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In this study, extracts from non-psychoactive Cannabis sativa L. varieties were characterized by means of ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) and their antiproliferative activity was assessed in vitro. The human chronic myelogenous leukaemia cell line K562 was chosen to investigate the mechanism of cell death. The effect on the cell cycle and cell death was analysed by flow cytometry. Proteins related to apoptosis were studied by western blotting. Mechanical properties of cells were assessed using the Micropipette Aspiration Technique (MAT). The results indicated that the cannabidiol (CBD)-rich extract inhibited cell proliferation of K562 cell line in a dose-dependent manner and induced apoptosis via caspase 3 and 7 activation. A significant decrease in the mitochondrial membrane potential was detected, together with the release of cytochrome c into the cytosol. The main apoptotic markers were not involved in the mechanism of cell death. The extract was also able to modify the mechanical properties of cells. Thus, this hemp extract and its pure component CBD deserve further investigation for a possible application against myeloproliferative diseases, also in association with other anticancer drugs.

Neuropathic pain (NP) is a chronic disease that affects the normal quality of life of patients. To date, the therapies available are only symptomatic and they are unable to reduce the progression of the disease. Many studies reported the efficacy of Cannabis sativa L. (C. sativa) on NP, but no Δ

Mechanobiology has nowadays acquired the status of a topic of fundamental importance in a degree in Biological Sciences. It is inherently a multidisciplinary topic where biology, physics and engineering competences are required. A course in mechanobiology should include lab experiences where students can appreciate how mechanical stimuli from outside affect living cell behaviour. Here we describe all the steps to build a cell stretcher inside an on-stage cell incubator. This device allows exposing living cells to a periodic mechanical stimulus similar to what happens in physiological conditions such as, for example, in the vascular system or in the lungs. The reaction of the cells to the periodic mechanical stretching represents a prototype of a mechanobiological signal integrated by living cells. We also provide the theoretical and experimental aspects related to the calibration of the stretcher apparatus at a level accessible to researchers not used to dealing with topics like continuum mechanics and analysis of deformations. We tested our device by stretching cells of two different lines, U87-MG and Balb-3T3 cells, and we analysed and discussed the effect of the periodic stimulus on both cell reorientation and migration. We also discuss the basic aspects related to the quantitative analysis of the reorientation process and of cell migration. We think that the device we propose can be easily reproduced at low-cost within a project-oriented course in the fields of biology, biotechnology and medical engineering.

The edible insect food chain represents a relatively novel food-producing system; hence, associated biological risks still need to be exhaustively evaluated. In the present study, the dynamics of Escherichia coli during the whole living period of Tenebrio molitor larvae (from eggs to pupae) were studied. To this end, a rearing substrate consisting of organic wheat middlings was spiked with E. coli cells at two initial contamination levels: 1 log cfu g

The present research reports the results of a long-term study (70 days) of the dynamics of Staphylococcus aureus artificially inoculated in a Tenebrio molitor rearing chain for human consumption. To this end, a rearing substrate consisting of organic wheat middlings was spiked with S. aureus to obtain three initial contamination levels, namely 1 (low level), 5 (medium level) and 7 (high level) Log colony forming unit per gram. Microbial viable counting coupled with metataxonomic analysis were performed to evaluate: i) the persistence and growth of S. aureus in the rearing substrate; ii) the colonization and growth of S. aureus in the insect larvae; iii) the occurrence and load of S. aureus in the frass (excrement from larvae mixed with substrate residues); iv) the presence of S. aureus enterotoxins in the rearing substrate, frass, and larvae. The results of the present study highlighted that wheat middlings contaminated with S. aureus do not represent a suitable environment for the multiplication of the pathogen, irrespective of the initial contamination level. Of note, frass originated from the larvae reared on contaminated wheat middlings might potentially represent a source of S. aureus, with cell loads depending on the initial contamination level. A complex resident microbiota was revealed by metataxonomic analysis. Interestingly, co-occurrence/co-exclusions analysis did not reveal associations between the target microorganism and the microbiota of wheat middlings, larvae, or frass. Considering safety aspects of larvae, the results overall collected suggested that, under the applied conditions, T. molitor represents an inhospitable or even hostile environment for S. aureus, with this latter showing counts below the detection limit in the larvae at the end of the 70-day rearing trial, irrespective of the initial contamination level. The results also suggested that a combination of bactericidal factors, including unfavorable environmental conditions (such as low a

