Peptides Derived from Soybean ß-Conglycinin Induce the Migration of Human Peripheral Polymorphonuclear Leukocytes.

Food-derived peptides have various biological activities. When food proteins are ingested orally, they are digested into peptides by endogenous digestive enzymes and absorbed by the immune cell-rich intestinal tract. However, little is known about the effects of food-derived peptides on the motility of human immune cells. In this study, we aimed to understand the effects of peptides derived from a soybean protein ß-conglycinin on the motility of human peripheral polymorphonuclear leukocytes. We illustrated that MITL and MITLAIPVNKPGR, produced by digestion using in-vivo enzymes (trypsin and pancreatic elastase) of ß-conglycinin, induces the migration of dibutyryl cAMP (Bt2 cAMP)-differentiated human promyelocytic leukemia 60 (HL-60) cells and human polymorphonuclear leukocytes in a dose- and time-dependent manner. This migration was more pronounced in Bt2 cAMP-differentiated HL-60 cells; mRNA expression of formyl peptide receptor (FPR) 1 increased significantly than in all-trans-retinoic acid (ATRA)-differentiated HL-60 cells. This migration was inhibited by tert-butoxycarbonyl (Boc)-MLP, an inhibitor of FPR, and by pretreatment with pertussis toxin (PTX). However, the effect was weak when treated with WRW4, a selective inhibitor of the FPR2. We then demonstrated that MITLAIPVNKPGR induced intracellular calcium responses in human polymorphonuclear leukocytes and Bt2 cAMP-HL60 cells. Furthermore, pre-treatment by fMLP desensitized the calcium response of MITLAIPVNKPGR in these cells. From the above, MITLAIPVNKPGR and MITL derived from soybean ß-conglycinin induced polymorphonuclear leukocyte migration via the FPR1-dependent mechanism. We found chemotactic peptides to human polymorphonuclear leukocytes, which are the endogenous enzyme digests of soybean protein.

Method for Preparing Recombinant Galectin-2 Protein without Escherichia coli-Specific Post-translational Modifications.

Galectin-2 (Gal-2) is an animal lectin with specificity for ß-galactosides. It is predominantly expressed and suggested to play a protective function in the gastrointestinal tract; therefore, it can be used as a protein drug. Recombinant proteins have been expressed using Escherichia coli and used to study the function of Gal-2. The recombinant human Gal-2 (hGal-2) protein purified via affinity chromatography after being expressed in E. coli was not completely homogeneous. Mass spectrometry confirmed that some recombinant Gal-2 were phosphogluconoylated. In contrast, the recombinant mouse Gal-2 (mGal-2) protein purified using affinity chromatography after being expressed in E. coli contained a different form of Gal-2 with a larger molecular weight. This was due to mistranslating the original mGal-2 stop codon TGA to tryptophan (TGG). In this report, to obtain a homogeneous Gal-2 protein for further studies, we attempted the following methods: for hGal-2, 1) replacement of the lysine (Lys) residues, which was easily phosphogluconoylated with arginine (Arg) residues, and 2) addition of histidine (His)-tag on the N-terminus of the recombinant protein and cleavage with protease after expression; for mGal-2, 3) changing the stop codon from TGA to TAA, which is commonly used in E. coli. We obtained an almost homogeneous recombinant Gal-2 protein (human and mouse). These results have important implications for using Gal-2 as a protein drug.

Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization.

