Synthetic cannabinoid CP-55,940 induces apoptosis in a human skeletal muscle model via regulation of CB1 receptors and L-type Ca2+ channels.

Rhabdomyolysis has been reported in patients who abuse synthetic cannabinoids. However, no studies have yet assessed whether these cases reflect the direct cytotoxicity of synthetic cannabinoids on skeletal muscle, a possibility that the present study sought to address. Specifically, this study investigated the cytotoxicity of the synthetic cannabinoid CP-55,940, a compound that acts equally on both types of cannabinoid receptors (CB1 and CB2), in a human embryonic rhabdomyosarcoma (RD) cell line. Exposure of these cells to CP-55,940 resulted in concentration-dependent decreases in cell viability. These effects were attenuated by pre-incubation with AM251 (30 µM), a selective CB1 receptor antagonist, but not by pre-incubation with AM630 (30 µM), a selective CB2 receptor antagonist. Following treatment with CP-55,940, RD cells exhibited apoptosis, as indicated by the accumulation of annexin-V, activation of caspase-3, and a loss of the mitochondrial membrane potential. Additionally, CP-55,940 treatment of RD cells led to increases in intracellular Ca2+ levels. CP-55,940-induced cell death was significantly attenuated in the absence of extracellular Ca2+, and was partially decreased by pre-incubation with verapamil (5 µM) or diltiazem (5 µM), compounds that block the L-type Ca2+ channel. Our results indicate that the cytotoxicity of CP-55,940 towards RD cells (skeletal muscle cells) is mediated by the CB1 receptor, but not by the CB2 receptor. Our results further suggest that calcium influx through the L-type channel may play an important role in the apoptosis induced by these compounds.

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

The characteristic smell emitted from two scale insects, Ceroplastes japonicus and Ceroplastes rubens.

The volatile components emitted from two scale insects, Ceroplastes japonicus and Ceroplastes rubens, were identified using GC-MS analysis. The major volatile components of the solvent extract from C. japonicus were α-humulene (35.8%) and δ-cadinene (17.0%), while those of C. rubens were ß-selinene (10.3%) and ß-elemene (5.1%). In GC/olfactometry, linalool, butyric acid, 3-methylbutyric acid, 2-methylbutyric acid, and vanillin were identified as the odor-active components of the extract from C. japonicus, in addition to trace amounts of trans-4,5-epoxy-(2E)-decenal, 4-methyl-(3E)-hexenoic acid, and phenylacetic acid. With regard to C. rubens, trans-4,5-epoxy-(2E)-decenal, 3-methylbutyric acid, and phenylacetic acid were identified as the odor-active components. Besides, decan-1,4-olide (γ-decalactone) with milky cherry-like note and 3-hydroxy-4,5-dimethylfuran-2(5H)-one (sotolone) with brown sugar-like note were also detected as the characteristic cherry-like sweet-and-sour note of these two scale insects. ABBREVIATIONS: GC: Gas chromatography; GC/O: gas chromatography/olfactometry.

Characteristics of three-dimensional prospectively isolated mouse bone marrow mesenchymal stem/stromal cell aggregates on nanoculture plates.

Three-dimensional (3-D) aggregate culturing is useful for investigating the functional properties of mesenchymal stem/stromal cells (MSCs). For 3-D MSC analysis, however, pre-expansion of MSCs with two-dimensional (2-D) monolayer culturing must first be performed, which might abolish their endogenous properties. To avoid the need for 2-D expansion, we used prospectively isolated mouse bone marrow (BM)-MSCs and examined the differences in the biological properties of 2-D and 3-D MSC cultures. The BM-MSCs self-assembled into aggregates on nanoculture plates (NCP) that have nanoimprinted patterns with a low-cellular binding texture. The 3-D MSCs proliferated at the same rate as 2-D-cultured cells by only diffusion culture and secreted higher levels of pro-angiogenic factors such as vascular endothelial growth factor and hepatocyte growth factor (HGF). Conditioned medium from 3-D MSC cultures promoted more capillary formation than that of 2-D MSCs in an in vitro tube formation assay. Matrigel-implanted 3-D MSC aggregates tended to induce angiogenesis in host mice. The 3-D culturing on NCP induced alpha-fetoprotein (AFP) expression in MSCs without the application of AFP- or endodermal-inducible factors, possibly via an HGF-autocrine mechanism, and maintained their differentiation ability for adipocytes, osteocytes, and chondrocytes. Prospectively isolated mouse BM-MSCs expressed low/negative stemness-related genes including Oct3/4, Nanog, and Sox2, which were not enhanced by NCP-based 3-D culturing, suggesting that some of these cells differentiate into meso-endodermal layer cells. Culturing of prospectively isolated MSCs on NCP in 3-D allows the analysis of the biological properties of more closely endogenous BM-MSCs and might contribute to tissue engineering and repair.

