Age-related changes in metabolic responses to chronic monoamine depletion in central dopaminergic and serotonergic systems of rats treated with reserpine.

Groups of 5, 15, and 27-month-old rats were treated with reserpine (5 mg/kg IP) and killed after 1, 7, 14, and 21 days. Reserpine's effects on dopamine (DA) and serotonin (5HT) metabolism were studied in the striata and limbic areas, on the basis of changes in metabolite concentrations and a calculated index [delta(C/M)] expressing changes in the molar ratio between metabolite and parent monoamine. Twenty-four hours after drug treatment, when the two monoamines reached a new steady-state, about 10% of the normal concentration, there was evident enhancement in the metabolism of striatal DA, indicated by HVA levels and delta(C/M) changes. This increase was significantly lower in the senescent rats. In contrast with HVA, the levels of DOPAC were lowered in reserpinized rats. This effect, present in all three age groups, lasted up to 21 days in young and 14 days in adult and old rats. However, for this metabolite too, the delta(C/M) indicated an increase in its formation which was lower in 27-month-old rats. Similar effects were observed in limbic areas, but here the age-related differences were less evident. Serotonin metabolism was also increased by reserpine as indicated by the delta(C/M). This parameter was lower in young than in adult and old rats, at least in limbic area. Similarly, 5HIAA increased only in the senescent rats. These age-related differences were not evident in striata. In this rat population, aging apparently modified the compensatory metabolic responses of some dopaminergic and serotonergic systems. The direction of these changes, however, depends on the system and the brain region considered.

Depletion and recovery of neuronal monoamine storage in rats of different ages treated with reserpine.

The effect of reserpine on dopamine, noradrenaline, adrenaline and serotonin concentrations in different brain regions, and the recovery of normal levels of these monoamines after such treatment were studied in rats aged 5, 15 and 27 months. In a preliminary experiment we found that distribution of the drug was not altered in the aged rats. Then we observed that a single dose of reserpine (5 mg/kg IP) had a similar depleting effect on all the brain monoamines, in all the brain regions considered in all three age groups. The curves expressing recovery of monoamine storage in all the nerve terminals, several days after treatment, were superimposable. These results suggest that in the rat, age does not influence the effect of reserpine on the storage mechanism of brain monoamines. Moreover, as restoration of this mechanism depends on the synthesis of new vesicles, the similarity in the rates of recovery in adult, old and very old rats indicated indirectly that synthesis of these neuronal organelles is not affected by aging.

Detection of HTLV-III-specific IgG bands in the CSF from a patient with AIDS and encephalitis.

Recent evidence would suggest that HTLV-III may be neurotropic. We have found oligoclonal IgG bands by isoelectric focusing in the CSF of a homosexual man with AIDS and encephalitis. Subsequent analysis revealed that such bands contained anti-HTLV-III activity, suggesting that neurologic symptoms in AIDS patients may be caused by replication of HTLV-III inside the CNS.

Enzymatically active forms of reverse transcriptase of the human immunodeficiency virus.

The reverse transcriptase of HIV-1 (AIDS virus) is characterized by the presence of two highly immunogenic proteins of 66 and 51 kD known to be enzymatically active as a complex p66/51. Using an activity gel procedure that allows identification of catalytic polypeptides in situ after PAGE in denaturing conditions, we visualized two major active bands of 66 and 51 kD of reverse transcriptase from highly purified preparations of HIV-1. We show that both p66 and p51 are enzymatically active. An additional active band was also associated with a 165 kD polypeptide, representing about 2-4% of total activity and possibly corresponding to the putative gag-pol precursor. In H9-infected cells the 66 kD active band became visible 70 hours after infection. These studies show that the two major forms of reverse transcriptase (66 and 51 kD) of HIV-1 are independently active and that a higher Mr form of 165 kD is also enzymatically active.

HIV type 1 intraperitoneal infection of rabbits permits early detection of serum antibodies to Gag, Pol, and Env proteins, neutralizing antibodies, and proviral DNA from peripheral blood mononuclear cells.

