A short synthetic MAR positively affects transgene expression in rice and Arabidopsis

Matrix Attachment Regions (MARs) are DNA elements that are thought to influence gene expression by anchoring active chromatin domains to the nuclear matrix. When flanking a construct in transgenic plants, MARs could be useful for enhancing transgene expression. Naturally occurring MARs have a number of sequence features and DNA elements in common, and using different subsets of these sequence elements, three independent synthetic MARs were created. Although short, these MARs were able to bind nuclear scaffold preparations with an affinity equal to or greater than naturally occurring plant MARs. One synthetic MAR was extensively tested for its effect on transgene expression, using different MAR orientations, plant promoters, transformation methods and plant species. This MAR was able to increase average transgene expression and produced integration patterns of lower complexity. These data show the potential of making well defined synthetic MARs and using them to improve transgene expression.     

Recovery of gravitropic response during regeneration of root caps does not require developed columella cells and sedimentation of amyloplasts

Correlations between regeneration of the root cap and recovery of a gravitropic response were studied using primary roots of Phaseolus vulgaris. After removal of various lengths of the root tip a gravistimulus was continuously given to the root. The statistical analysis of data showed that recovery of the gravitropic response was gradually delayed as the length of the tips removed increased. This suggested that the columella cells of the root cap were involved in gravitropism. When the root cap was completely removed, the roots did not respond to gravistimuli for the first 15 h and began to reorient their growth direction at 20 h. At this time, the columella cells had just begun to regenerate and had immature amyloplasts which did not sufficiently form a sediment. These results suggest that other systems of perception exist in plant cells in addition to the amyloplast-based model of graviperception.     

Photo-stable chlorophylls conjugated with montmorillonite

Summary Chlorophyll a was adsorbed to freeze-dried montmorillonite, bentonite, in benzene to form chlorophyll-bentonite conjugate. The adsorption proceeds into two steps; monolayer and multilayer adsorption. The absorption maximum in red region of chlorophyll a shifts to longer wavelength from 660 nm to 678 nm by increasing the amount of chlorophyll a conjugated with bentonite. Chlorophylls in the conjugated state was found to be photo-stable, although chlorophylls free from bentonite took place the marked bleaching by light-illumination.     

Induction of toxin‐specific neutralizing immunity by molecularly uniform rice‐based oral cholera toxin B subunit vaccine without plant‐associated sugar modification

Plants have been used as expression systems for a number of vaccines. However, the expression of vaccines in plants sometimes results in unexpected modification of the vaccines by N‐terminal blocking and sugar‐chain attachment. Although MucoRice‐CTB was thought to be the first cold‐chain‐free and unpurified oral vaccine, the molecular heterogeneity of MucoRice‐CTB, together with plant‐based sugar modifications of the CTB protein, has made it difficult to assess immunological activity of vaccine and yield from rice seed. Using a T‐DNA vector driven by a prolamin promoter and a signal peptide added to an overexpression vaccine cassette, we established MucoRice‐CTB/Q as a new generation oral cholera vaccine for humans use. We confirmed that MucoRice‐CTB/Q produces a single CTB monomer with an Asn to Gln substitution at the 4th glycosylation position. The complete amino acid sequence of MucoRice‐CTB/Q was determined by MS/MS analysis and the exact amount of expressed CTB was determined by SDS‐PAGE densitometric analysis to be an average of 2.35 mg of CTB/g of seed. To compare the immunogenicity of MucoRice‐CTB/Q, which has no plant‐based glycosylation modifications, with that of the original MucoRice‐CTB/N, which is modified with a plant N‐glycan, we orally immunized mice and macaques with the two preparations. Similar levels of CTB‐specific systemic IgG and mucosal IgA antibodies with toxin‐neutralizing activity were induced in mice and macaques orally immunized with MucoRice‐CTB/Q or MucoRice‐CTB/N. These results show that the molecular uniformed MucoRice‐CTB/Q vaccine without plant N‐glycan has potential as a safe and efficacious oral vaccine candidate for human use.     

