URF13, a ligand-gated,pore-forming receptor for T-toxin in the inner membrane ofcms-T mitochondria

URF13 is the product of a mitochondrial-encoded gene (T-urfl3) found only in maize plants containing the Texas male-sterile cytoplasm (cms-T), and it is thought to be responsible for both cytoplasmic male sterility and the susceptibility ofcms-T maize to the fungal pathogensBipolaris maydis race T andPhyllosticta maydis. Mitochondria isolated fromcms-T maize are uniquely sensitive to pathotoxins (T-toxin) produced by these fungi and to methomyl (a commercial insecticide). URF13 acts as a receptor that specifically binds T-toxin to produce hydrophilic pores in the inner mitochondrial membrane. When expressed inEscherichia coli cells, URF13 also forms hydrophilic pores in the plasma membrane if exposed to T-toxin or methomyl. Topological studies established that URF13 contains three membrane-spanning -helices, two of which are amphipathic and can contribute to pore formation. Chemical crosslinking of URF13 was used to demonstrate the existence of URF13 oligomers incms-T mitochondria andE. coli cells. The ability of the carboxylate-specific reagent,N,N-dicyclohexycarbodiimide, to cross-link URF13 was used in conjunction with site-directed mutagenesis to establish that the URF13 tetramer has a central core consisting of a four--helical bundle which undergoes a conformational change after interaction with T-toxin or methomyl. Overall, the experimental evidence indicates that URF13 functions as a ligand-gated, pore-forming T-toxin receptor incms-T mitochondria.     

Iodothyronine deiodinase activity in methionine-deficient rats fed selenium-deficient or selenium-sufficient diets

We examined the effect of methionine deficiency on iodothyronine 5’-deiodinase activity in selenium-deficient rats or selenium-sufficient rats fed sodium selenate or selenomethionine. Forty-two weanling male Wistar rats were divided into six groups and pair fed the respective purifiedl-amino acid-based diets for 4 wk.l-methionine concentrations in the diet were 8.0 g/kg for sufficient rats, and 2.0 g/kg for deficient rats. Selenium concentrations in the diet were 0.5 mg/kg (as sodium selenate or selenomethionine) for selenium-sufficient rats and less than 0.005 mg/kg for selenium-deficient rats. Type I 5’-deiodinase activities were significantly lower in liver and higher in kidney of methionine-deficient rats than in those of methionine-sufficient rats fed either the selenium-sufficient or the selenium-deficient diets. The type I 5’-deiodinase activity in brain was significantly lower in the methionine-deficient rats than in the methionine-sufficient rats fed the selenium-deficient diet. Type II 5’-deiodinase activity in brain was significantly higher in the methionine-deficient rats than in the methionine-sufficient rats fed selenium-sufficient diet as sodium selenate. Both thyroxine and 3,3’,5-triiodothyronine concentrations in plasma were significantly higher in the methionine-deficient rats than in the methionine-sufficient rats. It is suggested that the methionine deficiency affects the 5’-deiodinase activity and thyroid hormones level in the rats.     

Astroglial Cells Express Large Amounts of GABAA Receptor Proteins in Mature Brain

Abstract: GABA_A receptors were characterized in cellular fractions isolated from adult bovine brain. The fraction enriched in cortical astrocytes is very rich in high-affinity binding sites for [³H]flunitrazepam and other "central-type" benzodiazepine ligands. The amount of specific [³H]flunitrazepam binding was more than five times higher in the glial fraction than in synaptosomal and perikaryal fractions. [³H]Flunitrazepam was displaced by low concentrations of clonazepam and other specific ligands for central GABA_A receptors. Specific binding sites for GABA, flunitrazepam, barbiturates, and picrotoxin-like convulsants were characterized. Allosteric interactions between the different sites were typical of central-type GABA_A receptors. The presence of α-subunit(s), as revealed by [³H]flunitrazepam photoaffinity labeling, was demonstrated in all brain fractions at molecular mass 51–53 kDa. Photoaffinity labeling was highest in the glial fraction. However, in primary cultured astrocytes from neonate rat cortex, no photoaffinity labeling was detected. Information obtained from astrocytes in culture should thus be taken with caution when extrapolated to differentiated astroglial cells. Our results actually show that, in mature brain, most of the fully pharmacologically active GABA_A receptors are extrasynaptic and expressed in astroglia.     

