Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase

Ataxia telangiectasia mutated (ATM) kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR). The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression.     

Dose-dependent differential regulation of cytokine secretion from macrophages by fractalkine

Although expression of the fractalkine (CX3CL1, FKN) is enhanced in inflamed tissues, it is detected at steady state in various organs such as the intestine, and its receptor CX3CR1 is highly expressed in resident-type dendritic cells and macrophages. We hypothesized that FKN might regulate the inflammatory responses of these cells. Therefore, murine macrophages were pretreated with FKN and then stimulated with LPS. We found that macrophages pretreated with 0.03 nM FKN but not with 3 nM FKN secreted 50% less TNF-alpha than did cells treated with LPS alone. Cells treated with 0.03 nM FKN and LPS also showed reduced phosphorylation of ERK1/2 and reduced NF-kappaB p50 subunit. Interestingly, the p65 subunit of NF-kappaB was translocated to the nuclei but redistributed to the cytoplasm in the early phase by forming a complex with peroxisome proliferator-activated receptor (PPAR) gamma. Exogenous 15-deoxy-Delta(12,14)-prostaglandin J2, a natural ligand for PPAR-gamma, also induced redistribution of p65 with decreased TNF-alpha secretion after LPS challenge. Pretreatment with 0.03 nM but not 3 nM FKN increased the cellular levels of 15-deoxy-Delta(12,14)-prostaglandin J2 as well as mRNA of PPAR-gamma. Requirement of PPAR-gamma for the effect of 0.03 nM FKN was confirmed by small interfering RNA of PPAR-gamma. In contrast, pretreatment with 3 nM FKN induced higher levels of IL-23 compared with cells pretreated with 0.03 nM FKN and produced TNF-alpha in a CX3CR1-dependent manner. These dose-dependent differential effects of FKN establish its novel role in immune homeostasis and inflammation.     

Large scale deletions in the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> genome create strains with altered regulation of carbon metabolism

Saccharomyces cerevisiae, for centuries the yeast that has been the workhorse for the fermentative production of ethanol, is now also a model system for biological research. The recent development of chromosome-splitting techniques has enabled the manipulation of the yeast genome on a large scale, and this has allowed us to explore questions with both biological and industrial relevance, the number of genes required for growth and the genome organization responsible for the ethanol production. To approach these questions, we successively deleted portions of the yeast genome and constructed a mutant that had lost about 5% of the genome and that gave an increased yield of ethanol and glycerol while showing levels of resistance to various stresses nearly equivalent to those of the parental strain. Further systematic deletion could lead to the formation of a eukaryotic cell with a minimum set of genes exhibiting appropriately altered regulation for enhanced metabolite production. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structure and characteristics of an endo-beta-1,4-glucanase, isolated from Trametes hirsuta, with high degradation to crystalline cellulose

Trametes hirsuta produced cellulose-degrading enzymes when it was grown in a cellulosic medium such as Avicel or wheat bran. An endo-beta-1,4-glucanase (ThEG) was purified from the culture filtrate, and the gene and the cDNA were isolated. The gene consisted of an open reading frame encoding 384 amino acids, interrupted by 11 introns. The whole sequence showed high homology with that of family 5 glycoside hydrolase. The properties of the recombinant enzyme (rEG) in Aspergillus oryzae were compared with those of the En-1 from Irpex lacteus, which showed the highest homology among all the endoglucanases reported. The rEG activity against Avicel was about 8 times higher than that of En-1 when based on CMC degradation. A remarkable structural difference between the two enzymes was the length of the linker connecting the cellulose-binding domain to the catalytic domain.     

Obligate symbiont involved in pest status of host insect

The origin of specific insect genotypes that enable efficient use of agricultural plants is an important subject not only in applied fields like pest control and management but also in basic disciplines like evolutionary biology. Conventionally, it has been presupposed that such pest-related ecological traits are attributed to genes encoded in the insect genomes. Here, however, we report that pest status of an insect is principally determined by symbiont genotype rather than by insect genotype. A pest stinkbug species, Megacopta punctatissima, performed well on crop legumes, while a closely related non-pest species, Megacopta cribraria, suffered low egg hatch rate on the plants. When their obligate gut symbiotic bacteria were experimentally exchanged between the species, their performance on the crop legumes was, strikingly, completely reversed: the pest species suffered low egg hatch rate, whereas the non-pest species restored normal egg hatch rate and showed good performance. The low egg hatch rates were attributed to nymphal mortality before or upon hatching, which were associated with the symbiont from the non-pest stinkbug irrespective of the host insect species. Our finding sheds new light on the evolutionary origin of insect pests, potentially leading to novel approaches to pest control and management.     

