Interaction of opioid peptides and other drugs with multiple delta ncx binding sites in rat brain: further evidence for heterogeneity.

Recent pharmacological data strongly support the hypothesis of δ receptor subtypes as mediators of both supraspinal and spinal antinociception (δ₁ and δ₂ receptors). In vitro ligand binding data, which are fully supportive of the in vivo data, are still lacking. A previous study indicated that [³H][ -Ala², -Leu⁵]enkephalin labels two binding sites in membranes depleted of μ binding sites by pretreatment with the site-directed acylating agent, 2-(p-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimidazole-HCI (BIT). The main goal of the present study was to develop a ligand-selectivity profile of the two δ_ncx binding sites. The data indicated that naltrindole and oxymorphindole were relatively selective for site 1 (20-fold). [ -Ser²,Thr⁶]Enkephalin and deltorphin-II were only 2.7-fold and 2.2-fold selective for site 1. [ -Pen², -Pen⁵]Enkephalin and deltorphin-I were 80-fold and 38-fold selective for site 2.3-Iodo-Tyr- -Ala-Gly-Phe- -Leu was 52-fold selective for site 1. Morphine had moderate affinity for site 1 (K_i = 16 nM), and was about 11-fold selective for site 1. Thus, of the 10 drugs studied, only DPDPE and DELT-I were selective for site 2. Viewed collectively with other data, it is likely that the δ₁ receptor and the δ_ncx binding site are synonymous.     

Guanine nucleotide- and muscarinic agonist-dependent phosphoinositide metabolism in synaptoneurosomes from cerebral cortex of immature rats

Guanine nucleotide-, neurotransmitter-, and fluoride-stimulated accumulation of [³H]inositol phosphates ([³H]InsPs) was measured in [³H]inositol-labeled synaptoneurosomes from cerebral cortex of immature (7-day-old) and adult rats, in order to clarify the role of GTP-binding proteins (G-proteins) in modulating phosphoinositide (PtdIns) metabolism during brain development. GTP(S) [Guanosine 5-O-(3-thio)triphosphate] time- and concentration-dependently stimulated PtdIns hydrolysis. Its effect was potentiated by full (carbachol, metacholine) and partial (oxotremorine) cholinergic agonists through activation of muscarinic receptors. The presence of deoxycholate was required to demonstrate agonist protentiation of the guanine nucleotide effect. The response to GTP(S) was higher in adult than in immature rats, while the effect of cholinergic agonists was similar at the two ages examined. At both ages, histamine potentiated the effect of GTP(S), while norepinephrine was ineffective. At both ages, guanosine 5-O-(2-thio)diphosphate [GDP(S)] and pertussis toxin significantly decreased GTP(S)-induced [³H]InsPs formation. The phorbol ester phorbol 12-myristate 13-acetate (PMA), on the other hand, did not inhibit the guanine nucleotide response in synaptoneurosomes from immature rats. NaF mimicked the action of GTP(S) in stimulating PtdIns hydrolysis. Its effect was not affected by carbachol and was highly synergistic with that of AlCl₃, according to the concept that fluoroaluminate (AlF₄ ^–) is the active stimulatory species. No quantitative differences were found in the response to these salts between immature and adult animals. These results provide evidence that, in both the immature and adult rat brain, neuroreceptor activation is coupled to PtdIns hydrolysis through modulatory G-proteins.     

New host and locality records for two protozoan (Myxosporea, Coccidia) parasites of marine fishes

This is the first report of the myxosporean Ortholinea orientalis from Atlantic herring Clupea harrengus. It infects the kidney tubules and previously was known from Pacific herring Clupea pallasii and navaga Eleginus navaga in the White Sea and North Pacific Ocean. This is also the first report of the coccidian Eimeria raibauti from Norway pout Trisopterus esmarkii. It infects the epithelium of the pyloric ceca and previously was known only from poorcod Trisopterus minutus in the Mediterranean Sea. The new records are both from the northern North Sea.     

Differences in accumulation of blunt- and cohesive-ended double-strand breaks generated by restriction endonucleases in electroporated CHO cells.

Restriction endonucleases (RE) have been used in cytogenetic studies to mimic the DNA double-strand break (dsb)-inducing action of radiation. In the experiments presented here, we have treated electroporated CHO cells with RE and have measured the resulting dsb using the filter elution technique under non-denaturing conditions (pH 9.6). PvuII, which generates blunt-ended dsb, gave rise to a significant number of measurable dsb. The frequency of the dsb induced by PvuII is shown to increase over a 3-12-h post-treatment incubation period, which implies that the RE is active in the cell for a considerable length of time. We postulate that the accumulation of dsb reflects a competition between enzymatic incision and repair of the DNA. The presence of araA, a known inhibitor of DNA synthesis, did not affect the frequency of PvuII-induced breaks indicating a lack of an inhibitory effect of araA on the repair of RE-induced dsb. Two RE which cause cohesive-ended dsb, namely BamHI and EcoRI, were found to be ineffective in giving rise to measurable dsb. Our interpretation of this is that for cohesive-ended dsb (caused by BamHI and EcoRI) the rate at which these breaks are rejoined matches or exceeds the rate of enzymatic incision and hence no dsb were observed. In the case of PvuII, the possibly slower rate of repair of blunt-ended termini would on this hypothesis result in the observed net accumulation of dsb.     

