Functions of the COOH-terminal region of cyclodextrin glucanotransferase of alkalophilic Bacillus sp. #1011: relation to catalyzing activity and pH stability

Cyclodextrin glucanotransferase (beta-CGTase) of alkalophilic Bacillus sp. #1011 degrades starch to mainly beta-cyclodextrin (beta-CD). This enzyme is considered to contain an extra-polypeptide in its COOH-terminal region in addition to its NH2-terminal domain which exhibits the starch-degrading activity. To analyze the functions of this extra-polypeptide in the beta-CGTase, two mutated enzymes, in which DNA regions encoding 10 or 13 amino acids from the COOH-terminus were deleted, were obtained. The mutated enzymes degraded starch to glucose, maltooligosaccharides and alpha-CD, in addition to beta-CD. Furthermore, the pH stability of the mutated enzymes in the alkaline pH range (pH 9-11) was reduced.     

Unusual distribution of mitochondrial large subunit rRNA in the cytosol during conjugation in Tetrahymena thermophila

The distribution of mitochondria during conjugation of the ciliated protozoan Tetrahymena thermophila was surveyed using a mitochondrial stain and fluorescence in situ hybridization (FISH). When the mitochondria-specific stain, Mito-Tracker, was used, the majority of mitochondria were detected in the cortex; their distribution was not changed during conjugation. On the other hand, FISH using mitochondrial large subunit (LSU) rRNA as a probe showed an unusual distribution of signals during conjugation. Unexpectedly, the signals were detected throughout the cytoplasm of conjugating cells. These signals were not observed in pre-mating cells and in exconjugants. The cytosolic localization of mitochondrial rRNA was supported by northern blot analysis using post-mitochondrial RNA fraction at the later stages of conjugation. These observations suggest selective mitochondrial breakdown or transport of LSU rRNA into cytosol. The biological significance of the conjugation-specific appearance of the cytosolic mitochondrial rRNA is discussed.     

Phosphorylation of proteins and apoptosis induced by c-Jun N-terminal kinase1 activation in rat cardiomyocytes by H(2)O(2) stimulation

Cytokines and various cellular stresses are known to activate c-Jun N-terminal kinase-1 (JNK1), which is involved in physiological function. Here, we investigate the activation of JNK1 by oxidative stress in H9c2 cells derived from rat cardiomyocytes. H(2)O(2) (100 microM) significantly induces the tyrosine phosphorylation of JNK1 with a peak 25 min after the stimulation. The amount of JNK1 protein remains almost constant during stimulation. Immunocytochemical observation shows that JNK1 staining in the nucleus is enhanced after H(2)O(2) stimulation. To clarify the physiological role of JNK1 activation under these conditions, we transfected antisense JNK1 DNA into H9c2 cells. The antisense DNA (2 microM) inhibits JNK1 expression by 80% as compared with expression in the presence of the sense DNA, and significantly blocks H(2)O(2)-induced cell death. Consistent with the decrease in cell number, we detected condensation of the nuclei, a hallmark of apoptosis, 3 h after H(2)O(2) stimulation in the presence of the sense DNA for JNK1. The antisense DNA of JNK1 inhibits the condensation of nuclei by H(2)O(2). Under these conditions, the H(2)O(2)-induced phosphorylation of proteins with molecular masses of 55, 72, and 78 kDa is blocked by treatment with the antisense DNA for JNK1 as compared with the sense DNA for JNK1. These findings suggest that JNK1 induces apoptotic cell death in response to H(2)O(2), and that the cell death may be involved in the phosphorylations of 55, 72, and 78 kDa proteins induced by JNK1 activation.     

Assignment of the apolipoprotein A-I gene to 11q23 based on RFLP in a case with a partial deletion of chromosome 11, del(11)(q23.3→qter)

Summary The XmnI genotype at the apolipoprotein A-I locus was heterozygous in a boy with partial deletion of the long arm of chromosome 11, del(11)(q23.3qter). The apolipoprotein A-I gene, previously assigned to chromosome region 11q23q24, has been more specifically localized to 11q23 by excluding the region 11q24qter.     

Purification and properties of thiosulfate reductase from Desulfovibrio vulgaris, Miyazaki F

Thiosulfate reductase was purified to an almost homogeneous state from Desulfovibrio vulgaris, strain Miyazaki F, by ammonium sulfate precipitation, chromatography on DEAE-Toyopearl, Ultrogel AcA 34, and hydroxylapatite, and disc electrophoresis. The specific activity was increased 580-fold over the crude extract. The molecular weight was determined by gel filtration to be 85,000-89,000, differing from those reported for thiosulfate reductases from other Desulfovibrio strains. The enzyme had no subunit structure. When coupled with hydrogenase and methyl viologen, it stoichiometrically reduced thiosulfate to sulfite and sulfide with consumption of hydrogen. It did not reduce sulfite or trithionate. Cytochrome c3 was active as an electron donor. More than 0.75 mM thiosulfate inhibited the enzyme activity. o-Phenanthroline and 2,2'-bipyridine inhibited the enzyme and ferrous ion stimulated the reaction.     

