Analyses of spontaneous pink mutant events in the stamen hairs of Tradescantia clone BNL 4430 cultivated in a nutrient solution circulating growth chamber

In order to obtain more fundamental data on Tradescantia clone BNL 4430, one of the most suitable testers for environmental mutagens, the occurrences of spontaneous somatic pink mutations in the stamen hairs were scored for 52 weeks from 12 December 1998 to 10 December 1999, cultivating the young inflorescence-bearing shoots with roots in a nutrient solution circulating (NSC) growth chamber. The environmental conditions in the chamber were 22.0+/-0.5 degrees C during the 16h day with the light intensity of 7.5klx from white fluorescent tubes, and 20.0+/-0.5 degrees C at night. During the scoring period, 697,443 stamen hairs with an average cell number of 25.36 were observed and 2642 pink mutant events (PMEs) were detected. The overall spontaneous mutation frequency was 1.56+/-0.03 PMEs per 10(4) hair-cell divisions, and the frequency was significantly lower in May, July and August and significantly higher in November and December. By analyzing the sectoring patterns of 1856 PMEs (70.25% of PMEs detected), the most of 172 cases of multiple (two to five) pink sectors observed in the same hairs (scored as 232 PMEs for calculating mutation frequency) were found to be the results of events involving somatic recombinations occurred in single cells or cell lineages, rather than those of two or more independent somatic mutations occurred in different cells. This finding clearly shows the significance of somatic recombinations in producing such multiple sectors (382 sectors in total) which occupied 19.0% of the 2006 pink sectors in total analyzed. Somatic recombinations were considered to be playing a significant role also in producing single PMEs in the stamen hairs.     

Induction of adherent activity in mastocytoma P-815 cells by the cooperation of two prostaglandin E2 receptor subtypes,EP3 and EP4

In this study, we investigated the role of PGE(2) in mouse mastocytoma P-815 cell adhesion to extracellular matrix proteins (ECMs) in vitro. We report that PGE(2) accelerated ProNectin F(TM) (a proteolytic fragment of fibronectin)-mediated adhesion, which was abolished by addition of the GRGDS peptide, an inhibitor of the RDG binding site of ProNectin F(TM). We show that the cAMP level and cAMP-regulated protein kinase (PKA) activity are critical mediators of this PGE(2) effect, because the cell-permeable cAMP analogue 8-Br-cAMP accelerated P-815 cell adhesion to ProNectin F(TM) and the pharmacological inhibitor of PKA, H-89, blocked PGE(2)-mediated adhesion. Consistent with mRNA expression of the G(s)-coupled EP4- and G(i)-coupled EP3-PGE receptor subtypes, P-815 cell adhesion was accelerated by treatment with a selective EP4 agonist, ONO-AE1-329, but not a selective EP1/EP3 agonist, sulprostone. However, simultaneous treatment with ONO-AE1-329 and sulprostone resulted in augmentation of both the cAMP level and cell adhesion. The augmentation of EP3-mediated cAMP synthesis was dose-dependent, without affecting the half-maximal concentration for EP4-mediated G(s)-activity, which was inhibited by a G(i) inhibitor, pertussis toxin. In conclusion, these findings suggest that PGE(2) accelerates RGD-dependent adhesion via cooperative activation between EP3 and EP4 and contributes to the recruitment of mast cells to the ECM during inflammation.     

