Cloning,expression and purification of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum

Abstract

The antimalarial drugs are of fundamental importance in the control of malaria, especially for the lack of efficient treatments and acquired resistance to the existing drugs. For this reason, there is a continuous work in identifying novel, less toxic and effective chemotherapies as well as new therapeutic targets against the causative agents of malaria. In this context, a superfamily of metalloenzymes named carbonic anhydrases (CAs, EC 4.2.1.1) has aroused a great interest as druggable enzymes to limit the development of Plasmodium falciparum gametocytes. CAs catalyze a common reaction in all life domains, the carbon dioxide hydration to bicarbonate and protons (CO₂?+?H₂O ? HCO₃^-?+?H⁺). P. falciparum synthesizes pyrimidines de novo starting from HCO₃^-, which is generated from CO₂ through the action of the η-CA identified in the genome of the protozoan. Here, we propose a procedure for the preparation of a wider portion of the protozoan η-CA, named PfCAdom (358 amino acid residues), with respect to the truncated form prepared by Krungkrai et al. (PfCA1, 235 amino acid residues). The results evidenced that the recombinant PfCAdom, produced as a His-tag fusion protein, was 2.7 times more active with respect the truncated form PfCA1.     

Ameliorating effect of lipo-ATRA treatment on the expression of TIG3 and its suppressing effect on PPARγ gene expression in lung cancer animal model

Vitamin D₃ deficiency was found to be tightly linked to many health problems including metabolic syndrome, cancer, cardiovascular diseases, and type 2 diabetes mellitus. In our study, we tested the possible antidiabetic effects of one of vitamin D₃ analogs, alfacalcidol, solely or in a combination with metformin on type 2 diabetic rats. Type 2 diabetic model rats were induced by feeding high-fat diet for 4 weeks followed by intraperitoneal injection of streptozotocin. In addition to the control group, the diabetic rats were divided into four groups: untreated, metformin-treated, alfacalcidol-treated, and combination-treated group (metformin?+?alfacalcidol) for 4 weeks. The level of fasting blood glucose, fasting serum insulin, homeostatic model of insulin resistance, serum lipid profile, liver enzymes, calcium, phosphorus, and 25-hydroxyvitamin D₃ were also determined. Besides, sterol regulatory element binding protein-1c (SREBP-1c) and vitamin D receptors (VDR) gene expression at mRNA and protein levels were evaluated. The level of significance was fixed at P?≤?0.05 for all statistical tests. Alfacalcidol, solely or combined with metformin, significantly ameliorated glucose homeostasis and lipid profile parameters (P?<?0.001) with a neutral effect on calcium and phosphorus levels. Significant downregulation of mRNA expression of SREBP-1c in the liver, white as well as brown adipose tissues (P?<?0.001) and different patterns of mRNA expression of VDR gene in pancreas and white adipose tissue were observed in rats treated with alfacalcidol solely or in combination with metformin. Vitamin D₃ analogs can modulate glucose parameters and lipid metabolism in a diabetic rat model and it provides additional protective effects when combined with metformin.

     

PfeIK1, a eukaryotic initiation factor 2α kinase of the human malaria parasite Plasmodium falciparum, regulates stress-response to amino-acid starvation

Background

Plasmodium falciparum -parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy.

Methods

Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp 60/70–2/70–3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane.

Results

In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of antioxidant enzymes was also increased in oxidatively-stressed trophozoites.

Conclusion

Results indicated that mRNA expression of parasite antioxidant enzymes and HSPs was co-ordinated and stage-dependent. Secondly, both systems were redox-responsive and showed remarkably increased and co-ordinated expression in oxidatively-stressed parasites and in parasites growing in antioxidant blunted G6PD-deficient RBCs. Lastly, as important anti-malarials either increase oxidant stress or impair antioxidant defense, results may encourage the inclusion of anti-HSP molecules in anti-malarial combined drugs.     

