Heterologous expression of a plant arginine decarboxylase gene in Trypanosoma cruzi.

Wild-type Trypanosoma cruzi epimastigotes lack arginine decarboxylase (ADC) enzymatic activity. However, the transformation of these parasites with a recombinant plasmid containing the oat ADC cDNA coding region gave rise to the transient heterologous expression of the enzyme, suggesting the absence of endogenous mechanisms that could inhibit the expression of a hypothetical own ADC gene or the assay used to measure its enzymatic activity. The foreign ADC enzyme expressed in the transgenic T. cruzi was characterized by identification of the products, the stoichiometry of the catalysed reaction, the specific inhibition by alpha-difluoromethylarginine (DFMA) and the study of its metabolic turnover. The half-life of the heterologous ADC activity in T. cruzi was about 150 min. Bioinformatics studies and polymerase chain reaction (PCR) analyses seem to indicate the absence of ADC-like DNA sequences in the wild-type T. cruzi genome.

Porphyria-induced hepatic porphyrinogen carboxy-lyase inhibitor and its interaction with the active site(s) of the enzyme.

Porphyrinogen carboxy-lyase is an enzyme that sequentially decarboxylates uroporphyrinogen III (8-COOH) to yield coproporphyrinogen III (4-COOH). In mammals this enzyme activity is impaired by hexachlorobenzene treatment, through generation of an enzyme inhibitor. The interaction of porphyrinogen carboxy-lyase inhibitor, extracted from the liver of hexachlorobenzene-treated rats, with substrate decarboxylation sites on the enzyme, was studied using four different carboxylated substrates belonging to the isomeric III series of naturally-formed porphyrinogens containing 8-,7-,6- and 5-COOH. Similar inhibitor effects were elicited against all the substrates assayed, with the exception of pentacarboxyporphyrinogen III in which decarboxylation was not inhibited to same extent. Enzyme protection assays in the presence of the different substrates, indicated that each porphyrinogen protects its own decarboxylation from inhibitor action. Preincubation of the inhibitor with normal enzyme increased its inhibitory effect. On the other hand, preincubation of both enzyme and inhibitor with superoxide dismutase or mannitol, did not alter inhibitory activity. Preincubation of the inhibitor with a number of amino acids showed that only arginine and its derivative N alpha-Benzoyl-L-Arginine ethyl ester interact with the inhibitor, noticeably reducing its ability to inhibit porphyrinogen carboxy-lyase. Albumin, histidine, serine, cysteine and imidazol, were unable to quench inhibitor activity. The present results indicate that the inhibitor acts at the binding site of each porphyrinogen. Taking into account that arginine is related to enzyme activity, and that histidine is found at the binding site of the substrates, the results suggest that the inhibitor could bind to arginine residues, blocking the access of substrates to histidine and altering the adequate orientation for decarboxylation by masking the positively charged active site necessary for porphyrinogen binding to the enzyme. In addition an indirect effect of the inhibitor mediated through free radicals could be discarded.

Complicaciones motoras precoces en parkisonianos de novo tratados con L-Dopa más inhibidor periférico de la decarboxilasa / Early motor complications in parkisonians of novo treated with L-Dopa plus descarboxylase inhibitor

En una serie de 27 pacientes con enfermedad de Parkinson esencial de novo, en grado 2,7 de Hoehn y Yahr como promedio, con un rango de 1 a 4, tratados con levodopa más benserazida durante 16 semanas, se observaron efectos motores adversos leves a moderados en 12 casos (44,4 por ciento): 8 pacientes presentaron diskinesias, 7 distonías y un deterioro de final de dosis. La dosis máxima de levodopa ID utilizada fue de 750 mg los que alcanzaron a la tercera semana, previo ascenso paulatino desde 125 mg inicial. Posteriormente, se buscó la mínima dosis efectiva la cual correspondió a una cifra de 475 mg como media con un rango de 250 a 750 mg correspondiente a 49,7 por ciento de reducción, lográndose un efecto antiparkinsoniano similar al obtenido con la dosis máxima. Se discuten los posibles factores que hayan influido en el porcentaje alto de efectos motores adversos encontrados en esta serie

Polyamines in Trypanosoma cruzi.

Growth and polyamine content of epimastigotes of Trypanosoma cruzi were studied in the presence of precursors or inhibitors of putrescine synthesis. Arginine and agmatine turned out to be better precursors than ornithine. alpha-D-difluoromethylarginine, an inhibitor of arginine decarboxylase, inhibited cellular proliferation and decreased putrescine, spermidine and spermine contents while that of cadaverine remained unchanged. These effects were reversed both by agmatine and putrescine. alpha-D-difluoromethylornithine, an inhibitor of ornithine decarboxylase did not inhibit growth of parasites grown in a polyamine free medium. These results suggest that epimastigotes need polyamines to grow, and that the parasite are able to synthesize them mainly through the arginine decarboxylase pathway.

Studies on the active centre of rat liver porphyrinogen carboxylase in vivo effect of hexachlorobenzene.

