Viability of D283 medulloblastoma cells treated with a histone deacetylase inhibitor combined with bombesin receptor antagonists

Purpose

Medulloblastoma (MB) comprises four distinct molecular subgroups, and survival remains particularly poor in patients with Group 3 tumors. Mutations and copy number variations result in altered epigenetic regulation of gene expression in Group 3 MB. Histone deacetylase inhibitors (HDACi) reduce proliferation, promote cell death and neuronal differentiation, and increase sensitivity to radiation and chemotherapy in experimental MB. Bombesin receptor antagonists potentiate the antiproliferative effects of HDACi in lung cancer cells and show promise as experimental therapies for several human cancers. Here, we examined the viability of D283 cells, which belong to Group 3 MB, treated with an HDACi alone or combined with bombesin receptor antagonists.

Methods

D283 MB cells were treated with different doses of the HDACi sodium butyrate (NaB), the neuromedin B receptor (NMBR) antagonist BIM-23127, the gastrin releasing peptide receptor (GRPR) antagonist RC-3095, or combinations of NaB with each receptor antagonist. Cell viability was examined by cell counting.

Results

NaB alone or combined with receptor antagonists reduced cell viability at all doses tested. BIM-23127 alone did not affect cell viability, whereas RC-3095 at an intermediate dose significantly increased cell number.

Conclusion

Although HDACi are promising agents to inhibit MB growth, the present results provide preliminary evidence that combining HDACi with bombesin receptor antagonists is not an effective strategy to improve the effects of HDACi against MB cells.

     

Simultaneous seminoma and interstitial cell tumour in a rabbit with a previous cutaneous basal cell tumour.

The development of spontaneous multiple tumours is a rare event in domestic rabbits. The diagnosis of a cutaneous basal cell tumour and the successive development of simultaneous bilateral testicular tumours with dissimilar histology (a seminoma and an interstitial cell tumour) are described in a vasectomized, crossbred dwarf rabbit, aged 6 years. Two cases of basalioma associated with uterine adenocarcinoma have been previously described in rabbits. A similar association between basal cell neoplasia and development of tumours (e.g., testicular and breast cancer) at cutaneous and non-cutaneous sites has been reported in man.     

Methylmercury inhibits glutamate uptake by synaptic vesicles from rat brain

Methylmercury (MeHg) is an environmental contaminant that continues to cause risk to human health. The toxic effects of MeHg on the CNS implicate the involvement of glutamatergic system. In this study, we evaluated the effects of MeHg on [3H]glutamate uptake by synaptic vesicles. MeHg inhibited [3H]glutamate uptake in a concentration dependent manner. Since glutamate uptake by synaptic vesicles is driven by an electrochemical gradient, formed across the vesicle membrane by a bafilomycin A(1)-sensitive H+-ATPase, we further investigated the effect of MeHg on activity of this enzyme.MeHg inhibited the H+-ATPase activity and also dissipated the proton gradient (DeltapH), indicating that MeHg decreased [3H]glutamate uptake involving the H+-ATPase activity. Until now, the toxic effects of MeHg on CNS were attributed mainly to an impairment of glial glutamate transporters. These findings contribute for the understanding of the neurotoxicity by MeHg, pointing to the involvement of vesicular glutamate.     

Impaired hippocampal glucoregulation in the cannabinoid CB1 receptor knockout mice as revealed by an optimized in vitro experimental approach

Several techniques exist to study the rate of glucose uptake and metabolism in the brain but most of them are not sufficiently robust to permit extensive pharmacological analysis. Here we optimized an in vitro measurement of the simultaneous accumulation of the metabolizable and non-metabolizable ³H and ¹⁴C d-glucose analogues; permitting convenient large-scale studies on glucose uptake and metabolism in brain slices. Next, we performed an extensive pharmacological characterization on the putative glucoregulator role of the endocannabinoid system in the hippocampal slices of the rat, and the wild-type and the CB₁ cannabinoid receptor (CB₁R) knockout mice.We observed that ³H-3-O-methylglucose is a poor substrate to measure glucose uptake in the hippocampus. ³H-2-deoxyglucose is a better substrate but its uptake is still lower than that of ¹⁴C-U-d-glucose, from which the slices constantly metabolize and dissipate ¹⁴C atoms. Thus, uptake and the metabolism values are not to be used as standalones but as differences between a control and a treatment.The CB₁R knockout mice exhibited ∼10% less glucose uptake and glucose carbon atom dissipation in comparison with the wild-type mice. This may represent congenital defects as acute treatments of the rat and mouse slices with cannabinoid agonists, antagonists and inhibitors of endocannabinoid uptake/metabolism failed to induce robust changes in either the uptake or the metabolism of glucose.In summary, we report here an optimized technique ideal to complement other metabolic approaches of high spatiotemporal resolution. This technique allowed us concluding that CB₁Rs are at least indirectly involved in hippocampal glucoregulation.     

