A New Isoquinolinium Derivative, Cadein1, Preferentially Induces Apoptosis in p53-defective Cancer Cells with Functional Mismatch Repair via a p38-dependent Pathway

We screened a protoberberine backbone derivative library for compounds with anti-proliferative effects on p53-defective cancer cells. A compound identified from this small molecule library, cadein1 (cancer-selective death inducer 1), an isoquinolinium derivative, effectively leads to a G₂/M delay and caspase-dependent apoptosis in various carcinoma cells with non- functional p53. The ability of cadein1 to induce apoptosis in p53-defective colon cancer cells was tightly linked to the presence of a functional DNA mismatch repair (MMR) system, which is an important determinant in chemosensitivity. Cadein1 was very effective in MMR⁺/p53⁻ cells, whereas it was not effective in p53⁺ cells regardless of the MMR status. Consistently, when the function of MMR was blocked with short hairpin RNA in SW620 (MMR⁺/p53⁻) cells, cadein1 was no longer effective in inducing apoptosis. Besides, the inhibition of p53 increased the pro-apoptotic effect of cadein1 in HEK293 (MMR⁺/p53⁺) cells, whereas it did not affect the response to cadein1 in RKO (MMR⁻/p53⁺) cells. The apoptotic effects of cadein1 depended on the activation of p38 but not on the activation of Chk2 or other stress-activated kinases in p53-defective cells. Taken together, our results show that cadein1 may have a potential to be an anti-cancer chemotherapeutic agent that is preferentially effective on p53-mutant colon cancer cells with functional MMR.     

Effect of Ischemic Neuronal Insults on Amyloid Precursor Protein Processing

The nature of the association between ischemic stroke and Alzheimer’s disease (AD) at the cellular and molecular level is still unknown. We evaluated the effect of ischemic neuronal insults on the regulation of amyloid precursor protein (APP) processing. We used an in vitro model of cerebral ischemia (oxygen-glucose deprivation) to evaluate the effect of ischemic neuronal insults on the amyloidogenic and non-amyloidogenic pathways using human neuroblastoma cell line and primary cultured cells of transgenic mice which expressed human APP (Tg2576). Ischemic neuronal insults increased the production of Aβ in Tg2576 primary culture cells compared to controls. A disintegrin and metalloprotease 10 (ADAM 10) was markedly increased in early stage of ischemic insults, which was followed by decreased level of ADAM 10 expression in later stage. The protein and mRNA expression of β-site cleavage enzyme (BACE) and BACE activity was not significantly different between the group of ischemic insults and control. By contrast, the activity of γ-secretase was significantly increased after 4 h of ischemic insults, as compared to controls. The present study showed that the ischemic neuronal insults increased the production of Aβ by influencing APP metabolism, which may link the role of ischemic insults to the pathogenesis of AD.     

Differential Involvement of Intracellular Ca2+ in 1-Methyl-4-phenylpyridinium- or 6-Hydroxydopamine-Induced Cell Viability Loss in PC12 Cells

1-Methyl-4-phenylpyridinium (MPP⁺) or 6-hydroxydopamine (6-OHDA) caused a nuclear damage, the mitochondrial membrane permeability changes, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in PC12 cells. Nicardipine (a calcium channel blocker), EGTA (an extracellular calcium chelator), BAPTA-AM (a cell permeable calcium chelator) and calmodulin antagonists (W-7 and calmidazolium) attenuated the MPP⁺-induced mitochondrial damage and cell death. In contrast, the compounds did not reduce the toxicity of 6-OHDA. Treatment with MPP⁺ or 6-OHDA evoked the elevation of intracellular Ca²⁺ levels. Unlike cell injury, addition of nicardipine, BAPTA-AM and calmodulin antagonists prevented the elevation of intracellular Ca²⁺ levels due to both toxins. The results show that the MPP⁺-induced formation of the mitochondrial permeability transition seems to be mediated by elevation of intracellular Ca²⁺ levels and calmodulin action. In contrast, the 6-OHDA-induced cell death seems to be mediated by Ca²⁺-independent manner.     

