Upstream and downstream pathways of diacylglycerol kinase : Novel phosphatidylinositol turnover-independent signal transduction pathways.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). Mammalian DGK comprise ten isozymes (α-κ) that regulate a wide variety of physiological and pathological events. Recently, we revealed that DGK isozymes use saturated fatty acid (SFA)/monosaturated fatty acid (MUFA)-containing and docosahexaenoic acid (22:6)-containing DG species, but not phosphatidylinositol (PI) turnover-derived 18:0/20:4-DG. For example, DGKδ, which is involved in the pathogenesis of type 2 diabetes, preferentially uses SFA/MUFA-containing DG species, such as 16:0/16:0- and 16:0/18:1-DG species, in high glucose-stimulated skeletal muscle cells. Moreover, DGKδ, which destabilizes the serotonin transporter (SERT) and regulates the serotonergic system in the brain, primarily generates 18:0/22:6-PA. Furthermore, 16:0/16:0-PA is produced by DGKζ in Neuro-2a cells during neuronal differentiation. We searched for SFA/MUFA-PA- and 18:0/22:6-PA-selective binding proteins (candidate downstream targets of DGKδ) and found that SFA/MUFA-PA binds to and activates the creatine kinase muscle type, an energy-metabolizing enzyme, and that 18:0/22:6-PA interacts with and activates Praja-1, an E3 ubiquitin ligase acting on SERT, and synaptojanin-1, a key player in the synaptic vesicle cycle. Next, we searched for SFA/MUFA-DG-generating enzymes upstream of DGKδ. We found that sphingomyelin synthase (SMS)1, SMS2, and SMS-related protein (SMSr) commonly act as phosphatidylcholine (PC)-phospholipase C (PLC) and phosphatidylethanolamine (PE)-PLC, generating SFA/MUFA-DG species, in addition to SMS and ceramide phosphoethanolamine synthase. Moreover, the orphan phosphatase PHOSPHO1 showed PC- and PE-PLC activities that produced SFA/MUFA-DG. Although PC- and PE-PLC activities were first described 70-35 years ago, their proteins and genes were not identified for a long time. We found that DGKδ interacts with SMSr and PHOSPHO1, and that DGKζ binds to SMS1 and SMSr. Taken together, these results strongly suggest that there are previously unrecognized signal transduction pathways that include DGK isozymes and generate and utilize SFA/MUFA-DG/PA or 18:0/22:6-DG/PA but not PI-turnover-derived 18:0/20:4-DG/PA.

The significance of T-BET-positive CD8 T-cells with diminished CD5 expression in Kikuchi-Fujimoto disease.

Kikuchi-Fujimoto disease (KFD), also known as histiocytic necrotizing lymphadenitis, is a rare condition characterized by benign localized lymphadenopathy and clinical symptoms such as fever, sore throat, odynophagia, and leukopenia. Though the etiology of KFD is unknown, this condition is similar to viral infection, including increased infiltration of activated plasmacytoid dendritic cells. KFD exhibits three histological phases that reflect its progression status: proliferative, necrotic, and xanthomatous lesions. The expression loss of pan T-cell markers, such as CD2, CD5, and CD7, of infiltrating T-cells is observed in KFD cases, complicating the distinction from T-cell lymphoma. However, reports on the loss of their expression in KFD have been limited. Furthermore, the precise population of the T-cell subset in KFD is still unclear. Here, we focused on surface markers and transcription factors for T-cell differentiation and analyzed them immunohistochemically in 46 KFD cases. We observed diminished CD5 expression of CD8-positive (CD5dim CD8+) T-cells in the proliferative lesion of KFD cases. Furthermore, these CD5dim CD8+ T-cells expressed T-BET, a master regulator of type 1 helper T-cells. The upregulation of T-BET and downregulation of CD5 in CD8+ T-cells causes dysregulated activation and proliferation of CD8+ T-cells, potentially contributing to the unique histopathological features of KFD. Recognizing the frequent infiltration of T-BET-positive CD5dim CD8+ T-cells in KFD is important for distinguishing it from mature T-cell lymphoma. Our findings suggest that the immune response in KFD shares similarities with viral infections and highlight the importance of characterizing T-BET-positive CD5dim CD8+ T-cell populations for understanding KFD pathogenesis.

Impacts of tumor microenvironment during neoadjuvant chemotherapy in patients with esophageal squamous cell carcinoma.

