G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

We have recently reported that cannabinoid agonists can up-regulate and enhance the activity of serotonin 2A (5-HT_2A) receptors in the prefrontal cortex (PFCx). Increased expression and activity of cortical 5-HT_2A receptors has been associated with neuropsychiatric disorders, such as anxiety and schizophrenia. Here we report that repeated CP55940 exposure selectively up-regulates GRK5 proteins in rat PFCx and in a neuronal cell culture model. We sought to examine the mechanism underlying the regulation of GRK5 and to identify the role of GRK5 in the cannabinoid agonist-induced up-regulation and enhanced activity of 5-HT_2A receptors. Interestingly, we found that cannabinoid agonist-induced up-regulation of GRK5 involves CB₂ receptors, β-arrestin 2, and ERK1/2 signaling because treatment with CB₂ shRNA lentiviral particles, β-arrestin 2 shRNA lentiviral particles, or ERK1/2 inhibitor prevented the cannabinoid agonist-induced up-regulation of GRK5. Most importantly, we found that GRK5 shRNA lentiviral particle treatment prevented the cannabinoid agonist-induced up-regulation and enhanced 5-HT_2A receptor-mediated calcium release. Repeated cannabinoid exposure was also associated with enhanced phosphorylation of CB₂ receptors and increased interaction between β-arrestin 2 and ERK1/2. These latter phenomena were also significantly inhibited by GRK5 shRNA lentiviral treatment. Our results suggest that sustained activation of CB₂ receptors, which up-regulates 5-HT_2A receptor signaling, enhances GRK5 expression; the phosphorylation of CB₂ receptors; and the β-arrestin 2/ERK interactions. These data could provide a rationale for some of the adverse effects associated with repeated cannabinoid agonist exposure.     

Metabolism in orange fruits is driven by photooxidative stress in the leaves

In plants, stress signals propagate to trigger distant responses and thus stress acclimation in non‐exposed organs. We tested here the hypothesis that leaves submitted to photooxidative stress may influence the metabolism of nearby fruits and thus quality criteria. Leaves of orange trees (Citrus sinensis (L.) Osbeck cv. ‘Navelate’) were acclimated to shade for 1 week and then submitted to full (FL) and medium light (ML) conditions. As expected, photoinhibition was detected in leaves of both FL and ML treatments as revealed by stress indicators (F_v/F_m, Performance Index) for at least 99 h after treatments. In the fruits near the stressed leaves, we then determined the activities of enzymes related to oxidative stress, superoxide dismutase, catalase and the enzymes of the ascorbate (AA)/glutathione cycle, as well as the contents in sugars, organic acids and carotenoids. Ascorbate peroxidase and monodehydroascorbate reductase activities in the pulp of fruits were dramatically higher in both treatments when compared to the control. AA and total sugars were not affected by the photooxidative stress. However, the FL treatment resulted in a 16% increase in total organic acids, with succinic acid being the major contributor, a shift towards less glucose + fructose and more sucrose, and a 15% increase in total carotenoids, with cis‐violaxanthin being the major contributor. Our observations strongly suggest the existence of a signal generated in leaves in consequence of photooxidative stress, transmitted to nearby fruits. Exploiting such a signal by agronomic means promises exciting perspectives in managing quality criteria in fruits accumulating carotenoids.     

Collecting and tabulating race/ethnicity data with diverse and mixed heritage populations: A case-study with US high school students

The increasing diversity of the US coupled with the continuing need for information gathered about race/ethnicity require us to re-examine our practices of collecting and tabulating such data, particularly from individuals of mixed heritage. In the context of Census 2000, which allowed people for the first time to identify with multiple race groups, this article focuses on the context of education and looks at high school students’ self-­identification practices on forms. Survey data gathered from 638 freshmen during 1999–2000 at a diverse, public high school in California indicate: there can be high levels of inconsistency in students’ individual identifications depending on question format and response options provided; and, overall demographic counts can greatly vary depending on how multiple-response data are tabulated. Students’ responses raise questions about whether it is possible to attain a high level of measurement reliability when working with a diverse population that includes individuals of mixed ­heritage.     

Seasonal changes in bird assemblages of coastal woodlands in east-central Argentina

We studied seasonal variation in bird assemblages during two years in the coastal woodlands of the reserve, 'El Destino', Buenos Aires Province, Argentina. Higher bird density was recorded during spring, and lower one during autumn, but species richness did not show changes during the annual cycle. Seasonal variation in abundance and species composition was recorded for the frugivore-insectivore and the granivore guilds, however, not for the insectivores. As winter is not harsh, insectivorous species might not be faced with food shortage. In contrast, the abundance of frugivorous-insectivorous and granivorous species appeared to be related with fluctuations in their food resources.     

Biological activities of the LXRα and β agonist, 4β-hydroxycholesterol,and of its isomer, 4α-hydroxycholesterol,on oligodendrocytes: Effects on cell growth and viability,oxidative and inflammatory status