Age-dependent and -independent changes in attention-deficit/hyperactivity disorder (ADHD) during spatial working memory performance

Objectives: Attention-deficit/hyperactivity disorder (ADHD) has been associated with spatial working memory as well as frontostriatal core deficits. However, it is still unclear how the link between these frontostriatal deficits and working memory function in ADHD differs in children and adults. This study examined spatial working memory in adults and children with ADHD, focussing on identifying regions demonstrating age-invariant or age-dependent abnormalities. Methods: We used functional magnetic resonance imaging to examine a group of 26 children and 35 adults to study load manipulated spatial working memory in patients and controls. Results: In comparison to healthy controls, patients demonstrated reduced positive parietal and frontostriatal load effects, i.e., less increase in brain activity from low to high load, despite similar task performance. In addition, younger patients showed negative load effects, i.e., a decrease in brain activity from low to high load, in medial prefrontal regions. Load effect differences between ADHD and controls that differed between age groups were found predominantly in prefrontal regions. Age-invariant load effect differences occurred predominantly in frontostriatal regions. Conclusions: The age-dependent deviations support the role of prefrontal maturation and compensation in ADHD, while the age-invariant alterations observed in frontostriatal regions provide further evidence that these regions reflect a core pathophysiology in ADHD.     

Cardiovascular disease risk factor profiles in children with celiac disease on gluten-free diets

AIM:To describe the cardiovascular disease(CVD)risk factors in a population of children with celiac disease(CD)on a gluten-free diet(GFD).METHODS:This cross-sectional multicenter study was performed at Schneider Children’s Medical Center of Israel(Petach Tiqva,Israel),and San Paolo Hospital(Milan,Italy).We enrolled 114 CD children in serologic remission,who were on a GFD for at least one year.At enrollment,anthropometric measurements,blood lipids and glucose were assessed,and compared to values at diagnosis.The homeostasis model assessment-estimated insulin resistance was calculated as a measure of insulin resistance.RESULTS:Three or more concomitant CVD risk factors[body mass index,waist circumference,low density lipoprotein(LDL)cholesterol,triglycerides,blood pressure and insulin resistance]were identified in 14%of CD subjects on a GFD.The most common CVD risk factors were high fasting triglycerides(34.8%),elevated blood pressure(29.4%),and high concentrations of calculated LDL cholesterol(24.1%).On a GFD,four children(3.5%)had insulin resistance.Fasting insulin and HOMA-IR were significantly higher in the Italian cohort compared to the Israeli cohort(P<0.001).Children on a GFD had an increased prevalence of borderline LDL cholesterol(24%)when compared to values(10%)at diagnosis(P=0.090).Trends towards increases in overweight(from 8.8%to 11.5%)and obesity(from 5.3%to 8.8%)were seen on a GFD.CONCLUSION:This report of insulin resistance and CVD risk factors in celiac children highlights the importance of CVD screening,and the need for dietary counseling targeting CVD prevention.     

Performance Survey and Comparison Between Rapid Sterility Testing Method and Pharmacopoeia Sterility Test

The sterility test described in pharmacopoeial compendia requires a 14-day incubation period to obtain a valid analytical result. Therefore, the use of alternative methods to evaluate the sterility of pharmaceuticals, such as the BacT/Alert® 3D system, is particularly interesting, because it allows a reduced incubation period and lower associated costs. Considering that the BacT/Alert® 3D system offers several culture media formulations developed for this microbial detection system, the present study was aimed to evaluate and compare the performance of BacT/Alert® 3D with the pharmacopoeial sterility test. There was no significant difference between the ability of the culture media to allow detection of microbial contamination. However, the rapid sterility testing method allowed a more rapid detection of the challenge microorganisms, which indicates that the system is a viable alternative for assessing the sterility of injectable products.     

Exercise affects glutamate receptors in postsynaptic densities from cortical mice brain

Physical activity has been proposed as a behavior intervention that promotes mental health and some of the benefits induced by exercise have been related to the glutamatergic system. Indeed, glutamate is the most abundant excitatory neurotransmitter in brain. Thus, we evaluated if voluntary exercise in mice could modulate glutamatergic synapses at level of postsynaptic density (PSD). Through Western blot, we found that exercise during 1 month increased glutamatergic-related protein content in PSD from cortex of mice. Exercise increased the immunocontent of GluR1 (129%), SAP-97 (179%), GRIP-1 (129%), and in less extent, GluR2/3 (118%) and PSD-95 (112%) proteins. The overall content of NMDA subunits R1, R2A and R2B were not altered in mice that had exercised, however, the phosphorylated NMDA subunits, phospho-NMDAR1 (150%), and phospho-NMDAR2B (183%) showed a strong increase. Because exercise increased the content of phosphorylated forms of NMDA receptors, we evaluated the binding of MK-801, a specific ligand that binds to open NMDA channel. Exercise increased the binding of MK-801 in cortical cellular membranes in 51%. Altogether, our results point to a modulation of glutamatergic synapses by exercise with likely implications in the exercise-induced mental health.     

The Role of Ambient Gas and Pressure on the Structuring of Hard Diamond-Like Carbon Films Synthesized by Pulsed Laser Deposition

Hard carbon thin films were synthesized on Si (100) and quartz substrates by the Pulsed Laser Deposition (PLD) technique in vacuum or methane ambient to study their suitability for applications requiring high mechanical resistance. The deposited films’ surface morphology was investigated by scanning electron microscopy, crystalline status by X-ray diffraction, packing and density by X-ray reflectivity, chemical bonding by Raman and X-ray photoelectron spectroscopy, adherence by “pull-out” measurements and mechanical properties by nanoindentation tests. Films synthesized in vacuum were a-C DLC type, while films synthesized in methane were categorized as a-C:H. The majority of PLD films consisted of two layers: one low density layer towards the surface and a higher density layer in contact with the substrate. The deposition gas pressure played a crucial role on films thickness, component layers thickness ratio, structure and mechanical properties. The films were smooth, amorphous and composed of a mixture of sp³-sp² carbon, with sp³ content ranging between 50% and 90%. The thickness and density of the two constituent layers of a film directly determined its mechanical properties.