Molecular Characterization of Photomixotrophic Tobacco Cells Resistant to Protoporphyrinogen Oxidase-Inhibiting Herbicides

Peroxidizing herbicides inhibit protoporphyrinogen oxidase (Protox), the last enzyme of the common branch of the chlorophyll- and heme-synthesis pathways. There are two isoenzymes of Protox, one of which is located in the plastid and the other in the mitochondria. Sequence analysis of the cloned Protox cDNAs showed that the deduced amino acid sequences of plastidial and mitochondrial Protox in wild-type cells and in herbicide-resistant YZI-1S cells are the same. The level of plastidial Protox mRNA was the same in both wild-type and YZI-1S cells, whereas the level of mitochondrial Protox mRNA YZI-1S cells was up to 10 times the level of wild-type cells. Wild-type cells were observed by fluorescence microscopy to emit strong autofluorescence from chlorophyll. Only a weak fluorescence signal was observed from chlorophyll in YZI-1S cells grown in the Protox inhibitor N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide. Staining with DiOC6 showed no visible difference in the number or strength of fluorescence between wild-type and YZI-1S mitochondria. Electron micrography of YZI-1S cells showed that, in contrast to wild-type cells, the chloroplasts of YZI-1S cells grown in the presence of N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide exhibited no grana stacking. These results suggest that the herbicide resistance of YZI-1S cells is due to the overproduction of mitochondrial Protox.     

O-Fucose Monosaccharide of Drosophila Notch Has a Temperature-sensitive Function and Cooperates with O-Glucose Glycan in Notch Transport and Notch Signaling Activation

Notch (N) is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell fate decisions. N has many epidermal growth factor-like repeats that are O-fucosylated by the protein O-fucosyltransferase 1 (O-Fut1), and the O-fut1 gene is essential for N signaling. However, the role of the monosaccharide O-fucose on N is unclear, because O-Fut1 also appears to have O-fucosyltransferase activity-independent functions, including as an N-specific chaperon. Such an enzymatic activity-independent function could account for the essential role of O-fut1 in N signaling. To evaluate the role of the monosaccharide O-fucose modification in N signaling, here we generated a knock-in mutant of O-fut1 (O-fut1^{R245A knock-in}), which expresses a mutant protein that lacks O-fucosyltransferase activity but maintains the N-specific chaperon activity. Using O-fut1^{R245A knock-in} and other gene mutations that abolish the O-fucosylation of N, we found that the monosaccharide O-fucose modification of N has a temperature-sensitive function that is essential for N signaling. The O-fucose monosaccharide and O-glucose glycan modification, catalyzed by Rumi, function redundantly in the activation of N signaling. We also showed that the redundant function of these two modifications is responsible for the presence of N at the cell surface. Our findings elucidate how different forms of glycosylation on a protein can influence the protein''s functions.     

Accumulation of Japanese cedar pollen allergen,Cry j 1, in the protein body I of transgenic rice seeds using the promoter and signal sequence of glutelin GluB-1 gene

Recombinant Cry j 1, a Japanese cedar pollen allergen, was produced in rice seeds for potential use for oral immunotherapy. Cry j 1 cDNA was divided into two parts, an N-terminal half and a C-terminal half, and each was fused downstream to glutelin GluB-1 gene containing sequences of the promoter, 5 untranslated region and signal peptide. A gene for green fluorescent protein was also fused to the 3 end of the Cry j 1 fragment. Recombinant Cry j 1 of up to 16.6 g per mg total protein of the seeds was expressed in transgenic rice seeds. Although the recombinant Cry j 1 was expected to be accumulated in protein body II because of the employment of glutelin signal peptide, it was demonstrated to be accumulated exclusively in protein body I. The recombinant Cry j 1 was not shown to react with IgE of allergic patients, indicating the reduction of the risk of anaphylactic reaction. These results demonstrate that the transgenic rice seeds with the recombinant Cry j 1 would be useful for the study of oral immunotherapy.     

Mitochondrial reactive oxygen species reduce insulin secretion by pancreatic beta-cells

Pancreatic beta-cells exposed to hyperglycemia produce reactive oxygen species (ROS). Because beta-cells are sensitive to oxidative stress, excessive ROS may cause dysfunction of beta-cells. Here we demonstrate that mitochondrial ROS suppress glucose-induced insulin secretion (GIIS) from beta-cells. Intracellular ROS increased 15min after exposure to high glucose and this effect was blunted by inhibitors of the mitochondrial function. GIIS was also suppressed by H(2)O(2), a chemical substitute for ROS. Interestingly, the first-phase of GIIS could be suppressed by 50 microM H(2)O(2). H(2)O(2) or high glucose suppressed the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, and inhibitors of the mitochondrial function abolished the latter effects. Our data suggested that high glucose induced mitochondrial ROS, which suppressed first-phase of GIIS, at least in part, through the suppression of GAPDH activity. We propose that mitochondrial overwork is a potential mechanism causing impaired first-phase of GIIS in the early stages of diabetes mellitus.     

