Synthetic peptides derived from the C-terminal 6 kDa region of Plasmodium falciparum SERA5 inhibit the enzyme activity and malaria parasite development

Background

Plasmodium falciparum serine repeat antigen 5 (PfSERA5) is an abundant blood stage protein that plays an essential role in merozoite egress and invasion. The native protein undergoes extensive proteolytic cleavage that appears to be tightly regulated. PfSERA5 N-terminal fragment is being developed as vaccine candidate antigen. Although PfSERA5 belongs to papain-like cysteine protease family, its catalytic domain has a serine in place of cysteine at the active site.

Methods

In the present study, we synthesized a number of peptides from the N- and C-terminal regions of PfSERA5 active domain and evaluated their inhibitory potential.

Results

The final proteolytic step of PfSERA5 involves removal of a C-terminal ~ 6 kDa fragment that results in the generation of a catalytically active ~ 50 kDa enzyme. In the present study, we demonstrate that two of the peptides derived from the C-terminal ~ 6 kDa region inhibit the parasite growth and also cause a delay in the parasite development. These peptides reduced the enzyme activity of the recombinant protein and co-localized with the PfSERA5 protein within the parasite, thereby indicating the specific inhibition of PfSERA5 activity. Molecular docking studies revealed that the inhibitory peptides interact with the active site of the protein. Interestingly, the peptides did not have an effect on the processing of PfSERA5.

Conclusions

Our observations indicate the temporal regulation of the final proteolytic cleavage step that occurs just prior to egress.

General significance

These results reinforce the role of PfSERA5 for the intra-erythrocytic development of malaria parasite and show the role of carboxy terminal ~ 6 kDa fragments in the regulation of PfSERA5 activity. The results also suggest that final cleavage step of PfSERA5 can be targeted for the development of new anti-malarials.     

Plasmodium falciparum antioxidant protein as a model enzyme for a special class of glutaredoxin/glutathione-dependent peroxiredoxins

Background

Peroxiredoxins are important heterogeneous thiol-dependent hydroperoxidases with a variety of isoforms and enzymatic mechanisms. A special subclass of glutaredoxin/glutathione-dependent peroxiredoxins has been discovered in bacteria and eukaryotes during the last decade, but the exact enzymatic mechanisms of these enzymes remain to be unraveled.

Methods

We performed a comprehensive analysis of the enzyme kinetics and redox states of one of these glutaredoxin/glutathione-dependent peroxiredoxins, the antioxidant protein from the malaria parasite Plasmodium falciparum, using steady-state kinetic measurements, site-directed mutagenesis, redox mobility shift assays, gel filtration, and mass spectrometry.

Results

P. falciparum antioxidant protein requires not only glutaredoxin but also glutathione as a true substrate for the reduction of hydroperoxides. One peroxiredoxin cysteine residue and one glutaredoxin cysteine residue are sufficient for catalysis, however, additional cysteine residues of both proteins result in alternative redox states and conformations in vitro with implications for redox regulation. Our data furthermore point to a glutathione-dependent peroxiredoxin activation and a negative subunit cooperativity.

Conclusions

The investigated glutaredoxin/glutathione/peroxiredoxin system provides numerous new insights into the mechanism and redox regulation of peroxiredoxins.

General significance

As a member of the special subclass of glutaredoxin/glutathione-dependent peroxiredoxins, the P. falciparum antioxidant protein could become a reference protein for peroxiredoxin catalysis and regulation.     

Inhibition by polyamines of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid

Background

Viroids are the smallest pathogens known to date. They infect plants and cause considerable economic losses. The members of the Avsunviroidae family are known for their capability to form hammerhead ribozymes (HHR) that catalyze self-cleavage during their rolling circle replication.

Methods

In vitro inhibition assays, based on the self-cleavage kinetics of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid (CChMVd-HHR) were performed in the presence of various putative inhibitors.

Results

Aminated compounds appear to be inhibitors of the self-cleavage activity of the CChMVd HHR. Surprisingly the spermine, a known activator of the autocatalytic activity of another hammerhead ribozyme in the presence or absence of divalent cations, is a potent inhibitor of the CChMVd-HHR with K_i of 17 ± 5 μM. Ruthenium hexamine and TMPyP4 are also efficient inhibitors with K_i of 32 ± 5 μM and IC₅₀ of 177 ± 5 nM, respectively.

Conclusions

This study shows that polyamines are inhibitors of the CChMVd-HHR self-cleavage activity, with an efficiency that increases with the number of their amino groups.

