Effect of MI-D, a new mesoionic compound, on energy-linked functions of rat liver mitochondria.

MI-D (4-phenyl-5-(4-nitro-cinnamoyl)-1,3,4-thiadiazolium-2-phenylami ne chloride), a new mesoionic compound, depressed the phosphorylation efficiency of liver mitochondria as deduced from an accentuated decrease of the respiratory control coefficient and ADP/O ratio. Analysis of segments of the respiratory chain suggested that the MI-D inhibition site is further on than complex I and between complexes II and III. The transmembrane electrical potential (delta psi) was collapsed dependent on MI-D concentration. ATPase activity was dramatically increased by MI-D in intact mitochondria, but inhibited in carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)-uncoupled mitochondria. These results suggest that MI-D acts as an uncoupler agent, a property closely related to its structural characteristics.

Inhibition of Trypanosoma cruzi trypanothione reductase by crystal violet.

A trypanothione reductase activity is present in all the main differentiation stages of Trypanosoma cruzi, amastigotes having the highest activity, and trypomastigotes the lowest. Trypanothione reductase could not be induced in epimastigotes exposed to H2O2. The trypanocidal drug crystal violet was a potent inhibitor of T. cruzi trypanothione reductase in vitro. The inhibition was competitive with respect to trypanothione with a Ki of 5.3 +/- 0.5 microM, uncompetitive with NADPH, and increased below pH 7.0 and above pH 8.0. Crystal violet, however, was not able to decrease the level of total reduced thiols in intact cells. Dihydrotrypanothione but not reduced glutathione, protected the enzyme from inhibition by crystal violet.

Trypanothione-dependent peroxide metabolism in Trypanosoma cruzi different stages.

Different stages of Trypanosoma cruzi are able to metabolize low concentrations of H2O2. Trypomastigotes showed a higher initial rate per mg protein than amastigotes or epimastigotes derived from them. Amastigotes could metabolize H2O2 at a lower rate than the other developmental stages of T. cruzi. A peroxide-metabolizing activity was detected in extracts of T. cruzi epimastigotes. This 'NADPH peroxidase' activity was lost upon dialysis of the extracts and was probably due to a non-enzymatic reaction(s) with endogenous dihydrotrypanothione (T(SH)2) and/or other thiols, thus explaining the inhibition of H2O2 metabolism in intact cells by thiol inhibitors. An amount of non-protein thiols equivalent to an intracellular concentration of 2.0-3.0 mM was found in epimastigotes, which is sufficient to account for the rate of NADPH oxidation observed in the presence of high concentration of peroxides (> 100 microM). Addition of T(SH)2 increased this rate, implying that this thiol could be used as a substrate in that reaction. In addition, this activity was hardly detectable in the extracts in the presence of low concentration of peroxides (< 20 microM), indicating a high Km, which would be incompatible with a true peroxidase activity. Taking into account the high intracellular concentration of thiols measured, this activity probably accounted for the rates of H2O2 metabolism detected in intact T. cruzi. These results also confirm that T. cruzi is an organism with limited ability to detoxify H2O2.

Inhibition of contact hypersensitivity reaction to picryl chloride: effect of small molecular weight peptidomimetic compounds possessing inhibitory activity against metalloproteinases.

We have studied the effect of small molecular weight inhibitors of snake venom metalloproteinases (SVMP) and matrix metalloproteinases (MMPs) on the induction and effector phase of the contact hypersensitivity reaction (CHR) in a mouse model. Identification of nonsteroid small molecules is very important for the development of new anti-inflammatory drugs. The compounds that we tested were synthetically modified tripeptides (peptidomimetic compounds) POL-257, POL-509, POL-443, POL-491, and POL-647, with structures based on natural occurring peptides in snake venom. A well-known hydroxamate-based inhibitor of the MMPs, Batimastat (BB-94), was also used. We have shown that these peptidomimetics possess in vitro inhibitory activity against the MMP-2 (gelatinase-A), MMP-9 (gelatinase-B), and MMP-3 (stromelysin). They also inhibit metalloproteinases purified from the venom of Crotalus adamanteus and C. atrox snakes, which are very similar to the so-called A Desintegrine, A Metalloproteinase (ADAMs) enzymes. When injected intraperitonealy before the topical application of the contact sensitizer (picryl chloride) or before the challenge, these compounds significantly inhibited the development of CHR. BB-94 at doses 0.4 and 4 mg/kg before the sensitization or before the challenge almost completely abrogated the reaction. POL-257 and POL-443 were among the most active peptidomimetics tested. They inhibited the inflammatory reaction up to 70-80%, when applied either immediately before sensitization or before challenge. POL-509, a methylated derivative of POL-257, inhibited the CHR to 40-50% when administered at either challenge or sensitization. However, when applied 24 h before the challenge, it completely abrogated the inflammatory reaction. The results show that these small molecular weight peptidomimetic compounds, as well as BB-94, are able to significantly inhibit the CHR. This finding opens possibilities for using metalloproteinase inhibitors in the treatment of allergic contact dermatitis and other inflammatory diseases.

