Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation

Messenger RNA decay, which is a regulated process intimately linked to translation, begins with the deadenylation of the poly(A) tail at the 3' end. However, the precise mechanism triggering the first step of mRNA decay and its relationship to translation have not been elucidated. Here, we show that the translation termination factor eRF3 mediates mRNA deadenylation and decay in the yeast Saccharomyces cerevisiae. The N-domain of eRF3, which is not necessarily required for translation termination, interacts with the poly(A)-binding protein PABP. When this interaction is blocked by means of deletion or overexpression of the N-domain of eRF3, half-lives of all mRNAs are prolonged. The eRF3 mutant lacking the N-domain is deficient in the poly(A) shortening. Furthermore, the eRF3-mediated mRNA decay requires translation to proceed, especially ribosomal transition through the termination codon. These results indicate that the N-domain of eRF3 mediates mRNA decay by regulating deadenylation in a manner coupled to translation.     

Independent roles of XRCC1's two BRCT motifs in recovery from methylation damage

XRCC1 is known to be involved in base excision repair (BER)/single-strand break repair (SSBR) through interaction with other BER enzymes. Hypersensitivity of XRCC1-deficient cells against alkylating agents has been explained by loss of interaction with BER proteins. XRCC1 is a unique DNA repair protein containing two BRCT motifs, recently identified in several DNA repair and cell cycle regulating proteins. To study the function(s) of the two BRCT motifs of the XRCC1 protein, we established CHO EM9 (XRCC1-null) cells expressing XRCC1 protein altered in either one of the two BRCT motifs. Colony-forming ability after methyl methanesulfonate (MMS) treatment was dependent on the BRCT-a motif, but not on the BRCT-b motif. Surprisingly, reduced BER/SSBR rate in vivo, measured by an alkaline comet assay, was observed in the BRCT-b motif-deficient cells, while the BRCT-a motif-deficient cells showed the repair rate comparable with the wild-type (WT) cells. The BRCT-a motif-mutated cells, instead, showed deficiency in initiation of DNA replications after MMS treatment. Furthermore, we found that XRCC1 is multiply phosphorylated in vivo and hyperphosphorylation of XRCC1 after MMS treatment is dependent on the BRCT-a motif. These data suggest a new function dependent on the integrity of the BRCT-a motif of XRCC1 in recovery from MMS-induced damage.     

Identification of a mitochondrial nucleoside diphosphate kinase from the green alga Dunaliella tertiolecta

We isolated a full-length cDNA encoding a nucleoside diphosphate (NDP) kinase from a Dunaliella tertiolecta cDNA library by homology cloning and rapid amplification of cDNA ends-PCR. The cDNA sequence, consisting of 840 bp, contained an open reading frame coding for a 221-amino acid protein. The predicted 24-kDa protein was named DtNDK1. It possesses all the residues involved in nucleotide binding and catalysis and, in its long N-terminus, contains putative mitochondrial targeting peptides. The full-length pre-protein expressed in Escherichia coli as a recombinant N-terminally His-tagged protein was retained in inclusion bodies, totally devoid of NDP kinase activity. Upon expression in yeast cells, the full-length protein His-tagged at the C-terminus was found processed in a soluble form that was lacking the first 67 amino acids from the N-terminus. The mature protein, which was purified by affinity chromatography to near homogeneity, showed NDP kinase activity. Confocal microscopy on yeast cells expressing the recombinant protein revealed the specific mitochondrial localization of DtNDK1 labeled at the C-terminus with green fluorescent protein.     

Expansion of the cell plate in plant cytokinesis requires a kinesin-like protein/MAPKKK complex

The tobacco mitogen-activated protein kinase kinase kinase NPK1 regulates lateral expansion of the cell plate at cytokinesis. Here, we show that the kinesin-like proteins NACK1 and NACK2 act as activators of NPK1. Biochemical analysis suggests that direct binding of NACK1 to NPK1 stimulates kinase activity. NACK1 is accumulated specifically in M phase and colocalized with NPK1 at the phragmoplast equator. Overexpression of a truncated NACK1 protein that lacks the motor domain disrupts NPK1 concentration at the phragmoplast equator and cell plate formation. Incomplete cytokinesis is also observed when expression of NACK1 and NACK2 is repressed by virus-induced gene silencing and in embryonic cells from Arabidopsis mutants in which a NACK1 ortholog is disrupted. Thus, we conclude that expansion of the cell plate requires NACK1/2 to regulate the activity and localization of NPK1.     

Changes in the fluctuation of interbeat intervals in spontaneously beating cultured cardiac myocytes: experimental and modeling studies

 Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically, and have the properties of a non-linear oscillator. In this study, we have analyzed the relationship between the fluctuation of contraction rhythm of spontaneously beating cultured cardiac myocytes, and the coupling strength among them. The coefficient of variation of contraction intervals increased transiently in the early stages of incubation, and then decreased almost monotonically with time. The contraction rhythm of the myocytes became synchronized in the late stage of the culture. The day on which synchronization occurred almost coincided with the day when the coefficient of variation reached its lowest value. In addition, we have performed a mathematical analysis using interacting Bonhoeffer–van der Pol oscillators to clarify the mechanisms underlying the changes in the fluctuation of contraction rhythm with time. As the coupling strength among oscillators increased, the coefficient of variation of oscillation periods increased temporarily, but then decreased rapidly when the oscillators showed synchronization. These results suggest that the changes in the fluctuation of beating rhythm result from the increase in strength of electrical coupling among spontaneously beating cardiac myocytes. Received: 10 August 2000 / Accepted in revised form: 19 August 2001     

