Elevated extracellular inorganic phosphate inhibits ecto-phosphatase activity in breast cancer cells: Regulation by hydrogen peroxide.

For cells to obtain inorganic phosphate, ectoenzymes in the plasma membrane, which contain a catalytic site facing the extracellular environment, hydrolyze phosphorylated molecules. In this study, we show that increased Pi levels in the extracellular environment promote a decrease in ecto-phosphatase activity, which is associated with Pi-induced oxidative stress. High levels of Pi inhibit ecto-phosphatase because Pi generates H2 O2 . Ecto-phosphatase activity is inhibited by H2 O2 , and this inhibition is selective for phospho-tyrosine hydrolysis. Additionally, it is shown that the mechanism of inhibition of ecto-phosphatase activity involves lipid peroxidation. In addition, the inhibition of ecto-phosphatase activity by H2 O2 is irreversible. These findings have new implications for understanding ecto-phosphatase regulation in the tumor microenvironment. H2 O2 stimulated by high Pi inhibits ecto-phosphatase activity to prevent excessive accumulation of extracellular Pi, functioning as a regulatory mechanism of Pi variations in the tumor microenvironment.

Effect of drying temperature and storage time on the crispiness of homemade apple snacks.

BACKGROUND: The apple (Malus domestica Borkh.) plays an important role in the trendy market of dried snacks because of its exceptional flavor and texture. In addition to the health benefits, there is also a general disposition to consume organic and do-it-yourself products. RESULTS: Three different drying temperatures, 65, 75, and 85 °C, were tested using a commercial ventilated drying oven in 'Royal Gala' and 'Golden Delicious' cultivars. Physical changes, including texture, color, shrinkage ratio, and microstructure, were evaluated for the temperatures and cultivars considered. Based on the results, particularly in terms of shrinkage, hardness, and crispiness, a drying temperature of 75 °C was selected to perform texture profile analyses throughout the drying period. Storability conditions were evaluated to determine the best moment to maintain the physical properties of the dried snacks during storage. Considered the more important property related to consumer preferences, crispiness was followed with puncture tests. CONCLUSION: The storage of apple chips, dried at the various temperatures, that must be performed in 5-10 min after removing from the drying oven, was assessed over the course of a month. Both the drying process and the subsequent storage proved effective in preserving the desired texture of the apple snacks, regardless of the specific cultivar or drying temperature used. Through this study, with a refined understanding of the changes occurring during the drying process and the optimization of storage conditions, we can confidently offer consumers the best combination of crispy and healthy snacks that meet their expectations. © 2023 Society of Chemical Industry.

Ectophosphatase activity in the triple-negative breast cancer cell line MDA-MB-231.

Breast cancer is one of the most common cancers in the female population worldwide, and its development is thought to be associated with genetic mutations that lead to uncontrolled and accelerated growth of breast cells. This abnormal behavior requires extra energy, and indeed, tumor cells display a rewired energy metabolism compared to normal breast cells. Inorganic phosphate (Pi) is a glycolytic substrate of glyceraldehyde-3-phosphate dehydrogenase and has an important role in cancer cell proliferation. For cells to obtain Pi, ectoenzymes in the plasma membrane with their catalytic site facing the extracellular environment can hydrolyze phosphorylated molecules, and this is an initial and possibly limiting step for the uptake of Pi by carriers that behave as adjuvants in the process of energy harvesting and thus partially contributes to tumor energy requirements. In this study, the activity of an ectophosphatase in MDA-MB-231 cells was biochemically characterized, and the results showed that the activity of this enzyme was higher in the acidic pH range and that the enzyme had a Km = 4.5 ± 0.5 mM para-nitrophenylphosphate and a Vmax = 2280 ± 158 nM × h-1 × mg protein-1 . In addition, classical acid phosphatase inhibitors, including sodium orthovanadate, decreased enzymatic activity. Sodium orthovanadate was able to inhibit ectophosphatase activity while also inhibiting cell proliferation, adhesion, and migration, which are important processes in tumor progression, especially in metastatic breast cancer MDA-MB-231 cells that have higher ectophosphatase activity than MCF-7 and MCF-10 breast cells.

