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D Grunwald

Publications and source records attributed to D Grunwald.

At least 19 recordsLinked to original sources
PubMed2001

Tetrahydrofolate biosynthesis in plants: molecular and functional characterization of dihydrofolate synthetase and three isoforms of folylpolyglutamate synthetase in Arabidopsis thaliana.

Tetrahydrofolate coenzymes involved in one-carbon (C1) metabolism are polyglutamylated. In organisms that synthesize tetrahydrofolate de novo, dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) catalyze the attachment of glutamate residues to the folate molecule. In this study we isolated cDNAs coding a DHFS and three isoforms of FPGS from Arabidopsis thaliana. The function of each enzyme was demonstrated by complementation of yeast mutants deficient in DHFS or FPGS activity, and by measuring in vitro glutamate incorporation into dihydrofolate or tetrahydrofolate. DHFS is present exclusively in the mitochondria, making this compartment the sole site of synthesis of dihydrofolate in the plant cell. In contrast, FPGS is present as distinct isoforms in the mitochondria, the cytosol, and the chloroplast. Each isoform is encoded by a separate gene, a situation that is unique among eukaryotes. The compartmentation of FPGS isoforms is in agreement with the predominance of gamma-glutamyl-conjugated tetrahydrofolate derivatives and the presence of serine hydroxymethyltransferase and C1-tetrahydrofolate interconverting enzymes in the cytosol, the mitochondria, and the plastids. Thus, the combination of FPGS with these folate-mediated reactions can supply each compartment with the polyglutamylated folate coenzymes required for the reactions of C1 metabolism. Also, the multicompartmentation of FPGS in the plant cell suggests that the transported forms of folate are unconjugated.

Arabidopsis↗
PubMed2001

Nadine Dobrovolskaïa-Zavadskaïa and the dawn of developmental genetics.

In one of the first genetic screens aimed at identifying induced developmental mutants, Nadine Dobrovolskaïa-Zavadskaïa, working at the Pasteur Laboratory in the 1920s, isolated and characterized a mutation affecting Brachyury, a gene that regulates tail and axial development in the mouse. Dobrovolskaïa-Zavadskaïa's analysis of Brachyury and other mutations affecting tail development were among the earliest attempts to link gene action with a tissue-specific developmental process in a vertebrate. Her analyses of genes that interacted with Brachyury led to the discovery of the t-haplotype chromosome of mouse. After 70 years, Brachyury and the multiple genes with which it interacts continue to occupy a prominent focus in developmental biology research. A goal of this review is to identify the contributions that Dobrovolskaïa-Zavadskaïa made to our current thinking about Brachyury and how she helped to shape the dawn of the field of developmental genetics. BioEssays 23:365-371, 2001.

Animals↗
PubMed2001

Developmental expression of the calcium release channels during early neurogenesis of the mouse cerebral cortex.

The developmental changes of intracellular calcium release channels of mouse neocortex were studied at the onset of neurogenesis, which occurs between embryonic days E11 and E17. The three main isoforms of the two families of intracellular calcium release channels, namely the inositol trisphosphate receptors (IP3R) and the ryanodine receptors (RyR), were detected by their transcripts in the cerebral hemispheres, as early as stage E11. The major isoforms of each family, IP3R-1 and RyR-2, were found at the protein level by Western blot analysis. Expression of these proteins increases progressively throughout brain development. Their localization in coronal sections of cortex has been observed by immunodetection from E12, and compared to the TuJ1 (anti-class III beta-tubulin antibody) neuronal specific labelling. The expression of both channels is greatly enhanced after E12, and both were seen to be present in most of the proliferative and neuronal cells of the slice. Between E12 and E13, there is a striking transition in the pattern of calcium release elicited by specific agonists of these channels, thimerosal for IP3R and caffeine for RyR. The signals induced by thimerosal were not zone-specific, while the observed calcium release signals induced by caffeine were predominantly restricted out of the ventricular zone. This zone-specific caffeine sensitivity is consistent with the main RyR localization immunodetected at E13. Our results indicate that there is a time lag of several days between the molecular detection of calcium release channels and their functional expression, around the time of neuronal differentiation. Altogether, they provide a molecular basis for analyzing the developmental modulation of calcium signals useful for neurogenesis progression.

Animals↗
PubMed2000

Calmodulin and immunophilin are required as functional partners of a ryanodine receptor in ascidian oocytes at fertilization.

