Regulation of apoptosis by glutathione redox state in PC12 cells exposed simultaneously to iron and ascorbic acid

We previously reported that the levels of non-protein-bound iron (NPBI) and ascorbic acid (AA) are markedly increased in the cerebrospinal fluid of infants with perinatal asphyxia. The present study showed that FeSO4 and AA synergistically induced apoptosis of PC12 cells, which was prevented by alpha-tocopherol and glutathione (GSH) ethyl ester. Markers of free radical damage, such as ortho-tyrosine, meta-tyrosine, and F(2alpha)-isoprostane, showed a gradual increase. AA and ferrous NPBI disappeared rapidly from the culture medium, but exposure for only a few hours was sufficient to trigger apoptosis. Intracellular GSH decreased progressively along with a concomitant increase of glutathione disulfide (GSSG). The baseline half-cell reduction potential (Ehc) for GSSG, 2H+/2GSH couple was -246 mV and an Ehc of -200 mV was the critical level to switch on apoptosis, although some cells escaped this fate by transient increase of intracellular GSH. Once Ehc reached around -165 mV (81 mV oxidation from the baseline), all cells lost the ability to maintain an adequate intracellular GSH level and subsequently underwent apoptosis. These findings at least partly explain the mechanism of Fe-AA cytotoxicity, in that ferrous iron catalyzes hydroxyl radical generation and induces lipid peroxidation, after which subsequent depletion of GSH raises Ehc to the critical level for triggering or potentiating the apoptotic cascade.     

SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor

Sclerosteosis is an autosomal recessive disease that is characterized by overgrowth of bone tissue and is linked to mutations in the gene encoding the secreted protein SOST. Sclerosteosis shares remarkable similarities with "high bone mass" diseases caused by "gain-of-function" mutations in the LRP5 gene, which encodes a coreceptor for Wnt signaling proteins. We show here that SOST antagonizes Wnt signaling in Xenopus embryos and mammalian cells by binding to the extracellular domain of the Wnt coreceptors LRP5 and LRP6 and disrupting Wnt-induced Frizzled-LRP complex formation. Our findings suggest that SOST is an antagonist for Wnt signaling and that the loss of SOST function likely leads to the hyperactivation of Wnt signaling that underlies bone overgrowth seen in sclerosteosis patients.     

Purification and characterization of phospholipase B from Candida utilis

Phospholipase B (PLB) from the asporogenous yeast Candida utilis was purified to homogeneity from a culture broth. The apparent molecular mass was 90-110 kDa by SDS-PAGE. The enzyme had two pH optima, one acidic (pH 3.0) and the other alkaline (pH 7.5). At acidic pH the enzyme hydrolyzed all phospholipids tested without metal ions. On the other hand, the PLB showed substrate specificity and required metal ions for alkaline activity.The cDNA sequence of the PLB was analyzed by a combination of several PCR procedures. The PLB encoded a protein consisting of 643 amino acids. The amino acid sequence contained a lipase consensus sequence (GxSxG) and catalytic arginine and aspartic acid motifs which were identified as the catalytic triad in the PLB from Kluyveromyces lactis, suggesting that the catalytic mechanism of the PLB is similar to that of cytosolic phospholipase A(2) (cPLA(2)), found in mammalian tissues.     

Light reaches the very heart of the zebrafish clock

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early-stage zebrafish embryos contain clocks that are directly light-responsive. We describe recent experiments using single-cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light-responsiveness of early-stage zebrafish embryos.     

Inhibitory activity of 1-farnesylpyridinium on the spatial control over the assembly of cell wall polysaccharides in Schizosaccharomyces pombe

The modes of actions of 1-farnesylpyridinium (FPy) on yeast cell growth were investigated on the basis of its effects on cell cycle progression, morphogenesis and the related events for construction of cell wall architecture in Schizosacchromyces pombe. FPy predominantly inhibited the growth of the yeast cells after various cycles of cell division so that cells were arrested at the phase of separation into daughter cells accompanying morphological changes to swollen spherical cells at 24 h of incubation. FPy-treated cells were osmotically stable but were susceptible to the lytic action of (1, 3) beta-D-glucanases, and characterized by serious damages to the cell wall architecture as represented by a rough and irregular surface outlook. The isolated cell wall fraction gave a similar hexose composition with or without FPy treatment, suggesting that FPy did not inhibit the synthesis of each cell wall polysaccharide. FPy was permissive for the extracellular accumulation of amorphous cell wall materials and septum development in protoplasts, but absolutely interfered with the following morphogenetic process for construction of the rod-shaped cell wall architecture. Our results suggest the inhibitory activity of FPy on the spatial control over the assembly of cell wall polysaccharides.     

