Activation of AMP-activated Protein Kinase Suppresses Oxidized Low-density Lipoprotein-induced Macrophage Proliferation

Macrophage-derived foam cells play important roles in the progression of atherosclerosis. We reported previously that ERK1/2-dependent granulocyte/macrophage colony-stimulating factor (GM-CSF) expression, leading to p38 MAPK/ Akt signaling, is important for oxidized low density lipoprotein (Ox-LDL)-induced macrophage proliferation. Here, we investigated whether activation of AMP-activated protein kinase (AMPK) could suppress macrophage proliferation. Ox-LDL-induced proliferation of mouse peritoneal macrophages was assessed by [³H]thymidine incorporation and cell counting assays. The proliferation was significantly inhibited by the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and restored by dominant-negative AMPKα1, suggesting that AMPK activation suppressed macrophage proliferation. AICAR partially suppressed Ox-LDL-induced ERK1/2 phosphorylation and GM-CSF expression, suggesting that another mechanism is also involved in the AICAR-mediated suppression of macrophage proliferation. AICAR suppressed GM-CSF-induced macrophage proliferation without suppressing p38 MAPK/Akt signaling. GM-CSF suppressed p53 phosphorylation and expression and induced Rb phosphorylation. Overexpression of p53 or p27^kip suppressed GM-CSF-induced macrophage proliferation. AICAR induced cell cycle arrest, increased p53 phosphorylation and expression, and suppressed GM-CSF-induced Rb phosphorylation via AMPK activation. Moreover, AICAR induced p21^cip and p27^kip expression via AMPK activation, and small interfering RNA (siRNA) of p21^cip and p27^kip restored AICAR-mediated suppression of macrophage proliferation. In conclusion, AMPK activation suppressed Ox-LDL-induced macrophage proliferation by suppressing GM-CSF expression and inducing cell cycle arrest. These effects of AMPK activation may represent therapeutic targets for atherosclerosis.     

Visualization and Identification of IL-7 Producing Cells in Reporter Mice

Interleukin-7 (IL-7) is required for lymphocyte development and homeostasis although the actual sites of IL-7 production have never been clearly identified. We produced a bacterial artificial chromosome (BAC) transgenic mouse expressing ECFP in the Il7 locus. The construct lacked a signal peptide and ECFP (enhanced cyan fluorescent protein ) accumulated inside IL-7-producing stromal cells in thoracic thymus, cervical thymus and bone marrow. In thymus, an extensive reticular network of IL-7-containing processes extended from cortical and medullary epithelial cells, closely contacting thymocytes. Central memory CD8 T cells, which require IL-7 and home to bone marrow, physically associated with IL-7-producing cells as we demonstrate by intravital imaging.     

Phosphoproteomic analysis of distinct tumor cell lines in response to nocodazole treatment

Here, we report for the first time a comparative phosphoproteomic analysis of distinct tumor cell lines in the presence or absence of the microtubule‐interfering agent nocodazole. In total, 1525 phosphorylation sites assigned to 726 phosphoproteins were identified using LC‐MS‐based technology following phosphopeptide enrichment. Analysis of the amino acid composition surrounding the identified in vivo phosphorylation sites revealed that they could be classified into two motif groups: pSer‐Pro and pSer‐Asp/Glu. Phosphoproteomic change resulting from nocodazole treatment varied among cell lines in terms of the numbers of total phosphopeptides identified, motif groups, and functional annotation groups; however, the cell lines were equally sensitive to nocodazole. The identified phosphoproteome subset contained major signaling proteins and proteins known to be involved in mitosis, but did not always exhibit the same changes in the tumor cells from nocodazole treatment. In spite of the complex changes observed in the phosphorylation of many of the proteins, possible common features induced by nocodazole were found, including phosphorylation of nucleophosmin (NPM) S254 and coatomer protein complex, subunit α (COPA) S173, suggesting that the events are not cell‐type specific but events generally occurring in mitosis or induced by a microtubule‐interfering agent. Further, temporal analysis of phosphoproteome change revealed that phosphorylation of NPM S254 and COPA S173 was observed from the early (6 h) and late (24 h) time point after nocodazole treatment, respectively, suggesting that NPM S254 may be involved in the induction of M‐phase arrest by nocodazole, whereas COPA S173 may be caused as a result of M‐phase arrest.     