With the advent of immune checkpoint inhibitors (ICIs), a better understanding of tumor microenvironment (TME) is becoming crucial in managing esophageal squamous cell carcinoma (ESCC) patients. We investigated the survival impact of TME status and changes in patients with ESCC who underwent neoadjuvant chemotherapy (NAC) followed by surgery (n = 264). We examined immunohistochemical status (CD4+, CD8+, CD20+, Foxp3+, HLA class-1+, CD204+, and programmed death ligand-1 [PD-L1+]) on 264 pre-NAC and 204 paired post-NAC specimens. Patients were classified by their pre- and post-NAC immune cell status and their changes following NAC. Our findings showed that pre-NAC TME status was not significantly associated with survival outcomes. In contrast, post-NAC TME status, such as low level of T cells, CD4+ T cells, and high PD-L1 combined positive score (CPS), were significantly associated with poor overall survival (OS). Notably, TME changes through NAC exerted significant survival impacts; patients with consistently low levels of T cells, low levels of CD4+ T cells, or high levels of PD-L1 (CPS) had very poor OS (3-year OS: 35.5%, 40.2%, and 33.3%, respectively). Tumor microenvironment changes of consistently low T cells, low CD4+ T cells, and high PD-L1 were independent predictors of poor OS in multivariate Cox hazards analyses, while factors indicating post-NAC status (T cells, CD4+, and PD-L1 [CPS]) alone were not. Therefore, we suggest that the consistently low T/high PD-L1 group could benefit from additional therapies, such as ICIs, and the importance of stratification by the TME, which has recently been recognized.

[Early Duodenal Cancer Resected by Using Laparoscopic and Endoscopic Assistance Surgery in a Patient with Peutz-Jeghers Syndrome].

A 28-year-old female with a history of treatment for small intestinal polyps and characteristic pigmentation of her lip was clinically diagnosed with Peutz-Jeghers syndrome(PJS). Her sister had the pathogenic variant of STK11 upon genetic testing. A 20-mm polyp was identified in the second part the patient's duodenum on routine gastrointestinal surveillance, and biopsy revealed a well-differentiated adenocarcinoma. Laparoscopic partial duodenectomy with endoscopy was planned. After confirming the location of the tumor and Kocherization using a laparoscope, the polyp was resected via submucosal dissection under direct visualization with a small incision. The polyp was diagnosed as well-differentiated adenocarcinoma in situ and was resected without remnants. PJS is characterized by a high incidence of malignant tumors, and lifelong surveillance for gastrointestinal and extra-gastrointestinal tumors is necessary. The incidence of duodenal cancer is not high among patients with PJS. However, surgery for advanced cancer is highly invasive. It is desirable to detect the tumors at an early stage so that they can be resected via a less invasive treatment method such as endoscopic resection or laparoscopic surgery with an endoscope.

[Mesenchymal and Non-meningothelial Tumors Involving the Central Nervous System].

In the World Health Organization Classification of Brain Tumors Fifth Edition, mesenchymal non-meningothelial tumors involving the central nervous system are divided into three major categories: soft tissue tumors, chondro-osseous tumors, and notochordal tumors. Soft tissue tumors are classified into four groups: fibroblastic and myofibroblastic tumors, vascular tumors, skeletal muscle tumors, and tumors of uncertain differentiation. This article will focus on solitary fibrous tumors(SFTs), which are frequently encountered clinically and continue to undergo classification revisions in the 5th edition, and outline the three newly added histological diagnoses. Although SFTs and hemangiopericytomas occur throughout the body, including the central nervous system, nomenclatures have been different between the classifications of "Tumours of Soft Tissue and Bone" and "Tumours of the Central Nervous System." The latest nomenclature is "SFT" in accordance with the nomenclature of bone and soft-tissue tumors. In addition, three new diagnoses, which are intracranial mesenchymal tumor FET-CREB fusion-positive, CIC-rearranged sarcoma, and primary intracranial sarcoma DICER1-mutant, have been defined based on genetic abnormalities in tumors of uncertain differentiation.

Human sphingomyelin synthase 1 generates diacylglycerol in the presence and absence of ceramide via multiple enzymatic activities.

