Application of a new probabilistic model for recognizing complex patterns in glycans

MOTIVATION: The study of carbohydrate sugar chains, or glycans, has been one of slow progress mainly due to the difficulty in establishing standard methods for analyzing their structures and biosynthesis. Glycans are generally tree structures that are more complex than linear DNA or protein sequences, and evidence shows that patterns in glycans may be present that spread across siblings and into further regions that are not limited by the edges in the actual tree structure itself. Current models were not able to capture such patterns. RESULTS: We have applied a new probabilistic model, called probabilistic sibling-dependent tree Markov model (PSTMM), which is able to inherently capture such complex patterns of glycans. Not only is the ability to capture such patterns important in itself, but this also implies that PSTMM is capable of performing multiple tree structure alignments efficiently. We prove through experimentation on actual glycan data that this new model is extremely useful for gaining insight into the hidden, complex patterns of glycans, which are so crucial for the development and functioning of higher level organisms. Furthermore, we also show that this model can be additionally utilized as an innovative approach to multiple tree alignment, which has not been applied to glycan chains before. This extension on the usage of PSTMM may be a major step forward for not only the structural analysis of glycans, but it may consequently prove useful for discovering clues into their function.     

X-inactivation is stably maintained in mouse embryos deficient for histone methyl transferase G9a

One of the two X chromosomes becomes inactivated during early development of female mammals. Recent studies demonstrate that the inactive X chromosome is rich in histone H3 methylated at Lys-9 and Lys-27, suggesting an important role for these modifications in X-inactivation. It has been shown that in the mouse Eed is required for maintenance of X-inactivation in the extraembryonic lineages. Interestingly, Eed associates with Ezh2 to form a complex possessing histone methyltransferase activity predominantly for H3 Lys-27. We previously showed that G9a is one of the histone methyltransferases specific for H3 Lys-9 and is essential for embryonic development. Here we examined X-inactivation in mouse embryos deficient for G9a. Expression of Xist, which is crucial for the initiation of X-inactivation, was properly regulated and the inactivated X chromosome was stably maintained even in the absence of G9a. These results demonstrate that G9a is not essential for X-inactivation.     

Edaravone inhibits acute renal injury and cyst formation in cisplatin-treated rat kidney

Background: Although cis-diamminedichloroplatinum (II) (cisplatin) is an effective anticancer agent, its clinical use is highly limited predominantly due to its adverse effects on renal functions. The present work examined the therapeutic potential of edaravone, a free radical scavenger, for inhibiting cisplatin-induced renal injury.

Methods: Edaravone, 3-methyl-1-phenyl-pyrazolin-5-one, was administrated intravenously at a dose of 30 mg/kg of body weight to male Wistar rats (200-220 g). After 30 min, cisplatin was injected intraperitoneally at a dose of 5 mg/kg of body weight. At the indicated times after the treatment, functions and histological changes of the kidney were analyzed. To test the therapeutic potential of edaravone in chemotherapy, its effect on the anticancer action of cisplatin was examined in ascites cancer-bearing rats.

Results: We found that cisplatin rapidly impaired the respiratory function and DNA of mitochondria in renal proximal tubules, thereby inducing apoptosis of tubular epithelial cells within a few days and chronic renal dysfunction associated with multiple cysts one-year after the administration. Administration of edaravone inhibited the cisplatin-induced acute injury of mitochondria and their DNA and renal epithelial cell apoptosis as well as the occurrence of chronic renal dysfunction and multiple cyst formation. The anticancer effect of cisplatin remained unaffected by intravenous administrating of edaravone.

Conclusions: These results indicate that edaravone may have therapeutic potential for inhibiting the acute and chronic injury of the kidney induced by cisplatin.     

Comparative analysis of GFP(UV)-beta1,3-N-acetylglucosaminyltransferase 2 production in two insect-cell-based expression systems