General significance

This fundamental investigation is of interest in understanding the catalytic activity of HHR as it is now known that HHR are present in the three domains of life including in the human genome. In addition these results emphasize again the remarkable plasticity and adaptability of ribozymes, a property which might have played a role in the early developments of life and must be also of significance nowadays for the multiple functions played by non-coding RNAs.     

Turning up the heat: heat stress induces markers of programmed cell death in Plasmodium falciparum in vitro

Malaria is characterised by cyclical febrile episodes that result from the rupture of mature schizont-infected erythrocytes releasing merozoites. In patients infected with Plasmodium falciparum, fever may reach peak temperatures as high as 41 °C. Febrile episodes typically have a deleterious effect on parasites and probably benefit the host by aiding parasite clearance; however, the parasite may also gain advantage from limiting its burden on the host and prolonging infection to ensure development and transmission of slow-maturing gametocytes. Programmed cell death (PCD) may provide the parasite with a mechanism of self-limitation, although the occurrence and phenotype of PCD in the erythrocytic stages remain controversial due to conflicting data. This study aimed to characterise the cell death phenotype of P. falciparum in response to in vitro heat stress. A variety of biochemical markers of PCD, including DNA fragmentation, mitochondrial dysregulation and phosphatidylserine externalisation, as well as morphological studies of Giemsa-stained thin smears and real-time microscopy were utilised to characterise the phenotype. Heat stress decreased P. falciparum growth and development in vitro. Late-stage parasites were more susceptible, although early stages were more affected than expected. Early-stage parasites exposed to 41 °C exhibited markers of an apoptosis-like PCD phenotype, including DNA fragmentation and mitochondrial depolarisation. Heat-stressed late-stage parasites showed no significant DNA fragmentation or mitochondrial dysregulation; however, cytoplasmic vacuolisation was suggestive of an autophagy-like form of PCD. Our results therefore showed that biochemical and morphological markers of PCD varied with intra-erythrocytic parasite development and that P. falciparum exhibited facets of both apoptosis- and autophagy-like phenotypes after exposure to febrile temperatures, which may reflect a unique PCD phenotype.     

Interaction of fosfomycin with the Glycerol 3-phosphate Transporter of Escherichia coli

Background

Fosfomycin is widely used to treat urinary tract and pediatric gastrointestinal infections of bacteria. It is supposed that this antibiotic enters cells via two transport systems, including the bacterial Glycerol-3-phosphate Transporter (GlpT). Impaired function of GlpT is one mechanism for fosfomycin resistance.

Methods

The interaction of fosfomycin with the recombinant and purified GlpT of Escherichia coli reconstituted in liposomes has been studied. IC₅₀ and the half-saturation constant of the transporter for external fosfomycin (K_i) were determined by transport assay of [¹⁴C]glycerol-3-phosphate catalyzed by recombinant GlpT. Efficacy of fosfomycin on growth rates of GlpT defective bacteria strains transformed with recombinant GlpT was measured.

Results

Fosfomycin, externally added to the proteoliposomes, poorly inhibited the glycerol-3-phosphate/glycerol-3-phosphate antiport catalyzed by the reconstituted transporter with an IC₅₀ of 6.4 mM. A kinetic analysis revealed that the inhibition was completely competitive, that is, fosfomycin interacted with the substrate-binding site and the K_i measured was 1.65 mM. Transport assays performed with proteoliposomes containing internal fosfomycin indicate that it was not very well transported by GlpT. Complementation study, performed with GlpT defective bacteria strains, indicated that the fosfomycin resistance, beside deficiency in antibiotic transporter, could be due to other gene defects.

Conclusions

The poor transport observed in a reconstituted system together with the high value of K_i and the results of complementation study well explain the usual high dosage of this drug for the treatment of the urinary tract infections.

General significance

This is the first report regarding functional analysis of interaction between fosfomycin and GlpT.     

Mobility of the conserved glycine 155 is required for formation of the active plasmodial Pdx1 dodecamer

Background

Vitamin B6 synthesis requires a functional Pdx1 assembly that is dodecameric in vivo. We have previously shown that mutation of a catalytic lysine in the plasmodial Pdx1 protein results in a protein that is both inactive and hexameric in vitro.

Methods

Static and dynamic light scattering, circular dichroism, co-purification and enzyme assays are used to investigate the role of a glycine conserved in all Pdx1 family members.

Results

Static light scattering indicates that a glycine to alanine mutant is present as a hexamer in vitro. Subsequent circular dichroism experiments demonstrate that a significant change in secondary structure content is induced by this mutation. However, this mutant is still competent to bind and support Pdx2 activity.

Conclusions

As the mutated glycine occupies an unrestricted region of the Ramachandran plot the additional stereo-chemical restrictions imposed on alanine residues strongly support our hypothesis that significant structural rearrangement of Pdx1 is required during the transition from hexamer to dodecamer.