PLD2 forms a functional complex with mTOR/raptor to transduce mitogenic signals

Mammalian target-of-rapamycin (mTOR), which is a master controller of cell growth, senses a mitogenic signal in part through the lipid second messenger phosphatidic acid (PA), generated by phospholipase D (PLD). To understand further which isozymes of PLD are involved in this process, we compared the effect of PLD isozymes on mTOR activation. We found that PLD2 has an essential role in mitogen-induced mTOR activation as the siRNA-mediated knockdown of PLD2, not of PLD1, profoundly reduced the phosphorylations of S6K1 and 4EBP1, well-known mTOR effectors. Furthermore, exogenous PA-induced mTOR activation was abrogated by PLD2 knockdown, but not by PLD1 knockdown. This abrogation was found to be the result of complex formation between PLD2 and mTOR/raptor. PLD2 possesses a TOS-like motif (Phe-Glu-Val-Gln-Val, a.a. 265–269), through which it interacts with raptor independently of the other TOS motif-containing proteins, S6K1 and 4EBP1. PLD2-dependent mTOR activation appears to require PLD2 binding to mTOR/raptor with lipase activity, since lipase-inactive PLD2 cannot trigger mTOR activation despite its ability to interact with mTOR/raptor. Abrogation of mitogen-dependent mTOR activation by PLD2 knockdown was rescued only by wild type PLD2, but not by raptor binding-deficient and lipase-inactive PLD2. Our results demonstrate the importance of localized PA generation for the mitogen-induced activation of mTOR, which is achieved by a specific interaction between PLD2 and mTOR/raptor.     

Memory-enhancing effect of a supercritical carbon dioxide fluid extract of the needles of Abies koreana on scopolamine-induced amnesia in mice

Abies koreana Wilson (A. koreana) is a shrub or broadly pyramidal evergreen tree endemic in the mountainous regions of South Korea. We obtained the essential oil (EO) from alpine needle leaves of A. koreana by the supercritical fluid extraction (SFE) method. EO was analyzed by gas chromatography-mass spectrometry (GC-MS), and 68 compounds were identified constituting 95.66% of the oil. The major components were elemol (11.17%), terpinen-4-ol (9.77%), sabinene (8.86%), 10(15)-cadien-4-ol (7.16%), alpha-terpineol (6.13%), alpha-pinene (6.07%) and gamma-terpinene (4.71%). To investigate the memory-enhancing effects, we conducted a passive avoidance test using a scopolamine (1 mg/kg, ip)-induced amnesia mouse model. A peritoneal injection of EO from A. koreana (100 mg/kg) showed a memory enhancing effect of 72.7% compared with the control. These results suggest that EO of A. koreana may be a useful therapeutic agent against such amnesia-inducing diseases as Alzheimer and vascular dementia.     

Cytoprotective effect of phloroglucinol on oxidative stress induced cell damage via catalase activation

We investigated the cytoprotective effect of phloroglucinol, which was isolated from Ecklonia cava (brown alga), against oxidative stress induced cell damage in Chinese hamster lung fibroblast (V79-4) cells. Phloroglucinol was found to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydrogen peroxide (H(2)O(2)), hydroxy radical, intracellular reactive oxygen species (ROS), and thus prevented lipid peroxidation. As a result, phloroglucinol reduced H(2)O(2) induced apoptotic cells formation in V79-4 cells. In addition, phloroglucinol inhibited cell damage induced by serum starvation and radiation through scavenging ROS. Phloroglucinol increased the catalase activity and its protein expression. In addition, catalase inhibitor abolished the protective effect of phloroglucinol from H(2)O(2) induced cell damage. Furthermore, phloroglucinol increased phosphorylation of extracellular signal regulated kinase (ERK). Taken together, the results suggest that phloroglucinol protects V79-4 cells against oxidative damage by enhancing the cellular catalase activity and modulating ERK signal pathway.     

