Effect of the urease-derived peptide Jaburetox on the central nervous system of Triatoma infestans (Insecta: Heteroptera)

Background

Triatoma infestans is the main vector of Chagas'disease in Southern Cone countries. In triatomines, symptoms suggesting neurotoxicity were observed after treatment with Jaburetox (Jbtx), the entomotoxic peptide obtained from jackbean urease. Here, we study its effect in the central nervous system (CNS) of this species.

Methods

Immunohistochemistry, Western blots, immunoprecipitation, two-dimensional electrophoresis, tandem mass spectrometry and enzymatic assays were performed.

Results

Anti-Jbtx antibody labeled somata of the antennal lobe only in Jbtx-treated insects. Western blot assays of nervous tissue using the same antibody reacted with a 61 kDa protein band only in peptide-injected insects. Combination of immunoprecipitation, two-dimensional electrophoresis and tandem mass spectrometry identified UDP-N-acetylglucosamine pyrophosphorylase (UDP-GlcNAcP) as a molecular target for Jbtx. The activity of UDP-GlcNAcP increased significantly in the CNS of Jbtx-treated insects. The effect of Jbtx on the activity of nitric oxide synthase (NOS) and NO production was investigated as NO is a recognized messenger molecule in the CNS of T. infestans. NOS activity and NO levels decreased significantly in CNS homogenates of Jbtx-treated insects.

Conclusions

UDP-GlcNAcP is a molecular target of Jbtx. Jbtx impaired the activity of T. infestans nitrergic system, which may be related with early behavioral effects.

General Significance

We report that the CNS of Triatoma infestans is a target for the entomotoxic peptide and propose that a specific area of the brain is involved. Besides potentially providing tools for control strategies of Chagas' disease vectors our data may be relevant in various fields of research as insect physiology, neurobiology and protein function.     

Jack bean urease alters serotonin-induced effects on Rhodnius prolixus anterior midgut

Urease isoforms from jack bean seeds are toxic to insects, and this entomotoxic effect is mostly due to the release of a peptide by insect digestive enzymes. We previously demonstrated that jack bean urease (JBU) has antidiuretic effects on Rhodnius prolixus Malpighian tubules, decreasing the serotonin-stimulated secretion of fluid. Now, we evaluate the toxicity of the intact JBU and its effect on R. prolixus anterior midgut, to further elucidate the mechanism of action of JBU in insects. JBU decreases the serotonin-induced fluid transport by the anterior midgut in vitro when injected into the lumen. A decrease in the levels of cAMP is observed in tissues treated with JBU (in the presence of serotonin). JBU also causes a dose-dependent increase in the frequency of serotonin-induced contractions in the anterior midgut, but does not alter the frequency of spontaneous contractions. The cyclooxygenase inhibitor indomethacin and the prostaglandin antagonist AH6809 block JBU's potentiation of serotonin-induced contractions, indicating that prostaglandins might act as second messengers for JBU action. Prostaglandin E₂ (PGE₂) increases the frequency of serotonin-induced contractions, again supporting the role of prostaglandins as second messengers for JBU action. JBU and PGE₂ increase cGMP levels in the anterior midgut, indicating that this molecule might also be part of the JBU pathway.     

Biochemical and pharmacological properties of a new thrombin-like serine protease (Russelobin) from the venom of Russell's Viper (Daboia russelii russelii) and assessment of its therapeutic potential

Background

Snake venoms are rich sources of bioactive molecules, and several venom-derived proteins have entered clinical trials for use in ischemic disorders; however, late-stage failure of a recent drug candidate due to low in vivo efficacy demonstrated the need for new sources of fibrinogenolytic drug candidates.

Methods

A 51.3 kDa thrombin-like serine protease (Russelobin) purified from the venom of Russell's Viper (Daboia russelii russelii) was subjected to extensive biochemical characterization, including N-terminal sequencing, substrate specificity, kinetic and inhibitor assays, glycosylation analysis and stability assays. Toxicity and pathology analyses were conducted in NSA mice.

