Differential responses of Brassica napus and Petunia hybrida to leaf protoplast isolation stress

Changes in the response to abiotic stress during the isolation of leaf protoplasts were compared between a recalcitrant species of Brassica napus and regenerating species of Petunia hybrida . Initially, levels of soluble free putrescine (put), spermidine (spd) and spermine (spm) in leaves and protoplasts were determined. The sum of these three polyamines increased in petunia and B. napus leaf protoplasts by 1.6-fold and 1.1-fold, respectively. The soluble free fraction of spd and spm decreased in B. napus but not in petunia protoplasts. During the isolation of leaf protoplasts from B. napus , the ratio of soluble free put to the total PAs almost doubled, but that of spd and spm declined significantly. Petunia leaf protoplasts treated with cyclohexylamine (CHA), an inhibitor of spermidine synthase, accumulated ammonia and soluble putrescine, but lost the soluble spermidine. The soluble polyamine levels of CHA-treated petunia leaf protoplasts corresponded with those in B. napus . Leaves were subjected to abiotic stress during the isolation of protoplasts, namely wounding and osmotic stress which changed soluble free polyamine levels in B. napus and petunia, respectively. Both B. napus and petunia leaf protoplasts showed an increase in ammonia, but total free amino acid content and activation of proteases were only enhanced in B. napus leaf protoplasts. These results suggest that in B. napus wounding initiated senescence of leaf protoplasts during their isolation, leading to a constant production of ethylene early in the culture.     

A back-up glycosylase in Nth1 knock-out mice is a functional Nei (endonuclease VIII) homologue

Thymine glycol, a potentially lethal DNA lesion produced by reactive oxygen species, can be removed by DNA glycosylase, Escherichia coli Nth (endonuclease III), or its mammalian homologue NTH1. We have found previously that mice deleted in the Nth homologue still retain at least two residual glycosylase activities for thymine glycol. We report herein that in cell extracts from the mNth1 knock-out mouse there is a third thymine glycol glycosylase activity that is encoded by one of three mammalian proteins with sequence similarity to E. coli Fpg (MutM) and Nei (endonuclease VIII). Tissue expression of this mouse Nei-like (designated as Neil1) gene is ubiquitous but much lower than that of mNth1 except in heart, spleen, and skeletal muscle. Recombinant NEIL1 can remove thymine glycol and 5-hydroxyuracil in double- and single-stranded DNA much more efficiently than 8-oxoguanine and can nick the strand by an associated (beta-delta) apurinic/apyrimidinic lyase activity. In addition, the mouse NEIL1 has a unique DNA glycosylase/lyase activity toward mismatched uracil and thymine, especially in U:C and T:C mismatches. These results suggest that NEIL1 is a back-up glycosylase for NTH1 with unique substrate specificity and tissue-specific expression.     

Novel nuclear and mitochondrial glycosylases revealed by disruption of the mouse Nth1 gene encoding an endonuclease III homolog for repair of thymine glycols

Endonuclease III, encoded by nth in Escherichia coli, removes thymine glycols (Tg), a toxic oxidative DNA lesion. To determine the biological significance of this repair in mammals, we established a mouse model with mutated mNth1, a homolog of nth, by gene targeting. The homozygous mNth1 mutant mice showed no detectable phenotypical abnormality. Embryonic cells with or without wild-type mNth1 showed no difference in sensitivity to menadione or hydrogen peroxide. Tg produced in the mutant mouse liver DNA by X-ray irradiation disappeared with time, though more slowly than in the wild-type mouse. In extracts from mutant mouse liver, we found, instead of mNTH1 activity, at least two novel DNA glycosylase activities against Tg. One activity is significantly higher in the mutant than in wild-type mouse in mitochondria, while the other is another nuclear glycosylase for Tg. These results underscore the importance of base excision repair of Tg both in the nuclei and mitochondria in mammals.     

Classification of RNA structures based on hydrogen bond and base-base stacking patterns: application for NMR structures

A computational system, CSNA, for classifying RNA structures according to structural characters was developed. CSNA lists up all the hydrogen bonds and base-base stackings in the structures, and classifies the structures into sub-groups based on their patterns as the first step grouping. The frequency of each hydrogen bond or base-base stacking is calculated, the frequency score being defined as the sum of the frequency of existing hydrogen bonds or base-base stackings for each sub-group. Finally, the sub-groups are further classified into groups based on the frequency score defined in this study and the difference between the patterns. According to the frequency score, CSNA suggests a group that shares most frequently appearing hydrogen bonds and base-base stackings. CSNA was applied to the classification of the results of two individual simulated annealing calculations based on NMR information. It was found that CSNA could extract structures with lower energy without checking any energy term and could provide well converged groups as the lowest energy structures. Thus, CSNA could be a new tool for structural determination of nucleic acids.     

Efficient expression system of human recombinant laminin-5

Laminin-5, a heterotrimer of laminin alpha3, beta3, and gamma2 chains, is an essential component of various epithelial basement membranes, and it strongly promotes cellular adhesion and motility in vitro. In this study, we established an efficient expression system of human recombinant laminin-5 (rLN5), in which full-length cDNAs encoding the human laminin alpha3, beta3, and gamma2 chains were introduced into the human embryonic kidney cell line HEK293. rLN5 was purified from the conditioned medium of the HEK293 transfectant (LN5-HEK) by immuno-affinity chromatography in a yield of 1 mg protein/liter, about 10 times higher than that of a natural LN5 from human gastric cancer cells. rLN5 was indistinguishable from the natural LN5 in its protein composition and biological activity. In addition, analysis of HEK293 transfectants expressing two exogenous LN5 subunits showed that the alpha3/gamma2 chains and the beta3/gamma2 chains, but not the alpha3/beta3 chains, were secreted as heterodimers, suggesting an important role of the gamma2 chain in the association of the three LN5 subunits. The expression system of rLN5 can be used as an important tool to understand the biological functions of this laminin and may be applicable to future regenerative medicine.     

