Parallel and antiparallel dimers of magainin 2: their interaction with phospholipid membrane and antibacterial activity.

Magainin 2 (M2) forms pores by associating with several other M2 molecules in lipid membranes and shows antibacterial activity. To examine the effect of M2 dimerization on biological activity and membrane interaction, parallel and antiparallel M2 dimers were prepared from two monomeric precursors. Antibacterial and haemolytic activities were enhanced by dimerization. CD measurements showed that both dimers and monomers have an alpha-helical structure in the presence of lipid vesicles. Tryptophan fluorescence shift and KI quenching studies showed that all the peptides were more deeply embedded in acidic liposomes than in neutral liposomes. Experiments on dye-leakage activity and membrane translocation of peptides suggest that dimers and monomers form pores through lipid membranes, although the pore formation may be accompanied by membrane disturbance. Although dimerization of M2 increased the interaction activity with lipid membranes, no appreciable difference between the activities of parallel and antiparallel M2 dimers was observed.     

Alendronate suppresses tumor angiogenesis by inhibiting Rho activation of endothelial cells

We previously reported that alendronate inhibits intraperitoneal dissemination in an in vivo ovarian cancer model. Recently, nitrogen-containing bisphosphonates have been reported to have antiangiogenic activities. In this study, alendronate inhibited human umbilical vein endothelial cell (HUVEC) migration and capillary-like structure formation in vitro. These inhibitory effects were associated with reduced Rho activation and suppression of the formation of actin stress fibers and focal adhesions in HUVECs. Furthermore, the inhibition by alendronate was reversed by geranylgeraniol, which abrogated the inhibition of Rho geranylgeranylation. Next, we examined the effect of alendronate on angiogenesis in disseminated ovarian tumors of athymic immunodeficient mice. Alendronate treatment reduced the intra-tumor neoangiogenesis compared with that in the non-treated mice, although tumor-derived VEGF expression was not altered. In conclusion, the in vivo anti-tumor effect of alendronate might be derived, at least in part, from its direct antiangiogenic effects on intra-tumor endothelial cells by inhibiting Rho geranylgeranylation.     

Decrease of hepatic delta-aminolevulinate dehydratase activity in an animal model of fatigue

Fatigue can be defined physiologically as inability to maintain the expected power output. At present, no standard of fatigue are yet available. In order to find biomarkers of fatigue, we investigated the level of delta-aminolevulinic acid (ALA), the first intermediate metabolite in the heme biosynthetic pathway, in the plasma and urine of an animal model of fatigue. To prepare fatigued animals, we kept rats for 5 days in a cage filled with water to a height of 1.5 cm. As a result, the plasma and urinary ALA levels were increased in the fatigued animals as compared with those in the control animals. One day after the rats had been returned to their normal cages, these increased levels were restored to the control ones. We also examined the activity of the enzyme ALA dehydratase (ALAD), which is the second enzyme in the heme biosynthetic pathway, and ALAD gene expression during the fatigue and its recovery sessions. The ALAD activity, as well as its gene expression, in the liver of the fatigued animals was decreased as compared with those of the control animals. Both activity and gene expression of ALAD were recovered to their respective control levels after the rats had been allowed to rest in their normal cages for 1 day. Furthermore, the activity of ALA synthase (ALAS), the rate-limiting enzyme in the heme biosynthesis, in the liver was increased after the fatigue session for 5 days. Although this level of increase in the plasma concentration of ALA may not induce fatigue, increase in plasma and urinary ALA levels can be biomarkers of fatigue.     

Telomerase activation induces elongation of the telomeric single-stranded overhang, but does not prevent chromosome aberrations in human vascular endothelial cells

Chromosome aberrations such as loss of chromosome 13 were frequently observed in human endothelial cells from umbilical cord veins (HUVEC). A recent study showed that the length of telomeric single-stranded 3'-overhangs (G-tails) is more important as an essential structure for chromosome maintenance than the net telomere length in telomere t-loop formation. Here, we have examined G-tail length using G-tail telomere HPA in normal and hTERT-transduced HUVECs. We found that forced expression of hTERT in HUVEC induced G-tail as well as total telomere length elongation. G-tail length was well correlated with total telomere length. However, hTERT introduction did not prevent chromosome aberrations such as loss of chromosome 13. Normal characteristics such as morphology, up-regulation of vWF, and tube formation were observed in hTERT-HUVEC as in young normal HUVEC. These results show that chromosome aberrations in HUVEC are independent of telomere G-tail and total telomere attrition.     

Possible Role of Xanthobaccins Produced by Stenotrophomonas sp. Strain SB-K88 in Suppression of Sugar Beet Damping-Off Disease

Three antifungal compounds, designated xanthobaccins A, B, and C, were isolated from the culture fluid of Stenotrophomonas sp. strain SB-K88, a rhizobacterium of sugar beet that suppresses damping-off disease. Production of xanthobaccin A in culture media was compared with the disease suppression activities of strain SB-K88 and less suppressive strains that were obtained by subculturing. Strain SB-K88 was applied to sugar beet seeds, and production of xanthobaccin A in the rhizosphere of seedlings was confirmed by using a test tube culture system under hydroponic culture conditions; 3 μg of xanthobaccin A was detected in the rhizosphere on a per-plant basis. Direct application of purified xanthobaccin A to seeds suppressed damping-off disease in soil naturally infested by Pythium spp. We suggest that xanthobaccin A produced by strain SB-K88 plays a key role in suppression of sugar beet damping-off disease.     

