The Dietary Excitotoxins β-N-Methylamino-l-Alanine and β-N-Oxalylamino-l-Alanine Induce Necrotic- and Apoptotic-Like Death of Rat Cerebellar Granule Cells

Abstract: The neurotoxic properties of the dietary excitotoxins β- N -methylamino- l -alanine and β- N -oxalylamino- l -alanine have been studied in rat cerebellar granule cells and compared with those of glutamate. Glutamate caused dose-dependent death of cerebellar granule cells after a 30-min exposure when viability was assessed 24 h later. β- N -Methylamino- l -alanine and β- N -oxalylamino- l -alanine, however, were toxic only after 24 or 48 h of exposure. The neurotoxic effects of β- N -methylamino- l -alanine were blocked by d (−)-2-amino-5-phosphonopentanoic acid, and those of β- N -oxalylamino- l -alanine were blocked by kynurenic acid, which demonstrated that these excitotoxins caused cerebellar granule cell death through the activation of glutamate receptors. The features of this death were examined morphologically (fluorescent dyes, electron microscopy) and biochemically (conventional agarose gel electrophoresis, effect of aurintricarboxylic acid). Characteristics of apoptosis were identified by transferring cerebellar granule cells from a high K⁺ (30 m M )- to a low K⁺ (10 m M )-containing medium. In cerebellar granule cells exposed to β- N -methylamino- l -alanine or β- N -oxalylamino- l -alanine (3 m M ), hallmarks of necrotic- and apoptotic-like death were observed at various time points over a 72-h period. Therefore, in cerebellar granule cells, β- N -methylamino- l -alanine and β- N -oxalylamino- l -alanine induce death over 12–72 h of exposure via a mechanism that involves both necrotic- and apoptotic-like cell death.     

Molecular phylogenetic evidence that the phylum Haplosporidia has an alveolate ancestry

The phylogenetic position of the phylum Haplosporidia among other protists was investigated with the complete 16S-like rRNA gene sequences from two species in the phylum: Haplosporidium nelsoni, a parasite of oysters, and Minchinia teredinis, a parasite of shipworms. Because the lack of obvious morphological homologies with other protists hampered decisions regarding taxonomic composition for sequence alignment and phylogenetic analysis, the complete sequences for these two haplosporidians were directed as search queries to the blast/ncbi.nlm.nih.gov electronic mail server. The results of this heuristic similarity search provided a basis for constructing a preliminary higher-taxonomic-level analysis comparing the haplosporidians with species from the slime molds, fungi, algae, amoebae, ciliates, dinoflagellates, and apicomplexans. Maximum parsimony yielded equivocal results, whereas transversionally weighted parsimony suggested an affinity with the alveolates (i.e., the ciliates, dinoflagellates, and apicomplexans). Multiple alignment of the two haplosporidian sequences against 17 taxa in a secondary analysis focusing on the alveolates and subsequent parsimony analysis placed the phylum Haplosporidia as a monophyletic group within the Alveolata and as a taxon of equal rank with the other three alveolate phyla. The precise placement within the Alveolata was sensitive to weighting.      

Deep undercooling of tissue water and winter hardiness limitations in timberline flora

Deep undercooled tissue water, which froze near −40 C, was found in winter collected stem and leaf tissue of the dominant timberline tree species of the Colorado Rocky Mountains, Engelmann spruce (Picea engelmannii (Parry) Engelm.) and subalpine fir (Abies lasiocarpa (Hook.) Nutt.), and in numerous other woody species in and below the subalpine vegetation zone. Previous work on numerous woody plants indicates that deep undercooling in xylem makes probable a −40 C winter hardiness limit in stem tissue. Visual injury determinations and electrolyte loss measurements on stem tissue revealed injury near −40 C associated with the freezing of the deep undercooled stem tissue water. These results suggest that the winter hardiness limit of this woody flora is near −40 C. The relevance of deep undercooling in relation to timberline, the upper elevational limit of the subalpine forest, is discussed.     

Preferential nitration with tetranitromethane of a specific tyrosine residue in penicillinase from Staphylococcus aureus PCl. Evidence that the preferentially nitrated residue is not part of the active site but that loss of activity is due to intermolecular cross-linking.

