Evidence for sedative effects of low doses of morphine in mice involving receptors insensitive to naloxone

Low doses of morphine (0.30–2.5 mg/kg) decrease in a dose-dependent manner spontaneous climbing behaviour in mice. This effect is not modified by administration of naloxone at doses up to 1.25 mg/kg. These morphine doses do not modify the locomotor activity but, when they are associated with naloxone (0.5 mg/kg), an obvious inhibition occurs. In rats, a hyperactivity follows the akinesia produced by a morphine administration (10 mg/kg). This hyperactivity is changed into a significant hypokinesia when the animals are treated with naloxone (0.05 mg/kg). These results might reveal a dual effect of low doses of morphine, the excitatory effect of morphine being antagonized by naloxone whereas no action on the sedative effect is observed.     

Construction of hybrid plasmids containing the lysA gene of Escherichia coli: studies of expression in Escherichia coli and Saccharomyces cerevisiae

Summary The lysA gene of Escherichia coli has been cloned from a transducing phage on various plasmids, present in different copy numbers in bacterial cells. Synthesis of the product of this gene, diaminopimelate (DAP)-decarboxylase, and its regulation have been studied. Expression does not follow a simple gene dosage effect, maximal expression already being obtained with a six-copy plasmid. This result suggests that either a positive or an autogenous regulatory mechanism is involved. We also used one of the hybrid plasmids to look for expression of the bacterial lysA gene in Saccharomyces cerevisiae. The results indicate that the product of the E. coli gene is not actively translated in yeast.     

Retrograde Inhibition of Transmitter Release by ATP

Abstract: After labelling ACh tissue stores in Torpedo electric organ prisms with radioactive acetate, the release of ACh and ATP triggered by electrical stimulation or KCI depolarization was measured in the same perfusate samples. The luciferin-luciferase reaction for ATP was first counted, then the radioactive content of the sample determined. Further evidence showing that ATP release resulted from postsynaptic transmitter action was that carbachol could induce the release of ATP. A dose-response curve was obtained. Curare or α-bungarotoxin block the release of ATP elicited by carbachol. When triggered by KCI depolarization the increased efflux of ACh and ATP returned to low levels in spite of the maintained depolarization. After two successive KCI depolarizations, it was possible to dissociate the release of both substances. The efflux of ATP was exhausted while ACh release was maintained. If the second KCI depolarization was delayed ATP release recovered, but the release kinetics of ACh and ATP were sustained. The exhaustion of endogenous ATP release or the action of exogenous ATP had little or no effect on the release of ACh triggered by KCI depolarization. On the contrary, the release of ACh induced by electrical stimulation was sensitive to the action of adenine nucleotides, and a quantitative estimation of the inhibition of ACh release by ATP and adenosine could be made. At the onset of stimulation ATP release predominated, being gradually replaced by adenosine, which can be reuptaken. This would terminate the inhibitory action of the nucleotide. Carbachol inhibits evoked ACh release, while the effect of α-bungarotoxin was to increase spontaneous ACh release. These effects could be respectively mediated by an increased or a reduced release of ATP resulting from the postsynaptic action of ACh agonists or antagonists. However, a direct presynaptic effect of these substances is not excluded. It seems possible that the action of ATP on ACh release can be explained through its inhibition of the depolarization-evoked Ca²⁺ entry.     

The use of cytoplasmic streptomycin resistance: Chloroplast transfer from Nicotiana tabacum into Nicotiana sylvestris, and Isolation of their somatic hybrids

Summary Leaf protoplasts of Nicotiana tabacum SR1 (2n=4x=48) treated with iodoacetate (10 mM; 25 C; 30 min) and consequently unable to divide, and untreated leaf protoplasts of Nicotiana sylvestris (2n=2x=24) were fused using polyethylene glycol (PEG). The SR1 line is resistant to streptomycin because of a maternally inherited mutation, and has streptomycin-insensitive chloroplast ribosomes.After 1 month of growth in the absence of streptomycin protoplast-derived calli were plated into selective medium (1,000 g ml^-1 streptomycin) and the resistant clones were isolated. Out of 10⁶ PEG-treated protoplasts (1:1 mixture of parental types) 137 resistant (green) clones were obtained, whereas in the same number of parental cells, not subjected to fusion induction, no resistant callus was found.At least four plants were regenerated from each of the clones. The regenerates were identified as somatic hybrids (H), N. sylvestris (Ns) or N. tabacum (Nt) by looking at esterase and peroxidase isoenzymes and morphology. The three types of regenerates were distributed amongst the clones as follows: H only (105 clones); Ns (16 clones); Ns+H (6 clones); Nt only (3 clones); Nt+H (6 clones); Nt+Ns (1 clone). The high proportion of hybrid regenerates indicates that nuclear fusion has occured in the overwhelming majority of the heterokaryocytes. Cytoplasmic mutations in combination with inactivation by iodoacetate, therefore, are suitable markers to produce somatic hybrids. Segregation of nuclei after fusion resulted in new combinations of organelles and nuclei, the final outcome being the transfer of resistant chloroplasts into N. sylvestris, some of which have the original diploid (2n=24) chromosome number. Data suggest that segregants were in most cases obtained from multiple fusions. Streptomycin resistance was inherited maternally in the N. sylvestris (six clones) tested and the hybrid (three clones) regenerates.     

Synaptosomal uptake

Using purified synaptosomal preparations from rat brain, the uptake ofl-tryptophan and norepinephrine was studied. We were unable to replicate some of the results of the experiments obtained with crude mitochondrial, fractions (P₂). Thus we examined the validity of the results of uptake studies obtained with the crude synaptosomes and established conditions which would simulate the biochemical milieu in which the nerve terminals functionin vivo, such as active substrate-dependent respiration, respiratory coupling on addition of ADP, low impurity of noncharacteristic markers, exogenous added proteins (e.g. bovine serum albumin), and verification by electron microscopy. All uptake studies withl-TRP and NE were completed in a system designed for simultaneous recording of respiration and the effect of added ADP. This system was also employed in comparative studies with mitochondria purified by multiple density gradients derived either from the perikaryon or from synaptosomes. Synaptosomal or mitochondrial preparations which did not conform to the above criteria invariably showed significantly lowered ability of uptake ofl-TRP or NE. This was found to be related to impairment in their respiratory and coupling ability. When the experimental conditions of others were employed, the time course of uptake of TRP for crude synaptosomes (P₂) was 100 nmol/g/min and was linear for 2.5 min, while for the purified synaptosomes it was 20 nmol/g/min with a l-min linearity. The mitochondria purified from P₂ displayed 30 nmol/g/min uptake withl-TRP with a linearity of 2.5 min. Reconstituted system of purified synaptosomes and mitochondria gave 60 nmol/g/min ofl-TRP transport with 2.5 min linearity. Also examined was the effect of eight different media. It was found that Krebs-Ringer solution containing glucose (40 mM), pyruvate and malate (10 mM), and ADP (250 nmol) gave optimal uptake of TRP both for synaptosomes and for mitochondria, increasing it to 60 and 86 nmol/g/min. The above conditions also enhanced the uptake of NE by synaptosomes and mitochondria. Uptake of NE was not proportional to protein concentration when the protein content exceeded 0.4 mg. Purified synaptosomal mitochondria accumulated NE more actively than the purified nonsynaptic free mitochondria, albeit at the same rate. Synaptic and free mitochondria had an impaired uptake of NE in presence of DNP, antimycin A, and rotenone, and unlike withl-TRP, pyruvate and malate also reduced uptake of NE. Significant differences were noted for the cytochrome oxidase activity between the synaptosomal and free michondria when compared to that of the homogenate.