Antibodies against PfEMP1, RIFIN, MSP3 and GLURP are acquired during controlled Plasmodium falciparum malaria infections in naïve volunteers

Antibodies to polymorphic antigens expressed during the parasites erythrocytic stages are important mediators of protective immunity against P. falciparum malaria. Therefore, polymorphic blood stage antigens like MSP3, EBA-175 and GLURP and variant surface antigens PfEMP1 and RIFIN are considered vaccine candidates. However, to what extent these antibodies to blood stage antigens are acquired during naive individuals' first infections has not been studied in depth. Using plasma samples collected from controlled experimental P. falciparum infections we show that antibodies against variant surface antigens, PfEMP1 and RIFIN as well as MSP3 and GLURP, are acquired during a single short low density P. falciparum infection in non-immune individuals including strain transcendent PfEMP1 immune responses. These data indicate that the immunogenicity of the variant surface antigens is similar to the less diverse merozoite antigens. The acquisition of a broad and strain transcendent repertoire of PfEMP1 antibodies may reflect a parasite strategy of expressing most or all PfEMP1 variants at liver release optimizing the likelihood of survival and establishment of chronic infections in the new host.     

The effect of illumination on the pigment composition of the ζ-carotenic mutant,PG1, of Scenedesmus obliquus

Dark grown Scenedesmus obliquus strain PG1 accumulated -carotene and phytoene as its major carotenoids. On illumination of dark-grown cultures in air/CO₂, cyclic carotenes and xanthophylls were formed, apparently at the expense of the accumulated phytoene and -carotene. This interconversion of carotenoids was accompanied by chlorophyll synthesis. In an atmosphere of nitrogen/CO₂ the light-induced changes occurred more slowly and in nitrogen alone the changes were incomplete. No massive production of cyclic carotenes from the accumulated -carotene was observed in cultures illuminated under anaerobic conditions.     

The effect of TGF-β1 and Smad7 gene transfer on the phenotypic changes of rat alveolar epithelial cells

The aim of this study was to investigate whether transforming growth factor-β1 (TGF-β1) could induce alveolar epithelial-mesenchymal transition (EMT) in vitro, and whether Smad7 gene transfer could block this transition. We also aimed to elucidate the possible mechanisms of these processes. The Smad7 gene was transfected to the rat type II alveolar epithelial cell line (RLE-6TN). Expression of the EMT-associated markers was assayed by Western Blot and Real-time PCR. Morphological alterations were examined via phase-contrast microscope and fluorescence microscope, while ultrastructural changes were examined via electron microscope. TGF-β1 treatment induced a fibrotic phenotype of RLE-6TN with increased expression of fibronectin (FN), α-smooth muscle actin (α-SMA) and vimentin, and decreased expression of E-cadherin (E-cad) and cytokeratin19 (CK19). After transfecting the RLE-6TN with the Smad7 gene, the expression of the mesenchymal markers was downregulated while that of the epithelial markers was upregulated. TGF-β1 treatment for 48 h resulted in the separation of RLE-6TN from one another and a change into elongated, myofibroblast-like cells. After the RLE-6TN had been transfected with the Smad7 gene, TGF-β1 treatment had no effect on the morphology of the RLE-6TN. TGF-β1 treatment for 48 h resulted in an abundant expression of α-SMA in the RLE-6TN. If the RLE-6TN were transfected with the Smad7 gene, TGF-β1 treatment for 48 h could only induce a low level of α-SMA expression. Furthermore, TGF-β1 treatment for 12 h resulted in the degeneration and swelling of the osmiophilic multilamellar bodies, which were the markers of type II alveolar epithelial cells. TGF-β1 can induce alveolar epithelialmesenchymal transition in vitro, which is dependent on the Smads signaling pathway to a certain extent. Overexpression of the Smad7 gene can partially block this process     

Efficacy of amodiaquine and sulfadoxine/pyrimethamine in the treatment of malaria not complicated by Plasmodium falciparum in Nariño,Colombia, 1999-2002

