The pentylenetetrazol model of anxiety detects withdrawal from diazepam in rats

This experiment tested whether benzodiazepine withdrawal could be detected in an animal model of anxiety. Rats were trained in operant chambers using food reward to press one lever after pentylenetetrazol (PTZ), 20 mg/kg, injection and the other lever after saline injection. Previously, the PTZ cue has been shown to be simulated by anxiogenic drugs and blocked by anxiolytic drugs. After rats reliably performed this discrimination, they were injected with diazepam, 20 mg/kg, from 1 to 4 times a day for six days. For one group of subjects, on the third, fourth and sixth days, they were also injected with 40 mg/kg of RO 15-1788, a benzodiazepine receptor antagonist, and tested for lever selection: 50–80% of the subjects selected the PTZ lever; these results are in contrast to those obtained prior to chronic diazepam treatment in which RO 15-1788 did not generalize to PTZ. A second group of subjects was also injected for six days with diazepam and then allowed to withdraw spontaneously for eight days: PTZ lever selection over this period varied from 20 to 60% of rats. These data indicate that animals trained to discriminate a PTZ cue: 1) generalize the benzodiazepine withdrawal state to the PTZ cue, and 2) discriminate the withdrawal state for long periods of time, agreeing with clinical observations of long-lasting anxiety signs during benzodiazepine withdrawal.     

Discriminative stimulus properties of L-phenylisopropyl adenosine: blockade by caffeine and generalization to 2-chloroadenosine

Recent neurochemical data on the effects of activation and blockade of adenosine A1 receptors has suggested a direct role of adenosine in neurotransmission. The present research used a drug discrimination procedure to test the hypotheses that A1 adenosine receptor activation could serve as a discriminative stimulus and that caffeine, a drug believed to be an A1 receptor antagonist, could block the adenosine discrimination. Food-deprived rats were trained to press one of two levers on an FR 10 schedule of food-pellet delivery. Responses on one lever were reinforced following i.p. injection of N6 - (L-phenylisopropyl) adenosine (L-PIA); responses on the other lever were reinforced following i.p. injection of saline. L-PIA training dose was increased from 0.064 to 0.08 mg/kg L-PIA in the course of the study. Subjects required an average of 91 sessions to acquire this discrimination. Stimulus control by L-PIA was dose-dependent, with the ED-50 being approximately 0.03 mg/kg. 2-Chloroadenosine (2CA) generalized to L-PIA with a tenth the potency. Caffeine blocked L-PIA-induced lever selection. These results indicate that 1) rats can be trained to discriminate L-PIA from saline in a two-lever food-reinforced task and 2) the discriminative stimuli produced by L-PIA are based on its agonistic action at the adenosine A1 receptor.     

Regulation of Legumin Levels in Developing Pea Seeds under Conditions of Sulfur Deficiency: Rates of Legumin Synthesis and Levels of Legumin mRNA

It was shown previously that when peas (Pisum sativum L.) are grown with suboptimal sulfur supply the level of legumin (the more S-rich of the two major seed storage proteins) in the mature seed is selectively reduced (Randall, Thomson, Schroeder, 1979 Aust J Plant Physiol 6: 11-24). This paper reports a study of the cellular mechanisms involved in regulating legumin synthesis under these conditions. Pulse and pulse-chase labeling experiments were carried out with excised, immature cotyledons from normal and S-deficient plants. Legumin was isolated from cotyledon extracts by immunochromatography, and the proportion of legumin synthesis relative to total protein synthesis was determined. Results showed that reduced legumin accumulation could largely be accounted for by a greatly reduced level of legumin synthesis (80-88% reduction) rather than by a major increase in legumin breakdown.

Legumin mRNA levels were assayed by two methods. In vitro translation of polysomal RNA from cotyledons of normal and S-deficient plants indicated a reduction of 60 to 70% in synthesis of legumin-related products by preparations from S-deficient plants. A legumin cDNA clone was constructed, characterized, and used to measure the levels of legumin mRNA in polysomal and total RNA preparations from developing cotyledons. Legumin mRNA levels were reduced by 90% in preparations from S-deficient plants.

When restored to an adequate S supply, S-deficient plants (or pods taken from such plants) recovered normal levels of legumin synthesis (in vivo and in vitro) and of legumin mRNA. These results indicate that reduced legumin accumulation under conditions of S deficiency is primarily a consequence of reduced levels of legumin mRNA.

     

Sequence interrelationships of the subunits of vicilin from pea seeds

Serological studies and comparison of N-terminal amino acid sequences with the amino acid sequence deduced from a cDNA clone have been used to establish the sequence relationships between the subunits of the pea seed storage protein, vicilin. Subunits smaller than M_r～50 000 (i.e., M_r 34 000, 30 000, 25 000, 18 000, 14 000, 13 000 and 12 000) show extensive homology with molecules within M_r～50 000 group. Both the sequencing and serological data confirm earlier evidence from studies on vicilin synthesisin vivo andin vitro which indicated that the vicilin subunits smaller than M_r～50 000 arose by endoproteolytic cleavage of parent molecules within the M_r～50 000 group. Cleavage in different M_r 50 000 parent molecules containing either one or both of two susceptible processing sites accounts for the formation of all the vicilin subunits smaller than M_r～50 000, with the possible exception of the M_r34 000 polypeptide. The position of these sites in the putative parents were defined by reference to a complete amino acid sequence deduced from the sequence of DNA complementary to mRNA for one member of the M_r～50 000 group.     

