Expression of hepatic genes related to energy metabolism during the transition period of Holstein and F1 Holstein-Gir cows.

The aim of this study was to investigate the expression of genes encoding enzymes and other factors involved with carbohydrate and lipid metabolism in the liver of 2 genetic groups of dairy cows during the transition period. We analyzed the expression of glucose-6-phosphatase (G6PC), cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), methylmalonyl-CoA mutase (MUT), ß-hydroxybutyrate dehydrogenase-2 (BDH2), acetyl-CoA carboxylase (ACC), carnitine palmitoyltransferase-2 (CPT2), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), glucose transporter-2 (SLC2A2), and the transcription factor peroxisome proliferator-activated receptor α (PPARA). Blood concentrations of glucose, nonesterified fatty acids, and ß-hydroxybutyrate were also determined. Liver biopsies and blood samples were taken at d 15 prepartum and at d 6, 21, 36, 51, and 66 postpartum from Holsteins (n = 6) and F1 Holstein-Gir (n = 6) cows. Cows were kept under the same prepartum and postpartum management conditions. The results showed that the expression of G6PC, PEPCK-C, BDH2, ACC, CPT2, HMGCR, SLC2A2, and PPARA genes did not differ between genetic groups. Except for PEPCK-C, no interaction between genetic groups and the experimental period was observed. Within both groups of cows, G6PC and PEPCK-C gene expression decreased when comparing prepartum gene expression with 21 and 36 DIM, and increased in d 51 postpartum. MUT mRNA levels differed between the 2 genetic groups and displayed a significant increase after d 36 postpartum, whereas mRNA levels of HMGCR tended to increase when comparing d 21 and 36 to d 51 postpartum. Glucose concentrations also differed between genetic groups, being significantly higher in the plasma of F1 Holstein-Gir cows than in Holstein cows, but no differences were found within each group during the analysis period. ß-Hydroxybutyrate and nonesterified fatty acid concentrations did not differ between genetic groups, but displayed increased levels from prepartum to d 6 and 21 postpartum. Our results indicated that expression in the liver of genes involved with glucose and fatty acid metabolism were similar in both groups of cows and significant differences were observed between the 2 groups in the expression of MUT, a gene involved in propionate metabolism.

Repeat-enriched proteins are related to host cell invasion and immune evasion in parasitic protozoa.

Proteins containing repetitive amino acid domains are widespread in all life forms. In parasitic organisms, proteins containing repeats play important roles such as cell adhesion and invasion and immune evasion. Therefore, extracellular and intracellular parasites are expected to be under different selective pressures regarding the repetitive content in their genomes. Here, we investigated whether there is a bias in the repetitive content found in the predicted proteomes of 6 exclusively extracellular and 17 obligate intracellular protozoan parasites, as well as 4 free-living protists. We also attempted to correlate the results with the distinct ecological niches they occupy and with distinct protein functions. We found that intracellular parasites have higher repetitive content in their proteomes than do extracellular parasites and free-living protists. In intracellular parasites, these repetitive proteins are located mainly at the parasite surface or are secreted and are enriched in amino acids known to be part of N- and O-glycosylation sites. Furthermore, in intracellular parasites, the developmental stages that are able to invade host cells express a higher proportion of proteins with perfect repeats relative to other life cycle stages, and these proteins have molecular functions associated with cell invasion. In contrast, in extracellular parasites, degenerate repetitive motifs are enriched in proteins that are likely to play roles in evading host immune response. Altogether, our results support the hypothesis that both the ability to invade host cells and to escape the host immune response may have shaped the expansion and maintenance of perfect and degenerate repeats in the genomes of intra- and extracellular parasites.

Cell culture and animal infection with distinct Trypanosoma cruzi strains expressing red and green fluorescent proteins.