Phospholipase A1 (PLA1) hydrolyzes the fatty acids of glycerophospholipids, which are structural components of the cellular membrane. Genetic mutations in DDHD1, an intracellular PLA1, result in hereditary spastic paraplegia (HSP) in humans. However, the regulation of DDHD1 activity has not yet been elucidated in detail. In the present study, we examined the phosphorylation of DDHD1 and identified the responsible protein kinases. We performed MALDI-TOF MS/MS analysis and Phos-tag SDS-PAGE in alanine-substitution mutants in HEK293 cells and revealed multiple phosphorylation sites in human DDHD1, primarily Ser8, Ser11, Ser723, and Ser727. The treatment of cells with a protein phosphatase inhibitor induced the hyperphosphorylation of DDHD1, suggesting that multisite phosphorylation occurred not only at these major, but also at minor sites. Site-specific kinase-substrate prediction algorithms and in vitro kinase analyses indicated that cyclin-dependent kinase CDK1/cyclin A2 phosphorylated Ser8, Ser11, and Ser727 in DDHD1 with a preference for Ser11 and that CDK5/p35 also phosphorylated Ser11 and Ser727 with a preference for Ser11. In addition, casein kinase CK2α1 was found to phosphorylate Ser104, although this was not a major phosphorylation site in cultivated HEK293 cells. The evaluation of the effects of phosphorylation revealed that the phosphorylation mimic mutants S11/727E exhibit only 20% reduction in PLA1 activity. However, the phosphorylation mimics were mainly localized to focal adhesions, whereas the phosphorylation-resistant mutants S11/727A were not. This suggested that phosphorylation alters the subcellular localization of DDHD1 without greatly affecting its PLA1 activity.

Lysophosphatidylinositol Induced Morphological Changes and Stress Fiber Formation through the GPR55-RhoA-ROCK Pathway.

We previously reported that lysophosphatidylinositol (LPI) functions as an endogenous agonist of GPR55, a novel cannabinoid receptor. However, the physiological roles of LPI-GPR55 have not yet been elucidated in detail. In the present study, we found that LPI induced morphological changes in GPR55-expressing HEK293 cells. LPI induced the cell rounding of GPR55-expressing HEK293 cells but not of empty-vector-transfected cells. LPI also induced the activation of small GTP-binding protein RhoA and increased stress fiber formation in GPR55-expressing HEK293 cells. The inhibition of RhoA and Rho kinase ROCK by the C3 exoenzyme and the ROCK inhibitor reduced LPI-induced cell rounding and stress fiber formation. These results clearly indicated that the LPI-induced morphological changes and the assembly of the cytoskeletons were mediated through the GPR55-RhoA-ROCK pathway.

Potential Interaction between Galectin-2 and MUC5AC in Mouse Gastric Mucus.

Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. Of these, galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract. In the current study, we used a mouse gastric mucous fraction to investigate whether Gal-2 is secreted from epithelial cells and identify its potential ligands in gastric mucus. Gal-2 was detected in the mouse gastric mucous fraction and could be eluted from it by the addition of lactose. Affinity chromatography using recombinant mouse galectin-2 (mGal-2)-immobilized adsorbent and subsequent LC-MS/MS identified MUC5AC, one of the major gastric mucin glycoproteins, as a potential ligand of mGal-2. Furthermore, MUC5AC was detected in the mouse gastric mucous fraction by Western blotting, and recombinant mGal-2 was adsorbed to this fraction in a carbohydrate-dependent manner. These results suggested that Gal-2 and MUC5AC in mouse gastric mucus interact in a ß-galactoside-dependent manner, resulting in a stronger barrier structure protecting the mucosal surface.

Complex formation of sphingomyelin synthase 1 with glucosylceramide synthase increases sphingomyelin and decreases glucosylceramide levels.

Sphingolipids, including sphingomyelin (SM) and glucosylceramide (GlcCer), are generated by the addition of a polar head group to ceramide (Cer). Sphingomyelin synthase 1 (SMS1) and glucosylceramide synthase (GCS) are key enzymes that catalyze the conversion of Cer to SM and GlcCer, respectively. GlcCer synthesis has been postulated to occur mainly in cis-Golgi, and SM synthesis is thought to occur in medial/trans-Golgi; however, SMS1 and GCS are known to partially co-localize in cisternae, especially in medial/trans-Golgi. Here, we report that SMS1 and GCS can form a heteromeric complex, in which the N terminus of SMS1 and the C terminus of GCS are in close proximity. Deletion of the N-terminal sterile α-motif of SMS1 reduced the stability of the SMS1-GCS complex, resulting in a significant reduction in SM synthesis in vivo In contrast, chemical-induced heterodimerization augmented SMS1 activity, depending on an increase in the amount and stability of the complex. Fusion of the SMS1 N terminus to the GCS C terminus via linkers of different lengths increased SM synthesis and decreased GlcCer synthesis in vivo These results suggest that formation of the SMS1-GCS heteromeric complex increases SM synthesis and decreases GlcCer synthesis. Importantly, this regulation of relative Cer levels by the SMS1-GCS complex was confirmed by CRISPR/Cas9-mediated knockout of SMS1 or GCS combined with pharmacological inhibition of Cer transport protein in HEK293T cells. Our findings suggest that complex formation between SMS1 and GCS is part of a critical mechanism controlling the metabolic fate of Cer in the Golgi.