Sphingomyelin (SM) synthase 1 (SMS1), which is involved in lipodystrophy, deafness, and thrombasthenia, generates diacylglycerol (DG) and SM using phosphatidylcholine (PC) and ceramide as substrates. Here, we found that SMS1 possesses DG-generating activities via hydrolysis of PC and phosphatidylethanolamine (PE) in the absence of ceramide and ceramide phosphoethanolamine synthase (CPES) activity. In the presence of the same concentration (4.7 mol%) of PC and ceramide, the amounts of DG produced by SMS and PC-phospholipase C (PLC) activities of SMS1 were approximately 65% and 35% of total DG production, respectively. PC-PLC activity showed substrate selectivity for saturated and/or monounsaturated fatty acid-containing PC species. A PC-PLC/SMS inhibitor, D609, inhibited only SMS activity. Mn2+ inhibited only PC-PLC activity. Intriguingly, DG attenuated SMS/CPES activities. Our study indicates that SMS1 is a unique enzyme with PC-PLC/PE-PLC/SMS/CPES activities.

Diacylglycerol kinase ζ interacts with sphingomyelin synthase 1 and sphingomyelin synthase-related protein via different regions.

We previously reported that diacylglycerol (DG) kinase (DGK) δ interacts with DG-generating sphingomyelin synthase (SMS)-related protein (SMSr), but not SMS1 or SMS2, via their sterile α motif domains (SAMDs). However, it remains unclear whether other DGK isozymes interact with SMSs. Here, we found that DGKζ, which does not contain SAMD, interacts with SMSr and SMS1, but not SMS2. Deletion mutant analyses demonstrated that SAMD in the N-terminal cytosolic region of SMSr binds to the N-terminal half catalytic domain of DGKζ. However, the C-terminal cytosolic region of SMS1 interacts with the catalytic domain of DGKζ. Taken together, these results indicate that DGKζ associates with SMSr and SMS1 in different manners and suggest that they compose new DG signaling pathways.

Identification and characterization of diacylglycerol kinase ζ as a novel enzyme producing ceramide-1-phosphate.

Ceramide-1-phosphate (C1P) is a sphingolipid formed by the phosphorylation of ceramide; it regulates various physiological functions, including cell survival, proliferation, and inflammatory responses. In mammals, ceramide kinase (CerK) is the only C1P-producing enzyme currently known. However, it has been suggested that C1P is also produced by a CerK-independent pathway, although the identity of this CerK-independent C1P was unknown. Here, we identified human diacylglycerol kinase (DGK) ζ as a novel C1P-producing enzyme and demonstrated that DGKζ catalyzes the phosphorylation of ceramide to produce C1P. Analysis using fluorescently labeled ceramide (NBD-ceramide) demonstrated that only DGKζ among ten kinds of DGK isoforms increased C1P production by transient overexpression of the DGK isoforms. Furthermore, an enzyme activity assay using purified DGKζ revealed that DGKζ could directly phosphorylate ceramide to produce C1P. Furthermore, genetic deletion of DGKζ decreased the formation of NBD-C1P and the levels of endogenous C18:1/24:1- and C18:1/26:0-C1P. Interestingly, the levels of endogenous C18:1/26:0-C1P were not decreased by the knockout of CerK in the cells. These results suggest that DGKζ is also involved in the formation of C1P under physiological conditions.

Saturated fatty acid- and/or monounsaturated fatty acid-containing phosphatidic acids selectively interact with heat shock protein 27.

Diacylglycerol kinase (DGK) α, which is a key enzyme in the progression of cancer and, in contrast, in T-cell activity attenuation, preferentially produces saturated fatty acid (SFA)- and/or monounsaturated fatty acid (MUFA)-containing phosphatidic acids (PAs), such as 16:0/16:0-, 16:0/18:0-, and 16:1/16:1-PA, in melanoma cells. In the present study, we searched for the target proteins of 16:0/16:0-PA in melanoma cells and identified heat shock protein (HSP) 27, which acts as a molecular chaperone and contributes to cancer progression. HSP27 more strongly interacted with PA than other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate. Moreover, HSP27 is more preferentially bound to SFA- and/or MUFA-containing PAs, including 16:0/16:0- and 16:0/18:1-PAs, than PUFA-containing PAs, including 18:0/20:4- and 18:0/22:6-PA. Furthermore, HSP27 and constitutively active DGKα expressed in COS-7 cells colocalized in a DGK activity-dependent manner. Notably, 16:0/16:0-PA, but not phosphatidylcholine or 16:0/16:0-phosphatidylserine, induced oligomer dissociation of HSP27, which enhances its chaperone activity. Intriguingly, HSP27 protein was barely detectable in Jurkat T cells, while the protein band was intensely detected in AKI melanoma cells. Taken together, these results strongly suggest that SFA- and/or MUFA-containing PAs produced by DGKα selectively target HSP27 and regulate its cancer-progressive function in melanoma cells but not in T cells.