General significance

The presented results demonstrate that reduction in the mobility of this region in Pdx1 proteins is required for formation of the in vivo dodecamer, negatively affecting the activity of Pdx1, opening the possibility of allosteric Pdx1 inhibitors.     

The sedimentation properties of ferritins. New insights and analysis of methods of nanoparticle preparation

Background

Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.

Methods

Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.

Results

More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe²⁺ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ∼ 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν? = 0.735 mL/g, s_20,w = 17.0 S and D_20,w = 3.21 × 10⁻⁷ cm²/s; and for apoHuHF M = 506,266 g/mol, ν? = 0.724 mL/g, s_20,w = 18.3 S and D_20,w = 3.18 × 10⁻⁷ cm²/s.

Significance

The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals.     

Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions

Background

The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes.

Methods

We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-³²P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation.

Results

This activity was linear with time up to 20 min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5 mM MgCl₂ was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1 mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN₃) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN₃. The dose–response of ATP revealed a hyperbolic profile with maximal velocity of 25.2 ± 1.2 nmol Pi x mg^− 1 x min^− 1 and K_0.5 of 0.07 ± 0.01 mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60 min of ischemia.

Conclusion

Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia.

General Significance

This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.     

XRCC3 Thr241Met polymorphism and risk of acute myeloid leukemia in a Romanian population

Background

DNA repair systems have a critical role in maintaining the genome integrity and stability. DNA repair gene polymorphisms may influence the capacity to repair DNA damage, and thus lead to an increased cancer susceptibility. X-ray repair cross-complementing groups 3 (XRCC3), a DNA repair gene, may be involved in acute myeloid leukemia susceptibility. The objective of the current study was to investigate the association of Thr241Met polymorphism of XRCC3 gene with the risk of acute myeloid leukemia (AML).

Methods

This study included 78 AML patients and 121 healthy individuals without cancer. We used polymerase chain reaction-restriction fragment length polymorphism assay to determine XRCC3 genotypes.

Results

The XRCC3 variant genotype (Thr/Met+Met/Met) was more frequent in AML patients than in healthy controls (OR = 2.76, 95% CI: 1.52-4.98, P = 0.001). Our study revealed a statistically significant association between variant genotype (Thr/Met+Met/Met) and AML de novo compared to secondary AML (P = 0.007). No significant associations were found between any genotype and age at diagnosis, number of white blood cells and subtype of AML. Overall survival of patients with Thr/Thr genotype was better than those of variant Thr/Met and Met/Met genotypes.

Conclusions

Our findings indicate that the XRCC3 Thr241Met polymorphism may be a genetic risk factor for AML, particularly in male patients with de novo AML from the central part of Romania.     

Molecular characterization and localization of Plasmodium falciparum choline kinase

Generation of phosphocholine by choline kinase is important for phosphatidylcholine biosynthesis via Kennedy pathway and phosphatidylcholine biosynthesis is essential for intraerythrocytic growth of malaria parasite. A putative gene (Gene ID PF14_0020) in chromosome 14, having highest sequence homology with choline kinase, has been identified by BLAST searches from P. falciparum genome sequence database. This gene has been PCR amplified, cloned, over-expressed and characterized. Choline kinase activity of the recombinant protein (PfCK) was validated as it catalyzed the formation of phosphocholine from choline in presence of ATP. The K_m values for choline and ATP are found to be 145 ± 20 μM and 2.5 ± 0.3 mM, respectively. PfCK can phosphorylate choline efficiently but not ethanolamine. Southern blotting indicates that PfCK is a single copy gene and it is a cytosolic protein as evidenced by Western immunoblotting and confocal microscopy. A model structure of PfCK was constructed based on the crystal structure of choline kinase of C. elegans to search the structural homology. Consistent with the homology modeling predictions, CD analysis indicates that the α and β content of PfCK are 33% and 14%, respectively. Since choline kinase plays a vital role for growth and multiplication of P. falciparum during intraerythrocytic stages, we can suggest that this well characterized PfCK may be exploited in the screening of new choline kinase inhibitors to evaluate their antimalarial activity.     

Tetracycline nanoparticles loaded calcium sulfate composite beads for periodontal management

Background

The objective of this study was to fabricate, characterize and evaluate in vitro, an injectable calcium sulfate bone cement beads loaded with an antibiotic nanoformulation, capable of delivering antibiotic locally for the treatment of periodontal disease.

