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.

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.

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.

Similarities between the effects of dimethyl sulfoxide and calmodulin on the red blood cell Ca2(+)-ATPase.

The Ca2(+)-ATPase of the erythrocyte plasma membrane can be activated by calmodulin, acidic phospholipids, limited proteolysis and self-association. Recently, it has been shown that different organic solvents increase both the Vmax and the Ca2+ affinity of the enzyme (Benaim, G. and De Meis, L. (1989) FEBS Lett. 244, 484-486). In this report the effects of calmodulin and dimethyl sulfoxide (20%, v/v) on the Ca2(+)-ATPase are compared. Dimethyl sulfoxide also elicits the appearance of the low-affinity binding site, which in this enzyme is strictly dependent on calmodulin. Dimethyl sulfoxide increases the Ca2+ affinity of the enzyme in a manner similar to that observed with the use of calmodulin and of acidic phospholipids. This was tested using both native and partially trypsinized ATPase. When activated by calmodulin the enzyme is inhibited by compound 48/80, trifluoperazine and calmidazolium. When activated by dimethyl sulfoxide the enzyme is still inhibited by calmidazolium but is no longer inhibited by either compound 48/80 or trifluoperazine. Activation of the ATPase promoted by either calmodulin or dimethyl sulfoxide is abolished when the Ca2+ concentration is raised from 10 microM to 2 mM. The effect of dimethyl sulfoxide is also abolished by 20 mM Pi. In the presence of 1 to 10 mM Ca2+ the ATPase catalyzes an ATP in equilibrium Pi exchange. The rate of exchange increases several fold when dimethyl sulfoxide is included in the assay medium.

A calcium pump in plasma membrane vesicles from Leishmania braziliensis.

A subcellular fraction highly enriched in plasma membrane vesicles was prepared from Leishmania promastigotes. This fraction showed (Ca2+ + Mg2+)-ATPase activity. This, however, represented a small fraction (about 25%) of the overall ATPase activity. The Ca2(+)-ATPase showed general characteristics common to plasma membrane ATPases involved in Ca2+ transport. Thus, the Ca2(+)-ATPase was activated by Ca2+ with a high affinity (Km about 0.7 microM), saturating at about 5 microM Ca2+. Furthermore, it was stimulated by calmodulin (about 70-80% with 5 micrograms/ml) and almost fully inhibited by trifluoperazine (100 microM). The above vesicles accumulated Ca2+ against a concentration gradient and released it after the addition of A23187, as shown independently by 45Ca2+ and Arsenazo III studies. The transport mechanism showed the same kinetics parameters as described for the enzyme, indicating a single molecular entity. In addition, Ca2(+)-ATPase activity and Ca2+ uptake were completely inhibited by vanadate (20 microM), indicating that an E1-E2 type mechanism is involved. The results clearly demonstrate the presence of a Ca2+ pump in the plasma membrane of Leishmania which is capable of maintaining a low cytoplasmic Ca2+ concentration.

Ethanol stimulates the plasma membrane calcium pump from human erythrocytes.

The plasma membrane Ca(2+)-ATPase from human erythrocytes can be stimulated by different treatments such as addition of calmodulin or acidic phospholipids and controlled proteolysis. In this report we show that short chain alkyl alcohols also stimulated this enzyme. At 5% (v/v) ethanol, the maximal velocity of the enzyme was about 2.4-fold higher than in the control, and thus, was also higher than the maximal velocity obtained in the presence of calmodulin (about 2-fold). When ethanol and calmodulin were present simultaneously, the stimulatory effect was additive (3.4-fold stimulation). On the other hand, the stimulatory effect of ethanol was preserved after treatment of the enzyme with trypsin to stimulate the Ca(2+)-ATPase and render it independent of calmodulin, thus suggesting that the interaction of ethanol and calmodulin with the Ca(2+)-ATPase occurred through a different mechanism. Other short chain alkyl alcohols (methanol, n-propanol and n-butanol) stimulated the Ca(2+)-ATPase activity to the same extent than ethanol but with different efficacy. Thus, the larger the carbon number, the lower the concentration needed to get the same maximal stimulation. Ethanol also increased the affinity of the enzyme for ATP to a larger extent and additively, when compared to calmodulin. All the effects of ethanol mentioned above were identically observed on the membrane-bound enzyme (i.e., erythrocyte ghosts) ruling out any effect of the alcohols attributable to the solubilized purified enzyme. Furthermore, Ca2+ transport by inside-out vesicles was also stimulated by ethanol, showing both the same concentration-dependence as the Ca(2+)-ATPase activity and the additive effect observed when calmodulin was also present. The stimulatory effect of ethanol was significant at pharmacological concentrations, thus suggesting potential implications of toxicological relevance.

