Inhibition of the dopamine D1 receptor signaling by PSD-95

Dopamine D1 receptors play an important role in movement, reward, and learning and are implicated in a number of neurological and psychiatric disorders. These receptors are concentrated in dendritic spines of neurons, including the spine head and the postsynaptic density. D1 within spines is thought to modulate the local channels and receptors to control the excitability and synaptic properties of spines. The molecular mechanisms mediating D1 trafficking, anchorage, and function in spines remain elusive. Here we show that the synaptic scaffolding protein PSD-95 thought to play a role in stabilizing glutamate receptors in the postsynaptic density, interacts with D1 and regulates its trafficking and function. Interestingly, the D1-PSD-95 interaction does not require the well characterized domains of PSD-95 but is mediated by the carboxyl-terminal tail of D1 and the NH(2) terminus of PSD-95, a region that is recognized only recently to participate in protein-protein interaction. Co-expression of PSD-95 with D1 in mammalian cells inhibits the D1-mediated cAMP accumulation without altering the total expression level or the agonist binding properties of the receptor. The diminished D1 signaling is mediated by reduced D1 expression at the cell surface as a consequence of an enhanced constitutive, dynamin-dependent endocytosis. In addition, genetically engineered mice lacking PSD-95 show a heightened behavioral response to either a D1 agonist or the psychostimulant amphetamine. These studies demonstrate a role for a glutamatergic scaffold in dopamine receptor signaling and trafficking and identify a new potential target for the modulation of abnormal dopaminergic function.     

Identification of estrogen-regulated genes by microarray analysis of the uterus of immature rats exposed to endocrine disrupting chemicals

Environmental estrogenic compounds which bind to the estrogen receptor (ER) can block or alter endogenous functions of estrogen in reproductive and developmental stages. A microarray technology is a very valuable method for the prediction of hormone-responsive activities in various gene expressions. Thus, we investigated the altered gene expression by estrogen and endocrine disruptors (EDs) using microarray technology in the uterus of immature rats. In this study, the expression levels of only 555 genes (7.42%) among the 7636 genes spotted on microarray chips were enhanced by more than two-fold following treatment with estradiol (E2), suggesting that direct or rapid response to E2 is widespread at the mRNA levels in these genes. In addition, elevated expression levels of the genes (over 2-fold) were observed by diethylstilbestrol (DES; 9.01%), octyl-phenol (OP; 8.81%), nonyl-phenol (NP; 9.51%), bisphenol-A (BPA; 8.26%) or genistein (9.97%) in the uterus of immature rats. The expression levels of representative genes, i.e., calbindin-D9k (CaBP-9k; vitamin D-dependent calcium-binding protein), oxytocin, adipocyte complement related protein (MW 30 kDa), lactate dehydrogenase A and calcium binding protein A6 (S100a6; calcyclin), were confirmed in these tissues by real-time PCR. In addition, the mRNA levels of these genes by real-time PCR were increased at follicular phase when E2 level was elevated during estrous cycle of adult female rats. In conclusion, these results indicate distinct altered expression of responsive genes following exposure to E2 and estrogenic compounds, and implicate distinct effects of endogenous E2 and environmental endocrine disrupting chemicals in the uterus of immature rats.     

Phylogeny of Myrmeleontiformia based on larval morphology (Neuropterida: Neuroptera)

The suborder Myrmeleontiformia is a derived lineage of lacewings (Insecta: Neuroptera) including the families Psychopsidae, Nemopteridae, Nymphidae, Ascalaphidae and Myrmeleontidae. In particular, Myrmeleontidae (antlions) are the most diverse neuropteran family, representing a conspicuous component of the insect fauna of xeric environments. We present the first detailed quantitative phylogenetic analysis of Myrmeleontiformia, based on 107 larval morphological and behavioural characters for 36 genera whose larvae are known (including at least one representative of all the subfamilies of the suborder). Four related families were used as outgroups to polarize character states. Phylogenetic analyses were conducted using both parsimony and Bayesian methods. The reconstructions resulting from our analyses corroborate the monophyly of Myrmeleontiformia. Within this clade, Psychopsidae are recovered as the sister family to all the remaining taxa. Nemopteridae (including both subfamilies Nemopterinae and Crocinae) are recovered as monophyletic and sister to the clade comprising Nymphidae + (Myrmeleontidae + Ascalaphidae). Nymphidae consist of two well‐supported clades corresponding to the subfamilies Nymphinae and Myiodactylinae. Our results suggest that Ascalaphidae may not be monophyletic, as they collapse into an unresolved polytomy under the Bayesian analysis. In addition, the recovered phylogenetic relationships diverge from the traditional classification scheme for ascalaphids. Myrmeleontidae are reconstructed as monophyletic, with the subfamilies Stilbopteryginae, Palparinae and Myrmeleontinae. We retrieved a strongly supported clade comprising taxa with a fossorial habit of the preimaginal instars, which represents a major antlion radiation, also including the monophyletic pit‐trap building species.     