Fertilization of oocytes incites numerous changes relying on Ca(2+) signaling. In inseminated ascidian eggs, an increase in the egg surface membrane, monitored by a change in electrical capacitance, is recorded at the onset of meiosis resumption. This membrane addition to the cell surface is controlled by calcium release through a ryanodine receptor (RyR), sensitive to cyclic ADP-ribose. Using confocal microscopy analysis of ascidian oocytes immunostained with anti-RyR antibody, we show here that this calcium channel is asymmetrically located in the vegetal cortical zone. Interestingly, the increase in cell capacitance occurring at fertilization is correlated with a fluorescent signal, imaged by the marker of vesicle trafficking FM 1-43, located close to the RyR region. Two putative partners of RyR, namely an FKBP-like protein and a calmodulin, are identified in these oocyte extracts by detection of enzyme activity and PCR amplification. Both are necessary to sustain ryanodine receptor activity in these oocytes since the membrane insertion triggered by fertilization is inhibited by the FKBP ligand rapamycin and by a calmodulin antagonist peptide. These findings suggest that exocytosis in ascidian eggs is triggered at fertilization by a functional Ca(2+) release unit operating as a complex of several proteins, including a calmodulin and an immunophilin, around the intracellular calcium channel itself.

Amino Acid Sequence↗
PubMed2000

Cloning and characterization of a new isoform of skeletal muscle triadin.

We have shown that several isoforms of triadin, a protein involved in calcium release process through the ryanodine receptor, are expressed in rat skeletal muscle, and we have cloned two of these isoforms. One is the rat homolog of the 95-kDa triadin identified in rabbit skeletal muscle, and the second one, shorter, is a truncated form of the previous one, but with a new unique COOH-terminal end. We propose to name the two proteins identified here Trisk 95 and Trisk 51. We have produced antibodies specific to each isoform. Using these antibodies, we have shown that the newly identified protein, Trisk 51, is actually expressed in adult rat skeletal muscle and also in rat embryo skeletal muscle. Immunofluorescent labeling of rat skeletal muscle with anti-Trisk 95, anti-Trisk 51, or anti-ryanodine receptor antibodies shows a similar localization of these proteins, in the tissue. Transfection of L6 cells with cDNA of Trisk 51 or Trisk 95 leads to the expression of proteins with the expected molecular weight, identical to those detected in rat skeletal muscle. Both proteins appear during differentiation of satellite cells in myotubes which may indicate the involvement of these two isoforms in the building of a functional calcium release machinery.

Amino Acid Sequence↗
PubMed2000

Response of chemosensitive and chemoresistant leukemic cell lines to drug therapy: simultaneous assessment of proliferation, apoptosis, and necrosis.

BACKGROUND: The balance between cell proliferation and drug-induced cell death by apoptosis or necrosis plays a major role in determining response to chemotherapy. Commonly-used DNA analysis methods cannot study both parameters simultaneously. A new approach described here combines a green fluorescent membrane-intercalating dye (PKH67) with Hoechst 33342 or annexin V and propidium iodide, to allow simultaneous assessment of cell division, cell cycle status, apoptosis, and necrosis, respectively. METHODS: To test this approach, we used cultured K562 leukemic cell lines which are drug-sensitive (K562S) or drug-resistant (K562R) by virtue of whether they lack or exhibit expression, respectively, of the gp-170 (PGP) glycoprotein pump involved in multidrug resistance. RESULTS: We found that: 1) PKH67 fluorescence intensity decreases proportionately to number of cell divisions, 2) labeling with PKH67 does not alter either cell cycle distribution, as assessed by vital DNA staining with Hoechst 33342, or cell growth, and 3) using a simple threshold analysis method suitable for real-time sorting decisions, subpopulations of proliferating cells present at initial levels of >/= 10% can readily be detected after two cell division times, based on decreased PKH67 intensity. Finally, we demonstrated that after treatment of an admixture of K562S and K562R with vincristine, triple-labeling with PKH67, annexin V, and propidium iodide can be used to identify and sort those cells which remain not only viable (nonnecrotic, nonapoptotic) but actively dividing (decreased PKH67 intensity) in the presence of drug. CONCLUSIONS: Although the studies described here were carried out in a model system using cells having known drug resistance phenotypes, we expect that the methods described will be useful in ex vivo studies of clinical leukemic specimens designed to identify the role played by specific chemoresistance proteins and mechanisms in therapeutic outcomes for individual patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

A ryanodine-sensitive calcium store in ascidian eggs monitored by whole-cell patch-clamp recordings.

