Localization of Endo-Oligopeptidase (EC 3.4.22.19) in the Rat Nervous Tissue

The subcellular and regional distribution of endo-oligopeptidase (EC 3.4.22.19), an enzyme capable of generating enkephalin by single cleavage from enkephalin-containing peptides, was determined by an enzymatic assay using metorphamide and by immunochemical techniques in the CNS of the rat. The rat CNS contains a membrane-associated form of endo-oligopeptidase, an enzyme predominantly associated with the soluble fraction of brain homogenates. Subcellular fractionation showed that approximately 17% of the total activity of the enzyme is associated with membrane fractions including synaptosomes. Synaptosomal membranes were prepared from neocortex, striatum, hypothalamus, medulla, spinal cord, and cerebellum. The amount of EC 3.4.22.19 activity solubilized by 3-[( 3-cholamidopropyl]dimethylammonio)-1-propanesulfonate from synaptosomal membranes was similar in neocortex, striatum, and hypothalamus, being three- to 10-fold greater than in spinal cord, cerebellum, and medulla. A polyclonal antibody exhibiting high affinity for endo-oligopeptidase was raised in rabbits against the purified rat brain enzyme and used to localize endo-oligopeptidase by Western blotting and by immunoperoxidase techniques. A strong band corresponding to the Mr of EC 3.4.22.19 was found in solubilized proteins obtained from synaptosomal membranes prepared from hypothalamus, neocortex, and striatum when subjected to Western blotting. The immunohistochemical localization of endo-oligopeptidase indicated that the immunoreactivity was confined to gray matter in regions known to be rich in peptide-containing neurons such as the striatum. In the cerebellum, a region poor in peptides, no staining could be detected. The nonuniform distribution of endo-oligopeptidase in rat brain suggests a role in neurotransmitter processing in the CNS.     

PHYLOGENY AND SYSTEMATICS OF THE MARINE ALGAL FAMILY GRACILARIACEAE (GRACILARIALES,RHODOPHYTA) BASED ON SMALL SUBUNIT rDNA AND ITS SEQUENCES OF ATLANTIC AND PACIFIC SPECIES1

We sequenced the small subunit rDNA and internal transcribed spacer region of Gracilariaceae from the tropical Atlantic and Pacific, with emphasis on flattened or compressed species. Sequence comparisons confirmed three main lineages of Gracilariaceae: Curdiea/Melanthalia, Gracilariopsis/Gracilariophila, and Gracilaria. The Curdiea/Melanthalia diverged early in the family. Gracilariopsis was paraphyletic, because at least one Gracilariophila species evolved from it. The Atlantic Gracilariopsis were monophyletic and separated from the Pacific lineages. The Gracilaria included all species referable to its own species and to Hydropuntia, which was paraphyletic, formed by distantly related lineages. The new combination Gracilaria pauciramosa (N. Rodríguez Ríos) Bellorin, M. C. Oliveira et E. C. Oliveira is proposed for Polycavernosa pauciramosa N. Rodríguez Ríos. Recognition of subgenera within Gracilaria, based on spermatangial arrangement, was not supported. Instead, infrageneric groups were delineated by geographic origins and combinations of reproductive characters. Most Pacific species with either “textorii” or “verrucosa” type spermatangia were deeply separated from Atlantic species. Within the Atlantic Gracilaria, a lineage encompassing mostly tropical cylindrical species with “henriquesiana” type spermatangia and distinctive cystocarp anatomy was recognized. A lineage was also retrieved for cold water stringy species with verrucosa type spermatangia. Several species from the western Atlantic are closely related to Gracilaria tikvahiae McLachlan with nearly identical morphology. On the other hand, most flattened species from the tropical Atlantic were closely related despite their diverse morphologies. The interpretation of our data in addition to the literature indicates that more populations from the Indo‐Pacific must be studied before a general picture of Gracilariaceae evolution can be framed.     

Massive Seed Predation of Pseudobombax grandiflorum (Bombacaceae) by Parakeets Brotogeris versicolurus (Psittacidae) in a Forest Fragment in Brazil1

Here we report massive seed predation of Pseudobombax grandiflorum (Bombacaceae) by Botogeris versicolurus (Psittacidae) in a forest fragment in Brazil. The intensity of seed predation was very high when compared to other studies in continuous forest, perhaps resulting from a scarcity of resources in such areas. This scarcity may limit the range of parrot's diet to a few plant species. It suggests that studies of Psittacidae seed predation may be important for conservation of some plants in fragments.     

Induction of SOS and adaptive responses by alkylating agents in Escherichia coli mutants deficient in 3-methyladenine-DNA glycosylase activities

The induction of SOS and adaptive responses by alkylating agents was studied in Escherichia coli mutants tagA and alkA deficient in 3-methyladenine-DNA glycosylase activities. The SOS response was measured using an sfiA::lacZ operon fusion. The sfiA operon, in the double mutant tagA alkA, is induced at 5-50-fold lower concentrations of all tested methylating and ethylating compounds, as compared to the wild-type strain. In all cases, the tagA mutation, which inactivates the constitutive and specific 3-alkyladenine-DNA glycosylase I (TagI), sensitizes the strain to the SOS response. The sensitization effect of alkA mutation, which inactivates the inducible 3-alkyladenine-DNA glycosylase II (TagII), is observed under conditions which allow the induction of the adaptive response. We conclude that the persistence of 3-methyladenine and 3-ethyladenine residues in DNA most likely leads to the induction of the SOS functions. In contrast, the adaptive response, evaluated by O6-methylguanine-DNA methyltransferase activity in cell extracts, was not affected by either tagA or alkA mutations. The results suggest that the SOS and adaptive responses use different alkylation products as an inducing "signal". However, adaptation protein TagII inhibits the induction of the SOS response to some extent, due to its action at the level of signal production. Finally, we provide conditions to improve short-term bacterial tests for the detection of genotoxic alkylating agents.     

Ecology of bdelloids: how to be successful

Bdelloids inhabit many different environments. The entire taxon is an order belonging to the phylum Rotifera. In spite of its wide distribution, it has a very uniform morphology, suggesting that natural selection has had almost no effect on its morphological characteristics.This paper reviews the geographical and ecological distributions of bdelloids and their ability to tolerate different conditions is discussed. Two characteristics account for the wide distribution of bdelloids, parthenogenetic reproduction and their ability to withstand unfavorable conditions through anhydrobiosis. The former is an apomictic thelytoky which may ensure genetic homogeneity within clones. However, evidence which suggests variability among and within parthenogenetic clones is discussed. Some recent experimental evidence indicates that anhydrobiosis does not affect the life history of the surviving individuals. The effects of anhydrobiosis on bdelloid populations are considered.     

Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using omega-conotoxin from Conus magus venom

The omega-conotoxins from the venom of fish-hunting cone snails are probably the most useful of presently available ligands for neuronal Ca channels from vertebrates. Two of these peptide toxins, omega-conotoxins MVIIA and MVIIB from the venom of Conus magus, were purified. The amino acid sequences show significant differences from omega-conotoxins from Conus geographus. Total synthesis of omega-conotoxin MVIIA was achieved, and biologically active radiolabeled toxin was produced by iodination. Although omega-conotoxins from C. geographus (GVIA) and C. magus (MVIIA) appear to compete for the same sites in mammalian brain, in amphibian brain the high-affinity binding of omega-conotoxin MVIIA has narrower specificity. In this system, it is demonstrated that a combination of two omega-conotoxins can be used for biochemically defining receptor subtypes and suggested that these correspond to subtypes of neuronal Ca2+ channels.