Structural and Molecular Basis of the Peroxynitrite-mediated Nitration and Inactivation of Trypanosoma cruzi Iron-Superoxide Dismutases (Fe-SODs) A and B: DISPARATE SUSCEPTIBILITIES DUE TO THE REPAIR OF TYR35 RADICAL BY CYS83 IN Fe-SODB THROUGH INTRAMOLECULAR ELECTRON TRANSFER*

Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 10⁴ m⁻¹ s⁻¹ and 4.3 ± 0.4 × 10⁴ m⁻¹ s⁻¹ at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr³⁵. Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys⁸³ mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys⁸³ present in Fe-SODB acts as an electron donor that repairs Tyr³⁵ radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.     

Cadherin, alkaline phosphatase, and aminopeptidase N as receptors of Cry11Ba toxin from Bacillus thuringiensis subsp. jegathesan in Aedes aegypti

Cry11Ba is one of the most toxic proteins to mosquito larvae produced by Bacillus thuringiensis. It binds Aedes aegypti brush border membrane vesicles (BBMV) with high affinity, showing an apparent dissociation constant (K(d)) of 8.2 nM. We previously reported that an anticadherin antibody competes with Cry11Ba binding to BBMV, suggesting a possible role of cadherin as a toxin receptor. Here we provide evidence of specific cadherin repeat regions involved in this interaction. Using cadherin fragments as competitors, a C-terminal fragment which contains cadherin repeat 7 (CR7) to CR11 competed with Cry11Ba binding to BBMV. This binding was also efficiently competed by the CR9, CR10, and CR11 peptide fragments. Moreover, we show CR11 to be an important region of interaction with Cry11Ba toxin. An alkaline phosphatase (AaeALP1) and an aminopeptidase-N (AaeAPN1) also competed with Cry11Ba binding to Ae. aegypti BBMV. Finally, we found that Cry11Ba and Cry4Ba share binding sites. Synthetic peptides corresponding to loops α8, β2-β3 (loop 1), β8-β9, and β10-β11 (loop 3) of Cry4Ba compete with Cry11Ba binding to BBMV, suggesting Cry11Ba and Cry4Ba have common sites involved in binding Ae. aegypti BBMV. The data suggest that three different Ae. aegypti midgut proteins, i.e., cadherin, AaeALP1, and AaeAPN1, are involved in Cry11Ba binding to Ae. aegypti midgut brush border membranes.     

Effects of irrigation and air humidity preconditioning on water relations, growth and survival of Rosmarinus officinalis plants during and after transplanting

The effect of different irrigation and air humidity conditioning treatments on the morphological and physiological responses of Rosmarinus officinalis in nursery conditions was investigated in order to evaluate the degree of hardening resulting from these conditions. Rosmarinus officinalis seedlings were pot-grown during 4 months in two greenhouses (nursery period), in which two irrigation treatments were used (control and deficit). In one of these greenhouses, air humidity was controlled using a dehumidifying system (low humidity), in the other greenhouse the air conditions were not artificially modified (control humidity). After the nursery period, the plants of all treatments were transplanted and well watered (100% water holding capacity for 1 month, transplanting period). After this period, they received no water (establishment period). At the end of the nursery period it was seen that deficit irrigation had altered the morphology of the R. officinalis plants by reducing plant height, stem diameter, leaf area, total dry weight, and root length, while humidity influenced the parameters related with plant water relations. Low air humidity and deficit irrigation-induced tissue dehydration and lower stomatal conductance values (gs). The plants subjected to deficit irrigation developed leaf osmotic adjustment, which was maintained during the transplanting period. At that time, the plants that had been exposed to deficit irrigation and low humidity showed efficient stomatal regulation (lower gs values). After transplanting and during the establishment period, these plants showed a better water status (higher psil and gs values). Their post-planting survival rate improved as a result of acclimation processes.     

A mitochondrial kinase complex is essential to mediate an ERK1/2-dependent phosphorylation of a key regulatory protein in steroid biosynthesis

ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein -a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser(232). Directed mutagenesis of Ser(232) to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined.     

