Cytotoxic Alangium alkaloids from Alangium longiflorum

Seven alkaloids (1-7) were isolated from the stem bark of Alangium longiflorum. Compound 1, (-)-10-O-demethylisocephaeline, was isolated for the first time as a naturally occurring product from a plant source. All structures were elucidated by detailed spectroscopic analysis. Biological evaluation showed that 2, 10-O-demethylcephaeline, exhibited potent cytotoxic activity against human lung carcinoma (A549) and breast adenocarcinoma (MCF-7) with ED(50) values of 0.013 and 0.062 microM, respectively. The stereoisomer 1 was less potent than 2, and related compounds with different hydroxy/methoxy substitution patterns were also less potent or inactive. Thus, compound 2 merits attention as a cytotoxic lead for further study.     

Chara myosin and the energy of cytoplasmic streaming

Recently, it was found that myosin generating very fast cytoplasmic streaming in Chara corallina has very high ATPase activity. To estimate the energy consumed by this myosin, its concentration in the internodal cells of C. corallina was determined by quantitative immunoblot. It was found that the concentration of Chara myosin was considerably high (200 nM) and the amount of ATP consumed by this myosin would exceed that supplied by dark respiration if all myosin molecules were fully activated by the interaction with actin. These results and model calculations suggested that the energy required to generate cytoplasmic streaming is very small and only one-hundredth of the existing myosin is enough to maintain the force for the streaming in the Chara cell.     

Contribution of indirect action to radiation-induced mammalian cell inactivation: dependence on photon energy and heavy-ion LET

The contribution of indirect action mediated by OH radicals to cell inactivation by ionizing radiations was evaluated for photons over the energy range from 12.4 keV to 1.25 MeV and for heavy ions over the linear energy transfer (LET) range from 20 keV/microm to 440 keV/microm by applying competition kinetics analysis using the OH radical scavenger DMSO. The maximum level of protection provided by DMSO (the protectable fraction) decreased with decreasing photon energy down to 63% at 12.4 keV. For heavy ions, a protectable fraction of 65% was found for an LET of around 200 keV/microm; above that LET, the value stayed the same. The reaction rate of OH radicals with intracellular molecules responsible for cell inactivation was nearly constant for photon inactivation, while for the heavy ions, the rate increased with increasing LET, suggesting a reaction with the densely produced OH radicals by high-LET ions. Using the protectable fraction, the cell killing was separated into two components, one due to indirect action and the other due to direct action. The inactivation efficiency for indirect action was greater than that for direct action over the photon energy range and the ion LET range tested. A significant contribution of direct action was also found for the increased RBE in the low photon energy region.     

Miniature neurotransmission stabilizes synaptic function via tonic suppression of local dendritic protein synthesis

Activity deprivation in neurons induces a slow compensatory scaling up of synaptic strength, reflecting a homeostatic mechanism for stabilizing neuronal activity. Prior studies have focused on the loss of action potential (AP) driven neurotransmission in synaptic homeostasis. Here, we show that the miniature synaptic transmission that persists during AP blockade profoundly shapes the time course and mechanism of homeostatic scaling. A brief blockade of NMDA receptor (NMDAR) mediated miniature synaptic events ("minis") rapidly scales up synaptic strength, over an order of magnitude faster than with AP blockade alone. The rapid scaling induced by NMDAR mini blockade is mediated by increased synaptic expression of surface GluR1 and the transient incorporation of Ca2+-permeable AMPA receptors at synapses; both of these changes are implemented locally within dendrites and require dendritic protein synthesis. These results indicate that NMDAR signaling during miniature synaptic transmission serves to stabilize synaptic function through active suppression of dendritic protein synthesis.     

Cloning and expression of two crystal protein genes, cry30Ba1 and cry44Aa1, obtained from a highly mosquitocidal strain, Bacillus thuringiensis subsp. entomocidus INA288

Two novel crystal protein genes, cry30Ba and cry44Aa, were cloned from Bacillus thuringiensis subsp. entomocidus INA288 and expressed in an acrystalliferous strain. Cry44Aa crystals were highly toxic to second-instar Culex pipiens pallens (50% mortality concentration [LC50] = 6 ng/ml) and Aedes aegypti (LC50 = 12 ng/ml); however, Cry30Ba crystals were not toxic.     

