GPCR-induced dissociation of G-protein subunits in early stage signal transduction

G-protein coupled receptors (GPCRs) form a ternary complex of agonist, receptor and G-proteins during primary signal transduction at the cell membrane. Downstream signalling is thought to be preceded by the process of dissociation of Gα and Gβγ subunits, thus exposing new surfaces to interact with downstream effectors. We demonstrate here for the first time, the dissociation of heterotrimeric G-protein subunits (i.e., Gα and Gβγ) following agonist-induced GPCR (α_2A-adrenergic receptor; α_2A-AR) activation in a cell-free assay system. α_2A-AR membranes were reconstituted with the G-proteins (±hexahistidine-tagged) Gα_i1 and Gβ₁γ₂ and functional signalling was determined following activation of the reconstituted receptor:G-protein complex with the potent agonist UK-14304, and [³⁵S]GTPγS. In the presence of Ni²⁺-coated agarose beads, the activated his-tagged Gα_i1his-[³⁵S]GTPγS complex was captured on the Ni²⁺-presenting surface. When his-tagged Gβ₁γ₂ (Gβ₁γ₂his) was used with Gα_i1, the [³⁵S]GTPγS-bound Gα_i1 was not present on the Ni²⁺-coated beads, but rather, it was separated from the β₁γ₂(his)-beads, demonstrating receptor-induced dissociation of Gα and Gβγ subunits. Treatment of the reconstituted α_2A-AR membranes containing Gβ₁γ₂his:Gα_i1 with imidazole confirmed the specificity for the Ni²⁺:G-protein surface dissociation of Gα_i1 from Gβ₁γ₂his. These data demonstrate for the first time, the complete dissociation of the G-protein subunits and extend observations on the role of G-proteins in the assembly and disassembly of the ternary complex in the primary events of GPCR signalling.     

Fish oil supplementation for two generations increases insulin sensitivity in rats

We investigated the effect of fish oil supplementation for two consecutive generations on insulin sensitivity in rats. After the nursing period (21 days), female rats from the same prole were divided into two groups: (a) control group and (b) fish oil group. Female rats were supplemented with water (control) or fish oil at 1 g/kg body weight as a single bolus for 3 months. After this period, female rats were mated with male Wistar rats fed on a balanced chow diet (not supplemented). Female rats continued to receive supplementation throughout gestation and lactation periods. The same treatment was performed for the next two generations (G1 and G2). At 75 days of age, male offspring from G1 and G2 generations from both groups were used in the experiments. G1 rats did not present any difference with control rats. However, G2 rats presented reduction in glycemia and lipidemia and improvement in in vivo insulin sensitivity (model assessment of insulin resistance, insulin tolerance test) as well as in vitro insulin sensitivity in soleus muscle (glucose uptake and metabolism). This effect was associated with increased insulin-stimulated p38 MAP kinase phosphorylation and lower n-6/n-3 fatty acid ratio, but not with activation of proteins from insulin signaling (IR, IRS-1 and Akt). Global DNA methylation was decreased in liver but not in soleus muscle. These results suggest that long-term fish oil supplementation improves insulin sensitivity in association with increased insulin-stimulated p38 activation and decreased n-6:n-3 ratio in skeletal muscle and decreased global DNA methylation in liver.     

Diversity and abundance of climbers from the Atlantic Forest, southeastern Brazil

Climbing plant community composition and abundance was compared between the Alto da Serra de Paranapiacaba Biological Reserve (ASPRB) and the Nascentes de Paranapiacaba Municipal Natural Park (NPMNP), two protected areas in the largest Atlantic Forest remnant, southeastern Brazil. Climbing plants ≥1 cm in diameter were sampled in 52 quadrats of 10 × 20 m (1.04 ha). From these data we calculated the Shannon–Wiener diversity index, calculated abundance and importance values for families, performed a principal coordinate analysis, and created a similarity dendrogram (TWINSPAN). The composition and abundance of species between ASPRB and NPMNP differed significantly due to the successional state of the ASPRB forest, which was affected by more recent disturbance. There we found 34 species, while in the NPMNP, species richness was 72, with only 24 species in common. The two areas formed two groups corresponding to two distinct floristic subsets. These montane forests were characterized by a predominance of species of the Asteraceae. The high species richness, floristic composition, and community composition reflect the greater degree of conservation in the area of NPMNP. The high density and community composition of climbers in the more conserved area of rainforest may be due to the low intensity of disturbance.     