Pine needle growth and fine structure after prolonged acid rain treatment in the subarctic

The growth and morphology of Scots pine needles were studied in a long-term acid rain experiment in the far north of Finnish Lapland. Pine trees 5 m tall of age 50–70 years were exposed, by spraying the foliage and soil from a height of 2 m, to either clean water (IC) or acidified water over the period 1985–1992, the acidification site being divided into sub-areas in which the precipitation contained two levels of either sulphuric (Sm, Sh) or nitric (Nm, Nh) acid, or both (SNm, SNh). The treatments with medium and high sulphate-S over eight consecutive years yielded a total sulphur deposition of 3·4 and 17·1 gm⁻², respectively, and those with medium and high nitrate-N a total nitrogen deposition of 1·1 and 5·9 g m⁻². Needles were collected for light and electron microscopy, growth measurements and morphometry. Growth in branch height had decreased by about 40% after 6 years of SNm or SNh treatment, and needle growth by 15% in the SNh trees as compared with the irrigated control trees (IC), although decreases were statistically significant only with respect to the non-irrigated control trees (DC). Growth of branches and needles was slightly better in the Nh treatment than in the IC group. The areas of the whole needle, the mesophyll and the phloem decreased in response to SNh treatment as compared with IC or DC, and a statistically significant decrease of about 30–40% was seen in the area of the xylem in comparison with DC. Cellular damage was observed following the acid treatments, especially with a high acid load. The damage was manifested in collapse of the cellular compartments, increases in lipid accumulations and swelling or disorganization of the protoplast. Increased vacuolization of the cytoplasm, plasmalemma irregularities and chilling-type damage to the mitochondria were also observed.     

Influences on the antimicrobial activity of surface-adsorbed nisin

The efficacy of the antimicrobial peptide nisin was examined after adsorption to silica surfaces. Three protocols were used to evaluate nisin's activity against adhered cells ofListeria monocytogenes: bioassay usingPediococcus pentosaceous FBB 61-2 as the sensitive indicator strain; visualization and enumeration of cells by microscopic image analysis; and viability of adhered cells as determined by lodonitrotetrazolium violet uptake and crystallization. The activity of adsorbed nisin was highly dependent upon conditions of adsorption. The highest antimicrobial activity of adsorbed nisin occurred with high concentrations of nisin (1.0 mg ml^–1) and brief contact times (1 h) on surfaces of low hydrophobicity. Sequential adsorption of a second protein (-lactoglobulin or bovine serum albumin) onto surfaces consistently resulted in decreased nisin activity. These data provide direction for the development of applications to limit microbial attachment on food contact surfaces through the use of adsorbed antimicrobial peptides.     

RACK1 is a BKCa channel binding protein

The large conductance calcium-activated potassium channel, or BK_Ca channel, plays an important feedback role in a variety of physiological processes, including neurotransmitter release and smooth muscle contraction. Some reports have suggested that this channel forms a stable complex with regulators of its function, including several kinases and phosphatases. To further define such signaling complexes, we used the yeast two-hybrid system to screen a human aorta cDNA library for proteins that bind to the BK_Ca channel's intracellular, COOH-terminal "tail". One of the interactors we identified is the protein receptor for activated C kinase 1 (RACK1). RACK1 is a member of the WD40 protein family, which also includes the G protein -subunits. Consistent with an important role in BK_Ca-channel regulation, RACK1 has been shown to be a scaffolding protein that interacts with a wide variety of signaling molecules, including cSRC and PKC. We have confirmed the interaction between RACK1 and the BK_Ca channel biochemically with GST pull-down and coimmunoprecipitation experiments. We have observed some co-localization of RACK1 with the BK_Ca channel in vascular smooth muscle cells with immunocytochemical experiments, and we have found that RACK1 has effects on the BK_Ca channel's biophysical properties. Thus RACK1 binds to the BK_Ca channel and it may form part of a BK_Ca-channel regulatory complex in vascular smooth muscle. calcium-activated potassium channel; protein kinase C; smooth muscle     

ES cell-derived neuroepithelial cell cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells. Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).