Carboxyl-terminal Tail-mediated Homodimerizations of Sphingomyelin Synthases Are Responsible for Efficient Export from the Endoplasmic Reticulum.

Sphingomyelin synthase (SMS) is the key enzyme for cross-talk between bioactive sphingolipids and glycerolipids. In mammals, SMS consists of two isoforms: SMS1 is localized in the Golgi apparatus, whereas SMS2 is localized in both the Golgi and plasma membranes. SMS2 seems to exert cellular functions through protein-protein interactions; however, the existence and functions of quaternary structures of SMS1 and SMS2 remain unclear. Here we demonstrate that both SMS1 and SMS2 form homodimers. The SMSs have six membrane-spanning domains, and the N and C termini of both proteins face the cytosolic side of the Golgi apparatus. Chemical cross-linking and bimolecular fluorescence complementation revealed that the N- and/or C-terminal tails of the SMSs were in close proximity to those of the other SMS in the homodimer. Homodimer formation was significantly decreased by C-terminal truncations, SMS1-ΔC22 and SMS2-ΔC30, indicating that the C-terminal tails of the SMSs are primarily responsible for homodimer formation. Moreover, immunoprecipitation using deletion mutants revealed that the C-terminal tail of SMS2 mainly interacted with the C-terminal tail of its homodimer partner, whereas the C-terminal tail of SMS1 mainly interacted with a site other than the C-terminal tail of its homodimer partner. Interestingly, homodimer formation occurred in the endoplasmic reticulum (ER) membrane before trafficking to the Golgi apparatus. Reduced homodimerization caused by C-terminal truncations of SMSs significantly reduced ER-to-Golgi transport. Our findings suggest that the C-terminal tails of SMSs are involved in homodimer formation, which is required for efficient transport from the ER.

Sphingomyelin synthase 2, but not sphingomyelin synthase 1, is involved in HIV-1 envelope-mediated membrane fusion.

Membrane fusion between the viral envelope and plasma membranes of target cells has previously been correlated with HIV-1 infection. Lipids in the plasma membrane, including sphingomyelin, may be crucially involved in HIV-1 infection; however, the role of lipid-metabolic enzymes in membrane fusion remains unclear. In this study, we examined the roles of sphingomyelin synthase (SMS) in HIV-1 Env-mediated membrane fusion using a cell-cell fusion assay with HIV-1 mimetics and their target cells. We employed reconstituted cells as target cells that stably express Sms1 or Sms2 in Sms-deficient cells. Fusion susceptibility was ∼5-fold higher in Sms2-expressing cells (not in Sms1-expressing cells) than in Sms-deficient cells. The enhancement of fusion susceptibility observed in Sms2-expressing cells was reversed and reduced by Sms2 knockdown. We also found that catalytically nonactive Sms2 promoted membrane fusion susceptibility. Moreover, SMS2 co-localized and was constitutively associated with the HIV receptor·co-receptor complex in the plasma membrane. In addition, HIV-1 Env treatment resulted in a transient increase in nonreceptor tyrosine kinase (Pyk2) phosphorylation in Sms2-expressing and catalytically nonactive Sms2-expressing cells. We observed that F-actin polymerization in the region of membrane fusion was more prominent in Sms2-expressing cells than Sms-deficient cells. Taken together, our research provides insight into a novel function of SMS2 which is the regulation of HIV-1 Env-mediated membrane fusion via actin rearrangement.

