Molecular, immunological, and physiological evidences of a sphingosine-activated plasma membrane Ca2+-channel in Trypanosoma equiperdum.

The hemoparasite Trypanosoma equiperdum belongs to the Trypanozoon subgenus and includes several species that are pathogenic to animals and humans in tropical and subtropical areas across the world. As with all eukaryotic organisms, Ca2+ is essential for these parasites to perform cellular processes thus ensuring their survival across their life cycle. Despite the established paradigm to study proteins related to Ca2+ homeostasis as potential drug targets, so far little is known about Ca2+ entry into trypanosomes. Therefore, in the present study, the presence of a plasma membrane Ca2+-channel in T. equiperdum (TeCC), activated by sphingosine and inhibited by verapamil, is described. The TeCC was cloned and analyzed using bioinformatic resources, which confirmed the presence of several domains, motifs, and a topology similar to the Ca2+ channels found in higher eukaryotes. Biochemical and confocal microscopy assays using antibodies raised against an internal region of human L-type Ca2+ channels indicate the presence of a protein with similar predicted molar mass to the sequence analyzed, located at the plasma membrane of T. equiperdum. Physiological assays based on Fura-2 signals and Mn2+ quenching performed on whole parasites showed a unidirectional Ca2+ entry, which is activated by sphingosine and blocked by verapamil, with the distinctive feature of insensitivity to nifedipine and Bay K 8644. This suggests a second Ca2+ entry for T. equiperdum, different from the store-operated Ca2+ entry (SOCE) previously described. Moreover, the evidence presented here for the TeCC indicates molecular and pharmacological differences with their mammal counterparts, which deserve further studies to evaluate the potential of this channel as a drug target.

Species of Metarhizium anisopliae complex implicated in human infections: retrospective sequencing study.

OBJECTIVES: Fungi belonging to the Metarhizium anisopliae complex comprise ubiquitous arthropod pathogenic moulds used as mycopesticides. Rare cases of human infections due to M. anisopliae have been reported. We hypothesize misidentifications of fungal strains implicated in these cases or used in mycopesticides. METHODS: A review of the literature was conducted to identify previously published cases. We collected some of these previous described strains and reported new cases, and a French mycopesticide containing M. anisopliae. All identifications were performed based on elongation factor-1α gene sequencing. RESULTS: We report eight new cases of Metarhizium infection in humans (three from France and five from Australia). The strains isolated from these cases, and three others from already published cases and reported as M. anisopliae, were molecularly identified based on elongation factor-1α (Ef1-α) gene sequencing as follows: Metarhizium robertsii (six), Metarhizium guizhouense (three), Metarhizium brunneum (one) and Metarhizium pingshaense (one). CONCLUSIONS: In this study, we report new human cases of Metarhizium infections, and, based on Ef-1α gene sequencing, we demonstrate the misidentification of species in case reports. We also correct the species identification of a strain reported as M. anisopliae used in a commercially available mycopesticide. According to our results, none of the strains from the human infection reports reviewed belongs to the species M. anisopliae.

Ergosterone-coupled Triazol molecules trigger mitochondrial dysfunction, oxidative stress, and acidocalcisomal Ca2+ release in Leishmania mexicana promastigotes.

