Calcium-activated neutral protease and its endogenous inhibitor Activation at the cell membrane and biological function

The structures of calcium-activated neutral protease (CANP) and its endogenous inhibitor elucidated recently have revealed novel features with respect to their structure-function relationship and enzyme activity regulation. The protease is regarded as a proenzyme which can be activated at the cell membrane in the presence of Ca²⁺ and phospholipid, and presumably regulates the functions of proteins, especially membrane-associated proteins, by limited proteolysis. Protein kinase C is hydrolysed and activated by CANP at the cell membrane to a cofactor-independent form. These results are reviewed and the possible involvement of CANP in signal transduction is discussed.     

Study of insulin response to oral glucose load after acute and chronic glycemic control in type 2 diabetic subjects

To investigate whether correction of fasting hyperglycemia per se improves the insulin secretion in type 2 diabetic subjects, plasma insulin response to 75 g oral glucose load has been studied after acute and chronic normalization of fasting plasma glucose levels in 7 overt type 2 diabetic subjects. For the acute normalization of elevated fasting plasma glucose levels, an artificial endocrine pancreas was employed. Although fasting plasma glucose concentrations were normalized before the oral glucose challenge, insulin response to oral glucose was not improved compared to those without normalization of fasting plasma glucose levels. After 1-3 month control of hyperglycemia, the insulin response to glucose in the subjects was significantly improved compared to those without treatments. Results indicate that chronic metabolic control is essential for the improvement of insulin response to glucose in type 2 diabetic subjects, and also suggest that the impaired insulin secretion in type 2 diabetes is not due to hyperglycemia per se, but due to the metabolic derangements which lead to chronic hyperglycemia.     

The systematic position ofAsplenium cardiophyllum (Aspleniaceae)

Asplenium cardiophyllum is a morphologically unusual species with simple leaves and anastomosing venation, and is often placed in the segregate genusBoniniella. To determine its systematic position, character comparisons were made of vascular anatomy, raphides in leaf epidermis, chromosome number and perispore of this species and those ofAsplenium sect.Hymenasplenium. Asplenium cardiophyllum conforms with sect.Hymenasplenium in its dorsiventral dictyostele, the presence of raphides, a chromosome number of 2n=156 (x=39), and lophate peristore with spinulate projections on the lumina. We therefore propose to includeA. cardiophyllum in that section. Dedicated to the memory of the late Professor Kunio Mitui.     

Purification of GP57, and auxin-regulated extracellular glycoprotein of carrots,and its immunocytochemical localization in dermal tissues

A glycoprotein (GP57) was purified by ion-exchange and hydroxylapatite column chromatography from the 70%-ethanol precipitate of culture medium of non-embryogenic carrot cells (Daucus carota L.) grown with 2,4-dichlorophen-oxyacetic acid (2,4-D). Its apparent molecular mass (M _r) was estimated to be 57000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis and 50000 by gel filtration. GP57 contained 14% (w/w) carbohydrate; the M _r of the peptide portion was estimated to be 55000 after deglycosylation by trifluoromethanesulfonic acid. GP57 is composed of two polypeptides with the same M_r and with very similar amino-acid composition but different pI values, 8.8 and 9.5. Both are rich in aspartic acid, serine and threonine, and may possess N-linked oligosaccharide chains, including fucose and xylose. A monoclonal antibody (MAb) against the purified GP57 reacted with both the pI 8.8 and the 9.5 components, as well as the deglycosylated GP57. Immunoblotting with the MAb indicated that GP57 is synthesized in and released from cultured cells which have been supplied with auxin. In immunocytochemical studies, GP57 was found in the space between the embryo and the endosperm of dry seeds, and its content decreased during germination. GP57 was also found in the endodermis and epidermis of young roots, the periderm of mature taproots, and the epidermis of petioles and young leaves.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GP57 M _r-57000 glycoprotein - GP65 M _r-65000 glycoprotein - MAb monoclonal antibody - M _r apparent molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - TFMS trifluoromethanesulfonic acid     

Effects of monoenergetic X-rays with resonance energy of bromine K-absorption edge on bromouracil-labelled E. coli cells

In order to examine enhanced killing that might be induced by Auger cascades in the incorporated atoms in cells, bromouracil(BrU)-labelled E. coli cells were irradiated with monoenergetic X-rays at 13.49 and 12.40keV, just above and below the K-absorption edge of bromine. In both cases BrU-labelled cells were more sensitive for killing than were normal cells. However, when the degree of BrU-sensitization was compared between the two energies of X-rays, the enhanced killing at 13.49 keV was only small, 2 +/- 8 per cent based on the D0 value in saline. By the addition of DMSO, which is believed to suppress radical-mediated effects, killing of BrU-labelled cells was enhanced at 13.49 keV by 8 +/- 4 per cent as compared with 12.40 keV, based on D0. These results have been examined in terms of absorbed energy in BrU-labelled cells and in terms of the number of induced Auger events.     

The branching of the inflorescence and vegetative shoot and taxonomy of the genusKummerowia (Leguminosae)

A study of the branching of the inflorescence and the vegetative shoot of the genusKummerowia, consisting ofK. stipulacea (Maxim.) Makino andK. striata (Thunb.) Schindler, has led to the following conclusions: (1) the inflorescences of both species are reduced compound cymes, (2) the branching system of the inflorescence ofKummerowia is not clearly different from that of the vegetative shoot and there are some transitional forms between both systems, and (3) the inflorescence ofKummerowia is different from the racemose inflorescences ofLespedeza andCampylotropis. Based on the differences found in the branching system of the inflorescence,Kummerowia is distinctly separated fromLespedeza andCampylotropis and is more correctly treated as a distinct genus from the latter two.     

Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection

Fusion of red cell ghosts containing foreign materials with cells results in the introduction of the materials into the cells (red cell-mediated microinjection). Until now, 'two-step dialysis' has mainly been used for trapping proteins in the ghosts. Large-sized materials such as DNA, however, are rarely trapped in the ghosts, since the holes in the red cell membrane caused by osmotic shock are too small for such materials to pass through. In this study, we improved the trapping technique. Some of the Hind III fragments of lambda phage DNA as well as proteins could be trapped in the ghosts when the mixture of these materials and red cells were frozen at -80 degrees C for a short period followed by quick thawing. Red cell-mediated microinjection using ghosts containing plasmid pBR322 linked with a Herpes simplex viral thymidine kinase (tk) gene brought about transformation of tk-defective L cells, the efficiency of transformation was 1 out of 20 000-60 000 cells fused with the ghosts.     

Expression of a moderately anionic peroxidase is induced by aluminum treatment in tobacco cells: Possible involvement of peroxidase isozymes in aluminum ion stress

To clarify the mechanism of aluminum (Al) toxicity and Al tolerance, we isolated a new clone (pAL201) from a tobacco cDNA library. Northern blot hybridization analysis indicated that the expression of pAL201 is induced by Al treatment and phosphate (P₁) starvation. The complete cDNA sequence suggested that this clone encodes a moderately anionic peroxidase (EC 1.11.1.7). Analysis by isoelectric focussing indicated that a moderately anionic peroxidase (approximately pI 6.7) and two cationic peroxidases (pI 9.2 and 9.7) in the soluble fraction are activated by Al treatment and P₁ starvation, while two moderately anionic isozymes are repressed by these stresses. We suppose that Al ion stress can control the activity of some peroxidase isozymes, one of which is probably induced by enhanced gene expression of pAL201. There is a possibility that some of these isozymes have some functions in Al ion stress.