High degree of homology of the deduced protein structures of the tetracycline-resistance determinants between Bacillus subtilis plasmid pNS1981 and Staphylococcus aureus plasmid pTP5

The Protein Journal - The amino acid sequences of the tetracycline-resistance (Tcr) determinants of Bacillus subtilis plasmid pNS1981 and Staphylococcus aureus plasmid pTP5 have been deduced from...     

Enhancement of S1 nuclease-susceptibility of negatively superhelical DNA by netropsin

It was evidenced that the antibiotic netropsin enhances the single-strand-specific nuclease S1-susceptibility of negatively superhelical DNA. In contrast, an intercalating drug inhibited S1 action on the superhelical DNA. Negatively superhelical DNA is known to possess several (or a number of) unbasepaired sites sensitive to S1 cleavage. S1 cleaves generally the DNA once at these sites to result in production of the full-length linear form. However netropsin-bound DNA had a tendency to be cleaved by S1 simultaneously at plural sites producing several species of linear DNAs smaller than full-length size.     

Two linear plasmid-like DNA elements simultaneously maintained in Pleurotus ostreatus

Two linear plasmid-like DNA elements, designated pLP01 and pLP02, have been isolated from a strain of Pleurotus ostreatus, an edible basidiomycete. pLP01 (10.0 kb) and pLP02 (9.4 kb) were found in mitochondrial preparations of the fungus and appear to have 5' ends blocked by association of a protein. Proteinase K cleavability of the 5'-terminal protein of pLP01 was higher than that of pLP02, indicating that the terminal proteins of both plasmid-like elements are distinct from one another. pLP01 and pLP02 were estimated to be present to the extent of 1-2 copies each per mitochondrial genome equivalent. The two plasmid-like elements had no homology between them and also were not homologous with the mitochondrial and nuclear genomic DNAs of the fungus.     

Potential role for astroglial D-amino acid oxidase in extracellular D-serine metabolism and cytotoxicity

D-amino acid oxidase (DAO) is a flavoenzyme that catalyzes the oxidation of D-amino acids. In the brain, gene expression of DAO is detected in astrocytes. Among the possible substrates of DAO in vivo, D-serine is proposed to be a neuromodulator of the N-methyl-D-aspartate (NMDA) receptor. In a search for the physiological role of DAO in the brain, we investigated the metabolism of extracellular D-serine in glial cells. Here we show that after D-serine treatment, rat primary type-1 astrocytes exhibited increased cell death. In order to enhance the enzyme activity of DAO in cells, we established stable rat C6 glial cells overexpressing mouse DAO designated as C6/DAO. Treatment with a high dose of D-serine led to the production of hydrogen peroxide (H(2)O(2)) followed by apoptosis in C6/DAO cells. Among the amino acids tested, D-serine specifically exhibited a significant cell death-inducing effect. DAO inhibitors, i.e., sodium benzoate and chlorpromazine, partially prevented the death of C6/DAO cells treated with D-serine, indicating the involvement of DAO activity in d-serine metabolism. Overall, we consider that extracellular D-serine can gain access to intracellular DAO, being metabolized to produce H(2)O(2). These results support the proposal that astroglial DAO plays an important role in metabolizing a neuromodulator, D-serine.     

Short-term electroacupuncture at Zusanli resets the arterial baroreflex neural arc toward lower sympathetic nerve activity

Although electroacupuncture reduces sympathetic nerve activity (SNA) and arterial pressure (AP), the effects of electroacupuncture on the arterial baroreflex remain to be systematically analyzed. We investigated the effects of electroacupuncture of Zusanli on the arterial baroreflex using an equilibrium diagram comprised of neural and peripheral arcs. In anesthetized, vagotomized, and aortic-denervated rabbits, we isolated carotid sinuses and changed intra-carotid sinus pressure (CSP) from 40 to 160 mmHg in increments of 20 mmHg/min while recording cardiac SNA and AP. Electroacupuncture of Zusanli was applied with a pulse duration of 5 ms and a frequency of 1 Hz. An electric current 10 times the minimal threshold current required for visible muscle twitches was used and was determined to be 4.8 +/- 0.3 mA. Electroacupuncture for 8 min decreased SNA and AP (n = 6). It shifted the neural arc (i.e., CSP-SNA relationship) to lower SNA but did not affect the peripheral arc (i.e., SNA-AP relationship) (n = 8). SNA and AP at the closed-loop operating point, determined by the intersection of the neural and peripheral arcs, decreased from 100 +/- 4 to 80 +/- 9 arbitrary units and from 108 +/- 9 to 99 +/- 8 mmHg (each P < 0.005), respectively. Peroneal denervation eliminated the shift of neural arc by electroacupuncture (n = 6). Decreasing the pulse duration to <2.5 ms eliminated the effects of SNA and AP reduction. In conclusion, short-term electroacupuncture resets the neural arc to lower SNA, which moves the operating point toward lower AP and SNA under baroreflex closed-loop conditions.     

Intron-dependent accumulation of mRNA in Coriolus hirsutus of lignin peroxidase gene the product of which is involved in conversion/degradation of polychlorinated aromatic hydrocarbons

The homobasidiomycete Coriolus hirsutus coding sequences of a lignin peroxidase (LiP) gene (lip, containing six (I-VI) introns), a lip cDNA (lipc), and three lipc derivatives containing one (I), three (I-III), or five (I-V) introns were inserted into chromosome-integrating expression vector. These recombinant plasmids were introduced into C. hirustus monokaryotic strain. The transformant carrying the promoter-lipc-terminator cassette did not contain enough mRNA molecules to be detectable by Northern-blot analysis. On the other hand, all the transformants carrying cassettes of genomic lip and intron(s)-containing lipc sequences contained sufficient amounts of mRNAs to be easily detected by Northern-blot analysis. LiP activities in the culture supernatants of these transformants were found to be about five times as high as those of transformants carrying the lipc cassette (or no cassette). The culture supernatants of the transformants with high LiP activity showed remarkably high conversion activity toward pentachlorophenol (PCP) and degradation activity toward 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD). These results indicate that at least one intron (intron I) is required for accumulation of lip mRNA and its subsequent translational expression in C. hirsutus.     

A high-level expression vector containing selectable marker for continuous production of recombinant protein in insect cells

Insect cell expression systems are widely used to produce active recombinant proteins. Here, we have developed a high-level expression vector containing a selectable marker for continuous production of recombinant proteins in insect cells. The plasmid, pXIHAbla, developed in this study, established a polyclonal cell line 8 days shorter than pXINSECT-DEST38 and pBmAneo. In addition, pXIHAbla exhibited an approximately fivefold higher average enhanced GFP expression level and approximately a twofold higher bionanocapsule secretion level than pXINSECT-DEST38. Using this plasmid, insect cells that highly express active proteins have been easily established.     

Agrobacterium-mediated transformation of the ectomycorrhizal basidiomycete Tricholoma matsutake that produces commercially valuable fruit bodies, matsutake

Using agroinfection with a T-DNA vector carrying a hygromycin resistance marker, the recombinants were generated for the first time from the ectomycorrhizal basidiomycete Tricholoma matsutake, which produces commercially valuable fruit bodies, matsutake, during association with Pinus sp. plants. The transformation system may be useful in the genetic analysis of T. matsutake.