Wireless UV-A LEDs-driven AOP in the treatment of agro-industrial wastewaters.

A wireless UV-A LEDs lab-scale reactor powered by a resonant inductive coupling (RLC) system was built to maximize the UV photon absorption of agro-industrial wastewaters. The UV-A LEDs (λ = 365 nm) energy was supplied through a magnetic field generated inside of the photoreactor by induction coils placed on the external wall made of polyvinyl chloride. Immersing the light sources in the wastewater increases the photon transfer efficiency and the reaction rate. Maximum magnetic field and optical irradiance were obtained at 26.8 and 27.0 kHz, respectively. As proof-of-concept, elderberry wastewater (EW), olive washing wastewater (OWW) and white and red winery wastewaters (WWW and RWW) were treated combining the wireless UV-A LEDs with the Advanced Oxidation Process (AOP) - Fenton reagent. Fenton experiments were performed using [Fe2+] = 10 mg L-1, [H2O2] = 500 mg L-1, pH = 3 and a reaction time of 4 h. With EW a DOC removal of 35% (k = 0.0696 h-1) was achieved, whereas adding the wireless UV-A LEDs (f = 26.8 kHz) 53% was attained (k = 0.1722 h-1). The Electric Energy per Order (EEO) for the wireless UV-A LEDs consumption was calculated (EEO LEDs = 48.7 kWh m-3 order-1) and for all the remain equipment (air pump, RC box and power amplifier), EEO total = 495 kWh m-3 order-1. Experiments with OWW presented a DOC removal of 62% and a EEO LEDs = 40.5 kWh m-3 order-1; RWW shown 40% of DOC removal and a EEO LEDs = 68.4 kWh m-3 order-1, while with WWW 35% of DOC removal and a EEO LEDs = 79.8 kWh m-3 order-1 were obtained. This work shows that wireless UV-A LEDs can be a promising alternative to conventional UV lamps and wired LEDs in the treatment of real wastewaters. However, optimization of the induction system is still needed, as well as the number and wavelength of the LEDs (e.g. UV-C LEDs) to reduce the overall treatment costs.

Photocatalytic degradation of an agro-industrial wastewater model compound using a UV LEDs system: kinetic study.

An ultraviolet light emitting diode (UV-A LED) system was built to test the capability of performing heterogeneous photocatalysis using TiO2 P25. The LEDs maximum wavelength is 365 nm with an irradiance power of 85 W m-2. The device was tested in batch and continuous (CSTR) mode in a laboratorial scale reactor. The degradation of an agro-industrial wastewater model compound (p-hydroxybenzoic acid, pHBA) was investigated, assessing the effect of different experimental conditions such as pH, pHBA and TiO2 concentration keeping constant the UV-A LEDs power and temperature. The photodegradation of different concentrations of pHBA with [TiO2] = 500 mg L-1, IUV = 85 W m-2 and a T = 21 °C were analysed by pseudo-first order kinetics. The results were applied to the Langmuir-Hinshelwood model yielding kc = 0.885 mg L-1 min-1 and kLH = 0.217 L mg-1. In a comparative experiment the UV-A LEDs system showed faster kinetics (k = 0.0134 min-1) than solar radiation (IUV = 23 W m-2; k = 0.0077 min-1), with [pHBA] = 75 mg L-1 and [TiO2] = 500 mg L-1. The values of the Electric Energy per Order (EEO) = 115 kWh m-3 order-1 and the Specific Applied Energy (ESAE) = 318 kWh mol-1 order-1 were obtained with [TiO2] = 1000 mg L-1 and [pHBA] = 50 mg L-1. Analogous results were obtained ([TiO2] = 500 mg L-1) in a CSTR with a slight decrease in the first order kinetic constant due to the "non-ideal" reactor: from 0.0284 to 0.0158 min-1 and from 0.0143 to 0.00825 min-1 with [pHBA] = 50 mg L-1 and 75 mg L-1, respectively. This work shows that photocatalytic reactors with UV-A LEDs can advantageously replace conventional UV mercury lamps based reactors in the photodegradation of phenolic compounds.

