Nerve growth factor and bromocriptine: a sequential therapy for human bromocriptine-resistant prolactinomas.

Nerve growth factor (NGF) administration to athymic mice with transplanted human bromocriptine-resistant prolactinoma, results in the expression of dopamine D-2 receptors in the tumour and restores sensitivity to subsequent treatment with bromocriptine, which then produces normalisation of plasma prolactin and tumour regression. Sequential administration of NGF and bromocriptine thus may be a promising therapy for patients refractory to bromocriptine

Opposing Roles for NF-κB/Rel Factors p65 and c-Rel in the Modulation of Neuron Survival Elicited by Glutamate and Interleukin-1β

The nuclear transcription factors NF-kappaB/Rel have been shown to function as key regulators of either cell death or survival in neuronal cells. Here, we investigated whether selective activation of diverse NF-kappaB/Rel family members might lead to distinct effects on neuron viability. In both cultured rat cerebellar granule cells and mouse hippocampal slices, we examined NF-kappaB/Rel activation induced by two opposing modulators of cell viability: 1) interleukin-1beta (IL-1beta), which promotes neuron survival and 2) glutamate, which can elicit toxicity. IL-1beta produced a prolonged stimulation of NF-kappaB/Rel factors by inducing both IkappaBalpha and IkappaBbeta degradation. Glutamate produced a delayed and transient activation of NF-kappaB/Rel, which was associated with a brief loss of IkappaBalpha. Moreover, IL-1beta activated the p50, p65, and c-Rel subunits of NF-kappaB/Rel, whereas glutamate activated only the p50 and p65 proteins. The inhibition of NF-kappaB/Rel protein expression by antisense oligonucleotides in cerebellar granule cells showed that p65 was involved in glutamate-mediated cell death, whereas c-Rel was essential for IL-1beta-preserved cell survival. Furthermore, the depletion of c-Rel in cultured neurons as well as in the hippocampus from the c-Rel(-/-) mouse converted the IL-1beta effect into toxicity. These findings suggest that, within a single neuron, the balance between cell death and survival in response to external stimuli may rely on the activation of distinct NF-kappaB/Rel proteins

Lack of coupling of D-2 receptors to adenylate cyclase in GH-3 cells exposed to epidermal growth factor. Possible role of a differential expression of Gi protein subtypes.

Exposure of GH-3 cells to epidermal growth factor for 4 consecutive days induced the expression of both D-2(415) and D-2(444) dopamine-receptor isoforms. Epidermal growth factor also promoted a remarkable increase in the content of Gi3 protein, which is responsible for receptor-induced activation of potassium channels in GH-3 cells. D-2 receptors in this model apparently activate a specific transducing pathway, leading to opening of potassium channels and inhibition of prolactin release by cAMP-independent mechanisms. This is shown by: 1) the selective D-2 agonist quinpirole, while inactive on vasoactive intestinal peptide-induced prolactin release, strongly inhibited the hormone secretion induced by neurotensin; 2) quinpirole, up to 100 microM, did not inhibit cAMP production evoked by vasoactive intestinal peptide both in intact cells and in broken cell membrane preparations; and 3) quinpirole and other D-2 agonists strongly potentiated Rb+ efflux when measured in a nominally calcium-free reaction solution containing 100 mM potassium (voltage-dependent component), but did not modify Rb+ efflux if measured in a reaction solution containing 1 mM calcium and 5 mM potassium (calcium-activated, cAMP-dependent component)

Glutamatergic Reinnervation and Assembly of Glutamatergic Synapses in Adult Rat Skeletal Muscle Occurs at Cholinergic Endplates

After denervation of adult rat abdominal muscles, the postsynaptic apparatus of neuromuscular junctions (NMJs) retains its original architecture and clustering of acetylcholine receptors (AChRs). When descending fibers of the spinal cord are surgically diverted to this muscle by a nerve grafting procedure, supraspinal glutamatergic neurons can innervate muscle fibers and restore motor function; the newly formed NMJs switch from a cholinergic to a glutamatergic-type synapse. We show here that regenerating nerve endings contact the fibers in an area occupied by cholinergic endplates. These NMJs are morphologically indistinguishable from those in controls, but they differ in the subunit composition of AChRs. Moreover, by immunofluorescence and immunoelectron microscopy, new NMJs express glutamatergic synapse markers. The \u3b1-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1 partially colocalizes with AChRs, and vesicular glutamate transporter 2 is localized in the presynaptic compartment. Immunoprecipitation analysis of membranes from reinnervated muscle showed that AMPA receptor subunits GluR1 and GluR2 coimmunoprecipitate with rapsyn, the AChR-anchoring protein at the NMJ. Taken together, these results indicate that cholinergic endplates can be targeted by new glutamatergic projections and that the clustering of AMPA receptors occurs there

Severity of oxidative stress and inflammatory activation in end-stage heart failure patients are unaltered after 1 month of left ventricular mechanical assistance

