Biomedical subjects

Gustavo Benaim

Publications and source records attributed to Gustavo Benaim.

8 recordsLinked to original sources

PubMed2006

Amiodarone has intrinsic anti-Trypanosoma cruzi activity and acts synergistically with posaconazole.

There is no effective treatment for the prevalent chronic form of Chagas' disease in Latin America. Its causative agent, the protozoan parasite Trypanosoma cruzi, has an essential requirement for ergosterol, and ergosterol biosynthesis inhibitors, such as the antifungal drug posaconazole, have potent trypanocidal activity. The antiarrhythmic compound amiodarone, frequently prescribed for the symptomatic treatment of Chagas' disease patients, has also recently been shown to have antifungal activity. We now show here for the first time that amiodarone has direct activity against T. cruzi, both in vitro and in vivo, and that it acts synergistically with posaconazole. We found that amiodarone, in addition to disrupting the parasites' Ca(2+) homeostasis, also blocks ergosterol biosynthesis, and that posaconazole also affects Ca(2+) homeostasis. These results provide logical explanations for the synergistic activity of amiodarone with azoles against T. cruzi and open up the possibility of novel, combination therapy approaches to the treatment of Chagas' disease using currently approved drugs.

Acute Disease↗

PubMed2005

The plasma membrane Ca2+-ATPase protein from red blood cells is not modified in preeclampsia.

Plasma membrane Ca2+-ATPase activity diminishes by about 50% in red blood cells during preeclampsia. We investigated whether the number of Ca2+-ATPase molecules is modified in red cell membranes from preeclamptic pregnant women by measuring the specific phosphorylated intermediate of this enzyme. Also, we isolated the Ca2+-ATPase protein from both normotensive and preeclamptic pregnant women and estimated its molecular weight, and its cross-reactions with specific polyclonal and monoclonal (5F10) antibodies against it. We measured the Ca2+-ATPase activity in a purified state and the effect of known modulators of this ATPase. It was found that the phosphorylated intermediate associated with PMCA is similar for red cell ghosts from normotensive and preeclamptic women, suggesting a similar number of ATPase molecules in these membranes. The molecular weight of the Ca2+-ATPase is around 140 kDa for both normotensive and preeclamptic membranes, and its cross-reactions with specific antibodies is similar, suggesting that the protein structure remains intact in preeclampsia. Calmodulin, ethanol, or both calmodulin plus ethanol, stimulated the Ca2+-ATPase activity to the same extent for both normotensive and preeclamptic preparations. Our results showed that the reduced Ca2+-ATPase activity of the red cell membranes from preeclamptic women is not associated with a defective enzyme, but rather with a high level of lipid peroxidation.

Adolescent↗

PubMed2005

Ceramide-1-P induces Ca2+ mobilization in Jurkat T-cells by elevation of Ins(1,4,5)-P3 and activation of a store-operated calcium channel.

Sphingolipids comprise a very important class of second messengers involved in cell growth, differentiation, and apoptosis, among other different functions. Recently, these lipids have been implicated in calcium mobilization in different cell lines, including Jurkat T-lymphocytes. However, the effect of each particular sphingolipid appears to be cell-line specific. Among them, the least studied is ceramide-1-P (Cer-1-P). Here, we show that Cer-1-P increased the intracellular Ca(2+) concentration in Jurkat T-cells. Furthermore, laser-scanning confocal microscopy indicated that Ca(2+) is released from the endoplasmic reticulum. An effect on store-operated Ca(2+) channels was evidenced by whole-cell "patch clamp" measurements after Cer-1-P induced Ca(2+) store depletion. The mechanism of action of Cer-1-P resembles that of the Jurkat anti-TCR antibody, but differs from that of ceramide, since Cer-1-P induced an increase in Ins(1,4,5)-P(3).

Calcium↗

PubMed2005

Ceramide increase cytoplasmic Ca2+ concentration in Jurkat T cells by liberation of calcium from intracellular stores and activation of a store-operated calcium channel.

The effect of ceramide on the cytoplasmic Ca2+ concentration ([Ca2+]i) varies depending on the cell type. We have found that in Jurkat human T cells ceramide increases the [Ca2+]i from a thapsigargin-sensitive calcium pool and the subsequent activation of a capacitative Ca2+ entry. This effect occurs both in the presence and in the absence of extracellular calcium. Addition of ceramine, a non-hydrolysable analogue of ceramide, reproduced its effect on the [Ca2+]i ruling out that this is due to the conversion of ceramide to sphingosine. The effect of ceramide was additive to that obtained by sphingosine, but not to the Jurkat T cells specific antibody OKT3. However, different to the latter, ceramide do not induced an elevation of InsP3. The opening of a store operated Ca2+ channel by ceramide was corroborated by experiments of Fura-2 quenching, using Mn2+ as a surrogate for Ca2+ and confirmed by whole-cell recording patch clamp techniques.

Calcium↗

PubMed2005

Characterisation of tyrosine-phosphorylation-defective calmodulin mutants.