The protozoan parasite Leishmania causes a variety of sicknesses with different clinical manifestations known as leishmaniasis. The chemotherapy currently in use is not adequate because of their side effects, resistance occurrence, and recurrences. Investigations looking for new targets or new active molecules focus mainly on the disruption of parasite specific pathways. In this sense, ergosterol biosynthesis is one of the most attractive because it does not occur in mammals. Here, we report the synthesis of ergosterone coupled molecules and the characterization of their biological activity on Leishmania mexicana promastigotes. Molecule synthesis involved three steps: ergosterone formation using Jones oxidation, synthesis of Girard reagents, and coupling reaction. All compounds were obtained in good yield and high purity. Results show that ergosterone-triazol molecules (Erg-GTr and Erg-GTr2) exhibit an antiproliferative effect in low micromolar range with a selectivity index ~10 when compared to human dermic fibroblasts. Addition of Erg-GTr or Erg-GTr2 to parasites led to a rapid [Ca2+]cyt increase and acidocalcisomes alkalinization, indicating that Ca2+ was released from this organelle. Evaluation of cell death markers revealed some apoptosis-like indicators, as phosphatidylserine exposure, DNA damage, and cytosolic vacuolization and autophagy exacerbation. Furthermore, mitochondrion hyperpolarization and superoxide production increase were detected already 6 hours after drug addition, denoting that oxidative stress is implicated in triggering the observed phenotype. Taken together our results indicate that ergosterone-triazol coupled molecules induce a regulated cell death process in the parasite and may represent starting point molecules in the search of new chemotherapeutic agents to combat leishmaniasis.

Role of frozen section analysis in nodular thyroid pathology.

INTRODUCTION: Frozen section (FS) analysis used to be the principal examination guiding surgical strategy. The development and recent standardization of fine-needle aspiration cytology (FNAC) challenges it as a systematic attitude. The present study assessed the current contribution of FS, comparing it with FNAC as a diagnostic tool guiding surgery. MATERIAL AND METHODS: A retrospective diagnostic study analyzed 1515 thyroid samples over a 6-year period. Two hundred and fifty-two of the patients had undergone both FNAC (analyzed in our unit) and FS, revealing 69 cancers. RESULTS: The sensitivity and specificity of FS and FNAC were 75.36% and 100% versus 31.88% and 100%, respectively. In case of malignancy on FNAC (22 patients), FS did not influence indications for surgery. In case of non-malignant FNAC findings, FS diagnosed cancer in 13% of cases (30/230). In the subgroup of follicular lesions (Bethesda 3 and 4), FS modified surgical strategy in only 6.2% of cases (6/97), but diagnosed 13 of the 16 cancers (81.25%) in case of Bethesda 5 on FNAC (21 cases) and in 9 of the 13 cancers (69%) associated with non-diagnostic FNAC results (Bethesda 1: 70 cases). CONCLUSION: Although its contribution is small, FS optimizes surgery in certain cases. Systematic implementation may be economically justified, especially in follicular lesions diagnosed on FNAC, improving interpretation of a difficult and operator-dependent test, as is essential in certain FNAC results.

Usefulness of ancillary methods for diagnosis, prognosis and targeted therapy in thyroid pathology.

The development of molecular analyses for thyroid pathologies is on going. These analyses provide new diagnostic tools with the aim of accurately distinguishing malignant and benign thyroid tumors. They are particularly useful as most of them can be done preoperatively on thyroid fine-needle aspiration biopsy samples. Furthermore, molecular biomarkers may play a promising role since they are able to predict the prognosis of patients with thyroid tumors. Moreover, identification of molecular markers as well as a better understanding of thyroid carcinogenesis will help develop innovative targeted therapies, particularly in patients with metastatic iodo-resistant thyroid carcinoma. To date, four types of somatic genetic alterations are known to hold potential interest for the diagnosis and/or prognosis of follicular cell-derived thyroid carcinomas: BRAF and RAS mutations, and RET/PTC and PAX8/PPARγ rearrangements. Other recent molecular biomarkers have been investigated in thyroid oncology, in particular different microRNA signatures. This review describes the different aspects of ancillary methods, including those bassed on molecular biology, that are of current interest for the diagnosis, prognosis and treatment of follicular cell-derived thyroid carcinomas.

Successful treatment of Old World cutaneous leishmaniasis caused by Leishmania infantum with posaconazole.

Old World cutaneous leishmaniasis is a widespread and potentially disfiguring protozoal infection that is endemic in the Mediterranean basin, Africa, and parts of Asia. Human infection is caused by several species of Leishmania parasites, such as Leishmania infantum. Available systemic and topical treatments vary in efficacy and are often unjustified due to their toxicity. We report on a case that was treated with posaconazole, a drug typically considered an antifungal agent but which also targets specific metabolic pathways of the parasite.