1. Porphyrinogen carboxylase from the liver of normal and hexachlorobenzene porphyric rats was subjected to chemical modification using photo-oxidation with methylene blue, diethylpyrocarbonate, butane-2,3-dione, and phenylglyoxal. 2. All of these chemicals inactivated the enzyme from both sources. 3. Reversion of the diethylpyrocarbonate reaction with hydroxylamine as well as protection of the enzymes with uroporphyrinogen III indicated that histidine is involved at least in the first decarboxylation active site of the porphyrinogen carboxylyase, and perhaps in one or more sites where the removal of the other carboxyl groups take place. 4. Arginine seems not to be at the active site of the enzyme but at its environment since two diketones alter the enzyme activity, however the substrate did not protect the enzyme from the butane-2,3-dione modification. 5. Comparative studies between the enzyme from normal and porphyric animals suggest that the low enzyme activity from intoxicated animals could be due to alterations of its active centre environment produced by hexachlorobenzene treatment. This treatment seems to partially protect the active site of the porphyrinogen carboxylase from the modification reactions.

Long-term treatment with Madopar HBS in parkinsonians with fluctuations.

1. Of 22 parkinsonians with fluctuations under long-dating dopatherapy in whom standard Madopar was substituted by the HBS form, 16 who performed the trial longer than 1 year were particularly studied to evaluate some parameters of this long-term follow-up (30-36 months). 2. The outstanding beneficial effects were an enhancement of the antiparkinsonian response, improvement or disappearance of motor oscillations, longer "on" periods, less severe "off" periods, and a more sustained nocturnal antiparkinsonian effect with a reduction of dystonias and pain at night and decreased or absent early morning parkinsonism. 3. The decrease or disappearance of dystonia was observed since the early stages of the trial and can be explained by the more sustained dopaminergic effect. 4. Surprisingly, dyskinesias also decreased in spite of the higher dopaminergic effect. The avoidance of sharp and repeated plasmatic peaks and the lower levels of L-dopa under HBS could explain this phenomenon. 5. The negative aspects of Madopar HBS are a lower bioavailability that means a dosage increase and a longer latency for the therapeutic response in the morning. 6. The dosage increase went up by 80% to 100% in relation to standard Madopar during the long-term treatment. 7. As Madopar HBS is a sustained release preparation, we had to increase the initially reduced the number of intakes, again in order to obtain better results. In the most severe cases with poor or absent response, benefits were achieved only when administering the HBS intake every hour.(ABSTRACT TRUNCATED AT 250 WORDS)

Inactivation of chicken liver mevalonate 5-diphosphate decarboxylase by sulfhydryl-directed reagents: evidence of a functional dithiol.

Chicken liver mevalonate 5-diphosphate decarboxylase (ATP:(R)-5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33) is inactivated by methylmethanethiosulfonate and 5,5'-dithiobis(2-nitrobenzoate). The presence of the substrates ATP or mevalonate 5-diphosphate protect very effectively against inactivation. The inactivation is second order with respect to methylmethanethiosulfonate, with an inactivation rate constant of (7.6 +/- 0.1).10(-5) microM-2.s-1, implying that the modifier may be reacting with more than one thiol in the enzyme. The enzyme is also inactivated by a number of dithiol-specific reagents, suggesting the presence of a functional dithiol. The determined pKapp values for enzyme modification by methyl methanethiosulfonate and phenylarsine oxide are 7.3 +/- 0.1 and 7.6 +/- 0.3, respectively. From the data presented, it is concluded that the enzyme possesses a functional dithiol that is important for substrate binding.

Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase. A resonance energy transfer study.

Phosphoenolpyruvate carboxylase, purified from maize leaves, is rapidly inactivated by the fluorescence probe dansyl chloride. The loss of activity can be ascribed to the covalent modification of an R-NH2 group, presumably the epsilon-NH2 group of lysine. Analysis of the data by the statistical method of Tsou [Sci. Sin. 11, 1535-1558 (1962)] provides clear evidence that a pH 8 eight R-NH2 groups can be modified in the tetrameric form of the enzyme, four of which are essential for catalytic activity. Essential groups are modified about five times more rapidly than the non-essential ones. The enzyme was completely protected against inactivation by Mg2+ plus phosphoenolpyruvate and consequently binding of the modifier to the essential groups is completely abolished. Hence the four essential groups seemed to be located at or near the active site(s). One of the four essential groups was modified with dansyl chloride and the other three progressively with eosin isothiocyanate. In the doubly labeled protein non-radiative single-singlet energy transfer between dansyl chloride (donor) and eosin isothiocyanate (acceptor) was observed. The low variance (+/- 5%) in the efficiency of energy transfer obtained at a particular acceptor stoichiometry (0.8-1.1, 1.9-2.1, 2.9-3.1) in triplicate samples provided confidence that the measured transfer efficiency may be interpreted as transfer between specific sites. The distances calculated from the efficiency of resonance energy transfer revealed two acceptor sites, equally separated, 4.8-5.1 nm from the donor site and third site being 6.4 nm apart from the donor. Under conditions where the tetrameric enzyme dissociates into the monomers, no transfer of resonance energy between the protein-bound dansyl chloride and eosin isothiocyanate was observed. Most likely the four essential lysyl residues in the tetrameric enzyme are located in different subunits of the enzyme, hence each of the subunits would contain a substrate-binding site with one lysyl residue crucial for activity.

Current concepts in the management of Parkinson's disease

L-Dopa has now undoubtly proved itself the drug of choice in the vast majority of cases of Parkinson's Disease. When used in combination with a decarboxylase inhibitor it would appear that significant therapeutic advantages are gained (Lancet 5th May, 1973, Marsden et al 1973). The metabolism, use and side effects of L-Dopa are outlined and the role of other forms of treatment discussed (AU)