[Zinc- and tin-induced apoptotic mechanisms in immune system and cranial nerve system].

This review explains the mechanisms of apoptosis related to the impacts of zinc deficiency and organotin exposure on the immune and central nervous systems. In the immune systems, both zinc deficiency and trialkyltin exposure lead to severe thymic atrophy and affect T-lymphocyte development through apoptosis of double positive stage pre-T-cells(CD4+/CD8+) in the cortex region. Their apoptosis are caused mainly through decrease in Bcl-2 expression, activation of ROS production/release, oxidative stress, mitochondrial cytochrome c release and activation of caspase cascade, with increases in glucocorticoids in zinc deficiency, without the involvement of glucocorticoid in organotin exposure In the central nervous system, both zinc deficiency and trialkyltin exposure reduce learning, memory and sensory functions through neuronal apoptosis caused by activation of ROS production/release, release of pro-apoptotic factors such as cytochrome c or apoptosis-inducing factor(AIF), with Fe excessive accumulation leading to ROS production and with depletion of hippocampus Zn (mossy fiber Zn) causing various Ca2+ channel disorder of synapse in the hippocampus, and with excessive accumulation of Ca through cAMP-dependent Ca(2+)-channel disorder by excessive PTH and cAMP excessive production in the olfactory systems such as olfactory epithelium and olfactory bulb.

[Biological functions of tin and disease].

Tin generates a wide variety of biological functions due to its chemical character. In this article, the modes of the biological functions of tin(especially organotin compounds) are reviewed, with special emphasis on the connection with the immune system, brain nervous system and endocrine system, on the basis of our data. To sum up this article, the biological functions of organotin compounds appear to be due to the following several processes: (1) their incorporation into the cells in vesicle form through fusion or in a similar manner to their incorporation in cationic form; (2) transport to and accumulation in the regions of the Golgi apparatus and endoplasmic reticulum (ER), but not to or in the plasma membrane or nucleus because of their hydrophobicity; (3) inhibition of intracellular phospholipid transport between organelles due to impairment of the structures and functions of the Golgi apparatus and ER; (4) inhibition of the membrane-mediated signal transduction system leading to DNA synthesis via phospholipid turnover and Ca2+ mobilization, as in cell proliferation systems; (5) disturbance of the trace element balance and the localization of certain elements; (6) disorders of membrane-mediated Ca2+ homeostasis via various channel functions including Zn modulation on the plasma and organelle membranes, and protein phosphorylation, as in the signal transduction systems of memory and olfaction; (7) necrosis or apoptosis in vivo or toxic cell death in vitro.

Radiation increases the cellular uptake of exosomes through CD29/CD81 complex formation.

