Biomedical subjects

M Dasso

Publications and source records attributed to M Dasso.

30 records · Page 2Linked to original sources

PubMed1996

Rapid kinetics of second messenger production in bitter taste.

The tasting of bitter compounds may have evolved as a protective mechanism against ingestion of potentially harmful substances. We have identified second messengers involved in bitter taste and show here for the first time that they are rapid and transient. Using a quench-flow system, we have studied bitter taste signal transduction in a pair of mouse strains that differ in their ability to taste the bitter stimulus sucrose octaacetate (SOA); however, both strains taste the bitter agent denatonium. In both strains of mice, denatonium (10 mM) induced a transient and rapid increase in levels of the second messenger inositol 1,4,5-trisphosphate (IP3) with a maximal production near 75-100 ms after stimulation. In contrast, SOA (100 microM) brought about a similar increase in IP3 only in SOA-taster mice. The response to SOA was potentiated in the presence of GTP (1 microM). The GTP-enhanced SOA-response supports a G protein-mediated response for this bitter compound. The rapid kinetics, transient nature, and specificity of the bitter taste stimulus-induced IP3 formation are consistent with the role of IP3 as a second messenger in the chemoelectrical transduction of bitter taste.

Animals↗

PubMed1995

The RCC1 protein interacts with Ran, RanBP1, hsc70, and a 340-kDa protein in Xenopus extracts.

RCC1 is an abundant, highly conserved, chromatin-associated protein whose function is necessary for the preservation of a properly ordered cell cycle. RCC1 is also necessary for numerous nuclear processes, including nuclear transport and RNA metabolism; and it functions enzymatically as a guanine nucleotide exchange factor for a small, ras-related GTPase called Ran. Studies in several organisms suggest that RCC1 may be part of a large complex containing multiple proteins. There is also evidence that RCC1 associates with chromatin through other proteins and that the binding of the complex to chromatin varies within the cell cycle. In order to characterize this putative complex, we have identified a number of other proteins as candidate components of the complex by their association with a GST-RCC1 fusion protein. Three of these proteins have previously been identified (Ran, RanBP1, and hsc70). The fourth protein is novel and has a molecular mass of 340 kDa. In this report, we discuss a preliminary characterization of the interactions between these proteins.

Animals↗

PubMed1995

The role of the Ran GTPase pathway in cell cycle control and interphase nuclear functions.

Ran is a small, highly abundant, nuclear GTPase. Mutants in Ran and in proteins that interact with it disrupt the normal checkpoint control of mitosis with respect to the completion of DNA synthesis. Ran and other components of this pathway are also required for numerous nuclear functions such as RNA export, protein import, RNA processing and DNA replication. It will be important to understand how these facets of Ran's activities are linked and how they promote correct control of the cell cycle. This review examines recent progress in discovering other components of the Ran GTPase pathway and considers how this pathway may be required for the control of the cell cycle.

Cell Cycle↗

PubMed1994

Nuclear assembly is independent of linker histones.

The role of linker histones in the assembly of functional nuclei was examined with the use of a cell-free extract of Xenopus eggs that transforms condensed sperm chromatin into DNA-replication-competent pronuclei. When linker histones were removed from the extract, the resultant pronuclei were indistinguishable from those formed in the complete extract. The assembly of functional nuclear membrane, nuclear lamina, and prereplication centers allowed identical DNA replication efficiencies. Thus, linker histones are not required for the assembly of morphologically normal nuclei capable of DNA replication.

Animals↗

PubMed1994

A mutant form of the Ran/TC4 protein disrupts nuclear function in Xenopus laevis egg extracts by inhibiting the RCC1 protein, a regulator of chromosome condensation.

