Rapid increase of vacuolar volume in response to salt stress

Suspension-cultured cells of mangrove [Bruguiera sexangula (Lour.) Poir.] showed a rapid increase in vacuolar volume under salt stress, although there was no change in the cell volume. The rapid increase in the vacuolar volume was an active process, which followed the activation of the tonoplast H(+)-ATPase and the vacuolar acid phosphatase. The same phenomenon was observed in barley (Hordeum vulgare L. cv. Doriru) root meristematic cells under salt stress but not in pea ( Pisum sativum L.). Increases in vacuolar volume could potentially protect the cytoplasm by decreasing the cytoplasmic volume during the initial phases of salt stress.     

Superoxide and hydrogen peroxide production by Drosophila mitochondria

Drosophila melanogaster is a key model organism for genetic investigation of the role of free radicals in aging, but biochemical understanding is lacking. Superoxide production by Drosophila mitochondria was measured fluorometrically as hydrogen peroxide, using its dependence on substrates, inhibitors, and added superoxide dismutase to determine sites of production and their topology. Glycerol 3-phosphate dehydrogenase and center o of complex III in the presence of antimycin had the greatest maximum capacities to generate superoxide on the cytosolic side of the inner membrane. Complex I had significant capacity on the matrix side. Center i of complex III, cytochrome c, and complex IV produced no superoxide. Native superoxide generation by isolated mitochondria was also measured without added inhibitors. There was a high rate of superoxide production with sn-glycerol 3-phosphate as substrate; two-thirds mostly from glycerol 3-phosphate dehydrogenase on the cytosolic side and one-third on the matrix side from complex I following reverse electron transport. There was little superoxide production from any site with NADH-linked substrate. Superoxide production by complex I following reverse electron flow from glycerol 3-phosphate was particularly sensitive to membrane potential, decreasing 70% when potential decreased 10 mV, showing that mild uncoupling lowers superoxide production in the matrix very effectively.     

Pro-carboxypeptidase R is an acute phase protein in the mouse, whereas carboxypeptidase N is not

Carboxypeptidase R (EC 3.4.17.20; CPR) and carboxypeptidase N (EC 3. 4.17.3; CPN) cleave carboxyl-terminal arginine and lysine residues from biologically active peptides such as kinins and anaphylatoxins, resulting in regulation of their biological activity. Human proCPR, also known as thrombin-activatable fibrinolysis inhibitor, plasma pro-carboxypeptidase B, and pro-carboxypeptidase U, is a plasma zymogen activated during coagulation. CPN, however, previously termed kininase I and anaphylatoxin inactivator, is present in a stable active form in plasma. We report here the isolation of mouse proCPR and CPN cDNA clones that can induce their respective enzymatic activities in culture supernatants of transiently transfected cells. Potato carboxypeptidase inhibitor can inhibit carboxypeptidase activity in culture medium of mouse proCPR-transfected cells. The expression of proCPR mRNA in murine liver is greatly enhanced following LPS injection, whereas CPN mRNA expression remains unaffected. Furthermore, the CPR activity in plasma increased 2-fold at 24 h after LPS treatment. Therefore, proCPR can be considered a type of acute phase protein, whereas CPN is not. An increase in CPR activity may facilitate rapid inactivation of inflammatory mediators generated at the site of Gram-negative bacterial infection and may consequently prevent septic shock. In view of the ability of proCPR to also inhibit fibrinolysis, an excess of proCPR induced by LPS may contribute to hypofibrinolysis in patients suffering from disseminated intravascular coagulation caused by sepsis.     

Transglutaminase in sporulating cells of Bacillus subtilis

We screened various Bacillus species producing transglutaminase (TGase), measured as labeled putrescine incorporated into N,N-dimethylcasein. As a result, we detected TGase activity in sporulating cells of B. subtilis, B. cereus, B. alvei and B. aneurinolyticus, and found TGase activity related to sporulation. TGase activity of Bacillus subtilis was detected in lysozyme-treated sporulating cells during late sporulation, but not in cells without lysozyme treatment or the supernatant of the culture broth. TGase was found to be localized on spores. TGase was preliminarily purified by gel filtration chromatography for characterization. Its activity was eluted in the fractions indicating a molecular weight of approximately 23 kDa. TGase could cross-link and polymerize a certain protein. The enzyme was strongly suggested to form epsilon-(gamma-glutamyl)lysine bonds, which were detected in the spore coat proteins of B. subtilis. The activity was Ca(2+)-independent like the TGases derived from Streptoverticillium or some plants. It is suggested that TGase is expressed during sporulation and plays a role in the assembly of the spore coat proteins of the genus Bacillus.     

Cytotoxic cycloartane-type triterpenes from Combretum quadrangulare

Seven novel cycloartane-type triterpenes were isolated from Combretum quadrangulare, and their structures were elucidated on the basis of spectral analysis. All these compounds were tested for their cytotoxicity against murine colon 26-L5 carcinoma cells. Methyl quadrangularate B (2) and methyl quadrangularate D (4) exhibited potent cytotoxicity having ED₅₀ values 9.54 and 5.42 μM, respectively.