Exosomes mediate intercellular communication, and mesenchymal stem cells (MSC) or their secreted exosomes affect a number of pathophysiologic states. Clinical applications of MSC and exosomes are increasingly anticipated. Radiation therapy is the main therapeutic tool for a number of various conditions. The cellular uptake mechanisms of exosomes and the effects of radiation on exosome-cell interactions are crucial, but they are not well understood. Here we examined the basic mechanisms and effects of radiation on exosome uptake processes in MSC. Radiation increased the cellular uptake of exosomes. Radiation markedly enhanced the initial cellular attachment to exosomes and induced the colocalization of integrin CD29 and tetraspanin CD81 on the cell surface without affecting their expression levels. Exosomes dominantly bound to the CD29/CD81 complex. Knockdown of CD29 completely inhibited the radiation-induced uptake, and additional or single knockdown of CD81 inhibited basal uptake as well as the increase in radiation-induced uptake. We also examined possible exosome uptake processes affected by radiation. Radiation-induced changes did not involve dynamin2, reactive oxygen species, or their evoked p38 mitogen-activated protein kinase-dependent endocytic or pinocytic pathways. Radiation increased the cellular uptake of exosomes through CD29/CD81 complex formation. These findings provide essential basic insights for potential therapeutic applications of exosomes or MSC in combination with radiation.

Cytotoxicity of synthetic cannabinoids on primary neuronal cells of the forebrain: the involvement of cannabinoid CB1 receptors and apoptotic cell death.

The abuse of herbal products containing synthetic cannabinoids has become an issue of public concern. The purpose of this paper was to evaluate the acute cytotoxicity of synthetic cannabinoids on mouse brain neuronal cells. Cytotoxicity induced by synthetic cannabinoid (CP-55,940, CP-47,497, CP-47,497-C8, HU-210, JWH-018, JWH-210, AM-2201, and MAM-2201) was examined using forebrain neuronal cultures. These synthetic cannabinoids induced cytotoxicity in the forebrain cultures in a concentration-dependent manner. The cytotoxicity was suppressed by preincubation with the selective CB1 receptor antagonist AM251, but not with the selective CB2 receptor antagonist AM630. Furthermore, annexin-V-positive cells were found among the treated forebrain cells. Synthetic cannabinoid treatment induced the activation of caspase-3, and preincubation with a caspase-3 inhibitor significantly suppressed the cytotoxicity. These synthetic cannabinoids induced apoptosis through a caspase-3-dependent mechanism in the forebrain cultures. Our results indicate that the cytotoxicity of synthetic cannabinoids towards primary neuronal cells is mediated by the CB1 receptor, but not by the CB2 receptor, and further suggest that caspase cascades may play an important role in the apoptosis induced by these synthetic cannabinoids. In conclusion, excessive synthetic cannabinoid abuse may present a serious acute health concern due to neuronal damage or deficits in the brain.

Fentanyl-Type Antagonist of the µ-Opioid Receptor: Important Role of Axial Chirality in the Active Conformation.

In recent years, synthetic opioids have emerged as a predominant cause of drug-overdose-related fatalities, causing the "opioid crisis." To design safer therapeutic agents, we accidentally discovered µ-opioid receptor (MOR) antagonists based on fentanyl with a relatively uncomplicated chemical composition that potentiates structural modifications. Here, we showed the development of novel atropisomeric fentanyl analogues that exhibit more potent antagonistic activity against MOR than naloxone, a morphinan MOR antagonist. Derivatives displaying stable axial chirality were synthesized based on the amide structure of fentanyl. The aS- and aR-enantiomers exerted antagonistic and agonistic effects on the MOR, respectively, and each atropisomer interacted with the MOR by assuming a distinct binding mode through molecular docking. These findings suggest that introducing atropisomerism into fentanyl may serve as a key feature in the molecular design of future MOR antagonists to help mitigate the opioid crisis.

Relevant use of Klotho in FGF19 subfamily signaling system in vivo.