Gangrenous ischemic colitis localized to the cecum: a case report.

BACKGROUND: Ischemic colitis affects the left colon in elderly individuals and localization on the right side, especially in the cecum, is rare. We report a case of gangrenous ischemic colitis localized in the cecum of a patient undergoing hemodialysis. CASE PRESENTATION: A 73-year-old man had been undergoing hemodialysis for chronic renal failure caused by diabetic nephropathy. He experienced frequent vomiting, diarrhea, and abdominal pain. Contrast-enhanced computed tomography revealed thickening of the cecal wall, poor enhancement, dilation of the cecum, and intrahepatic portal emphysema. No obvious abnormal findings were observed in the appendix. The patient was diagnosed with cecal necrosis and ileocecal resection was performed. Histopathological examination revealed gangrenous ischemic colitis of the cecum. He was discharged 12 days after surgery without postoperative complications. CONCLUSION: It is important to consider the possibility of ischemic colitis of the right colon in the event of renal failure requiring dialysis, to ensure that opportunities for surgical intervention are not missed.

Distinct regions of Praja-1 E3 ubiquitin-protein ligase selectively bind to docosahexaenoic acid-containing phosphatidic acid and diacylglycerol kinase δ.

1-Stearoyl-2-docosahexaenoyl (18:0/22:6)-phosphatidic acid (PA) interacts with and activates Praja-1 E3 ubiquitin-protein ligase (full length: 615 aa) to ubiquitinate and degrade the serotonin transporter (SERT). SERT modulates serotonergic system activity and is a therapeutic target for depression, autism, obsessive-compulsive disorder, schizophrenia and Alzheimer's disease. Moreover, diacylglycerol kinase (DGK) δ2 (full length: 1214 aa) interacts with Praja-1 in addition to SERT and generates 18:0/22:6-PA, which binds and activates Praja-1. In the present study, we investigated the interaction of Praja-1 with 18:0/22:6-PA and DGKδ2 in more detail. We first found that the N-terminal one-third region (aa 1-224) of Praja-1 bound to 18:0/22:6-PA and that Lys141 in the region was critical for binding to 18:0/22:6-PA. In contrast, the C-terminal catalytic domain of Praja-1 (aa 446-615) interacted with DGKδ2. Additionally, the N-terminal half of the catalytic domain (aa 309-466) of DGKδ2 intensely bound to Praja-1. Moreover, the N-terminal region containing the pleckstrin homology and C1 domains (aa 1-308) and the C-terminal half of the catalytic domain (aa 762-939) of DGKδ2 weakly associated with Praja-1. Taken together, these results reveal new functions of the N-terminal (aa 1-224) and C-terminal (aa 446-615) regions of Praja-1 and the N-terminal half of the catalytic region (aa 309-466) of DGKδ2 as regulatory domains. Moreover, it is likely that the DGKδ2-Praja-1-SERT heterotrimer proximally arranges the 18:0/22:6-PA-producing catalytic domain of DGKδ2, the 18:0/22:6-PA-binding regulatory domain of Praja-1, the ubiquitin-protein ligase catalytic domain of Praja-1 and the ubiquitination acceptor site-containing SERT C-terminal region.

Quantification of the Swallowing Mechanism Through Muscle Synergy Analysis.

Decreased swallowing function increases the risk of choking and aspiration pneumonia. Videofluoroscopy and computed tomography allow for detailed observation of the swallowing movements but have radiation risks. Therefore, we developed a method using surface electromyography (sEMG) to noninvasively assess swallowing function without radiation exposure. A 44-channel flexible sEMG sensor was used to measure the sEMG signals of the hyoid muscles during swallowing in 14 healthy young adult and 14 elderly subjects. Muscle synergy analysis was performed to extract the muscle synergies from the sEMG signals, and the three synergies were extracted from the hyoid muscle activities during the swallowing experiments. The experimental results showed that the three synergies represent the oral, early pharyngeal, and late pharyngeal swallowing phases and that swallowing strength is tuned by the strength of the muscle activities, whereas swallowing volume is controlled by adjusting muscle activation timing. In addition, the timing of the swallowing reflex is slower in elderly individuals. The results confirm that the proposed approach successfully quantifies swallowing function from sEMG signals, mapping the signals to the swallowing phases.

