Pituitary adenylate cyclase-activating polypeptide induces translocation of its G-protein-coupled receptor into caveolin-enriched membrane microdomains, leading to enhanced cyclic AMP generation and neurite outgrowth in PC12 cells

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal peptide family expressed throughout the nervous system, binds to the PACAP-specific G-protein-coupled receptor family members to promote both neuronal differentiation and survival. Although the PACAP receptor is known to activate its effector protein, adenylate cyclase (AC), and thus enhance cAMP generation, the molecular mechanism utilized by the receptor to activate AC is lacking. Here, we show that PACAP induces neurite outgrowth in PC12 cells by induction of translocation of the PACAP type 1 receptor (PAC1R) into caveolin-enriched Triton X-100-insoluble microdomains, leading to stronger PAC1R-AC interaction and elevated cAMP production. Moreover, we demonstrate that translocation of PAC1R is blocked by various treatments that selectively disrupt caveolae. As a result, intracellular cAMP level is decreased and consequently the PACAP-induced neurite outgrowth retarded. In contrast, addition of exogenous ganglioside GM1 to the cells shows the opposite effects. These results therefore identify the PACAP-induced translocation of its G-protein-coupled receptor into caveolae, where both AC and the regulating G-proteins reside, as the key molecular event in activating AC and inducing cAMP-mediated differentiation of PC12 cells.

Heterotrimeric G protein γ subunits provide functional selectivity in Gβγ dimer signaling in arabidopsis

The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical Galpha and Gbeta subunit genes and two Ggamma subunit genes (AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Ggamma genes revealed specialized roles for each Ggamma subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.2, and decreased sensitivity to methyl jasmonate. By contrast, both AGG1- and AGG2-deficient mutants were hypersensitive to auxin-mediated induction of lateral roots, suggesting that Gbetagamma1 and Gbetagamma2 synergistically inhibit auxin-dependent lateral root initiation. However, the involvement of each Ggamma subunit in this root response differs, with Gbetagamma1 acting within the central cylinder, attenuating acropetally transported auxin signaling, while Gbetagamma2 affects the action of basipetal auxin and graviresponsiveness within the epidermis and/or cortex. This selectivity also operates in the hypocotyl. Selectivity in Gbetagamma signaling was also found in other known AGB1-mediated pathways. agg1 mutants were hypersensitive to glucose and the osmotic agent mannitol during seed germination, while agg2 mutants were only affected by glucose. We show that both Ggamma subunits form functional Gbetagamma dimers and that each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein-mediated signaling in plants.

Irreversible and bivalent ligands for selected G-protein coupled receptors

Derivatives of pharmaceutical compounds that could potentially bind irreversibly to a hypertension controlling receptor were synthesised to provide tools for studying this receptor. Also compounds that simultaneously activate two cardiovascular receptors with opposing cellular mechanisms were synthesised. This resulted in a desired partial reduction in the activity of one receptor.

G-CSF treatment can attenuate dexamethasone-induced reduction in C2C12 myotube protein synthesis

Granulocyte-colony stimulating factor (G-CSF) has been demonstrated to enhance skeletal muscle recovery following injury and increases muscle function in the context of neuromuscular disease in rodent models. However, understanding of the underlying mechanisms used by G-CSF to mediate these functions remains poor. G-CSF acts on responsive cells through binding to a specific membrane spanning receptor, G-CSFR. Recently identified, the G-CSFR is expressed in myoblasts, myotubes and mature skeletal muscle tissue. Therefore, elucidating the actions of G-CSF in skeletal muscle represents an important prerequisite to consider G-CSF as a therapeutic agent to treat skeletal muscle. Here we show for the first time that treatment with moderate doses (4 and 40ng/ml) of G-CSF attenuates the effects of dexamethasone in reducing protein synthesis in C2C12 myotubes. However, a higher dose (100ng/ml) of G-CSF exacerbates the dexamethasone-induced reduction in protein synthesis. In contrast, G-CSF had no effect on basal or dexamethasone-induced protein degradation, nor did G-CSF influence the phosphorylation of Akt, STAT3, Erk1/2, Src, Lyn and Erk5 in C2C12 myotubes. In conclusion, physiologically relevant doses of G-CSF may attenuate reduced skeletal muscle protein synthesis during catabolic conditions, thereby improving recovery.