Anti-VSG antibodies induce an increase in Trypanosoma evansi intracellular Ca2+ concentration.

Trypanosoma evansi and Trypanosoma vivax have shown a very high immunological cross-reactivity. Anti-T. vivax antibodies were used to monitor changes in the T. evansi intracellular Ca2+ concentration ([Ca2+]i) by fluorometric ratio imaging from single parasites. A short-time exposure of T. evansi parasites to sera from T. vivax-infected bovines induced an increase in [Ca2+]i, which generated their complete lysis. The parasite [Ca2+]i boost was reduced but not eliminated in the absence of extracellular Ca2+ or following serum decomplementation. Decomplemented anti-T. evansi VSG antibodies also produced an increase in the parasite [Ca2+]i, in the presence of extracellular Ca2+. Furthermore, this Ca2+ signal was reduced following blockage with Ni2+ or in the absence of extracellular Ca2+, suggesting that this response was a combination of an influx of Ca2+ throughout membrane channels and a release of this ion from intracellular stores. The observed Ca2+ signal was specific since (i) it was completely eliminated following pre-incubation of the anti-VSG antibodies with the purified soluble VSG, and (ii) affinity-purified anti-VSG antibodies also generated an increase in [Ca2+]i by measurements on single cells or parasite populations. We also showed that an increase of the T. evansi [Ca2+]i by the calcium A-23187 ionophore led to VSG release from the parasite surface. In addition, in vivo immunofluorescence labelling revealed that anti-VSG antibodies induced the formation of raft patches of VSG on the parasite surface. This is the first study to identify a ligand that is coupled to calcium flux in salivarian trypanosomes.

Evaluation of the presence of a thapsigargin-sensitive calcium store in trypanosomatids using Trypanosoma evansi as a model.

Ca2+ plays an important role in the regulation of several important activities in different trypanosomatids. These parasites possess a Ca2+ transport system in the endoplasmic reticulum (ER) involved in Ca2+ homeostasis, which has been reported to be insensitive to thapsigargin, a classical inhibitor of the sarcoplasmic-ER Ca2+ adenosine triphosphatase (ATPase) (SERCA) in most eukaryotic cells. However, currently there is a controversy regarding the existence of a thapsigargin-sensitive ER Ca2+ store in these parasites. Therefore, we decided to explore the effect of this inhibitor using different methodological approaches. First, we selected Trypanosoma evansi as a parasite model to warrant the homogeneity of the population because this parasite has only a single life cycle, i.e., bloodstream-form trypomastigotes. Second, we compared the thapsigargin effect on Ca2+ homeostasis by spectrophotometrical Ca2+ measurements using 3 different approaches: whole-cell populations, cells that have been permeabilized by treatment with digitonin, and intact single cells. Our results demonstrate that a low concentration of thapsigargin induces Ca2+ release from intracellular Ca2+ stores in this parasite, which can be observed independently of the method used. Furthermore, the addition of thapsigargin before or after nigericin did not abolish its effect, showing that thapsigargin acts specifically on the ER. In conclusion, our results indicate the presence of a nonmitochondrial thapsigargin-sensitive Ca2+ store in T. evansi.

Efecto del etanol y del fosfatidiletanol sobre la ca²+ Atpasa de la membrana plasmática in situ / Ethanol and phosphatidylethanol effect on the Ca²+ Atpasa of the plasmatic membrane in situ