The resurgence and spread of antimalarial drug resistance is one of the causes of the worldwide increase of malaria. In Colombia, uncomplicated Plasmodium falciparum malaria has been treated with a combination of amodiaquine (AQ) and sulfadoxine/pyrimethamine (SP) since 2000. The efficacy of these two antimalarials was evaluated after the implementation of the new malaria treatment scheme. In the municipalities of El Charco and Tumaco (Nari?o) on the Pacific Coast region, the standard PAHO protocol was used to evaluate antimalarial efficacy in areas of low to moderate malaria transmission. Patients were randomly allocated to treatment regime in two cities of Nari?o, El Charco (n = 48) and Tumaco (n = 50). After 14 days none of El Charco patients presented therapeutic failure to either antimalarial. However, in Tumaco after 28 days, 12 of 24 (95% CI: 30.6-69.4) patients presented AQ treatment failure while 4 of 26 (95% CI: 5.1-33.1) patients had SP treatment failure. The high level of AQ treatment failure in Tumaco was unexpected because it had been introduced only recently as an antimalarial treatment in Colombia. The results suggest that the use of the current dose of AQ in combination with SP will be therapeutically useful for less time than expected. Use of combined therapies is a key strategy to delay antimalarial resistance. Unfortunately, its success depends on the efficacy of antimalarial drugs individually.     

Effects of Age,Hemoglobin Type and Parasite Strain on IgG Recognition of Plasmodium falciparum–Infected Erythrocytes in Malian Children

Background

Naturally-acquired antibody responses to antigens on the surface of Plasmodium falciparum-infected red blood cells (iRBCs) have been implicated in antimalarial immunity. To profile the development of this immunity, we have been studying a cohort of Malian children living in an area with intense seasonal malaria transmission.

Methodology/Principal Findings

We collected plasma from a sub-cohort of 176 Malian children aged 3-11 years, before (May) and after (December) the 2009 transmission season. To measure the effect of hemoglobin (Hb) type on antibody responses, we enrolled age-matched HbAA, HbAS and HbAC children. To quantify antibody recognition of iRBCs, we designed a high-throughput flow cytometry assay to rapidly test numerous plasma samples against multiple parasite strains. We evaluated antibody reactivity of each plasma sample to 3 laboratory-adapted parasite lines (FCR3, D10, PC26) and 4 short-term-cultured parasite isolates (2 Malian and 2 Cambodian). 97% of children recognized ≥1 parasite strain and the proportion of IgG responders increased significantly during the transmission season for most parasite strains. Both strain-specific and strain-transcending IgG responses were detected, and varied by age, Hb type and parasite strain. In addition, the breadth of IgG responses to parasite strains increased with age in HbAA, but not in HbAS or HbAC, children.

Conclusions/Significance

Our assay detects both strain-specific and strain-transcending IgG responses to iRBCs. The magnitude and breadth of these responses varied not only by age, but also by Hb type and parasite strain used. These findings indicate that studies of acquired humoral immunity should account for Hb type and test large numbers of diverse parasite strains.     

β-Secretase (BACE-1) inhibitory effect of biflavonoids

Here, we describe amentoflavone-type biflavonoids, which were isolated from natural sources and were found to inhibit β-secretase (BACE-1). The structure–activity relationship was studied, and compounds 1–8, 10, 17, and 18 showed BACE-1 inhibitory activity. Among these compounds, 2,3-dihydroamentoflavone 17 and 2,3-dihydro-6-methylginkgetin 18 exhibited potent inhibitory effects with IC₅₀ values of 0.75 and 0.35 μM, respectively.     