We characterize poly(3-hydroxybutyrate

Coronavirus Disease 2019 (COVID-19) is upsetting the world and innovative therapeutic solutions are needed in an attempt to counter this new pandemic. Great hope lies in vaccines, but drugs to cure the infected patient are just as necessary. In the most severe forms of the disease, a cytokine storm with neuroinflammation occurs, putting the patient's life at serious risk, with sometimes long-lasting sequelae. Palmitoylethanolamide (PEA) is known to possess anti-inflammatory and neuroprotective properties, which make it an ideal candidate to be assumed in the earliest stage of the disease. Here, we provide a mini-review on the topic, pointing out phospholipids consumption in COVID-19, the possible development of an antiphospholipid syndrome secondary to SARS-CoV-2 infection, and reporting our preliminary single-case experience concerning to a 45-year-old COVID-19 female patient recently treated with success by micronized / ultramicronized PEA.

On March 11, 2020, the World Health Organization declared coronavirus disease 2019 (COVID-19) a pandemic; from that date, the vaccine race has begun, and many technology platforms to develop a specific and effective COVID-19 vaccine have been launched in several clinical trials (protein subunit, RNA-based, DNA-based, replicating viral vector, nonreplicating viral vector, inactivated virus, live attenuated virus, and virus-like particle). Among the next-generation strategies, nucleoside-modified messenger RNA vaccines appear the most attractive, not only to counteract emerging pathogens but also for the possible applications in regenerative medicine and cancer therapy. However, exactly as all innovative drugs, they deserve careful pharmacovigilance in the short and long term.

Background Coronavirus disease 2019 (COVID-19) is a complex disease with many clinicopathological aspects, including abnormal immunothrombosis, and the full comprehension of its pathogenetic mechanisms is urgently required. Methods/Results By means of a multidisciplinary approach, we here report a catastrophic COVID-19 in a 44-year-old Philippine male patient, discovered lupus anticoagulant (LAC)-positive shortly before death, occurred 8 days after hospitalization in a clinical scenario refractory to standard high acuity care recalling Asherson’s syndrome (catastrophic antiphospholipid syndrome). Conclusion A parallelism between this severe form of COVID-19 and Asherson’s syndrome can be so drawn. Both the diseases in fact exhibit hypercytokinemia, thrombotic microangiopathy, disseminated intravascular coagulation and multiple organ failure, they show a relationship with viral infections, and they are burdened by a high mortality rate. A genetic predisposition to develop these two overlapping conditions may be supposed.

Stress is a typical body's natural defense to a generic physical or psychic change. A specific linking mechanism between ulcer onset and psycho-physical stress prolonged exposure has been reported. We decided to investigate the possible effects of Borago officinalis L. (Borago) in preventing physical (stress)-induced gastric ulcers in a rat model. Eighty male Sprague-Dawley rats were randomly divided into 16 groups, pretreated with a control solution, omeprazole (20 mg/kg), Borago methanolic extract (25, 50, 100, 250, and 500 mg/kg), Borago organic extract (50, 100, 250, and 500 mg/kg), Borago aqueous extract (5, 10, 20, 30, and 40 mg/kg), and D(-)-2-Amino-5-phosphonovaleric acid (AP5) (25 mg/kg) and kept in stressful conditions such as water immersion and restraint-induced stress ulcers. The animals were sacrificed and their stomach scored for the severity and the number of gastric ulcers. Methanolic extract (500 mg/kg) significantly reduced both ulcer parameters (***p < 0.001 and **p < 0.01, respectively). Aqueous and organic extract significantly decreased severity score at 5 and 10 mg/kg (**p < 0.01 and ***p < 0.001, respectively), and at 250 and 500 mg/kg (***p < 0.001), respectively, while gastric ulcers' resulted number significantly reduced only at 10 mg/kg (*p < 0.05) and at 500 mg/kg (**p < 0.01), respectively. On the other hand, aqueous extract significantly increased the mucosal gastric content of cAMP (*p < 0.05) and NR2A and NR2B subunits (*p < 0.05 and **p < 0.01, respectively) at 5 mg/kg. Organic extract showed also a significant cytotoxic effect at 500 and 1,000 mg/kg with a 3T3 cell viability reduction of 43.6% (**p < 0.01) and 92.1% (***p < 0.001), respectively. Borago aqueous extract at 10 mg/kg could be considered as a potential protective agent against stress-induced ulcers, and it is reasonable to possibly ascribe such protective activity to a modulation of the NR2A and NR2B subunit expression.