Inhibition of uracil DNA glycosylase by an oxacarbenium ion mimic

We have investigated the inhibition of the DNA repair enzyme uracil DNA glycosylase (UDG) by an 11-mer oligonucleotide (AIA) containing a cationic 1-aza-deoxyribose (I) residue designed to be a stable mimic of the high-energy oxacarbenium ion reaction intermediate [Werner, R. M., and Stivers, J. T. (2000) Biochemistry 39, 14054-14064]. Inhibition kinetics and direct binding studies indicate that AIA binds weakly to the free enzyme (K(D) = 2 microM) but binds 4000-fold more tightly to the enzyme-uracil anion (EU) product complex (K(D) = 500 pM). The importance of the positive charge on the 1-nitrogen in binding is established by the observation that AIA binds >30 000-fold more tightly to the EU complex than the corresponding neutral tetrahydrofuran (F) abasic site product analogue (AFA). The unusual inhibition mechanism for AIA results in a time dependence that resembles slow-onset inhibition even though the apparent on-rate of the inhibitor for the EU(-) binary product complex is moderate (1 microM(-1) x s(-1)). Accordingly, the low K(D) of AIA for the EU complex is largely due its very slow off-rate (5 x 10(-4) x s(-1)). These results support previous kinetic isotope effect measurements that indicate UDG stabilizes a discrete oxacarbenium ion-uracil anion intermediate. This oxacarbenium ion mimic represents the tightest binding inhibitor of UDG yet identified.     

A yeast mitochondrial ATPase inhibitor interacts with three proteins that are easy to dissociate from the mitochondrial inner membrane

A mitochondrial ATPase inhibitor is a 7.4 kDa protein that regulates the catalytic activity of ATP synthase (F(1)F(o)-ATPase). In the present study, we examined the binding sites of the inhibitor on the mitochondrial membrane using chemical cross-linkers, disuccinimidyl suberate (DSS) and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Most of the inhibitors were recovered from the inner membrane fraction of mitochondria, indicating that the inhibitor binds to the membrane. Seven different cross-linked products that reacted with the antibody against the inhibitor were detected. The apparent molecular masses of the products were 61, 58, 47, 41, 28, 27, and 26 kDa. The 61 and 58 kDa products were attributed to the inhibitor+alpha and inhibitor+beta adducts on immunoblotting. The proteins cross-linked to the inhibitor in the 28, 27, and 26 kDa products were distinguished from subunit 4 (23 kDa), oligomycin sensitivity conferring protein (21 kDa), and subunit d (20 kDa) of F(1)F(o)-ATPase by analysis of the cross-linked products of mutant mitochondria in which the three proteins were replaced by hemagglutinin-tagged versions. The 28, 27, and 26 kDa products could be gradually dissociated from the mitochondrial membrane by increasing the salt concentration. These results shows that the endogenous inhibitor binds not only to the catalytic part of the enzyme, but also to the 19-21 kDa proteins that loosely associate with the mitochondrial inner membrane.     

Structural analysis of N-linked glycans in Caenorhabditis elegans

Caenorhabditis elegans is an excellent model for morphogenetic research. However, little information is available on the structure of cell-surface glycans in C. elegans, although several lines of evidence have suggested a role for these glycans in cell-cell interactions during development. In this study, we analyzed N-glycan structures. Oligosaccharides liberated by hydrazinolysis from a total membrane fraction were labeled by pyridylamination, and around 90% of the N-glycans were detected as neutral oligosaccharides. The most dominant structure was Man(alpha)1-6(Man(alpha)1-3)Man(beta)1-4GlcNAc(beta)1-4GlcNAc, which is commonly found in insects. Branching structures of major oligomannose-type glycans were the same as those found in mammals. Structures that had a core fucose or non-reducing end N-acetylglucosamine were also identified, but ordinary complex-type glycans with N-acetyllactosamine were not detected as major components.     

Lipoprotein(a) enhances advanced atherosclerosis and vascular calcification in WHHL transgenic rabbits expressing human apolipoprotein(a)

High lipoprotein(a) (Lp(a)) levels are a major risk factor for the development of atherosclerosis. The risk of elevated Lp(a) concentration is increased significantly in patients who also have high levels of low density lipoprotein (LDL) cholesterol. To test the hypothesis that increased plasma levels of Lp(a) may enhance the development of atherosclerosis in the setting of hypercholesterolemia, we generated Watanabe heritable hyperlipidemic (WHHL) transgenic (Tg) rabbits expressing human apolipoprotein(a) (apo(a)). We report here that Tg WHHL rabbits developed more extensive advanced atherosclerotic lesions than did non-Tg WHHL rabbits. In particular, the advanced atherosclerotic lesions in Tg WHHL rabbits were frequently associated with calcification, which was barely evident in non-Tg WHHL rabbits. To investigate the molecular mechanism of Lp(a)-induced vascular calcification, we examined the effect of human Lp(a) on cultured rabbit aortic smooth muscle cells and found that smooth muscle cells treated with Lp(a) showed increased alkaline phosphatase activity and enhanced calcium accumulation. These results demonstrate for the first time that Lp(a) accelerates advanced atherosclerotic lesion formation and may play an important role in vascular calcification.     