Effects of rice potassium level on the fecundity and expression of the vitellogenin gene of Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

Potassium (K) is a key component of plant nutrition, significantly influencing crop growth. Levels of this nutrient in plants can also influence a number of pest infestations. The brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is an important pest of rice in Asia. In this study, we examined K contents in rice grown in hydroponic solution, and its relationship to the fecundity and expression of the vitellogenin (Nlvg) gene of N. lugens which was reared on the rice. Our findings indicated that K contents in rice increased with the increasing K concentration within the hydroponic solution, but reduced at the highest K concentration (160 mg/L). The number of eggs laid by N. lugens which was reared on the rice varied significantly with K concentration, and increased by 0.12 and 0.22 fold under 20 mg/L and 160 mg/L K level compared to that of the control (40 mg/L), decreasing by 0.57 fold under 0 mg/L K. Nlvg mRNA expression increased 1.17 and 1.94 fold in individuals which were reared on rice grown in 20 mg/L and 160 mg/L K level, compared to that of the control before mating; and by 3.36 and 2.97 after mating, respectively. However, Nlvg mRNA expression fold decreased by 0.99 under 0 mg/L K level before mating and 0.91 after mating. The variation of eggs may be attributed to the change of Nlvg mRNA expression, because there was a positive correlation between the eggs and Nlvg mRNA expression. These results demonstrated low (20 mg/L) and highest K levels (160 mg/L) in hydroponic solution showed the lower K level in plants than the control, which facilitated the fecundity of N. lugens. The study of the effects of K levels on the fecundity should have significance for insect control.     

Influence of TGF-β1 on the expression of BSP,DSP, TGF-β1 receptor I and Smad proteins during reparative dentinogenesis

Reparative dentin has a wide variety of manifestations ranging from a regular, tubular form to an irregular, atubular form. However, the characteristics of reparative dentin have not been clarified. This study hypothesized that the level of bone sialoprotein (BSP) expression will increase if the newly formed reparative dentin is bone-like but the dentin sialophosphoprotein (DSPP) level will decrease. In order to test this hypothesis, the expression of BSP and DSP was examined by immunohistochemistry and the expression of BSP was measured by in situ hybridization in an animal model. The pulps of 12 maxillary right first molars from twelve male rats were exposed and capped with MTA. In addition, in order to understand the role of transforming growth factor-beta 1 (TGF-β1) during reparative dentinogenesis, the expression of BSP and DSPP mRNA was analyzed by RT-PCR in a human dental pulp cell culture, and the transforming growth factor-beta 1 receptors (TβRI) and Smad 2/3 were examined by immunofluorescence in an animal model. DSP was expressed in the normal odontoblasts and odontoblast-like cells of the reparative dentin. Interestingly, BSP was strongly expressed in the odontoblast-like cells of reparative dentin. The level of the TβRI and Smad 2/3 proteins was higher in the reparative dentin than in the normal dentin. TGF-β1 up-regulated BSP in the human pulp cell cultures. This suggests that reparative dentin has both dentinogenic and osteogenic characteristics that are mediated by TGF-β1.     

An evaluation of the effects of probiotics on tumoral necrosis factor (TNF-α) signaling and gene expression

The search for functional foods containing probiotics has been growing due to numerous benefits they provide to health, such as modulation of the immune system and of the anti-inflammatory activity by inhibiting the release of pro-inflammatory cytokines, such as TNF-α. However, the mechanisms of actions of the probiotics responsible for this inhibition have not been completely explained so far. A better understanding of the interaction between probiotics and cell signaling pathways related to inflammatory processes shall help to prevent inflammatory bowel diseases. Therefore, the aim of this revision is to help understand the mechanisms of action of probiotics in cell signaling pathways that regulate TNF-α expression. Probiotics might act at different points of the MAPK pathway, on NF-kB, on proteasome activity, on Toll-like receptors, and on their regulators and stimuli. The present revision reaches the conclusion that probiotics act through multiple mechanisms, especially by inhibiting IkB phosphorylation and degradation, thus preventing the translocation of NF-kB. Effects are also shown to be strain-specific, and probiotics of the genus Lactobacillus are proved to play and essential role in anti-inflammatory activity.