Indomethacin influences arginine vasotocin-induced parturition and oviposition in lizards (Sceloporus jarrovi and Sceloporus undulatus )

The effect of the prostaglandin blocker indomethacin on arginine vasotocin-induced birth was examined. Gravid female, oviparous (Sceloporus undulatus ) and viviparous (Sceloporus jarrovi ) lizards were pretreated with saline or indomethacin, a potent blocker of PG synthesis. Pretreatment was followed by an intraperitoneal injection of AVT. Pretreatment with indomethacin significantly delayed the onset of AVT-induced oviposition in S. undulatus , whereas it had no effect on latency to birth in S. jarrovi . Female S. jarrovi treated with indomethacin, however, gave birth to only part of the total litter, whereas control females gave birth to complete litters. In viviparous females, an interaction of embryonic age with pretreatment was evident; females having more developed embryos decreased birth latency significantly and increased the percentage of parturition when compared with females that had embryos at earlier stages of development. Our data suggest that although exogenous AVT can stimulate oviposition or parturition, these events occur more rapidly and completely when prostaglandin synthesis is not inhibited.     

Enhancement of β-Amyloid Peptide Aβ(1–40)-Mediated Neurotoxicity by Glutamine Synthetase

Abstract: The β-amyloid peptide (Aβ), a main constituent in both senile and diffuse plaques in Alzheimer's disease brains, was previously shown to be neurotoxic and to be able to interact with several macromolecular components of brain tissue. Previous investigations carried out in our laboratory demonstrated free radical species formation in aqueous solutions of Aβ(1–40) and its C-end fragment, Aβ(25–35). Toxic forms of Aβ rapidly inactivate the oxidation-sensitive cytosolic enzyme glutamine synthetase (GS). In this regard, we suggested and subsequently demonstrated that Aβ radicals can cause an oxidative damage of cell proteins and lipids resulting in disruption of membrane functions, enzyme inactivation, and cell death. Because GS can be a substrate for Aβ-derived oxidizing species, the present study was conducted to determine if GS could protect against Aβ neurotoxicity. In contrast to this initial hypothesis, we here report that GS significantly enhances the neurotoxic effects of Aβ(1–40). The Aβ-mediated inactivation of GS was found to be accompanied by the loss of immunoreactive GS and the significant increase of Aβ(1–40) neurotoxicity.     

Oxidatively Induced Structural Alteration of Glutamine Synthetase Assessed by Analysis of Spin Label Incorporation Kinetics: Relevance to Alzheimer's Disease

Abstract: The activity of the astrocytic enzyme glutamine synthetase (GS) is decreased in the Alzheimer's disease brain, which may have relevance to mechanisms of chronic excitotoxicity. The molecular perturbation(s) that results in GS inactivation is not known, although oxidative lesioning of the enzyme is one likely cause. To assess structural perturbation induced in GS by metal-catalyzed oxidation, a series of spin-labeling studies were undertaken. Ovine GS was oxidized by exposure to iron/hydrogen peroxide and subsequently labeled with the thiol-specific nitroxide probe MTS [(1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl)methanethiosulfonate]. The reaction of MTS with cysteine residues within GS was monitored in real time by electron paramagnetic resonance spectrometry. Structural perturbation of GS, manifested as decreased thiol accessibility, was inferred from an apparent decrease in the rate constant for the second-order reaction of MTS with protein thiols. A subsequent spin-labeling study was undertaken to compare the structural integrity of GS purified and isolated from Alzheimer's disease-afflicted brain (AD-GS) with that of GS isolated from nondemented, age-matched control brain (C-GS). The rate constant for reaction of MTS with AD-GS was markedly decreased relative to that for the reaction of spin label with C-GS. The kinetic data were partially corroborated by spectroscopic data obtained from circular dichroism analysis of control and peroxide-treated ovine GS. In an adjunct experiment, the interaction of GS with a synthetic analogue of the Alzheimer's-associated β-amyloid peptide, known to induce free radical oxidative stress, indicated strong interaction of the enzyme with the peptide as reflected by a decrease in the rate constant for MTS binding to reactive protein thiols.     

