Role of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> peroxiredoxins in mitochondrial bioenergetics

Trypanosoma cruzi cytosolic (TcCPx) and mitochondrial tryparedoxin peroxidase (TcMPx) play a fundamental role in H₂O₂ detoxification. Herein, mitochondrial bioenergetics was evaluated in cells that overexpressed TcCPx (CPx) and TcMPx (MPx) and in pTEX. In MPx, a higher expression was observed for TcCPx, and the same correlation was true for CPx. Differences in H₂O₂ release among the overexpressing cells were detected when the mitochondrial respiratory chain was inhibited using antimycin A or thenoyltrifluoroacetone. MPx had higher O₂ consumption rates than pTEX and CPx, especially in the presence of oligomycin. In all of the cells, the mitochondrial membrane potential and the ATP levels were similar. Because of the mild uncoupling that was observed in MPx, the presence or induction of a proton transporter in the mitochondrial membrane is suggested when TcMPx is expressed at higher levels. Our results show a possible interplay between the cytosolic and mitochondrial antioxidant systems in a trypanosomatid.     

Evaluation of protein <Emphasis Type="BoldItalic">in vitro</Emphasis> digestibility of <Emphasis Type="BoldItalic">Palmaria palmata</Emphasis> and <Emphasis Type="BoldItalic">Gracilaria verrucosa</Emphasis>

Palmaria palmata and Gracilaria verrucosa are edible red seaweeds and potential protein sources for human or animal nutrition, so studies were conducted on their in vitro protein digestibility. After 30 min predigestion by pepsin followed by 6 h digestion into a cell dialysis containing porcine pancreatin, the in vitro protein digestibility of P. palmata and G. verrucosa, expressed in regard to casein digestibility, was 4.9% and 42.1%, respectively. The level of protein digestibility seems to be related to the amount of soluble fibre, which was 45.3% and 30.5%, respectively.     

Repetitive DNA is associated with centromeric domains in <Emphasis Type="Italic">Trypanosoma brucei</Emphasis> but not <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

Trypanosomes are parasitic protozoa that diverged early from the main eukaryotic lineage. Their genomes display several unusual characteristics and, despite completion of the trypanosome genome projects, the location of centromeric DNA has not been identified.     

Invasion of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> into host cells is impaired by <Emphasis Type="Italic">N</Emphasis>-propionylmannosamine and other <Emphasis Type="Italic">N</Emphasis>-acylmannosamines

The etiologic agent of Chagas’ disease, Trypanosoma cruzi, is widely distributed in South America, affecting millions of people with thousands of deaths every year. Adherence of the infectious trypomastigote to host cells is mediated by sialic acid. T. cruzi cannot synthesize sialic acids on their own but cleave them from the host cells and link them to glycans on the surface of the parasites using the trans-sialidase, a GPI-anchored enzyme. The infectivity of the protozoan parasites strongly depends on the activity of this enzyme. In this report, we investigated whether the transfer of sialic acids from the host to the parasites can be attenuated using novel sialic acid precursors. The cell line 86-HG-39 was infected with T. cruzi and treated with defined N-acylmannosamine analogues bearing an elongated N-acyl side-chain. By treatment of these cells the number of T.cruzi infected cell was reduced up to 60%. We also showed that the activity of the bacterial sialidase C was reduced with N-glycan substrates with elongated N-acyl side chains of the terminal sialic acids. The affinity of this sialidase decreased with the length of the N-acyl side-chain. The data presented suggest that N-acyl modified sialic acid precursors can change the transfer of sialic acids leading to modification of infection. Since the chemotherapy of this disease is inefficient and afflicted by side effects, the need of effective drugs is lasting. These findings propose a new path to prevent the dissemination of T. cruzi in the human hosts. These compounds or further modified analogues might be a basis for the search of new agents against Chagas’ disease.     