Resveratrol Reverses Functional Chagas Heart Disease in Mice.

Chronic chagasic cardiomyopathy (CCC) develops years after acute infection by Trypanosoma cruzi and does not improve after trypanocidal therapy, despite reduction of parasite burden. During disease, the heart undergoes oxidative stress, a potential causative factor for arrhythmias and contractile dysfunction. Here we tested whether antioxidants/ cardioprotective drugs could improve cardiac function in established Chagas heart disease. We chose a model that resembles B1-B2 stage of human CCC, treated mice with resveratrol and performed electrocardiography and echocardiography studies. Resveratrol reduced the prolonged PR and QTc intervals, increased heart rates and reversed sinus arrhythmia, atrial and atrioventricular conduction disorders; restored a normal left ventricular ejection fraction, improved stroke volume and cardiac output. Resveratrol activated the AMPK-pathway and reduced both ROS production and heart parasite burden, without interfering with vascularization or myocarditis intensity. Resveratrol was even capable of improving heart function of infected mice when treatment was started late after infection, while trypanocidal drug benznidazole failed. We attempted to mimic resveratrol's actions using metformin (AMPK-activator) or tempol (SOD-mimetic). Metformin and tempol mimicked the beneficial effects of resveratrol on heart function and decreased lipid peroxidation, but did not alter parasite burden. These results indicate that AMPK activation and ROS neutralization are key strategies to induce tolerance to Chagas heart disease. Despite all tissue damage observed in established Chagas heart disease, we found that a physiological dysfunction can still be reversed by treatment with resveratrol, metformin and tempol, resulting in improved heart function and representing a starting point to develop innovative therapies in CCC.

Synthesis, spectroscopic characterization and biological evaluation of unsymmetrical aminosquarylium cyanine dyes.

New unsymmetrical aminosquarylium cyanine dyes were synthesized and their potential as photosensitizers evaluated. New dyes, derived from benzothiazole and quinoline, were prepared by nucleophilic substitution of the corresponding O-methylated, the key intermediate that was obtained by methylation with CF3SO3CH3 of the related zwitterionic unsymmetrical dye, with ammonia and methylamine, respectively. All three news dyes herein described displayed intense and narrow bands in the Vis/NIR region (693-714nm) and their singlet oxygen formation quantum yields ranged from 0.03 to 0.05. In vitro toxicity, in Caco-2 and HepG2 cells, indicated that dark toxicity was absent for concentrations up to 5µM (for the less active dye) or up to 1µM (for the two more active dyes). The three dyes present potential as photosensitizers, differing in irradiation conditions and period of incubation in the presence of irradiated dye. The less active dye needs a longer irradiation period to exhibit phototoxicity which is only evident after longer period of contact with cells (24h). However, the remaining two more active dyes produce higher phototoxicity, even at shorter incubation periods (1h), with shorter irradiation time (7min). Although in different extents, these dyes show promising in vitro results as photosensitizers.

Inorganic phosphate uptake in unicellular eukaryotes.

BACKGROUND: Inorganic phosphate (Pi) is an essential nutrient for all organisms. The route of Pi utilization begins with Pi transport across the plasma membrane. SCOPE OF REVIEW: Here, we analyzed the gene sequences and compared the biochemical profiles, including kinetic and modulator parameters, of Pi transporters in unicellular eukaryotes. The objective of this review is to evaluate the recent findings regarding Pi uptake mechanisms in microorganisms, such as the fungi Neurospora crassa and Saccharomyces cerevisiae and the parasite protozoans Trypanosoma cruzi, Trypanosoma rangeli, Leishmania infantum and Plasmodium falciparum. MAJOR CONCLUSION: Pi uptake is the key step of Pi homeostasis and in the subsequent signaling event in eukaryotic microorganisms. GENERAL SIGNIFICANCE: Biochemical and structural studies are important for clarifying mechanisms of Pi homeostasis, as well as Pi sensor and downstream pathways, and raise possibilities for future studies in this field.