Alpha-Klotho (alpha-Kl) and its homolog, beta-Klotho (beta-Kl) are key regulators of mineral homeostasis and bile acid/cholesterol metabolism, respectively. FGF15/ humanFGF19, FGF21, and FGF23, members of the FGF19 subfamily, are believed to act as circulating metabolic regulators. Analyses of functional interactions between alpha- and beta-Kl and FGF19 factors in wild-type, alpha-kl(-/-), and beta-kl(-/-) mice revealed a comprehensive regulatory scheme of mineral homeostasis involving the mutually regulated positive/negative feedback actions of alpha-Kl, FGF23, and 1,25(OH)(2)D and an analogous regulatory network composed of beta-Kl, FGF15/humanFGF19, and bile acids that regulate bile acid/cholesterol metabolism. Contrary to in vitro data, beta-Kl is not essential for FGF21 signaling in adipose tissues in vivo, because (i) FGF21 signals are transduced in the absence of beta-Kl, (ii) FGF21 could not be precipitated by beta-Kl, and (iii) essential phenotypes in Fgf21(-/-) mice (decreased expressions of Hsl and Atgl in WAT) were not replicated in beta-kl(-/-) mice. These findings suggest the existence of Klotho-independent FGF21 signaling pathway(s) where undefined cofactors are involved. One-to-one functional interactions such as alpha-Klotho/FGF23, beta-Klotho/FGF15 (humanFGF19), and undefined cofactor/FGF21 would result in tissue-specific signal transduction of the FGF19 subfamily.

[Empyema from spontaneous esophageal rupture treated by thoracoscopic surgery].

A 61-year-old man who had been admitted for vomiting and abdominal pain 5 days before at another hospital was transferred to our hospital. He was diagnosed as pyo-pneumothorax, and chest drainage was initiated. Seven days after admission, food residue was observed in the drainage tube. A subsequent gastrointestinal fiberscopic examination could not identify the esophageal injury, but the acute empyema caused by spontaneous esophageal rupture was suggested by clinical signs. Thoracoscopic surgery for curettage and irrigation of the pleural cavity was performed. Esophageal rupture could not be confirmed at surgery. Spontaneous closure of the fistula was observed 2 weeks post-surgery.

[Drug discrimination properties and cytotoxicity of the cannabinoid receptor ligands].

The worldwide distribution of smokable herbal mixtures called "Spice" that contain synthetic cannabinoids with a pharmacological activity similar to delta 9-tetrahydrocannabinol (delta 9-THC) has been reported. The synthetic cannabinoids induce behavior and have biochemical properties similar to naturally occurring cannabinoids such as delta 9-THC. In drug discrimination procedures, animal behavior is differentially reinforced depending on the presence or absence of specific drug stimuli. This review seeks to establish an animal model to serve as a discriminative stimulus of the synthetic cannabinoids, to determine whether this discriminative stimulus is identical to that of delta 9-THC. Much data have been obtained in drug discrimination experiments with various synthetic cannabinoids. In the discriminative study, synthetic cannabinoids such as CP-55,940 and WIN-55,212-2 were substituted for delta 9-THC in rats trained to discriminate delta 9-THC from the vehicle. These discriminative effects of synthetic cannabinoids were antagonized by CB1 antagonist SR-141,716A. The discriminative effects of synthetic cannabinoids may overlap with the delta 9-THC cue mediated by CB1 receptors. In in vitro study using NG 108-15 cell lines, synthetic cannabinoids have produced strong cytotoxicities that were suppressed by pretreatment with the CB1 receptor antagonist. Furthermore, pretreatment with caspase inhibitors suppressed these synthetic-cannabinoid-induced cytotoxicities in NG 108-15 cells. These findings indicate that the cytotoxicity of synthetic cannabinoids towards NG 108-15 cells is mediated by the CB1 receptors and further suggest that caspase cascades may play an important role in the cytotoxicities induced by these synthetic cannabinoids. In conclusion, synthetic cannabinoid abuse could be a health hazard for humans.

Analytical method for tributyltin and triphenyltin contained in household products-preparing for the revision of authorized analytical method-.

