Growth-promoting activity of desmopressin in murine leukemia cells treated in vitro

The synthetic vasopressin analogue, desmopressin (dDAVP), has been shown to influence membrane transport of melphalan in murine L5178Y lymphoblasts. Accordingly, the effect of dDAVP on the cytocidal activity of melphalan in L5178Y cells was evaluated. dDAVP did not affect the cytocidal activity of melphalan in these cells, but significantly affected the cloning efficiency of stationary phase or slowly dividing L5178Y cells over a range of concentrations. In particular, stationary phase cells showed an increase in cloning efficiency from 4.3 ± 0.5% in control cells to 7.0 ± 0.3% in cells treated with 25 nM dDAVP (P < 0.001), whereas cells doubling every 26 h showed an increase from 10.8 ± 1.2% in control cells to 21.0 ± 2.0% in cells treated with 150 nM dDAVP (P < 0.001). This phenomenon was associated with significant elevations of 1,2[3H] diacylglycerol after incubation with dDAVP for 9 min (P < 0.01) and total [3H]diacylglycerols after incubation for both 3 min (P < 0.05) and 9 min (P < 0.02). Within 10 s of treatment with 100 nM dDAVP, there was a marked decrease in the levels of inositol 1,4,5-trisphosphate and inositol 1-phosphate, but subsequently no change was observed for up to 9 min after treatment. We postulate that the increase of diacylglycerol content produced by dDAVP might be primarily from a phosphatidylcholine source and that the growth-promoting activity of desmopressin may be a consequence of activation of protein kinase C

Multifactorial resistance to adriamycin: relationship of DNA repair, glutathione transferase activity, drug efflux, and p-glycoprotein in cloned cell lines of adriamycin-sensitive and -resistant P388 Leukemia

Cloned lines of Adriamycin (ADR)-sensitive and -resistant P388 leukemia have been established, including P388/ADR/3 and P388/ADR/7 that are 5- and 10-fold more resistant than the cloned sensitive cell line P388/4 (Cancer Res., 46: 2978, 1986). A time course of ADR-induced DNA double-strand breaks revealed that in sensitive P388/4 cells, evidence of DNA repair was noted 4 h after removal of drug, whereas in resistant clone 3 and 7 cells repair was observed 1 h after drug removal. The earlier onset of DNA repair was statistically significant (p = 0.0154 for clone 3 cells, and p = 0.0009 for clone 7 cells). By contrast, once the repair process was initiated, the rate of repair was similar for all three cell lines. The level of glutathione transferase activity was determined in whole cell extracts. Enzyme activity (mean ± SE) in sensitive cells was 9.49 ± 1.00 nmol/min/mg protein, that in resistant clone 3 cells was 13.36 ± 1.03 nmol/min/mg, and that in clone 7 cells was 13.96 ± 1.44 nmol/min/mg; the 1.44-fold increase in enzyme activity in resistant cells was statistically significant (p = 0.01). Further evidence of induction of glutathione transferase was provided by Northern blot analysis using a 32P-labeled cDNA for an anionic glutathione transferase, which demonstrated approximately a twofold increase in mRNA in resistant clone 7 cells. Western blot analysis with a polyvalent antibody against anionic glutathione transferase also revealed a proportionate increase in gene product in resistant cells. Dose-survival studies showed that ADR-resistant cells were cross-resistant to actinomycin D, daunorubicin, mitoxantrone, colchicine, and etoposide, but not to the alkylating agent melphalan; this finding provided evidence that these cells are multidrug resistant. Using a cDNA probe for P-glycoprotein, a phenotypic marker for multidrug resistance, Northern blot analysis showed an increase in the steady state level of mRNA of approximately twofold in resistant clone 3 and 7 cells. Southern analysis with the same cDNA probe showed no evidence of gene amplification or rearrangement. Western blot analysis with monoclonal C219 antibody demonstrated a distinct increase in P-glycoprotein in resistant cells. Efflux of Adriamycin as measured by the efflux rate constant was identical in all three cell lines. Furthermore, the metabolic inhibitors azide and dinitrophenol did not augment drug uptake in either sensitive or resistant cells. These findings suggest that despite the increase in P-glycoprotein, an active extrusion pump was not operational in these cells. This and previous studies provide unequivocal evidence that resistance to Adriamycin is multifactorial. Decreased drug uptake, decreased formation of DNA single- and double-strand breaks, increased glutathione transferase activity, earlier onset of DNA repair, as well as elevated P-glycoprotein are all characteristic of multifactorial drug resistance