Trypanosoma cruzi-secreted vesicles have acid and alkaline phosphatase activities capable of increasing parasite adhesion and infection.

Trypanosoma cruzi virulence factors include molecules expressed on the cell surface as well as those secreted or shed into the extracellular medium. Phosphatase activities modulate different aspects of T. cruzi infection, although no studies to date addressed the presence and activity of phosphatases in vesicles secreted by this parasite. Here, we characterized acidic and alkaline secreted phosphatase activities of human-infective trypomastigote forms of T. cruzi from the Y strain and the CL-Brener clone. These are widely studied T. cruzi strains that represent "opposite ends of the spectrum" regarding both in vitro and in vivo behavior. Ecto-phosphatase activities were determined in live parasites, and secreted phosphatase activities were analyzed in soluble protein (SP) and vesicular membrane fractions (VFs) of parasite-conditioned medium. Our analysis using different phosphatase inhibitors strongly suggests that vesicles secreted by Y strain (VF(Y)) and CL-Brener (VF(CLB)) trypomastigotes are derived mostly from the cell surface and from exosome secretion, respectively. Importantly, our results show that the acid phosphatase activities in vesicles secreted by trypomastigotes are largely responsible for the VF-induced increase in adhesion of Y strain parasites to host cells and also for the VF-induced increase in host cell infection by CL-Brener trypomastigotes.

Leishmania amazonensis impairs DC function by inhibiting CD40 expression via A2B adenosine receptor activation.

Dendritic cells (DCs) play an essential role in the modulation of immune responses and several studies have evaluated the interactions between Leishmania parasites and DCs. While extracellular ATP exhibits proinflammatory properties, adenosine is an important anti-inflammatory mediator. Here we investigated the effects of Leishmania infection on DC responses and the participation of purinergic signalling in this process. Bone marrow-derived dendritic cells (BMDCs) from C57BL/6J mice infected with Leishmania amazonensis, Leishmania braziliensis or Leishmania major metacyclic promastigotes showed decreased major histocompatibility complex (MHC) class II and CD86 expression and increased ectonucleotidase expression as compared with uninfected cells. In addition, L. amazonensis-infected DCs, which had lower CD40 expression, exhibited a decreased ability to induce T-cell proliferation. The presence of MRS1754, a highly selective A(2B) adenosine receptor antagonist at the time of infection increased MHC class II, CD86 and CD40 expression in L. amazonensis-infected DCs and restored the ability of the infected DCs to induce T-cell proliferation. Similar results were obtained through the inhibition of extracellular ATP hydrolysis using suramin. In conclusion, we propose that A(2B) receptor activation may be used by L. amazonensis to inhibit DC function and evade the immune response.

Rhodnius prolixus lipophorin: lipid composition and effect of high temperature on physiological role.

Lipophorin is a major lipoprotein that transports lipids in insects. In Rhodnius prolixus, it transports lipids from midgut and fat body to the oocytes. Analysis by thin-layer chromatography and densitometry identified the major lipid classes present in the lipoprotein as diacylglycerol, hydrocarbons, cholesterol, and phospholipids (PLs), mainly phosphatidylethanolamine and phosphatidylcholine. The effect of preincubation at elevated temperatures on lipophorin capacity to deliver or receive lipids was studied. Transfer of PLs to the ovaries was only inhibited after preincubation of lipophorin at temperatures higher than 55 °C. When it was pretreated at 75 °C, maximal inhibition of phospholipid transfer was observed after 3-min heating and no difference was observed after longer times, up to 60 min. The same activity was also obtained when lipophorin was heated for 20 min at 75 °C at protein concentrations from 0.2 to 10 mg/ml. After preincubation at 55 °C, the same rate of lipophorin loading with PLs at the fat body was still present, and 30% of the activity was observed at 75 °C. The effect of temperature on lipophorin was also analyzed by turbidity and intrinsic fluorescence determinations. Turbidity of a lipophorin solution started to increase after preincubations at temperatures higher than 65 °C. Emission fluorescence spectra were obtained for lipophorin, and the spectral area decreased after preincubations at 85 °C or above. These data indicated no difference in the spectral center of mass at any tested temperature. Altogether, these results demonstrate that lipophorin from R. prolixus is very resistant to high temperatures.

