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M Poenie

Publications and source records attributed to M Poenie.

28 records · Page 2Linked to original sources
PubMed1986

Calcium rises abruptly and briefly throughout the cell at the onset of anaphase.

Continuous measurement and imaging of the intracellular free calcium ion concentration ([Ca2+]i) of mitotic and interphase PtK1 cells was accomplished with the new fluorescent Ca2+ indicator fura-2. No statistically significant difference between basal [Ca2+]i of interphase and mitotic cells was detected. However, mitotic cells showed a rapid elevation of [Ca2+]i from basal levels of 130 nM to 500 to 800 nM at the metaphase-anaphase transition. The [Ca2+]i transient was brief, lasting approximately 20 seconds and the elevated [Ca2+]i appeared uniformly distributed over the entire spindle and central region of the cell. The close temporal association of the [Ca2+]i transient with the onset of anaphase suggests that calcium may have a signaling role in this event.

Anaphase↗
PubMed1986

Large changes in intracellular pH and calcium observed during heat shock are not responsible for the induction of heat shock proteins in Drosophila melanogaster.

Heat shock caused significant changes in intracellular pH (pHi) and intracellular free calcium concentration [( Ca2+]i) which occurred rapidly after temperature elevation. pHi fell from a resting level value at 25 degrees C of 7.38 +/- 0.02 (mean +/- standard error of the mean, n = 15) to 6.91 +/- 0.11 (n = 7) at 35 degrees C. The resting level value of [Ca2+]i in single Drosophila melanogaster larval salivary gland cells was 198 +/- 31 nM (n = 4). It increased approximately 10-fold, to 1,870 +/- 770 nM (n = 4), during a heat shock. When salivary glands were incubated in calcium-free, ethylene glycol-bis(beta-aminoethyl ether)-N,N',N'-tetraacetic acid (EGTA)-buffered medium, the resting level value of [Ca2+]i was reduced to 80 +/- 7 nM (n = 3), and heat shock resulted in a fourfold increase in [Ca2+]i to 353 +/- 90 nM (n = 3). The intracellular free-ion concentrations of Na+, K+, Cl-, and Mg2+ were 9.6 +/- 0.8, 101.9 +/- 1.7, 36 +/- 1.5, and 2.4 +/- 0.2 mM, respectively, and remained essentially unchanged during a heat shock. Procedures were devised to mimic or block the effects of heat shock on pHi and [Ca2+]i and to assess their role in the induction of heat shock proteins. We report here that the changes in [Ca2+]i and pHi which occur during heat shock are not sufficient, nor are they required, for a complete induction of the heat shock response.

Animals↗
PubMed1985

A new generation of Ca2+ indicators with greatly improved fluorescence properties.

A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.

Aminoquinolines↗
PubMed1985

Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths.

Free Ca2+ concentrations in the cytosol of individual small cells can be recorded with a new fluorescent Ca2+ indicator, "fura-2", and a fluorescence microscope modified to chop rapidly between two wavelengths of excitation. Both fura-2 and its Ca2+ complex fluoresce strongly, but their excitation peaks differ in wavelength. Alternation between the two preferred wavelengths allows assessment of the ratio of Ca2+-bound dye to free dye and hence cytosolic free Ca2+. This ratio measurement largely cancels out the effects of cell thickness, dye content, or instrumental efficiency, uncertainties that can jeopardize measurements at single wavelengths. We describe instrumentation that supplies rapidly alternating excitation wavelengths to either a standard cuvet or a fluorescence microscope. Its use is illustrated by experiments showing changes in cytosolic [Ca2+] accompanying activation of human platelets in suspension or single mouse thymocytes on the microscope.

Animals↗
PubMedDate not supplied

Changes of free calcium levels with stages of the cell division cycle.

Although the regulation of events in the cell division cycle by calcium or other cations has been the subject of much interest and speculation, experimental studies have been hampered by the difficulty of measuring submicromolar intracellular free calcium concentrations ([Ca2+]i) over an entire cell cycle. We now describe experiments using a new fluorescent calcium chelator, fura-2 (see Fig. 1c for structure), for continuous measurement of [Ca2+]i from fertilization through the first cleavage of individual eggs of the sea urchin Lytechinus pictus. We also show for comparison the results of parthenogenetic activation by ammonia. In addition to the known transient rise of [Ca2+]i at fertilization, further peaks are now revealed during pronuclear migration, nuclear envelope breakdown, the metaphase/anaphase transition and cleavage. Parthenogenetic activation by ammonia also elicits a sustained rise starting at nuclear envelope breakdown.

