Sphingomyelin Deacylase, the Enzyme Involved in the Pathogenesis of Atopic Dermatitis, Is Identical to the ß-Subunit of Acid Ceramidase.

A ceramide deficiency in the stratum corneum (SC) is an essential etiologic factor for the dry and barrier-disrupted skin of patients with atopic dermatitis (AD). Previously, we reported that sphingomyelin (SM) deacylase, which hydrolyzes SM and glucosylceramide at the acyl site to yield their lysoforms sphingosylphosphorylcholine (SPC) and glucosylsphingosine, respectively, instead of ceramide and/or acylceramide, is over-expressed in AD skin and results in a ceramide deficiency. Although the enzymatic properties of SM deacylase have been clarified, the enzyme itself remains unidentified. In this study, we purified and characterized SM deacylase from rat skin. The activities of SM deacylase and acid ceramidase (aCDase) were measured using SM and ceramide as substrates by tandem mass spectrometry by monitoring the production of SPC and sphingosine, respectively. Levels of SM deacylase activity from various rat organs were higher in the order of skin > lung > heart. By successive chromatography using Phenyl-5PW, Rotofor, SP-Sepharose, Superdex 200 and Shodex RP18-415, SM deacylase was purified to homogeneity with a single band of an apparent molecular mass of 43 kDa with an enrichment of > 14,000-fold. Analysis by MALDI-TOF MS/MS using a protein spot with SM deacylase activity separated by 2D-SDS-PAGE allowed its amino acid sequence to be determined and identified as the ß-subunit of aCDase, which consists of α- and ß-subunits linked by amino bonds and a single S-S bond. Western blotting of samples treated with 2-mercaptoethanol revealed that, whereas recombinant human aCDase was recognized by antibodies to the α-subunit at ~56 kDa and ~13 kDa and the ß-subunit at ~43 kDa, the purified SM deacylase was detectable only by the antibody to the ß-subunit at ~43 kDa. Breaking the S-S bond of recombinant human aCDase with dithiothreitol elicited the activity of SM deacylase with ~40 kDa upon gel chromatography. These results provide new insights into the essential role of SM deacylase expressed as an aCDase-degrading ß-subunit that evokes the ceramide deficiency in AD skin.

[Two Cases of Non-Occlusive Mesenteric Ischemia That Developed after Chemotherapy].

Non-occlusive mesenteric ischemia(NOMI)causes intestinal necrosis due to irreversible ischemia of the intestinal tract despite the absence of organic obstruction in the mesenteric blood vessels. The disease has extremely poor prognosis. We encountered 2 cases of NOMI hypothesized to have developed after chemotherapy; thus, we report these cases considering the available literature. Case 1: A7 9-year-old man. The patient complained of abdominal pain during the first week after introducing docetaxel for local recurrence of prostate cancer. Abdominal computed tomography(CT)revealed mesenteric ischemia and intestinal emphysema. The patient was diagnosed with NOMI, and an emergency operation was performed. Upon laparotomy, the small intestine; ascending, transverse, and descending colon; recto sigmoid; and gall bladder appeared mottled necrotic. As such, all these were excised. He was admitted back to the hospital 3 weeks after surgery due to pneumonia. Case 2: A7 4-year-old man. Combination chemotherapy of docetaxel, cisplatin, and 5-FU was given for oropharyngeal cancer. After 1 week, fever and abdominal pain were noted. Abdominal contrast CT examination was performed, and mesenteric ischemia was confirmed as NOMI. Emergency surgery was performed on the same day. The entire ileum was discolored with mottling, and it was determined to be necrotic. Thus, it was excised. Postoperative course is good, and the patient was followed up after discharge from the hospital. Before NOMI onset in both cases, docetaxel was used to treat myelosuppression. Considering the patient conditions, the association between NOMI onset and docetaxel was suspected. In general, mesenteric ischemia after administration of anticancer drugs is rare, and only a few cases have been reported.

A new methodology for simultaneous quantification of total-Aß, Aßx-38, Aßx-40, and Aßx-42 by column-switching LC/MS/MS.

