Involvement of calyculin A-sensitive phosphatase(s) in the differentiation of Trypanosoma cruzi trypomastigotes to amastigotes

Differentiation of the non-dividing trypomastigote form of Trypanosoma cruzi, the causative agent of Chagas disease, to the dividing amastigote form normally occurs in cytoplasm of infected cells. Here we show that calyculin A. a potent inhibitor of protein phosphatases 1 and 2A, induces at pH 7.5 extracellular transformation of long slender trypomastigotes to round amastigote-like forms which acquire characteristic features observed after the normal differentiation process: repositioning and structural changes of the kinetoplast, release of surface neuraminidase, and expression of amastigote-specific epitopes. Calyculin A inhibits parasite phosphatases and changes in the phosphorylation of specific proteins occur during the transformation process. As an exposure of trypomastigotes to calyculin A concentrations as low as 1 nM and for only 1-2 h is sufficient to induce transformation, the inhibition of calyculin A-sensitive phosphatase(s) appears to play a major role in initiating the trypomastigote differentiation.     

A fast screening method for the detection of CERA in dried blood spots

Continuous erythropoietin receptor activator (CERA) is a third-generation erythropoiesis-stimulating agent that was developed for the treatment of anemia. However, misuse of CERA for doping in endurance sports has been reported. Previous studies have shown blood as the matrix of choice for the detection of CERA, due to its high molecular weight. The use of dried blood spots (DBSs) for anti-doping purposes constitutes a complementary approach to the standard urine and venous blood matrices and could facilitate sample collection and increase the number of blood samples available for analysis due to reduced costs of sample collection and transport. Here, we investigated whether CERA could be indirectly detected in extracts of single DBSs using an erythropoietin-specific immunoassay that is capable of providing results within approximately 2 h. Reconstituted DBS samples were prepared from mixtures of red blood cell pellets and serum samples. The samples were collected in a previous clinical study in which six healthy volunteers were injected with a single, 200 μg dose of CERA. Using a commercially available ELISA kit, CERA was detected in the DBSs with a detection window of up to 20 days post-injection. Furthermore, in order to demonstrate the fitness-for-purpose, three authentic doping control serum samples, which were identified as containing CERA, were analyzed by the presented methodological approach on DBS. The testing procedure described here could be used as a fast and cost-effective method for the detection of CERA abuse in sport.     

Sarcoidosis (Heerfordt syndrome): a case report

We report the case of a 22-year-old woman who is suspected of having primary Sj?gren s syndrome. She complaining of bilateral swelling of eyelids and the parotid glands of three weeks duration. Physical examination revealed a bilateral enlargement of both parotid glands, which were solid and painful. Sj?gren s syndrome was suspected at that stage, and the serologic and specific analysis were done. All these tests didn t find any autoimmune or visceral features typical of Sj?gren s syndrome and autoantibodies were negative. During follow-up time the right facial nerve palsy developed. Pulmonary radiography revealed bihilar lymphadenopathy and labial salivary gland biopsy revealed non-caseating granuloma. The patient was classified as having stage I sarcoidosis. This case demonstrates the importance of being aware of the leading clinical signs and symptoms in case of Heerfordt syndrome.     

Intracellular energetic units in healthy and diseased hearts

BACKGROUND:

The present review examines the role of intra-cellular compartmentation of energy metabolism in vivo.

OBJECTIVE:

To compare the kinetics of the activation of mitochondrial respiration in skinned cardiac fibres by exogenous and endogenous adenine nucleotides in dependence of the modulation of cellular structure and contraction.

METHODS:

Saponin-permeabilized cardiac fibres or cells were analyzed using oxygraphy and confocal microscopy.

RESULTS:

Mitochondria respiration in fibres or cells was upregulated by cumulative additions of ADP to the medium with an apparent K_m of 200 μM to 300 μM. When respiration was stimulated by endogenous ADP produced by intracellular ATPases, a near maximum respiration rate was achieved at an ADP concentration of less than 20 μM in the medium. A powerful ADP-consuming system, consisting of pyruvate kinase and phosphoenolpyruvate, that totally suppressed the activation of respiration by exogenous ADP, failed to abolish the stimulation of respiration by endogenous ADP, but did inhibit respiration after the cells were treated with trypsin. The addition of up to 4 μM of free Ca²⁺ to the actively respiring fibres resulted in reversible hypercontraction associated with a decreased apparent K_m for exogenous ADP. These changes were fully abolished in fibres after the removal of myosin by KCl treatment.

CONCLUSIONS:

Mitochondria and ATPases, together with cytoskeletal proteins that establish the structural links between mitochondria and sarcomeres, form complexes – intracellular energetic units (ICEUs) – in cardiac cells. Within the ICEUs, the mitochondria and ATPases interact via specialized energy transfer systems, such as the creatine kinase- and adenylate kinase-phosphotransfer networks, and direct ATP channelling. Disintegration of the structure and function of ICEUs results in dyscompartmentation of adenine nucleotides and may represent a basis for cardiac diseases.     