A correlation of reactive oxygen species accumulation by depletion of superoxide dismutases with age-dependent impairment in the nervous system and muscles of Drosophila adults.

The theory that accumulation of reactive oxygen species (ROS) in internal organs is a major promoter of aging has been considered negatively. However, it is still controversial whether overexpression of superoxide dismutases (SODs), which remove ROS, extends the lifespan in Drosophila adults. We examined whether ROS accumulation by depletion of Cu/Zn-SOD (SOD1) or Mn-SOD (SOD2) influenced age-related impairment of the nervous system and muscles in Drosophila. We confirmed the efficient depletion of Sod1 and Sod2 through RNAi and ROS accumulation by monitoring of ROS-inducible gene expression. Both RNAi flies displayed accelerated impairment of locomotor activity with age and shortened lifespan. Similarly, adults with nervous system-specific depletion of Sod1 or Sod2 also showed reduced lifespan. We then found an accelerated loss of dopaminergic neurons in the flies with suppressed SOD expression. A half-dose reduction of three pro-apoptotic genes resulted in a significant suppression of the neuronal loss, suggesting that apoptosis was involved in the neuronal loss caused by SOD silencing. In addition, depletion of Sod1 or Sod2 in musculature is also associated with enhancement of age-related locomotion impairment. In indirect flight muscles from SOD-depleted adults, abnormal protein aggregates containing poly-ubiquitin accumulated at an early adult stage and continued to increase as the flies aged. Most of these protein aggregates were observed between myofibril layers. Moreover, immuno-electron microscopy indicated that the aggregates were predominantly localized in damaged mitochondria. These findings suggest that muscular and neuronal ROS accumulation may have a significant effect on age-dependent impairment of the Drosophila adults.

The actions and metabolism of lysophosphatidylinositol, an endogenous agonist for GPR55.

Lysophosphatidylinositol (LPI) is a subspecies of lysophospholipid and is assumed to be not only a degradation product of phosphatidylinositol (PI), but also a bioactive lysophospholipid mediator. However, not much attention has been directed toward LPI compared to lysophosphatidic acid (LPA), since the receptor for LPI has not been identified. During screening for an agonist for the orphan G protein coupled receptor GPR55, we identified LPI, 2-arachidonoyl LPI in particular, as an agonist for GPR55. Our efforts to identify an LPI receptor facilitated research on LPI as a lipid messenger. In addition, we also found that DDHD1, previously identified as phosphatidic acid-preferring phospholipase A1, was one of the synthesizing enzymes of 2-arachidonoyl LPI. Here, we summarized the background for discovering the LPI receptor, and the actions/metabolism of LPI. We also referred to the biosynthesis of PI, a 1-stearoyl-2-arachidonoyl species, since the molecule is the precursor of 2-arachidonoyl LPI. Furthermore, we discussed physiological and/or pathophysiological processes involving LPI and GPR55, including the relevance of LPI-GPR55 and cannabinoids, since GPR55 was previously postulated to be another cannabinoid receptor. Although there is no doubt that GPR55 is the LPI receptor, we should re-consider whether or not GPR55 is in fact another cannabinoid receptor.

Inhibitors of cannabinoid receptor 1 suppress the cellular entry of Lujo virus.

Lujo virus (LUJV), which belongs to Mammarenavirus, family Arenaviridae, has emerged as a pathogen causing severe hemorrhagic fever with high mortality. Currently, there are no effective treatments for arenaviruses, including LUJV. Here, we screened chemical compound libraries of Food and Drug Administration (FDA)-approved drugs and G protein-coupled receptor-associated drugs to identify effective antivirals against LUJV targeting cell entry using a vesicular stomatitis virus-based pseudotyped virus bearing the LUJV envelope glycoprotein (GP). Cannabinoid receptor 1 (CB1) antagonists, such as rimonabant, AM251 and AM281, have been identified as robust inhibitors of LUJV entry. The IC50 of rimonabant was 0.26 and 0.53 µM in Vero and Huh7 cells, respectively. Analysis of the cell fusion activity of the LUJV GP in the presence of CB1 inhibitors revealed that these inhibitors suppressed the fusion activity of the LUJV GP. Moreover, rimonabant, AM251 and AM281 reduced the infectivity of authentic LUJV in vitro, suggesting that the antiviral activity of CB1 antagonists against LUJV is mediated, at least in part, by inhibition of the viral entry, especially, membrane fusion. These findings suggest promising candidates for developing new therapies against LUJV infections.