A case of aggressive pituitary neuroendocrine tumour with extremely rapid progression: possible diagnostic value of TERT promoter methylation.

Most pituitary adenoma/neuroendocrine tumours (PitNET) are histologically benign and grow slowly; however, a subset of these tumours exhibit a more aggressive clinical course characterized by local invasiveness and early recurrence. These high-risk PitNETs often require multiple surgeries and radiation over several years and may eventually acquire carcinomatous characteristics, such as metastasis in some cases. Herein, we report a rare case of PitNET causing oculomotor paresis with extremely rapid recurrence only 3 months after initial surgery, followed by lethal liver metastasis. Preoperative magnetic resonance imaging and intraoperative findings were consistent with typical PitNETs, other than moderate invasion of the cavernous sinus. Pathological examination of the specimen obtained from the initial transsphenoidal surgery revealed increased mitosis and elevated rates of cells positive for Ki-67 and p53. Based on the immunohistochemical assessment for transcription factors and pituitary hormones, the diagnosis was determined to be a silent sparsely granulated corticotroph PitNET with focal malignant transformation. Aggressive features represented by Ki-67 and p53 positivity were more robust in recurrent and metastatic specimens, but hormone immunostaining was decreased. Epigenetic analysis revealed methylation of the telomerase reverse transcriptase (TERT) promoter in the tumour, resulting in TERT upregulation. Despite extensive research, markers for distinguishing extremely aggressive PitNETs have not been determined. Although further analysis is needed, our case demonstrates the possible usefulness of assessing TERT promoter methylation status in the stratification of recurrence risk in extremely high-risk variants of PitNET.

Phosphatidylinositol 4,5-bisphosphate-specific phospholipase C ß1 selectively binds dipalmitoyl and distearoyl phosphatidic acids via Lys946 and Lys951.

Phospholipase C (PLC) ß1 hydrolyzes 1-stearoyl-2-arachidonoyl (18:0/20:4)-phosphatidylinositol (PtdIns) 4,5-bisphosphate to produce diacylglycerol, which is converted to phosphatidic acid (PtdOH), in the PtdIns cycle and plays pivotal roles in intracellular signal transduction. The present study identified PLCß1 as a PtdOH-binding protein using PtdOH-containing liposomes. Moreover, the comparison of the binding of PLCß1 to various PtdOH species, including 14:0/14:0-PtdOH, 16:0/16:0-PtdOH, 16:0/18:1-PtdOH, 18:0/18:1-PtdOH, 18:0/18:0-PtdOH, 18:1/18:1-PtdOH, 18:0/20:4-PtdOH, and 18:0/22:6-PtdOH, indicated that the interaction of PLCß1 with 16:0/16:0-PtdOH was the strongest. The PLCß1-binding activity of 18:0/18:0-PtdOH was almost the same as the binding activity of 16:0/16:0-PtdOH. Furthermore, the binding of PLCß1 to 16:0/16:0-PtdOH was substantially stronger than 16:0/16:0-phosphatidylserine, 16:0/16:0/16:0/16:0-cardiolipin, 16:0/16:0-PtdIns, and 18:0/20:4-PtdIns. We revealed that a PLCß1 mutant whose Lys946 and Lys951 residues were replaced with Glu (PLCß1-KE) did not interact with 16:0/16:0-PtdOH and failed to localize to the plasma membrane in Neuro-2a cells. Retinoic acid-dependent increase in neurite length and numbers was significantly inhibited in PLCß1-expressing cells; however, this considerable attenuation was not detected in the cells expressing PLCß1-KE. Overall, these results strongly suggest that PtdOHs containing only saturated fatty acids, including 16:0/16:0-PtdOH, which are not derived from the PtdIns cycle, selectively bind to PLCß1 and regulate its function.

Diacylglycerol kinase η regulates cell proliferation and its levels are elevated by glucocorticoids in undifferentiated neuroblastoma cells.

