Post-synaptic Density-95 (PSD-95) Binding Capacity of G-protein-coupled Receptor 30 (GPR30), an Estrogen Receptor That Can Be Identified in Hippocampal Dendritic Spines

The estrogen 17β-estradiol (E2) modulates dendritic spine plasticity in the cornu ammonis 1 (CA1) region of the hippocampus, and GPR30 (G-protein coupled estrogen receptor 1 (GPER1)) is an estrogen-sensitive G-protein-coupled receptor (GPCR) that is expressed in the mammalian brain and in specific subregions that are responsive to E2, including the hippocampus. The subcellular localization of hippocampal GPR30, however, remains unclear. Here, we demonstrate that GPR30 immunoreactivity is detected in dendritic spines of rat CA1 hippocampal neurons in vivo and that GPR30 protein can be found in rat brain synaptosomes. GPR30 immunoreactivity is identified at the post-synaptic density (PSD) and in the adjacent peri-synaptic zone, and GPR30 can associate with the spine scaffolding protein PSD-95 both in vitro and in vivo. This PSD-95 binding capacity of GPR30 is specific and determined by the receptor C-terminal tail that is both necessary and sufficient for PSD-95 interaction. The interaction with PSD-95 functions to increase GPR30 protein levels residing at the plasma membrane surface. GPR30 associates with the N-terminal tandem pair of PDZ domains in PSD-95, suggesting that PSD-95 may be involved in clustering GPR30 with other receptors in the hippocampus. We demonstrate that GPR30 has the potential to associate with additional post-synaptic GPCRs, including the membrane progestin receptor, the corticotropin releasing hormone receptor, and the 5HT1a serotonin receptor. These data demonstrate that GPR30 is well positioned in the dendritic spine compartment to integrate E2 sensitivity directly onto multiple inputs on synaptic activity and might begin to provide a molecular explanation as to how E2 modulates dendritic spine plasticity.     

Protein kinase C phosphorylation of PLCβ1 regulates its cellular localization

Activation of phospholipase Cβ (PLCβ) by G proteins leads to a chain of events that result in an increase in intracellular calcium and activation of protein kinase C (PKC). It has been found that PKC phosphorylates PLCβ1 on S887 in vitro without affecting its enzymatic activity or its ability to be activated by Gα(q) proteins. To understand whether S887 phosphorylation affects the enzyme’s activity in cells, we constructed two mutants that mimic the wild type and PKC-phosphorylated enzymes (S887A and S887D). We find that these constructs bind similarly to Gα(q) in vitro. When expressed in HEK293 cells, both mutants associate identically to Gα(q) in both the basal and stimulated states. Both mutants diffuse with similar rates and also interact identically with another known binding partner, translin-associated factor X (TRAX), which associates with PLCβ1 in the cytosol and nucleus. However, the two mutants localize differently in the cell. We find that S887A has a much higher nuclear localization than its S887D counterpart both in HEK293 cells and PC12 cells. Our studies suggest that PKC phosphorylation regulates the level of PLCβ1 cytosolic and nuclear activity by regulating its cellular compartmentalization.     

Lung injury caused by high tidal volume mechanical ventilation and hyperoxia is dependent on oxidant-mediated c-Jun NH2-terminal kinase activation

Both prolonged exposure to hyperoxia and large tidal volume mechanical ventilation can each independently cause lung injury. However, the combined impact of these insults is poorly understood. We recently reported that preexposure to hyperoxia for 12 h, followed by ventilation with large tidal volumes, induced significant lung injury and epithelial cell apoptosis compared with either stimulus alone (Makena et al. Am J Physiol Lung Cell Mol Physiol 299: L711-L719, 2010). The upstream mechanisms of this lung injury and apoptosis have not been clearly elucidated. We hypothesized that lung injury in this model was dependent on oxidative signaling via the c-Jun NH(2)-terminal kinases (JNK). We, therefore, evaluated lung injury and apoptosis in the presence of N-acetyl-cysteine (NAC) in both mouse and cell culture models, and we provide evidence that NAC significantly inhibited lung injury and apoptosis by reducing the production of ROS, activation of JNK, and apoptosis. To confirm JNK involvement in apoptosis, cells treated with a specific JNK inhibitor, SP600125, and subjected to preexposure to hyperoxia, followed by mechanical stretch, exhibited significantly reduced evidence of apoptosis. In conclusion, lung injury and apoptosis caused by preexposure to hyperoxia, followed by high tidal volume mechanical ventilation, induces ROS-mediated activation of JNK and mitochondrial-mediated apoptosis. NAC protects lung injury and apoptosis by inhibiting ROS-mediated activation of JNK and downstream proapoptotic signaling.     

Zwitterionic poly(carboxybetaine) hydrogels for glucose biosensors in complex media

Zwitterionic hydrogels based on poly(carboxybetaine) methacrylate (polyCBMA) were developed to protect implantable electrochemical glucose biosensors from biofouling in complex media. To enhance the linearity and sensitivity of the sensing profile, both physical and chemical adsorption methods were developed. Results show that glucose sensors coated with polyCBMA hydrogels via the chemical method achieve very high sensitivity and good linearity in response to glucose in PBS, 10%, 50%, and 100% human blood serum. Essentially identical glucose signals were observed even after prolonged exposure to blood samples for over 12 days. The excellent performance of polyCBMA hydrogel coating offers great promise for designing biocompatible implantable glucose biosensors in biological medium.     