Heart Rate Variability and Skin Conductance During Repetitive TMS Course in Children with Autism

Autism spectrum disorder (ASD) is a developmental disorder marked by difficulty in social interactions and communication. ASD also often present symptoms of autonomic nervous system (ANS) functioning abnormalities. In individuals with autism the sympathetic branch of the ANS presents an over-activation on a background of the parasympathetic activity deficits, creating an autonomic imbalance, evidenced by a faster heart rate with little variation and increased tonic electrodermal activity. The objective of this study was to explore the effect of 12 sessions of 0.5 Hz repetitive transcranial magnetic stimulation (rTMS) over dorsolateral prefrontal cortex (DLPFC) on autonomic activity in children with ASD. Electrocardiogram and skin conductance level (SCL) were recorded and analyzed during each session of rTMS. The measures of interest were time domain (i.e., R–R intervals, standard deviation of cardiac intervals, NN50-cardio-intervals >50 ms different from preceding interval) and frequency domain heart rate variability (HRV) indices [i.e., power of high frequency (HF) and low frequency (LF) components of HRV spectrum, LF/HF ratio]. Based on our prior pilot studies it was proposed that the course of 12 weekly inhibitory low-frequency rTMS bilaterally applied to the DLPFC will improve autonomic balance probably through improved frontal inhibition of the ANS activity, and will be manifested in an increased length of cardiointervals and their variability, and in higher frequency-domain HRV in a form of increased HF power, decreased LF power, resulting in decreased LF/HF ratio, and in decreased SCL. Our post-12 TMS results showed significant increases in cardiac intervals variability measures and decrease of tonic SCL indicative of increased cardiac vagal control and reduced sympathetic arousal. Behavioral evaluations showed decreased irritability, hyperactivity, stereotype behavior and compulsive behavior ratings that correlated with several autonomic variables.     

The Control of Sea Urchin Metamorphosis: Ionic Effects

Because the cascade of events which comprise sea urchin metamorphosis occur rapidly, regulatory mechanisms able to respond in minutes must function. Employing sea water solutions of altered ionic composition in the presence or absence of metamorphically active microbial films, we tested the ability of particular ions to inhibit or enhance metamorphosis in competent larvae of the sea urchin, Lytechinus variegatus . At 40 mM excess potassium maximally induces normal metamorphosis in the absence of a microbial film. In the presence of metamorphically active microbial films, 40 mM excess magnesium inhibits the process. Increasing concentrations of calcium up to an excess of 40 mM stimulates larvae to undergo metamorphosis but in smaller proportions than similar concentrations of potassium. Divalent cation-free sea water solutions are toxic to larvae. These studies support the hypothesis that ion fluxes are involved in the regulation of metamorphosis and reveal a complexity of response that parallels the histological complexity of competent echinoid larvae.     

Uptake of l-Phenylalanine in Synchronous Chlorella fusca. Characterization of the Uptake System

Phenylalanine uptake in Chlorella fusca was measured, using the membrane filter technique. The cells were synchronized, and harvested at specific points of the life cycle. Experiments with autospores showed that the uptake followed saturation kinetics, with a K_m= 5 μM. V_max, was 0.1 nmol/min × 10⁷ cells. The optimum temperature for the uptake was 40°C, and the activation energy was 1700 J/mol. The uptake showed a high specificity towards l -phenylalanine; presence of the unlabelled stereoisomer did not inhibit the uptake. Uptake of l -phenylalanine was inhibited in the presence of other analogues or other amino acids, but only if they were present in concentrations considerably higher than that of L-phenylalanine. Variations in the ratio of Na4⁺ to K⁺ in the external solution during uptake experiments did not have any influence upon the uptake rate of l -phenylalanine. The cells were able to take up the amino acid against a concentration gradient. At pool maximum the ratio between internal and external amino acid concentration was 1000/1. 2,4-Dinitro-phenol inhibited the uptake completely. Exchange between internal and external l -phenylalanine could not be demonstrated. The K_m value did not change during the life cycle of the cells. The uptake rate reached a maximum at the end of the light period, and fell to a minimum just before sporulation started. It is concluded that Chlorella fusca cells have a highly specific, active uptake system for l -phenylalanine. The system is constitutive, independent on the K or Na concentration, and the mechanism of uptake does not change during the life cycle of the cells.     