Involvement of the endogenous cannabinoid 2 ligand 2-arachidonyl glycerol in allergic inflammation.

BACKGROUND: Cannabinoid (CB) 2 is expressed on immune and inflammatory cells. Identification of 2-arachidonyl glycerol (2-AG) and anandamide as endogenous CB2 ligands has allowed investigations of the roles of CB2 and its endogenous ligand system in inflammatory cells. However, the roles of this receptor-ligand system in inflammatory and allergic immune responses in vivo have not been fully elucidated. METHODS: Two mouse allergy models, namely ear dermatitis induced by 2,4-dinitrofluorobenzene and allergic bronchitis induced by ovalbumin, were analyzed for 2-AG amounts in allergic tissues, with reference to allergic and inflammatory symptoms. To investigate the gene expression via CB2 in inflammatory cells, human promyelocytic HL-60 cells were stimulated by the CB2 ligand 2-AG ether and analyzed using a DNA microarray. RESULTS: In the ear dermatitis model, the 2-AG amount increased upon serial 2,4-dinitrofluorobenzene challenges and was correlated with ear weight gain. The increased ear thickness in this allergy model was clearly suppressed in CB2 knockout mice, suggesting that the generated endogenous CB2 ligands induce ear thickness through aberrant inflammatory responses and remodeling mediated via CB2. In the allergic bronchitis model, the 2-AG level in bronchoalveolar lavage was increased and sustained during the elevation of inflammatory cell infiltration. The DNA microarray analysis of human HL-60 cells revealed that 2-AG ether induced expressions of not only inflammatory chemokines/cytokines but also of cell growth factors. CONCLUSION: Our data strongly suggest that endogenous CB2 ligands upregulated upon disease progression in allergic models are involved in aberrant alterations of both inflammatory responses and tissue cell growth.

Phosphorylation and subcellular localization of human phospholipase A1, DDHD1/PA-PLA1.

Protein phosphorylation is the most common post-translational modification of proteins and functions as a molecular switch for their regulation. This modification is reversibly regulated by protein kinases and phosphatases. In most cases, the phosphorylation of enzymes positively or negatively regulates enzyme activity. However, we found that the phosphorylation of DDHD1 phospholipase A1 (PLA1) did not affect PLA1 activity. Integrated analyses, including phospho-proteomics, Phos-tag SDS-PAGE, PLA1 enzyme assays, and immunofluorescent microscopy, revealed the subcellular localization of DDHD1 without greatly affecting its PLA1 activity. Our findings may contribute to understanding rare clinical cases that concern the implications of protein phosphorylation.

Generation of lysophosphatidylinositol by DDHD domain containing 1 (DDHD1): Possible involvement of phospholipase D/phosphatidic acid in the activation of DDHD1.

GPR55 is a seven-transmembrane G-protein-coupled receptor that has been proposed as a novel type of cannabinoid receptor. Previously, we identified lysophosphatidylinositol (LPI), in particular 2-arachidonoyl-LPI, as an agonist for GPR55. In the present study, we examined whether intracellular phospholipase A1 (DDHD domain containing 1, or DDHD1), previously identified as phosphatidic acid (PA)-preferring PLA1 (PA-PLA1), is involved in the formation of 2-arachidonoyl-LPI. HEK293 cells expressing DDHD1 produced [(3)H]arachidonic acid-containing LPI after prelabeling with [(3)H]arachidonic acid and subsequent activation by ionomycin; the formation of [(3)H]LPI was inhibited by n-butanol and the overexpression of an inactive PLD1 mutant PLD1K898R. DDHD1 was translocated from the cytosol to membranes upon ionomycin treatment. A purified recombinant DDHD1 formed [(3)H]LPI when incubated with [(3)H]PI; the V(max) and apparent K(m) were 190 micromol/min/mg protein and 10 mol% PI, respectively. DDHD1 binds PA, and the addition of PA to DDHD1 increased the affinity for PI (K(m) ; 3 mol%) and augmented the PI-PLA1 activity. DDHD1 activated by PA was returned to a basal state by its own PA-hydrolytic activity. These results implicate DDHD1 in the formation of 2-arachidonoyl-LPI and indicate that the process is modulated by PA released by phospholipase D. Similar observations for the production of arachidonic acid-containing LPI in neuroblastoma cells suggest the DDHD1-LPI-GPR55 axis to be involved in functions in the brain.

