Intracellular pH in one-cell mouse embryo differs between subcellular compartments and between interphase and mitosis.

The pH-sensitive dual-emission fluorophore SNARF-1 coupled with a laser confocal microspectrofluorimeter was used to measure the internal pH (pHi) in different subcellular and subnuclear compartments of early mouse embryos. By this method we analysed the first cell cycle of naturally fertilised embryos in order to detect possible pHi changes correlated to cellular events, particularly the onset of replication or transcription and the first mitosis. Throughout interphase, significant differences of pHi were observed between cytoplasm and pronuclei, and, even more striking, between these compartments and nucleolus precursor bodies, whose pHi was systematically lower.

Functional and molecular reorganization of the nucleolar apparatus in maturing mouse oocytes.

In mammalian preovulatory oocytes, rRNA synthesis is down-regulated until egg fertilization and zygotic genome reactivation, but the underlying regulatory mechanisms of this phenomenon are poorly characterized. We examined the molecular organization of the rRNA synthesis and processing machineries in fully grown mouse oocytes in relation to ongoing rDNA transcription and oocyte progression throughout meiosis. We show that, at the germinal vesicle stage, the two RNA polymerase I (RNA pol I) subunits, RPA116 and PAF53/RPA53, and the nucleolar upstream binding factor (UBF) remain present irrespective of ongoing rDNA transcription and colocalize in stoichiometric amounts within discrete foci at the periphery of the nucleolus-like bodies.

Induction of early transcription in one-cell mouse embryos by microinjection of the nonhistone chromosomal protein HMG-I.

In the mouse embryo, the onset of zygotic transcription occurs at the end of the first cell cycle, upon completion of DNA replication. We show that the nonhistone chromosomal protein HMG-I, whose translocation into the pronuclei of one-cell embryos is linked to this first round of DNA synthesis, plays a critical role in the activation of zygotic transcription. Indeed, microinjection of purified HMG-I results in a higher nuclear accumulation of the protein and triggers an earlier activation of zygotic transcription, an effect which is abolished by the preincubation of the protein with a specific antibody directed against its AT-hook DNA-binding motifs.

Three distinct sub-nuclear populations of HMG-I protein of different properties revealed by co-localization image analysis.

We have studied the nuclear distribution of the non-histone HMG-I protein by indirect immunofluorescence in several human and murine somatic cell lines and in growing mouse oocytes. We show that HMG-I, a high mobility-group protein which interacts in vitro with the minor groove of AT-rich B-DNA, is found exclusively in the nucleus and that this localization corresponds to a complex distribution. By comparing the HMG-I-dependent fluorescence signal with the chromatin density determined by Hoechst 33342 or propidium iodide staining, we present evidence for the existence of three HMG-I sub-populations whose contribution to the total fluorescence can be determined using a newly developed quantitative co-localization image analysis program: foci that correspond to regions of heterochromatin, intense dots located within decondensed chromatin, and a more diffuse component extending throughout the nucleoplasm.

Characterization of a constitutive type III nitric oxide synthase in human U937 monocytic cells: stimulation by soluble CD23.

The soluble cleavage fragment of the low-affinity immunoglobulin E (IgE) receptor/CD23 (sCD23 25000 MW) and antibodies directed against their receptors on monocytes, CD11b and CD11c, stimulate the production of nitric oxide (NO) by these cells and we have suggested that the enzyme involved could be related to the endothelial constitutive type III nitric oxide synthase (ecNOS). In the present work, we have analysed the characteristic properties of this NOS isoform in the model of the human promonocytic cells U937 By reverse-transcription polymerase chain reaction (RT-PCR), the presence of an mRNA coding for type III NOS was found in U937 cells and the corresponding protein was detected by immunofluorescence in permeabilized cells with a specific anti-ecNOS monoclonal antibody (mAb). Membrane extracts displayed a NOS activity dependent on the presence of calcium and calmodulin in the reaction medium and that was abrogated in the presence of EGTA.

Photofrin-induced fluorescence in progressive and regressive murine colonic cancer cells: correlation with cell photosensitivity.