International audience; A novel 2,3-benzodiazepine-4 derivative, named 1g, has recently been shown to function as an anti-proliferative compound. We now show that it perturbs the formation of a functional mitotic spindle, inducing a spindle assembly checkpoint (SAC)-dependent arrest in human cells. Live analysis of individual microtubules indicates that 1g promotes a rapid and reversible reduction in microtubule growth. Unlike most anti-mitotic compounds, we found that 1g does not interfere directly with tubulin or perturb microtubule assembly in vitro The observation that 1g also triggers a SAC-dependent mitotic delay associated with chromosome segregation in Drosophila neural stem cells, suggests that it targets a conserved microtubule regulation module in humans and flies. Altogether, our results indicate that 1g is a novel promising anti-mitotic drug with the unique properties of altering microtubule growth and mitotic spindle organization.

Background Rhodiola rosea L. (Crassulaceae) has been used for years in the traditional medicine of several countries as an adaptogen drug, able to preserve homeostasis in response to stress stimuli. Currently R. rosea roots and rhizome are classified as a traditional herbal medicinal product for temporary relief of symptoms of stress, such as fatigue and sensation of weakness by the European Medicines Agency. Hypothesis/Purpose Increasing evidences suggest the involvement of neuroinflammation in response to stress. However, whether the modulation of neuroinflammatory parameters could be involved in the anti-stress effect of R. rosea has been barely studied. Thus, the aim of this work is to investigate the possible modulation of molecular inflammatory processes elicited by a R. rosea roots and rhizome ethanolic extract in an in vitro model of corticotropin releasing hormone (CRH)-stimulated BV2 microglial cells. Methods BV2 cells were stimulated with CRH 100 nM and changes in cell viability, cytokines production and heat shock protein 70 (HSP70) levels were evaluated. Intracellular pathways related to inflammation, such as nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) nuclear translocation and mitogen-activated protein kinases (MAPK) activation were also analyzed. Results We found that R. rosea extract (2.7% m/m rosavin and 1% m/m salidroside) 20 µg/ml was able to counteract the neuroinflammatory effect of CRH by inhibiting NF-κB nuclear translocation with a mechanism of action involving the modulation of mitogen-activated protein kinase-activated protein kinase 2 (MKK2), extracellular signal-regulated kinase 1/2 (ERK 1/2) and c-Jun n-terminal kinase (JNK), resulting in a reduction of HSP70 expression. Conclusion This work expands the knowledge of the intracellular mechanisms involved in R. rosea anti-stress activity and may be useful for the study of other adaptogen drugs.

Background: Fibre-type Cannabis sativa L. (hemp) usually contains cannabidiolic acid and cannabidiol as the main non-psychoactive cannabinoids. Even though there is evidence of the neuroprotective activity of pure cannabidiol, no in vitro studies have reported so far the role of hemp extracts on neuroprotection. The objective of this study was to evaluate the neuroprotective effect of hemp extracts in in vitro cellular models of neurotoxicity. Methods: One extract was obtained from raw hemp inflorescences, while the other was prepared from the same plant material submitted to a decarboxylation process. The composition of both these extracts was evaluated by HPLC-UV/DAD. Human neuroblastoma SH-SY5Y and microglial BV-2 cell lines treated with rotenone were selected as the model of neurodegeneration. The neuroprotection of hemp extracts was assessed also in serum-free conditions both in the presence and in the absence of rotenone as the toxic agent by using the same cell lines. The neuroprotective potential of cannabidiol was tested in parallel. Results: The decarboxylated hemp extract possesses a mild neuroprotective activity on BV-2 cells treated with rotenone, higher than that of pure cannabidiol. As regards serum-free experiments, the nondecarboxylated hemp extract was the most effective neuroprotective agent toward SH-SY5Y cells, while BV-2 cells were better protected from the toxic insult by the decarboxylated extract and cannabidiol. Conclusion: Both hemp extracts and pure cannabidiol displayed a moderate neuroprotective activity in the neurotoxicity models considered in this study; in addition, they showed a trophic effect on SHSY5Y cells.