Sed4p stimulates Sar1p GTP hydrolysis and promotes limited coat disassembly

The coat protein complex II (COPII) generates transport vesicles that mediate protein export from the endoplasmic reticulum (ER). The first step of COPII vesicle formation involves conversion of Sar1p-GDP to Sar1p-GTP by guanine-nucleotide-exchange factor (GEF) Sec12p. In Saccharomyces cerevisiae, Sed4p is a structural homolog of Sec12p, but no GEF activity toward Sar1p has been found. Although the role of Sed4p in COPII vesicle formation is implied by the genetic interaction with SAR1, the molecular basis by which Sed4p contributes to this process is unclear. This study showed that the cytoplasmic domain of Sed4p preferentially binds the nucleotide-free form of Sar1p and that Sed4p binding stimulates both the intrinsic and Sec23p GTPase-activating protein (GAP)-accelerated GTPase activity of Sar1p. This stimulation of Sec23p GAP activity by Sed4p leads to accelerated dissociation of coat proteins from membranes. However, Sed4p binding to Sar1p occurs only when cargo is not associated with Sar1p. On the basis of these findings, Sed4p appears to accelerate the dissociation of the Sec23/24p coat from the membrane, but the effect is limited to Sar1p molecules that do not capture cargo protein. We speculate that this restricted coat disassembly may contribute to the concentration of specific cargo molecules into the COPII vesicles.     

Characterization of marine X-family DNA polymerases and comparative analysis of base excision repair proteins

While mammalian DNA polymerase β (Pol β), which is a member of the Pol X family, play important roles in base excision repair (BER) that efficiently removes DNA base lesions arising from both endogenous and exogenous agents, this protein has been found only a subset of animals. To understand natural evolution of this enzyme, we isolated and characterized Pol β from jellyfish Aurelia sp.1. (AsPol β). Despite of phylogenetic distance and environmental differences between jellyfish and mammals, in vitro assays showed biochemical characteristics of AsPol β were very similar to those of a mammalian counterpart. We also searched two other homologs of mammalian genes that were involved in short patch (sp) BER in the nucleotide sequence database, and found that both of these homologs were encoded in the genomes of a lineage from Cnidarians through mammals and Arthropods. This study suggests that a DNA repair mechanism resembling mammalian sp-BER may be largely limited to a subset of animals. On the basis of our findings and previous reports, we discuss possible evolutional model of Pol β and the other members of the Pol X family.     

Effect of Oleic Acid Level under Constant n-6/n-3 and P/S Ratios of Dietary Fats on Lipid Metabolism in Rats

The effect of dietary oleate levels (18, 39, 57 and 74% of total fatty acids) on various lipid parameters was studied in rats given cholesterol-enriched diets containing fat with a constant P/S (3.1–3.2) and n-6/n-3 (5.4–6.2) ratio. High-oleic safflower oil was used as a source of oleic acid, and was replaced stepwise with a mixture of cotton seed and perilla seed oils. After three weeks of feeding, there were no significant differences in the concentrations of serum and liver cholesterol, although they tended to increase with an increasing dietary oleate level. A hypotriglyceridemic trend was observed toward an increasing proportion of oleic acid. The linoleate desaturation index, (dihomo-γ-linolenic acid + arachidonic acid)/linoleic acid, in tissue phosphatidylcholine tended to increase with an increasing proportion of oleate, whereas the production of prostacyclin by the aorta and thromboxane A₂ by platelets was independent of the dietary oleate level. These results indicate that dietary oleate did not significantly modify the effect of polyunsaturated fatty acids on various lipid parameters under dietary conditions at which the P/S and n-6/n-3 ratios of the dietary fat were kept at an appropriate level to prevent ischemic heart disease.     

On the Constitution of Tea Tannin

Abstract

Folgende Uutersuchungen wurden im Laboratorium für Bakteriologie der Biologischen Reichsanstalt für Land- und Forstwirtschart in Berlin-Dahlem und im Agrikulturchemischen Institut der Kaiserlichen Universität zu Kyoto, Japan, ausgeführt. Wir sprechen Herrn Oberregierungsrat Dr. C. Stapp und Herrn Dr. H. Bortels für die Anregung 7U diesen Untersuchungen und für viele Ratschläge unseren herzlichen Dank aus. Ebenso sind wir den Herren Prof. Osugi, Prof. Kimura, Dr. Iwamura, und Dr. Miyanisi für mancherlei Rat zu Dank verpflichtet.