Metabolism of l-β-lysine by a Pseudomonas: Purification and properties of 3-keto-6-acetamidohexanoate cleavage enzyme

Extracts of Pseudomonas B4 grown with l-β-lysine (3,6-diaminohexanoate) as the main energy source are shown to contain a 3-keto-6-acetamidohexanoate cleavage enzyme that converts 3-keto-6-acetamidohexanoate and acetyl · CoA reversibly to 4-acetamidobutyryl · CoA and acetoacetate. The enzyme catalyzes the third step in β-lysine degradation. In unfractionated extracts cleavage enzyme activity is generally assayed spectrophotometrically by coupling the forward reaction with excess 4-acetamidobutyryl · CoA thiolesterase, derived from the same organism, and measuring the rate of CoASH formation by reaction with 5,5-dithiobis(2-nitrobenzoic acid). Enzyme freed of thiolesterase is conveniently assayed by using 4-acetamidobutyryl · CoA and acetoacetate as substrates and measuring acetyl · CoA formation by means of citrate synthase reaction in the presence of 5,5-dithiobis(2-nitrobenzoic acid). The cleavage enzyme has been purified 38-fold to a specific activity of 237 mU/mg. The stoichiometry, equilibrium constant, molecular weight, and various kinetic properties of the enzymatic reaction have been determined. The substrate specificity of the Pseudomonas enzyme differs markedly from that of the analogous 3-keto-5-aminohexanoate cleavage enzyme of Clostridium subterminale strain SB4 and is broader. In the forward reaction 3-ketohexanoate can replace 3-keto-6-acetamidohexanoate, and propionyl · CoA can replace acetyl · CoA as a substrate. In the backward reaction, 4-acetamidobutyryl · CoA can be replaced by any of several CoA thiolesters including the butyryl, valeryl, 4-propionamidobutyryl, 3-acetamidopropionyl, and β-alanyl derivatives, and acetoacetate can be replaced by 2-methylacetoacetate. The products of these reactions have been characterized. Unlike the cleavage enzyme of Clostridium subterminale strain SB4, the Pseudomonas enzyme is not stimulated by Co²⁺ or Mn²⁺ and is not inhibited by EDTA, 5,5-dithiobis(2-nitrobenzoic acid), or p-chloromercuribenzoate. Tracer experiments indicate that carbon atoms 1 and 2 of acetoacetate are derived from carbon atoms 1 and 2 of 3-keto-6-acetamidohexanoate, and carbon atoms 3 and 4 of acetoacetate are derived from the acetyl group of acetyl · CoA. The cleavage enzyme is not formed in detectable amounts when Pseudomonas B4 is grown in a peptone-yeast extract medium.     

Associated bacterial flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus.

The growth, toxicity, and associated bacterial flora of 10 clonal cultures of the toxic benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus isolated from the coastal waters of southwest Puerto Rico have been examined. Clonal cultures of O. lenticularis grew more rapidly and at broader temperature ranges than those of G. toxicus. All five Ostreopsis clones were toxic, while only one of the five Gambierdiscus clones was poisonous. The degree of toxicity among poisonous clones was highly variable. The number of associated bacterial genera and their frequency of occurrence were quite variable among clones of both dinoflagellate genera. Bacterial isolates represented six genera (Nocardia, Pseudomonas, Vibrio, Aeromonas, Flavobacterium, and Moraxella) in addition to coryneform bacteria. Extracts of dinoflagellate-associated bacteria grown in pure culture were not toxic. Gambierdiscus clones were characterized by the frequent presence of Pseudomonas spp. (four of five clones) and the absence of coryneforms. In O. lenticularis, only one of five clones showed the presence of Pseudomonas spp., and Moraxella sp. was absent altogether. Detailed analyses of toxicity and associated microflora in a selected Ostreopsis clone, repeatedly cultivated (four times) over a period of 160 days, showed that peak cell toxicities developed in the late static and early negative culture growth phases. Peak Ostreopsis cell toxicities in the stationary phase of culture growth were correlated with significant increases in the percent total bacteria directly associated with these cells. Changes in the quantity of bacteria directly associated with microalgal cell surfaces and extracellular matrices during culture growth may be related to variability and degree of toxicity in these laboratory-cultured benthic dinoflagellates.     

