ATP hydrolysis and AMP kinase activities of nonstructural protein 2C of human parechovirus 1

The highly conserved picornavirus 2C proteins, thought to be involved in genome replication, contain three motifs found in NTPases/helicases of superfamily III. We report that human parechovirus 1 2C displays Mg2+-dependent ATP diphosphohydrolase activity in vitro, whereas other nucleoside triphosphates are not substrates for the hydrolysis. We also found that the 2C protein has an enzymatic activity that converts AMP to a corresponding diphosphate using ADP or ATP as a phosphate donor. In addition, we observed that ATP hydrolysis results in 2C autophosphorylation. These findings indicate that the parechovirus 2C protein has enzymatic activities, which may contribute to several functions in the viral replication cycle.     

Analyzing genes using closing and replicating circles

During the past two years, significant breakthroughs have been achieved in genetic analyses through the application of technologies based on analytical DNA-circularization reactions. Padlock probes and molecular inversion probes have enabled parallel, high-throughput single nucleotide polymorphism (SNP) genotyping at increased scales, whereas, at the other end of the analysis spectrum, DNA molecules in individual cells have been genotyped, in situ, using padlock probes and rolling-circle amplification (RCA). This review describes the recent developments in the technologies that use specific DNA circularization, coupled to DNA amplification through PCR or rolling-circle amplification, and addresses the great potential of these tools.     

High levels of Notch signaling down-regulate Numb and Numblike

Inhibition of Notch signaling by Numb is critical for many cell fate decisions. In this study, we demonstrate a more complex relationship between Notch and the two vertebrate Numb homologues Numb and Numblike. Although Numb and Numblike at low levels of Notch signaling negatively regulated Notch, high levels of Notch signaling conversely led to a reduction of Numb and Numblike protein levels in cultured cells and in the developing chick central nervous system. The Notch intracellular domain but not the canonical Notch downstream proteins Hes 1 and Hey 1 caused a reduction of Numb and Numblike. The Notch-mediated reduction of Numblike required the PEST domain in the Numblike protein and was blocked by the proteasome inhibitor MG132. Collectively, these observations reveal a reciprocal negative regulation between Notch and Numb/Numblike, which may be of relevance for stabilizing asymmetric cell fate switches and for tumor development.     

The Schizosaccharomyces pombe replication inhibitor Spd1 regulates ribonucleotide reductase activity and dNTPs by binding to the large Cdc22 subunit

Ribonucleotide reductase (RNR) is an essential enzyme that provides the cell with a balanced supply of deoxyribonucleoside triphosphates for DNA replication and repair. Mutations that affect the regulation of RNR in yeast and mammalian cells can lead to genetic abnormalities and cell death. We have expressed and purified the components of the RNR system in fission yeast, the large subunit Cdc22p, the small subunit Suc22p, and the replication inhibitor Spd1p. It was proposed (Liu, C., Powell, K. A., Mundt, K., Wu, L., Carr, A. M., and Caspari, T. (2003) Genes Dev. 17, 1130-1140) that Spd1 is an RNR inhibitor, acting by anchoring the Suc22p inside the nucleus during G1 phase. Using in vitro assays with highly purified proteins we have demonstrated that Spd1 indeed is a very efficient inhibitor of fission yeast RNR, but acting on Cdc22p. Furthermore, biosensor technique showed that Spd1p binds to the Cdc22p with a KD of 2.4 microM, whereas the affinity to Suc22p is negligible. Therefore, Spd1p inhibits fission yeast RNR activity by interacting with the Cdc22p. Similar to the situation in budding yeast, logarithmically growing fission yeast increases the dNTP pools 2-fold after 3 h of incubation in the UV mimetic 4-nitroquinoline-N-oxide. This increase is smaller than the increase observed in budding yeast but of the same order as the dNTP pool increase when synchronous Schizosaccharomyces pombe cdc10 cells are going from G1 to S-phase.     

Recovery and evolutionary analysis of complete integron gene cassette arrays from Vibrio

Background  

Integrons are genetic elements capable of the acquisition, rearrangement and expression of genes contained in gene cassettes. Gene cassettes generally consist of a promoterless gene associated with a recombination site known as a 59-base element (59-be). Multiple insertion events can lead to the assembly of large integron-associated cassette arrays. The most striking examples are found in Vibrio, where such cassette arrays are widespread and can range from 30 kb to 150 kb. Besides those found in completely sequenced genomes, no such array has yet been recovered in its entirety. We describe an approach to systematically isolate, sequence and annotate large integron gene cassette arrays from bacterial strains.     

Pannexin: to gap or not to gap, is that a question?

Vertebrates express two families of gap junction proteins: the well characterized connexins and the recently discovered pannexins. The latter are related to invertebrate innexins. Here we present the hypothesis that pannexins, rather than providing a redundant system to gap junctions formed by connexins, exert a physiological role as nonjunctional membrane channels. Specifically, we propose that pannexins can serve as ATP release channels. This function presumptively is also performed by innexins in invertebrates, in addition to their traditional gap junction role.     

Intranuclear 3′-phosphoinositide metabolism and Akt signaling: New mechanisms for tumorigenesis and protection against apoptosis?

Lipid second messengers, particularly those derived from the polyphosphoinositide metabolism, play a pivotal role in multiple cell signaling networks. Phosphoinositide 3-kinase (PI3K) generate 3'-phosphorylated inositol lipids that are key players in a multitude of cell functions. One of the best characterized targets of PI3K lipid products is the serine/threonine protein kinase Akt (protein kinase B, PKB). Recent findings have implicated the PI3K/Akt pathway in tumorigenesis because it stimulates cell proliferation and suppresses apoptosis. However, it was thought that this signal transduction network would exert its carcinogenetic effects mainly by operating in the cytoplasm. Evidence accumulated over the past 15 years has highlighted the presence of an autonomous nuclear inositol lipid cycle, and strongly suggests that lipid molecules are important components of signaling pathways operating at the nuclear level. PI3K, its lipid product phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P3), and Akt have been identified within the nucleus and recent data suggest that they counteract apoptosis also by operating in this cell compartment through a block of caspase-activated DNase and inhibition of chromatin condensation. In this review, we shall summarize the most updated and intriguing findings about nuclear PI3K/PtdIns(3,4,5)P3/Akt in relationship with tumorigenesis and suppression of apoptotic stimuli.