No abstract

We have prepared an mAb specific for a human cell surface component (termed anti-Fas mAb). Anti-Fas shows cell-killing activity that is indistinguishable from the cytolytic activity of TNF. Fas antigen was characterized by western blotting, indicating that Fas antigen is a cell surface protein with a molecular weight of 200,000, which is different from the molecular weight of TNF-R. Fas antigen, however, is co-downregulated with the TNF-R when cells sensitive to the cytolytic activity of TNF are incubated with either TNF or anti-Fas. In contrast, Fas antigen on cells insensitive to TNF is not co-downregulated with the TNF-R. We suggest that the cell-killing activity of TNF is mediated by Fas antigen associated with the TNF-R.

Semaphorin 3A is a chemorepulsive axonal guidance molecule that depolymerizes the actin cytoskeleton and collapses growth cones of dorsal root ganglia neurons. Here we investigate the role of LIM-kinase 1, which phosphorylates an actin-depolymerizing protein, cofilin, in semaphorin 3A-induced growth cone collapse. Semaphorin 3A induced phosphorylation and dephosphorylation of cofilin at growth cones sequentially. A synthetic cell-permeable peptide containing a cofilin phosphorylation site inhibited LIM-kinase in vitro and in vivo, and essentially suppressed semaphorin 3A-induced growth cone collapse. A dominant-negative LIM kinase, which could not be activated by PAK or ROCK, suppressed the collapsing activity of semaphorin 3A. Phosphorylation of cofilin by LIM-kinase may be a critical signaling event in growth cone collapse by semaphorin 3A.

Cdc7 kinase, conserved from yeasts to human, plays important roles in DNA replication. However, the mechanisms by which it stimulates initiation of DNA replication remain largely unclear. We have analyzed phosphorylation of MCM subunits during cell cycle by examining mobility shift on SDS-PAGE. MCM4 on the chromatin undergoes specific phosphorylation during S phase. Cdc7 phosphorylates MCM4 in the MCM complexes as well as the MCM4 N-terminal polypeptide. Experiments with phospho-amino acid-specific antibodies indicate that the S phase-specific mobility shift is due to the phosphorylation at specific N-terminal (S/T)(S/T)P residues of the MCM4 protein. These specific phosphorylation events are not observed in mouse ES cells deficient in Cdc7 or are reduced in the cells treated with siRNA specific to Cdc7, suggesting that they are mediated by Cdc7 kinase. The N-terminal phosphorylation of MCM4 stimulates association of Cdc45 with the chromatin, suggesting that it may be an important phosphorylation event by Cdc7 for activation of replication origins. Deletion of the N-terminal non-conserved 150 amino acids of MCM4 results in growth inhibition, and addition of amino acids carrying putative Cdc7 target sequences partially restores the growth. Furthermore, combination of MCM4 N-terminal deletion with alanine substitution and deletion of the N-terminal segments of MCM2 and MCM6, respectively, which contain clusters of serine/threonine and are also likely targets of Cdc7, led to an apparent nonviable phenotype. These results are consistent with the notion that the N-terminal phosphorylation of MCM2, MCM4, and MCM6 may play functionally redundant but essential roles in initiation of DNA replication.

Every cyanobacterial species possesses multiple genes encoding AbrB-like transcriptional regulators (cyAbrBs) distinct from those conserved among other bacterial species. In this study, two genes encoding cyAbrBs in Synechocystis sp. PCC 6803, sll0359 and sll0822, were insertionally disrupted in order to examine their physiological roles. A fully segregated disrupted mutant of sll0822 (Dsll0822 mutant) but not of sll0359 was obtained, although both mutants exhibited similar phenotypes (i.e. decreases in growth rate and pigment content). The growth rate of the Dsll0822 mutant was low under any condition, but the low pigment content could be partially recovered by nitrate supplementation of the medium. DNA microarray and RNA-blot analyses revealed that the level of expression of a part of the NtcA regulon, such as urtA, amt1, glnB, sigE, and the nrt operon, was significantly decreased in the Dsll0822 mutant, although the induction of these genes upon nitrogen depletion was still observed to some extent. Sll0822 seems to work in parallel with NtcA to achieve flexible regulation of the nitrogen uptake system. The Sll0822 protein exists mainly in a dimeric form in vivo, and the amount of the protein was not affected by nitrogen availability. This observation, together with the low binding specificity of the purified histidine-tagged Sll0822 protein, implies that the activity of Sll0822 may be posttranslationally modulated in Synechocystis cells.