GLUT12 functions as a basal and insulin-independent glucose transporter in the heart

Glucose uptake from the bloodstream is the rate-limiting step in whole body glucose utilization, and is regulated by a family of membrane proteins called glucose transporters (GLUTs). Although GLUT4 is the predominant isoform in insulin-sensitive tissues, there is recent evidence that GLUT12 could be a novel second insulin-sensitive GLUT. However, its physiological role in the heart is not elucidated and the regulation of insulin-stimulated myocardial GLUT12 translocation is unknown. In addition, the role of GLUT12 has not been investigated in the diabetic myocardium. Thus, we hypothesized that, as for GLUT4, insulin regulates GLUT12 translocation to the myocardial cell surface, which is impaired during diabetes. Active cell surface GLUT (-4 and -12) content was quantified (before and after insulin stimulation) by a biotinylated photolabeled assay in both intact perfused myocardium and isolated cardiac myocytes of healthy and type 1 diabetic rodents. GLUT localization was confirmed by immunofluorescent confocal microscopy, and total GLUT protein expression was measured by Western blotting. Insulin stimulation increased translocation of GLUT-4, but not -12, in the healthy myocardium. Total GLUT4 content of the heart was decreased during diabetes, while there was no difference in total GLUT12. Active cell surface GLUT12 content was increased in the diabetic myocardium, potentially as a compensatory mechanism for the observed downregulation of GLUT4. Collectively, our data suggest that, in contrast to GLUT4, insulin does not mediate GLUT12 translocation, which may function as a basal GLUT located primarily at the cell surface in the myocardium.     

Depletion of nucleoporins from HeLa nuclear pore complexes to facilitate the production of ghost pores for in vitro reconstitution

During cell division, Nuclear Pore Complexes (NPCs) are broken down into protein subcomplexes that are the basis for reassembly in daughter cells. This is the driving force for the establishment of an in vitro reconstitution system to study aspects of NPC reassembly. In this study, nuclear envelope (NE) was isolated from HeLa cells. NE was treated with increasing concentrations of heparin to extract nucleoporins (Nups) for the production of “ghost pores” which are pores severely deficient in Nups, while still containing Pore Membrane proteins (POM) needed to anchor the NPC. Ghost pores have been subjected to incubation with previously stripped Nups and some re-binding has been shown to occur by western blot analysis. This in vitro assay provides a powerful tool to investigate the protein–protein interactions of NPC reassembly from a human cell line. Through a better understanding of the process of NPC reassembly, we can continue to piece together the puzzle of this macromolecular structure. It is most advantageous to establish a straightforward reconstitution procedure at the mammalian level.     

Deposition of pathogenic Mycoplasma gallisepticum onto bird feeders: host pathology is more important than temperature-driven increases in food intake

Although ambient temperature has diverse effects on disease dynamics, few studies have examined how temperature alters pathogen transmission by changing host physiology or behaviour. Here, we test whether reducing ambient temperature alters host foraging, pathology and the potential for fomite transmission of the bacterial pathogen Mycoplasma gallisepticum (MG), which causes seasonal outbreaks of severe conjunctivitis in house finches (Haemorhous mexicanus). We housed finches at temperatures within or below the thermoneutral zone to manipulate food intake by altering energetic requirements of thermoregulation. We predicted that pathogen deposition on bird feeders would increase with temperature-driven increases in food intake and with conjunctival pathology. As expected, housing birds below the thermoneutral zone increased food consumption. Despite this difference, pathogen deposition on feeders did not vary across temperature treatments. However, pathogen deposition increased with conjunctival pathology, independently of temperature and pathogen load, suggesting that MG could enhance its transmission by increasing virulence. Our results suggest that in this system, host physiological responses are more important for transmission potential than temperature-dependent alterations in feeding. Understanding such behavioural and physiological contributions to disease transmission is critical to linking individual responses to climate with population-level disease dynamics.     

Evaluating the influence of selection markers on obtaining selected pools and stable cell lines in human cells

Selection markers are common genetic elements used in recombinant cell line development. While several selection systems exist for use in mammalian cell lines, no previous study has comprehensively evaluated their performance in the isolation of recombinant populations and cell lines. Here we examine four antibiotics, hygromycin B, neomycin, puromycin, and Zeocin™, and their corresponding selector genes, using a green fluorescent protein (GFP) as a reporter in two model cell lines, HT1080 and HEK293. We identify Zeocin™ as the best selection agent for cell line development in human cells. In comparison to the other selection systems, Zeocin™ is able to identify populations with higher fluorescence levels, which in turn leads to the isolation of better clonal populations and less false positives. Furthermore, Zeocin™-resistant populations exhibit better transgene stability in the absence of selection pressure compared to other selection agents. All isolated Zeocin™-resistant clones, regardless of cell type, exhibited GFP expression. By comparison, only 79% of hygromycin B-resistant, 47% of neomycin-resistant, and 14% of puromycin-resistant clones expressed GFP. Based on these results, we rank Zeocin™ > hygromycin B ∼ puromycin > neomycin for cell line development in human cells. Furthermore, this study demonstrates that selection marker choice does indeed impact cell line development.     