Active beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) was produced in the baculovirus expression system (BES) and in stably transformed insect Tn-5B1-4 cells. beta3GnT2 was expressed as a secreted fusion protein with GFP(UV) with three different types of signal sequence to enhance the secretion of the fusion protein. In the stably transformed cells, the maximal beta3GnT2 activity differed between isolates, but their secretion efficiencies were similar. The difference between the maximal beta3GnT activities of the isolates studied was considered to be due to the presence of a copy number of the fusion gene, as determined on the basis of the results of Southern blot analysis. The beta3GnT activities of the culture supernatant in BES (Tn-5B1-4 cells) without or with the addition of the protease inhibitor, leupeptin, were 0.68 and 2.01 mU/ml, respectively. The stably transformed Tn-5B1-4 cells (Tn-pXme11) exhibited a beta3GnT activity of 6.83 mU/ml, which was 3.4-fold higher than that observed for BES with the leupeptin addition. The purity of fusion protein purified from the culture supernatant of the Tn-pXme11 was higher than 95% on SDS-PAGE, in contrast with that purified from the culture supernatant of the baculovirus-infected cells which contained low-molecular-weight fragments of the fusion protein. The stably transformed cell line is more suitable than BES for the efficient production of the secretory protein, beta3GnT2.     

Molecular phylogeny of Vincetoxicum (Apocynaceae-Asclepiadoideae) based on the nucleotide sequences of cpDNA and nrDNA

Molecular phylogenetic analyses of Vincetoxicum and Tylophora (Apocynaceae-Asclepiadoideae) were conducted based on the nucleotide sequences of cpDNA (two intergenic spacers of trnL (UAA)-trnF (GAA) and psbA-trnH and three introns, i.e., atpF, trnG (UCC) and trnL (UAA)), and nrDNA (ITS and ETS regions). Our phylogenetic analysis revealed two monophyletic groups; one consisted of seven taxa of Tylophora and Vincetoxicum inamoenum, Vincetoxicum magnificum and Vincetoxicum macrophyllum (Clade I) and the other consisted of 17 accessions of Vincetoxicum (Clade II). The monophyly of the genus Vincetoxicum was not supported. Although many nucleotide substitutions were observed in Clade I, the genetic differentiation within Clade II was small. Low genetic diversification but considerable morphological divergence suggests that the species in Clade II had undergone rapid diversification. Although most species in Clade I have tiny flowers, those in Clade II have larger and more nectariferous ones. Thus, we hypothesized that the rapid morphological radiation in Clade II may have been due to the gaining of floral characters such as large flowers and large amounts of nectar corresponding to diverse pollinators.     

Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK-dependent pathway

Achondroplasia is the most common genetic form of human dwarfism, for which there is presently no effective therapy. C-type natriuretic peptide (CNP) is a newly identified molecule that regulates endochondral bone growth through GC-B, a subtype of particulate guanylyl cyclase. Here we show that targeted overexpression of CNP in chondrocytes counteracts dwarfism in a mouse model of achondroplasia with activated fibroblast growth factor receptor 3 (FGFR-3) in the cartilage. CNP prevented the shortening of achondroplastic bones by correcting the decreased extracellular matrix synthesis in the growth plate through inhibition of the MAPK pathway of FGF signaling. CNP had no effect on the STAT-1 pathway of FGF signaling that mediates the decreased proliferation and the delayed differentiation of achondroplastic chondrocytes. These results demonstrate that activation of the CNP-GC-B system in endochondral bone formation constitutes a new therapeutic strategy for human achondroplasia.     

Fission yeast Rhp51 is required for the maintenance of telomere structure in the absence of the Ku heterodimer

The Schizosaccharomyces pombe Ku70–Ku80 heterodimer is required for telomere length regulation. Lack of pku70⁺ results in telomere shortening and striking rearrangements of telomere-associated sequences. We found that the rearrangements of telomere-associated sequences in pku80⁺ mutants are Rhp51 dependent, but not Rad50 dependent. Rhp51 bound to telomere ends when the Ku heterodimer was not present at telomere ends. We also found that the single-stranded G-rich tails increased in S phase in wild-type strains, while deletion of pku70⁺ increased the single-stranded overhang in both G₂ and S phase. Based on these observations, we propose that Rhp51 binds to the G-rich overhang and promotes homologous pairing between two different telomere ends in the absence of Ku heterodimer. Moreover, pku80 rhp51 double mutants showed a significantly reduced telomere hybridization signal. Our results suggest that, although Ku heterodimer sequesters Rhp51 from telomere ends to inhibit homologous recombination activity, Rhp51 plays important roles for the maintenance of telomere ends in the absence of the Ku heterodimer.     