Methods

Tetracycline nanoparticles (Tet NPs) were prepared using an ionic gelation method and characterized using DLS, SEM, and FTIR to determine size, morphology, stability and chemical interaction of the drug with the polymer. Further, calcium sulfate (CaSO₄) control and CaSO₄-Tet NP composite beads were prepared and characterized using SEM, FTIR and XRD. The drug release pattern, material properties and antibacterial activity were evaluated. In addition, protein adsorption, cytocompatibility and alkaline phosphatase activity of the CaSO₄-Tet NP composite beads in comparison to the CaSO₄ control were analyzed.

Results

Tet NPs showed a size range of 130 ± 20 nm and the entrapment efficiency calculated was 89%. The composite beads showed sustained drug release pattern. Further the drug release data was fitted into various kinetic models wherein the Higuchi model showed higher correlation value (R² = 0.9279) as compared to other kinetic models. The composite beads showed antibacterial activity against Staphylococcus aureus and Escherichia coli. The presence of Tet NPs in the composite bead didn't alter its cytocompatibility. In addition, the composite beads enhanced the ALP activity of hPDL cells.

Conclusions

The antibacterial and cytocompatible CaSO₄-Tet NP composite beads could be beneficial in periodontal management to reduce the bacterial load at the infection site.

General significance

Tet NPs would deliver antibiotic locally at the infection site and the calcium sulfate cement, would itself facilitate tissue regeneration.     

A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity

Background

Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.

Methods

The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.

Results and conclusions

The peptide has an amino acid sequence N-Ile¹-Cys²-Glu³-Ala⁴-Glu⁵-His⁶-Lys⁷-Trp⁸-Gly⁹-Asp¹⁰-Tyr¹¹-Leu¹²-Asp¹³-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I₅₀ = 600 nM and K_i = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)₂]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k₅ = 8.7 ± 1 × 10^− 3 s^− 1 and k₆ = 7.3 ± 0.6 × 10^− 5 s^− 1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.

General significance

The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.     

Interaction of metal nanoparticles with recombinant arginine kinase from Trypanosoma brucei: Thermodynamic and spectrofluorimetric evaluation

Background

Trypanosoma brucei, responsible for African sleeping sickness, is a lethal parasite against which there is need for new drug protocols. It is therefore relevant to attack possible biomedical targets with specific preparations and since arginine kinase does not occur in humans but is present in the parasite it becomes a suitable target.

Methods

Fluorescence quenching, thermodynamic analysis and FRET have shown that arginine kinase from T. brucei interacted with silver or gold nanoparticles.

Results

The enzyme only had one binding site. At 25 °C the dissociation (K_d) and Stern–Volmer constants (K_SV) were 15.2 nM, 0.058 nM^− 1 [Ag]; and 43.5 nM, 0.052 nM^− 1 [Au] and these decreased to 11.2 nM, 0.041 nM^− 1 [Ag]; and 24.2 nM, 0.039 nM^− 1 [Au] at 30 °C illustrating static quenching and the formation of a non-fluorescent fluorophore–nanoparticle complex. Silver nanoparticles bound to arginine kinase with greater affinity, enhanced fluorescence quenching and easier access to tryptophan molecules than gold. Negative ΔH and ΔG values implied that the interaction of both Ag and Au nanoparticles with arginine kinase was spontaneous with electrostatic forces. FRET confirmed that the nanoparticles were bound 2.11 nm [Ag] and 2.26 nm [Au] from a single surface tryptophan residue.

Conclusions

The nanoparticles bind close to the arginine substrate through a cysteine residue that controls the electrophilic and nucleophilic characters of the substrate arginine–guanidinium group crucial for enzymatic phosphoryl transfer between ADP and ATP.

General significance

The nanoparticles of silver and gold interact with arginine kinase from T. brucei and may prove to have far reaching consequences in clinical trials.     

Inorganic phosphate uptake in unicellular eukaryotes

Background

Inorganic phosphate (P_i) is an essential nutrient for all organisms. The route of P_i utilization begins with P_i 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 P_i transporters in unicellular eukaryotes. The objective of this review is to evaluate the recent findings regarding P_i 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

P_i uptake is the key step of P_i homeostasis and in the subsequent signaling event in eukaryotic microorganisms.

General significance

Biochemical and structural studies are important for clarifying mechanisms of P_i homeostasis, as well as P_i sensor and downstream pathways, and raise possibilities for future studies in this field.     

A common polymorphism in pre-microRNA-146a is associated with lung cancer risk in a Korean population

Introduction

MicroRNAs (miRs) play important roles in the development and progression of human cancers. MiR-146a down-regulates epidermal growth factor receptor and the nuclear factor-κB regulatory kinase interleukin-1 receptor-associated kinase 1 genes that play important roles in lung carcinogenesis. This study was conducted to evaluate the association between rs2910164C>G, a functional polymorphism in the pre-miR-146a, and lung cancer risk.

