Dynamic changes in subnuclear NP95 location during the cell cycle and its spatial relationship with DNA replication foci

We determined the expression and subcellular localization of nuclear protein NP95 during the cell cycle in mouse 3T3 cells. The levels of NP95 mRNA and protein were extremely low in quiescent cells; however, stimulation with 10% serum increased their expressions in a time course similar to that of the late growth-regulated gene proliferating cell nuclear antigen (PCNA). Subnuclear location of NP95 dynamically changed during the cell cycle. Double immunostaining for NP95 and chromatin-bound PCNA, a marker of DNA replication sites, revealed that NP95 was almost exclusively colocalized with chromatin-bound PCNA throughout the nucleus in early S phase and partly in mid-S phase. Distinct localization of the two proteins, however, became evident in mid-S phase, and thereafter, many chromatin-bound PCNA foci not carrying NP95 foci could be detected. In G2 phase, nodular NP95 foci were still identified without any chromatin-bound PCNA foci. Chromatin-bound PCNA was observed as a pre-DNA replication complex at the G1/S boundary synchronized by hydroxyurea treatment, while NP95 was detected in nucleolar regions as unique large foci. There was no significant redistribution of NP95 foci shortly after DNA damage by gamma-irradiation. Nodular NP95 foci characteristically seen in G2 phase were also detected in G2-arrested cells following gamma-irradiation. Taken together, our results indicate that NP95 is assigned to a late growth-regulated gene and suggest that NP95 does not take a direct part in DNA replication as part of the DNA synthesizing machinery, like PCNA, but is presumably involved in other DNA replication-linked nuclear events.     

A novel protein-conjugating system for Ufm1, a ubiquitin-fold modifier

Several studies have addressed the importance of various ubiquitin-like (UBL) post-translational modifiers. These UBLs are covalently linked to most, if not all, target protein(s) through an enzymatic cascade analogous to ubiquitylation, consisting of E1 (activating), E2 (conjugating), and E3 (ligating) enzymes. In this report, we describe the identification of a novel ubiquitin-fold modifier 1 (Ufm1) with a molecular mass of 9.1 kDa, displaying apparently similar tertiary structure, although lacking obvious sequence identity, to ubiquitin. Ufm1 is first cleaved at the C-terminus to expose its conserved Gly residue. This Gly residue is essential for its subsequent conjugating reactions. The C-terminally processed Ufm1 is activated by a novel E1-like enzyme, Uba5, by forming a high-energy thioester bond. Activated Ufm1 is then transferred to its cognate E2-like enzyme, Ufc1, in a similar thioester linkage. Ufm1 forms several complexes in HEK293 cells and mouse tissues, revealing that it conjugates to the target proteins. Ufm1, Uba5, and Ufc1 are all conserved in metazoa and plants but not in yeast, suggesting its potential roles in various multicellular organisms.     

(+)-3-[2-(Benzo[b]thiophen-2-yl)-2-oxoethyl]-1-azabicyclo[2.2.2]octane as potent agonists for the alpha7 nicotinic acetylcholine receptor

A series of 3-substituted 1-azabicyclo[2.2.2]octanes was discovered as the alpha7 nicotinic acetylcholine (alpha7) receptor agonists. It was found that (+)-3-[2-(benzo[b]thiophen-2-yl)-2-oxoethyl]-1-azabicyclo[2.2.2]octane (+)-15b has potent agonistic activity for the alpha7 receptor.     

Cutting edge: profile of chemokine receptor expression on human plasma cells accounts for their efficient recruitment to target tissues

We systematically examined the repertoire of chemokine receptors expressed by human plasma cells. Fresh bone marrow plasma cells and myeloma cells consistently expressed CXCR4, CXCR6, CCR10, and CCR3. Accordingly, plasma cells responded to their respective ligands in chemotaxis and very late Ag-4-dependent cell adhesion to fibronectin. Immobilized CXC chemokine ligand (CXCL)16, a novel transmembrane-type chemokine and CXCR6 ligand, also directly induced adhesion of plasma cells without requiring G(alpha i) signaling or divalent cations. Furthermore, we revealed consistent expression of CXCL12 (CXCR4 ligand), CXCL16 (CXCR6 ligand), and CC chemokine ligand 28 (CCR10 and CCR3 ligand) in tissues enriched with plasma cells including bone marrow, and constitutive expression of CXCL12, CXCL16, and CC chemokine ligand 28 by cultured human bone marrow stromal cells. Collectively, plasma cells are likely to be recruited to bone marrow and other target tissues via CXCR4, CXCR6, CCR10, and CCR3. CXCR6 may also contribute to tissue localization of plasma cells through its direct binding to membrane-anchored CXCL16.     