Results

Russelobin has extensive N-terminus identity with a beta-fibrinogenase-like serine proteinase precursor from Daboia russelii siamensis venom, a mass of 51.3 kDa and contains extensive N-linked oligosaccharides. Serine protease inhibitors and heparin significantly decreased activity, with much lower inhibition by DTT, antithrombin-III and α₂-macroglobulin. Russelobin preferentially released FPA and slowly released FPB from human fibrinogen, forming a labile fibrin clot readily hydrolyzed by plasmin. The partially deglycosylated enzyme showed significantly lower activity toward fibrinogen and less resistance against neutralization by plasma α₂MG and antithrombin-III. Russelobin was non-cytotoxic, non-lethal and produced no histopathologies in mice, and it demonstrated in vivo dose-dependent defibrinogenating activity.

Conclusions

Russelobin is an A/B fibrinogenase with high specificity toward fibrinogen, both in vitro and in vivo. Extensive glycosylation appears to protect the molecule against endogenous protease inhibitors, prolonging its in vivo efficacy.

General significance

Due to its low toxicity, stability and activity as a defibrinogenating agent, Russelobin shows high potential for cardiovascular drug development.     

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies

Background

Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC₄ (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC).

Methods

We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC₄ and LTD₄ by competing their binding to their receptors.

Results

mAbLTC and scFvLTC inhibited their binding of LTC₄ or LTD₄ to CysLT₁ receptor (CysLT₁R) and CysLT₂ receptor (CysLT₂R) overexpressed in Chinese hamster ovary cells. The induction by LTD₄ of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT₁R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC₄- and LTD₄-induced aggregation of mouse platelets expressing CysLT₂R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT₂R antagonists but not to CysLT₁R antagonists.

Conclusions

These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT₁R and CysLT₂R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors.

General significance

mAbLTC can be used in the treatment of inflammatory diseases such as asthma.     

Molecular genetics reveal that silvatic Rhodnius prolixus do colonise rural houses

Background

Rhodnius prolixus is the main vector of Chagas disease in Venezuela. Here, domestic infestations of poor quality rural housing have persisted despite four decades of vector control. This is in contrast to the Southern Cone region of South America, where the main vector, Triatoma infestans, has been eliminated over large areas. The repeated colonisation of houses by silvatic populations of R. prolixus potentially explains the control difficulties. However, controversy surrounds the existence of silvatic R. prolixus: it has been suggested that all silvatic populations are in fact Rhodnius robustus, a related species of minor epidemiological importance. Here we investigate, by direct sequencing (mtcytb, D2) and by microsatellite analysis, 1) the identity of silvatic Rhodnius and 2) whether silvatic populations of Rhodnius are isolated from domestic populations.

Methods and Findings

Direct sequencing confirmed the presence of R. prolixus in palms and that silvatic bugs can colonise houses, with house and palm specimens sharing seven cytb haplotypes. Additionally, mitochondrial introgression was detected between R. robustus and R. prolixus, indicating a previous hybridisation event. The use of ten polymorphic microsatellite loci revealed a lack of genetic structure between silvatic and domestic ecotopes (non-significant F_ST values), which is indicative of unrestricted gene flow.

Conclusions

Our analyses demonstrate that silvatic R. prolixus presents an unquestionable threat to the control of Chagas disease in Venezuela. The design of improved control strategies is essential for successful long term control and could include modified spraying and surveillance practices, together with housing improvements.     

Displacement of disordered water molecules from hydrophobic pocket creates enthalpic signature: Binding of phosphonamidate to the S1'-pocket of thermolysin

Background

Prerequisite for the design of tight binding protein inhibitors and prediction of their properties is an in-depth understanding of the structural and thermodynamic details of the binding process. A series of closely related phosphonamidates was studied to elucidate the forces underlying their binding affinity to thermolysin. The investigated inhibitors are identical except for the parts penetrating into the hydrophobic S₁'-pocket.

Methods

A correlation of structural, kinetic and thermodynamic data was carried out by X-ray crystallography, kinetic inhibition assay and isothermal titration calorimetry.

Results and conclusions

Binding affinity increases with larger ligand hydrophobic P₁'-moieties accommodating the S₁'-pocket. Surprisingly, larger P₁'-side chain modifications are accompanied by an increase in the enthalpic contribution to binding. In agreement with other studies, it is suggested that the release of largely disordered waters from an imperfectly hydrated pocket results in an enthalpically favourable integration of these water molecules into bulk water upon inhibitor binding. This enthalpically favourable process contributes more strongly to the binding energetics than the entropy increase resulting from the release of water molecules from the S₁'-pocket or the formation of apolar interactions between protein and inhibitor.

