One step purification procedure of elastase from pancreatic powder by affinity chromatography

Pancreatic elastase was isolated from acetone powder of porcine pancreas by a one step purification procedure on a trialanyl CH-Sepharose 4B affinity column. This column exhibited affinity not only for active elastase but also for trypsin and chymotrypsin which were present in the same pancreatic powder. However, as the extent of affinity toward elastase is considerably higher, the proper conditions were determined with which the adsorbed elastase was isolated in a highly purified form. The yield of elastolytic activity ranged from 60–85% and the purified elastase was shown to be one component by polyacrylamide disc electrophoresis.     

In vitro construction of bacteriophage λ and plasmid DNA molecules containing DNA fragments from bacteriophage T4

Restriction endonucleases EcoRI and HindIII generated fragments of T4 cytosine-containing DNA were inserted into bacteriophage vector λgtSu_III and plasmid vectors pMB9 and pBR313. Resulting clones were screened for hybridization with ³²P labeled T4 tRNA. Recombinant bacteriophages and plasmids were isolated which contained a T4 fragment coding for T4 RNA species 1 and 2 and T4 tRNA^Arg. Selected λ-T4 hybrid bacteriophages were grown to high titer and their DNA analyzed by gel electrophoresis.     

Indirect evidence that guarded quiescent deutonymph females invest energy to attract conspecific males in the Kanzawa spider mite (Acari: Tetranychidae)?

Because only the first mating results in fertilization in Tetranychus kanzawai (Acari: Tetranychidae), adult males guard quiescent deutonymph females (i.e., precopulatory mate guarding). A previous study reported that quiescent deutonymph females guarded by a male attract more conspecific males than solitary females and then hypothesized that guarded females release more chemical signals than solitary ones to attract males. Quiescent deutonymph females do not feed. If the hypothesis is appropriate, guarded females should invest energy in attracting males at the expense of investment in other activities, such as egg production. Therefore, we compared oviposition rates immediately after adult emergence between guarded females and solitary females. On the first day, the oviposition rate of guarded females was lower than that of solitary females. On the second day, however, there was no significant difference between female groups. These results suggest that guarded females invest energy in activities other than egg production before adult emergence and that the energetic cost is easily recoverable. We believe that our finding indirectly supports the hypothesis that guarded females release more chemical signals than solitary females to attract conspecific males.     

4-Hydroxy hexenal derived from dietary n-3 polyunsaturated fatty acids induces anti-oxidative enzyme heme oxygenase-1 in multiple organs

It has recently been reported that expression of heme oxygenase-1 (HO-1) plays a protective role against many diseases. Furthermore, n-3 polyunsaturated fatty acids (PUFAs) were shown to induce HO-1 expression in several cells in vitro, and in a few cases also in vivo. However, very few reports have demonstrated that n-3 PUFAs induce HO-1 in vivo.     

Cloning and expression of subtilisin amylosacchariticus gene

The gene encoding subtilisin Amylosacchariticus from Bacillus subtilis var. amylosacchariticus was isolated and the entire nucleotide sequence of the coding sequence was determined. The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprising 275 residues. There were discrepancies in 10 amino acids between the sequence elucidated from the nucleotide sequence and the published protein sequence (Kurihara et al. (1972) J. Biol. Chem. 247, 5619-5631). The nucleotide sequence was highly homologous to that of subtilisin E gene from B. subtilis 168, with discrepancies at 12 nucleotides out of 1,426 nucleotides we sequenced. Ten of them were found in mature subtilisin coding sequence, which resulted in two amino acid changes and another one was in the putative promoter region between two genes. The productivity of subtilisin in culture broth of B. subtilis var. amylosacchariticus was much higher than that of B. subtilis 168. The enzyme gene was inserted in a shuttle vector pHY300PLK, with which B. subtilis ISW1214 was transformed. The proteolytic activity found in the culture broth of the transformed bacterium was 20- and 4-fold higher than those of the host strain and B. subtilis var. amylosacchariticus, respectively. Subtilisin Amylosacchariticus was easily purified to a crystalline form from culture filtrate of cloned B. subtilis, after a single step of chromatography on CM-cellulose.     