In the last decades, a lot of attention has been paid to the compounds present in medicinal Cannabis sativa L., such as Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), and their effects on inflammation and cancer-related pain. The National Cancer Institute (NCI) currently recognizes medicinal C. sativa as an effective treatment for providing relief in a number of symptoms associated with cancer, including pain, loss of appetite, nausea and vomiting, and anxiety. Several studies have described CBD as a multitarget molecule, acting as an adaptogen, and as a modulator, in different ways, depending on the type and location of disequilibrium both in the brain and in the body, mainly interacting with specific receptor proteins CB1 and CB2. CBD is present in both medicinal and fibre-type C. sativa plants, but, unlike Δ9-THC, it is completely nonpsychoactive. Fibre-type C. sativa (hemp) differs from medicinal C. sativa, since it contains only few levels of Δ9-THC and high levels of CBD and related nonpsychoactive compounds. In recent years, a number of preclinical researches have been focused on the role of CBD as an anticancer molecule, suggesting CBD (and CBD-like molecules present in the hemp extract) as a possible candidate for future clinical trials. CBD has been found to possess antioxidant activity in many studies, thus suggesting a possible role in the prevention of both neurodegenerative and cardiovascular diseases. In animal models, CBD has been shown to inhibit the progression of several cancer types. Moreover, it has been found that coadministration of CBD and Δ9-THC, followed by radiation therapy, causes an increase of autophagy and apoptosis in cancer cells. In addition, CBD is able to inhibit cell proliferation and to increase apoptosis in different types of cancer models. These activities seem to involve also alternative pathways, such as the interactions with TRPV and GRP55 receptor complexes. Moreover, the finding that the acidic precursor of CBD (cannabidiolic acid, CBDA) is able to inhibit the migration of breast cancer cells and to downregulate the proto-oncogene c-fos and the cyclooxygenase-2 (COX-2) highlights the possibility that CBDA might act on a common pathway of inflammation and cancer mechanisms, which might be responsible for its anticancer activity. In the light of all these findings, in this review we explore the effects and the molecular mechanisms of CBD on inflammation and cancer processes, highlighting also the role of minor cannabinoids and noncannabinoids constituents of Δ9-THC deprived hemp.

Hermetia illucens larvae are among the most promising insects for use as food or feed ingredients due to their ability to convert organic waste into biomass with high-quality proteins. In this novel food or feed source, the absence of antibiotic-resistant bacteria and their antibiotic resistance (AR) genes, which could be horizontally transferred to animal or human pathogens through the food chain, must be guaranteed. This study was conducted to enhance the extremely scarce knowledge on the occurrence of AR genes conferring resistance to the main classes of antibiotics in a rearing chain of H. illucens larvae and how they were affected by rearing substrates based on coffee silverskin supplemented with increasing percentages of Schizochytrium limacinum or Isochrysis galbana microalgae. Overall, the PCR and nested PCR assays showed a high prevalence of tetracycline resistance genes. No significant effect of rearing substrates on the distribution of the AR genes in the H. illucens larvae was observed. In contrast, the frass samples were characterized by a significant accumulation of AR genes, and this phenomenon was particularly evident for the samples collected after rearing H. illucens larvae on substrates supplemented with high percentages (&gt, 20%) of I. galbana. The latter finding indicates potential safety concerns in reusing frass in agriculture.

Dystrophin deficiency is associated with alterations in cell physiology. The functional consequences of dystrophin deficiency are particularly severe for muscle physiology, as observed in Duchenne muscle dystrophy (DMD). DMD is caused by the absence of a 427 kDa isoform of dystrophin. However, in addition to muscular dystrophy symptoms, DMD is frequently associated with memory and attention deficits and epilepsy. While this may be associated with a role for dystrophin in neuronal physiology, it is not clear what neuronal alterations are linked with DMD. Our work shows that CA1 pyramidal neurons from DBA/2J-mdx mice have increased afterhyperpolarization compared to WT controls. All the other electrotonic and electrogenic membrane properties were unaffected by this genotype. Finally, basal synaptic transmission, short-term and long-term synaptic plasticity at Schaffer collateral to CA1 glutamatergic synapses were unchanged between mdx and WT controls. These data show that the excitatory component of hippocampal activity is largely preserved in DBA/2J-mdx mice. Further studies, extending the investigation to the inhibitory GABAergic function, may provide a more complete picture of the functional, network alterations underlying impaired cognition in DMD. In addition, the investigation of changes in neuronal single conductance biophysical properties associated with this genotype, is required to identify the functional alterations associated with dystrophin deficiency and clarify its role in neuronal function.

