Zygocotyle lunata: proteomic analysis of the adult stage

The somatic extract of Zygocotyle lunata (Trematoda: Paramphistomidae) adults collected from experimentally infected mice was investigated using a proteomic approach to separate and identify tryptic peptides from the somatic extract of Z. lunata adult worms. A shot-gun liquid chromatography/tandem mass spectrometry procedure was used. We used the MASCOT search engine (Matrix-Science) and ProteinPilot software v2.0 (Applied Biosystems) for the database search. A total of 36 proteins were accurately identified from the worms. The largest protein family consisted of metabolic enzymes. Structural, motor and receptor binding proteins and proteins related to oxygen transport were identified in the somatic extract of Z. lunata. This is the first study that attempts to identify the proteome of Z. lunata. However, more work is needed to improve our knowledge of trematodiasis in general and more specifically to have a better understanding about host–parasite relationships in infections with paramphistomes.     

The chemical chaperones tauroursodeoxycholic and 4-phenylbutyric acid accelerate thyroid hormone activation and energy expenditure

Exposure of cell lines endogenously expressing the thyroid hormone activating enzyme type 2 deiodinase (D2) to the chemical chaperones tauroursodeoxycholic acid (TUDCA) or 4-phenylbutiric acid (4-PBA) increases D2 expression, activity and T3 production. In brown adipocytes, TUDCA or 4-PBA induced T3-dependent genes and oxygen consumption (∼2-fold), an effect partially lost in D2 knockout cells. In wild type, but not in D2 knockout mice, administration of TUDCA lowered the respiratory quotient, doubled brown adipose tissue D2 activity and normalized the glucose intolerance associated with high fat feeding. Thus, D2 plays a critical role in the metabolic effects of chemical chaperones.     

Labeling and enzyme studies of the central carbon metabolism in Metallosphaera sedula

