1-aminocyclopropane-1-carboxylate (ACC) deaminase induced by ACC synthesized and accumulated in Penicillium citrinum intracellular spaces

We have already described how 1-aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of the plant hormone ethylene, is synthesized in Penicillium citrinum through the same reaction by the catalysis of ACC synthase [EC 4.4.1.14] as in higher plants. In addition, ACC deaminase [EC 4.1.99.4], which degrades ACC to 2-oxobutyrate and ammonia, was also purified from this strain. To study control of induction of ACC deaminase in this organism, we have isolated and analyzed the cDNA of P. citrinum ACC deaminase and studied the expression of ACC deaminase mRNA in P. citrinum cells. By the analysis of peptides from the digests of the purified and modified ACC deaminase with lysylendopeptidase, 70 % of its amino acid sequences were obtained. These amino acid sequences were used to identify a cDNA, consisting of 1,233 bp with an open reading frame of 1,080 bp encoding ACC deaminase with 360 amino acids. The deduced amino acids from the cDNA are identical by 52% and 45% to those of enzymes of Pseudomonas sp. ACP and Hansenula saturnus. Through Northern blot analysis, we found that the mRNA of ACC deaminase was expressed in P. citrinum cells grown in a medium containing 0.05% L-methionine. These findings suggest that ACC synthesized by ACC synthase and accumulated in P. citrinum intracellular spaces can induce the ACC deaminase that degrades the ACC.     

Fatty acid hydroperoxide lyase in tomato fruits: cloning and properties of a recombinant enzyme expressed in Escherichia coli

Fatty acid hydroperoxide lyase (HPL) is a member of a novel subfamily of cytochrome P450 and catalyzes a cleavage reaction of fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids. A cDNA encoding tomato fruit HPL (LeHPL) was obtained. An active LeHPL was expressed in E. coli and purified. It showed highest activity against the 13-hydroperoxide of linolenic acid, followed by that of linoleic acid. 9-Hydroperoxides were poor substrates. The absorption spectrum of the purified LeHPL in the native form was similar to that of most P450s although a CO-adduct having a lambda max at 450 nm could not be obtained. LeHPL activity is reversibly inhibited by nordihydroguaiaretic acid, while salicylic acid irreversibly inhibited it. LeHPL is kinetically inactivated by fatty acid hydroperoxides, especially 9-hydroperoxides. The inactivation is prevented by inhibitors of LeHPL. Thus, HPL catalytic activity is thought to be essential to its inactivation. During the inactivation, an abolition of the Soret band was evident, indicating that inactivation is caused mainly by degradation of the prosthetic heme in LeHPL.     

Analysis of Tag1-like elements in Arabidopsis thaliana and their distribution in other plants.

Analysis of genomic DNA of Arabidopsis Columbia (Col.) ecotype using a transposon Tag1-specific primer showed the presence of Tag1 homologues which was confirmed by Southern hybridization with a Tag1 probe. Further analysis showed that the homologue, 0.75 kb in length, had inverted repeats at both ends, 8-bp duplicated sequences at the site at which it is located and about 80% homology with Tag1, and was randomly distributed in the Arabidopsis genome. Based on these results, we concluded that these elements are non-autonomous variants of Tag1 and we termed this element sTag1. Using the polymerase chain reaction fragment hybridization technique, we found the distribution of such homologues in other plant species.     

Inhibition of CX3CL1 (fractalkine) improves experimental autoimmune myositis in SJL/J mice

Idiopathic inflammatory myopathy is a chronic inflammatory muscle disease characterized by mononuclear cell infiltration in the skeletal muscle. The infiltrated inflammatory cells express various cytokines and cytotoxic molecules. Chemokines are thought to contribute to the inflammatory cell migration into the muscle. We induced experimental autoimmune myositis (EAM) in SJL/J mice by immunization with rabbit myosin and CFA. In the affected muscles of EAM mice, CX3CL1 (fractalkine) was expressed on the infiltrated mononuclear cells and endothelial cells, and its corresponding receptor, CX3CR1, was expressed on the infiltrated CD4 and CD8 T cells and macrophages. Treatment of EAM mice with anti-CX3CL1 mAb significantly reduced the histopathological myositis score, the number of necrotic muscle fibers, and infiltration of CD4 and CD8 T cells and macrophages. Furthermore, treatment with anti-CX3CL1 mAb down-regulated the mRNA expression of TNF-alpha, IFN-gamma, and perforin in the muscles. Our results suggest that CX3CL1-CX3CR1 interaction plays an important role in inflammatory cell migration into the muscle tissue of EAM mice. The results also point to the potential therapeutic usefulness of CX3CL1 inhibition and/or blockade of CX3CL1-CX3CR1 interaction in idiopathic inflammatory myopathy.     