Using whole cell patch clamp recordings on unfertilized eggs of the ascidian Ciona intestinalis, we are able to detect ryanodine receptors within the oocytes. Our approach is based on measurements of the voltage-activated inward calcium currents. Two types of Ca2+ currents have been described on the oocyte membrane of Ciona: a low threshold slowly activating current, and a high threshold faster one. We show here that caffeine induces a decrease in the intensity of the Ca2+ currents, when applied either externally or internally from the mouth of a patch pipette. Caffeine application mimics fertilization which transiently decreases the high threshold Ca2+ current density during density during the first meiotic cycle. Ryanodine (> 1 nM) has an effect similar to caffeine. This partial decrease in Ca2+ current density elicited by caffeine or ryanodine is prevented by intracellular application of the calcium chelator BAPTA, then imputable to calcium release. In summary, the depolarization-induced Ca2+ current intensity allows monitoring of an intracellular calcium store which is sensitive to low concentrations of ryanodine in Ciona oocytes. Further identification of a ryanodine receptor was obtained by immunological staining with antibodies against mammalian skeletal muscle ryanodine receptor. Ryanodine receptors were asymmetrically localized in the cortex of Ciona eggs. We discuss the methodological relevance of our patch-clamp approach, in connection with the possible biological role of such a ryanodine receptor in the early stages of development.

Animals↗
PubMed1996

Novel postfertilization inward Ca2+ current in ascidian eggs ensuring a calcium entry throughout meiosis.

The conductance change after fertilization in the oocyte of the ascidian Ciona intestinalis has been followed by the whole cell patch-clamp technique. Two new inward currents, which are absent in unfertilized eggs, are elicited by hyperpolarization from a holding potential of +20 mV, which is the resting potential soon after fertilization. These currents reach their maximum level during the first meiotic division cycle, and then decrease in intensity, becoming almost undetectable at the 2-cell stage. These currents are most easily seen at high concentrations of barium. At least one, and likely both, of these currents appears to be carried by Ca ions. One of the currents is blocked by low concentrations of gadolinium; the other one is blocked by higher concentrations, although gadolinium at these levels does not block fertilization and the associated early depolarizing jump of the eggs. Thus these currents are not carried by channels that mediate the fertilization current. However, gadolinium blocks normal transition to 2-cell stage and blocks current oscillations synchronous to free calcium oscillations that occur normally in eggs around meiosis II. The electrical signature of calcium-release activated currents, taken together with these findings, suggests that these inward currents ensure a calcium entry pathway throughout meiosis. A plausible function of these currents may be to refill the Ca stores that are depleted after fertilization and that are required to progress into mitotic cell division. This interpretation is reinforced by experiments on unfertilized eggs with intracellular Ca stores depleted by thapsigargin, where both the newly described currents are observed.

Animals↗
PubMed1995

A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein that specifically recognizes DNA strand breaks produced by various genotoxic agents. To study the biological function of this enzyme, we have established stable HeLa cell lines that constitutively produce the 46-kDa DNA-binding domain of human PARP (PARP-DBD), leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version of the DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNA-binding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.

Apoptosis↗
PubMed1995

Accumulation of histone H1(0) during early Xenopus laevis development.

It is known that a transition in the linker-histone variants takes place within chromatin during early development of Xenopus laevis; a cleavage-type H1 is replaced by the somatic type. Based on cytofluorimetric analysis of the distribution of the embryo cells in the cell cycle, we showed that this previously described transition occurs when significant modifications of the proliferative capacities of the cells occur. Moreover, this analysis allowed us to show that cell proliferation decreases gradually after the gastrula stage of development. This period terminates with the arrest of more than 90% of cells in the G0/G1 phase of the cell cycle at stage 45. We showed that the major accumulation of the differentiation-specific H1 subtype, histone H1(0), occurs at this time. H1(0), first detected in a restricted set of tissues, is then widely expressed during the later development at stage 45. Moreover, the double staining of nuclei isolated from embryo cells, for H1(0) and DNA, allowed us to show that this accumulation of H1(0) is not restricted to arrested cells. The example of the Xenopus early development shows that there may be an adaptation of the type of H1 expressed to the proliferative abilities of cells. This observation may provide insight into the significance of the expression of different H1 subtypes during development.

Animals↗
PubMed1995

Developmentally regulated chromatin acetylation and histone H1(0) accumulation.

There exists a close relationship between core histone acetylation and the induced expression of the histone H1(0) gene. We took advantage of this fact to evaluate the influence of chromatin hyperacetylation on the developmentally regulated expression of this specific gene. In this study, the in situ immunodetection approach has been used to analyze both the acetylated histone H4 isoforms and histone H1(0) accumulation during early Xenopus laevis development. We have chosen two stages of development, gastrula stage, when H1(0) is not expressed and not inducible by butyrate treatment, and stage 27 when H1(0) is not expressed but is inducible by butyrate. At stage 27 of development, the early induced accumulation of histone H1(0) under butyrate treatment, occurs mainly in tissues that express the protein normally during later development. These experiments suggest that histone acetylation may be part of a pathway which, in a specific set of cells, keeps H1(0) and probably a series of specific genes, competent for transcription, but cell-specific factors are involved in the induced expression of these genes.

Acetylation↗
PubMed1995

[Principles of flow cytometry].