TG1 integrase-based system for site-specific gene integration into bacterial genomes

Serine-type phage integrases catalyze unidirectional site-specific recombination between the attachment sites, attP and attB, in the phage and host bacterial genomes, respectively; these integrases and DNA target sites function efficiently when transferred into heterologous cells. We previously developed an in vivo site-specific genomic integration system based on actinophage TG1 integrase that introduces ～2-kbp DNA into an att site inserted into a heterologous Escherichia coli genome. Here, we analyzed the TG1 integrase-mediated integrations of att site-containing ～10-kbp DNA into the corresponding att site pre-inserted into various genomic locations; moreover, we developed a system that introduces ～10-kbp DNA into the genome with an efficiency of ～10⁴ transformants/μg DNA. Integrations of attB-containing DNA into an attP-containing genome were more efficient than integrations of attP-containing DNA into an attB-containing genome, and integrations targeting attP inserted near the replication origin, oriC, and the E. coli “centromere” analogue, migS, were more efficient than those targeting attP within other regions of the genome. Because the genomic region proximal to the oriC and migS sites is located at the extreme poles of the cell during chromosomal segregation, the oriC–migS region may be more exposed to the cytosol than are other regions of the E. coli chromosome. Thus, accessibility of pre-inserted attP to attB-containing incoming DNA may be crucial for the integration efficiency by serine-type integrases in heterologous cells. These results may be beneficial to the development of serine-type integrases-based genomic integration systems for various bacterial species.     

Microwounding is a pivotal factor for the induction of actin-dependent penetration resistance against fungal attack

Induced penetration resistance is triggered by failed penetration attempts of nonpathogenic fungi. The resistance mechanism is an important nonhost reaction in plants that can block the invasion of filamentous pathogens such as fungi and oomycetes. However, it remains unclear whether the mechanical stimuli accompanying fungal penetration play a role in induced penetration resistance, whereas the perforation of the cell wall may provide significant stimuli to plant cells. Here, we used microneedles or biolistic bombardment to mimic fungal penetration pegs and a micromanipulation transfer technique of the bio-probe, a germling of Blumeria graminis hordei, to the wounded cells to demonstrate that microwounds derived from fungal penetration attempts may trigger induced penetration resistance in plant cells. When preinoculated with the nonpathogenic fungi Erysiphe pisi and Colletotrichum orbiculare, which were unable to penetrate a barley cell, the penetration of a bio-probe that was transferred by micromanipulation onto the same cell was completely blocked. Fungal penetration was essential to the triggering of induced penetration resistance because a penetration-peg-defective mutant of C. orbiculare completely lacked the ability to trigger resistance. The artificial microwounds significantly, but not completely, blocked the penetration of the bio-probe. Treatment with the actin polymerization inhibitor cytochalasin A or expression of the actin depolymerizing protein HvPro1 caused complete ablation of the induced penetration resistance triggered by either failed fungal penetration or artificial microwounds. These results strongly suggest that microwounding may trigger actin-dependent induced penetration resistance. Manipulation of induced penetration resistance may be a promising target to improve basic disease resistance in plants.     

Streptococcal cytoplasmic pH is regulated by changes in amount and activity of a proton-translocating ATPase

The Streptococcus faecalis H+-ATPase (F1 X F0 complex) level was elevated when the cytoplasmic pH was shifted below 7.5. The elevated level was attained by the increase in functional unit (F1 X F0 complex) in membranes, but not by the activation of the enzyme. Our data strongly suggested that the increase in enzyme arises from stimulation of enzyme biosynthesis. When calls growing at pH 7.6 were transferred to an acid medium with a pH below 7, the amount of H+-ATPase increased. The amount of H+-ATPase decreased to the basal level when the medium was alkalized again. Cytoplasmic pH was not controlled normally in cells where a change in the amount of H+-ATPase was inhibited. Based on these findings and previous data (Kobayashi, H. (1985) J. Biol. Chem. 260, 72-76), we propose a model for the regulatory mechanism of streptococcal cytoplasmic pH: the pH is regulated by changes in amount and activity of the H+-ATPase, which are dependent on the cytoplasmic pH.