Using site-directed mutagenesis, we have produced three calmodulin (CaM) mutants in which one or the two tyrosine residues of native CaM were substituted by phenylalanine. The three variants, denoted CaM(Y99F), CaM(Y138F), and CaM(Y99F/Y138F), were highly expressed in transformed Escherichia coli BL21(DE3)pLysS and purified in high yield. The three CaM mutants were able to activate the cyclic nucleotide phosphodiesterase and the plasma membrane Ca(2+)-ATPase, and present the characteristic Ca(2+)-induced electrophoretic mobility shift of native CaM. CaM(Y138F) and CaM(Y99F/Y138F), however, showed a slightly higher electrophoretic mobility than CaM(Y99F) or wild type CaM. The molar extinction coefficient of native CaM at 276 nm decreases 50% in CaM(Y99F) and CaM(Y138F), while the 276nm peak disappears in CaM(Y99F/Y138F). Terbium fluorescence studies with the different CaM mutants indicate that Y99 (but not Y138) closely interacts with Ca(2+) in the III Ca(2+)-binding domain. The epidermal growth factor receptor (EGFR) and the non-receptor tyrosine kinase c-Src phosphorylate CaM(Y99F) and CaM(Y138F) at a lesser extent than wild type CaM, while they fail to phosphorylate CaM(Y99F/Y138F) as expected. All resulting phospho-(Y)CaM species present the characteristic Ca(2+)-induced electrophoretic mobility shift observed in non-phosphorylated CaM. Quantitative analysis of the different phospho-(Y)CaM species suggests that the relative phosphorylation of Y99 and Y138 in wild type CaM by both the EGFR and c-Src is different than the respective phosphorylation of either Y99 in CaM(Y138F) or Y138 in CaM(Y99F).

Amino Acid Substitution↗

PubMed2002

A proton pumping pyrophosphatase in the Golgi apparatus and plasma membrane vesicles of Trypanosoma cruzi.

The proton pumping pyrophosphatase (H(+)-PPase) is an enzyme that has been identified in membranes of plant vacuoles, in the Golgi complex of plants and Chlamydomonas reinhardtii, and more recently in acidocalcisomes of different trypanosomatids and apicomplexan parasites. Immunofluorescence and immunoelectron microscopy studies using antibodies against the plant enzyme also suggested a plasma membrane localization in different stages of Trypanosoma cruzi. In this report we provide immunogold electron microscopy evidence of the presence of the H(+)-PPase in the Golgi complex and plasma membrane of epimastigotes of T. cruzi. Pyrophosphate promoted acidification of plasma membrane vesicles as determined using acridine orange. This activity was stimulated by K(+) ions, inhibited by the pyrophosphate analogs imidodiphosphate (IDP) and aminomethylenediphosphonate (AMDP) by KF, NaF and DCCD, and it had different responses to ions and inhibitors as compared with the activity present in acidocalcisomes. Surface localization of the H(+)-PPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against H(+)-PPase. Taken together, these results are consistent with the presence of a functional H(+)-PPase in the plasma membrane of these parasites.

Animals↗

PubMed2002

Ceramide and sphingosine have an antagonistic effect on the plasma-membrane Ca2+-ATPase from human erythrocytes.

The plasma-membrane Ca(2+)-ATPase is a key enzyme in the regulation of the intracellular Ca(2+) concentration. On the other hand, sphingolipids have been recognized recently as important second messengers, acting in many systems in combination with Ca(2+). In view of the fact that the Ca(2+)-ATPase is stimulated by ethanol, and since sphingolipids possess free hydroxy groups, we decided to study the possible effect of ceramide and sphingosine on this calcium pump. Here we show that ceramide stimulates the Ca(2+)-ATPase in a dose-dependent manner and additively to the activation observed in the presence of calmodulin or ethanol, when compared with any of these effectors added alone. Ceramide affects both the affinity for Ca(2+) and the V(max) of the enzyme. Furthermore, this second messenger also stimulates Ca(2+) transport in inside-out plasma-membrane vesicles from erythro cytes. Conversely, sphingosine, which is reported to act in many systems antagonistically with ceramide, showed an inhibitory effect on Ca(2+)-ATPase activity. This inhibition was also observed on the calmodulin-stimulated enzyme. These results, taken together, suggest that ceramide and sphingosine act antagonistically on the plasma-membrane Ca(2+)-ATPase. This is in accordance with the frequently reported opposite effect of these sphingolipids on intracellular Ca(2+) concentration.

Calcium↗

PubMed2002

Phosphorylation of calmodulin. Functional implications.

Calmodulin (CaM) is phosphorylated in vitro and in vivo by multiple protein-serine/threonine and protein-tyrosine kinases. Casein kinase II and myosin light-chain kinase are two of the well established protein-serine/threonine kinases implicated in this process. On the other hand, within the protein-tyrosine kinases involved in the phosphorylation of CaM are receptors with tyrosine kinase activity, such as the insulin receptor and the epidermal growth factor receptor, and nonreceptor protein-tyrosine kinases, such as several members of the Src family kinases, Janus kinase 2, and p38Syk. The phosphorylation of CaM brings important physiological consequences for the cell as the diverse phosphocalmodulin species have differential actions as compared to nonphosphorylated CaM when acting on different CaM-dependent systems. In this review we will summarize the progress made on this topic as the first report on phosphorylation of CaM was published almost two decades ago. We will emphasize the description of the phosphorylation events mediated by the different protein kinases not only in the test tube but in intact cells, the phosphorylation-mediated changes of CaM activity, its action on CaM-dependent systems, and the functional repercussion of these phosphorylation processes in the physiology of the cell.

Animals↗