The Association of Ascorbate and Ascorbate Oxidase in the Apoplast with IAA-Enhanced Elongation of Epicotyls from Vigna angularis

The level of (ascorbic acid (AA) plus dehydroascorbic acid (DHA))and the ratio of the level of AA to that of AA plus DHA in intercellularwashing fluid (IWF) of epicotyl segments from Vigna angularisdecreased from 2.8±0.3 to 1.2±0.5nmol (g fr wt)^–1and from 0.23±0.03 to 0.13±0.01, respectively,after incubation of the segments without IAA for 20 h at 27°C.However, these values changed to 5.3±1.7 nmol (g fr wt)^–1and 0.07±0.05 after incubation with 0.1 mM IAA. The activityof cell wall-bound ascorbate oxidase increased by about 20%and 70% after incubation without IAA and with IAA, respectively.However, the activity of cell wall-bound peroxidase was notaffected by incubation with or without IAA. The activities ofascorbate oxidase and peroxidase in IWF decreased by about 85and 75% after incubation without IAA. IAA did not affect thesedecreases to any great extent. A lignin-like compound was formedduring the incubation of epicotyl segments in the absence ofIAA. Formation of this compound was inhibited by IAA. The resultssuggest that one of the causes of the enhancement of elongationgrowth by IAA is the inhibition of peroxidase-dependent lignificationas a result of increases in levels of AA and DHA and in ascorbateoxidase activity. (Received August 16, 1993; Accepted December 6, 1993)     

Prophages integrating into prophages: A mechanism to accumulate type III secretion effector genes and duplicate Shiga toxin-encoding prophages in Escherichia coli

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 prophages (up to 21 prophages), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present phages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the phages integrated into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages in a single cell. The identified attB sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different attB sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These phages integrating into prophages represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx phages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species.     

Characterization of translucent segments observed in an smbA mutant of Escherichia coli

Abstract The smbA gene of Escherichia coli is essential for cell proliferation. The smbA2 mutant shows cold-sensitive colony formation at 22°C. A novel morphological phenotype, formation of a translucent segment at midcell or at a cell pole, was observed by phase-contrastt microscopy at a high frequency in the smbA2 mutant cells incubated in L medium lacking NaCl at 22°C, but not observed in L medium containing 1% NaCl or 20% sucrose at the same temperature. No translucent segment was observed in the wild-type cells in any of the media used. Electron microscopic observation revealed that the translucent segments resulted from the enlargement of a periplasmic space by separation of the inner membrane from the peptidoglycan layer and the outer membrane.     

Inhibiting Effect of Auxin on Glucosamine Incorporation into Cell Walls of Rice Coleoptiles

Auxin induced growth and decreased the hexosamine content ofthe cell walls of rice coleoptile sections. Indole-3-aceticacid (IAA) at 10^–5 M inhibited the incorporation of ¹⁴C-glucosamineinto the cell walls. IAA did not affect the ¹⁴C-incorporationinto the cytoplasm, while inhibitors of glycoprotein synthesis,unicamycin and monensin, suppressed the incorporation into boththe cytoplasm and the cell walls. The radioactivity due to labeledglucosamine in the cell walls increased during the chase, butthis increase was inhibited by IAA. Among the cell wall fractions,the increase in radioactivity and its inhibition by IAA wereconspicuous in the hemicellulose I fraction. The inhibitoryeffect of IAA on glucosamine incorporation into the cell wallswas observed even in the presence of 0.15 M mannitol solutionwhich completely suppressed the IAA-induced growth. These resultssuggest that auxin induces growth at least partly by inhibitingthe transport of asparagine-linked glycoproteins from the cytoplasmto the cell walls. ¹ Present address: Department of Biology, Faculty of Science,Osaka City University, Sumiyoshi-ku, Osaka 558, Japan (Received July 23, 1986; Accepted December 22, 1986)     

Immunohistochemical techniques for detection of dermatan sulfate proteoglycan in tissue sections

Dermatan sulfate proteoglycan chains were detected in tissue sections treated with chondroitin B-lyase (0.01 units/ml) in 20 mM Tris-HCl (pH 8.0) for 1 hr, followed by staining with antibody 9A2 specific for unsaturated uronic acid coupled to N-acetylgalactosamine-4 sulfate. In contrast, after treatment with chondroitin B-lyase, no positive staining was observed with antibodies 3B3 and 1B5 which react to the unsaturated uronic acid coupled to N-acetylgalactosamine 6-sulfate and unsaturated uronic acid coupled to N-acetylgalactosamine, respectively. The distribution of dermatan sulfate thus revealed was confirmed by comparison with that found by monoclonal antibody 6B6 which reacts with small proteoglycans carrying dermatan sulfate side chains. The localization of positive staining in fibrous connective tissues was almost identical with these two procedures.     

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

Development of nephrotic ICGN mice--the origin, reproductive ability, and incidence of glomerulonephritis

ICGN is a strain of spontaneous nephrotic mice with nonproliferative glomerular lesions. It was derived from an outbred Yok: ICR colony in our laboratory. The renal disease constantly occurred in animals of the first to the tenth generations (greater than 13.0%; 70 days of age). When affected males were mated with unaffected females, the incidence of the disease in their offspring was 38.8% (n = 49) at 70 days after birth. When both parents were affected, their offspring were all affected (n = 12). The disease evenly progressed in both sexes. It usually began 40 to 150 days after birth and death occurred within two months after onset. The animals usually showed sufficient reproductive ability as long as unaffected females were used for mating.