Synthesis and mitochondrial complex I inhibition of dihydroxy-cohibin A, non-THF annonaceous acetogenin analogue

To elucidate the inhibitory action of acetogenins, we synthesized an acetogenin derivative which possesses tetraol in place of the tetrahydrofuran ring and examined its inhibitory activity against bovine heart mitochondrial complex I. Our results indicate that these hydroxy groups are an essential structural factor though it is not effective as bis-THF hydroxy groups combination.     

X-ray-induced mutations in Escherichia coli K-12 strains with altered DNA polymerase I activities

Spectra of ionizing radiation mutagenesis were determined by sequencing X-ray-induced endogenous tonB gene mutations in Escherichia coli polA strains. We used two polA alleles, the polA1 mutation, defective for Klenow domain, and the polA107 mutation, defective for flap domain. We demonstrated that irradiation of 75 and 50 Gy X-rays could induce 3.8- and 2.6-fold more of tonB mutation in polA1 and polA107 strains, respectively, than spontaneous level. The radiation induced spectrum of 51 tonB mutations in polA1 and 51 in polA107 indicated that minus frameshift, A:T-->T:A transversion and G:C-->T:A transversion were the types of mutations increased. Previously, we have reported essentially the same X-ray-induced tonB mutation spectra in the wild-type strain. These results indicate that (1) X-rays can induce minus frameshift, A:T-->T:A transversion and G:C-->T:A transversion in E. coli and (2) presence or absence of polymerase I (PolI) of E. coli does not have any effects on the process of X-ray mutagenesis.     

Chromatin-mediated cortical granule redistribution is responsible for the formation of the cortical granule-free domain in mouse eggs

A cortical granule-free domain (CGFD) overlies the metaphase chromatin in fully mature mouse eggs. Although a chromatin-induced localized release of cortical granules (CG) during maturation is thought to be a major contributing factor to its formation, there are indications that CG redistribution may also be involved in generating the CGFD. We performed experiments to determine the relative contributions of CG exocytosis and redistribution in generating the CGFD. We found that the CGFD-inducing activity was not specific to female germ cell chromatin and was heat stable but sensitive to DNase and protease treatment. Surprisingly, chelation of egg intracellular Ca(2+) levels did not prevent CGFD formation in response to microinjection of exogenous chromatin, suggesting that development of the CGFD was not a result of CG exocytosis. This finding was confirmed by the lack of CG exudate on the plasma membrane surface of the injected eggs and the absence of conversion of ZP2 to ZP2(f) during formation of the new CGFD. Moreover, clamping intracellular Ca(2+) did not prevent the formation of the CGFD during oocyte maturation, but did inhibit the maturation-associated release of CGs between metaphase I and II. Results of these experiments suggest that CG redistribution is the dominant factor in formation of the CGFD.     

Combined trisomy 9P and Shprintzen syndrome resulting from a paternal t(9;22)

The authors report on a female infant with partial trisomy 9 (pter-->q12) together with partial monosomy 22 (pter-->q11.23) that included DiGeorge critical region (DGCR), as a result of adjacent-2 disjunction. In addition to the clinical features characteristic of trisomy 9p syndrome, the patient had Truncus arteriosus type A2, bilateral hydronephrosis, palatal anomaly, retrognathia, and laryngeal hypotonia, which are likely to be attributed to 22q11.2 deletion. This patient appears to be the first reported case with such unbalanced translocation resulting from a paternal reciprocal translocation. For live birth, the risk for male carrier is 8.7-17.4%. It is important to consider this higher risk when counseling. Precise study concerning the presence of the DGCR can facilitate in the better understanding of the condition.     

Planarian pharynx regeneration revealed by the expression of myosin heavy chain-A