Ammonia↗
PubMedDate not supplied

Pleiotropic loss of activation pathways in a T-cell receptor alpha-chain deletion variant of a cytolytic T-cell clone.

Untransformed T-cell clones maintained in culture are dependent on signals transmitted through their antigen receptors (Ti; alpha and beta chains associated with the CD3 molecules) for growth and effector function. For cytolytic T cells (CTL), Ti stimulation also activates the killing machinery and induces synthesis of gamma interferon (IFN-gamma) messenger RNA and IFN-gamma secretion. The Thy-1 molecule, expressed on all murine cells of the T-cell lineage, has been suggested to function in transmembrane signalling, based on the ability of some anti-Thy-1 monoclonal antibodies (mAb) to activate T cells. Recently, it was suggested that Thy-1 could function as a signal-transduction molecule when expressed in B-cell lymphomas after transfection of the gene, leading to speculation that the molecule was part of an activation pathway independent of the Ti/CD3 structures. Here we report the immunoselection of a variant CTL clone which has lost expression of mRNA for the alpha-chain of the Ti. The Ti- variant was defective in lectin-mediated activation whether measured by increase in intracytoplasmic Ca2+, CTL effector function or IFN-gamma synthesis. The variant, which expressed normal levels of Thy-1, was also unresponsive to Thy-1 mAb activation as measured by IFN-gamma secretion, whereas it responded to calcium ionophore plus phorbol ester. These results indicate that in a non-transformed, functional mature T-cell, Thy-1 mediated signalling is not an alternative to, but might depend on elements associated with the Ti/CD3-mediated T-cell activation pathway.

Antibodies, Monoclonal↗
PubMedDate not supplied

Requirement for intrinsic protein tyrosine kinase in the immediate and late actions of the EGF receptor.

The epidermal growth factor (EGF) receptor is a transmembrane glycoprotein of relative molecular mass 170,000 with intrinsic ligand-dependent protein tyrosine kinase activity. Binding of EGF to its receptor activates a number of immediate biochemical processes, such as alterations of intracellular free calcium, pH, and increased transcription of several responsive genes, which usually culminate many hours later in DNA replication and cell division. Abolishing the tyrosine kinase activity of three related oncogenes, v-src, v-mos, and v-fps, eliminates their capacity to transform cell. Several reports have suggested that specific aspects of EGF receptor function are independent of the intrinsic tyrosine kinase activity; however, these studies used an antibody against EGF receptor which failed to activate phosphorylation of exogenous substrates and an insertional mutation in the EGF receptor tyrosine kinase domain which had not been shown to abolish protein kinase activity in cells. Because many transmembrane receptors interact with intrinsic membrane proteins to activate second messenger systems, it is important to resolve experimentally whether mechanisms, in addition to activation of the intrinsic tyrosine kinase activity, mediate some EGF actions. From functional analyses of an EGF receptor containing a single amino-acid mutation at a site required for phosphate transfer from ATP, we conclude that the tyrosine kinase activity of the EGF receptor is essential for the diverse biochemical effects of EGF, including rapid alterations in intracellular calcium, activation of gene transcription, receptor down-regulation and the ultimate stimulatory effects on cell proliferation.

Animals↗
PubMedDate not supplied

Expression and function of the CD3-antigen receptor on murine CD4+8+ thymocytes.

Mature T cells arise from progenitor cells by a complex and poorly understood process of differentiation in the thymus. Thymocytes can be divided into four major compartments on the basis of surface expression of the murine equivalents of CD8 (Lyt-2) and CD4 (L3T4) (refs 1,2). Functionally mature thymocytes express only CD4 or CD8. The CD4-8- subset contains progenitor cells capable of giving rise to all the phenotypic and functional classes of T cells on adoptive transfer. The function of the major population, the CD4+8+ cells, which carry both the CD4 and CD8 antigens, in thymic differentiation is controversial. It has been variously proposed that they represent terminally differentiated cells which die in the thymus or that they represent an intermediate stage and can differentiate into functionally and phenotypically mature single positive T cells. The CD3-antigen receptor complex is probably important in thymic differentiation. The receptor has two functions: recognition and transmembrane signalling. To help clarify the function of CD4+8+ thymocytes in thymic differentiation, the expression and function of the antigen receptor complex on these cells should be determined. We show here that most CD4+8+ thymocytes express CD3 which functions in transmembrane signalling. The consequences of this signalling differ from those in mature T cells, however, in that the CD4+8+ cells do not secrete IL-2, express IL-2 receptor, or proliferate.

Animals↗