Grass pollen immunotherapy induces an allergen-specific IgA2 antibody response associated with mucosal TGF-beta expression

Allergen immunotherapy (IT) has long-term efficacy in IgE-mediated allergic rhinitis and asthma. IT has been shown to modify lymphocyte responses to allergen, inducing IL-10 production and IgG Abs. In contrast, a putative role for IgA and local TGF-beta-producing cells remains to be determined. In 44 patients with seasonal rhinitis/asthma, serum IgA1, IgA2, and polymeric (J chain-containing) Abs to the major allergen Phl p 5 were determined by ELISA before and after a 2-year double-blind trial of grass pollen (Phleum pratense) injection IT. Nasal TGF-beta expression was assessed by in situ hybridization. Sera from five IT patients were fractionated for functional analysis of the effects of IgA and IgG Abs on IL-10 production by blood monocytes and allergen-IgE binding to B cells. Serum Phl p 5-specific IgA2 Abs increased after a 2-year treatment (approximately 8-fold increase, p = 0.002) in contrast to IgA1. Increases in polymeric Abs to Phl p 5 (approximately 2-fold increase, p = 0.02) and in nasal TGF-beta mRNA (p = 0.05) were also observed, and TGF-beta mRNA correlated with serum Phl p 5 IgA2 (r = 0.61, p = 0.009). Post-IT IgA fractions triggered IL-10 secretion by monocytes while not inhibiting allergen-IgE binding to B cells as observed with IgG fractions. This study shows for the first time that the IgA response to IT is selective for IgA2, correlates with increased local TGF-beta expression, and induces monocyte IL-10 expression, suggesting that IgA Abs could thereby contribute to the tolerance developed in IT-treated allergic patients.     

Fungal peptide Destruxin A plays a specific role in suppressing the innate immune response in Drosophila melanogaster

Destruxins are a class of insecticidal, anti-viral, and phytotoxic cyclic depsipeptides that are also studied for their toxicity to cancer cells. They are produced by various fungi, and a direct relationship has been established between Destruxin production and the virulence of the entomopathogen Metarhizium anisopliae. Aside from opening calcium channels, their in vivo mode of action during pathogenesis remains largely uncharacterized. To better understand the effects of a Destruxin, we looked at changes in gene expression following injection of Destruxin A into the fruit fly Drosophila melanogaster. Microarray results revealed reduced expression of various antimicrobial peptides that play a major role in the humoral immune response of the fly. Flies co-injected with a non-lethal dose of Destruxin A and the normally innocuous Gram-negative bacteria Escherichia coli, showed increased mortality and an accompanying increase in bacterial titers. Mortality due to sepsis was rescued through ectopic activation of components in the IMD pathway, one of two signal transduction pathways that are responsible for antimicrobial peptide induction. These results demonstrate a novel role for Destruxin A in specific suppression of the humoral immune response in insects.     

Differences in rheotactic responses contribute to divergent habitat use between parapatric lake and stream threespine stickleback

Migration among populations is widely thought to undermine adaptive divergence, assuming gene flow arises from random movement of individuals. If individuals instead differ in dispersal behavior, phenotype‐dependent dispersal can reduce the effective rate of gene flow or even facilitate divergence. For example, parapatric populations of lake and stream stickleback tend to actively avoid dispersing into the adjoining habitat. However, the behavioral basis of this nonrandom dispersal was previously unknown. Here, we show that lake and stream stickleback exhibit divergent rheotactic responses (behavioral response to currents). During the breeding season, wild‐caught inlet stream stickleback were better than lake fish at maintaining position in currents, faced upstream more, and spent more time in low‐current areas. As a result, stream fish expended significantly less energy in currents than did lake fish. These divergent rheotactic responses likely contribute to divergent habitat use by lake and stream stickleback. Although rheotactic differences were absent in nonbreeding fish, divergent behavior of breeding‐season fish may suffice for assortative mating by breeding location. The resulting reproductive isolation between lake and stream fish may explain the fine‐scale evolutionary differentiation in parapatric stickleback populations.     

The Role of Palmitoylation for Protein Recruitment to the Inner Membrane Complex of the Malaria Parasite

To survive and persist within its human host, the malaria parasite Plasmodium falciparum utilizes a battery of lineage-specific innovations to invade and multiply in human erythrocytes. With central roles in invasion and cytokinesis, the inner membrane complex, a Golgi-derived double membrane structure underlying the plasma membrane of the parasite, represents a unique and unifying structure characteristic to all organisms belonging to a large phylogenetic group called Alveolata. More than 30 structurally and phylogenetically distinct proteins are embedded in the IMC, where a portion of these proteins displays N-terminal acylation motifs. Although N-terminal myristoylation is catalyzed co-translationally within the cytoplasm of the parasite, palmitoylation takes place at membranes and is mediated by palmitoyl acyltransferases (PATs). Here, we identify a PAT (PfDHHC1) that is exclusively localized to the IMC. Systematic phylogenetic analysis of the alveolate PAT family reveals PfDHHC1 to be a member of a highly conserved, apicomplexan-specific clade of PATs. We show that during schizogony this enzyme has an identical distribution like two dual-acylated, IMC-localized proteins (PfISP1 and PfISP3). We used these proteins to probe into specific sequence requirements for IMC-specific membrane recruitment and their interaction with differentially localized PATs of the parasite.     

Infrared Spectroscopic Biosignatures from Hidden Cave,New Mexico: Possible Applications for Remote Life Detection

Subsurface environments are known to support and preserve diverse microbial communities. Giant pool fingers from Hidden Cave, New Mexico consist of mm-scale dark micritic calcite layers alternating with clear dogtooth spar crystals and contain morphological and geochemical evidence of past microbial communities. We used Fourier Transform infrared spectroscopy to identify fatty acids, proteins, PO₂-carrying compounds, and polysaccharides spatially related to morphological fossil filaments throughout the surface micritic laminations and central pool finger regions. These biomolecular signatures are important components that contribute to the biosignature suite under development that identify microbial involvement in carbonate precipitation on Earth and remotely.