Metallosphaera sedula (Sulfolobales, Crenarchaeota) uses the 3-hydroxypropionate/4-hydroxybutyrate cycle for autotrophic carbon fixation. In this pathway, acetyl-coenzyme A (CoA) and succinyl-CoA are the only intermediates that can be considered common to the central carbon metabolism. We addressed the question of which intermediate of the cycle most biosynthetic routes branch off. We labeled autotrophically growing cells by using 4-hydroxy[1-¹⁴C]butyrate and [1,4-¹³C₁]succinate, respectively, as precursors for biosynthesis. The labeling patterns of protein-derived amino acids verified the operation of the proposed carbon fixation cycle, in which 4-hydroxybutyrate is converted to two molecules of acetyl-CoA. The results also showed that major biosynthetic flux does not occur via acetyl-CoA, except for the formation of building blocks that are directly derived from acetyl-CoA. Notably, acetyl-CoA is not assimilated via reductive carboxylation to pyruvate. Rather, our data suggest that the majority of anabolic precursors are derived from succinyl-CoA, which is removed from the cycle via oxidation to malate and oxaloacetate. These C₄ intermediates yield pyruvate and phosphoenolpyruvate (PEP). Enzyme activities that are required for forming intermediates from succinyl-CoA were detected, including enzymes catalyzing gluconeogenesis from PEP. This study completes the picture of the central carbon metabolism in autotrophic Sulfolobales by connecting the autotrophic carbon fixation cycle to the formation of central carbon precursor metabolites.Sulfolobales (Crenarchaeota) comprise extreme thermoacidophiles from volcanic areas that grow best at a pH of around 2 and a temperature of 60 to 90°C (32, 33). Most Sulfolobales can grow chemoautotrophically on sulfur, pyrite, or H₂ under microaerobic conditions, which also applies to Metallosphaera sedula (31), the organism studied here. Its genome has been sequenced (2). Some species of the Sulfolobales secondarily returned to a facultative anaerobic or even strictly anaerobic life style (33), and some laboratory strains appear to have lost their ability to grow autotrophically (8). Autotrophic representatives of the Sulfolobales use a 3-hydroxypropionate/4-hydroxybutyrate cycle (in short, hydroxypropionate/hydroxybutyrate cycle) for autotrophic carbon fixation (Fig. ​(Fig.1)1) (6-8, 38). The enzymes of this cycle are oxygen tolerant, which predestines the cycle for the lifestyle of the aerobic Crenarchaeota (8). The presence of genes coding for key enzymes of the hydroxypropionate/hydroxybutyrate cycle in the mesophilic aerobic “marine group I” Crenarchaeota suggests that these abundant marine archaea use a similar autotrophic carbon fixation mechanism (6, 24, 68) (for a review of autotrophic carbon fixation in Archaea, see reference 7).Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle functioning in autotrophic carbon fixation in Sulfolobales and its relation to the central carbon metabolism, as studied in this work for Metallosphaera sedula. The situation may be similar in other Sulfolobales and possibly in autotrophic marine Crenarchaeota. Enzymes: 1, acetyl-CoA/propionyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonic semialdehyde reductase (NADPH); 4, 3-hydroxypropionate-CoA ligase (AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, acetyl-CoA/propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH); 11, succinic semialdehyde reductase (NADPH); 12, 4-hydroxybutyrate-CoA ligase (AMP forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14 and 15, crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 16, acetoacetyl-CoA β-ketothiolase; 17, succinyl-CoA synthetase (ADP forming); 18, succinic semialdehyde dehydrogenase; 19, succinate dehydrogenase (natural electron acceptor unknown); 20, fumarate hydratase; 21, malate dehydrogenase; 22, malic enzyme; 23, PEP carboxykinase (GTP); 24, pyruvate:water dikinase (ATP); 25, enolase; 26, phosphoglycerate mutase; 27, phosphoglycerate kinase; 28, glyceraldehyde 3-phosphate dehydrogenase; 29, triosephosphate isomerase; 30, fructose 1,6-bisphosphate aldolase/phosphatase; 31, (si)-citrate synthase; 32, aconitase; 33, isocitrate dehydrogenase.In the cycle, one molecule of acetyl-coenzyme A (CoA) is formed from two molecules of bicarbonate. The key carboxylating enzyme is a bifunctional biotin-dependent acetyl-CoA/propionyl-CoA carboxylase (10, 11, 36, 38, 48, 49). In Bacteria and Eukarya, acetyl-CoA carboxylase catalyzes the first step in fatty acid biosynthesis. However, archaea do not contain fatty acids, and therefore acetyl-CoA carboxylase obviously plays a different metabolic role. The hydroxypropionate/hydroxybutyrate cycle can be divided into two parts. The first transforms acetyl-CoA and two bicarbonate molecules via 3-hydroxypropionate to succinyl-CoA, and the second converts succinyl-CoA via 4-hydroxybutyrate to two acetyl-CoA molecules. In brief, the product of the acetyl-CoA carboxylase reaction, malonyl-CoA, is reduced via malonic semialdehyde to 3-hydroxypropionate, which is further reductively converted to propionyl-CoA. Propionyl-CoA is carboxylated to (S)-methylmalonyl-CoA by the same carboxylase as that that carboxylates acetyl-CoA (11, 36). (S)-Methylmalonyl-CoA is isomerized to (R)-methylmalonyl-CoA, followed by carbon rearrangement to succinyl-CoA catalyzed by coenzyme B₁₂-dependent methylmalonyl-CoA mutase.Succinyl-CoA then is converted into two molecules of acetyl-CoA via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA, crotonyl-CoA, 3-hydroxyacetyl-CoA, and acetoacetyl-CoA. This reaction sequence apparently is common to the autotrophic Crenarchaeota, as it also is used by autotrophic Crenarchaeota of the orders Thermoproteales and Desulfurococcales, which use a dicarboxylate/4-hydroxybutyrate cycle for autotrophic carbon fixation (8, 34, 55, 56) (also see the accompanying work [57]).From the list of intermediates of the hydroxypropionate/hydroxybutyrate cycle, acetyl-CoA and succinyl-CoA are the only intermediates considered common to the central carbon metabolism. In this work, we addressed the question of which intermediate of the cycle most biosynthetic routes branch off, and we came to the conclusion that succinyl-CoA serves as the main precursor for cellular carbon. This requires one turn of the cycle to regenerate the CO₂ acceptor and to generate one extra molecule of acetyl-CoA from two molecules of bicarbonate. Acetyl-CoA plus another two bicarbonate molecules are converted by an additional half turn of the cycle to succinyl-CoA. This strategy differs from that of the anaerobic pathways, in which acetyl-CoA is reductively carboxylated to pyruvate, and from there the other precursors for building blocks ultimately are derived (discussed in reference 7).     

Evolutionary implications of morphogenesis and molecular patterning of the blind gut in the planarian Schmidtea polychroa

