Antibacterial Effects of Glycyrrhetinic Acid and Its Derivatives on Staphylococcus aureus

Staphylococcus aureus is a major pathogen in humans and causes serious problems due to antibiotic resistance. We investigated the antimicrobial effect of glycyrrhetinic acid (GRA) and its derivatives against 50 clinical S. aureus strains, including 18 methicillin-resistant strains. The minimum inhibitory concentrations (MICs) of GRA, dipotassium glycyrrhizate, disodium succinoyl glycyrrhetinate (GR-SU), stearyl glycyrrhetinate and glycyrrhetinyl stearate were evaluated against various S. aureus strains. Additionally, we investigated the bactericidal effects of GRA and GR-SU against two specific S. aureus strains. DNA microarray analysis was also performed to clarify the mechanism underlying the antibacterial activity of GR-SU. We detected the antimicrobial activities of five agents against S. aureus strains. GRA and GR-SU showed strong antibacterial activities compared to the other three agents tested. At a higher concentration (above 2x MIC), GRA and GR-SU showed bactericidal activity, whereas at a concentration of 1x MIC, they showed a bacteriostatic effect. Additionally, GRA and GR-SU exhibited a synergistic effect with gentamicin. The expression of a large number of genes (including transporters) and metabolic factors (carbohydrates and amino acids) was altered by the addition of GR-SU, suggesting that the inhibition of these metabolic processes may influence the degree of the requirement for carbohydrates or amino acids. In fact, the requirement for carbohydrates or amino acids was increased in the presence of either GRA or GR-SU. GRA and GR-SU exhibited strong antibacterial activity against several S. aureus strains, including MRSA. This activity may be partly due to the inhibition of several pathways involved in carbohydrate and amino acid metabolism.     

Triphala,a formulation of traditional Ayurvedic medicine,shows protective effect against X-radiation in HeLa cells

Ayurveda is a holistic medical system of traditional medicine, and Triphala is one of the most popular formulations in Ayurveda. Triphala is composed of three kinds of herb, Terminalia chebula, Terminalia bellirica, and Emblica officinalis. Since Triphala is shown to exhibit a protective activity against ionizing radiation in mice, we investigated its activity in HeLa cells. We found that Triphala showed the protective effects against X-radiation and bleomycin, both of which generate DNA strand breaks, in HeLa cells. Further, Triphala efficiently eliminated reactive oxygen species (ROS) in HeLa cells. Thus, the antioxidant activity of Triphala would likely play a role in its protective actions against X-radiation and bleomycin because both agents damage DNA through the generation of ROS. These observations suggested that the radioprotective activity of Triphala can be, at least partly, studied with the cells cultured in vitro. The simple bioassay system with human cultured cells would facilitate the understanding of the molecular basis for the beneficial effects of Triphala.     

The MRX Complex Ensures NHEJ Fidelity through Multiple Pathways Including Xrs2-FHA–Dependent Tel1 Activation

Because DNA double-strand breaks (DSBs) are one of the most cytotoxic DNA lesions and often cause genomic instability, precise repair of DSBs is vital for the maintenance of genomic stability. Xrs2/Nbs1 is a multi-functional regulatory subunit of the Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex, and its function is critical for the primary step of DSB repair, whether by homologous recombination (HR) or non-homologous end joining. In human NBS1, mutations result truncation of the N-terminus region, which contains a forkhead-associated (FHA) domain, cause Nijmegen breakage syndrome. Here we show that the Xrs2 FHA domain of budding yeast is required both to suppress the imprecise repair of DSBs and to promote the robust activation of Tel1 in the DNA damage response pathway. The role of the Xrs2 FHA domain in Tel1 activation was independent of the Tel1-binding activity of the Xrs2 C terminus, which mediates Tel1 recruitment to DSB ends. Both the Xrs2 FHA domain and Tel1 were required for the timely removal of the Ku complex from DSB ends, which correlates with a reduced frequency of imprecise end-joining. Thus, the Xrs2 FHA domain and Tel1 kinase work in a coordinated manner to maintain DSB repair fidelity.     