The mechanism and biological role of calcium transport by plant mitochondria.

Using a tetraphenylphosphonium-sensitive electrode, a linear relationship was obtained between membrane depolarization during Ca2+ influx into plant mitochondria and the rate of respiration when the rate of succinate oxidation was gradually inhibited by increasing concentrations of malonate. These results are consistent with the existence of electrogenically-mediated Ca2+ transport in the mitochondrial membrane, driven by the protonmotive force. The data are discussed in terms of the biological role of such a Ca2+ transport system in plant mitochondria.

[Pneumocystomammography]. / La pneumocistomammografia.

The Authors re-examined a series of 50 female patients after 12 months to assess the diagnostic and therapeutic value of percutaneous evacuation of mammary cysts, followed by a radiological study using PNCMX.

[Mammographic screening of a random population. Evaluation of 16,500 cases]. / Screening mammografico su popolazione non selezionata. Valutazione su 16.500 casi.

After analysing data collected over a 5-year period, the Authors emphasise that screening for breast cancer is becoming increasingly common-place. This has led to a marked improvement of the prognosis of this pathology.

[Percutaneous localization of breast lesions. Personal experience]. / Localizzazione percutanea di lesioni mammarie. Esperienza personale.

A critical analysis is made of a series of percutaneous localisations of radiological breast lesions. The Authors underline the importance of close collaboration between the various specialists in order to obtain an early diagnosis which, in breast cancer, ensures a better prognosis and also avoids the necessity of radical surgery which affects the woman's attitude towards cancer.

A new specimen and dental microwear in Oreopithecus bambolii.

The authors describe a new specimen of Oreopithecus bambolii, a catarrhine primate from late Miocene sites of Tuscany and Sardinia (Italy). Microwears of 4 specimens of Oreopithecus bambolii have been analysed on standard surfaces of facet 9 to determine the diet. The microwear pattern confirms the hypothesis already put forward on the basis of its dental morphology that Oreopithecus bambolii had a leaf-based diet.

New Middle Pleistocene human remains from northern Italy.

The description of several teeth and a fragment of mandible found in the Visogliano rock-shelter (Northern Italy) is presented in this paper. The teeth have been analysed from a morphological and metrical point of view and compared with other specimens of Homo erectus, archaic Homo sapiens and Homo neanderthalensis. The MD and BL diameters and the peculiar dental features are archaic and support the antiquity of the specimens. Microwear analysis of Visogliano teeth show various dietary strategies adopted by the individuals of the same site.

Calcium transport by corn mitochondria : evaluation of the role of phosphate.

Mitochondria from some plant tissues possess the ability to take up Ca(2+) by a phosphate-dependent mechanism associated with a decrease in membrane potential, H(+) extrusion, and increase in the rate of respiration (AE Vercesi, L Pereira da Silva, IS Martins, CF Bernardes, EGS Carnieri, MM Fagian [1989] In G Fiskum, ed, Cell Calcium Metabolism. Plenum Press, New York, pp 103-111). The present study reexamined the nature of the phosphate requirement in this process. The main observations are: (a) Respiration-coupled Ca(2+) uptake by isolated corn (Zea mays var Maya Normal) mitochondria or carbonyl cyanide p-trifluoromethoxyphenylhydrazone-induced efflux of the cation from such mitochondria are sensitive to mersalyl and cannot be dissociated from the silmultaneous movement of phosphate in the same direction. (b) Ruthenium red-induced efflux is not affected by mersalyl and can occur in the absence of phosphate movement. (c) In Ca(2+)-loaded corn mitochondria, mersalyl causes net Ca(2+) release unrelated to a decrease in membrane potential, probably due to an inhibition of Ca(2+) cycling at the level of the influx pathway. It is concluded that corn mitochondria (and probably other plant mitochondria) do possess an electrophoretic influx pathway that appears to be a mersalyl-sensitive Ca(2+)/inorganic phosphate-symporter and a phosphate-independent efflux pathway possibly similar to the Na(2+)-independent Ca(2+) efflux mechanism of vertebrate mitochondria, because it is not stimulated by Na(+).