In recent years the possibility of observing by microscopy the dynamic activity of living cells has been largely pursued. We have developed a low-cost (~ 260 euros) on-stage cell incubator for inverted optical microscopes. This device allows to keep cells in good conditions for their survival and proliferation. The device is based on the use of the Arduino microprocessor interfaced with LabView. It can be connected to a computer via USB port allowing to monitor and register all the useful parameters of the measurements: temperature, CO2 concentration and relative humidity. It consists of a closed metallic and plastic (PMMA) chassis which provides optical transparency to the petri dish in order to use interference contrast imaging techniques. The system exploits also a second Arduino microprocessor to perform autofocus of the images and to automatically acquire images at defined time intervals. Cell biology laboratories could easily construct this device to allow also students to follow dynamic processes of living cells and to practice with the DIY (Do-It-Yourself) approach to biology. At the same time, students could become familiar with the use of low-cost microprocessors like Arduino.

Background:Worldwide antibiotic abuse represents a huge burden, which can have a deep impact on pet and human health through nutrition and medicalization representing another way of antibiotic resistance transmission.Objective:We aimed our research to determine a possible complex formation between biological bone substrates, such as proteins, and Oxytetracycline (OTC), an approved antibiotic for use in zootechny, which might determine a toxic effect on K562 cells.Method:Cell viability and HPLC-ESI/QqToF assays were used to assess potential toxicity of bone extract derived from OTC-treated chickens according to standard withdrawal times and from untreated chickens at 24, 48 and 72h of incubation.Results:Cell culture medium with ground bone from chickens reared in the presence of OTC (OTC-CCM) resulted significantly cytotoxic at every incubation time regardless of the bone concentration while cell culture medium with ground bone from chickens reared without OTC (BIO-CCM) resulted significantly cytotoxic only after 72h of incubation. HPLC-ESI/QqToF assay ruled out the possible presence of OTC main derivatives possibly released by bone within culture medium until 1 μg/mL.Conclusion:The presence of a protein complex with OTC is able to exert a cytotoxic effect once released in the medium after 24-48h of incubation.

This study was designed to assess the effect of the phytoestrogenic compound ferutinin, chronically administered in ovariectomized progesterone primed rats, alone or in combination with estradiol benzoate. After 2, 3 and 4 weeks of treatments, female rats were tested for receptive (lordosis) and proceptive behaviors (hops, darts and ear wigglings). Ferutinin given alone markedly increased the intensity of the lordotic response in ovariectomized rats but failed to significantly affect proceptivity. On the other hand estradiol benzoate significantly increased both receptive and proceptive behaviors. When administered in combination with estradiol, ferutinin reduced the increase in receptivity and proceptivity due to estrogen effects, acting as an antiestrogen. At the end of the behavioral experiments, animals were sacrificed and Western blot analysis of estrogen receptor alpha (ERalpha) levels was performed in the dissected hypothalami. Ferutinin increased ERalpha expression when administered alone, as estradiol did, but decreased the response to estradiol when administered in combination. These results suggest that ferutinin displays estrogenic or antiestrogenic activity through ERalpha in the hypothalamus, depending on the absence or the presence of estrogen priming.

Benzodiazepines of natural origin (NBZDs) have been found in human blood and brains as well as in medicinal plants and foods. In plasma and brain tissue there are i.e. diazepam and nordiazepam equal to commercial drugs but there are also other benzodiazepine-like compounds termed "endozepines", which act as agonists at the benzodiazepine receptors of central type (CBR). A synthetic pathway for the production of NBZDs has not yet been found, but it has been suggested that micro-organisms may synthesize molecules with benzodiazepine-like structures. Hence NBZDs could be of both endogenous and exogenous source and be considered as natural anxyolitic and sedative. Interestingly there are also natural compounds, such as the polypeptide Diazepam Binding Inhibitor (DBI) acting as an "inversive agonist" implicated in fair and panic disorders. It has been suggested that NBZDs may play a role in the pathogenesis of hepatic encephalopathy (HE). Multidirectional studies evaluated NBZDs levels (1) in the blood of normal subjects, of cirrhotic with or without HE and in commercial benzodiazepine consumers; (2) in the blood of cirrhotic treated or not with a non-absorbable antibiotic; (3) in several constituents of our diet. In conclusion, NBZDs increase sometime in cirrhotics with or without HE but they reach concentrations not higher than those found in commercial benzodiazepines consumers. Hence NBZDs must be considered as occasional precipitating factor of HE and benzodiazepine antagonists only symptomatic drugs. The finding that NBZDs may be in part synthesized by intestinal bacterial flora and in part constituent of our diet underlines the importance to feed cirrhotic patients with selected food.

