High affinity dopamine D2 receptor radioligands. 2. [125I]epidepride, a potent and specific radioligand for the characterization of striatal and extrastriatal dopamine D2 receptors.

Epidepride, (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenzamide+ ++, the iodine analogue of isoremoxipride (FLB 457), was found to be a very potent dopamine D2 receptor antagonist. Optimal in vitro binding required incubation at 25 degrees C for 4 h at pH 7.4 in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. Scatchard analysis of in vitro binding to striatal, medial frontal cortical, hippocampal and cerebellar membranes revealed a KD of 24 pM in all regions, with Bmax's of 36.7, 1.04, 0.85, and 0.37 pmol/g tissue, respectively. The Hill coefficients ranged from 0.91-1.00 in all four regions. The IC50's for inhibition of [125I]epidepride binding to striatal, medial frontal cortical, and hippocampal membranes for SCH 23390, SKF 83566, serotonin, ketanserin, mianserin, naloxone, QNB, prasozin, clonidine, alprenolol, and norepinephrine ranged from 1 microM to greater than 10 microM. Partial displacement of [125I]epidepride by nanomolar concentrations of clonidine was noted in the frontal cortex and hippocampus, but not in the striatum. Scatchard analysis of epidepride binding to alpha 2 noradrenergic receptors in the frontal cortex and hippocampus revealed an apparent KD of 9 nM. At an epidepride concentration equal to the KD for the D2 receptor, i.e. 25 pM, no striatal alpha 2 binding was seen and only 7% of the specific epidepride binding in the cortex or hippocampus was due to binding at the alpha 2 site. Correlation of inhibition of [3H]spiperone and [125I]epidepride binding to striatal membranes by a variety of D2 ligands revealed a correlation coefficient of 0.99, indicating that epidepride labels a D2 site. In vitro autoradiography revealed high densities of receptor binding in layers V and VI of prefrontal and cingulate cortices as well as in striatum. In vivo rat brain uptake revealed a hippocampal:cerebellar and frontal cortical:cerebellar ratio of 2.2:1 which fell to 1.1:1 following haloperidol pretreatment. These properties suggest that [125I]epidepride is a superior radioligand for the in vitro and in vivo study of striatal and extrastriatal dopamine D2 receptors.     

Partial depletion of endogenous zinc level by (D-Pen2, D-Pen5)enkephalin in the rat brain.

The effects of the potent delta opioid agonist (D-Pen2, D-Pen5)enkephalin (DPDPE) were studied on the endogenous levels and regional distribution of Zn2+ in rat central nervous system by means of flame atomic absorption spectrophotometry. The olfactory bulb exhibited the highest Zn2+ level, followed by the frontal and parietal cortices, striatum and hippocampus; the lowest ion levels were found in the medulla and thoracic spinal cord. Intracerebroventricular administration of DPDPE resulted in significant, time- and dose-dependent decreases in endogenous Zn2+ contents in the parietal cortex, hippocampus and striatum. The action of DPDPE was antagonized by a 30 min naloxone pretreatment. Naloxone alone was without effect in eliciting these responses. Thus, delta opioid receptors may regulate or modulate endogenous Zn2+ levels in the rat brain.     

The marine toxin okadaic acid is a potent neurotoxin for cultured cerebellar neurons.

The tumor promoter okadaic acid (OKA), is a marine toxin of algal origin, identified as a potent inhibitor of protein phosphatases 1 and 2A, and possibly enhancing calcium influx through voltage dependent calcium channels (VSSC). We now report that OKA at concentrations as low as 0.5 nM produced neurotoxicity, characterized first by the desintegration of the neurites and swelling of cell bodies, and later by cellular death. Non-neuronal cells viability and morphology were unaffected up to at least 5 nM OKA. Neurons sensitivity to the toxin changed with age in culture. Maximum neurotoxicity was observed in neurons at 9 DIC, when the OKA concentration producing half of the maximum reduction in neuronal survival (EC50) was approximately 0.65 nM. At 5 DIC or 19 DIC (EC50 approximately 2.5 nM and approximately 4.5 nM respectively), neurons appeared to be less sensitive to OKA. Neurotoxicity by OKA was not reduced by VSCC antagonists such as nifedipine and verapamil, nor by antagonists of excitatory aminoacid (EAA) receptors including APV, MK801 or CNQX. VSCC antagonists and EAA receptors antagonists fully protected from neurotoxicity induced by depolarization with KCl. These results suggest that OKA mechanism of neurotoxicity may not directly involve VSCC, endogenous EAA release and EAA receptors, but may depend upon other neurochemical events.     

Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera

To investigate whether live attenuated pseudorabies virus (PRV) can be used as a vaccine vector, PRV recombinants that expressed envelope glycoprotein E1 of hog cholera virus (HCV) were generated. Pigs inoculated with these recombinants developed high levels of neutralizing antibodies against PRV and HCV and were protected against both pseudorabies and hog cholera (classical swine fever).     

