Structural requirements of a human deoxyribonuclease II for the development of the active enzyme form, revealed by site-directed mutagenesis

Using site-directed mutagenesis, we eliminated three potential N-glycosylation sites (N86, N212, and N266) of human deoxyribonuclease II (DNase II), conserved in mammalian enzymes, and a proteolytic processing site (Q46-R47), forming a propeptide subunit of the enzyme. We expressed a series of these mutant DNase II constructs in COS-7 and Hep G2 cells. Liberation of each glycosylation site at N86 and N266 and the cleavage site interfered dramatically with expression of the intracellular and secreted DNase II activities, irrespective of cell line transfected. A chimeric mutant in which the signal peptide of the DNase II was replaced with that of human DNase I had no intracellular or secreted enzyme activity. Therefore, a simultaneous attachment of a carbohydrate moiety to N86 and N266, cleavage of the propeptide from the single DNase II precursor, and the inherent signal peptide might be required for subcellular sorting and proteolytic maturation of the enzyme.     

Evaluation of the CFP-substrate-YFP system for protease studies: advantages and limitations

A protease can be defined as an enzyme capable of hydrolyzing peptide bonds. Thus, characterization of a protease involves identification of target peptide sequences, measurement of activities toward these sequences, and determination of kinetic parameters. Biological protease substrates based on fluorescent protein pairs, which allow for use of fluorescence resonance energy transfer (FRET), have been recently developed for in vivo protease activity detection and represent a very interesting alternative to chemical substrates for in vitro protease characterization. Here, we analyze a FRET system consisting of cyan and yellow fluorescent proteins (CFP and YFP, respectively), which are fused by a peptide linker serving as protease substrate. Conditions for CFP-YFP fusion protein production in Escherichia coli and purification of proteins were optimized. FRET between CFP and YFP was found to be optimum at a pH between 5.5 and 10.0, at low concentrations of salt and a temperature superior to 25 degrees C. For efficient FRET to occur, the peptide linker between CFP and YFP can measure up to 25 amino acids. The CFP-substrate-YFP system demonstrated a high degree of resistance to nonspecific proteolysis, making it suitable for enzyme kinetic analysis. As with chemical substrates, substrate specificity of CFP-substrate-YFP proteins was tested towards different proteases and kcat/Km values were calculated.     

Novel lipoxygenase inhibitors, tetrapetalone B, C, and D from Streptomyces sp

Three novel lipoxygenase inhibitors, tetrapetalone B (2, C(28)H(35)NO(9)), C (3, C(26)H(34)NO(8)), and D (4, C(28)H(36)NO(10)), were isolated from a culture broth of Streptomyces sp. USF-4727 that produced a lipoxygenase inhibitor tetrapetalone A (1) simultaneously. Each chemical structure was revealed by spectroscopic evidence, this suggests that these three compounds are structurally related to 1. They had a tetracyclic skeleton and a beta-D-rhodinosyl moiety. Tetrapetalone B, C, and D inhibited soybean lipoxygenase with IC(50): 320, 360, and 340 microM respectively.     

Crystallization and preliminary X-ray analysis of plant class I chitinase from rice

We report here on crystallization and preliminary X-ray analysis of plant class I chitinase from rice (OsChia1b). Similar single crystals of full-length OsChia1b were obtained under two independent conditions. The crystals grown under these conditions diffracted up to 2.1 and 2.5 angstroms resolution, respectively, at a synchrotron beamline, and were found to belong to the tetragonal space group P4(3)2(1)2.     

Structure,function and assembly of Photosystem II and its light-harvesting proteins

Photosystem II (PSII) is a multisubunit chlorophyll–protein complex that drives electron transfer from water to plastoquinone using energy derived from light. In green plants, the native form of PSII is surrounded by the light-harvesting complex (LHCII complex) and thus it is called the PSII–LHCII supercomplex. Over the past several years, understanding of the structure, function, and assembly of PSII and LHCII complexes has increased considerably. The unicellular green alga Chlamydomonas reinhardtii has been an excellent model organism to study PSII and LHCII complexes, because this organism grows heterotrophically and photoautotrophically and it is amenable to biochemical, genetic, molecular biological and recombinant DNA methodology. Here, the genes encoding and regulating components of the C. reinhardtii PSII–LHCII supercomplex have been thoroughly catalogued: they include 15 chloroplast and 20 nuclear structural genes as well as 13 nuclear genes coding for regulatory factors. This review discusses these molecular genetic data and presents an overview of the structure, function and assembly of PSII and LHCII complexes.     