Material and methods

The rs2910164C>G genotypes were determined in 1094 patients with lung cancer and 1100 healthy controls who were frequency matched for age and gender.

Results

The rs2910164 CG or GG genotype was associated with a significantly decreased risk for lung cancer compared to that of the CC genotype (adjusted odds ratio = 0.80, 95% confidence interval = 0.66–0.96, P = 0.02). When subjects were stratified according to smoking exposure (never, light and heavy smokers), the effect of the rs2910164C>G genotype on lung cancer risk was significant only in never smokers (adjusted odds ratio = 0.66, 95% confidence interval = 0.45–0.96, P = 0.03, under a dominant model for the C allele) and decreased as smoking exposure level increased (P_trend < 0.001). In line with this result, the level of miR-146a expression in the tumor tissues was significantly higher in the GG genotype than in the CC or CG genotype only in never-smokers (P = 0.02).

Conclusions

These findings suggest that the rs2910164C>G in pre-miR-146a may contribute to genetic susceptibility to lung cancer, and that miR-146a might be involved in lung cancer development.     

Impaired cardiac mitochondrial function and contractile reserve following an acute exposure to environmental particulate matter

Background

It has been suggested that mitochondrial function plays a central role in cardiovascular diseases associated with particulate matter inhalation. The aim of this study was to evaluate this hypothesis, with focus on cardiac O₂ and energetic metabolism, and its impact over cardiac contractility.

Methods

Swiss mice were intranasally instilled with either residual oil fly ash (ROFA) (1.0 mg/kg body weight) or saline solution. After 1, 3 or 5 h of exposure, O₂ consumption was evaluated in heart tissue samples. Mitochondrial respiration, respiratory chain complexes activity, membrane potential and ATP content and production rate were assessed in isolated mitochondria. Cardiac contractile reserve was evaluated according to the Langendorff technique.

Results

Three hours after ROFA exposure, tissue O₂ consumption was significantly decreased by 35% (from 1180 ± 70 to 760 ± 60 ng-at O/min g tissue), as well as mitochondrial rest (state 4) and active (state 3) respiration, by 30 and 24%, respectively (control state 4: 88 ± 5 ng-at O/min mg protein; state 3: 240 ± 20 ng-at O/min mg protein). These findings were associated with decreased complex II activity, mitochondrial depolarization and deficient ATP production. Even though basal contractility was not modified (control: 75 ± 5 mm Hg), isolated perfused hearts failed to properly respond to isoproterenol in ROFA-exposed mice. Tissue O₂ consumption rates positively correlated with cardiac contractile state in controls (r² = 0.8271), but not in treated mice (r² = 0.1396).

General Significance

The present results show an impaired mitochondrial function associated with deficient cardiac contractility, which could represent an early cardiovascular alteration after the exposure to environmental particulate matter.     

In vivo formation of Plasmodium falciparum ribosomal stalk — A unique mode of assembly without stable heterodimeric intermediates

Background

The ribosomal stalk composed of P-proteins constitutes a structure on the large ribosomal particle responsible for recruitment of translation factors and stimulation of factor-dependent GTP hydrolysis during translation. The main components of the stalk are P-proteins, which form a pentamer. Despite the conserved basic function of the stalk, the P-proteins do not form a uniform entity, displaying heterogeneity in the primary structure across the eukaryotic lineage. The P-proteins from protozoan parasites are among the most evolutionarily divergent stalk proteins.

Methods

We have assembled P-stalk complex of Plasmodium falciparum in vivo in bacterial system using tricistronic expression cassette and provided its characteristics by biochemical and biophysical methods.

Results

All three individual P-proteins, namely uL10/P0, P1 and P2, are indispensable for acquisition of a stable structure of the P stalk complex and the pentameric uL10/P0-(P1-P2)₂ form represents the most favorable architecture for parasite P-proteins.

Conclusion

The formation of P. falciparum P-stalk is driven by trilateral interaction between individual elements which represents unique mode of assembling, without stable P1–P2 heterodimeric intermediate.

General significance

On the basis of our mass-spectrometry analysis supported by the bacterial two-hybrid assay and biophysical analyses, a unique pathway of the parasite stalk assembling has been proposed. We suggest that the absence of P1/P2 heterodimer, and the formation of a stable pentamer in the presence of all three proteins, indicate a one-step formation to be the main pathway for the vital ribosomal stalk assembly, whereas the P2 homo-oligomer may represent an off-pathway product with physiologically important nonribosomal role.