The association of cyclin A and cyclin kinase inhibitor p21 in response to gamma-irradiation requires the CDK2 binding region, but not the Cy motif

The cyclin kinase inhibitor p21 associates with and inhibits cyclin-CDKs to retard the progress of the cell cycle in response to DNA damage. The recognition sites for cyclin binding on the various cell cycle-related molecules have been identified as RXL motifs. In the case of p21, the dependence of the Cy1 (18CRRL) or Cy2 (154KRRL) motifs on cyclin E, but not on cyclin A has been demonstrated by in vitro experiments. In this study, to clarify the mechanism of p21 association with cyclin A, we constructed a p21 expression system in mammalian cells. After transfection with an expression vector containing cDNA of various p21-mutants, cells were irradiated with 10 Gy of gamma-rays to introduce DNA damage, followed by quantification of the p21-cyclin A association. The p21-mutant constructs were single or multiple deletions in Cy1, Cy2, and the CDK2 binding region, and a nonphosphorylatable alanine mutant of the C-terminal phosphorylation site. We demonstrated that the association of p21 and cyclin A in response to gamma-irradiation requires the CDK binding region, 49-71 aa, but not the Cy motifs. We believe the mechanism by which p21 inhibits cyclin-CDKs is distinct in each phase of the cell cycle. Furthermore, the increase in the association of p21 and cyclin A was not correlated with the levels of p21. This suggests that DNA damage triggers a signal to the p21 region between 21 and 96 aa to allow cyclin A association.     

Sequence analysis of 193.4 and 83.9 kbp of mouse and chicken genomic DNAs containing the entire Prkdc (DNA-PKcs) gene

The catalytic subunit of DNA-dependent protein kinase plays critical roles in nonhomologous end joining in repair of DNA double-strand breaks and V(D)J recombination. In addition to the SCID phenotype, it has been suggested that the molecule contributes to the polymorphic variations in radiosensitivity and susceptibility to cancer in mouse strains. Here we show the nucleotide sequence of approximately 193-kbp and 84-kbp genomic regions encoding the entire Prkdc gene (also known as DNA-PKcs) in the mouse and chicken, respectively. A large retroposon was found in intron 51 in the mouse but not in the human or chicken. Comparative analyses of the genome strongly suggested that the region contains only two genes for Prkdc and Mcm4; however, several conserved sequences and cis elements were also predicted.     

Cutting edge: a novel role for Fas ligand in facilitating antigen acquisition by dendritic cells

Fas ligand (FasL)-expressing tumor cells are found to effectively mediate rejection of the coinoculated FasL negative parental cells while having no effect on the growth of histologically distinct tumor cells. These observations indicate that FasL induces a specific immune response against Ag derived from FasL-bearing tumors and suggest a possible role for FasL in tumor Ag presentation. Indeed, tumor cells expressing FasL can efficiently interact with dendritic cells (DCs) and this interaction requires the expression of membrane-bound FasL on tumors and Fas on DCs. Moreover, DCs cocultured with FasL-expressing tumors are able to elicit a tumor-specific immune response in vivo, suggesting that DCs acquire tumor Ag during the Fas/FasL-mediated DC-tumor contact. These results identify a novel role for FasL in augmenting tumor-DC interactions and subsequent tumor Ag acquisition by DCs, and suggest that FasL-expressing tumor cells could be used to generate tumor-specific DC vaccines.     

Structure of genes for Hsp30 from the white-rot fungus Coriolus versicolor and the increase of their expression by heat shock and exposure to a hazardous chemical

We isolated and analysed two genomic DNAs that encode the heat-shock protein Hsp30 from Coriolus versicolor. The amino acid sequences substitute only three amino acid substitutions. The promoter regions contain the consensus heat-shock element, a xenobiotic-response element, a stress-response element, and a metal-response element. The levels of mRNAs for Hsp30 increased markedly after exposure of C. versicolor to pentachlorophenol and levels were higher than those after heat shock.     

Expression of a gene for Mn-peroxidase from Coriolus versicolor in transgenic tobacco generates potential tools for phytoremediation

In efforts aimed at the detoxification of contaminated areas, plants have many advantages over bacteria and fungi. We are attempting to enhance the environmental decontamination functions of plants by transferring relevant genes from microorganisms. When the gene for Mn-peroxidase (MnP) from Coriolus versicolor was expressed in transgenic tobacco plants, one line (designated fMnP21) expressed MnP activity at levels 54-fold higher than in control lines. When undamaged roots of transgenic plants were applied to liquid medium supplemented with 250 microM pentachlorophenol (PCP), the decrease in the level of PCP in fMnP21 (86% reduction) was about 2-fold higher than that in control lines (38% reduction). Expression of the gene for MnP in the transgenic plants had no obvious negative effects on their vegetative and sexual growth. Our system should contribute to the development of novel methods for the removal of hazardous chemicals from contaminated environments using transgenic plants.     

Molecular cloning of Chinese hamster ceramide glucosyltransferase and its enhanced expression in peroxisome-defective mutant Z65 cells

To clarify the metabolic bases of characteristic increases in the concentrations of glucosylceramide (CMH) and GM3 in peroxisome-defective mutant Chinese hamster ovary (CHO) cells (Z65), we measured the ceramide glucosyltransferase (CGT) and beta-glucosidase activities in Z65 and CHO-K1 cells, and found that the former enzyme was responsible for the accumulation of CMH in Z65 cells. Inhibition of CGT by D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) caused a marked reduction in a incorporation of [3-14C]serine to CMH in both CHO-K1 and Z65 cells, but resulted in the accumulation of ceramide in Z65 cells in a concentration higher than that in CHO-K1 cells. Then, we cloned the cDNA encoding CGT from CHO-K1 cells, which exhibited sequence homology with the human gene product (98.7%). Northern blot analysis of CGT revealed increased expression of it in Z65 cells compared with that in CHO-K1 cells, which probably caused the simultaneous increase in GM3. With an immunohistochemical procedure, GM3 was found to be more strongly expressed in the cell membrane of Z65 cells than in CHO-K1 cells.