El etanol estimula de una manera aditiva con la calmodulina a la Ca²+ATPasa de membrana plasmática de eritrocitos humanos, por lo que esta enzima ha sido objeto de estudio con el fin de caracterizar el mecanismo de acción de este alcohol. Sin embargo, la mayoría de estos estudios han sido enfocados sobre la actividad de la enzima purificada en su forma soluble. Es importante poder extrapolar las evidencias obtenidas sobre dicha forma solubilizada y libre de fosfolípidos naturales a la enzima en su ambiente lipídico natural, con el fin de establecer la posible relevancia farmacológica de su efecto. En este trabajo evidenciamos que el efecto del etanol y otros alcoholes alifáticos de cadena corta como metanol, n-propanol y n-butanol en la Ca²+ATPasa presente en fragmentos de membranas de eritrocitos humanos (fantasmas), es esencialmente idéntico al efecto reportado sobre la enzima purificada. También demostramos en este mismo sistema que la estimulación inducida por el etanol es reversible, al igual que ocurre "in vivo". Por otra parte, similar a lo que se observa con la enzima purificada, en este trabajo evidenciamos que el fosfatidiletanol, un fosfolípido acídico que se acumula en la membrana plasmática luego de la ingesta de etanol, estimula la Ca²+ ATPasa de fragmentos de la membrana, incrementando la afinidad por Ca²+ a niveles superiores a los inducidos por calmodulina y por etanol. Se observó además un efecto aditivo sobre la afinidad por Ca²+ y la Vmax de la enzima en presencia simultánea de etanol y fosfatidiletanol, lo cual permite postular que este efecto también podría ocurrir en la célula intacta

Comparative phosphorylation of calmodulin from trypanosomatids and bovine brain by calmodulin-binding protein kinases.

Calmodulin (CaM), a major intracellular Ca2+ receptor protein, has been identified and partially characterized in several trypanosomatids. The amino acid sequences of CaM from Trypanosoma cruzi and Trypanosoma brucei are known, while that from Leishmania mexicana is not. CaM from T. cruzi contains 18 amino acid substitutions, as compared with CaM from bovine brain. In addition, CaM from bovine brain contains two tyrosine residues (Tyr-99 and Tyr-138), while CaM from T. cruzi only contains Tyr-138. In the present work we show that a monoclonal antibody developed against the carboxyl-terminal region of bovine brain CaM fails to recognize CaM from both T. cruzi and L. mexicana. CaM from both parasites and from bovine brain were phosphorylated in vitro by a preparation of CaM-binding protein kinases enriched in the epidermal growth factor (EGF) receptor. Phosphoamino acids analysis demonstrated EGF-dependent phosphorylation of tyrosine residues in bovine brain CaM, while only trace amounts of tyrosine phosphorylation were detected in CaM from both trypanosomatids. These results demonstrate that the EGF receptor tyrosine kinase targets Tyr-99, but not Tyr-138, as the single major phosphorylatable residue of CaM. On the other hand, and in contrast to bovine brain CaM, there is a significant phosphorylation of serine residues in CaM from trypanosomatids which is activated by the EGF receptor via a protein-serine/threonine kinase cascade.

The effect of ethanol on the plasma membrane calcium pump is isoform-specific.

The effect of ethanol has been studied on four different isoforms of the plasma membrane Ca2+-ATPase expressed in Sf9 cells with the help of the baculovirus system. The PMCA2CI protein was maximally activated by 0.5% ethanol, a concentration 8-10 times lower than that needed to obtain the same effect on the PMCA4 protein or on the pump of erythrocyte membranes, which is a mixture of isoforms 1 and 4. Experiments performed with truncated pumps indicated that the stimulation by ethanol was lost if the C-terminal region between Lys1065 and Lys1161, encompassing the calmodulin binding domain, was removed. These observations indicate that the stimulation is the result of a direct interaction of ethanol with the C-terminal regulatory domain of the Ca2+ pump.

Iatrogenic external iliac artery disruption during open pelvic lymph node dissection: successful repair with hypogastric artery transposition.

We report the first case of a wide, iatrogenic, proximal disruption of the right external iliac artery, occurring during staging open lymph node dissection for prostate cancer, which was repaired by hypogastric artery transposition. The hypogastric artery was mobilized and rotated anteriorly, and sutured to the distal segment of the external iliac artery. This is a feasible, innovative and safe technique which permits, by a single anastomosis, the secure reconstruction of a vascular axis to the leg when other procedures are not accessible.