General significance

Displacement of highly disordered water molecules from a rather imperfectly hydrated and hydrophobic specificity pocket can reveal an enthalpic signature of inhibitor binding.     

LRRK2, but not pathogenic mutants,protects against H2O2 stress depending on mitochondrial function and endocytosis in a yeast model

Background

Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). Studies in the yeast Saccharomyces cerevisiae have provided valuable insights into the mechanisms of cellular dysfunction associated with the expression of faulty PD genes.

Methods

We developed a yeast model for full-length LRRK2 studies. We expressed wild-type (wt) LRRK2 and mutations and evaluated their role during oxidative stress conditions. The involvement of mitochondria was assessed by using rho-zero mutants and by evaluating reactive oxygen species (ROS) production and mitochondrial membrane potential by flow cytometry. The involvement of endocytosis was also studied by testing several endocytic mutants and by following the vacuolar delivery of the probe FM4-64.

Results

Expression of LRRK2 in yeast was associated to increased hydrogen peroxide resistance. This phenotype, which was dependent on mitochondrial function, was not observed for PD-mutants G2019S and R1441C or in the absence of the kinase activity and the WD40 repeat domain. Expression of the pathogenic mutants stimulated ROS production and increased mitochondrial membrane potential. For the PD-mutants, but not for wild-type LRRK2, endocytic defects were also observed. Additionally, several endocytic proteins were required for LRRK2-mediated protection against hydrogen peroxide.

Conclusions

Our results indicate that LRRK2 confers cellular protection during oxidative stress depending on mitochondrial function and endocytosis.

General significance

Both the loss of capacity of LRRK2 pathogenic mutants to protect against oxidative stress and their enhancement of dysfunction may be important for the development of PD during the aging process.     

In silico and in vitro characterization of anti-amyloidogenic activity of vitamin K3 analogues for Alzheimer's disease

Background

Aggregation of amyloid-beta (Aβ) has been proposed as the main cause of Alzheimer's disease (AD). Vitamin K deficiency has been linked to the pathogenesis of AD. Therefore, 15 synthesized vitamin K3 (VK3) analogues were studied for their anti-amyloidogenic activity.

Methods

Biological and spectroscopic assays were used to characterize the effect of VK3 analogues on amyloidogenic properties of Aβ, such as aggregation, free radical formation, and cell viability. Molecular dynamics simulation was used to calculate the binding affinity and mode of VK3 analogue binding to Aβ.

Results

Both numerical and experimental results showed that several VK3 analogues, including VK3-6, VK3-8, VK3-9, VK3-10, and VK3-224 could effectively inhibit Aβ aggregation and conformational conversion. The calculated inhibition constants were in the μM range for VK3-10, VK3-6, and VK3-9 which was similar to the IC₅₀ of curcumin. Cell viability assays indicated that VK3-9 could effectively reduce free radicals and had a protective effect on cytotoxicity induced by Aβ.

Conclusions

The results clearly demonstrated that VK3 analogues could effectively inhibit Aβ aggregation and protect cells against Aβ induced toxicity. Modified VK3 analogues can possibly be developed as effective anti-amyloidogenic drugs for the treatment of AD.

General significance

VK3 analogues effectively inhibit Aβ aggregation and are highly potent as anti-amyloidogenic drugs for therapeutic treatment of AD.     

Moderate concentrations of 4-O-methylhonokiol potentiate GABAA receptor currents stronger than honokiol

Background

Magnolia bark preparations from Magnolia officinalis of Asian medicinal systems are known for their muscle relaxant effect and anticonvulsant activity. These CNS related effects are ascribed to the presence of the biphenyl-type neolignans honokiol and magnolol that exert a potentiating effect on GABA_A receptors. 4-O-methylhonokiol isolated from seeds of the North-American M. grandiflora was compared to honokiol for its activity to potentiate GABA_A receptors and its GABA_A receptor subtype-specificity was established.

Methods

Different recombinant GABA_A receptors were functionally expressed in Xenopus oocytes and electrophysiological techniques were used determine to their modulation by 4-O-methylhonokiol.

Results

3 μM 4-O-methylhonokiol is shown here to potentiate responses of the α₁β₂γ₂ GABA_A receptor about 20-fold stronger than the same concentration of honokiol. In the present study potentiation by 4-O-methylhonokiol is also detailed for 12 GABA_A receptor subtypes to assess GABA_A receptor subunits that are responsible for the potentiating effect.