Isolation and partial characterization of crispacin A, a cell-associated bacteriocin produced by Lactobacillus crispatus JCM 2009

Crispacin A, a cell-associated bacteriocin produced by Lactobacillus crispatus JCM 2009, was purified from culture broth by ammonium sulfate precipitation, followed by ion exchange and reversed-phase chromatography. Crispacin A was also purified from the cells of L. crispatus JCM 2009 by acid extraction and reversed-phase chromatography. Purified crispacin A was determined to be 5393 Da by mass spectrometry and found not to show sequence homology with other bacteriocins from lactic acid bacteria.     

Identification and characterization of a brilliant yellow pigment produced by Bordetella pertussis

Culture supernatants of Bordetella pertussis are a brilliant yellow; however, the structure and biological role of the responsible pigment have not been investigated. In this study, a brilliant yellow‐colored fraction was extracted from culture supernatants of B. pertussis and analyzed by HPLC. UV–visible spectral analysis and mass spectrometry identified the brilliant yellow pigment as riboflavin. Riboflavin production was high in lag and early log phases and riboflavin was found to enhance growth of B. pertussis in low‐density cultures. Riboflavin production is not regulated by the BvgAS system. In addition, it was found that other Bordetella species, such as B. parapertussis , B. holmesii and B. bronchiseptica, also release riboflavin into their culture supernatants. This is the first report that B. pertussis secrets riboflavin to the extracellular space and that riboflavin may promote its growth. The mechanism may be associated with pathogenesis of B. pertussis .

     

Age-related increase of superoxide generation in the brains of mammals and birds

Oxidative stress, an imbalance between endogenous levels of oxygen radicals and antioxidative defense, increases with aging. However, it is not clear which of these two factors is the more critical. To clarify the production of oxygen radicals increases with age, we examined oxygen radical-dependent chemiluminescent signals in ex vivo brain slices using a novel photonic imaging method. The chemiluminescent intensity was significantly decreased by the membrane permeable superoxide dismutase (SOD)/catalase mimic, but not by Cu,Zn-SOD. Inhibitors for complex I, III, and IV of the mitochondrial electron transport chain transiently enhanced the chemiluminescent signal. The superoxide-dependent chemiluminescent intensity in senescence accelerated mouse (SAM) brain tissues increases with age. Moreover, the slope of the age-dependent increase was steeper in SAMP10, a strain characterized by a short lifespan and atrophy in the frontal cerebral cortex, than the senescence-resistant strain SAMR1, which has a longer lifespan. An increase in chemiluminescence with age was also observed in C57/BL6 mice, Wistar rats, and pigeons, although levels of chemiluminescence were lower in the pigeons than murines. The rate of age-related increases of superoxide-dependent chemiluminescence was inversely related to the maximum lifespan of the animals. The activity of superoxide dismutase was unchanged during the aging process in the brain. This suggested that superoxide production itself may increase with age. We speculated that reactive oxygen may be a signal to determine the aging process.     

Characterization of the {alpha}-helix region in domain 3 of the haemolytic lectin CEL-III: implications for self-oligomerization and haemolytic processes

CEL-III is a haemolytic lectin, which has two beta-trefoil domains (domains 1 and 2) and a beta-sheet-rich domain (domain 3). In domain 3 (residues 284-432), there is a hydrophobic region containing two alpha-helices (H8 and H9, residues 317-357) and a loop between them, in which alternate hydrophobic residues, especially Val residues, are present. To elucidate the role of the alpha-helix region in the haemolytic process, peptides corresponding to different parts of this region were synthesized and characterized. The peptides containing the sequence that corresponded to the loop and second alpha-helix (H9) showed the strongest antibacterial activity for Staphylococcus aureus and Bacillus subtilis through a marked permeabilization of the bacterial cell membrane. The recombinant glutathione S-transferase (GST)-fusion proteins containing domain 3 or the alpha-helix region peptide formed self-oligomers, whereas mutations in the alternate Val residues in the alpha-helix region lead to decreased oligomerization ability of the fusion proteins. These results suggest that the alpha-helix region, particularly its alternate Val residues are important for oligomerization of CEL-III in target cell membranes, which is also required for a subsequent haemolytic action.     

Aluminum distribution and reactive oxygen species accumulation in root tips of two Melaleuca trees differing in aluminum resistance

To elucidate the mechanism of the high aluminum (Al) resistance of a Myrtaceae tree, Melaleuca cajuputi Powell, we investigated the responses of root tips to Al and compared them with those of an Al-sensitive species, M. bracteata F. Muell. Roots of seedlings of both species were treated with a calcium solution (pH 4.0) containing 0 or 1 mM AlCl₃. After 3 h of Al treatment, inhibition of root elongation and deposition of callose and lignin in root tips, typical signs of Al injury, were induced in M. bracteata but not in M. cajuputi, yet Al accumulation in root tips was similar in both species. These results indicate that internal Al tolerance mechanisms, not Al exclusion mechanisms, are responsible for the Al resistance of M. cajuputi. After 3 h of Al treatment, amount of Al tightly bound to root tips, Al remaining after washing with a desorbing solution, was less in M. cajuputi than in M. bracteata. In M. bracteata, 6 h of Al treatment triggered the accumulation of hydrogen peroxide (H₂O₂) in root tips despite the upregulation of antioxidant mechanisms, activity of peroxidase and concentration of reduced glutathione. In M. cajuputi, 6 h of Al treatment did not affect the concentration of H₂O₂, but decreased activity of peroxidase, and increased concentration of reduced glutathione in root tips. These results suggest that the less Al tightly bound to root tips is involved in the suppressing the H₂O₂ accumulation and the internal Al tolerance in M. cajuputi, and that the H₂O₂ accumulation or changes in cellular environment that bring about H₂O₂ accumulation despite the upregulation of antioxidant mechanisms results in Al-induced inhibition of root elongation in M. bracteata.