The inhibitory action of SQDG (sulfoquinovosyl diacylglycerol) from spinach on Cdt1-geminin interaction

A human replication initiation protein, Cdt1, is a central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by direct binding. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance, for example, by geminin silencing with small interfering RNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. We established a high throughput screening system based on a modified enzyme linked immunosorbent assay to identify compounds that interfere with human Cdt1-geminin binding. Using this system, we screened inhibitors from natural materials containing food components, and found that a glycolipid, sulfoquinovosyl diacylglycerol (SQDG), from spinach can inhibit Cdt1-geminin interaction in vitro, with 50% inhibition observed at concentrations of 1.79mug/ml. Other major glycolipids, such as monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) from spinach, had no influence. Surface plasmon resonance analysis demonstrated that SQDG bound selectively to Cdt1, but did not interact with geminin. Using three-dimensional computer modeling analysis, SQDG was considered to interact with the geminin interaction interface on Cdt1, and the sulfate group of SQDG was assumed to make hydrogen bonds with the residue of Arg346 of Cdt1. These data could help to further understanding of the structure and function of Cdt1. In addition, SQDG could be a clue to developing more appropriate inhibitors of Cdt1-geminin interactions.     

Involvement of altered arginase activity, arginase I expression and NO production in accelerated intimal hyperplasia following cigarette smoke extract

In the present experiments, we tried to elucidate whether changes in arginase activity, protein expression of arginase-I and -II, and NO production are involved in accelerating the intimal hyperplasia following administration of cigarette smoke extract (CSE). The intimal hyperplasia was caused by removing endothelial cells with the aid of balloon embolectomy catheter in the right carotid artery of the male rabbit. The left carotid artery underwent sham operation and served as control. CSE was prepared by bubbling a stream of cigarette smoke into phosphate buffered saline. Rabbits were given subcutaneously with CSE once a day for 5 weeks from 1 week before to 4 weeks after the surgery. The specimens were assessed histologically and the intima/media ratio (%) was evaluated as an index of the intimal hyperplasia. The accelerated intimal hyperplasia with CSE was accompanied by the augmentation of the impaired cyclic GMP production, enhanced overall arginase activity and up-regulation of arginase-I. Pearson's correlation coefficient analyses revealed the close relationships among the arginase activities in endothelial cells and smooth muscle layer, the intimal/media ratio and cyclic GMP production. These results suggest that the enhanced arginase activity together with facilitated up-regulation of arginase-I with CSE, which was associated with the augmented impairment of NO production, shed a new light on the processes associated with accelerating the intimal hyperplasia in rabbit carotid arteries following CSE.     

Modulation of reactive oxygen species in endothelial cells by peroxynitrite-treated lipoproteins

Peroxynitrite has been implicated in the oxidative modification of low-density lipoprotein (LDL) particles, and nitrotyrosine residues in the LDL have been detected in atherosclerotic plaques. Studies have suggested that lipoproteins modified by peroxynitrite lead to the onset of atherosclerotic vascular disease. We therefore prepared in vitro lipoproteins oxidatively modified by peroxynitrite (NO(2)-lipoprotein) and investigated the effect of NO(2)-lipoprotein on the viability of cultured endothelial cells. After exposure of a high-density lipoprotein (HDL) to peroxynitrite, some intermolecular complexes of apolipoproteins in HDL were detected on immunoblotting with monoclonal antibodies against apolipoprotein AI and AII, suggesting that nitration of HDL by peroxynitrite causes intermolecular cross-linking of the apolipoproteins in the particles. Treatment with 1 mM peroxynitrite increased the 3-nitrotyrosine level to 28.5 mmol/mol of tyrosine residues in the prepared NO(2)-HDL, as quantitated by HPLC, and the amount in NO(2)-lipoprotein depended on the peroxynitrite concentration. HDL exhibited a shorter lag phase and the reaction plateaued more rapidly than that with LDL. To clarify whether or not NO(2)-lipoproteins affect the function of endothelial cells, we first examined the viability of cultured human aortic endothelial cells (HAECs) exposed to NO(2)-lipoproteins. Incubation with either NO(2)-HDL or NO(2)-LDL significantly reduced the HAEC viability at 72 h. The results of RT-PCR and Western blotting showed that NO(2)-HDL markedly suppressed at 48 h not only the expressed levels of mRNA and protein but also the activity of catalase in HAECs. In contrast, NO(2)-LDL significantly reduced the expression and activity of Cu(2+),Zn(2+)-superoxide dismutase (CuZn-SOD) in the cells. Neither NO(2)-HDL nor NO(2)-LDL interfered with nitric oxide production or expression of cyclooxygenases and NADPH oxidase in HAECs. Increased radical production in NO(2)-lipoprotein-treated HAECs implied that reactive oxygen species such as superoxide anions and hydroxyl radicals may contribute to the mechanism of the toxic effect induced in endothelial cells by NO(2)-lipoprotein. Overall, NO(2)-lipoprotein may lead to deterioration of the vascular function through these endothelial cell responses.     