GPR35 is a novel lysophosphatidic acid receptor.

GPR35 is a rhodopsin-like G protein-coupled receptor identified in 1998. It has been reported that kynurenic acid, a tryptophan metabolite, may act as an endogenous ligand for GPR35. However, the concentrations of kynurenic acid required to elicit the cellular responses are usually high, raising the possibility that another endogenous ligand may exist. In this study, we searched for another endogenous ligand for GPR35. Finally, we found that the magnitude of the Ca(2+) response induced by 2-acyl lysophosphatidic acid in the GPR35-expressing HEK293 cells was markedly greater than that in the vector-transfected control cells. Such a difference was not apparent in the case of 1-acyl lysophosphatidic acid. 2-Acyl lysophosphatidic acid also caused the sustained activation of RhoA and the phosphorylation of extracellular signal-regulated kinase, and triggered the internalization of the GPR35 molecule. These results strongly suggest that 2-acyl lysophosphatidic acid is an endogenous ligand for GPR35.

Feasibility study of ultrasound video bronchoscopy for sampling endobronchial lesions.

INTRODUCTION: Endobronchial biopsy using forceps is generally performed with a standard bronchoscope, while a needle is used with an endobronchial ultrasound (EBUS) bronchoscope. An EBUS video bronchoscope, such as the EB-530US instrument (Fujifilm, Tokyo, Japan), provides good visibility and may, therefore, enable both forceps biopsies and needle aspirations to be performed. OBJECTIVES: The aim of this study was to investigate the feasibility of performing forceps biopsies and needle aspirations using the EBUS video bronchoscope for diagnosing endobronchial lesions. METHODS: Seventy patients with suspected endobronchial lesions based on a computed tomography scan were recruited and underwent forceps biopsy using the EB-530US EBUS bronchoscope. If the result of a rapid on-site cytological evaluation was negative, an additional needle aspiration of the same lesion or other target lesions was performed. The primary outcome was the completion rate of bronchoscopy, using only the EBUS bronchoscope without removal. RESULTS: In the 70 patients, forceps biopsies and/or needle aspirations using the EBUS video bronchoscope were performed and completed without removing the EBUS bronchoscope in 67 patients (95.7%). The remaining three patients required the removal of the EBUS bronchoscope from the trachea (blood clot obstruction of the working channel in one patient and change to a thin bronchoscope to sample an EBUS bronchoscope-inaccessible lesion in two others). The EBUS bronchoscope provided diagnostic material in 66 patients (94.3%). One case each of bleeding and pneumonia were observed (1.4%). CONCLUSION: Both forceps biopsy and needle aspiration are feasible using a Fujifilm EB-530US EBUS video bronchoscope.

Solvent-based paclitaxel or nab-paclitaxel for heavily treated relapsed/refractory small cell lung cancer: Retrospective single-institution observational study.

Treatment options for patients with relapsed/refractory small cell lung cancer (R/R SCLC) are limited, and the efficacy of salvage therapies for heavily treated patients should be assessed. Here, we evaluated the efficacy of paclitaxel (PTX) in R/R SCLC patients.A single-institute retrospective chart review was conducted. The primary endpoint was overall survival (OS), whereas the secondary endpoints were progression-free survival (PFS), overall response rate, disease control rate (DCR), and safety.Thirty-one patients (median age, 69 [range, 56-80] years) were analyzed. The median follow-up period was 122 (range, 28-1121) days. The median OS and PFS were 4.4 and 2.2 months, respectively. Adverse events of grade 3 or higher, other than hematological toxicity, were febrile neutropenia and neuropathy. Multivariate analyses identified the following independent predictors of poor OS: performance status and lactate dehydrogenase at the upper limit of normal.PTX monotherapy showed moderate efficacy with acceptable toxicity in heavily treated patients with R/R SCLC patients.

