AXONAL TRANSPORT OF PHENYLETHANOLAMINE-N-METHYLTRANSFERASE IN TOAD SCIATIC NERVE

—The presence of phenylethanolamine-N-methyltransferase (EC 2.1.1.-) and dopamine-β-hydroxylase (EC 1.14.2.1) activities was demonstrated in the sciatic nerve of the toad, Bufo marinus. The rates of accumulation of phenylethanolamine-N-methyltransferase (PNMT) and dopamine-β-hydroxylase (DBH) proximal to a ligation of the sciatic nerve were studied. DBH accumulated proximal to the ligation at a more than 10-fold faster rate than PNMT. By measuring the rate of loss of enzyme activity distal to a ligation, an estimate of per cent clearance of each enzyme was made. Based on the per cent of enzyme activity free to move, the absolute transport rates for each enzyme were estimated to be: PNMT, 3.6 mm/24 h; DBH, 102 mm/24 h. PNMT activity (89 per cent) was recovered in the soluble fraction of sciatic nerve homogenates with no change occurring in the subcellular distribution of the enzyme proximal to ligations. In contrast, 43 per cent of DBH activity was found in the soluble fraction of sciatic nerve homogenates; but a disproportionate increase in paniculate DBH activity was found proximal to sciatic nerve ligations. Reduction of toad body temperature to 4°C resulted in a complete but totally reversible block of the axonal transport of both PNMT and DBH.     

ENZYMATIC ISOTOPIC ASSAY FOR AND PRESENCE OF β-PHENYLETHYLAMINE IN BRAIN

Abstract— An enzymatic isotopic assay for the measurement of β-phenylethylamine in brain, with a sensitivity of 100-200 pg, has been developed. With this assay, the endogenous β-phenylethylamine content (1.5 ng/g) in the rat brain has been determined. Phenylalanine administration increases the brain levels of this amine; inhibition of monoamine oxidase causes a 40-fold increase in brain β-phenylethylamine. After a combined treatment with a monoamine oxidase inhibitor and phenylalanine, the β-phenylethylamine content in the brain increases to about 400-fold. This increase can be blocked by the central decarboxylase inhibitor NSD-1055. p-Chlorophenylalanine also increases β-phenylethylamipe concentration in the brain, and this effect is potentiated by a simultaneous administration of phenylalanine.     

Studies on reactivated cilia : I. The utilization of various nucleoside triphosphates during ciliary movement

It has been found that the movement of reactivated cilia is prolonged, providing various nucleoside triphosphates (NTP) are present in the reactivation medium in addition to ATP. The results suggest the existence of an axoneme-bound phosphotransferase that regenerates ATP from NTP. A model is proposed for the enzymatic regulation of ATP levels in the cilium.     

A new case for a presynaptic role of dendrites: An immunocytochemical study of the N. Raphé Dorsalis

The distribution of serotonin (5-HT) was determined by the application of the prembedding peroxidase-anti-peroxidase (PAP) technique in vibratome and ultrathin sections of the brain stem. The antiserum stained the neuronal groups B₁ to B₉. Somata, dendrites and axons of multipolar and bipolar neurons were recognized in the usual locations. The most commonly found profiles in the area of the n.raphe dorsalis were dendrites. The search for axon terminals was unsuccesful. The labeled dendrites appear in synaptic contact with unlabeled endings containing round or pleomorphic vesicles, and occasionally some large dense core vesicles. Contacts between two labeled dendrites or processes were not found. Occasionally a dendrodendritic junction between a 5-HT labeled dendrite and an unlabeled dendrite has been found. There are areas of the dendritic membrane free of synaptic junctions and free of glial insulation. Results are discussed in relation with the previously proposed presynaptic role of the dendrites in the neuronal circuitry of then. raphé dorsalis.Special Issue dedicated to Prof. Eduardo De Robertis.Research supported by grants from the CONICET and SECYT, Argentina.     

High-Protein Carboxymethylase Activity and Low Endogenous Methyl Acceptor Proteins in Posterior Pituitary Lobe of Rats Lacking Neurophysin-Vasopressin (Brattleboro Rats)

Abstract: The activity of protein carboxymethylase and the endogenous protein methyl acceptor capacity were examined in the posterior, intermediate, and anterior lobes of the pituitaries of homozygous Brattleboro rats with diabetes insipidus and in heterozygous Brattleboro and Long-Evans control rats. Protein carboxyl methylation is selectively altered in the posterior pituitary lobes of homozygous Brattleboro rats. Protein carboxymethylase activity is higher (+40%) and endogenous methyl acceptor protein capacity is lower (-80%) with respect to heterozygous Brattleboro and Long-Evans control rats. This latter change is correlated with decreased methylation of proteins of a molecular weight of approximately 11K daltons, is selective for the posterior pituitary lobe, since it does not occur in the intermediate and anterior lobes, and probably reflects the absence of vasopressin-associated neurophysin in homozygous Brattleboro rats. Our results support a physiological role of protein carboxyl methylation in the neurosecretory process in the posterior pituitary gland.     

