Comparative sequence analysis of colinear barley and rice bacterial artificial chromosomes

Colinearity of a large region from barley (Hordeum vulgare) chromosome 5H and rice (Oryza sativa) chromosome 3 has been demonstrated by mapping of several common restriction fragment-length polymorphism clones on both regions. One of these clones, WG644, was hybridized to rice and barley bacterial artificial chromosome (BAC) libraries to select homologous clones. One BAC from each species with the largest overlapping segment was selected by fingerprinting and blot hybridization with three additional restriction fragment-length polymorphism clones. The complete barley BAC 635P2 and a 50-kb segment of the rice BAC 36I5 were completely sequenced. A comparison of the rice and barley DNA sequences revealed the presence of four conserved regions, containing four predicted genes. The four genes are in the same orientation in rice, but the second gene is in inverted orientation in barley. The fourth gene is duplicated in tandem in barley but not in rice. Comparison of the homeologous barley and rice sequences assisted the gene identification process and helped determine individual gene structures. General gene structure (exon number, size, and location) was largely conserved between rice and barley and to a lesser extent with homologous genes in Arabidopsis. Colinearity of these four genes is not conserved in Arabidopsis compared with the two grass species. Extensive similarity was not found between the rice and barley sequences other than within the exons of the structural genes, and short stretches of homology in the promoters and 3' untranslated regions. The larger distances between the first three genes in barley compared with rice are explained by the insertion of different transposable retroelements.     

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on *OH formation by the Fenton reaction

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.     

High activity of the calcineurin A subunit with a V314 deletion

A deletion mutant (V314) of the calcineurin A subunit was constructed using site-directed mutagenesis. Its phosphatase activity and function were then characterized. The V314 deletion significantly altered the phosphatase activity, which was more than ten times higher than that of wild-type calcineurin, the calcineurin-immunosuppressant/immunophilin interaction, the effect of metal ions and calcineurin subunit interaction. We propose that the change of the activity and function of V314 is due to conformational changes of calcineurin to benefit the binding of, or stimulation by, Mn2+, or to affect the interaction between the A and B subunits.     

mE10, a novel caspase recruitment domain-containing proapoptotic molecule

Apoptotic signaling is mediated by homophilic interactions between conserved domains present in components of the death pathway. The death domain, death effector domain, and caspase recruitment domain (CARD) are examples of such interaction motifs. We have identified a novel mammalian CARD-containing adaptor molecule termed mE10 (mammalian E10). The N-terminal CARD of mE10 exhibits significant homology (47% identity and 64% similarity) to the CARD of a gene from Equine Herpesvirus type 2. The C-terminal region is unique. Overexpression of mE10 in MCF-7 human breast carcinoma cells induces apoptosis. Mutational analysis indicates that CARD-mediated mE10 oligomerization is essential for killing activity. The C terminus of mE10 bound to the zymogen form of caspase-9 and promoted its processing to the active dimeric species. Taken together, these data suggest a model where autoproteolytic activation of pro-caspase-9 is mediated by mE10-induced oligomerization.     

Oligosaccharyltransferase: a complex multisubunit enzyme of the endoplasmic reticulum

The attachment of N-linked oligosaccharide chains to proteins is an important cotranslational process. These chains can, in some cases, serve to stabilize the protein, while in other cases they function as recognition elements. A key enzyme in the N-glycosylation process is oligosaccharyltransferase (OT). In yeast this enzyme, which is found in the endoplasmic reticulum, consists of nine different transmembrane protein subunits. Our general aim is to learn more about the functions of the multiple subunits of yeast OT and their mode of interaction with each other. Using a combination of biochemical and genetic techniques the subunit Ost1p has been shown to recognize Asn-X-Ser/Thr glycosylation sites. The principle tool used in the identification process was a benzophenone-based glycosylation site peptide that was shown to be crosslinked to Ost1p. Our current objective is to identify the domain in the primary structure that is involved in recognition of the glycosylation site sequence. By use of bifunctional crosslinkers, the possible interaction of Ost1p with other subunits of OT will be studied. This work and other studies on the OT subunits are concisely summarized.     

Altered levels of primary antioxidant enzymes in progeria skin fibroblasts

Free radicals are involved in the aging process. In this study, the profile of primary antioxidant enzymes that scavenge reactive oxygen species (ROS) was examined for the first time in human skin fibroblasts from progeria, a premature aging disease. Altered levels of antioxidant enzymes were found in progeria cells. Basal levels of MnSOD were decreased in progeria cells as well as a blunted induction in response to chronic stress. This change may contribute to the accelerated aging process in progeria cells. In contrast, the levels of CuZnSOD showed no progeria-related change. Two H2O2 removing enzymes demonstrated a significant reduction in progeria cells: only 50% of normal CAT activity and 30% of normal GPX activity can be detected in progeria cells. This diminished H2O2 removing capacity in progeria cells may lead to an imbalance of intracellular ROS and therefore may play an important role in the development of progeria.     

Solution structure of carnobacteriocin B2 and implications for structure-activity relationships among type IIa bacteriocins from lactic acid bacteria

Carnobacteriocin B2 (CbnB2), a type IIa bacteriocin, is a 48 residue antimicrobial peptide from the lactic acid bacterium Carnobacterium pisicola LV17B. Type IIa bacteriocins have a conserved YGNGVXC sequence near the N-terminus and usually contain a disulfide bridge. CbnB2 seemed to be unique in that its two cysteines (Cys9 and Cys14) could be isolated as free thiols [Quadri et al. (1994) J. Biol. Chem. 26, 12204-12211]. To establish the structural consequences of the presence or absence of a disulfide bridge and to investigate if the YGNGVXC sequence is a receptor-binding motif [Fleury et al. (1996) J. Biol. Chem. 271, 14421-14429], the three-dimensional solution structure of CbnB2 was determined by two-dimensional (1)H nuclear magnetic resonance (NMR) techniques. Mass spectroscopic and thiol modification experiments on CbnB2 and on model peptides, in conjunction with activity measurements, were used to verify the redox status of CbnB2. The results show that CbnB2 readily forms a disulfide bond and that this peptide has full antimicrobial activity. NMR results indicate that CbnB2 in trifluoroethanol (TFE) has a well-defined central helical structure (residues 18-39) but a disordered N terminus. Comparison of the CbnB2 structure with the refined solution structure of leucocin A (LeuA), another type IIa bacteriocin, indicates that the central helical structure is conserved between the two peptides despite differences in sequence but that the N-terminal structure (a proposed receptor binding site) is not. This is unexpected because LeuA and CbnB2 exhibit >66% sequence identity in the first 24 residues. This suggests that the N-terminus, which had been proposed [Fleury et al. (1996) J. Biol. Chem. 271, 14421-14429] to be a receptor binding site of type IIa bacteriocins, may not be directly involved and that recognition of the amphiphilic helical portion is the critical feature.