclear. This study mapped dynamic liver adaptation in males and females during prolonged training and identified underlying molecular programs. METHODS: Using publicly available time-resolved liver multi-omics data generated by the Molecular Transducers of Physical Activity Consortium (MoTrPAC), we established a computational pipeline for differential analysis of transcriptomic, proteomic, phosphoproteomic, and metabolomic data with FDR correction, followed by FGSEA pathway enrichment. Kinase activities were inferred through ortholog mapping and PhosphoSitePlus. Cross-omics co-expression networks were constructed using WGCNA and topological overlap to link omics features with physiological phenotypes. For experimental validation, liver tissues were collected from endurance-trained Sprague-Dawley rats, and key nodes were confirmed by Western blotting, qRT-PCR, and immunofluorescence/immunohistochemical staining. Public scRNA-seq data were further integrated to map multi-omics signals to single-cell resolution and assess functional changes in specific cell types. RESULTS: The hepatic response to exercise stress was stage-specific, shifting from early transcriptional activation to later proteomic and metabolic remodeling. Multi-omics integration revealed distinct sex-associated adaptive trajectories: males were more strongly associated with energy metabolism, redox-related programs, and amino acid/organic acid catabolism, whereas females showed prominent membrane lipid remodeling, proteostasis -related programs, and mitochondrial/ribosomal translational features. Single-cell analysis showed that tissue remodeling occurred without major lineage turnover, instead involving altered communication among pre-existing cell communities. Validation of PPP1R3G identified a protein-dominant exercise-responsive marker, supporting the contribution of post-transcriptional or protein-level regulation. CONCLUSIONS: Hepatic adaptation to endurance stress follows a cross-omics evolutionary pattern with sex-specific reprogramming of energy supply and homeostatic maintenance. This time-resolved framework clarifies how exercise improves liver function and supports sex-oriented metabolic interventions and therapeutic target discovery.

Animals↗
PubMed1999

Peripheral T lymphocyte cytokine profile (IFNgamma, IL-2, IL-4) and CD30 expression/release during measles infection.

BACKGROUND AND OBJECTIVE: Measles virus infection (MVI) has been reported to be characterized by an imbalanced Th(1/2)-type cytokine profile. CD30 has been proposed as a receptor preferentially associated with the Th(0/2)-type cytokine pattern. The aim of this study was therefore to define the peripheral T lymphocyte cytokine profile and to test which CD30 expression pattern it was associated with in MVI. DESIGN AND METHODS: The design of the study was a prospective evaluation with comparative analysis. The serum levels of the soluble form of CD30 (sCD30) were determined at diagnosis and at weekly intervals up to 4 weeks, using an ELISA, in 23 males (median age 19), who developed MVI while serving in the Italian army and who were admitted to the Infectious Disease Unit of the Military Hospital in Padua. In 10 of the patients at diagnosis we studied the lymphoid immunophenotype and, after non-specific ex vivo stimulation, the expression of IFNgamma, IL-2 and IL-4 by peripheral T cells using flow cytometry single cell analysis. In 3 patients such evaluations were also performed 7 weeks later. RESULTS: At diagnosis, we found (i) reduction of IFNgamma+/CD4+ T cells (p=0.048 vs controls) in the absence of substantial variation of IL-2+ and IL-4+ T cells (p=ns vs controls); (ii) expansion of CD30+/ CD4+ and CD30+/CD8+ T cell subsets (p<0.01 vs controls); (iii) high sCD30 values (median 61 U/mL; p<0.001 vs controls); (iiii) a context of lymphopenia (0. 728+/-0.292 lymph x10(9)/L). sCD30 remained elevated up to 4 weeks from MVI onset [median values 53, 49, 50, 34 U/mL after 1, 2, 3 and 4 weeks, respectively (p=ns between different time points)]. In 3 patients tested 7 weeks after diagnosis, we still observed decreased IFNgamma production by CD4+ and CD8+ T cells (p=0.05 and <0.01, respectively vs controls) and reduction of CD4+ and CD8+/IL-2+ T cells (p<0.01). INTERPRETATION AND CONCLUSIONS: MVI was characterized by featuresof inadequate Th/Tc(1) activation associated with increased circulating CD30+ T cells and elevated sCD30 levels, supporting a correlation between Th/Tc status and CD30 expression/release pattern in vivo.

