Characterization of mitochondrial electron-transfer in Leishmania mexicana.

Some general features of the respiratory chain and respiratory control were characterized in coupled mitochondrial preparations from Leishmania mexicana promastigotes. O2 uptake was sensitive to the electron-transfer inhibitors rotenone, flavone, malonate, 4,4,4-trifluoro-1-(2-thienyl) 1.3 butanedione (TTFA), antimycin A, 2n-nonyl-4-hydroxyquinoline-N-oxide (HQNO), myxothiazol, cyanide and azide. A high concentration of rotenone (60 microM) was required to inhibit O2 uptake effectively. Difference spectra revealed the presence of cytochromes (a + a3), b and c. Respiratory control was stimulated 2-fold by ADP with different exogenous oxidizable substrates. Calculated ADP/O ratios were consistent with the notion that ascorbate/N,N,N',N'-tetramethylphenylenediamine (TMPD)-linked and FAD-linked respiration proceeds, respectively, with one third and two thirds of the ATP producing capacity of NADH-linked respiration. State 3 was suppressed by the ATP synthase inhibitors oligomycin and aurovertin and by the adenine nucleotide translocator inhibitors atractyloside and carboxy atractyloside. The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) provoked state 3u respiration. The mitochondrial preparation was capable of Ca2+ uptake and Ca2+ stimulated respiration. Data obtained suggests strongly that mitochondrial complexes I, II, III and IV are present in a major pathway of electron-transfer and that oxidative phosphorylation might proceed with high bioenergetic efficiency.

Ouabain-sensitive Na+,K(+)-ATPase in the plasma membrane of Leishmania mexicana.

The mechanism responsible for the regulation of intracellular Na+ and K+ concentrations in trypanosomatids is unknown. In higher eukaryotes a ouabain-sensitive Na+,K(+)-ATPase located in the plasma membrane is the main mechanism for the regulation of the intracellular concentrations of Na+ and K+, while in trypanosomatids there are conflicting evidences about the existence of this type of ATPase. By the use of a highly enriched plasma membrane fraction, we showed that an ouabain-sensitive Na+,K(+)-ATPase is present in L. mexicana. The affinity of the enzyme for Na+ and K+ is similar to that reported for the mammalian Na+,K(+)-ATPase, showing also the same kinetic parameters regarding the relative concentration of those cations that give the optimal activity. Vanadate (10 microM) fully inhibits the ATPase activity, suggesting that the enzyme belongs to the P-type family of ionic pumps. The enzyme is sensitive to ouabain and other cardiac glycosides. These cardiac glycosides do not show any appreciable effect on the higher Mg(2+)-ATPase activity present in the same preparation. By the use of [3H]ouabain, we also show in this report that the binding of the inhibitor to the enzyme was specific. Taken together, these results demonstrate that an ouabain-sensitive Na+,K(+)-ATPase is present in the plasma membrane of Leishmania mexicana. Therefore, this Na+,K(+)-ATPase should participate in the intracellular regulation of these cations in Leishmania.

Isolation and characterization of the thermoresistant gluconokinase from Escherichia coli.

It is known that two gluconokinases are inducibly expressed during the utilization of gluconate by E. coli. One is thermoresistant (activity stable for 3 h at 30 degrees C) and the other thermosensitive (losses 75% or more of its activity under the above conditions). The thermoresistant gluconokinase (EC 2.7.1.12) was isolated, purified and characterized for the first time from the E. coli mutant Ca26, a K12 derivative which lacks the thermosensitive activity. The enzyme was purified 43 fold with a recovery of 11%. The M(r) of the enzyme was 100 kDa with three equal subunits of approximately 29.5 kDa. The enzyme exhibited Michaelis-Menten kinetics and the Km values for gluconate and ATP were 0.02 mM and 0.045 mM respectively.

[Evaluation of the nervous system in workers of a enolofos production department in chemical factories]. / Ocena stanu ukladu nerwowego u pracowników oddzialu produkcji enolofosu zakladów chemicznych.