Mechanism of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> death induced by <Emphasis Type="Italic">Cratylia mollis</Emphasis> seed lectin

Incubation of T. cruzi epimastigotes with the lectin Cramoll 1,4 in Ca²⁺ containing medium led to agglutination and inhibition of cell proliferation. The lectin (50 μg/ml) induced plasma membrane permeabilization followed by Ca²⁺ influx and mitochondrial Ca²⁺ accumulation, a result that resembles the classical effect of digitonin. Cramoll 1,4 stimulated (five-fold) mitochondrial reactive oxygen species (ROS) production, significantly decreased the electrical mitochondrial membrane potential (ΔΨ_m) and impaired ADP phosphorylation. The rate of uncoupled respiration in epimastigotes was not affected by Cramoll 1,4 plus Ca²⁺ treatment, but oligomycin-induced resting respiration was 65% higher in treated cells than in controls. Experiments using T. cruzi mitochondrial fractions showed that, in contrast to digitonin, the lectin significantly decreased ΔΨ_m by a mechanism sensitive to EGTA. In agreement with the results showing plasma membrane permeabilization and impairment of oxidative phosphorylation by the lectin, fluorescence microscopy experiments using propidium iodide revealed that Cramoll 1,4 induced epimastigotes death by necrosis.     

All <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> developmental forms present lysosome-related organelles

Trypanosoma cruzi epimastigote forms concentrate their major protease, cruzipain, in the same compartment where these parasites store macromolecules obtained from medium and for this ability these organelles were named as reservosomes. Intracellular digestion occurs mainly inside reservosomes and seems to be modulated by cruzipain and its natural inhibitor chagasin that also concentrates in reservosomes. T. cruzi mammalian forms, trypomastigotes and amastigotes, are unable to capture macromolecules by endocytosis, but also express cruzipain and chagasin, whose role in infectivity has been described. In this paper, we demonstrate that trypomastigotes and amastigotes also concentrate cruzipain, chagasin as well as serine carboxypeptidase in hydrolase-rich compartments of acidic nature. The presence of P-type proton ATPase indicates that this compartment is acidified by the same enzyme as epimastigote endocytic compartments. Electron microscopy analyzes showed that these organelles are placed at the posterior region of the parasite body, are single membrane bound and possess an electron-dense matrix with electronlucent inclusions. Three-dimensional reconstruction showed that these compartments have different size and shape in trypomastigotes and amastigotes. Based on these evidences, we suggest that all T. cruzi developmental stages present lysosome-related organelles that in epimastigotes have the additional and unique ability of storing cargo.     

Thermal-unfolding Reaction of Triosephosphate Isomerase from <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Thermal denaturation of triosephosphate isomerase from Trypanosoma cruzi was studied by circular dicrhoism and fluorescence spectroscopies. The unfolding transition was found to be highly irreversible even at the very early stages of the reaction. Kinetic studies, allowed us to identify consecutive reactions. Firstly, only the tryptophan environment is altered. Next, changes on the secondary structure and hydrophobic surface exposure measured by 1-anilino-8-naphthalenesulfonate (ANS) binding were observed. Further conformational changes imply additional modifications on the secondary and tertiary structures and release of the hydrophobic dye leading to the formation of the unfolded state that is prone to aggregate. Edgar Mixcoha-Hernández and Liliana M. Moreno-Vargas contributed equally to this work     

Isolation and characterization of the major histocompatibility complex <Emphasis Type="BoldItalic">DQA1</Emphasis> and <Emphasis Type="BoldItalic">DQA2</Emphasis> genes in gayal (<Emphasis Type="BoldItalic">Bos frontalis</Emphasis>)

