The in vivo treatment with the plant urease “Jack Bean Urease” impaired reproduction in females of Rhodnius prolixus (Hemiptera: Reduviidae)

Ureases are enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. In recent decades, it has been postulated that plant ureases are also defense proteins against phytophagous insect species with potential biotechnological application. Previous reports demonstrated that the injection of "Jack Bean Urease" (JBU), the major isoform of urease from the legume Canavalia ensiformis, into the hemocele of triatomine insects, resulted in several toxic effects including activation of the immune response. Although the insecticidal effect of JBU was described several years ago, many aspects of its mechanism of action as well as the target organs remains largely uncharacterized. In particular, the effects of JBU on the female reproductive system and the consequences of sublethal doses have not been studied.Para acceder a la videoconferencia completa, hacer clic en "Enlace externo".Sociedad Latinoamericana de Ecología de Vectore

DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima (Hemiptera: Reduviidae): Purification, bioinformatic analyses and the significance of its interaction with lipophorin in the internalization by developing oocytes

DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima, is synthesized by the fat body and the ovary and functions as yolk protein precursor. Functionally, DmCatD is involved in vitellin proteolysis. In this work, we purified and sequenced DmCatD, performed bioinformatic analyses and investigated the events involved in its targeting and storage in developing oocytes. By ion exchange and gel filtration chromatography, DmCatD was purified from egg homogenates and its identity was confirmed by mass spectrometry. Approximately 73% of the full-length transcript was sequenced. The phylogeny indicated that DmCatD has features which suggest its distancing from “classical” cathepsins D. Bioinformatic analyses using a chimeric construct were employed to predict post-translational modifications. Structural modeling showed that DmCatD exhibited the expected folding for this type of enzyme, and an active site with conserved architecture. The interaction between DmCatD and lipophorin in the hemolymph was demonstrated by co-immunoprecipitation. Colocalization of both proteins in developing oocyte membranes and yolk bodies was detected by immunofluorescence. Docking assays favoring the interaction DmCatD-lipophorin were carried out after modeling lipophorin of a related triatomine species. Our results suggest that lipophorin acts as a carrier for DmCatD to facilitate its further internalization by the oocytes. The mechanisms involved in the uptake of peptidases within the oocytes of insects have not been reported. This is the first experimental work supporting the interaction between cathepsin D and lipophorin in an insect species, enabling us to propose a pathway for its targeting and storage in developing oocytes.Fil: Leyria, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Fruttero, Leonardo Luis. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Pontificia Universidade Católica do Rio Grande do Sul; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Ligabue Braun, Rodrigo. Universidade Federal do Rio Grande do Sul; BrasilFil: Defferrari, Marina S.. University of Toronto; CanadáFil: Arrese, Estela L.. Oklahoma State University; Estados UnidosFil: Soulages, José L.. Oklahoma State University; Estados UnidosFil: Settembrini, Beatriz Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”; ArgentinaFil: Carlini, Célia Regina R S. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Canavoso, Lilian Etelvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentin

Design, Synthesis and Characterization of N-oxide-containing Heterocycles with In vivo Sterilizing Antitubercular Activity

Tuberculosis, caused by the Mycobacterium tuberculosis (Mtb), is the infectious disease responsible for the highest number of deaths worldwide. Herein, 22 new N-oxide- containing compounds were synthesized followed by in vitro and in vivo evaluation of their antitubercular potential against Mtb. Compound 8 was found to be the most promising compound, with MIC90 values of 1.10 and 6.62 μM against active and non- replicating Mtb, respectively. Additionally, we carried out in vivo experiments to confirm the safety and efficacy of compound 8; the compound was found to be orally bioavailable and highly effective leading to the reduction of the number of Mtb to undetected levels in a mouse model of infection. Microarray-based initial studies on the mechanism of action suggest that compound 8 blocks the process of translation. Altogether, these results indicated benzofuroxan derivative 8 to be a promising lead compound for the development of a novel chemical class of antitubercular drugs

Protein expression and activity levels of DmCatD in the hemolymph of <i>D</i>. <i>maxima</i>.

<p><b>(A)</b>, western blot was performed to evaluate the expression of pro-DmCatD and DmCatD. The hemolymph was obtained at pre-vitellogenesis, vitellogenesis, early and late follicular atresia (lanes 2–5 respectively, 40 μg each). In lane 1, 0.2 μg of whole cell lysate of MCF7 was used as a positive control. The arrow indicates pro-DmCatD (~43 kDa). The western blot shown was a representative experiment of three independent assays. <b>(B)</b>, specific activity of DmCatD in the hemolymph. Assays were performed as stated in Materials and Methods. Results expressed as relative units of fluorescence (RUF)/μg protein/min are the mean ± SEM (n = 3). *<i>P</i><0.05 vs. pre-vitellogenesis, early and late atresia.</p

Interaction of DmCatD and tyrosine phosphatase with vitellin in yolk bodies of vitellogenic oocytes.