Signal peptidase I overproduction results in increased efficiencies of export and maturation of hybrid secretory proteins in Escherichia coli

Summary The effects of 25-fold overproduction ofEscherichia coli signal peptidase I (SPase I) on the processing kinetics of various (hybrid) secretory proteins, comprising fusions between signal sequence functions selected from theBacillus subtilis chromosome and the mature part of TEM-β-lactamase, were studied inE. coli. One precursor (pre[A2d]-β-lactamase) showed an enhanced processing rate, and consequently, a highly improved release of the mature enzyme into the periplasm. A minor fraction of a second hybrid precursor (pre[Al3i]-β-lactamase), which was not processed under standard conditions of SPase I synthesis, was shown to be processed under conditions of SPase I overproduction. However, this did not result in efficient release of the mature β-lactamase into the periplasm. In contrast, the processing rates of wild-type pre-β-lactamase and pre(A2)-β-lactamase, already high under standard conditions, were not detectably altered by SPase I overproduction. These results demonstrate that the availability of SPase I can be a limiting factor in protein export inE. coli, in particular with respect to (hybrid) precursor proteins showing low (SPase I) processing efficiencies.     

2-Amino-N6-hydroxyadenine induces gene/point mutations and multiple-locus mutations, but not multilocus deletion mutations, in the ad-3 region of a two-component heterokaryon of Neurospora crassa.

The mutagenicity of 2-amino-N6-hydroxyadenine (AHA) has been studied in Neurospora crassa by treating a two-component heterokaryon (H-12) and recovering specific-locus mutations induced in the ad-3 region. This assay system permits the identification of ad-3A and/or ad-3B mutants resulting from gene/point mutations, multilocus deletion mutations, and multiple-locus mutations of various genotypes, involving one or both loci. Genetic characterization of the ad-3 mutants recovered from experiments with AHA in H-12 shows that 98.9% (270/273) of the ad-3 mutants are gene/point mutations (ad-3R), 1.1% (3/270) are unknowns, and none is a multilocus deletion mutation (ad-3IR). Among the gene/point mutations, 3.3% (9/273) are multiple-locus mutations (gene/point mutations with a closely-linked recessive lethal mutation [ad-3R + RLCL]). Another 25.3% (69/273) are multiple-locus mutations with a recessive lethal mutation located elsewhere in the genome [ad-3R + RL]. Heterokaryon tests for allelic complementation among the ad-3BR mutants showed that 90.8% (139/153) of the mutants were complementing, and 20.3% (31/153) were leaky. In addition, 32.5% (38/117) of the ad-3AR mutants were leaky. These data are consistent with the hypothesis that AHA produces specific-locus mutations in the ad-3 region of N. crassa by base-pair substitution. The data from the present experiments are compared with the data for 2-aminopurine (2AP)-induced ad-3 mutants in H-12 (de Serres and Brockman, 1991). Whereas, 2AP is a weak mutagen in H-12, AHA is extremely potent (Brockman et al., 1987). In contrast with 2AP, AHA induces ad-3 mutants exclusively by gene/point mutation in H-12. We conclude that whereas AHA induces ad-3 mutants predominantly by AT to GC base-pair transitions, 2AP induces ad-3 mutants by a wide variety of mechanisms including: (1) AT to GC and GC to AT base-pair transitions, (2) frameshift mutations, (3) other, as yet unidentified, intragenic alterations, (4) small multilocus deletion mutations, and (5) multiple-locus ad-3R mutations with closely linked recessive lethal mutations.     

Qualitative differences in the spectra of genetic damage in 2-aminopurine-induced ad-3 mutants between nucleotide excision-repair-proficient and -deficient strains of Neurospora crassa.