Trypanosoma rangeli: an alkaline ecto-phosphatase activity is involved with survival and growth of the parasite.

The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using ß-glycerophosphate (ß-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of ß-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of ß-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca(2+) present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. ß-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (Pi). Levamisole at the IC50 spared significantly parasite growth when ß-GP was the sole source of Pi and stopped it in the absence of ß-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.

Degradation of Agro-Industrial Wastewater Model Compound by UV-A-Fenton Process: Batch vs. Continuous Mode.

The degradation of a model agro-industrial wastewater phenolic compound (caffeic acid, CA) by a UV-A-Fenton system was investigated in this work. Experiments were carried out in order to compare batch and continuous mode. Initially, batch experiments showed that UV-A-Fenton at pH 3.0 (pH of CA solution) achieved a higher generation of HO•, leading to high CA degradation (>99.5%). The influence of different operational conditions, such as H2O2 and Fe2+ concentrations, were evaluated. The results fit a pseudo first-order (PFO) kinetic model, and a high kinetic rate of CA removal was observed, with a [CA] = 5.5 × 10−4 mol/L, [H2O2] = 2.2 × 10−3 mol/L and [Fe2+] = 1.1 × 10−4 mol/L (kCA = 0.694 min−1), with an electric energy per order (EEO) of 7.23 kWh m−3 order−1. Under the same operational conditions, experiments in continuous mode were performed under different flow rates. The results showed that CA achieved a steady state with higher space-times (θ = 0.04) in comparison to dissolved organic carbon (DOC) removal (θ = 0−0.020). The results showed that by increasing the flow rate (F) from 1 to 4 mL min−1, the CA and DOC removal rate increased significantly (kCA = 0.468 min−1; kDOC = 0.00896 min−1). It is concluded that continuous modes are advantageous systems that can be adapted to wastewater treatment plants for the treatment of real agro-industrial wastewaters.

Growth and Structural Characterization of h-LuMnO3 Thin Films Deposited by Direct MOCVD.

In this work, we investigated the MOCVD conditions to synthesize thin films with the hexagonal P63cm h-LuMnO3 phase as a potential low-band gap ferroelectric material. The main parameters investigated were the ratio of organometallic starting materials, substrate temperature, and annealing effect. Two different substrates were used in the study: fused silica (SiO2) glass and platinized silicon (Pt\Ti\SiO2\Si(100)). In order to investigate the thermodynamic stability and quality of the developed phases, a detailed analysis of the crystal structure, microstructure, morphology, and roughness of the films was performed by X-ray diffractometer, scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), Raman spectroscopy, and piezoelectric force microscopy (PFM). Molar compositions in the film within 0.93 < |Lu|/|Mn| < 1.33 were found to be suitable for obtaining a single-phase h-LuMnO3. The best films were obtained by depositions at 700 °C, followed by thermal treatments at 800 °C for long periods of up to 12 h. These films exhibited a highly crystalline hexagonal single phase with a relatively narrow direct band gap, around 1.5 eV, which is within the expected values for the h-LuMnO3 system.

Evaluation of Antifungal Activity and Potential Application as Fluorescent Probes of Indolenine and Benzo[e]Indole-Based Squarylium Dyes.

The antifungal performance and the possible use as fluorescent probes of a series of squarylium dyes derived from indolenine and benzo[e]indole previously synthesized was evaluated. Some photophysical properties were performed in ethanol and phosphate buffer, and the type of aggregates form in phosphate buffer was analyzed. Using the 1,3-diphenylisobenzofuran assay, a qualitative assessment of the capacity of dyes to produce singlet oxygen after irradiation was performed. Regarding the antifungal activity, this was studied through a broth microdilution assay using Saccharomyces cerevisiae PYCC 4072 as a biological model. The effect of irradiation of the dyes, with an appropriate light emitting diode system, on the antifungal activity was also evaluated, and it was verified that some of the dyes improve their activity after irradiation. Using fluorescence microscopy techniques, the colocalization of dyes in S. cerevisae cells was investigated and it was possible to verify that some of the squarylium dyes with a barbituric moiety in the four-membered central ring stained and accumulated preferentially in the mitochondrial web and perinuclear membrane of the cells. The possible use as a fluorescent probe for the detection of HSA was also evaluated for one of the dyes of the series, demonstrating a linear variation in the fluorescence intensity accompanied by the increase in the protein concentration.

