Transcriptional regulation of acetoacetyl-CoA synthetase by Sp1 in neuroblastoma cells.

Acetoacetyl-CoA synthetase (AACS) is the enzyme responsible for cholesterol and fatty acid synthesis in the cytosol. We have previously shown that AACS has an important role in normal neuronal development and that knockdown of SREBP-2, which orchestrates cholesterol synthesis, resulted in the downregulation of AACS mRNA levels. In this study, we investigated the transcriptional mechanism of AACS in Neuro-2a, neuroblastoma cells. Luciferase assay showed that the minimal core promoter of the mouse AACS gene is located in a region with 110 bps upstream from the transcription start site. Mutagenesis studies showed that the Sp1 binding site was crucial for AACS promoter activity. ChIP assay and DNA affinity precipitation assay showed that Sp1 binds to the Sp1 binding site on the promoter region of AACS. Moreover, overexpression of Sp1 increased AACS mRNA levels. Knockdown of AACS resulted in a decrease in histone deacetylase 9, associated with gene silencing. These results suggest that Sp1 regulates gene expression of AACS in Neuro-2a cells and ketone body utilization affects the balance of histone acetylation.

Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells.

It has recently been recognized that inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), upregulate the secretion of matrix metalloproteinase-9 (MMP-9) from cancer cells and thereby promote peritoneal dissemination. In this study, we found that TNF-α also stimulated peritoneal mesothelial cells to secrete MMP-9 as assessed by zymography. MMP-9 gene expression in mesothelial cells induced by TNF-α was confirmed by quantitative RT-PCR analysis. We then utilized the reconstituted artificial mesothelium, which was composed of a monolayer of mesothelial cells cultured on a Matrigel layer in a Boyden chamber system, to examine the effects of TNF-α on carcinoma cell invasion. The transmigration of MKN1 human gastric carcinoma cells through the reconstituted mesothelium was promoted by TNF-α in a dose-dependent manner. The increased MKN1 cell migration was partially inhibited by the anti-α3 integrin antibody, indicating that the invasion process involves an integrin-dependent mechanism. Finally, we observed that the invasion of MMP-9-knockdown MKN1 cells into Matrigel membranes was potentiated by the exogenous addition of purified proMMP-9. These results suggest that TNF-α-induced MMP-9 secretion from mesothelial cells plays an important role in the metastatic dissemination of gastric cancer.

High-fat diet-induced obesity stimulates ketone body utilization in osteoclasts of the mouse bone.

Previous studies have shown that high-fat diet (HFD)-induced obesity increases the acetoacetyl-CoA synthetase (AACS) gene expression in lipogenic tissue. To investigate the effect of obesity on the AACS gene in other tissues, we examined the alteration of AACS mRNA levels in HFD-fed mice. In situ hybridization revealed that AACS was observed in several regions of the embryo, including the backbone region (especially in the somite), and in the epiphysis of the adult femur. AACS mRNA expression in the adult femur was higher in HFD-fed mice than in normal-diet fed mice, but this increase was not observed in high sucrose diet (HSD)-induced obese mice. In addition, HFD-specific increases were observed in the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and interleukin (IL)-6 genes. Moreover, we detected higher AACS mRNA expression in the differentiated osteoclast cells (RAW 264), and found that AACS mRNA expression was significantly up-regulated by IL-6 treatment only in osteoclasts. These results indicate the novel function of the ketone body in bone metabolism. Because the abnormal activation of osteoclasts by IL-6 induces bone resorption, our data suggest that AACS and ketone bodies are important factors in the relationship between obesity and osteoporosis.

Degradation of acetoacetyl-CoA synthetase, a ketone body-utilizing enzyme, by legumain in the mouse kidney.