The Ran protein is a small GTPase that has been implicated in a large number of nuclear processes including transport. RNA processing and cell cycle checkpoint control. A similar spectrum of nuclear activities has been shown to require RCC1, the guanine nucleotide exchange factor (GEF) for Ran. We have used the Xenopus laevis egg extract system and in vitro assays of purified proteins to examine how Ran or RCC1 could be involved in these numerous processes. In these studies, we employed mutant Ran proteins to perturb nuclear assembly and function. The addition of a bacterially expressed mutant form of Ran (T24N-Ran), which was predicted to be primarily in the GDP-bound state, profoundly disrupted nuclear assembly and DNA replication in extracts. We further examined the molecular mechanism by which T24N-Ran disrupts normal nuclear activity and found that T24N-Ran binds tightly to the RCC1 protein within the extract, resulting in its inactivation as a GEF. The capacity of T24N-Ran-blocked interphase extracts to assemble nuclei from de-membranated sperm chromatin and to replicate their DNA could be restored by supplementing the extract with excess RCC1 and thereby providing excess GEF activity. Conversely, nuclear assembly and DNA replication were both rescued in extracts lacking RCC1 by the addition of high levels of wild-type GTP-bound Ran protein, indicating that RCC1 does not have an essential function beyond its role as a GEF in interphase Xenopus extracts.

Animals↗

PubMed1994

Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression.

TC4, a ras-like G protein, has been implicated in the feedback pathway linking the onset of mitosis to the completion of DNA replication. In this report we find distinct roles for TC4 in both nuclear assembly and cell cycle progression. Mutant and wild-type forms of TC4 were added to Xenopus egg extracts capable of assembling nuclei around chromatin templates in vitro. We found that a mutant TC4 protein defective in GTP binding (GDP-bound form) suppressed nuclear growth and prevented DNA replication. Nuclear transport under these conditions approximated normal levels. In a separate set of experiments using a cell-free extract of Xenopus eggs that cycles between S and M phases, the GDP-bound form of TC4 had dramatic effects, blocking entry into mitosis even in the complete absence of nuclei. The effect of this mutant TC4 protein on cell cycle progression is mediated by phosphorylation of p34cdc2 on tyrosine and threonine residues, negatively regulating cdc2 kinase activity. Therefore, we provide direct biochemical evidence for a role of TC4 in both maintaining nuclear structure and in the signaling pathways that regulate entry into mitosis.

Amino Acid Sequence↗

PubMed1993

Chromatin transitions during early Xenopus embryogenesis: changes in histone H4 acetylation and in linker histone type.

We describe major transitions in the type and modification of chromatin-associated proteins during the early development of Xenopus laevis. Histone H4 is stored in the diacetylated form in the egg and is progressively deacetylated during normal development. If histone deacetylases are inhibited with sodium butyrate, hyperacetylated histone H4 only accumulates after the mid-blastula transition. The type of linker histone in chromatin also changes during embryogenesis, from predominantly the B4 protein at the mid-blastula transition to predominantly histone H1 at the end of gastrulation. These transitions in chromatin composition correlate with major changes in the replicative and transcriptional activity of embryonic nuclei.

Acetylation↗

PubMed1993

RCC1 in the cell cycle: the regulator of chromosome condensation takes on new roles.

In the eukaryotic cell cycle, nuclear DNA replication (S phase) and mitosis (M phase) are linked such that replication must be complete before mitosis can begin. In order for this coupling to work, there must be some system for detecting unreplicated DNA and transducing an inhibitory signal to prevent the activation of mitotic factors. The DNA-bound protein RCC1 is involved in this regulatory process since mitosis initiates before DNA synthesis is finished in the absence of RCC1. This has led to the proposal that RCC1 is a signalling molecule, detecting unreplicated DNA and producing the inhibitory signal. However, mutants in RCC1 show defects beyond their inability to regulate the cell cycle, suggesting other roles for the RCC1 protein in the nucleus and thus hitherto unexplored relationships between cell cycle control and other cellular processes.

Amino Acid Sequence↗

PubMed1992

DNA replication and progression through the cell cycle.