The biochemical and biological properties of 4β-hydroxycholesterol and of its isomer, 4α-hydroxycholesterol, are not well known. So, we determined the ability of 4α- and 4β-hydroxycholesterol to react with LXRα and LXRβ, and we characterized the activities of these oxysterols on oligodendrocytes which are myelin synthesizing cells. The effects of 4α- and 4β-hydroxycholesterol were studied on 158N murine oligodendrocytes to assess their activities on cell growth and viability, oxidative and inflammatory status. To this end different parameters were used: cell counting with trypan blue; identification of dead cells and cell cycle analysis with propidium iodide; evaluation of mitochondrial depolarization, lysosomal membrane integrity, actin depolimerization, nuclear morphology, and superoxide anion production after staining with JC-1, acridine orange, rhodamine-phalloidin, Hoechst 33342, and dihydroethidium, respectively; evaluation of ultrastructural changes by transmission electron microscopy, and cytokine quantification with a cytometric bead array. Only 4β-hydroxycholesterol is a LXRα and β agonist. No cytotoxic effects were found with 4α-hydroxycholesterol except a slight inhibition of cell growth at elevated concentrations. At high concentrations, 4β-hydroxycholesterol was not only able to inhibit cell growth, but also to induce cell death associated with a loss of mitochondrial transmembrane potential, dysfunctions of lysosomal membrane integrity, and superoxide anion overproduction. These side effects were lower than those observed with 7-ketocholesterol and 25-hydroxycholesterol used as positive controls. On oligodendrocyte murine primary cultures, only lysosomal membrane integrity was slightly affected under treatment with 4α- and 4β-hydroxycholesterol. So, 4α- and 4β-hydroxycholesterol have different biological activities. Their ability to induce cytotoxic effects on oligodendrocytes can be considered as weak comparatively to 7-ketocholesterol and 25-hydroxycholesterol.     

Concomitant combination of active immunotherapy and carboplatin- or paclitaxel-based chemotherapy improves anti-tumor response

Recent preclinical evidence substantially supports the successful combination of chemotherapies and active immunotherapy for cancer treatment. These data sustain the effect of sequential combination schemes (vaccine plus chemotherapy or vice versa), which could be difficult to implement in clinical practice. Since chemotherapy is the standard treatment for most cancers, ethical issues forbid its delay and make difficult the evaluation of other treatments such as using an immunotherapeutic agent. Besides, vaccines must be applied as soon as possible to advanced cancer patients, in order to give them time to develop an effective immune response. Thus, a clinically attractive scenario is the concomitant application of treatments. However, little is known about the specific effect of different chemotherapeutic agents when combined with a cancer vaccine in such concomitant treatment. In this work, we analyze the influence of high-dose carboplatin or paclitaxel in the generation of a specific immune response when administered concomitantly with an OVA vaccine. Interestingly, neither carboplatin nor paclitaxel affects the humoral and CTL in vivo response generated by the vaccine. Moreover, an enhancement of the overall anti-tumor effect was observed in animals treated with OVA/CF vaccine combined with cytotoxic drugs. Moreover, the effect of the concomitant treatment was tested using a tumor-related antigen, the epidermal growth factor (EGF). Animals administered with EGF–P64k/Montanide and cytotoxic agents showed an antibody response similar to that from control animals. Therefore, our study suggests that carboplatin and paclitaxel can be concomitantly combined with active immunotherapies in the clinical practice of advanced cancer patients.     

Diphenyl Diselenide Prevents Cortico-cerebral Mitochondrial Dysfunction and Oxidative Stress Induced by Hypercholesterolemia in LDL Receptor Knockout Mice

Recent studies have indicated a causal link between high dietary cholesterol intake and brain oxidative stress. In particular, we have previously shown a positive correlation between elevated plasma cholesterol levels, cortico-cerebral oxidative stress and mitochondrial dysfunction in low density lipoprotein receptor knockout (LDLr^?/?) mice, a mouse model of familial hypercholesterolemia. Here we show that the organoselenium compound diphenyl diselenide (PhSe)₂ (1 mg/kg; o.g., once a day for 30 days) significantly blunted the cortico-cerebral oxidative stress and mitochondrial dysfunction induced by a hypercholesterolemic diet in LDLr^?/? mice. (PhSe)₂ effectively prevented the inhibition of complex I and II activities, significantly increased the reduced glutathione (GSH) content and reduced lipoperoxidation in the cerebral cortex of hypercholesterolemic LDLr^?/? mice. Overall, (PhSe)₂ may be a promising molecule to protect against hypercholesterolemia-induced effects on the central nervous system, in addition to its already demonstrated antiatherogenic effects.     

Evaluation of four Agrobacterium tumefaciens strains for the genetic transformation of tomato (Solanum lycopersicum L.) cultivar Micro-Tom

Key message

Agrobacterium tumefaciens strains differ not only in their ability to transform tomato Micro-Tom, but also in the number of transgene copies that the strains integrate in the genome.

Abstract

The transformation efficiency of tomato (Solanum lycopersicum L.) cv. Micro-Tom with Agrobacterium tumefaciens strains AGL1, EHA105, GV3101, and MP90, harboring the plasmid pBI121 was compared. The presence of the nptII and/or uidA transgenes in regenerated T₀ plants was determined by PCR, Southern blotting, and/or GUS histochemical analyses. In addition, a rapid and reliable duplex, qPCR TaqMan assay was standardized to estimate transgene copy number. The highest transformation rate (65 %) was obtained with the Agrobacterium strain GV3101, followed by EHA105 (40 %), AGL1 (35 %), and MP90 (15 %). The mortality rate of cotyledons due to Agrobacterium overgrowth was the lowest with the strain GV3101. The Agrobacterium strain EHA105 was more efficient than GV3101 in the transfer of single T-DNA insertions of nptII and uidA transgenes into the tomato genome. Even though Agrobacterium strain MP90 had the lowest transformation rate of 15 %, the qPCR analysis showed that the strain MP90 was the most efficient in the transfer of single transgene insertions, and none of the transgenic plants produced with this strain had more than two insertion events in their genome. The combination of higher transformation efficiency and fewer transgene insertions in plants transformed using EHA105 makes this Agrobacterium strain optimal for functional genomics and biotechnological applications in tomato.