Flow cytometry (FCM) allows the simultaneous measurement of different parameters in a population of cell or sub-cellular particles, which are individually analysed at a rate of 500 to 5,000 objects/second. Results are displayed as univariate or bivariate histograms, giving the distribution of the population in relation to the studied parameters. Moreover, FCM offers the possibility to physically sort the sub populations defined by the analysis.

Cell Cycle↗
PubMed1993

Flow cytometry and RNA studies.

RNA was less studied by flow cytometry (FCM) in comparison to other cellular components. Moreover, most of these studies were applied to the measurement of total RNA. Nevertheless, such a quantification, essentially by the use of acridine orange, but also other dyes or dye combinations, permitted the acquisition of important information about the general metabolism, and the characterization of different tumoral cell types. More recently, FCM has been used to quantify specific mRNA, in two different approaches: the first one employs the sorting followed by RNA extraction and/or detection by specific radiolabeled probes; in the second approach, the use of fluorescence in situ hybridization (FISH) on cells in suspension allowed the direct quantification of specific mRNA by FCM.

Acridine Orange↗
PubMed1993

p53-mediated cell death: relationship to cell cycle control.

M1 clone S6 myeloid leukemic cells do not express detectable p53 protein. When stably transfected with a temperature-sensitive mutant of p53, these cells undergo rapid cell death upon induction of wild-type (wt) p53 activity at the permissive temperature. This process has features of apoptosis. In a number of other cell systems, wt p53 activation has been shown to induce a growth arrest. Yet, wt 53 fails to induce a measurable growth arrest in M1 cells, and cell cycle progression proceeds while viability is being lost. There exists, however, a relationship between the cell cycle and p53-mediated death, and cells in G1 appear to be preferentially susceptible to the death-inducing activity of wt p53. In addition, p53-mediated M1 cell death can be inhibited by interleukin-6. The effect of the cytokine is specific to p53-mediated death, since apoptosis elicited by serum deprivation is refractory to interleukin-6. Our data imply that p53-mediated cell death is not dependent on the induction of a growth arrest but rather may result from mutually incompatible growth-regulatory signals.

Animals↗
PubMed1992

Characterization of the tumor suppressor protein p53 as a protein kinase C substrate and a S100b-binding protein.

We report here that the negative cell cycle regulator protein p53 is an in vivo and in vitro substrate for protein kinase C, a cellular receptor for the tumor-promoter phorbol esters. We also demonstrate that p53 interacts in a calcium-dependent manner with S100b, a member of the S100 protein family involved in cell cycle progression and cell differentiation, and that such an interaction inhibits in vitro p53 phosphorylation by protein kinase C. The interaction between p53 and S100b was utilized for the purification of cellular and recombinant murine p53 by affinity chromatography with S100b-Sepharose. Furthermore, and of particular interest, we have shown that purified p53 undergoes temperature-dependent oligomerization and that the interaction between S100b and p53 not only induces total inhibition of p53 oligomerization but also promotes disassembly of the p53 oligomers. We suggest that these effects result from the binding of S100b to the multifunctional basic C-terminal domain of p53 and propose that p53 may be a cellular target for the S100 protein family members involved in the control of the cell cycle at the G0-G1/S boundary.

Amino Acid Sequence↗
PubMed1992

Antisense RNA and p53 regulation in induced murine cell differentiation.

p53 expression is strongly modulated during the process of induced differentiation, at the same time as both cell cycle and genetic expression become modulated, giving rise to a commitment to terminal differentiation. We took advantage of two murine cell lines inducible for differentiation, an erythroleukemia and a melanoma cell line, to outline common features of the regulation of p53 expression during the differentiation process. We found that p53 mRNA decreased early after induced differentiation and that regulation was controlled at a posttranscriptional level. Our data showed that this regulation affects p53 pre-mRNA maturation. Because, in both systems used, actinomycin D treatment abolished the inducer-mediated decrease of p53 mRNA, we looked for induced RNAs potentially involved in this process. Using different parts of the p53 gene and flanking regions as probes, we identified three RNA species whose expression is modulated during induced differentiation. A first species is made of high molecular weight RNAs that accumulate in the nuclear compartment and seem to represent antisense transcripts of the p53 gene. A second species, 1.3-kb long, was found to accumulate in the nucleus of induced MEL cells and was homologous to a restricted part of the first intron of the p53 gene due to the presence of a B1 repetitive element in an antisense orientation with respect to the p53 pre-messenger RNA. Finally, a family of B2-containing RNAs was observed in both cytoplasmic and nuclear compartments. The variation in the amounts of sense and antisense RNAs, respectively, suggested an interesting speculative model for the maturation of B2-containing pre-messenger RNAs.

Animals↗