Alternatively spliced RNAs encode several isoforms of CD46 (MCP), a regulator of complement activation

Five alternative cDNA clones were isolated for CD46, also known as the membrane cofactor protein (MCP) for the factor I-mediated cleavage of the complement convertases. One of these cDNA clones (a) was identical to an earlier MCP clone. The other four CD46 clones 3ontained the four NH₂-terminanl short consensus repeat (SCR) units of MCP, but differed at the region encoding the carboxyl-terminal of the protein which includes an extracellular segment rich in Ser, Thr, and Pro residues, a hydrophobic membrane-spanning domain, and a 33 amino acid cytoplasmic tail. The different CD46 cDNAs have variously: (b) inserted a 93 base pair (bp) exon resulting in a new cytoplasmic tail of 26 amino acids; (c) deleted a 42 bp exon from the extracellular Ser/Thr rich region; (d) used a cryptic splice acceptor sequence to delete 37 bp from an exon encoding transmembrane sequence; or (e) failed to splice the intron after the four SCR units. These were shown by northern blot and polymerase chain reaction to arise by alternative splicing of CD46 RNA. Forms (a), (b), and (c) of CD46 RNA are common in placental RNA, but (d) was rare, and (e) was incompletely processed and therefore aberrant. The polymerase chain reaction (PCR) was used to map the sites of the intron/exon junctions and demonstrate further possible splice variants of CD46. The alternative RNAs for CD46 may correlate to the different isoforms of CD46 found in different tissues, tumors, and in serum.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M58050. Address correspondence and offprint requests to: D. F. J. Purcell.     

Neurofilament protein triplet immunoreactivity in the dorsal root ganglia of the guinea-pig

Summary Immunoreactivity for the neurofilament protein triplet was investigated in neurons of the dorsal root ganglia of the guinea-pig by using a battery of antibodies. In unfixed tissue, nearly all neurons in these ganglia demonstrated some degree of neurofilament protein triplet immunoreactivity. Large neurons generally displayed intense immunoreactivity, whereas most small to medium-sized neurons showed faint to moderate immunoreactivity. Double-labelling immunofluorescence demonstrated that most antibodies to the individual subunits of the neurofilament protein triplet had the same distribution and intensity of labelling in sensory neurons. Increasing durations of tissue fixation in aldehyde solutions selectively diminished neurofilament protein triplet immunoreactivity in small to medium-sized neurons. Double-labelling with neurofilament protein triplet antibodies in combination with antibodies to other neuronal markers, such as neuron-specific enolase, substance P and tyrosine hydroxylase, showed that tissue processing conditions affect the degree of co-localization of immunoreactivity to the neurofilament protein triplet and to these other neuronal markers. These results indicate that, with a judicious manipulation of the duration of tissue fixation, neurofilament protein triplet immunoreactivity can be used in combination with other neuronal markers to distinguish groups of neurons according to their size and chemical coding.     

Esterase-6 polymorphism inDrosophila melanogaster:Effects of temperature and methyl malonate on genotypic trajectories in polymorphic populations set up with highly inbred lines

It is generally difficult to identify possible effects of selection at a specific locus because of the heterogeneity of the genetic background. Geographical patterns ofEst-6 gene frequencies suggest that there is selection at this locus but selection on loci closely linked to it cannot be excluded. Differences in catalytic properties between allozymes have been shownin vitro; further, several laboratory studies have shown apparent fitness differences between allozymes. Our study used inbred lines highly homogeneous in the genetic background. Four populations were set up fromEst-6s andEst-6F homozygous females inseminated by males of the same genotype at each combination of three factors: temperature (18 and 25°C); methyl malonate (presence or absence); input gene frequencies [p(S) = 0.2 and 0.8]. The populations were sampled periodically for about 28 generations. Methyl malonate was chosen to exert pressure in the enzymatic function of esterase-6. Statistical analyses show that: there are no sex differences; gene frequencies change from input values to those of the first sampling, when only individuals of the first generation are present at 18^oC or individuals of the second generation just begin to appear at 25°C; gene frequencies do not change thereafter and Hardy-Weinberg equilibrium is established. The changes in gene frequencies observed in the first generations suggest thatEst-6 can under certain conditions be a target of selection. Such conditions may not, however, occur in natural populations.