Mitochondrial sensitivity to AZT.

The possibility of tissue-specific effects regarding mitochondrial sensitivity to AZT was evaluated in this study. When mitochondria isolated from liver, kidney, skeletal and cardiac muscle were oxidizing glutamate, a dose-dependent inhibition by AZT of state 3 respiration was observed; using succinate as substrate the inhibition occurred only in skeletal and cardiac muscle mitochondria. The same results were obtained with FCCP-uncoupled mitochondria. NADH oxidase of intact and disrupted mitochondria, isolated from all four tissues was strongly inhibited. Succinate oxidase activity was inhibited by AZT only in intact mitochondria from skeletal and cardiac muscles, suggesting the involvement of succinate transport systems. Similarly, inhibition by the drug of the hydrolytic activity of H(+)-ATPase was observed only in mitochondria of these tissues. These effects taken together, indicate a tissue/carrier-specific inhibition in vitro, although its precise mechanism requires further research.

Neutrality of amiodarone on the initiation and propagation of membrane lipid peroxidation.

Amiodarone is an iodinated benzofuran derivative largely used as an antiarrhythmic. Owing to the sensitivity of heart tissue to radicals, amiodarone was assayed for putative effects on lipid peroxidation studied in liposomes of soybean phosphatidylcholine and of bovine heart mitochondrial lipids used as model systems. Lipid peroxidations were initiated with Fe2+/ascorbic acid, and with peroxyl radicals generated from the azocompounds, AAPH and AMVN. These assays were carried out by following the quenching of the fluorescent probe cis-parinaric acid and by monitoring oxygen consumption. It has been ascertained that amiodarone does not protect or potentiate significantly the lipid peroxidation both lipidic systems. To fully ascertain the neutral behaviour of amiodarone in the lipid peroxidation process, the degradation of phospholipid acyl chains has been checked by GLC. These data confirm that amiodarone does not protect or potentiate lipid peroxidation to a significant extent. It is concluded that the limited effects of amiodarone might be related only indirectly with the lipid peroxidation. It is possible that the drug causes limited conformational and biophysical alterations in membrane phospholipid bilayers that can affect the process of peroxidation. Therefore, it is concluded that the therapeutic effects and benefits as a heart antiarrhythmic agent are independent of lipid peroxidation processes. Furthermore, the interaction of the drug with lipid bilayers does not induce significant conformational perturbations that could significantly favour or depress the peroxidation process.

[CT of the operated liver]. / La TC nel fegato operato.

The Authors examine retrospectively 93 CT exams performed on 60 patients submitted to hepatic surgery both for malignant and benign lesions. It is concluded that CT, if performed correctly, is helpful in recognizing both complications and recurrences.

Ca2+ induces a cyclosporin A-insensitive permeability transition pore in isolated potato tuber mitochondria mediated by reactive oxygen species.

Oxidative damage of mammalian mitochondria induced by Ca2+ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca2+ uniport, undergo inner membrane permeabilization when treated with Ca2+ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg2+ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis-trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional thiol reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca2+ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.

Methotrexate: pentose cycle and oxidative stress.

The effect of methotrexate (MTX) and leucovorin (LCV) on pentose cycle enzymes and the activity of enzymes involved in enzyme defence mechanisms against ROS in HeLa cells, were studied. The effect of MTX was also investigated on the cellular levels of glutathione. MTX inhibited the activity of glucose-6-phosphate and 6-phosphogluconate dehydrogenases. The activities of glutathione reductase and gamma-glutamylcysteine synthetase were also inhibited by the drug. No effect was observed on the activities of catalase, superoxide dismutase or transketolase. LCV had no effect on any of the enzymes studied. MTX decreased the cellular levels of glutathione (70 per cent), while the presence of LCV and glutamine did not interfere with the effect of MTX. The net results appear to show that the biological situation resulting from treatment with MTX leads to a reduction of effectiveness of the antioxidant enzyme defence system.

Citrinin-induced mitochondrial permeability transition.