Knockout mice of diacylglycerol kinase (DGK) η, which has been repeatedly suggested to be associated with bipolar disorder (BPD) by genome-wide association studies, exhibited abnormal behaviors similar to the manic phase of BPD. Chronic stress is also linked to changes in mood symptoms, including BPD. In the present study, we analyzed the effects of the glucocorticoid stress hormones, triamcinolone acetonide (TAA) and dexamethasone (DEX), on DGKη protein levels in neuroblastoma cell lines, Neuro-2a and SH-SY5Y. The protein levels of DGKη were significantly increased in the undifferentiated Neuro-2a and SH-SY5Y cells by TAA and DEX, but not in the differentiated neuroblastoma cells. To assess the functions of DGKη in undifferentiated neuroblastoma cells, we established DGKη-deficient SH-SY5Y cells using the clustered regularly interspaced palindromic repeat/caspase 9 system. Notably, proliferation of DGKη-deficient SH-SY5Y cells was markedly attenuated, concomitant with the decrease in levels of phosphorylated extracellular signal-regulated kinase. Taken together, these results suggest that DGKη levels are increased in undifferentiated neuroblastoma cells by glucocorticoid stress hormones and regulate cell proliferation.

Remimazolam decreased the incidence of early postoperative nausea and vomiting compared to desflurane after laparoscopic gynecological surgery.

PURPOSE: Postoperative nausea and vomiting (PONV) is a common adverse event after surgery. Remimazolam is a novel sedative agent recently approved for general anesthesia in Japan. This study evaluated the efficacy of remimazolam in the incidence of PONV after laparoscopic gynecological surgery under general anesthesia. METHODS: This prospective, randomized controlled trial included 64 women who underwent laparoscopic gynecological surgery. The patients were randomly assigned to undergo general anesthesia with either remimazolam (REM group) or desflurane (DES group, n = 30, each group). The primary outcome was the incidence of PONV in the two groups at 2 h and 24 h after the surgery. The incidence of vomiting, rescue antiemetic use, and severity of nausea were also evaluated. RESULTS: In the REM group, the incidence of PONV (27% versus 60%, respectively; P = 0.02), rescue antiemetic use (0 versus 7, respectively; P = 0.01), and nausea score (P = 0.01) were significantly decreased during the first 2 h after surgery. No parameters were significantly different 24 h after surgery between the two groups. CONCLUSION: Remimazolam can reduce the incidence of PONV after laparoscopic gynecological surgery compared to general anesthesia with desflurane during the early postoperative period.

Better method for detection of CD30: Immunohistochemistry or flow cytometry?

We compared the two methods of assessing CD30 protein expression in DLBCL and TCL specimens routinely employed at our hospital, immunohistochemistry (IHC) and flow cytometry (FCM), using the same clone of the anti-CD30 antibody (Ber-H2) in 123 patients with DLBCL and 28 patients with TCL. FCM was more sensitive than IHC, especially in cases with low expression. In three cases of TCL and two cases of DLBCL, there was discordance between these two methods. Two of these TCL cases were ALCL and one was peripheral T-cell lymphoma, NOS, but ALCL was unable to be excluded. One of two cases of DLBCL was an anaplastic variant of DLBCL. The data suggested that CD30 was undetectable, though rare, by FMC in several cases. Based on this study, a combination of IHC and FCM is recommended for the reliable and quantitative detection of CD30.

Diacylglycerol Kinase η Activity in Cells Using Protein Myristoylation and Cellular Phosphatidic Acid Sensor.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid (PtdOH) and regulates the balance between two lipid second messengers: diacylglycerol and PtdOH. Several lines of evidence suggest that the η isozyme of DGK is involved in the pathogenesis of bipolar disorder. However, the detailed molecular mechanisms regulating the pathophysiological functions remain unclear. One reason is that it is difficult to detect the cellular activity of DGKη. To overcome this difficulty, we utilized protein myristoylation and a cellular PtdOH sensor, the N-terminal region of α-synuclein (α-Syn-N). Although DGKη expressed in COS-7 cells was broadly distributed in the cytoplasm, myristoylated (Myr)-AcGFP-DGKη and Myr-AcGFP-DGKη-KD (inactive (kinase-dead) mutant) were substantially localized in the plasma membrane. Moreover, DsRed monomer-α-Syn-N significantly colocalized with Myr-AcGFP-DGKη but not Myr-AcGFP-DGKη-KD at the plasma membrane. When COS-7 cells were osmotically shocked, all DGKη constructs were exclusively translocated to osmotic shock-responsive granules (OSRG). DsRed monomer-α-Syn-N markedly colocalized with only Myr-AcGFP-DGKη at OSRG and exhibited a higher signal/background ratio (3.4) than Myr-AcGFP-DGKη at the plasma membrane in unstimulated COS-7 cells (2.5), indicating that α-Syn-N more effectively detects Myr-AcGFP-DGKη activity in OSRG. Therefore, these results demonstrated that the combination of myristoylation and the PtdOH sensor effectively detects DGKη activity in cells and that this method is convenient to examine the molecular functions of DGKη. Moreover, this method will be useful for the development of drugs targeting DGKη. Furthermore, the combination of myristoylation (intensive accumulation in membranes) and α-Syn-N can be applicable to assays for various cytosolic PtdOH-generating enzymes.