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.

Effect of several bicyclic peptide and cyclic pseudopeptide tachykinin NK2 receptor antagonists in the human isolated urinary bladder.

1. We have studied several tachykinin NK2 receptor antagonists, bearing a monocyclic pseudopeptide (MEN 10,508, MEN 10,573, MEN 10,581, MEN 10,612, MEN 10,619 and MEN 10,677), or bicyclic peptide (MEN 10,627, MEN 10,692, MEN 10,771, MEN 10,882 and MEN 10,993) structure, on the human isolated urinary bladder detrusor muscle against neurokinin A as an agonist, and compared their affinities in this preparation with those for NK2 receptors expressed in the rabbit isolated pulmonary artery and hamster isolated trachea. 2. In the human bladder, all the antagonists tested produced a concentration-dependent and competitive antagonism of neurokinin A-mediated contractions: among the cyclic pseudopeptides MEN 10,677 (pKB = 8.0) was the most potent antagonist, while among the bicyclic analogues it was MEN 10,993 (pKB = 8.8). 3. In general, the bicyclic peptide antagonists tested were more potent than the monocyclic pseudopeptide compounds, either in the human urinary bladder or in the rabbit pulmonary artery or hamster trachea, showing a nanomolar affinity for the human NK2 receptor. 4. A highly significant correlation was found between the estimated pKB values of all the antagonists tested in the human urinary bladder and rabbit pulmonary artery (r2 = 0.94, n = 12, P < 0.01), whereas no linear correlation was found between pKB values measured in the human urinary bladder and hamster trachea (r2 = 0.52, n = 12, P > 0.05): these observations provide further pharmacological evidence for receptor homology between the human and rabbit NK2 receptor. 5. The present results point out the class of NK2 receptor antagonists bearing a bicyclic peptide structure, like MEN 10,627, as candidates for testing in pathological conditions, such as bladder hyperactivity, for which preclinical evidence indicates that a therapeutic effect could result from the block of the tachykinin NK2 receptor.

Intravesical capsaicin for treatment of severe bladder pain: a randomized placebo controlled study.

PURPOSE: Present therapeutic approaches to control bladder pain are clinically and scientifically unsatisfactory, and pain in the lower urinary tract remains a challenge even to the skilled urologist. A randomized placebo controlled study was done to evaluate intravesical capsaicin for severe bladder pain. Followup was 6 months. MATERIALS AND METHODS: A total of 36 patients was prospectively randomized into those receiving 10 microM. intravesical capsaicin twice weekly for 1 month (group 1) or placebo (group 2). All patients had pelvic pain for at least 6 months, and had no urinary tract infection within the last 3 months, functional disorders of the lower urinary tract, or other vesical or urethral pathology. Pretreatment voiding pattern and pain score were recorded. Patients were evaluated immediately at the end of treatment (primary end point) and 6 months later (secondary end point). RESULTS: Both groups were adequately homogeneous with regard to age, sex ratio, duration of disease, voiding pattern and pain score. At both end points group 1 had significant improvement in frequency and nocturia but no improvement in urgency. No change was noted in group 2. A significant decrease in pain score was found in group 1 at the primary (mean plus or minus standard deviation 3.22 +/- 0.42, p < 0.01) and secondary (3.83 +/- 0.47, p < 0.01) end points compared to before treatment (5.61 +/- 0.40, chi-square with 2 degrees of freedom 29.25, p < 0.0001). A significant improvement was also observed in the placebo group, in which the pretreatment pain score (5.47 +/- 0.37) was decreased at the primary (4.47 +/- 0.36, p < 0.01) and secondary (4.48 +/- 0.34, p < 0.01, chi-square with 2 degrees of freedom 12.71, p < 0.002) end points. There were no statistically significant differences between the 2 groups. CONCLUSIONS: We confirmed the beneficial effect of intravesical instillation of capsaicin on voiding pattern in patients with hypersensitive disorders (frequency and nocturia). We could not confirm improvement in pain score after capsaicin treatment compared to placebo. Possibly a larger dose of capsaicin would be more effective in controlling pain and neurological disease of the bladder.