Control of cyclin-dependent kinase 5 (Cdk5) activity by glutamatergic regulation of p35 stability

Although the roles of cyclin-dependent kinase 5 (Cdk5) in neurodevelopment and neurodegeneration have been studied extensively, regulation of Cdk5 activity has remained largely unexplored. We report here that glutamate, acting via NMDA or kainate receptors, can induce a transient Ca(2+)/calmodulin-dependent activation of Cdk5 that results in enhanced autophosphorylation and proteasome-dependent degradation of a Cdk5 activator p35, and thus ultimately down-regulation of Cdk5 activity. The relevance of this regulation to synaptic plasticity was examined in hippocampal slices using theta burst stimulation. p35(-/-) mice exhibited a lower threshold for induction of long-term potentiation. Thus excitatory glutamatergic neurotransmission regulates Cdk5 activity through p35 degradation, and this pathway may contribute to plasticity.     

High throughput easy microinjection with a single-cell manipulation supporting robot

A single-cell manipulation supporting robot (SMSR) has been developed for the high throughput and easy microinjection. Its concept is to let an experimenter concentrate his/her attention only on the microinjection by facilitating other associated works. SMSR was applied to the microinjection into rice protoplasts and mouse embryonic stem (ES) cells. The microinjection into these cells is exceptionally difficult than usual animal cells such as fibroblasts. In the case of rice protoplast, for example, non-stop microinjection into 100 cells could be done within 1h that was 17-times faster than that of the robot-less work. The success rate was 7-8% that was same level obtained by the robot-less work. The present results indicate that SMSR is a useful machine for the microinjection of specific genes and proteins in living cells to analyze their respective functions, which is an urgent and important subject in the post-genome era.     

Improved production of L-lysine by disruption of stationary phase-specific rmf gene in Escherichia coli

Growth and rate, at which fermentation products are formed in cells, generally decreases during the stationary phase as a result of changes in gene expression. We focused on the rmf gene, which encodes the ribosome modulation factor protein, as a target for strain modification in order to improve the rate of L-lysine production in Escherichia coli. Increased expression of the rmf gene during the stationary phase was confirmed under various cultivation conditions using DNA macroarray analysis. Mutants with disrupted rmf were then generated from an L-lysine-producing E. coli strain. The rates of L-lysine accumulation and production were significantly increased in disruptants that were cultivated with excess phosphate. By contrast, a higher biomass was generated in disruptants that were grown under limited phosphate conditions. These results demonstrate that disruption of the rmf gene significantly affects L-lysine production and growth in E. coli.     

α-Glucosidase from a strain of deep-sea Geobacillus: a potential enzyme for the biosynthesis of complex carbohydrates

An α-glucosidase from Geobacillus sp. strain HTA-462, one of the deepest sea bacteria isolated from the sediment of the Mariana Trench, was purified to homogeneity and estimated to be a 65-kDa protein by SDS-PAGE. At low ion strength, the enzyme exists in the homodimeric form (130 kDa). It is a thermo- and alkaline-stable enzyme with a half-life of 13.4 h and a maximum hydrolytic activity at 60°C and pH 9.0 in 15 mM glycine–NaOH buffer. The enzyme exclusively hydrolyzed α-1,4-glycosidic linkages of oligosaccharides in an exo-type manner. The enzyme had an overwhelming transglycosylation activity and glycosylated various non-sugar molecules when maltose was used as a sugar donor. It converted maltose to isomaltose. The gene encoding the enzyme was cloned and sequenced. The recombinant enzyme could be extracellularly overproduced by Bacillus subtilis harboring its gene and preserved the primary properties of the native enzyme. Site-directed mutagenesis experiments showed that Asp98 is essential for the enzyme activity in addition to Asp199, Asp326, and Glu256.