Radioimmunoassay of met-enkephalin in microdissected areas of paraformaldehyde-fixed rat brain

We studied the effect of various sample preparation procedures on rat brain met-enkephalin content, measured by radioimmunoassay. Whole brain met-enkephalin content of rats killed by decapitation followed by immediate tissue freezing was similar to that of rats killed by microwave irradiation and to those of rats anesthetized with pentobarbital or halothane before killing, whether previously perfused with paraformaldehyde or not. In contrast, a decrease (up to 80%) in met-enkephalin concentrations was observed when brain samples were frozen and thawed to mimic the procedure utilized in the “punch” technique for analysis of discrete brain nuclei. This decrease was totally prevented by paraformaldehyde perfusion of the brain prior to sacrifice. Brain perfusion did not alter the amount of immunoassayable met-enkephalin extracted from tissue or its profile after Sephadex chromatography. Paraformaldehyde perfusion results in better morphological tissue preservation and facilitates the “punch” dissecting technique. Paraformaldehyde perfusion may be the procedure of choice for the measurement of neuropeptides in specific brain nuclei dissected by the “punch” technique.     

Quantitative in vitro autoradiographic characterization of [125I]angiotensin III binding sites in rat adrenal gland

A single class of high-affinity binding sites for [125I]angiotensin III and [125I]angiotensin II were found in rat adrenal medulla and zona glomerulosa by quantitative autoradiography. In the medulla, Kd were 1.46 and 1.16 nM, and Bmax 1700 and 1700 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. In the zona glomerulosa, Kd were 0.86 and 0.90 nM, and Bmax 790 and 560 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. Unlabeled angiotensin III and angiotensin II displaced [125I]angiotensin III with similar potency in both adrenal zona glomerulosa and medulla. Our findings suggest that angiotensin III and angiotensin II might share the same binding sites in adrenal gland and support the hypothesis of a role for angiotensin III in the adrenal medulla and zona glomerulosa.     

Combined effect of synthetic enterocin CRL35 with cell wall,membrane‐acting antibiotics and muranolytic enzymes against Listeria cells

Aims: To evaluate the inhibition effectiveness of enterocin CRL35 in combination with cell wall, membrane‐acting antibiotics and muranolytic enzymes against the foodborne pathogen Listeria. Methods and Results: Synthetic enterocin CRL35 alone and in combination with monensin, bacitracin, gramicidin, mutanolysin and lysozyme were used in this study. Minimal inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) index assays were performed using Listeria innocua 7 and Listeria monocytogenes FBUNT as sensitive strains. Antibiotics showed positive interactions with the bacteriocin in both strains tested. On the other hand, when mutanolysin and enterocin CRL35 were added to resting cells in a buffer system, the lytic effect of mutanolysin was enhanced. However, the addition of mutanolysin showed no effect on the growth of L. innocua 7 cells in a culture medium. Moreover, mutanolysin allowed the overgrowth of L. innocua 7 cells to an OD similar to control cells in the presence of inhibitory concentration of enterocin CRL35. In contrast, the combination of lysozyme and enterocin CRL35 resulted in a 50% inhibition of the L. innocua 7 growth. Conclusions: Based on our results, we conclude that the combination of synthetic enterocin CRL35 with some antibiotics is effective against L. innocua 7 and L. monocytogenes FBUNT cells, and more importantly the amount of these agents to be used was considerably reduced. The effectiveness of the combination of synthetic enterocin CRL35 with muramidases seems to depend on complex environments, and more detailed studies need to be performed to elucidate this issue. Significance and Impact of the Study: Enterocin CRL35 represents a promising agent that not only can ensure the quality and safety of food but it can also be combined with several antimicrobial agents important in the medical field.     

New differences between the Wistar rat and Octodon degus, a putative laboratory animal resistant to morphine

1. The effects of CNS depressants (methadone and alcohol) and natural neurotransmitters (NA and ACh) are studied in O. degus. 2. O. degus shows resistance to methadone in the formalin algesiometric test and EEG. 3. Ethanol elimination profile suggest the presence of an atypical alcohol dehydrogenase in O. degus 4. O. degus is extremely resistant to the pressor effects of noradrenaline 5. the isolated atrium of this rodent is 40 times more sensitive to the negative chronotropic effect of methadone, than the rat atrium. 6. These effects could be explained in terms of an important catecholamine and endorphin co-secretion from adrenal glands in O. degus.