The aim of this study is the development of an animal model useful for studying HIV-1 pathogenesis, candidate vaccines, and antiviral drugs. Aseptic thioglycolate peritonitis was induced in six rabbits. After 4 days, four rabbits were infected with 1 ml of HIV-1 stock containing 100 times the MID50. Blood samples were collected every 2 weeks for 8 months. Serum antibodies were tested by ELISA, using as antigen the recombinant protein p24; synthetic peptides of highly conserved regions of p31, gp41, and gp120; and a synthetic peptide of gp120 at the V3 loop region of HIV-1 strains IIIB and MN. Furthermore, neutralizing antibodies were tested by a microscale neutralization assay. Proviral DNA was detected by PCR, and virus isolation was performed by a cocultivation technique using primary rabbit peripheral blood mononuclear cells (PBMCs). All infected rabbits produced antibodies to HIV-1 proteins within 2 weeks and up to 8 months after virus infection. Serum antibodies were directed against the Env (gp120 and gp41), Gag (p24), and Pol (p31) proteins and against two synthetic peptides whose sequence corresponds to gp120 at the V3 loop region of HIV-1 strains IIIB and MN. Neutralizing antibodies were also detected in the sera of infected animals. Proviral DNA was detected in PBMCs by PCR within 4 weeks and up to 8 months after HIV-1 infection. HIV-1 was also isolated from PBMCs of infected animals at 30, 60, and 120 days after infection. Results obtained indicate that HIV-1 intraperitoneal infection of the rabbit permits the early detection of serum antibodies to Gag, Pol, and Env proteins, neutralizing antibodies, and proviral DNA sequences from PBMCs.

Dopaminergic effects of buspirone, a novel anxiolytic agent.

The novel anxiolytic drug buspirone raised striatal levels of the dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) 1 hr after oral administration. This effect was dose-dependent with a peak at 60 min. No changes were observed in the levels of 3-methoxytyramine (3MT), the extraneuronal metabolite of dopamine. Noradrenaline, serotonin and its metabolite 5-hydroxyindoleacetic acid (5HIAA) were not affected. Buspirone displaced [3H]spiroperidol from striatal binding sites, with an IC50 (1.8 x 10(-7) M), comparable to that of clozapine (IC50 = 1.4 x 10(-7) M) but considerably lower than that of haloperidol (4.7 x 10(-9) M). Buspirone was only a weak inhibitor of dopamine-stimulated adenyl cyclase. Buspirone was not active on the binding of trifluoperazine to calmodulin and did not modify calmodulin-induced activation of phosphodiesterase (PDE). Repeated administration of buspirone did not increase the number of DA receptors. These data show that, although buspirone has antidopaminergic activity, it can hardly be classified as a classic neuroleptic agent.

Central side effects of pentamethylmelamine: biochemical and behavioural studies.

The central side effects of pentamethylmelamine (PMM), an antitumoral agent, were studied on brain neurotransmitters from the biochemical and behavioural points of view. PMM causes a dose-related reduction in the body temperature and motility of mice. 100 mg/kg of PMM lowers the levels of noradrenaline (NA) and raises 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) in the telencephalon. A similar dose increased striatal levels of dopamine (DA) metabolites, homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC), at earlier times (30 min), reducing their levels at 2 hr. These effects disappear at longer times (4 hr). No changes were observed in the levels of 3-methoxytyramine (3-MT), the extraneuronal metabolite of DA. The serotonin metabolite 5-hydroxyindolacetic acid (5HIAA) was almost not affected. PMM and its metabolites do not displace [3H]-spiroperidol from mouse striatal binding sites. These data show that some of the neurological effects induced by PMM are associated with changes in the metabolism and/or release of brain catecholamines but are not mediated by direct action on DA receptors.

Does acute L-DOPA increase active release of dopamine from dopaminergic neurons?

L-DOPA is believed to be decarboxylated by the residual striatal dopaminergic presynaptic terminals with formation of the putative neurotransmitter dopamine (DA) and with increased availability of DA at post-synaptic receptors. However there is no direct evidence that the DA formed is released into the synaptic cleft. We therefore investigated the biochemical modifications occurring in the dopaminergic system after acute administration of L-DOPA. After acute L-DOPA (100 mg/kg plus 25 mg/kg of benserazide p.o.) the levels of 3-methoxytyramine (3-MT), a metabolite reflecting release of the neurotransmitter DA, were significantly raised, following the same pattern as DA levels, indicating that DA release from DA nerve terminals is increased after L-DOPA administration. The increased DA release and 3-MT formation were not reduced by pretreatment with direct DA agonists such as apomorphine (5 mg/kg i.p.) or piribedil (120 mg/kg p.o.). Thus in this case DA release is not under the control of the compensatory mechanisms induced by post-synaptic receptor hyperstimulation.

