Stress-Induced Antinociception in Fish Reversed by Naloxone

Pain perception in non-mammalian vertebrates such as fish is a controversial issue. We demonstrate that, in the fish Leporinus macrocephalus, an imposed restraint can modulate the behavioral response to a noxious stimulus, specifically the subcutaneous injection of 3% formaldehyde. In the first experiment, formaldehyde was applied immediately after 3 or 5 min of the restraint. Inhibition of the increase in locomotor activity in response to formaldehyde was observed, which suggests a possible restraint-induced antinociception. In the second experiment, the noxious stimulus was applied 0, 5, 10 and 15 min after the restraint, and both 3 and 5 min of restraint promoted short-term antinociception of approximately 5 min. In experiments 3 and 4, an intraperitoneal injection of naloxone (30 mg.kg⁻¹) was administered 30 min prior to the restraint. The 3- minute restraint-induced antinociception was blocked by pretreatment with naloxone, but the corresponding 5-minute response was not. One possible explanation for this result is that an opioid and a non-preferential μ–opioid and/or non-opioid mechanism participate in this response modulation. Furthermore, we observed that both the 3- and 5- minutes restraint were severely stressful events for the organism, promoting marked increases in serum cortisol levels. These data indicate that the response to a noxious stimulus can be modulated by an environmental stressor in fish, as is the case in mammals. To our knowledge, this study is the first evidence for the existence of an endogenous antinociceptive system that is activated by an acute standardized stress in fish. Additionally, it characterizes the antinociceptive response induced by stress in terms of its time course and the opioid mediation, providing information for understanding the evolution of nociception modulation.     

Genome of Herbaspirillum seropedicae strain SmR1, a specialized diazotrophic endophyte of tropical grasses

The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme--GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species.     

N-Glycosylation-dependent Control of Functional Expression of Background Potassium Channels K2P3.1 and K2P9.1

Two-pore domain potassium (K_2P) channels play fundamental roles in cellular processes by enabling a constitutive leak of potassium from cells in which they are expressed, thus influencing cellular membrane potential and activity. Hence, regulation of these channels is of critical importance to cellular function. A key regulatory mechanism of K_2P channels is the control of their cell surface expression. Membrane protein delivery to and retrieval from the cell surface is controlled by their passage through the secretory and endocytic pathways, and post-translational modifications regulate their progression through these pathways. All but one of the K_2P channels possess consensus N-linked glycosylation sites, and here we demonstrate that the conserved putative N-glycosylation site in K_2P3.1 and K_2P9.1 is a glycan acceptor site. Patch clamp analysis revealed that disruption of channel glycosylation reduced K_2P3.1 current, and flow cytometry was instrumental in attributing this to a decreased number of channels on the cell surface. Similar findings were observed when cells were cultured in reduced glucose concentrations. Disruption of N-linked glycosylation has less of an effect on K_2P9.1, with a small reduction in number of channels on the surface observed, but no functional implications detected. Because nonglycosylated channels appear to pass through the secretory pathway in a manner comparable with glycosylated channels, the evidence presented here suggests that the decreased number of nonglycosylated K_2P3.1 channels on the cell surface may be due to their decreased stability.     

A radioenzymatic assay to identify three groups of phospholipase A(2) in platelets

Phospholipases A(2) (PLA(2)) are key enzymes in membrane metabolism. The release of fatty acids and lysophospholipids by PLA(2) activates several intra-cellular second messenger cascades that regulate a wide variety of physiological responses. The aim of the present study is to describe a radioenzymatic assay to determine the activity of three main PLA(2) subtypes in platelets, namely extracellular calcium-dependent PLA(2) (sPLA(2)) and intracellular calcium-dependent (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)). The differentiation of these distinct PLA(2) subtypes was based on the enzyme substrate preference (arachdonic acid or palmitoyl acid) and calcium concentration. Our results indicate that this new assay is feasible, precise and specific to measure the activity of the aforementioned subtypes of PLA(2). Therefore, this protocol can be used to investigate modifications of PLA(2) homeostasis in distinct biological models addressing the pathophysiology of many medical and neuropsychiatric disorders such as schizophrenia and Alzheimer's disease.     