Participation of caspase-3-like protease in oxidation-induced impairment of erythrocyte membrane properties

Erythrocytes are very susceptible to oxidative stress, having a high content of intracellular oxygen and hemoglobin. In the present study, exposure to oxidative stress resulted in a significant impairment of erythrocyte membrane functions, such as deformability and anion exchange. Band 3 protein, also known as anion exchanger-1, plays an important role in these two functions. We show that oxidative stress activated caspase-3 inside the erythrocytes, which resulted in band 3 protein cleavage. Interestingly, inhibition of the caspase-3 with its specific inhibitor not only suppressed the digestion of band 3 protein, but also blunted the functional damage to erythrocytes, such as deformability and anion exchange, without changing the level of peroxidation of membrane lipids. These results provide experimental evidence that activation of caspase-3 plays an important role in the oxidative stress-induced impairment of membrane functions of erythrocytes.     

Neurturin-GFRalpha2 signaling controls liver bud migration along the ductus venosus in the chick embryo

During chick liver development, the liver bud arises from the foregut, invaginates into the septum transversum, and elongates along and envelops the ductus venosus. However, the mechanism of liver bud migration is only poorly understood. Here, we demonstrate that a GDNF family ligand involved in neuronal outgrowth and migration, neurturin (NRTN), and its receptor, GFRalpha2, are essential for liver bud migration. In the chick embryo, we found that GFRalpha2 was expressed in the liver bud and that NRTN was expressed in the endothelial cells of the ductus venosus. Inhibition of GFRalpha2 signaling suppressed liver bud elongation along the ductus venous without affecting cell proliferation and apoptosis. Moreover, ectopic expression of NRTN perturbed the directional migration along the ductus venosus, leading to splitting or ectopic branching of the liver. We showed that liver buds selectively migrated toward an NRTN-soaked bead in vitro. These data represent a new model for liver bud migration: NRTN secreted from endothelial cells functions as a chemoattractant to direct the migration of the GFRalpha2-expressing liver bud in early liver development.     

Solution structure of agelenin, an insecticidal peptide isolated from the spider Agelena opulenta, and its structural similarities to insect-specific calcium channel inhibitors

Agelenin, isolated from the Agelenidae spider Agelena opulenta, is a peptide composed of 35 amino acids. We determined the three-dimensional structure of agelenin using two-dimensional NMR spectroscopy. The structure is composed of a short antiparallel beta-sheet and four beta-turns, which are stabilized by three disulfide bonds. Agelenin has characteristic residues, Phe9, Ser28 and Arg33, which are arranged similarly to the pharmacophore of the insect channel inhibitor, omega-atracotoxin-Hv1a. These observations suggest that agelenin and omega-atracotoxin-Hv1a bind to insect calcium channels in a similar manner. We also suggest that another mode of action may operate in the channel inhibition by omega-agatoxin-IVA and omega-atracotoxin-Hv2a.     

Desmoglein versus non-desmoglein signaling in pemphigus acantholysis: characterization of novel signaling pathways downstream of pemphigus vulgaris antigens

Although it is accepted that pemphigus antibody binding to keratinocytes (KCs) evokes an array of intracellular biochemical events resulting in cell detachment and death, the triggering events remain obscure. It has been postulated that the binding of pemphigus vulgaris IgG (PVIgG) to KCs induces "desmosomal" signaling. Because in contrast to integrins and classical cadherins, desmoglein (Dsg) molecules are not known to elicit intracellular signaling, and because PV patients also produce non-Dsg autoantibodies, we investigated the roles of both Dsg and non-desmoglein PV antigens. The time course studies of KCs treated with PVIgG demonstrated that the activity of Src peaked at 30 min, EGF receptor kinase (EGFRK) at 60 min, and p38 MAPK at 240 min. The Src inhibitor PP2 decreased EGFRK and p38 activities by approximately 45 and 30%, respectively, indicating that in addition to Src, PVIgG evokes other triggering events. The shrinkage of KCs (cell volume reduction) became significant at 120 min, keratin aggregation at 240 min, and an increase of TUNEL positivity at 360 min. Pretreatment of KCs with PP2 blocked PVIgG-dependent cell shrinkage and keratin aggregation by approximately 50% and TUNEL positivity by approximately 25%. The p38 MAPK inhibitor PD169316 inhibited these effects by approximately 15, 20, and 70%, respectively. Transfection of KCs with small interfering RNAs that silenced expression of Dsg1 and/or Dsg3 proteins, blocked approximately 50% of p38 MAPK activity but did not significantly alter the PVIgG-dependent rise in Src and EGFRK activities. These results indicate that activation of p38 MAPK is a late signaling step associated with collapse of the cytoskeleton and disassembly of desmosomes caused by upstream events involving Src and EGFRK. Therefore, the early acantholytic events are triggered by non-Dsg antibodies.     