The effects of mycotoxin citrinin on Ca2+ efflux and membrane permeabilization were studied in isolated rat liver mitochondria. The efflux rate observed when in presence of ruthenium red was higher when citrinin was added. Swelling experiments demonstrated Ca(2+)-dependent membrane permeabilization by citrinin. Catalase, butylhydroxitoluene (BHT), and dithiothreitol (DTT) did not protect swelling caused by Ca2+ plus citrinin. The protection conferred by ATP-Mg2+ and cyclosporin A in the latter experiments are strong indications of pore formation. These results suggest that citrinin can induce permeability transition by a mechanism that does not involve oxidative damage.

Occurrence of the Crabtree effect in HeLa cells.

The occurrence of a Crabtree effect in HeLa cells was detected. Some properties of pyruvate kinase (PK) were also evaluated. Hexose phosphate, triose-phosphate and phosphoenolpyruvate (PEP) significantly decreased the oxygen consumption of digitonin-permeabilized HeLa cells, which were oxidizing succinate. The Crabtree effect promoted by PEP was concentration-dependent and was lowered by an increase of ADP concentration, suggesting a participation of PK. The dependence of fructose-1,6-bisphosphate (FDP) by HeLa cell PK was observed. The PK of HeLa cells was inhibited by L-alanine only in the absence of FDP, while in the presence of the metabolite, an increase in the activity was observed. PK was also inhibited in the presence of L-histidine and L-leucine, while L-serine promoted activation. L-Cysteine and L-phenylalanine also inhibited the PK of HeLa cells. This, together with the sigmoidal character in relation to substrate concentration, suggests the presence of the K-type of PK in HeLa cells.

Effect of methotrexate (MTX) on NAD(P)+ dehydrogenases of HeLa cells: malic enzyme, 2-oxoglutarate and isocitrate dehydrogenases.

The effects of methotrexate (MTX) on oxygen uptake by permeabilized HeLa cells were evaluated. MTX did not inhibit state III respiration when the oxidizable substrate was succinate, but when the substrates were 2-oxoglutarate or isocitrate the respiration decreased about 50 per cent at 1.0 mM concentration of the drug. This effect was explained by inhibition of 2-oxoglutarate and isocitrate dehydrogenases by MTX. No effect was observed on succinate dehydrogenase. An evaluation of the effects of MTX on malic enzyme activity as measured by pyruvate plus lactate production in intact cells supplied with malate showed a decrease of about 40 per cent in metabolite production using 0.4 mM MTX. HeLa cell malic enzyme, as observed for other tumour cells, is compartmentalized in mitochondria and cytosol, and is another example of a dehydrogenase inhibited by MTX.

Discrepancy between polymerase chain reaction assay and Western blot analysis in the assessment of CagA status in dyspeptic patients.

BACKGROUND: Infection with CagA-positive Helicobacter pylori may be diagnosed by detecting cagA gene by polymerase chain reaction assay (PCR) or serum antibodies against CagA by Western blot analysis. The aim of this study is to evaluate whether results of PCR and Western blot analysis are in agreement in CagA status assessment. PATIENTS AND METHODS: Thirty-six dyspeptic patients with unknown H. pylori status underwent upper gastrointestinal endoscopy to assess the presence of mucosal lesions and to collect six gastric biopsies (three from the antrum and three from the body) for evaluation of H. pylori infection (rapid urease test, histology and PCR for ureA gene) and gastritis. CagA status was assessed by PCR (cagA gene) on two biopsy specimens and by Western blot analysis of serum (CagA-antibodies) in each patient. RESULTS: At endoscopy, nine patients showed normal mucosa, 15 a duodenal ulcer and 12 antral erosions. Twenty-eight patients were found to be H. pylori-positive and eight H. pylori-negative. Of the 28 H. pylori-positive patients, 17 were CagA-positive and five were CagA-negative by both methods, five were CagA-positive by Western blot analysis but not by PCR and one was CagA-positive by PCR but not by Western blot analysis. Of the eight H. pylori-negative patients, none was CagA-positive by PCR, while six were CagA-positive by Western blot analysis. Therefore, the two tests agreed in only 24 patients (67%). In those patients in whom the PCR and Western blot analysis were not in agreement, the histological features appear to suggest that the results of the Western blot analysis should be considered false positives or false negatives. CONCLUSIONS: PCR and Western blot analysis failed to provide comparable data in many cases. Western blot analysis seems to be more likely to give misleading results than PCR. Thus, PCR seems to be the method of choice to assess CagA status.