Novel Pharmacologic Targeting of Tight Junctions and Focal Adhesions in Prostate Cancer Cells

Cancer cell resistance to anoikis driven by aberrant signaling sustained by the tumor microenvironment confers high invasive potential and therapeutic resistance. We recently generated a novel lead quinazoline-based Doxazosin® derivative, DZ-50, which impairs tumor growth and metastasis via anoikis. Genome-wide analysis in the human prostate cancer cell line DU-145 identified primary downregulated targets of DZ-50, including genes involved in focal adhesion integrity (fibronectin, integrin-α6 and talin), tight junction formation (claudin-11) as well as insulin growth factor binding protein 3 (IGFBP-3) and the angiogenesis modulator thrombospondin 1 (TSP-1). Confocal microscopy demonstrated structural disruption of both focal adhesions and tight junctions by the downregulation of these gene targets, resulting in decreased cell survival, migration and adhesion to extracellular matrix (ECM) components in two androgen-independent human prostate cancer cell lines, PC-3 and DU-145. Stabilization of cell-ECM interactions by overexpression of talin-1 and/or exposing cells to a fibronectin-rich environment mitigated the effect of DZ-50. Loss of expression of the intracellular focal adhesion signaling effectors talin-1 and integrin linked kinase (ILK) sensitized human prostate cancer to anoikis. Our findings suggest that DZ-50 exerts its antitumor effect by targeting the key functional intercellular interactions, focal adhesions and tight junctions, supporting the therapeutic significance of this agent for the treatment of advanced prostate cancer.     

Phylogeny and taxonomy of the Prenolepis genus‐group of ants (Hymenoptera: Formicidae)

Abstract. We investigated the phylogeny and taxonomy of the Prenolepis genus‐group, a clade of ants we define within the subfamily Formicinae comprising the genera Euprenolepis, Nylanderia, gen. rev. , Paraparatrechina, gen. rev. & stat. nov. , Paratrechina, Prenolepis and Pseudolasius. We inferred a phylogeny of the Prenolepis genus‐group using DNA sequence data from five genes (CAD, EF1αF1, EF1αF2, wingless and COI) sampled from 50 taxa. Based on the results of this phylogeny the taxonomy of the Prenolepis genus‐group was re‐examined. Paratrechina (broad sense) species segregated into three distinct, robust clades. Paratrechina longicornis represents a distinct lineage, a result consistent with morphological evidence; because this is the type species for the genus, Paratrechina is redefined as a monotypic genus. Two formerly synonymized subgenera, Nylanderia and Paraparatrechina, are raised to generic status in order to provide names for the other two clades. The majority of taxa formerly placed in Paratrechina, 133 species and subspecies, are transferred to Nylanderia, and 28 species and subspecies are transferred to Paraparatrechina. In addition, two species are transferred from Pseudolasius to Paraparatrechina and one species of Pseudolasius is transferred to Nylanderia. A morphological diagnosis for the worker caste of all six genera is provided, with a discussion of the morphological characters used to define each genus. Two genera, Prenolepis and Pseudolasius, were not recovered as monophyletic by the molecular data, and the implications of this result are discussed. A worker‐based key to the genera of the Prenolepis genus‐group is provided.