Adult↗
PubMed2005

Molecular diversity of voltage-gated sodium channel alpha subunits expressed in neuronal and non-neuronal excitable cells.

In order to investigate the role of molecular diversity of voltage-activated sodium channel alpha-subunits in excitability of neuronal and non-neuronal cells, we carried out patch-clamp recordings and single-cell RT-PCR on two different types of mammalian excitable cells i.e. hippocampal neurons and non-neuronal utricular epithelial hair cells. In each cell type, multiple different combinations of sodium channel alpha-subunits exist from cell to cell despite similar sodium current properties. The mRNA isoforms, Nav1.2 and Nav1.6, are the most frequently detected by single cell analysis in the two cell types while Nav1.3 and Nav1.7 are also moderately expressed in embryonic hippocampal neurons and in neonatal utricular hair cells respectively. By investigating the particular alternate splice isoforms of Nav1.6 occurring at the exon 18 of the mouse orthologue SCN8A, we revealed that this subunit co-exist in the two cell types under different alternative spliced isoforms. The expression of non-functional isoforms of Nav1.6 in utricular epithelial hair cells excludes the involvement of this subunit in supporting their excitability. Thus, from a functional point of view, the present results suggest that, at the single cell level, both neuronal and non-neuronal excitable cells expressed different and complex patterns of sodium channel gene transcripts but this diversity alone cannot explain the sodium current properties of these cell types.

Alternative Splicing↗
PubMed2001

Assessment of clonality of rosetting T lymphocytes in Hodgkin's disease by single-cell polymerase chain reaction: detection of clonality in a polyclonal background in a case of lymphocyte predominance Hodgkin's disease.

Rosetting of CD4+ T cells around the neoplastic Hodgkin and Reed-Sternberg (H&RS) cells is a characteristic feature of Hodgkin's disease (HD). To answer the question whether this phenomenon is solely due to chemokine-mediated attraction of T cells or whether the rosetting T cells in addition recognize antigens presented by the H&RS cells, we examined the T cells adherent to H&RS cells. Cells from five cases of HD [four classic HD and one lymphocyte-predominant (LP) HD] were examined by single-cell analysis for the T-cell receptor (TCR) gamma gene. Between 5 and 17 rosettes containing one to ten rosetting lymphocytes and the corresponding H&RS cells were amplified in separate plastic tubes. Of the resulting 119 TCRgamma polymerase chain reaction (PCR) products, 87 were sequenced. While no evidence of a clonal expansion was obtained in the lymph nodes from four of five patients with classic HD, clonal TCRgamma sequences were found in the lymph node from the patient within LPHD in two independent experiments analyzing seven and ten different rosetting complexes, respectively. Of 13 products, 11 showed identical Vgamma9 sequences. Unrelated products were found in all other TCRgamma family subgroups in this case. Single H&RS cells picked as controls were negative for TCRgamma rearrangements. Our results demonstrate that clonal proliferations on a polyclonal background can occur among the T cells forming rosettes with Hodgkin cells and lend support to the view that Hodgkin cells may also function as cells presenting antigens to the adhering T cells.

Adolescent↗
PubMed1998

Current knowledge about fetal blood cells in the maternal circulation.

The aim of this review is to summarize the current status and future prospects of noninvasive prenatal genetic analysis by the isolation of fetal cells from the maternal circulation. The presence of fetal cells in maternal blood is no longer considered controversial. A variety of cell separation methods recover fetal cells from maternal blood; these differ in cost-effectiveness and required expertise. Fetal cell types that are useful for prenatal diagnosis are terminally differentiated and are unlikely to persist post-partum. Most investigators are isolating trophoblast sprouts, nucleated erythrocytes, or both from maternal blood. Advances in the understanding of cell surface and cytoplasmic protein expression have translated into better and more specific fetal cell identification. Fetal cells, once identified, are being screened for the presence of aneuploidy using chromosome-specific probes on interphase nuclei. Significant progress in single gene and single cell analysis has expanded the diagnostic possibilities for noninvasive fetal testing. Although fetal cells are generally rare in maternal blood samples, they appear to be more common when the fetus has trisomy 21. This is beneficial for clinical diagnosis. Furthermore, a large fetomaternal transfusion occurs at the time of labor and delivery in all pregnant women. This may establish fetal cell microchimerism in the mother, which may be implicated in the subsequent development of diseases such as scleroderma that are more common in women. The study of fetal cells in maternal blood, while technically challenging, provides a unique opportunity to explore the immunobiology of pregnancy.