Pterygodermatites nycticebi (Nematoda: Rictulariidae): accidental detection of encapsulated third-stage larvae in the tissue of a white-fronted marmoset

Twin, white-fronted marmosets (Callithrix geoffroyi) born and raised in a zoo in Japan died at 7 mo of age. Several encapsulated nematode larvae were detected in the intestinal wall, as well as a few in the mesenteric lymph nodes of 1 of the twins. In the other marmoset, no encapsulated nematode larva was detected in the organs, but many adult Pterygodermatites nycticebi were found in the intestinal lumen. In the past 5 yr, 5 primates kept in the same zoo, i.e., 1 squirrel monkey (Saimiri sciureus), 2 Pygmy marmosets (Cebuella pygmaea), 1 Senegal galago (Galago senegalensis), and 1 cotton-top tamarin (Saguinus oedipus), died from heavy infestation with the same nematode. A few migrating larvae of the rictulariid were also identified histologically in the intestinal wall and liver of the cotton-top tamarin. Although no other primate currently held in the same zoo was infected with the rictulariid, German cockroaches (Blattella germanica) collected with traps near marmoset cages had encapsulated P. nycticebi larvae, indicating latent perpetuation of the life cycle of this rictulariid species in the zoo premises. Our results indicated that encapsulation or migration of third-stage larvae of P. nycticebi might occur accidentally in the organs of callithrichid primates.     

RNA helicase domain of tobamovirus replicase executes cell-to-cell movement possibly through collaboration with its nonconserved region

UR-hel, a chimeric virus obtained by replacement of the RNA helicase domain of tobacco mosaic virus (TMV)-U1 replicase with that from the TMV-R strain, could replicate similarly to TMV-U1 in protoplasts but could not move from cell to cell (K. Hirashima and Y. Watanabe, J. Virol. 75:8831-8836, 2001). It was suggested that TMV recruited both the movement protein (MP) and replicase for cell-to-cell movement by unknown mechanisms. Here, we found that a recombinant, UR-hel/V, in which the nonconserved region was derived from TMV-R in addition to the RNA helicase domain of replicase, could move from cell to cell. We also analyzed revertants isolated from UR-hel, which recovered cell-to-cell movement by their own abilities. We found amino acid substitutions responsible for phenotypic reversion only in the nonconserved region and/or RNA helicase domain but never in MP. Together, these data show that both the nonconserved region and the RNA helicase domain of replicase are involved in cell-to-cell movement. The RNA helicase domain of tobamovirus replicase possibly does not interact directly with MP but interacts with its nonconserved region to execute cell-to-cell movement.     

Construction of reporter yeasts for mouse aryl hydrocarbon receptor ligand activity

Aryl hydrocarbons such as dioxins, polychlorinated biphenyls and polyaromatic hydrocarbons bind to the cellular aryl hydrocarbon receptor (AhR) in the initial step of their metabolism. The activation of intracellular signaling subsequent to the AhR binding is highly correlated with the toxicity and carcinogenicity of these chemicals. We produced Saccharomyces cerevisiae coexpressing mouse AhR and aryl hydrocarbon receptor nuclear translocator (Arnt) protein in accordance with Miller III's method for constructing yeasts with human Ahr and Arnt [Toxicol. Appl. Pharmacol. 160 (1998) 297]. Ligand treatment induced a dose-dependent increase in beta-galactosidase activity from a reporter plasmid in the yeast. Then, we compared activities of several ligands in yeast having the mouse Ahr/Arnt genes with those in yeast having the human genes, both of which have the same genetic background. There was no significant difference in the EC50 values of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo[a]pyrene, 3-methylcholanthrene and beta-naphthoflavone between the mouse and human genes. However, indirubin, which was recently found in human urine as a potent AhR ligand [J. Biol. Chem. 276 (2001) 31475], had a 35-140 times higher EC50 value in the yeast with human genes than mouse genes. This difference might reflect species-specificity between mouse and human AhR/Arnt.