Phase II Study of Weekly Amrubicin for Refractory or Relapsed Non-small Cell Lung Cancer.

BACKGROUND: Amrubicin is usually administered on days 1-3 every 3 weeks by intravenous infusion. However, it causes severe hematological toxicity, especially febrile neutropenia. It was reported that weekly administration confers higher dose intensity, less severe adverse events, and anti-tumor activity that is as effective as that of treatment with a conventional schedule. PATIENTS AND METHODS: Weekly amrubicin was administered at a dose of 60 mg/m2 on days 1 and 8 every 3 weeks. The primary endpoint was overall response rate. RESULTS: A total of 33 patients were enrolled. The overall response rate was 6.1% (95% confidence interval(CI)=0.7-20.2%) and the disease control rate after 2 months was 51.5%. The median progression-free survival was 2.93 months. Febrile neutropenia was observed in only two patients. CONCLUSION: The primary endpoint was not met in this study. However, weekly amrubicin achieved a high disease control rate and good tolerability.

Biochemistry, pharmacology and physiology of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand.

2-Arachidonoylglycerol (2-AG) is a unique molecular species of monoacylglycerol isolated in 1995 from rat brain and canine gut as an endogenous ligand for the cannabinoid receptors. 2-AG is rapidly formed from arachidonic acid-containing phospholipids through increased phospholipid metabolism, such as enhanced inositol phospholipid turnover, in various tissues and cells upon stimulation. 2-AG binds to the cannabinoid receptors (CB1 and CB2) and exhibits a variety of cannabimimetic activities in vitro and in vivo. Notably, anandamide, another endogenous ligand for the cannabinoid receptors, often acts as a partial agonist at these cannabinoid receptors, whereas 2-AG acts as a full agonist in most cases. The results of structure-activity relationship studies suggested that 2-AG rather than anandamide is the true natural ligand for both the CB1 and the CB2 receptors. Evidence is gradually accumulating which shows that 2-AG plays physiologically essential roles in diverse biological systems. For example, several lines of evidence indicate that 2-AG plays an important role as a retrograde messenger molecule in the regulation of synaptic transmission. 2-AG has also been shown to be involved in the regulation of various types of inflammatory reactions and immune responses. In this review, we focused on 2-AG, and summarized information concerning its biosynthesis, metabolism, bioactions and physiological significance, including our latest experimental results.

Depolarization-induced rapid generation of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, in rat brain synaptosomes.

2-arachidonoylglycerol (2-AG) is an endogenous ligand for the cannabinoid receptors with a variety of potent biological activities. In this study, we first examined the effects of potassium-induced depolarization on the level of 2-AG in rat brain synaptosomes. We found that a significant amount of 2-AG was generated in the synaptosomes following depolarization. Notably, depolarization did not affect the levels of other molecular species of monoacylglycerols. Furthermore, the level of anandamide was very low and did not change markedly following depolarization. It thus appeared that the depolarization-induced accelerated generation is a unique feature of 2-AG. We obtained evidence that phospholipase C is involved in the generation of 2-AG in depolarized synaptosomes: U73122, a phospholipase C inhibitor, markedly reduced the depolarization-induced generation of 2-AG, and the level of diacylglycerol was rapidly elevated following depolarization. A significant amount of 2-AG was released from synaptosomes upon depolarization. Interestingly, treatment of the synaptosomes with SR141716A, a CB1 receptor antagonist, augmented the release of glutamate from depolarized synaptosomes. These results strongly suggest that the endogenous ligand for the cannabinoid receptors, i.e. 2-AG, generated through increased phospholipid metabolism upon depolarization, plays an important role in attenuating glutamate release from the synaptic terminals by acting on the CB1 receptor.