Dapper Antagonist of Catenin-1 Cooperates with Dishevelled-1 during Postsynaptic Development in Mouse Forebrain GABAergic Interneurons

Synaptogenesis has been extensively studied along with dendritic spine development in glutamatergic pyramidal neurons, however synapse development in cortical interneurons, which are largely aspiny, is comparatively less well understood. Dact1, one of 3 paralogous Dact (Dapper/Frodo) family members in mammals, is a scaffold protein implicated in both the Wnt/β-catenin and the Wnt/Planar Cell Polarity pathways. We show here that Dact1 is expressed in immature cortical interneurons. Although Dact1 is first expressed in interneuron precursors during proliferative and migratory stages, constitutive Dact1 mutant mice have no major defects in numbers or migration of these neurons. However, cultured cortical interneurons derived from these mice have reduced numbers of excitatory synapses on their dendrites. We selectively eliminated Dact1 from mouse cortical interneurons using a conditional knock-out strategy with a Dlx-I12b enhancer-Cre allele, and thereby demonstrate a cell-autonomous role for Dact1 during postsynaptic development. Confirming this cell-autonomous role, we show that synapse numbers in Dact1 deficient cortical interneurons are rescued by virally-mediated re-expression of Dact1 specifically targeted to these cells. Synapse numbers in these neurons are also rescued by similarly targeted expression of the Dact1 binding partner Dishevelled-1, and partially rescued by expression of Disrupted in Schizophrenia-1, a synaptic protein genetically implicated in susceptibility to several major mental illnesses. In sum, our results support a novel cell-autonomous postsynaptic role for Dact1, in cooperation with Dishevelled-1 and possibly Disrupted in Schizophrenia-1, in the formation of synapses on cortical interneuron dendrites.     

Ligands of second generation platinum analogs decrease both platinum-induced DNA cross-linking and its ability to interact with 1-beta-D-arabinofuranosyl cytosine to potentiate cytotoxic efficacy

We evaluated the cytotoxic and DNA cross-linking (CL) ability of four second generation platinum coordination complexes (TNO-6, JM-89, JM-8 and JM-9) delivered alone or in combination with 1-beta-D-arabinofuranosyl cytosine (ara-C) to human colon cancer cells (LoVo). Cell survival varied markedly as a function of the particular substitution moiety. JM-8 and JM-9 were virtually ineffective, even at concentrations as high as 50 micrograms/ml. At that concentration cis-diamminedichloroplatinum(II) (cis-DDP) killed greater than 99.99% of the cells. JM-82 was slightly more active while TNO-6 was the only derivative with appreciably higher cytotoxic activity due to an abrogation of the shoulder region of the type C survival curve. The highest CL effect was observed for cis-DDP followed closely by TNO-6. Very little CL effects were demonstrated for the other three analogs JM-82, JM-8 and JM-9 when measured 6 h after treatment. The combination of cis-DDP and ara-C augmented 10-fold the cytotoxic activity of cis-DDP alone, an effect accompanied by an almost 2-fold increase in CL; every other analog failed to interact in a potentiating manner (either cytotoxicity, or CL at 6 h) with the antimetabolite. Thus, it appears clear that the associated moieties of the Pt coordination complex play a fundamental role in reducing the interaction of the analogs with DNA (as reflected by the decreased CL and cytotoxic effects produced by each agent alone) and in totally preventing their interaction with ara-C to yield a potentiating lethal effect.     

Role of the modulator protein in the interconversion of rabbit skeletal muscle protein phosphatase

The major active protein phosphatase present in a rabbit skeletal muscle extract is associated with the glycogen particle and migrates in sucrose density gradient centrifugation as a Mr = 70,000 protein and contains modulator activity. Addition of extra modulator protein causes a time- and concentration-dependent conversion of the enzyme to an inactive FA-ATP, Mg-dependent form. The intrinsic modulator in the active phosphatase is destroyed by limited proteolysis without an appreciable change in the phosphatase activity. The proteolyzed active enzyme has a lower molecular weight (Mr = 40,000) and it reassociates with the modulator producing a FA-ATP, Mg-dependent enzyme form (Mr = 60,000). The modulator protein is used stoichiometrically in the activation of the ATP, Mg-dependent phosphatase. This is in agreement with the presence of one unit of modulator activity per unit of native spontaneously active phosphatase.     