Facile Synthesis of Highly Stretchable, Tough, and Photodegradable Hydrogels.

Recently, highly stretchable and tough hydrogels that are photodegradable on-demand have been reported. Unfortunately, the preparation procedure is complex due to the hydrophobic nature of the photocrosslinkers. Herein, a simple method is reported to prepare photodegradable double-network (DN) hydrogels that exhibit high stretchability, toughness, and biocompatibility. Hydrophilic ortho-nitrobenzyl (ONB) crosslinkers incorporating different poly(ethylene glycol) (PEG) backbones (600, 1000, and 2000 g mol-1 ) are synthesized. These photodegradable DN hydrogels are prepared by the irreversible crosslinking of chains by using such ONB crosslinkers, and the reversible ionic crosslinking between sodium alginate and divalent cations (Ca2+ ). Remarkable mechanical properties are obtained by combining ionic and covalent crosslinking and their synergistic effect, and by reducing the length of the PEG backbone. The rapid on-demand degradation of these hydrogels is also demonstrated by using cytocompatible light wavelength (λ = 365 nm) that degrades the photosensitive ONB units. The authors have successfully used these hydrogels as skin-worn sensors for monitoring human respiration and physical activities. A combination of excellent mechanical properties, facile fabrication, and on-demand degradation holds promise for their application as the next generation of substrates or active sensors eco-friendly for bioelectronics, biosensors, wearable computing, and stretchable electronics.

Iron Uptake Controls Trypanosoma cruzi Metabolic Shift and Cell Proliferation.

(1) Background: Ionic transport in Trypanosoma cruzi is the object of intense studies. T. cruzi expresses a Fe-reductase (TcFR) and a Fe transporter (TcIT). We investigated the effect of Fe depletion and Fe supplementation on different structures and functions of T. cruzi epimastigotes in culture. (2) Methods: We investigated growth and metacyclogenesis, variations of intracellular Fe, endocytosis of transferrin, hemoglobin, and albumin by cell cytometry, structural changes of organelles by transmission electron microscopy, O2 consumption by oximetry, mitochondrial membrane potential measuring JC-1 fluorescence at different wavelengths, intracellular ATP by bioluminescence, succinate-cytochrome c oxidoreductase following reduction of ferricytochrome c, production of H2O2 following oxidation of the Amplex® red probe, superoxide dismutase (SOD) activity following the reduction of nitroblue tetrazolium, expression of SOD, elements of the protein kinase A (PKA) signaling, TcFR and TcIT by quantitative PCR, PKA activity by luminescence, glyceraldehyde-3-phosphate dehydrogenase abundance and activity by Western blotting and NAD+ reduction, and glucokinase activity recording NADP+ reduction. (3) Results: Fe depletion increased oxidative stress, inhibited mitochondrial function and ATP formation, increased lipid accumulation in the reservosomes, and inhibited differentiation toward trypomastigotes, with the simultaneous metabolic shift from respiration to glycolysis. (4) Conclusion: The processes modulated for ionic Fe provide energy for the T. cruzi life cycle and the propagation of Chagas disease.

Functional characterization of maternally accumulated hydrolases in the mature oocytes of the vector Rhodnius prolixus reveals a new protein phosphatase essential for the activation of the yolk mobilization and embryo development.