The pharynx is a distinctive organ in the center of the body of planarians. Although the process of pharynx regeneration has been studied previously, the details and mechanism of the process remain controversial. We examined the process of regeneration of the pharynx in the planarian Dugesia japonica in detail by in situ hybridization and immunohistochemistry for myosin heavy chain-A (DjMHC-A), which is mainly expressed in the pharynx muscles and pharynx-anchoring muscles. We also monitored the behavior of the neoblasts in this process. In the regenerating posterior body fragment, the pharyngeal rudiment was formed by accumulation of cells that were probably undifferentiated cells derived from the neoblasts. The pharynx muscles appeared to differentiate in the rudiment in a manner that was coordinated with the differentiation of the pharynx-anchoring muscles in the region surrounding the rudiment. During this process, all cells containing mRNA for DjMHC-A also contained the DjMHC-A protein. These results argue against a previously proposed hypothesis that in the mesenchyme, 'pharynx-forming cells', which are committed to differentiate into the pharyngeal cells but have not yet differentiated, gather in the rudiment to form the pharynx (Agata and Watanabe, 1999). Rather, the present observations suggest that regeneration of the planarian pharynx proceeds by accumulation of cells that are probably undifferentiated cells derived from neoblasts in the rudiment, followed by their differentiation into the pharyngeal cells there.     

CD27 and CD40 inhibit p53-independent mitochondrial pathways in apoptosis of B cells induced by B cell receptor ligation

B cells in the germinal center are known to undergo apoptosis after B cell receptor (BCR) ligation, a process relevant to immunological tolerance. Human CD27 is a B cell co-stimulatory molecule. The aim of this study was to compare the effects of CD27 and CD40 signals on BCR-mediated apoptosis of B cells. BCR ligation activated mitochondrial apoptotic pathways including down-regulation of Bcl-X(L), dissipation of mitochondrial transmembrane potential, release of cytochrome c, and activation of caspase-9. Each of these effects was significantly inhibited by CD27 and CD40. Bik expression was weakly but significantly down-regulated by CD27 but up-regulated by CD40. BCR ligation resulted in p53 activation including its phosphorylation at Ser(15), nuclear translocation, and target gene p53AIP1 induction. CD27 and CD40 clearly suppressed these processes. Analyses that used dominant-negative p53 variants revealed a low but still substantial level of BCR-mediated apoptosis and intact mitochondria-mediated apoptotic pathway. These pathways were further inhibited by CD27 and CD40, although the cells showed no p53 phosphorylation or p53AIP1 expression. Our results suggested that, at the mitochondrial level, CD27 and CD40 co-stimulatory signals regulated the p53-amplified apoptotic pathway in B cells through the inhibition of p53-independent apoptotic pathway primarily induced by BCR ligation.     

New plant growth promoters,repraesentins A,B and C,from Lactarius repraesentaneus

Three new plant growth regulatory sesquiterpenoids were isolated from the Lactarius repraesentaneus fungus. Their structures were elucidated to be a protoilludene sesquiterpene, namely repraesentin A (1), and two related sesquiterpenes, namely repraesentins B (2) and C (3). Compounds 1-3 showed promotion activities toward the radicle elongation of lettuce seedlings by 136%, 118% and 184% at 67 ppm, respectively.     

The repair of UV-irradiated plasmids transfected into cultured fish cells

UV-irradiated plasmids (pNPV B and pBSCATSV) were transfected into RBCF-1 cells derived from a goldfish (Carassius auratus) and into a xeroderma pigmentosum (group A) cell line, XP20SSV. The frequency of stable neor transformation by pNPV B decreased in a dose-dependent manner. However, in spite of large differences in UV sensitivity detected in the colony formation assay, the dose-response curves of RBCF-1 cells and XP20SSV cells were almost the same. The photorecovery (PR) of transforming activity of UV-irradiated plasmids was confirmed in RBCF-1 cells but its extent was much smaller than that observed in the survival assay. The expression of the transfected cat (chloramphenicol acetyltransferase; CAT) gene after 24-h incubation in the dark was much more sensitive to UV irradiation when compared with the stable transformation assay. The extent of PR of cat gene expression in RBCF-1 cells was high and comparable with that of the survival assay. The CAT value of RBCF-1 cells transfected with UV-irradiated plasmids relative to that of unirradiated controls increased as incubation time in the dark after transfection became longer. This suggests that the UV lesions on the plasmids transfected in the RBCF-1 cells were repaired in the dark. The cat gene expression of UV-irradiated plasmids in XP20SSV was very low and independent of incubation time after transfection.     

EPR-Spin Trapping and Flow Cytometric Studies of Free Radicals Generated Using Cold Atmospheric Argon Plasma and X-Ray Irradiation in Aqueous Solutions and Intracellular Milieu

Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M₄PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H₂O₂, which is the recombination product of ·OH, and OCl^- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an intracellular milieu is discussed.