In preparing for the revision of the authorized analytical method for tributyltin (TBT) and triphenyltin (TPT), which are banned from using according to the "Act on the Control of Household Products Containing Harmful Substances", an examination was conducted on the detection method of these substances using gas chromatography/mass spectrometry (GC/MS), after derivatizing them (ethyl-derivatizing method and hydrogen-derivatizing method). Ethyl-derivatized compounds had stability, which enabled the detection of TPT with a higher sensitivity. In addition, a preparation suitable for the following analytical objects was established: (1) textile products, (2) water-based products (such as water-based paint), (3) oil-based products (such as wax), and (4) adhesives. Addition-recovery experiments were conducted using the prescribed pretreatment method, when each surrogate substances (TBT-d27, TPT-d15) were added and the data were corrected, good recovery rates (94.5-118.6% in TBT, and 86.6-110.1% in TPT) were obtained. When TBT and TPT in 31 commercially available products were analyzed based on the developed analytical method, an adhesive showed 13.2 microg/g of TBT content, which exceeded the regulatory criterion (1 microg/g as tin). Next, when the same products with different manufacturing date were analyzed, TBT (10.2-10.8 microg/g), which exceeded the regulatory criterion, was detected in 4 products among 8 products, and simultaneously, a high concentration (over 1000 microg/g) of dibutyltin (DBT) was detected. It was suggested that TBT as an impurity of DBT remained, and the manufacturer chose the voluntary recall of the product. The new method is considered sufficiently applicable as a revised method for the conventionally authorized method.

[A case of complete resection of a squamous cell lung cancer invading diaphragm after neoadjuvant chemotherapy of nedaplatin and irinotecan].

The treatment for squamous cell lung cancer has remained unclear, while that for lung cancer according to each pathology type has advanced. This is a case of complete response of a squamous cell lung cancer invading the diaphragm which could be resected completely after neoadjuvant chemotherapy of nedaplatin (CDGP) and irinotecan (CPT-11). CDGP and CPT-11 might be effective for squamous cell lung cancer.

[Present Conditions of Marijuana Regulation in USA: Medical and Recreational Use].

In most countries marijuana is regulated by the Single Convention on Narcotic Drugs. In Japan marijuana use is illegal under the Marijuana Control Law. In USA, marijuana is also classified as a schedule I drug, which is the most stringent regulation category under federal law. On the other hand, California became the first state to legalize marijuana for medical uses in 1996. Since then, several other US states have approved marijuana for medical or recreational use. However, marijuana remains completely illegal in most states, while some allow only cannabidiol (CBD) extracted from marijuana for medical use. In June 2018, the US Food and Drug Administration approved Epidiolex, the first marijuana-derived drug, containing purified CBD, to treat certain rare childhood seizure syndromes. Therefore the situation surrounding control of marijuana in USA is complex. Recently, a definite trend toward reconsidering marijuana regulation has been seen around the world, which could have a major impact on marijuana policy in Japan. In this review, we investigated existing medical and recreational marijuana laws in various US states, with a focus on California, which approved recreational use in 2018. Here, we describe the current state of marijuana regulation in terms of both medical and recreational use. In addition, we discuss public safety issues associated with marijuana, including crime, traffic accidents, and emergency department visits from possible marijuana exposure, as well as generated tax revenues, from official marijuana-related reports in Colorado, which legalized marijuana use in 2012.

[Dependence and Cytotoxicity of Components of Cannabis].

Cannabis use among the younger population in Japan has been steadily increasing. The aim of the present review is to highlight recent knowledge regarding the molecular mechanisms of action and health risks associated with cannabis and synthetic cannabinoid consumption. We investigated the effects of Δ9-tetrahydrocannabinol (THC) and synthetic cannabinoids on place conditioning in ICR mice. Both Δ9-THC and synthetic cannabinoids produce a significant conditioned place preference. These rewarding effects were completely suppressed by the cannabinoid CB1 receptor type antagonist AM251. The cytotoxicological effects of Δ9-THC and synthetic cannabinoids were also characterized in the limbic forebrain of mice in primary culture in vitro. Δ9-THC and synthetic cannabinoids caused cell death in a dose-dependent manner. The rank order of cytotoxicological potency was synthetic cannabinoids>Δ9-THC and related to the agonistic activities of the CB1 receptor. A recent review on the harmful effects of cannabis use in humans reported that behavioral impairments, especially in terms of attention, memory, and complex information-processing ability, can last for many weeks after cessation of cannabis use among heavy users. In addition, cannabis use could be a risk factor for drug dependence and later psychosis among adolescents. The results of animal and human studies suggest that CB1 receptors play an important role in the expression of harmful effects of cannabis and synthetic cannabinoid use. Moreover, concern regarding increasing concentrations of Δ9-THC in cannabis in many countries has been noted, because more potent cannabis may be associated with worse adverse effects.