Polycystin-2 is an intracellular calcium release channel

Polycystin-2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease (ADPKD), is the prototypical member of a subfamily of the transient receptor potential (TRP) channel superfamily, which is expressed abundantly in the endoplasmic reticulum (ER) membrane. Here, we show by single channel studies that polycystin-2 behaves as a calcium-activated, high conductance ER channel that is permeable to divalent cations. Epithelial cells overexpressing polycystin-2 show markedly augmented intracellular calcium release signals that are lost after carboxy-terminal truncation or by the introduction of a disease-causing missense mutation. These data suggest that polycystin-2 functions as a calcium-activated intracellular calcium release channel in vivo and that polycystic kidney disease results from the loss of a regulated intracellular calcium release signalling mechanism.     

Two forms of membrane-bound sphingosine kinase in Tetrahymena and activity changes during growth and the cell cycle

Sphingosine kinase is responsible for the formation of sphingosine-1-phosphate, a sphingolipid mediator with important roles in numerous physiological processes. The sphingosine kinase activity of Tetrahymena pyriformis was recovered predominantly in the particulate fraction and it could be solubilised in 1% beta-octylglucoside. Anion-exchange chromatography resolved the beta-octylglucoside-solubilised sphingosine kinase activity into two peaks corresponding to proteins of Mr 140,000 and 80,000 respectively, as determined by subsequent size exclusion chromatography on Superdex 200. N,N-dimethylsphingosine did not inhibit the sphingosine kinase activity in either fraction, whereas D,L-threo-dihydrosphingosine inhibited sphingosine phosphorylation by the Mr 80,000 kinase but had no effect on the Mr 140,000 kinase activity. The activities also showed different stimulatory responses to Triton X-100 or NaCl. Overall, the results suggest the existence in Tetrahymena of two distinct membrane-associated sphingosine kinases. The kinase activity determined at the different culture stages showed a transient elevation at the mid-logarithmic phase. Further, the sphingosine kinase activity was examined during the synchronous cell division induced by cyclic heat treatments in T. pyriformis. We report for the first time that the sphingosine kinase activity greatly increased at 30 to 45 min after the end of heat treatment prior to the synchronous cell division (75 min), suggesting that the activity changes were associated with the cell cycle and that the up-regulated sphingosine kinase activity would be required for the initiation of the oncoming synchronous cell division in Tetrahymena cells.     

Extrachromosomal transposition of the transposable element Minos in embryos of the cricket Gryllus bimaculatus

Effective germline transformation of insects has been shown to depend on the right choice of transposon system and selection marker. In this study the promoter region of a Gryllus cytoplasmic actin (GbA3/4) gene was isolated and characterized, and was used to drive the expression of Minos transposase in embryos of the cricket Gryllus bimaculatus. Active Minos transposase was produced in these embryos as monitored through established transposon excision and interplasmid transposition assays. In contrast, Drosophila melanogaster hsp70 promoter, previously used to express Minos transposase in a number of insect species and insect cell lines, failed to produce any detectable Minos transposase activity, as recorded by using the very sensitive transposon excision assay. In addition, the GbA3/4 promoter was found to drive expression of enhanced green fluorescent protein (eGFP) predominantly in vitellophages of the developing Gryllus eggs when a plasmid carrying a GbA3/4 promoter-eGFP fusion gene was transiently injected into embryos. These results strongly support the use of Minos transposons marked with the GbA3/4 promoter-eGFP for the genetic transformation of this emerging model insect species.     

Mast cell chymase regulates dermal mast cell number in mice

Chymase inhibitor reduced the increase in the number of dermal mast cells in 2,4-dinitrofluorobenzene-induced dermatitis in a dose-dependent manner. Intradermal injection of human chymase to mouse ear significantly increased histamine content, the marker for mast cell number in the skin. These results suggest that chymase released by mast cells may participate in local mast cell accumulation in a positive feedback fashion. Immunohistochemical analysis revealed that the intradermal injection of chymase reduces expression of stem cell factor (SCF) on surface of the skin keratinocytes. In addition, incubation of human keratinocytes with chymase in vitro resulted in release of SCF into the culture medium. Since soluble SCF is thought to regulate mast cell number, the chymase-induced mast cell accumulation may occur via the ability of chymase to process membrane-bound SCF on the epidermal keratinocytes.     

Salts and glycine increase reversibility and decrease aggregation during thermal unfolding of ribonuclease-A

Ribonuclease-A (RNase-A) has been a model for studying protein folding and unfolding. However, we show here that its unfolding at neutral pH is complicated by aggregation. Circular dichroism thermal scans showed that reversibility of RNase-A after heating is only about 63%. In accordance with this observation, native-polyacrylamide gel electrophoresis of the sample heated at 75 degrees C showed formation of soluble oligomers. Ammonium sulfate at 0.4 M caused about a 3 degrees C higher melting temperature and nearly complete reversibility, while glycine and NaCl at 0.4 M significantly increased reversibility and decreased aggregation without affecting melting temperature. These results demonstrate that aggregation makes thermal unfolding of RNase-A at least partially irreversible and salts and glycine increase reversibility and decrease aggregation.