Yolk biogenesis and consumption have been well conserved in oviparous animals throughout evolution. Most egg-laying animals store yolk proteins within the oocytes' yolk granules (Ygs). Following fertilization, the Ygs participate in controlled pathways of yolk breakdown to support the developing embryo's anabolic metabolism. While the unfolding of the yolk degradation program is a crucial process for successful development in many species, the molecular mechanisms responsible for yolk mobilization are still mysterious and have mostly not been explored. Here, we investigate the functional role of the oocyte maternally accumulated mRNAs of a protein phosphatase (PP501) and two aspartic proteases (cathepsin-D 405, CD405 and cathepsin-D 352, CD352) in the yolk degradation and reproduction of the insect vector of Chagas disease Rhodnius prolixus. We found that PP501 and CD352 are highly expressed in the vitellogenic ovary when compared to the other organs of the adult insect. Parental RNAi silencing of PP501 resulted in a drastic reduction in oviposition and increased embryo lethality whereas the silencing of CD352 resulted only in a slight decrease in oviposition and embryo viability. To further investigate the PP501-caused high reproduction impairment, we investigated the Ygs biogenesis during oocyte maturation and the activation of the yolk degradation program at early development. We found that the Ygs biogenesis was deficient during oogenesis, as seen by flow cytometry, and that, although the PP501-silenced unviable eggs were fertilized, the Ygs acidification and acid phosphatase activity were affected, culminating in a full impairment of the yolk proteins degradation at early embryogenesis. Altogether we found that PP501 is required for the oocyte maturation and the activation of the yolk degradation, being, therefore, essential for this vector reproduction.

Surface phosphatase in Rhinocladiella aquaspersa: biochemical properties and its involvement with adhesion.

Rhinocladiella aquaspersa is an etiologic agent of chromoblastomycosis, a subcutaneous chronic infectious disease. In the present work, we found that the three morphological forms of this fungus (conidia, mycelia and sclerotic bodies) expressed different levels of ecto-phosphatase activity. Our results demonstrated that surface conidial enzyme is an acid phosphatase, inhibited by sodium salts of molybdate, orthovanadate and fluoride and that the inhibition caused by orthovanadate and molybdate was irreversible. The conidial ecto-phosphatase efficiently released phosphate groups from different phosphorylated substrates, causing a higher rate of phosphate removal when p-nitrophenylphosphate was used as substrate. This ecto-enzyme of R. aquaspersa is modulated by Co(2 +) ions and inorganic phosphate (Pi). Accordingly, removal of Pi from the culture medium resulted in a marked (121-fold) increase of ecto-phosphatase activity. Surface phosphatase activity is apparently involved in fungal adhesive properties, since the attachment of R. aquaspersa to epithelial cells was reversed by the pre-treatment of the conidia with orthovanadate, molybdate and anti-phosphatase antibody. Corroborating this finding, conidia with greater ecto-phosphatase activity (grown in Pi-depleted medium) showed higher adherence to epithelial cells than fungi cultivated in the presence of Pi.

Gene identification and enzymatic properties of a membrane-bound trehalase from the ovary of Rhodnius prolixus.

Trehalose represents the main hemolymph sugar in most insects and its metabolic availability is regulated by trehalase. In this study, trehalase activity associated with the reproductive system was investigated in the insect Rhodnius prolixus, a hematophagous hemipteran vector of Chagas' disease. A single-copy gene that encodes a membrane-bound trehalase (RpTre-2) was identified in the genome of R. prolixus. RpTre-2 deduced amino acid sequence is closely related to other insect membrane-bound trehalases. The expression of this gene was detected in all analyzed organs, including ovary, where total trehalase enzymatic activity was determined, and was highest at day 7 after blood meal. Ovary membranes showed a major trehalase specific activity, which confirmed the presence of a membrane-bound trehalase in this insect. This trehalase activity seemed not to be regulated at transcriptional level, as the expression of RpTre-2 gene in the ovary did not change over the days after feeding. Similarly, ovarian follicles at different developmental stages did not show any variation in the transcription level of this gene. The RpTre-2 kinetic parameters were also investigated. Activity was highest at pH 5.5 and followed Michaelis-Menten kinetics, with an apparent K(m) = 1.42 ± 0.36 mM and Vmax = 167.90 ± 12.91 nmol/mg protein/h. These data reveal the presence of a membrane-bound trehalase in R. prolixus that is active in ovary and probably takes part in the insect carbohydrate metabolism associated with the reproductive process.