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Autophagy is an evolutionarily conserved intracellular degradation system that is involved in cell survival and activated in various diseases, including cancer. Beclin 1 is a central scaffold protein that assembles components for promoting or inhibiting autophagy. Association of Beclin 1 with its interacting proteins is regulated by the phosphorylation of Beclin 1 by various Ser/ Thr kinases, but the Ser/Thr phosphatases that regulate these phosphorylation events remain unknown. Here we identify Ser-90 in Beclin 1 as a regulatory site whose phosphorylation is markedly enhanced in cells treated with okadaic acid, an inhibitor of protein phosphatase 2A (PP2A). Beclin 1 Ser-90 phosphorylation is induced in skeletal muscle tissues isolated from starved mice. The Beclin 1 S90A mutant blocked starvationinduced autophagy. We found association of PP2A B55␣ with Beclin 1, which dissociate by starvation. We also found that death-associated protein kinase 3 directly phosphorylates Beclin 1 Ser-90. We propose that physiological regulation of Beclin 1 Ser-90 phosphorylation by PP2A and death-associated protein kinase 3 controls autophagy.

Background and Purpose-To evaluate the mechanisms of failure of blood oxygenation level-dependent (BOLD) imaging in stroke, we compared the evoked cerebral blood oxygenation (CBO) responses and activation volumes (AVs) of BOLD functional MRI (fMRI) in chronic stroke patients with moderate and severe cerebral ischemia. Methods-We measured the evoked CBO responses in the primary sensorimotor cortex (PSMC) by means of near-infrared spectroscopy during contralateral motor tasks. We compared the AV of BOLD-functional MRI in the PSMC on the nonlesion and lesion sides. Single-photon emission computed tomography was used to classify ischemic status as moderate (slight reduction of regional cerebral blood flow and cerebrovascular reserve capacity [CVRC]) or severe (marked reduction of regional cerebral blood flow and CVRC; ie, misery perfusion). Results-In age-matched controls, deoxyhemoglobin concentration decreased with concomitant increases in oxyhemoglobin and total hemoglobin concentrations during activation. The PSMC on the nonlesion side exhibited a normal CBO response pattern. On the lesion side, moderate cerebral ischemia did not affect the CBO response pattern, but severe cerebral ischemia caused an increase of deoxyhemoglobin during the task, associated with increases of oxyhemoglobin and total hemoglobin. Moderate cerebral ischemia induced only a slight reduction of the AV on the lesion side; however, severe cerebral ischemia markedly reduced the AV on the lesion side. The BOLD signal did not change in some areas of the PSMC on the lesion side in severe cerebral ischemia, whereas it tended to decrease in other areas during the tasks. Conclusions-Misery perfusion caused a marked reduction of the AV on BOLD imaging, associated with an increase of deoxyhemoglobin concentration during activation. BOLD-fMRI investigations of stroke patients should be performed while giving consideration to baseline circulatory status. Functional near-infrared spectroscopy could be an alternative means to assess the CVRC. (Stroke. 2006;37:2514-2520.)

The calcitonin (CT)/CT gene‐related peptides (CGRPs) constitute a large peptide family in vertebrates. However, no CT/CGRP superfamily members have so far been identified in invertebrates, and the evolutionary process leading to the diverse vertebrate CT/CGRP superfamily members remains unclear. In this study, we have identified an authentic invertebrate CT, Ci‐CT, in the ascidian Ciona intestinalis, which is the phylogenetically closest invertebrate chordate to vertebrates. The amino acid sequence of Ci‐CT was shown to display high similarity to those of vertebrate CTs and to share CT consensus motifs, including the N‐terminal circular region and C‐terminal amidated proline. Furthermore, the Ci‐CT gene was found to be the only Ciona CT/CGRP superfamily gene. Ci‐CT also exhibited less potent, but significant, activation of the human CT receptor, as compared with salmon CT. Physiological analysis revealed that Ci‐CT reduced the osteoclastic activity that is specific to vertebrate CTs. CD analysis demonstrated that Ci‐CT weakly forms an α‐helix structure. These results provide evidence that the CT/CGRP superfamily is essentially conserved in ascidians as well as in vertebrates, and indicate that Ci‐CT is a prototype of vertebrate CT/CGRP superfamily members. Moreover, expression analysis demonstrated that Ci‐CT is expressed in more organs than vertebrate CTs in the cognate organs, suggesting that an original CT/CGRP superfamily member gene was also expressed in multiple organs, and each CT/CGRP superfamily member acquired its current specific tissue distribution and physiological role concomitantly with diversification of the CT/CGRP superfamily during the evolution of chordates. This is the first report on a CT/CGRP superfamily member in invertebrates.

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Tumor microenvironment has been implicated in tumor development and progression. As a three-dimensional tumor microenvironment model, air liquid interface (ALI) organoid culture from oncogene transgenic mouse gastrointestinal tissues was recently produced. However, ALI organoid culture system from tissues of colorectal cancer patients has not been established. Here, we developed an ALI organoid model from normal and tumor colorectal tissues of human patients. Both organoids were successfully generated and showed cystic structures containing an epithelial layer and surrounding mesenchymal stromal cells. Structures of tumor organoids closely resembled primary tumor epithelium. Expression of an epithelial cell marker, E-cadherin, a goblet cell marker, MUC2, and a fibroblast marker, vimentin, but not a myofibroblast marker, α-smooth muscle actin (SMA), was observed in normal organoids. Expression of E-cadherin, MUC2, vimentin, and α-SMA was observed in tumor organoids. Expression of a cancer stem cell marker, LGR5 in tumor organoids, was higher than that in primary tumor tissues. Tumor organoids were more resistant to toxicity of 5-fluorouracil and Irinotecan than colorectal cancer cell lines, SW480, SW620, and HCT116. These findings indicate that ALI organoid culture from colorectal cancer patients may become a novel model that is useful for examining resistance to chemotherapy in tumor microenvironment.

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