Affinity selection of DNA-binding proteins from yeast genomic DNA libraries by improved lambda phage display vector

Phage display is a useful means of identifying and selecting proteins of interest that bind specific targets. In order to examine the potential of phage display for the genome-wide screening of DNA-binding proteins, we constructed yeast genomic libraries using lambda foo-based vectors devised in this work. After affinity selection using GAL4 UAS(G) as a probe, phages expressing GAL4 were enriched approximately 5 x 10(5)-fold from the library. Approximately 90% of polypeptides encoded in correct translation reading frames by the selected phages were known or putative polynucleotide-binding proteins. This result clearly indicates that the modified lambda phage display vector in combination with our enrichment technique has great potential for the enrichment of DNA-binding proteins in a sequence-specific manner.     

Isolation of three main sericin components from the cocoon of the silkworm,Bombyx mori

To characterize the sericin components of the cocoon of silkworm Bombyx mori, fresh cocoon shells were dissolved in saturated aqueous lithium thiocyanate containing 2-mercaptoethanol, and fractionated by ethanol precipitation. Cocoon sericin was found to mainly consist of three polypeptides having molecular masses of the 400, 250, and 150 kDa estimated by SDS-PAGE, which corresponds to the sericin present in the middle, anterior, and posterior part of the middle silk gland. The amino acid compositions of the 400 and 150 kDa components were similar to each other, but that of the 250 kDa component was different. This suggests differences in the coding gene and properties of the 250 kDa sericin from the other two.     

Effect of the polyoxyethylene chain length of triton X surfactants on the adsolubilization of reconstituted wax model compounds

The effect of the surfactant, alpha-[4-(1,1,3,3-tetramethylbutyl) phenyl]-omega-hydroxypolyoxy-1,2-ethanediyl, on the adsolubilization of cholesterol and/or dotriacontane as model compounds of the epicuticular wax of mature tomato (Lycopersicon esculentum Mill.) fruit was investigated. Cholesterol as a model compound of such triterpenols as alpha- and beta-amyrins was solubilized in a concentration-dependent manner above the critical micelle concentration (cmc), while non-detectable quantities of the saturated hydrocarbon, dotriacontane, was solubilized at any concentration used. However, the surfactants solubilized more cholesterol from mixed than single membranes. The surfactants with a shorter polyoxyethylene (POE) chain length solubilized greater quantities than those with longer POE chains, suggesting that the microenvironment of micelles related to the polyoxyethylene moiety had an important effect on surfactant solubilization and that the octylphenol moiety must be capable of adsorbing to a specific region of the reconstituted membrane like dotriacontane.     

Expression of Apc2 during mouse development

APC2 (previously known as APCL), a molecule closely related to the adenomatous polyposis coli (APC) tumor suppressor, can deplete cytoplasmic beta-catenin, like APC itself. Recently, it has been shown that APC2 may regulate the localization of p53 and the microtubule stability and/or extension. Although it has been reported that APC2 mRNA is expressed in human brain, the anatomical and ontogenic expression patterns remain unclear. The purpose of this study was to investigate the distribution of mouse Apc2 during mouse development. In the adult brain, Apc2 is expressed predominantly in neurons and throughout the brain. Northern blot analysis demonstrated a high level of Apc2 expression in embryonic and early postnatal brain. Ontogenic analysis has indicated that Apc2 is expressed in neural tissue, including the peripheral nervous system. During development of cortex, retina and cerebellum, Apc2 is expressed in post-mitotic cells. These findings suggest that Apc2 may contribute to the development of neuronal cells.     

cAMP modulation of Ca(2+)-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig

Effects of cAMP accumulation on ATP-dependent priming and Ca(2+)-dependent fusion in Ca(2+)-regulated exocytosis were examined in antral mucous cells of guinea pigs by using video-enhanced contrast microscopy. The Ca(2+)-regulated exocytosis activated by 1 microM ACh consisted of two phases, an initial transient phase followed by a sustained phase, which were potentiated by cAMP accumulation. Depletion of ATP by 100 microM dinitrophenol (uncoupler of oxidative phosphorylation) or anoxia induced the sustained phase without the initial transient phase in Ca(2+)-regulated exocytosis. However, accumulation of cAMP before depletion of ATP induced and potentiated an initial transient phase followed by a sustained phase in Ca(2+)-regulated exocytosis. This suggests that the initial transient phase of Ca(2+)-regulated exocytosis is induced by fusion of all primed granules maintained by ATP and that accumulation of cAMP accelerates ATP-dependent priming of the exocytotic cycle. Moreover, ACh and Ca(2+) dose-response studies showed that accumulation of cAMP shifted the dose-response curves to the low concentration side, suggesting that it increases Ca(2+) sensitivity in the fusion of the exocytotic cycle. In conclusion, cAMP accumulation increases the number of primed granules and Ca(2+) sensitivity of the fusion, which potentiates Ca(2+)-regulated exocytosis in antral mucous cells.