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.

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.

ATPase activity and Ca2+ transport by reconstituted tryptic fragments of the Ca2+ pump of the erythrocyte plasma membrane.

The purified Ca2+ ATPase of the erythrocyte plasma membrane has been submitted to controlled trypsin proteolysis under conditions that favor either its (putative) E1 or E2 configurations. The former configuration has been forced by treating the enzyme with Ca2+-saturated calmodulin, the latter with vanadate and Mg2+. The E1 conformation leads to the accumulation of a polypeptide of Mr 85 KDa which still binds calmodulin, the E2 conformation to the accumulation of one of Mr 81 KDa which does not. Both fragments arise from the hydrolysis of a transient 90 KDa product which has Ca2+-calmodulin dependent ATPase activity, and which retains the ability to pump Ca2+ in reconstituted liposomes. Highly enriched preparations of the 85 and 81 KDa fragments have been obtained and reconstituted into liposomes. The former has limited ATPase and Ca2+ transport ability and is not stimulated by calmodulin. The latter has much higher ATPase and Ca2+ transport activity. It is proposed that the Ca2+ pumping ATPase of erythrocytes plasma membrane contains a 9 KDa domain which is essential for the interaction of the enzyme with calmodulin and for the full expression of the hydrolytic and transport activity. This putative 9 KDa sequence contains a 4 KDa "inhibitory" domain which limits the activity of the ATPase. In the presence of this 4 KDa sequence, i.e., when the enzyme is degraded to the 85 KDa product, calmodulin can still be bound, but no longer stimulates ATPase and Ca2+ transport.

Activation of the purified erythrocyte plasma membrane Ca2+- ATPase by organic solvents.

In this report it is shown that organic solvents mimic the stimulatory effects of calmodulin and acidic phospholipids on the erythrocyte plasma membrane Ca2+-ATPase. The solvents used were dimethyl sulfoxide (20%, v/v), glycerol (20% v/v), ethylene glycol (20%, v/v) and polyethylene glycol (Mr 6000-8000) (10%, w/v). These solvents increased both the affinity for Ca2+ and the turnover number of the enzyme. The increase in Ca2+ affinity is additive to that achieved with calmodulin. The calcium cooperativity observed in the presence of calmodulin disappears after the addition of dimethyl sulfoxide to the medium. The present data support the proposal that activation of the erythrocyte plasma membrane Ca2+-ATPase is promoted by hydrophobic interactions along the enzyme molecule.

Increased calcium permeability is not responsible for the rapid lethal effects of amphotericin B on Leishmania sp.

The mode of action of the polyene antibiotic amphotericin B (AmB), the drug of choice for the treatment of systemic fungal infections and visceral leishmaniasis, is still unclear. An increase in intracellular Ca2+ concentration [( Ca2+]i), toxic in many cases, has been postulated as a possible lethal mechanism for AmB. Cell permeabilization to ethidium bromide (EB) was used as a criterion of viability. Kinetics of the DNA-EB fluorescent complex formation was studied in ergosterol-containing Leishmania promastigotes. Intracellular Ca2+ concentration was measured using quin-2 fluorescence in parallel aliquots. It is shown in this work that AmB can act as an efficient Ca2+ ionophore. However, the rapid permeabilization effect induced by AmB on these cells was not dependent on an increase in [Ca2+]i. On the contrary, it was found that leishmanicidal effect of AmB was enhanced in the absence of external calcium. Furthermore, A23187 a Ca2+ ionophore did not provoke cell permeabilization to EB.