Constituents of Burasaïa madagascarensis: a new clerodane-type diterpene

From an ethanol extract of the stems of Burasa?a madagascarensis Thouars (Menispermaceae) were isolated N-acetylnornuciferine and two clerodane-type diterpenes, one of them, epicordatine, being new. The structures were established by the interpretation of the spectral data. All the described compounds exhibited weak antimalarial activity.     

Long-term outcome of patients with claudication after balloon angioplasty of the femoropopliteal arteries

PURPOSE: To report the long-term outcome of patients with lifestyle-limiting claudication after percutaneous transluminal angioplasty (PTA) of the femoropopliteal arteries. MATERIALS AND METHODS: Between 1989 and 1992, 173 consecutive claudicant patients (mean age, 65 years; age range, 41-90 years) underwent PTA in 218 limbs; all interventions included femoral and/or popliteal arterial segments, and additional iliac (n = 27) and infrapopliteal (n = 11) arterial lesions were also treated. Patients were followed up for 7-10 years. Altogether, 37 (17%) limbs were classified as Fontaine class 2A, and 181 (83%) were class 2B. Average length of the primary lesion was 5.2 cm. Reinterventions were analyzed. Patency rates and patient survival were assessed by means of life table analysis. Cox-Mantel tests and Cox proportional hazards models were used to define associated independent determinants. Development of chronic critical ischemia (CCI) and its determinants was assessed by using the Pearson chi(2) test and multiple logistic regression analysis. RESULTS: The primary and secondary patencies (+/- standard error of the estimate), respectively, were 46% +/- 3 and 63% +/- 3 at 1 year, 25% +/- 3 and 41% +/- 4 at 5 years, and 14% +/- 3 and 22% +/- 4 at 10 years. One-third (71 of 218) of the limbs required repeat interventions, including surgical revascularization in 35 limbs. Fourteen (6.4%) limbs developed CCI, resulting in a 0.8% incidence per year. In multivariate analysis, poor postinterventional peripheral runoff was an indicator of increased risk of CCI development (P =.03). CONCLUSION: Although the long-term patency rates of PTA of the femoropopliteal arteries in claudicant patients were poor, the acceptable number of reinterventions and the low frequency of development of CCI imply the long-term benefits achievable with this treatment.     

Flavonoids from Dalbergia louvelii and their antiplasmodial activity

Four new flavonoids (1-4), along with 13 known compounds, were isolated from the heartwood of Dalbergia louvelii by following their potential to inhibit in vitro the growth of Plasmodium falciparum. Of the isolated compounds, four known compounds showed antiplasmodial activity with IC(50) values ranging from 5.8 to 8.7 microM, namely, (R)-4' '-methoxydalbergione (5), obtusafuran (6), 7,4'-dihydroxy-3'-methoxyisoflavone (7), and isoliquiritigenin (8). The structures of the new compounds were determined using spectroscopic techniques as 1-(3-hydroxyphenyl)-3-(4-hydroxy-2,5-dimethoxyphenyl)propane (1), spirolouveline (2), (3R)-7,2'-dihydroxy-4',5'-dimethoxyisoflavanone (3), and 3-(2,4-dihydroxy-5-methoxy)phenyl-7-hydroxycoumarin (4), respectively.     

New synthesis of benzo-delta-carbolines, cryptolepines, and their salts: in vitro cytotoxic, antiplasmodial, and antitrypanosomal activities of delta-carbolines, benzo-delta-carbolines, and cryptolepines

The paper describes, in its first part, a new synthesis of benzo-delta-carbolines, cryptolepines, and their salts. The strategy is based on the association between halogen-dance and hetero-ring cross-coupling. It is fully convergent and regioselective with interesting overall yields from 27% to 70%. A halogen-dance mechanism in quinoline series is also proposed. The formal synthesis of potential antimalarial compounds and the first total synthesis of 11-isopropylcryptolepine are also described. In the second part, cytotoxic activity against mammalian cells and activities against Plasmodium falciparum and Trypanosoma cruzi of benzo-delta-carbolines and delta-carbolines were evaluated in vitro to study the structure-activity relationships. For benzo-delta-carbolines, methylation at N-5 increases the cytotoxic and antiparasitic activities. A further alkylation on C-11 generally increases the cytotoxic activity but not the antiparasitic activity, cryptolepine and 11-methylcryptolepine being the most active on both parasites. Taking advantage of the fluorescence of the indoloquinoline chromophore, cryptolepine was localized by fluorescence microscopy in parasite DNA-containing structures suggesting that these compounds act through interaction with parasite DNA as proposed for cryptolepine on melanoma cells. For delta-carbolines, methylation at N-1 is essential for the antimalarial activity. 1-Methyl-delta-carboline specifically accumulates in the intracellular parasite. It has weak cytotoxic activity and can be considered as a potential antimalarial compound.