Serotonin 1a receptor and associated G-protein activation in schizophrenia and bipolar disorder

Abnormalities in the serotonergic signalling system, including the serotonin 1a receptor, have been implicated in the pathogenesis of schizophrenia and bipolar 1 disorder. However, there is no consensus on whether the density of the serotonin 1a receptor and/or the activity of the G-proteins linking the receptor to the intracellular cascade are altered in these disease states. To address these issues, tissue obtained postmortem from four cortical regions was used to measure [3H] 8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) binding and 8-OH-DPAT-stimulated guanosine 5′-[γ-thio]triphosphate (GTPγS) binding to determine if either parameter is altered in schizophrenia or bipolar I disorder. There was an effect of diagnosis on the level of [3H] 8-OH-DPAT binding that may indicate a global change in the density of serotonin 1a receptors, although this effect did not reach significance in any individual brain region. The activation of serotonin 1a receptors did not differ significantly with diagnoses. However, in the outer cortical layers, there appeared to be a dissociation between the number of receptors available and the extent of ligand-induced GTPγS binding, suggesting considerable receptor reserve. In addition, comparing gender independent of diagnoses, a decrease in the levels of serotonin 1a receptors was observed in the cortex of female subjects. These data indicates that there may be subtle changes in serotonin 1a receptors across the cortex in schizophrenia or bipolar I disorder and suggests a gender discordance in receptor levels.

Creatine transporter protein content, localization, and gene expression in rat skeletal muscle

The present study examined the gene expression and cellular localization of the creatine transporter (CreaT) protein in rat skeletal muscle. Soleus (SOL) and red (RG) and white gastrocnemius (WG) muscles were analyzed for CreaT mRNA, CreaT protein, and total creatine (TCr) content. Cellular location of the CreaT protein was visualized with immunohistochemical analysis of muscle cross sections. TCr was higher (P <= 0.05) in WG than in both RG and SOL, and was higher in RG than in SOL. Total CreaT protein content was greater (P <= 0.05) in SOL and RG than in WG. Two bands (55 and 70 kDa) of the CreaT protein were found in all muscle types. Both the 55-kDa (CreaT-55) and the 70-kDa (CreaT-70) bands were present in greater (P <= 0.05) amounts in SOL and RG than in WG. SOL and RG had a greater amount (P <= 0.05) of CreaT-55 than CreaT-70. Immunohistochemical analysis revealed that the CreaT was mainly associated with the sarcolemmal membrane in all muscle types. CreaT mRNA expression per microgram of total RNA was similar across the three muscle types. These data indicate that rat SOL and RG have an enhanced potential to transport Cr compared with WG, despite a higher TCr in the latter.

ORP-3, a human oxysterol-binding protein gene differentially expressed in hematopoietic cells

Using differential display polymerase chain reaction, a gene was identified in CD34+-enriched populations that had with low or absent expression in CD34- populations. The full coding sequence of this transcript was obtained, and the predicted protein has a high degree of homology to oxysterol-binding protein. This gene has been designated OSBP-related protein 3 (ORP-3). Expression of ORP-3 was found to be 3- to 4-fold higher in CD34+ cells than in CD34- cells. Additionally, expression of this gene was 2-fold higher in the more primitive subfraction of hematopoietic cells defined by the CD34+38- phenotype and was down-regulated with the proliferation and differentiation of CD34+ cells. The ORP-3 predicted protein contains an oxysterol-binding domain. Well-characterized proteins expressing this domain bind oxysterols in a dose-dependent fashion. Biologic activities of oxysterols include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, among them hematopoietic cells. Characterization and differential expression of ORP-3 implicates a possible role in the mediation of oxysterol effects on hematopoiesis.