Conclusion

The much higher potentiation of GABA_A receptors at identical concentrations of 4-O-methylhonokiol as compared to honokiol parallels previous observations made in other systems of potentiated pharmacological activity of 4-O-methylhonokiol over honokiol.

General significance

The results point to the use of 4-O-methylhonokiol as a lead for GABA_A receptor potentiation and corroborate the use of M. grandiflora seeds against convulsions in Mexican folk medicine.     

Antibacterial performance of nanoscaled visible-light responsive platinum-containing titania photocatalyst in vitro and in vivo

Background

Traditional antibacterial photocatalysts are primarily induced by ultraviolet light to elicit antibacterial reactive oxygen species. New generation visible-light responsive photocatalysts were discovered, offering greater opportunity to use photocatalysts as disinfectants in our living environment. Recently, we found that visible-light responsive platinum-containing titania (TiO₂–Pt) exerted high performance antibacterial property against soil-borne pathogens even in soil highly contaminated water. However, its physical and photocatalytic properties, and the application in vivo have not been well-characterized.

Methods

Transmission electron microscopy, energy dispersive spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction, ultraviolet–visible absorption spectrum and the removal rate of nitrogen oxides were therefore analyzed. The antibacterial performance under in vitro and in vivo conditions was evaluated.

Results

The apparent quantum efficiency for visible light illuminated TiO₂–Pt is relatively higher than several other titania photocatalysts. The killing effect achieved approximately 2 log reductions of pathogenic bacteria in vitro. Illumination of injected TiO₂–Pt successfully ameliorated the subcutaneous infection in mice.

Conclusions

This is the first demonstration of in vivo antibacterial use of TiO₂–Pt nanoparticles. When compared to nanoparticles of some other visible-light responsive photocatalysts, TiO₂–Pt nanoparticles induced less adverse effects such as exacerbated platelet clearance and hepatic cytotoxicity in vivo.

General significance

These findings suggest that the TiO₂–Pt may have potential application on the development of an antibacterial material in both in vitro and in vivo settings.     

Internal charge transfer in cytochrome c oxidase at a limited proton supply: Proton pumping ceases at high pH

Background

In the membrane-bound enzyme cytochrome c oxidase, electron transfer from cytochrome c to O₂ is linked to proton uptake from solution to form H₂O, resulting in a charge separation across the membrane. In addition, the reaction drives pumping of protons across the membrane.

Methods

In this study we have measured voltage changes as a function of pH during reaction of the four-electron reduced cytochrome c oxidase from Rhodobacter sphaeroides with O₂. These electrogenic events were measured across membranes containing purified enzyme reconstituted into lipid vesicles.

Results

The results show that the pH dependence of voltage changes (primarily associated with proton transfer) during O₂ reduction does not match that of the previously studied absorbance changes (primarily associated with electron transfer). Furthermore, the voltage changes decrease with increasing pH.

Conclusions

The data indicate that cytochrome c oxidase does not pump protons at high pH (10.5) (or protons are taken from the “wrong” side of the membrane) and that at this pH the net proton-uptake stoichiometry is ∼ 1/2 of that at pH 8. Furthermore, the results provide a basis for interpretation of results from studies of mutant forms of the enzyme.

General significance

These results provide new insights into the function of cytochrome c oxidase.     

Molecular targeting of Akt by thymoquinone promotes G1 arrest through translation inhibition of cyclin D1 and induces apoptosis in breast cancer cells

Aim

Thymoquinone (TQ), the predominant bioactive constituent of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against multifarious tumors. However, the meticulous mechanism of TQ on Akt mediated survival pathway is still unrevealed in breast cancer. Here, we investigated TQ's mechanism of action against PI3K/Akt signaling and its downstream targets by modulating proteins translational machinery, leading to apoptosis in cancer cells.

Main methods

MDA-MB-468 and T-47D cells were treated with TQ and evaluated for its anticancer activity through phase distribution and western blot. Modulatory effects of TQ on Akt were affirmed through kinase and drug potential studies.