This study investigates the impact of early left ventricular (LV)-mechanical unloading on systemic oxidative stress and inflammation in terminal heart failure patients and their impact both on multi organ failure and on intensive care unit (ICU) stay. Circulating levels of urinary 15-isoprostane-F2t (8-epi-PGF2a) and pro-inflammatory markers [plasma interleukin (IL)-6, IL-8, and urinary neopterin, a monocyte activation index] were analyzed in 20 healthy subjects, 22 stable end-stage heart failure (ESHF) patients and in 23 LV assist device (LVAD) recipients at pre-implant and during first post-LVAD (PL) month. Multiorgan function was evaluated by total Sequential Organ Failure Assessment (tSOFA) score. In LVAD recipients the levels of oxidative-inflammatory markers and tSOFA score were higher compared to other groups. After device implantation 8-epi-PGF2a levels were unchanged, while IL-6, and IL-8 levels increased during first week, and at 1 month returned to pre-implant values, while neopterin levels increased progressively during LVAD support. The tSOFA score worsened at 1 PL-week with respect to pre-implant value, but improved at 1 PL-month. The tSOFA score related with IL-6 and IL-8 levels, while length of ICU stay related with pre-implant IL-6 levels. These data suggest that hemodynamic instability in terminal HF is associated to worsening of systemic inflammatory and oxidative milieu that do not improve in the early phase of hemodynamic recovery and LV-unloading by LVAD, affecting multi-organ function and length of ICU stay. This data stimulate to evaluate the impact of inflammatory signals on long-term outcome of mechanical circulatory support

Revealing new perspectives on the regulation of gene expression by the trans-splicing mechanism in Shistosoma mansoni.

Schistosomiasis is the second most prevalent neglected tropical disease caused by a Trematode flatworm from the genus Schistosoma. Schistosomes possess a complex life cycle requiring numerous and intricate mechanisms to rule transcriptional and post-transcriptional gene regulation. Thus, it is believed that spliced leader (SL) trans-splicing could play an important role in the parasite biology. The purpose of this study was is to shed light on the function of the trans-splicing mechanism in S. mansoni by searching gene categories that could be target of this process and attempting to silence transcripts harboring the SL sequence. Here, we analyze different life stages cDNA libraries enriched on SL-transcripts of S. mansoni. Our results show that, contrary to previous hypothesis, trans-spliced transcripts are associated to specific gene categories, such as carbohydrate metabolic process, RNA metabolism and DNA repair. Several trans-spliced transcripts were verified in at least two different stages of the S. mansoni life cycle. The marjority of the SL-transcripts are shared among males, females and mixed-sex adults, being followed by SL-transcripts shared by all adults and schistosomula. Among the analyzed life stages, the one presenting the lower number of shared genes is the egg. The results also reveal a set of SL-transcripts that are observed only in one stage of the life cycle and could represent transcripts that undergo stage-specific trans-splicing. Finally, we demonstrated that trans-splicing knockdown in sprocysts caused a reduction on the levels of all tested trans-spliced transcripts, but surprisingly the only phenotypic effect observed was a diminished larvae size. Financial support: CNPq, FAPEMIGX-MEETING 2011

Association of pre-operative interleukin-6 levels with Interagency Registry for Mechanically Assisted Circulatory Support profiles and intensive care unit stay in left ventricular assist device patients

BACKGROUND: Inflammatory mechanisms are associated with worse prognosis in end-stage heart failure (ESHF) patients who require left ventricular assist device (LVAD) support. Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) profiles describe patient condition at pre-implant and outcome. This study assessed the relationship among inflammation patterns and INTERMACS profiles in LVAD recipients. METHOD: Thirty ESHF patients undergoing LVAD implantation as bridge to transplant were enrolled. Blood and urine samples were collected pre-operatively and serially up to 2 weeks post-operatively for assessment of inflammatory markers (plasma levels of interleukin [IL]-6, IL-8, IL-10, and osteopontin, a cardiac inflammatory-remodeling marker; and the urine neopterin/creatinine ratio, a monocyte activation marker). Multiorgan function was evaluated by the total sequential organ failure assessment (tSOFA) score. Outcomes of interest were early survival, post-LVAD tSOFA score, and intensive care unit (ICU) length of stay. RESULTS: Fifteen patients had INTERMACS profiles 1 or 2 (Group A), and 15 had profiles 3 or 4 (Group B). At pre-implant, only IL-6 levels and the IL-6/IL-10 ratio were higher in Group A vs B. After LVAD implantation, neopterin/creatinine ratio and IL-8 levels increased more in Group A vs B. Osteopontin levels increased significantly only in Group B. The tSOFA score at 2 weeks post-LVAD and ICU duration were related with pre-implant IL-6 levels. CONCLUSIONS: The INTERMACS profiles reflect the severity of the pre-operative inflammatory activation and the post-implant inflammatory response, affecting post-operative tSOFA score and ICU stay. Therefore, inflammation may contribute to poor outcome in patients with severe INTERMACS profile

Automating multimodal microscopy with NanoJ-Fluidics

Fluorescence microscopy can reveal all aspects of cellular mechanisms, from molecular details to dynamics, thanks to approaches such as super-resolution and live-cell imaging. Each of its modalities requires specific sample preparation and imaging conditions to obtain high-quality, artefact-free images, ultimately providing complementary information. Combining and multiplexing microscopy approaches is crucial to understand cellular events, but requires elaborate workflows involving multiple sample preparation steps. We present a robust fluidics approach to automate complex sequences of treatment, labelling and imaging of live and fixed cells. Our open-source NanoJ-Fluidics system is based on low-cost LEGO hardware controlled by ImageJ-based software and can be directly adapted to any microscope, providing easy-to-implement high-content, multimodal imaging with high reproducibility. We demonstrate its capacity to carry out complex sequences of experiments such as super-resolved live-to-fixed imaging to study actin dynamics; highly-multiplexed STORM and DNA-PAINT acquisitions of multiple targets; and event-driven fixation microscopy to study the role of adhesion contacts in mitosis