Differential effects of certain dopaminergic drugs on the striatal concentration of dopamine metabolites, with special reference to 3-methoxytramine.

Studies were undertaken to evaluate the effect of certain dopamine (DA) agonists and antagonists on DA metabolite concentrations in rat striatum, with special regard to 3-methoxytyramine (3-MT). Quipazine, nomifensine and piribedil act as dopaminergic agonist drugs. However piribedil, which is a dopaminergic receptor agonist, reduced the concentrations of all 3 metabolites considered, while nomifensine produced an increase, and quipazine caused an early rise in 3-MT followed by a lowering of the concentration of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Haloperidol and clozapine are both DA antagonists. Haloperidol, which blocks DA receptors, caused an increase in the formation of all 3 metabolites while clozapine raised DOPA and HAV but not 3-MT. The importance of simultaneous determination of these 3 metabolites is discussed with relation to evaluation of the mechanism of action of these drugs and - more generally - of drugs acting on the dopaminergic system.

In vivo and in vitro evidence of dopaminergic system down regulation induced by chronic L-DOPA.

The biochemical modifications which occur in the dopaminergic system after chronic administration of L-DOPA are investigated. Levels of DA and of its metabolite 3-methoxytyramine (3-MT), an expression of the amount of DA released, were raised to the same extent in controls given a single dose of 1-DOPA and in chronically treated rats given 100 mg/kg of 1-DOPA plus 25 mg/kg of benserazide twice a day for 24 days. However, the reduction in neuronal function expressed by the decrease in 3-MT which follows treatment with DA agonists such as piribedil and apomorphine was less pronounced in the chronically L-DOPA treated rats. This suggests that such treatment causes a down regulation of DA receptors. These in vivo results were confirmed by in vitro analysis of DA receptor activity after chronic L-DOPA. Under these conditions there was a significant reduction in the number of [3H]-spiperone and [3H]-ADTN binding sites with no changes in their affinity. The in vivo and in vitro findings both suggest the involvement of a subsensitive compensatory mechanism or down regulation of dopaminergic neurons after chronic treatment with L-DOPA.

Determination of illicit drugs and related substances by high-performance liquid chromatography with an electrochemical coulometric-array detector.

A general method for the simultaneous determination of fifteen common drugs (6-acetylmorphine, 3,4-methylenedioxymetamphetamine, buprenorphin, cocaine, codeine, dihydrocodeine, ethylmorphine, heroin, hydrocodone, lidocaine, methadone, morphine, naloxone, procaine and thebaine) was developed using reversed-phase HPLC and electrochemical detection. The separation of the drugs was achieved by using as the mobile phase 20 mM monobasic sodium phosphate-acetonitrile (90:10) with a gradient to 50% of the organic modifier, on a silica based C18 column (150 x 4.6 mm I.D.) of 3 microns particle size and by the selectivity supplied by an array of eight coulometric electrodes at increasing potential. It was possible to identify and to determine fifteen different drugs in the same chromatographic run in 50 min. The method was tentatively applied to the determination of drugs in extracts of human hair.

Screening of homocysteine from newborn blood spots by high-performance liquid chromatography with coulometric array detection.

Homocystinuria, due to a deficiency of cystationine-beta-synthase, refers to the rare inborn error of the metabolism of homocysteine. The identification and prompt treatment of homocystinuria during the neonatal period can prevent or greatly reduce the severity of the clinical consequences. We report a new method for homocystinuria diagnosis from dried blood spots on newborn screening cards, based on high-performance liquid chromatography with electrochemical coulometric array detection. This method shows an excellent linearity (y=10.36x+0.04; r=0.999), precision (RSDs ranged from 2.7 to 5.8%), recovery (87%) and appears to be a cost-effective approach, being simple, rapid, sensitive and cheap.

Quantification of HIV-1 proviral DNA in patients with undetectable plasma viremia over long-term highly active antiretroviral therapy.