GABAB Receptor-Mediated Effects in Synaptosomes of Lethargic (lh/lh) Mice

Abstract: Previously, we have shown a significant increase in number of GABA_B receptor binding sites in neocortex and thalamus of lethargic ( lh/lh ) mice, a mutant strain exhibiting absence seizures. This study was performed to test our hypothesis that presynaptic GABA_B receptors would inhibit [³H]GABA release to a greater degree in lh/lh mice compared with their nonepileptic littermates (designated +/+). Synaptosomes isolated from neocortex and thalamus of age-matched male lh/lh and +/+ mice were similar in uptake of [³H]GABA. In the neocortical preparation, baclofen dose-dependently inhibited [³H]GABA release evoked by 12 m M KCl, an effect mediated by GABA_B receptors. The maximal inhibition ( I _max) value was significantly greater (80%) in lh/lh than +/+ mice, whereas the IC₅₀ (3 µ M ) was unchanged. In the thalamic preparation, the effect of baclofen (50 µ M ) was 58% less robust in lh/lh mice. Other effects mediated by GABA_B receptors (inhibitions in Ca²⁺ uptake and cyclic AMP formation) were also significantly reduced in thalamic synaptosomes from lh/lh mice. These data suggest a greater presynaptic GABA_B receptor-mediated effect in neocortex and a reduced effect in thalamic nuclei of lh/lh mice. It is possible that selective effects of presynaptic GABA_B receptors on GABA release in neocortex and thalamic nuclei of lh/lh mice may contribute to mechanisms underlying absence seizures.     

Characterisation of the Binding of [3H]WAY-100635, a Novel 5-Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

Abstract: The specific binding of [³H]WAY-100635 {N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [³H]WAY-100635 association (k₊₁ = 0.069 ± 0.015 nM^?1 min^?1) and dissociation (k_?1 = 0.023 ± 0.001 min^?1) followed monoexponential kinetics. Saturation binding isotherms of [³H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (B_max) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [³H]WAY-100635 was ～36% higher compared with that of 8-hydroxy-2-(di-n-[³H]-propylamino)tetralin ([³H]8-OH-DPAT). The binding affinity of [³H]WAY-100635 was significantly lowered by the divalent cations CaCl₂ (2.5-fold; p < 0.02) and MnCl₂ (3.6-fold; p < 0.05), with no effect on B_max. Guanyl nucleotides failed to influence the K_D and B_max parameters of [³H]WAY-100635 binding to 5-HT_1A receptors. The pharmacological binding profile of [³H]WAY-100635 was closely correlated with that of [³H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine_1A (5-HT_1A) sites in rat hippocampus. [³H]WAY-100635 competition curves with 5-HT_1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT_1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites.     

THE FUCUS (PHAEOPHYCEAE) SPERM RECEPTOR FOR EGGS. II. ISOLATION OF A BINDING PROTEIN WHICH PARTIALLY ACTIVATES EGGS1

An in vitro binding assay involving egg plasma membrane vesicles (PMVs) of Fucus serratus L. and proteins contained in a KCl extract of sperm has been used to identify a sperm protein involved in egg binding. High-performance gel filtration (HPGF) separated the sperm KCl extract into several major fractions, and a protein (apparent M, 60 kDa) was identified as being involved in binding to the egg PMVs. This protein ran on denaturing sodium dodecyl sulfate (SDS)gels with an apparent molecular weight of 27 kDa. This suggests that either the native form of the protein is a dimer or the molecular weight on HPGF is an artifact caused by high ionic strength buffer promoting hydrophobic interactions. When KCl-sol-uble proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE), blotted onto nitrocellulose, and incubated with biotinylated egg PMVs, these bound to a band at 27 kDa, confirming the role of this protein. Addition of the Fucus sperm extract or HPGF fractions containing the binding protein to eggs in the absence of sperm induced the release of polysaccharides onto the egg cell surface. This labeling was patchy, in contrast to the uniform release of polysaccharides observed when sperm were added to eggs. The monoclonal antibody (MAb) FS17 was raised against the 27-kDa sperm protein. It labeled the sperm body and both flagella by immunofluorescence, though the sperm had to he permeabilized to observe labeling, suggesting that the epitope recognized is not exposed at the cell surface. Addition of FS17 to the KCl extract in the binding assay reduced subsequent binding of egg PMVs. Removal of the 27-kDa protein recognized by FS17 from the sperm extract prevented the binding of egg PMVs in the binding assay and the triggering of the patchy release of polysaccharides when added to eggs. Overall the results suggest that the 27-kDa sperm protein is involved in binding to the egg plasma membrane and can trigger partial activation of the egg .     