Growth of Australian shortfin eel (Anguilla Australis) elvers given different dietary protein and lipid levels

The Australian shortfin eel, Anguilla australis is a potential candidate for intensive aquaculture. The present study was undertaken to evaluate the growth of elvers (5.4 g ± 0.1 initial weight) fed with diets of varying protein and lipid content, and to assess the potential of using soya-bean meal as a dietary ingredient. A 10 week experiment was conducted at 24 (±1.0) &deg;C by rearing fish, in 60 L conical fibre glass tanks using a closed recirculation system. Diets having protein concentrations of 40 or 50% (by dry weight) were tested at three lipid levels (15, 20, 25%); diets being designated P₄₀L₁₅, P₄₀L₂₀, P₄₀L₂₅, P₅₀L₁₅, etc. All these diets contained 5% soya-bean meal. In addition P₅₀L₂₀ diets were formulated to contain 10 and 20% soya-bean meal in the diet (Diets S1 & S2). Shortfin eel grew best on the P₅₀L₁₅ diet, with an average specific growth rate of 2.26%. Food conservation ratio (FCR) and Protein efficiency ratio (PER) ranged from 1.21 (P₅₀L₁₅) to 2.12 (P₄₀L₂₅), and 0.92 (P₅₀L₂₅) to 1.65 (P₅₀L₁₅), respectively. Based on all criteria the best growth performance of shortfin eel was on the P₅₀L₁₅ diet, followed by P₄₀L₂₀ and P₄₀L₁₅. At both protein levels fish reared on diets with 25% lipid performed poorly. The performance of shortfin eel was not affected by the amount of soya-bean meal in the diet, up to a maximum of 20% dietary inclusion. No significant differences in muscle protein were evident in shortfin eel reared on different dietary treatments, nor was the lipid content of muscle related to dietary lipid level.

Performance of juvenile Murray cod, Maccullochella peelii peelii (Mitchell), fed with diets of different protein to energy ratio

The results of a 56-day experiment on juvenile Murray cod, Maccullochella peelii peelii, an Australian native fish with a high aquaculture potential, of mean weight 14.9 ± 0.04 g, fed with five experimental diets, one a series of 40% protein content and lipid levels of 10, 17 and 24% (P40L10, P40L17 and P40L24), and another of 50% protein and 17 and 24% (P50L17 and P50L24) lipid are presented. The specific growth rate (SGR) (% day−1) of fish maintained on different diets ranged from 1.18 to 1.41, and was not significantly different between dietary treatments, except P40L10 and the rest. However, there was a general tendency for SGR to increase with increasing dietary lipid content at both protein levels. The food conversion ratio (FCR) for the 40% protein series diets were poorer compared with those of the 50% protein diets, and the best FCR of 1.14 was observed with the P50L17 diet. The protein efficiency ratio (PER), however, was better in fish reared on low protein diets. The net protein utilization (NPU) also did not differ significantly (P &gt; 0.05) in relation to dietary treatment. As in the case of PER the highest NPU was observed in Murray cod reared on diet P40L24 and the lowest in fish fed with diet P50L24. The carcass lipid content reflected that of the diets, when significant increases in the lipid content was observed in relation to dietary lipid content at both protein levels. However, body muscle lipid content did not increase with increasing dietary lipid content, and was significantly lower than in the whole body. The fatty acids found in highest concentration amongst the saturates, monoenes and polyunsaturates (PUFAs) were 16 : 0, 18 : 1n-9 and 22 : 6n-3, respectively, and each of these accounted for more than 60% of each of the group's total. The muscle fatty acid content was affected by the dietary lipid content; for example the total amount (in μg mg−1 lipid) of monoenes ranged from 72 ± 5.1 (P40L10) to 112 ± 10 (P40L24) and 112 ± 2.8 (P50L17) to 132 ± 11.8 (P50L24) and the n-6 series fatty acids increased with increasing dietary lipid content, although not always significant. Most notably, 18 : 2n-6 increased with the dietary lipid level in both series of diets.