The genetic structure of endangered indigenous populations of the amago salmon, <Emphasis Type="Italic">Oncorhynchus</Emphasis><Emphasis Type="Italic">masou</Emphasis><Emphasis Type="Italic">ishikawae</Emphasis>, in Japan

The amago salmon, Oncorhynchus masou ishikawae, is an endemic subspecies of O. masou in Japan. Owing to the extensive stocking of hatchery fish throughout Japan, indigenous populations of O. m. ishikawae are now on the verge of extinction. We examined the genetic effects of stocking hatchery fish on wild populations in the River Koza, Japan, using microsatellite and mitochondrial DNA (mtDNA) markers. For mtDNA, haplotype mt1, which is common in wild populations, was present exclusively in isolated wild populations assumed to be unaffected by previous stocking, while it was never observed in hatchery fish. Genetic diversity was much higher in wild populations in the stocked area, which shared many mtDNA haplotypes with hatchery fish, than in isolated wild populations with haplotype mt1. Pairwise F _ST estimates based on microsatellites showed significant differentiation among the isolated populations with many microsatellite loci monomorphic. Significant deviation from Hardy–Weinberg equilibrium was observed in wild populations in the area subject to stocking, where a Bayesian-based assignment test showed a high level of introgression with hatchery fish. These results suggest that wild populations with haplotype mt1, which became isolated through anthropogenic environmental change in the 1950–1960s, represent indigenous populations of O. m. ishikawae in the River Koza. They have low genetic diversity, most likely caused by genetic bottlenecks following damming and environmental deterioration, while stocking of hatchery fish over the past 30 years apparently had a large impact on the genetic structure of wild populations in the main channel of the River Koza.     

Heterozygous loss of Zbtb38 leads to early embryonic lethality via the suppression of Nanog and Sox2 expression

ObjectivesMammalian DNA methyltransferases are essential to re‐establish global DNA methylation patterns during implantation, which is critical for transmitting epigenetic information to the next generation. In contrast, the significance of methyl‐CpG binding proteins (MBPs) that bind methylated CpG remains almost unknown at this stage. We previously demonstrated that Zbtb38 (also known as CIBZ)—a zinc finger type of MBP—is required for mouse embryonic stem (ES) cell proliferation by positively regulating Nanog expression. However, the physiological function of Zbtb38 in vivo remains unclear.Materials and MethodsThis study used the Cre‐loxP system to generate conditional Zbtb38 knockout mice. Cell proliferation and apoptosis were studied by immunofluorescence staining. Quantitative real‐time PCR, immunoblotting and immunofluorescence were performed to investigate the molecular mechanisms.ResultsGermline loss of the Zbtb38 single allele resulted in decreased epiblast cell proliferation and increased apoptosis shortly after implantation, leading to early embryonic lethality. Heterozygous loss of Zbtb38 reduced the expression of Nanog, Sox2, and the genes responsible for epiblast proliferation, differentiation, and cell viability. Although this early lethal phenotype, Zbtb38 is dispensable for ES cell establishment and identity.ConclusionsThese findings indicate that Zbtb38 is essential for early embryonic development via the suppression of Nanog and Sox2 expression.

Heterozygous loss of Zbtb38 leads to aberrant epiblast cell proliferation and apoptosis shortly after implantation. Heterozygous loss of Zbtb38 reduced the expression of Nanog and Sox2 in ICM and epiblast.