N-[18F]fluoroethyl-4-piperidyl acetate ([18F]FEtP4A): A PET tracer for imaging brain acetylcholinesterase in vivo

N-[(18)F]Fluoroethyl-4-piperidyl acetate ([(18)F]FEtP4A) was synthesized and evaluated as a PET tracer for imaging brain acetylcholinesterase (AchE) in vivo. [(18)F]FEtP4A was previously prepared by reacting 4-piperidyl acetate (P4A) with 2-[(18)F]fluoroethyl bromide ([(18)F]FEtBr) at 130 degrees C for 30 min in 37% radiochemical yield using an automated synthetic system. In this work, [(18)F]FEtP4A was synthesized by reacting P4A with 2-[(18)F]fluoroethyl iodide ([(18)F]FEtI) or 2-[(18)F]fluoroethyl triflate ([(18)F]FEtOTf in improved radiochemical yields, compared with [(18)F]FEtBr under the corresponding condition. Ex vivo autoradiogram of rat brain and PET summation image of monkey brain after iv injection of [(18)F]FEtP4A displayed a high radioactivity in the striatum, a region with the highest AchE activity in the brain. Moreover, the distribution pattern of (18)F radioactivity was consistent with that of AchE in the brain: striatum>frontal cortex>cerebellum. In the rat and monkey plasma, two radioactive metabolites were detected. However, their presence might not preclude the imaging studies for AchE in the brain, because they were too hydrophilic to pass the blood-brain barrier and to enter the brain. In the rat brain, only [(18)F]fluoroethyl-4-piperidinol ([(18)F]FEtP4OH) was detected at 30 min postinjection. The hydrolytic [(18)F]FEtP4OH displayed a slow washout and a long retention in the monkey brain until the PET experiment (120 min). Although [(18)F]FEtP4A is a potential PET tracer for imaging AchE in vivo, its lower hydrolytic rate and lower specificity for AchE than those of [(11)C]MP4A may limit its usefulness for the quantitative measurement for AchE in the primate brain.     

Preferential paternal origin of microdeletions caused by prezygotic chromosome or chromatid rearrangements in Sotos syndrome

Sotos syndrome (SoS) is characterized by pre- and postnatal overgrowth with advanced bone age; a dysmorphic face with macrocephaly and pointed chin; large hands and feet; mental retardation; and possible susceptibility to tumors. It has been shown that the major cause of SoS is haploinsufficiency of the NSD1 gene at 5q35, because the majority of patients had either a common microdeletion including NSD1 or a truncated type of point mutation in NSD1. In the present study, we traced the parental origin of the microdeletions in 26 patients with SoS by the use of 16 microsatellite markers at or flanking the commonly deleted region. Deletions in 18 of the 20 informative cases occurred in the paternally derived chromosome 5, whereas those in the maternally derived chromosome were found in only two cases. Haplotyping analysis of the marker loci revealed that the paternal deletion in five of seven informative cases and the maternal deletion in one case arose through an intrachromosomal rearrangement, and two other cases of the paternal deletion involved an interchromosomal event, suggesting that the common microdeletion observed in SoS did not occur through a uniform mechanism but preferentially arose prezygotically.     

Oxidized LDL through LOX-1 modulates LDL-receptor expression in human coronary artery endothelial cells

Experimental studies have shown that oxidized low-density lipoprotein (ox-LDL) up-regulates its receptor LOX-1. Both ox-LDL and LOX-1 are expressed in atherosclerotic plaques. Native LDL concentrations are elevated in atherosclerosis, suggesting a reduction in LDL-receptors. We hypothesized that ox-LDL via LOX-1 could influence the expression of LDL-receptors. This study was designed to examine the interaction between ox-LDL, LOX-1, and LDL-receptors in human coronary artery endothelial cells (HCAECs). HCAECs were incubated with ox-LDL (10-80 microg/ml) for 3-24h. Ox-LDL decreased the expression of LDL-receptor in a concentration- and time-dependent fashion. The effects of ox-LDL were mediated by its endothelial receptor LOX-1, since pretreatment of HCAECs with a blocking antibody to LOX-1 (JTX92, 10 microg/ml) prevented the effect of ox-LDL on LDL-receptor expression. The role of LOX-1 was further confirmed by the use of an antisense to LOX-1 mRNA, which also blocked the effect of ox-LDL in LDL-receptor expression. In other experiments, ox-LDL as expected induced superoxide anion generation; and pretreatment of HCAECs with the anti-oxidants trolox and alpha-tocopherol (each 10 microM) inhibited the formation of superoxide anions as well as the down-regulation of LDL-receptor in response to ox-LDL. These studies provide the first evidence that ox-LDL via LOX-1 modulates LDL-receptor expression in HCAECs. The generation of free radicals elicited by ox-LDL may be a key step in this process.