Microspectrofluorometry and fluorescence imaging were used to investigate the intracellular fluorescence of two murine colonic cancer cell lines--a progressive cell line (PROb) and a regressive cell line (REGb)--incubated with Photofrin. These two cell lines, which were initially cloned from the same chemically induced colonic murine cancer, differ in their metastatic properties and have been considered as models to mimic the tumoral cell heterogeneity. The fluorescence from cytoplasmic area of cells incubated with Photofrin appeared as a complex emission, with two maxima at 632 and 695 nm assigned to monomer species, and a poorly resolved band around 665 nm assigned to aggregates.

IL-4 induces cAMP and cGMP in human monocytic cells.

Human monocytes, preincubated with IFN-gamma respond to IL-4 by a cGMP increase through activation of an inducible NO synthase. Here, IL-4 was found to induce an accumulation of cGMP (1 - 3 min) and cAMP (20 - 25 min) in unstimulated monocytes. This was impaired with NOS inhibitors, but also with EGTA and calcium/calmodulin inhibitors.

Asymmetrical DNA and AT/GC base content of differential sector of Pleurodeles waltl sexual bivalent: a quantitative fluorescence imaging analysis in lampbrush chromosomes.

The mitotic Z and W sex chromosomes in Pleurodeles seem to be identical. Earlier morphological and molecular analyses of lampbrush paired chromosomes in the female meiosis showed clearly that 20% of the chromosomal length located in the middle part of the sex bivalent (bivalent IV) is heteromorphic. We investigated here the base content and composition of the DNA axes in the heteromorphic region by quantitative fluorescence imaging using various base-specific (DAPI, Hoechst 33342 and chromo-mycin A3) or base-nonspecific (ethidium bromide) fluorescent DNA probes.

Biochemical and functional alterations induced by CD23 ligation in the human promonocytic cell line U937.

The early events triggered in interleukin-4 (IL-4)-stimulated U937 cells by ligation of CD23/Fc epsilon RII with specific monoclonal antibodies (mAb) were analysed, as a model of the action of this molecule on the differentiation of promonocytic cells. As well as IL-4-activated human monocytes, addition of anti-CD23 mAb to IL-4-treated U937 cells triggered cAMP accumulation but did not evoke significant polyphosphoinositide hydrolysis. However, by a microspectrofluorometric technique allowing single cell analysis, anti-CD23 mAb was found to elicit calcium mobilization in these cells.

Interactions of iron-anthracycline complexes with living cells: a microspectrofluorometric study.

The interaction of iron-anthracycline complexes with tumor cells has been studied using microspectrofluorometry. The anthracyclines used were adriamycin, 4'-O-tetrahydropyranyladriamycin and daunorubicin. In every case, a 1:3 Fe(III)-anthracycline complex is formed.

Activation pathways triggered by interleukin-4 in the human plasmacytoma cell line RPMI-8226--differences with resting B lymphocytes.

The early events following the ligation of interleukin-4 (IL-4) to the plasmacytoma cell line RPMI-8226 were analysed as a model of action for this interleukin on differentiated cells of the B lymphocyte lineage. The addition of recombinant IL-4 to these cells resulted in an increase of the intracytoplasmic free calcium concentration [Ca2+]i, but in contrast to normal B cells, this increase was mostly due to a calcium influx rather than to a mobilization from endoplasmic reticulum stores. IL-4 was also found to trigger cAMP accumulation in RPMI-8226 cells, with kinetics similar to that which has been described for normal resting human B lymphocytes.

Intracellular signaling events associated with the induction of DNA synthesis in human B lymphocytes. I. Stimulation of PKC-dependent and -independent pathways by LMW-BCGF.

The low molecular weight B cell growth factor (LMW-BCGF) induces the G1 --> S transition in human B lymphocytes activated by a first signal, Staphylococcus aureus Cowan (SAC) or anti-mu antibody. It also stimulates proliferation of normal long-term B cell lines and some B cell tumors. We have previously reported that LMW-BCGF induces the hydrolysis of polyphosphoinositides (PI) and a rise in intracellular free calcium concentration, through the generation of inositol trisphosphate (InsP3) (Renard et al.

Microspectrofluorimetric study of the kinetics of cellular uptake and metabolization of benzo(a)pyrene in human T 47D mammary tumor cells: evidence for cytochrome P1450 induction.

The kinetics of penetration, activation and detoxification of benzo(a)pyrene were determined by near U.V. microspectrofluorimetric measurements on single living cells.