Method for purification of fluorescence-labeled oligosaccharides by pyridylamination

We developed a convenient method for purification of PA-oligosaccharides to remove contaminants originating from natural fluorescent materials, and excess reagents as well as by-products of tagging reactions in glycan analysis. The method, using a C18-cartridge, is simple and powerful to remove them. Several examples of experiments that showed the usefulness of this purification method are described in this report.     

Mechanism of the Escherichia coli ADP-ribose pyrophosphatase,a Nudix hydrolase

Escherichia coli ADP-ribose (ADPR) pyrophosphatase (ADPRase), a Nudix enzyme, catalyzes the Mg(2+)-dependent hydrolysis of ADP-ribose to AMP and ribose 5-phosphate. ADPR hydrolysis experiments conducted in the presence of H(2)(18)O and analyzed by electrospray mass spectrometry showed that the ADPRase-catalyzed reaction takes place through nucleophilic attack at the adenosyl phosphate. The structure of ADPRase in complex with Mg(2+) and a nonhydrolyzable ADPR analogue, alpha,beta-methylene ADP-ribose, reveals an active site water molecule poised for nucleophilic attack on the adenosyl phosphate. This water molecule is activated by two magnesium ions, and its oxygen contacts the target phosphorus (P-O distance of 3.0 A) and forms an angle of 177 degrees with the scissile bond, suggesting an associative mechanism. A third Mg(2+) ion bridges the two phosphates and could stabilize the negative charge of the leaving group, ribose 5-phosphate. The structure of the ternary complex also shows that loop L9 moves fully 10 A from its position in the free enzyme, forming a tighter turn and bringing Glu 162 to its catalytic position. These observations indicate that as part of the catalytic mechanism, the ADPRase cycles between an open (free enzyme) and a closed (substrate-metal complex) conformation. This cycling may be important in preventing nonspecific hydrolysis of other nucleotides.     

Identification of genes regulating colorectal carcinogenesis by using the algorithm for diagnosing malignant state method

We studied the expression profiles of various stages of colorectal tumors (adenoma (AD), seven samples; carcinoma (CA), 16 samples) by using cDNA microarrays and developed ADMS (algorithm for diagnosing malignant state) method, selecting 335 clones characteristic of CA state. We, then, applied ADMS to 12 additional samples (five from primary lesions with metastasis and seven metastases); all 16 CAs and 12 metastatic tumors were diagnosed correctly as cancerous states. Although three of the seven ADs were diagnosed as "cancerous," the large size of two of these tumors suggested their potential malignancy. Our strategy for selecting clones characteristic of the malignant state is widely applicable to diagnosis and for predicting the stage of progression during multistep carcinogenesis. Of the 335 clones we selected, 135 were known genes. Included in the 135 genes were tumor suppressor and growth factor-related genes and were consistent with the literature. ADMS is a reliable means for identifying genes useful for the diagnosis of cancer.     

Paternal expression of a novel imprinted gene, Peg12/Frat3, in the mouse 7C region homologous to the Prader-Willi syndrome region.

Paternally expressed imprinted genes (Pegs) were systematically screened by comparing gene expression profiles of parthenogenetic and normal fertilized embryos using an oligonucleotide array. A novel imprinted gene, Peg12/Frat3, was identified along with 10 previously known Pegs. Peg12/Frat3 is expressed primarily in embryonic stages and might be a positive regulator of the Wnt signaling pathway. It locates next to the Zfp127 imprinted gene in the mouse 7C region, which has syntenic homology to the human Prader-Willi syndrome region on chromosome 15q11-q13, indicating that this imprinted region extends to the telomeric side in the mouse.