1. Nitration of tyrosine residues of staphylococal penicillinase was accompanied by a partial loss of enzymic activity, which was not readily explained by nitration of a single residue. 2. Loss of activity correlated with low recovery of tyrosine plus nitrotyrosine, which was consistent with cross-linking. 3. The fraction of treated enzyme that was eluted from Sephadex G-75 earlier than native penicillinase was similar to the fraction of enzyme activity lost. Protein eluted in positions corresponding to monomer, dimer and higher oligomers respectively showed major bands in corresponding positions in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicating that the increase in molecular weight was due to intermolecular cross-linking. Monomeric enzyme containing up to 4 mol of nitrotyrosine/mol retained full catalytic activity. Dimeric enzyme retained 50% of normal activity, whereas higher oligomers retained an average of 8-15% of normal activity. 4. Monomeric enzyme isolated after treatment with equimolar tetranitromethane was nitrated predominantly at tyrosine-72.5. Reaction of reduced nitrated monomer with 1,5-difluoro-2,4-dinitrobenzene gave a monomeric, apparently cross-linked product with full catalytic activity. 6. It is concluded that tyrosine-72 plays no part in the active site. Its preferential nitration may be due to its being insufficiently exposed to be available for intermolecular cross-linking. This poperty may make it useful for attachment of a reporter group.     

Soil fungistasis: role of the microbial nutrient sink and of fungistatic substances in two soils.

Sensitivity of conidia of Cochliobolus victoriae to fungistasis decreased markedly following incubation on moist sand for at least 1 h. Germination was greater on Conover loam or on sand being leached with water than on an alkaline clay loam soil known to produce a volatile fungistatic substance. Evolution of 14CO2 began within 3 min after [14C]glucose was applied to the soils; the rate of 14CO2 evolution was faster with Conover loam. Germination of Thielaviopsis basicola conidia per unit of glucose remaining in agar discs initially containing 0-1% glucose, was lower for discs incubated on the clay loam soil than on Conover loam, and was greatest on a bed of sand undergoing aqueous leaching. Germination of ascospores of Neurospora tetrasperma and conidia of C. victoriae was suppressed on discs of washed, Purified Agar or polyacrylamide gel incubated on or over the clay loam soil, but no suppression resulted when discs were incubated on Conover loam. Extensive aeration of either soil did not remove its fungistatic effect. Fungistasis in Conover loam appears to be caused primarily by nutrient deprivation, whereas volatile fungistatic substances may play a major role in the clay loam soil.     

Superpotency and superaffinity phenomena in the stimulation of steroidogenesis in adrenocortical cells by adrenocorticotropin-tobacco mosaic virus conjugates

Summary Adrenocorticotropin-(1-24)-tetracosapeptide was covalently attached to tobacco mosaic virus in two different manners: (i) through a handle near the C-terminus on tyrosine-(23) and (ii) through a handle at the N-terminus on serine-(1). Compounds of type (i) with their N-terminal message sequence freely exposed on the virion surface were considerably more potent for stimulating steroidogenesis in isolated adrenocortical cells than those of type (ii) with a more congested message. Conjugates with 50 or less hormone molecules per virion were less potent per peptide unit than the free handle-substituted hormones, whereas conjugates with 150 ACTH units exhibited superpotency effects. Superpotency disappeared when the substituted virions were disaggregated into (substituted) capsomers, suggesting influences of hormone clustering and virion geometry on biological activity. Superpotent stimulation was irreversible under conditions that immediately inhibited steroidogenesis by ACTH (dilution, addition of a peptide antagonist). Thus, superpotency might be caused by superaffinity arising from a slow rate of dissociation of the conjugates from the target cell receptors. The reason for the slow dissociation rate is still unclear: possible explanations include cooperative affinity, rapid internalization of the conjugate-receptor complexes, or decreased rates of peptide degradation at the receptor site.Abbreviations ACTH_1–39 adrenocorticotropin-(1–39)-nonatriacontapeptide - ACTH_1–24 adrenocorticotropin-(1–24)-tetracosapeptide - ACTH_6–24 adrenocorticotropin-(6–24)-nonadecapeptide - TMV tobacco mosaic virus wild strain To whom reprints should be addressed.     

Effects of ACTH (corticotropin) analogues on steroidogenesis and cyclic AMP in rat adrenocortical cells. Evidence for two different steroidogenically responsive receptors.

Comparative studies on the mechanism of action of ACTH1-39 and ACTH5-24 [corticotropin-(1-39)- and corticotropin-(5-24)-peptides] on isolated rat adrenal cells were performed. The relationship between stimulated steroidogenesis and cyclic AMP was very different, suggesting that cyclic AMP does not play the same role in mediating the action of the two agonists. Data from three separate experiments showed that the competitive antagonist ACTH6-24 [corticotropin-(6-24)-peptide] had mean inhibitor constants of 13.4 +/- 3.1 nM against ACTH1-39 and 3.4 +/- 1.0 nM against ACTH5-24, indicating that the steroidogenic actions of these two agonists are mediated by two different receptor types. We conclude that there are two possible mechanisms for corticotropin action, only one of which involves the necessary production of cyclic AMP.