A Novel Anti-Inflammatory and Pro-Resolving Role for Resolvin D1 in Acute Cigarette Smoke-Induced Lung Inflammation

Introduction

Cigarette smoke is a profound pro-inflammatory stimulus that contributes to acute lung injuries and to chronic lung disease including COPD (emphysema and chronic bronchitis). Until recently, it was assumed that resolution of inflammation was a passive process that occurred once the inflammatory stimulus was removed. It is now recognized that resolution of inflammation is a bioactive process, mediated by specialized lipid mediators, and that normal homeostasis is maintained by a balance between pro-inflammatory and pro-resolving pathways. These novel small lipid mediators, including the resolvins, protectins and maresins, are bioactive products mainly derived from dietary omega-3 and omega-6 polyunsaturated fatty acids (PUFA). We hypothesize that resolvin D1 (RvD1) has potent anti-inflammatory and pro-resolving effects in a model of cigarette smoke-induced lung inflammation.

Methods

Primary human lung fibroblasts, small airway epithelial cells and blood monocytes were treated with IL-1β or cigarette smoke extract in combination with RvD1 in vitro, production of pro-inflammatory mediators was measured. Mice were exposed to dilute mainstream cigarette smoke and treated with RvD1 either concurrently with smoke or after smoking cessation. The effects on lung inflammation and lung macrophage populations were assessed.

Results

RvD1 suppressed production of pro-inflammatory mediators by primary human cells in a dose-dependent manner. Treatment of mice with RvD1 concurrently with cigarette smoke exposure significantly reduced neutrophilic lung inflammation and production of pro-inflammatory cytokines, while upregulating the anti-inflammatory cytokine IL-10. RvD1 promoted differentiation of alternatively activated (M2) macrophages and neutrophil efferocytosis. RvD1 also accelerated the resolution of lung inflammation when given after the final smoke exposure.

Conclusions

RvD1 has potent anti-inflammatory and pro-resolving effects in cells and mice exposed to cigarette smoke. Resolvins have strong potential as a novel therapeutic approach to resolve lung injury caused by smoke and pulmonary toxicants.     

Acute SIV Infection in Sooty Mangabey Monkeys Is Characterized by Rapid Virus Clearance from Lymph Nodes and Absence of Productive Infection in Germinal Centers

Lymphoid tissue immunopathology is a characteristic feature of chronic HIV/SIV infection in AIDS-susceptible species, but is absent in SIV-infected natural hosts. To investigate factors contributing to this difference, we compared germinal center development and SIV RNA distribution in peripheral lymph nodes during primary SIV infection of the natural host sooty mangabey and the non-natural host pig-tailed macaque. Although SIV-infected cells were detected in the lymph node of both species at two weeks post infection, they were confined to the lymph node paracortex in immune-competent mangabeys but were seen in both the paracortex and the germinal center of SIV-infected macaques. By six weeks post infection, SIV-infected cells were no longer detected in the lymph node of sooty mangabeys. The difference in localization and rate of disappearance of SIV-infected cells between the two species was associated with trapping of cell-free virus on follicular dendritic cells and higher numbers of germinal center CD4⁺ T lymphocytes in macaques post SIV infection. Our data suggests that fundamental differences in the germinal center microenvironment prevent productive SIV infection within the lymph node germinal centers of natural hosts contributing to sustained immune competency.     

TGF-b Superfamily Cytokine MIC-1/GDF15 Is a Physiological Appetite and Body Weight Regulator

The TGF-b superfamily cytokine MIC-1/GDF15 circulates in all humans and when overproduced in cancer leads to anorexia/cachexia, by direct action on brain feeding centres. In these studies we have examined the role of physiologically relevant levels of MIC-1/GDF15 in the regulation of appetite, body weight and basal metabolic rate. MIC-1/GDF15 gene knockout mice (MIC-1^−/−) weighed more and had increased adiposity, which was associated with increased spontaneous food intake. Female MIC-1^−/− mice exhibited some additional alterations in reduced basal energy expenditure and physical activity, possibly owing to the associated decrease in total lean mass. Further, infusion of human recombinant MIC-1/GDF15 sufficient to raise serum levels in MIC-1^−/− mice to within the normal human range reduced body weight and food intake. Taken together, our findings suggest that MIC-1/GDF15 is involved in the physiological regulation of appetite and energy storage.