Solution structure of human growth hormone-releasing factor fragment (1-29) by CD: characteristic conformational change on phospholipid membrane

The conformations of synthetic human growth hormone-releasing factor fragment (1-29) in the presence and the absence of 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol liposome as well as in aqueous 2,2,2-trifluoroethanol solution were investigated by CD spectroscopy. The secondary structure of the peptide in each solution was analyzed by two methods. Both results show that the peptide has an unordered structure in the aqueous solution, whereas it folds into helical structure in the aqueous alcohol and in the phospholipid solution. In addition, although the peptide exists as almost complete helix in the 50 vol% aqueous alcohol (80-90% helicity), it does not reach full helicity even in the solution containing excess amount of phospholipid liposome (maximum 65-70% helicity). The conformational difference is explained by the characteristic amphipathy of the peptide, i.e., the necessity to twist the separated amphipathic helical parts in the interaction with the phospholipid membrane probably makes the helicity of the peptide decrease.     

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.     

Unique requirements for template primers of DNA polymerase beta from rat ascites hepatoma AH130 cells.

The optimal condition for the rat DNA polymerase beta activity with (rA)n . (dT)12-18 as a template-primer was determined. The activity was remarkably affected by the concentration of the primer, (dT)12-18' and the mixing ratio of (dT)12-18 to (rA)n. DNA polymerase beta requires higher primer concentration (Km = 11.1 microM with respect to 3'-OH of the primer) than DNA polymerase gamma (Km = 0.04 microM) or oncornaviral DNA polymerase (Km = 0.08 microM) and the enzyme represented the maximum activity in the base ratio of 2:1 with (dT)12-18 and (rA)n suggesting the difference in reaction mechanisms of these enzymes. Under the optimized conditions, the specific activity of the near homogeneous preparation of DNA polymerase beta was 1,000,000 units per mg protein.     

Aphidicolin: a specific inhibitor of DNA polymerases in the cytosol of rat liver.

Aphidicolin, a tetracyclic diterpenoid, is known to be antiviral and to inhibit the incorporation of thymidine into DNA of cultured human embryonic lung cells. We examined effects of the compound on the activity of several DNA polymerases obtained from subcellular fractions of rat liver. Aphidicolin at a concentration of 15 μg/ml caused a 85% reduction in level of the activity of crude and partially purified DNA polymerases from the cytosol. However, aphidicolin even at a concentration of 75 μg/ml failed to affect the activity of crude and partially purified DNA polymerases from nuclear and mitochondrial fractions.     

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.     

Purification and identification of intermediate catabolic products in the in vivo degradation of pig liver phosphofructokinase

Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was purified to homogeneity from pig livers. Polyclonal antibody against the enzyme was induced in a rabbit, and the IgG fraction was obtained by chromatography on a Protein A-Sepharose CL-4B column. The specific antibody was purified further by immunoaffinity chromatography on a phosphofructokinase-conjugated affinity column. Intermediate catabolic products of phosphofructokinase were extracted from fresh pig livers under conditions of inhibition of proteinases, concentrated by chromatography on an anti-phosphofructokinase IgG-conjugated affinity column, and purified by two-dimensional polyacrylamide gel electrophoresis. Their cross-reactivities to the purified phosphofructokinase were assessed by an immunoelectrotransfer blot method. The intact form of phosphofructokinase in pig liver was demonstrated as the major spot of 84 kDa on the blot. Polypeptides of 68, 64, 56, and 51 kDa showed apparent cross-reactivities to phosphofructokinase. The structural homology among them was confirmed by proteinase V8 digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The possibility of artifacts in preparation was ruled out by an internal tracer method. Thus, it is concluded that the predominant isozyme of phosphofructokinase in pig liver (84 kDa) is in vivo degraded via intermediate catabolic products of 68, 64, 56, and 51 kDa.     

Designed and real working situations in machine systems operation.

New work situations designed at the stage when new machine systems are introduced are realized on the assumption that the new systems can maintain their designed functions consistently, generally eliminating previous work habits and without sufficient knowledge about real working processes and skills. This may produce differences between designed and real working situations. Some examples are presented from observations on influence of modern design of cargo ships on their crews. It was difficult for crews to maintain stable working conditions, especially when machine systems deviated from their designed functions. Often the crew had to work in off-duty hours giving up private freetime activities. Among various factors contributing to the discrepancies between designed and real work, lack of availability of the new systems is the most important factor. Also important is lack of back-up systems which would function either when the machine systems are out of order or when previous working skills and habits must be applied. A need for developing methods of evaluation of these two factors from ergonomic points of view is pointed out.