The species origin of Yunnan gayal has been controversial since many years. However, few recent genetic studies have suggested that it has perhaps originated from the hybridization between male Bos frontalis and female B. taurus or B. indicus. Being an important semi-wild bovid species, this has also been listed under the red list of International Union of Conservation of Nature and Natural Resources. However, there is limited information available about the immunogenicity of this precarious species of Bos. Major histocompatibility complex (MHC) plays a pivotal role in immune response to infectious diseases in vertebrates. In the present study, we have investigated the structural and functional characteristics and possible duplication of the MHC-DQA genes in gayal (B. frontalis). Two full-length cDNA clones of the MHC-DQA genes were amplified and designated as Bofr-DQA1 (DQA*0101) and Bofr-DQA2 (DQA*2001) with GenBank accession numbers KT318732 and KT318733, respectively. A comparison between Bofr-DQA1, Bofr-DQA2 and to other MHC-DQA molecules from different animal species showed that nucleotide and encoded amino acid sequences of these two identified MHC-DQA genes have more similarity to alleles of specific DQA1 and DQA2 molecules from other Ruminantia species than to each other. The phylogenic investigation also demonstrated a large genetic distance between these two genes than to homologous from the other species. The large genetic distance between Bofr-DQA1 and Bofr-DQA2, and the presence of different bovine DQA putative motifs clarify that these sequences are nonallelic type. These results could suggest that duplication of the DQA genes has also occurred in gayal. The findings of the present study have strengthened our understanding to MHC diversity in rare ruminants and mutation of immunological functions, selective and evolutionary forces that affect MHC variation within and between species.     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.     

Intra-allelic variation in introns of the <Emphasis Type="Italic">S</Emphasis><Subscript><Emphasis Type="BoldItalic">13</Emphasis></Subscript><Emphasis Type="Italic">-RNase</Emphasis> allele distinguishes sweet,wild and sour cherries

The cherry (Prunus avium), a self-incompatible diploid species, and the sour cherry (Prunus cerasus), a self-incompatible or self-compatible allotetraploid species derived from P. avium and Prunus fruticosa, share several S-RNase alleles, including S ₁₃ . An inactive form, S ₁₃ °, is found in some sour cherries. Two (AT) microsatellites are associated with allele S ₁₃ -RNase, one in the first intron and one in the second. Their length polymorphisms were studied in 14 sweet and 17 wild cherries (both P. avium) and in 42 sour cherries. Fluorescent primers amplifying each microsatellite were designed and amplification products sized on an automated sequencer. Variants ranged from 247 to 273 bp for the first intron microsatellite and from 308 to 322 bp for the second. There were 34 combinations and, surprisingly, the lengths of the two microsatellites were correlated. Generally, the sweet, wild and sour cherries had different combinations, and the four examples of S ₁₃ °-RNase were associated with three different combinations. Certain sequences associated with the microsatellites match footprints of transposons. The distribution of combinations indicated little overlap between the three populations analysed and provided useful insights into relationships of some of the accessions allowing some parentages to be checked. In the diploid sweet and wild cherries, S ₁₃ variants presumably resulted from slippage during replication, but in the tetraploid sour cherries, which can have more than one copy of S ₁₃ or S ₁₃ °, intra-allelic crossing over may have generated new variants. The possible involvement of transposable elements in the origin of these microsatellites is considered.     

Marker-assisted pyramiding of <Emphasis Type="BoldItalic">Thinopyrum-</Emphasis>derived leaf rust resistance genes <Emphasis Type="BoldItalic">Lr19</Emphasis> and <Emphasis Type="BoldItalic">Lr24</Emphasis> in bread wheat variety HD2733

This study was undertaken to pyramid two effective leaf rust resistance genes (Lr19 and Lr24) derived from Thinopyrum (syn. Agropyron), in the susceptible, but agronomically superior wheat cultivar HD2733 using marker-assisted selection. In the year 2001, HD2733 was released for irrigated timely sown conditions of the north eastern plains zone (NEPZ) of India became susceptible to leaf rust, a major disease of the region. Background selection helped in developing near-isogenic lines (NILs) of HD2733 with Lr19 and Lr24 with 97.27 and \(98.94\%\), respectively, of genomic similarity with the parent cultivar, after two backcrossing and one generation of selfing. NILs were intercrossed to combine the genes Lr19 and Lr24. The combination of these two genes in the cultivar HD2733 is expected to provide durable leaf rust resistance in farmers’ fields.     