Isolation and identification of lactic acid bacteria with effect of immune protection to Eschericia coli in mice

Lactic acid bacteria were isolated from an alcohol fermentation broth, and the activity as a probiotic was examined using pathogenic E. coli. Thirty-six strains exhibiting good growth were isolated in the medium of concentrated mush which was a residue resulted in the alcohol distillation process. One of these strains, Lactobacillus paracasei subsp. paracasei I-5, could be grown in the medium containing 8 vol% ethanol and at 45 degrees C. The characteristics were different from the type strain, L. paracasei subsp. paracasei NBRC 15889. L. paracasei I-5 showed an excellent growth in the concentrated mush, which just diluted two-fold and adjusted the pH. ICR mice were fed with a standard germ-free feed (CMF) and the strain I-5 (7 x 10(9) cells/day) was orally administrated for 11 days prior to the intraperitoneal challenge with pathogenic E. coli Juhl. After the challenge, mice administrated the strain I-5 exhibited a high survival rate and survival extension days (p < 0.01) compared with the control. The results suggested that the strain might enhance the animal resistance against microbial pathogens. Neonatal diarrhea caused by E. coli is a serious disease in calf breeding. The strain might be practically valuable to prevent diarrhea in calves.     

Mechanisms for the apoptosis of small cell lung cancer cells induced by anti-GD2 monoclonal antibodies: roles of anoikis

Anti-GD2 ganglioside antibodies could be a promising, novel therapeutic approach to the eradication of human small cell lung cancers, as anti-GD2 monoclonal antibodies (mAbs) induced apoptosis of small cell lung cancer cells in culture. In this study, we analyzed the mechanisms for the apoptosis of these cells by anti-GD2 mAbs and elucidated the mechanisms by which apoptosis signals were transduced via reduction in the phosphorylation levels of focal adhesion kinase (FAK) and the activation of a MAPK family member, p38, upon the antibody binding. Knock down of FAK resulted in apoptosis and p38 activation. The inhibition of p38 activity blocked antibody-induced apoptosis, indicating that p38 is involved in this process. Immunoprecipitation-immunoblotting analysis of immune precipitates with anti-FAK or anti-integrin antibodies using an anti-GD2 mAb revealed that GD2 could be precipitated with integrin and/or FAK. These results suggested that GD2, integrin, and FAK form a huge molecular complex across the plasma membrane. Taken together with the fact that GD2+ cells showed marked detachment from the plate during apoptosis, GD2+ small cell lung cancer cells seemed to undergo anoikis through the conformational changes of integrin molecules and subsequent FAK dephosphorylation.     

Efflux of sphingomyelin, cholesterol, and phosphatidylcholine by ABCG1

Cholesterol and phospholipids are essential to the body, but an excess of cholesterol or lipids is toxic and a risk factor for arteriosclerosis. ABCG1, one of the half-type ABC proteins, is thought to be involved in cholesterol homeostasis. To explore the role of ABCG1 in cholesterol homeostasis, we examined its subcellular localization and function. ABCG1 and ABCG1-K120M, a WalkerA lysine mutant, were localized to the plasma membrane in HEK293 cells stably expressing ABCG1 and formed a homodimer. A stable transformant expressing ABCG1 exhibited efflux of cholesterol and choline phospholipids in the presence of BSA, and the cholesterol efflux was enhanced by the presence of HDL, whereas cells expressing ABCG1-K120M did not, suggesting that ATP binding and/or hydrolysis is required for the efflux. Mass and TLC analyses revealed that ABCG1 and ABCA1 secrete several species of sphingomyelin (SM) and phosphatidylcholine (PC), and SMs were preferentially secreted by ABCG1, whereas PCs were preferentially secreted by ABCA1. These results suggest that ABCA1 and ABCG1 mediate the lipid efflux in different mechanisms, in which different species of phospholipids are secreted, and function coordinately in the removal of cholesterol and phospholipids from peripheral cells.