Site-specific isotope labeling of long RNA for structural and mechanistic studies

A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, 'isotopically labeled guanosine 5'-monophosphate' (5'-GMP), steadily gave a 5'-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5'-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with (15)N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry.     

A novel function of Noc2 in agonist-induced intracellular Ca2+ increase during zymogen-granule exocytosis in pancreatic acinar cells

Noc2, a putative Rab effector, contributes to secretory-granule exocytosis in neuroendocrine and exocrine cells. Here, using two-photon excitation live-cell imaging, we investigated its role in Ca(2+)-dependent zymogen granule (ZG) exocytosis in pancreatic acinar cells from wild-type (WT) and Noc2-knockout (KO) mice. Imaging of a KO acinar cell revealed an expanded granular area, indicating ZG accumulation. In our spatiotemporal analysis of the ZG exocytosis induced by agonist (cholecystokinin or acetylcholine) stimulation, the location and rate of progress of ZG exocytosis did not differ significantly between the two strains. ZG exocytosis from KO acinar cells was seldom observed at physiological concentrations of agonists, but was normal (vs. WT) at high concentrations. Flash photolysis of a caged calcium compound confirmed the integrity of the fusion step of ZG exocytosis in KO acinar cells. The decreased ZG exocytosis present at physiological concentrations of agonists raised the possibility of impaired elicitation of calcium spikes. When calcium spikes were evoked in KO acinar cells by a high agonist concentration: (a) they always started at the apical portion and traveled to the basal portion, and (b) calcium oscillations over the 10 μM level were observed, as in WT acinar cells. At physiological concentrations of agonists, however, sufficient calcium spikes were not observed, suggesting an impaired [Ca(2+)](i)-increase mechanism in KO acinar cells. We propose that in pancreatic acinar cells, Noc2 is not indispensable for the membrane fusion of ZG per se, but instead performs a novel function favoring agonist-induced physiological [Ca(2+)](i) increases.     

Association between risk of myopathy and cholesterol-lowering effect: a comparison of all statins

In the present study, we examined the mechanisms underlying the cytotoxicity of pitavastatin, a new statin, and we compared the in vitro potencies of muscle cytotoxicity using a prototypic embryonal rhabdomyosarcoma cell line (RD cells), a typical side effect of statins and compared the cholesterol-lowering effects of statins using Hep G2 hepatoma cells. Pitavastatin reduced the number of viable cells and caused caspase-9 and -3/7 activation in a time- and concentration-dependent manner. The comparison of cytotoxities of statins showed that statins significantly reduced cell viability and markedly enhanced activity of caspase-3/7 in concentration-dependent manner. On the other hand, the effects of hydrophilic statins, pravastatin, rosuvastatin were very weak. The rank order of cytotoxicity was cerivastatin > simvastatin acid> fluvastatin > atorvastatin > lovastatin acid > pitavastatin > rosuvastatin, pravastatin. Statin-induced cytotoxicity is associated with these partition coefficients. On the other hand, the cholesterol-lowering effect of statins did not correlate with these partition coefficients and cytotoxicity. Thus, it is necessary to consider the association between risk of myopathy and cholesterol-lowering effect of a statin for precise use of statins.     

Detection and changes in levels of testosterone during spermatogenesis in the freshwater planarian Bdellocephala brunnea

It was reported recently that vertebrate-type steroids exist and control reproduction in several groups of invertebrates, including molluscs. Sexually reproductive freshwater planarians of the species Bdellocephala brunnea have a limited breeding season in their natural habitat. This phenomenon suggests that some endogenous reproductive hormones might play a role in vivo. However, to date, sex steroids such as androgen, estrogen, and progesterone have not been found in planarians. The goal of the present study was to determine whether androgen is present in sexual planarians such as B. brunnea. The presence of testosterone was detected by high-pressure liquid chromatography and, in sexually reproductive individuals in which no seminal vesicles were visible, the level of testosterone was about twice than that in individuals with visible seminal vesicles. An enzyme-linked immunosorbent assay revealed that the levels of testosterone during terminal spermatogenesis were three times higher than during the spermatocyte-building phase. Our results indicate that sexually reproductive freshwater planarians such as B. brunnea might have vertebrate-type steroids and show variation in testosterone levels during spermatogenesis.