The role of a H(+)-ATPase in the regulation of cytoplasmic pH in Trypanosoma cruzi epimastigotes.

Cytoplasmic pH (pHi) regulation was studied in Trypanosoma cruzi epimastigotes using fluorescent probes. Steady-state pHi was maintained even in the absence of extracellular Na+ or K+, but was significantly decreased in the absence of Cl-. Acid-loaded epimastigotes regained normal pHi by a process that was ATP-dependent and sensitive to N-ethylmaleimide, dicyclohexyl-carbodi-imide and diethylstiboestrol, suggesting involvement of a H(+)-pumping ATPase. Recovery from an acid load was independent of extracellular Na+ or K+ and insensitive to omeprazole, vanadate and low concentrations of bafilomycin A1. Using the fluorescent probe bisoxonol to measure the membrane potential of intact cells, acid loading of epimastigotes was shown to result in a dicyclohexylcarbodi-imide-sensitive hyperpolarization, which suggests electrogenic pumping of protons across the plasma membrane. Addition of glucose, but not of 6-deoxyglucose, produced a transient cellular acidification of possible metabolic origin, and increased the rate of recovery from an acid load. Taken together, these results are consistent with an important role of a H(+)-ATPase in the regulation of pHi homoeostasis in T. cruzi.

Phosphatidylethanol stimulates the plasma-membrane calcium pump from human erythrocytes.

Phosphatidylethanol is formed by "transphosphatidylation' of phospholipids with ethanol catalysed by phospholipase D and can be accumulated in the plasma membrane of mammalian cells after treatment of animals with ethanol. In the present work we show that phosphatidylalcohols, such as phosphatidylethanol and phosphatidylbutanol, produced a twofold stimulation of the Ca(2+)-ATPase activity of human erythrocytes. This stimulation occurs with the purified, solubilized enzyme as well as with ghost preparations, where the enzyme is in its natural lipidic environment and is different to that obtained with other acidic phospholipids such as phosphatidylserine. Addition of either phosphatidylserine, phosphatidylethanol or phosphatidylbutanol to the purified Ca(2+)-ATPase, or to ghosts preparations, increased the affinity of the enzyme for Ca2+ and the maximal velocity of the reaction as compared with controls in the absence of acidic phospholipids. However, in contrast with what occurs with phosphatidylserine, simultaneous addition of phosphatidyl-alcohols and calmodulin increased the affinity of the enzyme for Ca2+ to a greater extent than each added separately. When ethanol was added to either the purified erythrocyte Ca(2+)-ATPase or to erythrocyte-ghost preparations in the presence of acidic phospholipids, an additive effect was observed. There was an increase in the affinity for Ca2+ and in the maximal velocity of the reaction, well above the values obtained with ethanol or with the acidic phospholipids tested separately. These findings could have pharmacological importance. It is conceivable that the decrease in the intracellular Ca(2+) concentration that has been reported in erythrocytes as a result of ethanol intoxication could be due to the stimulation of the Ca(2+)-ATPase by the accumulated phosphatidylethanol, to a direct effect of ethanol on the enzyme or to an additive combination of both.

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.

Vesical dysfunction in systemic sclerosis (scleroderma)

There have been only a few reports on the involvement of the urinary tract in patients with systemic sclerosis, a disease of the connective tissue characterized by thickening and fibrosis of the skin, abnormality of the small arteries, and involvement of the gastrointestinal tract, heart, lung and kidney. We report the urodynamic assessment and histological examination of 9 women with scleroderma. Three patients voided less than 100 ml. with a significant residual volume and 4 presented with detrusor areflexia during a filling cystometrogram. Histopathological examination in all patients with detrusor areflexia demonstrated the presence of arterial lesions and derangement of the capillary bed of the detrusor musculature. Our data provide evidence for the functional and histological involvement of the bladder in patients with systemic sclerosis.