The Microbial <Emphasis Type="BoldItalic">Phyllo</Emphasis>geography of the Carnivorous Plant <Emphasis Type="BoldItalic">Sarracenia alata</Emphasis>

Carnivorous pitcher plants host diverse microbial communities. This plant–microbe association provides a unique opportunity to investigate the evolutionary processes that influence the spatial diversity of microbial communities. Using next-generation sequencing of environmental samples, we surveyed microbial communities from 29 pitcher plants (Sarracenia alata) and compare community composition with plant genetic diversity in order to explore the influence of historical processes on the population structure of each lineage. Analyses reveal that there is a core S. alata microbiome, and that it is similar in composition to animal gut microfaunas. The spatial structure of community composition in S. alata (phyllogeography) is congruent at the deepest level with the dominant features of the landscape, including the Mississippi river and the discrete habitat boundaries that the plants occupy. Intriguingly, the microbial community structure reflects the phylogeographic structure of the host plant, suggesting that the phylogenetic structure of bacterial communities and population genetic structure of their host plant are influenced by similar historical processes.     

Comparing EST-based genetic maps between <Emphasis Type="BoldItalic">Pinus sylvestris</Emphasis> and <Emphasis Type="BoldItalic">Pinus taeda</Emphasis>

A genetic map of Pinus sylvestris was constructed using ESTP (expressed sequence tag polymorphism) markers and other gene-based markers, AFLP markers and microsatellites. Part of the ESTP markers (40) were developed and mapped earlier in Pinus taeda, and additional markers were generated based on P. sylvestris sequences or sequences from other pine species. The mapping in P. sylvestris was based on 94 F₁ progeny from a cross between plus-tree parents E635C and E1101. AFLP framework maps for the parent trees were first constructed. The ESTP and other gene sequence-based markers were added to the framework maps, as well as five published microsatellite loci. The separate maps were then integrated with the aid of AFLPs segregating in both trees (dominant segregation ratios 3:1) as well as gene markers and microsatellites segregating in both parent trees (segregation ratios 1:1:1:1 or 1:2:1). The integrated map consisted of 12 groups corresponding to the P. taeda linkage groups, and additionally three and six smaller groups for E1101 and E635C, respectively. The number of framework AFLP markers in the integrated map is altogether 194 and the number of gene markers 61. The total length of the integrated map was 1,314 cM. The set of markers developed for P. sylvestris was also added to existing maps of two P. taeda pedigrees. Starting with a mapped marker from one pedigree in the source species resulted in a mapped marker in a pedigree of the other species in more than 40% of the cases, with about equal success in both directions. The maps of the two species are largely colinear, even if the species have diverged more than 70 MYA. Most cases of different locations were probably due to problems in identifying the orthologous members of gene families. These data provide a first ESTP-containing map of P. sylvestris, which can also be used for comparing this species to additional species mapped with the same markers.Communicated by C. Möllers     

Cells of <Emphasis Type="BoldItalic">Candida utilis</Emphasis> for <Emphasis Type="BoldItalic">in vitro</Emphasis> (<Emphasis Type="BoldItalic">R</Emphasis>)-phenylacetylcarbinol production in an aqueous/octanol two-phase reactor

(R)-Phenylacetylcarbinol (PAC), a pharmaceutical precursor, was produced from benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) of Candida utilis in an aqueous/organic two-phase emulsion reactor. When the partially purified enzyme in this previously established in vitro process was replaced with C. utilis cells and the temperature was increased from 4 to 21 °C, a screen of several 1-alcohols (C4–C9) confirmed the suitability of 1-octanol as the organic phase. Benzyl alcohol, the major by-product in the commercial in vivo conversion of benzaldehyde and sugar to PAC by Saccharomyces cerevisiae, was not formed. With a phase volume ratio of 1:1 and 5.6 g C. utilis l⁻¹ (PDC activity 2.5 U ml⁻¹), PAC levels of 103 g l⁻¹ in the octanol phase and 12.8 g l⁻¹ in the aqueous phase were produced in 15 h at 21 °C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 °C, PAC was produced at a 4-times increased specific rate (1.54 versus 0.39 mg U⁻¹ h⁻¹) with simplified catalyst production and reduced cooling cost. Compared to traditional in vivo whole cell PAC production, the yield on benzaldehyde was 26% higher, the product concentration increased 3.9-fold (or 6.9-fold based on the organic phase), the productivity improved 3.1-fold (3.9 g l⁻¹ h⁻¹) and the catalyst was 6.9-fold more efficient (PAC/dry cell mass 10.3 g g⁻¹).*Dedicated with gratitude to Prof. Dr. Franz Lingens – “Theo”.     