Antibodies, Monoclonal↗
PubMed1990

Quantitative amino acid analysis of individual snail neurons by open tubular liquid chromatography.

A method is described for the determination of amino acids in individual cells. The amino acids are derivatized with naphthalene-2,3-dicarboxaldehyde and then analyzed by open tubular liquid chromatography with amperometric detection. The total volume present after derivatization is approximately 25 nL. It was possible to quantitatively determine 17 amino acids in three different neurons of the land snail Helix aspersa. Quantitation was accomplished through the use of two internal standards and a calibration curve. Alanine was found to be the most abundant amino acid by about a factor of 2 over glutamine in all three types of neurons. This method has the advantages of sensitivity in the attomole range (5 X 10(-9) M) and selectivity for a specific class of compounds and is at least as reliable as other methods used for single-cell analysis.

Amino Acids↗
PubMed2004

Early single cell bifurcation of pro- and antiapoptotic states during oxidative stress.

In a population of cells undergoing oxidative stress, an individual cell either succumbs to apoptotic cell death or maintains homeostasis and survives. Exposure of PC-12-D(2)R cells to 200 microm hydrogen peroxide (H(2)O(2)) induces apoptosis in about half of cells after 24 h. After 1-h exposure to 200 microm H(2)O(2), both antiapoptotic extracellular regulated kinase (ERK) phosphorylation and pro-apoptotic Ser-15-p53 phosphorylation are observed. Microarray and real-time PCR assays of gene expression after H(2)O(2) exposure identified several transcripts, including egr1, that are rapidly induced downstream of ERK. Single cell analysis of egr1 induction and of phospho-ERK and phospho-p53 formation revealed the presence of two distinct cellular programs. Whereas the proportion of cells activating ERK versus p53 at 1 h depended on H(2)O(2) concentration, individual cells showed exclusively either phospho-p53 formation or activation of ERK and egr1 induction. Exposure to H(2)O(2) for 1 h also elicited these two non-overlapping cellular responses in both dopaminergic SN4741 cells and differentiated postmitotic PC-12-D(2)R cells. Repressing p53 with pifithrin-alpha or small interfering RNA increased ERK phosphorylation by H(2)O(2), indicating that p53-dependent suppression of ERK activity may contribute to the bi-stable single cell responses observed. By 24 h, the subset of cells in which ERK activity was suppressed exhibit caspase 3 activation and the nuclear condensation characteristic of apoptosis. These studies suggest that the individual cell rapidly and stochastically processes the oxidative stress stimulus, leading to an all-or-none cytoprotective or pro-apoptotic signaling response.

Animals↗
PubMed2006

Protein transduction as a means of effective manipulation of Cdc42 activity in primary T cells.

The Rho family GTPase Cdc42 is a critical regulator of cellular polarization from yeast to man. An analysis of its function in T cell activation is therefore of interest. This analysis poses two substantial challenges, similar to the analysis of many other critical T cell signaling intermediates. First, Cdc42 is required for development and cell survival, necessitating short-term manipulation of its activity. Second, Cdc42 is likely involved in multiple signaling pathways, requiring approaches to distinguish multiple roles. To address these challenges, we first determined and quantified spatio-temporal patterns of Cdc42 activity using live cell video fluorescence microscopy. This generates hypotheses at which times and locations Cdc42 might play possibly distinct roles. Second and as the focus of this manuscript, we employed protein transduction to manipulate Cdc42 activity for the generation of causality. Protein transduction allows such manipulation to be short-term, quantitative, and with multiple reagents. Here, we characterize uptake, retention, and subcellular distribution of protein transduction reagents. We describe how a more quantitative single cell analysis of Cdc42 activity provides superior distinction between experimental conditions. And we show how we have used dose responses of the protein transduction reagents to minimize side effects while retaining efficacy. We suggest that our strategy is an important complement to more established techniques to study protein function in primary T cells, in particular in the investigation of signaling intermediates that are essential for cell survival and regulate multiple aspects of T cell activation.

Animals↗