Acetoacetyl-CoA synthetase (AACS) is a ketone body-utilizing enzyme, which is responsible for the synthesis of cholesterol and fatty acids from ketone bodies in lipogenic tissues, such as the liver and adipocytes. To explore the possibility of AACS regulation at the protein-processing level, we investigated the proteolytic degradation of AACS. Western blot analysis showed that the 75.1kDa AACS was cleaved to form a protein of approximately 55kDa in the kidney, which has considerable high activity of legumain, a lysosomal asparaginyl endopeptidase. Co-expression of AACS and legumain in HEK 293 cells generated the 55kDa product from AACS. Moreover, incubation of recombinant AACS with recombinant legumain resulted in the degradation of AACS. Knockdown of legumain with short-hairpin RNA against legumain using the hydrodynamics method led to a decrease in the 55kDa band of AACS in mouse kidney. These results suggest that legumain is involved in the processing of AACS through the lysosomal degradation pathway in the kidney.

Acetoacetyl-CoA synthetase is essential for normal neuronal development.

Cholesterol and fatty acids are essential, abundant components of neuronal tissue. Acetoacetyl-CoA synthetase (AACS) is a ketone body-utilizing enzyme for the synthesis of cholesterol and fatty acids and is highly expressed in the brain. In this study, we investigated the regulation of AACS during neurite outgrowth to clarify the physiological role of AACS in neurogenesis. Messenger RNA levels and the expression of AACS were increased during neurite outgrowth in Neuro-2a cells. The expression of HMG-CoA reductase, a key enzyme of cholesterol biosynthesis, was also increased. ChIP assays showed that the amount of SREBP-2, a key transcription factor of cholesterol synthesis, interacted with the AACS promoter was increased during neurite outgrowth, and knockdown of SREBP-2 down-regulated the mRNA levels of AACS in Neuro-2a cells. The expression of AACS in the brains of mouse embryos was dramatically increased between E16.5 and E18.5. Moreover, knockdown of AACS in primary neurons caused decreases in the expression of MAP-2 and NeuN, which are markers of neuronal differentiation, as well as synaptopodin, a marker of spine apparatus. These results suggest that AACS is regulated by SREBP-2 and involves in the normal development of neurons.

Acetoacetyl-CoA synthetase, a ketone body-utilizing enzyme, is controlled by SREBP-2 and affects serum cholesterol levels.

Ketone bodies have been regarded as an energy source that is mainly produced in the liver, and exported to extrahepatic tissues. However, ketone bodies have also been suggested to be used during the lipogenesis by the ketone body-utilizing enzyme, acetoacetyl-CoA synthetase (AACS). To elucidate the physiological role of AACS in the liver, we investigated the mechanism of transcription of the AACS gene and performed knockdown experiments. We showed that SREBP-2 regulates the expression of AACS and that knockdown of AACS in vivo, by the hydrodynamics method, resulted in the reduction of total blood cholesterol. These results suggest that ketone body metabolism via AACS activity plays an important role in cholesterol homeostasis.

The role of acetoacetyl-CoA synthetase, a ketone body-utilizing enzyme, in 3T3-L1 adipocyte differentiation.

Acetoacetyl-CoA synthetase (AACS) is a ketone body-utilizing enzyme that converts acetoacetate to acetoacetyl-CoA in the cytosol and consequently provides acetyl units as the precursors for lipogenesis. To clarify the role of AACS in adipogenesis, we investigated the expression and localization of the AACS protein and the effect of AACS knockdown on 3T3-L1 differentiation. The protein expression of AACS is dramatically induced during 3T3-L1 differentiation and is localized in the cytoplasm of differentiated 3T3-L1 cells. Moreover, knockdown of AACS inhibits differentiation of 3T3-L1 cells and suppresses expression of the adipocyte markers, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα). These results suggest that AACS has a crucial role in the mechanism of 3T3-L1 differentiation.

An extremely rare case of anterior mediastinal leiomyosarcoma: a case report.

We herein report an extremely rare case of leiomyosarcoma found in the anterior mediastinum. A 79-year-old man presented to our hospital with an anterior mediastinal mass incidentally found by chest computed tomography (CT) scan. Percutaneous needle biopsy revealed the presence of an undifferentiated sarcoma. Transsternal resection of the tumor with adjacent left mediastinal pleura was performed, and pathological analysis revealed a leiomyosarcoma, which was 11 cm in diameter, with bare margins. He was followed up on an outpatient basis with no adjuvant therapy. Although mediastinal lymph node recurrence was suspected on chest CT scan 18 months after surgery, the patient remained asymptomatic and rejected any additional antitumor treatments. He died of respiratory failure after incidental traumatic spinal injury about 30 months after tumor resection.