Preparation and regeneration of spheroplasts from <Emphasis Type="BoldItalic">Arthrospira</Emphasis><Emphasis Type="BoldItalic">platensis</Emphasis> (Spirulina)

Ultrasonic pretreatment, lysozyme, inorganic osmotics and bovine albumin were used to prepare the spheroplasts of Arthrospira platensis (Spirulina platensis). The average cell number of the fragments from the filaments of strain A9 was about 2.2 cells after 80-s ultrasonic pretreatment. These fragments could regenerate and were suitable material for isolating spheroplasts, so the optimum conditions for doing this were investigated. The best enzymolysis parameters were designed. During the isolation process, gentle shaking of the enzymolysis sample for several times greatly enhanced the proportion of spheroplasts. However, no spheroplasts were obtained when organic compounds were used as osmotics. The spheroplasts could form typical colonies on plate of inorganic medium, with a regeneration rate of about 3%. These spheroplasts might be used as competence cells to carry on the research of genetic transformation.     

Ammonium chloride: a novel effective and inexpensive salt solution for phycocyanin extraction from <Emphasis Type="BoldItalic">Arthrospira</Emphasis> (<Emphasis Type="BoldItalic">Spirulina</Emphasis>) <Emphasis Type="BoldItalic">platensis</Emphasis>

Phycocyanin, a blue pigment, is a type of phycobiliproteins. Because of its various potential properties, phycocyanin is applied to various fields, such as nutraceutical, pharmaceutical, medicine, cosmetics, and biotechnological research. The cost and application of phycocyanin are highly dependent on its purity index. In this study, ammonium chloride is presented as a novel, effective, and inexpensive salt for phycocyanin extraction. Compared with sodium phosphate, which is commonly used during phycocyanin extraction process, ammonium chloride solution efficiently extracted phycocyanin with high purity from Arthrospira platensis FACHB-314. In addition, ammonium phosphate solution is also presented as an alternative precipitation agent in phycocyanin purification that may replace the widely used ammonium sulfate. Statistical analysis shows that there is no significant difference in phycocyanin concentration between crude extracts (overall mean of 0.208 and 0.215 for extraction using sodium phosphate and ammonium chloride, respectively). However, the difference in phycocyanin purity ratio (A₆₂₀/A₂₈₀) between these two extractions is significant (overall mean of 0.742 and 1.428 for extraction using sodium phosphate and ammonium chloride, respectively). With ammonium chloride, the purity indexes of phycocyanin are 1.5 and 2.81 after the optimum extraction step, and precipitation used as the primary purification step, respectively. The present study describes a novel purification method to achieve phycocyanin with analytical grade without multiple purification steps.     

Immunocytochemical localisation of calreticulin in<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis>

Calreticulin, a Ca²⁺ chaperone, is found in many different locations in various eukaryotic cells, including lumen of the endoplasmic reticulum, the cell surface, perinuclear areas and cytosolic granules. In the present study, a polyclonal antibody against calreticulin was used for the immunocytochemical localisation of the protein in Trypanosoma cruzi. Labelling was observed in the endoplasmic reticulum, Golgi complex, reservosomes, flagellar pocket, cell surface, cytosol, nucleus and kinetoplast. Significant differences in labelling were observed among the three evolutive forms of the protozoan. The functional role of calreticulin in T. cruzi is discussed.     

<Emphasis Type="Bold">Report from the 33rd</Emphasis><Emphasis Type="BoldItalic">Mycotoxin-Workshop</Emphasis><Emphasis Type="Bold">(Freising,Germany)</Emphasis>

Society for Mycotoxin Research – News and Announcements 2011/II

Report from the 33rd Mycotoxin-Workshop (Freising, Germany)