Many reports have shown promising beneficial effects of long-chain polyunsaturated fatty acids (L-PUFAs) of the omega 3 series in several brain diseases. In the present study, we tested the hypothesis that omega 3 fatty acids supplement reduced pro-inflammatory functions in vitro and in vivo. We demonstrated that a supplement rich in PUFAs (SRP) increased cell viability in a dose-dependent manner suggesting its protective role against lipopolysaccharide (LPS)-induced cell death in BV2 microglial cell line. In the same cultures, the supplement rich in PUFAs reduced the reactive oxygen species (ROS) and nitric oxide (NO) production. A most prominent target for ROS management is the family of peroxisome proliferator-activated receptors (PPARs). The co-treatment with SRP and LPS increased significantly the nuclear immunoreactivity of PPAR-γwhen compared the LPS treatment alone. Moreover, the chronic administration of the SRP in rats, increased the immunoreactivity of the PPAR-γ1 protein confirming its potential neuroprotective effect.

PBR is involved in numerous biological functions, including steroid biosynthesis, mitochondrial oxidative phosphorylation and cell proliferation. The presence of PBR at the perinuclear/nuclear subcellular level has been demonstrated in aggressive breast cancer cell lines and human glioma cells where it seems to be involved in cell proliferation. In our study we investigated the presence of perinuclear/nuclear PBR in different hepatic tumor cell lines with regard to binding to [3H] PK 11195 and protein analysis. The results obtained by saturation binding experiments and scatchard analysis of perinuclear/nuclear PBR density in parallel with the results on the growth curves of the cell lines tested, indicate that the perinuclear/nuclear PBR density correlates inversely with cell doubling time. Moreover, the cell line with high perinuclear/nuclear PBR proliferated in response to PBR ligand, whereas that with low perinuclear/nuclear PBR did not. Our results reinforce the idea that the subcellular localisation of PBR defines its function and that this receptor could be a possible target for new strategies against cancer.

Extracts from pods and leaves of carob (Ceratonia siliqua L.) were tested for their ability to inhibit cell proliferation of mouse hepatocellular carcinoma cell line (T1). The two extracts showed a marked alteration of T1 cell proliferation in a dose-related fashion reaching the maximal effect at 1 mg/ml. Moreover, we demonstrated that leaf and pod extracts were able to induce apoptosis in T1 cell lines after 24-h treatment mediating a direct activation of the caspase 3 pathway. HPLC analysis revealed the presence of gallic acid, (-) epigallocatechin-3-gallate and (-) epicatechin-3-gallate in pod and leaf extracts, compounds well known to exert antiproliferative effects. Their concentration reached 6.28 mg/g in carob leaves and 1.36 mg/g in carob pods extract. The discovery that carob pod and leaf extracts contained antiproliferative agents could be of practical importance in the development of functional foods and/or chemopreventive drugs.

We have shown that the vertebrate neuropeptide N-acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short-term treatment of cerebellar granule cells with NAAG (30 microM) results in the transient increase in content of GABA(A) alpha6 subunit mRNA. Using quantitative PCR, this increase was determined to be up to 170% of control values. Similar effects are seen following treatment with trans-1-aminocyclopentane-1,3-dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl) glycine and (2S)-alpha-ethylglutamic acid. The effect is pertussis toxin-sensitive. The increase in alpha6 subunit mRNA level can be simulated by activation of other receptors negatively linked to adenylate cyclase activity, such as adenosine A1, alpha2-adrenergic, muscarinic, and GABA(B) receptors. Forskolin stimulation of cyclic AMP (cAMP) levels abolished the effect of NAAG. The change in alpha6 levels induced by 30 microM NAAG can be inhibited in a dose-dependent manner by simultaneous application of increasing doses of the beta-adrenergic receptor agonist isoproterenol. The increase in alpha6 mRNA content is followed by a fourfold increase in alpha6 protein level 6 h posttreatment. Under voltage-clamped conditions, NAAG-treated granule cells demonstrate an increase in the furosemide-induced inhibition of GABA-gated currents in a concentration-dependent manner, indicating an increase in functional alpha6-containing GABA(A) receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABA(A) alpha6 subunit via a cAMP-mediated pathway and that cAMP-coupled receptors for other neurotransmitters may similarly influence GABA(A) receptor subunit composition.