Essential function of linoleic acid esterified in acylglucosylceramide and acylceramide in maintaining the epidermal water permeability barrier. Evidence from feeding studies with oleate, linoleate, arachidonate, columbinate and alpha-linolenate

Essential fatty acid-deficient rats were supplemented with 300 mg per day of pure fatty acid esters: oleate (O), linoleate (L), arachidonate (A), and columbinate (C) for 10 days. During this period, the rats in groups L, A, and C all showed a decrease in their initially high trans-epidermal water loss, a classical essential fatty acid-deficiency symptom, to a level seen in non-deficient rats (group N). The trans-epidermal water loss in rats of group O was unaffected by the supplementation. Fatty acid composition of two epidermal sphingolipids, acylglucosylceramide and acylceramide, from the skin were determined. The results indicate that re-establishment of a low trans-epidermal water loss was associated with incorporation of linolenate into the two epidermal sphingolipids. Supplementation with columbinate resulted in relatively high amounts of this fatty acid in the investigated epidermal sphingolipids. Analysis of pooled skin specimens from a previous study in which weanling rats were fed a fat-free diet and supplemented orally with pure alpha-linolenate for 13 weeks (Hansen, H.S. and Jensen, B. (1983) Lipids 18, 682-690) revealed very little polyunsaturated fatty acid in the two sphingolipids. These rats showed increased evaporation which was comparable to that of essential fatty acid-deficient rats. We interpret these results as strong evidence for a very specific and essential function of linoleic acid in maintaining the integrity of the epidermal water permeability barrier. This function of linoleate is independent of its role as precursor for arachidonate and icosanoids.     

Determination of oxalyl thiolesters, N-oxalylcysteine and N-oxalylcysteamine in biological materials

A convenient method is described for the quantitative analysis of oxalyl thiolesters (OTEs), a newly discovered class of mammalian metabolites, in biological samples. By this particular technique the total concentration of all OTEs in the sample is determined. The method involves first reacting the biological material with cysteamine (2-aminoethanethiol) or cysteine under conditions that convert OTEs quantitatively to N-oxalylcysteamine (or N-oxalylcysteine), followed by reaction with monobromobimane to give a highly fluorescent derivative that is analyzed by reversed-phase ion-pair chromatography, with tetrabutylammonium ion as the counterion and N-(2-mercaptopropionyl)glycine as an internal standard. The method is capable of detecting as little as 0.6 pmol of the bimane derivative of the N-oxalyl compound in a single HPLC injection. The application of this method has led to the discovery that not only OTEs but also N-oxalylcysteine and N-oxalylcysteamine are normal mammalian metabolites. In various rat tissues the OTE concentration ranges up to 65 nmol/g (wet wt), the N-oxalylcysteine concentration is approximately 10 nmol/g, and the N-oxalylcysteamine concentration is 0-3 nmol/g.     

Eye size variation reflects habitat and daily activity patterns in colubrid snakes

The functioning of the vertebrate eye depends on its absolute size, which is presumably adapted to specific needs. Eye size variation in lidless and spectacled colubrid snakes was investigated, including 839 specimens belonging to 49 genera, 66 species and subspecies. Variations of adult eye diameters (EDs) in both absolute and relative terms between species were correlated with parameters reflecting behavioral ecology. In absolute terms, eye of arboreal species was larger than in terrestrial and semiaquatic species. For diurnal species, EDs of terrestrial species do not differ from semiaquatic species; for nocturnal species the ED of terrestrial species is larger than fossorial species but not different from semiaquatic species. In relative terms, ED did not differ significantly by habitat for diurnal species. Although the ED of terrestrial species is larger than fossorial species there were no differences for nocturnal species between semiaquatic and fossorial snakes. In contrast to other vertebrates studied to date, colubrid EDs in absolute and relative terms are larger in diurnal than in nocturnal species. These observations suggest that among colubrid snakes, eye size variation reflects adaptation to specific habitats, foraging strategies and daily activities, independently of phylogeny. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Soluble, prolonged-acting insulin derivatives. II. Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B13, B27 and B30