Overexpression of the yeast MCK1 protein kinase suppresses conditional mutations in centromere-binding protein genes CBF2 and CBF5

We find that overexpression in yeast of the yeast MCK1 gene, which encodes a meiosis and centromere regulatory kinase, suppresses the temperature-sensitive phenotype of certain mutations in essential centromere binding protein genes CBF2 and CBF5. Since Mck1p is a known serine/threonine protein kinase, this suppression is postulated to be due to Mck1p-catalyzed in vivo phosphorylation of centromere binding proteins. Evidence in support of this model was provided by the finding that purified Mck1p phosphorylates in vitro the 110 kDa subunit (Cbf2p) of the multimeric centromere binding factor CBF3. This phosphorylation occurs on both serine and threonine residues in Cbf2p.     

Mechanisms for species differences in receptor-mediated carcinogenesis

Species differences resulting from a number of mechanisms are common in receptor-mediated chemical carcinogenesis. In this review, examples of possible mechanisms underlying these differences are discussed, including ligand metabolism, receptor polymorphisms, receptor isoforms, receptor levels, and crosstalk between signal transduction pathways. In addition, a number of other mechanisms also are likely to be important. The developmental state of the animal will determine the expression of receptors in different tissues. The regulatory pathways for cell proliferation and cell death and cell cycle check point controls can vary among species and tissues. Adaptation or potentiation of responses during chronic exposures to chemicals can greatly influence species differences. The mechanisms of adaptive processes are poorly understood but probably highly important for chronic toxicities such as cancer. Finally, different species may have different stem cell populations that are the targets for neoplastic transformation, and this will influence receptor-mediated carcinogenic responses. The implications of species differences in receptor-mediated responses for risk assessment are discussed.     

Effects of plant nutrition on the balance of insect relevant cardenolides and glucosinolates in Erysimum cheiranthoides

The possible effects of environmental stress on plant chemistry that are important to herbivorous insects were examined by growing a wild crucifer, Erysimum cheiranthoides, under different nutrient regimes. Oviposition by the cabbage butterfly, Pieris rapae, is thought to be affected by the balance of glucosinolates (stimulants) and cardenolides (deterrents) at the surface of leaves. E. cheiranthoides seedlings were provided with three levels of nitrogen and two levels of sulfur for a period of 15 days before analysis of semiochemicals in whole leaf tissue and at the surface of the foliage. The ratio of cardenolides to glucosinolates in the plants at elevated C/N ratios followed the carbon/nutrient balance hypothesis. However, a high nitrogen supply enhanced biomass production to the extent that concentrations of secondary compounds were unchanged or reduced. The concentration of glucosinolates (glucoiberin and glucocheirolin) at the surface was positively related to whole tissue levels. However, cardenolide (erysimoside and erychroside) concentrations, which were highest in leaf tissue of nitrogen-deficient plants, had the lowest surface levels on foliage of these plants. Possible reasons for differential expression of cardenolides and glucosinolates in a plant as a result of nutrient deficiency are discussed.     

Effects of nucleotide analogs on ATP hydrolysis by P-type ATPases. Comparison between the canine kidney (Na++ K+) ATPase and maize root H+-ATPase

The mechanism by which chemical energy is converted into an electrochemical gradient by P-type ATPase is not completely understood. The effects of ATP analogs on the canine kidney (Na⁺+ K⁺) ATPase were compared to effects of the same analogs on the maize (Zea mays L. cv. W7551) root H⁺-ATPase in order to identify probes for the ATP binding site of the maize root enzyme and to determine potential similarities of ATP hydrolysis mechanisms in these two enzymes. Six compounds able to modify the ATP binding site covalently were compared. These compounds could be classed into three distinct groups based on activity. The first group had little or no effect on catalytic activity of either enzyme and included 7-chloro-4-nitrobenz-2-oxa-1.3-diazole. The second group, which included azido adenine analogs. fluorescein isothiocyanate and 5′-p-fluorosulfonylbenzoyladenine, were inhibitors of ATP hydrolysis by both enzymes. However, the sensitivity of the (Na⁺+ K⁺) ATPase to inhibition was much greater than that exhibited by the maize root enzyme. The third group, which included periodate treated nucleotide derivatives and 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate. inhibited both enzymes similarly. This initial screening of these covalent modifiers indicated that 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate was the optimal covalent modifier of the ATP binding site of the maize root enzyme. Certain reagents were much more effective against the (Na⁺+ K⁺) ATPase than the maize root enzyme, possibly indicating differences in the ATP binding and hydrolysis pathway for these two enzymes. Two ATP analogs that are not covalent modifiers were also tested: the trinitrophenyl derivatives of adenine nucleotides were better than 5′-adenylylimidodiphosphate for use as an ATP binding probe.