The formation of a through-gut was a key innovation in the evolution of metazoans. There is still controversy regarding the origin of the anus and how it may have been either gained or lost during evolution in different bilaterian taxa. Thus, the study of groups with a blind gut is of great importance for understanding the evolution of this organ system. Here, we describe the morphogenesis and molecular patterning of the blind gut in the sexual triclad Schmidtea polychroa. We identify and analyze the expression of goosecoid, commonly associated with the foregut, and the GATA, ParaHox and T-box genes, members of which commonly are associated with gut regionalization. We show that GATA456a is expressed in the blind gut of triclads, while GATA456b is localized in dorsal parenchymal cells. Goosecoid is expressed in the central nervous system, and the unique ParaHox gene identified, Xlox, is detected in association with the nervous system. We have not isolated any brachyury gene in the T-box complement of S. polychroa, which consists of one tbx1/10, three tbx2/3 and one tbx20. Furthermore, the absence of genes like brachyury and caudal is also present in other groups of Platyhelminthes. This study suggests that GATA456, in combination with foxA, is a gut-specific patterning mechanism conserved in the triclad S. polychroa, while the conserved gut-associated expression of foregut, midgut and hindgut markers is absent. Based on these data and the deviations in spiral cleavage found in more basal flatworms, we propose that the lack of an anus is an innovation of Platyhelminthes. This may be associated with loss of gut gene expression or even gene loss.     

Ca2+ binding alters the interdomain flexibility between the two cytoplasmic calcium-binding domains in the Na+/Ca2+ exchanger

The Na(+)/Ca(2+) exchanger (NCX) is a membrane protein, which catalyzes the counter transport of Na(+) and Ca(2+) ions across the plasma membrane, playing a key role in the maintenance of the intracellular Ca(2+) homeostasis in various cell types. NCX consists of a transmembrane part and a large intracellular loop. The activation of the NCX transport function requires the binding of Ca(2+) to two tandem C2 domains, CBD1 and CBD2, which are an integral part of the exchanger's intracellular loop. Although high-resolution structures of individual CBD1 and CBD2 are available, their interdomain structure and dynamics and the atomic level mechanism of allosteric Ca(2+)-regulation remains unknown. Here, we use solution NMR spectroscopy to study the interdomain dynamics of CBD12, a 32 kDa construct that contains both the CBD1 and CBD2 domains connected by a short linker. Analysis of NMR residual dipolar couplings shows that CBD12 assumes on average an elongated shape both in the absence and in the presence of Ca(2+). NMR (15)N relaxation data of the Apo state indicate that the two domains sample a wide range of relative arrangements on the nanosecond time scale. These arrangements comprise significantly non-linear interdomain orientations. Binding of Ca(2+) to CBD1 significantly restricts the interdomain flexibility, stabilizing a more rigid elongated conformation. These findings suggest a molecular mechanism for the role of CBD12 in the function of NCX.     

Savanna soil fertility limits growth but not survival of tropical forest tree seedlings

Background and Aims

Cerradão (Brazilian woodland savannas) and seasonally dry forests (SDF) from southeastern Brazil occur under the same climate but are remarkably distinct in species composition. The objective of this study was to evaluate the role of soil origin in the initial growth and distribution of SDF and Cerradão species.

Methods

We conducted a greenhouse experiment growing Cerradão and SDF tree seedlings over their soil and the soil of the contrasting vegetation type. We evaluated soil nutrient availability and seedling survivorship, growth and leaf functional traits.

Results

Despite the higher nutrient availability in SDF soils, soil origin did not affect seedling survivorship. The three SDF species demonstrated home-soil advantage, enhanced growth with increasing soil nutrient availability and had higher growth rates than Cerradão species, even on Cerradão soils. Growth of Cerradão seedlings was not higher on Cerradão soil and, overall, was not positively correlated with soil nutrient availability.

Conclusions

SDF species are fast-growing species while Cerradão trees tend to be slow-growing species. Although savanna soil reduces growth of forest species, our findings suggest that soil chemical attributes, alone, does not exclude the occurrence of SDF seedlings in Cerradão and vice-versa.     

A new species of Heliocephala from Mexico with an assessment of the systematic positions of the anamorph genera Heliocephala and Holubovaniella

During a study of the microfungi on decaying leaves collected in México we encountered a fungus sharing features of the anamorph genera Heliocephala and Holubovaniella. This hyphomycete resembled Hol. elegans in possessing obclavate to fusiform, three-septate, rostrate phragmoconidia, but it can be distinguished by its much shorter, branched, determinate conidiophores. Unlike members of genus Heliocephala, which also possess determinant conidiophores, the phragmoconidia of this taxon are short-rostrate and have not been observed to germinate iteratively. Prompted by the discovery of this hyphomycete, we investigated the phylogenetic relationships among species of Heliocephala and Holubovaniella. Analysis of large subunit rDNA gene sequences positioned species of Heliocephala and Holubovaniella in the Dothideomycetes and identified Stomiopeltis betulae (Micropeltididaceae) as their closest relative. These results also indicated that Heliocephala and Holubovaniella are closely related taxa. Based on these findings and the similarity of the developmental and morphological characters of species of Heliocephala and Holubovaniella, we recognize these genera as synonymous and describe a new species, Heliocephala triseptata. A dichotomous key to the species of Heliocephala is provided.