Recordings of NMDA-activated currents from cerebellar granule neurons in culture revealed a developmental increase in current density accompanied by a slight decrease of the half-maximal effective concentration. At the same time, a decrease of NMDA receptors comprising NR2B subunits was demonstrated by the reduction in the antagonism of NMDA currents by ifenprodil. Ifenprodil antagonism increased after treatment for 24 h with KN93- and KN62-selective inhibitors of the Ca2+/calmodulin-dependent protein kinases (CaM kinases), indicating a selective increase of receptor containing NR2B subunit. This increase was observed at all ages tested: 4 days in vitro (DIV4), DIV6, and DIV13. Western blot analysis with specific NMDA receptor antibodies performed at DIV6 confirmed the electrophysiological data. At this age, the negative control KN92 was ineffective. The increasing ifenprodil antagonism after KN93 treatment was proportionally greater in cells at DIV13 than at DIV4. Treatment with NMDA (100 microM) of cerebellar cultures for 24 h produced a decrease in the NMDA-induced current density by almost 50% at all ages tested. Ifenprodil antagonism, however, was unchanged. We propose that the expression of NR2B subunits in cerebellar granule cells is selectively stimulated by the inhibition of CaM kinases.

Metal-based nanoparticles (NPs), are currently used in many application fields including consumer products, pharmaceuticals, and biomedical treatments. In spite to their wide applications, an in-depth study of their potential toxic effects is still lacking. The aim of the present research was to investigate the potential initiator or promoter-like activity of different metallic NPs such as gold, iron, cobalt, and cerium using the Balb/3T3 two-stage transformation assay. The results indicated that all the selected metallic NPs, except for cobalt, when used as initiators did not induce any transformation in Balb/3T3 cell line. Moreover, Au and Fe3O4 NPs, when used in place of the tumor promoter treatment TPA, increased significantly the number of Foci/dish as compared to the MCA treatment alone. The number of Foci/dish was 2.6 for Au NPs and 2.13 for Fe3O4 ones, similar to those obtained by the positive control treatment (MCA + TPA), whereas 1.27 for MCA treatment alone. On the contrary, CeO2 NPs did not show any difference in the number of Foci/dish, as compared to MCA alone, but it decreased the number of foci by 65% in comparison to the positive control (MCA + TPA). As expected, cobalt NPs showed an increased cytotoxicity and only a few surviving cells were found at the time of analysis showing a number of Foci/dish of 0.13. For the first time, our data clearly showed that Au and Fe3O4 NPs act as promoters in the two stage transformational assay, suggesting the importance to fully investigate the NPs carcinogenic potential with different models. © 2014 Wiley Periodicals, Inc. Environ Toxicol, 2014.

Aims The aim of this study is to investigate the effect of hemin in colonic epithelial cells (Caco-2) cell proliferation and if this effect was due to a direct modulation of 18-kDa translocator protein (TSPO) and/or heme oxygenase type 1 (HO-1). Main methods The main methods are as follow: cell proliferation and cell cytotoxic assays on Caco-2 cell lines treated with hemin in the presence or not of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195) and Sn-protoporphyrin IX (Sn-PPIX), and immunoblotting for TSPO and HO-1 protein analysis, siRNA directed against TSPO. Key findings Hemin was shown to be toxic for the Caco-2 cell line in a concentration and time dependent manner. Although hemin was able to induce HO-1 in a dose dependent manner, a specific HO-1 inhibitor, Sn-PPIX, was unable to interfere with the effect of hemin on Caco-2 cells. Instead, PK 11195, a specific TSPO ligand, was able to counteract the effect of hemin, suggesting an important role of TSPO in the hemin activity. Cell viability assay further confirms the high cytotoxic effects exerted by hemin on Caco-2 cells expressing TSPO compared to the siRNA-TSPO targeted cells. In addition, hemin was able to decrease significantly the TSPO protein density in a dose dependent manner after 24 h of incubation. Significance The interaction and the consecutive down regulation of TSPO by hemin played an important role in the control of Caco-2 cell viability. The presented data suggest that TSPO might contribute to protect cells from potential toxic compounds such as free tetrapyrroles, candidating this receptor as a survival receptor protein.

Objectives We investigated the ability of a novel low-molecular-weight free-radical scavenger, bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate (IAC), to protect the gastric mucosa from indometacin-induced ulceration. Methods The pharmacological effects of IAC, administered orally or by intraperitoneal injection, on the gastric mucosa were assessed in a rat model of gastric ulceration induced by indometacin. The effect of IAC on the level of prostaglandin PGE2 in the gastric mucosa was also investigated. Results IAC, which has no ulcerative activity per se, had a preventive and protective activity against indometacin-induced gastric ulceration. This effect could be only partially attributed to a modulatory effect of IAC on PGE2 levels; it is more likely to be due to the antioxidant activity of the compound. Conclusions Taking into account the properties of IAC and the mechanisms underlying gastric inflammation elicited by non-steroidal anti-inflammatory drugs, IAC may represent a novel anti-ulcer agent.