Sphingomyelin synthase-related protein generates diacylglycerol via the hydrolysis of glycerophospholipids in the absence of ceramide.

Diacylglycerol (DG) is a well-established lipid second messenger. Sphingomyelin synthase (SMS)-related protein (SMSr) produces DG and ceramide phosphoethanolamine (CPE) by the transfer of phosphoethanolamine from phosphatidylethanolamine (PE) to ceramide. We previously reported that human SMSr overexpressed in COS-7 cells significantly increased DG levels, particularly saturated and/or monounsaturated fatty acid-containing DG molecular species, and provided DG to DG kinase (DGK) δ, which regulates various pathophysiological events, including epidermal growth factor-dependent cell proliferation, type 2 diabetes, and obsessive-compulsive disorder. However, mammalian SMSr puzzlingly produces only trace amounts of CPE/DG. To clarify this discrepancy, we highly purified SMSr and examined its activities other than CPE synthase. Intriguingly, purified SMSr showed a DG-generating activity via hydrolysis of PE, phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylcholine (PC) in the absence of ceramide. DG generation through the PA phosphatase (PAP) activity of SMSr was approximately 300-fold higher than that with PE and ceramide. SMSr hydrolyzed PI ten times stronger than PI(4,5)bisphosphate (PI(4,5)P2). The PAP and PC-phospholipase C (PLC) activities of SMSr were inhibited by propranolol, a PAP inhibitor, and by D609, an SMS/PC-PLC inhibitor. Moreover, SMSr showed substrate selectivity for saturated and/or monounsaturated fatty acid-containing PA molecular species, but not arachidonic-acid-containing PA, which is exclusively generated in the PI(4,5)P2 cycle. We confirmed that SMSr expressed in COS-7 cells showed PAP and PI-PLC activities. Taken together, our study indicated that SMSr possesses previously unrecognized enzyme activities, PAP and PI/PE/PC-PLC, and constitutes a novel DG/PA signaling pathway together with DGKδ, which is independent of the PI(4,5)P2 cycle.

CNS Low-grade Diffusely Infiltrative Tumors With INI1 Deficiency, Possessing a High Propensity to Progress to Secondary INI1-deficient Rhabdoid Tumors.

Atypical teratoid/rhabdoid tumors (AT/RTs) are highly malignant tumors of the central nervous system that predominantly occur in infants, and are characterized by the presence of rhabdoid cells and inactivation of INI1 or (rarely) BRG1. Most AT/RT are identified as primary tumors; however, rare AT/RT or INI1-deficient RTs arising from other primary tumors have been reported. Here, we report 3 cases of hitherto unclassifiable low-grade tumors with loss of INI1 nuclear expression, for which we propose the designation of central nervous system low-grade diffusely infiltrative tumors with INI1 deficiency (CNS LGDIT-INI1), 2 of which progressed to secondary RT. All 3 CNS LGDIT-INI1 exhibited a similar histology: diffusely distributed small tumor cells with round to oval or irregular nuclei and scant cytoplasm were admixed with degenerative neurons and large reactive astrocytes in an edematous, myxoid, or collagenous background. Mitotic figures were absent. Immunohistochemistry revealed that the tumor cells in all 3 CNS LGDIT-INI1 and 2 RT were negative for INI1. Genetically, total or partial homozygous deletions of the INI1 gene were detected in all CNS LGDIT-INI1 and RT excluding 1 CNS LGDIT-INI1 without sufficient DNA quality and quantity. Despite the loss of INI1 expression, these low-grade lesions were clearly distinguishable from AT/RT by their low proliferative activity, diffusely infiltrative growth pattern, and lack of rhabdoid cells and polyphenotypic immunoreactivity. In conclusion, CNS LGDIT-INI1 may represent a rare group of tumors that are clinically indolent but have a high propensity to progress to RT.