Characteristic Odor of the Japanese Liverwort (Leptolejeunea elliptica).

The volatile components produced by Leptolejeunea elliptica (Lejeuneaceae), which is a liverwort grown on the leaves of tea (Camellia sinensis), were collected and analyzed using headspace solid-phase microextraction-gas chromatography/mass spectrometry (HS-SPME-GC/MS). 1-Ethyl-4-methoxybenzene (1), 1-ethyl-4-hydroxybenzene (2), and 1-acetoxy-4-ethylbenzene (3) were identified as the major components together with several other phenolic compounds, including 1,2-dimethoxy-4-ethylbenzene, and 4-ethylguaiacol in addition to sesquiterpene hydrocarbons, such as α-selinene, ß-selinene, ß-elemene, and ß-caryophyllene. GC/Olfactometry showed the presence of linalool, acetic acid, isovaleric acid, trans-methyl cinnamate, and trans-4,5-epoxy-(2E)-decenal, as the volatile components produced by L. elliptica.

Oesophagobronchial perforations after placement of an oesophageal self-expanding metallic stent.

An oesophageal fully covered self-expanding metallic stent (SEMS) was placed in a 54-year-old Japanese man to relieve dysphagia owing to a stage cT1bN3M1c lung adenocarcinoma. High expression of programmed cell death-ligand 1 was microscopically confirmed, and pembrolizumab was subsequently administered. Several days later, the patient was hospitalized with septic shock, and severe mediastinitis and pneumonia caused by oesophageal SEMS-induced oesophageal and bronchial perforations were observed. Thoracoscopic surgery was performed to drain the mediastinal abscess, and an additional oesophageal SEMS was placed to close the oesophageal perforation. The patient gradually recovered from the potentially fatal infection, and the SEMS was retrieved after confirming perforation closure. We re-initiated pembrolizumab administration, and the patient responded well. The present report reveals the potential risk and effectiveness of SEMS, especially when administered with immune checkpoint inhibitors.

Characteristic Scent from the Tahitian Liverwort, Cyathodium foetidissimum.

The volatile components of the Tahitian liverwort Cyathodium foetidissimum was analyzed using headspace solid phase micro-extraction (SPME) and GC-MS. Three volatile components, 4-methoxystyrene (24.4%), 3,4-dimethoxystyrene (28.7%), and skatole (15.9%) were identified as the major components from the fresh C. foetidissimum, along with several aliphatic aldehydes, n-octanal, n-nonanal, and n-decanal. However, (E)-2-nonenal recognized as aged malodor was not identified. In GC-O analysis, 2-aminoacetophenone was detected as one of the minor components with a strong aging note. In fact, C. foetidissimum showed the characteristic aging odor reminiscent the damp smell from old chest of drawers, or the civet like note with very strong feces and urine odor. The mixture consisted of 4-methoxystyrene, 3,4-dimethoxystyrene, and skatole in the detected ratio showed the sedative effect on CNV (contingent negative variation) measurement.

Comparative Study on Volatile Compounds of Alpinia japonica and Elettaria cardamomum.

The volatile compounds obtained from the ether extracts, headspace gases and steam distillates of Alpinia japonica and Elettaria cardamomum were analyzed by GC/MS. Both species were rich sources of naturally rare fenchane-type monoterpenoids, fenchene, fenchone, fenchyl alcohol and its acetate, together with 1,8-cineole. The distributions of volatile sesquiterpenoids were very poor in both species. Chiralities of fenchone in A. japonica and E. cardamomum were 99% of (1S,4R)-(+)-form. Camphor in A. japonica is composed of a mixture of (1R,4R)-(+)-form (94.3%) and (1S,4S)-(-)-form (5.7%). On the other hand, E. cardamomum produced only (1R,4R)-(+)-camphor (99%).