Genetic analysis of the mode of interplay between an ATPase subunit and membrane subunits of the lipoprotein-releasing ATP-binding cassette transporter LolCDE

The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the inner membrane. Thus, functional interaction between LolD and LolC/E is critically important for coupling of ATP hydrolysis to the lipoprotein release reaction. LolD contains a characteristic sequence called the LolD motif, which is highly conserved among LolD homologs but not other ABC transporters of E. coli. The LolD motif is suggested to be a region in contact with LolC/E, judging from the crystal structures of other ABC transporters. To determine the functions of the LolD motif, we mutagenized each of the 32 residues of the LolD motif and isolated 26 dominant-negative mutants, whose overexpression arrested growth despite the chromosomal lolD(+) background. We then selected suppressor mutations of the lolC and lolE genes that correct the growth defect caused by the LolD mutations. Mutations of the lolC suppressors were mainly located in the periplasmic loop, whereas ones of lolE suppressors were mainly located in the cytoplasmic loop, suggesting that the mode of interaction with LolD differs between LolC and LolE. Moreover, the LolD motif was found to be critical for functional interplay with LolC/E, since some LolD mutations lowered the ATPase activity of LolCDE without affecting that of LolD.     

Effects of restricted feeding on daily fluctuations of hepatic functions including p450 monooxygenase activities in rats

Hepatic P450 monooxygenase activities, assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) activities, show obvious daily fluctuations in male rats with high values during the dark period and low values during the light period. We have already confirmed that the ACD activities are controlled by the suprachiasmatic nucleus (SCN), which is well known as the oscillator of circadian rhythm. Recently, it is reported that circadian oscillators exist not only in the SCN but also in peripheral organs. To date, it is unclear which circadian oscillators predominantly drive the daily fluctuations of hepatic ACD activities. To address this question, we examined the effects of restricted feeding, which uncouples the circadian oscillators in the liver from the central pacemaker in the SCN, on the daily fluctuations in hepatic ACD activities in male rats. Here we show that restricted feeding inverts the oscillation phase of the daily fluctuations in hepatic ACD activities. Regarding the hepatic P450 content, there were no fluctuations between the light and dark periods under ad libitum and restricted feeding conditions. Therefore, it is considered that the daily fluctuations in hepatic ACD activities are predominantly driven by the circadian factors in peripheral organs rather than by the oscillator in the SCN directly.     

A novel phosphatidylcholine which contains pentadecanoic acid at sn-1 and docosahexaenoic acid at sn-2 in Schizochytrium sp. F26-b

Docosahexaenoic acid (DHA, 22:6n-3)-containing phospholipids are a ubiquitous component of the central nervous system and retina, however their physiological and pharmacological functions have not been fully elucidated. Here, we report a novel DHA-containing phosphatidylcholine (PC) in a marine single cell eukaryote, Schizochytrium sp. F26-b. Interestingly, 31.8% of all the fatty acid in F26-b is DHA, which is incorporated into triacylglycerols and various phospholipids. In phospholipids, DHA was found to make up about 50% of total fatty acid. To identify phospholipid species containing DHA, the fraction of phospholipids from strain F26-b was subjected to normal phase high-performance liquid chromatography (HPLC). It was found that DHA was incorporated into PC, lyso-PC, phosphatidylethanolamine, and phosphatidylinositol. The major DHA-containing phospholipid was PC in which 32.5% of the fatty acid was DHA. The structure of PC was analyzed further by phospholipase A2 treatment, fast atom bombardment mass spectrometry, and 1H- and 13C-NMR after purification of the PC with reverse phase HPLC. Collectively, it was clarified that the major PC contains pentadecanoic acid (C15:0) at sn-1 and DHA at sn-2; the systematic name of this novel PC is therefore "1-pentadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine."     

Hypoxia-inducible factor-1 mediates the expression of DNA polymerase iota in human tumor cells

Hypoxia generated in tumors has been shown to contribute to mutations and genetic instability. However, the molecular mechanisms remain incompletely defined. Since reactive oxygen species (ROS) are overproduced immediately after reoxygenation of hypoxic cells and generate oxidized guanine, we assumed that the mechanisms might involve translesion DNA polymerases that can bypass oxidized guanine. We report here that hypoxia as well as hypoxia mimetics, desferrioxamine, and CoCl(2), enhanced the expression of DNA polymerase iota (pol iota) in human tumor cell lines. Searching the consensus sequence of hypoxia response element to which HIF-1 binds revealed that it locates in the intron 1 of the pol iota gene. These results suggest that HIF-1-mediated pol iota gene expression may be involved in the generation of translesion mutations during DNA replication after hypoxia followed by reoxygenation, thereby contributing to the accumulation of genetic changes in tumor cells.