Energy, protein, calcium, vitamin D and fibre intakes from meals in residential care establishments in Australia

Residents from high level (nursing homes) and low-level care facilities (hostel) being served the three common diet texture modifications (full diet, soft-minced diet and pureed diet) were assessed. Individual plate waste was estimated at three meals on one day. Fifty-six males and 156 females, mean age 82.9+/-9.5 (SD) years, of which 139 lived in nursing homes (NH) and 76 in hostels (H) were included. Mean total energy served from meals was 5.3 MJ/day, 5.1 to 5.6 MJ/day, 95% confidence intervals (CI), in NH which was less than in H, 5.9 MJ/day (CI 5.6 to 6.2 MJ/day) (P=0.007). Protein and calcium intakes were lower in NH, 44.5g (CI 41.5 to 47.5g), 359.0mg (CI 333.2 to 384.8mg), versus 50.5g (CI 46.6 to 54.3g), 480.5mg (CI 444.3 to 516.7mg) in H (P=0.017, P<0.001 respectively). There was no difference in nutrient/energy ratios, except for protein/energy, which was higher in NH 11.7 (CI 11.3 to 12.2) than in H 9.8 (CI 9.4 to 10.3) (P<0.001). Ability to self-feed had no significant effect on nutrient intakes in NH. The self fed group (N=63) had the following nutrient intakes: energy 4.0 MJ (CI 3.6 to 4.3 MJ), protein 44.6g (CI 40.3 to 48.9g), calcium 356.9mg (CI 316.3 to 397.4mg), fibre 14.9g (CI 13.2 to 16.5g). The assisted group (N=64) had the following nutrient intakes: energy 3.9MJ (CI 3.6 to 4.2MJ), protein 46.0g (CI 40.7 to 49.6), calcium 361.9mg (CI 327.8 to 396.1mg), fibre 14.9g (CI 13.2 to 16.1g). Of NH classified as eating impaired, 36% received no assistance with feeding and had lower intakes of protein 37.8g (CI 33.0 to 42.1g) compared to those receiving some assistance 46.1g (CI 41.3 to 50.9g) (P=0.026). Reduced energy intake accounted for the differences in nutrient intakes between nursing homes and hostels, except for protein. Strategies to effectively monitor nutrient intakes and to identify those with eating impairment are required in order to ensure adequate nutrition of residents in nursing homes and hostels.

Predicting prosocial personality from attachment facets: are some facets more critical than others?

Research across various countries and relationship contexts suggests that attachment anxiety and avoidance are associated with people’s prosocial feelings, tendencies, and behaviors (e.g., Gillath et al., 2005; Karantzas, Evans, & Foddy, 2007). In the present paper we extend the two dimensional model of attachment to include a series of nested facets. Doing so allowed us to examine whether the multifaceted nested factor model provides a better explanation of the associations between attachment and the components of prosocial personality as compared to the bi-factor model (attachment anxiety and avoidance). Three hundred and eighty participants, aged 18 to 33 years completed self-report measures of adult attachment and prosocial personality. Data were fitted to various models – as expected the nested model provided a better fit to the data and explained a significantly larger proportion of the variance in prosocial tendencies than the bi-factor model. The attachment facets were found to make distinct contributions to prosocial personality beyond the broad attachment dimensions (e.g., the preoccupied facet was uniquely associated with personal distress). Implications for the revised attachment structure across various prosocial contexts are discussed, as are the limitations of using the Experience in Close Relationships Scale (ECR; Brennan et al., 1998) to test a multifaceted attachment model.