Background/Aim—Despite some controversy, it has been suggested that endogenous benzodiazepine plays a role in the pathogenesis of hepatic encephalopathy. The aim of the present study was to evaluate the concentrations of endogenous benzodiazepines and the peptide, diazepam binding inhibitor, in the blood of patients with liver cirrhosis with and without overt encephalopathy, and to compare these levels with those of consumers of commercial benzodiazepines.Subjects—Normal subjects (90), benzodiazepine consumers (14), and cirrhotic patients (113) were studied.Methods—Endogenous benzodiazepines were measured by the radioligand binding technique after high performance liquid chromatography (HPLC) purification. The presence of diazepam andN-desmethyldiazepam was assayed by HPLC-electrospray tandem mass spectrometry. Diazepam binding inhibitor was studied in serum by radioimmunoassay.Results—Endogenous benzodiazepines were below the limit of detection in 7% of patients with encephalopathy. When detectable, their levels were at least comparable with those of benzodiazepine consumers and correlated with the liver dysfunction but not the stage of encephalopathy. Serum levels of diazepam binding inhibitor tended to decrease when endogenous benzodiazepines levels increased.Conclusions—Endogenous benzodiazepines may accumulate in patients with liver cirrhosis during the course of the disease, and the phenomenon appears to be independent of the presence or absence of encephalopathy.

Immunoblot analysis, using antibodies against distinct N-methyl-d-aspartic acid (NMDA) receptor subunits, illustrated that the NR2A and NR2B subunit proteins have developmental profiles in cultured cortical neurons similar to those seen in vivo. NR1 and NR2B subunits display high levels of expression within the first week. In contrast, the NR2A subunit is barely detectable at 7 days in vitro (DIV) and then gradually increased to mature levels at DIV21. Immunocytochemical analysis indicated that NMDA receptor subunits cluster in the dendrites and soma of cortical neurons. Clusters of NR1 and NR2B subunits were observed as early as DIV3, while NR2A clusters were rarely observed before DIV10. At DIV18, NR2B clusters partially co-localize with those of NR2A subunits, but NR2B clusters always co-localize with those of NR1 subunits. Synapse formation, as indicated by the presence of presynaptic synaptophysin staining, was observed as early as 48–72 h after plating. However, in several neurons at ages less than DIV5 where synapses were scarce, NR2B and NR1 clusters were abundant. Furthermore, while NR2B subunit clusters were seen both at synaptic and extrasynaptic sites, NR2A clusters occurred almost exclusively in front of synaptophysin-labelled boutons. This result was supported by electrophysiological recording of NMDA-mediated synaptic activity [NMDA-excitatory postsynaptic currents (EPSCs)] in developing neurons. At DIV6, but not at DIV12, CP101, 606, a NR1/NR2B receptor antagonist, antagonized spontaneously occurring NMDA-EPSCs. Our data indicate that excitatory synapse formation occurs when NMDA receptors comprise NR1 and NR2B subunits, and that NR2A subunits cluster preferentially at synaptic sites.

Cholesterol is used by cells for biosynthetic processes and for steroid synthesis. Although the role of cholesterol in tumorigenesis is not clear it is known that steroids are important factors in human carcinogenesis. A polypeptide, diazepam binding inhibitor (DBI), which is an endogenous ligand for peripheral benzodiazepine receptors enhances steroidigenesis by promoting cholesterol delivery to the inner mitochondrial membrane which represents the rate-limiting step of steroid biosynthesis. We have assayed the total cholesterol (TC) and the DBI plasma concentrations in patients with liver cirrhosis complicated by hepatocellular carcinoma (HCC) in comparison with those of uncomplicated liver cirrhosis. TC and DBI levels have been studied in 73 cirrhotic patients and in 23 patients with HCC. Both TC and DBI levels were higher in HCC patients when compared with age, sex and Child-Pugh class matched cirrhotic controls. The values (mean+/-S.D.) in patients in Child-Pugh class B and C with and without HCC were respectively 128+/-30 mg/dl vs. 106+/-27 mg/dl (P < 0.01) and 2.05+/-0.78 pmol/ml vs. 0.78+/-0.84 pmol/ml (P < 0.0001). The data may be the result of the metabolic influence of tumors that enhances steroid biosynthesis during tumor proliferation.