Somatic cells possess control mechanisms which monitor DNA replication and assure that it is complete before mitosis is initiated. We have been investigating these mechanisms in Xenopus egg extracts. Using in vitro cycling extracts, which spontaneously alternate between interphase and mitosis, we found that the onset of mitosis is inhibited by the presence of unreplicated DNA, demonstrating that the completion of DNA replication and the initiation of mitosis are coupled in these extracts. As in somatic cells, this coupling is sensitive to caffeine and to okadaic acid. In Xenopus extracts unreplicated DNA increases the tyrosine phosphorylation of p34cdc2, thereby maintaining MPF (mitosis-promoting factor) in an inactive state and preventing the onset of mitosis. The block to mitosis in the presence of unreplicated DNA can be reversed by the addition of bacterially expressed cdc25 protein. The extent of MPF activation by cdc25 protein under these conditions depends on the number of nuclei present. We have developed an assay to examine the rate of tyrosine phosphorylation on p34cdc2. It is increased by unreplicated DNA, in a manner consistent with unreplicated DNA up-regulating the kinase that phosphorylates p34cdc2. We have begun to examine how unreplicated DNA generates the signal that inhibits MPF activation by testing the ability of naked single- and double-stranded DNA templates to inhibit mitosis, and by investigating the role of RCC1, a chromatin-associated protein required for the coupling of DNA replication and mitosis.

Animals↗

PubMed1992

RCC1, a regulator of mitosis, is essential for DNA replication.

Temperature-sensitive mutants in the RCC1 gene of BHK cells fail to maintain a correct temporal order of the cell cycle and will prematurely condense their chromosomes and enter mitosis at the restrictive temperature without having completed S phase. We have used Xenopus egg extracts to investigate the role that RCC1 plays in interphase nuclear functions and how this role might contribute to the known phenotype of temperature-sensitive RCC1 mutants. By immunodepleting RCC1 protein from egg extracts, we find that it is required for neither chromatin decondensation nor nuclear formation but that it is absolutely required for the replication of added sperm chromatin DNA. Our results further suggest that RCC1 does not participate enzymatically in replication but may be part of a structural complex which is required for the formation or maintenance of the replication machinery. By disrupting the replication complex, the loss of RCC1 might lead directly to disruption of the regulatory system which prevents the initiation of mitosis before the completion of DNA replication.

Animals↗

PubMed1990

Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus.

During cell division complete DNA replication must occur before mitosis is initiated. Using a cell-free extract derived from Xenopus eggs that oscillates between S phase and mitosis, we have investigated how completion of DNA synthesis is coupled to the initiation of mitosis. We find that Xenopus eggs contain a feedback pathway which suppresses mitosis until replication is completed and that activation of this inhibitory system is dependent on the presence of a threshold concentration of unreplicated DNA. We demonstrate that in the presence of unreplicated DNA the active feedback system inhibits initiation of mitosis by blocking the activation of MPF, a regulator of mitosis found in all eukaryotic cells. Our results demonstrate that the feedback system does not inhibit MPF activation by blocking the synthesis or accumulation of cyclin protein, a subunit of MPF, or by blocking association of cyclin with the cdc2 subunit of MPF. We propose that the feedback system blocks mitosis by maintaining MPF in an inactive state by modulating posttranslational modifications critical for MPF activation.

Animals↗

PubMed1989

On the coupling between DNA replication and mitosis.

The rapid, early cell divisions in Xenopus laevis embryos are driven by an inflexible oscillator that is not influenced by the state of the DNA. In contrast, mitosis in somatic cells can be prevented by blocking replication or by damaging the DNA through irradiation. We have investigated the transition from the rapid, early cell cycle to the slower, more somatic-like cell cycle that occurs after division twelve in developing Xenopus embryos, a stage called the mid-blastula transition (MBT). When aphidicolin, an inhibitor of DNA synthesis, was added to embryos just post-fertilization, the embryos continued to divide despite incomplete replication. Also, embryos incubated with aphidicolin from early times did not slow their cell cycles after division twelve as control embryos did, indicating a connection between the accumulation of DNA and the post-MBT timing of the cell cycle. However, incubation with hydroxyurea, an inhibitor of ribonucleotide reductase, resulted in an S phase arrest when the pools of dNTPs became depleted after division twelve. These experiments showed that the embryos had acquired the ability to arrest in S phase some time after the early divisions and before division thirteen. The acquisition of the ability to arrest in S phase did not depend upon new transcription. These experiments suggested that the number of nuclei present could be responsible for the extension of the cell cycle observed after the MBT. To investigate this, we added increasing concentrations of nuclei to an in vitro cell cycle system. We have shown that at high concentrations of nuclei the in vitro cycle is extended.

Animals↗