Different strains of Trypanosoma cruzi were transfected with an expression vector that allows the integration of green fluorescent protein (GFP) and red fluorescent protein (RFP) genes into the beta-tubulin locus by homologous recombination. The sites of integration of the GFP and RFP markers were determined by pulse-field gel electrophoresis and Southern blot analyses. Cloned cell lines selected from transfected epimastigote populations maintained high levels of fluorescent protein expression even after 6 months of in vitro culture of epimastigotes in the absence of drug selection. Fluorescent trypomastigotes and amastigotes were observed within Vero cells in culture as well as in hearts and diaphragms of infected mice. The infectivity of the GFP- and RFP-expressing parasites in tissue culture cells was comparable to wild type populations. Furthermore, GFP- and RFP-expressing parasites were able to produce similar levels of parasitemia in mice compared with wild type parasites. Cell cultures infected simultaneously with two cloned cell lines from the same parasite strain, each one expressing a distinct fluorescent marker, showed that at least two different parasites are able to infect the same cell. Double-infected cells were also detected when GFP- and RFP-expressing parasites were derived from strains belonging to two distinct T. cruzi lineages. These results show the usefulness of parasites expressing GFP and RFP for the study of various aspects of T. cruzi infection including the mechanisms of cell invasion, genetic exchange among parasites and the differential tissue distribution in animal models of Chagas disease.

Analysis of DNA polymerase activity in vitro using non-radioactive primer extension assay in an automated DNA sequencer.

Although different DNA polymerases have distinct functions and substrate affinities, their general mechanism of action is similar. Thus, they can all be studied using the same technical principle, the primer extension assay employing radioactive tags. Even though fluorescence has been used routinely for many years for DNA sequencing, it has not been used in the in vitro primer extension assay. The use of fluorescence labels has obvious advantages over radioactivity, including safety, speed and ease of manipulation. In the present study, we demonstrated the potential of non-radioactive in vitro primer extension for DNA polymerase studies. By using an M13 tag in the substrate, we can use the same fluorescent M13 primer to study different substrate sequences. This technique allows quantification of the DNA polymerase activity of the Klenow fragment using different templates and under different conditions with similar sensitivity to the radioactive assay.

Characterization of a Trypanosoma cruzi antigen with homology to intracellular mammalian lectins.

Two cDNAs, isolated from a Trypanosoma cruzi amastigote library immunoscreened with sera from patients with Chagas disease, encode proteins with sequence homology to eukaryotic components of the cellular sorting and recycling machinery. These proteins, denominated TcAGL, present an N-terminal lectin domain and a C-terminal region containing repetitive amino acids and a poly-glutamine tract. They are products of polymorphic alleles of a single copy gene constitutively expressed during the parasite life cycle. Polyclonal antibodies obtained from mice immunized with the recombinant antigen recognize proteins with apparent molecular weight ranging from 95 to 120 kDa in cell lysates from all three life stages and in various strains of the parasite. Sera from Chagas disease patients recognize the recombinant antigen in ELISA and immunoprecipitation assays but not in Western blot assays under denaturing conditions. Consistent with its proposed role in the glycoprotein secreting pathway, immunofluorescence analyses and expression of a green fluorescent protein-tagged TcAGL protein indicate a sub-cellular localization in the vicinity of the flagellar pocket membrane and the Golgi complex of the parasite.

Control of gene expression in Trypanosomatidae

The study of mechanisms which control gene expression in trypanosomatids has developed at an increasing rate since 1989 when the first successful DNA transfection experiments were reported. Using primarily Trypanosoma brucei as a model, several groups have begun to elucidate the basic control mechanisms and to define the cellular factors involved in mRNA transcription, processing and translation in these parasites. This review focuses on the most recent studies regarding a subset of genes that are expressed differentially during the life cycle of three groups of parasites. In addition to T. brucei, I will address studies on gene regulation in a few species of Leishmania and the results obtained by a much more limited group of laboratories studying gene expression in Trypanosoma cruzi. It is becoming evident that the regulatory strategies chosen by different species of trypanosomatids are not similar, and that for these very successful parasites it is probably advantageous to employ multiple mechanisms simultaneously. In addition, with the increasing numbers of parasite genes that have now been submitted to molecular dissection, it is also becoming evident that, among the various strategies for gene expression control, there is a predominance of regulatory pathways acting at the post-transcriptional level