Ca2+ transport in isolated mitochondrial vesicles from Leishmania braziliensis promastigotes.

Leishmania braziliensis maintained very low (50 +/- 20 nM) intracellular concentrations of calcium ions under normal conditions, as shown by the fluorimetric indicator QUIN2. Digitonin-permeabilized cells liberated large amounts of calcium ions in the presence of the ionophore A23187, indicating the presence of a large intracellular reservoir for this ion. Given the extraordinary extension of the single giant mitochondrion of Kinetoplastida and the known capacity of mitochondria from other sources to accumulate calcium, we tested the capacity of this organelle to accumulate calcium ions in Leishmania. Coupled mitochondrial vesicles, five-fold enriched in succinate-cytochrome c oxidoreductase, were obtained from promastigotes by gentle grinding (45 s) with glass beads in hypertonic buffer solution, followed by differential centrifugation. These vesicles had a respiratory control ratio of 1.82 +/- 0.15, and two phosphorylation sites (sites II and III) using succinate as electron donor, and were capable of calcium uptake in the presence of several respiratory substrates; this uptake was enhanced in the presence of ADP and Pi and was blocked by classical electron transport inhibitors. Uncouplers such as carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) and the calcium ionophore A23187 released previously accumulated calcium ions, suggesting that the driving force for the calcium uptake by the vesicles is the respiratory generated electrochemical potential gradient of protons. A study of the affinity of this system for calcium showed that even at 90 microM free calcium, succinate-induced calcium uptake is not saturated while approaching a level of 200 nmol min-1 (mg protein)-1, indicating a low-affinity, large-capacity system.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

The behavioral response induced by intravesical instillation of capsaicin rats is mediated by pudendal urethral sensory fibers.

The behavioral response induced by the intravesical instillation of capsaicin has been investigated in catheter-implanted, freely moving, conscious male and female rats. Intravesical instillation of capsaicin (25 nmol/rat in 0.5 ml) evoked an intense licking directed toward the lower abdominal and perineal skin which lasted for 15 min. Capsaicin-induced licking was reduced by systemic capsaicin pretreatment at a dose of 150 mg/kg but not at 50 mg/kg, administered 4 days before the intravesical instillation. Both doses of capsaicin effectively abolished the eye-wiping response to local capsaicin application. The capsaicin-induced licking was preserved (and prolonged) in both male and female animals subjected to bilateral ablation of the pelvic ganglia, 48 h before the intravesical instillation of the irritant. The capsaicin-induced licking was almost abolished by placing a ligature around the proximal urethra (24 h before), in order to avoid the contact of the irritant with the urethra or by previous section of the pudendal nerves (48 h before). One hour intravesical infusion with tetrodotoxin (1 and 10 microM) reduced the amplitude of micturition contractions but did not prevent the licking response evoked by capsaicin. On the contrary, the intravesical infusion of lidocaine (100 mM) abolished both micturition contractions and the capsaicin-induced licking. We conclude that the behavioral response induced by the intravesical instillation of capsaicin is mainly due to the stimulation of urethral afferents travelling within pudendal nerves.

Fluorimetric quantification of cell death in monolayer cultures and cell suspensions.

A fluorimetric assay using ethidium bromide (EB) was employed to quantify cell death in monolayer cell cultures (MA-104 cells) in situ and isolated cell suspensions (isolated colonic cells and Leishmania). Fluorescence of EB stained cells was measured with a photometer coupled to an inverted microscope for cell monolayers or in a spectrofluorometer for cell suspensions. Dead cells stained with trypan blue were fluorescent with EB in all preparations studied, but the latter gave an unequivocal signal. Staining with EB and fluorescein diacetate was mutually exclusive. The relationship between the number of EB fluorescent cells and the intensity of fluorescence measured in the microphotometer was linear for a large range of cell numbers (1-14000) from different types of preparations. Applicability of the method for measuring living and dead cells in two different time scales (minutes and hours) is shown using MA-104 cell monolayers infected with rotavirus and Leishmania suspensions treated with amphotericin B. The method is fast, simple, sensitive and reliable, enabling quantification of living and dead cells in monolayers and suspensions.

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.

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.

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.