This article details the development of a novel method that overcomes the drawbacks of sandwich ELISA (sELISA) and allows reliable evaluation of simultaneous quantification of the amyloid (Aß)-peptides, total-Aß, Aßx-38, Aßx-40, and Aßx-42, in rat brain by optimized sample purification and column-switching liquid chromatographic-tandem mass spectrometry (LC/MS/MS). This method provides accurate analyses of total-Aß, Aßx-38, Aßx-40, and Aßx-42 with a linear calibration range between 0.05 and 45 ng/mL. Verification for accuracy and precision of biological samples were determined by a standard addition and recovery test, spiked with synthetic Aß1-38, Aß1-40, and Aß1-42 into the rat brain homogenate. This method showed <20% relative error and relative standard deviation, indicating high reproducibility and reliability. The brain concentrations of total-Aß, Aßx-38, Aßx-40, and Aßx-42 after oral administration of flurbiprofen in rats were measured by this method. Aßx-42 concentrations (4.57 ± 0.69 ng/g) in rats administered flurbiprofen were lower than those in untreated rats (6.48 ± 0.93 ng/g). This was consistent with several reports demonstrating that NSAIDs reduced the generation of Aß. We report here a method that allows not only the quantification of specific molecular species of Aß but also simultaneous quantification of total-Aß, Aßx-38, Aßx-40, and Aßx-42, thus overcoming the drawbacks of sELISA.

Nuclear localization signal and phosphorylation of Serine350 specify intracellular localization of DRAK2.

DAP kinase-related apoptosis-inducing kinase 2 (DRAK2) is a serine/threonine kinase of the death-associated protein kinase family. DRAK2 mediates apoptosis induced by extracellular stimuli, including UV irradiation and interleukin-2, and also regulates T-cell receptor sensitivity in developing thymocytes. During these events, the subcellular localization of DRAK2 changes between the nucleus and cytoplasm. We found that DRAK2 has a putative nuclear-localization signal (NLS) sequence. Mutations in this sequence interfered with DRAK2 localization to the nucleus. Furthermore, green fluorescence protein fused to the putative NLS accumulated in the nucleus, indicating that the putative sequence functions as an NLS. We also found that the function of the NLS was regulated by phosphorylation. Phorbol myristate acetate (PMA) induced the accumulation of DRAK2 in the cytoplasm of NIH3T3 cells, whereas in the absence of PMA, DRAK2 was localized to the nucleus. Ectopic expression of PKC-gamma induced cytoplasmic localization of DRAK2 and PKC-gamma phosphorylated Ser350 flanking the NLS. DRAK2, but not the Ser350Asp mutant, accumulated in the nuclei of ACL-15 cells in response to UV-irradiation. These results suggest that phosphorylation of Ser350 plays an essential role in regulating translocation of DRAK2 to the nucleus from the cytoplasm, possibly by affecting the activity of the NLS.

The apoptosis-inducing protein kinase DRAK2 is inhibited in a calcium-dependent manner by the calcium-binding protein CHP.

Calcineurin homologous protein (CHP) is an EF-hand Ca(2+)-binding protein capable of interacting with various cellular proteins including Na(+)/H(+) exchangers, kinesin-related proteins, and apoptosis-inducing protein kinase DRAK2. We investigated the role of CHP on the DRAK2 protein kinase in vitro. CHP significantly reduced (approximately 85% inhibition) the kinase activity of DRAK2 for both autophosphorylation and phosphorylation of exogenous substrate (myosin light chain). The inhibitory effect of CHP was dependent on the presence of Ca(2+), whereas the interaction between CHP and DRAK2 was not Ca(2+)-dependent. These observations suggest that CHP negatively regulates the apoptosis-inducing protein kinase DRAK2 in a manner that depends on intracellular Ca(2+)-concentration.

Nuclear localization of the serine/threonine kinase DRAK2 is involved in UV-induced apoptosis.

DAP kinase-related apoptosis-inducing kinase 2 (DRAK2), a member of the DAP kinase family, is a serine/threonine kinase capable of inducing apoptosis. Here we studied the relationship between DRAK2 intracellular localization and apoptosis, and found that UV light acts as a stimulus for apoptosis induced by DRAK2. The intracellular location of DRAK2 depended on the cell line: DRAK2 was found primarily in the nuclei of NRK, NIH3T3, and Caco-2 cells while it was present primarily in the cytoplasm of ACL-15, HeLa, and WI-38 cells. Overexpression of Myc-tagged DRAK2 led to apoptosis-like cell death in NRK cells, but not in ACL-15 cells. A GFP fusion protein of DRAK2 was spontaneously localized to the nucleus of ACL-15 cells and resulted in cell death. Nuclear localization and cell death were also observed with DRAK2(1-293) fused to the NLS of SV40 but not with DRAK2(1-293) alone. These results suggested that nuclear accumulation of DRAK2 and the resulting increase in the endogenous level of its kinase activity are required for cell death. UV irradiation caused nuclear accumulation of endogenous DRAK2 in ACL-15 cells, which was followed by apoptosis-like cell death. Knockdown of DRAK2 expression by siRNA partially suppressed UV-induced apoptosis. These results suggest that DRAK2 plays a role in UV induced apoptosis.