Trypanosoma rangeli Transcriptome Project: Generation and analysis of expressed sequence tags

Trypanosoma rangeli is an important hemoflagellate parasite of several mammalian species in Central and South America, sharing geographical areas, vectors and reservoirs with T. cruzi, the causative agent of Chagas disease. Thus, the occurrence of single and/or mixed infections, including in humans, must be expected and are of great importance for specific diagnosis and epidemiology. In comparison to several Trypanosomatidae species, the T. rangeli biology and genome are little known, reinforcing the needs of a gene discovery initiative. The T. rangeli transcriptome initiative aims to promote gene discovery through the generation of expressed sequence tags (ESTs) and Orestes (ORF ESTs) from both epimastigote and trypomastigote forms of the parasite, allowing further studies of the parasite biology, taxonomy and phylogeny

A high-throughput cloning system for reverse genetics in Trypanosoma cruzi

<p>Abstract</p> <p>Background</p> <p>The three trypanosomatids pathogenic to men, <it>Trypanosoma cruzi</it>, <it>Trypanosoma brucei </it>and <it>Leishmania major</it>, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.</p> <p>Results</p> <p>We constructed a platform with a flexible structure allowing the exchange of various elements, such as promoters, fusion tags, intergenic regions or resistance markers. This platform is based on Gateway^®technology, to ensure a fast and efficient cloning system. We obtained plasmid vectors carrying genes for fluorescent proteins (green, cyan or yellow), and sequences for the <it>c-myc </it>epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (<it>Tc</it>Rab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed.</p> <p>Conclusions</p> <p>We constructed plasmids with an efficient cloning system and suitable for use across various applications, such as protein localization and co-localization, protein partner identification and protein expression. This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements. The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.</p

The characterization of RNA-binding proteins and RNA metabolism-related proteins in fungal extracellular vesicles

RNA-binding proteins (RBPs) are essential for regulating RNA metabolism, stability, and translation within cells. Recent studies have shown that RBPs are not restricted to intracellular functions and can be found in extracellular vesicles (EVs) in different mammalian cells. EVs released by fungi contain a variety of proteins involved in RNA metabolism. These include RNA helicases, which play essential roles in RNA synthesis, folding, and degradation. Aminoacyl-tRNA synthetases, responsible for acetylating tRNA molecules, are also enriched in EVs, suggesting a possible link between these enzymes and tRNA fragments detected in EVs. Proteins with canonical RNA-binding domains interact with proteins and RNA, such as the RNA Recognition Motif (RRM), Zinc finger, and hnRNP K-homology (KH) domains. Polyadenylate-binding protein (PABP) plays a critical role in the regulation of gene expression by binding the poly(A) tail of messenger RNA (mRNA) and facilitating its translation, stability, and localization, making it a key factor in post-transcriptional control of gene expression. The presence of proteins related to the RNA life cycle in EVs from different fungal species suggests a conserved mechanism of EV cargo packing. Various models have been proposed for selecting RNA molecules for release into EVs. Still, the actual loading processes are unknown, and further molecular characterization of these proteins may provide insight into the mechanism of RNA sorting into EVs. This work reviews the current knowledge of RBPs and proteins related to RNA metabolism in EVs derived from distinct fungi species, and presents an analysis of proteomic datasets through GO term and orthology analysis, Our investigation identified orthologous proteins in fungal EVs on different fungal species