Neurological examination including EEG, ENG and measurement of nerve motor conduction velocity were performed in 41 male workers, exposed to enolofos. 43% of the population examined developed subjective neurological symptoms 34%--discrete lesions of CNS and in 31% polineuropathy was observed. Motor nerve conduction velocity was decreased in 10 workers (24%) in peroneus nerves and in 1 worker in ulnaris nerve. Comparison of EEG and ENG with the control group was not statistically significant.

A new method for the partial purification of leishmania amastigotes from cutaneous lesionS.

Amastigotes from Leishmania braziliensis and L. mexicana were isolated from cutaneous lesions in infected animals using the plant lectin Concanavalin A as a specific agglutination agent. Amastigotes were collected in preparations of up to 95% purity as determined by cell count. The parasites obtained by this method showed no apparent loss of viability as measured by growth rated and DNA replication, or pathogenicity as measured by routine passages in hamsters or mice.

Protein synthesis by synaptosomes from rat brain: the effect of centrifugal forces.

1. Synaptosomal protein synthesis has been found to differ depending on whether synaptosomes are recovered from the sucrose gradients by centrifuging at 20,000 g or at 150,000 g. 2. The fraction obtained at 20,000 g incorporated 60% more amino acid into proteins after 30 min of incubation than the one obtained in the same period of time at 150,000 g. 3. The respiratory capacity of synaptosomes obtained at 20,000 g in the presence or absence of glucose as substrate, is also better preserved than that of those obtained at 150,000 g. 4. It is shown that the yield, enzymic content, and nucleic acid composition is the same in both fractions. 5. These results cannot be explained by the presence of inhibitory factors in the 150,000 g fraction or by the presence of better preserved free mitochondria in the 20,000 g fraction. 6. Our findings are best explained by postulating that the synaptosomal metabolic activity is damaged when synaptosomes are pelleted at too high a speed.

In vitro infection of murine macrophage by Leishmania brasiliensis. Mechanism of penetration.

The in vitro infection of peritoneal murine macrophages by a cultured pathogenic strain of Leishmania brasiliensis was studied. By phase contrast and light microscopy it could be observed that L. brasiliensis is rapidly endocytosed by the macrophages and localized in the cytoplasm in a vacuole inside which it multiplies. By phase contrast and electron microscopy it was observed that L. brasiliensis enters the macrophage by the anterior end through which it first attaches to the cell membrane. This penetration involves invagination of the cell membrane around the entering flagellum and parasite body. A mechanism of entry involving and active participation of the parasite membrane through receptor sites and membrane activity by the microfilament system is presented.

The surface membrane of Leishmania. I. The effects of lectins on different stages of Leishmania braziliensis.

The infective stages of Leishmania braziliensis, amastigotes and promastigotes subcultured a limited number of times, were agglutinated by Ricinus communis agglutinin and Concanavalin A. These results suggest that terminal ligands similar or identical with alpha-D mannose, alpha-D glucose (specific receptors for Con A), and alpha-D galactose (specific receptor for RCA) are present in the surface membrane of L. braziliensis. Noninfective promastigotes from the same stock, but subcultured approximately 500 times, were not agglutinated by RCA suggesting either the absence of the alpha-D galactose groups in the surface membrane or their presence in a very reduced number. Agglutination with soybean agglutinin, wheat germ agglutinin, or phytohemagglutinin P was not observed in any of the L. braziliensis forms tested. The difference in polysaccharide residues on the surface membrane of L. braziliensis may be related to the different pathogenic properties of the cell.

Autonomy of mitochondria in Saccharomyces cerevisiae in their production of messenger RNA.

In ts(-)136, a temperature-sensitive mutant of Saccharomyces cerevisiae, nuclear and mitochondrial RNA production can be inhibited selectively by exposure to 36 degrees and ethidium bromide, respectively. Using the programming of mitochondrial polysomes, as measured by their ability to form nascent polypeptide chains, as an assay for functional messenger RNA, we have determined its response to temperature shifts and ethidium bromide. Only ethidium bromide produced a measurable effect; in contrast the cell-sap system responded exclusively to temperature shifts. We conclude that transcription of mitochondrial DNA is sufficient and that import of messenger RNA transcribed from nuclear chromosomes makes no measurable contribution to intramitochondrial protein synthesis.