The Pro to glycine mutation of staphylococcal nuclease simplifies the unfolding—folding kinetics

Kinetics of unfolding and refolding of a staphylococcal nuclease mutant, in which Pro¹¹⁷ is replaced by glycine, have been investigated by stopped-flow circular dichroism, and the results are compared with those for the wild-type protein. In contrast to the biphasic unfolding of the wild-type nuclease, the unfolding of the mutant is represented by a single-phase reaction, indicating that the biphasic unfolding for the wild-type protein is caused by cis-trans isomerization about the prolyl peptide bond in the native state. The proline mutation also simplifies the kinetic refolding. Importance of the results in elucidating the folding mechanism is discussed.     

Growth and reproduction of asexual konjak plants (Amorphophalus konjac K. koch)

To examine the differences of the growth and reproduction of different-aged plants, 0-, 1-, and 2-year plants ofAmorphophalus konjac were investigated. RGR and daily net production per unit productive part, relative net-production rate (α′), of the 0-year plant were largest, although NAR was highest in the 2-year plant. This was due to the large LAR of the 0-year plant, owing to its large SLA. With increase in age, LAR decreased and NAR increased. Thus, it appeared that the age of plant exerts two opposite effects on dry-matter production. Since these effects cancel each other out, differences in RGR and α′ between the two older plants were not significant. We estimated that plant size appears to be primarily responsible for these effects. The 0-year plant showed the least distribution ratio of net production into reproductive (storage) organs, and the highest productivity of the reproductive part. The ratio of the production of corm to total reproductive-part production, the D-reproduction index, was independent of age, and critical size in vegetative propagation could not be detected.     

Alteration of plasmid DNA-mediated transformation and mutation induced by covalent binding of benzo[a]pyrene- 7,8-dihydrodiol-9,10-oxide in Escherichia coli

Plasmid-mediated transformation and mutagenesis induced by (±)-trans- benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide (BP-DEI) in recipient Escherichia coli (E. coli) have been studied. Because plasmid DNA is used, the system is entirely free from direct toxic effects of BP-DEI on the recipient cells. Plasmid pK0482 DNA, which has two dominant genes, β-lactamase (amp-r) and galactokinase (galK) was modified with BP-DEI prior to its transformation of E. coli N99, AB1157, AB2463(recA^?) and AB1886(uvrA^?). Transformants were selected by ampicillin resistance and mutations were analyzed simultaneously by the altered expression of the galK gene. (1) Approx. 3 molecules of BP-DEI per molecule of pK0482 DNA decreased the transformation efficiency to 37% in AB1157 and the mutation frequency in this strain was proportional to the amount of BP-DEI covalently bound to pK0482 DNA. (2) In AB1886(uvrA^?) a 37% transformation efficiency was produced by only 1 molecule of BP-DEI per molecule of pK0482 DNA, and the mutation frequency in this strain was higher than in AB1157. (3) In AB2463(recA^?), the transformation efficiency was similar to that obtained with AB1157, but mutagenesis was clearly suppressed. (4) Polyacrylamide gel patterns of restriction digests of the pK0482 mutated at the galK gene were indistinguishable from those of the unmutated plasmid DNA.     

Establishment of Autoantibody-Producing Cell Lines from Peripheral Blood Lymphocytes of Patients with Systemic Lupus Erythematosus

Autoantibody-producing B cell lines were established from peripheral blood lymphocytes of patients with systemic lupus erythematosus. Peripheral blood lymphocytes obtained from five of seven patients were successfully transformed by Epstein-Barr virus. Two of four established B lymphoblastoid cell lines examined in this study produced anti-nuclear factor antibodies and one of them produced anti-single-stranded DNA and anti-double-stranded DNA antibodies. These results indicate that B cell clones committed to self antigens are transformed by Epstein-Barr virus and continue to produce autoantibodies. In order to establish a monoclonal autoantibody-producing B cell line, the cells were cloned by a limiting dilution method. The data suggest that it is possible to establish a monoclonal autoantibody-producing B cell line by the combination of transformation of B cells by Epstein-Barr virus and extensive cloning.     

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of α-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that α-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that α-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.     

Proline in the Osmoregulation of Brevibacterium lactofermentum

Osmoregulation in Brevibacterium lactofermentum was studied. Proline was accumulated up to approximately 35mg/g dry cell weight in the cells of a wild strain of the bacterium grown under osmotic stress. The osmotic tolerance of a proline auxotroph mutant obtained from the bacterium was lower than that in the wild strain. The activity of pyrroline-5-carboxylate reductase, one of the enzymes in the proline biosynthetic pathway, increased about 3-fold when the cells of B. lactofermentum were grown under osmotic stress. These data indicated that proline is important in osmoregulation in the bacterium.