Jorge Moraes

Binding and storage of heme by vitellin from the cattle tick, Boophilus microplus

We have previously shown (, Curr. Biol. 9, 703-706) that the cattle tick Boophilus microplus does not synthesize heme, relying solely on the recovery of the heme from the diet to make all its hemeproteins. Here we present evidence that Vitellin (VN(1)), the main tick yolk protein, is a reservoir of heme for embryo development. VN was isolated from eggs at different days throughout embryogenesis. Immediately after oviposition, Boophilus VN contains approximately one mol of heme/mol of protein. During embryo development about one third of egg VN is degraded. The remaining VN molecules bind part of the heme released. These results suggest that VN functions as a heme reservoir, binding any free heme that exceeds the amount needed for development. In vitro measurement of the binding of heme to VN showed that each VN molecule binds up to 31 heme molecules. The association of heme with VN strongly inhibits heme-induced lipid peroxidation, suggesting that binding of heme is an important antioxidant mechanism to protect embryo cells from oxidative damage. This mechanism allows this hematophagous arthropod to safely store heme obtained from a blood meal inside their eggs for future use. Taken together our data suggest that, besides its known roles, VN also plays additional functions as a heme deposit and an antioxidant protective molecule.

Germ band retraction as a landmark in glucose metabolism during Aedes aegypti embryogenesis

BackgroundThe mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown.ResultsGlucose metabolism was investigated throughout Aedes aegypti (Diptera) embryonic development. Both cellular blastoderm formation (CBf, 5 h after egg laying - HAE) and germ band retraction (GBr, 24 HAE) may be considered landmarks regarding glucose 6-phosphate (G6P) destination. We observed high levels of glucose 6-phosphate dehydrogenase (G6PDH) activity at the very beginning of embryogenesis, which nevertheless decreased up to 5 HAE. This activity is correlated with the need for nucleotide precursors generated by the pentose phosphate pathway (PPP), of which G6PDH is the key enzyme. We suggest the synchronism of egg metabolism with carbohydrate distribution based on the decreasing levels of phosphoenolpyruvate carboxykinase (PEPCK) activity and on the elevation observed in protein content up to 24 HAE. Concomitantly, increasing levels of hexokinase (HK) and pyruvate kinase (PK) activity were observed, and PEPCK reached a peak around 48 HAE. Glycogen synthase kinase (GSK3) activity was also monitored and shown to be inversely correlated with glycogen distribution during embryogenesis.ConclusionsThe results herein support the hypothesis that glucose metabolic fate changes according to developmental embryonic stages. Germ band retraction is a moment that was characterized as a landmark in glucose metabolism during Aedes aegypti embryogenesis. Furthermore, the results also suggest a role for GSK3 in glycogen balance/distribution during morphological modifications.

Glycogen and Glucose Metabolism Are Essential for Early Embryonic Development of the Red Flour Beetle Tribolium castaneum

Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.

Trypanosoma cruzi : IgG1 and IgG2b are the main immunoglobulins produced by vaccinated mice

Abstract Mice vaccinated with CL-14, a non-infective and non-pathogenic clone isolated from Trypanosoma cruzi CL strain, become protected against lethal challenge by infective trypomastigotes. It has been shown that animals infected with T. cruzi show polyclonal activation of B lymphocytes with an early production of several non-specific immunoglobulins. Vaccinated mice, however, have an early production of antigen-specific IgG1 and IgG2b. Considering the lack of infectivity of CL-14, our data strongly suggest a role for IgG1 and IgG2b in protection to T. cruzi.

The embryogenesis of the Tick Rhipicephalus (Boophilus) microplus: The establishment of a new chelicerate model system

Summary: Chelicerates, which include spiders, ticks, mites, scorpions, and horseshoe crabs, are members of the phylum Arthropoda. In recent years, several molecular experimental studies of chelicerates have examined the embryology of spiders; however, the embryology of other groups, such as ticks (Acari: Parasitiformes), has been largely neglected. Ticks and mites are believed to constitute a monophyletic group, the Acari. Due to their blood‐sucking activities, ticks are also known to be vectors of several diseases. In this study, we analyzed the embryonic development of the cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). First, we developed an embryonic staging system consisting of 14 embryonic stages. Second, histological analysis and antibody staining unexpectedly revealed the presence of a population of tick cells with similar characteristics to the spider cumulus. Cumulus cell populations also exist in other chelicerates; these cells are responsible for the breaking of radial symmetry through bone morphogenetic protein signaling. Third, it was determined that the posterior (opisthosomal) embryonic region of R. microplus is segmented. Finally, we identified the presence of a transient ventral midline furrow and the formation and regression of a fourth leg pair; these features may be regarded as hallmarks of late tick embryogenesis. Importantly, most of the aforementioned features are absent from mite embryos, suggesting that mites and ticks do not constitute a monophyletic group or that mites have lost these features. Taken together, our findings provide fundamental common ground for improving knowledge regarding tick embryonic development, thereby facilitating the establishment of a new chelicerate model system. genesis 51:803–818.

Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus.

Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 μmol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.

Vaccination with cyclin-dependent kinase tick antigen confers protection against Ixodes infestation.

Among arthropods, ticks lead as vectors of animal diseases and rank second to mosquitoes in transmitting human pathogens. Cyclin-dependent kinases (CDK) participate in cell cycle control in eukaryotes. CDKs are serine/threonine protein kinases and these catalytic subunits are activated or inactivated at specific stages of the cell cycle. To determine the potential of using CDKs as anti-tick vaccine antigens, hamsters were immunized with recombinant Ixodes persulcatus CDK10, followed by a homologous tick challenge. Though it was not exactly unexpected, IpCDK10 vaccination significantly impaired tick blood feeding and fecundity, which manifested as low engorgement weights, poor oviposition, and a reduction in 80% of hatching rates. These findings may underpin the development of more efficacious anti-tick vaccines based on the targeting of cell cycle control proteins.

Non-classical gluconeogenesis-dependent glucose metabolism in Rhipicephalus microplus embryonic cell line BME26.

In this work we evaluated several genes involved in gluconeogenesis, glycolysis and glycogen metabolism, the major pathways for carbohydrate catabolism and anabolism, in the BME26 Rhipicephalus microplus embryonic cell line. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by alterations in energy resource availability (primarily glucose). BME26 cells in media were investigated using three different glucose concentrations, and changes in the transcription levels of target genes in response to carbohydrate utilization were assessed. The results indicate that several genes, such as glycogen synthase (GS), glycogen synthase kinase 3 (GSK3), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6 phosphatase (GP) displayed mutual regulation in response to glucose treatment. Surprisingly, the transcription of gluconeogenic enzymes was found to increase alongside that of glycolytic enzymes, especially pyruvate kinase, with high glucose treatment. In addition, RNAi data from this study revealed that the transcription of gluconeogenic genes in BME26 cells is controlled by GSK-3. Collectively, these results improve our understanding of how glucose metabolism is regulated at the genetic level in tick cells.

Inhibition of Enzyme Activity of Rhipicephalus (Boophilus) microplus Triosephosphate Isomerase and BME26 Cell Growth by Monoclonal Antibodies

In the present work, we produced two monoclonal antibodies (BrBm37 and BrBm38) and tested their action against the triosephosphate isomerase of Rhipicephalus (Boophilus) microplus (RmTIM). These antibodies recognize epitopes on both the native and recombinant forms of the protein. rRmTIM inhibition by BrBm37 was up to 85% whereas that of BrBrm38 was 98%, depending on the antibody-enzyme ratio. RmTIM activity was lower in ovarian, gut, and fat body tissue extracts treated with BrBm37 or BrBm38 mAbs. The proliferation of the embryonic tick cell line (BME26) was inhibited by BrBm37 and BrBm38 mAbs. In summary, the results reveal that it is possible to interfere with the RmTIM function using antibodies, even in intact cells.

Inorganic polyphosphates regulate hexokinase activity and reactive oxygen species generation in mitochondria of Rhipicephalus (Boophilus) microplus embryo.

The physiological roles of polyphosphates (poly P) recently found in arthropod mitochondria remain obscure. Here, the possible involvement of poly P with reactive oxygen species generation in mitochondria of Rhipicephalus microplus embryos was investigated. Mitochondrial hexokinase and scavenger antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione reductase were assayed during embryogenesis of R. microplus. The influence of poly P3 and poly P15 were analyzed during the period of higher enzymatic activity during embryogenesis. Both poly Ps inhibited hexokinase activity by up to 90% and, interestingly, the mitochondrial membrane exopolyphosphatase activity was stimulated by the hexokinase reaction product, glucose-6-phosphate. Poly P increased hydrogen peroxide generation in mitochondria in a situation where mitochondrial hexokinase is also active. The superoxide dismutase, catalase and glutathione reductase activities were higher during embryo cellularization, at the end of embryogenesis and during embryo segmentation, respectively. All of the enzymes were stimulated by poly P3. However, superoxide dismutase was not affected by poly P15, catalase activity was stimulated only at high concentrations and glutathione reductase was the only enzyme that was stimulated in the same way by both poly Ps. Altogether, our results indicate that inorganic polyphosphate and mitochondrial membrane exopolyphosphatase regulation can be correlated with the generation of reactive oxygen species in the mitochondria of R. microplus embryos.