<p>Vitellogenic ovaries were processed for immunofluorescence as stated in Materials and Methods. Images at the left of the panels <b>(A)</b> and <b>(B)</b> show the Z-projections calculated from maximum intensity signals derived from all focal planes of the image stacks for vitellin (Vt, green) and DmCatD or tyrosine phosphatase (both red). Donor images (Vt signal) pre- and post-photobleaching of the corresponding acceptors, DmCatD and tyrosine phosphatase, show FRET. The white boxes indicate the pre- and post-bleaching areas. Each of the arrows point out the increase in the fluorescence intensity of Vt. A gray scale image pseudocolored with the color gradient is shown within each image to enhance the visualization of the increase in the fluorescence intenstity. Similar results were obtained in 3 separate experiments. Bars: 5 μm for image on the left panel (B) and 2 μm for the other pictures.</p

Activity of acid phosphatase in the ovarian tissue of <i>D</i>. <i>maxima</i>.

<p><b>(A)</b>, the enzymatic assays were performed using 30 μg of ovarian homogenates from females at representative days of the reproductive cycle and measuring the amount of <i>p</i>-nitrophenol (<i>p</i>NP). The results are expressed as specific activity (nmol of <i>p</i>NP/mg protein/min) and each point represents the mean ± SEM (n = 3). *<i>P</i><0.05 vs. pre-vitellogenesis, vitellogenesis and early atresia; **<i>P</i><0.001 vs. pre-vitellogenesis and vitellogenesis. <b>(B),</b> the effect on acid phosphatase activity of several inhibitors was analyzed by pre-incubating protein homogenates of ovaries at early atresia with Na₃VO₄ (inhibitor of tyrosine phosphatase), NaF (inhibitor of serine/threonine phosphatase) or Na⁺/K⁺ tartrate (a broad spectrum phosphatase inhibitor). *<i>P</i><0.05 vs. Na₃VO₄ and Na⁺/K⁺ tartrate; **<i>P</i><0.001 vs. Na₃VO₄, NaF and Na⁺/K⁺ tartrate. Specific activity of tyrosine phosphatase <b>(C)</b> and serine/threonine phosphatase <b>(D)</b> in ovarian homogenates from females at early and late atresia. The assays were performed using specific phosphopeptides as substrates either in the presence or in the absence of NaF, Na₃VO₄ or Na⁺/K⁺ tartrate. The specific activities are expressed as pmol phosphate/μg protein/min. Each point represents the mean ± SEM (n = 3). (C), activity in early atresia: **<i>P</i><0.001 vs. Na₃VO₄ and Na⁺/K⁺ tartrate; activity in late atresia: *<i>P</i><0.05 vs. Na₃VO₄ and Na⁺/K⁺ tartrate. (D), activity in early atresia: *<i>P</i><0.01 vs. NaF and Na⁺/K⁺ tartrate; activity in late atresia: *<i>P</i><0.01 vs. NaF and Na⁺/K⁺ tartrate; **<i>P</i><0.001 vs. NaF and Na⁺/K⁺ tartrate.</p

Relative DmCatD mRNA quantification evaluated by RT-qPCR.

<p>Total RNA was extracted from fat bodies <b>(A)</b> and ovaries <b>(B)</b> of females at different stages of the reproductive cycle. Quantification of 18S ribosomal RNA was used as internal control. Results expressed as mean ± SEM indicate the fold of change respect to the mRNA levels at pre-vitellogenesis (n = 3). (A), *<i>P</i><0.01 vs. early and late atresia. (B), *<i>P</i><0.05 vs. pre-vitellogenesis and early atresia; **<i>P</i><0.001 vs. pre-vitellogenesis, early and late atresia.</p

Co-localization of DmCatD and tyrosine phosphatase with vitellin in yolk bodies of vitellogenic oocytes.

<p>Vitellogenic ovaries were processed for immunofluorescence as stated in Materials and Methods. <b>(A-B),</b> the vitellin (Vt) signal is displayed in green and the enzyme signal (DmCatD or tyrosine phosphatase), in red. In merged images, the partial co-localization of Vt/DmCatD (A) and Vt/tyrosine phosphatase (B) is indicated with arrowheads. Similar results were obtained in three separate experiments. Bars: 2 μm.</p

pH dependence and effect of DmCatD and acid phosphatase inhibitors on vitellin (Vt) proteolysis.

<p>Homogenates from atretic ovaries were used as the enzyme source and purified Vt as substrate. <b>(A),</b> left panel, purified Vt and homogenates of ovarian tissues without incubation (lane 1 and 2, respectively). Right panel, <i>in vitro</i> pH dependence of Vt proteolysis. The reaction was carried out for 12 h at 37°C in different reaction buffers, according to the pH evaluated (3.0 to 7.0). In all cases, Vt degradation was evidenced by western blot using an anti-Vt antibody, after fractionation of proteins by 7.5% SDS-PAGE. <b>(B),</b> Vt and ovarian homogenates were incubated for 12 h at 37°C in a reaction medium at pH 4.0 either in the absence (lane 3) or in the presence of pesptatin A (inhibitor of aspartic peptidases, lane 4), Na₃VO₄ (inhibitor of tyrosine phosphatases, lane 5) or Na⁺/K⁺ tartrate (a broad spectrum phosphatase inhibitor, lane 6). Purified Vt and ovarian homogenate (lanes 1–2, respectively) were incubated in the same conditions and showed as controls. The arrows in the panels indicate the main subunits of purified Vt (Mr ~170 kDa and 174 kDa) visualized as a single immunoreactive band.</p