It has previously been found that insulins, to which positive charge has been added by substitutions in position B30, thus raising the isoelectric point towards pH 7, had a prolonged action when injected as slightly acidic solutions because such derivatives crystallize very readily upon neutralization. Positive charge has now been added by substituting the B13 and A17 glutamic acid residues with glutamines and B27 threonine with lysine or arginine. These substitutions were introduced by site-specific mutagenesis in a gene coding for a single-chain insulin precursor. By tryptic transpeptidation the single-chain precursors were transformed to the double-chain insulin structure, concomitantly with incorporation of residue B30. Thus insulins combining B13 glutamine, A17 glutamine and B27 lysine or arginine with B30 threonine, threonine amide or lysine amide were synthesized. The time course of blood glucose lowering effect and the absorption were studied after subcutaneous injection in rabbits and pigs. The prolonged action of B30-substituted insulins was markedly enhanced by B27 lysine or arginine substitutions and by B13 glutamine. The B27 residue is located on the surface of the hexamer, so a basic residue in this position presumably promotes the packing of hexamers at neutral pH. The B13 residues cluster in the centre of the hexamer. When the electrostatic repulsive forces from six glutamic acid residues are abolished by substitution with glutamine, a stabilization of the hexamer can be envisaged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intra- and inter-specific effects of mast seeding on seed fates of two sympatric <Emphasis Type="Italic">Corylus</Emphasis> species

We released seeds of two sympatric tree species, Corylus mandshurica (seed with thinner seed hull, higher nutrition) and C. heterophylla (seeds with thicker seed hull, lower nutrition) in the masting year of C. mandshurica in 2008, and C. heterophylla in 2009, respectively, to investigate how seed masting of the two sympatric Corylus species affects seed removal and dispersal fitness of the two species differently at both intra- and inter-specific levels. At intra-specific level, the authors found mast seeding of both C. mandshurica and C. heterophylla significantly reduced seed removal, seed consumption, but increased seed dispersal distance and seed dispersal fitness of the released seeds. Mast seeding of C. mandshurica increased seed caching of C. mandshurica. At inter-specific level, the authors found mast seeding of C. mandshurica reduced seed removal of C. heterophylla, but mast seeding of C. heterophylla did not significantly reduce seed removal of C. mandshurica. Mast seeding of C. mandshurica reduced seed consumption of C. heterophylla, while mast seeding of C. heterophylla reduced seed consumption of C. mandshurica. We found mast seeding of C. mandshurica significantly reduced seed dispersal distance of C. heterophylla, while mast seeding of C. heterophylla significantly increased seed dispersal distance of C. mandshurica. We found that mast seeding of C. mandshurica significantly increased seed dispersal fitness of C. heterophylla, while mast seeding of C. heterophylla did not significantly increase seed dispersal fitness of C. mandshurica. More studies are needed to reveal the ecological consequences of mast seeding at inter-specific or community-level. Seed traits may attribute the differences of mast seeding at inter-specific level. Because seeds with thinner seed hull and higher nutrition were more harvested and eaten by rodents, mast seeding of C. mandshurica might have reduced seed removal and seed consumption, but increased dispersal fitness of C. heterophylla (seeds with thicker seed hull, lower nutrition). Therefore, synchrony among species is, or is not, selectively beneficial to the focus species depends on seed traits which determine gains from mast seeding at inter-specific level.