Analysis of Scc1-deficient cells defines a key metaphase role of vertebrate cohesin in linking sister kinetochores

Cleavage of the cohesin subunit Scc1p/Mcd1p/Rad21 permits sister chromatid separation and is considered to trigger anaphase onset. It has also been suggested that the cohesin complex is essential for chromosome condensation and for assembling fully functional kinetochores. Here, we used vertebrate cells conditionally deficient in Scc1 to probe cohesin function in mitosis. Cells lacking cohesin arrest in prometaphase, with many chromosomes failing to align at a metaphase plate and high levels of the spindle assembly checkpoint protein, BubR1, at all kinetochores. We show that the structural integrity of chromosomes is normal in the absence of Scc1. Furthermore, specific inhibition of topoisomerase II, which is required for decatenation of replicated chromosomes, can bypass the cohesin requirement for metaphase chromosome alignment and spindle checkpoint silencing. Since the kinetochore effects of Scc1 deficiency can be compensated for by topoisomerase II inhibition, we conclude that Scc1 is not absolutely required for kinetochore assembly or function, and that its principal role in allowing the onset of anaphase is the establishment of sufficient inter-sister tension to allow biorientation.     

Papain protects papaya trees from herbivorous insects: role of cysteine proteases in latex

Many plants contain latex that exudes when leaves are damaged, and a number of proteins and enzymes have been found in it. The roles of those latex proteins and enzymes are as yet poorly understood. We found that papain, a cysteine protease in latex of the Papaya tree (Carica papaya, Caricaceae), is a crucial factor in the defense of the papaya tree against lepidopteran larvae such as oligophagous Samia ricini (Saturniidae) and two notorious polyphagous pests, Mamestra brassicae (Noctuidae) and Spodoptera litura (Noctuidae). Leaves of a number of laticiferous plants, including papaya and a wild fig, Ficus virgata (Moraceae), showed strong toxicity and growth inhibition against lepidopteran larvae, though no apparent toxic factors from these species have been reported. When the latex was washed off, the leaves of these lactiferous plants lost toxicity. Latexes of both papaya and the wild fig were rich in cysteine-protease activity. E-64, a cysteine protease-specific inhibitor, completely deprived the leaves of toxicity when painted on the surface of papaya and fig leaves. Cysteine proteases, such as papain, ficin, and bromelain, all showed toxicity. The results suggest that plant latex and the proteins in it, cysteine proteases in particular, provide plants with a general defense mechanism against herbivorous insects.     

Mass-tag technology for monitoring of protein kinase activity using mass spectrometry

Monitoring of intracellular protein kinase activity is very important for fields involving diagnosis and drug screening. However, current methods, such as radiometry using (32)P, or ELISA, are laborious and time-consuming. We have developed high-throughput assay system of protein kinase activity using mass-tagged substrate peptide probes and mass spectrometry. This assay system can easily evaluate target kinase activity and will potentially be able to simultaneously profile many protein kinase activities.     