Sequential steps and checkpoints in the early exocytic compartment during secretory IgM biogenesis

The biogenesis of secretory IgM occurs stepwise under stringent quality control, formation of μ₂L₂ preceding polymerization. How is efficiency of IgM secretion coupled to fidelity? We show here that ERp44, a soluble protein involved in thiol-mediated retention, interacts with ERGIC-53. Binding to this hexameric lectin contributes to ERp44 localization in the ER-golgi intermediate compartment. ERp44 and ERGIC-53 increase during B-lymphocyte differentiation, concomitantly with the onset of IgM polymerization. Both preferentially bind μ₂L₂ and higher order intermediates. Their overexpression or silencing in non-lymphoid cells promotes or decreases secretion of IgM polymers, respectively. In IgM-secreting B-lymphoma cells, μ chains interact first with BiP and later with ERp44 and ERGIC-53. Our findings suggest that ERGIC-53 provides a platform that receives μ₂L₂ subunits from the BiP-dependent checkpoint, assisting polymerization. In this process, ERp44 couples thiol-dependent assembly and quality control.     

Multi-transgenic pigs expressing three fluorescent proteins produced with high efficiency by sperm mediated gene transfer

Multi-gene transgenic pigs would be of benefit for large animal models in medical, agricultural, and pharmaceutical applications; in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We used the sperm mediated gene transfer (SMGT) method to produce with high efficiency multi-gene transgenic pigs using three genes coding for fluorescent proteins: enhanced blue (EBFP), green (EGFP), and red (DsRed2). All three fluorescent proteins were expressed in 171 out of 195 normally developed morula/blastocysts examined at day 6 post insemination (88%). Genomic DNA of 18 piglets born from two litters was screened by PCR, showing that all piglets were transgenic with at least one gene, 7/18 piglets were triple transgenic, 7/18 double transgenic, and 4/18 single transgenic. Fluorescence in situ hybridization (FISH) analysis revealed multiple sites of integration of the transgenes. RNA and protein expression was found in muscle, heart, liver, hair, and peripheral blood mononuclear cells (PBMCs). These results show that SMGT is an effective method for introducing multiple genes into pigs as shown by the simultaneous expression of three fluorescent proteins.     

CD8+ T cells are not required for vaccine-induced immunity against Leishmania amazonensis in IL-12/23P40(-/-) C57BL/6 mice

Vaccine-induced protection against leishmaniasis is largely dependent on cell-mediated type 1 response and IL-12-driven IFN-gamma production. Surprisingly, our previous data showed that IL-12/23p40(-/-) mice could be vaccinated against L. amazonensis and were able to produce limited amounts of IFN-gamma. Since the role of CD8+ T in immunization against L. amazonensis is obscure, the aim of this study was to evaluate the effects of CD8+ cells in protection against L. amazonensis in IL-12/23p40(-/-) mice. In order to deplete CD8+ cells, one group of vaccinated animals was treated with anti-CD8 mAb. Infection was followed for 8 weeks. The vaccinated CD8+ -depleted group developed smaller lesions than the non-depleted group. CD8 depletion did not affect tissue parasitism or antibody response against the parasite, and treated animals displayed milder inflammation and better tissue integrity. IFN-gamma production in spleen and draining lymph node was impaired in the depleted group, suggesting that CD8+ cells produced this cytokine in IL-12-independent vaccination. Such results suggest that this T cell subset contributes to augmented pathology in IL12/23p40(-/-) mice vaccinated and challenged with L. amazonensis. Although these cells could produce some IFN-gamma the in the absence of IL-12, they do not affect the parasite tissue load.