Congenital syphilis in Argentina: Experience in a pediatric hospital

In spite of being preventable, Congenital syphilis (CS) is still an important, and growing health problem worldwide. Fetal infection can be particularly aggressive, but newborns can be asymptomatic at birth and, if left untreated, develop systemic compromise afterwards with poor prognosis. We analyzed 61 CS diagnosis cases between 1987–2019 presenting at the Buenos Aires Children’ Hospital. The distribution of cases showed a bimodal curve, with a peak in 1992–1993 and in 2014–2017. Median age at diagnosis was 2 months (IQ 1–6 months). The main clinical findings were: bone alterations (59%); hepatosplenomegaly (54.1%); anemia (62.8%); skin lesions (42.6%) and renal compromise (33.3%). Cerebrospinal fluid (CSF) was abnormal in 5 patients, normal in 45 and was not available for 11 patients. Remarkably, spinal lumbar puncture did not modify therapeutic decisions in any case. Between mothers, only 46% have been tested for syphilis during pregnancy and 60.5% patients had non-treponemal titers equal to or less than fourfold the maternal titer. Intravenous penicillin G was prescribed for all except one patient, who received ceftriaxone with good therapeutic response. During follow-up, 1.6% infants died, 6.5% had persistent kidney disorders and 1.6% showed bone sequelae damage. RPR titers decreased after treatment, reaching negative seroconversion in 43% subjects at a median of 26.4 months. Low adherence to follow up was observed due to inherent vulnerable and low-income population characteristics in our cohort. Our results highlight a rising tendency in cases referred for CS in our population with high morbidity related to delayed diagnosis. A good therapeutic response was observed. CS requires a greater effort from the health system to adequately screen for this disease during pregnancy, and to detect cases earlier, to provide an adequate diagnosis and treatment.     

Alterations in cerebrospinal fluid glycerophospholipids and phospholipase A2 activity in Alzheimer's disease

Our aim is to study selected cerebrospinal fluid (CSF) glycerophospholipids (GP) that are important in brain pathophysiology. We recruited cognitively healthy (CH), minimally cognitively impaired (MCI), and late onset Alzheimer''s disease (LOAD) study participants and collected their CSF. After fractionation into nanometer particles (NP) and supernatant fluids (SF), we studied the lipid composition of these compartments. LC-MS/MS studies reveal that both CSF fractions from CH subjects have N-acyl phosphatidylethanolamine, 1-radyl-2-acyl-sn-glycerophosphoethanolamine (PE), 1-radyl-2-acyl-sn-glycerophosphocholine (PC), 1,2-diacyl-sn-glycerophosphoserine (PS), platelet-activating factor-like lipids, and lysophosphatidylcholine (LPC). In the NP fraction, GPs are enriched with a mixture of saturated, monounsaturated, and polyunsaturated fatty acid species, while PE and PS in the SF fractions are enriched with PUFA-containing molecular species. PC, PE, and PS levels in CSF fractions decrease progressively in participants from CH to MCI, and then to LOAD. Whereas most PC species decrease equally in LOAD, plasmalogen species account for most of the decrease in PE. A significant increase in the LPC-to-PC ratio and PLA₂ activity accompanies the GP decrease in LOAD. These studies reveal that CSF supernatant fluid and nanometer particles have different GP composition, and that PLA₂ activity accounts for altered GPs in these fractions as neurodegeneration progresses.     

Sex pheromone and period gene characterization of Lutzomyia longipalpis sensu lato (Lutz & Neiva) (Diptera: Psychodidae) from Posadas, Argentina

Lutzomyia longipalpis s.l. is the primary vector of Leishmania (L.) infantum in the New World. In this study, male Lutzomyia longipalpis specimens from Posadas, Argentina were characterized for two polymorphic markers: the male sex pheromone and the period (per) gene. The male sex pheromone was identified as (S)-9-methylgermacrene-B, the same compound produced by Lu. longipalpis from Paraguay and many populations from Brazil. The analysis of per gene sequences revealed that the population from Argentina is significantly differentiated from previously studied Brazilian populations. Marker studies could contribute to the understanding of the distribution and spread of urban American visceral leishmaniasis, thus aiding in the design of regional surveillance and control strategies.     

Lipid packing determines protein-membrane interactions: Challenges for apolipoprotein A-I and high density lipoproteins

Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction, and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. Fluorescence Correlation Spectroscopy (FCS) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan Generalized Polarization (GP) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis and offer a methodological design suited to different biological systems.     

Culture of bovine hepatocytes: a non-perfusion technique for cell isolation

In this work we have studied the isolation and culture of mature bovine hepatocytes on plastic dishes without exogenous matrix. The liver has been disaggregated in a collagenase solution instead of undergoing a perfusion step. After a few days in culture, the plates showed several clusters of different cell types. Although the average yield was 1.60±0.57×10⁸ viable liver cells per gram of tissue, these cultures were formed by non-parenchymal cells and only very few or none by parenchymal cells. In these cultures, actin structures used as a marker for Stellate (Ito) cells have been visualized by immunocytochemical techniques. In order to increase the proportion of parenchymal cells a centrifugation on Percoll, which separates cell sub-populations, has been introduced. Though the yield was lower than in the previous method, these pre-purified cultures were only composed of hepatocytes. It has been shown that these cells exhibited albumin synthesis, which is a specific hepatocytes function. In addition, these cultures were capable of producing metabolites of 7-ethoxycoumarin at a higher rate than non purified cell cultures. Therefore this simplified procedure for the isolation and culture of functional and viable hepatocytes may be applied for in vitro studies in bovine.