Inhibition by local anaesthetics of adenine nucleotide translocation in rat liver mitochondria

1. The mechanism of adenine nucleotide translocation in mitochondria isolated from rat liver was further examined by using the local anaesthetics procaine, butacaine, nupercaine and tetracaine as perturbators of lipid-protein interactions. Each of these compounds inhibited translocation of ADP and of ATP; butacaine was the most effective with 50% inhibition occurring at 30mum for 200mum-ATP and at 10mum for 200mum-ADP. The degree of inhibition by butacaine of both adenine nucleotides was dependent on the concentration of adenine nucleotide present; with low concentrations of adenine nucleotide, low concentrations of butacaine-stimulated translocation, but at high concentrations (greater than 50mum) low concentrations of butacaine inhibited translocation. Butacaine increased the affinity of the translocase for ATP to a value which approached that of ADP. 2. Higher concentrations of nupercaine and of tetracaine were required to inhibit translocation of both nucleotides; 50% inhibition of ATP translocation occurred at concentrations of 0.5mm and 0.8mm of these compounds respectively. The pattern of inhibition of ADP translocation by nupercaine and tetracaine was more complex than that of ATP; at very low concentrations (less than 250mum) inhibition ensued, followed by a return to almost original rates at 1mm. At higher concentrations inhibition of ADP translocation resulted. 3. That portion of ATP translocation stimulated by Ca(2+) was preferentially inhibited by each of the local anaesthetics tested. In contrast, inhibition by the anaesthetics of ADP translocation was prevented by low concentrations of Ca(2+). 4. The data provide further support for our hypothesis that lipid-protein interactions are important determinants in the activity of the adenine nucleotide translocase in mitochondria.     

Production and utilization of acetate in mammals

1. In an attempt to define the importance of acetate as a metabolic precursor, the activities of acetyl-CoA synthetase (EC 6.2.1.1) and acetyl-CoA hydrolase (Ec 3.1.2.1) were assayed in tissues from rats and sheep. In addition, the concentrations of acetate in blood and liver were measured, as well as the rates of acetate production by tissue slices and mitochondrial fractions of these tissues. 2. Acetyl-CoA synthetase occurs at high activities in heart and kidney cortex of both species as well as in rat liver and the sheep masseter muscle. The enzyme is mostly in the cytosol fraction of liver, whereas it is associated with the mitochondrial fraction in heart tissue. Both mitochondrial and cytosol activities have a K(m) for acetate of 0.3mm. Acetyl-CoA synthetase activity in liver was not altered by changes in diet, age or alloxan-diabetes. 3. Acetyl-CoA hydrolase is widely distributed in rat and sheep tissues, the highest activity being found in liver. Essentially all of the activity in liver and heart is localized in the mitochondrial fraction. Hepatic acetyl-CoA hydrolase activity is increased by starvation in rats and sheep and during the suckling period in young rats. 4. The concentrations of acetate in blood are decreased by starvation and increased by alloxan-diabetes in both species. The uptake of acetate by the sheep hind limb is proportional to the arterial concentration of acetate, except in alloxan-treated animals, where uptake is impaired. 5. Acetate is produced by liver and heart slices and also by heart mitochondrial fractions that are incubated with either pyruvate or palmitoyl-(-)-carnitine. Liver mitochondrial fractions do not form acetate from either substrate but instead convert acetate into acetoacetate. 6. We propose that acetate in the blood of rats or starved sheep is derived from the hydrolysis of acetyl-CoA. Release of acetate from tissues would occur under conditions when the function of the tricarboxylic acid cycle is restricted, so that the circulating acetate serves to redistribute oxidizable substrate throughout the body. This function is analogous to that served by ketone bodies.     

Effect of Lytic Enzymes of Acanthamoeba castellanii on Bacterial Cell Walls

Extracts of Acanthamoeba castellanii (Neff) contain alpha- and beta-glucosidase, beta-galactosidase, beta-N-acetylglucosaminidase, amylase, and peptidase. All of these activities are optimal between pH 3 and 4. These extracts also were found to clarify suspensions of cell walls from nine different gram-positive bacteria, including Micrococcus lysodeikticus. The pH optimum for the lytic activity was between 3 and 4. The extent of lysis of the various cell walls did not correlate with the release of free amino groups and of free N-acetylated sugars from the walls during digestion with these extracts. Suspensions of cell walls of Escherichia coli (a gram-negative bacterium), Cordiceps militaris (a fungus), and Acanthamoeba cysts, as well as of colloidal chitin, were not clarified by incubation with these extracts, although reducing sugars were released from each of these materials. Exhaustive digestion of M. lysodeikticus walls by lysozyme released no free N-acetylglucosamine. The products of exhaustive digestion of this cell wall with Acanthamoeba extracts were free N-acetylglucosamine, free N-acetylmuramic acid, glycine, alanine, glutamic acid, lysine, and N-acetylmuramic acid peptide fragments. These results suggest that the amoeba extracts contain endo- and exo-hexosaminidases, in addition to beta-hexosaminidase and peptide hydrolases.