Design and synthesis of long-acting inhibitors of dipeptidyl peptidase IV

A series of (4-substituted prolyl)prolinenitriles were synthesized and evaluated as inhibitors of dipeptidylpeptidase IV (DPP-IV). Among those tested, the 4beta-[4-(hydroxyphenyl)prolyl]prolinenitriles showed a potent inhibitory activity with a long duration of action. Metabolic formation of the corresponding phenol glucuronates was found to contribute to their long duration of action. The activity profiles of the synthesized compounds are reported and structure-activity relationships are also presented.     

TGF-beta-neutralizing antibodies improve pulmonary alveologenesis and vasculogenesis in the injured newborn lung

Pulmonary injury is associated with the disruption of alveologenesis in the developing lung and causes bronchopulmonary dysplasia (BPD) in prematurely born infants. Transforming growth factor (TGF)-beta is an important regulator of cellular differentiation and early lung development, and its levels are increased in newborn lung injury. Although overexpression of TGF-beta in the lungs of newborn animals causes pathological features that are consistent with BPD, the role of endogenous TGF-beta in the inhibition of the terminal stage of lung development is incompletely understood. In this investigation, the hypothesis that O(2)-induced injury of the maturing lung is associated with TGF-beta-mediated disruption of alveologenesis and microvascular development was tested using a murine model of BPD. Here we report that treatment of developing mouse lungs with TGF-beta-neutralizing antibodies attenuates the increase in pulmonary cell phospho-Smad2 nuclear localization, which is indicative of augmented TGF-beta signaling, associated with pulmonary injury induced by chronic inhalation of 85% oxygen. Importantly, the neutralization of the abnormal TGF-beta activity improves quantitative morphometric indicators of alveologenesis, extracellular matrix assembly, and microvascular development in the injured developing lung. Furthermore, exposure to anti-TGF-beta antibodies is associated with improved somatic growth in hyperoxic mouse pups and not with an increase in pulmonary inflammation. These studies indicate that excessive pulmonary TGF-beta signaling in the injured newborn lung has an important role in the disruption of the terminal stage of lung development. In addition, they suggest that anti-TGF-beta antibodies may be an effective therapy for preventing some important developmental diseases of the newborn lung.     

Identification of Anhydrobiosis-related Genes from an Expressed Sequence Tag Database in the Cryptobiotic Midge Polypedilum vanderplanki (Diptera; Chironomidae)

Some organisms are able to survive the loss of almost all their body water content, entering a latent state known as anhydrobiosis. The sleeping chironomid (Polypedilum vanderplanki) lives in the semi-arid regions of Africa, and its larvae can survive desiccation in an anhydrobiotic form during the dry season. To unveil the molecular mechanisms of this resistance to desiccation, an anhydrobiosis-related Expressed Sequence Tag (EST) database was obtained from the sequences of three cDNA libraries constructed from P. vanderplanki larvae after 0, 12, and 36 h of desiccation. The database contained 15,056 ESTs distributed into 4,807 UniGene clusters. ESTs were classified according to gene ontology categories, and putative expression patterns were deduced for all clusters on the basis of the number of clones in each library; expression patterns were confirmed by real-time PCR for selected genes. Among up-regulated genes, antioxidants, late embryogenesis abundant (LEA) proteins, and heat shock proteins (Hsps) were identified as important groups for anhydrobiosis. Genes related to trehalose metabolism and various transporters were also strongly induced by desiccation. Those results suggest that the oxidative stress response plays a central role in successful anhydrobiosis. Similarly, protein denaturation and aggregation may be prevented by marked up-regulation of Hsps and the anhydrobiosis-specific LEA proteins. A third major feature is the predicted increase in trehalose synthesis and in the expression of various transporter proteins allowing the distribution of trehalose and other solutes to all tissues.