Key findings

Studies revealed G₁ phase arrest till 24 h incubation with TQ while extended exposure showed phase shift to subG₁ indicating apoptosis, supported by suppression of cyclin D1, cyclin E and cyclin dependent kinase inhibitor p27 expression. Immunoblot and membrane potential studies revealed mitochondrial impairment behind apoptotic process with upregulation of Bax, cytoplasmic cytochrome c and procaspase-3, PARP cleavage along with Bcl-2, Bcl-xL and survivin downregulation. Moreover, we construed the rationale behind mitochondrial dysfunction by examining the phosphorylation status of PDK1, PTEN, Akt, c-raf, GSK-3β and Bad in TQ treated cells, thus ratifying the involvement of Akt in apoptosis. Further, the consequential effect of Akt inhibition by TQ is proven by translational repression through deregulated phosphorylation of 4E-BP1, eIF4E, S6R and p70S6K.

Significance

Our observations for the first time may provide a new insight for the development of novel therapies for Akt overexpressed breast cancer by TQ.     

Synthesizing and staining manganese oxide nanoparticles for cytotoxicity and cellular uptake investigation

Background

For decades, contrast agents have been used to reduce longitudinal (T₁) or transverse (T₂) relaxation times. High toxicity of gadolinium-based contrast agents leads researchers to new T₁ contrast agents. Manganese oxide (MnO) nanoparticle (NP) with the lower peril and good enough signal change ability has been offered as a new possibility for magnetic resonance imaging (MRI).

Methods

The synthesized NPs were investigated for physicochemical and biological properties by X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscope, dynamic light scattering (DLS), inductively coupled plasma, enzyme-linked immunosorbent assay, and 3 T magnetic resonance imaging.

Results

Due to physical contact importance of T₁ contrast agents with tissues' protons, extremely thin layer of the surfactant, less than 2 nm, was coated on NPs for aqueous stabilizing. The hydrophilic gentisic acid with low Dalton, around 154, did that role truly. Moreover, decreasing NP size to 5 nm which increases available surface for the proton relaxation is another important parameter to reach an appropriate longitudinal relaxation rate. The NPs didn't reveal any side effects on the cells, and cellular uptake was considerable.

Conclusions

The synthesized NPs represented a promising result in comparison to clinical gadolinium chelates, due to higher r₁ relaxivity and lower toxicity.

General significance

In addition to considerable signal change and cellular uptake, Prussian blue was tried on MnO NPs for the initial time, which can be observed within cells by pale blue color.     

Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli

Background

F₁F_O ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli.

Methods

We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy.

Results

We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F₁F_O ATP synthases.

Conclusions

Our system now allows performing previously impossible structural and functional studies on A. aeolicus F₁F_O ATP synthase.

General significance

More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.     

Crystal structure of a Bombyx mori sigma-class glutathione transferase exhibiting prostaglandin E synthase activity

Background

Glutathione transferases (GSTs) are members of a major family of detoxification enzymes. Here, we report the crystal structure of a sigma-class GST of Bombyx mori, bmGSTS1, to gain insight into the mechanism catalysis.

Methods

The structure of bmGSTS1 and its complex with glutathione were determined at resolutions of 1.9 Å and 1.7 Å by synchrotron radiation and the molecular replacement method.

Results

The three-dimensional structure of bmGSTS1 shows that it exists as a dimer and is similar in structure to other GSTs with respect to its secondary and tertiary structures. Although striking similarities to the structure of prostaglandin D synthase were also detected, we were surprised to find that bmGSTS1 can convert prostaglandin H₂ into its E₂ form. Comparison of bmGSTS1 with its glutathione complex showed that bound glutathione was localized to the glutathione-binding site (G-site). Site-directed mutagenesis of bmGSTS1 mutants indicated that amino acid residues Tyr8, Leu14, Trp39, Lys43, Gln50, Met51, Gln63, and Ser64 in the G-site contribute to catalytic activity.

Conclusion

We determined the tertiary structure of bmGSTS1 exhibiting prostaglandin E synthase activity.

General significance

These results are, to our knowledge, the first report of a prostaglandin synthase activity in insects.     

Structural basis of phospholipase A2-like myotoxin inhibition by chicoric acid,a novel potent inhibitor of ophidian toxins

Background

Specific compounds found in vegetal species have been demonstrated to be efficient inhibitors of snake toxins, such as phospholipase A₂-like (PLA₂-like) proteins. These particular proteins, present in several species of vipers (Viperidae), induce a severe local myotoxic effect in prey and human victims, and this effect is often not efficiently neutralized by the regular serum therapy. PLA₂-like proteins have been functionally and structurally studied since the early 1990s; however, a comprehensive molecular mechanism was proposed only recently.