Combination of RET siRNA and irinotecan inhibited the growth of medullary thyroid carcinoma TT cells and xenografts via apoptosis.

Medullary thyroid carcinoma (MTC) is a rare endocrine tumor that frequently metastasizes, and treatment with irinotecan (CPT-11) is limited because of side effects. Mutations in the Rearranged during transfection (RET) proto-oncogene are considered the causative event of MTC. The objective of this study was to examine whether small interfering RNA (siRNA) and its combined treatment with CPT-11 could inhibit MTC cell growth in vitro and in vivo. The transfection of RET siRNA suppressed RET expression, reduced proliferation, and increased caspase-3/7 activity via the down-regulation of Bcl-2 expression. Combined treatments with CPT-11 or SN-38 significantly increased caspase 3/7 activity compared with RET siRNA, CPT-11 or SN-38 treatment alone. Importantly, intratumoral injection of RET siRNA along with intravenous injection of CPT-11 significantly inhibited the tumor growth of MTC xenografts via an increased apoptotic effect. These findings that RET siRNA enhanced sensitivity for CPT-11 will provide a novel strategy for the treatment of MTC with RET mutation.

A fourth scale sensitive to the magnetic field; intermolecular frequency symmetry in a specific interaction between protein and low-molecular compound.

We found a new method that a specific interaction between prion, i.e., high-molecular compound, and Cp-60, i.e., low-molecular one, could be successfully elucidated with intermolecular frequency symmetry (IFS). To accomplish this, the former sequence is analyzed with a sequence Fourier analysis used average nuclear (N) resonant frequency scale as a fourth one, and the latter structure with a ¹³C-NMR software. Further, such the symmetry could be observed in a specific interaction between a segment of human immunodeficiency virus (HIV)gag and PA-457 or between 1918 neuraminidase and peramivir. Therefore, the IFS rule seems to be evolutionarily conserved as a necessary condition even in a specific protein-organic compound interaction.

Thoracoscopic surgery under local anesthesia for high-risk intractable secondary spontaneous pneumothorax.

OBJECTIVES: To evaluate the outcomes of thoracoscopic surgery for intractable secondary spontaneous pneumothorax (SSP) under local anesthesia in high-risk patients and report intraoperative findings useful for identifying air leakage points. METHODS: We analyzed outcomes of 14 consecutive thoracoscopic operations under local anesthesia for high-risk SSP from 2015 to 2019. Suspicious lesions were determined based on intraoperative direct or indirect detections. Direct detection involved identifying pleural fistulas or air bubbles. Indirect detection involved finding thin and transparent bullae without any other suspicious lesions. Identifications of culprit lesions were confirmed by arrest or significant decrease in air leakage after surgical repair. All surgical repairs were followed by immediate single pleurodesis for a definitive cure and prevention of recurrence. Success was defined as the removal of the thoracic tube by surgical repair combined with immediate postoperative single pleurodesis. RESULTS: The main underlying pulmonary diseases were emphysema (n = 7), carcinoma (n = 3), interstitial pneumonia (IP) (n = 3), and nontuberculous mycobacterial infection (n = 1). A leakage point was identified in 13 cases (six on direct and seven on indirect detections). Success was achieved in nine cases (four on direct and five on indirect detections). Adverse events included one case of acute exacerbation of IP and one case of carbon dioxide narcosis. CONCLUSION: Thoracoscopic surgery under local anesthesia can be the worthwhile definitive modality, among few remaining treatments, for highly fragile patients with SSP. Detecting air leakage directly and the presence of thin and transparent bullae without any other suspicious lesions can be clues for identifying culprit lesions.

Transcriptional regulation of ketone body-utilizing enzyme, acetoacetyl-CoA synthetase, by C/EBPalpha during adipocyte differentiation.

