Recombinant sea urchin flagellar adenylate kinase

Adenylate kinase (AK) is localized in sea urchin sperm flagella and embryonic cilia. To investigate sea urchin Strongylocentrotus purpuratus AK (SpAK) enzymatic characteristics, the full-length recombinant protein of 130 kDa (SpAKr) and each of its three catalytic domains were expressed in Escherichia coli. Although the full-length SpAK had high enzymatic activity, each of the three catalytic domains had no activity. The Km for ATP synthesis from ADP was 0.23 mM and the Vmax was 4.51 mumol ATP formed per minute per milligram of protein. The specific AK inhibitor, Ap5A, blocks SpAKr enzymatic activity with an IC50 of 0.53 microM. The pH optimum for SpAKr is 8.1, as compared to 7.7 for the natural SpAK. Calcium inhibits SpAKr activity in a dose-dependent manner. Although SpAKr has three cAMP-dependent protein kinase phosphorylation sites, and can be phosphorylated in vitro, the enzymatic kinetics after phosphorylation are not significantly altered. SpAK and Chlamydomonas flagellar AKs are the only AKs with three catalytic sites. Further study of the SpAKr will aid in understanding the active site of this interesting and important ATP synthase.     

Chromosome instability and kinetochore dysfunction

Chromosomal instability (CIN) has been recognized as a hallmark of human cancer and is caused by continuous chromosome missegregation during mitosis. Proper chromosome segregation requires a physical connection between spindle microtubules and centromeric DNA and this attachment occurs at proteinaceous structures called kinetochore. Thus, defect in kinetochore function is a candidate source for CIN and the generation of aneuploidy. Recently, a number of kinetochore components have been shown to be mutated and/or aberrantly expressed in human cancers, which suggests an important role of kinetochore for CIN and carcinogenesis. In this article, we will discuss about how kinetochore dysfunction causes CIN and might lead to the development of cancer.     

Involvement of aldolase A in X-ray resistance of human HeLa and UV(r)-1 cells

To find novel proteins involved in radio-resistance of human cells, we searched for nuclear proteins, whose expression levels alter after X-ray irradiation in HeLa cells, using agarose fluorescent two-dimensional differential gel electrophoresis following mass spectrometry. We identified 6 proteins, whose levels were increased in nuclei 24 h after irradiation at 5 Gy, including aldolase A. Nuclear aldolase A levels increased twofold after the irradiation, however, total aldolase A levels did not change. When the expression of aldolase A was suppressed by its specific siRNA, sensitization of the suppressed cells to X-ray-induced cell death was observed. In addition, UV^r-1 cells with higher aldolase A expression exhibited lower sensitivity to X-ray-induced cell death than the parental RSa cells with lower aldolase A expression. These results suggest that aldolase A may play a role in the radio-response of human cells, probably in nuclei, in addition to its glycolytic role in the cytosol.     

Novel and orally bioavailable inducible nitric oxide synthase inhibitors: synthesis and evaluation of optically active 4,5-dialkyl-2-iminoselenazolidine derivatives

We have previously reported that (4R,5R)-5-ethyl-2-imino-4-methylthiazolidine (3) strongly inhibits inducible nitric oxide synthase (iNOS). In a successive search for strong and selective iNOS inhibitors, we, herein, describe the synthesis of the selenium analogue of 3 (4: ES-2133) and its related optically active compounds and examine their in vitro and in vivo inhibitory activity against iNOS. In addition, an alternative synthetic method to the selected compound 4 and its pharmacokinetic profile is also reported.     

Molecular characterization of ENU mouse mutagenesis and archives

The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.     

Upregulation of thromboxane synthase in human colorectal carcinoma and the cancer cell proliferation by thromboxane A2

Tumor growth of colorectal cancers accompanies upregulation of cyclooxygenase-2, which catalyzes a conversion step from arachidonic acid to prostaglandin H(2) (PGH(2)). Here, we compared the expression levels of thromboxane synthase (TXS), which catalyzes the conversion of PGH(2) to thromboxane A(2) (TXA(2)), between human colorectal cancer tissue and its accompanying normal mucosa. It was found that TXS protein was consistently upregulated in the cancer tissues from different patients. TXS was also highly expressed in human colonic cancer cell lines. Depletion of TXS protein by the antisense oligonucleotide inhibited proliferation of the cancer cells. This inhibition was rescued by the direct addition of a stable analogue of TXA(2). The present results suggest that overexpression of TXS and subsequent excess production of TXA(2) in the cancer cells may be involved in the tumor growth of human colorectum.