Comparison of an oxidative stress biomarker "urinary8-hydroxy-2'-deoxyguanosine," between smokers and non-smokers

A great deal of effort has been made on the effect of oxidative stress for smokers. What seems to be lacking, however, is its evidence. Analyzing 1076 participants (age 35.9 +/- 12.9, urinary8-OHdG Mean +/- S.D., 11.4 +/- 6.7, n = 1076), our study found the significant increase in a biomarker of DNA damage urinary 8-OHdG/creatinine among smokers (7.75 +/- 2.8 ng/ml x CRE (n = 154) and 7.36 +/- 2.5 ng/ml x CRE (n = 627) (p < 0.05), Relative Risk = 2.9 (1.4-6.2) sex and age +/- 2 matching 105 male smokers and non-smokers. There was no significance on the comparison between female smokers and non-smokers. Smokers have significantly decreased serum alpha-tocopherol (1012 +/- 455, 1152 +/- 857, p < 0.03). The amount of serum ascorbate did not change. Smokers lowered serum HDL-cholesterol compared to non-smokers (59.3 +/- 11.8, 63.9 +/- 13.3, p < 0.05). The result of oxidative stress profile (OSP) also indicated that the increase of oxidative stress to smokers (p < 0.05). The calculated value of oxygen radical absorbance capacity (ORAC) of the meal for subjects was 1600 ORAC units.     

Localization of NAD(P)H dehydrogenase in the cyanobacterium Synechocystis sp. strain PCC 6803

Antibodies raised against NdhH and NdhB detected these proteins in the thylakoid membrane of Synechocystis sp. strain PCC 6803, but not in a purified cytoplasmic membrane. We conclude that NAD(P)H dehydrogenase is largely, if not exclusively, confined to the thylakoid membrane.     

Availability of oncogene activated production system for mass production of light chain of human antibody in CHO cells

We previously established a ras-oncogene amplified Chinesehamster ovary (CHO) cell line, named ras clone I, as anuniversal host cell line for oncogene activated production(OAP) system to mass-produce recombinant protein by activationof the cytomegalovirus immediate early (CMV) promoter with ras protein. The light chain(C5) of human monoclonal antibody HB4C5 is expected tobe potentially useful for lung cancer targeting. We generated aC5 hyper-producing cell line by transfecting ras cloneI with the C5 gene expression plasmid regulated by theCMV promoter, of which productivity was 5.3 times greater thanthe hyper productive CHO cell line generated by using conventional CHO cells. Introduction of the adenovirus E1A geneinto the hyper-producing cell line derived from ras clone I resulted in further 9.5 times enhancement of the productivity,suggesting the synergistic effect of E1A and ras oncogenes on the recombinant protein production driven by the CMV promoter. In addition, intracellular accumulation of C5 andupregulation of BiP was found in hyper-producing cell lineswhich were introduced E1A and ras oncogene. This resultsuggests that excessive intracellular accumulation ofC5 protein, which might be caused by that the amount of produced C5 in ER is beyond the ability of CHO cells to secrete, might signal the BiP promoter. Our data imply that ras clone I is available as a general host cell for establishing the recombinant protein hyper-producing CHOcells by the OAP system, and suggest that further mass production of recombinant proteins in the OAP system can be possible by clarifying the accurate role of upregulated BiP protein.     

Genetic selection for and molecular dynamic modeling of a protein transmembrane domain multimerization motif from a random Escherichia coli genomic library

In order to identify new transmembrane helix packing motifs in naturally occurring proteins, we have selected transmembrane domains from a library of random Escherichia coli genomic DNA fragments and screened them for homomultimerization via their abilities to dimerize the bacteriophage lambda cI repressor DNA-binding domain. Sequences were isolated using a modified lambda cI headpiece dimerization assay system, which was shown previously to measure transmembrane helix-helix association in the E. coli inner membrane. Screening resulted in the identification of several novel sequences that appear to mediate helix-helix interactions. One sequence, representing the predicted sixth transmembrane domain (TM6) of the E. coli protein YjiO, was chosen for further analysis. Using site-directed mutagenesis and molecular dynamics, a small set of models for YjiO TM6 multimerization interface interactions were generated. This work demonstrates the utility of combining in vivo genetic tools with computational systems for understanding membrane protein structure and assembly.