Two genes that encode Ca2+-dependent protein kinases are induced by drought and high-salt stresses in Arabidopsis thaliana

Two cDNA clones, AATCDPK1 and cATCDPK2, encoding Ca²⁺-dependent, calmodulin-independent protein kinases (CDPK) were cloned from Arabidopsis thaliana and their nucleotide sequences were determined. Northern blot analysis indicated that the mRNAs corresponding to the ATCDPK1 and ATCDPK2 genes are rapidly induced by drought and high-salt stress but not by low-temperature stress or heat stress. Treatment of Arabidopsis plants with exogenous abscisic acid (ABA) had no effect on the induction of ATCDPK1 or ATCDPK2. These findings suggest that a change in the osmotic potential of the environment can serve as a trigger for the induction of ATCDPK1 and ATCDPK2. Putative proteins encoded by ATCDPK1 and ATCDPK2 which contain open reading frames of 1479 and 1488 bp, respectively, are designated ATCDPK1 and ATCDPK2 and show 52% identity at the amino acid sequence level. ATCDPK1 and ATCDPK2 exhibit significant similarity to a soybean CDPK (51 % and 73%, respectively). Both proteins contain a catalytic domain that is typical of serine/threonine protein kinases and a regulatory domain that is homologous to the Ca²⁺-binding sites of calmodulin. Genomic Southern blot analysis suggests the existence of a few additional genes that are related to ATCDPK1 and ATCDPK2 in the Arabidopsis genome. The ATCDPK2 protein expressed in Escherichia coli was found to phosphorylate casein and myelin basic protein preferentially, relative to a histone substrate, and required Ca²⁺ for activation.     

Effects of inner solution viscosity and membrane stiffness on erythrocyte deformability on passing through a microchannel: prediction by two-dimensional simulation

We studied erythrocyte deformability in an effort to develop diagnostic methods based on its measurement and thus aid in the development of therapies for circulatory diseases. In the reported work, we performed two-dimensional numerical simulations of blood flow through a microchannel (MC) to evaluate erythrocyte deformability, applying the immersed boundary method to simulate erythrocyte movement and deformation. To evaluate deformability, MC transit capacity and shape recoverability were considered, defined as the time required to pass through the MC and the time constant during the shape-recovery process after exiting the MC, respectively. The simulation results showed that the erythrocyte MC transit time increased when the viscosity of the inner solution or the stiffness of the membrane increased. The time constant for erythrocyte shape recovery increased as the inner solution viscosity increased. In contrast, the time constant decreased as the erythrocyte membrane stiffness increased. These time-constant trends were in agreement with a theoretical equation derived using the Kelvin model and with previous experimental results. This diagnostic method of measuring erythrocyte shape recoverability and MC transit capacity is anticipated to have clinical application.     

Relationships among tributary length,census population size,and genetic variability of white-spotted charr, <Emphasis Type="Italic">Salvelinus leucomaenis</Emphasis>, in the Lake Biwa water system

The relationships between census population size and tributary length and between haplotype diversity of the mitochondrial DNA and census population size in ten white-spotted charr populations in the Lake Biwa water system and its adjacent basins were investigated. The census population size (number of fish with ≥100 mm in standard length) significantly increased with the tributary length. In the eastern part of the Lake Biwa water system, haplotype diversity increased with the census population size. On other hand, in the western part of the water system and adjacent basins, haplotype diversity was zero irrespective of the census population size. These results suggest that white-spotted charr populations in the eastern and western part of the Lake Biwa water system have undergone different levels of bottlenecks related to the habitat size in the postglacial warming.     

Identification of minimal sequence for HIV-1 fusion inhibitors

Emergence of multi-drug resistant HIV-1 is a serious problem for AIDS treatment. Recently, the virus-cell membrane fusion process has been identified as a promising target for the development of novel drugs against these resistant variants. In this study, we identified a 29-residue peptide fusion inhibitor, SC29EK, which shows activity comparable to the previously reported inhibitor SC35EK. Some residues in SC29EK not required for interaction with virus gp41 heptad repeat 1 (HR1) were replaced with a non-proteinogenic amino acid, 2-aminoisobutyric acid (Aib), to stabilize the alpha-helix structure and to provide resistance to peptidases.     

Origins of mouse inbred strains deduced from whole-genome scanning by polymorphic microsatellite loci

Microsatellite loci are uniformly distributed at approximately 100-kbp intervals on all chromosomes except the chromosome Y, and genetic information about more than 9000 loci and high-throughput polymorphism analysis are now available. Taking advantage of these properties, we carried out whole-genome scanning using eight common inbred strains (CIS) of laboratory mice, including A/J, C57BL/6J, CBA/J, DBA/2J, SM/J, SWR/J, NC/Nga, and 129/SvJ, and eight wild-derived inbred strains (WIS), BGL2/Ms, CAST/Ei, JF1/Ms, MSM/Ms, NJL/Ms, PGN2/Ms, SK/CamEi, and SWN/Ms. We selected and located 1226 informative loci at 1.2-cM average intervals on all of the chromosomes of the 16 strains and compared the polymorphisms of the eight CIS with those from the eight WIS as subspecies representatives. More than 50% of the loci can be identified as WIS (therefore, subspecies-specific) alleles in the CIS genomes. We also discovered that the CIS chromosomes form a mosaic structure with an average ratio of domesticus to non-domesticus alleles of 3:1. Furthermore, the domesticus alleles were present much more frequently on the CIS chromosome X than on their autosomes, suggesting that successive backcrossing of non-domesticus stocks to domesticus stocks had been undergone at the beginning of CIS history.