Acetoacetyl-CoA synthetase (AACS), an essential enzyme for the synthesis of fatty acid and cholesterol from ketone bodies, was found to be highly expressed in mouse adipose tissue, and GC box and C/EBPs motif were crucial for AACS promoter activity in 3T3-L1 adipocytes. Moreover, we found that AACS promoter activity was controlled mainly by C/EBPalpha during adipogenesis.

Immunotherapy for advanced lung cancer combined with surgery for mediastinal myxofibrosarcoma: a case report.

BACKGROUND: It is unclear whether simultaneous primary neoplasm resection and immunotherapy for advanced lung cancer is safe. We report a case of an elderly man with advanced lung cancer and myxofibrosarcoma. CASE PRESENTATION: The advanced lung cancer was treated with pembrolizumab, and partial response was achieved in 3 months. However, the mediastinal cyst enlarged rapidly. We resected the mediastinal tumor and diagnosed it as myxofibrosarcoma. The postoperative course was uneventful. Immunotherapy was resumed after the operation without any adverse effects. No recurrence of mediastinal sarcoma or progression of lung cancer was found until the patient died in an accident 8 months after surgery. CONCLUSION: Surgery for mediastinal sarcoma could be performed safely in combination with immunotherapy for advanced lung cancer.

Simple chest closure of open window thoracostomy for postpneumonectomy empyema: a case report.

BACKGROUND: Management of postpneumonectomy empyema requires comprehensive strategies, especially when the condition is associated with large bronchopleural fistulae. We report a case involving the simple chest closure of open window thoracostomy with remaining residual space. CASE PRESENTATION: We performed open window thoracostomy for empyema with a huge bronchial stump dehiscence after right pneumonectomy for a large lung cancer. We definitively closed the chest window infected with chronic persistent Pseudomonas aeruginosa via a simple chest closure technique with the remaining residual space, after repairing the bronchial dehiscence using an omental flap and the appearance of healthy granulation tissue throughout the cavity. The patient died of recurrent cancer 10 months after the definitive chest closure. Until the patient died, there were no symptoms or signs suggestive of recurrent empyema. CONCLUSION: This simple chest closure technique allows "silent empyema" to be observed carefully, is less invasive, and can even be applied to cases of recurrent cancer.

Different localization in rat brain of the novel cytosolic ketone body-utilizing enzyme, acetoacetyl-CoA synthetase, as compared to succinyl-CoA:3-oxoacid CoA-transferase.

In lipogenic tissue cytosol, ketone bodies are known to be activated by acetoacetyl-CoA synthetase (AACS) and incorporated into cholesterol and fatty acids. In order to investigate the physiological role of AACS in the brain, we examined the localization of AACS mRNA in rat brain by in situ hybridization using a labeled probe. High labeling was observed in the midbrain, pons/medulla, cerebral cortex, hippocampus and cerebellum, and the localization profile of AACS mRNA was different from that of succinyl-CoA:3-oxoacid CoA-transferase (SCOT), a mitochondrial ketone body-activating enzyme. In addition, the expression of AACS mRNA in the cerebellum was restricted primarily to glial cells, while in the cerebral cortex, it was restricted to neuronal cells. Streptozotocin treatment caused remarkable decreases in AACS mRNA levels in all regions where expression was observed, but changes in SCOT mRNA levels were not observed. These results suggest that the physiological role of AACS is different from that of SCOT and varies depending upon its localization in the brain.

Site-specific cleavage of acetoacetyl-CoA synthetase by legumain.

Acetoacetyl-CoA synthetase (AACS) is a ketone body-utilizing enzyme and is responsible for the synthesis of cholesterol and fatty acids. We have previously shown that AACS is cleaved by legumain, a lysosomal asparaginyl endopeptidase. In this study, we attempted to determine the cleavage site of AACS. Mutagenesis analysis of AACS revealed that Asn547 is the specific cleavage site of AACS in mouse livers. The cleaved form of AACS (1-547) lost the ability to convert acetoacetate to acetoacetyl-CoA. Moreover, hydrodynamics-based gene transduction showed that overexpression of AACS (1-547) increases the protein expression of caveolin-1, the principal component of the caveolae. These results suggest that cleavage of AACS by legumain is critical for the regulation of enzymatic activity and results in gain-of-function changes.

