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.     

Oxidation of myosin heavy chain and reduction in force production in hyperthyroid rat soleus.

We tested the hypothesis that a force reduction in hyperthyroid rat soleus muscle would be associated with oxidative modification in myosin heavy chain (MHC). Daily injection of thyroid hormone [3,5,3'-triiodo-L-thyronine (T3)] for 21 days depressed isometric forces of whole soleus muscle across a range of stimulus frequencies (P < 0.01). In fiber bundles, hyperthyroidism also led to pronounced reductions (P < 0.01) in both K+ - and 4-chloro-m-cresol-induced contracture forces. The degrees of the reductions were similar between these two contractures that were induced by distinct reagents. Treatment with T3 elicited a significant decrease ( approximately 14%; P < 0.05) in the relative content of MHC contained in myofibrillar proteins. The content of carbonyl groups in myofibrillar protein extracts was elevated (P < 0.05) by approximately 50% in T3-treated muscles. Immunoblot analyses on T3-treated muscles showed a greater increase (106%; P < 0.05) of the carbonyl content in MHC than in myofibrillar protein extracts. These data suggest that in hyperthyroidism the decrease in force production of skeletal muscles may stem primarily from failure in myofibrillar protein function resulting from oxidative modification of MHC.     

Metabolic control of vesicular glutamate transport and release

Fasting has been used to control epilepsy since antiquity, but the mechanism of coupling between metabolic state and excitatory neurotransmission remains unknown. Previous work has shown that the vesicular glutamate transporters (VGLUTs) required for exocytotic release of glutamate undergo an unusual form of regulation by Cl(-). Using functional reconstitution of the purified VGLUTs into proteoliposomes, we now show that Cl(-) acts as an allosteric activator, and the ketone bodies that increase with fasting inhibit glutamate release by competing with Cl(-) at the site of allosteric regulation. Consistent with these observations, acetoacetate reduced quantal size at hippocampal synapses and suppresses glutamate release and seizures evoked with 4-aminopyridine in the brain. The results indicate an unsuspected link between metabolic state and excitatory neurotransmission through anion-dependent regulation of VGLUT activity.     

Prognostic value of CD208-positive cell infiltration in gastric cancer

Background and aim

A new marker, CD208, was recently explored as a mature interdigitating dendritic cell (DC), and the correlation between the infiltration of CD208-positive cells and clinical factors has been reported in various types of cancers. In this study, we tried to clarify the clinical implication of CD208-positive cell infiltration in gastric cancer immunohistochemically.

Patients and methods

A total of 128 gastric cancer patients who underwent a curative operation were enrolled. DCs in tumor nests were identified with two DC markers, CD208 and S-100 protein (S100), by immunohistochemistry. The correlation between clinicopathological features and the CD208- or S100-positive cell infiltration degree was analyzed.

Results

Infiltration of S100-positive cells did not correlate with the degree of CD208-positive cell infiltration. Patients with high CD208-positive cell infiltration in the peritumor had a poorer surgical outcome than those with low CD208 infiltration (p < 0.05). Multivariate analysis revealed that CD208-positive cell infiltration was not an independent prognostic factor.

Conclusion

We showed that intratumoral CD208-positive cells, as mature DCs, had an inverse correlation to patients’ postoperative outcome in gastric cancer, unlike a conventional DC marker. Evaluation of CD208-positive cell infiltration with S100-positive cell infiltration in gastric cancer is useful to predict antitumor immunological conditions in gastric cancer.     

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.