The mutagenic effects of 2-aminopurine (2AP) have been compared in the adenine-3 (ad-3) region of two-component heterokaryons of Neurospora crassa: nucleotide excision repair-proficient (uvs-2+/uvs-2+) heterokaryon 12 (H-12) and nucleotide excision repair-deficient (uvs-2/uvs-2) heterokaryon 59 (H-59). This forward-mutation, morphological and biochemical, specific-locus assay system permits the recovery of ad-3A and/or ad-3B mutants in 3 major classes: gene/point mutations, multilocus deletion mutations, and unknowns, and 3 different subclasses of multiple-locus mutations. Previous studies (Brockman et al., Mutation Res., 218 (1989) 1-11) showed that 2AP treatment of growing cultures of H-12 and H-59 gave no difference between ad-3 forward-mutation frequencies over a wide range of 2AP concentrations in each strain. In the present experiments, genetic analyses of ad-3 mutants recovered from these experiments has demonstrated qualitative differences between the spectra of the 3 main classes of ad-3 mutations. In H-12, 84.2% (203/241) resulted from gene/point mutation, 11.6% (28/241) from multilocus deletion mutation, and 4.1% (10/241) were unknowns. In contrast, in H-59, 43.0% (99/230) resulted from gene/point mutation, 55.7% (128/230) from multilocus deletion mutation, and 1.3% (3/230) were unknowns. In addition, quantitative differences were also found between the spectra of ad-3 mutations in 1 subclass of multiple-locus mutations, but not 2 additional subclasses. The first subclass consisted of 1.7% (4/241) and 9.6% (22/230) gene/point mutations with a closely linked recessive lethal mutation, in H-12 and H-59, respectively. The second two subclasses consisted of (a) 0.4% (1/241) and 0.4% (1/230) multilocus deletion mutations with a closely linked recessive lethal mutation, and (b) 13.3% (32/241) and 15.2% (35/230) gene/point mutations with a separate recessive lethal mutation elsewhere in the genome, in H-12 and H-59, respectively. Data from studies by others have shown that 2AP inhibits adenosine deaminase, resulting in nucleotide precursor pool inbalance, and that 2AP can saturate the mismatch repair system. As a consequence of either effect of 2AP, the spectrum of 2AP-induced mutation could include frameshift mutations and chromosome aberrations such as multilocus deletions in addition to base-pair substitutions. The defect in DNA repair due to the uvs-2 allele, which has been shown to be a deficiency in pyrimidine dimer excision (Worthy and Epler, 1974), most probably has some other excision-repair deficiency (Macleod and Stadler, 1986; Baker et al., 1991).(ABSTRACT TRUNCATED AT 400 WORDS)     

The genetic toxicology of 2-amino-N6-hydroxyadenine in eukaryotic organisms: significance for genetic risk assessment

A collaborative study was designed to assess the mutagenicity of 2-amino-N6-hydroxylaminopurine (AHA) in a wide variety of eukaryotic assays systems in terms of potency and specificity. Earlier studies in Salmonella and Neurospora had shown that AHA was an extremely potent mutagen which appeared to cause predominantly AT to GC base-pair transitions. This discovery was viewed as an unusual opportunity to explore the general utility of different eukaryotic assay systems for genetic risk assessment. The objective was to determine whether AHA would show comparable potency and specificity in those eukaryotic organisms used to evaluate mutagenic potential of environmental chemicals for the human population. The data presented in this report show that AHA was mutagenic in all the eukaryotic assays utilized; however, the level of effect was found to be assay system-dependent. In addition, in assays where other base analogs were used as positive controls, differences in relative potency were observed from those obtained in the earlier studies with Salmonella and Neurospora. When alkylating agents were used as positive controls in the higher eukaryotic assays, AHA was found to have a mutagenic potency comparable to ethylnitrosourea (ENU), ethyl methanesulfonate (EMS) or methyl methanesulfonate (MMS) for many of the assays. With regard to mutagenic specificity, AHA appears to induce gene/point mutations in eukaryotic organisms, resulting predominantly from base-pair substitutions, predominantly AT to GC base-pair transitions; however, there was some unexplained variation in the ratio of these base-pair transitions and other transitions and transversions as a function of assay system. In addition, studies on the induction of micronuclei have shown that AHA induces chromosomal damage at high concentrations and low levels of survival.     

On the opportunity cost of the photosynthate invested in stem elongation reactions mediated by phytochrome

Summary Seedlings of shade-intolerant species react to alterations of the light climate caused by their neighbors with morphological changes that may influence the pattern of resource acquisition and utilization at the whole-canopy level. One such change, the increased stem elongation rate that is triggered by low red (R, 660 nm) to far-red (FR, 730 nm) ratios (R:FR) in dense canopies, might reduce the amount of assimilates available for leaf area expansion or root growth, and in that way affect resource capture by the canopy. We have tested this hypothesis by comparing the growth of both isolated individuals and canopies of the weed Amaranthus quitensis under conditions differing only in the spectral distribution of the incident light. When canopies received the full spectrum of sunlight, the stems were a large proportion (40–57%) of total biomass. Filtering the FR waveband (and hence raising the R:FR ratio to eliminate the neighbors' proximity-signal) resulted in shorter canopies with lighter stems. However, the growth of leaves and roots was not promoted by this treatment, indicating that the opportunity cost of the assimilates invested in the stems was nil or very small. Filtering the FR had no effect on biomass accumulation when plants were grown as isolated individuals. The higher growth of the canopics under full spectrum could be due to a higher light interception or to a higher efficiency of light conversion into biomass. The first possibility is weakened by the observation that filtering the FR had no effect on the dynamics of soil covering by the crops. The second is indirectly strengthened by results of an experiment with isolated plants showing that stem elongation, stem growth, and total plant biomass can be increased by reducing the flux of R light received by the stems without affecting the light climate of the leaves. Further work is needed to distinguish between these two possibilities; whatever the cause, our results show that the elongation responses to decreased R:FR may lead to a net increase in canopy productivity, and do not necessarily have a negative impact on the growth of resource-harvesting organs.