The platypus is in its place: nuclear genes and indels confirm the sister group relation of monotremes and Therians

Morphological data supports monotremes as the sister group of Theria (extant marsupials + eutherians), but phylogenetic analyses of 12 mitochondrial protein-coding genes have strongly supported the grouping of monotremes with marsupials: the Marsupionta hypothesis. Various nuclear genes tend to support Theria, but a comprehensive study of long concatenated sequences and broad taxon sampling is lacking. We therefore determined sequences from six nuclear genes and obtained additional sequences from the databases to create two large and independent nuclear data sets. One (data set I) emphasized taxon sampling and comprised five genes, with a concatenated length of 2,793 bp, from 21 species (two monotremes, six marsupials, nine placentals, and four outgroups). The other (data set II) emphasized gene sampling and comprised eight genes and three proteins, with a concatenated length of 10,773 bp or 3,669 amino acids, from five taxa (a monotreme, a marsupial, a rodent, human, and chicken). Both data sets were analyzed by parsimony, minimum evolution, maximum likelihood, and Bayesian methods using various models and data partitions. Data set I gave bootstrap support values for Theria between 55% and 100%, while support for Marsupionta was at most 12.3%. Taking base compositional bias into account generally increased the support for Theria. Data set II exclusively supported Theria, with the highest possible values and significantly rejected Marsupionta. Independent phylogenetic evidence in support of Theria was obtained from two single amino acid deletions and one insertion, while no supporting insertions and deletions were found for Marsupionta. On the basis of our data sets, the time of divergence between Monotremata and Theria was estimated at 231-217 MYA and between Marsupialia and Eutheria at 193-186 MYA. The morphological evidence for a basal position of Monotremata, well separated from Theria, is thus fully supported by the available molecular data from nuclear genes.     

Comparative analysis of two biliproteins, BP1 and BP2, from haemolymph of cabbage white butterfly, Pieris rapae

Two blue-pigment binding proteins, BP1 and BP2, are present in larval and pupal haemolymph of cabbage white butterfly, Pieris rapae, and fluctuate in expression during development. Both BP1 and BP2 are found in pupal haemolymph in varying proportions as well as in adult haemolymph, while only small amounts of BP2 are found in larval haemolymph. BPs are separated by 75% ammonium sulfate, and then purified effectively by ion exchange column chromatography and preparative gel electrophoresis. It was shown that BP1 and BP2 have molecular masses of 20,244 and 19,878 Da, and isoelectric points of 7.0 and 6.8, respectively. Considering their amino acid compositions and N-terminal amino acid sequences, the two proteins are almost identical except the first N-terminal amino acid. The first amino acid of BP1 is asparagine, whereas the initial residue of BP2 is aspartic acid. Anti-BP1 cross-reacts with BP2, indicating that they have immunological homogeneity. Western blotting analyses revealed that only BP1 was present in the larval tissues such as fat body, integument, muscle, and hindgut. However, BP1 was not found in midgut, Malphigian tubules, and silk gland. BP1 was also present in the protein bodies, and both cuticle and hemocoel sides of larval epidermis cells by the transmission electron microscopic observation. The information in this report will facilitate studies on the molecular biology and biological significance of insect BPs.     

Evolutionary movement of centromeres in horse, donkey, and zebra

Centromere repositioning (CR) is a recently discovered biological phenomenon consisting of the emergence of a new centromere along a chromosome and the inactivation of the old one. After a CR, the primary constriction and the centromeric function are localized in a new position while the order of physical markers on the chromosome remains unchanged. These events profoundly affect chromosomal architecture. Since horses, asses, and zebras, whose evolutionary divergence is relatively recent, show remarkable morphological similarity and capacity to interbreed despite their chromosomes differing considerably, we investigated the role of CR in the karyotype evolution of the genus Equus. Using appropriate panels of BAC clones in FISH experiments, we compared the centromere position and marker order arrangement among orthologous chromosomes of Burchelli's zebra (Equus burchelli), donkey (Equus asinus), and horse (Equus caballus). Surprisingly, at least eight CRs took place during the evolution of this genus. Even more surprisingly, five cases of CR have occurred in the donkey after its divergence from zebra, that is, in a very short evolutionary time (approximately 1 million years).These findings suggest that in some species the CR phenomenon could have played an important role in karyotype shaping, with potential consequences on population dynamics and speciation.