Placental extracts induce the expression of antioxidant enzyme genes and suppress melanogenesis in B16 melanoma cells.

One of the activities of placental extracts (PEs) is skin-whitening effect, but the physiological and genetic mechanism for this effect has not yet been clarified. Here, we focus on PE as a regulator of antioxidant enzyme genes. Porcine PE was prepared, and its activity was investigated in B16 melanoma cells. PE treatment decreased the melanin content of UV-irradiated B16 cells in a dose-dependent manner. PE directly reduced the enzyme activity of tyrosinase in a cell-free assay. In addition, PE treatment increased the gene expression of cytosolic superoxide dismutase (SOD-1), extracellular SOD (SOD-3) and catalase but did not affect the expression of tyrosinase. Moreover, PE protected the B16 cells from H2O2-induced cell death. Taken together, our data suggest that PEs could play a role not only as a suppressor of melanin synthesis but also as a regulator of antioxidant genes and might protect the skin against oxidative stress.

Expression of acetoacetyl-CoA synthetase, a novel cytosolic ketone body-utilizing enzyme, in human brain.

Acetoacetyl-CoA synthetase (AACS, acetoacetate-CoA ligase, EC 6.2.1.16) is a ketone body-utilizing enzyme, the physiological role of which remains unclear yet in mammals, particularly has never been studied in human. In order to investigate the tissue distribution of AACS in human, cDNA encoding AACS was isolated from HepG2 cells. Amino acid sequence of human AACS deduced from the open reading frame showed high homology (89.3%) with that of rat AACS and much less homology (43.7%) with that of bacterial AACS. The expression level of the AACS mRNA was high in kidney, heart and brain, but low in liver, and the expression profile of AACS in the human brain was quite similar to that of 3-hydroxy-3-methylglutaryl-CoA reductase.

Effects of streptozotocin-induced diabetes on acetoacetyl-CoA synthetase activity in rats.

In order to investigate the physiological role of acetoacetyl-CoA synthetase (acetoacetate-CoA ligase, EC 6.2.1.16), a cytosolic acetoacetate-activating enzyme, effects of streptozotocin (STZ)-induced diabetes on the enzyme activity was investigated in rats. At 72 hr of the STZ administration (80 mg/kg body weight, injected intravenously), hepatic enzyme specific activity decreased to 23% of its initial activity. However, the enzyme activities in non-hepatic tissues were not significantly affected by the STZ treatment. Feeding of rats with both 4% cholestyramine and 0.4% pravastatin for 3 days remarkably increased the hepatic acetoacetyl-CoA synthetase activity and decreased the plasma ketone bodies level in the diabetic rats. These results suggest that acetoacetyl-CoA synthetase has important roles in the regulation of ketone body utilization in rat liver and that these hypocholesterolemic agents have the ability to remedy the impaired utilization of ketone bodies under the diabetic condition.

Antioxidative collagen-derived peptides in human-placenta extract.

Human-placenta extract (PLx), has been widely used in clinical and cosmetic fields, and is known to possess marked antioxidant activity. We have attempted to identify the antioxidative components in PLx. Initially, we purified PLx using Sephadex G-10 column. The first eluted peak which had optical density at 280nm and exhibited approximately 20 per cent of antioxidant activity of the applied PLx, did not contain uracil, tyrosine, phenylalanine, or tryptophan which are the previously identified antioxidants from PLx. This fraction was further purified by reverse-phase high performance liquid chromatography. All eluted peaks containing antioxidant activity exhibited optical density at 280nm. Six separate fractions of eluent having antioxidant activity were analyzed by an amino acid sequencer, and each turned out to contain Glycine(G)-XY amino acid repeats, which appear to be derived from collagen. These results suggest that peptides produced from collagen are also antioxidative components of PLx.