OBJECTIVES: To assess the prognostic role of proviral DNA in peripheral blood mononuclear cells (PBMC) of patients with undetectable viremia over long-term highly active antiretroviral therapy (HAART). METHODS: Eighty-two human immunodeficiency virus (HIV)-1-infected patients, free of acquired immunodeficiency syndrome (AIDS), received zidovudine plus lamivudine plus indinavir. Levels of plasma HIV-RNA, and PBMC proviral DNA and RNA unspliced (US) transcripts were evaluated by using competitive polymerase chain reaction (cPCR) assays, every 3 months over 1 year. RESULTS: Among patients with undetectable viremia at baseline, 13 of 18 with CD4 cell count 350/mm3 or less and 12 of 16 with CD4 between 351 and 700/mm3, constantly maintained undetectable RNA levels; in these patients, a mean proviral DNA decrease of 0.67 6 0.7 and 1.03 6 0.53 log (P < 0.001), respectively, a significant decrease of RNA-US transcripts (P < 0.001), and significant correlations between decreases of proviral DNA and RNA-US transcripts (P = 0.008 and P < 0.001, respectively) were observed. CONCLUSIONS: Proviral DNA quantitation permits the continued monitoring of HAART in patients with undetectable viremia.

Amineptine: its effect on the dopaminergic system of rats.

3-Methoxytyramine formation was enhanced by amineptine (40 mg kg-1 i.p.) in the striatum and limbic area, indicating an increase in the concentration of dopamine in the extraneuronal space. Since dopamine turnover, determined as the rate of L-dopa accumulation, is reduced by the drug at short (10 min) times, the enhanced extraneuronal dopamine concentration seems mainly related to amineptine-induced inhibition of its uptake.

[Distribution of HB s Ag subtypes in a sample of acute viral hepatitis of type B in Northern Italy]. / Distribuzione di sottotipi di HBsAg in un campione di epatiti virali acute di tipo B del Nord Italia

82 HBsAg from acute hepatitis B patients living in Northern Italy were subtyped. HBsAg has been detected by immunodiffusion (ID), electrosyneresis (ES), complement fixation (CF) and radioimmunoassay (RIA, Kit Ausria I and II, Abbott). Subtyping was performed by ES in 50 and by ID in 30 of the 82 assayed sera. An higher frequency of ayw subtype (83,1%) in comparison with adw subtype (16,9%) has been observed. No difference was found between adw and ayw subtypes analyzed as far as geographic-ethnic characters, incubation time, course and prognosis of the disease or the biohumoral indices. No patient was found to have both y and d specificities and so r determinant was never found.

[Hypotension following maximum physical exercise. Evaluation of hemodynamic and humoral mechanisms]. / Ipotensione dopo esercizio fisico massimale. Valutazione dei meccanismi emodinamici ed umorali.

The period after exercise has received little attention although there are rapid and arge changes in the loading conditions of the heart and circulation which may precipitate hypotension or arrhythmias. Little is known of the time course of the recovery of cardiac output and humoral changes occurring during this periods. After a single bout of prolonged muscular exercise, systolic and diastolic blood pressure decrease, sometimes for several hours. In a recent controlled study for the possible effects of the expecting of the exercise, a reduction in diastolic blood pressure was observed particularly in the first 10 min and lasting to 60 min. The mechanisms of the acute hypotensive effect of upright dynamic exercise have not yet been clarified. Little is known of the time course of the recovery of cardiac output, humoral and autonomic changes occurring during this period. Conflicting data are presented by different authors. The aim of the present study was to study the role of the haemodynamic and humoral changes in the modifications in blood pressure occurring in the hour of recovery after maximal exercise in normal subjects. Nine normal male volunteers (age: 28:34 years) have been studied on 2 separate days. Subjects were studied on a non-exercise (control) day (the subjects maintained the upright position for 30 min, followed by 60 min supine) and an exercise day (maximal upright bicycle exercise followed by supine rest for 60 min), in a random order. The following data have been recorded before the test and serially during 60 minute supine: systolic and diastolic blood pressure, heart rate, haemodynamic changes (by suprasternal aortic Doppler), and humoral changes (renin).(ABSTRACT TRUNCATED AT 250 WORDS)