TATA Box-Binding Protein gene is associated with risk for schizophrenia, age at onset and prefrontal function

Schizophrenia is a common polygenic disease in distinct populations, while spinocerebellar ataxia type 17 (SCA17) is a rare autosomal dominant neurodegenerative disorder. Both diseases involve psychotic symptoms. SCA17 is caused by an expanded polyglutamine tract in the TATA box-binding protein ( TBP ) gene. In the present study, we investigated the association between schizophrenia and CAG repeat length in common TBP alleles with fewer than 42 CAG repeats in a Japanese population (326 patients with schizophrenia and 116 healthy controls). We found that higher frequency of alleles with greater than 35 CAG repeats in patients with schizophrenia compared with that in controls ( p = 0.042). We also examined the correlation between CAG repeats length and age at onset of schizophrenia. We observed a negative correlation between the number of CAG repeats in the chromosome with longer CAG repeats out of two chromosomes and age at onset of schizophrenia ( p = 0.020). We further provided evidence that TBP genotypes with greater than 35 CAG repeats, which were enriched in patients with schizophrenia, were significantly associated with hypoactivation of the prefrontal cortex measured by near-infrared spectroscopy during the tower of Hanoi, a task of executive function (right PFC; p = 0.015, left PFC; p = 0.010). These findings suggest possible associations of the genetic variations of the TBP gene with risk for schizophrenia, age at onset and prefrontal function.     

Determination of partial genomic sequences and development of a CAPS system of the S-RNase gene for the identification of 22 S haplotypes of apple (Malus × domestica Borkh.)

Information about self-incompatibility (S) genotypes of apple cultivars is important for the selection of pollen donors for fruit production and breeding. Although S genotyping systems using S haplotype-specific PCR of S-RNase, the pistil S gene, are useful, they are sometimes associated with false-positive/negative problems and are unable to identify new S haplotypes. The CAPS (cleaved amplified polymorphic sequences) system is expected to overcome these problems, however, the genomic sequences needed to establish this system are not available for many S-RNases. Here, we determined partial genomic sequences of eight S-RNases, and used the information to design new primer and to select 17 restriction enzymes for the discrimination of 22 S-RNases by CAPS. Using the system, the S genotypes of three cultivars were determined. The genomic sequence-based CAPS system would be useful for S genotyping and analyzing new S haplotypes of apple.     

Nest intrusion and infanticidal attack on nestlings in great cormorants Phalacrocorax carbo: why do adults attack conspecific chicks?

Infanticide, the killing of young animals by conspecifics, has been observed in diverse taxa. The function of infanticide has been classified as exploitation, sexual selection, parental manipulation or resource competition. We observed infanticidal behavior and its reproductive results at five breeding colonies of great cormorants from January to August 2008. Eighteen cases of nest intrusions and/or attacks toward a chick by conspecific non-nest-owners were observed, and two of them were filmed. In both attacks, perpetrators pecked the necks of chicks several times with display. The chicks bent their necks down onto the nest and remained stationary. Our data did not support the exploitation hypothesis because adult cormorants did not use chicks as food. In addition, the perpetrators were not true parents and did not mate with the female nest owner, indicating that parental manipulation and sexual selection hypotheses were unlikely explanations. On the other hand, concurrent presence of adults during prelaying and chick-rearing periods at a particular colony affected the occurrence of nest takeovers and intrusions and/or attacks, suggesting that some conflicts over nests arise between individuals that are at different stages of the breeding cycle. Digital videos relating to this article are available at and .     

Changes in Extracellular Kynurenic Acid Concentrations in Rat Prefrontal Cortex After <Emphasis Type="SmallCaps">d</Emphasis>-Kynurenine Infusion: An In vivo Microdialysis Study

Using a microdialysis technique, we continuously infused d-kynurenine (KYN) (0, 50, and 100 μM) into the prefrontal cortices (PFCs) of male Sprague–Dawley rats. We then used column-switching high-performance liquid chromatography to assess the alterations in the concentration of kynurenic acid (KYNA)—an antagonist of N-methyl-d-aspartate and α7 nicotinic acetylcholine receptors—in the extracellular fluid in the PFC. Local infusion of d-KYN into the PFC remarkably increased the extracellular KYNA concentration, indicating that d-KYN is metabolized to KYNA in the PFC. The d-KYN-induced increase in KYNA levels was significantly attenuated by the co-administration of 3-methylpyrazole-5-carboxylic acid (AS057278)—a specific inhibitor of d-amino acid oxidase (DAAO). These results suggest that DAAO may be involved in the production of KYNA from d-KYN in the PFC in vivo.